Overview of Transcriptome Sequencing
To compare the transcriptomes of the P. wutunensis drought-stress and rehydration groups, we performed transcriptional profiling of leaves after drought and rehydration treatments. The three libraries (T1, T2, and T4) produced 45 GB of raw data, and the Q20 and Q30 values were higher than 90%. A total of 354,109 transcripts and 171,998 unigenes were identified (Table 1). The number of N50/N90 accounts for only three thousandths of the whole transcript sequence. The GC content of the transcripts was relatively balanced, indicating that the transcriptome splicing quality was good and could be further analyzed (Table S2).
The assembled unigenes were annotated and classified using the Nucleotide Sequence Database (NT), Non-Redundant Protein Sequence Database (NR), Gene Ontology (GO), Swissprot, EuKaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases (Table 2), producing 129,468 unigene annotations from NT (75.27%), 104,273 from NR (60.62%), 87,581 from GO (50.92%), 78,135 from Swissprot (45.43%), 42,940 from KOG (24.97%), and 6861 from KEGG (3.99%).
Table 1
Summary of transcriptome assembly quality.
| Number | ≥ 500 bp | ≥ 1,000 bp | N50 | N90 | Max Length | Min Length | Total Length | Average Length |
Transcript | 354,109 | 164,348 | 91,699 | 1,273 | 293 | 12,017 | 201 | 271,244,089 | 765.99 |
Unigene | 171,998 | 47,159 | 22,578 | 813 | 233 | 12,017 | 201 | 93,567,101 | 544 |
Table 2
Success rate of gene annotation.
Database | Number of genes | Percentage (%) |
Annotated in NT | 129,468 | 75.27 |
Annotated in NR | 104,273 | 60.62 |
Annotated in GO | 87,581 | 50.92 |
Annotated in Swissprot | 78,135 | 45.43 |
Annotated in KOG | 42,940 | 24.97 |
Annotated in KEGG | 6,861 | 3.99 |
Differential Expression Analysis Of Genes Responding To Drought Stress And Rehydration
To study DEGs in P. wutunensis under drought stress and rehydration, differential expression between the control (T1) and the drought group (T2) and rehydration group (T4) was determined (Table S3). Gene expression was calculated based on the fragments per kilobase of transcript per million mapped reads (TPM) value, and DEGs were screened based on the following criteria: two or more changes in the expression level and P < 0.05. A total of 5,102 DEGs were screened, among which 1,259 were up-regulated and 3,843 were down-regulated (Fig. S1). Most of the genes were consistently up-regulated or down-regulated during both drought and rehydration. However, a few genes that responded to drought showed the opposite regulatory role during the rehydration process (Fig. 1A-F). No common up-regulated genes were observed between drought and rehydration (Fig. 1A). Of the 694 up-regulated genes under drought conditions, 19.7% (178) of them were down-regulated upon rewatering (Fig. 1E-F). These genes are the key stress genes in response to drought. Among the 306 genes up-regulated in rewatering, 251 are newly up-regulated in rewatering, which indicates that these genes play a key role in rewatering recovery. (Fig. 1C-D).
Go Enrichment Of Degs
GO enrichment analysis was performed to further clarify the functions of the DEGs (Table S4, Fig. S2). We found most significant GO enrichment of up-regulated and down-regulated DEGs at drought stress and rehydration (Table 3). The up-regulated DEGs under drought stress were significantly enriched in response to hormone, response to acid chemical, response to abscisic acid, response to water, and hormone-mediated signaling pathways. The down-regulated DEGs under drought stress were mainly enriched in cell wall organization, external encapsulating structure organization, cell wall organization or biogenesis, cell wall, and apoplast. However, after rehydration, recovery was mainly by up-regulating chloroplast and plastid. The up-regulated, significantly enriched DEGs under drought stress began to decline under rehydration.
