Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

Faiza Tawab; Iqbal Munir; Zeeshan Nasim; Mohammad Sayyar Khan; Saleha Tawab; Adnan Nasim; Aqib Iqbal; Mian Afaq Ahmad; Waqar Ali; Raheel Munir; Maria Munir; Noreen Asim

doi:10.1371/journal.pone.0244030

. 2020 Dec 17;15(12):e0244030. doi: 10.1371/journal.pone.0244030

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

Faiza Tawab ¹, Iqbal Munir ^1,^*, Zeeshan Nasim ¹, Mohammad Sayyar Khan ², Saleha Tawab ³, Adnan Nasim ³, Aqib Iqbal ¹, Mian Afaq Ahmad ¹, Waqar Ali ⁴, Raheel Munir ¹, Maria Munir ¹, Noreen Asim ²

Editor: Keqiang Wu⁵

PMCID: PMC7746274 PMID: 33332435

Abstract

Abiotic stresses especially salinity, drought and high temperature result in considerable reduction of crop productivity. In this study, we identified AT4G18280 annotated as a glycine-rich cell wall protein-like (hereafter refer to as GRPL1) protein as a potential multistress-responsive gene. Analysis of public transcriptome data and GUS assay of pGRPL1::GUS showed a strong induction of GRPL1 under drought, salinity and heat stresses. Transgenic plants overexpressing GRPL1-3HA showed significantly higher germination, root elongation and survival rate under salt stress. Moreover, the 35S::GRPL1-3HA transgenic lines also showed higher survival rates under drought and heat stresses. GRPL1 showed similar expression patterns with Abscisic acid (ABA)-pathway genes under different growth and stress conditions, suggesting a possibility that GRPL1 might act in the ABA pathway that is further supported by the inability of ABA-deficient mutant (aba2-1) to induce GRPL1 under drought stress. Taken together, our data presents GRPL1 as a potential multi-stress responsive gene working downstream of ABA.

Introduction

Being sessile in nature the plants are continuously exposed to different kinds of environmental stresses, including elevated level of salinity, drought and intense temperature [1] that causes reduction in crop productivity and pose a major threat to global food security [2]. The adverse effects of these abiotic stresses are further worsening by climate change, persistent reduction in the arable land and limiting water resources [3,4]. Extensive research in model plants and crops has aimed to understand the responses of plants to various biotic and abiotic stresses [5]. To meet the demands of growing population and varying climatic conditions of the globe there is a great urge to increase the global food production, consequently the demand of stress-tolerant crop varieties has been increased than in the past [6,7].

To cope with stress condition, plants have developed numerous adoptive strategies which include physiological, morphological, molecular and biochemical responses [8]. Plants have evolved several molecular mechanisms for adaptation and subsequent survival under stress conditions as revealed by stress induced transcriptomics. Hundreds of plant genes are differentially regulated in response to abiotic stresses, as demonstrated by RNA-seq analyses. A variety of stresses has been extensively studied in Arabidopsis thaliana [9]. RNA-seq has been proven as a powerful method for understanding the complex regulation networks and gene expression in many plant species responding to several kinds of biotic and abiotic stresses, such as Chinese cabbage [10], chickpea [11], maize [12], potato [13], Ammopiptanthus mongolicus [14] and soybean [15].

To mitigate the negative effects of these stresses, plants have evolved a sophisticated system of stress sensors, signaling transduction pathways and transcription factors (TFs) [16,17]. Over the past several years, the functional roles of glycine-rich proteins (GRPs) in the stress response have been investigated, and some members of this family in Arabidopsis have been shown to enhance seed germination and seedling tolerance to cold stress [18]. A large number of glycine rich proteins (GRPs) are also modulated by biotic and abiotic factors [19]. GRPs are also involved in responses to water stress, including drought and water logging. The grain yield of rice (Oryza sativa) during drought stress was improved following the incorporation of AtGRP2 and AtGRP7 into the rice genome [20].

In this study, we identified a novel gene GRPL1 (AT4G18280) that is annotated as a glycine-rich cell wall protein-like protein in Arabidopsis thaliana by using publicly available transcriptomic data of plants exposed to different abiotic stresses. The co-regulation of AT4G18280 with high-affinity K+ 5 (HAK5) under K⁺ deficiency and salt stress has been reported [21]; however, no attempts were made to functionally characterize this gene. In this study, we characterized GRPL1by generating overexpression lines and its performance analyses under different abiotic stresses. Overpopulation of GRPL1 results in increased tolerance to salt, drought and heat stresses in Arabidopsis thaliana. Manipulating the orthologues of this gene in economical important crops can be helpful to ensure the global food security under changing environmental conditions.

Materials and methods

RNA-seq public datasets retrieval and analysis using Tuxedo protocol

To gain insights of transcriptome wide genes expression, we selected publicly available transcriptome datasets of Arabidopsis thaliana plants based on the criteria that the samples used should be of similar developmental stage and ideally the level and duration of the specific abiotic stress should be similar. Based on this criterian, SRA009031 [22], GSE72806 [23], and SRP035234 [24] of Arabidopsis thaliana (Col-0) were downloaded and analyzed with the Tuxedo protocol [25]. Briefly, the raw reads were quality assessed, adopter trimmed and then aligned with TOPHAT2 followed by the transcript assembly with Cufflinks and differential gene expression analysis with Cuffdiff2. Downstream analysis and visualization of differentially expressed genes (DEGs) were done with custom R and Python scripts. Common target genes of different stresses were determined using Venny2 (available at: bioinfogp.cnb.csic.es/tools/venny).

Plant material, growth conditions and stress response analyses

For all experiments Columbia accession (Col-0) of Arabidopsis thaliana was used. Two weeks-old plants grown under controlled conditions (16:8h day/night) were used for stress response experiments.

