Transcriptomic and proteomic analyses of far-red light effects in inducing shoot elongation in the presence or absence of paclobutrazol in Chinese pine

Yingtian Guo·Shihui Niu·Yousry A.El-Kassaby·Wei Li

Abstract In angiosperms,low red (R)/far-red (FR) ratio light increases gibberellin (GA) levels.GA signaling in conifer seedlings requires FR to promote shoot elongation and reduce the inhibition of shoot elongation induced by paclobutrazol (PAC),yet the effects of far-red light in inducing shoot elongation in the presence or absence of PAC is poorly understood.In this study,transcriptomic and proteomic analyses was used to examine the molecular mechanism of FR regulation of shoot elongation in Chinese pine(Pinus tabuliformis Carr.) seedlings in the presence of PAC.Four treatments were compared:white light+water (WW),FR+water (RW),white light+PAC (WP),and FR+PAC(RP),and 1436 differentially expressed genes (DEGs) and 450 differentially expressed proteins (DEPs) were identified in RW_WW (RW contrast WW),and 1862 DEGs and 481 DEPs in RP_WP (RP contrast WP).Metabolic and signal transduction pathway analyses of DEGs and DEPs in RW_WW and RP_WP,indicated that the former required more energy than the latter.Moreover,gibberellic acid,auxin,and brassinolide are equally important in RW_WW and RP_WP for shoot elongation,except for the ethylene pathway.Amino acid metabolism and cell wall organization were significantly enriched in RW_WW and RP_WP,respectively.In summary,RW_WW and RP_WP had different effects in secondary metabolism,energy metabolism,amino acid metabolism,cell wall organization,and hormone response.These results provide an important theoretical and reference basis for studying the regulatory effect of low R/FR and PAC in conifer shoot elongation.

Keywords Low R/FR ratio·Pinus tabuliformis ·Paclobutrazol·Proteome·Shoot elongation·Transcriptome

Introduction

Light is essential for plant life and affects all levels of organization from visible phenotype to gene expression (Quai 2002).In shade-intolerant plants,red (R)/far-red (FR) ratio light reduction is an indication of neighbor proximity,triggering a series of developmental responses known as shade avoidance syndrome (SAS) (Ballare and Pierik 2017;Wille et al.2017).Plants react shade through changes in hormone content and signaling,which regulate adaptive responses(Yang and Li 2017).Gibberellins (GAs) are hormones that regulate plant growth and development;additionally,in combination with light,they mediate many important and partially overlapping developmental processes (Feng et al.2008).Through phytochrome B (PHYB),FR light regulates the expression ofGA20ox2,GA3ox1,andGA3ox2genes as well as the deactivation of theGA2ox2gene (Hisamatsu et al.2005;Yamauchi et al.2007),which together,increase the content of active GAs.The downstream DELLA family of proteins (a family of putative transcriptional regulators that inhibits cell proliferation and expansion controlling organ growth),play key roles in the GA signal transduction pathway.Shade can increase the production of active GAs and accelerate the degradation of DELLA proteins to release the inhibition of phytochrome-interacting factors(PIFs),thereby increasing stem and hypocotyl elongation(Feng et al.2008;Harberd et al.2009).Thus,DELLA proteins constrain shade avoidance syndrome in angiosperms(Djakovic-Petrovic et al.2007).

Compared with angiosperms,the molecular mechanisms of shade avoidance in gymnosperms are relatively unknown.Prior to the development of high-throughput sequencing,several studies reported that FR light affected cotyledons,hypocotyls,and stem growth,and regulated the expression of photosynthesis-related genes (Fernbach and Mohr 1990;Alosi and Neale 1992;Mukai et al.1992;De La Rosa et al.1998;Burgin et al.1999).Advances in high-throughput technologies,including transcriptomics and proteomics,have created a system biology approach facilitating the acquisition of in-depth information,including the identity and abundance of ribonucleic acid (RNA) and proteins and their related biological processes,gene functions,and physiological stress responses (Voelckel et al.2017).Differences between gymnosperms and angiosperms in terms of FR light regulation of GA synthesis and SAS have been reported for several conifers,leading to a significant improvement of our understanding of conifer adaptive diversity (Ouyang et al.2015;Hu et al.2016;Ranade et al.2019;Li et al.2020).

