Metabolic Response of Larix Olgensis A. Henry to Polyethylene Glycol-simulated Drought Stress


 BackgroundDrought stress in trees limits their growth, survival, and productivity, and it negatively affects the afforestation survival rate. Molecular responses to drought stress have been extensively studied in broad-leaved species, but studies on coniferous species are limited.ResultsOur study focused on the molecular responses to drought stress in a coniferous species, Larix olgensis A. Henry. Drought stress was simulated in one-year-old seedlings using 25% polyethylene glycol 6000. The drought stress response in these seedlings was assessed by analyzing select biochemical parameters, along with gene expression and metabolite profiles. The soluble protein content, peroxidase activity, and malondialdehyde content of L. olgensis were significantly changed during drought stress. Quantitative gene expression analysis identified a total of 8172 differentially expressed genes in seedlings processed after 24 h, 48 h, and 96 h of drought stress treatment. Compared with the gene expression profile of the untreated control, the number of up-regulated genes was higher than that of down-regulated genes, indicating that L. olgensis mainly responded to drought stress through positive regulation. Metabolite analysis of the control and stress-treated samples showed that under drought stress, the increased abundance of linoleic acid was the highest among up-regulated metabolites, which also included some saccharides. A combined analysis of the transcriptome and metabolome revealed that genes dominating the differential expression profile were involved in glutathione metabolism, galactose metabolism, and starch and sucrose metabolism. Moreover, the relative abundance of specific metabolites of these pathways was also altered. Thus, our results indicated that L. olgensis prevented free radical-induced damage through glutathione metabolism and responded to drought through sugar accumulation.ConclusionsThe soluble protein content, peroxidase activity, and malondialdehyde content of L. olgensis were significantly changed during drought stress. A total of 8172 differentially expressed genes in seedlings processed after drought stress treatment. A combined analysis of the transcriptome and metabolome revealed that genes dominating the differential expression profile were involved in glutathione metabolism, galactose metabolism, and starch and sucrose metabolism. Our results indicated that L. olgensis prevented free radical-induced damage through glutathione metabolism and responded to drought through sugar accumulation.


Background
Drought is a main limiting factor for tree growth, survival, and productivity, with a negative effect on the afforestation survival rate [1]. The response to drought stress is complex in the whole tree as well as at the tissue and cellular level. Drought resistance mechanisms vary among tree species with substantial differences between angiosperms (broad-leaved plants) and gymnosperms (coniferous plants). Molecular responses to drought stress have been extensively studied in broad-leaved species, but studies on coniferous species are limited [2]. Larix olgensis A. Henry is a more drought-tolerant conifer [3], which is mainly distributed in the Changbai Mountain and Laoyeling Mountain area of Northeast China. The lack of research on basic drought stress mechanisms in L. olgensis has hindered the improvement of larch yield and wood quality in arid environments to some extent. However, to potentially improve drought resistance in L. olgensis, it is necessary to explore drought stress-induced molecular mechanisms in this conifer.
With the rapid development of sequencing technology, molecular biology has entered the era of "big data" by facilitating research on physiological and biochemical changes in plants and animals through omicsbased approaches, which provides explanations for some scienti c problems at the genetic level that expand our understanding of certain physiological and biochemical processes. Speci cally, transcriptome research can deepen the analysis of physiological processes based on the level of genes [4]. However, using a single-omics approach has failed to fully reveal the mechanism of the plant response to drought stress. Metabolomics is an emerging omics technique, adding a new level to the established approaches in genomics, proteomics, and transcriptomics. Data sets generated by measuring changes in the gene expression and metabolite levels in the transcriptome and metabolome, respectively, can be used for association analyses. This approach has been utilized in drought resistance studies in plants, such as ryegrass [5], poplar [6], Astragalus membranaceus [7], and Panicum virgatum L. [8]. However, there are no relevant reports on conifers.
To maintain normal growth and development under drought stress, plants respond by activating a series of physiological and biochemical regulatory mechanisms [9]. The drought stress response is a complex biochemical process that diminishes the effects of drought through molecular mechanisms that ensure osmotic adjustment, along with proper levels of antioxidants and scavenger compounds.
