Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Comparative transcriptome analyses in contrasting onion (Allium cepa L.) genotypes for drought stress

Comparative transcriptome analyses in contrasting onion (Allium cepa L.) genotypes for drought stress

  • Pranjali Ghodke, 
  • Kiran Khandagale, 
  • A. Thangasamy, 
  • Abhijeet Kulkarni, 
  • Nitin Narwade, 
  • Dhananjay Shirsat, 
  • Pragati Randive, 
  • Praveen Roylawar, 
  • Isha Singh, 
  • Suresh J. Gawande
PLOS
x

Abstract

Onion (Allium cepa L.) is an important vegetable crop widely grown for diverse culinary and nutraceutical properties. Being a shallow-rooted plant, it is prone to drought. In the present study, transcriptome sequencing of drought-tolerant (1656) and drought-sensitive (1627) onion genotypes was performed to elucidate the molecular basis of differential response to drought stress. A total of 123206 and 139252 transcripts (average transcript length: 690 bases) were generated after assembly for 1656 and 1627, respectively. Differential gene expression analyses revealed upregulation and downregulation of 1189 and 1180 genes, respectively, in 1656, whereas in 1627, upregulation and downregulation of 872 and 1292 genes, respectively, was observed. Genes encoding transcription factors, cytochrome P450, membrane transporters, and flavonoids, and those related to carbohydrate metabolism were found to exhibit a differential expression behavior in the tolerant and susceptible genotypes. The information generated can facilitate a better understanding of molecular mechanisms underlying drought response in onion.

Introduction

Bulb onion (Allium cepa L.) is an economically important vegetable crop cultivated worldwide in a diverse range of climatic conditions varying from temperate to semi-arid. India is one of the largest producers and exporters of onion globally. During 2017–2018, India produced 232 lakh tonnes of onion, of which 15.8 lakh tonnes was exported (http://agricoop.gov.in/). Asia contributes 67.5% of total world production, followed by Africa (12.9%), America (10.1%), and Europe (9.3%) (http://www.fao.org/faostat/en/#data/QC/visualize). However, drought stress causes approximately 30% yield losses in onion [1]. Stress due to biotic and abiotic factors is among the major constraints in exploiting the yield potential of the onion crop. In addition to biotic stress, onions are highly vulnerable to abiotic stresses such as extreme temperature injuries, drought, and waterlogging [2, 3]. In India, the majority of onions are produced during the post-monsoon season. Being a shallow-rooted crop, in the post-monsoon season, onion is highly susceptible to mid-season drought due to low moisture resulting from inadequate rainfall and the shallowness of soil, which is insufficient to cater to the crop’s water demand [4, 5]. Furthermore, the majority of available high-yielding modern onion varieties are developed for their best performance under optimum irrigation conditions. Therefore, genetic improvement of the existing genetic stock for drought tolerance is key to overcome the problem of drought-related yield losses in onion.

Drought tolerance is a complex phenomenon governed by numerous genes. Drought induces a vast array of plant responses that include a change in the gene expression pattern, accumulation of metabolites such as abscisic acid (ABA) or osmotically active compounds, and synthesis of specific proteins, namely largely hydrophilic proteins, oxygen radical scavenging proteins, and chaperones. Moreover, transcriptome analyses using microarray technology, along with conventional approaches, have identified many drought stress-responsive transcription factors (TFs) in plants [6, 7].

In recent years, plant transcriptome analysis using next-generation sequencing (NGS) technology has proven to be a robust and cost-effective tool for high-throughput sequence determination. NGS-based transcriptome data analysis facilitates differential gene expression (DGE) analysis at a global level, even when the plant genome sequence is unknown. This technology thus has been widely used in different economically important crops to identify DGE under various stresses; these genes are associated with different metabolic pathways and phenotypic traits [810].

In Allium, key metabolites and genes were identified through targeted metabolome and transcriptome profiling of dihaploid A. cepa and dihaploid A. cepa var. aggregatum under normal conditions and various stresses [11]. Han et al. [12] identified the genes that are differentially expressed during cold acclimation of onion genotypes and revealed the freezing tolerance mechanisms in onion crop. Zheng et al. [13] identified 39 CepNAC TFs (the NAC family of genes, particularly NAC-IV and NAC-V groups) that are likely to be involved in stress response in onion.

However, no study to date has determined the changes at the transcriptome level, DGE profiling, and alteration in the biochemical pathways of onion under drought stress. Therefore, in the present study, the transcriptome of two contrasting onion genotypes (accession nos. 1656 and 1627) subjected to drought stress was sequenced using Illumina paired-end sequencing technology. Due to the lack of the onion genome sequence and annotation information, the generated sequence data were de novo assembled to yield the transcriptome, which was then annotated using publicly available resources. To the best of our knowledge, this is the first report on the drought stress transcriptome of bulb onion. The study findings suggest the differential molecular behavior of the selected varieties toward drought stress. The study also provides a basis for elucidating the further understanding of transcriptional changes underlying the drought stress response in the onion crop.

Materials and methods

Plant material and drought treatment

The experiment was conducted at the ICAR-Directorate of Onion and Garlic Research (ICAR-DOGR), Pune, Maharashtra, India (N 18°84′, E 73°88′, H 553.8 m) under an automated rainout shelter. The identified drought-tolerant (Acc. 1656) and drought-senstitive (Acc. 1627) onion genotypes were selected from the germplasm collection of ICAR-DOGR. Initially, seedlings were raised in the nursery on raised beds. Then, 6-week-old seedlings from the nursery were transplanted in a plastic pot (height: 25 cm and diameter: 25 cm) of 12-kg capacity filled with field soil. The seedlings were raised under ambient growth conditions and irrigated at 100% field capacity until they reached the 5–6 leaf stage. Each treatment comprised 10 replicates (i.e., 10 pots/treatment). For drought stress treatment, 60% field capacity was maintained by withholding irrigation for 25–50 days after transplanting the seedling (drought stress-sensitive phase), and thereafter, normal 100% field capacity was retained. For the control treatment, 100% field capacity was maintained throughout the experiment. The recommended package of practices for onion was followed to raise a good crop. The leaf samples were harvested from each genotype for both the control and drought treatments. These samples were immediately frozen in liquid nitrogen and stored at −80 °C until use. The soil moisture level was monitored using the gravimetric method after every 24-h interval during the treatment. Additionally, to confirm the impact of drought stress, the plant water status was recorded by measuring relative water content (RWC) [14].

Growth and physiological analyses

The growth of the plant was monitored by measuring plant height, number of leaves per plant, leaf area, and leaf length and width under the control and drought stress treatments at an interval of 6 days throughout the experiment. Total chlorophyll content of the onion leaves was estimated using the method described by Hiscox and Israelsta [15]. Total chlorophyll content was determined using the equation proposed by Arnon [16]. The membrane stability index (MSI %) was periodically measured (at 6-day interval) throughout the experiment by following the procedure described by Sairam et al. [17]. Total antioxidant capacity was estimated through ferric reducing antioxidant power (FRAP) assays, according to Benzie and Strain [18]. Total antioxidant capacity (TAC) was expressed as microgram ascorbic acid equivalents per milligram of fresh weight (FW). Total phenols were determined colorimetrically using the Folin-Ciocalteu reagent as described by Pinelo et al. [19]. The phenol content reported as gallic acid equivalent per gram FW of the sample. Proline accumulation was estimated according to the method given by Bates et al. [20]. The proline content was estimated from the standard curve using L-proline and expressed as μmol/g of FW.

