The Plant Cell, Vol. 7, 373-384, March 1995 O 1995 American Society of Plant Physiologists zyxw Pollen Specificity Elements Reside in 30 bp of the Proximal Promoters of Two Pollen-Expressed Genes Yoram Eyal,' Catherine Curie,' and Sheila McCormick2 Plant Gene Expression Center, United States Department of Agriculture-Agricultura1 Research Service, and University of California-Berkeley, 800 Buchanan Street, Albany, California 94710 Functional analyses previously identified minimal promoter regions required for maintaining high-leve1 expression of the late anther tomato zyxwvutsrq LAT52 and LAT59 genes in tomato pollen. Here, we now define elementsthat direct pollen specific- ity. We used a transient assay system consisting of two cell types that differentially express the LAT genes and both "loss-of-function"and "gain-of-function"approaches. Linker substitution mutants analyzed in the transient assay and in transgenic plants identified 30-bp proximal promoter regionsof LAT52 and LAT59 that are essential for their expression in pollen and that confer pollen specificity when fused to the heterologous cauliflower mosaic virus 35s core promoter. In vivo competition experiments demonstrated that a common trans-acting factor interacts with the pollen specificity region of both LAT gene promoters and suggested that a common mechanism regulates their coordinate expression. Adjacent upstream elements, the 52/56 box in LAT52 and the 56/59 box in LAT59, are involved in modulating the leve1 of expression in pollen. The 52/56 box may be a target for the binding of a member of the GT-1 transcriptionfactor family. INTRODUCTION Reproduction in angiosperms involves a double fertilization event in which two male gametes (sperm cells) combine with the egg and the central cell to yield a diploid embryo and triploid endosperm. Pollen, the male gametophyte, serves as a special- ized vector for the delivery of the male gametes into the female gametophyte. This highly specific role undoubtedly involves proteins that are specifically expressed in pollen and that are necessary for pollen maturation, pollen-pistil communication, and growth of the pollen tube through the style for delivery of the sperm cells. Indeed, during the process of pollen matu- ration, severa1 mRNAs have been found to be expressed at specific developmental stages (reviewed in Mascarenhas, 1990; McCormick, 1993). Some of these mRNAs exhibit over- lapping expression in sporophytic tissue, and others have been found to be pollen specific. A number of so-called late genes, active at a late stage of pollen development, have been iso- lated in different species (reviewed in McCormick, 1991a, 1993). However, the mechanism by which their coordinate develop- mental expression is achieved is not yet understood. In this work, we have focused our studies on two such late pollen- specific genes, late anther tomato zyxwvutsrq LAT52 and LAT59 (Twell et al., 1990), to help elucidate the mechanism(s)directing pollen specificity. We chose to study these two genes concomitantly to determine whether a common mechanism could be respon- sible for their coordinate expression. 1 Yoram Eyal and Catherine Curie contributed equally to this work and are considered joint first authors. zyxwvutsr To whom correspondence should be addressed. Based on sequence homology with pectate lyases (Wing et al., 1989; McCormick, 1991a), the LAT59 gene product is suggested to function during pollen germination, whereas re- cent results show that the LAT52 gene product affects pollen hydration (Muschiettiet al., 1994). RNA gel blot analyses (Twell et al., 1989a; Wing et al., 1989) as well as reporter gene anal- yses with the promoters of LAT52 and LAT59 (Twell et al., 1990) revealed that transcriptional activity in pollen was correlated with the onset of microspore mitosis and increased progres- sively until anthesis. A functional analysis of the promoters of both genes in transgenic plants revealed that high levels of expression and pollen specificity are maintained in the prox- imal regions: -115 bp for LAT59 and -100 bp for LAT52 (Twell et al., 1991). Although sequence comparisons of LAT52, LAT59, and other pollen-specific gene promoters yielded no common conserved regions (reviewed in McCormick, 1991a), conserved sequences at the 5'end of the proximal promoter regions were noted between LAT52 and an additional pollen-specific gene, LAT56 (52/56 box), and between LAT59 and LAT56 (56/59 box) (Twell et al., 1991). The 52/56 box was found to be redundant in sequences farther upstream in LAT52, whereas the 56/59 box occurred only once in the LAT56 and LAT59 promoters. Mutagenesis of these boxes and analysis in transient assays confirmed that these sequences modulated expression levels in pollen. Promoter interna1deletion experimentsdemonstrated that redundant upstream sequences can partly compensate for the loss of the LAT52 proximal region 52/56 box and that constructs with upstream LAT52 promoter sequences (-225 to -129) can somewhat activate a -89 cauliflower mosaic virus