Abstract
Background and aims
MYB transcription factors play critical roles in plant development and stress responses. Our objective was to characterize a role of AtMYB20 (AT1G66230) in regulating the ABA-dependent adaptive response to desiccation stress in Arabidopsis.
Methods
Sequencing analysis revealed that there is a site located on the AtMYB20 transcript which is potentially base-paired by miR858. To avoid the possible cleavage, a vector with a miR858-resistant version of AtMYB20 under the CaMV 35S promoter (35S:m5AtMYB20) was constructed. The AtMYB20 knock-out mutant myb20 was applied to identifying AtMYB20 functions.
Results
While AtMYB20 was induced by high levels of NaCl, its expression was suppressed by desiccation and cold stresses and abscisic acid (ABA) treatment. Compared with wild-type, AtMYB20 over-expression (35S:m5AtMYB20) seedlings were susceptible to desiccation, whereas MYB20 loss of function mutant myb20 plants displayed resistance to desiccation stress. 35S:m5AtMYB20 plants were less sensitive to ABA, but myb20 mutants were hypersensitive to ABA. This could be validated by the experiment in which treatment with 10 μM ABA for 2 h resulted in constant stomatal opening on leaves of 35S:m5AtMYB20 plants but stomatal closure on myb20 mutant plants. Expression of ABA- and drought stress-responsive marker genes (e.g. ABI3-5 and ABF3-4) was up-regulated in myb20 plants but down-regulated in 35S:m5AtMYB20 plants.
Conclusions
AtMYB20 acts as a negative regulator of plant response to desiccation stress in an ABA-dependent manner.
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Introduction
Plants grow in changing environments and their growth and development are constantly influenced by environmental stresses such as desiccation (or drought), salt, cold, high or low temperature, and even heavy metals (Khraiwesh et al. 2012; Yang and Chen 2013). Water deficit is one of the most serious environmental problems that influence the worldwide plant growth and crop production. Under drought stress, biochemical and physiological metabolisms are altered, and cellular aqueous and ionic equilibriums are disrupted. Furthermore, hundreds of genes at transcriptional and post-transcriptional levels are reprogrammed (Sreenivasulu et al. 2007; Chen et al. 2012; Zhou et al. 2013). Plants have evolved delicate mechanisms to cope with drought stress, and their physiological and molecular changes are usually considered as adaptive responses to the adverse environment (Zhu 2002; Shinozaki et al. 2003). To date, a number of drought-inducible genes have been characterized, and most of them are found to be involved in synthesis of dehydrins and osmolytes, enzymes for antioxidation, and components for ABA synthesis or response (Bartels and Sunkar 2005). In addition, a large group of specific genes coding for transcription factors (TFs) are shown to be activated by drought stress (Singh et al. 2002; Zhu 2002). In plants, the TFs have been classified into different superfamilies, e.g. MYB, bZIP, WRKY, NAC and AP2, and some of members from the families participate in various plant responses to abiotic stresses (Riechmann et al. 2000).
Plant MYB TFs belong to one of the largest TFs families (Chen et al. 2006). Currently, more than 100 MYB TFs have been found in Arabidopsis, rice (Oryza sativa), and other plant species (Cedroni et al. 2003; Shinozaki et al. 2003; Rahaie et al. 2010; Liu et al. 2011). According to the number of adjacent imperfect repeats (51–53 amino acids) in their DNA-binding domain, MYB transcription factors can be classified into several subfamilies including R1/2-MYB, R2R3-MYB (3R-MYB) and 4R-MYB (with four R1/R2-like repeats) (Dubos et al. 2010). Recently, several MYB TFs have been reported to involve plant response to drought stress. R2R3-MYB proteins such as AtMYB60 and AtMYB96 (subgroup 1) are involved in this response. AtMYB60 and AtMYB96 regulate plant drought stress response through the ABA signaling cascade (Cominelli et al. 2005; Seo et al. 2009). Also, both AtMYB2 and AtMYB44 were identified as regulators of ABA-dependent salt and drought stress responses (Abe et al. 2003; Jung et al. 2008). Despite the existence of numerous R2R3-MYB protein-encoding genes, only a few of them have been functionally identified, particularly with regard to the plant drought stress response. In this study, we genetically identified an Arabidopsis gene coding for AtMYB20 (R2R3-MYB protein) transcription factor and show that AtMYB20 is involved in plant response to desiccation stress in the plant. AtMYB20 over-expression (35S:m5AtMYB20) plants show sensitivity to desiccation, whereas MYB20 loss of function myb20 mutant plants display resistance to desiccation stress. 35S:m5AtMYB20 plants showed ABA resistance phenotypes, but myb20 mutant plants were hyper-susceptible to ABA during the process of desiccation. These results indicate that AtMYB20 is negatively involved in plant adaptive response to drought stress.
