[Show abstract][Hide abstract] ABSTRACT: Relative little is known about the functions of the so-called Dof zinc factors in plants. Here we report on the analysis of OsDof25 and show a function in regulation of the important C4 photosynthesis gene, OsC4PPDK in rice. Over-expression of OsDof25 enhanced the expression of OsC4PPDK in transient expression experiments by binding in a specific way to a conserved Dof binding site which was confirmed by yeast and in vitro binding studies. Expression studies using promoter GUS plants as well as qPCR experiments showed that OsDof25 expressed in different tissues including both photosynthetic and non-photosynthetic organs and that expression of OsDof25 was partially overlapping with the OsC4PPDK gene. Conclusive evidence for a role of OsDof25 in regulation of C4PPDK came from loss-of-function and gain-of-function experiments with transgenic rice, which showed that down-regulation or over-expression of OsDof25 correlated with OsC4PPDK expression and that OsDof25 has functions as transcriptional activator.
[Show abstract][Hide abstract] ABSTRACT: Fruits are complex plant structures that nurture seeds and facilitate their dispersal. The Arabidopsis fruit is termed silique. It develops from the gynoecium, which has a stigma, a style, an ovary containing the ovules, and a gynophore. Externally, the ovary consists of two valves, and their margins lay adjacent to the replum, which is connected to the septum that internally divides the ovary.In this work, we describe the role for the zinc finger transcription factor NO TRANSMITTING TRACT (NTT) in replum development. NTT loss-of-function leads to reduced replum width and cell number, whereas increased expression promotes replum enlargement. NTT activates the homeobox gene BP, which, together with RPL, is important for replum development. In addition, the NTT protein is able to bind the BP promoter in yeast, and when this binding region is not present, NTT fails to activate BP in the replum. Furthermore, NTT interacts with itself and different proteins involved in fruit development: RPL, STM, FUL, SHP1, and SHP2 in yeast and in planta. Moreover, its genetic interactions provide further evidence about its biological relevance in replum development.This article is protected by copyright. All rights reserved.
The Plant Journal 07/2014; 80:69-81. DOI:10.1111/tpj.12617 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Jasmonates are important phytohormones regulating reproductive development. We used two recessive rice Tos17 alleles of OsJAR1, osjar1-2 and osjar1-3, to study the biological function of jasmonates in rice anthesis. The florets of both osjar1 alleles stayed open during anthesis because the lodicules, which control flower opening in rice, were not withering on time. Furthermore, dehiscence of the anthers filled with viable pollen, was impaired, resulting in lower fertility. In situ hybridization and promoter GUS transgenic analysis confirmed OsJAR1 expression in these floral tissues. Flower opening induced by exogenous applied methyl jasmonate was impaired in osjar1 plants and was restored in a complementation experiment with transgenics expressing a wild type copy of OsJAR1 controlled by a rice actin promoter. Biochemical analysis showed that OsJAR1 encoded an enzyme conjugating jasmonic acid (JA) to at least Ile, Leu, Met, Phe, Trp and Val and both osjar1 alleles had substantial reduction in content of JA-Ile, JA-Leu and JA-Val in florets. We conclude that OsJAR1 is a JA-amino acid synthetase that is required for optimal flower opening and closing and anther dehiscence in rice.
[Show abstract][Hide abstract] ABSTRACT: KNOX genes are important regulators of meristem function and a complex network of transcription factors ensures tight control of their expression. Here we show that members of the GROWTH-REGULATING FACTOR (GRF) family act as players in this network. A yeast one-hybrid screen with the upstream sequence of the KNOX gene Oskn2 from rice (Oryza sativa) resulted in isolation of OsGRF3 and OsGRF10. Specific binding to a region in the untranslated leader sequence of Oskn2 was confirmed by yeast and in vitro binding assays. ProOskn2:GUS reporter expression was downregulated by OsGRF3 and OsGRF10 in vivo, suggesting that these proteins function as transcriptional repressors. Likewise, we found that the GRF protein BGRF1 from barley could act as a repressor on an intron sequence in the KNOX gene Hooded/Bkn3 and that AtGRF4, -5 and -6 from Arabidopsis thaliana could repress KNAT2 promoter activity. OsGRF overexpression phenotypes in rice were consistent with aberrant meristematic activity, showing reduced formation of tillers and internodes and extensive adventitious root/shoot formation on nodes. These effects were associated with downregulation of endogenous Oskn2 expression by OsGRF3. Conversely, RNAi silencing of OsGRF3, -4 and -5 resulted in dwarfism, delayed growth and inflorescence formation, and upregulation of Oskn2. These data demonstrate conserved interactions between the GRF and KNOX families of transcription factors in both monocot and dicot plants.
[Show abstract][Hide abstract] ABSTRACT: Glutelins are the most abundant storage proteins in rice grain and can make up to 80 % of total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been intensively used as a model to understand regulation of seed-storage protein accumulation. In this study, we describe a zinc finger gene of the Cys3His1 (CCCH or C3H) class, named OsGZF1, which was identified in a yeast one-hybrid screening using the core promoter region of GluB-1 as bait and cDNA expression libraries prepared from developing rice panicles and grains as prey. The OsGZF1 protein binds specifically to the bait sequence in yeast and this interaction was confirmed in vitro. OsGZF1 is predominantly expressed in a confined domain surrounding the scutellum of the developing embryo and is localised in the nucleus. Transient expression experiments demonstrated that OsGZF1 can down-regulate a GluB-1-GUS (β-glucuronidase) reporter and OsGZF1 was also able to significantly reduce activation conferred by RISBZ1 which is a known strong GluB-1 activator. Furthermore, down-regulation of OsGZF1 by an RNAi approach increased grain nitrogen concentration. We propose that OsGZF1 has a function in regulating the GluB-1 promoter and controls accumulation of glutelins during grain development.