Previously,we reported on low R/FR regulation of GA accumulation through controlling the expression of kaurenoic acid oxidase (KAO),thereby influencingPinus tabuliformisshoot elongation (Li et al.2020).These findings clearly indicated that angiosperms and conifers have different targets in FR/R light mediated GA synthesis (Li et al.2020).Furthermore,we also found that FR reduced or removed shoot elongation inhibition induced by paclobutrazol (PAC);however,the properties of FR in inducing shoot elongation in the presence or absence of PAC remained poorly understood.In the present study,four different light treatments were applied toP.tabuliformisseedlings.RNA sequencing (RNA-Seq)-based transcriptome and iTRAQbased proteomics analyses were carried out to resolve the biological processes associated with low R/FR and PAC treatments.This study had the following objectives:(1) to analyze the biological processes related to low R/FR in the presence or absence of PAC;and,(2) to propose molecular pathways for shoot elongation in seedlings under RW_WW and RP_WP treatment combinations.The metabolic and signal transduction pathway activities were examined on shoot elongation in seedlings grown under RW_WW and RP_WP treatments.The results may provide an important theoretical basis and reference for future studies of the regulation of conifer shoot elongation by low R/FR and PAC.

Materials and methods

Plant material and treatment

P.tabuliformisseeds were collected from a clonal Chinese pine seed orchard in Pingquan City,Hebei Province,China(40° 99’ N,118° 45’ E,560 m a.s.l.).The seeds were sown in 8-cm-diameter plastic pots and cultured in a mixture of peat and black soil (2:1,v/v) at 22 °C under a 14-h/10-h light/dark photoperiod.The fully developed seedlings were irrigated weekly with water and 50 μmol/L PAC.Lightemitting diode (LED) lamps provided 170 μmol m–2s–1FR(730 nm) and R (660 nm) light.The seedlings were divided into four groups:white light+water (WW),FR+water(RW),white light+PAC (WP),and FR+PAC (RP).Needles were sampled two months after treatment,and immediately frozen and stored in liquid nitrogen prior to analysis.All analyses were conducted using three biological replicates per treatment.Each treatment contained eight seedlings with similar growth rates.

RNA extraction and cDNA library construction

Total RNA was isolated using TRIzol (Invitrogen,USA)according to the manufacturer’s protocol.RNA integrity was detected using the RNA Nano6000 detection kit and the Bioanalyzer 2100 system (Agilent Technologies,USA).A NanoPhotometer spectrophotometer was used to verify RNA purity.Library construction and sequencing were performed by Gene Denovo Biotechnology using the HiSeq X platform(Illumina,USA).

Data processing and analysis

Low-quality regions and adaptors were trimmed through pre-processing,and reads were aligned to the transcriptome in the absence of a reference genome.Kallisto software (Bray et al.2016) was used to estimate transcription abundance and Sleuth software (Pimentel et al.2017) for differential expression analysis.Genes were regarded as differentially expressed when the effect size [analogous to foldchange (FC)] values were ≥1 andP<0.05.Gene ontology(GO) analysis was performed for functional classification of differentially expressed genes (DEGs) and pathway analysis performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG).MapMan software was used to elucidate DEG functions.

iTRAQ-based proteome analysis

Needle samples were ground in liquid nitrogen,vortexed with a lysis buffer,extracted for 30 min and centrifuged.The supernatant was collected and stored at?80 °C.Protein yield was measured using the Bradford method.A 100 μg protein solution was placed in a centrifuge tube following quantification and protein digestion according to the filteraided sample preparation (FASP) procedure.The resulting peptide mixture was labeled using iTRAQ Reagent 8 Plex(PN:4,390,812;AB Sciex,USA).Each iTRAQ reagent was dissolved in 150 μL isopropyl alcohol and added to the sample.The sample (two replications for analysis) were labelled with the iTRAQ tags as follows:RP,iTRAQ 113 and 114;RW,iTRAQ 115 and 116;WP,iTRAQ 117 and 118;WW,iTRAQ 119 and121.

Labeled samples were dissolved and loaded onto a Durashell-C18 column (Agela,China).The fractions obtained were re-dissolved and separated by liquid chromatography-mass spectrometry (LC–MS) using the EASYSpray column (Thermo Fisher,USA).An ABI-5600 (AB Sciex) mass spectrometer was used for protein analysis with the following parameters:2.1 kv spray voltage;EASY-Spray ion source (Thermo);350–1800 m/z full MS scan range.Proteome Discoverer 1.4 software (Thermo) was used to search databases and quantitatively analyze the raw data acquired by MS.Proteins were identified and quantified using the MASCOT search engine (Matrix Science,USA)and the reference transcriptome.

