Ethylene increases the NaHCO3 stress tolerance of grapevines partially via the VvERF1B-VvMYC2-VvPMA10 pathway

Abstract

Here, we evaluated the role of ethylene in regulating the NaHCO₃ stress tolerance of grapevines and clarified the mechanism by which VvERF1B regulates the response to NaHCO₃ stress. The exogenous application of ACC and VvACS3 overexpression in grapevines and grape calli revealed that ethylene increased NaHCO₃ stress tolerance, and this was accompanied by increased plasma membrane H⁺-ATPase (PMA) activity. The expression of VvERF1B was strongly induced by ACC, and overexpression of this gene in grapevines conferred increased NaHCO₃ stress tolerance and enhanced PMA activity and H⁺ and oxalate secretion. Additionally, the function of VvERF1B was also verified using mutant transgenic grape calli and overexpression in Arabidopsis plants. The expression of VvPMA10 was strongly induced following the overexpression of VvERF1B in grapevine roots, and VvPMA10 was shown to regulate PMA activity, oxalate and H⁺ secretion, and NaHCO₃ stress tolerance via its overexpression and mutation in grapevine roots, calli, and/or Arabidopsis. However, VvPMA10 was not a direct target gene of VvERF1B but was directly transactivated by VvMYC2. The function of VvMYC2 was shown to be similar to that of VvPMA10 via its overexpression and mutation in grape calli. Additional experiments revealed that the interaction of VvERF1B with VvMYC2 increased its ability to activate VvPMA10 expression and that VvMYC2 played a role in the VvERF1B-mediated pathway. Overall, the VvERF1B-VvMYC2-VvPMA pathway played a role in regulating ethylene-induced NaHCO₃ stress tolerance in grapevines, and this process contributed to increases in PMA activity and H⁺ and oxalate secretion.

Keywords: NaHCO3 tolerance; VvERF1B; VvMYC2; VvPMA10; ethylene.

Figure 1

Effects of the exogenous application of ACC and 1‐MCP and VvACS3 overexpression on the NaHCO₃ stress tolerance of grapevines and calli. (a) Phenotypes of grapevines grown in a greenhouse under natural conditions and under various treatments (0, 14, and 21 DAT). The vines were treated with 100 mm NaHCO₃ and 50 μm ACC for 3 h, followed by 100 mm NaHCO₃ (ACC + NaHCO₃) and 0.2 μm 1‐MCP for 3 h, and 100 mm NaHCO₃ (1‐MCP + NaHCO₃). (b–d) Root activity, MDA content, and relative conductivity of grapevines under various treatments at 0 and 14 DAT. (e, f) Expression and ethylene release rate in VvACS3‐overexpressing calli. (g–i) Phenotypes, MDA content, and growth increase of VvACS3‐overexpressing calli at 21 days after 2.5 mm NaHCO₃ treatment. DAT, days after treatment. Data represent means ± standard deviation (SD) of three biological experiments. Significant differences between WT and transgenic lines were calculated using Student's t‐test. ** P‐value <0.01, and different lowercase letters represent significant differences at P < 0.05.

Figure 2

Changes in PMA activity and H⁺ and oxalate secretion in roots and calli under NaHCO₃ stress. (a) PMA activity in roots under NaHCO₃ treatment. (b) Rhizosphere acidification is indicated by pH‐sensitive bromocresol purple. Vine roots were divided into two equal parts; one part was placed in a medium containing 100 mm NaHCO₃, and another part was placed in a medium containing 0.006% bromocresol purple. For NaHCO₃ + ACC treatment, the roots were pretreated with 50 μm ACC for 3 h before being covered by the medium. (c) Oxalate content in root exudates. (d–f) PMA activity and H⁺ and oxalate secretion in VvACS3‐overexpressing calli at 2 days after subculture in the medium; calli were treated with 100 mm NaHCO₃ for 6 h before subculture. Values represent the means ± SD of three replicates. * Significant difference, P < 0.05; ** highly significant difference, P < 0.01.

Figure 3

The role of VvERF1B in increasing NaHCO₃ stress tolerance and modifying PMA activity, H⁺ efflux, and oxalate secretion in grapevines and calli. (a, b) The expression of VvERF1B in roots treated with different concentrations of ACC at 3 h (a) and with 10 mm ACC at different time points (b). (c) The expression of VvERF1B in the four transgenic grapevine lines. (d–h) Phenotypes of WT and VvERF1B‐overexpressing grapevines (d) and corresponding physiological parameters, including the MDA content (e), PMA activity (f), H⁺ efflux rate, and oxalate content in their roots (h) under 100 mm NaHCO₃ treatment for 5 days. (i–m) Phenotype evaluation (i), growth increase (j), PMA activity (k), and levels of oxalate (l) and H⁺ (m) secretion in WT, VvERF1B‐overexpressing, and mutant grape calli. The calli in panels i–l were treated with 2.5 mm NaHCO₃ for 21 days; the calli in panel m were treated with 100 mm NaHCO₃ for 6 h and then were subcultured on medium containing 0.006% bromocresol purple. ns, not significant. Values are the means of three replicates, and error bars denote the SD. ** highly significant difference, P < 0.01.

