The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter-GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.

The Arabidopsis thaliana MALE STERILITY 2 (MS2) gene product is involved in male gametogenesis. The first abnormalities in pollen development of ms2 mutants are seen at the stage in microsporogenesis when microspores are released from tetrads. Expression of the MS2 gene is observed in tapetum of wild-type flowers at, and shortly after, the release of microspores from tetrads. The MS2 promoter controls GUS expression at a comparable stage in the tapetum of transgenic tobacco containing an MS2 promoter-GUS fusion. The occasional pollen grains produced by mutant ms2 plants have very thin pollen walls. They are also sensitive to acetolysis treatment, which is a test for the presence of an exine layer. The MS2 gene product shows sequence similarity to a jojoba protein that converts wax fatty acids to fatty alcohols. A possible function of the MS2 protein as a fatty acyl reductase in the formation of pollen wall substances is discussed.

Male sterility in a petunia cytoplasmic male sterile line has been attributed to the early appearance of active callase, a P-1,3-glucanase, in the anther locule. This leads to premature dissolution of the callose walls surrounding the microsporogenous cells. We have mimicked this aspect of the petunia line in transgenic tobacco by engineering the secretion of a modified pathogenesis-related vacuolar P-1,3-glucanase from the tapetum prior to the appearance of callase activity in the locule. Plants expressing the modified glucanase from tapetum-specific promoters exhibited reduced male fertility, ranging from complete to partia1 male sterility. Callose appearance and distribution are normal in the male sterile transgenic plants up to prophase I, whereupon callose is prematurely degraded. Meiosis and cell division occur normally. The resultant microspores have an abnormally thin cell wall that lacks sculpturing. The tapetum shows hypertrophy. Male sterility is probably caused by bursting of the aberrant microspores at a time corresponding to microspore release. These results demonstrate that premature callose degradation is sufficient to cause male sterility and suggest that callose is essential for the formation of a normal microspore cell wall.

The Brassica napus cDNA clone A9 and the corresponding Arabidopsis thaliana gene have been sequenced. The B. napus cDNA and the A. thaliana gene encode proteins that are 73% identical and are predicted to be 10.3 kDa and 11.6 kDa in size respectively. Fusions of an RNase gene and the reporter gene beta-glucuronidase to the A. thaliana A9 promoter demonstrated that in tobacco the A9 promoter is active solely in tapetal cells. Promoter activity is first detectable in anthers prior to sporogenous cell meiosis and ceases during microspore premitotic interphase. The deduced A9 protein sequence has a pattern of cysteine residues that is present in a superfamily of seed plant proteins which contains seed storage proteins and several protease and alpha-amylase inhibitors.

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An anther-specific Brassica napus cDNA, A6, and two corresponding Arabidopsis thaliana genes have been isolated. Sequence analyses of A6 revealed similarity to beta-1,3-glucanases. The deduced A6 protein differs from other beta-1,3-glucanases in the possession of a long C-terminus. Immunoblotting using an antibody raised to the A6 protein detects a temporal 60 kDa protein in B. napus buds, suggesting that the long C-terminal region is present in the mature protein. A6 promoter-GUS and RNase fusions demonstrate that the A6 gene is tapetum-specific and temporally expressed with a peak in activity when the plant normally expresses callase (a complex of endo- and exo-beta-1,3-glucanase activities). The sequence similarity of A6 to other beta-1,3-glucanases, coupled with the temporal and spatial expression data, suggests that A6 may be part of the callase enzyme complex.

Southern blot analysis of cloned K5-and K7-antigen genes, using DNA fragments from cloned Kl genes as radiolabeled probes, demonstrated that each K-antigen gene cluster is organized in a manner similar to that shown for the Ki antigen. That is, a central DNA segment unique for a given antigen type is flanked by DNA sequences that encode common functions for the management of intracellular polymer. This has been confirmed by transposon and deletion mutagenesis of plasmids carrying the KS and K7 genes. We also describe a series of complementation experiments in which transport or postpolyIlerizational modification functions for one K antigen are used to complement mutations in the corresponding regions of a different K-antigen gene cluster. Thus, postpolymerizational modification of polysaccharide and transport of mature polysaccharide from the periplasmic space are comon mechanisms and are independent of polysaccharide structure.

The relationship between bud length, anther length and stage of anther development has been investigated in Brassica napus using a series of cytological markers that define steps in the process of male gametogenesis. It was determined that bud length is directly related to anther length and that anther or bud length is tightly linked to the stage of male gametogenesis within the anther. This simple correlation has enabled the construction of cDNA libraries representing transcripts expressed in defined stages of anther development, and the detailed examination of the developmental pattern of expression of anther RNAs. Two anther cDNA libraries were constructed, one from anthers of 1.2-1.8 mm long buds (sporogenesis library) and one from anthers of 1.8-4.0 mm long buds (microspore development library). A total of 19 independent cDNAs have been isolated by differential screening whose temporal expression patterns overlap and which together cover the stages of anther development from pre-meiotic microsporocytes to tri-nucleate pollen grains. The pattern of expression of each of these clones is unique and indicates that stages of anther development which cannot be easily distinguished by light microscopy can be recognised by virtue of the absence or presence of certain RNAs. Three cDNAs isolated from the sporogenesis library have been shown by in situ hybridisation to be tapetum-specific. In contrast, five clones isolated from the microspore development library are microspore-specific. These clones exhibit a pattern of expression different to those previously described in that their transcripts are absent in mature pollen grains. Thus these RNAs are probably required in microspore development rather than for the growth of the germinating pollen grain.