SummaryAll four members of a gene family, which are highly expressed in the cells of the female gametophyte (ZmES1±4: Zea mays embryo sac), were isolated from a cDNA library of maize egg cells. High expression of ZmES genes in the synergids around the micropylar region was detected in thin sections of maize ovaries. Single-cell RT±PCR analyses with the various cells of the female gametophyte con®rmed the expression in synergids and also showed expression in the egg cell and central cell, and low expression in the antipodals. The expression of the whole gene family is suppressed after fertilization of the embryo sac, and expression in two-cell or later embryo stages or other tissues of maize could not be detected. In order to investigate ZmES mRNA gradients in the highly polarized and vacuolized cells of the maize embryo sac, a whole-mount in situ protocol with isolated single cells was developed: as for total RNA, ZmES transcripts are uniformly distributed in the cytoplasm of egg cell, synergids and central cell. ZmES genes encode small, cysteine-rich proteins with an N-terminal signal peptide, probably for translocation into the embryo sac cell wall. The four ZmES proteins display high sequence identity with each other, and the proposed tertiary structure of the mature peptides is similar to that of plant and animal defensins. The function of ZmES1±4 during the fertilization process is discussed.

MADS box genes represent a large gene family of transcription factors with essential functions during flower development and organ differentiation processes in plants. Addressing the question of whether MADS box genes are involved in the regulation of the fertilization process and early embryo development, we have isolated two novel MADS box cDNAs, ZmMADS1 and ZmMADS3, from cDNA libraries of maize (Zea mays) pollen and egg cells, respectively. The latter gene is allelic to ZAP1. Transcripts of both genes are detectable in egg cells and in in vivo zygotes of maize. ZmMADS1 is additionally expressed in synergids and in central and antipodal cells. During early somatic embryogenesis, ZmMADS1 expression is restricted to cells with the capacity to form somatic embryos, and to globular embryos at later stages. ZmMADS3 is detectable only by more sensitive reverse transcriptase-PCR analyses, but is likewise expressed in embryogenic cultures. Both genes are not expressed in nonembryogenic suspension cultures and in isolated immature and mature zygotic embryos. During flower development, ZmMADS1 and ZmMADS3 are co-expressed in all ear spikelet organ primordia at intermediate stages. Among vegetative tissues, ZmMADS3 is expressed in stem nodes and displays a gradient with highest expression in the uppermost node. Transgenic maize plants ectopically expressing ZmMADS3 are reduced in height due to a reduced number of nodes. Reduction of seed set and male sterility were observed in the plants. The latter was due to absence of anthers. Putative functions of the genes during reproductive and vegetative developmental processes are discussed.

The maize (Zea mays) late pollen gene ZmMADS2 belongs to the MIKC type of MADS box transcription factor genes. Here, we report that ZmMADS2, which forms a homodimer in yeast (Saccharomyces cerevisiae), is required for anther dehiscence and pollen maturation. Development of anthers and pollen was arrested at 1 d before dehiscence in transgenic plants expressing the ZmMADS2-cDNA in antisense orientation. Temporal and spatial expression analyses showed high amounts of ZmMADS2 transcripts in endothecium and connective tissues of the anther at 1 d before dehiscence and in mature pollen after dehiscence. Transient transformation of maize and tobacco (Nicotiana tabacum) pollen with the luciferase reporter gene under the control of different ZmMADS2 promoter deletion constructs demonstrated the functionality and tissue specificity of the promoter. Transgenic maize plants expressing a ZmMADS2-green fluorescent protein fusion protein under control of the ZmMADS2 promoter were used to monitor protein localization during anther maturation and pollen tube growth. High amounts of the fusion protein accumulate in degenerating nuclei of endothecial and connective cells of the anther. A possible function of ZmMADS2 during anther dehiscence and pollen maturation and during pollen tube growth is discussed.In higher plants, development of the haploid male gametophyte (pollen) is closely related to maturation of the surrounding sporophytic tissues of the anther. In angiosperms, anther tissues arise from three "germ" layers, designated L1 to L3 (Satina and Blakeslee, 1941). In maize (Zea mays), tapetal initials and pollen mother cells are generated by the division of a diploid sporophytic cell. The tapetal initial cells give rise to the outer, middle, and inner (tapetal) layers of the sporangium wall (Kiesselbach, 1999). The sporogenous cells (pollen mother cells) undergo meiosis, giving rise to a tetrad of haploid cells, which are released as free microspores (McCormick, 1993). During microgametogenesis, microspores develop into mature pollen by two mitotic divisions. The first mitotic division results in a vegetative and a generative cell. The latter divides either in the pollen grain or the pollen tube to generate the two haploid sperm cells. In maize, the generative cell within the young pollen divides before maturation of the anther is completed to generate a tricellular mature pollen grain (Bedinger, 1992).During gametogenesis, the innermost cell layer of the anther, the tapetum, plays a crucial role for the release and nutrition of the microspores. Microspores are supplied with nutrients from the tapetum; therefore, mutations affecting tapetal development lead to abortion of microgametogenesis and male sterility (Cheng et al., 1979; Chaudhury, 1993;Okada et al., 1999;Wilson et al., 2001;Kapoor et al., 2002). Tapetal cells secrete callase to release the meiotic tetrad from an enclosing callose wall. The exact timing and proper function of callase has been shown to be essential for pollen development. Precursors for the biosynth...

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.35× larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (BIII hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin:: β -glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.