Under drought stress, plants produce many metabolites to maintain the osmotic pressure and balance, thus, maintaining the normal physiological and biochemical processes and preventing drought stressinduced damage. Under drought stress, plants regulate the expression of many synthetase genes, leading to increased concentrations of soluble metabolites, such as free proline, soluble sugars, organic acids, and betaine [10]. Osmotic regulation by accumulating solutes is a main mechanism in plants to adapt to drought stress. During drought, these compounds maintain the osmotic balance between the cytoplasmic matrix and the environment by preventing water loss and protecting membrane integrity [11]. Similar to other stress conditions, such as high temperature, drought stress is accompanied by increased reactive oxygen species (ROS) production. Plants prevent ROS production and oxidative stress through complex antioxidant defense systems involving multiple enzymes and antioxidants. The main non-enzymatic antioxidants are L-ascorbic acid (AsA) and glutathione (GSH), and critical enzymes are glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione S-transferase (GST) [12]. Thus, combining transcriptomics and metabolomics methods should generate results that will expand our knowledge of the mechanism of drought stress resistance in plants.
In recent years, "spring drought" has frequently occurred in the northeastern part of China, which has severely slowed the growth and development of forest trees. As a native tree species in Northeast China, L. olgensis has evolved drought resistance through "spring drought" cycles. However, the response mechanisms against drought stress in L. olgensis are still unclear. By performing a combined analysis of the physiological, gene expression, and metabolite pro le changes under drought stress, we aimed to identify the main metabolic pathways for regulating the response to drought stress, which are critical for studying the mechanism of the drought resistance in L. olgensis.

Results
Analysis of biochemical changes under PEG-simulated drought stress SPs functioning as metabolic enzymes under drought stress are involved in regulating the osmotic potential of plant cells. During drought stress in L. olgensis, the SP content generally increased. In the early stage of drought stress (0-24 h), the SP content increased from 55 to 77 mg g − 1 . During drought stress from 24 h to 48 h, the SP content reached a peak value of 137 mg g − 1 (Fig. 1a).
POD is involved in various physiological and metabolic processes, including scavenging excessive ROS in cells, controlling the growth and development of cells, and maintaining the stability of the cell membrane. POD is a key enzyme in the antioxidant enzyme protection system under unfavorable conditions and stress. In the early stage of drought stress, POD activity increased from 15.0 to 26.8 U min − 1 g − 1 . During drought stress, POD activity increased to 31.2 U min − 1 g − 1 at 48 h, reaching a peak value, and subsequently decreasing to 23.2 U min − 1 g − 1 at 96 h. Over the whole drought stress period, the POD activity showed a "rise-rise-decline" change, but the overall POD activity showed an upward trend (Fig. 1b).
MDA is produced by lipid peroxidation of the tissue or organ membrane when damaged under adverse conditions. The MDA content increased from 10.9 to 12.7 nmol g − 1 , displaying a "decline-rise" change over the whole drought stress period (Fig. 1c). Thus, the monitoring of select biochemical parameters indicated that the SP content, MDA content, and POD activity varied signi cantly throughout the consecutive drought stress periods, revealing some changes in L. olgensis associated with drought stress, which were related to gene expression levels and variations in metabolite content, indicating the need to conduct a transcriptomics and metabolomics analysis.
Transcriptomic analysis of L. olgensis under drought stress The cDNA library of drought stress-treated L. olgensis was sequenced using an Illumina Hiseq highthroughput sequencing platform based on sequencing by synthesis. The transcriptome sequencing of all samples yielded a clean data set of 36.88 Gb. The high-quality sequence data were assembled using the Trinity software. A total of 59 710 unigenes with an N50 of 1709 bp were obtained. Their assembly integrity was high; 21 907 unigenes had a length of ≥1000 bp. The complete transcriptional pro le of co-expressed transcripts at different stages of drought stress was obtained by hierarchical clustering. Clustering spectra indicated that drought stress signi cantly affected the transcriptional pro le of the co-expressed transcripts. Compared with the control expression levels, there were more up-regulated genes than down-regulated genes among the co-expressed transcripts (Fig. 3).