Materials and methods
Plant materials and treatments
Arabidopsis ecotype Col-0 was used throughout the study. The myb20 T-DNA insertion mutant (SALK_CS304073) seeds were obtained from ABRC (Ohio State University, Columbus, OH). Seeds were germinated on Murashige and Skoog (MS) solid medium with 1– 3 % Suc and 0.8 % phytoagar (pH 5.7) in a growth chamber at 22 °C with 100 μE m−2 s−1 photosynthetically active radiation and a 16 h light/8 h dark cycle for 7 days. After that, the seedlings were transferred to a black polyvinylchloride pot (1 L) containing the 1/2-strength Hoagland nutrient solution. Each pot contained six seedlings, which grew for another 7 days under the same condition. Normally, 2 weeks-old seedlings were used for stress treatments. For water loss determination, rosette leaves of the 2 weeks-old plants were detached, placed on an open-lid Petri-dish (dehydrated on filter paper) at room temperature and weighed at a certain period of time after their excision. Water loss was calculated as the percentage of initial fresh weight.
To identify the response of transgenic plants to drought stress, approximate 40 wild type seeds were sowed on the half surface of a pot with substrate, and the same number of myb20 or 35S::m5MYB20 seeds were sowed on the left site of the pot. The substrate used to culture Arabidopsis composed of humus soil, vermiculite and perlite in the ratio of 2:1:1 (Du et al. 2012). The experiments were repeated thrice. Each experiment was carried out with three treatments and each treatment contained at least three pots. After germination, seedlings grew for 2 weeks. Before the experiment was initiated, the substrate was saturated with water (with the maximum water-hold capacity). The seedlings were then not watered for 21 days. Photographs were taken 3 days after rewatering (Zhang et al. 2007; Li et al. 2012). Seedlings were irrigated through the bottom of a pot. Drought tolerance was assayed as the ability of plants to resume growth when returned to normal conditions.
For salt treatment, seedlings were transferred to the half-strength Hoagland nutritional solution with NaCl. The high concentration of NaCl (300 mM) used in this study was set based on the method described previously (Zhang et al. 2007; Li et al. 2012). For cold treatment, seedlings were moved to a chamber with temperature at 4 °C. ABA treatment was undertaken by spraying solution on leaves. The harvested plants were immediately frozen in liquid nitrogen and stored at −80 °C for analysis.
Plant transformation
The plasmid construction and plant transformation were performed based on the method described previously (Clough and Bent 1998). To construct the 35S:m5AtMYB20 transgenic plants, two sequences were PCR-amplified from AtMYB20 cDNA with the pairs of primers M5-s/T-a and T-s/M5-a (Supplementary Data 1A), respectively. With the two PCR products as common template, the miR858-resistant version of AtMYB20 was amplified by overlapping extension PCR using the primer pair of M5-s/M5-a, and cloned into the BglII/SpeI sites of the binary vector pCAMBIA1304. This vector was used as the plant expression vector with CaMV35S as a promoter and NOS terminator as transcriptional termination sequences (Shen et al. 2011). All amplified DNA was sequenced and confirmed. The vector was transformed into Agrobacterium tumefaciens strain LBA4404 and transformed into Arabidopsis using the floral dip method (Song et al. 2012). In this study, all homozygous transgenic lines (T4 generation) were used.
