Molecular analysis of an acid invertase gene family in Arabidopsis

Invertases can influence plant biology in at least two ways; first, by affecting resource allocation among plant parts through cleavage of the transport sugar, sucrose, and second, by indirectly altering expression of genes sensitive to shifts in sucrose/hexose availability. To initiate a test of roles and regulation for these invertases, their gene families in Arabidopsis thaliana were subjected to gene expression analyses. A search of predicted protein sequences in the Arabidopsis database indicated two vacuolar- and six cell-wall invertases in this species. Clues to their functions were obtained by analyzing mRNA levels for individual genes using quantitative RT-PCR. The two vacuolar invertase mRNAs, AtvacINV1 and AtvacINV2, were predominated in vegetative tissues. Sugar responses of the vacuolar invertases were reciprocal at the mRNA levels, with AtvacINV1 being up-regulated and AtvacINV2 being down-regulated. Analysis of mRNA turnover indicated that AtvacINV2 mRNAs were destabilized in glucose medium despite glucose induction of the AtvacINV2 promoter (as observed for the other invertases). Reciprocal responses of the two vacuolar invertases at the mRNA level may be responsible for a precise balance of expression across different conditions. An additional approach to identifying functional significance of these genes was to test the impact of their inactivation in single and double mutants. Activity in the vacuolar (soluble) fraction was markedly decreased in each of the single-gene knockouts, and undetectable in the double mutant. Data confirm that these two genes encode functionally active soluble acid invertases. Extensive phenotypic analysis revealed significant decreases in root length of single and double mutants on wet filter paper. However, roots of single mutants consistently outgrew those of wild-type seedlings in agar medium, where abundant osmolytes were supplied from agar medium for cell expansion. In addition, vacuolar invertase knockouts had markedly altered molecular phenotypes that included changes in mRNA levels for cell wall invertases and sucrose synthases. Mutants defective in the sugar-inducible AtvacINV1 showed diverse gene responses consistent with predicted reductions in signal-active hexoses. Although effects of the AtvacINV2 mutation were more complex, evidence from the molecular phenotype of AtvacINV1 knockouts was consistent with an upstream role of this vacuolar invertase in sugar signaling.