A novel endosperm transfer cell-containing region-specific gene and its promoter in rice

The endosperm of cereal grains is an important resource for both food and feed. It contains three major types of tissue: starchy endosperm, the aleurone layer, and transfer cells. To improve grain quality and quantity using molecular methods, control of transgene expression directed by distinct temporal and spatial promoter activity is necessary. To identify aleurone layer-specific and/or transfer cell-specific promoters in rice, microarray analyses were performed, comparing the aleurone layer containing transfer cells and the other reproductive and vegetative tissues. After confirmation by RT-PCR analysis, we identified two putative aleurone layer and/or transfer cell-specific genes, AL1 and AL2. The promoter regions of these genes and β-glucuronidase (GUS) fusion constructs were stably transformed into rice. The GUS expression patterns indicated that the AL1 promoter was active exclusively in the dorsal aleurone layer adjacent to the main vascular bundle. In rice, transfer cells are differentiated in this region. Therefore, the promoter of the AL1 gene exhibits transfer cell-containing region-specific activity. The AL1 gene encodes a putative anthranilate N-hydroxycinnamoyl/benzoyltransferase. The promoter of this gene will be useful for enhancing uptake of nutrients from the mother cells and protecting filial seeds from pathogen attack.     

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式,将UidA基因分别置于水稻谷蛋白GluA—2基因750bp和2．3kb上游序列下游,利用农杆菌转化法导人栽培稻品种中花8号并获得转基因植株。Southern blot检测表明,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northern blot检测表明,开花后13～15d和11～13d,UidA基因和水稻内源的GluA—2基因的表达量分别达到最高,随后逐渐降低。对转基因植株种子的GUS染色表明,UidA基因仅在胚乳中表达,在糊粉层中GUS表达量最高。测定了2．3kb和750bp转基因植株种子的GUS活性,结果表明前者的GUS活性是后者的2～3倍。序列分析表明,位于GluA—2基因转录启始位点上游2170bD的G-box可能是一个与表达量相关的顺式调控元件。     

籼稻蜡质基因5＇上游区与GUS基因编码区融合基因的构建和在水稻中的瞬间表达

为了鉴定水稻蜡质基因５＇上游区的顺式作用因子与研究它们在组织专一性表达中的作用,我们对籼稻品种２３２的蜡质基因５＇上游－１１５至－２１２０的区域进行了顺序测定,并将此基因的５＇上游区同报告基因ＧＵＳ构建成融合基因,用基因枪粒子轰击的方法将此融合基因导入水稻未成熟种子的幼胚和糊粉层细胞中,瞬间表达检测的结果表明,我们构建的融合基因中蜡质基因５＇上游区的长度已足以使ＧＵＳ基因在上述组织中表达。     

Metabolic regulation of α-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the -amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice -amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Localizing a-Amylase Gene Expression in Germinated Rice Grains

In situ hybridization was used to localize the sites of expressionfor two a-amylase genes (RAmy1A and RAmy3D) in rice (Oryza sativaL.) over five days of germination. Messenger RNAs from bothgenes were initially detected in the scutellar epithelium andappeared at later stages of germination in the aleurone layer.RAmy3D mRNA reached its peak of accumulation 2 days(d) earlierin the scutellar epithelium than RAmy1A mRNA. Both mRNAs continuedto accumulate in the aleurone layer up to 5 d of germination,although RAmy1A mRNA reached significantly higher levels thanRAmy3D mRNA. Overall, the aleurone layer was responsible forproducing the majority of the total grain a-amylase mRNA. (Received July 27, 1991; Accepted November 6, 1991)     

Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice

The spatial and temporal expression patterns of the rice VP1 (OSVP1) gene, as well as the OSEM gene which it controls, were studied during seed development by in situ hybridization and immuno-localization techniques. The expression of OSVP1 could be detected in embryos as early as 2-3 d after pollination (DAP) and thereafter became preferentially localized to shoot, radicle and vascular tissues during the embryo development at both the mRNA and protein levels. In the aleurone layers, OSVP1 mRNA and protein were detected after 6 DAP. OSEM mRNA was detectable after 6 DAP in the embryo and aleurone tissue. The spatial distribution within the embryo of OSEM mRNA and OSVP1 mRNA/protein was very similar after 6 DAP. Transgenic rice carrying a beta-glucuronidase (GUS) gene transcribed from a chimeric promoter consisting of the CaMV 35S minimal promoter (-46) and the 55-bp promoter fragment of OSEM, minimally required for ABA and VP1 regulation, also exhibited a spatial pattern of GUS expression similar to that of OSEM and OSVP1. These results suggest that (OS)VP1 is a major determinant not only of the seed specificity but also of the spatial pattern of OSEM expression in the developing seed.     

