Physiological adaptation and transcriptome changes in the plateau plant common vetch (Vicia sativa L.) in response to the plain environment

the important factors in plant growth. The variety common vetch was grown under the special of and the Qinghai-Tibet the the Qinghai-Tibet airlifted the regulatory are not clear.


Results
The samples collected the day before transportation were used as a control. After being transported back to the plain, the morphological structure and physiological indexes were determined on days one, three, ve and seven. Compared with the control group, the chlorophyll content in the experimental group changed signi cantly. Chlorophyll uorescence increased signi cantly on the fth day. The number of stomatal openings decreased signi cantly on the rst day and then increased gradually and exceeded that of the control. Compared with the control sample, the intercellular space of the experimental sample was larger and then did not return to the control level. The physiological indexes gradually returned to the control level on the 7th day. Transcriptome analysis showed that 3251 genes were upregulated and 3317 genes were downregulated on the third day, while 1359 genes were upregulated and 1648 genes were downregulated on the seventh day. These differentially expressed genes were signi cantly enriched in photosynthesis, photosynthetic antenna protein synthesis, carbon dioxide xation and chlorophyll synthesis pathways, and almost all of the genes involved in these pathways were downregulated. In addition, MYB, NAC, AP2-EREBP and the Orphans family of transcription factors (TFs) regulating the light response were more abundant on day 3 and day 7 than in the control group.

Conclusion
The physiological and gene expression levels of common vetch transported to the plain environment were analyzed. To reveal the adaptation mechanism of common vetch to plain environments, the key metabolic pathways of differential gene enrichment were analyzed. These ndings are helpful for introducing plateau suitable varieties into plain environments.

Background
The Qinghai-Tibet Plateau, known as the "third pole of the world" and "Asian water tower", has an average altitude of more than 4000 meters, including vast alpine grasslands [1]. It has the characteristics of low temperature, low oxygen, poor soil and a strong ultraviolet radiation environment. High solar radiation and its effects on secondary metabolites of higher plants in high-altitude areas have been con rmed in Under the plain environment, the chlorophyll content increased signi cantly on the rst and fth days.
Although the control level was restored on day 3, it was lower than that of the control on day 7 (Fig. 1a). Chlorophyll uorescence (Fv/Fm) was signi cantly higher on the 5th day and lower on the 7th day than on the day before relocation (Fig. 1b).
Changes in the number of stomata and in the anatomical structure of leaves Compared with the control, the number of stomatal openings decreased signi cantly on the rst day, increased signi cantly on the third day, and reached a relatively stable state on the fth and seventh days (Fig. 2 a-f). Common vetch is a double-sided leaf with palisade tissue and spongy tissue. On the rst day of returning to the plain, the arrangement of upper and lower epidermal cells, palisade cells and spongy cells was loose, and the intercellular space was larger than that of the control. In the next few days, the intercellular space and spongy tissue gradually recovered (Fig. 3 a-e). Therefore, the changes in stomata and anatomical structure showed that the change in common vetch was obvious after it was transported back to the plain, and it was di cult to return to the control state.
Physiological response of common vetch to the plain environment Compared with common vetch in the suitable plateau environment (J0), common vetch transported back to the plain showed higher MDA and soluble sugar contents; these contents decreased signi cantly on the rst day (J1) and then increased signi cantly. The content of soluble protein decreased signi cantly and then gradually recovered to the same level as on the third day (J3). The levels of H 2 O 2 and O 2 did not change signi cantly but increased. The activities of POD and GSH decreased signi cantly on the 5th day (J5) and the 1st day (J1), respectively, but remained the same as the control. The activity of SOD increased signi cantly; although it decreased signi cantly on the 5th day (J5), it was still higher than that of the control. In summary, these results show that the damage to the cell membrane and osmotic regulation system of high-altitude adapted species is serious in the plain environment, but it has no signi cant effect on ROS levels.

RNA sequencing and de novo assembly
After the seedlings were transported to the plain, the photosynthetic pigment changed signi cantly on the third day and gradually recovered to stability on the seventh day. Therefore, to obtain the gene expression pro le under plain and plateau conditions, the transcriptomes of the leaves of plateau control samples Compared with the annotation results of the NR database, it can be seen that the most homologous species with common vetch is Medicago truncatula, with 8391 species (33.3%). In addition, 18.4% of unigenes were similar to those of Trifolium pratense (Additional le 1: Figure S1.). In accordance with GO analysis, 105146 single genes were classi ed according to the three major categories of GO. There were 25 groups of biological processes (BPs), 20 groups of cellular components (CCs) and 10 groups of molecular functions (MFs) (Fig. 5b, additional le 2: Table S1). The most abundant GO terms in the unigenes of common vetch were "metabolic process" (10131), "cellular process" (11068) and "singleorganism process" (8056) for BP; "cell part" (6127) and "cell" (6127) for CC; and "catalytic activity" (8350) and "binding" (10,690) for MF.