RNA isolation

Total RNA was isolated from the leaves of plants under the control and drought treatments in triplicate by using the modified CTAB and lithium chloride method [21]. The quantity and quality of RNA was determined using the NanoDrop® ND-1000 spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis. The RNA samples were then treated with DNase I to avoid possible DNA contamination. Before cDNA synthesis, the integrity of RNA was determined using Bioanalyzer 2100 (Agilent technologies, Singapore). High-quality RNA having RIN values higher than 7 were pooled in equal quantities from three replicates of the control and drought-treated samples and were used for library construction and RNA-Seq analyses.

Library preparation and RNA-Seq

The RNA samples that passed the quality check were used to prepare RNA-Seq paired-end sequencing libraries by using the Illumina TruSeq Stranded mRNA Sample Prep kit as per the manufacturer’s protocol. In brief, the mRNA was enriched using poly-T beads and then fragmented enzymatically. First-strand cDNA synthesis was then performed using SuperScript II and ActD mix. The single-stranded cDNA was converted to double-stranded cDNA by using the second strand mix. The cDNA was then purified using AMpure XP beads, and poly(A)-tailing, adaptor ligation, and enrichment were performed through PCR. The PCR-enriched libraries were analyzed in the Agilent 4200 TapeStation system (Agilent Technologies, USA) using a high-sensitivity D1000 Screen Tape as per the manufacturer’s guidelines. The mean sizes of fragments in various libraries were 543, 524, 485, and 544 bp for 1656C, 1656D, 1627C, and 1627D, respectively. The libraries were then sequenced in the paired-end mode on NextSeq500 using 2 × 75 bp platform chemistry.

De novo transcriptome assembly and annotation

Quality of the captured high-throughput sequencing data was assessed using FastQC Toolkit v0.11.7 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The low-quality reads and adapters were removed from the raw reads using NGSQC Toolkit v2.3.3 (http://www.nipgr.ac.in/ngsqctoolkit.html) wherever necessary. The obtained high-quality raw reads (≤Q20) were subjected to the Trinity assembler v2.2.0 (https://github.com/trinityrnaseq/trinityrnaseq/wiki) to build the merged transcriptome (control and treated) for the 1656 and 1627 genotypes in independent attempts. To obtain representative transcripts, we clustered the merged transcriptome using CD-HIT v4.7 (http://weizhongli-lab.org/cd-hit/download.php). The whole transcriptome quantitation was performed using Kallisto v0.44.0 (https://pachterlab.github.io/kallisto/download). DESeq, an R package, was used for DGE profiling. To assess DGE between two conditions, we used 2 as a log2fold change cut-off by selecting significant (p value ≤ 0.05) transcripts.

The final version of the transcriptome (CD-HIT clustered) was annotated using DIAMOND BLASTX v0.8.22 (https://ab.inf.uni-tuebingen.de/software/diamond) utility against NCBI’s non-redundant protein database (NRDB) (ftp://ftp.ncbi.nlm.nih.gov/blast/db/), UniProt/SwissProt database (https://www.uniprot.org/), plantTFDB (http://planttfdb.cbi.pku.edu.cn/) with an e-value of ≤10−5. The gene ontology (GO) and pathway annotation fetched using the online UniProt/SwissProt ID mapping functionality. The Transeq utility is available under the EMBOSS v6.6.0 (http://emboss.sourceforge.net/download/) package used to convert the transcripts into the longest possible open reading frame. Such converted protein sequences were then scanned for the Clusters of Orthologous Group (COG) categories using a standalone version of emapper v1.0.3 against eggNOG v4.5.1 (http://eggnogdb.embl.de/#/app/downloads). The presence of various functional and/or conserved domains, protein families, and other important sequence signatures was determined by scanning the whole transcriptome against different databases such as Pfam, TIGRFAM, and SUPERFAMILY implemented under a standalone version of InterProScan v5.33–72.0 (https://www.ebi.ac.uk/interpro/download.html).

To study drought-responsive genes, we mapped the HQ reads on droughtDB (protein sequences) using the DIAMOND BLASTX utility with an e-value of ≤10−5 in an independent attempt for both samples of both genotypes. To capture the raw read count and coverage per gene, we used a pileup.sh program implemented under the BBMap suite (https://sourceforge.net/projects/bbmap/).

Validation of DGE under drought stress using qRT-PCR

Transcripts that showed the differential expression behavior and encoded drought stress-responsive proteins were selected and validated using qRT-PCR. Transcripts such as methylmalonyl-CoA epimerase, Ninja-family protein AFP3-like, vacuolar amino acid transporter, beta-galactosidase, WALLS ARE THIN1 (WAT1)-related protein, malate synthase, 21-kDa protein, NAC transcription factor 29-like, ABC transporter G (ABCG), protein STAY-GREEN, chaperone, L-ascorbate oxidase, superoxide dismutase, WRKY transcription factor 70, and aquaporin 1 were selected (S5 File). RNA was isolated from the same samples that were used for RNA-Seq analyses and treated with DNase I (Thermo Scientific, Lithuania) to remove possible DNA contamination. cDNA was synthesized using 1 μg of RNA from each sample using a cDNA synthesis kit (Thermo Scientific, Lithuania). Three biological and three technical replicates of each sample were used. Primers for the selected genes were designed using the Primer-BLAST program at NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The actin gene was used as an internal control of the experiment. Expression analyses of selected genes was performed in LightCylcer 480 II (Roche, Germany) using the LightCycler 480 SYBR Green I Master Mix kit (Roche, Germany). The relative expression and fold changes were calculated using the 2−ΔΔCt method [22].

