An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

Abstract

The HUB2 gene encoding histone H2B monoubiquitination E3 ligase is involved in seed dormancy, flowering timing, defence response and salt stress regulation in Arabidopsis thaliana. In this study, we used the cauliflower mosaic virus (CaMV) 35S promoter to drive AtHUB2 overexpression in cotton and found that it can significantly improve the agricultural traits of transgenic cotton plants under drought stress conditions, including increasing the fruit branch number, boll number, and boll-setting rate and decreasing the boll abscission rate. In addition, survival and soluble sugar, proline and leaf relative water contents were increased in transgenic cotton plants after drought stress treatment. In contrast, RNAi knockdown of GhHUB2 genes reduced the drought resistance of transgenic cotton plants. AtHUB2 overexpression increased the global H2B monoubiquitination (H2Bub1) level through a direct interaction with GhH2B1 and up-regulated the expression of drought-related genes in transgenic cotton plants. Furthermore, we found a significant increase in H3K4me3 at the DREB locus in transgenic cotton, although no change in H3K4me3 was identified at the global level. These results demonstrated that AtHUB2 overexpression changed H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus, leading the GhDREB gene to respond quickly to drought stress to improve transgenic cotton drought resistance, but had no influence on transgenic cotton development under normal growth conditions. Our findings also provide a useful route for breeding drought-resistant transgenic plants.

Keywords: HISTONE MONOUBIQUITINATION 2 (HUB2); Gossypium hirsutum Linn.; drought; histone methylation; histone monoubiquitination; transgenic plants.

Figure 1

Response of Athub2 mutants to osmotic stress. (a) Seeds of the indicated genotypes were germinated on 1/2 MS medium or 1/2 MS previously infused with 25% PEG. Photographs were taken 7 days after germination. Black arrows indicate un‐germinated seeds. (b) Percentages of seeds that germinated in different concentrations of PEG. (c) Root growth of the indicated genotypes. Six‐day‐old seedlings were transferred from 1/2 MS to 1/2 MS medium previously infused with different concentrations of PEG. Photographs were taken 4 and 7 days after transfer, bar = 0.5 cm. (d) Quantification of relative root length in (c). Vertical bars represented standard deviation (SD). (n ≥ 30, *P < 0.05; ***P < 0.001).

Figure 2

Agronomic traits of different lines grown in a waterproof shed. (a) Drought tolerance assay in the field under a waterproof shed in Anyang, Henan province, China. The photographs were taken 1 month after re‐watering. (b) Plant height, fruit branching, boll‐setting rates and abscission rates of controls (transgenic acceptor CCRI24 and drought‐resistant ZR409) and AtHUB2‐expressing cotton lines (line2, line4 and line6) in the field under drought stress conditions (n ≥ 30). Vertical bars represented SD. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

AtHUB2 significantly enhances the drought tolerance of transgenic cotton in the greenhouse. (a) Drought tolerance of control plants and AtHUB2‐expressing cotton plants. Photographs were taken at 4 weeks after germination, after 20, 28 and 35 days of drought stress, and after a 1‐week recovery period. Bar = 7.5 cm. (b) Leaf relative water content (RWC), proline content and soluble sugar content of transgenic cotton and control plants with or without drought treatment (n ≥ 30). (c) Survival rates of plants after 1 week of recovery (n ≥ 30). Vertical bars represent SD. (*P < 0.05; **P < 0.01).

Figure 4

RT‐qPCR analyses of the expression of stress‐responsive genes in control, AtHUB2 overexpression lines (a) and GhHUB2‐knockdown lines (b). The RNA extracted from the leaves of the indicated genotypes (4 weeks old) before or after air‐drying for 4 h. Values determined via RT‐qPCR were normalized to the expression of GhUBI1. Vertical bars represent the SD. (*P < 0.05; **P < 0.01, ***P < 0.001).

Figure 5

Response of GhHUB2‐knockdown lines to drought stress. (a) Drought tolerance of control CCRI24 and GhHUB2‐knockdown cotton plants. Photographs were taken at 4 weeks after germination, after 25 days of drought stress and re‐watering for 3 days. Bar = 7.5 cm. (b) Survival rates of plants after 25 days of drought stress and 3 days of recovery (n ≥ 30). (c) The volume of water lost from detached leaves of the indicated genotypes (4 weeks old) via air‐drying was measured as the percentage of change in the fresh weight (FW) of the leaves (n ≥ 30). Vertical bars represent SD. (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 6

AtHUB2 is a functional E3 ligase. (a) Yeast two‐hybrid (Y2H) assays. GhH2B1‐pGADT7 was transformed into yeast cells with either the pGBKT7 vector alone (BDB) or AtHUB2‐pGBKT7, and growth was monitored on selective medium plates. (b) Pull‐down assay. GST‐HUB2 and GST alone were incubated with GhH2B1‐His. And detected with an anti‐GST antibody and anti‐His antibody. (c) Split‐luciferase assay. GhH2B1‐LUC ⁿ and LUC ^c‐AtHUB2 co‐injection tobacco. The combinations of LUC ⁿ and LUC ^c, LUC ⁿ and LUC ^c‐ AtHUB2, and GhH2B1‐LUC ⁿ and LUC ^c were used as negative controls. (d) AtHUB2 possesses E3 ubiquitin ligase activity in vitro. GST‐AtHUB2 and the point mutant GST‐HUB2 ^C346A/H348 were tested for E3 ubiquitin ligase activity. Anti‐Ub and anti‐GST antibodies were used to detect ubiquitinated proteins and GST‐AtHUB2 variants respectively. The asterisk indicates the polyubiquitinated proteins. (e) Global histone modification in the control (CCRI24) and transgenic lines. H2B monoubiquitination was detected with an anti‐H2Bub1 antibody, and H3 was used as a loading control. ImageJ software was used to analyse the greyscale value of each band. Vertical bars represent SD (**P < 0.01). (f) Detection of H3K4 methylation on a global scale in the control (CCRI24) and transgenic lines. And an anti‐H3K4me2 and anti‐H3K4me3 antibodies were used to detect target band, respectively. H3K9me2 and H3K9me3 were used as unchanged controls, and H3 was used as a loading control. The number below the lane indicates the greyscale value of each band.

Figure 7

Detection of global histone H2Bub1 and H3K4me3 levels at the GhDREB chromatin locus. (a) Schematic diagram of the GhDREB gene. The exons are shown in boxes, and the indicated PCR fragments analysed in the ChIP assay are indicated with short black lines. P1: 2000 bp upstream of the ATG; P2: promoter region; P3: transcription start site (TSS) region; P4: gene body region. (b) H2Bub1 at the GhDREB locus in the CCRI24 and transgenic lines. Immunoprecipitated DNA was analysed via RT‐qPCR, and enrichment was determined as a percentage of the input. (c) H3K4me3 at the GhDREB locus in the CCRI24 and transgenic lines. (d) The GhUBI gene was selected as a housekeeping gene that was not differentially expressed in the transgenic lines. (e) Expression of GhDREB at different time points of air drought treatment in control and AtHUB2‐overexpressing lines. Real‐time RT‐qPCR quantification was normalized to the expression of GhUBI . Vertical bars represent SD. (*P < 0.05, **P < 0.01, ***P < 0.001).