Transcriptome analysis reveals the differential regulatory effects of red and blue light on nitrate metabolism in pakchoi (Brassica campestris L.)

FAN Xiao-xue,BlAN Zhong-hua,SONG Bo,XU Hai

1 Institute of Vegetable Crops/Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,P.R.China

2 Photobiology Research Center,Institute of Urban Agriculture,Chinese Academy of Agricultural Sciences,Chengdu 610200,P.R.China

Abstract Pakchoi (Brassica campestris L.ssp.chinensis) is an important leafy vegetable.Various light spectra,especially red and blue light,play vital roles in the regulation of nitrate metabolism.Information on the effects of red and blue light on nitrate metabolism at the transcriptome level in pakchoi is still limited,so this study used RNA sequencing technology to explore this molecular mechanism.Through pairwise comparisons with white LED light,3 939 and 5 534 differentially expressed genes (DEGs) were identified under red and blue light,respectively.By Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses,these unigenes were found to be involved in nitrate assimilation,plant-pathogen interaction,biosynthesis of secondary metabolites,and phenylpropanoid biosynthesis.The differential effects of light spectra on the nitrate concentration and metabolism-related enzyme activities were also confirmed at the physiological level.Several signal transduction modules,including Crys/Phys-COP1-HY5/HY5-like,were found to be involved in red and blue light-induced nitrate metabolism,and the transcript levels for this complex were consistent with the observed degree of nitrate assimilation.The expression patterns of 15 randomly selected DEGs were further validated using qPCR.Taken together,the results of this study could help improve our understanding of light spectrumregulated nitrate metabolism in pakchoi at the transcriptome level.

Keywords:nitrate metabolism,light spectra,transcriptome,gene expression,pakchoi

1.lntroduction

Pakchoi (BrassicacampestrisL.ssp.chinensis) is one of the most important leafy vegetable species because of its high concentrations of healthy nutrients (Kim Y Jet al.2015).However,pakchoi readily accumulates excessive nitrates during production,especially under hydroponic cultivation (Chenet al.2004).Some studies have reported the positive function of short-term high nitrate levels in the human diet in alleviating agingrelated diseases,such as high blood pressure (Ashworthet al.2015) and cardiovascular disease (Machha and Schechter 2011).In recent years,increasing numbers of studies have demonstrated that consuming vegetables with high nitrates every day increases the risk of gastric cancer (Ghaffariet al.2019;Zhanget al.2019) and methemoglobinemia (Bondonnoet al.2016).Therefore,to maintain human health,keeping the nitrate concentration below allowable limits is essential.

Nitrate metabolism in plants is interactively controlled by internal gene expression and related enzyme activities and external environmental cues (Bianet al.2020).Light is one of the most important environmental factors in the regulation of nitrate metabolism,especially the effects of specific light spectra which is a key factor in the regulation of the nitrate concentration in plants (Bianet al.2015,2020).With the development of light-emitting diode (LED) technology,the effects of LED light spectra on nitrate regulation in leafy vegetables have generated increasing concern among researchers and farmers (Bianet al.2015,2018;Liet al.2017).Among the different light spectra,red and blue light spectra are the most efficient in driving photosynthesis and regulating phytochemical metabolism in higher plants (Hanet al.2017;Monostoriet al.2018).

