University of Groningen

Copper toxicity and sulfur metabolism in Chinese cabbageShahbaz, Muhammad

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COPPER TOXICITY AND SULFUR METABOLISM INCHINESE CABBAGE

The studies described in this thesis were carried out in the Laboratory of Plant Physiology, Faculty of Mathematics and Natural Sciences, University of Groningen, Nijenborgh 7, 9700 CC, Groningen, The Netherlands. The candidate was financially supported by a scholarship from the Higher Education Commission (HEC), Pakistan with the collaboration of Netherlands organisation for international coorporation in higher education (Nuffic)

Cover design: Dick Visser

Printed by: Grafimedia, Facilitair Bedrijf RUG

ISBN: 978-90-367-5094-3 (book)ISBN: 978-90-367-5093-6 (digital)

RIJKSUNIVERSITEIT GRONINGEN

COPPER TOXICITY AND SULFUR METABOLISM INCHINESE CABBAGE

Proefschrift

ter verkrijging van het doctoraat in deWiskunde en Natuurwetenschappenaan de Rijksuniversiteit Groningen

op gezag van deRector Magnificus, dr. E. Sterken,in het openbaar te verdedigen op

vrijdag 14 oktober 2011om 12:45 uur

door

Muhammad Shahbaz

geboren op 2 april 1981te Sahiwal, Pakistan

Promotor: prof. dr. J.T.M. Elzenga

Copromotor: dr. L.J. de Kok

Beoordelingscommissie: prof. dr. M.J. Hawkesfordprof. dr. S.H. Haneklausprof. dr. M. Tausz

In memory of my beloved mother and fatherand

dedicated to my brother Ashfaq Ahmad

Contents

Chapter 1. General introduction 9

Chapter 2. Material and methods 23

Chapter 3. Copper exposure interferes with the regulation ofthe uptake, distribution and metabolism of sulfate in Chinese cabbage

33

Chapter 4. UV radiation increases the toxicity and impact of copper on sulfur metabolism in Chinese cabbage

51

Chapter 5. Sulfate deprivation overrules the toxicity and impact of copper on sulfur metabolism in Chinese cabbage

71

Chapter 6. Atmospheric H2S nutrition hardly affects the toxicity and impact of copper on sulfur metabolism in Chinese cabbage

81

Chapter 7. General discussion 101

References 111

Summary 129

Samenvating (Summary in Dutch) 135

Khulasa (Summary in Urdu) 141

Acknowledgements 147

General introduction

Chapter 1.

Chapter 1

11

Copper (Cu) is an essential redox-active transition metal for normal plant growth and development and a cofactor in many metalloproteins (Kopsell and Kopsell 2007; Burkhead et al. 2009; Yreula 2005, 2009). Prior to its identification as a plant nutrient, Cu was used in agriculture for chemical weed control and as a fungicide (Kopsell and Kopsell 2007). Despite its essentiality, Cu is phytotoxic at enhanced concentrations (Kopsell and Kopsell 2007; Burkhead et al. 2009; Yruela 2005, 2009). The typical Cu content in plants ranges from 0.08 to 0.24 µmol g-1 dry weight, and Cu toxicity in cabbage generally occurs when the leaf tissue level exceeds 0.4 µmol g-1 dry weight (Macnicol and Beckett 1985; Krämer 2010).

The Cu levels in agricultural soil may strongly be enhanced as a consequence of anthropogenic activities through the application of organic fertilizers, use of sewage sludge as a fertilizer, application of sewage water for crop production and the use of Cu-containing fungicides (Dach and Starmans 2005; Zhou et al. 2005; Yruela 2005, 2009). The Cu content in pig slurries is 10-40 times higher than in soil, and the amount of copper excreted via feces corresponds to 72-80% of the amount ingested by the animals (Mantovi et al. 2003).

Table 1. Input and output of heavy metals for arable land in the Netherlands in 1980 and 2003 (adapted from Dach and Starmans 2005). Values in 105

moles.

Total inputBy animalmanure

By mineralfertilizers

Bydeposition

Othersources

Total output

1980 2003 1980 2003 1980 2003 1980 2003 1980 2003 1980 2003

Cu 214 78 165 67 24 6 13 3 13 2 22 15Zn 367 232 275 191 23 8 43 12 29 21 107 86Cd 1.42 0.45 0.53 0.27 0.27 0.09 0.18 0.09 0.62 0 0.27 0.27

In the Netherlands arable soil contains high levels of Cu and other heavy metals as the consequence of intensive agriculture and the use of animal manure as organic fertilizers. Currently, the input of Cu and other heavy metals for arable land in the Netherlands still exceeds the output, despite strict regulatory legislation, which has resulted in a strongly decreased input as compared to that at the beginning of the eighties (Table 1).

Cu and other heavy metals are not very mobile in most of the soils, which is quite alarming, since presumably it will take several

12

hundreds or even thousands of years for the natural re-equilibration of heavy metals. For the Netherlands it has been estimated that the natural re-equilibration of Cd and Zn will only be reached after 100-1,000 years, whereas that of Pb and Cu may even take 3,000 and 4,000 years, respectively (Dach and Starmans 2005).

Table 2. Heavy metal concentration (µmol kg-1 dry weight) in harvested vegetables irregated with canal or sewage water in the vicinity of Faisalabad, Pakistan (Butt et al. 2005).

In developing countries Brassica and other vegetable crops are often grown in the vicinity of big cities and industrial areas, where they may be subjected to air and heavy metals pollution (Yang et al. 2006). The latter may originate from sewage, which is directly applied to vegetable crops. Sewage is not only the source of many nutrients,but it is often contaminated with high levels of Cu and other heavy metals. As a consequence of untreated sewage application, heavy metals not only accumulate in the soil but also in vegetables (Table 2; Younas et al. 1998; Butt et al. 2005). Enhanced Cu levels in crop plants might not only negatively affect plant growth and functioning,but will also enter the food chain (Brun et al. 2001).

Uptake and distribution of Cu in plants

Copper is taken up by plants with the lowest rate among all the essential elements (Kabata-Pendias and Pendiase 1992; Kopsell and Kopsell 2007). In the soil, Cu can be inaccessible due to its tendency to be present predominantly in an insoluble form because of its

Chapter 1

13

adsorption to clay, CaCO3 or organic matter and high pH (McBride et al. 1997; Sauvé et al. 1997; Kopsell and Kopsell 2007; Burkhead et al. 2009, Yruela 2005, 2009; Palmer and Guerinot 2009). Plants may mobilize essential metals by the secretion of chelators by the root and/or by acidification of the rhizosphere (Clemens et al. 2002). Cu is found predominantly as Cu2+ in the soil, which might be reduced by FRO2 (ferric reductase oxidase), prior to its uptake by high affinity COPT transporters (Robinson et al. 1999; Palmer and Guerinot 2009; Fig. 1).

Fig. 1. Scheme of Cu uptake and distribution in plant cells. Copper proteins are indicated in open rectangles; (CSD1, cytosolic Cu/Zn superoxide dismutase; CSD2, chloroplastic Cu/Zn superoxide dismutase; CSD3, peroxisomal Cu/Zn superoxide dismutase; ER, endoplasmic reticulum), transporters in light gray rectangles (COPT, Cu transporter; HMA, heavy metal P-type ATPase; RAN1, responsive-to-antagonist 1; PAA, P-type ATPase of Arabidopsis; ZIP, Zn transporters), Cu-metallochaperones in closed rectangles (CCH, Cu chaperone; ATX1, antioxidant 1; CCS, Cu chaperone for Cu/Zn superoxide dismutase; COX, cytochrome-c oxidase; SCO, synthesis of cytochrome-c oxidase), FRO (Fe(III) reductase oxidase) and possible metal-binding compounds, viz. metallothioneins (MTs) and phytochelatins (PCs). Adapted from Pilon et al. (2006) and Yruela (2009).

14

The uptake and transport of Cu in plants and its subcellular distribution and homeostasis are mediated by high affinity Cu transporter proteins (COPT transporters), other metallotransporter proteins (e.g. Cu-transporting P-type ATPases) and metallochaperone proteins (ATX, CCS and COX17 like Cu chaperone; Sancenon et al. 2003, 2004; Yruela 2005, 2009; Burkhead et al. 2009; Palmer and Guerinot 2009; Fig. 1). Plants may also take up Cu as the more abundant Cu2+ via a member of the ZIP family, a transporter family known to preferentially transport divalent cations. Both ZIP2 and ZIP4 are upregulated upon Cu deficiency (Clemens 2001; Wintz et al. 2003; Palmer and Guerinot 2009). Cu is removed from the cytosol by the HMA5 transporter (Andrés-Colás et al 2006) and excessive metal ions may complex with viz. glutathione (GSH), phytochelatins (PCs) and metallothioneins (MTs) and may be sequestered in the vacuole (Clemens et al. 2002; Pilon et al. 2006; Yruela 2009; Fig. 1).

Cu has limited mobility in plants and excessive Cu taken up is often found in the root tissue (Brun et al. 2001; Quartacci et al. 2003; Kopsell and Kopsell 2007; Guerra et al. 2009; Andrade et al. 2010). Excessive Cu taken up by roots is bound to the cell walls due to its affinity to carbonylic, carboxylic, phenolic and sulfydryl groups, ionic bonds with negatively charged sites and N, O and S bonds in cell membranes (Quartacci et al. 2003; Kopsell and Kopsell 2007). The translocation of Cu to above-ground parts seems to be well regulated, as it is not correlated to the Cu concentration in the root environment (Ginocchio et al. 2002; Kopsell and Kopsell 2007).

Functions and toxicity of Cu in plants

Copper is essential for plant growth and activity of many enzymes, with a requirement amongst the lowest of all elements (Kopsell and Kopsell, 2007). Cu is an integral component of photosynthetic electron transport (plastocyanin), mitochondrial respiration (cytochrome c oxidase), cell wall metabolism (amine oxidase), hormone signaling (ethylene receptors), thylakoids (polyphenol oxidase) and reactive oxygen metabolism (superoxide dismutase; Clemens 2001; Hall and Williams 2003; Yruela 2005, 2009; Burkhead et al. 2009). The reversible oxidation-reduction of Cu makes it very valuable as a cofactor in enzymes such as Cu/Zn-superoxide

Chapter 1

15

dismutase (Cu/ZnSOD), cytochrome c oxidase, ascorbate oxidase, amino oxidase, laccase, plastocyanin (PC), and polyphenol oxidase (Yruela 2005, 2009; Pilon et al. 2006).

Plants like all other organisms possess homeostatic mechanisms to maintain the correct concentration of essential metal ions in different cellular compartments and to minimize the damage from exposure to enhanced metal ions (Clemens 2001; Palmer and Guerinot 2009). Once inside the cell, Cu has to be distributed in proper amounts to cellular compartments and to proteins. Enhanced levels have to be rendered innocuous either by timely secretion, compartmentation and/or complexation in order to avoid their interference with the normal cellular metabolism (Hall 2002; Palmer and Guerinot 2009).

The toxic effects of Cu depend on the concentration of Cu accumulated, growth stage of plants and the duration of the exposure(Clemens 2001; Medoza-Cózatl et al. 2005). In living organisms the redox-active metal Cu is present in both the Cu2+ and Cu+ form. However, excessive free Cu ions may catalyze the formation of the highly toxic hydroxyl radicals (OH.) from hydrogen peroxide (H2O2) or superoxide anions (O2

-) via the Haber-Weiss reaction, which may induce oxidative stress and cause lipid peroxidation, proteindenaturation and DNA mutation (Pinto et al. 2003; Morelli and Scarano 2004). Excessive ionic Cu is phytotoxic and generally results in stunted root growth and shoot development and in leaf chlorosis (Yruela 2005; Kopsell and Kopsell 2007). The phytotoxicity of Cu is generally ascribed to its possible reaction with thiol groups of proteins and glutathione (De Vos et al. 1993; Yruela 2009). Cu toxicity may also be due to a reduced and/or altered uptake and distribution of other essential nutrients (Schiavon et al. 2007), e.g. a decrease in Feuptake essential in protein functioning (Pätsikkä et al. 1998, 2002; Kopsell and Kopsell 2007; Yruela 2009). Enhanced levels of Cu may injure the plasma membrane, resulting ion leakage from roots (Hall 2002). Moreover, Cu may hinder the biosynthesis of photosynthetic apparatus, resulting in an altered pigment and protein composition of photosynthetic membranes. It has been observed that especially photosystem II is a sensitive site to enhanced Cu levels (Barón et al. 1995; Yruela 2005, 2009).

16

Sulfate uptake and metabolism in plants

Plants depend on their roots for the uptake of sulfur and other nutrients. Generally sulfate taken up by the roots is the primary sulfur source for plant growth. The sulfate uptake by the roots is energy dependent (driven by a proton gradient generated by ATPases) through a proton/sulfate (presumably 3H+/SO4

2-) co-transport and the sulfate transporter-mediated uptake is likely one of the primary regulatory sites of sulfur metabolism (De Kok et al. 2002; Saito 2004; Hawkesford and De Kok 2006). Distinct sulfate transporter proteins mediate the uptake, transport and sub-cellular distribution of sulfate and their expression and activity are highly modulated by the sulfur status of the plant (Glass et al. 2002; Hawkesford and De Kok 2006; Koralewska et al. 2007, 2008, 2010, 2011; De Kok et al. 2011; Fig. 3).

Sulfate needs to be reduced prior to its metabolism into organic sulfur compounds (Fig. 2). The reduction of sulfur to sulfide may take place in the plastids in the root as well as in the chloroplasts in the shoot, since both root plastids and chloroplasts contain all sulfate reduction enzymes (De Kok et al. 2002, 2011; Hawkesford and De Kok 2006). However, on basis of the shoot to root partitioning in herbaceous species the main proportion of sulfate will be reduced in the shoot. Sulfate needs to be activated to adenosine 5'-phosphosulfate (APS) prior to its reduction to sulfite by ATP sulfurylase (Fig. 2). APS is subsequently reduced to sulfite by APS reductase with likely glutathione as a reductant (Leustek and Saito 1999; Kopriva and Koprivova 2003; Saito 2004).

Sulfite is reduced with high affinity by sulfite reductase to sulfide with ferredoxin as a reductant. Sulfide is incorporated into cysteine by O-acetylserine(thiol)lyase, with O-acetylserine as substrate. O-acetylserine is synthesized by serine acetyltransferase and together with O-acetylserine(thiol)lyase it is associated as enzyme complex called cysteine synthase (Hell 2003; Saito 2004). The formation of cysteine is a direct coupling step between sulfur and nitrogen assimilation in plants, and it is the precursor of reduced sulfur andsulfur donor for most other organic sulfur compounds present in plant tissue (Brunold 1993; De Kok et al. 2002).

Chapter 1

17

Fig. 2. Sulfate reduction and assimilation, and metabolism of H2S in plants. APS reductase, adenosine 5’-phosphosulfate reductase; Fdred, Fdox, reduced and oxidized ferredoxin; GSH, GSSG, reduced and oxidized glutathione (adapted from De Kok et al. 2007).

Different sulfate transporters are involved in the uptake and distribution of sulfate in plants. According to their cellular and subcellular expression and possible functioning, the sulfate transporters gene family has been classified in up to 5 different

18

groups (Buchner et al. 2004; Saito 2004; Hawkesford and De Kok 2006; Koralewska et al. 2007, 2008, 2009a, b, 2010; Fig. 3). Different isoforms are specifically expressed in the root or in the shoot or expressed both in root and shoot. The Group 1 transporters are 'high affinity sulfate transporters' (Km 10 μM), which are involved in the uptake of sulfate by the roots. The Group 2 transporters are vascular transporters and are 'low affinity sulfate transporters'. The role of Group 3 transporters is not well known yet. The Group 4 transporters may be involved in the transport of sulfate from the vacuole (Kataoka et al. 2004; Hawkesford 2007, 2008). The Group 5 transporters is a distinct group and one of the isoforms (Sultr5;2) in Arabidopsis is characterized as a molybdenum transporter (Tomatsu et al. 2007; Baxter et al. 2008). It is still unresolved, whether sulfate itself or metabolic products of the sulfur assimilation (viz. O-acetylserine, cysteine, glutathione) act as signals in the regulation of the expression and activity of the sulfate transporters (Buchner et al. 2004; Hawkesford and De Kok 2006; Koralewska et al. 2007, 2008, 2009a, b, 2010).

The majority of genes encoding the enzymes of sulfate assimilation, except sulfite reductase, are present in plants in multiple isoforms (Kopriva and Koprivova 2003; Takahashi et al. 2011). The in situ sulfate concentration in the plastid/chloroplast might be the limiting/regulatory step in the sulfur reduction pathway, since the affinity of ATP sulfurylase for sulfate is rather low (Km approximately 1 mM; Stulen and De Kok 1993). Regulation of the activity of APS reductase is presumably the primary regulation point in the sulfate reduction, since the activity of APS reductase is the lowest of the enzymes of the sulfate reduction pathway and it has a fast turnover rate (Brunold 1993; Leustek and Saito 1999; Kopriva and Koprivova 2003; Saito 2004). The sulfate fraction, which is not directly reduced, is present in the vacuoles. The rate of remobilization of sulfate from the vacuole may be rather slow and sulfur-deficient plants often contain detectable levels of sulfate (Davidian et al. 2000; Hawkesford and Wray 2000; Buchner et al. 2004).

Chapter 1

19

Fig. 3. Phylogenetic neighbour-joining tree of the coding cDNAs of the Arabidopsis and Brassica oleracea sulfate transporter family (adapted from Buchner et al. 2004).

In addition to sulfate taken up by the roots, plants are able to utilize foliarly absorbed sulfurous air pollutants (viz. SO2, H2S) as a sulfur source for growth. It has been well established that foliarly absorbed H2S is directly metabolized with high affinity into cysteine

20

and subsequently into other organic sulfur compounds (De Kok et al. 1998, 2002, 2007, 2009, 2011). There is a clear evidence for interaction between atmospheric and pedospheric sulfur utilization, as H2S exposure resulted in downregulation of the uptake and assimilation of sulfate (Westerman et al. 2000, 2001; Buchner et al. 2004; De Kok et al. 2007; Koralewska et al. 2008).

Interaction of Cu with the sulfur metabolism

Glutathione (GSH) is the predominant water-soluble non-protein thiol compound present is plant tissue and its concentration may be as high as 10 mM, though it varies with plant development and plant nutrition (Leustek et al. 2000). GSH is synthesized in both the cytosol and chloroplast and its level and redox state (GSH/GSSG ratio) may be affected by environmental conditions, e.g. temperature, drought, salinity, air pollution, irradiation (Tausz 2001). Moreover, the level of glutathione and other non-protein thiols increased upon exposure to heavy metals (Nocito et al. 2002, 2006; Sun et al. 2007). It has been observed that enhanced subcellular Cu levels (and other heavy metals) may induce the synthesis of phytochelatins (PCs), which are derived from glutathione (and in some plant species from related compounds, such as homo-glutathione; Cobbett 2003; Verkleij et al. 2003). PCs are synthesized enzymatically by inducible -glutamylcysteine dipeptidyltranspeptidases (PC synthases) with GSH as substrate:

(GluCys)nGly + (GluCys)nGly (GluCys)n+1Gly + (GluCys)n-1Gly

The number of -glutamylcysteine residues (GluCys)n in the phytochelatins is reported to be as high as 11, but normally in the range from 2-5. The induction of PCs synthesis upon Cu exposure is a rapid process (Grill et al. 1987; Zenk 1996). The length of PCs chain shows metal-specific differences with Cu2+ favoring PC2 (Ernst et al. 2008). Phytochelatins may complex with Cu, and together with cysteine-rich metallothionein proteins (at least in some plant species)they may buffer the Cu concentration in the cytosol (De Vos et al. 1992; Rauser 2001; Cobbett and Goldsbrough 2002; Ernst et al. 2008; Burkhead et al. 2009). However, the significance of

Chapter 1

21

phytochelatins in Cu detoxification appears to be restricted (De Vos et al. 1992; Hall 2002; Schat et al. 2002; Cobbett 2003; Cobbett and Goldsbrough 2002; Verkleij et al. 2003; Lee and Kang 2005; Ernst et al. 2008; Yruela 2005, 2009).

In addition to the sulfur-rich PCs also GSH itself (or even free cysteine), metallothionins and low molecular weight cysteine-rich proteins may be involved in Cu binding and detoxification in plants, which would imply a direct interference with sulfur metabolism (Cobbett and Goldsbrough 2002; Cobbett 2003; Verkleij et al. 2003; Lee and Kang 2005; Clemens and Peršoh 2009; Sirko and Gotor 2007). There is evidence that sulfur nutrition and its metabolism may be involved in tolerance and detoxification of heavy metals (Nocito et al. 2006; Sirko and Gotor 2007; Sun et al. 2007).