RT-PCR analysis
Real time-PCR and semi-quantitative PCR were performed to analyze gene transcripts based on the methods described previously (Guo et al. 2008; Shen et al. 2011). Briefly, total RNA was isolated by the method indicated above, and 1.0 μg RNA was used as templates for cDNA synthesis. Quantitative RT-PCR (qRT-PCR) was conducted on CFX96 Real-Time PCR Detection System (Bio-Rad). Amplification reaction was performed in a 25 μL mixture containing 5 ng template, 12.5 μL SYBR-Green PCR Mastermix (Toyoba, Japan) and 10 pmol primers. The temperature profile was 98 °C for 30s, followed by 40 cycles at 98 °C for 2 s, 60 °C for 5 s and melt curve at 65 °C for 5 s. Data were analyzed using CFX Data Analysis Manager Software. The relative expression level was normalized to ACTIN2, which was used as the internal control, with the 2-△CT method representing the relative quantification of gene expression. The primers used for analysis are presented in Supplementary Data 1B.
Stomatal aperture measurement
Stomatal apertures were determined in the focal planes of the outer edges of guard cells in epidermis (Lemichez et al. 2001). Detached leaves of 4 weeks-old seedlings were incubated in the stomatal opening solution with 10 mM KCl, 100 mM CaCl2, and 10 mM MES (pH 6.1) for 2 h, and transferred to the same solution with ABA at 0, 1 and 10 μM for 2 h. Subsequently, the adaxial surface of each leaf was applied to 3 M clear tape to peel off the epidermal layer. The epidermal strips were mounted on glass slides and observed with a microscope (YS100, Nikon). Photographs were taken with a digital camera (P5000 COOLPIX, Nikon) attached to the microscope. The ratio of width to length of the stomata was measured using Multigauge version 3.1 soft ware (Fuji Film). More that 60 guard cells from each sample were monitored.
Statistical analysis
All experiments in the study were independently performed three times.Each result shown in the figures was the mean of at least three replicated treatments and each treatment contained at least 12-40 seedlings. Unless indicated, the equal amount of mixed transgenic line seeds was used and samples for analysis were randomly selected from all transgenic lines. The significant differences between treatments were statistically evaluated by standard deviation and one-way analysis of variance (ANOVA). The data between differently treated groups were compared statistically by ANOVA, followed by the least significant difference (LSD) test if the ANOVA result was significant at P < 0.05.
Results
Expression of AtMYB20 under abiotic stresses and ABA treatment
To identify whether AtMYB20 expression responded to abiotic stresses, 2 weeks-old Arabidopsis seedlings were exposed to desiccation, salt and cold for 1–12 h, depending on the treatments. Total RNA was isolated from seedlings. We first tested the expression of RD29A, a stress responsive gene (Shinozaki et al. 2003), as a positive control. Semi-quantitative RT-PCR analysis revealed that AtMYB20 could be induced by NaCl, but suppressed by desiccation and cold (Fig. 1a). To validate the observation, a quantitative real-time RT-PCR experiment was carried out. AtMYB20 had a similar expression pattern in response to the stresses (Fig. 1b, c). Expression of AtMYB20 was induced 3.5-fold by NaCl, while expression of AtMYB20 in response to desiccation and cold was only 37–47 % and 22–55 % of the control, respectively. The phytohormone ABA mediates the plant response to various abiotic stresses (Zhu 2002). Examination of AtMYB20 response to ABA treatment (100 μM) demonstrated that AtMYB20 was also depressed. Its expression was 23–27 % of the control (Fig. 1b). The expression of control genes (RD29 and RAB18) was confirmed for AtMYB20 expression. These results indicate that AtMYB20 can be differently regulated by abiotic stresses.
Identification of 35S::m5AtMYB20 and myb20 mutant lines
To identify the role of AtMYB20 in desiccation response, we constructed transgenic plants over-expressing AtMYB20 driven by the cauliflower mosaic virus (CaMV) 35S promoter. Because AtMYB20 was identified as one of the potential targets of miR858 (German et al. 2008), a miR858-resistant version (35S::m5AtMYB20) was generated by introducing five silent mutations in the miR858 binding site but without changing the protein sequences (Fig. 2a). The vector was transformed into Arabidopsis. The transgenic lines can over-express AtMYB20 (35S::m5AtMYB20) without cleavage of its mRNA by miR858. The 35S::m5AtMYB20 transgenic lines were screened and identified from T0 to T4 generation. Finally, a total of three homozygous 35S::m5AtMYB20 transgenic lines have been obtained. The transgenic plants carrying 35S::m5AtMYB20 have 4.6 to 6.8-fold AtMYB20 expression levels over the wild-type (WT) (Fig. 2b).