Expression analysis

For transcript level expression of genes and validation of RNA-seq data, RNA was extracted from Arabidopsis seedlings using TRI Reagent (Sigma), and cDNA was synthesized from total RNA (2 μg) using Superscript-II reverse transcriptase (Invitrogen). Quantification was performed using LightCycler® 480 instrument. qPCR experiments were performed in three technical and biological replicates each. The average Ct values obtained from the qPCR reaction were used to calculate the relative quantity (RQ) for reference and gene of interest using the equation given below:

Relative Quantity (RQ) = E^{(Ct[control]–Ct[treatment])}
Where: E = Primer efficiency
T = Target gene
The relative quantities were then used to estimate the relative expression of the target gene using the equation:
Normalized Expression = RQ_sample(GOI)/RQ_sample(REF)
Where: GOI = Gene of interest
REF = Reference gene

Gene expression was normalized to Ubiquitin. Whereas, the protein gel blot was used for detection of HA-epitope-tagged proteins in the overexpression lines. For protein gel blot, anti- HA monoclonal antibody (Agrisera) was used. Ponceau staining was used as loading control.

PGRPL1::GUS cloning and GUS assay

To check the promoter activity of GRPL1 promoter, we cloned 1,758bp upstream region of GRPL1 translation start site using Infusion cloning system. Briefly, the pBI101 vector was linearized using SmaI restriction enzyme whereas the promoter region was amplified using a high fidelity pfu DNA polymerase using the primers mentioned in (S1 Table). The GUS assay was performed on the stable T₃ transgenic lines as described earlier [26]. Briefly, the samples were fixed with 90% acetone for 20min, washed and treated with staining buffer (containing X-Gluc) and incubated at 37°C for 2h. After the removal of staining buffer and washing with ethanol the samples were observed under light microscope.

Generation of over expression lines

The potential multi-stress tolerance AT4G18280 gene was amplified with gene specific primers (S1 Table) and cloned into a pUC19 background based binary vector containing a 35S Cauliflower Mosaic fused Virus (CaMV) promoter. Overexpression of GRPL1 was confirmed at transcript level via qPCR (S1 Table) and at protein level by western blot of the stable transgenic lines.

Stress assays

To access the functionality of AT4G18280 as a potential multi stress tolerance candidate, performance of the overexpression lines of T₄ generation under salt, drought and heat stress was determined in comparison with wild-type plants. Salt stress was induced by addition of 100 and 200mM NaCl to the MS plates. Wild type and over expression lines were evaluated for fresh weight, germination percentage, relative root elongation, chlorophyll contents, proline content, melondialdehyde content (MDA) and survival rate (%). After completion of stress period, seedlings (10 plants/genotype) were taken from control and stressed pots and their fresh weights (mg/plant) were determined using a digital balance. For germination test, differences in the seeds germination under salt stress was recorded on the third day of transfer to growth chamber (22°C). Relative root elongation (%) was measured as the percentage of root elongation under salt stress normalized to the control condition. Chlorophyll contents from leaves of the plants treated with 100 and 200 mM NaCl were quantified using a spectrophotometer (U-2810, Hitachi). After extraction of the supernatant from samples homogenized in acetone the chlorophyll a and b were determined in totality in mg/g FW using the methodology described earlier [27]. Proline contents (μg/g FW) were quantified using the method reported earlier with minor modifications [28]. Melondialdehyde (MDA) content was measured by obtaining the supernatant from leaf samples of control and stressed plants through series of steps and then the obtained supernatant was used to measure the OD at 532, 600, and 450 nm and the MDA content was calculated using the method described by Zhang et al. [29].Survival rate (%) was determined by subjecting two week-old seedlings for NaCl (200 mM) treatment followed by calculating the alive plants percentage as described earlier [30].

Drought stress tolerance analysis was done by calculating the survival rate and water loss. Survival percentage was measures by subjecting 14-day old soil grown plants (WT and over-expression lines) to dehydration by withholding water supply for 20 days and then re-watered. Survival percentages were recorded 6 days after the recovery and experiment was repeated thrice [31]. Water loss analysis from detached leaves was performed as described earlier [32]. Briefly, ~0.5 g rosette leaves of three weeks old plants were weighed (FW₀) and incubated at 23°C. The samples were then reweighed at 1, 2, 3, 4, and 5 hours (FW_i), whereas the dry weight (DW) was measured by drying the detached leaves at 80°C for 5 hr. The water loss was calculated based on the initial fresh weight of plants using the formula as:

[(FW_i-DW)/(FW₀-DW)] ×100 Where FW₀ and FW_i are fresh weight for original and any given interval fresh weight, respectively, and DW is dry weight.

For heat stress tolerance analysis, the survival rate was measured after subjecting the plants to heat stress of 45°C for 1 hr.

Statistical analysis

Data of the physiological and biochemical parameters between the transgenic and non-transgenic lines were analyzed in triplicate through analysis of variance (ANOVA) using student t-test.

Results

Identification of GRPL1 as a multi-abiotic stress-responsive gene

Adverse environmental conditions have been a major threat to global food security. The condition got worsens with the persistent reduction in the arable land, limiting water resources and climate change trends [33]. Therefore, we aimed to utilize the publicly available transcriptome data i.e. SRA009031 [22], SRP035234 [24] and GSE72806 [23] of plants exposed to different abiotic stresses in order to identify potential stress tolerance genes. Our analyses of these public transcriptome datasets identified a total of 760 genes that were commonly up regulated in response to heat, drought, salt and the combined heat and drought stress (Fig 1A and S2 Table). The top 10 multi-stress-induced genes of that list contained some heat shock proteins along with other known abiotic stress-responsive genes (Fig 1A). However, we selected GRPL1 i.e. AT4G18280 as a candidate because it showed strong induction in response to these abiotic stresses and was not studied earlier in the context of multi abiotic stress tolerance (Fig 1A). The expression level of GRPL1 in response to different abiotic stresses is shown in Fig 1B. Though, all tested abiotic stresses showed induction of GRPL1, the PEG-induced osmotic and salt stress showed comparably higher levels of GRPL1 mRNA than heat stress (Fig 1B). Consistent with the RNA-seq results, our qPCR analyses showed similar expression pattern for the given abiotic stresses (Fig 1C). Precisely, the mRNA levels of GRPL1gene was up-regulated by ~5.8 folds under salt stress, ~6.1 folds in the drought stress and ~4.1 folds in heat stress (Fig 1C). To further confirm this induction of GRPL1 by abiotic stress-induced, we generated promoter GRPL1-driven GUS transgenic lines (pGRPL1::GUS). pGRPL1::GUS seedlings showed weak GUS staining under control conditions, indicating the weak promoter activity of GRPL1 (Fig 1D), however, under abiotic stresses, we observed strong induction of pGRPL1 promoter activity represented by the strong GUS staining. Overall, these results show that transcription of GRPL1is strongly induced under abiotic stresses, suggesting a potential role of GRPL1in modulating plant’s response to abiotic stresses.