Overall analysis of DEGs
In this experiment, we used deseq software to analyze the differential expression among the sample groups, with FDR < 0.05. Compared with the control (J0), samples collected on the third day (J3) had 3251 upregulated genes and 3317 downregulated genes, and 1359 upregulated genes and 1648 downregulated genes were observed on the seventh day (Fig. 6a) The pathways photosynthesis (ko00195) and photosynthesis-antenna proteins (ko00196) were identi ed in the J3 vs J0 and J7 vs J0 comparisons. The pathways identi ed in only the J3 vs J0 comparison were carbon xation in photosynthetic organisms (ko00710), phenylalanine metabolism (ko00360), porphyrin and chlorophyll metabolism (ko00860), and tyrosine metabolism (ko00350). Pathways speci cally in J7 vs J0 are ribosome (ko03010) and ribosome biogenesis in eukaryotes (ko03008).

Differential expression of photosynthesis-related genes
To study the adaptability of plateau plants to the plain environment, genes related to environmental effects were screened out from the ltered DEGs for further study. In J3 and J7, DEGs related to the reaction center of the PSI and PSII, photosynthetic antenna protein, electron transfer and CO 2 xation were identi ed. The main pathways were photosynthesis (ko00195), photosynthesis antenna protein (ko00196) and photosynthesis biological carbon xation (ko00710). When the plants were transported to the plain environment, the carbon xation of photosynthetic organisms was inhibited. The main feature is that the core cytochrome b 6 /f (k02636), which connects PSII and PSI, is downregulated during electron transfer. During photophosphorylation, the genes encoding FD-NADP + reductase (k02641) and ATP synthase (k02113, k02109) were downregulated (Fig. 8b).

Discussion
Chlorophyll synthesis, stomata and leaf structure of common vetch in the plain environment Light is the direct energy source of plant growth. Among them, light intensity is one of the most important factors that affect many physiological processes of plants, such as photosynthesis [17,18]. There is a threshold for the effect of light intensity on the growth of plants; light intensity that is too high or too low will inhibit the growth of plants [19]. The results showed that chlorophyll content was positively correlated with CO 2 concentration and light intensity [20]. Higher chlorophyll content, higher stomatal density and thicker leaves were bene cial for increasing the photosynthetic rate. Under shading conditions, the stomatal density of soybean decreased signi cantly [21,22]. In this study, the chlorophyll content in the leaves of common vetch decreased signi cantly in the plain environment compared with the plateau environment, which indicated that photosynthesis was inhibited under low light conditions in the plain. At rst, when stimulated by the environment (J1), the stomata closed, and the intercellular space became larger, representing the response of common vetch to a stressful environment. To adapt to the plain environment, the above indexes gradually recovered to stable levels. In addition, there is a close relationship between the activity of the chlorophyll cycle and the construction and destruction of photosynthetic organs. The lack of chlorophyll b resulted in a signi cant decrease in lhcb1, Lhcb6 and lhca1 in LHC subunits [23,24]. ChlH is a porphyrin-binding subunit and a key enzyme in chlorophyll synthesis [25]. Mg-chelatase makes protoporphyrin IX participate in chlorophyll biosynthesis, and its activity decreases with decreasing ChlH transcription levels [26]. In this study, ChlH and other genes involved in chlorophyll synthesis (HemA, HemC, HemE) were downregulated. The above results were consistent with the chlorophyll content results (Fig. 11c).

