Citrullus colocynthis is a very drought tolerant species, closely related to watermelon (C. lanatus var. lanatus), an economically important cucurbit crop. Drought is a threat to plant growth and development, and the discovery of drought inducible genes with various functions is of great importance. We used high throughput mRNA Illumina sequencing technology and bioinformatic strategies to analyze the C. colocynthis leaf transcriptome under drought treatment. Leaf samples at four different time points (0, 24, 36, or 48 hours of withholding water) were used for RNA extraction and Illumina sequencing. qRT-PCR of several drought responsive genes was performed to confirm the accuracy of RNA sequencing. Leaf transcriptome analysis provided the first glimpse of the drought responsive transcriptome of this unique cucurbit species. A total of 5038 full-length cDNAs were detected, with 2545 genes showing significant changes during drought stress. Principle component analysis indicated that drought was the major contributing factor regulating transcriptome changes. Up regulation of many transcription factors, stress signaling factors, detoxification genes, and genes involved in phytohormone signaling and citrulline metabolism occurred under the water deficit conditions. The C. colocynthis transcriptome data highlight the activation of a large set of drought related genes in this species, thus providing a valuable resource for future functional analysis of candidate genes in defense of drought stress.
Citation: Wang Z, Hu H, Goertzen LR, McElroy JS, Dane F (2014) Analysis of the Citrullus colocynthis Transcriptome during Water Deficit Stress. PLoS ONE 9(8): e104657. https://doi.org/10.1371/journal.pone.0104657
Editor: Girdhar K. Pandey, University of Delhi South Campus, India
Received: April 28, 2014; Accepted: July 6, 2014; Published: August 13, 2014
Copyright: © 2014 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Raw sequence data is available for download at NCBI Sequence Read Archive under the accession numbers SAMN02769576, SAMN02769577, SAMN02769578 and SAMN02769579.
Funding: Funding or sources of support for the study were the Alabama Agricultural Experiment Station and the State of Alabama. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Water is essential for plant growth in modern agriculture . Drought delays the development of crops, and strongly affects morphology, as well as physiological processes like transpiration, photosynthesis, respiration and translocation of assimilates . Drought avoidance can be achieved through morphological changes in plants, such as decreased stomatal conductance, reduced leaf area, and extensive root systems . Drought tolerance is achieved by physiological and molecular mechanisms, including osmotic adjustment, antioxidant and scavenger compounds . Both strategies involve the induction of specific genes and proteins, such as dehydrins (dehydration-induced proteins), key enzymes for osmolyte biosynthesis, and detoxification enzymes , .
Plant species have developed diverse strategies to adapt and thrive in all kinds of climates and terrains to deal with extreme changes in the environment. These strategies are supported by rich and complex metabolic networks that enable the plant to synthesize a wide range of compounds. Plant responses to abiotic stresses involve interactions and crosstalk between many molecular pathways. High throughput screening techniques such as transcriptome sequencing have been used to study the adaptability of plants to drought . This led to the discovery of many drought related genes. For example, PIP aquaporins were found to fine-tune the environment in response to declining water availability . However, few natural allelic variants have been cloned for drought related traits, so QTL, RNA sequencing, and other trait isolation methods are needed to improve methodology for exploring complex multivariate data , .
The cucurbit family is a large family with several economically important species, such as watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (Cucumis sativus) and several Cucurbita species with edible fruits . Citrullus colocynthis (L.) Schrad (2n = 2x = 22), the bitter apple, closely related to domesticated watermelon (Citrullus lanatus var. lanatus), is a very drought-tolerant perennial herbaceous species in the Cucurbitaceae family . It can survive arid environments by maintaining its water content under severe stress conditions. C. colocynthis is an important medicinal plant and a source of valuable oil . Its seeds were found in several early Egyptian, Libyan and Near Eastern sites from about 4000 BC. This species grows in sandy areas throughout northern Africa, southwestern Asia and the Mediterranean region , . The species has been used as a model to elucidate the function of genes implicated in the stress response ultimately leading to enhancement of stress tolerance in cucurbit crops through genetic manipulation. Si et al.  found dynamic gene expression changes in C. colocynthis root tissues using cDNA amplified fragment length polymorphism (cDNA-AFLP) technique.
Several research groups have used next generation sequencing technologies to study gene expression profiles in species of the cucurbit family. Guo et al  used 454 sequencing technology to study the comprehensive profile for watermelon fruit flesh tissues, Grassi et al  studied carotenoid pathway regulators in ripening watermelon fruit. The draft genome of watermelon (C. lanatus, 2n = 2x = 22, ∼425 Mb) was analyzed by Guo et al  using three different watermelon subspecies. Comparative genomic analysis provided an evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. The genome sequence of cucumber (Cucumis sativus, 2n = 2x = 14) has been completed, and the genome of melon (Cucumis melo, 2n = 2x = 24) is being sequenced under the Spanish Genomics Initiative (MELONOMICS) , . Liu et al  used sequencing techniques to identify conserved and novel miRNA in watermelon, while Wincker  used comparative analysis of genomes between watermelon and sweet orange to detect the traits related to their domestication.
Here, high-throughput sequencing of the leaf transcriptome from C. colocynthis provides a glimpse at drought related genes in this uniquely drought tolerant cucurbit species. The lack of extensive genomic and functional genomic resources in Citrullus species has hampered research and breeding of the cultivated and economically important C. lanatus. This study should facilitate the identification of valuable multiple genes, needed for complex interactions of plant species with the environment.
