Phenotype Analysis Of Aquaporin Mutant Pip2;5 In Arabidopsis Thaliana

It is a major goal for plant biologists to understand the physiological and molecular bases of the pollen-pistil interaction. Before fertilization, the pollen-pistil cellular and molecular interaction consist of six stages:1) pollen capture and adhesion,2) pollen hydration,3) germination of the pollen to produce a pollen tube,4) penetration of the stigma by the pollen tube,5) growth of the pollen tube through the stigma and style,6) entry of the pollen tube into the ovule and discharge of the sperm cells. It is necessary that these six steps are complete for the success of double fertilization. To identify the key genes involved in pollination recognition of Arabidopsis, we screened a large number of mutants from ABRC. A T-DNA insertion mutant in Col-0 background, plasma membrane intrinsic protein2;5 (pip2;5), was identified that the hydration of pollen was delayed on its stigmas compared with on wild type stigmas. But pip2;5 pollen activity, hydration rate, germination percentage and the growth speed of pollen tubes had no significant difference compared with the wild type pollen in vivo or vitro. It was confirmed the T-DNA was inserted into the second intron of PIP2;5 gene by analyzing the flanking sequence of T-DNA insertion sites. The PIP2;5 gene encodes a aquaporin protein PIP2;5, which belongs to plasma membrane intrinsic protein subfamily PIP2s, with water channel activity.Through observing and analyzing transformed plants with pPIP2;5:PIP2;5-GFP and pPIP2;5':PIP2;5-GUS respectivly, we obtained the following results and conclusions:(1) The hydration of wild type pollen grains were delayed on homozygous pip2;5 mutant stigma. In the complementary plants the rate of pollen hydration could be restored. In addition, the growth of pip2;5 plants were normal.(2) PIP2;5 was located in the cell plasma membrane. In vegetative tissue, PIP2;5 expressed in shoot apex, young leaves and root vascular bundles. In reproductive organs, the expression of PIP2;5 was focused on the style of stage 9-16 flower and the papilla of stage 9-14 flower. The expression level of PIP2;5 on the papilla and style was reduced from stage 15 until it disappeared.(3) After the cold treatment, the expression levels and ranges of PIP2;5 in seedling and flowers were increased.In summary, PIP2;5 mutations may affect the transport activity or density of aquaporin in stigma papillae of Arabidopsis, thereby leading to the delayed pollen hydration. The molecular mechanisms of this process still need to be further studied.