Nitrate reductase (NR),which reduces nitrate into nitrite,is the rate-limiting enzyme in nitrate assimilation in plants (Sivasankar and Oaks 1996).The resulting nitrite will be rapidly converted into the less toxic ammonia by nitrite reductase (NiR) (Leiet al.2018).The activity of NR and related gene (NR) expression are profoundly affected by red and blue light (Sakuraba and Yanagisawa2018;Fanet al.2019).Phytochromes(Phys) and cryptochromes (Crys) play pivotal roles in red and blue light-induced NR activity through transcriptional and posttranslational regulation (Castillonet al.2009;Sakuraba and Yanagisawa 2018).In higher plants,the basic leucine zipper (bZIP) transcription factors ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG (HYH) are the dominant regulators of nitrate metabolism (Gangappa and Botto 2016).The ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) participates in the light-related regulation of nitrate assimilation through interactions with HY5/HYH(Gangappa and Botto 2016).It is assumed that the cascade of phys-COP1-HY5/HYH may be involved in red light-dependent nitrate assimilation (Sakuraba and Yanagisawa 2018).In addition,the red light was shown to promote nitrate reduction through a red light signalingmediated reversion of NR dephosphorylation (Qiet al.2007).However,the mechanism by which red light regulates nitrate assimilation mediated by phytochromes remains unclear.The regulation of nitrate metabolism by blue light seems more complex than the regulation by red light.Crys and Phys are both involved in blue-lightmediated NR activity andNRexpression (Osterlundet al.2000;Castillonet al.2009).Nitrate metabolism in higher plants is not only regulated by NR but also significantly affected by other nitrogen metabolism enzymes,including nitrite reductase (NiR),glutamine synthetase (GS),and glutamate synthase (GOGAT).Blue light was reported to participate in regulating the expression of GS-related genes (GS) and GOGAT-related genes (GOGAT)viaPhys at both the transcriptional and translational levels(Elmlingeret al.1994;Kauret al.2013).Our previous study reported that blue and red light has different effects on the nitrate metabolites and phytochemical accumulation in hydroponically grown pakchoi (Fanet al.2019).However,the molecular mechanism(s) of red and blue light signaling in the regulation of nitrate metabolism in pakchoi remain elusive.

In this study,RNA-seq analysis was used to elucidate the mechanisms by which red and blue light affect nitrate metabolism in hydroponic pakchoi at a genome-wide level.This study provides a global view of the pakchoi responses to monochromatic red and blue light at the transcriptome level.Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology(GO) enrichment analyses,the identified unigenes were found to be involved in nitrate assimilation,plantpathogen interactions,and phenylpropanoid biosynthesis.The results of our study present an overall view of the transcriptomic response to light spectra in pakchoi.Thus,not only can our study further expand our understanding of light spectra-regulated nitrate metabolism in pakchoi at the transcriptome level but it may also provide useful information on producing high-quality leafy vegetables through light spectral management under controlled environments.

2.Materials and methods

2.1.Plant materials and growth conditions

Pakchoi (BrassicacampestrisL.) seeds were provided by the Institute of Vegetable Crops of Jiangsu Academy of Agricultural Sciences,China.The seeds were soaked in purified water for 8 h and then sown in a seedling sponge and germinated under white LED light with an intensity of 150 μmol m-2s-1in an environmentally controlled growth chamber (Fanet al.2019).The day/night temperatures,relative humidity,photoperiod,and CO2level were set at (20±2)/(25±2)°C,60-80%,12 h and 400 μmol mol-1,respectively.When the cotyledons were fully expanded,seedlings were transplanted into a 40-L container with Hoagland nutrient solution (pH (6.5±0.2),electrical conductivity (1.9±0.1) dS m-1) and grown in the same growth chamber.When these seedlings developed a sixth true leaf,similarly sized pakchoi seedlings were selected and randomly treated with different light spectra.There were three light treatments:white LED light (400-700 nm,White),monochromatic red LED light (660 nm,Red),and monochromatic blue LED light (450 nm,Blue).The white LED light was used as a control in this study.Every other day,the light intensity and light spectra of the white,red,and blue light were monitored using a spectroradiometer(AvaSpec-2048CL,Avantes,Apeldoorn,The Netherlands).The light intensities at the plant canopies were maintained at 150 μmol m-2s-1.Throughout the experiment,the other environmental conditions in the growth chamber were maintained as described previously.After 14 d of light treatment,nine plants were randomly selected from each light treatment.The second fully expanded leaf from the top of each plant was sampled and immediately frozen with liquid nitrogen before being stored at -80°C for further analysis.

2.2.Nitrate and nitrite concentration determinations

The nitrate concentration was measured according to the method described by Bianet al.(2016),with some modifications.Briefly,the leaf sample (1.0 g) was pulverized in 10 mL deionized water and placed into a boiling water bath for 20 min.The supernatant was diluted and mixed with 5% salicylic acid-sulfuric acid.The mixture was kept at room temperature for 20 min before adding 8% NaOH.The absorbance of the extracted solution measured at 410 nm was used to calculate the nitrate concentration.

