SummaryWe have used reverse-genetic analysis to investigate the function of MAP3Ke1 and MAP3Ke2, a pair of closely related Arabidopsis thaliana genes that encode protein kinases. Plants homozygous for either map3ke1 or map3ke2 displayed no apparent mutant phenotype, whereas the double-mutant combination caused pollen lethality. Transmission of the double-mutant combination through the female gametophyte was normal. Tetrad analysis performed using the Arabidopsis quartet mutation demonstrated that the pollen-lethal phenotype segregated at meiosis with the map3ke1;map3ke2 genotype. We used transmission electron microscopy to determine that double-mutant pollen grains develop plasma membrane irregularities following pollen mitosis I. Analysis of the subcellular localization of a yellow fluorescent protein (YFP):MAP3Ke1 fusion protein using confocal microscopy and biochemical fractionation indicated that a substantial portion of the MAP3Ke1 present in Arabidopsis cells is localized to the plasma membrane. Taken together, our results suggest that MAP3Ke1 is required for the normal functioning of the plasma membrane in developing Arabidopsis pollen.

Summary Arabidopsis cngc2 plants are hypersensitive to external calcium and exhibit reduced plant size and fertility, especially when they are treated with elevated but physiologically relevant levels of calcium. This report focuses on the role of cyclic nucleotide‐gated channel 2 (CNGC2) in plant fertility. To determine the cause of the reduced fertility, we investigated the flower structure and growth potential of both male and female reproductive organs in cngc2 plants grown in high‐calcium conditions. cngc2 mutants had short stamens that may limit pollen deposition and pistils that were not conducive to pollen tube growth. Our data indicate that sporophytic, but not gametophytic, defects are the main cause of the observed reduction in seed yield in cngc2 plants, and suggest that correct cyclic nucleotide and calcium signaling are important for cell elongation and pollen tube guidance.

MAP3Kε1 and MAP3Kε2 are a pair of Arabidopsis thaliana genes that encode protein kinases related to cdc7p from Saccharomyces cerevisiae. We have previously shown that the map3kε1;map3kε2 double-mutant combination causes pollen lethality. In this study, we have used an ethanol-inducible promoter construct to rescue this lethal phenotype and create map3kε1-/-;map3kε2-/- double-mutant plants in order to examine the function of these genes in the sporophyte. These rescued double-mutant plants carry a yellow fluorescent protein (YFP)-MAP3Kε1 transgene under the control of the alcohol-inducible AlcA promoter from Aspergillus nidulans. The double-mutant plants were significantly smaller and had shorter roots than wild-type when grown in the absence of ethanol treatment. Microscopic analysis indicated that cell elongation was reduced in the roots of the double-mutant plants and cell expansion was reduced in rosette leaves. Treatment with ethanol to induce expression of YFP-MAP3Kε1 largely rescued the leaf phenotypes. The double-mutant combination also caused embryos to arrest in the early stages of development. Through the use of YFP reporter constructs we determined that MAP3Kε1 and MAP3Kε2 are expressed during embryo development, and also in root tissue. Our results indicate that MAP3Kε1 and MAP3Kε2 have roles outside of pollen development and that these genes affect several aspects of sporophyte development.

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