Results

Physiological and biochemical analyses

Physiological and biochemical parameters such as chlorophyll content, MSI, RWC, and antioxidant, phenol, and proline content were found to be higher in the tolerant genotype (1656) than in the susceptible genotype under drought stress (Fig 1; S1 File). The two genotypes significantly varied in plant height under drought stress. The tolerant genotype significantly maintained higher number of leaves and leaf area during stress treatment than the susceptible genotype (Fig 2), reflecting its ability to maintain higher photosynthesis activity under stressful conditions. RWC and MSI were directly proportional to drought tolerance and differed significantly among the studied genotypes as the stress increased. After 25 days of stress treatment, the tolerant genotype (1656) maintained higher plant RWC (>60%) and less membrane damage as reflected by their higher membrane stability (75%). Conversely, the sensitive genotype (1627) showed more membrane damage and lower tissue water content in response to water stress. The leaf chlorophyll content also followed the same pattern, that is, it differed among the genotypes subjected to drought stress. The tolerant genotype retained significantly higher chlorophyll content as stress severity increased, whereas the leaf senescence rate became more pronounced in the susceptible genotype in response to water stress. Total phenol content, which is directly linked to onion pungency, was found to be elevated in response to drought stress. The tolerant genotype had 10% more phenol content than the susceptible genotype under water stress. Proline is a crucial drought stress indicator that plays a major role in cell osmoregulation. After 25 days of water stress treatment, proline content increased in the contrasting genotypes [204% in the tolerant genotype (1656) and 137% in the susceptible genotype (1627)]. This higher increase in the proline level reflects the drought adaptive mechanism present in the tolerant genotype (1656). Similarly, antioxidant enzyme activity, which is involved in scavenging reactive oxygen species (ROS) during oxidative or water stress, was found to be significantly higher in the tolerant genotype (1656) and lower in the sensitive genotype (1627). Thus, the tolerant genotype exhibited a drought adaptive mechanism that enabled it to survive in the water scarce environment.

thumbnail
Fig 1. Differential physiological and biochemical response in drought sensitive (1627) and tolerant (1656) onion genotypes.

A. Total antioxidant, B. Proline content, C. Relative water content and D. Total chlorophyll content.

https://doi.org/10.1371/journal.pone.0237457.g001

thumbnail
Fig 2. Diffrential morphological response in drought sensitive (1627) and tolerant (1656) onion genotypes.

A. Plant height, B. Leaf area, C. Number of leaves, D. Leaf length.

https://doi.org/10.1371/journal.pone.0237457.g002

RNA-Seq and de novo transcriptome assembly

A total of 150.92 million raw reads generated from the control and drought-treated samples of drought-sensitive (1627) and drought-tolerant (1656) genotypes. In both the samples, we got sufficient HQ reads required for the transcriptome expression analysis, that is, on an average >30 million reads. The de novo transcriptome assembly yielded a total of 144668 and 164956 transcripts for samples 1656 and 1627, respectively. Then, these primary transcripts were clustered at 80% identity and 80% coverage cut-off to obtain the representative and non-redundant transcript set for both the samples in an independent attempt. A total of 123206 and 139252 non-redundant transcripts for 1656 and 1627, respectively, were clustered and resulted in the final transcriptome. The average transcript length was 690 bp with N50 statistics 1110 bp, and the maximum transcript length was 15436 bases while the GC content of the transcripts was 37.7% (1656) and 37.5% (1627) (Table 1). This set of transcripts was used for further downstream analysis. The raw sequencing data have been submitted to NCBI (BioProject: PRJNA595061).

thumbnail
Table 1. Primary and final assembly statistics of 1656 and 1627 in control and drought stress treated onion libraries.

https://doi.org/10.1371/journal.pone.0237457.t001

Differential gene expression

We performed de novo DGE analyses using aligned reads of the drought-tolerant (1656C vs 1656D) and drought-sensitive (1627C vs 1627D) onion cultivars. DGE analyses of 1656C versus 1656D resulted in the upregulation of 1189 genes and downregulation of 1180 genes, whereas in the case of 1627C versus 1627D, 872 genes were upregulated and 1292 genes were downregulated (Fig 3, S2 and S3 Files).

thumbnail
Fig 3. Differential expression pattern showed by drought sensitive (1627) and tolerant (1656) onion genotypes under drought stress.

https://doi.org/10.1371/journal.pone.0237457.g003

Functional annotation of transcripts expressed in onion under drought stress

In total, 26428 (i.e., 21.45%) and 28109 (i.e., 20.18%) transcripts were successfully annotated with NCBI’s non-redundant protein database (NRDB) from samples 1656D and 1627D, respectively, at an e-value of ≤10−5 and a query coverage of ≥50%. The reference database identifiers from the annotated transcripts were used for obtaining the GO and KEGG pathways along with other relevant information.

The transcripts were grouped into three categories based on the GO annotation: cellular components (CC), biological processes (BP), and molecular functions (MF). For sample 1627D, 255 unique CC categories were reported for 2252 transcripts (5.57%), whereas 3034 transcripts (7.51%) were annotated with 715 unique BP terms and 4864 transcripts (12.04%) showed hits against 757 unique MF terms. On the other hand, for sample 1656D, 255 unique CC categories were reported for 3442 transcripts (9.19%), 707 unique BP terms were reported for 4012 transcripts (10.77%), and 751 non-redundant MF terms were assigned to 6582 transcripts (17.68%). In both the samples, the MF terms were abundant, followed by the BP and CC categories. In the CC category, integral components of the membrane (GO:0016021) and nucleus (GO:0005634) were overrepresented, followed by the cytoplasm (GO:0005737), ribosome (GO:0005840), plasma membrane (GO:0005886), chloroplast (GO:0009507), and mitochondrion (GO:0005739). Similarly, the BP category indicates the high abundance of vital cellular processes such as photosynthesis, carbohydrate metabolism, cell wall organization, and transcription–translation. Among them, DNA integration (GO:0015074) and transcription (GO:0006451) were adequately represented. On the other hand, the MF category showed a high influence of MF such as nucleic acid binding, ATP binding, and protein kinase activity. In the MF category, nucleic acid binding (GO:0003676) and ATP binding (GO:0005524) were abundantly represented. (Fig 4A and 4B). In the KEGG pathway analysis, the highest percentage of the transcripts was reported to be involved in glycan metabolism (6%–7%), lipid metabolism (5%–6%), pectin degradation (4%–5%), and carbohydrate degradation (3%–5%) (Fig 4C and 4D). Of the total annotated transcripts, most transcripts matched with the Asparagus officinalis proteome (46%– 48%), followed by the proteomes of Elaeis guineensis (6.5%) and Phoenix dactylifera (5.3%) (Fig 4E and 4F).

thumbnail
Fig 4. Functional annotation of transcripts expressed in drought sensitive (1627) and tolerant (1656) onion genotypes under drought stress.