The nitrite concentration was determined spectrophotometrically (Fanet al.2019).Leaf samples (1.0 g)were homogenized with 2.0 mL extraction buffer.The extracts were diluted with water to reach a final volume of 50 mL before adding 1.0 mL of ferrocyanide and 1 mL of ZnSO4.The reaction mixture containing 5 mL extraction,5 mL sulfanilamide,and 5 mL 1-naphthylamine was incubated at 30°C for 30 min.The absorbance monitored at 530 nm was used to calculate nitrite concentration.

2.3.Measurements of nitrogen metabolism enzyme activities

The method described by Hageman and Reed(1980) was used to determine the activity of NR.The absorbance measured at 540 nm was used to calculate NR activity.The activity of NiR was determined spectrophotometrically using the method of Leiet al.(2018).The activities of GS and GOGAT were measured as described by Shapiroet al.(1970) and Jiaoet al.(2002),respectively.

2.4.RNA extraction,RNA-seq library construction,and sequencing

Total RNA was extracted using an RNAprep Pure Plant Kit (Tiangen,China) according to the manufacturer’s instructions.The quantity and purity of total RNA were determined using a Nanodrop 2000 spectrophotometer(Thermo Scientific,USA).The RNA integrity was checked using the Agilent Bioanalyzer 2100 System(Agilent Technologies,USA) with an RNA Nano 6000 Assay Kit.

Total RNA extracted from each sample was used for Illumina sequencing at Genepioneer Biotechnologies Co.,Ltd.(Nanjing,China).The enriched cDNA libraries were assessed using the Agilent Bioanalyzer 2100 System before being prepared for sequencing analysis on the HiSeq 2000 sequencing platform (Illumina,USA) with a paired-end (PE150) strategy.

2.5.Transcription analysis

The reference genome and gene model annotation files forBrassicawere obtained from the database website(http://brassicadb.org/brad/) (Chenget al.2011).The raw reads were cleaned by discarding the reads with adaptor contamination and low-quality reads (those with a quality score of Q＜20) and filtering the reads with unknown nucleotide percentages greater than 10%.HISAT v2.0.5 was used to count the number of reads for each gene (Kim Det al.2015).Gene expression levels were calculated by Fragments Per Kilobase of Transcript Per Million Mapped Reads (FPKM) using the RSEM (RNAseq by expectation maximization) module provided within the Trinity package (Trapnellet al.2009).Differential expression analysis of pairs of treatments (three biological replicates) was performed using the DESeq2 R package(v.1.16.1).Benjamini and Hochberg’s (1995) approach was used to adjust the resultingP-values for controlling the false discovery rate.Genes with an adjustedP-value＜0.05 found by DESeq2 were assigned as differentially expressed between pairs of treatments.

The cluster Profiler R package was used to carry out the analysis of GO and KEGG pathway enrichment of the DEGs (Tarazonaet al.2011).The GO and KEGG terms with correctedP＜0.5 were considered to be significantly enriched by DEGs between the two treatments.

2.6.Validation of RNA-seq data by qPCR analysis

Fifteen DEGs were randomly selected for validating the RNA-seq results by qPCR.Total RNA (1 μg),which was the same as that used for the RNA-seq sequence,was treated with RNA-free DNase I before cDNA synthesis was performed using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific,USA).The primers for these selected genes were designed by Primer Premier 6.0,and their sequences are summarized in Appendix A.Actin1was employed as an internal reference gene (Qiet al.2010).The qPCR was performed on a Light Cycler 480 (Roche Diagnostics,Germany) with SYBR PremixExTaq (TaKaRa Bio,Beijing,China).The thermocycling conditions were set to 95°C for 30 s,then 40 cycles of 95°C for 5 s,60°C for 20 s,and 72°C for 30 s,followed by a melting curve (65-95°C).The qPCR was performed in triplicate,with three total RNA samples extracted from nine plants.The relative gene expression levels of these selected genes were calculated using the 2-ΔΔCtmethod(Livak and Schmittgen 2001).