Functional analysis of DEGs
GO is an internationally standardized gene function classi cation system with three main GO categories, cellular component, molecular function, and biological process. Among these categories, the DEGs were enriched under the GO term metabolic process, followed by the terms cellular process and singleorganism process (Fig. 4a). After 24 h of drought stress, metabolic, cellular, and single-organism processes were associated with more up-regulated than down-regulated DEGs (Fig. 4b), but after 48 h of drought stress, the same GO process terms had more down-regulated DEGs (Fig. 4b, c). After 96 h of drought stress, metabolic, cellular, and single-organism processes were more abundant among downregulated DEGs than among up-regulated DEGs (Fig. 4b,  Gene expression response to drought Based on the gene expression ratio between 24 h drought stress and the control, an analysis of the 50 genes with the highest differential expression found that a gene annotated with the Jacalin-like lectin domain had the highest expression, and there were a total of three annotations for the Jacalin-like lectin domain and one annotation for peroxidase among the top 50 DEGs. After 48 h of drought stress, the top 50 DEGs included two genes with an annotation for heat shock protein (HSP), and two other genes were associated with ATP. Among the top 50 DEGs at 96 h of drought stress, the highest expressed gene annotation was a bidirectional sugar transporter, and three genes had the annotation HSP20/α crystallin family; two other genes were annotated as late embryogenesis abundant (LEA) genes (Supporting information: The top 50 differentially expressed genes and their predicted functions.).

DEGs during drought stress
Non-speci c lipid transfer proteins (nsLTPs) are a class of small, basic proteins in plants. Our results showed that three genes annotated as nsLTPs were among the DEGs that were up-regulated throughout three drought stress periods ( Table 2). LEA proteins are small, hydrophilic proteins with a protective function associated with water de cit. A total of 11 drought stress-associated DEGs were annotated as LEA protein genes, which were largely expressed during the last drought stress period, indicating that they are late response proteins during drought stress in L. olgensis (Table 3).  Metabolic changes in L. olgensis during drought stress Samples were evaluated for repeated biological correlations using the Spearman rank correlation.
Repeated correlations between samples were above 0.7, indicating that the entire metabolome assay was reliable. Prior to the difference analysis, the different groups were subjected to a Principal component analysis (PCA). Twelve samples were divided into four components by principal component PC1 (34.13%) and principal component PC2 (18.21%), wherein the main component PC1 clearly separated the control group from each treatment group, indicating a large difference between the treatment and control after drought stress treatment (Fig. 5).
The screen for differential metabolites was performed using a combination of the P value from Student's t-test and the variable in uence on projection (VIP) value from the orthogonal projections to latent structures discriminant analysis (OPLS-DA) model. The screening criteria were P < 0.05 and VIP > 1. After 24 h of drought stress, 11 differentially expressed metabolites were increased, and after 96 h of drought stress, there was an increase in 42 differentially expressed metabolites (Table 4). A total of 12 metabolites were differentially expressed throughout the entire drought stress treatment (Fig. 5). The level of metabolite expression changed in a time-dependent manner throughout the 96 h of drought stress treatment. Speci cally, among the differentially expressed metabolites, linoleic acid and dimidazoleglycerol-phosphate dehydratase had the most pronounced change in expression level compared with that in the control. Under long-term drought stress, the abundance of linoleic acid and dimidazoleglycerol-phosphate dehydratase increased 5.36 times and 4.63 times, respectively. During the drought stress treatment, the abundance of galactose, maltose, mannose, ra nose, D-glucose 6phosphate, and inositol was increased after 24 h, slightly reduced after 48 h, and increased again after long-term drought stress that lasted for 96 h. The expression of some alkaloids, such as betaine, acetylcholine, and glycerophosphocholine, was continuously up-regulated in a time-dependent manner during drought stress. Most amino acids, such as isoleucine, arginine, lysine, and glutamic acid, were initially up-regulated in the early drought stress period but gradually decreased during the remainder of the drought stress treatment. However, the level of proline was elevated throughout the whole drought stress treatment, compared with that in the control, and the maximum level was reached after 96 h longterm drought treatment (Table 5). Comprehensive analysis of gene expression and metabolite levels To better assess transcriptional regulation mechanisms in L. olgensis under drought stress, a correlation analysis was performed between the metabolome and the transcriptome data sets. Using the analysis results for differential metabolites and transcriptome-based differential genes, the same groups of differential metabolites and genes were simultaneously mapped on the KEGG pathway map, and 20 KEGG pathways were obtained. The results indicated that drought stress signi cantly affected fructose and mannose metabolism, galactose metabolism, biosynthesis of keratin, berberine, and wax, starch and sucrose metabolism, amino acid sugar and nucleotide sugar metabolism, inositol phosphate metabolism, glyceryl phosphatide metabolism, and GSH metabolism. The relationship between metabolite abundance and gene expression in GSH metabolism, galactose metabolism, and starch and sucrose metabolism was determined (Table 6, 7) (Fig. 6).  The GSH metabolic pathway had two annotated GPX and two annotated GR genes, all of which were upregulated during drought stress, compared with the control transcription pro le. From the four genes annotated as glucose-6-phosphate 1-dehydrogenase, two were up-regulated, but the other two were downregulated during drought stress. Three genes were annotated as 6-phosphogluconate dehydrogenase, and two enzymes were involved in the conversion of NADP/NADPH. There were four genes annotated as glutathione S-transferase, which showed up-regulated expression under drought stress. One gene was annotated as L-ascorbate peroxidase, and in addition, one gene was annotated as leucyl aminopeptidase, which is involved in cysteine and glycine synthesis.