The uptake of sulfate by the root and its assimilation are modulated by both the plant sulfur status and the sulfur demand for growth and is affected by environmental conditions (Hawkesford and De Kok 2006; Haneklaus et al. 2007; De Kok et al. 2011). Synthesis of PCs, metallothionins and glutathione upon heavy metal exposure might require an increased sulfate uptake and assimilation (Anderson and McMahon 2001; Nocito et al. 2006; Sun et al. 2007; Ernst et al. 2008). For instance, Cd exposure induced an upregulation of expression and activity of the sulfate transporters and several enzymes of the sulfur assimilation pathway and glutathione synthesis, viz. ATP sulfurylase, APS sulfotransferase, -glutamylcysteine synthetase and glutathione synthetase (Anderson and McMahon 2001). Exposure of plants to enhanced levels of heavy metals (e.g. Cd, Cu) resulted in an enhanced expression and activity of high affinity sulfate transporters (Sultr1;1, Sultr1;2; Nocito et al. 2006; Sun et al. 2007). Moreover, the expression of Sultr4;1, which is involved in the efflux of sulfate from the vacuole was upregulated upon exposure to Cu (Schiavon et al. 2007).

Aim and outline of the thesis

Anthropogenic activities have resulted in a widespread pollution of soils with Cu and other heavy metals. Cu and other heavy metal pollution will presumably further increase due to an increasing global

22

population and the subsequent intensification of plant and animal production (Dach and Starmans 2005). Enhanced Cu levels in plants may interact with and disturb metabolism in several ways and sulfur metabolites might have potential in its detoxification. The general aim of this project was to get more insight in the physiological basis of the phytotoxicity of Cu and the significance of sulfur metabolism in itsdetoxification. In this thesis the followings topics were investigated: і)the impact of enhanced Cu concentrations on physiological functioning and its interaction with the regulation of uptake, distribution and metabolism of sulfate, іі) the impact of light quality (UV radiation) on Cu toxicity, iii) the interaction between enhanced Cu2+ levels and sulfate deprivation, iv) the significance of pedospheric (sulfate) andatmospheric (H2S) sulfur nutrition in Cu detoxification.

Chinese cabbage [Brassica pekinensis (Lour.) Rupr.] belongs to the Brassicaceae (the mustard family; Schranz et al. 2006). Brassicaspecies are characterized by a high sulfur requirement for growth, and the impact of sulfur nutrition on its physiology has extensively been studied (De Kok et al. 2000; Buchner et al. 2004; Koralewska et al. 2007, 2008, 2009a, b, 2011; Parmar et al. 2007; Stuiver et al. 2009). Exceptionally, plant species belonging to the family Brassicaceae are not able to evolve Cu tolerance on Cu-enriched soils (Walker and Bernal 2004).

Chapter 2 describes the material and methods used in the present study. In Chapter 3 the interference of Cu exposure with the regulation of the uptake, distribution and metabolism of sulfate, and the mineral nutrient composition was studied. In Chapter 4 the impact of UV radiation on Cu toxicity and sulfur metabolism was investigated. In Chapter 5 the impact of enhanced Cu concentrations and sulfate deprivation on the uptake and metabolism of sulfate was studied. In Chapter 6 the significance of pedospheric (sulfate) and atmospheric sulfur nutrition (H2S) on Cu toxicity was evaluated. In Chapter 7 the phytotoxicity of enhanced Cu concentrations and possible significance of sulfur metabolism in its detoxification in Chinese cabbage is discussed.

Material and methods

Chapter 2.

Chapter 2

25

Plant material and growth conditions

Chinese cabbage (Brassica pekinensis (Lour.) Rupr. cv. Kasumi F1 (Nickerson-Zwaan, Made, The Netherlands) was used in the present studies. Seeds were germinated in vermiculite in a climate controlled room. Day and night temperatures were 22 and 18°C, respectively, relative humidity was 60-70% and the photoperiod was 14 h at a photon fluence rate of 300 10 μmol m–2 s–1 (within the 400-700 nm range) at plant height, supplied by Philips Master 58W/83 and 84 fluorescent tubes in a ratio of 1:1.

10–14 day-old seedlings were transferred to an aerated 25% Hoagland nutrient solution, pH 5.9-6.0, consisting of 1.25 mM Ca(NO3)2.4H2O, 1.25 mM KNO3, 0.25 mM KH2PO4, 0.5 mM MgSO4.7H2O, 11.6 μM H3BO3, 2.4 μM MnCl2.4H2O, 0.24 μM ZnSO4.7H2O, 0.08 μM CuSO4.5H2O, 0.13 μM Na2MoO4.2H2O and 22.5 μM Fe3+-EDTA, containing supplemental CuCl2 concentrations ranging from 1-15 μM in 13 l stainless steel containers (10 sets of plants per container, 3 plants per set) or in 30 l containers (20 sets of plants per container, 3 plants per set) in a climate-controlled room. For the experiments on sulfate deprivation all sulfate salts were replaced by chloride salts.

H2S fumigation

Two 13 l containers (10 sets of plants per container, 3 plants per set)were placed in a 150 l cylindrical stainless steel cabinets (0.6 m diameter) with a poly(methyl methacrylate) top. Day and night temperatures were 22 and 18 °C ( 1 °C), respectively, and the relative humidity was 60-70%. The photoperiod was 14 h at a photon fluence rate of 320 20 μmol m–2 s–1 (within the 400-700 nm range) at a UV radiation level of 1.0 mW cm-2 (UV-A+B; within the 280-400 nm range) at plant height, supplied by Philips HPL(R)N (400 W) lamps as light source. The temperature was controlled by adjusting the cabinet wall temperature; the air exchange was 40 l min-1 and the air inside the cabinets was stirred continuously by two ventilators. Pressurized H2S diluted with N2 (1 ml l-1) was injected into the incoming air stream and adjusted to the desired level by ASM electronic mass flow controllers (Bilthoven, The Netherlands). H2S

26

levels in the cabinets were controlled by an SO2 analyzer (model 9850) equipped with a H2S converter (model 8770, Monitor Labs, Measurement Controls Corporation, Englewood, CO, USA). Sealing of the lid of the containers and plant sets prevented the absorption of atmospheric H2S by the solution. UV-A+B radiation was measured with a Digital Ultraviolet Radiometer model 5.0 (Solartech Inc. Fenton, MI 48430, USA).

Plant harvest

Plants were harvested 3 h after start of the light period, and for anion determination, roots were rinsed in ice-cold de-mineralized water (for 3 x 20 s) in order to remove sulfate from the free space (Kronzucker et al. 1995). Roots were separated from the shoots, weighed, and for pigment, phytochelatins and RNA isolation plant material was frozen immediately in liquid N2 and stored at -80 °C until further use. For analysis of the water-soluble non-protein thiol content freshly harvested plant material was used. For determination of dry matter, total sulfur, nitrogen and Cu content, plant tissue was dried at 80 °C for 48 h. For data on dry matter, total sulfur and Cu content,presented in Chapter 6, plant material was freeze dried at -60 °C for 48-72 h.

Pigment content

Shoots or leaf discs were homogenized in 100% acetone using an Ultra Turrax (10 ml per g fresh weight) and centrifuged at 800 g for 20 minutes. The chlorophyll a, b and total carotenoids content were determined according to Lichtenthaler (1987).

Chlorophyll a fluorescence

Chlorophyll a fluorescence was measured on the adaxial side of the youngest fully developed leaves using a modulated fluorometer (PAM 2000, H. Walz GmbH, Effeltrich, Germany). Measurements were performed on intact plants in a dark room under dim green light at

Chapter 2

27

room temperature after dark adaptation for at least 30 min. The maximum chlorophyll a fluorescence level of dark-adapted leaves (Fm) was determined using a saturating flash of white light during 1 s at a photon fluence rate of 4,000 µmol photons m-2 s-1. The initial chlorophyll a fluorescence level of dark-adapted leaves (F0) was sensitized with red light at a photon fluence rate of about 0.1 µmol m-2 s-1 at a modulation of 1.6 kHz. The quantum yield of photosystem II photochemistry (Fv/Fm) was calculated from the ratio of variable (Fv

= Fm - F0) to maximum chlorophyll a fluorescence.

Specific leaf area

Leaf area was measured by sealing the leaves in plastic sheets. The projected leaf area was measured by scanning and leaf area was determined by ImageJ (see: http://rsb.info.nih.gov/ij/). The specific leaf area ratio (cm-2 g-1 FW) was calculated.

Photosynthesis and dark respiration

Net photosynthesis and dark respiration rates were recorded in the climate room using a portable, open-flow gas exchange analyzer (TPS-2, PP systems Inc., Amesbury, MA, USA). Measurements were taken of a 2.5 cm2 area of the youngest fully expanded leaf attached to the plant. The flow rate of air through the leaf chamber was set at300 ml min–1 and the measurement conditions were similar to the ambient growth conditions. The photosynthesis rate was measured at a photon fluence rate of 340 20 µmol m–2 s–1 (within the 400-700 nm range). Dark respiration was measured after dark adaptation for at least 10 min.

Cu and mineral nutrient content

Dried plant material was pulverized by a Retsch Mixer-Mill (type MM2; Haan, Germany) and Cu and mineral nutrient content were analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES). 5 ml of a 85:15 (v/v) mixture of nitric acid (Primar, Aristar s.g

28

1.42, 70%) and perchloric acid (Aristar/Primar, 70%) were added to 250 mg of oven dried plant material and allowed to stand at room temperature for at least two hours. Samples were digested in a heating block (Carbolite, London, UK) for 3 h at 60 °C, 1 h at 100 °C, 1 hour at 120 °C and 2.5 h at 200 °C. Temperatures were ramped at either 1 or 2 °C min-1 for the first and last or 2nd and 3rd periods. Subsequently 5 ml of 9.25% (v/v) HCl (AR grade) was added to the dried samples and heated for 1 h at 80 °C. After a further addition of 20 ml of water, the samples were heated for 30 min at 80 °C. Finally,extract were made up to 25 ml with water and subjected to ICP-OES (Applied Research Laboratories, Vallaire, Ecublens, Switzerland).

Total nitrogen and sulfur content

The total nitrogen content was determined with a modified Kjeldahl method (Barneix et al. 1988). 50 mg of dried, pulverized plant tissue (by a Retsch Mixer-Mill, type MM2; Haan, Germany) was extracted in 2 ml extraction solution (33.3 g Na salicylate l-1 concentrated H2SO4) in presence of 0.33 g Na2S2O3.5H2O and a small amount of catalyst (K2SO4:CuSO4:Na2SeO3, 15:5:0.085 (w/w/w) folded in a small ash free filter until the samples were completely clear. The samples were made up to 50 ml with de-mineralized water and NH4

+ was measured colorimetrically at 410 nm after reaction with Nessler’s reagent.

The total sulfur content was determined according to a modification of the method of Jones (1995). Dried plant tissue was pulverized and drop by drop, a 50% Mg(NO3)2.6H2O (w/v) solution was added to 100-250 mg dry material just to saturate the tissue and the samples were dried overnight in an oven at 100 °C. Subsequently, the trays were covered with lids and the samples were ashed in an oven (Carbolite BWF 11/13, Hope Valley, England) at 650 °C until the residue had become completely white. The residue was dissolved in 10 ml 20% aqua regia (50 ml conc. HNO3 and 150 ml conc. HCl in 1 l distilled water), quantitatively transferred to a measuring flask and added up to 50 or 100 ml with de-mineralized water. To 10 or 25 ml of extract, one SulfaVer®4 Reagent Powder Pillow (HACH, Permachem® reagents, Loveland, USA) containing BaCl2 was added, and the turbidity was measured with a spectrophotometer (HACH DR/400V, Loveland, USA) at 450 nm.

Chapter 2

29

Nitrate and sulfate content

Frozen material was homogenized in de-mineralized water (10 ml per g fresh weight) with an Ultra Turrax for 30 s (0 °C), the homogenate was filtered through one layer of Miracloth and incubated at 100 °C in a boiling water bath for 10 min. The filtrate was centrifuged at 30,000 g for 15 min (0 °C). The anions were separated by HPLC on an IonoSpher A anion exchange column (250 x 4.6 mm; Varian Benelux, Bergen op Zoom, The Netherlands) and determined refractometrically according to Maas et al. (1986) using a Knauer differential refractometer (model 98.00, Bad Homburg, Germany). 25 mM potassium biphthalate (pH 4.3) containing 0.02% NaN3 (w/v) was used as a mobile phase. The HPLC apparatus consisted of a Separations high precision pump (model 300; H.I. Ambacht, The Netherlands) provided with a Rheodyne sample injector (model 7175; loop volume 20 l; Cotati, CA). The flow rate was 1 ml min-1 and the detector temperature was kept at 25 °C by a water bath. Peak analysis was performed with a Shimadzu Chromatopac C-R8A data processor (Kyoto, Japan).

Amino acid content

The amino acids were extracted as described for nitrate and sulfate,and after deproteinization of the extracts, its content was determined with the ninhydrin color reagent according to Rosen (1957) and measured colorimetrically at 578 nm.

Water-soluble non-protein thiols

For analysis of the water-soluble non-protein thiols, fresh plant material was homogenized in an extraction medium containing 80 mM sulfosalicylic acid, 1 mM EDTA, and 0.15% (w/v) ascorbic acid with an Ultra Turrax at 0 °C (10 ml per g fresh weight). Oxygen was removed from the solution by aspiration with N2. The homogenate was filtered through one layer of Miracloth and the filtrate was centrifuged at

30

30,000 g for 15 min (0 °C). Total water-soluble non-protein thiol content was determined colorimetrically at 413 nm after reaction with 5,5'-dithiobis[2-nitrobenzoic acid] according to De Kok et al. (1988).

Phytochelatins

Frozen plant material was dried in a freeze dryer (Heto Lyolab 3000; Holten, The Netherlands) for 48-72 hours. The freeze-dried plant material was pulverized by a Retsch Mixer-Mill (type MM2; Haan, Germany) and phytochelatins were analyzed according to Wojas et al. (2008).

Sulfate and nitrate uptake and sulfate uptake capacity

For measurement of sulfate and nitrate uptake, seedlings were grown on a 25% Hoagland solution at various CuCl2 concentrations, and subsequently transferred to pots containing 1 l freshly prepared, aerated 25% Hoagland solution in presence of CuCl2 in a climate-controlled room, for conditions see above. At the beginning of the uptake measurement and after 24 h, aliquots were taken from the nutrient solution (adjusted to the original volume with de-mineralized water by weighing) and analyzed for sulfate and nitrate content by HPLC. Sulfate uptake capacity was determined as described by Koralewska et al. (2007). Three sets of plants (3 plants per set) per treatment were transferred to a 25% Hoagland solution labeled with 35S-sulfate (2 MBq l-1) and incubated for 30 min at 25 °C at at a photon fluence rate of 300 20 μmol m–2 s–1 (within the 400-700 nm range). Subsequently, plants were removed and roots rinsed in ice-cold non-labeled nutrient solution (3 x 20 s). Roots and shoots were separated and digested in 1 N HCl at room temperature for 7 days. The extracts were filtered through one layer of Miracloth (Calbiochem Corporation, La Jolla, CA, USA) and 100 μl of the filtrate was mixed with 1 ml Emulsifier Scintillator Plus (Perkin Elmer, Boston, MA, USA). Radioactivity was measured with a liquid scintillation counter (TRI-CARB 2,000 CA Liquid Scintillation Analyzer, Perkin Elmer, Waltham, MA, USA).

Chapter 2

31

Total RNA isolation

Total RNA from root and shoot was isolated by using TRI REAGENTTM

(Sigma), a mixture of guanidine thiocyanate and phenol in a mono-phase solution, which involved an additional phenol-chloroform-isoamyl alcohol extraction of the aqueous phase after the first centrifugation (Verwoerd et al. 1989). The final air-dried pellet was dissolved in an appropriate volume of diethyl pyrocarbonate-treated water. The quality of the RNA samples was checked by electrophoresis of a 2 µg aliquot on a 1% (w/v) Tris-acetate/agarosegel. The RNA concentration was measured at 260 nm using a NanoDrop ND1000 UV/VIS Spectrophotometer (Wilmington, USA).

Northern analysis

Northern hybridization of the sulfate transporters and APS reductasetranscripts was performed according to Church and Gilbert (1984),with pre-hybridization/hybridization at 65 and 60 °C, respectively. 10 μg of total RNA per slot was separated on an agarose/formaldehydegel and blotted onto a positively charged nylon membrane (Hybond-N+). Sequence diversity, especially in the 3' non-coding region, allowed the use of partial cDNA fragments for gene-specifichybridization to the respective Brassica sulfate transporter mRNA (Buchner et al. 2004). For APS reductase, a cDNA fragment of APS reductase 2 (APR2) from Arabidopsis thaliana (L.) Heynh was used as a probe (Durenkamp et al. 2007). cDNA fragments were labeled with 32P-dCTP and used as hybridization probes. After hybridization with probes for sulfate transporters membranes were washed at 65 °Ctwice with 2 x SSC, 0.1% SDS for 5 and 30 min, once with 1 x SSC, 0.1% SDS and twice with 0.1 x SSC, 0.1% SDS for at least 30 min each, and exposed to a Cyclone MultiPurpose Phosphor Screen (Perkin Elmer, U.K.). After hybridization with a probe specific for APS reductase, membranes were washed at 60 °C twice with 2 x SSC, 0.1% SDS for 10 min and twice with 1 x SSC, 0.1% SDS for 15 min, and exposed to a Cyclone MultiPurpose Phosphor Screen (Perkin Elmer, U.K.). The signal was visualized and the relative expression estimated by using OptiQuant Acquisition and Analysis (Packard

32

Instrument Co.) software in case of exposure to a Cyclone MultiPurpose Phosphor Screen (Perkin Elmer, U.K.).

Statistics

For the statistical analysis of the data an unpaired Student’s t-test (p ≤ 0.01) was used. Further details on the number of experiments, measurements per experiment and number of plants are given in the legends of the tables and figures.

Copper exposure interferes with theregulation of the uptake, distribution andmetabolism of sulfate in Chinese cabbage

Chapter 3.

Chapter 3

35

Abstract

Exposure of Chinese cabbage (Brassica pekinensis) to enhanced Cu2+

concentrations (1-10 μM) resulted in leaf chlorosis, a loss of photosynthetic capacity and lower biomass production at ≥ 5 µM. The decrease in pigment content was likely not the consequence of degradation, but due to a hindered chloroplast development upon Cu exposure. The Cu content of the root increased with the Cu2+

concentration (up to 40-fold), though only a minor proportion (4%) of it was transferred to the shoot. Enhanced Cu2+ levels substantially affected the overall mineral nutrient composition at ≥ 5 µM Cu2+. The nitrate uptake by the root was substantially reduced at ≥ 5 µM Cu2+. The nitrogen content of the root was affected little at lower Cu2+

levels, whereas that in the shoot was decreased at ≥ 5 µM Cu2+. Cu affected the uptake, distribution and metabolism of sulfate in Chinese cabbage. The total sulfur content of the shoot was increased at ≥ 2 µM Cu2+, which could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in water-soluble non-protein thiol content in the root and to a lesser extent in the shoot at ≥ 1 µM, which could only partially be ascribed to a Cu-induced enhancement of the phytochelatin content. The nitrate uptake by the root was substantially reduced at ≥ 5 µM Cu2+, coinciding with the decrease in biomass production. However, the activity of the sulfate transporters in the root was slightly enhanced at 2 and 5 µM Cu2+, accompanied by an enhanced expression of the Group 1 high affinity transporter Sultr1;2, and the Group 4 transporters Sultr4;1 and Sultr4;2. In the shoot there was an induction of expression of Sultr4;2 at 5 and 10 µM Cu2+. The expression of APS reductase was affected little in the root and shoot up to 10 µM Cu2+. The upregulation of the sulfate transporters may be due not only to a greater sulfur demand at higher Cu levels but also the consequence of interference of Cu with the signal transduction pathway regulating the expression and activity of the sulfate transporters.

36

Introduction

Copper is a nutrient essential for plant functioning, with a requirement amongst the lowest of all elements. However, at elevated levels, it may rapidly become phytotoxic (Sheldon and Menzies 2005; Xiong et al. 2006; Kopsell and Kopsell 2007; Han et al. 2008). Excessive ionic Cu is phytotoxic and generally results in stunted root growth and shoot development and in leaf chlorosis (Yruela 2005; Kopsell and Kopsell 2007). The phytotoxicity of Cu is generally ascribed to its possible reaction with thiol groups of proteins and glutathione, and its potential to induce the production of reactive oxygen species via the metal-catalyzed Haber-Weiss reaction, and the subsequent lipid peroxidation, protein denaturation and DNA mutation(De Vos et al. 1993; Pinto et al. 2003; Morelli and Scarano 2004; Yruela 2009). Furthermore, Cu toxicity may be caused by antagonistic effects with other cations, such as Fe, in protein functioning (Pätsikkä et al. 1998, 2002; Kopsell and Kopsell 2007; Yruela 2009).