A, B: Gene ontology, C,D: KEGG pathway analyses, E,F: Species hit distribution.

https://doi.org/10.1371/journal.pone.0237457.g004

The COG category distribution revealed that the major transcripts may contribute to signal transduction mechanisms (~7%), post-translational modification/protein turnover/chaperones (~6.8%), carbohydrate transport (4.92%), and metabolism/energy production and conversion (4.43%) COG categories in both the samples (S1 Fig, S4 File). The InterProScan analysis acknowledged the presence of various crucial conserved and functional signatures in our transcriptome such as membrane topology, signal peptides, and various functional domains (S1 Table). The homology-based sequence search against plantTFDB revealed the strong association of TFs extensively involved in plant growth promotion in both the samples such as MYB, bHLH, and ERF. The detailed distribution of TFs is presented in Fig 5. The aforementioned TFs were found to be upregulated in onion under drought stress.

thumbnail
Fig 5. Transcription factor distribution in assembled transcriptome of A. drought sensitive (1627) and B. tolerant (1656) onion genotypes under drought stress.

https://doi.org/10.1371/journal.pone.0237457.g005

Drought-related gene expression analysis

The average gene sequence coverage (≥50%) was considered to prune down the significant hits in the homology-based sequence search performed against droughtDB. The overall expression pattern of the significant genes from droughtDB was visualized in the form of a divergent plot by using fold change calculated by comparing the drought treatment with respective controls of the variety (Fig 6). TFs, membrane transporters, ABC transporters, cytochrome P450, antioxidants, and heat shock proteins were upregulated in onion cultivars under drought stress.

thumbnail
Fig 6. Differential expression pattern (fold change) of the genes from droughtDB in A. drought sensitive (1627) and B. drought tolerant (1656) onion genotypes under drought stress.

https://doi.org/10.1371/journal.pone.0237457.g006

Among TFs, NAC, MYB, and WRKY families were highly upregulated in the tolerant cultivar. From 1656D, 10 members of NAC were differentially expressed and 9 were upregulated (up to 5.3-fold). While in the case of 1627D, 7 members of NAC were found to be differentially expressed and only 1 was upregulated. NAC29 was upregulated 4.8-fold in the tolerant cultivar (1656D). Similarly, in 1656D, of the 10 MYB family members, 8 were upregulated (up to 4-fold), whereas in 1627D, all 3 MYBs were downregulated in response to drought. WRKY TFs were also upregulated up to 5.5-fold in 1656D, whereas all WRKYs were downregulated in 1627D under drought.

Cytochrome P450 (CYP) genes were also found to be differentially expressed in response to drought stress in onion. CYP81, CYP71A, and CYP85A (7-, 6.1-, and 3.2-fold, respectively) showed high upregulation in the tolerant cultivar (1656) than in the susceptible cultivar (1627) (2.6-, −4-, and −2.5-fold, respectively). None of the P450 members were downregulated under drought stress in the tolerant cultivar, but several CYPs were downregulated in the susceptible cultivar.

Aquaporins occur in multiple isoforms in both plasmalemma and tonoplast membranes of plants. They regulate water transport in plants. In the present study, several aquaporins were differentially expressed in onion under drought stress such as aquaporin NIP1-1-like (5-fold) and aquaporin TIP3-2 (3.9-fold). Amino acid transporters such as vacuolar amino acid transporter (6.1-fold) and cationic amino acid transporter (3.6-fold) were upregulated in onion under drought stress. Similarly, the GABA transporter showed 4.8-fold upregulation in response to drought stress in onion.

ABA biosynthesis from carotenoids is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). A 3-fold increase was observed in the transcript level of NCED in the tolerant genotype (1656). Genes encoding ABA transporters such as the ABC subfamily G and NRT1/PTR family genes were also upregulated (3.9- and 6.2-fold, respectively) in the tolerant genotype. SNF1-related protein kinases were also upregulated (4.1-fold) in the tolerant genotype under drought stress.

Onion RNA-Seq data in the present study showed upregulation of flavonoid biosynthesis genes such as those encoding UDP-glycosyltransferase (4.9-fold), anthocyanidin 5,3-O-glucosyltransferase (3.2-fold), flavonoid glucosyltransferase (3.5-fold), and flavonol synthase (2.4-fold) in the drought-tolerant genotype (1656).

Moreover, genes for detoxification and ROS-scavenging enzymes (peroxidase, superoxide dismutase, and ascorbate oxidase) were found to be differentially expressed in onion under drought stress. Several genes related to carbohydrate metabolism, such as α-galactosidase (3.7-fold), β-galactosidase (7.3-fold), galactinol synthase (2.5-fold), galactinol—sucrose galactosyltransferase (4.9-fold), sucrose synthase (2.3-fold), and UDP-glucose 6-dehydrogenase (3.8-fold), were upregulated in the tolerant genotype (1656) under drought stress. Upregulation of these sugar metabolism genes in 1656 indicated their role in drought tolerance.

Overall, 21-kDA protein (9-fold), cytochrome P450 CYP81 (7-fold), RING-H2 finger protein (6.3-fold), momilactone A synthase-like (8.3-fold), peroxygenase 4 (6.7-fold), BAT-1 (DEAD Box BAT-1-like RNA helicase 15 isoform) (7.03-fold), and NAC29 (4.8-fold) are promising candidate genes that were upregulated manifold in the tolerant lines than in the sensitive counterpart in response to drought stress.

Validation of DGE under drought stress using qRT-PCR

We validated the results of transcriptome analyses by using qRT-PCR of 15 randomly selected drought-related genes. The fold changes varied in RNA-Seq and qPCR analyses. However, the overall qPCR expression profile of most of the genes was in agreement with the RNA-Seq profile, which indicated the reliability of RNA-Seq data (Fig 7).

thumbnail
Fig 7. Validation of few selected genes using qRT-PCR in A. drought sensitive (1627) and B. tolerant (1656) onion genotypes under drought stress.

https://doi.org/10.1371/journal.pone.0237457.g007

Discussion

Two contrasting onion genotypes under drought stress were employed: drought-tolerant genotype (1656) and drought-susceptible genotype (1627). Their ability to tolerate drought stress for consecutive 25 days was evaluated on the basis of their morphological and biochemical performance. Being a shallow-rooted crop, onion requires frequent irrigation to maintain the desired yield and bulb quality [23]. According to a previous report, higher accumulation of compatible solutes such as proline and soluble sugars and an increase in antioxidant enzyme activity play a substantial role in osmoregulation, thus improving cellular turgidity and membrane stability in the tolerant wheat cultivar under water stress [24]. Similar results were recorded in our earlier study with the well-known short-day onion cultivar Bhima Kiran, where the decline in overall physiological and biochemical parameters was recorded in response to drought stress, which affected the entire plant growth and yield [2]. Wakchaure et al. [25] reported that limited irrigation or water deficit stress in onion severely affects the crop growth rate as indicated by a reduction in plant height, leaf area index, and chlorophyll content and other important physiological parameters contributing to the overall bulb quality and yield.

RNA-Seq and functional annotation

RNA-Seq is rapid, inexpensive, and independent of genome complexity, and thus, NGS has emerged as a method of choice for expression analyses, discovery of new genes, and development of molecular markers in crops where genome sequence information is not available. In the present study, we used the Illumina Next 500 platform and generated 150.92 million raw reads from the control and drought-treated samples of drought-sensitive (1627) and drought-tolerant (1656) genotypes. Similar transcriptome statistics were also reported in other RNA-Seq analyses in onion; Zhang et al. [13] generated 72.53 million 100-bp paired-end reads, while Shemesh-Mayer et al. [26] sequenced six libraries from 100-bp one-end reads. These RNA-Seq data suggested that de novo assembly was effective and captured the majority portion of the onion transcriptome.