Materials and Methods
Plant Materials and RNA Extraction
C. colocynthis seedlings were grown in Sunshine Mix #8 under a 16 h light/8 h dark photoperiod at 26°C day, 22°C night temperature. Seedlings with 2–3 true leaves (2–3 week old) in 50 ml containers were exposed to drought by withholding water for 0 (day 1, D1), 24 (day 2, D2), 36 (day 3, D3) or 48 hours (day 4, D4). True leaf samples were collected each day at noon, flash frozen in liquid nitrogen and stored at −80°C. RNA was subsequently extracted using the TRIzol method .
Preparation of cDNA Library and Sequencing
Illumina sequencing was performed at the HudsonAlpha Institute of Biotechnology (Huntsville, AL) following manufacturer's instructions. RNA-Seq reads were first processed to remove rRNA sequence contamination. First strand cDNA was synthesized with reverse transcriptase and random primers using the small fragment RNAs as template. Second strand cDNA was then synthesized followed by phosphorylation by T4 DNA polymerase. The cDNA fragments were 3′ adenylated and ligated to the Illumina's paired-end adapters. Enrichment of cDNA templates was conducted following fifteen cycles of PCR amplification. In total, over 20*4Mp were sequenced for mapping assembly and differential expression analysis. Raw sequence data are available for download at NCBI Sequence Read Archive (SAMN02769576, SAMN02769577, SAMN02769578, and SAMN02769579).
Raw sequencing data were filtered Z (0.05) using the CLC Genomics workbench (CLC Bio, Aarhus, Denmark). Paired–end sequences from the four samples were used to construct the de novo C. colocynthis transcriptome assembly with default parameters. Assembly reads were also assembled against the watermelon (C. lanatus) genome sequences, which were downloaded from the Cucurbit Database (http://www.icugi.org/cgi-bin/ICuGI/index.cgi). Reads were filtered and assembled using the CLC genomics workbench. The parameters used were as follows: 2 points of mismatch cost, 2 points of insertion cost, 2 points of deletion cost, 0.5 as length fraction, 0.95 as similarity fraction. After the de novo assembly and watermelon mapping assembly, we used Trinity to assemble all the contigs with the default parameters.
Identification of Full Length cDNAs
Two methods were used to identify full length cDNAs. First, BLASTX searching (E value: 1e−10) was used to detect the matched cDNAs in SwissProt database; second, ESTScan 2.0 was used to identify the translated sequences. Sequences with either start codon (ATG) and stop codon (TAG/TGA/TAA), or sequences with start codon (ATG) and homologue to a known protein with ≥80% similarity, were chosen as full length cDNAs.
Expression Analysis Using Transcriptome Reference
Pair-end sequencing reads of the four libraries were filtered using CLC Genomics workbench (0.05) before mapping to the references sequences from assembled cDNAs. First, read counts of each unigene were converted to reads per million (RPM). Secondly, statistical analysis using Kal's test  provided in CLC Genomics workbench (P<0.05 and fold change ≥1.5) was conducted. These transcripts were annotated against the reference sequences.
Gene Ontology Analysis
The functional annotation software BLAST2GO (http://www.blast2go.com/b2ghome) was used to conduct gene ontology (GO) analysis of C. colocynthis genes in this study. The databases used were SwissProt and NCBI. BLAST E-value was set at 1.0e−3. The major GO analysis was determined by BLAST, mapping, and annotation. Results are presented as a bar chart showing the percent of genes belonging to each group.
For cDNA synthesis, 500 ng of the total RNA for each sample (the same RNA was used for RNA-seq analysis) was used in reverse transcription with ProtoScript First Strand cDNA synthesis kit from New England BioLabs (Ipswich, MA). qRT-PCR was performed with SYBR-Green Supermix from Bio-Rad (Hercules, CA) in an Eppendorf Mastercycler ep realplex (Hauppauge, NY). Table S1 contains gene specific primer sequences. Each reaction contained 10 µl of SYBR-Green supermix, 1 µl of cDNA template, 1 µl forward primer (4 µm), 1 µl reverse primer (4 µm), and 7 µl ddH2O. The qRT-PCR program consisted of one cycle at 95°C for 15 sec, followed 40 cycles of 15 sec at 95°C, 15 sec at 55°C, and 30 sec at 72°C. The relative expression data was compared with actin  from C. colocynthis. Quantification of the relative transcript levels was performed using the comparative CT method. The induction ratio (IR) was calculated as recommended by the manufacturer and corresponds to 2−ΔΔCT, where ΔΔCT = (CT, target gene, -CT, actin)treatment- (CT, target-CT, actin)control. All experiments were replicated three times.
Results and Discussion
Transcriptome Assembly Results
The transcriptome of C. colocynthis leaves following 4 days of drought stress was assembled and assessed following paired-end (2*50 bp) Illumina sequencing. The Illumina platform yielded an average of 24 million high-quality reads per sample (Table 1). All sample reads were used to construct a de novo assembly. A reference assembly was constructed using the completely sequenced watermelon genome. A total of 20,581 contigs were generated (Table 2). The contigs had an average length of 1350 bp and N50 of 1870 bp. BLASTX was used against SwissProt and ESTscan of translated protein sequences to detect full length cDNAs. A total of 5,038 full length cDNAs were detected in our sequencing assembly (Table 2).
Principle component analysis (PCA) was implemented in the CLC workbench. The results (Figure 1) illustrate differential gene expression patterns in C. colocynthis seedlings following four days of withholding water. Gene expression patterns on D1 and D2 were nearly similar, while results from D3 and D4 showed substantial differences as compared to D1 and D2, and gene expression patterns detected on D4 were very different from all other time points as a result of drought stress.