In galactose metabolism (Table 7), the abundance of several metabolites, ra nose, inositol galactose, Dglucose, and D-fructose, were increased. A total of 114 genes were obtained by analyzing galactose metabolism-related genes, and the respective gene annotations identi ed 12 enzymes. Two annotated inositol 3-α-galactosyltransferase genes were up-regulated during the three drought stress periods. There were six annotated ra nose synthase genes and one annotated stachyose synthetase gene, the latter being a key enzyme in stachyose synthesis. These genes were also up-regulated during drought stress.
One gene was annotated as α-glucosidase, and two genes were annotated as α-galactosidase. The changes in the expression level of these genes increased the ra nose content by 1.24 times during the drought.
In sucrose metabolism (Fig. 6a), the gene encoding sucrose synthase was up-regulated after 24 h and 48 h of drought stress but slightly down-regulated under long-term drought stress lasting for 96 h. The αglucosidase gene was up-regulated throughout the drought stress treatment, and the UTP-glucose-1phosphate uridine acyltransferase gene was up-regulated during the early drought period. Compared with the sucrose pro le in the control, the sucrose content was signi cantly increased during the early drought stress period but decreased after the long-term treatment, which may be related to the large amount of sucrose decomposed to D-fructose during the last drought stress period; speci cally, the D-fructose content (1.18620722, 0.245392272, 1.45427517) was increased relative to that in the control.
In starch metabolism (Fig. 6b), the glucose content (2.416205353, 2.180226375, 2.741899739) was increased by the drought stress treatment. The β-amylase and α-amylase genes were down-regulated after 24 h of drought stress but up-regulated after 48 h and 96 h of drought stress. Additionally, the 1,4-αglucan branching enzyme gene was up-regulated during the initial 24 h of drought stress but downregulated after the 48 h and 96 h drought stress periods. The expression level changes in these genes caused starch accumulation during the early drought stress period compared with the starch content in the control. Moreover, the α-glucosidase gene was up-regulated during the last drought stress period.

Discussion
Select biochemical parameters of L. olgensis were determined under PEG-simulated drought stress before treatment initiation (0 h) and after 24 h, 48 h, and 96 h. Based on transcriptomics and metabolomics data, the effects of drought stress were studied, and the metabolic pathways and related genes that changed during the drought stress treatment were analyzed.
Under normal environmental conditions, the metabolic, physiological, and biochemical processes in plants are relatively stable; however, various metabolic activities in the plant can change in response to adverse conditions and stress. Osmotic adjustment is a critical physiological mechanism for plants to endure and resist drought and an important physiological indicator for selecting drought-tolerant crops.
Osmotic adjustment typically occurs when the degree of drought stress is mild or moderate. During environmental stress, various organic and inorganic substances accumulate in the plant, increasing their cytoplasmic concentration and reducing the osmotic potential, all of which maintain the water balance in the plant for adaptation to the adverse environment. When the degree of water stress is severe, the osmotic adjustment ability is weakened or lost. Under PEG stress, changes in the SP content showed a "rise-rise-decline" trend over the entire stress period. The SP content (106.726 mg/g) after 96 h of PEG stress treatment was 93.61% higher than that of the control (55.125 mg/g), indicating that the plant underwent a strong osmotic adjustment during PEG stress treatment.
