No abstract

The homeobox gene knottedl (knl) was first isolated by transposon tagging a dominant leaf mutant in maize. Related maize genes, isolated by virtue of sequence conservation within the homeobox, fall into two classes based on sequence similarity and expression patterns. Here, we report the characterization of two genes, KNATl and KNAT2 (forbotted-like from Arabidopsis thaliana) that were cloned from Arabidopsis using the k n l homeobox as a heterologous probe. The homeodomains of KNATl and KNAT2 are very similar to the homeodomains of proteins encoded by ClaSS 1 maize genes, ranging from 78 to 95% amino acid identity. Overall, the deduced KNATl and KNAT2 proteins sham dmino acid identities of 53 and 4096, respectively, with the KN1 protein. lntron positions are also fairly well conserved among KNAT7, KNAT2, and knl. Based on in situ hybridization analysis, the expression pattern of KNATl during vegetative development is similar to that of class 1 maize genes. In the shoot apex, KNAT7 transcript is localized primarily to the shoot apical meristem; down-regulation of expression occurs as leaf primordia are initiated. In contrast to the expression of class 1 maize genes in floral and inflorescence meristems, the expression of KNATl in the shoot meristem decreases during the floral transition and is restricted to the cortex of the inflorescence stem. Transgenic Arabidopsis plants carrying the KNAT7 cDNA and the kn7 cDNA fused to the cauliflower mosaic virus 35s promoter were generated. Misexpression of KNATl and k n l resulted in highly abnormal leaf morphology that included severely lobed leaves. The expression pattern of KNATl in the shoot meristem combined with the results of transgenic overexpression experiments supports the hypothesis that class 1 knl-like genes play a role in morphogenesis.

We have investigated the functional organization and properties of cis regulatory elements in the promoter regions of two genes from tomato (LAT52 and LAT59) that are preferentially and coordinately expressed during pollen maturation. Promoter deletion analysis in transgenic plants demonstrated that only minimal (~200 bp) promoter proximal regions are required for developmentally regulated expression in pollen and in specific cell types of the sporophyte. Cis-acting regulatory regions of these two promoters and of a third pollen-expressed promoter (LAT56) were characterized in detail using a transient expression assay. We identified two upstream activator regions in the LAT52 promoter and further showed that a 19-bp segment from one of those regions enhanced expression of the heterologous CaMV35S promoter in pollen. Similarities in sequence between crucial cis elements provide evidence that shared regulatory elements are involved in the coordinate expression of the LAT genes during microsporogenesis.[Key Words: Tomato; tobacco; male gametophyte; transcriptional control; enhancer; cell-specific gene regulation]Received September 28, 1990; revised version accepted January 4, 1991.Identifying the molecular components that control the spatial and temporal patterns of gene expression accompanying gametogenesis is an important step in understanding sexual reproduction in eukaryotes. We are interested in the development of the male gametophyte (pollen) of angiosperms because it provides a unique system with which to study cell-specific gene regulation and cellular differentiation during reproductive development in plants. Major cytological and biochemical events that occur during pollen development are well characterized, the mRNA populations in pollen have been analyzed, pollen-specific isoenzymes have been identified, and several genes that are preferentially expressed in pollen in a stage-specific manner recently have been cloned and sequenced (for reviews, see Mascarenhas 1975Mascarenhas , 1989. Of particular interest is the asymmetric mitotic division of the haploid microspore (microspore mitosis), which results in the formation of two dimorphic cells with different developmental fates. The Present addresses: ~Leicester Biocentre, University of Leicester, Leicester LE1 7RH UK;

The homeobox of the knottedl (knl) gene was used to isolate 12 related sequences in maize. The homeodomains encoded by the knl-like genes are very similar, ranging from 55 to 89% amino acid identity. Differences outside the precisely conserved third helix allowed us to group the genes into two classes. The homeodomains of the seven class 1 genes share 73 to 89% identical residues with knl. The four class 2 genes share 55 to 58% identical residues with knl, although the conservation within the class is greater than 87%. Expression patterns were analyzed by RNA gel blot analysis. Class 1 genes were highly expressed in meristem-enriched tissues, such as the vegetative meristem and ear primordia. Expression was not detectable in leaves. The class 2 genes were expressed in all tissues, although one was abundantly expressed in roots. The genes were mapped using recombinant inbred populations. We determined that clusters of two to three linked genes are present on chromosomes 1 and 8; otherwise, the genes are distributed throughout the genome. Four pairs of genes, similar in both sequence and expression patterns, mapped within duplicated regions of the genome.

We have isolated and sequenced an anther-specific cDNA clone and a corresponding genomic clone from tomato. The gene (LAT52) encodes an 800-nucleotide-long transcript that is detectable in pollen, anthers and at 20- to 50-fold lower levels in petals. LAT52 mRNA is not detectable in pistils, sepals or non-reproductive tissues. Steady-state levels of LAT52 mRNA are detectable in immature anthers containing pollen at the tetrad stage and increase progressively throughout microsporogenesis until anthesis (pollen shed). The LAT52 gene contains 5' and 3' untranslated regions of 110 and approximately 150 nucleotides, respectively, and a single intron with a highly repetitive sequence. A TATA box motif is located 28 nucleotides upstream of the transcription start site. The gene encodes a putative protein of 18 kDa that is cysteine rich and has an N-terminal hydrophobic region with characteristics similar to eucaryotic secretory signal sequences. LAT52 is a single or low copy gene in tomato and shares homology with sequences in tobacco.