For further functional analysis,differentially expressed proteins (DEPs) were selected based on FC ≥ 1.2 or ≤ 0.833(P<0.05).GO analysis of DEPs was performed using tools available from OmicShare,a free online data analysis platform ( www.omics hare.com/ tools);KEGG was used to predict the biological and functional properties of DEPs.

Results

Changes in seedling morphology induced by FR and PAC treatments

FR and PAC affected shoot elongation.During the 2-month treatment,PAC treatment resulted in a dwarf phenotype under normal light treatment (WL),and FR removed growth inhibition by PAC (Fig.1).These findings indicate that FR can regulate shoot elongation in ways different from the known GA signaling pathway.To identify the FR response networks in the presence or absence of PAC and to determine how FR removed shoot elongation inhibition by PAC,samples from the four treatments were collected for iTRAQbased proteomics and RNA-Seq-based transcriptomics analyses.Additionally,in order to explicitly determine the role of FR on seedling morphology in the presence or absence of PAC,treatment comparisons are restricted to the RW_WW and RP_WP combinations.

Fig.1 Shoot elongation changes induced by far-red (FR) light and paclobutrazol (PAC) in Pinus tabuliformis seedlings; a changes in seedlings treated with RP,WP,RW and WW for 2 months respectively,needles were removed before photographs taken; b average shoot length of seedlings under different treatments for two months.Bars are means (n=8) ± standard deviation (SD),different letters indicate significant differences between treatments (Student’s t-test;** P <0.01,*** P <0.001)

Overview of transcriptomics and proteomics data

Transcriptome analysis identified 64,518 genes in all treatment combinations (Table S1);among these,1436 and 1862 were differentially expressed genes (DEGs) (fold change ≥ 1 andP<0.05) in the RW_WW and RP_WP treatment combinations,respectively (Table S2).A total of 809 and 627 DEGs were identified and down-and up-regulated in RW_WW,and 771 and 1091 DEGs were down-and 1091 upregulated in RP_WP (Fig.2 a).A total of 11,188 and 3358 unique peptides and proteins were identified via iTRAQ(Table S3).The molecular weights of the identified proteins,sequence coverage of the detected peptides,and unique peptide number generated from the iTRAQ-LC–MS/MS data are presented in Fig.S1.Among all proteins detected,450 were DEPs (FC >1.2 or 0.85 andP<0.05),of which 228 and 222 were up-and down-regulated in RW_WW,respectively (Table S4 and Fig.2 b).Additionally,481 DEPs(FC >1.2 or 0.85 andP<0.05) were detected,with 263 and 218 were up-and down-regulated in RP_WP,respectively(Table S4 and Fig.2 b).

Fig.2 Venn diagrams of P.tabuliformis DEGS and DEPs.a Numbers of up-down DEGs in RW_WW and RP_WP.b Numbers of up-down DEPs in RW_WW and RP_WP

To evaluate the relationship between protein and transcript levels,DEPs and their cognate mRNAs were compared (Fig.3).Pearson product-moment correlation analysis showed that protein abundance was positively (r=0.0409)and negatively (r=? 0.0183) correlated with the corresponding gene expression in RW_WW and RP_WP,respectively,suggesting inconsistent transcription and protein translation processes.

Fig.3 Correlation between DEPs and DEGs in P.tabuliformis

GO and KEGG pathway analysis for DEGs and DEPs

The FR response networks in the presence of PAC were determined by observing the differences between DEGs and DEPs in the resulting Venn diagrams (Fig.2).The results showed 1188 DEGs and 299 DEPs were detected in RW_WW,while 1614 DEGs and 330 DEPs were detected in RP_WP (Fig.2).Additionally,37 DEGs and 38 DEPs showed opposite expression in RW_WW and RP_WP.The different DEGs and DEPs detected by the Venn diagrams were enriched via GO analysis (Fig.4).The highly expressed DEGs presented in RW_WW were enriched in two GO terms related to amino acid,"leucine biosynthetic process" and "leucine metabolic process" (Fig.4 a).Additionally,the DEPs related to "small molecule metabolic process" and "organonitrogen compound metabolic process" were enriched in RW_WW (Fig.4 b).Furthermore,"cell wall organization or biogenesis" and "cell wall organization","protein import into mitochondrial outer membrane" and "outer mitochondrial membrane organization"were enriched in DEGs and DEPs of RP_WP,respectively(Fig.4 c,d).In addition,pathway enrichment analysis was carried out of DEGs and DEPs in RW_WW and RP_WP based on the KEGG database (Fig.5).In RW_WW,DEGs were mainly enriched in secondary metabolites and in pyruvate metabolism,while DEPs were chiefly enriched in metabolic pathways and in the biosynthesis of amino acids (Fig.5 a,b).In RP_WP,DEGs were mainly enriched in cutin,suberine,and in wax biosynthesis,while DEPs were predominantly enriched in pyruvate metabolism and in carbon metabolism (Fig.5 c,d).Thus,secondary metabolic pathways,energy metabolism,cell wall organization,and amino acid metabolism were further investigated as candidate pathways related to the FR response networks in the presence of PAC.