Fig 1 — (A) Commonly up-regulated genes in response to multiple abiotic stresses. Table showing the top 10 upregulated genes among the total 760 genes. (B)Expression of *GRPL1* under drought, heat, salt and combined stress of heat and salinity, derived from public RNA-seq datasets. (C) qPCR validation of *GRPL1* induction under abiotic stresses (D) *pGPRL1*::*GUS* transgenic lines under control, drought, heat and salt stresses. Scale bar = 0.1 cm.

Cloning and expression analysis of AT4G18280

To validate the potential role of GRPL1 in abiotic stress response, we generated transgenic plants overexpressing HA-tagged GRPL1.First, the overexpression was analyzed at the transcript level by RT-PCR and at protein level by protein gel blot. Indeed, the three independent transgenic lines 1–4, 4–3 and 6–2 showed higher mRNA (Fig 2A) and protein accumulation (Fig 2B). Also, the transgenic plants overexpressing GRPL1 showed better growth and had significantly higher fresh weight compared to the WT plants (Fig 2C and 2D). These results confirmed the overexpression of GPRL1 at both transcript and protein levels. The confirmed overexpression lines were tested under salt, drought and heat stress conditions to confirm their tolerance level.

Fig 2 — Confirmation of *GRPL1* overexpression through RT-PCR (A) and western blot (B). (C) Phenotype of *35S*::*GRPL1-3HA* transgenic lines. Scale bar = 1 cm. (D) Fresh weight comparison of WT and *35S*::*GRPL1-3HA* lines. 10 plants from each genotype were weighed.

Function of AT4G18280 overexpression under salt, drought and heat stress

To assess the effect of GRPL1overexpression, the transgenic lines were tested under salt, drought and heat stress conditions. As salinity is one of the major abiotic stresses that compromises crop productivity [34], we evaluated the performance of overexpression lines under 100 mM and 200 mM salt stress. Under control conditions, all lines showed 96–100% germination (Fig 3A). However, the WT seeds grown on MS media supplemented with 100 mM and 200 mM NaCl showed reduced germination of ~55% and ~33%, respectively. Whereas, the overexpression lines showed better germination under both concentrations of salt stress i.e. about 75–85% of seeds successfully germinated under low (100 mM) while ~50–60% seeds germinated under high concentration (200 mM NaCl) salt stress (Fig 3A).

Fig 3 — (A) Germination of three independent overexpression lines and WT seeds under control and under 100 and 200mM salt stressed conditions. (B) Relative root elongation and (C) Chlorophyll contents of *35S*::*GRPL1-3HA* lines in response to salt stress. (D) Higher accumulation of proline contents in *GRPL1* overexpression lines upon exposure to salinity stress. (E) Quantification of MDA contents and (F) Survival percentage of WT and transgenic plants exposed to salt stress.

Consistent with the previous reports [35], we observed about 65% relative root elongation in WT plants whereas the overexpression lines showed significantly higher root elongation under stress condition i.e. up to 80% relative root elongation (Fig 3B). Salt stress induces production of reactive oxygen species (ROS), which damage the cellular components [36] including chlorophyll [37]. WT plants showed over 50% reduction in the overall chlorophyll contents under salt stress (Fig 3C). However, the GRPL1 overexpressing plants showed significant tolerance towards salt stress as indicated by the reduced degradation of chlorophyll contents (~30–45%), highlighting the tolerance potential of the overexpression lines (Fig 3C). To further test the salt tolerance of GRPL1 overexpression plants, we performed proline and MDA contents quantification. Proline acts as an osmolyte plays important roles in stabilizing macromolecules and membranes in cells by a higher accumulation [38]. The WT plants under salt stress accumulated ~2-fold higher proline contents whereas the overexpression lines showed ~4-fold higher proline accumulation (Fig 3D) implying that the transgenic plants are more tolerant to salt stress. However, performance of the transgenic line 1–4 was somewhat comparable with WT plants. The Melondialdehyde (MDA) level is an indicator of the cellular damage caused by stress conditions [39]. Although the GRPL1-mediated tolerance mechanism is still elusive, there is a possibility that overexpression of GRPL1 may do so by the accumulation of proline and the efficient scavenging of ROS as represented by reduced quantities of MDA in transgenic plants (Fig 3D and 3E). Salinity is a serious threat to plants, disturbing all the physiological processes and even causes death of the plants [34]. Our results demonstrated that the overexpression of GRPL1 enhanced the survival rate of plants under higher dose of salinity (Fig 3F). All of the overexpression lines showed significantly higher survival rates.

Drought stress is also one of the biggest threats to food productivity. To examine the response of overexpression lines to drought stress, we subjected 14 days old seedlings to dehydration as no water was given to the plants for 20 days, followed by re-watering and checking the survival rates. Under water-deficient conditions for 20 days, only 22–27% of WT plants survived, whereas the overexpression lines showed significantly higher survival rates of 34–42% (Fig 4A and 4B). Furthermore, we also found the overexpression plants tend to retain more water contents compared to WT plants. After three hours of dehydration, the overexpression lines lost significantly lower amounts of water compared to WT plants and the difference between water loss got more significant with the increase in dehydration time (Fig 4C). These results suggest that overexpression of GRPL1 results in reduced water loss under drought stress compared to WT plants and hence showed higher survival rates.