Enhanced subcellular Cu levels generally induce the synthesis of phytochelatins, which are derived from glutathione (and in some plant species from related compounds, such as homo-glutathione). Phytochelatins may complex with Cu, and together with cysteine-rich metallothionein proteins (at least in some plants species) and maybuffer the Cu concentration in the cytosol (De Vos et al. 1992; Rauser 2001; Cobbett and Goldsbrough 2002; Ernst et al. 2008; Burkhead et al. 2009). Still, the significance of phytochelatins in Cu detoxification appears to be very restricted (De Vos et al. 1992; Cobbett and Goldsbrough 2002; Schat et al. 2002; Cobbett 2003; Verkleij et al. 2003; Yruela 2005, 2009; Ernst et al. 2008).

The uptake and assimilation of sulfur in plants is generally demand driven and is regulated by the actual sulfur requirement for growth (Hawkesford and De Kok 2006). Brassica species are characterized by a high sulfur requirement for growth and the impact of sulfur nutrition on expression and activity of the sulfate transporters and its assimilation has extensively been studied (Buchner et al. 2004; Koralewska et al. 2007, 2008, 2009a,b; Parmar et al. 2007; Stuiver et al. 2009). Exposure of plants to excessive toxic metals might affect the regulation of uptake and assimilation of sulfate, due to their reaction with sulfur metabolites and the possible induction of synthesis of sulfur-rich phytochelatins and metallothioneins (Ernst et

Chapter 3

37

al. 2008; Sirko and Gotor 2007). In the present study, therefore, the impact of Cu2+ (up to 10 µM) in the root environment on physiological functioning of Chinese cabbage (Brassica pekinensis) was studied and the consequences for the uptake, distribution and metabolism of sulfate were evaluated.

Plant growth conditions

10–14 day-old seedlings were transferred to a 25% Hoagland nutrient solution, containing supplemental concentrations of 0, 1, 2, 5 and 10μM CuCl2, in 13 l stainless steel containers (10 sets of plants per container, 3 plants per set) in a climate controlled room for 7 days. Day and night temperatures were 25 and 20 °C ( 1 °C), respectively, and the relative humidity was 60-70%. The photoperiod was 14 h at a photon fluence rate of 340 ± 20 µmol m–2 s–1 (within the 400-700 nm range) and 1.5 mW cm-2 UV-A+B radiation (within the 280-400 nm range) at plant height, supplied by Philips HPI-T (400 W) lamps as light source. UV-A+B radiation was measured with a Digital Ultraviolet Radiometer model 5.0 (Solartech Inc. Fenton, MI 48430, USA).

Results

Impact of Cu on growth, pigments and content of Cu and othermineral nutrients

Exposure of Chinese cabbage to enhanced Cu2+ concentrations (≥ 2 µM) in the nutrient solution resulted in chlorosis of the shoot which began in young developing leaves, and in a decreased biomass production of both root and shoot (Fig. 1, Table 1 and 2). A 3 day exposure resulted in a significant decrease in pigment content at ≥ 2 µM Cu2+. In addition, concentrations ≥ 5 µM Cu2+ affected plant biomass production as demonstrated by the increase in the shoot to root ratio, indicating that at the onset of exposure, root growth was relatively more reduced than shoot growth (Fig. 1).

38

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3 days 5 days 7 days

Fig. 1. Impact of Cu2+ on biomass production and pigment content of Chinese cabbage. 10 to 14 day-old seedlings were grown on a 25% Hoagland solution containing 0, 1, 2, 5 and 10 µM CuCl2 for 3, 5 and 7 days. The initial fresh weight of shoot and root was 0.17 0.03 and 0.04 0.01 g, respectively. The initial chlorophyll content was 0.42 0.08 mg g-1 fresh weight, the chlorophyll a/b and chlorophyll/carotenoid ratios were 3.77 0.13 and 2.89 0.06, respectively. All data represent the mean of 3 measurements on 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Chapter 3

39

After 5 and 7 days the toxic effects of Cu2+ concentrations of ≥ 5 µM became more pronounced and both shoot and root biomass production were strongly reduced, whereas the pigment content of the shoot was substantially decreased (Fig. 1, Table 1 and 2). At ≥ 5 µM Cu2+ there was an increase in dry matter content in both root and shoot (Table 2). It was evident from the chlorophyll a/b and chlorophyll/carotenoid ratios that the content of the different pigments decreased similarly with time upon exposure to enhanced Cu2+ concentrations (Fig. 1). Upon exposure to 10 µM Cu2+ shoot chlorosis started to develop rapidly and chlorophyll a content decreased significantly faster than that of chlorophyll b and carotenoids, resulting in a significantly decreased chlorophyll a/b and chlorophyll/carotenoid ratio (Fig. 1, Table 2), whereas after 7 days leaf injury (leaf edge necrosis) started to develop.

The Cu content of both root and shoot increased with the Cu2+

concentration in the nutrient solution (Table 2). However, the level of increase was quite different in root and shoot. At 10 µM Cu2+ the Cu content of roots increased 40-fold upon 7 days of exposure, where in the shoot it only increased up to 4.6-fold (Table 2). It was evident that the excessive Cu taken up by the root was rather immobile. From the root and shoot biomass and their Cu content it could be estimated that the translocation of Cu from root to the shoot decreased with increasing Cu concentrations from 28% in control to 4% at 10 µM Cu2+.

The overall mineral nutrient compositions of root and shoot were also substantially affected at ≥ 5 µM Cu2+ (Table 3). Exposure of Chinese cabbage to Cu2+ resulted in a decrease in the content of Ca and Mg in root and that of Fe in the shoot, and a decrease in the content of Mn, P, K in root and shoot, whereas that of Na and Znincreased (Table 3).

Impact of Cu on chlorophyll fluorescence, photosynthesis anddark respiration

Despite the decrease in pigment content upon exposure to high Cu2+

concentrations, the quantum yield of photosystem II photochemistry (Fv/Fm) in leaves was affected little at ≥ 2 µM (Table 1). The Fv/Fm

ratio was slightly decreased only at 10 µM Cu2+, whereas the pigment content had decreased to 26% of that of control plants. On a leaf area

40

basis, the decrease in pigment content was accompanied by a decrease in the rate of photosynthesis. However, when expressed on a chlorophyll basis, the rate of photosynthesis was hardly affected at levels up to 10 µM Cu2+ (Table 1). The rate of dark respiration was significantly decreased at toxic Cu2+ levels (≥ 5 µM; Table 1).

Table 1. Impact of Cu2+ on chlorophyll content, chlorophyll a fluorescence, photosynthesis and dark respiration in leaves of Chinese cabbage. 10 to 14 day-old seedlings were grown on a 25% Hoagland nutrient solution containing 0, 1, 2, 5 and 10 µM CuCl2 for 7 days. Photosynthesis and dark respiration (µmol m-2 s-1) of the youngest fully expanded leaves (3rd or 4th) were measured and represent the mean of 3 to 4 measurements on 3 to 4 plants ( SD). Chlorophyll content (mg m-2) of leaf discs punched from the same leaves was measured and represents the mean of 3 measurements (SD). Chlorophyll a fluorescence (Fv/Fm ratio) represents the mean of 5 to 10 measurements ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Impact of Cu on nitrogen and sulfur metabolite content

The total nitrogen content of the root of Chinese cabbage was affected little upon exposure to enhanced Cu2+ concentrations, whereas that in the shoot was decreased at ≥ 5 µM Cu2+ (Table 2). Furthermore, the nitrate and amino acid contents of the root were hardly affected by Cu2+; their contents were only substantially enhanced at 10 µM. Cu2+ concentrations of ≥ 2 µM had a substantial effect on the total sulfur content of the shoot, whereas the content of the root remained unaltered (Table 2). The enhancement in total sulfur of the shoot could be attributed to an increase in sulfate content at ≥ 2 µM Cu2+. Moreover, upon exposure to ≥ 1 µM Cu2+, there was a strong increase in water-soluble non-protein thiol content in the root and, to a lesser extent in the shoot (at ≥ 2 µM Cu2+). However, this increase in thiol content could only partially be ascribed

Chapter 3

41

Table 2. Impact of Cu2+ on biomass production, dry matter content and content of pigments, copper, total nitrogen and sulfur, nitrate, sulfate, amino acids, water-soluble non-protein thiols and phytochelatins. For experimental details see legends Table 1. The initial fresh weight of shoot and root was 0.13 0.03 and 0.03 0.01 g, respectively. Data on biomass (g FW) and dry matter content (DMC; %) represent the mean of 6 and 3 independent experiments, respectively, with 3 measurements on 6 and 3 plants in each, respectively ( SD). Pigment content (mg g-1 FW) represents the mean of 2 independent experiments with 3 measurements on 3 shoots in each ( SD). Copper content (µmol g-1 DW) represents the mean of 3 measurements with 9 plants in each ( SD). Total nitrogen (mmol g-1 DW) and sulfur content (µmol g-1 DW) represent the mean of 1 and 2 independent experiments, respectively, with 3 to 6 measurements on 3 plants in each ( SD). Nitrate, sulfate and water-soluble non-protein thiol content (µmol g-1 FW) are the mean of 2 independent experiments with 3 measurements on 3 plants in each ( SD). Phytochelatins (PC2 + PC3) and amino acids (µmol g-1 FW) represent the mean of 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

42

to a Cu-induced enhancement of the phytochelatin content, of which thiol residues in the root accounted for 42% of the accumulated water-soluble non-protein thiols (Table 2). In the root, PC2 and PC3

phytochelatins were induced in nearly equal amounts, whereas in the shoot PC2 was the pre-dominant phytochelatin present (data not presented).

Table 3. Impact of Cu2+ levels on mineral content of Chinese cabbage. For experimental details see legends Table 1. Data are expressed as μmol g-1 dry weight and represent the mean of 3 measurements with 9 plants in each (SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Impact of Cu on nitrate and sulfate uptake, and expression ofsulfate transporters and APS reductase

The uptake of sulfate and nitrate were affected differently upon exposure to enhanced Cu2+ concentrations. The uptake of sulfate was slightly enhanced at 2 and 5 µM Cu2+, respectively, whereas it was decreased at 10 µM Cu2+ (Fig. 2). The uptake of nitrate by Chinese cabbage was substantially reduced at ≥ 5 µM Cu2+ (Fig. 2). From the sulfate to nitrate uptake ratio, it was obvious that the sulfate uptake increased at ≥ 2 µM, but was most pronounced at 5 µM Cu2+ (Fig. 2).

Chapter 3

43

The increase in sulfate uptake upon Cu2+ exposure was also evident from sulfate uptake capacity measurements, which showed that the activity of the sulfate transporters in the root was upregulated at 2 and 5 µM Cu2+ (Fig. 3). At 10 µM Cu2+ the sulfate uptake capacity did not significantly differ from that of the control plants (Fig. 3).

The Group 1 high affinity sulfate transporters are involved in the primary uptake of sulfate by the root, and Sultr1;2 was highly expressed in the root of Chinese cabbage, whereas Sultr1;1 was hardly expressed (Fig. 4). The impact of Cu2+ on the expression of Sultr1;2 was ambiguous, since there was a 1.8-fold increase in expression at 5 µM Cu2+, whereas it decreased by approx. 50% at 10 µM Cu2+ (Fig. 4). The Group 4 sulfate transporters are involved in vacuolar efflux of sulfate and Sultr4;1 was highly expressed in both root and shoot. The expression of Sultr4;1 was hardly affected at 5 µM Cu2+, but it was approx. 60% decreased in the root and increased 1.5-fold in the shoot at 10 µM Cu2+ (Fig. 4). Sultr4;2 was slightly expressed in both the root and shoot, though its expression increased more than 2-fold and 4-fold in the shoot at 5 and 10 µM Cu2+, respectively (Fig. 4). The expression of APS reductase was not affected in either root or shoot at < 10 µM Cu2+, however, at 10 µM expression was approx. 50% decreased in the root and increased 1.5-fold in the shoot (Fig. 4).

Discussion

Cu toxicity is generally characterized by an inhibition of shoot and root biomass production, leaf chlorosis, a loss of photosynthetic activity (Mocquot et al. 1996; Yruela 2005, 2009; Xiong et al. 2006; Han et al. 2008; Burkhead et al. 2009) and an altered root morphology (Panou-Filotheou and Bosabalidis 2004; Sheldon and Menzies 2005). In Chinese cabbage, the decrease in biomass production upon exposure to high Cu levels was preceded by a decrease in pigment content of the shoot. The Cu-induced decrease in the pigment content was accompanied with a decrease in photosynthetic activity and the rate of dark respiration; however, expressed on a chlorophyll basis, the rate of photosynthesis was not noticeably affected. The latter indicated that chlorosis upon high Cu concentrations was most likely not the consequence of pigment

44

degradation, but due to hindered chloroplast development upon Cu exposure, as has also been observed in leaves of oregano plants (Panou-Filotheou et al. 2001). This was also supported by chlorophyll fluorescence measurements, which showed that the Fv/Fm ratio, representing the quantum yield of photosystem II, was only slightly reduced at 10 µM Cu2+. This result indicates that the remaining chloroplasts were functional and either did not or only marginally suffered from photoinhibition. The impact of enhanced Cu levels on chloroplast development and/or functioning may be attributed to a Cu-induced Fe deficiency or the exchange of Mg ion in its chlorin ring by Cu (Pätsikkä et al. 2002; Küpper et al. 2003).

High Cu levels resulted in a substantial increase in the Cu content of both root and shoot of Chinese cabbage. Despite the relatively strong enhancement of the Cu content in the root upon exposure to high levels (up to 40-fold), the excessive Cu taken up by the root was relatively immobile, since only a minor proportion was transferred to the shoot. However, root growth was only slightly more affected than shoot growth at the higher Cu2+ concentrations (≥ 5 µM), though the root morphology was altered and more undeveloped side roots were formed (data not shown). The nature of these alterations needs to be studied further. On the basis of the current results, it is difficult to assess the critical Cu concentration, though evidently a more than 3-fold increase in Cu content of the shoot of Chinese cabbage already initiated toxic effects.

Alterations in mineral composition at excessive Cu levels have also been observed in other plant species (Ebbs and Kochian 1997; Ouzounidou et al. 1998; Pätsikkä et al. 1998, 2002; Ali et al. 2002; Fageria 2002; Alaoui-Sossé et al. 2004; Sheldon and Menzies 2005;Kováčik et al. 2009). The interactions of Cu with micro and macronutrients are either synergistic, antagonistic or have no effects, differ with the crop species and plant growing conditions (Fageria 2002). To what extent the changes in mineral composition are the consequence of an altered competition in the uptake and transport of the minerals or an altered root architecture at the occurrence of root injury at toxic levels of Cu2+ levels needs to be further evaluated (Sheldon and Menzies 2005). It is not yet established to what extent a change in mineral composition is involved in the onset of the phytotoxity of Cu.

Chapter 3

45

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Fig. 2. Impact of Cu2+ on the uptake of sulfate and nitrate by Chinese cabbage and sulfate/nitrate uptake ratio. For experimental details seelegends Table 1. Data on sulfate and nitrate uptake (μmol g-1 FW root 24 h-1) represent the mean of 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

46

Exposure of Chinese cabbage to enhanced Cu concentrations did not markedly affect nitrogen content. Only at 10 µM Cu2+, where shoots had become severely chlorotic and visible injury symptoms (leaf edge necrosis) started to develop, there was a substantial increase in nitrate and amino acid content, indicating that nitrogen metabolism was disturbed.

Exposure of Chinese cabbage to enhanced Cu concentrations substantially affected the content and distribution of sulfur compounds in root and shoot. There was an accumulation of water-soluble non-protein thiols in the root and, to lesser extent, in the shoot. However, this enhancement could only partially be ascribed to Cu-induced accumulation of phytochelatins (PC2 and PC3). The enhanced thiol level was most likely due to an accumulation of reduced glutathione (and maybe for a lesser part to cysteine), regularly the pre-dominant thiol compound present in plant tissue (De Kok and Stulen 1993; De Kok et al. 2007; Rennenberg 2001). The possible impact of high Cu levels on the glutathione redox status, as has been observed upon exposure of Brassica napus to acute toxic levels of Cu (100 µM; Russo et al. 2007), needs to be studied further. Under normal conditions, the uptake, distribution and assimilation of sulfur are under strict regulatory control and the rate of sulfate uptake by the root will equal the overall sulfur demand to maintain plant growth (Hawkesford and De Kok 2006; Koralewska et al. 2007, 2008; Rouached et al. 2009). Brassica species are characterized by a high sulfur requirement for growth. However, the greater proportion of the sulfate taken up by the root may not directly be metabolized, but stored as sulfate in the shoot (Castro et al. 2004; Koralewska et al. 2007, 2008, 2009a, b). It has been presumed that the synthesis of sulfur-rich metal-chelating compounds upon exposure of plants to potentially toxic metals might require an enhanced sulfur demand, viz. the rate of uptake and assimilation of sulfate (Sirko and Gotor 2007; Ernst et al. 2008). Indeed, exposure of Chinese cabbage to high Cu2+ concentrations resulted in upregulated expression of Sultr1;2 and Sultr4;1 sulfate transporters accompanied with slightly upregulated sulfate uptake capacity at 2 and 5 µM Cu2+. In contrast, the uptake of nitrate was reduced at 5 µM Cu2+, which coincided with the decrease in biomass production. In general, the net nitrate uptake is closely linked to the relative growth rate (Ter Steege et al. 1999; Westerman et al. 2000).

Chapter 3

47

0 1 2 5 100

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Fig. 3. Impact of Cu2+ on the sulfate uptake capacity of Chinese cabbage. For experimental details see legends Table 1. Data on sulfate uptake capacity (μmol g-1 FW root h-1) represents the mean of two independent experiments with 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

However, it was doubtful as to whether the observed upregulation of the sulfate transporters in Chinese cabbage was solely due to a higher sulfur demand at higher Cu tissue contents. It is unlikely that the observed enhancement of the thiol compounds in Chinese cabbage at high Cu2+ concentrations would require substantial upregulation of the sulfate uptake, since this sulfur proportion never exceeded more than 5% of the total sulfur content. The upregulation of the sulfate transporters in the root was accompanied with an enhanced total sulfur content of the shoot, which could be attributed to sulfate accumulation. It may be that this accumulation of sulfate was the consequence of a Cu-induced disturbance in the signal transduction pathway regulating the expression and activity of the sulfate transporters. Evidently, Cu exposure affected the regulation of sulfate transporters in the root in such a way that more sulfate was taken up than could be metabolized and utilized for growth, resulting in enhanced sulfate content of the shoot.

48

Fig. 4. Impact of Cu2+ on mRNA abundance of sulfate transporters (Sultr) and APS reductase (APR) (Northern-blot analysis) in root and shoot of Chinese cabbage. Equal RNA loading was determined by ethidium bromide staining of gels (shown in the bottom panels). For experimental details see legends Table 1. A representative set of data from two independent experiments is presented.

Regulation of sulfate uptake and its distribution may be controlled at a transcriptional, translational and/or post-translational level. Both external and internal sulfate concentration and that of thiol compounds viz. glutathione, have been presumed to be involved in the control of the expression and activity of sulfate transporters (Hawkesford and De Kok 2006; Rouached et al. 2009). The high affinity sulfate transporters Sultr1;1 and Sultr1;2 are responsible for the primary uptake of sulfate by the root, though their expression and activity are differently regulated (Hawkesford and De Kok 2006; Koralewska et al. 2008; Rouached et al. 2008, 2009). The expression and activity of the constitutive sulfate transporter Sultr1;2 appeared to be related to metabolic demand (Koralewska et al. 2008; Rouached et al. 2008, 2009). This transporter was only upregulated at sulfate

Chapter 3

49

concentrations in the root environment close to the Km value (5-10 µM) or upon sulfate deprivation, presumably triggered by a low in situthiol concentration. Sultr1;1 is an inducible sulfate transporter, which is generally almost not expressed in sulfate-sufficient plants, as was also obvious from the current study. However, the expression of Sultr1;1 is highly upregulated in the root upon sulfate deprivation and is triggered by the in situ (internal or external) sulfate concentration rather than the thiol concentration (Buchner et al. 2004; Koralewska et al. 2007, 2008, 2009a, b; Parmar et al. 2007; Stuiver et al. 2009; Rouached et al. 2008, 2009). The constitutive sulfate transporter Sultr4;1 and the inducible sulfate transporter Sultr4;2 are generally only upregulated upon sulfur deficiency, when plants try to remobilize and redistribute all possible available sulfate from, for instance, the vacuoles (Buchner et al. 2004; Parmar et al. 2007; Koralewska et al. 2009a,b; Stuiver et al. 2009). The expression and activity of APS reductase is generally downregulated at high thiol levels (Westerman et al. 2001; Durenkamp et al. 2007; Koralewska et al. 2008).