The genomic sequences of AtMYB20 contain 925 bp, comprising two exons (849 bp), which are interrupted by an intron towards 5′ end; the CDS contains an open reading frame coding for a protein with 282 amino acid residues (Fig. 3a). A T-DNA insertion mutant, SALK_CS304073 (myb20) was obtained from the Arabidopsis Biological Resource Center. The mutant myb20 has a T-DNA insertion in the second exon towards 3′ UTR. The mutant was verified by diagnostic PCR using AtMYB20 gene-specific and T-DNA border primers. So there was no full length of AtMYB20 transcripts to be expressed (Fig. 3b).
AtMYB20 regulates plant response to dehydration stress
Two weeks-old wild-type, myb20 and 35S:m5AtMYB plants cultured in soil were placed under desiccation treatment for 3 weeks, and most of the plants were withered (Fig. 4a). After a 3-day re-watering, 85.7 % of myb20 mutant plants displayed continued survival and growth, whereas only 45.4 % of the wild-type plants survived (Fig. 4b). By contrast, the 35S::m5AtMYB20 plants showed a more severe dehydrated phenotype. After re-watering for 3 days, there was only 42.2 % survival (Fig. 4c). These results indicated that the 35S::m5AtMYB20 plants were more sensitive to desiccation stress than myb20 mutant plants.
We further examined the plant response to desiccation stress using the method of water loss rate (Bouchabke et al. 2008). The detached rosette leaves of 2 weeks-old wild-type, myb20, 35S::m5AtMYB20 plants were placed on the open-lid petri dishes under the dim light at room temperature. The reduced fresh weight was measured over time (0–330 min). Rosette leaves from all plants showed progressive loss of weight (Fig. 4d). To the end of the experiment, the fresh weight for myb20 mutant leaves was 50.4 %. The fresh weight for 35S::m5AtMYB20 plant leaves was only 28.7 % of their starting weight, and the wild-type had an intermediate value of 34.6 %. Although the 35S::m5AtMYB20 plants contained less water in leaves than wild-type at the end of experiment, no significant difference was found between them.
AtMYB20 is involved in ABA-dependent stomatal closure
Leaves of 4 weeks-old plants were submerged in a stomatal opening solution and treated with 10 μM ABA for 2 h. In the absence of ABA, all guard cells on the leaves of wild-type, 35S::m5AtMYB20 and myb20 plants were fully opened in the stomatal opening solution (Fig. 5a). However, when ABA was added to the solution, the stomata on the leaves of myb20 plants were closed, whereas those on 35S::m5AtMYB20 plant leaves were still open. Quantitative analysis using stomatal aperture (the ratio of width to length) showed that the much stronger stomatal closure occurred in the myb20 plants than in the wild-type with ABA (Fig. 5b). Conversely, stomata on the 35S::m5AtMYB20 leaves were not closed but a little more open than those of WT.
Expression of desiccation- and ABA responsive genes in 35S::m5AtMYB20 and myb20 plants
To investigate further whether the response of desiccation stress could be regulated by AtMYB20, several abiotic stress and ABA responsive genes were analyzed. RD22, RD29A, KIN1 and COR47 belong to the DRE/CRT (drought responsive/C-repeat) elements-containing class of stress- and ABA-responsive genes (Yamaguchi-Shinozaki and Shinozaki 1993; Shinozaki et al. 2003). In the presence of ABA, RD22, RD29A, KIN1 and COR47 were expressed at higher levels in myb20 mutant than in wild type plants (Fig. 6a–d). Compared to wild type, expression of RD22, RD29A, KIN1 and COR47 was increased 5.8, 2.5, 2.4 and 1.6 folds, respectively. By contrast, expression of these genes was lower in 35S::m5AtMYB20 plants. We further analyzed three Ser/Thr protein phosphatase 2C genes ABI1, ABI2 and AtPP2CA. These genes have been characterized as negative regulators of ABA signal (Schweighofer et al. 2004) and respond to salt stress (Cui et al. 2013). Our analysis showed that expression of ABI1, ABI2 and AtPP2CA was significantly reduced in 35S::m5AtMYB20 plants but significantly increased in myb20 mutant plants with ABA treatment (Fig. 6e–g). This pattern was very similar to RD22, RD29A, KIN1 and COR47 in AtMYB20 over-expression and myb20 mutant plants, respectively. Finally, we analyzed the genes ABI3, ABI4, ABI5, ABF3, and ABF4. This group of genes encodes ABA-responsive basic leucine zipper (bZIP) transcription factors which bind to the ABA response element (ABRE) of their targets and function during the plant stress responses (Hauser et al. 2011). After treatment with 100 μM ABA for 5 h, all the genes were induced in myb20 mutant plants, although the degree of the gene induction varied differently (Fig. 6h–l). Also, expression of the genes was lower in 35S::m5AtMYB20 plants. Overall, AtMYB20 over-expression resulted in the transcriptional modulation of desiccation, salt- and ABA-responsive genes.