3.Results

3.1.The effects of red and blue light on nitrate metabolism in pakchoi

The nitrate and nitrite concentrations in pakchoi were significantly affected by the light spectra (Fig.1-A).The lowest nitrate and nitrite concentrations were both observed in pakchoi under Blue.Compared with the values under White and Red,the nitrate concentration under Blue was lower by 165.4 and 296.3 mg g-1,while the nitrite concentration was lower by 1.69 and 0.98 mg g-1.The differences in nitrate concentration between those light treatments differed significantly,while the marked difference in nitrite concentration was only obtained in the comparison between Blue and White.In addition,the activities of NR,NiR,and GS were the highest under Blue,followed by Red and then White(Fig.1-B).However,the activity of GOGAT,another important enzyme in the nitrogen metabolism pathway,presented the opposite trend under the studied light treatments,as shown by the lowest and highest values of this parameter under Blue and White,respectively(Fig.1-B).

Fig.1 The effects of red and blue light on nitrate metabolism in pakchoi.A,nitrate and nitrite concentrations.B,the activities of important enzymes in the nitrogen metabolism pathway.NR,nitrate reductase;NiR,nitrite reductase;GS,glutamine synthetase;GOGAT,glutamate synthase.

3.2.ldentification of differentially expressed genes(DEGs)

To further reveal the mechanism of different light spectra in the regulation of nitrate metabolism,transcriptome analysis was performed to determine the genes that may be involved in spectral-dependent nitrate metabolism.A total of 10 939 DEGs were identified in pakchoi through pairwise comparisons,of which 4 577,5 534,and 1 466 DEGs were obtained in the three comparisons of Redvs.White,Bluevs.White,and Redvs.Blue,respectively(Fig.2-A).Furthermore,12 and 26 DEGs were commonly up-and downregulated,respectively,by red,blue,and white light (Fig.2-B).A total of 511,1 258,and 391 DEGs were upregulated uniquely,while the numbers of unique downregulated DEGs were 391,1 585,and 691 in the comparisons between Redvs.White,Bluevs.White,and Redvs.Blue,respectively.

Fig.2 Identification of differentially expressed genes (DEGs).A,number of DEGs.B,number of upregulated and downregulated DEGs.C,hierarchical clustering analysis of the expression pattern of the DEGs.

In addition,a hierarchical clustering analysis was carried out to present a general overview of the expression pattern of the DEGs (Fig.2-C).Most of the genes with higher levels under White displayed lower levels of expression under Blue,andvice versa.Furthermore,the expression profiles of most genes showed great differences between Red and Blue(Fig.2-C).

3.3.Functional classification of DEG responses to different light spectra

To reveal the monochromatic red and blue lightinduced transcriptomes,GO enrichment analysis was conducted to categorize the DEGs.Compared with White,a total of 32 263 and 48 953 DEGs regulated by Red and Blue,respectively,were annotated into three major GO categories (Appendix B).The GO terms markedly enriched in the biological process category included ‘metabolic process’,‘cellular process’,‘singleorganism process’,and ‘response to stimulus’;while‘cell’,‘organelle’,and ‘cell part’ in the cellular component category were significantly enriched in the comparisons of Redvs.White,Bluevs.White,and Redvs.Blue (Fig.3).In the molecular function category,the markedly enriched GO terms were observed in ‘catalytic activity’ and ‘binding’in the comparisons of Redvs.White,Bluevs.White,and Redvs.Blue (Fig.3).

Fig.3 Functional classification of differentially expressed genes (DEGs) responses to different light spectra.