Fig.4 GO enrichment results of DEGs and DEPs in RW_WW and RP_WP.a and b represent DEGs and DEPs in RW_WW,respectively; c and d represent DEGs and DEPs in RP_WP,respectively

Fig.5 KEGG enrichment results of DEGs and DEPs in RW_WW and RP_WP.a and b represent DEGs and DEPs in RW_WW,respectively; c and d represent DEGs and DEPs in RP_WP,respectively

Secondary metabolism of DEGs and DEPs involved in RW_WW and RP_WP

Through KEGG enrichment analysis of DEGs in RW_WW and RP_WP,it was found that both were significantly enriched in secondary metabolic processes but the pathways involved were different.In RW_WW,the DEGs related to flavonoids (i.e.,CHS,ANR,LAR) and terpenoids (terpene synthase 03,terpene synthase 21) had been enriched and were down-regulated,suggesting some secondary metabolic processes were inhibited (Table 1).While in RP_WP,DEGs were primarily enriched in cutin,suberine,and wax biosynthesis,and genes related to cytochrome P450 superfamily proteins were up-regulated,indicating that cutin,suberine and wax biosynthesis had important roles in RP_WP treatment (Table 2).These results suggest that different secondary metabolic processes are induced in RW_WW and RP_WP.

Energy metabolism of DEGs and DEPs involved in RW_WW and RP_WP

In addition to secondary metabolism,the glycolysis cycle was enriched by KEGG analysis.In RW_WW,one DEGs(Pita_unigene22911) encoded phosphoenolpyruvate carboxylase (PEPC) was down-regulated,and five DEGs(Pita_unigene3047,Pita_unigene3046,Pita_unigene13047,Pita_unigene62636,Pita_unigene18120) encoded phosphoenolpyruvate carboxykinase (PECK) were up-regulated (Table 3).While in RP_WP,two DEPs (Pita_unigene40479,Pita_unigene63143) related to the glycolysis cytosolic branch were up and down regulated,respectively,and two DEPs (Pita_unigene45809,Pita_unigene2392) ofhexokinase (HXK) were down-regulated.Additionally,two DEPs (Pita_unigene20045,Pita_unigene7139) of pyruvate orthophosphate dikinase (PPDK) were also down-regulated(Table 4).These results indicated that different glycolysis cycle of energy metabolism processes are induced in RW_WW and RP_WP.

Table 1 Differentially expressed genes (DEGs) involved in secondary metabolism in P.tabuliformis under RW_WW treatment

Table 2 Differentially expressed genes (DEGs) involved in secondary metabolism in P.tabuliformis under RP_WP treatment

Table 3 DEGs involved in energy metabolism in P.tabuliformis under RW_WW treatment

Amino acid metabolism of DEGs and DEPs involved in RW_WW

According to the results of GO and KEGG enrichment,many DEGs and DEPs were involved in amino acid metabolism (Table 5).DEPs related to methionine (Pita_unigene47134,Pita_unigene1427,Pita_unigene60392),DEGs related to leucine (Pita_unigene17647,Pita_unigene40345,Pita_unigene45957,Pita_unigene6964),DEPs related to cysteine (Pita_unigene63891,Pita_unigene60155),DEPs related to phenylalanine (Pita_unigene4659),DEPs related to tyrosine (Pita_unigene59567),DEPs related to arginine(Pita_unigene15438),and DEPs related to tryptophan (Pita_unigene58805,Pita_unigene58119) metabolism were up regulated (Table 5;Fig.6),suggesting that the metabolism of these amino acids play different roles in RW_WW processing.

Fig.6 DEGs and DEPs involved in amino acid metabolism in P.tabuliformis under RW_WW treatment

Cell wall organization by DEGs and DEPs involved in RP_WP

Cell walls are thicker in blue and white light than in farred and dark light (Falcioni et al.2020).In our study,"cell wall organization" were enriched in RP_WP.Most DEGs belonged to the pectin lyase-like superfamily,which is involved in cell wall degradation,were up-regulated expression (Table 6).Also,three DEGs related to cell wall modification genes (EXPA16,EXGT-A3,XTH9) were induced.These results indicate shoot elongation is controlled by regulating cell elongation and expansion in RP_WP.