Plants produce low concentrations of ROS under normal growth conditions. However, under drought stress, ROS production is increased, which causes oxidative damage in plants [13]. Plants act through an enzymatic protection system that uses POD to scavenge ROS, thereby protecting the membrane system from damage [14]. The POD activity (23.2) after 96 h of PEG stress treatment was 54.67% higher than that of the control (15.0), and the overall trend was upward.
In arid environments, cell membrane permeability changes, resulting in an increase in relative conductivity and triggering membrane lipid peroxidation, which generates MDA, causing damage to cells due to increased MDA content [15]. Therefore, the MDA content is an indicator for the degree of damage to the plant under drought stress, which indirectly re ects the plant's drought resistance; that is, the higher the MDA content, the greater the plant damage and the worse the drought resistance [16]. The MDA content (12.67 nmol·g − 1 ) under stress treatment for 96 h was increased by 18.05% compared with that in the control (10.73 nmol·g − 1 ). The MDA content was decreased after 24 h of PEG stress treatment, which may be due to the increase in the POD content to remove a portion of MDA during the initial 24 h.
However, with the prolongation of stress time, the regulation ability decreased, and the MDA content continued to increase.
Due to the signi cant differences in biochemical indicators between the stress periods, the same four treatment periods were used for analyzing the DEGs and metabolites. The total number of droughtresponsive genes was related to the length of the drought stress period. Speci cally, the total number of DEGs after 24 h of stress treatment was lower than that after 96 h. HSPs are responsible for protein folding, assembly, and translocation, and the degradation of damaged proteins. They play a key role in plants, conferring protection against stress by stabilizing proteins and membranes [17]. During long-time drought stress, several genes related to HSPs were up-regulated, such as LEA proteins genes (8.69 times higher than the control value at 0 h). The LEA genes encode hydrophilic proteins that strongly bind to water, thus retaining moisture, preventing the crystallization of important cellular proteins and other molecules in the absence of water, and stabilizing the cell membrane [18]. In addition, the expression of genes annotated with the Jacalin-like lectin domain was up-regulated. Plant lectins, which reversibly bind to carbohydrates, are associated with plant stress resistance [19].
Similar to the DEGs analysis, we identi ed many metabolites associated with drought stress. Trigonelline (TG) is an alkaloid found in the leguminous plant Trigonella foenum-graecum (Fabaceae). As a penetrant, its accumulation reduces the osmotic potential of cells. Thus, TG plays an important role in protecting the cell membrane stability in plants under environmental stress [20]. In our study, TG accumulated 4.46 times in L. ogensis seedlings due to drought stress. Proline is widely recognized as a critical drought-induced metabolite that protects cell membranes by reducing the cell osmotic potential. In addition, it is also a regulator of the cellular redox state and an ROS scavenger [21]. The results showed that proline was only up-regulated after long-term drought stress, with an upregulation of 2.69 times. Under drought stress, soluble sugar usually accumulates and functions as a signal and osmotic adjustment [22]. The content of monosaccharides (glucose and fructose) also increased in this study, and the genes involved in the decomposition of polysaccharides were up-regulated.
After a comprehensive analysis of the metabolomics and transcriptome data, we obtained more information about metabolic pathways. Among the DEGs and metabolites, GSH and soluble sugars have signi cant regulatory properties; further analysis focused on GSH metabolism, galactose metabolism, and sucrose and starch metabolism pathways.
GSH, a tripeptide widely present in organisms, has important physiological functions [23]. The active sulfhydryl group in the GSH structure binds to free radicals, which plays an important role in the cellular capacity for scavenging ROS. In this process, GST plays an important catalytic role. GST is mainly present in the cytoplasm and functions in oxidative stress resistance under biotic or abiotic stress. GST acts as a detoxifying agent for some extracellular substances that are harmful in cells, and it is also known to inhibit lipid peroxidation in vitro [24]. In our study, four GST-related genes were up-regulated to different degrees, indicating that GST was actively involved in the drought stress response and helped to maintain normal growth and development of the plants.
