Functional analysis of plasma membrane H+-ATPases in response to alkaline stress in blueberry

Abstract

• At pH 8.0, H + influx and H + efflux were restricted in blueberry roots. • In blueberry roots and leaves, most VcHAs had lowest transcript levels at pH 8.0. • ATP proton pump activity was increased in Arabidopsis over-expressing (OE) VcHAs . • A. thaliana OE lines tolerated alkaline stress due to increased H + pumping. Blueberry ( Vaccinium spp.) favors acid soils (pH 4.0–5.5), but the reasons for this strong preference are unclear. Root plasma membrane proton pumps (PM H + -ATPases - HAs) promote the efflux of hydrogen ions (H + ) and thus promote nutrient absorption, thereby reducing stress associated with alkaline soils. In this study, ‘Jewel’ blueberry ( V. corymbosum ) was grown in media with a range of pH values (pH 5.0, pH 7.0, pH 8.0). After 30 days of growth, the physiological responses and the transcript levels of VcHAs (PM H + -ATPases) were analyzed. Eight VcHA genes were genetically transformed into Arabidopsis thaliana for functional verification. The results showed that (1) blueberry plants grew best at pH 5.0. The root activity at pH 5.0 was more than 1.6 times that at pH 8.0. In the pH 8.0 treatment, H + influx and efflux from blueberry roots were limited. (2) Different VcHA genes showed diverse expression patterns. Among them, VcHA8 was only expressed in the root at pH 5.0. Except for VcHA11.2 in the roots, the transcript levels of VcHAs in roots and leaves were lowest in the pH 8.0 treatment. (3) Transgenic Arabidopsis thaliana plants over-expressing VcHA4.1, VcHA4.2, VcHA4.3, VcHA5, VcHA10.1, VcHA10.2, VcHA11.1, or VcHA11.2 showed stronger root activity than that of wild-type in all pH treatments. The root activity of the line overexpressing VcHA5 was 96 times that of the control. Compared with control plants, the phenotypic analyses of the VcHAs transgenic plants revealed enhanced levels of plant height, leaf size and chlorophyll content. The H + -ATPase activity of Arabidopsis overexpressing VcHA10.2 was 44 times that of the control. VcHAs not only significantly increased the hydrolytic activity and ATP proton pump activity in Arabidopsis but also increased H + efflux, leading to cytoplasmic alkalinization in root epidermal cells. Over-expression of VcHAs significantly increased H + pumping in Arabidopsis roots under alkaline rhizosphere stress, confirming that these genes encode functional proteins that are capable of acidifying the rhizosphere. VcHA10.2 and VcHA5 had the strongest ability to pump H + . The expression of VcHAs was inhibited in blueberry plants growing in high-pH media. This may explain why blueberry grows best in acid soils. These results help to explain why blueberry favors acid soils and may be useful for developing strategies to cultivate this crop in a wider range of soil types.