ORIGINAL ARTICLE

Physiological and transcriptional analysis reveals pathwaysinvolved in iron deficiency chlorosis in fragrant citrus

Long-Fei Jin1,2& Yong-Zhong Liu1,2

& Wei Du1,2& Li-Na Fu1,2

& Syed Bilal Hussain1,2&

Shu-Ang Peng1,2

Received: 17 October 2016 /Revised: 2 March 2017 /Accepted: 20 March 2017# Springer-Verlag Berlin Heidelberg 2017

Abstract Iron (Fe) deficiency chlorosis is a yield-limitingproblem in citrus production regions with calcareous soils.Physiological and transcriptional analyses of fragrant citrus(Citrus junos Sieb. ex Tanaka) leaves from Fe-sufficient (IS)and Fe-deficient (ID) plants were investigated in this study.The physiological results showed that Fe, potassium, and ni-trogen levels decreased by 12, 15, and 41% in ID leaves,respectively. However, zinc and copper levels increased by49 and 35% in ID leaves, respectively. The chlorophyll(Chl) content, photosynthesis rate, stomatal conductance,and transpiration rate in ID leaves decreased by 55, 33, 38,and 42%, respectively, compared with IS leaves. Moreover,transcriptional profiling analysis showed that genes associatedwith Chl metabolism, photosynthesis, and nitrogen metabo-lism were dramatically downregulated by Fe deficiency. Theexpression of glutamyl-tRNA reductase 1, chlorophyll(ide) breductase, and geranylgeranyl diphosphate reductase in IDleaves was 0.26–0.37 times that in IS leaves. The expressionlevels of 16 photosynthesis-related genes were severely

downregulated by Fe deficiency. In addition, the transcriptionlevels of nitrate transporter, nitrate reductase, and ferredoxin-nitrite reductase genes in ID leaves were 0.38–0.45 timesthose in IS leaves. Taken together, these results indicated thatthe block of Chl biosynthesis, the reduction of photosynthesis,and the repression of nitrogen absorption resulted in the chlo-rosis symptoms observed in fragrant citrus leaves.

Keywords Fragrant citrus . Iron deficiency chlorosis .

Chlorophyll . Photosynthesis . Nitrogenmetabolism

Introduction

Iron (Fe) is an essential nutrient for plant multiple functionsincluding photosynthesis, respiration, chlorophyll (Chl) bio-synthesis, and redox reactions in plants, and is a structuralcomponent in heme, the Fe-sulfur cluster, and Fe-binding sites(Briat et al. 2007; Hänsch and Mendel 2009; Nouet et al.2011). Although total Fe content in most soils is much higherthan is required by plants, Fe is often unavailable for plantsdue to the low solubility of its oxidized form, Fe(III)(Kobayashi and Nishizawa 2012). Fe deficiency leads to de-velopmental defects, including chlorosis, growth retardation,and reductions in productivity (Briat et al. 2007). Particularlyin fruit crops, Fe deficiency causes serious decreases in treevegetative growth, fruit yield, and quality (Tagliavini et al.2000). Fe deficiency chlorosis (IDC) occurs in interveinaltissue of young leaves when a plant is unable to utilize Fe inthe soil. IDC was first reported in 1843 in grapes grown incalcareous soil where it is difficult to release ferrous Fe fromsoil particles and Fe availability is limited. Approximately30% of the world’s soils are considered calcareous with lowFe availability, which results in extensive areas of Fe deficien-cy in plants (Takahashi 2003).

Communicated by W.-W. Guo

Electronic supplementary material The online version of this article(doi:10.1007/s11295-017-1136-x) contains supplementary material,which is available to authorized users.

* Yong-Zhong Liuliuyongzhong@mail.hzau.edu.cn

* Shu-Ang Pengshuangpeng428@126.com

1 Key Laboratory of Horticultural Plant Biology, Ministry ofEducation, Huazhong Agricultural University, Wuhan 430070,People’s Republic of China

2 Key Laboratory of Horticultural Crop Biology and GeneticImprovement (Central Region), Ministry of Education,Wuhan 430070, People’s Republic of China