Differentially Expressed Genes Involved in Response to Drought Stress in C. colocynthis
The paired-end reads were mapped to the reference genomes after filtration. Read counts of each unigene were converted to reads per million (RPM). The read number of each cDNA was divided by the total number of reads per day (1, 2, 3, or 4) from the data set, and multiplied by 106. Statistical analysis was conducted using Kal's test (p<0.05 and fold change ≥1.5). Genes showing non-significant and significant changes in read counts are shown in Table S2. Each sample was compared to the day 1 sample reads for analysis of their significance level of gene expression. The read results indicated that 59 genes showed significant changes at D2, D3, and D4 as compared to D1; 13 genes showed significant differential expression at D2 and D3 as compared to D1; 30 genes showed significant differential expression early, at D2, as compared to D1; 897 genes showed changes at D3 and D4 as compared to D1; 341 genes were only differentially expressed at D3; 1191 genes were regulated late, at D4 only, under drought stress. In conclusion, the C. colocynthis gene expression patterns showed dramatic changes with 2545 genes showing significant changes, mostly occurring late under drought conditions (D3 and D4 of withholding water).
The heat map depicted in Figure 2 corresponded to the principal component analysis. D3 and D4 transcripts were clustered together, and D1 and D2 transcripts were clustered. Also significant changes were seen at D3 and D4 suggesting that the transcriptional response of many genes was up-regulated during drought. Strong effects were especially observed on D3 and D4.
Heatmap was produced using R. Each red or green block represents normalized expression value of a gene detected at 0 (D1), 24 (D2), 36 (D3) and 48 (D4) hours of withholding water. Red indicates higher gene expression values across treatment, while green indicates lower expression values across treatment.
Gene Ontology (GO) Classification
To functionally categorize significantly changed genes in C. colocynthis under drought treatment, gene ontology analysis by BLAST2GO was performed. C. colocynthis unigenes were categorized in three main GO categorizes: biological process (2672), molecular function (1368) and cellular component (1053). These GO terms were further divided into several sub-categories (Figure 3). In the biological process category, single organism process genes accounted for more than 20% of the biological process genes. In the molecular function category, more than 40% of genes were associated with a catalytic activity. In the cellular category, more than 35% of the genes were associated with the cellular component.
Based on high-score BLASTX matches in the NR (non-redundant) plant protein database, C. colocynthis genes were classified into three main GO categories. The y-axis indicates the percentage of a specific category of genes in each main category.
Validation of Illumina Expression Patterns by qRT-PCR Analysis
To confirm the reliability of the Illumina sequencing read analysis, 8 candidate genes were selected and their expression was compared at D4 and D1 using qRT-PCR. The expression patterns resulting from qRT-PCR showed general agreement with those from the Illumina sequencing analysis (Table 3). Discrepancies with respect to ratio of fold changes between sequencing and qRT-PCR analysis can be attributed to the essentially different algorithm and sensitivity of the two techniques . In the deep-sequencing method the absolute expression rather than relative expression as in qRT-PCR analysis is used. Transcriptional qRT-PCR analyses of 16 genes during the drought treatments are shown in Figures 4 and 5.
The y-axis indicates relative expression compared with Day 1 expression of each gene. The x-axis shows days of water-withholding time points.
The y-axis indicates relative expression compared with Day 1 expression of each gene. The x-axis shows days of water-withholding time points.
Gene Comp5873 is homeobox-leucine zipper protein, with significant upregulation during all days of drought in C. colocynthis. It is known that the expression of several homeobox-leucine zipper proteins is correlated to stress. Athb-12, a homeobox-leucine zipper domain protein from Arabidopsis, is functionally involved in salt tolerance in yeast . Hahb-4, a homeobox-leucine zipper gene is potentially involved in water stress in sunflower . Comp862 belongs to the glutathione S-transferase (GST) family, which contains heterogeneous, multifunctional dimeric proteins. This gene is highly up-regulated (60x) during drought in C. colocynthis. It is thought that GSTs are involved in cellular detoxification . Comp1108 is a member of the NAC (NAM/ATAF1, 2/CUC2) gene family and NACs are known to be involved in numerous biological processes, including drought stress .
Comp10156 is a member of the GID1 (GIBBERELLIN INSENSITIVE DWARF1) family in C. colocynthis with high expression under drought conditions. GID1 is a soluble GA receptor in rice . GA-GID1 complex interacts with DELLA proteins, which are negative regulators of GA signaling pathway. RD22, a known dehydration responsive gene in Arabidopsis, which is mediated by abscisic acid (ABA), may have physiological and molecular significance for processes underlying memory functions of plants in response to ABA and light pulses , . One RD22-like protein from soybean can alleviate salinity and osmotic stress . RD22-like genes (Comp 6528) with significant up-regulation (>160x) under drought in C. colocynthis were confirmed by qRT-PCR, especially after days of withholding water.
NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE1) plays an important role in coordinating broader cellular processes in response to stress and bacterial pathogen infection . The chemical β-aminobutyric acid, which is known to induce resistance in plants, primed the expression of many genes, and NDR1/NHL10 was one of them . Comp7317 gene, which is a member of NDR1/NH10 showed significant changes at D2 only during the early stage of water deficit stress.