To further identify the biological function of DEGs,KEGG pathway analysis was performed based on the KEGG database,and these data are summarized in Appendix C.The top 20 pathways for the prominent enriched DEGs in different light comparisons are listed in Fig.4.In the Redvs.White comparison,872 DEGs were annotated to 106 KEGG pathways.These DEGs were significantly enriched in ‘monoterpenoid biosynthesis’,‘glucosinolate biosynthesis’,‘zeatin biosynthesis’,‘a(chǎn)lphalinolenic acid metabolism’,‘tryptophan metabolism’,‘plant-pathogen interaction’,‘linoleic acid metabolism’,and ‘nitrogen metabolism’ (Fig.4-A),while a total of 1 134 DEGs were assigned to 114 KEGG pathways in the comparison of Bluevs.White.These DEGs were significantly enriched in ‘monoterpenoid biosynthesis’,‘flavone and flavonol biosynthesis’,‘glucosinolate biosynthesis’,‘flavonoid biosynthesis’,‘plantpathogen interaction’,‘phenylpropanoid biosynthesis and phenylalanine metabolism’ (Fig.4-B).In the comparison of Redvs.Blue,435 DEGs were identified and annotated to 92 KEGG pathways.These DEGs were mostly and significantly enriched in ‘flavone and flavonol biosynthesis’,‘flavonoid biosynthesis’,‘nitrogen metabolism’,‘phenylpropanoid biosynthesis’,‘phenylalanine metabolism’ and ‘terpenoid backbone biosynthesis’ (Fig.4-C).

Fig.4 The top 20 KEGG pathways for the prominent enriched differentially expressed genes (DEGs) in the different light comparisons.

3.4.Expression profiles of nitrate metabolic structure gene responses to different light spectra

To explore the effect of light on nitrogen metabolism,the DEGs involved in nitrogen metabolism are summarized in Table 1.These genes mainly encode proteins that are involved in the nitrogen metabolic pathway,including glutamate dehydrogenase,aminomethyltransferase,nitrilase,glutamine synthetase,asparagine synthase,and phenylalanine ammonia-lyase.The transcript profiles of these genes differed significantly under the comparisons of Redvs.White and Bluevs.White (Table 1).Furthermore,one phenylalanine ammonia-lyase related gene was upregulated in the comparison of Redvs.White.However,most of the genes encoding phenylalanine ammonia-lyase were downregulated.Compared with white light,the proposed red and blue light-induced nitrate metabolic pathways are presented in Fig.5,based on the DEGs annotated in the KEGG pathway analysis and related enzyme activities obtained from the physiological determination.Compared with white light,no significant differences were found in the transcripts of NR-or NiRrelated genes under red or blue light,but the activities of NR and NiR were higher than those under white light (Fig.1-A and B).In the present study,four DEGs involved in ammonia reduction were identified under Red in comparison to White.Two putative GS-related genes (LOC103839002 and LOC103859729) and the asparagine synthase-related gene (LOC103873584) were upregulated,while one of the related genes encoding phenylalanine ammonia-lyase (LOC103870475) was downregulated under Red compared with White (Fig.5-A).In contrast,the expression level of one GS-related gene(LOC103863734) was significantly decreased,while most of the genes encoding phenylalanine ammonialyase (LOC103865574,LOC103867229,LOC103841251,LOC103863433,and LOC10384932) were upregulated under Blue compared with White (Fig.5-B).

Fig.5 The proposed red and blue light-induced nitrate metabolic pathways.NR,nitrate reductase;NiR,nitrite reductase;GS,glutamine synthetase;GOGAT,glutamate synthase;PLA,phenylalanine ammonia-lyase.

Table 1 Differentially expressed genes (DEGs) invoved in nitrogen metabolism in red and blue light exposure compared to that under white light exposure condition1)

3.5.The expression profile of light signaling involved in nitrate metabolism-related signaling pathways

Higher plants perceive light signals through variouslight receptors,including Phys,Crys,and phototropins(Phots).Phys mainly mediates red and far-red light signal transduction,while Crys and Phots are involved in blue light signal transduction.In this study,the expression levels ofPHYB(LOC103869720) andPHYC(LOC103868296) were higher under Red than under Blue and White,while the transcripts ofPHYA(LOC103871728)andPHYE(LOC103856075) were upregulated under Blue when compared with Red and White (Fig.6-A).Two genes (CRY1and LOC103836576) encodingCRY1andCRY2bwere increased under Red,while oneCRY2arelated and oneCRY2b-related (LOC103874351 and LOC 103837069) were higher under Blue than White(Fig.6-B).Furthermore,the transcripts of all the identified related genes encoding HY5 (LOC103850763 and HY5)and HY5-like (LOC103869673),and COP1-related genes(LOC103867588),were upregulated under Blue compared with White;however,the expression levels of these genes were lower under Red than under White (Fig.6-C).