For the functional annotation, we searched the final transcriptome against NCBI’s NRDB using DIAMOND BLASTX utility. Very few curated protein sequences are available for A. cepa in the database; therefore, we got the annotation across various organisms. Of all other organisms, we found A. officinalis as the major contributor. Recently, Mehra et al. [27] performed a transcriptome analysis of snow mountain garlic and reported the highest homology with A. officinalis. Han et al. [12] conducted a similar study investigating the effect of cold stress on onion by using the transcriptome of A. fistulosum. Sun et al. [28] reported the highest match with Vitis vinifera, while Zhang et al. [13] reported the highest match with E. guineensis, followed by P. dactylifera. A. officinalis is closely related to the alliums, and the recent availability of its genome sequence resulted in the highest similarity with onion transcriptome data. Several TF families (MYB, WRKY, NAC, and bHLH) were identified by searching plantTFDB. These TFs are known to play a role in molecular regulation in response to biotic as well as abiotic stresses in several plants [29], including onion under the cold [12] and heat stress [30]. InterProScan analysis was performed for functional analysis of transcripts by classifying them into families and predicting domains and important sites. Transmembrane domains, conserved domains, and protein superfamilies were predicted from the present transcriptome data by using InterProScan. Similar analyses were performed for domain and motif information in garlic [27] and giant reed [31]. Almost 30% of transcripts in the present study were unknown or not annotated, and they might be unique to onion.

Drought-related gene expression analysis

NAC29 is known for its role in abiotic stress tolerance, and transgenic Arabidopsis expressing NAC29 from wheat showed increased tolerance to salinity and dehydration stress by delaying senescence [32]. When overexpressed in Arabidopsis, MYB44 helps in ABA-mediated stomatal closure upon salt and drought stress [33]. MYB108 was reported to be highly upregulated under drought stress in poplar [34]. MYB108 and MYB39 were also upregulated in response to heavy metal stress [35]. WRKY41 increased tolerance to salinity and drought stress when expressed in transgenic tobacco [36]. Similarly, along with other WRKYs, WRKY75 is known to modulate abiotic stress response [37] and is a modulator for phosphate uptake and root development in Arabidopsis [38]. Such a significant difference in the expression pattern of NAC, MYB, and WRKY suggests their crucial role in molecular reconfiguration at the RNA level, which ultimately imparted drought stress tolerance to 1656.

CYPs are a large and diverse family of genes in plants; several P450 genes are reported to increase abiotic stress tolerance [39]. CYP85A is involved in brassinosteroid biosynthesis, and overexpression of spinach CYP85A in tobacco led to an increase in drought tolerance as well as root development [40]. The upregulation of several CYPs was also reported in the transcriptome data of perennial ryegrass in response to heat stress [41]. Thus, the upregulation of P450 genes in 1656 might contribute to its drought tolerance.

NIPs have an essential role in maintaining water balance during drought and salinity stress [42]. RNA-Seq analyses of potato revealed the upregulation of aquaporin TIP3-2 under drought stress [43]. Thus, aquaporins can be considered potential drought tolerance-inducing proteins in onion and other Allium crops. Drought tolerance is partially associated with amino acid accumulation [44]. These amino acids serve as osmolytes, and ROS-scavenging and signaling molecules in plant stress response [45]. GABA is a well-known molecule involved in enhancement of abiotic stress tolerance [46, 47]. Moreover, few ABC transporters were upregulated under drought stress in onion. They are involved in stomatal closure during water stress.

ABA is known as a stress hormone because of its central role in response to various stresses. To cope up with stress with the help of ABA signaling, transcriptional upregulation of NCED occurs under drought stress [48]. ABA is generally transported by passive diffusion to guard cells. However, ABA can be transported by a few transporters such as members of the ABC subfamily G and NRT1/PTR family [49, 50]. They might facilitate stomatal closure in 1656 and assist in minimizing water loss. SNF1-related protein kinases are the subfamily of serine/threonine kinases that play a crucial role in ABA and sugar signaling [51]. SNF1-related protein kinases that are upregulated in onion under drought stress might phosphorylate the various TFs by regulating the ABA-dependent signaling cascade to enhance drought tolerance.

Flavonoids have antioxidant properties and are known to have a role in conferring abiotic stress tolerance to plants [52, 53]. Integrated RNA-Seq and metabolomics studies have revealed the upregulation of flavonoids and flavonoid biosynthesis genes in shallot doubled haploid [11].

Carbohydrate metabolism also plays a vital role in abiotic stress tolerance [54, 55]. Several sugar metabolism-associated genes were upregulated in onion under drought stress. These genes were reported to be upregulated in shallot doubled haploid and might have imparted stress tolerance to shallot than to onion [11]. Carbohydrate metabolism-associated genes were also upregulated in onion under cold stress [12] and in Camellia sinensis under drought stress [9].

Few important genes in abiotic stress response such as 21-kDa protein, cytochrome P450 CYP81, RING-H2 finger protein, momilactone A synthase-like, peroxygenase 4, and BAT-1 (DEAD Box BAT-1-like RNA helicase 15 isoform) were upregulated several fold in 1656 under drought stress. DEAD-box RNA helicases are proteins of a category that play a crucial role in maintaining cell genome integrity during stress conditions [56]. OsBAT1 was upregulated under abiotic stress in rice and showed unique characteristics such as unwinding of both DNA and RNA duplexes; bipolar translocation and its transcript upregulation under abiotic stresses, indicated that it is a multifunctional protein [57]. ROS signaling plays a critical role in plant responses to abiotic stress such as drought and salinity. CYPs are associated with protection of plants from harsh environmental conditions by increasing the activity of compounds such as flavonoids that have an increased antioxidant activity [58]. However, the cytochrome P450 gene cluster member TaCYP81D5 conferred salinity tolerance in wheat by ROS scavenging [59]. Peroxygenases are invloved in oxylipin metabolism and are important in plant stress response. In response to drought stress, peroxygenase 4 was upregulated (10.1-fold) in creeping bent grass (Agrostis stolonifera) [60]. Furthermore, RING-H2 finger proteins are a special type of zinc finger proteins known to increase stress tolerance by modulating the hormonal profile of tomato [61] and Arabidopsis [62] to cope up with adverse environmental conditions. Momilactones are allopathic phytoalexins involved in disease and weed resistance in rice. Xuan et al. [63] reported that momilactone A was more efficient in conferring salinity and drought stress tolerance than resistance to weed. Momilactone need to be characterized in onion for a better understanding of their role in conferring stress tolerance.