Tree Genetics & Genomes (2017) 13:51 DOI 10.1007/s11295-017-1136-x

In leaves, the major symptom of Fe deficiency is the inhi-bition of chloroplast development, which results in interveinalchlorosis (Miller et al. 1984). Fe deficiency also induces mor-phological, physiological, and molecular changes in roots inorder to adapt to low-Fe stress. Fe deficiency inhibits rootelongation and increases the diameter of apical root zonesand the density of root hair (Graziano and Lamattina 2007).These morphological changes are often associated with theformation of cells with a distinct wall labyrinth typical oftransfer cells in the rhizodermis or hypodermis (Landsberg1989). The Fe deficiency-induced formation of rhizodermaltransfer cells enhances net excretion of protons and reducingcapacity as well as the release of phenolic compounds, whichenhance Fe uptake (Kramer et al. 1980). An increase in citrateaccumulation in leaves and roots under low Fe stress was alsoobserved (Abadia et al. 2002). In recent years, impressiveprogress has been made characterizing the molecular path-ways underlying root responses to Fe deficiency in strategy Iplants, which occur in three stages: (1) phenolic compoundsand protons are exuded in order to acidify the soil and increaseFe(III) solubility (Kobayashi and Nishizawa 2012; Santi andSchmidt 2009); (2) ferric chelate reductase 2 (FRO2) reducesFe(III) to Fe(II) (Robinson et al. 1999); and (3) Fe(II) istransported into roots mainly by the Fe transporter, iron-regulated transporter 1 (IRT1), in plant roots (Varotto et al.2002; Vert et al. 2002). In contrast, graminaceous plants usestrategy II (a chelation-based) to improve Fe acquisition. Indetail, the mugineic acid family phytosiderophore (PS) is re-leased to chelate Fe(III). Fe(III)-mugineic acid chelate com-plexes are then transported into plant roots via a specific trans-port system (Mori 1999; Walker and Connolly 2008). Overthe past decade, various transporter genes have been identifiedand described in maize (Curie et al. 2001), barley (Murataet al. 2006), and rice (Lee et al. 2009), for the root uptake,shoot transport, and seed deposition of Fe. In addition,nicotianamine has been suggested to play important roles inphloem transport and seed deposition of Fe (Haydon andCobbett 2007).

Citrus is one of the most important fruit crops in the world.However, Fe deficiency occurs universally when citrus growsin calcareous soils (Tagliavini and Rombolà 2001). To date,studies on IDC in citrus have mainly been related to field-based and physiological descriptions (Cimen et al. 2014;Martinez-Cuenca et al. 2016; Wulandari et al. 2014). Themolecular mechanisms underlying IDC occurrence remainunclear. Fragrant citrus (Citrus junos) is a special citrus germ-plasm native to China, which belongs to an Fe deficiency-tolerant rootstock (Tan et al. 2015). In this study, in combina-tion with measurements of Chl content, photosynthesis rate,and mineral nutrition, we used RNA-Seq technology to ex-plore the transcriptional changes in fragrant citrus leaves sub-jected to Fe-deficient stress in order to investigate the possiblemolecular mechanisms of IDC in citrus.

Materials and methods

Plant materials and Fe treatments

This study was conducted at Huazhong AgricultureUniversity (30° 289′ N, 114° 219′ E), Wuhan, China. Plantculture was performed according to a previous study (Wanget al. 2014). Briefly, 15-week-old seedlings of fragrant citrusin Hoagland’s No. 2 nutrient solution were grown in a green-house under a natural photoperiod. The nutrient solutioncontained 2.5 mM KNO3, 2.5 mM Ca(NO3)2, 0.5 mMKH2PO4, 10 μM H3BO3, 2 μM MnCl2, 2 μM ZnSO4,0.5 μM CuSO4, 0.065 μM (NH4)6Mo7O24, 1 mM MgSO4,and 0.5 μM (Fe-deficiency) or 50 μM (Fe-sufficiency) Fe-EDTA. Each treatment contained three pots, and each potcontained 15 seedlings. The solution was ventilated for20 min every 2 h and replaced twice a week. The pH of allthe nutrient solutions was adjusted to 6.0 with 0.1 M KOH.After 40 days when the chlorosis leaves occurred clearly onnew shoots of Fe-deficient treatment, the leaves (leaf lengthwas about 7–8 cm) of Fe-deficient and Fe-sufficient treat-ments were collected, immediately frozen in liquid nitrogen,and then stored at −80 °C for later use.