From the present study, however, it was evident that upon exposure of plants to enhanced Cu2+ levels, the presumed signal transduction pathway of the regulation of the expression and the activity of the sulfate transporters and that of APS reductase were bypassed or overruled. Here the upregulated expression of Sultr1;2 (root), Sultr4;1 (shoot) and the Sultr4;2 (shoot) sulfate transporter genes resulted in a modest increase in sulfate uptake capacity, and net uptake occurred both at normal or even enhanced thiol levels (root, shoot) and normal (root) or even enhanced sulfate levels (shoot). Moreover, the strongly enhanced thiol levels in the root and to lesser extent in the shoot at high Cu levels did not result in a downregulation of the expression of APS reductase. The majority of cells in both shoot and root have the capacity to reduce and assimilate sulfate in their plastids, enabling local signaling of sulfate uptake, distribution and reduction/assimilation at a cellular level. The possible significance of the direct reaction of Cu with sulfide and/or other metabolites involved in the sulfur metabolism signal transduction pathway needs further to be evaluated.

50

Conclusions

High Cu2+ concentrations in the root environment were toxic for Chinese cabbage, which may in part be ascribed to a Cu-induced disturbed development of the chloroplasts, resulting in chlorosis and a hindered biomass production. Furthermore, enhanced Cu contents in the root affected the regulation of the uptake and distribution of sulfate, which could not be ascribed to an impact on the presumed signal transduction pathway therein involved. It remains unclear to what extent the observed upregulation of the sulfate transporters was the consequence of a higher sulfur demand at higher Cu concentration or the result of an interference/reaction of Cu with the signal compounds regulating the expression and activity of the sulfate transporters.

UV radiation increases the toxicity andimpact of copper on sulfur metabolism in

Chinese cabbage

Chapter 4.

Chapter 4

53

Abstract

Biomass production, dry matter content, specific leaf area and pigment content of Chinese cabbage were all quite similar whether plants were grown with HPI-T (2.2 mW cm-2, UV-A+B) or SON-T (0mW cm-2, UV-A+B) lamps as a light source. Exposure of plants to enhanced Cu2+ concentrations (2–10 μM) resulted in a more rapid and stronger decrease in plant biomass production and pigment content in presence of UV radiation (HPI-T) compared to the absence of UV radiation (SON-T). The Fv/Fm ratio was only decreased at ≥ 5 μM Cu2+

in the presence of UV radiation, when leaf tissue started to become necrotic. The content of Cu in both root and shoot of Chinese cabbage was strongly affected by the level of UV radiation, both in absence and presence of enhanced Cu2+ concentrations in the root environment. The observed enhanced Cu toxicity in presence of UV is probably due largely to a higher accumulation of Cu in both root and shoot. The total sulfur content of the shoot was increased at ≥ 2 µMCu2+ in presence of UV and at ≥ 5 µM Cu2+ in absence of UV, which was mainly attributed to an increase in sulfate content. Moreover, there was a strong increase in water-soluble non-protein thiol content upon Cu2+ exposure in the root and, to a lesser extent in the shoot,both in presence and absence of UV. The expression and activity of the high affinity sulfate transporter, Sultr1;2, was enhanced at ≥ 2 µM in presence of UV, and at ≥ 5 µM Cu2+ in absence of UV. Furthermore, in the shoot, the expression of vacuolar sulfate transporter, Sultr4;1, was upregulated at ≥ 5 µM Cu2+ in presence and absence of UV, whilst the expression of second vacuolar sulfate transporter, Sultr4;2, was upregulated at 10 µM Cu2+ in presence of UV. The expression of APS reductase in the root was hardly affected and it was slightly downregulated at 2 µM in presence of UV and at 10 µM, in absence of UV. The expression and activity of sulfate transporters were enhanced upon exposure at enhanced Cu2+

concentrations; this may be due not only to a greater sulfur demand at higher Cu levels, but more likely was the consequence of Cu toxicity, since it occurred more rapidly in presence of compared to the absence of UV.

54

Introduction

Exposure of Chinese cabbage (Brassica pekinensis) to ≥ 5 µM Cu2+

resulted in leaf chlorosis and a decrease in plant biomass production and pigment content (Chapter 3). The decrease in pigment content was accompanied by a loss of photosynthetic capacity, however, expressed on chlorophyll basis, the rate of photosynthesis was hardly affected at levels up to 10 µM Cu2+ (Chapter 3). Leaf chlorosis, whichwas first visible in young developing leaves, was probably not the consequence of pigment degradation, but due to a hindered chloroplast development at high Cu2+ concentrations (Chapter 3). Enhanced Cu concentrations affected the uptake, distribution and assimilation of sulfate in Chinese cabbage. Both the expression and activity of the sulfate transporters were enhanced at approx. 2-5 µM Cu2+. At ≥ 1 µM Cu2+ there was an increase in water-soluble non-protein thiol content of the root and to a lesser extent of the shoot of Chinese cabbage. This increase could only be partially attributed to a Cu-induced accumulation of the phytochelatins (Chapter 3). Cu2+

exposure hardly affected the total sulfur content of the root of Chinese cabbage, whereas that of the shoot was increased at ≥ 1 µM, the latter mainly due to an increase in sulfate content (Chapter 3). It remains to be resolved to what extent changes in sulfate uptake andmetabolism upon Cu2+ exposure were the consequence of a higher sulfur demand in order to detoxify the excess Cu, or the result of a direct interference of Cu with the signal transduction pathway involved in sulfur metabolism.

It has been predicted that there will be an increase in UV radiation on the earth surface due to ozone depletion in the stratosphere and reduction of aerosols and clouds (McKenzie et al. 2007). UV-B (280-320 nm) is the most harmful part of the UV spectrum for plants reaching the surface of the earth. UV-B absorption by the foliage may result in the formation of reactive oxygen species, especially in the chloroplasts (Tausz 2001). Consequently, high UV-B levels may alter thylakoid integrity, induce pigment degradation and decrease chlorophyll a/b ratio, Rubisco activity and stomatal conductivity(Jansen et al. 1998; Larsson et al. 1998) and substantially affect plant performance. The level of pigment degradation was dependent on the length and intensity of the UV-B radiation (Kakani et al. 2003). Plant species differ in their ability to tolerate UV-B radiation and

Chapter 4

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pigment degradation occurred more rapidly in dicotyledons than in monocotyledons (Kakani et al. 2003).

Cu has potential also to accelerate the formation of reactive oxygen species in plant tissue (Pinto et al. 2003) and the high light intensities which may enhance the toxicity of Cu to chloroplasts functioning, was presumably due to an enhanced production of hydroxyl radicals (Yruela et al. 1996; Yruela 2009). The impact of Cu exposure and UV radiation on plants may be synergistic. For instance,both growth and chlorophyll content were reduced to a greater extent when the aquatic plant Lemna gibba was simultaneously exposed to Cu and UV radiation (Babu et al. 2003). Moreover, Cu toxicity wasincreased upon exposure to high light intensities and/or UV radiationin chloroplasts (Pätsikkä et al. 1998), green alga (West et al. 2003, Lupi et al. 1998), cyanobacteria (Rai et al. 1998; Gouvêa et al. 2008) and duck weed (Babu et al. 2003), with symptoms indicative of synergistic oxidative damage.

The interactive effects of Cu and UV radiation have only beenstudied in cyanobacteria and aquatic plant species. There is little known about the combined effects of enhanced Cu levels and light quality (viz. UV radiation) in plants. The present study was undertaken to investigate (і) the impact of light quality (UV radiation) on Cu toxicity, and (іі) the interaction of Cu toxicity with the regulation of the uptake and metabolism of sulfate in Chinese cabbage (Brassica pekinensis).

Plant growth conditions

10-day-old seedlings were transferred to a 25% Hoagland nutrient solution, containing supplemental concentrations of 0, 2, 5 and 10 μM CuCl2 in 13 l stainless steel containers (10 sets of plants per container, 3 plants per set) in a climate-controlled room for 7 days. Day and night temperatures were 25 and 20 °C ( 1 °C), respectively, and the relative humidity was 60-70%. The photoperiod was 14 h at a photon fluence rate of 500 25 µmol m–2 s–1 (within the 400-700 nm range) at plant height. For the different light quality and UV-A+B radiation studies, the climate-controlled room was split into two parts by a non-transparent thermopore sheet. In one part light was supplied by Philips HPI-T lamps (400 W) and in the other part by

56

Philips SON-T lamps (600 W). HPI-T lamps had 2.2 mW cm-2 UV-A+B(within the 280-400 nm range) and 0.06 mW cm-2 UV-B radiation (within the 280-325 nm range), whereas SON-T lamps had almost no UV radiation, measured with a Digital Ultraviolet Radiometer model 5.0 and model SM 6.0, respectively (Solartech Inc. Fenton, MI 48430, USA). The spectrum of light from both the lamps is shown in figure 1. In experiments with UV filtered out, light was provided by HPI-Tlamps and the UV (A+B) radiation was filtered out with a Perspex sheet (ISPA Plastics Groningen, The Netherlands).

Fig. 1. Spectrum of light at plant height from Philips HPI-T and Philips SON-T lamps. UV (A+B), ultraviolet irradiation; PAR, photosynthetically active radiation.

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Chapter 4

57

Results

Impact of Cu and light quality on growth, dry matter content,pigment content and Cu content

Irrespective of whether Chinese cabbage was grown with HPI-T and SON-T lamps as the light source, plant biomass production, dry matter content, shoot pigment content and the specific leaf area were all quite similar, however, shoot to root ratio was slightly lower withHPI-T (Fig. 2 and 3). However, if plants were exposed to enhanced Cu2+ concentrations in the nutrition solution, biomass production was more rapidly affected with the HPI-T compared to SON-T light source. With HPI-T, Cu2+ exposure resulted in a significant decrease in plant biomass production at ≥ 2 µM (up to 70% at 10 µM) accompanied by a decrease in specific leaf area, whereas the shoot to root ratio was only slightly increased (Fig. 2). However, with SON-T, plant biomass production was only slightly decreased at ≥ 5 µM Cu2+ (up to 34% at 10 µM), whereas shoot to root ratio only significantly increased at 10 µM (Fig. 2). At ≥ 5 µM Cu2+ there was an increase in dry matter content in both root and shoot with HPI-T, whereas it was hardly affected at all with SON-T (Fig. 2).

The pigment content started to decrease with both HPI-T and SON-T at ≥ 2 µM Cu2+, but similarly to the observations on biomass production, the decrease occurred more rapidly with HPI-T compared to SON-T (Fig. 3). For example, at 2 µM Cu2+, pigment content had decreased to 55 and 21% with HPI-T and SON-T, respectively (Fig. 3). If plants were exposed to ≥ 5 µM Cu2+ and HPI-T, not only the pigment content was decreased, but there was also a change in pigment composition. Here, chlorophyll a content decreased significantly faster than that of chlorophyll b and carotenoids, resulting in a significantly decreased chlorophyll a/b and chlorophyll/carotenoid ratio (Fig. 3). With SON-T, the chlorophyll a/band chlorophyll/carotenoid ratios were only slightly decreased at ≥ 5 µM Cu2+ (Fig. 3). The quantum yield of photosystem ІІ photochemistry (Fv/Fm) only decreased in Chinese cabbage leaves upon exposure to ≥ 5 µM Cu2+ with HPI-T whereas, with SON-T it was not affected (Fig. 3).

58

Fig. 2. Impact of Cu2+ and light quality on biomass production, dry matter content and specific leaf area of Chinese cabbage. 10-day-old seedlings of Chinese cabbage were grown to 25% Hoagland solution containing 0, 2, 5 and 10 µM CuCl2 for 7 days. Plants were grown with HPI-T (black bars) andSON-T lamps as a light source (grey bars). The initial shoot and root weight was 0.045 0.004 and 0.009 0.001 g fresh weight, respectively. The data on biomass production (g FW), dry matter content (DMC; %) and shoot/root ratio represent the mean of 6 experiments with 9 measurements with 3 plants in each ( SD), and specific leaf area (SLA; cm-1 g-1 FW) represents the mean of 6 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Chapter 4

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Fig. 3. Impact of Cu2+ and light quality on pigment content, pigment composition and chlorophyll a fluorescence. For experimental details see legends Fig. 2. Plants were grown with HPI-T (black bars) and SON-T lamps as a light source (grey bars). The pigment content (mg g-1 FW) represents the mean of 2 experiments with 3 measurements in each ( SD) and chlorophyll a fluorescence (Fv/Fm ratio) the mean of 9 measurements ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Chinese cabbage grown with HPI-T had a 40% higher Cu content in both root and shoot compared to plants grown with SON-T (Fig. 4).With both HPI-T and SON-T, the Cu content of both root and shootincreased with the Cu2+ concentration in the nutrient solution, however, plants grown with HPI-T accumulated more Cu than withSON-T upon exposure to Cu2+ (Fig. 4) For example, at 2 µM Cu2+, the Cu content in root of HPI-T and SON-T was 4.18 and 1.54 μmol g-1

DW, respectively. The accumulation of Cu upon exposure of plants to high Cu concentrations in the nutrient solution differed strongly between root and shoot, since the excessive Cu taken up by the root appeared to be rather immobile. For instance, at 10 µM Cu2+ the Cucontent of root increased 11-fold after 7 days of exposure, whereas in

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the shoot it was only increased up to 1.3-fold with HPI-T. With SON-T the content of Cu increased in root up to 14-fold and in the shoot, up to 2.8-fold with 10 µM Cu2+ (Fig. 4).

In order to determine the significance of UV radiation on the impact of enhanced Cu concentration on Chinese cabbage, plants were grown under conditions where the UV-A+B radiation of the HPI-T lamps was filtered out by a UV-absorbing Perspex sheet (HPI-T*). Biomass production and pigment content of Chinese cabbage grown with HPI-T and HPI-T* were quite similar (Fig. 5). However, the observed decrease in plant biomass production, pigment content and chlorophyll fluorescence at ≥ 2 µM Cu2+ with HPI-T was largely diminished with HPI-T*, demonstrating that UV-A+B radiation strongly enhanced the phytotoxicity of enhanced Cu2+ concentrations (Fig. 5).

Fig. 4. Impact of Cu2+ and light quality on Cu content. For experimental details see legends Fig. 2. Plants were grown with HPI-T (black bars) and SON-T lamps as a light source (grey bars). The Cu content (µmol g-1 DW) represents the mean of 3 measurements with 9 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Impact of Cu and light quality on nitrogen and sulfur metabolitecontent

The nitrate, amino acid and sulfur metabolite contents of Chinese cabbage were quite similar for both HPI-T and SON-T –grown plants. Upon Cu2+ exposure, the nitrate content was slightly decreased in the

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Chapter 4

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root at ≥ 2 µM and at 10 µM Cu2+ in the shoot with both HPI-T and SON-T (Fig. 6 and 7). There was a strong increase in amino acidcontent of the shoot upon Cu2+ exposure at ≥ 2 µM and to a lesser extent in the root with HPI-T, whereas it was hardly affected withSON-T (Fig. 6). At 2 µM Cu2+ there was a substantial increase in total sulfur content of the shoot and at ≥ 5 µM in the root, with HPI-T. However, with SON-T, Cu2+ concentrations of ≥ 5 µM had a substantial effect on total sulfur content of the shoot whereas the content of the root was only increased at 10 µM Cu2+. The increase in total sulfur content upon Cu2+ exposure could for the greater part be attributed to enhance sulfate content (Fig. 7). Moreover, upon Cu2+

exposure at ≥ 2 µM, there was a strong increase in water-soluble non-protein thiol content in the root and to a lesser extent in the shoot (at ≥ 5 µM Cu2+) for both HPI-T and SON-T (Fig. 7).

Impact of Cu and light quality on sulfate uptake capacity andexpression of sulfate transporters and APS reductase

Both the expression of the high affinity sulfate transporter Sultr1;2 and the sulfate uptake capacity of the root of Chinese cabbage were slightly higher with SON-T compared to HPI-T illumination (Fig. 8 and 9). The expression of the Group 2 sulfate transporter Sultr2;2, which is involved in vascular transport, was also slightly more expressed with SON-T compared to HPI-T –grown plants, in root (Fig. 9). The expression of the Group 4 sulfate transporter Sultr4;1, which is involved in vacuolar efflux of sulfate, was quite similar for both HPI-Tand SON-T illumination, but in the shoot expression was 40% lower with SON-T (Fig. 9).

Upon Cu2+ exposure of HPI-T grown plants, there was a strong increase in sulfate uptake capacity at ≥ 2 µM (about 2-fold), whereas it was slightly decreased at 10 µM Cu2+ (Fig. 8). With SON-Tillumination, sulfate uptake capacity was increased at ≥ 5 µM Cu2+

(Fig. 8). Both for HPI-T and SON-T illumination, the increase in sulfate uptake capacity upon Cu2+ exposure was accompanied with an enhanced expression of Sultr1;2 in the root, whereas Sultr1;1 was hardly expressed (Fig. 8 and 9). With HPI-T illumination, the expression of Sultr1;2 in roots was increased by 159 and 77% at 2 and 5 µM Cu2+, respectively, whereas with SON-T illumination, expression was increased by 40% at ≥ 5 µM Cu2+ (Fig. 9). Upon Cu2+

62

exposure with HPI-T illumination, the expression of Sultr2;2 was slightly upregulated in root at 2 µM Cu2+, however with SON-T there was no responce (Fig. 9). Upon Cu2+ exposure, the expression of Sultr4;1 was hardly affected in the root under both HPI-T and SON-Tillumination. However, for HPI-T illumination, the expression of Sultr4;1 was increased by 53 and 166% at 5 and 10 µM Cu2+

respectively, and for SON-T illumination, its expression was increased by 50% at 10 µM Cu2+ in shoot (Fig. 9). Sultr4;2 was slightlyexpressed in root with SON-T illumination, but upon Cu2+ exposureexpression was strongly decreased. However, in shoot, the expression of Sultr4;2 was increased 1.4-fold and 3.6-fold at 5 and 10 µM Cu2+, respectively, under HPI-T illumination, but was hardly expressed under SON-T illumination (Fig. 9). The expression of APS reductase in both root and shoot was quite similar for both HPI-T and SON-Tgrown plants, and expression was slightly enhanced in root upon Cu2+

exposure. However, in shoot the expression of APS reductase was slightly decreased at 2 µM Cu2+ under HPI-T, and at 10 µM Cu2+ underSON-T illumination (Fig. 9).

Discussion

A simultaneous exposure of plants to enhanced Cu levels and UV radiation may result in synergistic negative effects on plant growth and functioning (Lupi et al. 1998; Babu et al. 2003). From the current study it was evident that growth and metabolite content of Chinese cabbage was hardly affected upon illumination with HPI-T and SON-T lamps as light sources, despite the large differences in light spectrum (viz. UV level). However, if Chinese cabbage was exposed to enhanced Cu2+ concentrations in the root environment, toxic effects occurred more rapidly with HPI-T compared to SON-T illumination. The differences in the development of toxic effects of Cu2+ at different light conditions, could be solely ascribed to differences in the level of UV radiation upon illumination with HPI-T (UV-A+B) and SON-T (no UV-A+B) lamps. The negative effects of enhanced Cu2+ concentrations on biomass production, pigment content and the Fv/Fm ratio in Chinese cabbage was strongly decreased in absence of UV-A+B (SON-T lamps) and when the UV-A+B of the HPI-T lamps was filtered out. Moreover, with HPI-T lamps, enhanced Cu2+ concentrations also

Chapter 4

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resulted in a change in leaf morphology of Chinese cabbage, illustrated by a decrease in specific leaf area (SLA).

Fig. 5. Impact of Cu2+ and UV radiation on biomass production, pigment content and chlorophyll a fluorescence. 10-day-old seedlings of Chinese cabbage were grown on 25% Hoagland solution containing 0, 2, and 5 µM CuCl2 for 7 days. Plants were grown with HPI-T (black bars) and UV radiation filtered out from HPI-T* lamps by a Perspex sheet (white bars). The initial shoot and root weight was 0.050 0.002 and 0.008 0.001 g fresh weight, respectively. The data on biomass production (g FW) and shoot/root ratio represent the mean of 9 measurements with 3 plants in each ( SD). The pigment content (mg g-1 FW) represents the mean of 2 experiments with 3 measurements in each ( SD) and chlorophyll a fluorescence (Fv/Fm ratio) the mean of 9 measurements ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Fig. 6. Impact of Cu2+ and light quality on the content of nitrate and amino acids. For experimental details see legends Fig. 2. Plants were grown withHPI-T (black bars) and SON-T lamps as a light source (grey bars). The nitrate and amino acid content (µmol g-1 FW) represent the mean of 2 experiments with 3 measurements in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

The content of Cu in both root and shoot of Chinese cabbage was strongly affected by the level of UV, both in absence and presence of enhanced Cu2+ concentrations in the root environment. In absence of enhanced Cu2+ concentrations, Chinese cabbage grown with HPI-Tillumination had a 40% higher Cu content in root and shoot compared to SON-T grown plants. Moreover, the accumulation of Cu at enhanced Cu2+ concentrations was always substantially higher in both root and shoot of Chinese cabbage grown with HPI-T rather thanSON-T illumination (in root even up 2.5-fold). The observation that Cu was more toxic in presence than in absence of UV is probably largely due to the observed differences in accumulation of Cu in root and shoot of Chinese cabbage. Similar to previous observations (Chapter

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3), the excessive Cu taken up by the root was rather immobile and on a whole plant basis not more than 20% was transferred to the shoot.