As the most abundant antioxidant in the cell, GSH protects DNA, proteins, and other biomolecules against oxidative damage and converts ROS and free radicals in general into metabolites that are easily eliminated in vivo [25]. Reduced GSH can reduce H 2 O 2 to H 2 O by GPX in the process of scavenging ROS, and GSH itself is oxidized to glutathione disul de (GSSG), while GSSG is mainly re-reduced to GSH by GR [26]. Through the conversion between reduced and oxidized forms, active oxygen is continuously removed, and the effect of ROS on the plant is reduced [27]. Our transcriptome analysis identi ed two genes annotated as GPX and one annotated as GR, all of which were up-regulated during drought stress. This result indicated that these two key enzymes actively respond to drought stress and play an important role in the process of scavenging ROS. Glucose-6-phosphate 1-dehydrogenase and 6phosphogluconate dehydrogenase participate in the conversion of NADP/NADPH and use NADP as an electron acceptor to catalyze the formation of NADPH, which provides hydrogen reduction during the conversion of oxidized GSSG to reduced GSH [28]. The genes involved in the regulation of these enzymes were also up-regulated in our study, indicating that under the drought stress-induced action of reductase, the coenzyme with hydrogen reduction activity also responds positively to drought stress.
AsA, as an important antioxidant in cells, plays an important role in scavenging ROS. In addition, AsA can act as a cofactor for some antioxidant enzymes under stress [29]. Under drought stress, the AsA content increased signi cantly, and the ribonucleoside-diphosphate reductase associated with ascorbate synthesis was up-regulated, and the ascorbate reductase-related gene associated with AsA-mediated scavenging of hydrogen peroxide was also up-regulated. Under drought stress, both GSH and AsA are the main antioxidants that play a major role in scavenging ROS.
The L. olgensis seedlings also responded positively to drought stress through antioxidant metabolism. GSH can increase the expression level of the upstream metabolite directly related to GSH by increasing the expression of the leucyl aminopeptidase gene. Additionally, 5-L-glutamyl-L-alanine, a downstream metabolite of GSH, was detected and down-regulated during drought stress. Thus, both effects combined, the expression level increase of upstream metabolites directly related to GSH and the expression level decrease of downstream metabolites, elevated the synthesis yield of GSH and decreased its degradation rate, which was responsible for maintaining a high intracellular GSH concentration. In the scavenging of ROS, the increased activity of GR and GPX ensured that the conversion of GSSG and GSH was accomplished using the reduced hydrogen provided by glucose-6-phosphate 1-dehydrogenase and 6phosphate dehydrogenase involved in the conversion of NADP/NADPH, thereby achieving the clearing of active oxygen. In addition, under drought stress, GSH metabolism was actively regulating the expression of GST-related enzymes, which improved the ability for scavenging ROS. Therefore, the regulation of the differential expression of key enzyme genes in GSH metabolism provides drought tolerance in larch trees.
Ra nose series oligosaccharides act as osmotic adjustment substances to stabilize the photosynthetic system under abiotic stress and protect cells as protective agents [30]. In addition, ra nose also scavenges free radicals. UDP-galactose synthesizes inositol galactoside involving the activity of 3-αgalactosyltransferase [31]. The 3-α-galactosyltransferase gene was up-regulated after each of the three drought stress periods, and ra nose was synthesized from inositol galactosides by ra nose synthase.
Thus, the 3-galactosyltransferase gene was highly expressed in the galactose metabolic pathway to increase the upstream galactose metabolites and reduce the consumption of stachyose synthase and αgalactosidase, all of which maintained a high intracellular concentration of galactose. Hence, ra nose acted as an osmotic regulator substance and maintained the osmotic pressure in plants to persist under drought.
Studies have shown that drought-tolerant soybean varieties have more starch decomposition than drought-sensitive varieties [32]. Our results were consistent with this conclusion, indicating that the drought resistance mechanism in L. olgensis was similar to that in other plants. The starch content of the treatment group increased rapidly within 24 h and then decreased. The latter effect might have been caused by the decomposition of starch into soluble sugar molecules to counter the pressure of long-term drought stress. Speci cally, the decomposition of maltose into glucose by α-glucosidase increased the osmotic potential of plant cells, preventing excessive water loss due to glucose accumulation.