Leaf ionomic analysis

Leaves were dried at 70 °C for 48 h. Leaf P, S, K, Ca, Mg, Fe,Mn, Zn, Cu, and B content was assayed by inductivelycoupled plasma-atomic emission spectrometry (ICP-AES,IRIS-Advan type, Thermo, USA) after digestion with 1 MHCl. Leaf total nitrogen was determined by an automaticazotometer (KT8200, FOSS, Sweden), through alkaline hy-drolysis diffusion.

Leaf gas exchange and chlorophyll measurement

Leaf gas exchange parameter measurements were carried outusing a LI-6400 portable photosynthesis system (LI-COR6400XT system; LI-COR, Lincoln, NE, USA) connected toa red-blue LED light source. The parameters included thephotosynthetic rate (Pn, μmol CO2 m

−2 s−1), stomatal conduc-tance (Gs, mol H2O m−2 s−1), intercellular CO2 concentration(Ci, μmol CO2 mol−1), and transpiration rate (Tr, mmolH2O m−2 s−1). Measurements were performed on healthyand developed leaves between 09:00 a.m. and 11:00 a.m. withsteady light intensity (1000 μmol m−2 s−1). The detection ofChl content was followed by previous research (Holm 1954).Fresh leaf tissue (200 mg) was cut into small pieces, andpigment was extracted by grinding with a mortar and pestlefor 5 min in 10 mL of 80% acetone/95% ethanol (1:1). Thesupernatant was then transferred into a new 15-mL centrifugetube and incubated with 80% acetone/95% ethanol (1:1) for24 h in the dark. Optical density (OD) of the extracted samples

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was measured at both 663 and 644 nm using ultraviolet spec-trophotometry (UV-1800, Shimadzu, Japan).

Leaf RNA library construction and Illumina sequencing

Equal amounts of frozen Fe-deficient (ID) or Fe-sufficient (IS)leaves from five plants were combined for RNA extraction.Total RNA was extracted using TRIzol reagent (Invitrogen,Carlsbad, CA) following the manufacturer’s instructions.Total RNA quantity and purity were analyzed with a 2100Bioanalyzer and RNA 6000 Nano LabChip Kit (Agilent,CA, USA), respectively. ID and IS leaf RNAs with a RINnumber >7.0 were used for library construction according toa previously described method (Zhao et al. 2015). Sequencingwas performed on an Illumina HiSeq™ 2500 sequencer by theGene Denovo Biotechnology Company (Guangzhou, China).

RNA-Seq data analysis

To obtain high-quality clean reads, raw reads were filteredaccording to the following rules: (1) reads containing adapterswere removed; (2) reads containing more than 10% of un-known nucleotides (N) were removed; and (3) low-qualityreads containing more than 50% of low-quality (Q-value≤20) bases were removed. Furthermore, the short-read align-ment tool Bowtie 2 (Langmead and Salzberg 2012) was usedfor mapping reads to a ribosome RNA (rRNA) database. TherRNA-mapped reads were then removed. Clean reads of eachsample were subsequently mapped to the reference genomeby TopHat2 (Kim et al. 2013). Reference genome sequencesand annotations were downloaded from the Orange (Citrussinensis) Genome Annotation Project (http://citrus.hzau.edu.cn/orange/). The alignment parameters were as follows: (1)maximum read mismatch was 2; (2) the distance betweenmate-pair reads was 50 bp; and (3) the error of distance be-tween mate-pair reads was ±80 bp. After alignment with areference genome, unmapped reads were re-aligned withBowtie 2 (Langmead and Salzberg 2012) to identify newgenes. Novel genes were then aligned to the Nr database toobtain protein functional annotations.

Gene abundances were quantified by software RSEM (Liand Dewey 2011). Gene expression levels were normalized byusing the fragments per kilobase of transcript per millionmapped reads (FPKM) method using the following formula:

FPKM ¼ 106CNL=103

;

where C represents the number of reads that uniquelyaligned to a gene, N represents the total number of reads thatuniquely aligned to all genes, and L represents the number ofbases in a gene. To identify differentially expressed genesacross samples or groups, the edgeR package (http://www.

rproject.org/) was used. A fold change ≥2 and a falsediscovery rate (FDR) <0.05 were used to screen significantdifferent-expression genes (DEGs). DEGs were then subject-ed to enrichment analysis of GO functions and KEGG path-ways. All DEGs were mapped to GO terms in the GeneOntology database (http://www.geneontology.org/). Genenumbers were calculated for every term, and significantlyenriched GO terms in DEGs compared to the genomebackground were defined by a hypergeometric test. KEGG(http:/ /www.kegg.jp/kegg/pathway.html) pathwayenrichment analysis was used to identify significantlyenriched metabolic pathways or signal transductionpathways in DEGs compared with the whole genomebackground.

