Acta Physiol Plant (2017) 39:130 DOI 10.1007/s11738-017-2429-2

ORIGINAL ARTICLE

Transcriptome profiling of cucumber genome expression in response to long-term low nitrogen stress Ming Xin1 • Lei Wang2 • Yunping Liu1 • Zhuo Feng1 • Xiuyan Zhou1 Zhiwei Qin1

•

Received: 14 July 2016 / Revised: 17 January 2017 / Accepted: 7 May 2017 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2017

Abstract The present study aimed to delineate the genes mediating nitrogen metabolism in cucumber (Cucumis sativus L.) and elucidate the mechanisms underlying the response to long-term nitrogen limitation. As an economically important crop, cucumber is strongly nitrogen dependent. The mechanisms underlying nitrogen metabolism in cucumber are not fully known. This study found that cucumber developed to a 3.5-leaf stage with reduced plant size and biomass under chronic low nitrogen stress condition. Gene expression profiling and analysis of cucumber roots and leaves under nitrogen-starved condition identified a total of 2991 unigenes as reliable differentially expressed genes (DEGs). A comprehensive Communicated by T. K. Mondal.

Electronic supplementary material The online version of this article (doi:10.1007/s11738-017-2429-2) contains supplementary material, which is available to authorized users. & Zhiwei Qin [email protected] Ming Xin [email protected] Yunping Liu [email protected] Zhuo Feng [email protected] Xiuyan Zhou [email protected] 1

Horticultural Department, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture, Northeast Agricultural University, Harbin, China

2

School of Resource and Environment, Northeast Agricultural University, Harbin, China

analysis of the transcriptome revealed that the mechanisms underlying the response of cucumber roots to nitrogendeficient stress were considerably different from those of its leaves. Importantly, the DEGs involved in the photosynthesis were almost downregulated, suggesting that the photosystem was sensitive to nitrogen starvation. Otherwise, the nitrate metabolic pathway of cucumber was suppressed by nitrogen deficiency, which was further confirmed by quantitative reverse transcription–polymerase chain reaction. This study represents a comparative analysis of the transcriptome levels of roots and leaves of cucumber, which possibly provides a valuable resource for further investigating the mechanism underlying plant response to long-term nitrogen limitation stress along with the candidate genes controlling the nitrogen metabolism. Keywords Cucumis sativus L.  DEGs  Low nitrogen stress  Transcriptome

Background Nitrogen is an important component of fertilizers and pesticides. In the last several decades, nitrogen has been widely used in agriculture to improve yield worldwide (Chen et al. 2011). Thus, reduction in nitrogen fertilizer consumption and breeding of new plant varieties with high nitrogen utilization efficiency has become the strategy worldwide. A number of studies have been performed to identify the molecular mechanisms underlying plant response to nitrogen deficiency. In aerobic soil conditions, nitrate is the major nitrogen source for most plants. High input of fertilizer gaining high yield in traditional agriculture has resulted in severe pollution. China is the largest fertilizer consumer in the world.

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The fertilizer consumption of China has reached approximately 60 million tons, more than one-third of the total fertilizer usage of the world, but with lower utilization. In 2014, the amount of nitrogen fertilizer application in agriculture had reached 24 million tons in China (National Bureau of Statistics of the People’s Republic of China). Although many nitrogen metabolism genes have been identified and cloned, the effect of the expression of these functional genes on nitrogen assimilation varies in different species. The nitrogen transporter gene, ZmDof1, which was reported to play an important role in accelerating nitrogen uptake, has been transferred to many species to promote nitrogen assimilation and growth in nitrogen-starved conditions (Kurai et al. 2011). However, the overexpression of ZmDof1 gene in Populus did not improve nitrogen assimilation under low nitrogen limitation conditions (Lin et al. 2013). Cucumber (Cucumis sativus L.) is one of the most important vegetable crops worldwide with increased nitrogen dependence (Wu et al. 2014). Although the transcriptome of cucumber leaves was analyzed by Zhao et al. (2015) under nitrogen-deficient conditions, cucumbers were treated with low nitrogen for a short time (12 h) in a hydroponic system. How does the transcriptome of cucumber respond to the long-term low nitrogen stress condition (LN)? Is the expression of genes in leaves and roots consistent? The present study sought to delineate the genes mediating nitrogen metabolism in cucumber and elucidate the mechanisms underlying the response to long-term nitrogen limitation. The cucumber cultivar ‘‘Jinyan No. 4’’ was treated with either adequate or inadequate nitrate levels from the seedling stage. The roots and leaves were harvested for transcriptome sequencing and analysis at the 3.5-leaf stage, yielding some transcripts in response to nitrogen-starved condition. Therefore, a number of candidate genes and functional elements determining major metabolic pathways of chronic low nitrogen-deficient stress response could be identified. It is of increasing importance to enhance efforts for genetic improvement of the cultivated cucumber.

