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. 2017 Aug 24;18(9):1841.
doi: 10.3390/ijms18091841.

Arabidopsis E3 Ubiquitin Ligases PUB22 and PUB23 Negatively Regulate Drought Tolerance by _targeting ABA Receptor PYL9 for Degradation

Jinfeng Zhao  1 , Linlin Zhao  2 , Ming Zhang  3   4   5 , Syed Adeel Zafar  6 , Jingjing Fang  7 , Ming Li  8 , Wenhui Zhang  9 , Xueyong Li  10
Affiliations

Arabidopsis E3 Ubiquitin Ligases PUB22 and PUB23 Negatively Regulate Drought Tolerance by _targeting ABA Receptor PYL9 for Degradation

Jinfeng Zhao et al. Int J Mol Sci. .

Abstract

Drought causes osmotic stress and rapidly triggers abscisic acid (ABA) accumulation in plants. The roles of various ABA receptors in drought tolerance and molecular mechanisms regulating ABA receptor stability needs to be elucidated. Here, we report that Arabidopsis plants overexpressing PYL9, one of the 14 pyrabactin resistance (PYR)/pyrabactin resistance-like (PYL)/regulatory component of ABA receptors (RCAR) family ABA receptors, gained drought tolerance trait. Osmotic stress induced accumulation of the PYL9 protein, which was regulated by the 26S proteasome. PYL9 interacted with two highly homologous plant U-box E3 ubiquitin ligases PUB22 and PUB23. In the cell-free degradation assay, the degradation of GST-PYL9 was accelerated in protein extract from plants overexpressing PUB22 but slowed down in protein extract from the pub22 pub23 double mutant. The in vivo decay of Myc-PYL9 was significantly reduced in the pub22 pub23 double mutant as compared with the wild-type. Additionally, PUB22 also interacted with other ABA receptors such as PYL5, PYL7 and PYL8. Considering the improved drought tolerance in the pub22 pub23 double mutant in previous studies, our results suggest that PUB22 and PUB23 negatively regulate drought tolerance in part by facilitating ABA receptors degradation.

