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. 2017 Aug 22;17(1):142.
doi: 10.1186/s12870-017-1078-3.

GaMYB85, an R2R3 MYB gene, in transgenic Arabidopsis plays an important role in drought tolerance

Hamama Islam Butt  1 , Zhaoen Yang  1 , Qian Gong  1 , Eryong Chen  1 , Xioaqian Wang  1 , Ge Zhao  1 , Xiaoyang Ge  1 , Xueyan Zhang  2 , Fuguang Li  3
Affiliations

GaMYB85, an R2R3 MYB gene, in transgenic Arabidopsis plays an important role in drought tolerance

Hamama Islam Butt et al. BMC Plant Biol. .

Abstract

Background: MYB transcription factors (TFs) are one of the largest families of TFs in higher plants and are involved in diverse biological, functional, and structural processes. Previously, very few functional validation studies on R2R3 MYB have been conducted in cotton in response to abiotic stresses. In the current study, GaMYB85, a cotton R2R3 MYB TF, was ectopically expressed in Arabidopsis thaliana (Col-0) and was functionally characterized by overexpression in transgenic plants.

Results: The in-silico analysis of GaMYB85 shows the presence of a SANT domain with a conserved R2R3 MYB imperfect repeat. The GaMYB85 protein has a 257-amino acid sequence, a molecular weight of 24.91 kD, and an isoelectric point of 5.58. Arabidopsis plants overexpressing GaMYB85 exhibited a higher seed germination rate in response to mannitol and salt stress, and higher drought avoidance efficiency than wild-type plants upon water deprivation. These plants had notably higher levels of free proline and chlorophyll with subsequent lower water loss rates and higher relative water content. Germination of GaMYB85 transgenics was more sensitive to abscisic acid (ABA) and extremely liable to ABA-induced inhibition of primary root elongation. Moreover, when subjected to treatment with different concentrations of ABA, transgenic plants with ectopically expressed GaMYB85 showed reduced stomatal density, with greater stomatal size and lower stomatal opening rates than those in wild-type plants. Ectopic expression of GaMYB85 led to enhanced transcript levels of stress-related marker genes such as RD22, ADH1, RD29A, P5CS, and ABI5.

Conclusions: Our results indicate previously unknown roles of GaMYB85, showing that it confers good drought, salt, and freezing tolerance, most probably via an ABA-induced pathway. These findings can potentially be exploited to develop improved abiotic stress tolerance in cotton plants.

Keywords: Abiotic stress tolerance; Abscisic acid; MYB transcription factor.

