Functional studies of Arabidopsis annexins AnnAt1 and AnnAt2 in primary root growth

Annexins are a multigene family of calcium-dependent membrane-binding proteins that play important roles in plant cell signaling. Annexins are multifunctional proteins, and their function in plants is not comprehensively understood. Arabidopsis (Arabidopsis thaliana) annexins ANN1 and ANN2 are 64% identical in their primary structure, and both are highly expressed in seedlings. Here, we showed that null mutants of AnnAt1 or AnnAt2 grown as seedlings in the absence of sugar show decreased primary root growth and altered columella cells in root caps; but these mutant defects are rescued by sucrose, glucose, or fructose. In seedlings grown without sugar, significant up-regulation of genes encoding proteins needed for photosynthesis and increased chlorophyll accumulation was found in the cotyledons of the null mutants compared to in wild-type, which indicates potential sugar starvation in the roots of the mutant seedlings. Unexpectedly, the overall sugar content of primary roots was significantly higher in roots of the mutants compared to wild-type when the seedlings were grown without sugar for a week. To examine the diffusion of sugar along the entire root to the root tip, we examined the unloading pattern of carboxyfluorescein dye and found that post-phloem sugar transport was impaired in the mutant root tips compared to that in wild type. Also, in the root tips of the mutant seedlings grown without sucrose we detected increased levels of ROS and callose, the latter of which might restrict plasmodesmal sugar transport to root tips. These results indicate that ANN1 and ANN2 play an important role in post-phloem sugar transport to the root tip, which in turn might indirectly influence photosynthesis in cotyledons, as suggested by observed changes in chlorophyll content and expression levels of photosynthetic genes. Using transmission electronic microscopy, we observed structural alterations in the plasmodesmata of post-phloem root cells of the null mutants, and using RNA-seq and gene ontology analyses we found that knocking out ANN1 and ANN2 induced global transcriptomic changes in Arabidopsis roots. This study expands our understanding of the function of annexins in plants and sheds light on the role of plasmodesmata in post-phloem transport and intercellular signaling