Functional activity analysis of plasma membrane H+-ATPase gene promoter and physiological functional identification of interactive transcription factor in blueberry

Blueberry plant (Vaccinium spp.) roots are sensitive to pH changes (> 5.5) in the rhizosphere. In China, the soil pH in blueberry planting areas is typically above 5.5, consequently restricting industry expansion. Root plasma membrane proton pumps (PM H+-ATPase - HAs) promote the efflux of H+ during nutrient absorption, thereby reducing stress associated with alkaline rhizosphere soils. However, the regulatory mechanisms governing expression of blueberry VcHAs genes are unknown. We isolated promoter fragments upstream of the translational start of nine VcHA genes from Vaccinium spp. Based on the strength of transient promoter::luc expression in tobacco leaves, VcHA11.1-prom was selected for further analyses of cis-acting elements and transcription factors (TFs). In silico and in vitro (yeast one-hybrid - Y1H) studies with a 5-prime deletion VcHA11.1-prom variant (B) were used to identify and verify binding of two blueberry TFs, namely VcDof2.1 and VcbHLH104. Ensuing transient Luc assays in transgenic tobacco leaves revealed that VcbHLH104 upregulated and VcDof2.1 downregulated expression from VcHA11.1-B-prom. qRT-PCR analysis of RNA isolated from WT blueberry roots confirmed that plants propagated at pH 5.0 resulted in high VcDof2.1 but low VcHA11.1 transcripts. VcbHLH104 transcript levels significantly increased (ca. 242.0%) and paralleled those of VcHA11.1 when plants were cultivated at pH 7.0. Transgenic Arabidopsis seedlings overexpressing VcbHLH104 demonstrated improved H+ efflux and alleviated stress at pH ≥ 7.0 with no distinguishable plant etiolation. Conversely, transgenic plants expressing VcDof2.1 displayed weak H+ release, poor growth, leaf sparsity and chlorosis at pH ≥ 7.0. We conclude that expression of VcHA11.1 is pH dependent and that promoter transcription is counter regulated by VcDof2.1 and VcbHLH104 at pH ≤ 5.0 and pH ≥ 7.0, respectively.