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Figures

Fig. 1
Fig. 1
The Phylogenetic analysis and predicted structure of R2R3 MYB GaMYB85. a Homologous sequences retrieved by Blast p were aligned with GaMYB85 showing R2 and R3 repeats represented by black and red lines respectively. b The NJ tree analysis of GaMYB85 proteins with homologous and known R2R3 MYB monocots and dicots sequences, along with scale bar that shows the calculated distance by multiple sequence alignment (MSA). The MSA of the respective sequences were provided in Additional file 1
Fig. 2
Fig. 2
Gel and qRT-PCR analysis of GaMYB85 transgenic plants a The confirmation of GaMYB85 gene CDS (771 bp) integration in T0 generated overexpressed Arabidopsis plants. The DNA of WT (Col-0) was used as negative control (lane 1) and 35S:GaMYB85 DNA was used in (lanes 1–10, 11–16). M; DNA marker III, 1 kb. b Relative expression of GaMYB85 gene in T2 transgenic lines by qRT-PCR having segregating ratio of 3:1 on the selective medium, three cDNA preparations were used and error bars represented with SD value. AtUBQ10 gene (Accession no: AT4G05320) was used as an internal standard in qRT-PCR
Fig. 3
Fig. 3
Overexpressing of GaMYB85 modulates hypersensitivity to ABA-elicited root inhibition and seed germination rate. a 35S:GaMYB85 plants and WT root elongation comparisons on MS with and without varied ABA conc. (μM). The seedlings were scored and photographed after 7 days. Assay was run in triplicates, Bar line = 1 cm. b Quantitative comparisons of root elongation assay on ABA MS with (0, 0.3, 0.5, 1 and 2 μM). The three replicates used with 30 seedlings each, * P < 0.05 of mean value represented by ± SE. c 35S:GaMYB85 plants and WT seeds germination rates with and without ABA, the results were scored at 10th day by using 50 seeds each, ± SE (n = 3). d ABA 1(μM) supplemented plates used for time course analysis, the error bars represent SE of 3 replicates
Fig. 4
Fig. 4
Overexpressing of GaMYB85 enhances germination and root length in response to NaCl stress. a 35S:GaMYB85 plants and WT seed germination rates on MS and MS supplemented with 0-150 mM salt were photographed at 10th day of seed germination. b The seed germination rates plot scored at 10th day for both transgenic and WT on salt medium, 50 seeds each were used in three biological replicates (n = 3), *P < 0.05 calculated by t-test with means values ± SE. c 35S:GaMYB85 and WT seedlings fresh weights, grown on MS media with 0-150 mM NaCl, scored at 10th day of germination. 10 seedlings each used with three technical repeats and error bars with mean values ±SE. d Root elongation comparisons of 35S:GaMYB85 and WT on 0-150 mM NaCl MS for 6 days. The data scored from three independent growth assays. Line bar: 1 cm. e Quantitative comparison of root elongation of 35S:GaMYB85 and WT seedlings on MS supplemented with 0-150 mM NaCl. 30 seedlings each, (*P < 0.05) calculated by t-test and mean value ± SE (n = 3)
Fig. 5
Fig. 5
Plants overexpressing GaMYB85 perform well in response to mannitol stress a 35S:GaMYB85 and WT seed germination performance on MS supplemented with 0, 100, 200, and 300 mM mannitol. Germination rates were scored on the 10th day. b A plot of the germination rates scored on the 10th day for 35S:GaMYB85 and WT seeds germinated on the different mannitol media. Data are the means ± SE for three replicates of 50 seeds each (*P < 0.05). c Fresh weights of seedlings grown on 0–300 mM mannitol MS medium scored on the 10th day. Each treatment with 10 seedlings was performed in triplicate. Data are the means ± SE (*P < 0.05). d Comparison of root elongation of 5-day-old seedlings of 35S:GaMYB85 and WT plants transferred to 0, 100, 200, and 300 mM mannitol MS for 6 days. Values represent the data from three independent growth assays. Scale bar: 1 cm. e Quantitative comparison of root elongation of 35S:GaMYB85 and WT seedlings on MS supplemented with 0–300 mM mannitol (30 seedlings each). Data are the means ± SE (n = 3). P < 0.05 determine by the t-test
Fig. 6
Fig. 6
The characterization of 35S:GaMYB85 under drought stress. a The phenotypes of 35S:GaMYB85 and WT at the initial and late stages of dehydration and 3 days after re-watering b Survival percentages are the mean values ± SE of three separate assays (n = 18). *P < 0.05 and **P < 0.01 determined by the t test. c Water loss rates of detached leaves of 3-week-old 35S:GaMYB85 and WT plants, expressed as percentages of initial fresh weight ± SE (n = 10) and (*P < 0.05). d Relative water content in detached leaves of 4-week-old 35S:GaMYB85 and WT plants. Data are the means ± SD (n = 10). Significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001) were determined by Student’s t-test. e Proline content of 35S:GaMYB85 and WT plants under normal conditions and after 14 days of water deprivation. For concentration determinations, absorbances were measured spectrophotometrically and Duncan’s multiple range test was used for comparing means. Data are biological replicate means ± SE (n = 3)
Fig. 7
Fig. 7
Freezing stress responses of 35S:GaMYB85 and WT plants. 35S:GaMYB85 and WT plants at −10 °C treatment, the photographs were taken after 7 days of plants revival. The survival rate percentage evaluated from three separate assays, mean values ± SD (n = 18) and significant difference *P < 0.05, calculated by t-test
Fig. 8
Fig. 8
ABA induces positive stomatal modulation in 35S:GaMYB85-overexpressing lines. a The stomatal pores of 35S:GaMYB85 and WT were photographed following treatment with 0, 5, and 10 μM ABA. Five views from three replicate plants were observed at ×40 magnification b Quantitative comparisons of the stomatal pores of 35S:GaMYB85 and WT plants following treatment with 0, 5, and 10 μM ABA. The data shown are the mean values of three replicates ± SD (n = 10). Scale bars: approximately 1 μm c Leaf stomatal density of 35S:GaMYB85 and WT plants photographed under an OLYMPUS Bx51 microscope at ×40 magnification. d Quantitative comparisons of 35S:GaMYB85 and WT stomatal density e Width and length of guard cells measured using ImageJ software. Measurements were taken from the leaves of three plants (10 stomata from five microscopic views). Data are the means ± SD. *P < 0.05; ** P < 0.01
Fig. 9
Fig. 9
Transcript levels of ABA signaling and ABA stress-responsive marker genes in 35S: GaMYB85 and WT. (a–e) Transcript level of ABA signaling and ABA stress-responsive marker genes in two week old seedlings on (100 μM) ABA treatment for 6 h, using qRT-PCR. AtUQB10 was used as internal control gene, with error bars of 3 biological replicates with ± SD. *P < 0.05; ** P < 0.01 and *** P < 0.001
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