Gene expression validation

To verify RNA-Seq results, qRT-PCR with β-actin as an in-ternal control was used to detect mRNA expression levels.qRT-PCR was performed with a SYBR Green Master kit(TaKaRa, Dalian, China) according to the manufacturer’s pro-tocol. qRT-PCR primers are listed in Table S1. The experi-ments were carried out in triplicate with a total volume of20 μL in ABI StepOnePlus™ (Applied Biosystems,Waltham, MA, USA), containing 10 μL of SYBRGreen mas-ter, 4 μL of complementary DNA (cDNA) (500 ng), and 3 μLof forward and reverse primers (2μmol/L). The qRT-PCRwasprogrammed at 95 °C for 10 min, followed by 40 cycles of95 °C for 15 s and 55 °C for 1 min. The expression level wascalculated by the 2−ΔΔCT method (Livak and Schmittgen2001).

Fig. 1 Iron-deficient chlorosis in developing leaves. IS refers to Fesufficiency, ID refers to Fe deficiency. The white scale represents 2 cm

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Statistical analysis

The significant differences of nutrient element content, Chlcontent, gas exchange parameter, and qRT-PCR results be-tween IS and ID treatment were analyzed with a t test (LSD)using SAS software version 8 (SAS Institute, Cary, NC,USA). Differences were considered significant at P < 0.05.

Results

Fe deficiency caused leaves chlorosis, disorderedthe assimilation of mineral nutrients, and changed leaf gasexchange parameters

Leaves under sufficient Fe condition were green and glossyand plants displayed vigorous growth, whereas leaves underID conditions showed clear interveinal chlorosis (Fig. 1). Fedeficiency also disordered the assimilation of mineral nutri-ents. The Fe content in ID leaves (80.1 mg/kg DW) was sig-nificantly lower than that in IS leaves (135.2 mg/kg DW).Nitrogen and potassium levels decreased by 12 and 15%, re-spectively, whereas zinc and copper levels increased by 49and 54%, respectively, in ID leaves compared with the ISleaves (Table 1).

Leaf Chl content in ID leaves was lower than that inIS leaves. In detail, chlorophyll a (Chl a), chlorophyll b(Chl b), and Chl in ID leaves deceased by 67, 36, and55% compared with ID leaves, respectively (Fig. 2a–c).Moreover, Fe deficiency also reduced the ratio of Chl a/b (Fig. 2d).

Pn, Gs, and Tr were severely depressed as a consequence ofFe deficiency. Namely, Pn (Fig. 3a), Gs (Fig. 3b), and Tr(Fig. 3d) in ID leaves decreased by 33, 38, and 42%, respec-tively, compared with those in IS leaves. However, no signif-icant difference in Ci levels was found between ID and ISleaves (Fig. 3c).

Transcriptomic analysis of fragrant citrus in responseto Fe deficiency

In order to identify specific transcripts responding to Fe defi-ciency in the leaves of fragrant citrus, two cDNA librariesrepresenting the ID and IS, respectively, were constructedand subjected to Illumina deep sequencing. After discardinglow-quality raw reads, 33,299,870 and 34,562,102 reads wereobtained, respectively. After removing ribosome reads,24,402,734 (73.28%) and 25,263,013 (73.09%) reads wereleft and mapped to the Orange (Citrus sinensis) AnnotationProject, respectively. In the end, a total of 20,795 (70.12%)known genes and 289 new genes were obtained (Table S2).Randomness assessments showed that the reads of each refer-ence gene distributed evenly and demonstrated highly similartendencies between the two libraries (Fig. S1). The sequenc-ing saturation was analyzed in either the two libraries. Whenthe number of reads reached five million reads or higher, thegrowth rate of detected genes became flattened, which sug-gested that the number of detected genes became saturated(Fig. S2). Moreover, 913 genes were differentially expressedbetween the IS and ID library according to the criteria of a foldchange ≥2 and a FDR <0.05. Among these genes, 598(65.50%) were upregulated and 315 (34.50%) were downreg-ulated in response to Fe deficiency (Fig. S3). In addition, a

