This proposal seeks to identify an integrin-like protein in the plant, Arabidopsis and to demonstrate a role of this integrin-like protein in gravitropism. Previous work has identified a 58 kD and an 80 kD protein, both associated with membranes, which cross-reacts with an antibody to chick beta-integrin. Immunostaining using this same antibody shows enriched activity in the root tips in both Arabidopsis and the lower plant, Chara, the anatomical site of the gravitropic response. Furthermore, gravitropism is inhibited by the tetrapeptide RGDS (Arg-Gly-Asp-Ser), which is known to inhibit integrin functions. In addition, the root cap cells have been shown to contain cytoskeletal proteins which are involved in linking integrins to the cytoskeleton in animals. The first part of this proposal is designed to isolate and purify integrin-like proteins from plant cells. The first approach to this problem will be to use degenerate primers from the most conserved portion of animal integrins to try to amplify an integrin-like protein. Toward this end, primers have already been designed and have been used to generate a number of fragments from genomic and cDNA libraries. These cloned fragments are under study. The second large scale approach to the identification and purification of integrin-like proteins is to use an RGD-column to identify proteins capable of binding the RGD (arg-gly-asp) sequence. Proteins eluted from the column will be tested using the antibody against the chick-derived integrin. Partial protein sequence will be compared to sequences in Genbank and used to design primers to amplify full-length cDNAs. Once the gene(s) and/or protein(s) have been isolated, the full-length cDNA will be used to overproduce protein in E. coli and the overproduced protein will be used to generate new mono-clonal antibody. these newly- raised specific antibodies will be used to study any changes in the distribution or level of production during gravitropism. The antibody will be used to determine the location of these integrin-like proteins both within the root-cap tissue as well as at a subcellular level. Antisense constructs will be used to gain insight into the function of these proteins in vivo.
| Yamamoto, Kazuyoshi; Kiss, John Z (2002) Disruption of the actin cytoskeleton results in the promotion of gravitropism in inflorescence stems and hypocotyls of Arabidopsis. Plant Physiol 128:669-81 |
| Kiss, John Z; Miller, Kelley M; Ogden, Lisa A et al. (2002) Phototropism and gravitropism in lateral roots of Arabidopsis. Plant Cell Physiol 43:35-43 |
| Correll, Melanie J; Kiss, John Z (2002) Interactions between gravitropism and phototropism in plants. J Plant Growth Regul 21:89-101 |
| Weise, S E; Kuznetsov, O A; Hasenstein, K H et al. (2000) Curvature in Arabidopsis inflorescence stems is limited to the region of amyloplast displacement. Plant Cell Physiol 41:702-9 |
| Kiss, J Z (2000) Mechanisms of the early phases of plant gravitropism. CRC Crit Rev Plant Sci 19:551-73 |