GRAS (for GA Insensitive, REPRESSOR of gal-3 (RGA), SCARECROW (SCR)) transcription factors, have a major function in plant development and environmental adaption. These TFs are particularly implicated in the modulation of plant tolerance to stressors as cold, drought, salinity by crosstalks via GA to ABA-dependent and ABA-independent pathways . For example, SCL7 confers salt and drought tolerance in Arabidopsis . SCL14 is involved in the detoxification of xenobiotics and possibly endogenous harmful metabolites . Comp20554, which belongs to the GRAS transcription factor family, showed significant up-regulation especially on D2.
The expression profile of Comp3048, which codes for a methyltransferase, maintained high levels during drought stress, especially on D2. It was found that myo-inositol-O-methyltransferase (Imt1) responded to low temperature stress in transgenic Arabidopsis . Trithorax-like H3K4 methyltransferase from barley is drought inducible . The methylation of myo-inositol catalyzed by myo-inositol methyltransferase (IMT) occurs when plants are under abiotic stress. Over-expression of IMT resulted in improved tolerance to dehydration and salt stress treatment in Arabidopsis .
Heat-shock proteins (HSPs) are environmentally induced proteins that enable plants to cope with heat and other environmental stresses. For example, Trichoderma harzianum Hsp70 transgenic Arabidopsis is abiotic stress tolerant . Similarly HSP22 was found to be highly upregulated in C. colocynthis roots during drought conditions . Overexpression of GmHsp90S can decrease damage of abiotic stresses in Arabidopsis . Comp14675 belongs to the HSP family, and showed up-regulation at later stages of D3 and D4 (Figure 5).
Plant cold shock proteins (CSP) are very conserved among various plant genera . The first CSP identified was WCSP1 from winter wheat, which did accumulate in response to low-temperature stress . Similarly in C. colocynthis CSP (Comp13927) was up-regulated during later stages of water deficit.
Comp2553, one MA3 domain-containing protein gene, showed marked changes on D3 and D4 under drought conditions (Figure 5). Loss-of-function of ECIP1 (one MA3 domain-containing protein) resulted in enhanced ethylene response but altered salt response  in Arabidopsis.
Overexpression of plant BL-1 in Arabidopsis resulted in the attenuation of cell death induced by biotic stresses (pathogens) and abiotic stresses such as heat, cold, drought, salt and chemical-induced oxidative stresses . BL-1 might function to control the level of the “pro-survival and pro-death” signals under multiple stress conditions in plants . For example, cucumber BAX inhibitor-1 is a conserved cell death suppressor induced by cold stress and a negative regulator of programmed cell death (PCD) . Comp372 encodes one BL-1 gene, which was up-regulated by drought at later stages (D3 and D4).
Comp8117, which is a homologue of a Populus EST (CU233481.1), is a drought stress related gene, up-regulated at D4. Comp10586, which encodes a member of the MYB transcription factor family, is up-regulated during the late stage of drought stress. Some MYB members have been shown to regulate plant responses to biotic and abiotic stress conditions . For example, AtMYB96 acts through the ABA signaling pathway to induce pathogen resistance by promoting salicylic acid biosynthesis, and thus regulating stomata movement, drought tolerance and disease resistance in Arabidopsis , . MYB88 might function directly or indirectly, as positive regulator of stress-responsive genes . TaMYB30-B from wheat did improve drought stress tolerance in transgenic Arabidopsis . Another family of transcription factors, the WRKY family, named for the WRKY domain of about 60 amino acids, contains a highly conserved WRKYGQK heptapeptide at its N-terminus and a zinc-finger-like motif at its C-terminus . WRKY transcription factors are involved in multiple aspects of plant growth, development and stress . Several TaWRKY in wheat with roles in the abiotic stress response acted in an ABA-dependent manner . Here, gene Comp19751encodes a WRKY gene, which showed remarkable up-regulation at D4.
MADS-box family members function in reproductive development and stress . For example, OsMADS25 and OsMADS27 transcripts accumulate in response to osmotic stress . Comp6823, which encodes for a MADS-box gene in C. colocynthis, is up-regulated markedly at the last stage of drought (D4; Figure 5).
Analysis of the Drought Stress Signaling Transcriptome in C. colocynthis
Drought stress signal transduction consists of several pathways including ionic and osmotic homeostasis signaling, detoxification response and growth regulation pathways . Genes detected in C. colocynthis leaves during water deficit are listed in Table 4.
In the ion signaling pathway, calcium binding proteins are well known for their involvement in both biotic and abiotic stress response pathways . The calcium ion (Ca2+) as a secondary messenger in plants is sensed by calmodulins (CaMs)/CaM-like protein (CMLs), the caldineurin B-like proteins (CBLs) and Ca2+-dependent protein kinases (CDPKs). CaM binds to CaM-binding proteins (CBPs), which function in different pathways under biotic and biotic stresses . A total of 10 Ca2+ binding proteins were detected in the C. colocynthis transcriptome.
Protein phosphorylation is a central theme in the cell's response to stress. The MAP kinase cascade in transcript levels consist of a number of protein kinases, such as two-component histidine kinase, MAPKKK, MAPKK, MAPK etc. . Here we detected 16 MAP kinases in the C. colocynthis transcriptome.
Membrane phospholipids can activate several types of phospholipases that cleave certain phospholipids to generate lipid messengers (eg. PA, DAG, IP3), which further regulate stress tolerance through modulation of stress-responsive gene expression . Several members in this pathway, such as phospholipase C (PLC), diacylglycerol (DAG) and phosphotidylinositol 4,5-bisphosphate (PIP2)-like aquaporin were detected (Table 4).