Materials and methods Plant material and culture conditions The seeds of cucumber variety ‘‘Jinyan No. 4’’ were germinated and grown in plastic pots containing clean sand. The plants were grown in a growth chamber under a 14/10-h photo period at 25 °C (day) and 18 °C (night), and 65% humidity. The Yamazaki cucumber nutrient solution was used in this study according to the method proposed by Wu et al. (2014). The same nutrient composition was supplied excluding nitrogen. Control (CK) plants were treated with a complete Yamazaki cucumber nutrient solution. Under the chronic LN,

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the plants were treated with one-fifth of nitrogen solution, while some plants were watered with a solution containing no nitrogen (NN) source. The plants were also treated with a 1 mM nitrogen solution [0.8 mM KNO3, 0.1 mM Ca(NO3)2, 0.25 mM KH2PO4, 0.25 mM MgSO4, 1.2 mM K2SO4, 0.8 mM KCl, 0.4 mM CaCl2, and 0.2 mM NaCl]. The CK plants were treated with a complete nutrient solution containing 5 mM nitrate as the nitrogen source [4 mM KNO3, 0.5 mM Ca(NO3)2, 0.25 mM KH2PO4, 0.25 mM MgSO4, 1.2 mM K2SO4, 0.8 mM KCl, 0.4 mM CaCl2, and 0.2 mM NaCl]. Under the NN condition, the plants were watered with Yamazaki solution containing NN (0.25 mM KH2PO4, 0.25 mM MgSO4, 1.2 mM K2SO4, 0.8 mM KCl, 0.4 mM CaCl2, and 0.2 mM NaCl). All the nutrient solutions contained microelements [24 lM H3BO3, 10 lM MnSO4, 3 lM ZnSO4, 0.9 lM CuSO4, 0.04 lM (NH4)6Mo7O24] and 10 mg/ L iron–EDTA. Each plant was irrigated with 25 mM nutrient solution every 3 days. The plants under CK, LN, and NN conditions were monitored, and leaves and roots of CK and LN plants were used for total RNA extraction after 28 days of treatment. The present study adopted random sampling with three replicates. Solexa sequencing and statistical analysis The total RNA preparations from 15 representative individual plants of each type were pooled for further analysis (Miller et al. 2007). The cDNA libraries for Solexa sequencing were prepared following the Illumina protocol for digital gene expression tag profiling with NlaIII. Oligo(dT) beads were used to enrich mRNA from 6 lg of total RNA, which was then converted into double-stranded cDNA through reverse transcription (RT) and second cDNA strand synthesis using random primers. The cDNA library was generated according to the manufacturer’s protocol (Illumina HiSeq 2000 system). In brief, the four-base recognition enzyme NlaIII was used to digest cDNA at the cathepsin G (CatG) site, and Illumina adaptor 1 was ligated. The Mmel was used to digest at 17 bp downstream of CATG site, followed by ligation of Illumina adaptor 2. After polymerase chain reaction (PCR) purification with primers GX1 and GX2, the 95-bp cDNA products were separated on 6% total binding energy- polyacrylamide gel electrophoresis (TBE-PAGE) and purified for sequencing on an Illumina HiSeq 2000 platform. The leaves and roots of CK and LN plants were run in separate lanes for Solexa sequencing. Reads were exported in FASTQ format and deposited at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus under the accession number GSE46678. The raw sequences were converted into clean tags after removing the 30 adaptor sequence. Reads of low quality and those shorter than 16 nt were excluded from further analysis. All clean tags were mapped onto the assembled

