Post-pollination gene expression: a methodological approach and preliminary results

--Carla Frova and Gloria Padoani

Gene expression in the male gametophytic phase has been detected and analysed essentially at or before anthesis. Post-anthesis stages, i. e. germination, tube growth and fertilization, have been characterized to a limited extent for two main reasons: (a) even the best in vitro germination procedures currently available do not promote tube elongation to the extent to which it occurs in vivo (few mm versus 20-25cm); moreover in the in vitro system pollen-style interactions are excluded. (b) in vivo analysis of isolated pollen tubes is not feasible since they cannot be separated from the stylar tissues they are growing through.

To overcome these limitations we developed an indirect approach to the problem, based on the comparative analysis of pollinated and non-pollinated silks. This method allows the expression of those genes which code for an easily identifiable gene product to be detected. By choosing appropriate genotypes as female and pollen source, the pollen tube and stylar contribution to the gene products found in pollinated versus non-pollinated silks can be discriminated. The approach appears susceptible to further applications also for the study of pollen-style interactions.

Silks are cut 0.5cm above emergence from the husks and heavily pollinated; 3.5 hours are then left for germination and tube growth. After this time it is expected that tube tips have reached a distance of 2-2.5cm under the silk emergence level. This region of the silks is called "base". The portion of the silks where the pollen grains are deposited is called "tip" and is analysed separately.

In this study we considered 4 enzymatic systems: ADH, GOT, CAT and B-GLU. For all of them a pronounced enzymatic activity was detected in mature non-germinating pollen and in the early stages of germination (in vitro and tip region of pollinated silks), while the elongating tubes (base region of pollinated silks) showed activity only in the case of GOT-1.

In the course of this analysis we found additional enzymatic activities, expressed in pollinated and non-pollinated silks but not in mature germinating pollen or in other sporophytic tissues: ADH-2, whose gene is usually transcribed only under anaerobiosis, and an as yet unidentified CAT. The nature of this catalase isozyme and the possible role of these additional activities in pollen-style interactions are currently under investigation.


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