Detoxification signaling can ameliorate the damage in plants under stresses , as noted in many other plant species. A total of 32 detoxification proteins were detected in the C. colocynthis transcriptome.
Molecular mechanisms regulating gene expression in response to drought stress have been studied by analyzing the functional transcription factors in ABA-dependent and ABA-independent pathways . Several regulatory proteins in ABA-dependent or –independent pathway were detected, among which NAC, MYB/C and leucine-rich repeat proteins (LRR). These regulatory proteins can further modulate many responsive transcription factors. Several functional proteins such as heat shock protein (HSP) 70, HSP22, grpE like protein, respiratory burst oxidase D (RBOHD), VIRE2-Interacting protein2 (VIP2), ABA transporter-like protein, synaptobrevin-related protein, translocon outer envelope of chloroplast (Toc34-1), beta-amylase, TIP1 (TIP GROWTH DEFECTIVE 1), and RD22 were detected.
Analysis of Phytohormone Signaling Mediators in C. colocynthis
Phytohormones play important roles in regulating plant responses under biotic and abiotic stress. Elaborate phytohormone signaling networks mediate the adaptability of plants to different environmental conditions . Many phytohormones such as ABA, salicylic acid (SA), jasmonic acid (JA), auxin, ethylene and gibberellic acid (GA) are being studied for their role in abiotic stress responses , , . It is known that downstream signaling proteins for auxin, GA, JA, and ABA are subjected to ubiquitin-dependent degradation . Putative phytohormone signaling genes detected in C. colocynthis during the drought response are listed in Table 5.
Ethylene signaling pathway components were ordered into a hypothetical linear pathway based on both genetic (epistasis) analysis and biochemical interactions . Almost all of the ethylene signaling homologous members (118 ethylene response factors or ERFs) were detected in the C. colocynthis transcriptome. APETALA2/ethylene responsive factor (AP2/ERF) transcription factors are well-known for mediating stress responses and development in plants .
The auxin response factor (ARF) family contains transcription factors that bind to auxin-responsive elements (AREs) in the promoters of primary auxin-responsive genes. Aux/IAAs are early auxin-response proteins that bind ARFs, therefore inhibiting ARE-mediated gene transcription. Aux/IAAs are involved in ubiquitin-mediated degradation, which is catalyzed by SCF E3 ubiquitin ligase. TIR1 can stimulate Aux/IAA proteolysis by binding auxin to this protein . All of the major auxin signaling related transcription factors found in the C. colocynthis transcriptome are shown in Table 5. Similar to the auxin pathway, a novel family of transcriptional regulators, the jasmonate ZIM-domain (JAZ) proteins play a target part as Aux/IAA. In addition, COI1 plays a similar role as TIR1, while MYC and R2R3-MYB transcription factors work as ARFs . Several JA signaling pathway related genes exist in the C. colocynthis transcriptome.
In the SA pathway, NPR1 (non-specific disease resistance 1) is a key regulator in SA-dependent defense signaling . Similarly WRKY and TGA play major roles as transcriptional regulators in the SA pathway. We detected 2 NPR1 related proteins, 46 WRKYs and 6 TGAs in C. colocynthis, which might function in the C. colocynthis SA pathway.
Drought triggers the production of the phytohormone ABA, which in turn causes stomatal closure and induces expression of stress-related genes. The soluble PYR/PYL/RCAR receptors function at the apex of a negative regulatory pathway to directly regulate PP2C phosphatases, which in turn directly regulate SnRK2 kinases . Several of the core transcription factors in the ABA pathway are listed in Table 5.
We also detected 11 GA receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1), 4 DELLA growth inhibitors (DELLAs) and 2 F-box proteins (SLY1) and SNEEZY (SNZ), which play important roles in GA signaling pathways , .
Cellular Metabolism under Drought Stress in C. colocynthis
Global gene expression analyses have shown substantial down-regulation of many photosynthetic genes under drought not only in Arabidopsis , but also several other species such as indica rice , . Similarly, many photosystem I and II, chlorophyll a, b binding protein, and oxygen evolving enhancer protein genes , showed down-regulation during water deficit stress in C. colocynthis (Table 6).
Citrulline, a non-protein amino acid intermediate in the arginine biosynthetic pathway, has been found to accumulate in leaves of drought tolerant watermelon under water deficit conditions , , . Thus several factors related to the response of this model drought tolerant species to stress have been identified (Table 7, Figure S1). Citrulline metabolic genes (carbamoyl-phosphate synthetase, acetyl glutamate synthase, acetylornithine aminotransferase, aminoglutamate decarboxylase, acetylornithine deacetylase, and glutamate dehydrogenase) were found to be significantly up-regulated during drought.
One of the major research goals is to understand the molecular mechanisms underlying drought tolerance in plants. It is clear that drought triggers a wide variety of responses in C. colocynthis. Down regulation of many photosynthetic genes was observed especially at the later stages of drought. Up regulation of many transcription factors, stress signaling factors, detoxification genes, and genes involved in phytohormone signaling occurred throughout the water deficit experiment. Systematic approaches using genomic analyses should lead to the discovery of additional stress factors and provide us with a better understanding of the stress tolerance mechanism of this drought tolerant plant species.
Citrulline metabolism pathway in C. colocynthis.
qRT-PCR primer sequence information.
Conceived and designed the experiments: ZW FD. Performed the experiments: ZW FD. Analyzed the data: ZW FD HH LG. Contributed reagents/materials/analysis tools: FD LG SM. Contributed to the writing of the manuscript: ZW FD.