Fig 4 — (A) Phenotype of WT and transgenic lines in response to dehydration. (B) Bar graph representing the survival percentage of *GRPL1* overexpression lines in response in drought stress. (C) Line graph showing the water loss comparison between WT and transgenic lines upon exposure to dehydration. (D) Phenotype of WT and *GRPL1* overexpression lines under heat stress. Scale bar = 0.5 cm. (E) Survival percentage of WT and transgenic plants under heat stress.

Re-analysis of public transcriptome data and qPCR analysis revealed induction of GRPL1 under heat stress (Fig 1) which prompted us to check the performance of these overexpression lines under heat stress. Interestingly, the overexpression lines showed significantly higher survival rates compared to WT plants (Fig 4D and 4E) suggesting the possible role of GRPL1 in heat stress response too. However, the role of GRPL1 in heat stress response requires further study.

Tolerance to abiotic stresses in plants is often mediated by ABA [39]. In order to check if GRPL1 acts in the same pathway, we checked the expression of GRPL1 in a collection of public microarray data. Interestingly, GRPL1 showed similar expression patterns with the ABA INSENSITIVE 1 (ABI1), 2, and 5 (Fig 5A), suggesting a possibility that GRPL1 might act in the ABA pathway. To further support this hypothesis, we used public transcriptome dataset (GSE75933) of ABA-deficient (aba2-1) mutant [40]. Reanalysis of RNA-seq data revealed GRLP1 to be induced (~2.4-fold) by drought stress in WT background (Fig 5B), however, such stress treatment induction of GRPL1was compromised in the ABA-deficient mutants (aba2-1). Taken together, these results suggest that GRPL1 may act downstream of ABA signaling.

Fig 5 — (A) Profile of ABA pathway genes and *GRPL1* from a number of public microarray data sets (using ePLANT), *PP2A* was used as a control. (B) Expression of *GRPL1* in WT and *aba2-1* mutants under control and drought stress from the public RNA-seq data under the accession no GSE75933.

Discussion

Plants are continuously exposed to a wide range of biotic and abiotic stresses in their native environment. Extensive research in model plants and crops has aimed to understand the plant responses to a range of biotic and abiotic stresses, as these stresses reduce harvest yields [41]. In response to abiotic stresses, hundreds of plant genes are differentially regulated as demonstrated by RNA-seq analyses. In this study, our basic understanding of the plant acclimation to abiotic stresses was extended through whole transcriptome (RNA-Seq) analysis based on the utilization of the publicly available transcriptome data sets. A total of 760 genes were commonly up regulated in response to heat, drought, salt and the combined heat and drought stress based on our analysis of the selected transcriptomic datasets. In order to identify the potentially multi stress responsive genes, we sorted the genes based on their fold-change values. The top 10 multi-stress-induced genes contained some heat shock proteins along with other known abiotic stress-responsive genes. However, we selected GRPL1 i.e.AT4G18280 as a candidate because it showed strong induction in response to these abiotic stresses and was not studied earlier in the context of multi abiotic stress tolerance. The RNA-seq predicted expression of the selected gene was confirmed by qRT-PCR, which showed that the results of both are significantly comparable to each other, and RNA-Seq results are reliable and can be used for further analysis. These results are consistent with the previous finding that the mRNA levels of glycine rich proteins (GRPs) are affected by different abiotic stresses, in a number of plant species [42].

The selected multi-stress induced gene was over-expressed in Arabidopsis thaliana, and its expression pattern was confirmed at transcript and protein level. The overexpression transgenic Arabidopsis lines with AT4G18280 functional attributes were studied to confirm their role in different stress conditions. Significant differences in the AT4G18280 overexpression lines and wild-type plants were observed under salt, drought and heat stresses. To confirm the tolerance level of over-expressed transgenic plants with AT4G18280 gene under salt stress it was tested for some of the physiological parameters. The biological role of transgenic plants over-expressing the AT4G18280 gene under salt stress was confirmed by measuring the fresh weight (mg/plant). Salt stress causes the reduction in fresh weights due to the relative increase in Na+ concentration by means of ionic stress. In our study, highest fresh weight was shown by all of the overexpression lines as compared to the wild type (Col-0) plants.

Germination is the most feasible approach used for selecting salt tolerance in plants [43]. A number of scientific studies demonstrated the negative effect of increasing dose of salinity on germination percentage of plants [44,45]. In our study, the overexpression lines evaluated for germination rate (%) under control and stress condition. The overexpression lines showed significantly high germination rate under salt stress condition than the wild type plants suggesting that the overexpression lines are able to germinate under salt stress and hence tolerant to reduce the detrimental effect on germination under increase dose of salt stress. This reduction in germination of WT seeds is consistent with the previous reports of salt stress negatively affecting germination rates, most probably by repressing biosynthesis of two phytohormones, gibberellic acid (GA) and salicylic acid (SA) [46,47]. Overexpression of GRPL1 exaggerated germination most probably through biosynthetic regulation of these two phytohormones. Being in direct contact with the soil, root length is considered one of the most important parameters to determine the salt stress tolerance of plants. It is well known that salt stress inhibits root meristem activity, cell cycle, and elongation of root cells in Arabidopsis and other plant species [48,49], resulting in retarded primary root growth [50,51]. It has been demonstrated that high dose of salt stress may reduce the root length by decelerating the water uptake by plants [52]. The relative root lengths determined in our study revealed that under stress condition (100 mM and 200 mM NaCl), the overexpression lines showed more root growth as compared to wild type plants which implies that the roots of overexpression lines are capable to grow even in high dose of salinity and hence are more tolerant. While similar pattern of root length was observed for overexpression lines as well as wild type plants under normal growth conditions. Soil salinity is a serious threat to plants, disturbing all the physiological processes and even causes death of the plants [34]. To know if the AT4G18280 overexpression can rescue the low survival rate, survival rates were recorded for both the overexpression transgenic lines and wild-type plants after exposure to a salt stress of 200 mM NaCl. Our results showed that the overexpression lines were able to survive under higher dose of salinity as compared to wild type control plants and hence they are more tolerant to salinity.