Validation of DGE under drought stress using qRT-PCR

The expression trends of genes from qRT-PCR corresponded with those of transcriptome analyses, thus validating the RNA-Seq data. NAC29 from wheat enhanced drought and salt tolerance in Arabidopsis by delaying senescence and boosting primary root elongation [32]. STAY-GREEN is a well-known protein involved in drought tolerance in a number of crops; it acts by slowing down chlorophyll degradation [64]. In the present experiment, chlorophyll content in the drought-tolerant genotype (1656) was more under drought stress than in its susceptible counterpart (1627). This might be linked to the upregulation of the aforementioned genes. Ascorbate oxidases are involved in plant stress tolerance through ROS scavenging [65], which is upregulated in the drought-tolerant genotype (1656). Malate synthase, a marker for the glyoxylate cycle, was upregulated >15-fold under drought stress in the drought-tolerant genotype (1656). Malate synthase is also found to be upregulated by ABA treatment [66]. The 21-kDa protein was one of the prominent proteins to be upregulated in salinity stress in finger millet [67]. However, in the current study, the 21-kDa protein gene was upregulated 9.33-fold in the drought-tolerant genotype under drought stress. WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis during drought stress [68]. WAT1 was upregulated in the drought-tolerant line of maize [69]. The TF ABA-Insensitive 5 (ABI5) is a key regulator of ABA signaling and stress response. ABI5-binding proteins are induced by ABA and/or dehydration stresses in Arabidopsis [70, 71]. ABA biosynthesis is highly induced by dehydration in the vascular parenchyma cells of roots and shoots. However, the plant ABCG was shown to transport terpenoids, and because ABA is a tetraterpene-derived sesquiterpene, ABCG proteins are strong candidates for ABA transporters [72].

Conclusion

Onion is a shallow-rooted plant, and thus is likely susceptible to drought stress. Here, we performed transcriptome sequencing of drought-tolerant and susceptible onion genotypes. More than 1100 differentially expressed genes were identified from these genotypes under drought stress. These genes were functionally annotated using various standard bioinformatics programs. Several drought-responsive genes were upregulated in the tolerant genotype (1656) such as those encoding TFs, cytochrome 450, and membrane transporters, and those associated with carbohydrate metabolism and flavonoid biosynthesis. These genes might confer drought tolerance in this onion genotype at the molecular level. Physiological and biochemical parameters also indicated the better performance of the 1656 genotype over the 1627 genotype under drought stress conditions. To our best knowledge, the present study is the first to report the transcriptomic analysis of drought response in onion. The study findings will help researchers have an improved understanding of the molecular basis of drought response in onion.

Supporting information

S1 Fig. COG analyses of A: 1627; B: 1656.

https://doi.org/10.1371/journal.pone.0237457.s002

(TIF)

S1 File. Data of physiological and biochemical analyses.

https://doi.org/10.1371/journal.pone.0237457.s003

(XLSX)

S2 File. Differential gene expression of 1627.

https://doi.org/10.1371/journal.pone.0237457.s004

(XLSX)

S3 File. Differential gene expression of 1656.

https://doi.org/10.1371/journal.pone.0237457.s005

(XLSX)