- 1. Guo Y, Tan J (2013) A biophotonic sensing method for plant drought stress. Sensor Actuat B-Chem 188: 519–524.
- 2. Do PT, Degenkolbe T, Erban A, Heyer AG, Kopka J, et al. (2013) Dissecting rice polyamine metabolism under controlled long-term drought stress. PLOS ONE 8: e60325.
- 3. Levitt J (1980) Responses of plants to environmental stress: chilling, freezing and high temperature stresses, 2nd Ed New York: Academic Press.
- 4. Bartels D, Sunkars R (2005) Drought and salt tolerance in plants. Cr Rev Plant Sci 24: 23–58.
- 5. Reddy AR, Chaitanya KV, Vivekanandan M (2004) Drought induced responses of photosynthesis and antioxidant metabolism in higher plants. Plant Physiol 161: 1189–1202.
- 6. Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular response and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57: 781–803.
- 7. Zhao J, Gao Y, Zhang Z, Chen T, Guo W, et al. (2013) A receptor-like kinase gene (GbRLK) from Gossypium barbadense enhances salinity and drought-stress tolerance in Arabidopsis. BMC Plant Biol 13: 110–125.
- 8. Sŭrbanovski N, Sargent DJ, Else MA, Simpson DW, Zhang H, et al. (2013) Expression of Fragaria vesca PIP aquaporins in response to drought stress: PIP down-regulation correlates with the decline in substrate moisture content. PLOS ONE 8: e74945.
- 9. Caccamo M, Grotewold E (2013) Turning over a new leaf in plant genomics. Genome Biol 14: 403.
- 10. Juenger TE (2013) Natural variation and genetic constraints on drought tolerance. Curr Opin Plant Biol 16: 274–281.
- 11. Jeffrey C (2008) A review of Cucurbitaceae. Bot J Linn Soc 81: 233–24.
- 12. Dane F, Liu J, Zhang C (2006) Phylogeography of the bitter apple, Citrullus colocynthis. Genet Resour Crop Ev 54: 327–336.
- 13. Si Y, Zhang C, Meng S, Dane F (2009) Gene expression changes in response to drought stress in Citrullus colocynthis. Plant Cell Rep 28: 997–1009.
- 14. Guo S, Liu J, Zheng Y, Huang M, Zhang H, et al. (2011) Characterization of the transcriptome dynamics during watermelon fruit development: sequencing, assembly, annotation and gene expression profiles. BMC Genomics 12: 454.
- 15. Grassi S, Piro G, Lee JM, Zheng Y, Fei Z, et al. (2013) Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit. BMC Genomics 14: 781.
- 16. Guo S, Zhang J, Sun H, Salse J, William JL, et al. (2013) The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet 45: 51–58.
- 17. Gonzalez VM, Garcia-Mas J, Arus P, Puigdomenech P (2010) Generation of a BAC-based physical map of the melon genome. BMC Genomics 11: 339.
- 18. Huang S, Li R, Zhang Z, Li L, Gu X, et al. (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet 41: 1275–1281.
- 19. Liu N, Yang J, Gao S, Xu Y, Zhang M (2013) Genome-wide identification and comparative analysis of conserved and novel microRNAs in grafted watermelon by high-throughput sequencing. PLOS ONE 8: e57359.
- 20. Wincker P (2013) Genomics and fruit crop selection. Nature Genetics 45: 9–10.
- 21. Chomczynski P, Sacchi N (1987) Signal-step method of RNA isolation by acid guanidinium thiocyanate–phenol-chloroform extraction. Anal Biochem 162: 156–159.
- 22. Kal AJ, van Zonneveld AJ, Benes V, van den Berg M, Koerkamp MG, et al. (1999) Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources. Mol Biol Cell 10: 1859–1872.
- 23. Shi T, Gao Z, Wang L, Zhang Z, Zhuang W, et al. (2012) Identification of differentially expressed genes associated with pistil abortion in Japanese apricot by genome-wide transcriptional analysis. Plos ONE 7: e47810.
- 24. Shin D, Koo YD, Lee J, Lee H, Baek D, et al. (2004) Athb-12, a homeobox-leucine zipper domain protein from Arabidopsis thaliana, increases salt tolerance in yeast by regulating sodium exclusion. Biochem Biophys Res 323: 534–540.
- 25. Gago GM, Almoguera C, Jordano J, Gonzalez DH, Chan RL (2002) Hahb-4, a homeobox-leucine zipper gene potentially involved in abscisic acid-dependent responses to water stress in sunflower. Plant Cell Environ 25: 633–640.
- 26. Park H, Choung Y (2010) Evaluation of the biodegradation feasibility of antibiotics by three bacteria involving glutathione S-transferases. Can J Civ Eng 37: 814–819.
- 27. Shaminuzzaman M, Vodkin L (2013) Genome-wide identification of binding sites for NAC and YABBY transcription factors and co-regulated genes during soybean seedling development by ChIP-Seq and RNA-Seq. BMC Genomics 14: 477.
- 28. Ueguchi-Tanaka M, Ashikari M, Nakajima M, Itoh M, Katoh E, et al. (2005) GIBBERELLIN INSENSITIVE DWARFS encodes a soluble receptor for gibberellin. Nature 437: 693–69.
- 29. Goh CH, Nam HG, Park YS (2003) Stress memory in plants: a negative regulation of stomatal response and transient induction of rd22 gene to light in abscisic acid-entrained Arabidopsis plants. Plant J 36: 240–255.
