Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

Molecular characterization of mature pollen-specific genes encoding novel small cysteine-rich proteins in rice (Oryza sativa L.)

In our previous cDNA microarray analysis, we identified 53 mature anther-specific genes, whose function was unknown, in rice. We reanalyzed these genes from the viewpoint of the specific amino acid motif. Out of 53 genes, three genes, Os-26, Os-32, and Os-169 (renamed as OsSCP1, OsSCP2, and OsSCP3), encoded cysteine-rich motif (Cys-X₃-Cys-X₁₃-Cys-X₃-Cys), indicating that they were novel small cysteine-rich proteins. From the search of specific elements in promoter regions, several pollen-specific elements were found. In order to determine whether three promoters were functional in pollen or not, the gene constructs with promoter regions fused to the β-glucuronidase gene were transformed into tobacco. Histochemical analysis showed that these promoters were active in the mature pollen grains and pollen tubes. Furthermore, OsSCP1 and OsSCP3 formed a multigene family tandemly in the rice genome. From the results, OsSCPs might have important roles in mature pollen development and pollen tube growth.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.     

A Novel <Emphasis Type="Italic">PhLRR</Emphasis> Gene Promoter is Sufficient for Engineering Male Sterility in Petunia

Tissue-specific promoters can drive genes specifically expressed in the target organs and have been widely used in plant molecular breeding. In this study, a 1.2-kb promoter region of an anther-specific gene PhLRR from Petunia hybrida “Fantasy” was isolated and fused to the β-glucuronidase (GUS) gene. The pPhLRR::GUS vector was heterogeneously transformed into tobacco in which the GUS staining was only detected in the early development stage of anthers and no GUS expression in any other three floral whirls or vegetative organs was observed. It is very different from other well-studied anther-specific promoters which drive genes specifically expressed in the later development stage of anthers or only in the pollens. Furthermore, the pPhLRR::Barnase was introduced into petunia and induced complete male sterility without influencing the ornamental characteristics or the female fertility in transformed plants. These results indicate that PhLRR promoter is a new kind of petunia anther-specific promoter and could be taken as a valuable tool in ornamental plant breeding.     

Tapetum-specific expression of theOsg6B promoter-β-glucuronidase gene in transgenic rice

The promoter of an anther tapetum-specific gene,Osg6B, was fused to a-glucuronidase (GUS) gene and introduced into rice byAgrobacterium-mediated gene transfer. Fluorometric and histochemical GUS assay showed that GUS was expressed exclusively within the tapetum of anthers from the uninucleate microspore stage (7 days before anthesis) to the tricellular pollen stage (3 days before anthesis). This is the first demonstration of an anther-specific promoter directing tapetum-specific expression in rice.Abbreviations GUS ßGlucuronidase     

Suppression of OsMADS7 in rice endosperm stabilizes amylose content under high temperature stress

High temperature significantly alters the amylose content of rice, resulting in mature grains with poor eating quality. However, only few genes and/or quantitative trait loci involved in this process have been isolated and the molecular mechanisms of this effect remain unclear. Here, we describe a floral organ identity gene, OsMADS7, involved in stabilizing rice amylose content at high temperature. OsMADS7 is greatly induced by high temperature at the early filling stage. Constitutive suppression of OsMADS7 stabilizes amylose content under high temperature stress but results in low spikelet fertility. However, rice plants with both stable amylose content at high temperature and normal spikelet fertility can be obtained by specifically suppressing OsMADS7 in endosperm. GBSSI is the major enzyme responsible for amylose biosynthesis. A low filling rate and high expression of GBSSI were detected in OsMADS7 RNAi plants at high temperature, which may be correlated with stabilized amylose content in these transgenic seeds under high temperature. Thus, specific suppression of OsMADS7 in endosperm could improve the stability of rice amylose content at high temperature, and such transgenic materials may be a valuable genetic resource for breeding rice with elite thermal resilience.     

Intragenic control of expression of a rice MADS box gene OsMADS1

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.     

<Emphasis Type="Italic">OsMADS22</Emphasis>, an <Emphasis Type="Italic">STMADS11</Emphasis>-like MADS-box gene of rice,is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy

We report the cDNA sequence and gene expression patterns of OsMADS22, a novel member of the STMADS11-like family of MADS-box genes, from rice. In contrast to previously reported STMADS11-like genes, whose expression is detected in vegetative tissues, OsMADS22 is mainly expressed during embryogenesis and flower development. In situ hybridization analysis revealed that OsMADS22 expression is localized in the L1 layer of embryos and in developing stamen primordia. Ectopic expression of OsMADS22 in transgenic rice plants resulted in aberrant floral morphogenesis, characterized by a disorganized palea, an elongated glume, and a two-floret spikelet. The results are discussed in terms of rice spikelet development and a novel non-vegetative role for a STMADS11-like gene.     

Conservation of the E-function for Floral Organ Identity in Rice Revealed by the Analysis of Tissue Culture-induced Loss-of-Function Mutants of the <Emphasis Type="Italic">OsMADS1</Emphasis> Gene

Rapid progress in studies on flower development has resulted in refining the classical ‘ABC model’ into a new ‘ABCDE model’ to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicotyledonous (dicot) plants has been revealed. However, its conservation in monocotyledonous (monocot) plants remains largely unknown. Here, we show the conservation of E-function in rice (Oryza sativaL.) by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123(SEPALLATA1/2/3) and the petunia FBP2(Floral Binding Protein 2), suggesting that OsMADS1play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice, a monocot model plant.     

Defensin promoters as potential tools for engineering disease resistance in cereal grains

Engineering of plant protection in cereals requires well characterized tissue-specific and wounding/pathogen-inducible promoters for targeted expression of pathogen responsive and resistance genes. We describe the isolation of seven wheat and rice defensin genes expressed in early developing grain and during grain germination, two developmental stages that are particularly vulnerable to pathogens and insects. Comparison of three-dimensional (3D) models of these rice and wheat PRPI defensins indicated variations in spatial architectures that could reflect their functional diversities. Wheat and rice were stably transformed with promoter–GUS fusion constructs and the spatial and temporal activities of four promoters were studied using whole-mount and histological assays. PRPI promoters were active before and at anthesis in both transgenic wheat and rice with activity mainly in the ovary. In rice, GUS activity was also observed in vascular tissue of the lemma, palea and anthers. After fertilization, GUS was strongly expressed in the outer cell layers of the pericarp and in the main vascular bundle of the grain. During, and a short time after, seed germination, wheat promoters were active in transgenic rice embryos, roots and/or coleoptiles. All wheat and rice promoters were strongly induced by wounding in leaf, stem and grain of transgenic rice plants. These results suggest that PRPI promoters will be useful for specific targeting and accumulation of proteins conferring resistance to pathogens in vulnerable tissues of developing and germinating grain.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.