CkREV Enhances the Drought Resistance of Caragana korshinskii through Regulating the Expression of Auxin Synthetase Gene CkYUC5

Abstract

As a common abiotic stress, drought severely impairs the growth, development, and even survival of plants. Here we report a transcription factor, Caragana korshinskii REVOLUTA(CkREV), which can bidirectionally regulate the expression of the critical enzyme gene CkYUC5 in auxin synthesis according to external environment changes, so as to control the biosynthesis of auxin and further enhance the drought resistance of plants. Quantitative analysis reveals that the expression level of both CkYUC5 and AtYUC5 is down-regulated after C. korshinskii and Arabidopsis thaliana are exposed to drought. Functional verification of CkREV reveals that CkREV up-regulates the expression of AtYUC5 in transgenic A. thaliana under common conditions, while down-regulating it under drought conditions. Meanwhile, the expression of CkYUC5 is also down-regulated in C. korshinskii leaves instantaneously overexpressing CkREV. We apply a dual-luciferase reporter system to discover that CkREV can bind to the promoter of CkYUC5 to regulate its expression, which is further proved by EMSA and Y1H esxperiments. Functional verification of CkREV in C. korshinskii and transgenic A. thaliana shows that CkREV can regulate the expression of CkYUC5 and AtYUC5 in a contrary way, maintaining the equilibrium of plants between growth and drought resisting. CkREV can positively regulate the expression of CkYUC5 to promote auxin synthesis in favor of growth under normal development. However, CkREV can also respond to external signals and negatively regulate the expression of CkYUC5, which inhibits auxin synthesis in order to reduce growth rate, lower water demands, and eventually improve the drought resistance of plants.

Keywords: Caragana korshinskii; HD-ZIP III; auxin synthesis; drought; stress resistance.

Figure 1

Phylogenetic analysis of HD-ZIP III family between C. korshinskii, G. max, C. arietinum, M. truncatula, C. cajan, C. sinensis, and A. thaliana. Bootstrap support (1000 repetitions) is shown for each node.

Figure 2

Conserved domain analysis of HD-ZIP III family between C. korshinskii, G. max, C. arietinum, M. truncatula, C. cajan, C. sinensis, and A. thaliana.

Figure 3

CkREV subcellular localization observation. Scale bars, 50 μm.

Figure 4

qRT–PCR analysis on the expression level of relevant genes. (a) Analysis of the expression level of CkYUC5 in C. korshinskii leaves after natural drought treatment; (b) Analysis of the expression level of AtYUC5 in A. thaliana seedlings after cultured on 1/2 MS medium for 7 days and transferred to PEG medium for simulated drought treatment; (c) Analysis of the expression level of CkREV in hydroponic C. korshinskii after drought simulation on PEG medium; (d) Analysis of the expression level of AtYUC5 in A. thaliana CkREV–OE lines at the age of 4 weeks; (e) Analysis of the expression level of CkYUC5 in C. korshinskii leaves instantaneously overexpressing CkREV; (f) Analysis of the expression level of AtYUC5 in wild type and transgenic A. thaliana after drought treatment. (a,d–f) Data are shown as the mean ± SD of three independent experiments. Student’s t–test is employed to measure statistical significance between two samples with confidence level at 0.95 (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (b,c) Data are shown as the mean ± SD of three independent experiments. One–way ANOVA was performed for the statistical analysis, where different letters represent significant differences (p < 0.05).

Figure 5

CkREV bidirectionally regulates the expression of CkYUC5 and inhibits the A. thaliana root length under drought stress. (a–d) Observe the expression changes of CkYUC5 under different treatment conditions through tobacco leaves cultured for 28 days (n = 3 biologically independent samples); (a) Localization of CkYUC5 in the tip of tobacco leaf under normal culture conditions; (b) Localization of CkYUC5 in the tip of tobacco leaf after CkREV overexpression; (c) Localization of CkYUC5 in the tip of tobacco leaf after PEG treatment; (d) Localization of CkYUC5 in the tip of tobacco leaf after overexpression of CkREV treated with PEG; (e–g) Four days after germination on 1/2MS plates, the phenotype of root length change of A. thaliana CkREV–OE strain and wild type under normal culture conditions, PEG treatment and PEG treatment with NAA added for 3 days (n = 5 biologically independent samples); (h) GUS staining statistics of A. thaliana CkREV–OE strain and wild type under different treatments; (i) Root length statistics of A. thaliana CkREV–OE strain and wild type under different treatments; (j) IAA content determination. Scale bars in (a–g), 10 mm. (i) Data are shown as the mean ± SD of five independent experiments; Student’s t–test is employed to measure statistical significance between two samples with a confidence level of 0.95 (**, p < 0.01). (h,j) Data are shown as the mean ± SD of three independent experiments. One–way ANOVA was performed for the statistical analysis, where different letters represent significant differences (p < 0.05).

Figure 6

Interaction proof between CkREV and CkYUC5. (a) Recognition site of downstream genes of REV; (b) Dual–LUC experimental mode diagram; (c) Results of Dual–LUC experiment CkREV interacted with the promoter region of CkYUC5 and negatively regulated its expression compared with GFP control group; (d) Sketch map of probe binding site in EMSA experiment; (e) Results of EMSA experiment indicated that CkREV–GST could directly bind to ATGAT element in the promoter region of critical enzyme gene CkYUC5 in auxin synthesis; (f) The results of yeast one–hybrid further verified the binding of CkREV to the promoter region of CkYUC5; (g) qRT–PCR detection on CkAS1 expression level in C. korshinskii leaves under different treatments; (h) Dual–LUC experimental mode diagram; (i) The Dual–LUC experiment was used to detect the effect of CkAS1 as an effector on the regulation of CkREV on CkYUC5. (c,e,g,i) Data are shown as the mean ± SD of three independent experiments. (c,i) Data are shown as the mean ± SD of three independent experiments. Student’s t–test is employed to measure statistical significance between two samples with a confidence level of 0.95 (**, p < 0.01; ***, p < 0.001). (g) Data are shown as the mean ± SD of three independent experiments. One–way ANOVA was performed for the statistical analysis, where different letters represent significant differences (p < 0.05).

Figure 7

Phenotype analysis on transgenic A. thaliana. (a) DAB staining on rosette leaves at the same position after natural drought treatment for 0, 5, and 10 days (n = 3 biologically independent samples); (b) NBT staining on rosette leaves at the same position after natural drought treatment for 0, 5, and 10 days (n = 3 biologically independent samples); (c) Results of proline content determination; (d) Results of relative water content determination. (c,d) Data are shown as the mean ± SD of three independent experiments. Scale bars in (a,b), 5 mm. (c,d) Data are shown as the mean ± SD of three independent experiments. Student’s t–test is employed to measure statistical significance between two samples with a confidence level of 0.95 (**, p < 0.01; ***, p < 0.001).

Figure 8

Model of regulation of CkREV on the expression of CkYUC5. The model concludes our research and exhibits that CkREV bidirectionally regulates the expression of CkYUC5 critical in auxin synthesis depending on changes in external environmental signals, balancing the growth and drought-resistance of plants by influencing auxin synthesis.