- 30. Xu YH, Liu R, Yan L, Liu ZQ, Jiang SC, et al. (2012) Light-harvesting chlorophyll a/b-binding proteins are required for stomatal response to abscisic acid in Arabidopsis. J Exp Bot 63: 1095–1106.
- 31. Wang H, Zhou L, Fu Y, Cheung M, Wong F, et al. (2012) Expression of an apoplast-localized BURP-domain protein from soybean (GmRD22) enhances tolerance towards abiotic stress. Plant Cell Environ 35: 1932–1947.
- 32. Knepper C, Savory EA, Brad D (2011) Arabidopsis NDR1 is an integrin-like protein with a role in fluid loss and plasma membrane-cell wall adhesion. Plant Physiol 156: 286–300.
- 33. Chen P, Parshant S, Laurent Z (2011) Priming of the Arabidopsis pattern-triggered immunity response upon infection by necrotrophic Pectobacterium carotovorum bacteria. Mol Plant Pathol 14: 58–70.
- 34. Golldack D, Li C, Mohan H, Probst N (2013) Gibberelins and abscisic acid signal crosstalk: living and developing under unfavorable conditions. Plant Cell Rep 32: 1007–1016.
- 35. Ma H, Liang D, Shuai P, Xia X, Yin W (2010) The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana. J Exp Bot 61: 4011–4019.
- 36. Fode B, Siemsen T, Thurow C, Weigel R, Gatz C (2008) The Arabidopsis GRAS protein SCL14 interacts with Class II TGA transcription factors and is essential for the activation of stress-inducible promoters. Plant Cell 20: 3122–3135.
- 37. Zhu B, Peng RH, Xiong AS, Xu J, Fu XY, et al. (2012) Transformation with a gene for myo-inositol O-methyltransferase enhances the cold tolerance of Arabidopsis thaliana. Biol Plantarum 56: 135–139.
- 38. Papaefthimiou D, Tsataris AS (2012) Characterization of a drought inducible trithorax-like H3K4 methytransferase from barley. Biol Plantarum 56: 683–692.
- 39. Ahn C, Park U, Park PB (2011) Increased salt and drought tolerance by D-ononitol production in transgenic Arabidopsis thaliana. Biochem Biophys Res 415: 669–674.
- 40. Montero-Barrientos M, Hermosa R, Cardoza RE, Gutiérrez S, Nicolás C, et al. (2010) Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses. Plant Physiol 167: 659–665.
- 41. Xu J, Xue C, Xue D, Zhao J, Gai J, et al. (2013) Overexpression of GmHsp90s, a heat shock protein (Hsp90) gene family form soybean, decreases damage of abiotic stresses in Arabidopsis thaliana. PLOS ONE 8: e69810.
- 42. Karlson D, Imai R (2003) Conservative of the cold shock domain protein family in plants. Plant Physiol 131: 12–15.
- 43. Karlson D, Nakaminami K, Toyomasu T, Imai R (2002) A cold-regulated nucleic acid-binding protein of winter wheat shares a domain with bacterial cold shock proteins. J Biol Chem 277: 35248–35256.
- 44. Lei G, Shen M, Li Z, Zhang B, Duan K, et al. (2011) EIN2 regulates salt stress response and interacts with a MA3 domain-containing protein ECIP1 in Arabidopsis. Plant Cell Environ 34: 1678–1692.
- 45. Ishikawa T, Watanabe N, Nagano N, Kawai-Yamada M, Lam E (2011) Bax inhibitor-1: a highly conserved endoplasmic reticulum-resident cell death suppressor. Cell Death Differ 18: 1271–1278.
- 46. Watanabe N, Lam E (2009) Bax Inhibitor-1, a conserved cell death suppressor, is a key molecular switch downstream from a variety of biotic and abiotic stress signals in plants. Int. J. Mol. Sci 10: 3149–3167.
- 47. Chen XH, Yu H, Deng HJ, Chen JX, MI HB, et al. (2013) Cucumber BAX inhibitor-1, a conserved cell death suppressor and a negative programmed cell death regulator under cold stress. Biol Plantarum 57: 684–690.
- 48. Cao ZH, Zhang SZ, Wang RK, Zhang RF, Hao YJ (2013) Genome wide analysis of the apple MYB transcription factor family allows the identification of MdoMYB121 gene conferring abiotic stress tolerance in plants. PLOS ONE 8: e69955.
- 49. Reyes JL, Chua NH (2007) ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J 49: 592–606.
- 50. Seo PJ, Xiang F, Qiao M, Park JY, Lee YN, et al. (2009) The MYB96 transcription factor mediates abscisic acid signaling during drought stress response in Arabidopsis. Plant Physiol 151: 275–289.
- 51. Xie Z, Li D, Wang L, Sack FD, Grotewold E (2010) Role of the stomatal development regulators FLP/MYB88 in abiotic stress response. Plant J 64: 731–739.
- 52. Zhang L, Zhao G, Xia C, Jia J, Liu X, et al. (2012) A wheat R2R3-MYB gene, TaMYB30-B, improves drought stress tolerance in transgenic Arabidopsis. J Exp Bot 63: 5873–5885.
- 53. Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15: 247–258.
- 54. Niu CF, Wei W, Zhou QY, Tian AG, Hao YJ, et al. (2012) Wheat WRKY genes TaWRKY2 and TaWRKY19 regulate abiotic stress tolerance in transgenic Arabidopsis plants. Plant Cell Environ 35: 1156–1170.