Figure 4

The role of VvPMA10 in increasing NaHCO₃ stress tolerance and modifying PMA activity in grapevines and calli. (a) Relative expression of the eight PMA genes in VvERF1B‐overexpressing grapevines. (b) Expression of VvPMA10 in VvERF1B‐overexpressing and mutant grape calli. (c) Identification of VvPMA10‐overexpressing grape roots via Agrobacterium rhizogenes‐mediated gene transformation using qRT‐PCR. (d–h) Phenotypes of WT and transgenic roots and corresponding physiological parameters, including the MDA content (e), PMA activity (f), H⁺ efflux rate (g), and oxalate secretion (h) under 50 mm NaHCO₃ treatment for 3 days. (i) Expression of VvPMA10 in WT and VvPMA10‐overexpressing grape calli. (j–o) Phenotype (j), growth increase (k), MDA content (l), PMA activity (m), and oxalate (n) and H⁺ (o) secretion of WT, VvPMA10‐overexpressing, and mutant grape calli under 2.5 mm NaHCO₃ for 21 days. The calli in panel m were treated with 100 mm NaHCO₃ for 6 h and then were subcultured on medium containing 0.006% bromocresol purple. Values represent the means ± SD of three replicates. * Significant difference, P < 0.05; ** highly significant difference, P < 0.01.

Figure 5

Characterization of the transcriptional activation of VvPMA10 by VvMYC2 and the role of VvMYC2 in increasing NaHCO₃ stress tolerance of calli. (a) Y1H assays. AD+ProG‐box indicates empty pGADT7 + ProG‐box::pHIS2, and AD‐VvMYC2 + ProG‐box indicates VvMYC2‐pGADT7 + ProG‐box::pHIS2. (b) Interaction of the VvMYC2 fusion protein with the DNA probes for the G‐box and mutant G‐box within the VvPMA10 promoter in an EMSA. (c) Representative images of tobacco leaves at 48 h after infiltration. (d) Expression of VvMYC2 in grapevine roots under 100 mm NaHCO₃. (e) Identification of VvMYC2‐overexpressing grape calli using qRT‐PCR. (f–k) Phenotypes (f), growth (g), expression of VvPMA10 (h), PMA activity (i), and oxalate (j) and H⁺ secretion level (k) of WT, VvMYC2‐overexpressing, and mutant grape calli under 2.5 mm NaHCO₃ for 21 days. The calli in panel k were treated with 100 mm NaHCO₃ for 6 h and then were subcultured on a medium containing 0.006% bromocresol purple. Values represent the means ± SD of three replicates. ** highly significant difference, P < 0.01.

Figure 6

Interaction of VvERF1B with VvMYC2 in vitro and in vivo. (a) The VvERF1B‐pGADT7 interaction with VvMYC2‐pGBKT7 in a Y2H system. (b) Confirmation of the interaction of VvERF1B with VvMYC2 by BiFC in N. benthamiana leaf epidermal cells, as indicated by a yellow fluorescent signal. (c) VvERF1B interacted with VvMYC2 in pull‐down assays. “+” and “−” indicate the presence and absence of the indicated protein, respectively. (d) Luciferase complementarity assay showing that VvERF1B interacts with VvMYC2.

Figure 7

Effects of the VvERF1B–VvMYC2 complex on VvPMA10 expression and NaHCO₃ stress tolerance in grape calli. (a) LUC experiments revealing the enhanced transcriptional activation of VvPMA10 by VvMYC2 in the presence of VvERF1B. The number in brackets indicates the ratio of Agrobacterium tumefaciens containing different expression vectors. (b) Phenotypes of the WT and transgenic calli under normal and 2.5 mm NaHCO₃ conditions for 21 days. (c–h) Growth (c), MDA content (d), VvPMA10 expression (e), and PMA activity (f) from the same calli shown in panel b. (g) The oxalate content in the extrudate of calli from the same materials as shown in panel b. (h) H⁺ secretion level. The calli in panel k were treated with 100 mm NaHCO₃ for 6 h and then were subcultured on medium containing 0.006% bromocresol purple. Different lowercase letters represent significant differences at P < 0.05.

Figure 8

Model of the regulation of NaHCO₃ stress tolerance by ethylene via the VvERF1B‐VvMYC2‐VvPMA10 pathway. NaHCO₃ stress induces ethylene production and up‐regulates VvERF1B expression. VvERF1B interacts with VvMYC2 to activate VvPMA10 expression, which increases PMA activity. The enhanced PMA activity promotes H⁺ efflux and generates an electrochemical gradient, which provides energy for the transport of oxalate, Na⁺, and ⁻HCO₃ across the plasma membrane.