References

  1. 1. Potopova V, Stepanek P, Farda A, Turkott L, Zahradnicek P, Soukup J. Drought stress impact on vegetable crop yields in the Elbe River Lowland between 1961 and 2014. Cuadernos De Investigacion Geografica. 2016 Jun 27;42(1):127–43.
  2. 2. Ghodke PH, Andhale PS, Gijare UM, Thangasamy A, Khade YP, Mahajan V, et al. Physiological and biochemical responses in onion crop to drought stress. Int J Curr Microbiol App Sci. 2018;7(1):2054–62.
  3. 3. Ghodke PH, Shirsat DV, Thangasamy A, Mahajan V, Salunkhe VN, Khade Y, et al. Effect of water logging stress at specific growth stages in onion crop. Int. J. Curr. Microbiol. Applied Sci. 2018;7:3438–48.
  4. 4. Pelter GQ, Mittelstadt R, Leib BG, Redulla CA. Effects of water stress at specific growth stages on onion bulb yield and quality. Agricultural water management. 2004 Aug 1;68(2):107–15.
  5. 5. El Balla MD, Hamid AA, Abdelmageed AH. Effects of time of water stress on flowering, seed yield and seed quality of common onion (Allium cepa L.) under the arid tropical conditions of Sudan. Agricultural Water Management. 2013 Apr 1;121:149–57.
  6. 6. Vinocur B, Altman A. Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Current opinion in biotechnology. 2005 Apr 1;16(2):123–32. pmid:15831376
  7. 7. Bartels D, Sunkar R. Drought and salt tolerance in plants. Critical reviews in plant sciences. 2005 Feb 23;24(1):23–58.
  8. 8. Rashmi D, Barvkar VT, Nadaf A, Mundhe S, Kadoo NY. Integrative omics analysis in Pandanus odorifer (Forssk.) Kuntze reveals the role of Asparagine synthetase in salinity tolerance. Scientific reports. 2019 Jan 30;9(1):932. pmid:30700750
  9. 9. Parmar R, Seth R, Singh P, Singh G, Kumar S, Sharma RK. Transcriptional profiling of contrasting genotypes revealed key candidates and nucleotide variations for drought dissection in Camellia sinensis (L.) O. Kuntze. Scientific reports. 2019 May 16;9(1):7487. pmid:31097754
  10. 10. Xie Z, Zhou Z, Li H, Yu J, Jiang J, Tang Z, et al. High throughput sequencing identifies chilling responsive genes in sweetpotato (Ipomoea batatas Lam.) during storage. Genomics. 2019 Sep 1;111(5):1006–17. pmid:29792923
  11. 11. Abdelrahman M, Sawada Y, Nakabayashi R, Sato S, Hirakawa H, El-Sayed M, et al. Integrating transcriptome and target metabolome variability in doubled haploids of Allium cepa for abiotic stress protection. Molecular breeding. 2015 Oct 1;35(10):195.
  12. 12. Han J, Thamilarasan SK, Natarajan S, Park JI, Chung MY, Nou IS. De novo assembly and transcriptome analysis of bulb onion (Allium cepa L.) during cold acclimation using contrasting genotypes. PloS one. 2016 Sep 14;11(9):e0161987. pmid:27627679
  13. 13. Zhang C, Li X, Zhan Z, Cao L, Zeng A, Chang G, et al. Transcriptome Sequencing and Metabolism Analysis Reveals the role of Cyanidin Metabolism in Dark-red Onion (Allium cepa L.) Bulbs. Scientific reports. 2018 Sep 20;8(1):14109. pmid:30237461
  14. 14. Barrs HD, Weatherley PE. A re-examination of the relative turgidity technique for estimating water deficits in leaves. Australian journal of biological sciences. 1962;15(3):413–28.
  15. 15. Hiscox JD, Israelstam GF. A method for the extraction of chlorophyll from leaf tissue without maceration. Canadian journal of botany. 1979 Jun 15;57(12):1332–4.
  16. 16. Arnon DI. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiology. 1949 Jan;24(1):1. pmid:16654194
  17. 17. Sairam RK, Shukla DS, Saxena DC. Stress induced injury and antioxidant enzymes in relation to drought tolerance in wheat genotypes. Biologia Plantarum. 1997 Nov 1;40(3):357–64.
  18. 18. Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Analytical biochemistry. 1996 Jul 15;239(1):70–6. pmid:8660627
  19. 19. Pinelo M, Rubilar M, Sineiro J, Nunez MJ. Extraction of antioxidant phenolics from almond hulls (Prunus amygdalus) and pine sawdust (Pinus pinaster). Food Chemistry. 2004 Apr 1;85(2):267–73.
  20. 20. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant and soil. 1973 Aug 1;39(1):205–7.
  21. 21. Rubio-Piña JA, Zapata-Pérez O. Isolation of total RNA from tissues rich in polyphenols and polysaccharides of mangrove plants. Electronic Journal of Biotechnology. 2011 Sep;14(5):11–11.
  22. 22. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods. 2001 Dec 1;25(4):402–8. pmid:11846609
  23. 23. Rao NS, Shivashankara KS, Laxman RH, editors. Abiotic stress physiology of horticultural crops. New Delhi: Springer; 2016 Apr 8.
  24. 24. Abid M, Ali S, Qi LK, Zahoor R, Tian Z, Jiang D, et al. Physiological and biochemical changes during drought and recovery periods at tillering and jointing stages in wheat (Triticum aestivum L.). Scientific reports. 2018 Mar 15;8(1):4615. pmid:29545536
  25. 25. Wakchaure GC, Minhas PS, Meena KK, Singh NP, Hegade PM, Sorty AM. Growth, bulb yield, water productivity and quality of onion (Allium cepa L.) as affected by deficit irrigation regimes and exogenous application of plant bio–regulators. Agricultural Water Management. 2018 Feb 1;199:1–0.
  26. 26. Shemesh-Mayer E, Ben-Michael T, Rotem N, Rabinowitch HD, Doron-Faigenboim A, Kosmala A, et al. Garlic (Allium sativum L.) fertility: transcriptome and proteome analyses provide insight into flower and pollen development. Frontiers in plant science. 2015 Apr 28;6:271. pmid:25972879
  27. 27. Mehra R, Jasrotia RS, Mahajan A, Sharma D, Iquebal MA, Kaul S, et al. Transcriptome analysis of Snow Mountain Garlic for unraveling the organosulfur metabolic pathway. Genomics. 2020 Jan 1;112(1):99–107. pmid:31356969
  28. 28. Sun XD, Yu XH, Zhou SM, Liu SQ. De novo assembly and characterization of the Welsh onion (Allium fistulosum L.) transcriptome using Illumina technology. Molecular genetics and genomics. 2016 Apr 1;291(2):647–59. pmid:26515796
  29. 29. Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, et al. Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Current opinion in plant biology. 2006 Aug 1;9(4):436–42. pmid:16759898
  30. 30. Galsurker O, Doron-Faigenboim A, Teper-Bamnolker P, Daus A, Lers A, Eshel D. Differential response to heat stress in outer and inner onion bulb scales. Journal of experimental botany. 2018 May 18;69(16):4047–64. pmid:29788446
  31. 31. Evangelistella C, Valentini A, Ludovisi R, Firrincieli A, Fabbrini F, Scalabrin S, et al. De novo assembly, functional annotation, and analysis of the giant reed (Arundo donax L.) leaf transcriptome provide tools for the development of a biofuel feedstock. Biotechnology for biofuels. 2017 Dec;10(1):138.
  32. 32. Huang Q, Wang Y, Li B, Chang J, Chen M, Li K, et al. TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis. BMC plant biology. 2015 Dec;15(1):268.
  33. 33. Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, et al. Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis. Plant physiology. 2008 Feb 1;146(2):623–35. pmid:18162593
  34. 34. Jia J, Zhou J, Shi W, Cao X, Luo J, Polle A, et al. Comparative transcriptomic analysis reveals the roles of overlapping heat-/drought-responsive genes in poplars exposed to high temperature and drought. Scientific reports. 2017 Feb 24;7:43215. pmid:28233854
  35. 35. Yuan J, Bai Y, Chao Y, Sun X, He C, Liang X, et al. Genome-wide analysis reveals four key transcription factors associated with cadmium stress in creeping bentgrass (Agrostis stolonifera L.). PeerJ. 2018 Jul 30;6:e5191. pmid:30083437
  36. 36. Chu X, Wang C, Chen X, Lu W, Li H, Wang X, et al. The cotton WRKY gene GhWRKY41 positively regulates salt and drought stress tolerance in transgenic Nicotiana benthamiana. PLoS One. 2015 Nov 12;10(11):e0143022. pmid:26562293
  37. 37. Van Aken O, Zhang B, Law S, Narsai R, Whelan J. AtWRKY40 and AtWRKY63 modulate the expression of stress-responsive nuclear genes encoding mitochondrial and chloroplast proteins. Plant physiology. 2013 May 1;162(1):254–71. pmid:23509177
