Molecular framework under water deficit
Considering that drought-resistant cultivars are not readily available for commercial cotton production, more effort is required to identify or generate traits to withstand soil water deficit stress. In cotton production systems, rain-free periods occurring during reproductive growth are especially damaging. In this study, our objective was to understand a molecular architecture of water deficit stress at the level of gene expression by analyzing differentially expressed transcripts in the field during reproductive growth. Therefore, differentially expressed TDFs identified here can be considered genes functionally active (in case of up-regulation) or inactive (in case of down-regulation) at the stage of adaptation to naturally occurring water deficit stress. Our study revealed more than 500 transcripts with altered gene expression levels. The number of down-regulated genes was 1.5X higher than the number of up-regulated genes. Down-regulated genes occurred more frequently in root tissues. The majority of TDFs with altered expression belonged to functional categories including metabolism, stress/defense, signal transduction, and gene regulation as well as the category unclassified. This result corresponds well with previous findings of other species demonstrating transcriptome alteration under water deficit stress [12, 13].
In terms of cotton, a recent microarray expression profiling experiment conducted with plants in a greenhouse reported a total of 2,106 stress responsive-transcripts . The majority of transcripts showed tissue specific expression and a higher number of stress responsive-transcripts were identified in leaf compared to root. In comparison, our study with field grown plants identified fewer stress responsive-transcripts (519), of which, 215 were leaf specific, 293 were root specific, and 11 were expressed in both leaf and root. The candidate gene targets identified in both studies require future work to determine their potential application to improve cotton water use.
Our expression data suggests the involvement of the ROS related defensive pathway since we identified 10 TDFs related to anti-oxidation mechanisms. The increased level of ROS, driven by water deficit stress, can affect cellular components that are oxidized partially or severely [43, 44]. Therefore, it is critical that plants protect themselves from harmful oxidations with detoxifying mechanisms by using antioxidants and scavenging agents . DST (Drought and Salt Tolerance), a previously unknown zinc finger protein, was found as a negative regulator of drought and salt stress by repressing H2O2 accumulation and stomatal closure in an abscisic acid (ABA)-independent manner . In addition, recent studies on developmental and stress-induced cellular processes suggest that ROS and callose deposition are co-regulated, thereby controlling the cell wall matrix adjacent to the plasmodesmata for intercellular redox signal transduction . In our study, one TDF (17-1A09) encoding a callose synthase-like family protein was isolated as a down-regulated gene and many ROS-related TDFs showed up regulation (seven TDFs) or down-regulation (three TDFs). It is of interest to note that this plasmodesmata-related co-regulation of ROS and callose represents a cellular mechanism in the cotton fiber elongation process leading to water uptake following decreased osmotic potential in elongating fiber cells . These findings highlight a possible interconnection between ROS and callose deposition in the areas adjacent to plasmodesmata that cotton employs in response to water deficit stress.
Previous studies have shown that phosphoethanolamine N-methyltransferase, vacuolar invertase, and aldo/keto reductase (as identified in this study as TDFs 07B12, A05D02, and A04A09, respectively) are involved in water deficit stress-related defense mechanisms [48–50]. When silenced, the decrease of phosphoethanolamine N-methyltransferase produced not only multiple growth defects but also temperature sensitive male sterility in Arabidopsis. The vacuolar invertase was shown to be related to water deficit stress in maize . Aldo/keto reductase catalyzes the detoxification reaction of reactive aldehyde groups generated by abiotic stresses thereby providing plants with stress tolerance .
It is well known in many plant species that ABA acts as a key hormone in the abiotic plant stress response [51, 52]. Upon water deficit stress, stomata in leaves are closed and prevent water loss through transpiration. This process is believed to be regulated by ABA [53, 54]. Interestingly, we did not identify any gene related to biosynthesis or action mechanisms of ABA nor genes involved in the regulation of stomata . However, besides the function of ABA in stomatal closure, it was recently shown in Arabidopsis and tobacco that interactions possibly occur between water deficit-responsive proteins and HSP chaperones [56, 57]. Under water deficit conditions, both HSP70 and HSP90 chaperones were recruited to control stomatal closure, thereby serving as machinery important for stomatal gating. The finding of HSP-coding TDFs also suggests the existence of the HSP related machinery in water deficit stress in cotton. In our study, five HSP-coding TDFs were up-regulated while only one showed down-regulation. Previously, a drought-related alpha-crystalline HSP was identified by differential screening from 10-d drought-stressed G. arboreum cotton seedlings and was effective in providing tetraploid cotton plants with drought stress tolerance when over-expressed [58, 59]. The potential involvement of HSP-related drought tolerance was also suggested recently in a microarray transcriptome analysis in drought-stressed cotton plants .
In accordance with transpiration, molecular mechanisms underlying water uptake and transport are pivotal throughout the plant body. There is accumulating evidence that addresses the relationship between aquaporins and transport of water in various physiological conditions including water deficit stress [60–64]. Aquaporins are frequently identified as water deficit stress responsive genes in diverse plant species [65–67]. In roots of drought stressed chickpea plants, members of the aquaporin gene family appeared up- and down-regulated suggesting that a complex regulation of water status governs plant growth and development through aquaporin activity under water deficit . The identification of two homologous TDFs (06 C03 and 06E09) with similarity to members of the aquaporin water-channel protein family in this study (Table 2 and Additional file 1) indicate the possible involvement of cotton aquaporins under water deficit stressed leaf and root tissues. Recently, the identification of the large cotton aquaporin family illustrated their diverse function . In addition to their fundamental role in intercellular transport of water molecules across the plant body, reports have shown the significance of aquaporins in facilitating leaf CO2 conductivity relevant to plant photosynthetic capacity [69–71]. However, the function of aquaporins in water deficit stress tolerance remains unclear as aquaporin genes have not been identified in water deficit stress QTL studies to date . Therefore, more supportive and quantitative data need to follow.
After sequencing 366 excised differentially expressed fragments, we functionally annotated 147 TDFs following sequence verification. Of the 147, we described a number of water deficit stress-responsive genes functionally relevant to metabolism, signal transduction, gene regulation, and stress/defense mechanisms in root and leaf tissues (Figure 3). In addition, homology searches using BlastX did not classify 24.5% of TDFs due to lack of sequence similarity to known proteins. Among those, twenty-seven unclassified TDFs were differentially expressed in root tissue. The abundance of unclassified TDFs identified in this study provide additional transcriptome coverage not represented in EST populations commonly used in microarray experiments. It was reported that unknown genes such as proteins with obscure functions (POF) cover more than 20% of each new genome sequenced with many being species specific .
In maize seedlings with water shortage, 5 – 11% of genes were differentially expressed across an array of genetically diverse inbred lines. Also, while many of the genes were not repeatedly identified in different maize lines, more than 40% of the cellular pathways were shared across all the lines examined . In our study, a similar percentage of genes (13%) showed expression level changes upon water deficit stress. Since the genotype in this study is believed to be drought tolerant, it would be interesting to determine if biological pathways highlighted in this study (for example, ROS-, or HSP-related defense mechanisms) would appear in common across an array of cotton genotypes. Transcripts involving the HSP-containing functional group were also identified in a microarray based, water deficit stress response gene expression study using another cotton cultivar, FiberMax 989 . The few water deficit stress-related cotton genes identified in the current study could be used in candidate gene-focused association mapping approaches to identify QTL under drought stress. This approach was previously shown to identify SNPs associated with ABA and sugar levels under water deficit .