Table 3
GO analysis of the most significant up- and downregulated DEGs
Comparison | Up-regulated | Down-regulated |
GO ID | Description | p value | DEGs | GO ID | Description | p value | DEGs |
T2vsT1 | GO: 0009725 | response to hormone | 2.40E-17 | 69 | GO: 0071555 | cell wall organization | 1.00E-30 | 64 |
GO: 0001101 | response to acid chemical | 4.70E-17 | 53 | GO: 0045229 | external encapsulating structure organization | 1.00E-30 | 64 |
GO: 0009737 | response to abscisic acid | 9.60E-17 | 32 | GO: 0071554 | cell wall organization or biogenesis | 1.70E-30 | 67 |
GO: 0009415 | response to water | 3.00E-16 | 26 | GO: 0005618 | cell wall | 2.80E-24 | 54 |
GO: 0009755 | hormone-mediated signaling pathway | 3.40E-16 | 44 | GO: 0048046 | apoplast | 9.80E-23 | 38 |
T4vsT2 | GO: 0009536 | plastid | 1.00E-30 | 80 | GO: 0001101 | response to acid chemical | 3.60E-29 | 63 |
GO: 0044434 | chloroplast part | 1.00E-30 | 63 | GO: 0009755 | hormone-mediated signaling pathway | 2.10E-26 | 52 |
GO: 0009507 | chloroplast | 1.00E-30 | 79 | GO: 0009725 | response to hormone | 7.40E-24 | 71 |
GO: 0044435 | plastid part | 1.00E-30 | 63 | GO: 0009415 | response to water | 7.30E-23 | 30 |
GO: 0009941 | chloroplast envelope | 1.00E-30 | 47 | GO: 0009414 | response to water deprivation | 4.30E-21 | 28 |
T4vsT1 | GO: 0005840 | ribosome | 1.00E-30 | 64 | GO: 0009506 | plasmodesma | 1.00E-30 | 178 |
GO: 0003735 | structural constituent of ribosome | 1.00E-30 | 62 | GO: 0055044 | symplast | 1.00E-30 | 178 |
GO: 0022626 | cytosolic ribosome | 1.10E-27 | 46 | GO: 0005618 | cell wall | 1.00E-30 | 147 |
GO: 0044391 | ribosomal subunit | 9.60E-27 | 48 | GO: 0030312 | external encapsulating structure | 1.00E-30 | 147 |
GO: 0009522 | photosystem I | 2.40E-26 | 21 | GO: 0071554 | cell wall organization or biogenesis | 1.00E-30 | 218 |
Kegg Pathway Enrichment Analysis
To identify the pathways involved in the P. wutunensis drought stress and rehydration response, the KEGG metabolic pathway of the DEGs was analyzed (Table 4, Table S5, Fig. S3). Among the significantly enriched KEGG pathways, Plant hormone signal transduction has enriched the most DEGs. These up-regulated and down-regulated DEGs are involved throughout the process of drought and rehydration. MAPK signaling pathway–plant shows similarity with plant hormone signal transduction. Photosynthesis-antenna proteins, which can resist drought and recover through photosynthesis, are up-regulated in both drought and rehydration. These results are similar to those of the GO analysis. Interestingly, DEGs enriched in Galactose metabolism were only up-regulated in drought stress and down-regulated in rehydration. Streptomycin biosynthesis and Cysteine and methionine metabolism were up-regulated in drought stress. Flavonoid biosynthesis, Ascorbate and aldarate metabolism, Vitamin B6 metabolism, Carbon fixation in photosynthetic organisms, and Lysosome play important roles in rehydration recovery (Table 4).
