The U-box E3 ubiquitin ligase PalPUB79 positively regulates ABA-dependent drought tolerance via ubiquitination of PalWRKY77 in Populus

Abstract

The abscisic acid (ABA) signalling pathway is involved in the plant response to osmotic stress caused by drought and/or salinity. Although the ABA signalling pathway has been elucidated in Arabidopsis, it remains elusive in woody poplars. In this study, genome-wide analyses of U-box genes in poplars revealed that a U-box E3 ubiquitin ligase gene, PalPUB79, is significantly induced following drought, salinity and ABA signalling. PalPUB79 overexpression enhanced drought tolerance in transgenic poplars, while PalPUB79 RNAi lines were more sensitive to drought. PalPUB79 positively regulated ABA signalling pathway. Furthermore, PalPUB79 interacted with PalWRKY77, a negative transcriptional regulator of ABA signalling, and mediated its ubiquitination for degradation, therefore counteracting its inhibitory effect on PalRD26 transcription. However, the finding that PalWRKY77 negatively regulates PalPUB79 expression was indicative of a negative feedback loop between PalWRKY77 and PalPUB79 during ABA signalling in poplar. These findings provide novel insight into the mechanism through which PalPUB79 enhances the ABA-mediated stress response in woody poplars.

Keywords: Populus; PalPUB79; U-box type E3 ubiquitin ligases; abscisic acid; drought.

Figure 1

The transcriptome of Populus alba var. pyramidalis under salt stress, along with the expression pattern of PalPUB79 under mannitol and ABA treatments. (a) The gene ontology (GO) enrichment analysis of the genes with up‐regulated and (b) with down‐regulated expression under salt treatment. All genes analysed based on GO enrichment show differential expression at a significance level of P < 0.05. (c–d) Heatmaps of the differential expression (P < 0.05) of 478 genes with or without salt treatment. (e) The expression pattern of PalPUB79 under mannitol, salt and ABA treatments.

Figure 2

Characteristics of PalPUB79. (a) Phylogenetic tress constructed from the full‐length peptide sequences of PalPUB79 and AtPUB19 homologues from multiple plants, including basal angiosperms. The PalPUB79 is highlighted in red while AtPUB19 is denoted by red star. (b) The tissue expression profile of PalPUB79 in Populus alba var. pyramidalis, based on relative PalPUB79 levels, in young leaf (YL), mature leaf (ML) stem (S), primary root (PR), lateral root (LR) and petiole (P). Error bars indicate SD (n = 3). (c) The subcellular localization of PalPUB79 in P. alba var. pyramidalis protoplasts. The pBI221 empty vector served as a control, while 4′,6‐diamidino‐2‐phenylindole (DAPI) staining was used to identify the nucleus. (d) The E3 ubiquitin (Ub) ligase activity of PalPUB79 was detected using the GST‐PalPUB79 fusion protein, His‐Ub UBCH5C (E2) and E1 (R&D Systems, E‐300‐050). An anti‐Ub antibody was used to detect the ubiquitinated proteins (top panel) and ubiquitin monomers (bottom panel).

Figure 3

Drought tolerance among PalPUB79 overexpression and RNAi poplars. (a) The transgenic Populus tomentosa overexpressing PalPUB79 (PalPUB79‐OE‐L14 and PalPUB79‐OE‐L18) and WT plantlets at a similar status were completely dehydrated for 4 days, then rehydrated for 2 days. Three independent plants of both overexpression line were examined. (b) The RNAi poplars (PalPUB79‐Ri‐L2 and PalPUB79‐Ri‐L12) were more sensitive to drought stress than WT plants. (c–e) Physiological indices, including malondialdehyde (MDA) content (c), electrolyte leakage (EL) (d), chlorophyll content (e), among the overexpression poplars under dehydration conditions. (f–h) MDA content (f), EL (g) and chlorophyll content of RNAi lines under drought stress. Error bars indicate SD (n = 5), **P < 0.01. The experiment was repeated three times with similar results.

Figure 4

The enhanced drought tolerance of PalPUB79 overexpression poplars was removed by fluridone treatment. (a) PalPUB79 overexpression and WT plantlets were transplanted into the solid WPM containing 100 mM mannitol and 10 μM fluridone for 1 month, with all poplar plantlets were at a similar growth status before treatment. (b) PR length of plantlets. (c) The MDA content in the plantlets. The independent experiment was repeated three times and similar results were obtained.

