Identification of microspore-active promoters that allow targeted manipulation of gene expression at early stages of microgametogenesis in Arabidopsis

Abstract

Background: The effective functional analysis of male gametophyte development requires new tools enabling the spatially and temporally controlled expression of both marker genes and modified genes of interest. In particular, promoters driving expression at earlier developmental stages including microspores are required.

Results: Transcriptomic datasets covering four progressive stages of male gametophyte development in Arabidopsis were used to select candidate genes showing early expression profiles that were male gametophyte-specific. Promoter-GUS reporter analysis of candidate genes identified three promoters (MSP1, MSP2, and MSP3) that are active in microspores and are otherwise specific to the male gametophyte and tapetum. The MSP1 and MSP2 promoters were used to successfully complement and restore the male transmission of the gametophytic two-in-one (tio) mutant that is cytokinesis-defective at first microspore division.

Conclusion: We demonstrate the effective application of MSP promoters as tools that can be used to elucidate gametophytic gene functions in microspores in a male-specific manner.

Figure 1

Expression profiles of candidate genes selected for promoter analyses. Expression profiles of seven genes were compared in available male gametophytic and sporophytic transcriptomic datasets. Individual transcriptomic experiments are described in Material and methods.

Figure 2

Verification of microarray gene expression data by RT-PCR. The expression of three genes selected for further GUS expression assays was examined in microspores (MS); bicellular (BC), tricellular (TC) and mature pollen (MP); whole flowers (FW); leaves (LF); stems (ST) and roots (RT).

Figure 3

In situ GUS expression driven by three promoters, MSP1 (A, D, G, J, M, P, S and V), MSP2 (B, E, H, K, N, Q, T and W) and MSP3 (C, F, I, L, O, R, U and X). GUS staining in whole inflorescences (A, B, C); transverse sections of whole anthers (D, E, F); five-day (S, T, U) and ten-day old (V, W, X) seedlings. Light and DAPI-stained fluorescence images of mature pollen (G, H, I), immature tricellular pollen (J, K, L) bicellular pollen (M, N, O) and uninucleate microspores (P, Q, R) are shown.

Figure 4

Mature pollen tetrad phenotype after DAPI staining. (A) Tetrad from +/tio-3;qrt1/qrt1 plant showing 2:2 segregation of wild type and tio mutant pollen. (B) Tetrad from a transformant containing MSP1-TIO in the +/tio-3;qrt1/qrt1 background, showing three pollen grains with a wild type phenotype and a single mutant tio pollen grain.