Discussion
With recent advance in high-throughput sequencing technologies and availability of genomic sequences of various plant species, a large number of genes responding at the transcriptional level to abiotic stresses have been identified (Kreps et al. 2002; Khraiwesh et al. 2012; Zhou et al. 2012, 2013). Amongst these, a group of stress-responsive genes encoding transcription factors (e.g. MYB, bHLH, bZIP, WRKY, and DREB families) are of particular interest (Shinozaki et al. 2003), because they are involved in many signaling and transcriptional regulatory pathways. R2R3-MYB TFs belong to the largest subfamily of MYB and are involved in various plant abiotic stress responses (Chen et al. 2006; Dubos et al. 2010). Expression of AtMYB15 was regulated by drought and salinity stresses; AtMYB15 over-expression plants were able to confer drought tolerance (Ding et al. 2009). Ecotopic expression of the Chrysanthemum R2R3-MYB transcription factor CmMYB2 enhanced tolerance to drought and saline stresses (Shan et al. 2012). Moreover, AtMYB96 transcription factor was shown to mediate abscisic acid signaling during drought stress response in Arabidopsis (Seo et al. 2009). In this study, a R2R3-MYB transcription factor AtMYB20 has been identified as a regulator of desiccation stress response in Arabidopsis. Like other MYB TFs, AtMYB20 can be differently regulated by abiotic stresses. However, while AtMYB20 was induced by high levels of NaCl, its expression was suppressed by desiccation and cold (Fig. 1). This expression pattern with salinity is in accordance with the most recent report; but the AtMYB20 response to desiccation in this study was slightly different from that to drought stress (Cui et al. 2013).
To identify whether AtMYB20 was able to regulate plant response to desiccation stress, transgenic lines over-expressing AtMYB20 were constructed. We applied the AtMYB20 knock-out mutant myb20 to identifying AtMYB20 function. Because AtMYB20 transcript may be cleavaged by miR858, a cleavage-resistance version to eradicate the interference by miR858 was constructed. Our analysis showed that 35S::m5AtMYB20 plants have a higher level of AtMYB20 expression than wild-type (4.6 to 6.8-folds, Fig. 2). These transgenic lines plus myb20 could allow us to demonstrate that AtMYB20 is critical for plant response to desiccation stress. Whereas AtMYB20 over-expression resulted in plant sensitivity to desiccation, the myb20 mutant plants displayed enhanced resistance to water loss.