Table 4 DEPs involved in energy metabolism in P.tabuliformis under RP_WP treatment

Table 5 DEGs and DEPs involved in amino acid metabolism in P.tabuliformis under RW_WW treatment

Table 6 DEGs involved in cell wall organization in P.tabuliformis under RP_WP treatment

Hormone-related DEGs and DEPs involved in RW_WW and RP_WP

The shade avoidance syndrome (SAS) is closely associated with the interaction between light signaling and hormones,and is related to the basic functions of the photosensitivepigment B photoreceptor and GA (Arana et al.2014).GA and low R/FR may be integrated with signals from other hormones.In the present study,DEGs involved in hormones pathways,including abscisic acid (ABA),brassinosteroids (BR),cytokinin (CKT),ethylene (Eth),gibberellic acid (GA),auxin (IAA),jasmonic acid (JA),and salicylic acid (SA) were analyzed.The results show that most DEGs related to BR,GA,and IAA were up-regulated in RW_WW and RP_WP,while genes involved in ABA,CKT,JA were down-regulated.Furthermore,DEGs involved in the Eth pathway were up-regulated in RW_WW and were recovered in RP_WP (Fig.7).These results indicate that,with the exception of ethylene and salicylic acid (too few genes involved),other hormone regulation activities did not change significantly in RW_WW and RP_WP treatments.

Fig.7 Expression patterns of genes involved in different hormone pathways of RW_WW and RP_WP;the size effect is analogous to the fold change value

Discussion

Shade can affect several stages of plant growth and development,including inhibition of seed germination,promotion of hypocotyl elongation,and early flowering (Djakovic-Petrovic et al.2007).Numerous studies have reported that various hormones participate in shade regulation,of which GA,as an important growth factor,is an essential component of plant shade response (Yang and Li 2017).Shade increases the production of active GA,accelerates the degradation of DELLA proteins,and removes PIF inhibition,which increases stem and hypocotyl elongation (Jing and Lin 2020).Our ongoing research has recently documented the necessity of GA signaling for low R/FR-induced shoot elongation in pine seedlings (Li et al.2020).We also reported that low R/FR can remove shoot elongation inhibition by PAC (Fig.1);however,the underlying molecular mechanisms remain poorly understood.In this study,we have further demonstrated transcription and translation changes in response to WW,RW,RP,and WP treatments in pine seedlings.

In the present study,transcriptome and proteome analyses identified 1436 DEGs and 450 DEPs in RW_WW,and 1862 DEGs and 481 DEPs in RP_WP (Fig.2).However,Pearson’s product-moment correlation analysis showed that protein abundance was weakly negatively correlated with gene expression.A possible explanation is the rapid fluctuation of mRNA levels,such that transcription stabilizes before changes in the protein levels are detected (Luo et al.2018).GO and KEGG pathway enrichment and MapMan functional analyses revealed differences in secondary metabolism,energy and amino acid metabolism,cell wall organization and hormone response under RW_WW and RP _WP.

Light quality affects the production of secondary metabolites,especially flavonoids (Jaakola and Hohtola 2010).In addition to light,the application of GA3and PAC also can affect secondary metabolites such as xylem development(Abbasi et al.2012;Guo et al.2015;Wang et al.2017).In this study,upstream genes encoding CHS enzymes that synthesize flavonoids were downregulated,suggesting that flavonoid biosynthesis was suppressed in RW_WW.Two DEGs (Pita_unigene49176andPita_unigene9896) associated with procyanidin synthesis were down-regulated,suggesting flavonols were also repressed (Table 1).Additionally,four terpene synthase genes (Pita_unigene7876,Pita_unigene43485,Pita_unigene62550,andPita_unigene46399)were also down-regulated.While in RP_WP,many DEGs enriched with cytochrome P450 superfamily proteins are related to the biosynthesis of cutin,suberine,and wax(Table 2),essential compounds for plant protection against other environmental stresses,including desiccation and UV radiation (Lewandowska et al.2020).Previous studies have reported that low R/FR ratios reduce flavonoid levels,thus limiting the energy cost of shade avoidance responses (Cagnola et al.2012).Therefore,we speculate that pine seedlings may reduce flavonoids and terpene biosynthesis so that more energy is allocated towards growth under RW_WW,but not under RP_WP,in a plant growth-defense trade-off.