Adaptive regulation of photosynthesis in the plain environment
Photosynthesis usually refers to the process by which green plants absorb light energy, synthesize carbon dioxide and water into energetic organic matter, and release oxygen at the same time. It is also an important factor that affects plant growth, yield and fruit quality. The differences in genes among different varieties result in differences in photosynthesis. Therefore, more attention should be paid to improving the photosynthesis of plants in actual agricultural production [27,28]. The electron transport system of water molecule oxidation and release is transferred to NADP+ and reduced to NADPH, and the cycle of electron transport is driven by light reactions. The electrons produced by water splitting in PSII are transferred to ferritin (Fd) through cytochrome (Cyt) b 6 /f and PSI; thus, NADPH is produced. Cyt-b 6 /f plays a unique role in photosynthetic electron transport. It can act on both linear electron transport (production of ATP and NADPH) and cyclic electron transport (production of ATP only) [29]. The cytochrome b 6 /f complex and chloroplast ATPase are the main sites that control photosynthetic ux. The ability of protons to enter the lumen is controlled by the electron ow of the Cyt-b 6 /f complex, and the proton ow through ATPase is balanced. ATP synthases drive ATP synthesis [30]. It has been reported that the content of the Cyt-b 6 /f complex in Arabidopsis thaliana is positively correlated with an increase in the light intensity for constant growth, that is, high light > normal light > low light [31]. It can be inferred that the content of the Cyt-b 6 /f complex was increased in common vetch to adapt to the strong light of the Qinghai-Tibet Plateau. After moving to the plain, the light intensity decreased, and the content of the Cyt-b 6 /f complex decreased. This is consistent with the transcriptome sequencing results. The decrease in the Cyt-b 6 /f complex reduces the electron transport rate of PSII, which leads to a decrease in the CO 2 assimilation rate [32]. The results show that Cyt-b 6 /f has a high level of control over the electronic transmission rate of Setaria viridis in C4 plants [33], and high Cyt-b 6 /f abundance in C4 plants may lead to a high CO 2 assimilation rate and high yield [34]. In plants, NADP+ reductase (FNR) receives electrons from FD at the end of the electron transport chain of photosynthesis and converts NADP+ to NADPH for CO 2 xation and chloroplast metabolism [35]. In pea leaf experiments, it was suggested that the activation of FNR and the increase in electron ow between PSII and PSI could lead to the acceleration of NADP+ reduction by FD [36]. ATPase plays a key role in the coregulation of light and dark reactions in photosynthesis. Experiments have suggested that photosynthesis can be controlled by regulating ATPase in tobacco under the conditions of high CO 2 and low O 2 [37] (Fig. 11a-b).
Ribulose 1,5-diphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) xes carbon dioxide in the atmosphere to participate in the Calvin cycle of photosynthesis. Rubisco immobilizes carbon dioxide to form two 3-phosphoglycerate (PGA) molecules. The formed 3-PGA forms glyceraldehyde-3-phosphate through two reactions of ATP consumption and NADPH consumption. The regeneration stage of the cycle includes a series of reactions to form phosphorylation. Finally, ribose-5-phosphate kinase (Ru5PK) catalyzes the formation of RuBP by consuming 1 molecule ATP. The whole process requires the consumption of two molecules of NADPH and three molecules of ATP [38,39]. common vetch belongs to the C4 plant of the NAD-ME type, which uses aspartic acid as the main transport metabolite. Aspartic acid is converted to malic acid by reductive deamination [40]. It has been con rmed that the Rubisco content of sun ower (Helianthus annuus L.) decreased under shading [41] (Fig. 11d).
The Rubisco content of rice decreased signi cantly under high temperature [42]. However, in this experiment, compared with the control, 19 genes in J3 and J7 were involved in carbon xation in photosynthetic organisms, and all of them were downregulated, including six downregulated genes encoding rbcL and rbcS. This is suitable for highland common vetch in environments of low light intensity and low CO 2 concentrations in the plain. These genes limit the photosynthesis and carbon-xing capacity of common vetch by reducing the e ciency of electron transport.

Regulation of antioxidant capacity in the plain environment
TFs are proteins that can regulate gene expression [43]. TF gene expression regulation can make plants respond to changes in the external environment in a highly speci c and exible way [44]. Furthermore, the environment can promote the activity of photosynthetic genes and improve the photosynthetic e ciency of plants [45]. To better understand the regulation process of common vetch under low light and low CO 2 concentrations, we identi ed a large number of candidate TFs that directly or indirectly participate in the transcription of genes related to the light response. It is noteworthy that the TF family related to MYB, NAC, AP2-EREBP and Orphans was differentially expressed.

Conclusions
The results showed that the plateau plant common vetch could adapt by changing its morphological and physiological characteristics when it was transported to the plain environment by air at the seedling stage. A large number of DEGs were identi ed by RNA-Seq in pea. These DEGs were mainly downregulated and involved in photosynthesis and carbon dioxide xation pathways. TFs regulate the expression of light-responsive genes to allow plants to enter the plain environment.