- 55. Zhu X, Liu S, Meng C, Qin L, Kong L, et al. (2013) WRKY transcription factors in wheat and their induction by biotic and abiotic stress. Plant Mol Biol Rep 31: 1053–1067.
- 56. Arora R, Agarwal P, Ray S, Singh AK, Singh VP, et al. (2007) MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics 8: 242.
- 57. Puig J, Meynard D, Khong GN, Pauluzzi G, Guiderdoni E, et al. (2013) Analysis of the expression of the AGL17-like clade of MADS-box transcription factors in rice. Gene Expr Patterns 13: 160–170.
- 58. Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53: 247–273.
- 59. Wan D, Li R, Zou B, Zhang X, Cong J, et al. (2012) Calmodulin-binding protein CBP60g is a positive regulator of both disease resistance and drought tolerance in Arabidopsis. Plant Cell Rep 31: 1269–1281.
- 60. Ranty B, Aldon D, Galaud JP (2006) Plant calmodulins and calmodulin-related proteins: multifaceted relays to decode calcium signals. Plant Signal Behav 1: 96–104.
- 61. Morris PC (2001) MAP kinase signal transduction pathways in plants. New Phytol 151: 67–89.
- 62. Singh A, Kanwar P, Pandey A, Tyagi AK, Sopory SK, et al. (2013) Comprehensive genomic analysis and expression profiling of phospholipase C gene family during abiotic stresses and development in rice. PLOS ONE 8: e62494.
- 63. Triantaphylidès C, Havaux M (2009) Singlet oxygen in plants: production, detoxification and signaling. Cell 14: 1360–1385.
- 64. Kohli A, Sreenivasulu N, Lakshmanan P, Kuman PP (2013) The phytohormone crosstalk paradigm takes center stage in understanding how plants respond to abiotic stresses. Plant Cell Rep 32: 945–957.
- 65. Peleg Z, Blumwald E (2011) Hormone balance and abiotic stress tolerance in crop plants. Curr Opin Plant Biol 14: 290–295.
- 66. Santner A, Calderon-Villalobos LIA, Estelle M (2009) Plant hormones are versatile chemical regulators of plant growth. Nature Chem Biol 5: 301–307.
- 67. Santino A, Taurino M, De Domenico S, Bonsegna S, Poltronieri P, et al. (2013) Jasmonate signaling in plant development and defense response to multiple (a)biotic stresses. Plant Cell Rep 32: 1085–1098.
- 68. Bleecker AB (2000) Ethylene: A gaseous signal molecule in plants. Annu Rev Cell Dev Biol 16: 1–18.
- 69. Licausi F, Ohme-Takagi M, Perata P (2013) APETALA2/Ethylene responsive factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs. New Phytol 199: 639–649.
- 70. Teale WD, Paponov IV, Palme K (2006) Auxin in action: signaling, transport and the control of plant growth and development. Nature Rev 7: 847–859.
- 71. Pérez AC, Goossens A (2013) Jasmonate signaling: a copycat of auxin signaling? Environ 36: 2071–2084.
- 72. Boatwright J, Pajerowska-Mukhtar K (2013) Salicylic acid: an old hormone up to new tricks. Mol Plant Pathol 14: 623–634.
- 73. Cutler SR, Rodriguez PL, Finkelstein RR, Abrams SR (2010) Abscisic acid: emergence of a core signaling network. Annu Rev Plant Biol 61: 651–679.
- 74. Davière JM, Achard P (2013) Gibberellin signaling in plants. Dev 140: 1147–1151.
- 75. Richards DE, King KE, Ait-ali T, Harberd NP (2001) How gibberellin regulates plant growth and development: a molecular genetic analysis of gibberellin signaling. Annu Rev Plant Physiol 52: 67–88.
- 76. Harb A, Krishnan A, Ambavaram MMR, Pereira A (2010) Molecular and physiological analysis of drought stress in Arabidopsis reveals early responses leading to acclimation in plant growth. Plant Phys 154: 1254–1271.
- 77. Damarajua S, Schledeb S, Eckhardta U, Loksteinb H (2011) GrimmaB (2011) Functions of the water soluble chlorophyll-binding protein in plants. J Plant Phys 168: 1444–1451.
- 78. Gorantla M, Babu PR, Reddy Lachagari VB, Reddy AMM, Wusirika R, et al. (2007) Identification of stress-responsive genes in an indica rice (Oryza sativa L.) using ESTs generated from drought-stressed seedlings. J Exp Bot 58: 253–265.
- 79. Guha A, Sengupta D, Reddy AR (2013) Polyphasic chlorophyll a fluorescence kinetics and leaf protein analyses to track dynamics of photosynthetic performance in mulberry during progressive drought. J Photochem Photobiol B 119: 71–83.
- 80. Kawasaki S, Miyake C, Kohchi T, Fujii S, Uchida M, et al. (2000) Responses of wild watermelon to drought stress: accumulation of an ArgE homologue and citrulline in leaves during water deficits. Plant Cell Physiol 41: 864–873.
- 81. Kusvuran S, Dasgan HY, Abak K (2013) Citrulline is an important biochemical indicator in tolerance to saline and drought stresses in melon. Scientific World J. 2013 ID:253414.
- 82. Dasgan HY, KusvuranS, Abak K, Leport L, Larher F, et al. (2009) The relationship between citrulline accumulation and salt tolerance during the vegetative growth of melon (Cucumis melo L.). Plant Soil Environ 55: 51–57.