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.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Tissue-specific expression in transgenic maize of four endosperm promoters from maize and rice

The tissue-specific, developmental, and genetic control of four endosperm-active genes was studied via expression of GUS reporter genes in transgenic maize plants. The transgenes included promoters from the maize granule-bound starch synthase (Waxy) gene (zmGBS), a maize 27 kDa zein gene (zmZ27), a rice small subunit ADP-glucose pyrophosphorylase gene (osAGP) and the rice glutelin 1 gene (osGT1). Most plants had a transgene expression profile similar to that of the endogenous gene: expression in the pollen and endosperm for the zmGBS transgene, and endosperm only for the others. Histological analysis indicated expression initiated at the periphery of the endosperm for zmGBS, zmZ27 and osGT1, while osAGP transgene activity tended to start in the lower portion of the seed. Transgene expression at the RNA level was proportional to GUS activity, and did not influence endogenous gene expression. Genetic analysis showed that there was a positive dosage response with most lines. Activity of the zmGBS transgene was threefold higher in a low starch (shrunken2) genetic background. This effect was not seen with zmZ27 or osGT1 transgenes. The expression of the transgenes is discussed relative to the known behaviour of the endogenous genes, and the developmental programme of the maize endosperm     

Evidence for factors regulating transfer cell-specific expression in maize endosperm

In maize, a layer of basal endosperm cells adjacent to the pedicel is modified for a function in solute transfer. Three genes specifically expressed in this region, termed the basal endosperm transfer layer (BETL-2 to -4), were isolated by differential hybridization. BETL-2 to -4 are coordinately expressed in early and mid-term endosperm development, but are absent at later stages. BETL-2 to -4 coding sequences all predict small (<100 amino acids), secreted, cysteine-rich polypeptides which lack close relatives in current database accessions. BETL-3 and BETL-1 display some sequence similarities with each other and to plant defensins. BETL-2 to -4 promoter regions were isolated and compared, revealing the presence of a promoter-proximal microsatellite repeat as the most highly conserved sequence element in each sequence. Electrophoretic mobility shift assays (EMSA) showed that specific BETL-2 to -4 promoter fragments competed for binding to the same DNA-binding activity in nuclear extracts prepared from maize endosperm. Although BETL-2 to -4 are only expressed in basal endosperm cells, the DNA-binding activities detected were of two types: distal endosperm-specific, or present in both basal and distal endosperm extracts. On the basis of these findings, a model to account for the coordinate regulation of BETL genes in endosperm cells is proposed.     

The structure of the seed in some Western Australian species of the genus Hibbertia (Dilleniaceae)

The internal structure of the seed was studied for four Western Australian species of the genus Hibbertia (Fam.: Dilleniaceae). The principal mechanical layer of the seed coat is the endotesta. The large cells of the endotesta are heavily cutinized in potentially viable seeds of all examined species. However, results for H. hypericoides show that the cells of the endotesta are distinctly less cutinized in non-viable seeds. The tegmen consists of only one layer which is spirally thickened. Remains of the nucellus enclose the endosperm. The endosperm is rich in both starch and lipids, and the embryo is underdeveloped and minute.     

Mechanisms of hormonal regulation of endosperm cap-specific gene expression in tomato seeds

The micropylar region of endosperm in a seed, which is adjacent to the radicle tip, is called the 'endosperm cap', and is specifically activated before radicle emergence. This activation of the endosperm cap is a widespread phenomenon among species and is a prerequisite for the completion of germination. To understand the mechanisms of endosperm cap-specific gene expression in tomato seeds, GeneChip analysis was performed. The major groups of endosperm cap-enriched genes were pathogenesis-, cell wall-, and hormone-associated genes. The promoter regions of endosperm cap-enriched genes contained DNA motifs recognized by ethylene response factors (ERFs). The tomato ERF1 (TERF1) and its experimentally verified targets were enriched in the endosperm cap, suggesting an involvement of the ethylene response cascade in this process. The known endosperm cap enzyme endo-β-mannanase is induced by gibberellin (GA), which is thought to be the major hormone inducing endosperm cap-specific genes. The mechanism of endo-β-mannanase induction by GA was also investigated using isolated, embryoless seeds. Results suggested that GA might act indirectly on the endosperm cap. We propose that endosperm cap activation is caused by the ethylene response of this tissue, as a consequence of mechanosensing of the increase in embryonic growth potential by GA action.     

Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype

The titan (ttn) mutants of Arabidopsis exhibit striking alterations in chromosome dynamics and cell division during seed development. Endosperm defects include aberrant mitoses and giant polyploid nuclei. Mutant embryos differ in cell size, morphology and viability, depending on the locus involved. Here we demonstrate that three TTN genes encode chromosome scaffold proteins of the condensin (SMC2) and cohesin (SMC1 and SMC3) classes. These proteins have been studied extensively in yeast and animal systems, where they modulate chromosome condensation, chromatid separation, and dosage compensation. Arabidopsis contains single copies of SMC1 and SMC3 cohesins. We used forward genetics to identify duplicate T-DNA insertions in each gene. These mutants (ttn7 and ttn8) have similar titan phenotypes: giant endosperm nuclei and arrested embryos with a few small cells. A single SMC2 knockout (ttn3) was identified and confirmed by molecular complementation. The weak embryo phenotype observed in this mutant may result from expression of a related gene (AtSMC2) with overlapping functions. Further analysis of titan mutants and the SMC gene family in Arabidopsis should provide clues to chromosome mechanics in plants and insights into the regulation of nuclear activity during endosperm development.     

miRNA参与植物胚和胚乳发育调控的研究进展

籽粒性状是构成产量的重要基础,是粮食产量的最终体现,也是育种最复杂的性状之一。籽粒主要由胚和胚乳组成,胚乳是积累和贮藏营养物质的场所,主要为胚的萌发和生长提供营养。胚乳细胞的发育、增殖和充实情况决定了籽粒的重量和品质。籽粒发育是一个非常复杂的生物过程,涉及许多基因的时空表达以及转录水平和转录后水平的调控。微小RNA（microRNAs,miRNAs）是一类内源性的非编码小RNA（21～24 nt）,可通过靶向降解和翻译抑制在转录后水平调控植物基因表达。miRNA及其靶基因组成精密的调控网络参与籽粒的发育。基于此,概述了植物miRNAs的生成及作用机制,综述了miRNAs在植物胚和胚乳发育中的调控功能研究进展,以期为进一步鉴定与玉米籽粒发育相关的miRNAs并解析其调控功能提供更好的研究方向。     

The dynamics of protein body formation in developing wheat grain

Wheat is a major source of protein in the diets of humans and livestock but we know little about the mechanisms that determine the patterns of protein synthesis in the developing endosperm. We have used a combination of enrichment with ¹⁵N glutamine and NanoSIMS imaging to establish that the substrate required for protein synthesis is transported radially from its point of entrance in the endosperm cavity across the starchy endosperm tissues, before becoming concentrated in the cells immediately below the aleurone layer. This transport occurs continuously during grain development but may be slower in the later stages. Although older starchy endosperm cells tend to contain larger protein deposits formed by the fusion of small protein bodies, small highly enriched protein bodies may also be present in the same cells. This shows a continuous process of protein body initiation, in both older and younger starchy endosperm cells and in all regions of the tissue. Immunolabeling with specific antibodies shows that the patterns of enrichment are not related to the contents of gluten proteins in the protein bodies. In addition to providing new information on the dynamics of protein deposition, the study demonstrates the wider utility of NanoSIMS and isotope labelling for studying complex developmental processes in plant tissues.