Growth and water relations of salinised tomato plants Growth and water relations of salinised tomato plants overexpressing overexpressing LeNHX2LeNHX2 and and SlSOS2SlSOS2

Jurado-Lavado O.1, Huertas, R.2., Olías R.2, Venema K.2, Rodríguez-Rosales M.P.2, Belver A.2, and Romero-Aranda, R.1 1Departament of Plant Breeding, Estación Experimental La Mayora, C.S.I.C., 29760 Algarrobo-Costa, Málaga, Spain

2Department of Biochemistry, Molecular and Cell Biology of Plants, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain

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INTRODUCTION

High external NaCl concentrations limit plant growth by exerting both osmotic stress and toxic stress. Osmotic stress is caused primarily by salt-induced reduction of root water uptake and toxic stress is caused mainly by Na+ accumulation in the cytoplasm, leading to the inhibition of many metabolic processes including photosynthesis. Under salt stress. Na+ extrusion out of the cell across plasma membrane and/or compartmentalization of Na+ into the vacuoles are related with salt tolerance. At the same time high K+ concentration in plant tissues is related with a better plant water status to sustain stomatal conductance and net photosynthesis. Compartmentalization of Na+ is suggested to be achieved through the operation of Na+,K+ /H+ transporters codified by the NHX genes, while Na+ extrusion is achieved through the operation of the SOS pathway (Zhu 2003). In this pathway a calcium-binding protein, SOS3, senses cytosolic calcium changes elicited by salt stress. SOS3 physically interacts with and activates the protein kinase, SOS2. The SOS3/SOS2 kinase complex phosphorylates and activates the transport activity of the plasma membrane Na+/H+ exchanger encoded by the SOS1 gene. Both Na+/H+ and K+/H+ antiporters have been related with K+ homeostasis under salt stress. Therefore, the aim of this study was to determine possible benefits of the overexpression of the endosomal (K+,Na+)/H+ antiporter LeNHX2 (Venema et al. 2003) as well as the regulatory protein in the SOS pathway, SlSOS2 (Huertas et al. 2008, Olías et al. 2008) in mitigating physiological disturbances induced by salinity in tomato plants.

MATERIALS AND METHODS

The study was developed in a growth chamber that allowed to set cycles of air temperature, relative humidity and radiation with a daily evolution similar to that of natural days. A set of environmental conditions typical of spring season was designed using as reference the climatic data recorded inside a Mediterranean-greenhouse during this time in the last 10 years. Tomato plants (Solanum lycopersicum, cv. Microtom) overexpressing (K+,Na+)/H+ antiporter LeNHX2 (L-452) (Rodríguez-Rosales et al. 2008) and SlSOS2 (Olías et al. 2008) (L-742, L-821) were grown hydroponically with Hoagland nutrient solution supplied with 0 and 80 mM NaCl. Plant water consumption, leaf relative water content (RWC), net photosynthesis (A), stomatal conductance (gs), transpiration (E), and fresh and dry weight of plants were measured after 8 weeks of salt treatment. Nutrient solution was changed every week and water consumption was estimated weekly as the difference of pots’ weight. Water use efficiency was estimated as the ratio between A/E and as the ratio between cumulative water consumption and plant dry weight. RESULTS

Fig. 1: Cumulative water consumption over a period of six

weeks (ml plant-1).

CONCLUSIONS

Fig. 2: Fresh and dry weight (roots+shoots+fruits) of 55 days-old plants.

Fig. 4: Water use efficiency as plant dry weight/cumulative water consumption.

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Fig. 3: Plant water content calculated as the difference between plant fresh and plant dry

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Table 1: Leaf relative water content (RWC), net photosynthesis rate (A), stomatal conductance (gs), transpiration rate, and instantaneous water use efficiency measured on 50 days-old plants, after 8 weeks of salt treatments.

Significant reduction in plant water consumption, gas exchange parameters and plant growth were observed in response to NaCl in all plants. However, the three transgenic lines overexpressing LeNHX2 (L-452) and SlSOS2 (L-742, L-821) showed values slightly lower than wild-type plants (Figs, 1,2,3 and table 1). No difference in water use efficiency was observed between wild-type and transgenic plants, under non-saline and saline growth conditions.

Huertas R, Belver A, Li J, Venema K y Rodríguez-Rosales M P (2008) IX Biologia Molecular de Plantas, Santiago, p26

Olías R, Eljakaoui Z, Alvarez-de-Morales P, Li J, Huertas R, Marín-Manzano MC, Rodriguez-Rosales P, and Belver A (2008) XVI FESPB 2008 Tampere, Finland P09-021

Rodríguez-Rosales MP, Jiang XJ, Gálvez FJ, Aranda MN, Cubero B, Venema K (2008). New Phytol, 179: 366-377.

Venema K, Belver A, Rodríguez-Rosales MP, Marín MC, Donaire JP (2003).. J Biol Chem 278: 22453-22459.

Zhu, J.K. 2003. Current Op. Plant Biol. 6: 441-445

- Tomato plants overexpressing the (K+, Na+)/H+ antiporter LeNHX2 and SlSOS2 Na+/H+ antiporters show values of plant water consumption slightly lower that those recorded on wild-type plants.

- Both under non-saline and saline growth conditions, tomato plants overexpressing the (K+,Na+)/H+ antiporter LeNHX2 and SlSOS2 show lower values of fresh and dry weight than wild-type plants.

- The overexpression of the (K+,Na+)/H+ antiporter LeNHX2 and SlSOS2 of K+/H+ and Na+/H+ antiporters does not alter gas exchange parameters and water use efficiency estimated as the ratio between A/E or as the ratio between biomass production and volume of water consumption.

RWC (%)

A (µmol CO2/m2s)

Gs (mol H2O/m2s)

E (mmol H2O/m2s)

WUE (A/E)

WT 94.0±2.6 8.4±1.5 0.116±0.040 2.6±0.5 4.0±0.5 0 mM Na Cl L-452 95.3±1.5 7.6±2.0 0.107±0.033 2.1±0.4 3.6±0.5 L-742 95,1±3.6 6.6±1.9 0.071±0.019 1.5±0.35 4.5±0.6 L-821 95.5±3.2 7.8±2.2 0.116±0.056 2.1±0.7 3.9±0.6 WT 91.5±3.1 5.6±1.3 0.069±0.024 1.4±0.40 4.0±0.6 80 mM NaCl L-452 95.3±4.6 5.7±1.6 0.057±0.017 1.3±0.3 4.5±0.5 L-742 98,3±1.2 3.7±1.4 0.055±0.016 1.2±0.3 3.5±1.0 L-821 93.2±4.3 6.8±0.9 0.077±0.024 1.6±0.4 4.5±1.0