Figure 5

The RNA‐seq and qPCR analysis of PalPUB79 overexpression and RNAi poplars. (a) The Venn diagram indicates which differentially expressed genes overlapped in the PalPUB79 overexpression and RNAi poplars. (b) The doughnut chart showed the proportion of these genes that were related to plant drought response. (c) A heatmap of the expression levels of drought stress‐related genes in WT, PalPUB79 overexpression and RNAi poplars. (d) qPCR verified the expression levels of OSM34, SUS3, RLK7, PRX64, RD26, HIS1‐3 and LEA4‐5 across the different poplar lines. The gene‐specific primers used in the qPCR assay are presented in Table S6. The error bars represent the SD of mean values (n = 3). The internal reference was the ubiquitin (UBQ) gene.

Figure 6

PalPUB79 interacts with PalWRKY77. (a) The list of proteins that interact with PalPUB79 that were screened using a yeast two‐hybrid screening assay. (b) A point‐by‐point yeast two‐hybrid assay provided evidence for the interaction between PalPUB79 and PalWRKY77. The BD and AD empty plasmids with AD‐PalWRKY77 or BD‐PalPUB79 constructs, respectively, served as the negative controls. (c) A bimolecular fluorescence complementation (BiFC) assay was used to demonstrate the in vivo interaction between PalWRKY77 and PalPUB79. The nVenus‐PalPUB79 and cCFP‐PalWRKY77 constructs were co‐transformed into the mesophyll protoplasts of poplar. The empty plasmids with nVenus or cCFP were co‐transformed with cCFP‐PalWRKY77 or nVenus‐PalPUB79, respectively, to serve as negative controls.

Figure 7

PalPUB79 mediates PalWRKY77 degradation via ubiquitination. (a) 45‐day‐old transgenic poplars expressing the HA‐PalWRKY77 fused protein were irrigated with 300 mM mannitol for 0, 1 and 2 h, after which leaves of the second node were collected for Western blot analysis. The anti‐HA antibody was used to detect the abundance of HA‐PalWRKY77. Actin content, detected using the anti‐actin antibody, served as the internal reference. (b) The 35S: HA‐PalWRKY77 construct, alone or in conjunction with the 35S:PalPUB79 construct, was introduced into Nicotiana benthamiana leaves through agrobacterium‐mediated transformation. Immunoblotting was performed with anti‐HA and anti‐Ub antibodies. (c) PalPUB79‐mediated degradation of PalWRKY77 in tobacco leaves, which involved UPS recruitment, was inhibited by MG132. (d) The cell‐free extract from PalPUB79 overexpression poplar leaves mediated degradation of the bacterially expressed MBP‐PalWRKY77 recombinant protein, and this process was inhibited by MG132. (e) Results of the dual‐luciferase reporter assay, shown as the expression level of firefly luciferase gene (LUC) driven by a 1.5 kb‐length promoter of PalRD26 with or without PalWRKY77 and PalPUB79. Renilla luciferase (REN) activity served as the internal reference. ANOVA was performed in SPSS. Error bars represent the SD of mean values (n = 3), while significant differences (P < 0.05) between groups are indicated by ‘a’ and ‘b’.

Figure 8

PalWRKY77 negatively regulates PalPUB79 transcription. (a) The results of the dual‐luciferase reporter assay, shown as the expression level of LUC driven by a 1.5 kb‐length promoter of PalPUB79 with or without PalWRKY77. The experiments were performed under normal conditions, mannitol treatment and ABA treatment. Error bars indicate SD (n = 3), *: P < 0.05, **: P < 0.01. (b) The W‐box in the promoter of PalPUB79 and the ChIP‐qPCR results demonstrated that PalWRKY77 binds to the W‐box of the PalPUB79 promoter. WT plants served as the negative controls. P1 to P5 indicate the different regions of the PalPUB79 promoter. Error bars indicated SD (n = 3), **: P < 0.01. All of the primers used in ChIP‐qPCR are listed in Table S6. (c) The yeast one‐hybrid assay results showed how PalWRKY77 binds to the W boxes (PW1 and PW2, the mutant mPW1 and mPW2) in the promoter of PalPUB79. (d) The results of the dual‐luciferase reporter assay, shown as the expression level of LUC driven by a 1.5 kb‐length promoter of PalPUB79 with or without PalWRKY77 and PalPUB79. REN activity served as the internal reference. ANOVA was performed in SPSS. Error bars represent the SD of mean values (n = 3), and significant differences (P < 0.05) between groups are indicated by ‘a’ and ‘b’.

Figure 9

A proposed model for the action mechanism of PalPUB79. (a) PalWRKY77 inhibited PalPUB79 and PalRD26 transcription under normal condition. (b) The feedback regulation of PalPUB79 and PalWRKY77 under drought conditions, i.e., the expression of PalPUB79 removes the PalWRKY77‐mediated inhibition of PalPUB79. (c) PalWRKY77‐mediated inhibition of PalRD26 was relieved by PalPUB79‐mediated ubiquitination of PalWRKY77.