3.6.Validation of differentially expressed genes by qPCR

To verify the RNA-seq data,15 DEGs were randomly selected and the expression profiles were validated by qPCR (Appendix D).In the qPCR analysis,the expression levels of the selected genes showed similar patterns as those identified by the FPKM from RNAseq under the corresponding treatments.These results confirm the reliability of the RNA-seq data.

4.Discussion

For non-model plants with limited omics data in public databases,thedenovoassembly of sequencing reads of the transcriptome is an effective method which has been widely applied in the analysis of gene expression (Unambaet al.2015).In the present study,an RNA-seq-enabled deep transcriptome analysis was performed to reveal the potential molecular mechanisms of light spectra on nitrate metabolism inB.campestris,a popular vegetable crop species in East Asia.Light is one of the most important environmental cues in regulating relevant gene expression.In the present study,a higher number of DEGs was observed under the comparison Redvs.White(5 534 DEGs) than under Bluevs.White (3 939 DEGs),and most of the DEGs under Red were downregulated compared with Blue (Fig.1-A).These results indicate that monochromatic blue light was more efficient in triggering gene expression in pakchoi than monochromatic red light.

In plants,NR is the rate-limiting enzyme that catalyzes nitrate to nitrite conversion in the cytoplasm,and the resulting nitrite is further converted into ammonia in chloroplasts by NiR under light conditions (Tegeder and Masclaux-Daubresse 2018).Light spectra play pivotal roles in the regulation of NR activity and relevant gene expression (Kim Det al.2015;Bianet al.2020).In the present study,the low nitrate and nitrite concentrations under red and blue light contributed to the higher NR and NiR activities induced by monochromatic red and blue light compared with white light (Fig.1-A and B).Similar results were also reported by Lillo and Appenroth(2001) and Qiet al.(2007).However,no significant differences were found in the transcripts of NR-and NiRrelated genes among Red,Blue,and White (Fig.2).These results indicate that NR and NiR are regulated at the posttranslational level by red and blue light through different light receptors (Sakuraba and Yanagisawa2018).HY5/HYH,an important member of the bZIP transcription factor family,is necessary forNRexpression and NR activity (Jonassenet al.2009).Previous studies reported that the cascade of PhyB-COP1-HY5/HYH plays an important role in nitrate assimilation (Appenrothet al.2000;Sakuraba and Yanagisawa 2018).In this study,the higher expression ofPHYB(LOC103869720) and oneHY5-likegene (LOC103869673) under Red compared with White (Fig.6-A and B) suggests that the red lightinduced PhyB-COP1-HY5/HYH signal transduction pathway of nitrate assimilation is conserved in pakchoi(Fig.7).Similar to Phys,Crys is involved in nitrate assimilation by limiting the COP-dependent degradation of HY5 upon blue light exposure (Osterlundet al.2000).Furthermore,the red and far-red light receptor Phys can perceive blue light to a limited extent,so it may also participate in the regulation of blue light-induced NR activity (Castillonet al.2009).Thus,compared with Red and White,the higher NR activity and concomitantly lower nitrate concentration under Blue could be explained by Phys-and Crys-involved posttranslational regulation of NR activity under blue light conditions through the cascade of COP1-HY5/HYH (Fig.7-B) (Sakuraba and Yanagisawa 2018),as shown by the blue lightinduced higher expression levels ofPHYA/E,CRY2a/2b(LOC103837069 and LOC103874351),HY5/HY5-like,andCOP1(Fig.6-A-C).

Fig.6 The expression of genes for different light receptors,including phytochromes (Phys) (A),cryptochromes (Crys) (B),and phototropins (Phots) (C).