Previous studies have shown that far-red light can influence respiratory metabolism and photosynthesis in order that plants can regulate energy needed under shade conditions (Ding et al.2016;Zhen and van Lersel 2017).Under RW_WW,five DEGs (Pita_unigene3047,Pita_unigene3046,Pita_unigene13047,Pita_unigene62636,Pita_unigene18120) encoding phosphoenolpyruvate carboxykinase (PEPCK) were up-regulated,and down-regulation of PEPCK negatively affected seedling growth (Huang et al.2015).Furthermore,one gene PPC3 (Pita_unigene22911)associated with the cytosolic branch of glycolysis and two genes (Pita_unigene5459,Pita_unigene476) involving the TCA cycle were up-regulated (Table 3).It is postulated that more energy is needed in RW_WW.While in RP_WP,three proteins (Pita_unigene20045,Pita_unigene7139,Pita_unigene63143) associated with glycolysis and two DEPs (Pita_unigene45809,Pita_unigene2392) related to sucrose degradation were down-regulated (Table 4).Furthermore,two DEPs (Pita_unigene43232,Pita_unigene64085)involved in TCA cycle and one DEPs (Pita_unigene11370)associated with photorespiration were up-regulated,implying that less energy is needed in RP_WP than in RW_WW.

According Go and KEGG enrichment,amino acid metabolism was significantly enriched in RW_WW,and cell wall organization in RP_WP.In RW_WW,DEGs and DEPs related to methionine (Met),cysteine (Cys),leucine(Leu),phenylalanine (Phe),arginine (Arg),tryptophan (Trp),branched-chain and aromatic amino acids were induced(Table 5;Fig.6).Among,DEPs,tryptophan (Trp) and cysteine (Cys) were up-regulated,while the remaining ones were down-regulated.Several amino acids serving as precursors for secondary metabolite synthesis,such as methionine(Met),and branched-chain and aromatic amino acids (Hildebrandt et al.2015),were found to be negatively regulated in RW_WW,implying that plants can regulate amino acid metabolism in response to far-red light.Cell wall-modifying mechanisms are vital regulatory points for controlling shoot elongation (Sasidharan et al.2008;Fan et al.2018).Expansins and xyloglucan endotransglucosylase/hydrolases(XTHs) belong to cell wall-modifying proteins and are essential for shade-induced growth (Sasidharan and Pierik 2010).In RP_WP,expansin A16 (Pita_unigene11581),endoxyloglucan transferase A3 (Pita_unigene12450),and XTH9 (Pita_unigene22488) were induced (Table 6),suggesting wall modifying activities are needed in RP_WP.Furthermore,most DEGs encoded pectin lyase-like superfamily proteins were up-regulated,which can reduce pectin contents to thin cell walls (Falcioni et al.2020).Thus,amino acid metabolism and cell wall organization contribute to shoot elongation in RW_WW and RP_WP,respectively.

Plant hormones play important roles in plant responses to low R/FR signals.GAs,IAA,and BR are required for low R/FR-induced petiole or hypocotyl elongation (Yang and Li 2017).Studies have shown that GAs activate PIF accumulation,and that PIF,the BR-responsive TF heterodimerize,and active IAA response genes promote hypocotyl elongation.In turn,the IAA response increases GA levels(Kozuka et al.2010;Keuskamp et al.2011;Liu et al.2011).In this experiment,numerous DEGs were up-regulated and were related to GAs,IAA,and BR,confirming that these are equally important in RW_WW and RP_WP for shoot elongation (Fig.7).Additionally,DEGs involved in the Eth pathway were up-regulated in RW_WW and were recovered in RP_WP.DEGs in RP_WP were involved in metabolic pathways,but in RW_WW they were related to signal transduction pathways (Fig.8),suggesting the different responses to ethylene in RW_WW and RP_WP.

Fig.8 Heatmap of DEPs related to ethylene pathway in P.tabuliformis under RW_WW and RP_WP

According to iTRAQ-based proteomics and RNA-Seqbased transcriptomics analyses in the four treatments (WW,RW,RP,and WP),RW_WW and RP_WP had different responses in secondary metabolism and energy metabolism,amino acid metabolism,cell wall organization,and hormone response,which clarified understanding of FR and PAC treatment-induced changes in seedling morphology.This study provided an important theoretical basis and reference for further investigations of the regulation of shoot elongation in coniferous species under low R/FR and PAC.