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cucumber contigs (http://www.phytozome.net/cucumber. php) using the Bowtie algorithm with only a 1-nt mismatch. Clean tags mapping to the reference contigs assembly from multiple genes were filtered. The remaining clean tags were designed as unambiguous clean tags, which were calculated and normalized to the RPKM (reads per kilobase transcript per million reads) value for each gene to represent the gene expression level. The differentially expressed genes (DEGs) were screened using the edgeR package with a P value cutoff of 0.01 and fold change cutoff of 2. The Arabidopsis homologous genes of DEGs were searched using the Basic Local Alignment Search Tool program, and functional enrichment was assessed using the DAVID database (http://david.abcc. ncifcrf.gov/). The MapMan metabolism overview maps were drawn as suggested by Yu et al. (2016). Real-time quantitative RT-PCR (qRT-PCR) analysis The first-strand cDNA fragment was synthesized from total RNA using the RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). The primers for qRT-PCR were designed using the Primer Premier 5.0 (Premier Biosoft International, CA, USA) and synthesized commercially (Sangon, Shanghai, China). The primers are listed in Table S1. Samples of 20 lL volume were run in triplicate on Bio-Rad iQ5 cycler (BioRad Laboratories, Hercules, CA) using 1 lL first-strand cDNAs and SYBR Green PCR Master Mix (Applied Biosystems). Thermal cycling was performed at an initial denaturation step at 95 °C for 1 min followed by 40 cycles at 95 °C for 15 s, and annealing at 72 °C for 45 s. The results were analyzed using the software accompanying the Bio-Rad iQ5 machine. The relative quantitation of gene expression was performed and normalized to b-actin (accession number NM_007393, forward: 50 -CCACCAATCTTGTACACATCC-30 and reverse: 50 -AGACCACCAAGTACT ACTGCAC-30 ) (Chen et al. 2011). The normalized expression value was calculated as follows: Difference in the gene expression ¼ 2DCðtÞSample Ave  DCðtÞCalibrator Ave: where DC(t) is sample (Cttarget gene-Cthousekeeping Each RNA sample was tested in three replications.

gene).

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Result and discussion Effect of nitrogen stress on growth performances of cucumber seedlings The growth of cucumber seedlings was evaluated under adequate and inadequate nitrogen conditions to test the changes in plant growth response to a nitrogen-deficient condition. As shown in Table 1, after 28 days of treatment, the plant height, leaf area, and total root biomass were significantly reduced under nitrogen-deficient conditions. Especially, the plant dry mass decreased by approximately 43.39 and 72.88% with LN and NN treatment, respectively, compared with the normal-nitrogen exposure. Although the cucumber development was not delayed and reached the 3.5-leaf stage, inadequate nitrogen triggered severe developmental retardation (Fig. 1a). Under nitrogen-deficient stress, the cucumber stalk was slender (Fig. 1b), the cotyledon was yellowish (Fig. 1c), and leaves were significantly smaller (Fig. 1d). Furthermore, the samples cultured without nitrogen addition were too small and wilting to extract sufficient RNA and verify the differentially expressed genes by qRT-PCR. Abundant nitrogen was involved in protein synthesis, nutrient accumulation, and plant metabolism. Consequently, nitrogen starvation severely affected plant growth, leading to reduced yield (Wang et al. 2011), poor germination (Welch et al. 1973), and smaller and abnormal leaves (Yang et al. 2013). The small and yellowish leaf phenotype caused by nitrogen deficiency could result in a decline in photosynthesis (Paul and Driscoll 2008). Transcriptome expression profile of cucumber in response to nitrogen starvation stress Leaves and roots of cucumber plants grown under adequate-nitrogen (CK) and LN conditions were harvested at 28 days after sowing to unravel the molecular basis of the response of cucumber to long-term nitrogen stress. A total of 4.8, 4.7, 3.7, and 3.5 million clean sequencing tags were obtained for the sampling of CK root, LN root, CK leaf, and LN leaf, respectively. Over 99% of the tags were successfully mapped to the published cucumber