The phytohormone abscisic acid (ABA) regulates numerous developmental processes and stress responses in plants; under adverse conditions, ABA serves as a signal molecule to sense abiotic stresses; ABA regulates stomatal closure in plants to avoid the water loss during the water stress (Cutler et al. 2010). Heterologous expression in Arabidopsis of Craterostigma plantagineum MYB10 has been shown to increase ABA hypersensitivity and enhance drought tolerance (Shan et al. 2012). Similarly, over-expression of miR159-resistant MYB33 and MYB101 resulted in ABA hypersensitivity (Reyes and Chua 2007). The present study demonstrated that expression of AtMYB20 was reduced during the first several hours of ABA treatment (Fig. 1). Our result is in a good agreement with the recent report, in which expression of AtMYB20 in guard cells was found to be reduced by ABA (50 μM) treatment (Winter et al. 2007; http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi?dataSource=Guard_Cell). While AtMYB20-overexpressing plants displayed insensitivity to ABA-responsive stomatal closure, AtMYB20 knock-out mutation intensified the ABA-promoted stomatal closure in the presence of ABA (Fig. 5). Furthermore, expression of ABA-responsive genes such as ABI3, ABI4, ABI5, ABF3, and ABF4 was more evident in myb20 plants than in wild-type, whereas expression of these genes in 35S::AtMYB20 plants was depressed. The elevated expression of ABI3, ABI4, ABI5, ABF3, and ABF4 in myb20 plants suggests that AtMYB20 can negatively interact with the ABA synthesis or responsive genes under desiccation stress. In this regard, our result is very similar to the recent study (Cui et al. 2013), in which AtMYB20 over-expression transgenic lines showed a reduced expression of ABA-responsive genes (e.g. ABI1 and ABI2); simultaneously, the salt-induced expression of AtPP2CA, a negative regulator of ABA signaling (Kuhn et al. 2006; Yoshida et al. 2006), was found to be significantly depressed in the AtMYB20 over-expression plants. These results suggest that salt stress would induce AtMYB20 expression by accumulating ABA, and AtMYB20 in turn may directly bind to the promoters of ABI1 and AtPP2CA to negatively regulate ABA response and ultimately improve the plant salt tolerance. Although the AtMYB20-regulated salt and desiccation responses in AtMYB20 over-expression plants appear different, the two phenotypes should be under the control of ABA signal. The molecular mechanisms underlying the response of plants to desiccation and salt stresses are complicated. Some genes that execute concomitantly positive or negative regulation of both salt and desiccation stress responses have been reported (Shan et al. 2012; Zhao et al. 2013). However, if ABA-dependent signal transduction cascades are involved, genes that depend on ABA signals usually display the contrast regulation of salt and desiccation responses (Zhang et al. 2007; Li et al. 2012). The AtMYB20-regulated desiccation stress response could be also confirmed with myb20 mutants, in which knock-out of AtMYB20 resulted in plant more sensitivity to ABA (Fig. 5). In the desiccation experiment, the myb20 mutant plants always contained a higher level of water in rosette leaves than wild-type (Fig. 4d). Although the 35S::m5AtMYB20 plants showed less water content in leaves than wild-type at the end of the experiment, no significant difference was found between them. It is unclear for the reason, but it was possible that under the desiccation, both wild-type and 35S::m5AtMYB20 leaves underwent a severe lose of water. This could be explained by the observation that guard cells on the leaves of wild-type and 35S::m5AtMYB20 were always open in the presence of ABA (Fig. 5a), which finally may result in an equivalent amount of water loss.
We analyzed the stress-responsive genes RD22, RD29A, KIN1 and COR47. These genes are up-regulated when plants are exposed to salinity, drought, cold or exogenous ABA (Shinozaki et al. 2003). A group of Ser/Thr protein phosphatase 2C genes, such as ABI1, ABI2, and AtPP2CA were additionally analyzed because these genes were reported to involve the plant salt stress response (Cui et al. 2013). The elevated expression of these genes is considered to be advantageous for plants resistance to abiotic stress (Zhu 2002). In response to ABA, these genes were expressed at higher level in myb20 mutants than in wild type plants. By contrast, their expression was lower in 35S::AtMYB20 plants. We also found a group of genes (ABI3, ABI4, ABI5, ABF3, and ABF4) coding for ABA-responsive basic leucine zipper (bZIP) transcription factors had a similar expression pattern in the 35S::AtMYB20 plants and myb20 mutants. The results indicate that AtMYB20-regulated desiccation phenotype is involved in the ABA response and AtMYB20 is most likely to function as a negative regulator of ABA-mediated stomatal closure.
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This research was supported by The National Research Foundation for the Doctoral Program of Higher Education of China under Grant No B0201100671.
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Responsible Editor: John McPherson Cheeseman.
Shuai Gao and Yong Li Zhang made equal contribution to the study
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Supplementary Data 1A
Primer sequences used for PCR and semi-quantitative RT-PCR (DOC 34 kb)
Supplementary Data 1B
Primer sequences used for quantitative real-time RT-PCR (DOC 37 kb)
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Gao, S., Zhang, Y.L., Yang, L. et al. AtMYB20 is negatively involved in plant adaptive response to drought stress. Plant Soil 376, 433–443 (2014). https://doi.org/10.1007/s11104-013-1992-6
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DOI: https://doi.org/10.1007/s11104-013-1992-6