Fig. 7. Impact of Cu2+ and light quality on the content of total sulfur, sulfate and water-soluble non-protein thiols. For experimental details see legends Fig. 2. Plants were grown with HPI-T (black bars) and SON-T lamps as a light source (grey bars). The sulfate and water-soluble non-protein thiol content (µmol g-1 FW) represent the mean of 2 experiments with 3 measurements in each ( SD). Total sulfur content (µmol g-1 DW) represents the mean of 3 measurements ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Enhanced Cu levels as well as UV radiation have the potential to induce oxidative stress by the formation of reactive oxygen species (Lupi et al. 1998; Tausz 2001; Babu et al. 2003), which might also have contributed the development of leaf necrosis at ≥ 5 µM Cu2+

with HPI-T illumination. Exposure of Lemna gibba to UV radiation and enhanced Cu concentrations resulted in an enhanced activity of Cu/ZnSOD (Babu et al. 2003). Cu/ZnSOD performs a significant role in the detoxification of reactive oxygen species and expression in the cytosol and chloroplasts was upregulated at high light and enhanced Cu levels (Tausz 2001; Pilon et al. 2011). This enzyme is the most abundant Cu protein along with plastocyanin in green plants (Yruela 2005, 2009, Cohu and Pilon 2007). To what extent the observed enhancement of Cu content in Chinese cabbage with HPI-Tillumination was the consequence of UV radiation-induced enhanced Cu uptake for synthesis of Cu/ZnSOD is not known.

Similar to previous observations (Chapter 3), the decrease in pigment content at enhanced Cu2+ concentrations in the root environment with HPI-T (and UV-filtered out HPI-T) and SON-T illumination was not accompanied with a change quantum yield of photosystem II photochemistry (Fv/Fm ratio) indicating that the remaining chloroplasts were functional. Only at ≥ 5 μM Cu2+ upon HPI-T illumination, where leaf tissue started to become necrotic, there was a decrease in Fv/Fm ratio, confirming higher toxicity of Cu in presence of UV.

Exposure of Chinese cabbage to enhanced Cu concentrations substantially affected the content and distribution of sulfur compounds in the root and shoot of Chinese cabbage (Chapter 3). There was a strong accumulation of water-soluble non-protein thiols in the root and, to a lesser extent in the shoot. However, increase in thiol content in Chinese cabbage could only partially be ascribed to a Cu-induced synthesis of phytochelatins (Chapter 3). There was enhanced total sulfur content in both root and shoot which could be attributed to sulfate accumulation. The enhanced sulfur content upon Cu2+ exposure was most likely the consequence of upregulation of the expression and activity of sulfate transporters in the root of Chinese cabbage. Expression and activity of sulfate transporters in Chinese cabbage were slightly affected by HPI-T and SON-T illumination, however, expression was strongly increased at enhanced Cu2+

concentrations. Expression and activity of Sultr1;2 in the root of

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Chinese cabbage were slightly enhanced with SON-T compared toHPI-T illumination, which was most likely due to a slightly higher shoot biomass production. It was evident that the rate of sulfate uptake capacity by the root was determined by the sink capacity of the shoot (Koralewska et al. 2009b).

Fig. 8. Impact of Cu2+ and light quality on sulfate uptake capacity. For experimental details see legends Fig. 2. Plants were grown with HPI-T (black bars) and SON-T lamps as a light source (grey bars). Data on sulfate uptake capacity (µmol g-1 FW root h-1) represent the mean of 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

The uptake, distribution and assimilation of sulfur at a whole plant level are controlled by the plant sulfur status and the sulfur demand to maintain the plant growth (Hawkesford and De Kok 2006; Rouached et al. 2009). It has been proposed that both low and high-affinity sulfate transporters may play key roles in sulfate uptake, translocation and distribution in higher plants (Buchner et al. 2004; Hawkesford and De Kok 2006). However, the mechanism by which Cu influences the sulfur status of the plants and the altered gene expression of both sulfate transporters and APS reductase is not clear. It may be that Cu itself interferes/reacts with the regulatory

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Fig. 9. Impact of Cu2+ and light quality on mRNA abundance of sulfate transporters (Sultr) and APS reductase (APR; Northern-blot analysis) in the root and shoot of Chinese cabbage. Equal RNA loading was determined by ethidium bromide staining of gels (shown in the bottom panels). For experimental details see legends Fig. 2. A representative set of data from two independent experiments is given.

The expression of the constitutively expressed sulfate transporter,Sultr4;1, and the wholly inducible sulfate transporter, Sultr4;2, was upregulated in the shoot upon Cu2+ exposure at enhanced levels (≥ 5 μM); generally these sulfate transporters only upregulated upon sulfate deficiency (Buchner et al. 2004; Parmar et al. 2007; Koralewska et al. 2009a,b; Stuiver et al. 2009). The expression and activity of APS reductase is generally downregulated at enhanced thiol levels (Westerman et al. 2001; Durenkamp et al. 2007; Koralewska et al. 2008). However, similarly to previous observations, the strongly enhanced thiol levels in the root and to a lesser extent in the shoot did not result in downregulation of the expression of APS reductase(Chapter 3). Whereas, the expression of APS reductase was slightly

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downregulated at 2 and 10 µM Cu2+ with HPI-T and with SON-Tillumination respectively, which may be due to an enhanced sulfate content of the shoot. It may be that the sulfate content rather than the thiol concentration was of greater significance in the regulation of the expression of APS reductase in the shoot.

From the present data it was evident that the upregulated expression and activity of sulfate transporters upon Cu2+ exposure at enhanced levels was likely not due to a higher sulfur need at highertissue Cu contents, since plants took up more sulfate than wasmetabolized, resulting in an enhanced sulfate content of the shoot (Chapter 3). Furthermore, the water-soluble non-protein thiols which are presumed to be involved in binding/chelating excessive Cu in plant tissue, accumulated at quite similar levels with HPI-T and SON-T illumination irrespective of the development of Cu toxicity. However, the observed enhancement in expression and activity of sulfate transporters, total sulfur content and sulfate content upon Cu2+

exposure, occurred more rapidly in presence of UV radiation (HPI-T) than in absence of UV radiation (SON-T). Therefore, increases in sulfate uptake, thiol content and total sulfur content may not only be due to a higher sulfur need, but may be specifically a response to the occurrence of Cu toxicity, as the increases appear more rapidly inpresence of UV radiation.

Conclusions

Enhanced Cu2+ concentrations in the root environment and UV radiation had negative synergistic effects in Chinese cabbage. The negative effects of enhanced Cu2+ concentrations on biomass production, pigment content and the Fv/Fm ratio in Chinese cabbage were strongly increased in presence of UV illumination. Enhanced Cu toxicity in presence of UV was largely due to a UV-induced enhanced accumulation of Cu content in both root and shoot of Chinese cabbage. Enhanced Cu content in the root affected the regulation of the uptake and distribution of sulfate, which was due not only to higher sulfur need for its detoxification. It is suggested that the regulation also responded to the occurrence of Cu toxicity directly, since this occurred more rapidly in presence of UV radiation compared to the absence of UV radiation. Furthermore, it is suggested that the

70

enhanced Cu content in the roots interferes/reacts with the signal compounds involved in the regulation of expression and activity of sulfate transporters.

Sulfate deprivation overrules the toxicity andimpact of copper on sulfur metabolism in

Chinese cabbage

Chapter 5.

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Abstract

Exposure of Chinese cabbage (Brassica pekinensis) to an enhanced Cu2+ level (4 µM) resulted in a reduced plant biomass production and an increased shoot to root ratio at both sulfate-sufficient and sulfate-deprived conditions. However, sulfate deprivation had a more rapid negative effect on plant biomass production than enhanced Cu2+ level.The expression and activity of the sulfate transporters and the expression of APS reductase in Chinese cabbage were rapidly upregulated (already after 1 or 2 days) upon exposure to 4 μM Cu2+, sulfate deprivation and their combination. Though, the impact of sulfate deprivation on the expression and activity of the sulfate transporters was hardly further affected by Cu2+.

74

Introduction

The uptake, distribution and assimilation of sulfur are modulated by the plant sulfur status and the sulfur demand for growth (Hawkesford and De Kok 2006). Sulfate deprivation of Chinese cabbage resulted in a rapidly induced expression of Sultr1;1 and an enhanced expression of the constitutively expressed Sultr1;2 in the root, accompanied with an increased sulfate uptake capacity (Koralewska et al. 2008; Stuiver et al. 2009). It has also been recognized that enhanced Cu levels interact with sulfate metabolism. For example, a one week exposure of Chinese cabbage to enhanced Cu levels (≥ 5 μM), resulted in a decreased plant biomass production, enhanced expression of the Group 1 high affinity sulfate transporters and enhanced sulfate uptake capacity (Chapter 3 and 4). The upregulation of the sulfate transporters in Chinese cabbage upon Cu2+ exposure was likely not only due to a higher sulfur demand necessary for the synthesis of metal-binding compounds (viz. phytochelatins), but might also be the consequence of a direct interference/reaction of Cu with the signal transduction pathway involved in the regulation of the sulfate transporters (Chapter 3). In the present chapter, the interaction between an enhanced Cu2+ level and sulfur nutrition was studied.

Plant growth conditions

10-day-old seedlings were transferred to 30 l containers and grown on a 25% Hoagland nutrient solution containing 0.5 mM sulfate for 7days and subsequently transferred to a fresh nutrient solution containing 0.5 mM sulfate (+S) or 0 mM sulfate (-S) at 0 and 4 µM CuCl2 for 1, 2, 3 and 4 days. Day and night temperatures were 25 and 20 °C ( 1 °C), respectively, and the relative humidity was 60-70%. The photoperiod was 14 h at a photon fluence rate of 400 20 µmol m–2 s–1 (within the 400-700 nm range) and 2.2 mW cm-2 UV-A+B radiation (within the 280-400 nm range) at plant height, supplied by Philips HPI-T (400 W) lamps as light source. UV-A+B radiation was measured with a Digital Ultraviolet Radiometer model 5.0 (Solartech Inc. Fenton, MI 48430, USA).

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Results and discussion

Exposure of Chinese cabbage to 4 µM Cu2+ resulted in a decreased plant biomass production, an increased dry matter content and an increased shoot to root ratio, as it has been observed previously (Fig. 1; Chapter 3 and 4). Root growth was more rapidly affected than shoot growth, resulting in an increased shoot to root ratio already after 2 days (Fig. 1). Similarly to previous observations (Koralewska et al. 2008; Stuiver et al. 2009), sulfate deprivation of Chinese cabbage resulted in a decreased plant biomass production and in a change in shoot to root biomass partitioning in favor of that of the root, as illustrated by a decrease in shoot to root ratio (Fig. 1).

Fig. 1. Impact of Cu2+ and sulfate deprivation on plant biomass production (gFW), dry matter content (%) and shoot to root ratio of Chinese cabbage. Seedlings were grown on a 25% Hoagland nutrient solution containing 0.5 mM sulfate in a climate-controlled room for 7 days and subsequently transferred to a fresh nutrient solution at 0.5 mM sulfate (+S) or 0 mM sulfate (-S) containing supplemental 0 or 4 µM CuCl2 for 1, 2, 3 and 4 days. Data represent the mean of 15 measurements with 3 plants in each (± SD). Different letters indicate significant differences between treatments within the day of exposure (p < 0.01, Student’s t-test).

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Fig. 2. Impact of Cu2+ and sulfate deprivation on nitrate, sulfate and thiol content (µmol g-1 FW) of Chinese cabbage. For experimental details see legends Fig. 1. Data represent the mean of 3 measurements with 3 plants in each (± SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

A simultaneous exposure of plants to sulfate deprivation and 4 μM Cu2+ resulted in an almost similar decrease of plant biomass production as observed upon Cu2+ exposure of sulfate-sufficient plants. The shoot to root biomass partitioning, however, was quite

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similar to that observed for Cu2+-exposed sulfate-sufficient plants (Fig. 1). There was an increase in dry matter content of the root of the Cu2+-exposed sulfate-deprived plants after 3 days, whereas that of the shoot remained unaffected (Fig. 1). Upon sulfate deprivation the biomass production at an enhanced Cu2+ level was only reduced after 4 days of exposure.

At sulfate-sufficient conditions, Cu2+ exposure resulted in a rapid increase in water-soluble non-protein thiol content in both root and shoot. However, the thiol accumulation was much more pronounced in the root than in the shoot; after 4 days its content had increased 4-fold and 1.5-fold in root and shoot, respectively (Fig. 2). It was evident that in Chinese cabbage only a small proportion of the increase in thiol content could be ascribed to a Cu2+-induced synthesis of phytochelatins (Chapter 3). The sulfate content of the root was hardly affected upon Cu2+ exposure, but its content in the shoot started to increase after 2 days, up to 1.5-fold after 4 days of exposure (Fig. 2). In contrast, the nitrate content decreased in both root and shoot upon Cu2+ exposure (Fig. 2).

Upon sulfate deprivation the thiol and sulfate content were strongly decreased in both root and shoot. However, the nitrate content of the shoot was increased, whereas that of the root was not affected upon sulfate deprivation (Fig. 2; Koralewska et al. 2008; Stuiver et al.2009). The thiol content in the root of plants simultaneously exposed to sulfate deprivation and 4 μM Cu2+ was increased after 1 day, thereafter it remained unaltered (Fig. 2). In the shoot, however, the thiol content was increased after 1 day and remained higher than that of the sulfate-deprived plants up to 4 days of exposure (Fig. 2). The strong decrease in thiol content in both root and shoot upon sulfate deprivation was most likely due to growth dilution and/or metabolism of thiol compounds to support the synthesis of other essential organic sulfur-containing compounds (e.g. proteins). The thiol content of plants simultaneously exposed to sulfate deprivation and 4 μM Cu2+

remained unaltered/higher in the root and shoot than in sulfate-deprived plants. This might indicate that part of the accumulated thiols upon Cu2+ exposure, presumably the phytochelatins fraction could not be re-metabolized. The nitrate content of Chinese cabbage simultaneously exposed to sulfate deprivation and Cu2+ was not substantially affected in the both root and shoot (Fig. 2).

78

Fig. 3. Impact of Cu2+ and sulfate deprivation on sulfate uptake capacity (µmol g-1 FW root h-1) of Chinese cabbage. For experimental details see legends Fig. 1. Data represent the mean of 3 measurements with 3 plants in each (± SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Exposure of Chinese cabbage to 4 µM Cu2+ resulted in an enhanced expression and activity of the sulfate transporters in the root (Fig. 3 and 4). The expression of the constitutively abundant sulfate transporter Sultr1;2 and the sulfate uptake capacity were already upregulated after 1 day of exposure (Fig. 3 and 4). In both root and shoot, also the Group 4 sulfate transporter Sultr4;1 was already upregulated upon 1 day of Cu2+ exposure, whereas in the root also Sultr4;2 was slightly upregulated upon 4 days of exposure (Fig. 4). The expression of APS reductase, the key regulating enzyme in sulfate reduction pathway, was first upregulated upon 1 day of Cu2+

exposure in the root, but after 2 days it was downregulated again to a level similar to that of non-exposed control plants (Fig. 4). Theexpression of APS reductase in the shoot, however, was not affected upon Cu2+ exposure (Fig. 4).

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Fig. 4. Impact of Cu2+ and sulfate deprivation on mRNA abundance of sulfate transporters (Sultr) and APS reductase (APR; Northern-blot analysis) in the root and shoot of Chinese cabbage. Equal RNA loading was determined by ethidium bromide staining of gels (shown in the bottom panels). For experimental details see legends Fig. 1.

The transient upregulation of APS reductase in the root may illustrate a temporary upregulation of the sulfate reduction pathway possibly needed for a Cu-induced synthesis of thiols (presumably glutathione and for a lesser part phytochelatins; Chapter 3), in response to excessive Cu taken up by the root (Fig. 3 and 4). Sulfate deprivation also resulted in a strongly enhanced expression of Sultr1;1 and Sultr1;2 in the root, an enhanced expression of Sultr2;2, Sultr4;1, Sultr4;2 and APS reductase in both root and shoot, and in

80

an increased sulfate uptake capacity of the root (Fig. 3 and 4; Koralewska et al. 2008; Stuiver et al. 2009). A simultaneous exposure of plants to sulfate deprivation and 4 μM Cu2+ hardly affected the observed upregulated expression of the sulfate transporters and APS reductase, but after 2 days it resulted in a slightly less upregulation of Sultr1;1 in the root along with the decrease in the sulfate deprivation-induced upregulation of the sulfate uptake capacity of the root (Fig. 3 and 4).

Conclusions

From the current results it was evident that sulfate deprivation had a more rapid negative effect on plant biomass production than an enhanced Cu2+ level. Cu2+ exposure disturbed the sulfate metabolism very rapidly and already after one day the expression and the activity of the sulfate transporters were upregulated. It is unlikely that the upregulation of the sulfate uptake could solely be attributed to a higher sulfur need upon Cu2+ exposure, since Cu2+ exposure also resulted in enhanced sulfate levels in the shoot. The consequences of sulfate deprivation on plant growth and the expression and activity of the sulfate transporters, such as the induction and upregulation of sulfate transporters and APS reductase were hardly further affected by an enhanced Cu2+ level. This indicated that sulfate deprivation more or less surpassed the development of Cu toxicity.

Atmospheric H2S nutrition hardly affects thetoxicity and impact of copper on sulfur

metabolism in Chinese cabbage

Chapter 6.

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Abstract

H2S exposure did not affect plant biomass production, dry matter, pigment and total sulfur content, if plants were grown at an ample sulfate supply to the root. There was a direct interaction between atmospheric H2S and pedospheric sulfate utilization and H2S exposure resulted in a downregulation of the sulfate uptake capacity and the expression of Sultr1;2 in the root. H2S exposure did not affect the toxicity of Cu2+, indicating that a higher sulfur status of the plant did not have any significance in Cu toxicity. There was a substantial increase in thiol content of the shoot (2-fold) and to a lesser extent of that in the root in H2S exposed plants. If plants were simultaneously exposed to H2S and enhanced Cu2+ concentrations, the thiol accumulation in the root even further increased. Both in absence and presence of H2S, high Cu2+ concentrations (> 10 µM) resulted in an upregulation of the sulfate uptake capacity, however, a H2S-induced partial downregulation of the sulfate uptake capacity also occurred at all Cu2+ concentrations. The H2S-induced downregulation of the expression of Sultr1;2 was hardly affected at increasing Cu2+

concentrations. Sulfate deprivation of Chinese cabbage resulted in a decreased biomass production, a decreased shoot to root ratio, an increased dry matter content (especially in the shoot), and in a decreased pigment and sulfur metabolite content, and in a strongly increased sulfate uptake capacity of the root. The Group 1, 2 and 4 sulfate transporters were all upregulated in both root and shoot. The impact of sulfate deprivation on growth and the expression and activity of the sulfate transporters were hardly further affected at enhanced Cu2+ levels. If sulfate-deprived plants were simultaneously exposed to H2S, biomass production was quite similar to that of sulfate-sufficient plants, though the shoot to root ratio remained lower. The upregulation of the expression of the sulfate transporters upon sulfate deprivation and that of APS reductase in absence and presence of enhanced Cu2+ in the shoot was largely or completely alleviated upon H2S exposure. At all conditions, the regular signal transduction pathway of sulfate transporters and APS reductase was overruled at high Cu tissue levels.

84

Introduction

Enhanced Cu2+ concentrations (≥ 5 μM) in the root environment were toxic for Chinese cabbage, which could in part be ascribed to a Cu-induced disturbed development of the chloroplasts, resulting in chlorosis and a reduced biomass production (Sheldon and Menzies 2005; Yruela 2005; Chapter 3 and 4). Upon UV radiation, enhanced Cu2+ concentrations had negative synergistic effects in Chinese cabbage, which was probably largely due to a higher accumulation of Cu in both root and shoot (Chapter 4). Sulfate deprivation had a more rapid negative effect on plant biomass production than an enhanced Cu2+ level in Chinese cabbage (Chapter 5). There was a strong accumulation of water-soluble non-protein thiols in the root and, to a lesser extent in the shoot upon Cu2+ exposure. The increase in thiolcontent in Chinese cabbage could only partially be ascribed to a Cu-induced synthesis of phytochelatins (Chapter 3). Phytochelatins and other thiols may be involved in the binding of excessive free Cu andits detoxification, and the Cu-induced synthesis of these thiols might require an enhanced sulfate uptake and assimilation (Inouhe 2005;Lee and Kang 2005; Sirko and Gotor 2007). Indeed, enhanced Cu contents in the root affected the regulation of the uptake and distribution of sulfate; there was an increase in expression and activity of the high affinity sulfate transporter Sultr 1;2. This increase was accompanied with an increase in total sulfur content in the shoot, which was mainly due to an accumulation of sulfate (Chapter 3 and 4). However, it needs still to be resolved to what extent the upregulation of the sulfate transporters was the consequence of a higher sulfur demand at higher Cu concentrations or the result of an interference/reaction of Cu with the signal compounds regulating the expression and activity of the sulfate transporters (Chapter 3).