In addition to being regulated by NR activity andNR,nitrate assimilation in the leaves of higher plants is subjected to the negative feedback regulation of ammonium (Leiet al.2018).The resulting ammonia from nitrite reduction will be further assimilated either by enhancing the principal assimilatory route (GS/GOGAT)(Forde and Lea 2007) or by activating other accessory pathways involving related enzymes,including asparagine synthetase (AS) and glutamate dehydrogenase (GDH)(Vega-Maset al.2019).Therefore,the assimilation of nitrate is indirectly modulated by the activities of GS,GOGAT,and AS (Barneix 2007;Quet al.2019).In the present study,monochromatic red light exposure induced higher expression of GS (LOC103839002 and LOC103859729)-and AS (LOC103873584)-related genes and concomitantly higher GS activity (Table 1;Fig.5),suggesting that the regulation of the GS/AS pathway plays a dominant role in red light-induced nitrate assimilation (Fig.7).In contrast,monochromatic blue light exposure showed different regulatory pathways in pakchoi,as indicated by the downregulation of one GSrelated gene (LOC103863734),and lower GOGAT activity compared with white light exposure (Table 1;Fig.5).In higher plants,phenylalanine ammonia-lyase (PLA) is the first enzyme of phenylpropanoid metabolism and the key enzyme in the biosynthesis of many secondary metabolites (e.g.,flavonoids,anthocyanins and phenolic acids) (Mattet al.2002).It is involved in recycling of ammonia usingtrans-cinnamic acid to generate other nitrogen compounds (Razalet al.1996;Zhang and Liu 2015).In this study,compared with red and white light,most of the DEGs were significantly enriched in ‘flavone and flavonol biosynthesis’,‘flavonoid biosynthesis’,‘nitrogen metabolism’,‘phenylpropanoid biosynthesis’,and ‘phenylalanine metabolism’ (Fig.4).Additionally,most PLA-related gene expression was upregulated in combination with a low nitrate concentration under monochromatic blue light exposure (Fig.2;Table 1).These results suggest that PLA plays a dominant role in the regulation of nitrate metabolism and the biosynthesis of secondary metabolites under blue light conditions.Previous studies reported that UV-B exposure upregulated PLA-related gene expression and enzyme activity,as well as the transcription of the UVR8-COP1-HY5 transduction system,to induce photoprotective flavonoid accumulation(Tevini and Teramura 1989;Contreraset al.2019).In this study,blue light induced the expression of Crys-,COP1-,and HY5/HY5-like-related genes (Fig.6).Similar results were also reported by Taoet al.(2018).InArabidopsis,Crys interacts with COP1 to minimize the COP1 Ubmediated degradation of HY5 under blue light conditions(Maieret al.2013).Taken together,the results in this study indicate that a different blue light signal transduction module,Crys/Phys-COP1-HY5/HY5-like,may contribute to nitrate metabolism in pakchoi (Fig.7-B),but this needs to be confirmed by further studies.

Fig.7 The red (A) and blue (B) light-induced signal transduction pathway of nitrate assimilation in pakchoi.Phys,phytochromes;Crys,cryptochromes;NR,nitrate reductase;NiR,nitrite reductase;GS,glutamine synthetase;AS,asparagine synthetase;COP1,constitutive photomorphogenesis;PLA,phenylalanine ammonia-lyase.

5.Conclusion

This study reveals that different regulation pathways are involved in red and blue light-induced nitrogen metabolism,and an optimal composition of red and blue light could be an efficient way of reducing nitrate accumulation and concomitantly increasing secondary metabolite contents in pakchoi.A total of 10 939 DEGs were identified through pairwise comparisons among three different spectral treatments.Based on GO and KEGG analyses,most of the key DEGs were found to be involved in nitrate assimilation,plant-pathogen interaction,biosynthesis of secondary metabolites,and phenylpropanoid biosynthesis.The relevant genes encoding light signal transduction,nitrate metabolism pathway structure genes,and related signaling pathways under monochromatic red and blue light were identified.This study provides a better understanding of light spectra-regulated nitrate metabolism in pakchoi at the transcriptome level and valuable information for the production of high-quality vegetables with low nitrate concentrations under controlled environments.

Acknowledgements

This research was financially supported by the National Key Research and Development Program of China(2017YFB0403903) and the Agricultural Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (ASTIP-CAAS,34-IUA-03).The authors thank Genepioneer Biotechnologies (Nanjing,China) for assistance in the analysis of RNA-seq data.We should also give our thanks to Dr.Xu Gang and Dr.Chen Longzheng from Jiangsu Academy of Agricultural Sciences,China for their suggestions and help throughout the experiments and manuscript revision.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on http://www.ChinaAgriSci.com/V2/En/appendix.htm