ZmEBE genes show a novel,continuous expression pattern in the central cell before fertilization and in specific domains of the resulting endosperm after fertilization

Two novel maize genes expressed specifically in the central cell of the female gametophyte and in two compartments of the endosperm (the basal endosperm transfer layer and the embryo surrounding region) were characterized. The ZmEBE (embryo sac/basal endosperm transfer layer/embryo surrounding region) genes were isolated by a differential display between the upper and the lower half of the kernel at 7 days after pollination (DAP). Sequence analysis revealed ORFs coding for two closely related proteins of 304 amino acids (ZmEBE-1) and 286 amino acids (ZmEBE-2). This size difference was due to differences in the splicing of the two genes. Both protein sequences showed significant similarity to the DUF239 family of Arabidopsis, a group of 22 proteins of unknown function, a small number of which are putative peptidases. ZmEBE genes had a novel cell type-specific expression pattern in the central cell before and the resulting endosperm after fertilization. RT-PCR analysis showed that the expression of both genes started before pollination in the central cell and continued in the kernel up to 20 DAP with a peak at 7 DAP. In situ hybridization revealed that the expression in the kernel was restricted to the basal transfer cell layer and the embryo surrounding region of the endosperm. The expression of ZmEBE-1 was at least 10 times lower than that of ZmEBE-2. Similarly to other genes expressed in the endosperm, ZmEBE-1 expression was subject to a parent-of-origin effect, while no such effect was detected in ZmEBE-2. Sequence analysis of upstream regions revealed a potential cis element of 33 bp repeated 7 times in ZmEBE-1 and ZmEBE-2 between positions -900 and -100. The 1.6 kb ZmEBE-2 upstream sequence containing the seven R7 elements was able to confer expression in the basal endosperm to a Gus reporter gene. These data indicate that ZmEBE is potentially involved in the early development of specialized domains of the endosperm and that this process is possibly already initiated in the central cell, which is at the origin of the endosperm.     

Molecular characterization of BET1, a gene expressed in the endosperm transfer cells of maize.

A cDNA clone, BET1 (for basal endosperm transfer layer), was isolated from a cDNA bank prepared from 10-days after pollination (DAP) maize endosperm mRNA. BET1 mRNA was shown to encode a 7-kD cell wall polypeptide. Both the mRNA and protein were restricted in their distribution to the basal endosperm transfer layer and were not expressed elsewhere in the plant. BET1 expression commenced at 9 DAP, reached a maximum between 12 and 16 DAP, and declined after 16 DAP. The initial accumulation of the BET1 polypeptide reached a plateau by 16 DAP and declined thereafter, becoming undetectable by 20 DAP. The antibody raised against the BET1 protein reacted with a number of polypeptides of higher molecular mass than the BET1 monomer. Most of these were present in cytosolic fractions and were found in nonbasal cell endosperm extracts, but three species appeared to be basal cell specific. This result and the reactivity of exhaustively extracted cell wall material with the BET1 antibody suggest that a fraction of the protein is deposited in a covalently bound form in the extracellular matrix. We propose that the BET1 protein plays a role in the structural specialization of the transfer cells. In addition, BET1 provides a new molecular marker for the development of this endosperm domain.     

Analysis of ZmAE3 upstream sequences in maize endosperm and androgenic embryos

The single copy gene ZmAE3 (androgenic embryo) has been isolated from early androgenic embryos of maize. It codes for a small hydrophilic protein with partial similarity to basal layer antifungal proteins. During normal reproductive development ZmAE3 is specifically expressed in the embryo-surrounding region, a specialised region of the endosperm. Here we report the cloning, sequencing and functional analysis of upstream sequences of ZmAE3. The fusion of a 1.1 kb potential promoter fragment of ZmAE3 to a uidA reporter gene resulted in g-glucuronidase activity in transiently transformed androgenic embryos and in Black Mexican Sweet maize suspension cells. The AE box, an 18 bp sequence present in the ZmAE3 promoter and in other endosperm-specific promoters, was shown to bind proteins present specifically in nuclear extracts of the lower half of the maize kernel. The potential role of this and other cis elements in the binding of trans-acting factors will be discussed.     

maternally expressed gene1 Is a novel maize endosperm transfer cell-specific gene with a maternal parent-of-origin pattern of expression

Growth of the maize (Zea mays) endosperm is tightly regulated by maternal zygotic and sporophytic genes, some of which are subject to a parent-of-origin effect. We report here a novel gene, maternally expressed gene1 (meg1), which shows a maternal parent-of-origin expression pattern during early stages of endosperm development but biallelic expression at later stages. Interestingly, a stable reporter fusion containing the meg1 promoter exhibits a similar pattern of expression. meg1 is exclusively expressed in the basal transfer region of the endosperm. Further, we show that the putatively processed MEG1 protein is glycosylated and subsequently localized to the labyrinthine ingrowths of the transfer cell walls. Hence, the discovery of a parent-of-origin gene expressed solely in the basal transfer region opens the door to epigenetic mechanisms operating in the endosperm to regulate certain aspects of nutrient trafficking from the maternal tissue into the developing seed.     

Characterization of a barley gene coding for an α-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment

A gene coding for a barley CMd protein was isolated from a genomic library using a cDNA probe encoding the wheat CM3 protein. Promoter sequence analysis reveals motifs found in genes specifically expressed in endosperm and aleurone cells, as well as TATA and other putative functional boxes. 720 bp of the Hv85.1 CMd protein gene promoter, when fused to a gus coding region, were unable to direct GUS activity in the seeds of transgenic tobacco plants. In contrast, the same construction delivered into immature maize kernels by microprojectile bombardment was able to direct expression of GUS in the outermost cell layers of maize endosperm in both a tissue-specific and a developmentally determined manner.