In addition to sulfate taken up by the root, plants are able to utilize the foliarly-absorbed sulfurous air pollutants (SO2, H2S) as sulfur source for growth (De Kok et al. 2002, 2007, 2009; Yang et al. 2006; Koralewska et al. 2008). Atmospheric H2S is directly metabolized with high affinity into cysteine and subsequently into other organic sulfur compounds (De Kok et al. 1998, 2002, 2007, 2009). There was a direct interaction between atmospheric and pedospheric sulfur utilization in Brassica, and H2S exposure resulted in downregulation of sulfate uptake by the root and its and assimilation in the shoot

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(Westerman et al. 2000, 2001; Buchner et al. 2004; De Kok et al. 2007; Koralewska et al. 2008). In absence of sulfate in the root environment, an atmospheric level of 0.2 µl l-1 H2S appeared to be sufficient to cover the sulfur demand for growth of Brassica(Koralewska et al. 2008).

In the present chapter, the interaction between pedospheric sulfate and atmospheric H2S nutrition, and enhanced Cu2+ levels was studied, in order to get insight into the significance of the plant sulfur status inCu toxicity, and the interference of Cu with the signal transduction pathway regulating the expression and activity of the sulfate transporters (and APS reductase).

Plant growth conditions

10-day-old seedlings of Chinese cabbage were transferred to a 25% Hoagland solution at 0.5 mM sulfate (+S, sulfate-sufficient) or 0 mM sulfate (-S, sulfate-deprived), containing supplemental concentrations of 0, 5, 10 and 15 μM CuCl2 in 13 l containers (10 sets of plants per container, 3 plants per set) in fumigation cabinets and fumigated with0 or 0.2 µl l-1 H2S for 11 days.

Results

Impact of Cu, H2S and sulfate deprivation on growth, pigmentcontent and Cu content

Exposure of Chinese cabbage to 0.2 µl l-1 H2S for 11 days did not affect plant biomass production, shoot to root ratio, dry matter content and pigment content at sulfate-sufficient condition (Fig. 1 and 2). However, the Cu content was slightly higher in root and slightly lower in shoot at sulfate-sufficient conditions upon H2S exposure (Fig. 4). If plants were exposed to enhanced Cu2+ concentrations (≥ 5 µM) at sulfate-sufficient conditions, the biomass production of both root and shoot was decreased in both absence (+S) and presence of H2S (+S, H2S), whereas the shoot to root ratio was hardly affected (Fig. 1). At ≥ 10 µM Cu2+ there was an increase in dry matter content inboth root and shoot (Fig. 2).

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Fig. 1. Impact of Cu2+ and H2S exposure and sulfate deprivation on biomass production of Chinese cabbage. 10-day-old seedlings of Chinese cabbage were grown on a 25% Hoagland solution containing 500 µM sulfate (+S, light grey bars), fumigated with 0.2 µl l-1 H2S (+S,H2S, black bars) or 0 µM sulfate (-S, white bars), fumigated with 0.2 µl l-1 H2S (-S,H2S, dark grey bars) for 11 days. Plants were exposed to 0 to 15 µM Cu2+ in the root environment. The initial shoot and root weight was 0.059 0.004 and 0.009 0.003 g fresh weight, respectively. Data on biomass production (g FW) and shoot/root ratio represent the mean of 2 experiments with 9 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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At sulfate-sufficient conditions pigment content was only decreased at 15 µM Cu2+, whereas the chlorophyll a/b and chlorophyll/carotenoid ratio were hardly affected in both absence and presence of H2S (Fig. 3). The Cu content in both root and shoot of Chinese cabbage increased with the Cu2+ concentration (Fig. 4). At sulfate-sufficient conditions, the Cu content was increased up to 40-fold in root and up to 5-fold in shoot at 15 µM Cu2+. H2S exposure of Cu2+-exposed plants resulted in a lower Cu content at 15 µM and in root, and in shoot at all Cu2+ concentrations (Fig. 4).

The plant biomass production was strongly decreased upon a 11-day sulfate deprivation, whereas the shoot to root ratio was decreased and the dry matter content substantially increased (Fig. 1 and 2). Exposure of sulfate-deprived plants to enhanced Cu2+

concentrations resulted in a decreased biomass production of both root and shoot at 15 µM, whereas the shoot to root ratio was increased at ≥ 5 µM, but dry matter content of both root and shoot was hardly affected (Fig. 1 and 2). Sulfate deprivation resulted in a decreased pigment content, and upon exposure to Cu2+ the chlorophyll content and the chlorophyll a/b and chlorophyll/carotenoid ratio were hardly further affected (Fig. 3). The Cu content of sulfate-deprived plants was rather high in both root and shoot; it increased up to 56-fold in root and up to 5.5-fold in shoot at 15 µM Cu2+ (Fig. 4). H2S exposure of sulfate-deprived (-S, H2S) plants alleviated the development of sulfur deficiency symptoms. The plant biomassproduction, dry matter and pigment content were quite similar to that of sulfate-sufficient (+S) plants, however, the chlorophyll a/b was significantly higher in the presence of H2S (Fig. 1, 2 and 3). A simultaneous exposure of sulfate-deprived plants to Cu2+ and H2S resulted in a decreased plant biomass production at ≥ 5 µM, an increased shoot to root ratio at 15 µM, and an increased dry matter content of root at ≥ 10 µM (Fig. 1 and 2). There was a decrease in pigment content at ≥ 10 µM Cu2+, however, the chlorophyll a/b and chlorophyll/carotenoid ratio were hardly affected (Fig. 3). The Cu content of H2S-fumigated, sulfate-deprived plants was higher in root, and at enhanced Cu2+ concentrations, its content increased in both root and shoot. However, the Cu content of H2S-fumigated, sulfate-deprived plants remained significantly lower than that of sulfate-sufficient plants at all Cu2+ concentrations (Fig. 4). The excessive Cu taken up by the root was relatively immobile and only a minor

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proportion was transferred to the shoot (Fig. 4). The latter further decreased with increasing Cu2+ concentrations in the root environment at both sulfate-sufficient and sulfate-deprived conditions (Fig. 4).

Fig. 2. Impact of Cu2+ and H2S exposure and sulfate deprivation on dry matter content of Chinese cabbage. For experimental details see legends Fig. 1. Data on dry matter content (DMC; %) represent the mean of 2 experiments with 9 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Impact of Cu, H2S and sulfate deprivation on nitrogen andsulfur metabolite content

The content of nitrate, amino acids and total sulfur was not affected in sulfate-sufficient, H2S-exposed plants (Fig. 5 and 6). However, H2S exposure resulted in a slightly lower sulfate content of both root and shoot, a strong increase in the thiol content of the shoot, and to a

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lesser extent in the root (Fig. 6 and 7). Exposure to Cu2+ at enhanced concentrations (≥ 10 µM) resulted in a decreased nitrate content of both root and shoot in absence (+S) and presence of H2S (+S, H2S).The amino acid content was hardly affected upon Cu2+ exposure in both root and shoot in absence and presence of H2S (Fig. 5). The total sulfur content of the shoot, both in absence and presence of H2S, was substantially increased at ≥ 10 µM Cu2+, whereas that of the root remained unaltered (Fig. 6). The increase in total sulfur content upon exposure to Cu2+ could for the greater part be attributed to an enhance sulfate content (Fig. 6). There was an increase in water-soluble non-protein thiol content in the root and to a lesser extent in the shoot at ≥ 5 µM Cu2+ at sulfate-sufficient conditions. The increase in thiol content of the root was further increased upon a simultaneous exposure to Cu2+ and H2S, the thiol content of shoot was hardly further affected (Fig. 7).

Sulfate-deprived plants (-S) contained very low levels of total sulfur, sulfate and thiols in both root and shoot. Moreover, there was a strong increase in amino acid content, whereas the nitrate content was hardly affected (Fig. 5 and 6). If sulfate-deprived plants were exposed to enhanced Cu2+ concentrations, the total sulfur and thiol content in shoot and sulfate content in both root and shoot were hardly affected (Fig. 5, 6 and 7). However, the total sulfur and thiol content in the root was increased at ≥ 5 µM and the nitrate content decreased at 15 µM, and the nitrate content in the shoot increased at ≥ 5 µM Cu2+ (Fig. 5, 6 and 7). H2S exposure of sulfate-deprived plants (-S, H2S) resulted in a substantial increase in total sulfur and thiol content of both root and shoot, whereas the sulfate content remained low (Fig. 6). The amino acid content was quite similar to that of sulfate-sufficient plants (Fig. 5). A simultaneous exposure of sulfate-deprived plants to Cu2+ and H2S resulted in a slight decrease in nitrate content in both root and shoot at ≥ 15 µM, whereas the amino acid, total sulfur and sulfate contents were hardly affected. Moreover, there was a strong increase in thiol content in the root at ≥ 5 µM, and a slight increase in the shoot at ≥ 10 µM upon a simultaneous Cu2+ and H2S exposure of sulfate-deprived plants (Fig. 5, 6 and 7).

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Fig. 3. Impact of Cu2+ and H2S exposure and sulfate deprivation on pigment content of Chinese cabbage. For experimental details see legends Fig. 1. Data on chlorophyll content (chl a+b; mg g-1 FW), chlorophyll a to chlorophyll bratio (Chl a/b), and chlorophyll to carotenoid ratio (Chl/Car) represent the mean of 2 experiments with 3 measurements in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Fig. 4. Impact of Cu2+ and H2S exposure and sulfate deprivation on copper content of Chinese cabbage. For experimental details see legends Fig. 1. Data represent the mean of 3 measurements with 9 plants each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

Impact of Cu, H2S and sulfate deprivation on sulfate uptakecapacity and expression of sulfate transporters and APSreductase

Exposure of Chinese cabbage to H2S resulted in a downregulation of the sulfate uptake capacity at sulfate-sufficient conditions (+S; Fig. 8). Both in absence and presence of H2S, concentrations > 10 µM Cu2+ resulted in an upregulation of the sulfate uptake capacity, however, the H2S-induced partial downregulation of the sulfate uptake capacity also occurred at all Cu2+ concentrations. There was an increase in sulfate uptake capacity at enhanced Cu2+ concentrations in both absence (+S) and in presence of H2S (+S, H2S) at ≥ 10 µM

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Cu2+ (Fig. 8). This increase in sulfate uptake capacity was accompanied with an enhanced expression of Sultr1;2 in root, whereas Sultr1;1 was hardly expressed at sulfate-sufficient conditions. The expression of Sultr1;2 was downregulated by 70% upon H2S exposure and its expression was increased by 190% upon exposure to 15 µM Cu2+ in root (Fig. 9). H2S exposure to sulfate-sufficient Chinese cabbage resulted in a downregulation of expression of the sulfate transporters and that of APS reductase in both root and shoot (Fig. 9). The expression of Sultr2;2 was enhanced by 120% at ≥ 5 µM Cu2+ in root, whereas Sultr2;2 was hardly expressed in the shoot of Chinese cabbage. The Cu-induced increased expression of Sultr2;2 was absent upon H2S exposure (Fig. 9). The expression of Sultr4;1 increased with 50% in the root at 15 µM Cu2+ in absence and presence of H2S, whereas, its expression in the shoot was slightly decreased (30%). The expression of Sultr4;2 was increased by 70% in root at 15 µM Cu2+ in absence and presence of H2S, whereas, its expression was hardly expressed in the shoot (Fig. 9). The expression of APS reductase was increased by 60% in root at 15 µM Cu2+ in absence and presence of H2S, whereas its expression was hardly affected by Cu2+ exposure and upon H2S exposure down regulated by 70% in shoot (Fig. 9).

Sulfate deprivation (-S) resulted in an increased sulfate uptake capacity of the root of Chinese cabbage, which was even higher upon H2S exposure (Fig. 8). Exposure of sulfate-deprived plants to enhanced Cu2+ concentrations did not affect the sulfate uptake capacity. A simultaneous Cu2+ and H2S exposure of sulfate-deprived plants resulted in an increased sulfate uptake capacity only at 15 µM (Fig. 8). The expression of the sulfate transporters and APS reductase was strongly enhanced upon sulfate deprivation in both root and shoot (Fig. 9). The expression of Sultr1;1 was decreased by 80% at 15 µM Cu2+ in root and was hardly affected upon H2S exposure at sulfate-deprived conditions. Sultr2;2 was slightly expressed at sulfate-deprived conditions but it was hardly affected by Cu2+

exposure in both root and shoot. Upon H2S exposure Sultr1;2 and Sultr2;2 were hardly expressed in shoot in both absence and presence of enhanced Cu2+ concentrations in the root environment (Fig. 9).

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Fig. 5. Impact of Cu2+ and H2S exposure and sulfate deprivation on nitrate and amino acids content of Chinese cabbage. For experimental details see legends Fig. 1. Data on nitrate and amino acids content (µmol g-1 FW) represent the mean of 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Fig. 6. Impact of Cu2+ and H2S exposure and sulfate deprivation on total sulfur and sulfate content of Chinese cabbage. For experimental details see legends Fig. 1. Data on total sulfur (µmol g-1 DW), and sulfate content (µmol g-1 FW) represent the mean of 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Upon sulfate deprivation, the expression of Sultr4;1 and Sultr4;2 in root was hardly affected by Cu2+ and by a simultaneous Cu2+ and H2S exposure. The expression of these transporters was decreased by80% upon H2S exposure in shoot in absence and presence of enhanced Cu2+ concentrations. The expression of APS reductase was hardly affected in root upon Cu2+ and upon a simultaneous Cu2+ and H2S exposure at sulfate-deprived conditions. However, its expression was downregulated by 75-90% in shoot upon H2S exposure in absence and presence of enhanced Cu2+ concentrations in the root environment (Fig. 9).

Fig. 7. Impact of Cu2+ and H2S exposure and sulfate deprivation on thiol content of Chinese cabbage. For experimental details see legends Fig. 1. Data on thiol content (µmol g-1 FW) represent the mean of 3 measurements with 3 plants in each ( SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Discussion

Similar to previous observations, Chinese cabbage was able to utilize foliarly absorbed H2S and replace sulfate, taken up by the root, as sulfur source for growth (Koralewska et al. 2008). At sulfate-sufficient conditions, H2S exposure did not affect plant biomass production, dry matter, pigment and total sulfur content, but it resulted in a substantial increase in thiol content of the shoot (2-fold) and to a lesser extent of that in the root. If sulfate-sufficient plants were simultaneously exposed to H2S and enhanced Cu2+ concentrations in the root environment, the thiol accumulation in the root was further increased. Exposure of sulfate-sufficient Chinese cabbage to H2S did not affect the toxicity of Cu2+ and the Cu-induced decrease in plant biomass production was quite comparable, even though the Cu content of the shoot was significantly lower upon H2S exposure. This indicated that a higher sulfur status of the plant did not have any significance in Cu toxicity.

Fig. 8. Impact of Cu2+ and H2S exposure and sulfate deprivation on sulfate uptake capacity of Chinese cabbage. For experimental details see legends Fig. 1. Data on sulfate uptake capacity (µmol g-1 FW root h-1) represent the mean of two independent experiments with 3 measurements with 3 plants each (SD). Different letters indicate significant differences between treatments (p < 0.01, Student’s t-test).

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Fig. 9. Impact of Cu2+ and H2S exposure and sulfate deprivation on mRNA abundance of sulfate transporters (Sultr) and APS reductase (APR) (Northern-blot analysis) in the root and shoot of Chinese cabbage. Equal RNA loading was determined by ethidium bromide staining of gels (shown in the bottom panels). A representative set of data from two independent experiments is given. For experimental details see legends Fig. 1.

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Similar to previous observations, there was a direct interaction between atmospheric H2S and pedospheric sulfate utilization in Chinese cabbage (Westerman et al. 2000, 2001; Buchner et al. 2004; De Kok et al. 2007; Koralewska et al. 2008). At sulfate-sufficient conditions, H2S exposure resulted in a downregulation of the sulfate uptake capacity and the expression of Sultr1;2 in the root. Both in absence and presence of H2S, increasing Cu2+ concentrations resulted in an upregulation of the sulfate uptake capacity. However, a H2S-induced partial downregulation of the sulfate uptake capacity occurred at all Cu2+ concentrations. The H2S-induced downregulation of the expression of Sultr1;2 was hardly affected at increasing Cu2+

concentrations and it even depressed the Cu-induced upregulation of this transporter. Moreover, the Cu-induced upregulation of Sultr1;1 and Sultr2;2 were alleviated upon H2S exposure, the latter transporter is involved in the vascular transport of sulfate (Buchner et al. 2004; Parmar et al. 2007; Koralewska et al. 2010).

The expression of Sultr4;1 and Sultr4;2 was upregulated at 15 μM Cu2+ in root at sulfate-sufficient conditions. Generally, Group 4 sulfate transporters are only upregulated upon sulfur limitation, when plants remobilize and/or redistribute available sulfate, for instance from vacuoles (Buchner et al. 2004; Parmar et al. 2007; Koralewska et al. 2009a,b, 2010; Stuiver et al. 2009; Chapter 5).

Sulfate deprivation of Chinese cabbage resulted in a decreased biomass production, a decrease in shoot to root ratio, an increased dry matter content (especially in the shoot) and in a decrease in pigment and sulfur metabolite content. As observed before, sulfate deprivation resulted in a strongly increased sulfate uptake capacity of the root, and the Group 1, 2 and 4 sulfate transporters were all upregulated in both root and shoot (De Kok et al. 1997; Westerman et al. 2000; Buchner et al. 2004; Yang et al. 2006; Koralewska et al. 2007, 2008; Chapter 5). Similar to previous observations (Chapter 5), the consequences of sulfate deprivation on growth and the expression and activity of the sulfate transporters were hardly further affected byenhanced Cu2+ levels.

If sulfate-deprived plants were simultaneously exposed to H2S, plant biomass production was quite similar to that of sulfate-sufficient plants. However, the shoot to root ratio remained lower, demonstrating that upon H2S exposure in the absence of sulfate in the root environment, plants still invested relatively more biomass in

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root, even though the sulfur supply was sufficient to cover the sulfur demand for growth (Buchner et al. 2004; Koralewska et al. 2007, 2008). If plants were grown with H2S as the sole sulfur source, the impact of enhanced Cu2+ concentrations on biomass production was comparable to that observed for sulfate-sufficient plants and the degree of Cu toxicity was similar. However, the upregulation of the expression of the sulfate transporters upon sulfate-deprivation and APS reductase in absence and presence of enhanced Cu2+ in the shoot was largely or completely alleviated upon H2S exposure.

It is generally presumed that metabolic products of sulfate assimilation, viz. thiols as sulfide, cysteine, and glutathione act as signals in the regulation of the expression and activity of the sulfate transporters (Hawkesford and De Kok 2006). However, at enhanced Cu2+ concentrations, there was hardly any relation between the abundant thiol levels and the expression and activity of the sulfate transporters at sulfate-sufficient, sulfate-deprived conditions, both in presence and absence of H2S. Apparently, the regular signal transduction pathway of the sulfate transporters was overruled at high Cu tissue levels. The expression and activity of APS reductase is generally downregulated at enhanced thiol levels (Westerman et al. 2001; Durenkamp et al. 2007; Koralewska et al. 2008). However, in sulfate-sufficient plants its expression was hardly affected in the root in presence of H2S and enhanced Cu2+ concentrations, irrespective of the high thiol levels present under these conditions. Only in the shoot the increase in thiol content upon H2S exposure was accompanied with a slight decrease in expression of APS reductase.

Despite the observations that enhanced Cu2+ concentrations interfered with the regulation of the uptake and assimilation of sulfur in plants, there was no direct relation between their sulfur status and Cu toxicity. Evidently, total sulfur, sulfate and thiol content, and the level of activity and expression of the sulfate transporters and of APS reductase, which all strongly differed in presence and absence of sulfate and/or H2S as sulfur source for growth, had hardly impact on the development of Cu toxicity in Chinese cabbage.