Table 4
KEGG analysis of the most significant upregulated and downregulated DEGs
Comparison | Up-regulated | Down-regulated |
KEGG ID | Description | p value | DEGs | KEGG ID | Description | p value | DEGs |
T2vsT1 | ko00052 | Galactose metabolism | 2.45E-07 | 8 | ko04075 | Plant hormone signal transduction | 6.51E-07 | 14 |
ko04075 | Plant hormone signal transduction | 7.22E-05 | 10 | ko04016 | MAPK signaling pathway - plant | 0.000801 | 7 |
ko00196 | Photosynthesis - antenna proteins | 9.57E-05 | 4 | ko00910 | Nitrogen metabolism | 0.001395 | 4 |
ko04016 | MAPK signaling pathway - plant | 0.001313 | 6 | ko00520 | Amino sugar and nucleotide sugar metabolism | 0.013767 | 6 |
ko00521 | Streptomycin biosynthesis | 0.011537 | 2 | ko00053 | Ascorbate and aldarate metabolism | 0.020228 | 3 |
ko00270 | Cysteine and methionine metabolism | 0.038079 | 4 | ko00592 | alpha-Linolenic acid metabolism | 0.022692 | 3 |
| | | | | ko00942 | Anthocyanin biosynthesis | 0.025777 | 1 |
| | | | | ko00944 | Flavone and flavonol biosynthesis | 0.038419 | 1 |
T4vsT2 | ko04075 | Plant hormone signal transduction | 9.16E-06 | 9 | ko04075 | Plant hormone signal transduction | 6.95E-06 | 11 |
ko00941 | Flavonoid biosynthesis | 0.00077 | 3 | ko00052 | Galactose metabolism | 0.000373 | 5 |
ko00053 | Ascorbate and aldarate metabolism | 0.003087 | 3 | ko00073 | Cutin, suberine and wax biosynthesis | 0.000771 | 3 |
ko00750 | Vitamin B6 metabolism | 0.004761 | 2 | ko00500 | Starch and sucrose metabolism | 0.001305 | 7 |
ko00196 | Photosynthesis - antenna proteins | 0.010593 | 2 | ko04016 | MAPK signaling pathway - plant | 0.005711 | 5 |
ko00710 | Carbon fixation in photosynthetic organisms | 0.024658 | 3 | ko00450 | Selenocompound metabolism | 0.036418 | 2 |
ko04142 | Lysosome | 0.043505 | 3 | ko00906 | Carotenoid biosynthesis | 0.047835 | 2 |
| | | | | ko04080 | Neuroactive ligand-receptor interaction | 0.047896 | 1 |
T4vsT1 | ko03010 | Ribosome | 3.80E-15 | 39 | ko04075 | Plant hormone signal transduction | 6.75E-10 | 41 |
| ko00196 | Photosynthesis - antenna proteins | 1.38E-09 | 8 | ko02010 | ABC transporters | 4.27E-07 | 12 |
| | | | | ko00500 | Starch and sucrose metabolism | 7.00E-07 | 30 |
| | | | | ko04016 | MAPK signaling pathway - plant | 0.000256 | 19 |
Drought-responsive Genes
Under drought stress, DEGs related to Plant hormone signal transduction, Galactose metabolism and Ribosome metabolism were involved in drought response (Fig. 2). Plant hormone signal transduction-related DEGs showed different trends in response to drought. Compared with normal watering, GA, ABA, and Ethylene were up-regulated under drought stress, such as: GID1, PP2C,ABF,EBF1/2,ERF. The down-regulated pathways include Auxin, Cytokinine, Brassinolide, Jasmonic acid and Salicylic acid; the DEGs involved include IAA, GH3, A-ARR, TCH4 and NPR1(Fig. 2A). The main transcription factors involved in signal transduction are MYC2, WRKY22, and WRKY33(Fig. 2A). DEGs that up-regulate galactose metabolism mainly include GOLS, RAFS, GALM, USP, and HK(Fig. 2B). The majority of the DEGs of the photosynthesis - antenna proteins and ribosome are up-regulated(Fig. 2D-E).
Degs After Rehydration
Compared with drought stress, most of the genes showed the opposite trend after rehydration (Table S3), Up-regulated genes include ARR-A,ARR-B,GID1,TF,PYL,OXI1,MEKK1,WRKY33,CHS, CYP75, and F3H, which are mainly involved in Plant hormone signal transduction and Flavonoid metabolism recovery. Galactose-related DEGs are down-regulated after rehydration, and these genes no longer function after rehydration. After rehydration, DEGs of the photosynthesis-antenna proteins and ribosome were higher than those of drought and control, and the up-regulated genes LHCA, LHCB, RPS, and RPL may be beneficial to compensatory growth after recovery.
Verification Of Rna-sequence Data By Qrt-pcr
To validate the transcriptome data sets, real-time qPCR technology was applied to analyze transcription levels of nine genes which were randomly selected from drought or rehydration induced pathways. The relative expression levels of the genes were measured and calculated using PtRG1 as the internal reference gene. These nine genes included: galactinol synthase 3 (GOLS3), galactinol synthase 6 (GOLS6), galactinol synthase 7 (GOLS7), ankyrin repeat family protein (ANKRA), phaseolin G-box binding protein (PG2), chalcone synthase (CHS), purple acid phosphatase 3 (PAP3), beta-galactosidase 1 (BGAL1), and two-component response regulator (ARR5). The RT-PCR results confirmed that the transcription level changes of these 9 genes were comparable with the fold-changes observed in our transcriptome analysis.