Table 1 Effects of low nitrogen treatment on the total plant height, leaf area, total biomass, and root biomass of cucumber after Nitrogen treatment

Plant height (cm)

Leaf area (cm2 plant-1)

Total dry weight (g plant-1)

Root dry weight (g plant-1)

No nitrogen added

5.16 ± 0.98a

18.83 ± 3.22a

0.46 ± 0.11a

0.16 ± 0.09a

Low nitrogen

9.67 ± 1.22ab

24.37 ± 2.98a

0.75 ± 0.14b

0.31 ± 0.07ab

66.38 ± 7.62b

1.268 ± 0.31c

0.59 ± 0.12b

Normal-nitrogen

14.53 ± 2.07b

Each value is the mean ± SD Significant differences are marked with different letters with P \ 0.05

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Fig. 1 Phenotypes of nitrogen sufficient (CK), low nitrogen (LN) and nitrogen starved (NN) cucumber seedlings. a Cucumber seedlings cultured in pot containing clean sand; b stem of cucumber seedlings; c cotyledon of cucumber seedlings; d leaf of cucumber seedlings and

e root of cucumber seedlings. The right: sufficient nitrogen (CK), the middle: low nitrogen (LN) and the left: no nitrogen added (NN) in each figure

genome sequence (Huang et al. 2009). In roots, 827 and 641 genes were upregulated and downregulated, respectively, in response to the long-term nitrogen starvation. Also, 667 genes were upregulated and 856 were downregulated in the leaves, accounting for 7.04% of DEGs. Only a small number of regulated genes overlapped in these two different organs (Fig. 2). For experimental confirmation, qRT-PCR was performed to detect the expression of main candidate genes (Fig. 6). The qRT-PCR results were in agreement with the RNAseq, indicating that the Illumina results were reliable (Fig. 6).

As presented in Fig. 2, 827 DEGs exhibiting elevated expression between CK and long-term nitrogen-deficient stress were divided into seven cluster groups in the roots. The Gene Ontology biological process enrichment indicated that glucosyltransferase activity, membrane, transmembrane, calcium, and uridine 50 -diphospho-glucuronosyltransferase (UDP)-glucosyltransferase activityrelated processes were overrepresented. Moreover, 641 downregulated DEGs were classified into 7 clusters according to their expression patterns in the roots, and mainly involved in external encapsulating structure, inorganic anion transport, cell wall, inorganic anion transmembrane transporter activity, ion transport, and cellular response to phosphate starvation-related processes. The sucrose and starch metabolic pathway genes were upregulated in the roots under LN stress (AT1G12240, AT2G44450, AT2G44480, AT5G42260). The majority of genes mediating glucosyltransferase and UDP-glucosyltransferase activity were upregulated in the roots under LN treatment (Table 2). Steryl glycosides—the abundant constituents in the membranes of higher plants—were synthesized by membrane-bound UDP-glucose. Additionally, UDP-glucosyltransferase involved in starch synthesis was

Identification of biological processes involved in response of cucumber to long-term nitrogendeficient conditions Hierarchical clusters were performed through the heat map analysis representing the transcription levels and metabolic pattern changes in the roots (Fig. 3) and leaves (Fig. 4), respectively, to further explore the regulatory processes between the normal and long-term nitrogen-deficient conditions. Multiple metabolic-related pathways responding to long-term nitrogen-deficient have been identified.

