Role Of Rice Bean V-ATPase Subunit E (VuVHAE1) In Plant Aluminum Stress Resistance

Aluminum stress is one of the most important limiting factors of crop production in acidic soils. And understanding of the molecular mechanisms of plant Al resistance and improving plant Al tolerance ability by genetic manipulation is crucial to solve it. Mounting evidence suggests that cell metabolic changes play an important role in plant aluminum stress response; however, the molecular mechanisms still remains vague. ATP is a cardinal energy and signal molecule in plants, and H+-ATPase can regulate cell metabolism by affecting ATP homeostasis. H+-ATPase has long been hypothesized to be involved in the Al stress response, but the role of specific H+-ATPase in aluminum stress is still scarcely reported. In this study,6 genes relating to ATP homeostasis were cloned, and their functions in Al stress were studied by heterologous overexpression. The main results are as follows:1. Full-length cDNA sequences of the 6 genes were obtained by RACE technique. Expression patterns analysis showed that their transcription levels of were not affected by Al stress. However, Al3+ can decrease the gross plasma membrane H+- ATPase activity, it can immediately increase the gross mitochondria H+-ATPase activity(<4h), but has little impact on the gross vacuolar H+-ATPase activity. So there must be some post-transcriptional regulation mechanisms to be operated. To identify whether these genes are responsible for plant Al resistance, transgenic Arabidopsis lines harboring over-expression (OE) structure of these genes were generated. Among the 6 genes, V-ATPase subunit E (VuVHAE1) OE lines showed enhanced Al resistance while others not. Thus the following efforts were mainly dedicated to the role of VuVHAEl in plant Al resistance.2 VuVHAE1 encodes a plasma membrane and nuclear localized V-ATPase. The OE plants showed significant rhizosphere acidification, indicating that VuVHAE1 can augment plasma membrane H+-ATPase activity. In addition, OE plants absorbed less Al, especially in root apex and root hairs as visualized by hematoxylin staining. Evans Blue staining also showed that the plasma membrane integrity were better in OE plants. These evidences indicate that the external exclusion mechanism may count for their Al tolerance. Expression level analysis of Arabidopsis Al resistance genes indicated that the transcription level of AtALMTl in the OE lines was higher, while the differences of other genes were not as significant; the subsequent test also proved that Al induced malate secretion was higher in the OE lines when juxtaposed to WT plants, which suggests that the enhanced malate secretion is an important mechanism to reduce Al accumulation in plants. However, other roles of VuVHAEl in Al tolerance requires further investigation.