Under drought stress, sucrose is an important soluble sugar in plants, which diminishes the stressinduced damage [33]. Moreover, sucrose directly acts as an osmotic adjustment substance because under the action of sucrose synthase or α-glucosidase, glucose and fructose are produced, which increase the osmotic potential of plant cells. However, in plants, sucrose also acts as a signal substance, regulates transporters, and induces the expression of resistance genes. In addition, sucrose functions as an antioxidant [3]. Sucrose synthase is an extremely important soluble enzyme in sucrose metabolism, which is found in the cytoplasm. Sucrose synthase not only catalyzes the synthesis of sucrose but also catalyzes its decomposition. This dual property of sucrose synthase may play a critical role in sucrose metabolism. UTP-glucose-1-phosphate uridine acyltransferase catalyzes the synthesis of UDP-glucose from α-D-glucose-1P. UDP-glucose is used for sucrose production by sucrose synthase, but sucrose can also be decomposed into UDP-glucose by the same enzyme.
UTP-glucose-1-phosphate uridine acyltransferase was initially up-regulated under drought stress, which increased the UDP-glucose content. During the rst 24 h of drought stress, the sucrose content increased.
Sucrose synthase might have been involved in the synthesis of sucrose. After 48 h and 96 h of drought stress, the sucrose content was decreased, and again, sucrose synthase might have been involved in the decomposition of sucrose to produce glucose and fructose. Our observations indicated that sucrose was an important soluble sugar component in the plants during the early drought period. Thus, sucrose limited the damage caused by drought stress and participated in the drought stress response of L. olgensis. During long-term drought stress, sucrose decomposed into glucose and fructose to adjust the osmotic pressure, which diminished the effects of drought stress.

Conclusions
The drought transcriptome of this study included a high proportion of transcripts without annotation.

Data analysis
Quality control Raw data (raw reads) in Fastq format were initially processed using in-house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N, and low-quality reads from raw data. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. All downstream analyses were based on clean, high-quality data.

Transcriptome assembly
The left read les (read1 les) and the right read les (read2 les) from all libraries/samples were pooled into one large left.fq le and one large right.fq le, respectively. Transcriptome assembly was accomplished based on the left.fq and right.fq using Trinity v2.5.1 [37]with 2 as default for min_kmer_cov and default settings for all other parameters.
Quanti cation of gene expression levels Gene expression levels were estimated for each sample using RSEM v1.2.19 [38]: Clean data sets were mapped back onto the assembled transcriptome, and the read count for each gene was obtained from the mapping results.

Differential gene expression analysis
Differential expression analyses of two conditions/groups were performed using the DESeq R package, which provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini-Hochberg method for controlling the false discovery rate. Genes with an adjusted P value < 0.05 by DESeq were assigned as differentially expressed. Gene ontology (GO) annotations were obtained using Blast2GO (v2.5) based on Unigene NR annotation results. The background set of topGO analysis included all Unigene GO annotations. GO enrichment [39]analysis of differentially expressed genes (DEGs) was implemented by the topGO R package-based Kolmogorov-Smirnov test. We used KOBAS software[41]to test the statistical enrichment of DEGs in the pathways of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Metabolite extraction
Samples were thawed at 4 °C on ice. Then, 100 µL of each sample was transferred into a microcentrifuge tube, extracted with 300 µL of methanol, and prepared for analysis by adding 20 µL of an internal standard substance. Samples were vortexed for 30 s, ultrasound treated for 10 min (in ice water), and incubated for 1 h at -20 °C to precipitate proteins. Then, the samples were centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant (200 µL) was transferred to a fresh 2 mL glass vial for ultra-high performance liquid chromatography-quadrupole time-of-ight mass spectrometry (UHPLC/Q-TOF-MS) analysis, and 20 µL was retained from each sample and pooled as quality control samples.
The UHPLC/Q-TOF-MS analyses were performed using a UHPLC system (1290,Agilent Technologies ·). MS raw data (.d) les were converted to the mzXML format using ProteoWizard and processed using the Ethics approval and consent to participate Not applicable. Neither human or animal subjects, human or animal materials nor human or animal data were used on this manuscript. Collections of plant material comply with institutional, national, and international guidelines.