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Fig. 2 Distribution of differentially expressed genes comparing control plants versus plants treated with low nitrogen. a Root of cucumber seedling; b leaf of cucumber seedling

Fig. 3 DEGs ontology categories and clustering analysis of upregulated genes in root of cucumber seedlings responding to long-term nitrogen deficiency. DEGs ontology categories (a) and clustering analysis (b) of upregulated genes in sufficient nitrogen (CK) root of cucumber seedlings; DEGs ontology categories (c) and clustering analysis (d) of upregulated genes in low nitrogen (LN) root of

cucumber seedlings; diagrams in a and c depict the top functional enriched clusters between the nitrogen deficiency and control samples; the horizontal bars depict P values for the overall enrichment levels. Diagrams in b and d, heat map of the genes in top functional enrichment clusters. Scaled gene expression values are color-coded according to the legend on the left

documented to be indispensable to metabolic processes (DeBolt et al. 2009). These DEGs involved in ion transport were discovered to be mainly downregulated in the roots. The nutritional status, such as nitrogen, phosphorus, and sulfur, can influence the expression of ion transport systems. Conversely, the specific feedback regulation of ion transporters in the roots by nutrition assimilation or the products of their metabolism probably played a prominent role in some other metabolism pathway under different nutritional conditions, including carbon and phosphorus metabolism (Lejay et al. 2008). Likewise, transcripts related to the cellular response to phosphorus-deficient conditions were downregulated in the cucumber roots. In the present study, nitrogen starvation to suppress the root cell wall formation

was found to be in agreement with previous studies regarding the effect of nitrogen fertilization on cell wall composition (Sindelar et al. 2015). However, the genes associated with the cell wall were upregulated in the leaves (Fig. 3). A large proportion of DEGs upregulated in the leaves were found to be involved in the regulation of plant growth and development, such as chromosome, cell cycle, cell division, postembryonic development, shoot development, leaf development, and reproductive structure development, whereas most of the transcription factors involved in the photosynthesis were found to be notably inhibited under long-term nitrogen deficiency. Functional clustering of the genes downregulated by nitrogen starvation in cucumber leaves showed a strong enrichment of genes regulating the

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Fig. 4 DEGs ontology categories and clustering analysis of upregulated genes in leaf of cucumber seedlings responding to long-term nitrogen deficiency. DEGs ontology categories (a) and clustering analysis (b) of upregulated genes in sufficient nitrogen (CK) leaf of cucumber seedlings; DEGs ontology categories (c) and clustering analysis (d) of up-regulated genes in low nitrogen (LN) leaf of

cucumber seedlings; diagrams in a and c depict the top functional enriched clusters between the nitrogen deficiency and control samples, the horizontal bars depict P values for the overall enrichment levels. Diagrams in b and d, heat map of the genes in top functional enrichment clusters. Scaled gene expression values are color-coded according to the legend on the left

chloroplast, chloroplast thylakoid membrane, photosynthesis, light reaction, photosystem, and carbohydrate biosynthesis. Nitrogen metabolism requires large amounts of energy, lots of equivalents (NADH and NADPH), and numerous organic carbon intermediates. The tricarboxylic acid (TCA) cycle and glycolysis are very important metabolic pathways that provide energy, several organic carbon intermediates, and reducing equivalents (NADH and NADPH). The nitrogen starvation could obviously suppress plant development, including nitrogenous macromolecule synthesis, energy consumption, hydrocarbon synthesis, and so on. In the present study, the genes involved in glycolysis and TCA cycle in the leaves were repressed by long-term nitrogen starvation. However, the impact of nitrogen starvation on the expression levels of genes involved in both glycolysis and TCA cycle in the roots was less than those in the leaves, suggesting differences among organs of cucumber in response to long-term nitrogen deficiency (Figs. 3, 4, 5). Actually, genes related to photosynthesis, such as light reactions and photorespiration, were strongly downregulated. Otherwise, reduced photosynthesis possibly explained the yellow cotyledons, smaller leaves (Fig. 1), and plant biomass suppression (Table 1). Bi et al. (2007) previously demonstrated significantly reduced (*30% reduction) chlorophyll content and downregulation of genes involved in chlorophyll metabolism in Arabidopsis under severe nitrogen-limiting conditions. Starch—the

main reserve deposit in plants—was consequently inhibited. Noticeably, the genes related to phenylpropanoid pathways were evidently upregulated in the leaves, but downregulated in the roots under nitrogen-starved conditions (Fig. 5). The secondary metabolites, phenylpropanoids, participated in resisting photooxidative stress, playing the complementary role of antioxidant enzymes under severe conditions (Tattini et al. 2015). Soluble sugars and phenylpropanoids were inversely correlated under heat treatment (Xue et al. 2013). Consistently, in this study, genes associated with sucrose mobilization were downregulated in the leaves.