Free proline content can increase upon exposure of plants to drought, salinity, cold, heavy metals or certain pathogens. Determination of free proline levels is a useful assay to monitor physiological status and to assess stress tolerance of higher plants [53]. Our results demonstrated the increase in proline content by all of the overexpression lines as compared to the wild type plants suggesting that they are more tolerant to salt stress. A large number of scientific studies have been conducted in Arabidopsis thaliana by overexpressing the native or antonym genes that resulted in higher proline content accumulation [54]. Different plants produce higher free proline levels in response to salinity and many scientists have cited the potential roles of proline such as stabilizing proteins, osmolyte, scavenging of hydroxyl radicals and regulating cytosolic pH [55–57]. It is known that salt stress causes membrane lipid peroxidation which is used as an indicator for salt stress induced oxidative membranes damage [58]. It is therefore used as an important criterion for plant stress tolerance evaluation under stress condition. In our study the melondialdehyde (MDA) content was determined both in control and salt stress conditions. Under salt stress condition all of the overexpression lines showed significantly lower MDA content as compared to the wild type plants. This imply that the transgenic plants over-expressing the AT4G18280 gene confers tolerance to salt stress as they provide better protection against the oxidative damage. Plant tolerant to salt stress would accumulate less MDA contents compared to the susceptible plants [59]. Our results are consistent with the previous reports of reduced MDA accumulation in tolerant transgenic plants under salt and osmotic stresses [59]. Salt stress has been demonstrated to damage the chlorophyll contents of plants and hence reduces the photosynthesis of plants in different plant species [60,61]. In this study the overexpression lines and control plants were evaluated for chlorophyll content (mg/g FW) under control and salt stress condition. The overexpression lines maintain the chlorophyll content at high dose (200 mM NaCl) of salinity which means that they can withstand in saline environment by maintaining its chlorophyll content and hence more tolerant. Prolonged drought stress causes dehydration and a result death of the plants. To know the role of AT4G18280 overexpression under drought stress, the overexpression lines with AT4G18280 gene were tested for the survival rate. Significantly higher survival rate was recorded for all of the overexpression lines as compared to wild type control plants under drought stress. These results imply that the overexpression of Arabidopsis with AT4G18280 gene results in greater tolerance to drought stress than the wild-type (Col-0) plants. Further, to support that AT4G18280 overexpression is indeed involved in providing drought tolerance to transgenic plants, they were tested for water loss at different intervals of dehydration. Our results showed that all of the overexpression lines were able to maintain its water content over increasing hours of dehydration. Significantly less water loss was recorded for the overexpression lines as compared to wild-type control plants; suggests that they are more tolerant to drought conditions.

After knowing the potential role of the over-expressed transgenic Arabidopsis plants with AT4G18280 gene in salt and drought stress, it was evaluated to confirm its role in heat stress as well. As the heat stress is lethal to plants, causing a number of changes in plant’s metabolism and even causes the mortality in plants. For this, the survival rate of the overexpression lines and wild type plants were recorded after subjected to heat stress of 45°C for 1hr. The survival rate recorded for all the overexpression lines was significantly higher than the wild type plants which means that they are able to withstand heat stress and hence more tolerant.

The expression of GRPL1 was checked in a collection of public microarray data and interestingly, GRPL1 showed similar expression pattern with the ABA INSENSITIVE 1 (ABI1), 2, and 5, suggesting a possibility that GRPL1 might act in the ABA pathway. This hypothesis was further supported by analyzing public transcriptome dataset (GSE75933) of ABA-deficient (aba2-1) mutant [40]. GRPL1 was highly induced under drought stress in WT background, however, such stress treatment induction of GRPL1was compromised in the ABA-deficient mutants (aba2-1). ABA2 gene is an alcohol dehydrogenase and mutation in this gene results in blockage of xanthoxin to ABA aldehyde, a key step in ABA-biosynthesis resulting in low ABA production. ABA positively regulates the induction of drought-inducible genes. The signaling mechanism is well-known [62]. Briefly, ABA inhibits the PP2C-dependent negative regulation of the SnRK2 protein kinase [63], as a result the SnRK2 can phosphorylate its downstream target transcription factors (for instance ABFs) which in turn induces the target genes. The fact that the ABA-deficient mutant is unable to induce the expression of GRPL1 in response to drought stress suggests that ABA is required for the induction of GRPL1 and that it acts downstream of ABA. Taken together; these results suggest that GRPL1 may act downstream of ABA signaling, however, further experiments are required to validate this hypothesis.

Conclusions

In conclusion, using a number of publicly available transcriptomic datasets and transgenic approaches, we identified and characterized a previously unknown GRPL1 as an abiotic stress-responsive gene. Constitutive expression of GRPL1 in Arabidopsis increased plant’s tolerance to salt, drought and heat stresses. Further understanding of the GRPL1function could be helpful in the devising breeding strategies for abiotic stress tolerance and can contribute to the global food security.

Supporting information

S1 Fig

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S2 Fig

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S1 Table. Primers used for RT-PCR.

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S2 Table. List of commonly up-regulated genes in the selected datasets.

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Click here for additional data file.^{(39.9KB, docx)}

Acknowledgments

Authors are thankful to the contributions of each member of the Biochemical and Analytical Division in experiments, data analysis and manuscript preparation.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0244030.r001

Decision Letter 0

Keqiang Wu

25 Sep 2020

PONE-D-20-26347

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

PLOS ONE

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Reviewer #1: Partly

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors carried out re-analysis of three publically available RNA seq datasets and zeroed in on At4g18280 (here named as GRPL1). The manuscript described the physiological analysis of GRPL1 over expression lines under salt, drought and heat stress. The study does add to knowledge on relatively less explored and functionally uncharacterized protein. However there are several points were this manuscript needs improvements.