Plant materials
The experiment was carried out in the eld of Sangzhuzi District, Tibet Autonomous Region in 2019. The seeds of common vetch (V. sativa), that is, XiMu432, by Qinghai Academy of Animal Husbandry and Veterinary Sciences, were seeded in a round plastic basin (diameter: 10 cm and height: 15 cm). The soil was a uniform nutrient soil. The plants were transported in the basin during the seedling stage to the outdoor experimental site of Northeast Agricultural University of Harbin City by air (Additional le 8: Table  S7). Samples were collected on the rst, third, fth and seventh days (J1, J3, J5, and J7, respectively).
The samples collected before high-altitude transportation were set as the control (J0). After sampling from 9:00 to 11:00, the plants were frozen in liquid nitrogen and stored in a refrigerator at -80°C, and three independent samples were collected daily for physiological index measurements. The samples collected on the 3rd and 7th days were used for transcriptome sequencing.

Measurement of stomatal density and leaf structure
Fresh leaves were cut into 2× 5 mm strips and stored in glutaraldehyde. The leaves were dehydrated with a series of solutions. The stomatal opening and closing of leaves were observed with an S-3400N (Hitachi, China) scanning electron microscope. Five eld images were saved for each sample, and the number of stomata was calculated.
The veinless leaves of common vetch were xed in FAA solution to prepare para n patches (3 × 5 mm) and stained with solid green and safranin. The cross sections of 10 μm thick leaves were analyzed under a 10-fold microscope (Nikon Eclipse 50i, Japan) to observe the state of palisade tissue and spongy tissue.

Measurement of chlorophyll uorescence and physiological index
Chlorophyll uorescence (Fv/Fm) was measured by a portable photosynthetic apparatus after 30 minutes of leaf shading. After the samples were transported back to the laboratory, physiological indexes, namely, the osmotic regulation system (soluble sugar, soluble protein), antioxidant system (SOD, POD, CAT), oxidation system (O 2 -, H 2 O 2 ) and MDA, were determined with a test kit (Comin, Suzhou, China).

RNA extraction and library preparation for transcriptome sequencing
Total RNA was isolated from common vetch by using the NEBNext® Ultra™ RNA Library Prep Kit (NEB, USA). Agarose gel electrophoresis (1% gel concentration) and a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) were used to detect whether RNA was degraded and RNA purity. The cDNA library was prepared with the NEBNext® Ultra™ RNA Library Prep Kit for the Illumina sequencing system (NEB, USA). The mRNA was isolated and puri ed from total RNA by magnetic beads linked with poly-T oligosaccharides. The rst cDNA was synthesized from the interrupted mRNA, and then the second strand cDNA was synthesized by DNA Polymerase I and RNase H. An Ampure XP system (Beckman Coulter, Beverly, USA) was used to select the fragment size (250-300 bp), and PCR was used to enrich the cDNA library. The quality of the constructed library was evaluated by an Agilent Bioanalyzer 2100 system. Finally, the Illumina HiSeq platform was used for high-throughput sequencing. Transcriptome datasets can be used in the NCBI Sequences Read Archive under accession number PRJNA688111.

Functional annotation and differentially expressed gene (DEG) analysis
Use the FASTX toolkit to read the clean sequence. The Trinity program was used for the assembly of single genes. The NR, NT (http://www.ncbi.nlm.nih.gov/), PFAM (http://pfam.sanger.ac.uk/), GO (http://www.geneontology.org/), and KOG (http://www.ncbi.nlm.nih.gov/COG/) databases were used to annotate gene functions to obtain gene annotation functions. Differential expression analysis of two samples was performed using the DEGseq (2010) R package. P value was adjusted using q value. q value < 0.005 & |log2 fold change |>1 was set as the threshold for signi cantly differential expression. All the DEGs were subjected to enrichment analysis according to GO functions and KEGG pathways.

Quantitative real-time PCR (qRT-PCR) analysis
To con rm the DEG results, 8 transcripts were randomly selected from the photosynthesis pathways (ko00195) and carbon xation in photosynthetic organisms pathways (ko00710) for qRT-PCR veri cation. Primer 5 software was used to design gene-speci c qRT-PCR primers (Attached le 9:

Declarations
Ethics approval and consent to participate Not applicable.

Consent to publication
Not applicable.

Competing interests
The authors declare that they have no competing interests.

Availability of data and materials
Raw reads of one Illumina RNA-seq library generated in this study are available from BioProject at NCBI (https://www.ncbi. nlm.nih.gov/bioproject/) under accession number PRJNA688111.

Funding
This research was funded by the National Natural Science Foundation of China (31872998). The funding organizations provided nancial support to the research projects but were not involved in the design of the study, data collection, analysis of the data, or writing of the manuscript.
Authors' contributions GW designed the research. JL carried out the experiments with the help of ZL, JQ, BL, XY, YY and HY. JL collected the experimental data and drafted the manuscript. GW reviewed the manuscript and part of the data analysis. All authors read and approved the nal manuscript.        Supplementary Files