100

Conclusions

In Chinese cabbage there was a direct interaction between atmospheric H2S and pedospheric sulfate nutrition and H2S exposure resulted in a downregulation of sulfate uptake by the root. There was an upregulation of the sulfate uptake capacity at high Cu2+

concentrations (> 10 µM) in both absence and presence of H2S, though the H2S-induced partial downregulation of the sulfate uptake capacity occurred at all Cu2+ concentrations. Upon sulfate deprivation, foliarly absorbed H2S could replace sulfate as sulfur source for growth, however, the toxicity of Cu2+ was similar to that observed in sulfate-sufficient plants both in absence and presence of H2S, indicating that the sulfur status of the plant did not have any significance in Cu tolerance. Moreover, the presumed signal transduction pathway involved in the regulation of the expression and activity of the sulfate transporters (APS reductase) appeared to be overruled at high Cu tissue levels.

General discussion

Chapter 7.

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The physiological basis for the toxicity of Cu in Chinesecabbage

Cu is an essential nutrient for plant growth and development, but at enhanced levels in the root environment it may rapidly become phytotoxic (Chapter 3, 4, 5 and 6). The toxicity of Cu depends on the concentration of Cu accumulated, growth stage of plants and the duration of the exposure (Clemens 2001; Medoza-Cózatl et al. 2005; Chapter 3, 4, 5 and 6) and on the light quality, viz. UV abundance (Chapter 4). Generally, Cu toxicity resulted in an inhibition of root and shoot biomass production, leaf chlorosis, a loss of photosynthetic activity (Mocquot et al. 1996; Yruela 2005, 2009; Xiong et al. 2006; Han et al. 2008; Burkhead et al. 2009; Chapter 3, 4, 5 and 6) and an altered root morphology (Panou-Filotheou and Bosabalidis 2004; Sheldon and Menzies 2005). Root growth of Chinese cabbage was slightly more affected than shoot growth at high Cu2+ concentrations (≥ 5 µM; Chapter 3, 4 and 5).

Exposure of Chinese cabbage to enhanced Cu2+ concentrations in the root environment resulted in a substantial increase in the Cu content of both root and shoot. The excessive Cu accumulated in the root was relatively immobile and only a minor proportion was transferred to the shoot (Chapter 3, 4 and 6). Cu has a high affinity for carbonylic, carboxylic, phenolic and sulfydryl groups and therefore it is strongly bound to the cell wall, which presumably limits both influx of Cu into the symplast and its translocation to the shoot (Quartacci et al. 2003). A 3- to 5-fold increase in Cu content of the shoot of Chinese cabbage already resulted in a 50% decrease in plant biomass production (Table 1). Under normal conditions the Cu content in plants ranges from 0.08 to 0.24 µmol g-1 dry weight, whereas incabbage Cu already became toxic when the leaf tissue level exceeded0.4 µmol g-1 dry weight (Macnicol and Beckett 1985; Marschner 1995;Krämer 2010).

A simultaneous exposure of plants to enhanced Cu levels and UV radiation may result in synergistic negative effects on plant growth and functioning (Lupi et al. 1998; Babu et al. 2003). If Chinese cabbage was exposed to enhanced Cu2+ concentrations in the root environment, its toxic effects occurred more rapidly in presence than in absence of UV radiation (Chapter 4). The content of Cu in both root and shoot of Chinese cabbage was strongly enhanced upon UV

104

radiation (up 40%). The higher Cu toxicity in presence of UV may largely be attributed to the observed higher Cu content in root and shoot (Chapter 4).

Table 1. Cu content in plant tissue at control and EC50 (half maximal effective concentration at which 50% plant biomass production was decreased).

Cu content (μmol g-1 FW)Control EC50

Root 0.02 – 0.10 0.7 – 1.0Shoot 0.01 – 0.03 0.05 – 0.09Plant 0.01 – 0.04 0.1 – 0.3

The physiological basis for the phytotoxicity of Cu is still ambivalent. Cu has the potential to accelerate the formation of reactive oxygen species in plant tissue (Pinto et al. 2003). For instance, the toxicity of Cu for chloroplasts functioning was enhanced at high light intensities, which was presumed to be due to an enhanced production of hydroxyl radicals (Yruela et al. 1996; Yruela 2009). The toxicity of Cu may also be ascribed to its possible reaction with thiol groups of proteins, thereby inhibiting enzyme activity andprotein functioning; moreover it may replace other essential metals in proteins (De Vos et al. 1993; Yruela 2009).

Enhanced Cu concentrations may interfere with the biosynthesis of photosynthesis machinery and modify the pigments and proteins of the thylakoid membrane network (Pätsikkä et al. 2002; Yruela 2005, 2009). From the current study it became evident that chlorosis of the shoot of Chinese cabbage at high Cu concentrations was likely not due to pigment degradation, but the consequence of hinderedchloroplast development (Chapter 3 and 4). The Cu-induced chlorosis in Chinese cabbage was accompanied by a decrease in photosynthetic activity, however, both the rate of photosynthesis, expressed on chlorophyll basis, and the quantum yield of photosystem II (Fv/Fm

ratio) were hardly affected up to 10 µM Cu2+. The Fv/Fm ratio was only affected when leaves of Chinese cabbage had started to become necrotic (Chapter 3 and 4). The impact of enhanced Cu levels on chloroplast development and/or functioning may be attributed to a Cu-induced Fe deficiency or by the substitution of the central Mg ion of chlorophyll by Cu (Pätsikkä et al. 2002; Küpper et al. 2003).

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The overall mineral nutrient composition of Chinese cabbage was affected upon Cu2+ exposure at enhanced concentrations in the root environment. Cu accumulation at enhanced levels in the roots may result in an unbalanced mineral nutrient uptake and distribution in Chinese cabbage. It is not yet well characterized to what extent a change in mineral composition is involved in the onset of the phytotoxicity of Cu (Chapter 3).

Interaction between Cu and sulfur metabolism

Similar to all other organisms, plants possess homeostatic mechanisms to control the concentrations of essential metal ions in different cellular compartments, in order to prevent the occurrence of toxic effects of excessive levels of free metal ions in the cytoplasm (Clemens 2001; Palmer and Guerinot 2009). Cu is a strong activator of phytochelatins biosynthesis, and can form a complex with phytochelatins. However, it is still doubtful to what extent phytochelatin-Cu complexes are sequestered in the vacuole (Cobbett and Goldbrough 2002; Yruela 2009). In addition to the sulfur-rich phytochelatins, glutathione itself (or even free cysteine) and low molecular weight cysteine-rich proteins may be involved in Cu binding and its detoxification in plants. This would imply a direct interference of enhanced tissue Cu levels with the regulation of sulfate uptake and its assimilation in the plant (Cobbett and Goldsbrough 2002; Cobbett2003; Verkleij et al. 2003; Lee and Kang 2005; Clemens 2001; Sirko and Gotor 2007).

Exposure of Chinese cabbage to enhanced Cu2+ concentrations in the root environment resulted in an accumulation of water-soluble non-protein thiols in the root, and to lesser extent in the shoot (Chapter 3, 4, 5 and 6). However, this enhancement could only partially be ascribed to a Cu-induced synthesis of phytochelatins (PC2

and PC3; Chapter 3). The enhanced thiol levels were most likely due the accumulation of reduced glutathione and may be for a lesser part to cysteine (De Kok et al. 2007; Chapter 3).

The high affinity sulfate transporters Sultr1;1 and Sultr1;2 are involved in the primary uptake of sulfate by the root (Hawkesford 2003, 2007; Koralewska et al. 2008; Takahashi and Saito 2008). From the present observations it was evident, that at ample sulfate

106

supply Sultr1;2 was the principal sulfate transporter responsible for sulfate uptake by Chinese cabbage (Chapter 3, 4, 5 and 6). Exposure of Chinese cabbage to enhanced Cu2+ concentrations resulted in upregulation of expression of Sultr1;2, accompanied with an increased sulfate uptake capacity of the root (Fig. 1; Table 2; Chapter 3, 4, 5 and 6).

Fig. 1. Relationship between Cu content in the root tissue and sulfate uptake capacity of Chinese cabbage (derived from Chapter 3, 4 and 6).

It was doubtful whether the observed upregulation of the expression and activity of sulfate transporters in Chinese cabbage was solely due to a higher sulfur demand at higher Cu tissue contents. Since, it is unlikely that the observed enhancement of the thiol compounds in Chinese cabbage at high Cu2+ concentrations would require substantial upregulation of the sulfate uptake, since this sulfur proportion never exceeded more than 5 % of the total sulfur content(Chapter 3). Moreover, the toxicity of Cu was hardly affected by the plant sulfur nutritional status. Biomass production of Chinese cabbage upon sulfate deprivation to Chinese cabbage was severely affected and the simultaneous exposure of sulfate-deprived plants to enhanced

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Cu2+ concentrations had hardly additional effects. It was obvious, that the development of sulfur deficiency was more rigorously and more or less overruled the occurrence of Cu toxicity (Chapter 5 and 6). Moreover, if Chinese cabbage was grown at sulfate, at H2S or at both simultaneously as sulfur source, the Cu-induced decrease in plant biomass production was quite comparable at all conditions (Chapter 6). From the relationship between Cu content of root tissue and sulfate uptake capacity it was obvious, that the activity of the sulfate transporter Sultr1;2 increased with the Cu content up to 0.7–1.0 μmol g-1 FW root, at which visible injury symptoms started to develop (chlorosis and necrosis; Fig. 1). At higher concentrations is decreased again.

Table 2. Sulfur metabolite content and regulation of sulfur metabolism as affected by enhanced Cu2+ concentrations in the root environment. , upregulation; , downregulation; , not affected.

Upon Cu2+ exposure Root ShootSulfur metabolite contentTotal sulfur Sulfate Thiols Regulation of sulfur metabolismSulfate uptake Sultr1;1 Sultr1;2 APS reductase

It is generally presumed that sulfate itself or metabolic products of the sulfate assimilation, viz. sulfide, cysteine, glutathione, would act as signals in the regulation of the expression and activity of the sulfate transporters (Hawkesford and De Kok 2006). At an ample sulfur supply the levels of these compounds would act as repressors of the sulfate transporters. Likewise, high levels of reduced sulfur compounds (sulfide, cysteine and glutathione) would downregulate the expression and activity of APS reductase (Westerman et al. 2001; Durenkamp et al. 2007; Koralewska et al. 2008). Whereas, upon sulfate deprivation, low tissue levels of these sulfur metabolites are generally accompanied by an upregulated (de-repressed) expression and activity of the sulfur transporters and APS reductase (Chapter 5

108

and 6; Buchner et al. 2004; Parmar et al. 2007; Koralewska et al. 2009a,b; Stuiver et al. 2009). However, if Chinese cabbage was exposed to enhanced Cu2+ concentrations, the expression and activity of Sultr1;2 in roots and the expression of Sultr4;1 and Sultr4;2, and of APS reductase in both root and shoot were upregulated, even at an enhanced sulfate and thiol content in root and shoot (Chapter 3, 4, 5 and 6, Table 2).

Fig. 2. The possible significance of H2S in the cross-talk between the sulfate reduction pathway in the chloroplasts/plastids and the transcription of sulfate transporters in the nucleus.

The majority of plant cells in both root and shoot are able to reduce and assimilate sulfate, enabling local cellular signaling of the uptake, subcellular distribution and reduction of sulfate (Chapter 3). However, a key unresolved issue is the signal transduction in the cross-talk between the sulfate reduction pathway in the chloroplasts/plastids and the transcription of sulfate transporters/sulfate reducing enzymes in

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the nucleus (Fig. 2). From human and animal physiology it has become evident that in addition to nitric oxide (NO) and carbon monoxide (CO), also H2S might function as an endogenous gaseous transmitter, where it may target KATP channel proteins and the cAMP-dependent protein kinase pathway (Wang 2002; Mancardi et al. 2009). In prokaryotes is has been shown that sulfide is involved in transcriptional regulation of the cys-operon, for genes involved in sulfur uptake and assimilation (Kredich 1993). H2S might have a similar function in plants - it is the first product of the sulfatereduction pathway - as an endogenous gaseous transmitter in the cross-talk between the sulfate reduction pathway in chloroplast/plastid and the transcription of sulfate transporters/sulfate reducing enzymes in the nucleus (De Kok et al. 2011; Fig. 2).

At the cellular pH, H2S is largely undissociated and in this form it may easily pass membranes (De Kok et al. 2007). Evidently, plants grown under normal conditions emit minute levels of H2S, which emission has been presumed to be a regulatory step in the homeostasis of the sulfur pools in plants (Rennenberg 1984; Schröder1993; Bloem et al. 2007). At a whole plant level, is has been shown that H2S exposure may diminish the activity of sulfate reduction in the shoot by a downregulation of the expression and activity of APS reductase (Durenkamp et al. 2007; Chapter 6), the key-regulating enzyme in the sulfate reduction pathway, and it may result in a downregulation of the expression of sulfate transporters in the shoot (Westerman et al. 2000, 2001; Buchner et al. 2004; Durenkamp et al. 2007; Koralewska et al. 2008; Chapter 6). From the current study it was evident that the presumed signal transduction pathway in the regulation of expression and the activity of the sulfate transporters and APS reductase (sulfate and thiols) was by-passed or overruled, if Chinese cabbage was exposed to enhanced Cu2+ in the root environment. H2S is rapidly reacting with free Cu2+ ions yielding in the formation of CuS as precipitate (Davidson et al. 2001; Tran et al. 2003; Yaşşyerli et al. 2003). If H2S would function as an endogenous gaseous transmitter in the cross-talk between the sulfate reduction pathway in chloroplast/plastid and the transcription of sulfate transporters/sulfate reducing enzymes in the nucleus, a Cu-induced decrease in H2S concentration might be responsible for the observed upregulation of the expression and activity of the sulfate transporters and the expression of APS reductase.

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References

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Summary

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Copper is an essential nutrient for plants, however, at enhanced concentrations in the root environment it may rapidly becomephytotoxic (Chapter 1). The toxic effects of Cu depend on the growth stage of the plant, duration of the exposure and the level of Cu accumulated. The typical Cu content in plants ranges from 0.08 to 0.24 µmol g-1 dry weight, and Cu toxicity generally occurs when the leaf tissue content exceeds 0.4 µmol g-1 dry weight. Arable soils may contain high levels of Cu and/or other toxic metals as the consequence of intensive agricultural practice, the use of animal manure as organic fertilizers, and of Cu-containing fungicides. Enhanced Cu levels in crop plants might not only negatively affect plant growth and functioning, but might also enter the food chain.

Sulfur is a macronutrient, essential for plant growth and its adaptation to the environment. Generally, sulfate taken up by the root is the primary sulfur source for plants. Moreover, plants are also able to utilize foliarly absorbed sulfurous air pollutants (viz. SO2, H2S) as sulfur source. Distinct sulfate transporter proteins mediate the uptake, transport and sub-cellular distribution of sulfate. Their expression and activity are modulated by the sulfur status of the plant. The synthesis of sulfur-rich metabolites (viz. PCs, GSH) is generally accelerated upon Cu exposure and may be involved in Cu binding, implying a direct significance of sulfur metabolism in Cu detoxification. In this thesis the physiological basis of the phytotoxicity of Cu and significance of sulfur metabolism in its detoxification have been investigated in Chinese cabbage.

Chapter 2 gives an overview of materials and methods used in this thesis. In all experiments Chinese cabbage (Brassica pekinensis(Lour.) Rupr. cv. Kasumi F1 (Nickerson-Zwaan, Made, The Netherlands) was used. In all experiments plants were cultivated on nutrient solution.

In Chapter 3 the toxicity of enhanced Cu concentrations and its interference with the regulation of the uptake, distribution and metabolism of sulfate was studied. Exposure of Chinese cabbage to enhanced Cu2+ concentrations resulted in leaf chlorosis, a loss of photosynthetic capacity and lower biomass production at ≥ 5 µM. The decrease in pigment content was likely not the consequence of degradation, but due to a hindered chloroplast development upon Cu exposure. The Cu content of the root increased with the Cu2+

concentration, though only a minor proportion of it was transferred to

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the shoot. Enhanced Cu2+ concentrations substantially affected the overall mineral nutrient composition at ≥ 5 µM Cu2+. A high Cu content in the root affected the uptake, distribution and metabolism of sulfate in Chinese cabbage. At ≥ 1 µM Cu2+ there was a strong increase in water-soluble non-protein thiol content in the root and to a lesser extent in the shoot, which could only partially be ascribed to a Cu-induced enhancement of the phytochelatins content. The activity of the sulfate transporters in the root was slightly enhanced at 2 and 5 µM Cu2+, accompanied by an enhanced expression of the Group 1 high affinity transporter Sultr1;2, and the Group 4 transporters Sultr4;1 and Sultr4;2. The expression of APS reductase was hardly affected in root and shoot at enhanced Cu2+ concentrations. The upregulation of the sulfate transporters was likely not only due to ahigher sulfur demand at higher Cu concentrations, but also the consequence of interference of Cu itself with the regulatory signal transduction pathway involved in the expression and activity of the sulfate transporters.

In Chapter 4 the impact of UV radiation on Cu toxicity and its interaction with the regulation of the uptake and metabolism of sulfate was investigated. The negative effects of enhanced Cu2+

concentrations on biomass production and pigment content of Chinese cabbage were strongly increased in presence of UV. Chlorophyll afluorescence (Fv/Fm) was only decreased when leaf tissue started to become necrotic. The expression and activity of the high affinity sulfate transporter Sultr1;2 were enhanced at ≥ 2 µM in presence of UV, and at ≥ 5 µM Cu2+ in absence of UV. Furthermore, in the shoot, the expression of Sultr4;1 was upregulated at ≥ 5 µM Cu2+ in presence and absence of UV and the expression of Sultr4;2 was upregulated at 10 µM Cu2+ in presence of UV. The enhanced Cu toxicity in presence of UV was largely due to a UV-induced enhanced accumulation of Cu in both root and shoot.

In Chapter 5 the impact of enhanced Cu concentrations and sulfate deprivation on the uptake and metabolism of sulfate in Chinese cabbage was studied. Exposure of plants to 4 µM Cu2+ resulted in a reduced plant biomass production and an increased shoot to root ratio at both sulfate-sufficient and sulfate-deprived conditions. However, sulfate deprivation had a more rapid negative effect on plant biomass production than enhanced Cu2+ concentrations. The expression and activity of the sulfate transporters and the expression of APS

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reductase in Chinese cabbage were rapidly up-regulated (already after 1 or 2 days) upon exposure to 4 μM Cu2+, sulfate deprivation and their combination. The impact of sulfate deprivation on the expression and activity of the sulfate transporters was hardly further affected by enhanced Cu2+ concentrations, which indicated that occurrence of sulfur deficiency symptoms upon sulfate deprivation more or less overruled the development of Cu toxicity.

In order to get more insight into the significance of the plant sulfur status in Cu toxicity, the interaction between pedospheric sulfate and atmospheric H2S nutrition, and enhanced Cu2+ concentrations wasstudied in Chapter 6. There was a direct interaction between atmospheric and pedospheric sulfur nutrition in Chinese cabbage and H2S exposure resulted in a downregulation of sulfate uptake by the root. Exposure to H2S resulted in an increased thiol content in the shoot and to a lesser extent in the root. The thiol content in the root was further increased upon a simultaneous exposure to H2S and enhanced Cu2+ concentrations. There was an upregulation of the sulfate uptake capacity at high Cu2+ concentrations (> 10 µM) in both absence and presence of H2S, though the H2S-induced partial downregulation of the sulfate uptake capacity occurred at all Cu2+

concentrations. The presumed signal transduction pathway involved in the regulation of the expression and activity of the sulfate transporters (APS reductase) appeared to be overruled at high Cu tissue levels. Upon sulfate deprivation, foliarly absorbed H2S could replace sulfate a sulfur source for growth, however, the toxicity of Cu2+ was quite similar to that observed in sulfate-sufficient plants, also in presence of H2S. Evidently, the plant sulfur status did not have any significance in Cu tolerance.

From the research presented in this thesis it can be concluded (Chapter 7) that enhanced Cu2+ concentrations in the root environment are toxic for Chinese cabbage. The presence of UV strongly enhanced Cu toxicity. Enhanced Cu2+ concentrations may interfere with the regulation of the uptake and assimilation of sulfur in Chinese cabbage, but there was no direct relation between the plant sulfur status and Cu toxicity. It is generally presumed that sulfate itself or metabolic products of the sulfate assimilation, viz. thiols (glutathione), would act as signals in the regulation of the expression and activity of the sulfate transporters. From the present study it was evident that the presumed signal transduction pathway in regulation

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of expression and activity of the sulfate transporters and APS reductase (sulfate and thiols) were by-passed or overruled, if Chinese cabbage was exposed to enhanced Cu2+ in the root environment. It is suggested that undissociated H2S may function as an endogenous gaseous transmitter in the cross-talk between the sulfate reduction pathway in chloroplasts/plastids and the transcription of sulfate transporters/sulfate reducing enzymes in the nucleus. A Cu-induced decrease in subcellular H2S concentration might be responsible for the observed upregulation of the expression and activity of the sulfate transporters and the expression of APS reductase.