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Nitrogen metabolism change in cucumber under long-term nitrogen-deficient stress Plant nitrogen metabolism involves a complicated network, which includes nitrogen uptake, reduction, amino acid metabolism and transport, and translocation and remobilization of nitrogen (Masclaux-Daubresse and Chardon 2011). In higher plants, the conversion of mineral nitrogen into organic nitrogen is primarily initiated through the uptake of nitrate by root cells followed by reduction and assimilation into other tissues. Compared with other species, cucumber carries a much smaller set of nitrate transporter (NRT) genes (Bouguyon et al. 2015). The changes in the expression of cucumber genes involved in nitrogen uptake, assimilation, and transport were investigated in this study. After retrieving the sequence tags from the NCBI using cDNA sequences of cucumber genes, a

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Table 2 Summary of DEGs encoding proteins related to nitrogen metabolism Cucumber ID

Arabidopsis ID

Root CK

Root LN

Leaf CK

Leaf LN

Gene symbol

Function

AT1G12110

235.42

11.03

0

0

NRT1.1a

Nitrate transporter

AT1G12110

31.49

29.08

34.63

40.28

NRT1.1

Nitrate transporter

Cucsa.313420L

AT1G32450

25.76

7.9

0

0.14

NRT1.5a

Nitrate transporter

Cucsa.257150H,R

AT1G32450

148.04

47.9

2.83

6.35

NRT1.5

Nitrate transporter

Cucsa.321620D

AT1G32450

50.65

44.92

2.24

0.48

NRT1.5

Nitrate transporter

Cucsa.362690H

AT1G32450

0.11

0.83

0.16

0.17

NRT1.5

Nitrate transporter

Cucsa.285460D,R

AT1G69870

2.32

2.04

2.16

5.69

NRT1.7

Nitrate transporter

Cucsa.268720L

AT5G60770

48.63

22.06

0

0

NRT2.4

Nitrate transporter

Cucsa.286270H

AT1G12940

0.15

19.57

0

0

NRT2.5a

Nitrate transporter

Cucsa.302000L Cucsa.320700

D,R

a

Nitrate reductase

Cucsa.274720

AT1G77760

4.53

0.08

0

0

NR1

Cucsa.026240 Cucsa.244280

AT1G37130 AT2G15620

82.85 150.49

79.43 43.85

179.91 178.51

425.17 101.92

NR2 NiR1

Nitrate reductase Nitrite reductase

Cucsa.034720

AT5G37600

18.58

4.35

12.85

18.37

GS1a

Glutamine synthetase

Cucsa.375220

AT5G37600

502.3

612.62

72.07

92.03

GS1

Glutamine synthetase

Cucsa.253880

AT5G35630

35.87

28.6

655.44

752.65

GS2

Glutamine synthetase

Cucsa.140600

AT4G28700

0.16

0.27

0.24

0

AMT1;4

Ammonia transporter

Cucsa.194400

AT4G13510

50.83

62.62

44.32

63.21

AMT1;1

Ammonia transporter

Cucsa.255930

AT4G13510

2.51

3.17

0

1.21

AMT1;1

Ammonia transporter

Cucsa.178210

AT1G64780

11.05

33.67

0.85

0.86

AMT1;2a

Ammonia transporter

Cucsa.178240

AT1G64780

0

0

6.88

9.04

AMT1;2

Ammonia transporter

Cucsa.044000

AT2G38290

0.18

0

0

0.55

AMT2

Ammonia transporter

Cucsa.176640

AT2G38290

16.56

44.74

0.73

0.94

AMT2

Ammonia transporter

Cucsa.024080

AT5G18170