My observation and comments are as follows.

• There is too much of redundancy in introduction section. There are many sentences with similar message or meaning, these needs to be removed. The aim of the study needs to be clearly mentioned.

• There is one study on identification and characterization of At4g18280 (here named as GRPL1) ‘Overexpression of a Novel Component Induces HAK5 and Enhances Growth in Arabidopsis’. Adams et al. 2013. The study suggested the role of this gene in pot deficiency and salt stress response. This ref is missing in the whole manuscript.

• It would be better to mention the plant /crop to which the SRA data sets belong. The reason or criteria for selecting only these three data sets out of many available for analysis should also be mentioned.

• Line 159-160. “leaves of three weeks old were excised to analyse the standardized water content”. What is the meaning of standardized water content??

• Line 187-192: As floral dip method is well known method, instead of detailed description just reference is sufficient to mention the transformation method used. Infact the very ref for floral dip is missing. The details of promoter cloning are also absent.

• There is repetition of methods in “Plant Material, Growth Conditions and Stress Treatment” and “Measurement of Physiological and Biochemical Indexes under Salt, Drought and Heat

Stress”. These can be combined under one heading.

• There is discrepancy in the number of days for drought stress imposition (water with holding) in Line no 156 (20 days), 231 (32 days) and 358 (28 days) .

• Result section:

• The authors described GO analysis of DEGs and next section started analyzing expression of AT4G18280 (GRPL1) which is too abrupt and requires explanation as to how the authors zeroed in on AT4G18280 (GRPL1). Moreover the first section “Global Gene …..Abiotic Stresses” details can be made shorter.

• Again there is redundancy in few lines of section 1 and section 2 of results.

• Line no. 277 PEG induces osmotic stress not drought stress. Moreover there is no distinction in the Table (Fig.1a) which is drought and PEG imposed stress.

• Line 295: First, the overexpression was analyzed at the…

• Fig. 2C should have scale bar for comparison of size. For Fig. 2D data from how many plants was recorded (replicates)? In general no of plants /replicates should be mentioned in all experiments

• Instead of “Over-expressed Transgenic Lines”, transgenic lines overexpressing GRPL1 or overexpression lines may be a better term.

• In Fig 3 B and C, over expression line 1-4 did not perform better than Wild type. so this should be mentioned instead of generalizing the observation. After how many days the survival % under salt stress was recorded? Should be mentioned in text.

• Line 350-353: without understanding of the biological function of GRPL1 , these are too presumptive statements.

• Lines 397 -406 these lines should be shifted to section 1 “Global Gene …..Abiotic Stresses” of results.

• Lines 410 -459: Discussion of DEGs is not required as these results might have been discussed by the authors of SRA study. Here authors should focus on how many genes they short listed infact a list of genes should have been more appropriate (in results). And then they can discuss validation of their selected gene GRPL1 by qRT-PCR (lines 459-462).

• Line 474-476: Authors have discussed propidium iodide staining for recording no of cells however there is no data presented. Similarly Line 528-529 electrolyte leakage observation under salt stress is mentioned however no data is given.

• Conclusion can be made more specific and short.

Reviewer #2: Reviewer 1

The author identified a gene GRPL1 which was induced by multiple stresses by querying transcriptome results of multiple stresses. The transgenic Arabidopsis was obtained by overexpression of GRPL1. It was proved that GRPL1 can improve the resistance to high salt, drought and heat stress through resistance identification and analysis of various physiological indexes. It is a new idea to identify genes involved in multiple stress resistance processes. However, the quality of the data in this paper is not high, especially the pictures are not standardized. It is difficult to understand that GRPL1 is regulated by ABA2 gene. Therefore, this paper can not be published in the PLOS ONE at present, and we hope to revise it. Suggestions for revision are as follows:

1. Why didn't we use GRPL1 mutants for phenotypic analysis.

2. When analyzing transcriptome data, the reason why GRPL1 gene was chosen is not clear.

3. Paragraph in P303-308 is too short, so it is suggested to combine with other results.

4. The discussion is too long.

5. There are conjectured results in the conclusion part, which are not supported by data, so it is suggested to remove them.

6. Fig2C is lack of scale, the size of pictures is different, and there is no clear picture of salt tolerance.

7. Fig.4A is not clear.

8. The primers of Tables 1 were incomplete and there were no QRT PCR primers.

9. ABA2 gene is alcohol dehydrogenase. It is difficult to understand why ABA2 mutation affects GRPL1 expression, because ABA2 is not a transcription factor.

**********

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Dec 17;15(12):e0244030. doi: 10.1371/journal.pone.0244030.r002

Author response to Decision Letter 0

13 Nov 2020

Reviewer 1:

1. There is too much of redundancy in introduction section. There are many sentences with similar message or meaning, these needs to be removed. The aim of the study needs to be clearly mentioned.

We thank Reviewer 1 for highlighting the redundancy in introduction section. We revised introduction section of the manuscript and also clearly included the aim of this study.

2. There is one study on identification and characterization of At4g18280 (here named as GRPL1) ‘Overexpression of a Novel Component Induces HAK5 and Enhances Growth in Arabidopsis’. Adams et al. 2013. The study suggested the role of this gene in pot deficiency and salt stress response. This ref is missing in the whole manuscript.

We thank Reviewer 1 for his suggestion, we cited the work of Adams et al., 2013 in the revised version.

3. It would be better to mention the plant /crop to which the SRA data sets belong. The reason or criteria for selecting only these three data sets out of many available for analysis should also be mentioned.

