Demonstration in vivo of the role of Arabidopsis PLIM2 actin-binding proteins during pollination.

In plant reproduction, pollination is the initial key process in bringing together the male and female gametophytes. When a pollen grain lands on the surface of the stigma, information is exchanged between the pollen and stigmatic cell to determine whether the pollen grain will be accepted or rejected. If it is accepted, the stigmatic papilla cell supplies water and other resources to the pollen for germination and pollen tube elongation. Cellular processes involving actin are essential for pollen germination and tube growth, and actin-binding proteins regulate these processes by interacting with actin filaments to assemble cytoskeletal structures and actin networks. LIM proteins, which belong to a subfamily of cysteine-rich proteins, are a family of actin-binding proteins in plants, and are considered to be important for formation of the actin cytoskeleton and maintenance of its dynamics. Although the physiological and biochemical characteristics of LIMs have been elucidated in vitro in a variety of cell types, their exact role in pollen germination and pollen tube growth during pollination remained unclear. In this manuscript, we focus on the pollen-specific LIM proteins, AtPLIM2a and AtPLIM2c, and define their biological function during pollination in Arabidopsis thaliana. The atplim2a/atplim2c double knockdown RNAi plants showed a reduced pollen germination, approximately one-fifth of wild type, and slower pollen tube growth in the pistil, that is 80.4 µm/hr compared to 140.8 µm/hr in wild type. These defects led to an occasional unfertilized ovule at the bottom of the silique in RNAi plants. Our data provide direct evidence of the biological function of LIM proteins during pollination as actin-binding proteins, modulating cytoskeletal structures and actin networks, and their consequent importance in seed production.

UDP-glucose pyrophosphorylase is rate limiting in vegetative and reproductive phases in Arabidopsis thaliana.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme in the metabolism of UDP-glucose, a precursor for the synthesis of carbohydrate cell wall components, such as cellulose and callose. The Arabidopsis thaliana genome contains two putative genes encoding UGPase, AtUGP1 and AtUGP2. These genes are expressed in all organs. In order to determine the role of UGPase in vegetative and reproductive organs, we employed a reverse genetic approach using the T-DNA insertion mutants, atugp1 and atugp2. Despite a significant decrease in UGPase activity in both the atugp1 and atugp2 single mutants, no decrease in normal growth and reproduction was observed. In contrast, the atugp1/atugp2 double mutant displayed drastic growth defects and male sterility. At the reproductive phase, in the anthers of atugp1/atugp2, pollen mother cells developed normally, but callose deposition around microspores was absent. Genes coding for enzymes at the subsequent steps in the cellulose and callose synthesis pathway were also down-regulated in the double mutant. Taken together, these results demonstrate that the AtUGP1 and AtUGP2 genes are functionally redundant and UGPase activity is essential for both vegetative and reproductive phases in Arabidopsis. Importantly, male fertility was not restored in the double knockout mutant by an application of external sucrose, whereas vegetative growth was comparable in size with that of the wild type. In contrast, an application of external UDP-glucose recovered male fertility in the double mutant, suggesting that control of UGPase in carbohydrate metabolism is different in the vegetative phase as compared with the reproductive phase in A. thaliana.