38.15

90.02

5.79

9.02

GDH1a

Ammonia assimilation

Cucsa.093950

AT5G07440

15.31

12.49

0.19

0.21

GDH2

Ammonia assimilation

Cucsa.129100

AT5G07440

3.74

2.75

1.04

2

GDH2

Ammonia assimilation

These data come from the Solexa transcript count data, the genes expression numbers units were RPKM (number of sequence reads per million clean tags per kilo base), more than two-fold expression difference to be formatted in bold a

Genes for qRT-PCR confirmation of the accuracy of the Solexa sequencing, CK control plant, LN plant treated with low nitrogen

L

Genes belong to low-affinity transport system

D

Genes encoded dual-affinity nitrate transporter

H

Genes belong to high-affinity transport system

R

Genes involved in nitrogen remobilization

total of 25 DEGs involved in nitrogen metabolism were identified, including 9 NRT genes, 3 nitrate reductase genes, 3 glutamine synthetase genes, 7 ammonia transporter genes, and 3 ammonia assimilation genes (Table 2). The NRT1 and NRT2 families are involved in N translocation and utilization for plant growth. Nine cDNA sequences related to NRT1 and NRT2 families were identified (Table 2). The NRT genes encoded by the cucumber genome included two homologs of NRT1.1, four of NRT1.5, and one each of NRT1.7, NRT 2.4, and NRT 2.5. NRT1.1 has been identified as a significant NRT, controlling several nitrate assimilation-related genes and signaling pathways in Arabidopsis (Bouguyon et al. 2015). Early studies have shown that NRT1.1 mediated nitrate uptake from the soil by root cells in many plants (Migocka

et al. 2013) and played a significant role in nitrogen use efficiency and yield (Hu et al. 2015). In the present study, a copy of NRT1.1 encoded by Cucsa.320700 was expressed predominately in the leaves, while another copy encoded by Cucsa.32000 was found only in the roots (Table 2). According to previous studies (Parker and Newstead 2014), NRT1.1 could switch a low-affinity transporter to a highaffinity state following phosphorylation of an intracellular threonine under low-nitrate conditions. However, nitrogen starvation triggered about a 20-fold decrease in the expression of Cucsa.302000 in the roots, while little difference in the expression of Cucsa.320700 was noted between CK and LN roots in this study (Table 2). Under nitrogen-starved conditions, NRT1.1 was dramatically downregulated in the roots without any significant changes

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b Fig. 5 Metabolism overview maps of root and leaf in cucumber

seedlings at long time nitrogen deficiency. Each small square represents a gene, in which the red is upregulated genes in low nitrogen conditions, blue is downregulated genes and the deeper the color shows the greater difference, black circle highlights DEGs gathering in metabolic pathway. a Leaf and b root