All the RNA-seq datasets belong to Arabidopsis thaliana, this information was added to the material and methods section (line83). A number of facts influenced the selection of these datasets, for instance, we wanted to have datasets that contain at least two or three biological replicates, the samples used should be of similar developmental stage, ideally the level and duration of the specific abiotic stress should be similar, and also our limited computational resources. We added this information in the methodology section (line 83-85).

4. Line 159-160. “leaves of three weeks old were excised to analyse the standardized water content”. What is the meaning of standardized water content??

We thank Reviewer 1 for pointing out this confusion. By standardized water content we meant “initial water content”. However, to make it clearer, we corrected and modified the water loss analysis methodology (line 152-158).

5. Line 187-192: As floral dip method is well known method, instead of detailed description just reference is sufficient to mention the transformation method used. Infact the very ref for floral dip is missing. The details of promoter cloning are also absent.

We agree with Reviewer 1, as the floral dip is a commonly used and well-known procedure, we excluded the detailed description (line 187-191). We added details of promoter length and cloning strategy in the methodology (line 110-113).

6. There is repetition of methods in “Plant Material, Growth Conditions and Stress Treatment” and “Measurement of Physiological and Biochemical Indexes under Salt, Drought and Heat

Stress”. These can be combined under one heading.

As suggested by the Reviewer 1, we combined both “Plant Material, Growth Conditions and Stress Treatment” and “Measurement of Physiological and Biochemical Indexes under Salt, Drought and Heat Stress” sections under one heading of “Plant Material, Growth Conditions and Stress Response Analyses”.

7. There is discrepancy in the number of days for drought stress imposition (water with holding) in Line no 156 (20 days), 231 (32 days) and 358 (28 days) .

We thank Reviewer 1 for pointing out these typos, the correct number of days is 20. We did corrections in line no 231 (line 254 now and 156 (line 150 now).

Result section:

8. The authors described GO analysis of DEGs and next section started analyzing expression of AT4G18280 (GRPL1) which is too abrupt and requires explanation as to how the authors zeroed in on AT4G18280 (GRPL1). Moreover the first section “Global Gene …..Abiotic Stresses” details can be made shorter.

We thank Reviewer 1 for this valuable recommendation, we modified this section and also added more detailed description of selecting GRPL1 as a candidate gene (line 173-180).

9. Again there is redundancy in few lines of section 1 and section 2 of results.

As recommended by Reviewer 1, we combined section 1 and 2 of results

10. Line no. 277 PEG induces osmotic stress not drought stress. Moreover there is no distinction in the Table (Fig.1a) which is drought and PEG imposed stress.

Correction was made to line no 277 (182 now) by changing “drought” to “osmotic”. Distinction was made to the table which now shows the drought and PEG imposed stress.

11. Line 295: First, the overexpression was analyzed at the…

We modified the sentence as suggested by Reviewer 1 (now line no 198)

12. Fig. 2C should have scale bar for comparison of size. For Fig. 2D data from how many plants was recorded (replicates)? In general no of plants /replicates should be mentioned in all experiments

We put the scale bar on each picture. For Fresh weight experiment, we weighed 10 plants for each genotype. We added this information in the figure legend and methodology section.

13. Instead of “Over-expressed Transgenic Lines”, transgenic lines overexpressing GRPL1 or overexpression lines may be a better term.

As per the recommendation of Reviewer 1, “Over-expressed Transgenic Lines” was replaced with “overexpression lines” throughout the text.

14. In Fig 3 B and C, over expression line 1-4 did not perform better than Wild type. so this should be mentioned instead of generalizing the observation. After how many days the survival % under salt stress was recorded? Should be mentioned in text.

AsReviewer 1recommended, we added the information of line 1-4 not performing better than WT plants (232-233). The survival percentage was recorded after two weeks of exposure to salt stress. We modified the text as well (line no 199).

15. Line 350-353: without understanding of the biological function of GRPL1, these are too presumptive statements.

We agree with Reviewer 1 and toned-down our statement as below:

“Although the GRPL1-mediated tolerance mechanism is still elusive, there is a possibility that” (line 234-237)

16. Lines 397 -406 these lines should be shifted to section 1 “Global Gene …..Abiotic Stresses” of results.

We removed the redundant and out of the context part of discussion, to give more focus to significance of GRPL1 in stress tolerance

17. Lines 410 -459: Discussion of DEGs is not required as these results might have been discussed by the authors of SRA study. Here authors should focus on how many genes they short listed infact a list of genes should have been more appropriate (in results). And then they can discuss validation of their selected gene GRPL1 by qRT-PCR (lines 459-462).

We thank Reviewer 1 for this suggestion and modified the discussion part accordingly.

18. Line 474-476: Authors have discussed propidium iodide staining for recording no of cells however there is no data presented. Similarly Line 528-529 electrolyte leakage observation under salt stress is mentioned however no data is given.

We thank Reviewer 1 for pointing out this mistake. We removed the description related to propidium iodide and electrolyte leakage under salt stress.

19. Conclusion can be made more specific and short.

As suggested by Reviewer 1, we modified and shortened the discussion part (413-418)

Reviewer #2:

1. Why didn't we use GRPL1 mutants for phenotypic analysis.

We did check two mutant lines (SALK_112442 and SALK_15781) for GRPL1, but unfortunately both mutant lines only showed a slight knock-down (~5-10%) of GRPL1. These lines have T-DNA insertion in 5′ and 3′ UTRs, respectively. Unfortunately, there are no available lines that has T-DNA insertion in the coding region. Therefore, we excluded them from further experiments.

2. When analyzing transcriptome data, the reason why GRPL1 gene was chosen is not clear.

We thank Reviewer 2 for highlighting this point. We modified the section of results focusing on shortlisting of GRPL1 (line 173-180)

3. Paragraph in P303-308 is too short, so it is suggested to combine with other results.

We thank Reviewer 2 for this valuable suggestion. We combined this section of results with the previous heading.

4. The discussion is too long.

As recommended by the Reviewer 2, we modified the discussion part to make it more concise.