Table 1 A comparison of some mineral element levels between iron-sufficient (IS) and iron-deficient (ID) leaves

Nmg/g

Pmg/g

Smg/g

Kmg/g

Gamg/g

Mgmg/g

Femg/kg

Mnmg/kg

Znmg/kg

Cumg/kg

Bmg/kg

IS 30.1 ± 2.3a 2.50 ± 0.3a 3.0 ± 0.3a 29.2 ± 0.9a 16.3 ± 0.9a 4.2 ± 0.7a 135.2 ± 2.4a 13.8 ± 0.7a 30.3 ± 3.5b 9.8 ± 0.9b 35.6 ± 3.2a

ID 26.4 ± 1.9b 2.2 ± 0.4a 2.8 ± 0.1a 24.8 ± 0.7b 16.7 ± 1.0a 4.1 ± 0.5a 80.1 ± 2.0b 12.5 ± 0.9a 59.4 ± 4.0a 15.1 ± 0.5a 31.6 ± 2.6a

Different lowercase letters within each column indicate significant differences between IS and ID at P < 0.05 based on the t test (LSD)

Fig. 2 Chlorophyll contents indeveloping leaves. a Chl acontent. b Chl b content. c Thecontent of total chlorophyll. d Theratio of total Chl a/b. The barsrepresent SE (n = 3). The asterisk(*) on bars indicates significantdifferences between control andtreatment at P < 0.05 based on a ttest (LSD)

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total of 16 DEGs were selected randomly for qRT-PCR toconfirm the results obtained through DGE analysis. Despitesome quantitative differences at expression level, the qRT-PCR results showed the same expression tendency as DGEresults (Fig. 4a–p), of which the overall correlation coefficientwas 0.916 (Fig. 4q).

According to the GO classification system, 913 genes wereclassified into three major functional categories including bi-ological process (511), cellular component (312), and molec-ular function (558) (Fig. S4). KEGG pathway enrichmentanalysis showed that a total of 13 pathways were significantlyenriched (P value <0.05). Among these pathways,

Bphotosynthesis-antenna proteins,^ Bphenylpropanoidbiosynthesis,^ Bphotosynthesis,^ and Bnitrogen metabolism^were the four most commonly represented subclasses (Fig. 5).However, Chl metabolism, which has a dialectical relationwith chlorosis, was not significantly enriched.

In the Chl metabolism pathway, four DEGs were enriched.Of these, the expression of three genes, namely, Glu-tRNAreductase (HEMA1), chlorophyll(ide) b reductase (NYC1),and geranylgeranyl diphosphate reductase (chlP), decreasedsubstantially in chlorosis leaves, whereas the expression ofthe chlorophyllide a oxygenase (CAO) gene in chlorosisleaves increased to five times that of green leaves. In the

Fig. 3 Comparison of photosynthetic rate (a, Pn), conductance to H2O(b, Gs), intercellular CO2 concentration (c, Ci) and transpiration rate (d,Tr) between iron-sufficient (IS) and iron-deficient (ID) leaves. The bars

represent SE (n = 10). The asterisk (*) on bars indicates significantdifferences between control and treatment at P < 0.05 based on a t test(LSD)

Fig. 4 qRT-PCR confirmationfor differentially expressed genes(DEGs) from digital gene expres-sion analysis. a–p The transcriptlevels of 16 randomly selectedDEGs, including 9 upregulatedand 7 downregulated transcripts.The bars represent SE (n = 4).The numbers on top of thehistogram are FPKM. q Thecomparison between the log2 ofgene expression ratios obtainedfrom RNA-Seq and qRT-PCRdata