Keywords: ABA receptor; U-box E3 Ubiquitin ligases; cell-free degradation assay; degradation; drought tolerance; protein extract.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Overexpression of PYL9 enhanced drought tolerance in plants. (a) Relative transcript level of PYL9 in two transgenic lines (#14 and #16) harboring Myc-PYL9. Data are the means ± standard errors (n = 3). (b) Overexpression of PYL9 confers drought tolerance in Arabidopsis. Four-week-old transgenic plants (upper) were treated by withholding water for 30 days (middle) and then rewatered. The representative images were taken after the plants were re-watered for 10 days (lower). (c) The percentage of surviving plants after re-watering for ten days. Data are the means ± standard errors (n = 24).
Figure 2
Figure 2
PYL9 was degraded by the 26S proteasome and osmotic stress increased PYL9 accumulation. (a) The proteasome inhibitor MG132 stabilized Myc-PYL9. Ten-day-old transgenic seedlings were treated with or without (mock) 50 μM MG132 for different time period. The bands detected by anti-Actin were used as loading control [44,45]. (b) Quantitative evaluation of the Myc-PYL9 protein level in (a). Data are the means ± standard errors (n = 3). (c) Osmotic stress increased the accumulation of Myc-PYL9. Seedlings of two T3 transgenic lines (#14 and #16) were grown on −1.2 MPa PEG-infused plates for three hours. The bands detected by anti-Actin were used as loading control. (d) Quantitative evaluation of the Myc-PYL9 protein level in (c). Independent experiments were repeated three times with the similar results. Data are the means ± standard errors (n = 3). Asterisks indicate significant differences (p < 0.05).
Figure 3
Figure 3
ABA receptor PYL9 interacts with two highly homologous plant U-box E3 ligases, PUB22 and PUB23. (a) PYL9 interacts with PUB22 and PUB23 in yeast two-hybrid assays. The growth of yeast transformed with the indicated constructs on selective plate is shown. Empty vectors (BD or AD) were used as negative controls. (b) PYL9 interacts with PUB22 and PUB23 in pull-down assays. Five micrograms MBP-PUB22, MBP-PUB23, or MBP bound with amylose resin were incubated with 1 μg GST-PYL9 for one hour at room temperature. Five percent of the input proteins were run on the PAGE gel and visualized via coomassie brilliant blue staining (upper panel). After washing five times, the protein complex pulled down by resin was detected with anti-GST antibody (lower panel). (c) PYL9 interacts with PUB22C13A by firefly luciferase complementation imaging assay. The indicated combinations were co-transfected with the CaMV35S:GUS construct into tobacco leaves and incubated for five days. LUC activity was normalized with the GUS activity. Data are the means ± standard errors (n = 3). (d) The transcript level of NLUC in the combinations in (c). Data are the means ± standard errors (n = 3). (e) The transcript level of CLUC in the combinations in (c). Data are the means ± standard errors (n = 3).
Figure 4
Figure 4
PUB22 and PUB23 regulate GST-PYL9 stability in cell-free degradation assay. (a) The degradation of GST-PYL9 in the extract from the wild-type at indicated time. MG132, a specific 26S proteasome inhibitor, was used as a proteasomal activity control. Coomassie brilliant blue (CBB) staining of Rubisco was used as loading control. (b) The degradation of GST-PYL9 in the wild-type, the pub22 pub23 double mutant and the transgenic plants overexpressing PUB22 (OEPUB22). The total protein isolated from ten-day-old seedlings were incubated with GST-PYL9 for the indicated hours. GST-PYL9 was detected with anti-GST antibody. CBB staining of Rubisco was used as loading control. Asterisk may represent truncated GST-PYL9 or unspecific band. (c) Quantitative evaluation of the GST-PYL9 protein level in (b). Independent experiments were repeated three times with the similar results. Data are the means ± standard errors (n = 3). Statistical significance was determined by a Student’s t-test; significant differences (p ≤ 0.05) are indicated by different lowercase letters.
Figure 5
Figure 5
Myc-PYL9 is stabilized in the pub22 pub23 double mutant. (a) The transcript level of Myc-PYL9 in the transgenic plants in Col-0 (#2) or pub22 pub23 (#42) background. Data are the means ± standard errors (n = 4). (b,c) Degradation rate of Myc-PYL9 in Col-0 (b); and the pub22 pub23 double mutant (c). Ten-day-old transgenic seedlings were incubated in liquid MS medium containing 50 μM MG132 for 16 h and washed five times before being transferred to liquid medium with 100 μM cycloheximide (CHX). Proteins were isolated at the indicated time points and detected with anti-Myc antibody. Anti-Actin was used as loading control. (d) Quantitative evaluation of the Myc-PYL9 protein level in (b). (e) Quantitative evaluation of the Myc-PYL9 protein level in (c). Independent experiments were repeated three times with the similar results. Data are the means ± standard errors (n = 3).
Figure 6
Figure 6
Expression patterns of PUB22, PUB23 and PYL9. (a–c) Quantitative real-time PCR was used to determine the expression of PUB22 (a); PUB23 (b); and PYL9 (c), in root, stem, cauline leaf, rosette leaf, flower, and silique of the Arabidopsis wild-type Col-0. Data are the means ± standard errors (n = 4).
Figure 7
Figure 7
PUB22 interacts with PYL5, PYL7, PYL8, and PYL9. (a) PUB22 interacts with PYL5, PYL7, PYL8, and PYL9 in yeast two-hybrid assay. The combinations of PUB22 with different PYLs were grown on the SC medium lacking Tryptophan and Leucine (SC-T/L) or Tryptophan, Leucine and Histidine (SC-H/T/L). (b) PUB22 interacts with PYL5, PYL7, PYL8, and PYL9 in the pull-down assay. MBP-PUB22 and MBP bound with Amylose resin were incubated with GST-PYL5, GST-PYL7, GST-PYL8, or GST-PYL9, separately. Five percent of the input proteins were run on the PAGE gel and visualized via coomassie brilliant blue (CBB) staining (upper panel). The protein complex pulled down by resin was detected with anti-GST antibody (lower panel).
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