The wheat VIH2-3B, a functional PPIP5K controls the localization of fasciclin-like arabinogalactan protein

Inositol pyrophosphates (PP[InsPs) are important signalling molecules, that participate in multiple physiological processes across a wide range of eukaryotes. Metabolic pathway kinases (VIP1/VIHs) leading to the production of PP-InsPs are now well characterized in yeast and plants. Previously, the wheat inositol pyrophosphate kinase ( TaVIH2 ) was shown to encode a catalytic active kinase domain. Heterologousexpression of TaVIH2 in Arabidopsis thaliana showed enhanced drought tolerance by modulating the cell composition. In this study, we attempted to identify the interacting protein targets of wheat VIH2-3B using a yeast two-hybrid (Y2H) cDNA library screen, identifying 52 putative interactors that are primarily involved in cell wall-related functions. Notably, fasciclin-like arabinogalactan protein (FLA7), a glycosylphosphatidylinositol (GPI)-anchored protein emerged as the most frequently interacting partner in the screen. Further analysis using pull-down assays validated the interaction between TaVIH2-3B and TaFLA7 in vivo . Using the reporter fusion studies, we observed the localization of TaFLA7 to be a plasma membrane. We also observed that this localization of the FLA7 was perturbed in the yeast vip1 Δ strain. The expression of TaVIH2-3B bearing PP-InsP5K enzymatic activity in yeast mutants rescued the localisation of the FLA7 to the membrane. Expression analysis of TaFLA7 showed differential expression response under drought in wheat shoot tissues. TaFLA7 was also found to be highly expressed during grain development, particularly in the endosperm and seed coat during grain maturation. Taken together, these findings highlight the potential role of TaVIH2 in cell wall remodelling and stress response pathways, offering new insights into the functional roles of VIH proteins in plants.

Key word: Inositol pyrophosphates, kinases, cell wall maintenance.