in the leaves compared with the control, according to the qRT-PCR results (Fig. 6). A similar phenomenon of NRT 1.5 was also found in the present study (Table 2). Cucsa.313420 encoding a copy of NRT1.5 showed a significant decrease in response to nitrogen stress, while the expression of Cucsa.257150 encoding a copy of NRT1.5 was strongly stimulated by nitrogen starvation in the roots. However, NRT 1.5, which was not significantly altered in leaf tissues (Table 2), showed dramatically decreased expression under nitrogen-starved conditions in both roots and leaves (Fig. 6). Consistent with the present study, Lin et al. (2008) reported that NRT1.5 was a low-affinity, pHdependent bidirectional NRT and was downregulated by stress. NRT1.7 has not been found in cucumber before. While, the expression of Cucsa.285460 encoding a copy of NRT1.7 was investigated for changed little in root (Guo et al. 2014), and slightly increased under nitrogen-deficient condition in this research. The nitrogen starvation-induced expression of NRT2.5 and NRT2.4 in the roots was reported in the 10-day stressed Arabidopsis (Krapp et al. 2011). Nevertheless, the expression of Cucsa.268720-encoded NRT 2.4 declined and that of Cucsa.286270-encoded NRT 2.5 increased in the roots of cucumber under LN condition (Table 2), which might be attributed to specific differences among plants. These results are similar to those reported by Guo and others (2014) that the expression of NRT2.5 gene exhibited a rapid increase while NRT2.4 with a decline in nitrogen starvation stress. Nitrate reductases (e.g., NR1, NR2, and NiR1) exhibited a significant decline under chronic nitrogen-starved conditions in the wheat root (Reid et al. 2016). Consistently, the expression of NR1 and NiR1 in the roots significantly decreased under nitrogen-limiting stress conditions, while NR2 levels increased in the leaves (Table 2). The expression of these three nitrate reductases decreased under chronic nitrogen-limiting stress in Arabidopsis (Bi et al. 2007). These genes mediating nitrogen transport and reduction were expressed differentially among various nitrogen applications in the tissues of cucumber. The ammonia transporters (AMT) were demonstrated to be responsive to nitrogen starvation at different developmental time points (Beatty et al. 2009). In Arabidopsis, AMT genes could be induced under nitrogen deficiency (Yuan et al. 2007). However, under low-nitrate stress, the

Fig. 6 Comparison of relative transcript abundance of candidate genes of root and leaf in cucumber seedlings by real-time quantitative RT-PCR (qRT-PCR). Normalized expression level normalized to bactin, error bars represent the gene transcript abundance ratios between the leaf and root in plants treated with sufficient nitrogen (CK) and low nitrogen (LN). NRT nitrate transporter, AMT ammonia transporter, NR1 nitrate reductase 1, GS1 glutamine synthetase 1, GDH1 glutamate dehydrogenase, ERF1 ethylene response factor 1, FZR3 FIZZY-related 3, NFA2 nucleosome/chromatin assembly factor group 2, RAP2.3 related to AP2.3, HB40 homeobox protein 40, SPX2 SPX domain gene 2, MGD2 monogalactosyldiacylglycerol synthase 2

expression of AMT gene showed to change little in the current study, indicating that the expression of AMT genes is unaffected by nitrogen levels in cucumber. Consistent with the present study, Loque´ et al. (2006) mentioned that AMT genes in different plant species might have a variety of expression patterns, and did not serve as a universal biomarker in the nitrogen-deficient environment. These results indicated that different members of the same family might have differential expression pattern with plant species-specific in cucumber under low nitrogen condition.

Conclusion N starvation greatly inhibited the growth and decreased the dry weight of cucumber seedlings. In the present study, a large-scale transcriptome analysis of the cucumber roots and leaves was performed to characterize common different transcripts in response to nitrogen-deficient treatment during the seedling stage. The DEG analysis suggested that

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low nitrogen significantly affects photosynthesis, and energy metabolism in cucumber. The expression analysis of DEGs validated that nitrogen starvation evidently influenced translation and metabolism of nitrogen in cucumber. NRT1.7 was first detected in cucumber seedlings, which was more sensitive to nitrogen starvation in leaves than that in root. The present study provides novel insights into the molecular mechanisms underlying the response to LN conditions in cucumber. Author contribution statement ZQ and MX conceived the study, designed the experiments, and contributed to the manuscript. MX and YL were responsible for sequence annotation and data analysis. ZF contributed to the experiments, and MX contributed to the data analysis. All authors read and approved the final manuscript. Acknowledgements This study was funded by the National Natural Science Foundation of China (31401863), Young University Innovative Talent Training Program of Heilongjiang Province (UNPYSCT-2016007), Supporting Certificate of Heilongjiang Postdoctoral Scientific Research Developmental Fund (LBHQ16021), the Open Project of Heilongjiang Provincial Key University Laboratory of Cold Area Vegetable Biology (CVB2012-001), Certificate of China Postdoctoral Science Foundation (2013M540265) and Certificate of Heilongjiang Postdoctoral Fund (LBH-Z12037). And there are no financial competing interests. Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests.

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