Tree Genetics & Genomes (2017) 13:51 Page 5 of 10 51

photosynthesis (including antenna proteins) pathway, 16genes were found to be downregulated in ID conditions com-pared with IS conditions. In the nitrogen metabolism pathway,five genes were differentially expressed between IS and IDleaves. Of these, the nitrate transport (Nrt) gene transcripts aswell as the levels of nitrate reductase (NR) and ferredoxin-nitrite reductase (nirA) genes were downregulated by Fe defi-ciency (Table 2). Moreover, the expression of two genes(Cs5g05410 and Cs8g16230) encoding the carbonicanhydrase for nitrogen utilization was downregulated: theFPKM of Cs8g16230 was very low, and the FPKM ofCs5g05410 in ID leaves was 0.37 times that of IS leaves. Inthe carotenoid biosynthesis pathway, genes encoding abscisicacid 8′-hydroxylase 4-like (CYP), beta-carotene 3-hydroxy-lase (crtZ), lycopene beta-cyclase (lcyB), and zeta-carotenedesaturase (ZDS) were differentially expressed. With the ex-ception of crtZ, in which expression level was over five timeshigher in ID leaves than IS leaves, the expression of the otherfour genes in ID leaves was approximately one third that of ISleaves (Table 2).

Discussion

Chlorosis is a typical symptom of Fe deficiency in developingleaves and has been found in many crops including rice, soy-bean (Lin et al. 1998), peach (Belkhodja et al. 1998), andcitrus (Martínez-Cuenca et al. 2013). In the present study,young leaves subjected to Fe starvation also showed visibleinterveinal chlorosis (Fig. 1). Reports in sugar beet, pear, andpeach indicated that the occurrence of chlorosis is due to adecrease in Chl (Larbi et al. 2006). Here, we found that Chl,Chl a, and Ch b levels decreased markedly in chlorosis leaves(Fig. 2), in agreement with a similar result reported in grapes(Chen et al. 2004a). It is clear that Chl content is determinedby its biosynthesis and degradation. HEMA1, which catalyzesthe reduction of glutamyl-tRNA to glutamate-1-semialdehyde(Pontoppidan and Kannangara 1994), the first committed stepin Chl biosynthesis, was markedly downregulated in chlorosisleaves (Table 2). The transcription of NYC1, which plays animportant role in Chl a formation (Scheumann et al. 1998),was reduced 0.27 times in chlorosis leaves compared with

Fig. 5 KEGG pathwayenrichment analysis of 913different expression genes indeveloping leaves

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green leaves (Table 2). Furthermore, expression of the CAOgene, which plays a role in the conversion of Chl a to Chl b(Eggink et al. 2004), was upregulated by 4.3 times by Fedeficiency (Table 2), which resulted in the reduction of Chla/b (Fig. 2d). These results implied that the chlorosis in devel-oping leaves that occurs under ID conditions is due to thereduction of Chl biosynthesis due to decreases in the expres-sion of key Chl biosynthesis-related genes.

Chlorosis is not only the inhibition of Chl accumulation butalso relates to carotenoid levels. Indeed, carotenoid biosynthe-sis is also affected by Fe deficiency. In sugar beets, Fe defi-ciency has been shown to decrease neoxanthin andβ-carotenelevels, but increased lutein and the xanthophyll cycle pool(Morales et al. 1990). In this study, although we did not assay

carotenoid levels, we found that two genes, ZDS and lcyB,involved in the biosynthesis of lycopene were downregulatedby Fe deficiency. Additionally, the beta-carotene 3-hydroxy-lase (crtZ) gene, which is involved in the pathways for luteinand zeaxanthin formation, was five times higher in chlorosisleaves (Table 2), suggesting that the characteristic chlorosis indeveloping leaves is partly a consequence of this relative en-richment of carotenoids.

Similar to studies in sugar beets (Morales et al. 1991) andgrapes (Chen et al. 2004a), the present study also showed thatFe deficiency significantly decreased Pn, Gs, and Tr levels,which reflected a decrease in the photochemical efficiency ofyoung leaves (Fig. 2). Fe is located in three major complexesof the photosynthetic apparatus: photosystem II (PSII),

Table 2 A list of different expression genes (DEGs) between iron-sufficient (IS) and iron-deficient (ID) libraries

Pathway Gene ID Annotation FPKM(IS)

FPKM(ID)

FC Log2(FC)

P value FDR

Chlorophyllmetabolism

Cs4g15890 Chlorophyll(ide) b reductase NYC1 63.71 16.94 0.27 −1.91 1.37E−167 4.32E−165Cs3g19690 Chlorophyllide a oxygenase 23.97 104.45 4.36 2.12 3.42E−276 1.68E−273Cs5g10740 Geranylgeranyl diphosphate reductase 410.3 151.76 0.37 −1.43 0.00E+00 0.00E+00