5. There are conjectured results in the conclusion part, which are not supported by data, so it is suggested to remove them.

We modified the conclusion part as recommended.

6. Fig2C is lack of scale, the size of pictures is different, and there is no clear picture of salt tolerance.

We thank Reviewer 2 for pointing this out, we added a scale bar on each picture of Figure 2C. We also updated the picture of salt tolerance (Fig. 4D)

7. Fig.4A is not clear.

We understand the concern of Reviewer 2 but the objective of showing this picture was to show the difference in color. As from the figure one can see that the transgenic lines were still alive (greenish) compared to the wild-type plants (brownish).

8. The primers of Tables 1 were incomplete and there were no QRT PCR primers.

We thank Reviewer 2 for pointing out this mistake, we updated the list of primers in supplementary Table 1.

9. ABA2 gene is alcohol dehydrogenase. It is difficult to understand why ABA2 mutation affects GRPL1 expression, because ABA2 is not a transcription factor.

ABA2 gene is an alcohol dehydrogenase and mutation in this gene results in blockage of xanthoxin to ABA aldehyde, a key step in ABA-biosynthesis resulting in low ABA production. ABA positively regulates the induction of drought-inducible genes. The signaling mechanism is well-known. Briefly, ABA inhibits the PP2C-dependent negative regulation of the SnRK2 protein kinase (Umezawa et al., 2010, PCP), as a result the SnRK2 can phosphorylate its downstream target transcription factors (for instance ABFs) which in turn induces the target genes.

The fact that the ABA-deficient mutant is unable to induce the expression of GRPL1 in response to drought stress suggests that ABA is required for the induction of GRPL1 and that it acts downstream of ABA.

We added this information in Discussion section (line 402-409).

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PLoS One. doi: 10.1371/journal.pone.0244030.r003

Decision Letter 1

Keqiang Wu

23 Nov 2020

PONE-D-20-26347R1

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

PLOS ONE

Dear Dr. Munir,

Please submit your revised manuscript by Jan 07 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

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Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: Line 58 full form of GRPs should be mentioned (when it is mentioned first time)

Ilalics for gene names

Line 65-67 may be deleted

Method of calculation for qRT-PCR should be included in the expression analysis section

Mention vector background for overexpression construct, Generation at which the transgenic lines were analysed

Line 151 the word dessication should be replaced by dehydration or drought

FIG 2C the legend mentions scale bar as 1 cm however the picture does not appear to be in same scale, hence should be otherwise removed

Reviewer #2: (No Response)

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PLoS One. 2020 Dec 17;15(12):e0244030. doi: 10.1371/journal.pone.0244030.r004

Author response to Decision Letter 1

1 Dec 2020

Response to Reviewers

Reviewer #1

Line 58 full form of GRPs should be mentioned (when it is mentioned first time)

We thank Reviewer 1 for highlighting this; the full form of GRPs is now mentioned in line 58.

Ilalics for gene names

All gene names have been italicized.

Line 65-67 may be deleted

Lines 65-67 have been removed.

Method of calculation for qRT-PCR should be included in the expression analysis section.

The qPCR quantification is mentioned in lines 101-111.

Mention vector background for overexpression construct

The vector background is mentioned in line 129.

Generation at which the transgenic lines were analysed

It is mentioned in line 136.

Line 151 the word dessication should be replaced by dehydration or drought

The dessication word is replaced by dehydration in the manuscript.

FIG 2C the legend mentions scale bar as 1 cm however the picture does not appear to be in same scale, hence should be otherwise removed

Although the scale bar represents 1cm, their lengths are different in both images, i.e. the WT’s scale bar of 1cm has an actual size of 0.489cm whereas the one for transgenic plants is 0.351cm.

Attachment

Submitted filename: Response to Reviewer R1.docx

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PLoS One. doi: 10.1371/journal.pone.0244030.r005

Decision Letter 2

Keqiang Wu

2 Dec 2020

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

PONE-D-20-26347R2

Dear Dr. Munir,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Keqiang Wu, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0244030.r006

Acceptance letter

Keqiang Wu

9 Dec 2020

PONE-D-20-26347R2

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

Dear Dr. Munir:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

(TIFF)

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S2 Fig

(TIFF)

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S1 Table. Primers used for RT-PCR.

(DOCX)

Click here for additional data file.^{(13.7KB, docx)}

S2 Table. List of commonly up-regulated genes in the selected datasets.

(DOCX)

Click here for additional data file.^{(39.9KB, docx)}

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Identification and characterization of a novel multi-stress responsive gene in Arabidopsis

Faiza Tawab

Iqbal Munir

Zeeshan Nasim

Mohammad Sayyar Khan

Saleha Tawab

Adnan Nasim

Aqib Iqbal

Mian Afaq Ahmad

Waqar Ali

Raheel Munir

Maria Munir

Noreen Asim

Roles

Abstract

Introduction

Materials and methods

RNA-seq public datasets retrieval and analysis using Tuxedo protocol

Plant material, growth conditions and stress response analyses

Expression analysis

PGRPL1::GUS cloning and GUS assay

Generation of over expression lines

Stress assays

Statistical analysis

Results

Identification of GRPL1 as a multi-abiotic stress-responsive gene

Fig 1. The GRPL1 expression is induced by abiotic stresses.

Cloning and expression analysis of AT4G18280

Fig 2. Generation of GRPL1 overexpression lines.

Function of AT4G18280 overexpression under salt, drought and heat stress

Fig 3. Performance of 35S::GRPL1-3HA transgenic lines under salt stress.

Fig 4. Overexpression of GRPL1 increases drought and heat-tolerance in transgenic plants.

Fig 5. Expression analysis.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Keqiang Wu

Roles

Author response to Decision Letter 0

Decision Letter 1

Keqiang Wu

Roles

Author response to Decision Letter 1

Decision Letter 2

Keqiang Wu

Roles

Acceptance letter

Keqiang Wu

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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