Evidence for a role of β-1,3-glucanase in dicot seed germination

Class I β-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The expression was studied in germinating tobacco seeds of a chimeric β-glucuronidase (GUS) reporter gene fused to 1.6 kb of the 5' flanking sequence of the tobacco class I β-1,3-glucanase B (GLB) promoter. Histological staining for GUS activity showed that expression of the GLB promoter is highly localized in a specific zone of the endosperm in germinating seeds. The temporal and spatial patterns of GUS and β-1,3-glucanase activity found, suggest a novel function for class I β-1,3-glucanases during seed germination in a dicotyledonous plant.     

Sugar Repression of a Gibberellin-Dependent Signaling Pathway in Barley Embryos

Increasing evidence shows that sugars can act as signals affecting plant metabolism and development. Some of the effects of sugars on plant growth and development suggest an interaction of sugar signals with hormonal regulation. We investigated the effects of sugars on the induction of [alpha]-amylase by gibberellic acid in barley embryos and aleurone layers. Our results show that sugar and hormonal signaling interact in the regulation of gibberellic acid-induced gene expression in barley grains. The induction of [alpha]-amylase by gibberellic acid in the aleurone layer is unaffected by the presence of sugars, but repression by carbohydrates is effective in the embryo. [alpha]-Amylase expression in the embryo is localized to the scutellar epithelium and is hormone and sugar modulated. The effects of glucose are independent from the effects of sugars on gibberellin biosynthesis. They are not due to an osmotic effect, they are independent of abscisic acid, and only hexokinase-phosphorylatable glucose analogs are able to trigger gene repression. Overall, the results suggest the existence of an interaction between the hormonal and metabolic regulation of [alpha]-amylase genes in barley grains.     

水稻Xa1基因启动子的克隆及功能分析

Xa1是一个能对日本白叶枯病优势小种(小种1号)产生专化性抗性的R基因,虽已有该基因克隆、表达和功能方面的研究,但对其表达调控分子机制还不很清楚。本研究利用Xa1启动子与GUS报告基因的转基因T1株系,研究了Xa1启动子的时空表达及对不同外源激素的应答特征。结果表明,Xa1启动子驱动的GUS基因在水稻根中的表达量明显高于茎和叶,且在根部的中柱区GUS的表达量明显高于周围组织;在外源MeJA作用下GUS的表达显著增强,在SA和ABA处理下也有一定程度的增强,这些结果暗示Xa1的抗病作用与其在根系中柱的组织特异性表达存在一定的相关性,MeJA对Xa1启动子的活性起重要的调控作用。     

The alpha-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3beta-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909-8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3beta-hydroxylase genes and also an alpha-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and alpha-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3beta-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3beta-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for alpha-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for alpha-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced alpha-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not.     

Multiple mode regulation of a cysteine proteinase gene expression in rice

In many plants, cysteine proteinases play essential roles in a variety of developmental and physiological processes. In rice (Oryza sativa), REP-1 is a primary cysteine proteinase responsible for the digestion of seed storage proteins to provide nutrients to support the growth of young seedlings. In the present study, the gene encoding REP-1 was isolated, characterized, and designated as OsEP3A. An OsEP3A-specific DNA probe was used to study the effect of various factors on the expression of OsEP3A in germinating seeds and vegetative tissues of rice. The expression of OsEP3A is hormonally regulated in germinating seeds, spatially and temporally regulated in vegetative tissues, and nitrogen-regulated in suspension-cultured cells. The OsEP3A promoter was linked to the coding sequence of the reporter gene, gusA, which encodes beta-glucuronidase (GUS), and the chimeric gene was introduced into the rice genome. The OsEP3A promoter is sufficient to confer nitrogen regulation of GUS expression in suspension-cultured cells. Histochemical studies also indicate that the OsEP3A promoter is sufficient to confer the hormonal regulation of GUS expression in germinating seeds. These studies demonstrate that in rice the REP-1 protease encoded by OsEP3A may play a role in various physiological responses and processes, and that multiple mechanisms regulate the expression of OsEP3A.     

Differential in Situ Expression of Three ABA-Regulated Genes of Rice, RAB16A, REG2 and OSBZ8, during Seed Development

The spatial and temporal expression patterns of three ABA-regulatedgenes of rice in the developing seeds of wild type and embryonicmutants were studied by in situ hybridization. By the use ofan embryo-less mutant, we found that the expression of thesegenes in the aleurone layers was independent of embryonic tissues. (Received August 24, 1998; Accepted January 23, 1999)