Cs3g16730 Glutamyl-tRNA reductase 1 114.34 30.15 0.26 −1.92 0.00E+00 0.00E+00

Carotenoidbiosynthesis

Cs8g05940 Abscisic acid 8′-hydroxylase 4-like 3.72 1.38 0.37 −1.43 8.61E−07 9.19E−06Cs5g03200 Beta-carotene 3-hydroxylase 2 1.07 5.36 5.01 2.32 6.51E−08 8.24E−07orange1.1t00772 Lycopene cyclase 14.06 6.59 0.47 −1.09 5.08E−18 1.58E−16Cs3g11180 Zeta-carotene desaturase 59.14 21.42 0.36 −1.47 6.01E−80 9.61E−78Cs3g11170 Zeta-carotene desaturase 38.75 13.42 0.35 −1.53 1.71E−34 1.12E−32

Photosynthesis Cs2g19680 Chlorophyll A/B binding protein (LHCB2) 2526.79 1129.08 0.45 −1.16 0.00E+00 0.00E+00

Cs1g06360 Chlorophyll a-b binding protein 13 (LHCB3) 159.43 46.39 0.29 −1.78 7.65E−312 0.00E+00

Cs2g03780 Chlorophyll a-b binding protein 3C (LHCB1) 8019.51 3987.35 0.50 −1.01 0.00E+00 0.00E+00

Cs3g06180 Chlorophyll a-b binding protein 8 (LHCA3) 691.48 343.92 0.50 −1.01 3.42E−270 1.60E−267orange1.1t03504 Chlorophyll a-b binding protein CP24 10B

(LHCB6)837.96 368.37 0.44 −1.19 0.00E+00 0.00E+00

Cs5g18620 Chlorophyll a-b binding protein CP26 (LHCB5) 1777.08 845.73 0.48 −1.07 0.00E+00 0.00E+00

Cs6g08260 Chlorophyll a-b binding protein CP29.2 (LHCB4) 1192.21 214.75 0.18 −2.47 0.00E+00 0.00E+00

Cs5g10800 Chlorophyll a-b binding protein P4 (LHCA4) 530.73 223.68 0.42 −1.25 0.00E+00 0.00E+00

Cs9g06950 Ferredoxin 18.33 8.69 0.47 −1.08 1.52E−05 1.28E−04Cs3g03670 Ferredoxin-2 391.41 86.55 0.22 −2.18 4.17E−222 1.66E−219Cs3g03680 Ferredoxin-2 5.84 0.18 0.03 −5.02 1.57E−08 2.17E−07Cs2g10620 Photosystem I reaction center subunit III (psaF) 1187.75 206.21 0.17 −2.53 0.00E+00 0.00E+00

Cs1g09140 Photosystem I reaction center subunit N (psaN) 460.11 84.03 0.18 −2.45 0.00E+00 0.00E+00

Cs2g09520 Photosystem I reaction center subunit X (psaK) 780.17 336.65 0.43 −1.21 5.03E−307 2.65E−304Cs6g12390 Photosystem I subunit O (psaO) 58.97 25.5 0.43 −1.21 1.08E−40 8.90E−39Cs8g13660 Photosystem II reaction center W protein (psaW) 271.7 124.3 0.46 −1.13 1.64E−129 3.81E−127

Nitrogenmetabolism

Cs8g16230 Bifunctional monodehydroascorbate reductase andcarbonic anhydrase nectarin-3-like

0.001 0.77 770.00 9.59 7.82E−03 3.20E−02

Cs5g05410 Carbonic anhydrase, chloroplastic-like 42.9 15.76 0.37 −1.44 3.52E−32 2.14E−30Cs8g05970 Ferredoxin-nitrite reductase, chloroplastic-like 83.32 31.45 0.38 −1.41 6.84E−131 1.62E−128Cs7g09040 High-affinity nitrate transporter 2.5-like 16.86 7.61 0.45 −1.15 2.67E−20 9.37E−19Cs3g19060 Nitrate reductase [NAD(P)H]-like 29.09 11.87 0.41 −1.29 1.84E−61 2.25E−59