Samenvatting (Summary in Dutch)

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Koper (Cu) is een essentiële voedingsstof voor planten, maar bij hoge concentraties in het wortelmilieu kan het ook toxisch worden (Hoofdstuk 1). De toxische effecten van Cu zijn afhankelijk van het groeistadium van de plant, de duur van de blootstelling en de concentratie van Cu in de plant. Het Cu gehalte in planten varieert van 0,08 tot 0,24 µmol g-1 drooggewicht, en Cu toxiciteit treedt in het algemeen op wanneer het bladweefsel meer dan 0,4 µmol g-1

drooggewicht bevat. Landbouwgronden kunnen verontreinigd zijn met Cu en andere giftige metalen, als gevolg van intensieve landbouw, het gebruik van dierlijke meststoffen en van Cu-houdende fungiciden. Te hoge Cu concentraties hebben niet alleen een negatieve invloed op de groei en fysiologie van planten, maar vormen ook een risico voor de voedselketen.

Zwavel is een macronutriënt die essentieel is voor de groei van planten en hun aanpassing aan het milieu. Onder normale omstandigheden wordt sulfaat, dat wordt opgenomen door de wortels, gebruikt als zwavelbron voor de plant. Planten kunnen ook zwavelgassen (m.n. SO2, H2S), welke door het blad (via de huidmondjes) worden opgenomen, gebruiken als zwavelbron voor de groei. Verschillende sulfaattransporter eiwitten zijn betrokken bij de opname, het transport en de sub-cellulaire verdeling van sulfaat in de plant. Hun expressie en activiteit zijn afgestemd op de zwavelbehoefte van de plant. Indien planten worden blootgesteld aan hoge Cu concentraties in het wortelmilieu dat leidt dit in het algemeen tot een verhoogde synthese van zwavelrijke verbindingen (m.n. fytochelatinen en glutathion), die toxische metalen zoals Cu kunnen binden, en zodanig mogelijk een rol spelen in de Cu ontgifting. In dit proefschrift is enerzijds de fysiologische basis van de toxiciteit van Cu en anderzijds het belang van het zwavelmetabolisme hierbij onderzocht in Chinese kool.

In Hoofdstuk 2 wordt een overzicht gegeven van materiaal en methoden die in dit proefschrift zijn gebruikt. In alle experimenten werd Chinese kool (Brassica pekinensis (Lour.) Rupr. cv. Kasumi F1 (Nickerson-Zwaan, Made, Nederland) gebruikt als proefplant en de proeven werden uitgevoerd met planten gekweekt op voedingsoplossing.

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In Hoofdstuk 3 werd de toxiciteit van verhoogde Cu concentraties en de invloed van dit metaal op de regulatie van de opname, de verdeling en het metabolisme van sulfaat bestudeerd. Het blootstellen van Chinese kool aan verhoogde Cu2+ concentraties leidde tot bladvergeling (chlorose), een verlies van fotosynthetische capaciteit en een verminderde groei (biomassaproductie) bij concentraties ≥ 5 μM. De daling van het pigmentgehalte was waarschijnlijk niet het gevolg van afbraak maar van een verstoorde chloroplastontwikkeling onder invloed van Cu. Het Cu gehalte in de plant nam toe met de Cu2+ concentratie in het wortelmilieu. Slechts een klein deel van het opgenomen Cu door de wortels werd naar de spruit getransporteerd. Verhoogde Cu2+ concentraties (≥ 5 μM) beïnvloedden sterk het gehalte van de andere mineralen in de plant. Er was een sterke stijging in het gehalte van water-oplosbare niet-eiwit gebonden thiolverbindingen in de wortel, en in mindere mate in de spruit bij ≥ 1 μM Cu2+. Deze stijging kon slechts gedeeltelijk worden toegeschreven aan een verhoging van het fytochelatinen gehalte. De activiteit van de sulfaattransporters in de wortel was iets verhoogd bij 2 en 5 μM Cu2+. Er was tevens een verhoging van de (gen)expressie van de Groep 1 “hoge affiniteit sulfaattransporter” Sultr1;2 (verantwoordelijk voor de opname van sulfaat), en de Groep 4 sulfaat transporters Sultr4;1 en Sultr4;2 (spelen een rol in het sulfaattransport vanuit de vacuole). Verhoogde Cu2+ concentraties hadden nauwelijks invloed op de expressie van APS reductase (een belangrijk enzym in de reduktie van sulfaat), zowel in de wortel als in de spruit. De verhoogde expressie en activiteit van de sulfaattransporters werden niet alleen veroorzaakt door een hogere zwavelbehoefte van de plant bij verhoogde Cu concentraties, maar was mogelijk ook het gevolg van interactie/reactie van Cu met verbindingen die een signaalfunctie vervullen in de regulatie van de expressie en activiteit van sulfaattransporters.

In Hoofdstuk 4 werd de invloed van UV op de Cu toxiciteit en de regulatie van de opname, de verdeling en het metabolisme van sulfaat in Chinese kool bestudeerd. De negatieve effecten van verhoogde Cu2+ concentraties op de biomassaproductie en het

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pigmentgehalte werden versterkt in aanwezigheid van UV. De chlorofyl a fluorescentie (Fv/Fm) werd alleen negatief beïnvloed nadat het bladweefsel zichtbaar beschadigd (necrotisch) was. De expressie en activiteit van sulfaattransporter Sultr1;2 werden verhoogd bij ≥ 2 μM in aanwezigheid van UV, en bij ≥ 5 μM Cu2+ in afwezigheid van UV. Bovendien werd in de spruit de expressie van Sultr4;1 sterk verhoogd bij ≥ 5 μM Cu2+ in aanwezigheid en afwezigheid van UV, en de expressie van Sultr4;2 werd verhoogd ij 10 μM Cu2+ in aanwezigheid van UV. De verhoogde Cu toxiciteit in aanwezigheid van UV kon grotendeels worden toegeschreven aan een door UV geïnduceerde verhoging van de Cu accumulatie in zowel de wortel als de spruit.

In Hoofdstuk 5 werd de invloed van de combinatie van verhoogde Cu2+ concentraties in afwezigheid van sulfaat in het wortelmilieu op de opnamecapaciteit en het metabolisme van sulfaat in Chinese kool onderzocht. Blootstelling van planten aan 4 μM Cu2+ leidde tot een lagere biomassaproductie van de plant en een verhoogde spruit/wortel verhouding zowel in aan- als afwezigheid van sulfaat. De afwezigheid van sulfaat had sneller een negatief effect op de biomassaproductie van de plant dan de verhoogde Cu2+

concentraties. De expressie en activiteit van de sulfaatransporters en de expressie van APS reductase in de Chinese kool werden al na 1 of 2 dagen verhoogd na blootstelling aan 4 μM Cu2+, bij aan- en afwezigheid van sulfaat. De invloed van sulfaatonttrekking op de expressie en activiteit van de sulfaattransporters werd nauwelijks verder beïnvloed door verhoogde Cu2+ concentraties. Hieruit bleek dat het onstaan van de negatieve effecten van zwaveldeficiëntie sneller verliep dan dat van Cu toxiciteit.

Om meer inzicht te krijgen in de rol die de zwavelvoorziening van de plant speelt in de ontwikkeling van Cu toxiciteit, werden de interacties tussen sulfaatvoeding en H2S begassing enerzijds, en verhoogde Cu2+ concentraties anderzijds, bestudeerd in Chinese kool (Hoofdstuk 6). Het blootstellen van planten aan H2S leidde tot een verminderde sulfaatopname door de wortel. De opnamecapaciteit van sulfaat werd verhoogd door Cu2+ (>10 μM), zowel in afwezigheid als aanwezigheid van H2S. Echter, H2S begassing leidde bij alle Cu2+

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concentraties tot een verminderde opnamecapaciteit van sulfaat. H2S begassing resulteerde in een verhoogd gehalte van water-oplosbare niet-eiwit gebonden thiol verbindingen, m.n. in de spruit, en in mindere mate in de wortels. Het thiolgehalte in de wortels steeg verder bij een gelijktijdige blootstelling aan H2S en verhoogde Cu2+

concentraties. Er bestond geen enkel verband tussen het thiolgehalte en de expressie en activiteit van de sulfaattransporters (en APS reductase). Planten waren in staat om in afwezigheid van sulfaat, H2S te gebruiken als zwavelbron voor de groei, maar de toxiciteit van Cu2+

was vergelijkbaar met die in aanwezigheid van sulfaat (met of zonder H2S). Blijkbaar speelde de zwavelvoorziening geen rol van betekenis in de Cu tolerantie van de plant.

Uit het onderzoek gepresenteerd in dit proefschrift kan geconcludeerd worden (Hoofdstuk 7), dat verhoogde Cu2+

concentraties (> 2 μM) in het wortelmilieu toxisch zijn voor Chinese kool. De aanwezigheid van UV leidde tot een sterk verhoogde Cu toxiciteit in de plant. Verhoogde Cu2+ concentraties verstoorden de regulatie van de opname en assimilatie van zwavel, maar er was geen directe relatie tussen de zwavelvoorziening van de plant en de Cu toxiciteit. Het wordt algemeen aangenomen dat sulfaat zelf, of producten van de zwavelassimilatie, o.a. glutathion, fungeren als signaalverbindigen in de regulatie van de expressie en activiteit van de sulfaattransporters (en APS reductase). Echter bij verhoogde Cu concentraties bleek er geen enkel verband te bestaan tussen de gehaltes van deze verbindingen en expressie en activiteit van de sulfaattransporters (en APS reductase). Mogelijk vervult H2S als gas een signaalfunctie in de interactie tussen de sulfaatreductie in de chloroplast/plastide en de transcriptie van de sulfaattransporters in de celkern. Een Cu-geïnduceerde afname van de subcellulaire H2S concentratie, als gevolg van een direkte reactie van Cu met H2S, zou verantwoordelijk kunnen zijn voor de waargenomen veranderingen in de expressie en activiteit van de sulfaattransporters (en APS reductase) in Chinese kool.

Khulasa (Summary in Urdu)

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Acknowledgements

With great pleasure I would like to express my gratitude to all who have contributed to this thesis. First and foremost I would like to thank Allah Almighty for His immense blessings and help provided to me throughout my life.

I would like to thank the Higher Education Commission (HEC), Pakistan for providing me financial support during my whole Ph.D. period, in collaboration with the Netherlands organisation for international cooporation in higher education (Nuffic).

I am heartily thankful to my supervisor, Luit J. De Kok, whose encouragement, guidance and support from the beginning to the end,enabled me to develop an understanding of the subject. I would like to thank him for teaching me scientific writing and for the opportunity of participation in international workshops and symposia. One simply could not wish a better or friendlier supervisor.

Bep and Freek, thank you very much for your excellent technical help. You both contributed a lot to this thesis, and without your help it would have not been possible to do some very big and extensive experiments. I would also like to thank Bep for the critical reading and language corrections of my manuscripts and thesis, and for accepting to be my paranymph.

I would like to thank Ineke Stulen for her interest in my project and her valuable contribution at our weekly work discussion. I would also like to thank Ger for helping me to arrange and change lamps and the tops of the fumigation cabinets, and for the financial matters.

I would like to thank Prof. Dr. Theo Elzenga, my promotor, for accepting me as a Ph.D. student. Thank you for your support and for the arrangement of paper work.

I am very grateful to Prof. Dr. Malcolm Hawkesford for his strong interest in my research and his visits to our lab during my Ph.D. period. Thank you for the valuable discussions and your encouragement. Peter Buchner, thank you for the sulfate transporter cDNA probes and for your valuable suggestions. I would also like to thank Mrs. Saroj Parmar for the mineral nutrient analysis. I am grateful to Dr. Ather Nadeem, for his favor and help to finish my master in time.

Aleksandra, thank you for teaching me molecular techniques and for the wonderful time we have had. I enjoyed all the friendly conversation on science and social aspects with you. I would like to thank Mei for being a nice colleague and for publishing together. I would also like to thank Jan Henk for his help with the photosynthesis measurements. Jannie, thank you for your help for arranging all documents and sending letters. Dick Visser, I am very grateful for designing my thesis cover page.

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I would like to show my gratitude to all the other members of Plant Physiology for creating a nice atmosphere at work and during coffee/tea breaks. Thanks to Fatma, Wouter, Ika, Cordula, Jacqueline, Martin Staal, Heba, Tahereh, Sumithra, Jan, Eelco, Sicco, Maria, Erica, Leen and Martin Reich for being such nice colleagues and for yourhelp during this period. I would like to thank Ijaz, Kamran and Sujeeth for their kind help and advice on molecular techniques and for sharing lunch. I would also like to thank Arjo Bunskoeke (Isotope Laboratory) for his kind help and support during Northern hybridization and sulfate uptake measurements.

I owe my deepest gratitude to Tayyab Akhtar for being such a caring and wonderful friend since we met 10 years ago and for accepting to become one of my paranymphs. I would like to thank Naeem Mushtaq for translating my thesis summary in Urdu. I would also like to thank my housemates Ehsan and Rashid for their great cooking abilities and for being so nice. Ehsan, thank you for your hospitality during my homeless period here in Groningen. Munir, thank you for your help in finding a house and for your guidance.

I should not forget to acknowledge Younas and Bhabhi for their kindness and all their affection. During the last 4 years whenever I met Younas or contacted him, he always offered me to have dinner with him and most of the time I had no choice than accepting it. I would also like to thank Zia for his support and entertainment he provided. It is a pleasure to thank my Pakistani colleagues Bilal Niazi, Ali, Arslan, Sulman, Samin and Imran Fazal here in Groningen and Muhammad Khurshid, Fraz, Mazhar, Imran Gujar, Shakeel, Sajid, Shujaa, Junaid and Aamir outside of Groningen for all their support, help and for the nice time we spent together.

Finally, I want to thank my family. Unfortunately, my parents are not here to share the Ph.D. celebration with me, but I am sure that they keep an eye on me from heaven. I am really grateful to all my brothers and sisters for their continuous love, support, wishes and prayers. Abdul Ghafoor, thank you for your support and guidance in the beginning of my university education. I would also like to thank to my elders and my friends back home in Pakistan, Samad, Zeeshan, Safdar, Asif for their continuous love, support and for all the good wishes.

If I did not mention someone’s name here, it does not mean that I do not acknowledge your support and help. Again, I would like to thank everyone who supported and helped me during my Ph.D. study.

Curriculum vitae

Muhammad Shahbaz was born on April 2, 1981 in Sahiwal, Pakistan. In 2001 he finished high school and obtained a Bachelor degree in Agronomy at the University of Agriculture, Faisalabad in 2005. He finished his master entitled “Comparative efficiency of different weed control practices in autumn planted maize (Zea mays)” at The University of Agriculture, Faisalabad in 2007. In September 2007 he started his Ph.D. study in the Laboratory of Plant Physiology, University of Groningen, The Netherlands. During this time he participated in several conferences and courses, listed below. He is permitted to defend his thesis in October, 2011.

Conferences and meetings

Symposium of the Task Force on Sustainable Agriculture of the Agenda 21 for the Baltic Sea Region (Baltic 21). Institute for Crop and Soil Science. Braunschweig, Germany, 28-30 April, 2008.

7th International Workshop on Sulfur in Plants: Advances in plant sulfur research including links to agriculture and environment, significance of sulfur in food chain and regulatory aspects of sulfur metabolism. Warszawa, Polland, 13-17 May, 2008. “Impact of copper on growth, sulfate uptake and assimilation in Brassica pekinensis “(Poster presentation).

7th APGC Symposium, Plant Functioning in a Changing Global Environment. The University of Melbourne, Creswick, Victoria, Australia, 7-11 December, 2008. “Atmospheric sulfur nutrition and copper toxicity in Chinese cabbage” (Oral presentation). “Impact of supra-optimal copper levels and UV irradiation on Chinese cabbage” (Poster presentation).

2nd Sulphyton meeting on Plant Sulfur Research. The University of Anglia, Norwich UK, 13–15 September, 2009. “Interaction between

pedospheric and atmospheric sulfur nutrition and copper toxicity in Chinese cabbage” (Oral presentation).

18th Symposium of the International Scientific Centre of Fertilizers. More Sustainability in Agriculture: New Fertilizers and Fertilization Management. Rome, Italy, 8–12 November, 2009. “Interaction between atmospheric and pedosperic sulfur nutrition and copper toxicity in Chinese cabbage” (Poster presentation). “Whole plant regulation of sulfate uptake and distribution in Chinese cabbage (Poster presentation).

8th International Workshop on Sulfur Metabolism in Higher Plants. “Sulfur Metabolism in Plants: Mechanisms and Application to Food Security and Responses to Climate Change” Department of Forest and Ecosystem Science, University of Melbourne, Creswick, Victoria 3363, Australia, 22-27 November, 2010. “Enhanced copper levels interfere with the regulation of the uptake and distribution of sulfate in Chinese cabbage” (Oral presentation).

8th APGC Symposium: Plant Fucntioning in a Changing Global and Polluted Environment Groningen, The Netherlands, 5–9 June, 2011. “Impact of enhanced copper levels and light quality (UV irradiation) on the physiological functioning and the regulation of the uptake and distribution of sulfate in Chinese cabbage” (Oral presentation).

EPS meeting, Lunteren, The Netherlands, 19-20 April, 2010.

Courses

Mass Spectrometry Course, 25–26 October, 2007

The Radiation Safety Course Level 5B, 22-25 January, 2008.

Dutch Language Course, February–May, 2008

The following publications are included in the thesis:

Koralewska A., Stuiver C.E.E., Posthumus F.S., Shahbaz M., Buchner P., Hawkesford M.J. and De Kok L.J. 2010. Whole plant regulation of sulfate uptake and distribution in Brassica species. In: More Sustainability in Agriculture: New Fertilizers and Fertilizer Management. (eds. Sequi P., Ferri D., Rea E., Montemurro A.V. and Fornado, F.), 18th International Symposium of CIEC, Fertilitas Agrorum pp. 274-280. ISSN 1971-0755 (Chapter 1).

Shahbaz M., Tseng M.H., Stuiver C.E.E., Koralewska A., PosthumusF.S., Venema J.H., Parmar S., Schat H., Hawkesford M.J. and De KokL.J. 2010a. Copper exposure interferes with the regulation of the uptake, distribution and metabolism of sulfate in Chinese cabbage. Journal of Plant Physiology 167: 438-446 (Chapter 3).

Shahbaz M., Tseng M.H., Stuiver C.E.E., Posthumus F.S., Parmar S., Koralewska A., Hawkesford M.J. and De Kok L.J. 2010b. Impact of copper exposure on physiological functioning of Chinese cabbage (Brassica pekinensis). In: More Sustainability in Agriculture: New Fertilizers and Fertilizer Management. (eds. Sequi P., Ferri D., Rea E., Montemurro A.V. and Fornado, F.), 18th International Symposium of CIEC, Fertilitas Agrorum pp. 318-324. ISSN 1971-0755 (Chapter 3).

Shahbaz M., Stuiver C.E.E., Posthumus F.S. and De Kok, L.J. 2011. Impact of enhanced copper levels and sulfate deprivation on the uptake and metabolism of sulfate in Chinese cabbage (Brassica pekinensis). In: Sulfur in Plants: Indication, Mechanism and Application to Food Security, and Responses to Climate Change (eds. De Kok L.J., Tausz M., Hawkesford M.J., Höfgen R., McManus M.T., Norton R.M., Rennenberg H., Saito K., Schnug E. and Tabe L.). Kluwer Academic Publishers, Dordrecht, In press (Chapter 5).

De Kok L.J., Stuiver C.E.E., Shahbaz M. and Koralewska A. (2011) Regulation of expression of sulfate transporters and APS reductase in leaf tissue of Chinese cabbage (Brassica pekinensis). In: Sulfur in Plants: Indication, Mechanism and Application to Food Security, and Responses to Climate Change (eds. De Kok L.J., Tausz M., Hawkesford M.J., Höfgen R., McManus M.T., Norton R.M., Rennenberg

H., Saito K., Schnug E. and Tabe L.). Kluwer Academic Publishers, Dordrecht, In press (Chapter 7).

Koralewska A., Posthumus F.S., Stuiver C.E.E., Shahbaz M., Stulen I. and De Kok L.J. (2011) Regulation of the uptake of sulfate by Chinese cabbage (Brassica pekinensis) at various sulfate concentrations in the root environment. In: Sulfur in Plants: Indication, Mechanism and Application to Food Security, and Responses to Climate Change (eds. De Kok L.J., Tausz M., Hawkesford M.J., Höfgen R., McManus M.T., Norton R.M., Rennenberg H., Saito K., Schnug E. and Tabe L.). Kluwer Academic Publishers, Dordrecht, In press (Chapter 1)

Shahbaz M., Parmar S., Stuiver C.E.E., Hawkesford M.J. and De Kok L.J. 2011. Copper toxicity and sulfur metabolism in Chinese cabbage are affected by UV radiation. Environmental and Experimental Botany, Submitted (Chapter 4).