  38. 38. Devaiah BN, Karthikeyan AS, Raghothama KG. WRKY75 transcription factor is a modulator of phosphate acquisition and root development in Arabidopsis. Plant physiology. 2007 Apr 1;143(4):1789–801. pmid:17322336
  39. 39. Magwanga RO, Lu P, Kirungu JN, Dong Q, Cai X, Zhou Z, et al. Knockdown of Cytochrome P450 Genes Gh_D07G1197 and Gh_A13G2057 on Chromosomes D07 and A13 Reveals Their Putative Role in Enhancing Drought and Salt Stress Tolerance in Gossypium hirsutum. Genes. 2019 Mar;10(3):226.
  40. 40. Duan F, Ding J, Lee D, Lu X, Feng Y, Song W. Overexpression of SoCYP85A1, a spinach cytochrome p450 gene in transgenic tobacco enhances root development and drought stress tolerance. Frontiers in plant science. 2017 Nov 9;8:1909. pmid:29209339
  41. 41. Tao X, Wang MX, Dai Y, Wang Y, Fan YF, Mao P, et al. Identification and Expression Profile of CYPome in Perennial Ryegrass and Tall Fescue in Response to Temperature Stress. Frontiers in plant science. 2017 Nov 20;8:1519. pmid:29209335
  42. 42. Kapilan R, Vaziri M, Zwiazek JJ. Regulation of aquaporins in plants under stress. Biological research. 2018 Dec;51(1):4.
  43. 43. Gong L, Zhang H, Gan X, Zhang L, Chen Y, Nie F, et al. Transcriptome profiling of the potato (Solanum tuberosum L.) plant under drought stress and water-stimulus conditions. PLoS One. 2015 May 26;10(5):e0128041. pmid:26010543
  44. 44. Ramanjulu S, Sudhakar C. Drought tolerance is partly related to amino acid accumulation and ammonia assimilation: a comparative study in two mulberry genotypes differing in drought sensitivity. Journal of Plant Physiology. 1997 Jan 1;150(3):345–50.
  45. 45. Pratelli R, Pilot G. Regulation of amino acid metabolic enzymes and transporters in plants. Journal of Experimental Botany. 2014 Aug 11;65(19):5535–56. pmid:25114014
  46. 46. Yong B, Xie H, Li Z, Li YP, Zhang Y, Nie G, et al. Exogenous application of GABA improves PEG-induced drought tolerance positively associated with GABA-shunt, polyamines, and proline metabolism in white clover. Frontiers in physiology. 2017 Dec 22;8:1107. pmid:29312009
  47. 47. Zhu X, Liao J, Xia X, Xiong F, Li Y, Shen J, et al. Physiological and iTRAQ-based proteomic analyses reveal the function of exogenous γ-aminobutyric acid (GABA) in improving tea plant (Camellia sinensis L.) tolerance at cold temperature. BMC plant biology. 2019 Dec;19(1):43. pmid:30700249
  48. 48. Osakabe Y, Yamaguchi-Shinozaki K, Shinozaki K, Tran LS. ABA control of plant macroelement membrane transport systems in response to water deficit and high salinity. New Phytologist. 2014 Apr;202(1):35–49. pmid:24283512
  49. 49. Kang J, Hwang JU, Lee M, Kim YY, Assmann SM, Martinoia E, et al. PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid. Proceedings of the National Academy of sciences. 2010 Feb 2;107(5):2355–60.
  50. 50. Kanno Y, Hanada A, Chiba Y, Ichikawa T, Nakazawa M, Matsui M, et al. Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor. Proceedings of the National Academy of Sciences. 2012 Jun 12;109(24):9653–8.
  51. 51. Jossier M, Bouly JP, Meimoun P, Arjmand A, Lessard P, Hawley S, et al. SnRK1 (SNF1-related kinase 1) has a central role in sugar and ABA signalling in Arabidopsis thaliana. The Plant Journal. 2009 Jul;59(2):316–28. pmid:19302419
  52. 52. Nakabayashi R, Yonekura-Sakakibara K, Urano K, Suzuki M, Yamada Y, Nishizawa T, et al. Enhancement of oxidative and drought tolerance in Arabidopsis by overaccumulation of antioxidant flavonoids. The Plant Journal. 2014 Feb;77(3):367–79. pmid:24274116
  53. 53. Ahmed NU, Park JI, Jung HJ, Hur Y, Nou IS. Anthocyanin biosynthesis for cold and freezing stress tolerance and desirable color in Brassica rapa. Functional & integrative genomics. 2015 Jul 1;15(4):383–94.
  54. 54. Basu PS, Sharma A, Garg ID, Sukumaran NP. Tuber sink modifies photosynthetic response in potato under water stress. Environmental and Experimental Botany. 1999 Aug 1;42(1):25–39.
  55. 55. Gupta AK, Kaur N. Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants. Journal of biosciences. 2005 Dec 1;30(5):761–76. pmid:16388148
  56. 56. Nidumukkala S, Tayi L, Chittela RK, Vudem DR, Khareedu VR. DEAD box helicases as promising molecular tools for engineering abiotic stress tolerance in plants. Critical reviews in biotechnology. 2019 Apr 3;39(3):395–407. pmid:30714414
  57. 57. Tuteja N, Tarique M, Trivedi DK, Sahoo RK, Tuteja R. Stress-induced Oryza sativa BAT1 dual helicase exhibits unique bipolar translocation. Protoplasma. 2015 Nov 1;252(6):1563–74. pmid:25772680
  58. 58. Pandian BA, Sathishraj R, Djanaguiraman M, Prasad PV, Jugulam M. Role of Cytochrome P450 Enzymes in Plant Stress Response. Antioxidants. 2020 May;9(5):454.
  59. 59. Wang M, Yuan J, Qin L, Shi W, Xia G, Liu S. Ta CYP 81D5, one member in a wheat cytochrome P450 gene cluster, confers salinity tolerance via reactive oxygen species scavenging. Plant biotechnology journal. 2020 Mar;18(3):791–804. pmid:31472082
  60. 60. Ma Y, Shukla V, Merewitz EB. Transcriptome analysis of creeping bentgrass exposed to drought stress and polyamine treatment. PloS one. 2017 Apr 26;12(4):e0175848. pmid:28445484
  61. 61. Song J, Xing Y, Munir S, Yu C, Song L, Li H, et al. An ATL78-Like RING-H2 finger protein confers abiotic stress tolerance through interacting with RAV2 and CSN5B in tomato. Frontiers in plant science. 2016 Aug 29;7:1305.
  62. 62. Li H, Jiang H, Bu Q, Zhao Q, Sun J, Xie Q, et al. The Arabidopsis RING finger E3 ligase RHA2b acts additively with RHA2a in regulating abscisic acid signaling and drought response. Plant physiology. 2011 Jun 1;156(2):550–63. pmid:21478367
  63. 63. Xuan TD, Minh TN, Khanh TD. Allelopathic momilactones A and B are implied in rice drought and salinity tolerance, not weed resistance. Agronomy for sustainable development. 2016 Sep 1;36(3):52.
  64. 64. Tian FX, Gong JF, Wang GP, Wang GK, Fan ZY, Wang W. Improved drought resistance in a wheat stay-green mutant tasg1 under field conditions. Biologia plantarum. 2012 Sep 1;56(3):509–15.
  65. 65. Batth R, Singh K, Kumari S, Mustafiz A. Transcript profiling reveals the presence of abiotic stress and developmental stage specific ascorbate oxidase genes in plants. Frontiers in plant science. 2017 Feb 17;8:198. pmid:28261251
  66. 66. Ebeed HT, Stevenson SR, Cuming AC, Baker A. Conserved and differential transcriptional responses of peroxisome associated pathways to drought, dehydration and ABA. Journal of experimental botany. 2018 Jul 19;69(20):4971–85. pmid:30032264
  67. 67. Aarati P, Krishnaprasad BT, Savitha M, Gopalakrishna R, Ramamohan G, Udayakumar M. Expression of an ABA responsive 21 kDa protein in finger millet (Eleusine coracana Gaertn.) under stress and its relevance in stress tolerance. Plant Science. 2003 Jan 1;164(1):25–34.
  68. 68. Ranocha P, Dima O, Nagy R, Felten J, Corratgé-Faillie C, Novák O, et al. Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis. Nature communications. 2013 Oct 16;4(1):1–9.
  69. 69. Zhang X, Liu X, Zhang D, Tang H, Sun B, Li C, et al. Genome-wide identification of gene expression in contrasting maize inbred lines under field drought conditions reveals the significance of transcription factors in drought tolerance. PLoS One. 2017 Jul 12;12(7):e0179477. pmid:28700592
  70. 70. Garcia ME, Lynch T, Peeters J, Snowden C, Finkelstein R. A small plant-specific protein family of ABI five binding proteins (AFPs) regulates stress response in germinating Arabidopsis seeds and seedlings. Plant molecular biology. 2008 Aug 1;67(6):643–58. pmid:18484180
  71. 71. Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Pérez AC, et al. NINJA connects the co-repressor TOPLESS to jasmonate signalling. Nature. 2010 Apr;464(7289):788–91. pmid:20360743
  72. 72. Kang J, Park J, Choi H, Burla B, Kretzschmar T, Lee Y, et al. Plant ABC transporters. The Arabidopsis book/American Society of Plant Biologists. 2011;9.