FPKM fragments per kilobase of transcript per million, FC fold change

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photosystem I (PSI), and light harvesting complex (LHC)(Eberhard et al. 2008), which are essential for the structureand function of the photosynthetic electron transfer chain.Two Fe atoms are present in the PSII complex: one non-heme Fe is coordinated by four histidines and a bicarbonate,whereas the second heme Fe is present in cytochrome b599(Eberhard et al. 2008). The expression level of PSII subunitPsbW protein gene in IS leaves was reduced 0.43 times(Table 2). Additionally, one [2Fe-2S] cluster is in soluble fer-redoxin (Fd) and three [4Fe-4S] clusters (i.e., 12 Fe atoms)locate in the PSI complex (Eberhard et al. 2008). Fd plays animportant role in electron transporting from PSI to Fd-NADP+

oxidoreductase (Tagawa et al. 1963). The present study indi-cated that three Fd genes were severely downregulated inchlorosis leaves (Table 2). As an important component ofPSI, Fe limitation results in collapse of structural integrity.The expression of four PSI subunit genes including PsaF,PsaK, PsaN, and PsaO was decreased by Fe deficiency(Table 2). The two LHC associated with the two photosystemscontain Chl, whose synthesis is also Fe-dependent (Eberhardet al. 2008). LHC contain five Lhca- and seven Lhcb-bindingproteins, which play a pivotal role in light absorption andenergy conversion (Thornber 1975). In chlorosis leaves, twoLhca-binding protein genes and six Lhcb-binding proteingenes were downregulated by Fe deficiency (Table 2). Theseresults indicated that the transcript levels of some importantgenes involved in the photosynthesis (including antenna pro-teins) pathway were severely inhibited by Fe deficiency,which possibly accounted for the decrease of Pn, Gs, and Trin young leaves caused by Fe deficiency.

Ionic antagonism and synergistic effect play a part in min-eral element absorption. For example, Fe deficiency has been

shown to induce Cu uptake and accumulation in Commelinacommunis (Chen et al. 2004b). Additionally, a decrease inNO3

− uptake was observed in ID cucumber plants (Agnolonet al. 2002). In this study, Fe deficiency decreased the N con-tent (Table 1). It is well known that Fe is a metal cofactor ofenzymes of ferredoxin-nitrite reductase (Jack et al. 1978) andnitrogen uptake relates to nitrate transport, nitrate reductase,and ferredoxin-nitrite reductase (Wang et al. 2012). Similar toa report on cucumber plants (Borlotti et al. 2012), we alsofound that Nrt, NR, and nirA gene transcript levels declineddramatically in developing leaves under ID conditions(Table 2), which possibly explained the reason for the ob-served decrease in N uptake. We also found that Fe deficiencydecreased the uptake of K and increased the uptake of Zn andCu, consistent with previous reports (Chen et al. 2004b;Marschner 2012). However, possible molecular mechanismsstill require further investigation.

In conclusion, the present results revealed that Fe deficien-cy induced chlorosis in developing leaves by affecting Chland carotenoid metabolism, photosynthesis pathway, N ho-meostasis, and so on (Fig. 6). Fe deficiency reduced the tran-script levels of some key genes involved in Chl metabolismand two carotenoid-related genes (ZDS and lcyB), but in-creased crtZ gene transcript levels. Additionally, Fe deficiencydecreased the transcript levels of many important genes in thephotosynthesis pathway, including PSII subunit, PSI subunit,Fd, and LHC chlorophyll a/b binding protein genes. Finally,Fe deficiency reducedNrt,NR, and nirA gene transcript levels,which play a role in N homeostasis. Although other pathwaysmay have also been altered by Fe deficiency, these resultsdeepen our understanding of the molecular mechanisms un-derlying the development of IDC in citrus leaves.

Fig. 6 A sketch of the possiblepathways affected by irondeficiency for the occurrence ofchlorosis in developing citrusleaves. Red and blue colorsrepresent the upregulation anddownregulation of physiologicalchanges or gene expression levels

51 Page 8 of 10 Tree Genetics & Genomes (2017) 13:51

Acknowledgements This work was supported by the National NaturalScience Foundation of China (Grant No. 31272121) and the earmarkedfund for the China Agriculture Research System (CARS-27).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict ofinterest.

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