[Show abstract][Hide abstract] ABSTRACT: Rice among other cereals is key to food security for at least half the world population. Since the 1960s, productivity of rice has largely been improved during the Green Revolution, which included development of new cultivars, irrigation infrastructure, new management techniques, and synthetic fertilizers and pesticides. Nowadays, scientists and breeders are more and more focused on improving the quality of rice for different purposes and markets. For instance, people in the Far East prefer sticky and soft rice, while in India, a non-sticky type is preferred. Consumers from developed countries ask mainly for grain with good cooking quality and eating characteristics, but in many developing regions, nutritional value is crucial as rice is the most consumed staple food. Grain quality is a general concept which covers many characteristics ranging from physical to biochemical and physiological properties. Starch and protein are the two main components of rice endosperm and therefore are key to quality. The knowledge of how starch and protein are synthesized, sorted, and stored in starch granules and protein bodies (PB) is important for rice breeding. Besides that, grain quality has been shown to be affected significantly by growing and environmental conditions, such as water availability, temperature, fertilizer application, drought, and salinity stresses. However, the signal transduction pathways controlling grain quality still remain largely unclear. In the following sections, we first briefly review the four main aspects of grain quality, followed by a discussion of the molecular and genetic basis of starch and seed-storage protein biosynthesis and the effects of environmental factors. Obviously, rice grain is also an important source of mineral micronutrients, as well as important vitamins. Storage of these also plays crucial roles in grain quality and nutritional value, but we will only discuss these aspects briefly in this review.
[Show abstract][Hide abstract] ABSTRACT: Oshox22 belongs to the homeodomain-leucine zipper (HD-Zip) family I of transcription factors, most of which have unknown functions. Here we show that the expression of Oshox22 is strongly induced by salt stress, abscisic acid (ABA), and polyethylene glycol treatment (PEG), and weakly by cold stress. Trans-activation assays in yeast and transient expression analyses in rice protoplasts demonstrated that Oshox22 is able to bind the CAAT(G/C)ATTG element and acts as a transcriptional activator that requires both the HD and Zip domains. Rice plants homozygous for a T-DNA insertion in the promoter region of Oshox22 showed reduced Oshox22 expression and ABA content, decreased sensitivity to ABA, and enhanced tolerance to drought and salt stresses at the seedling stage. In contrast, transgenic rice over-expressing Oshox22 showed increased sensitivity to ABA, increased ABA content, and decreased drought and salt tolerances. Based on these results, we conclude that Oshox22 affects ABA biosynthesis and regulates drought and salt responses through ABA-mediated signal transduction pathways.
[Show abstract][Hide abstract] ABSTRACT: In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.
[Show abstract][Hide abstract] ABSTRACT: In this study, we show that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis-element located in the 5' promoter region of the pathogen-induced Ep5C gene, which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knockdown mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence, our results substantiate that defense-related signaling pathways and cell wall integrity are interconnected and that MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense.
[Show abstract][Hide abstract] ABSTRACT: The yeast one-hybrid system is widely recognized as a valuable and straightforward technique to study interactions between transcription factors and DNA. By means of one-hybrid screens, transcription factors or other DNA-binding proteins, expressed from cDNA expression libraries, can be identified due to the interactions with a DNA sequence-of-interest that is linked to a reporter gene, such as the yeast HIS3 gene. Usually, the library is constructed in an E. coli-yeast shuttle vector designed for production of hybrid proteins consisting of a library protein and the trans-activating domain (AD) from the yeast GAL4 transcription factor. Here, we describe an optimized system of vectors for one-hybrid screenings together with detailed step-wise protocols, an elaborate trouble-shooting guide and many technical tips to conduct successful screenings. This system and other yeast genetic selection procedures derived from one-hybrid methodology proved highly useful to help understanding the regulatory networks controlling expression of the genome.
[Show abstract][Hide abstract] ABSTRACT: Heterosis is a biological phenomenon whereby the offspring from two parents show improved and superior performance than either inbred parental lines. Hybrid rice is one of the most successful apotheoses in crops utilizing heterosis. Transcriptional profiling of F(1) super-hybrid rice Liangyou-2186 and its parents by serial analysis of gene expression (SAGE) revealed 1183 differentially expressed genes (DGs), among which DGs were found significantly enriched in pathways such as photosynthesis and carbon-fixation, and most of the key genes involved in the carbon-fixation pathway exhibited up-regulated expression in F(1) hybrid rice. Moreover, increased catabolic activity of corresponding enzymes and photosynthetic efficiency were also detected, which combined to indicate that carbon fixation is enhanced in F(1) hybrid, and might probably be associated with the yield vigor and heterosis in super-hybrid rice. By correlating DGs with yield-related quantitative trait loci (QTL), a potential relationship between differential gene expression and phenotypic changes was also found. In addition, a regulatory network involving circadian-rhythms and light signaling pathways was also found, as previously reported in Arabidopsis, which suggest that such a network might also be related with heterosis in hybrid rice. Altogether, the present study provides another view for understanding the molecular mechanism underlying heterosis in rice.
[Show abstract][Hide abstract] ABSTRACT: In the barley (Hordeum vulgare) Hooded (Kap) mutant, the duplication of a 305-bp intron sequence leads to the overexpression of the Barley knox3 (Bkn3) gene, resulting in the development of an extra flower in the spikelet. We used a one-hybrid screen to identify four proteins that bind the intron-located regulatory element (Kap intron-binding proteins). Three of these, Barley Ethylene Response Factor1 (BERF1), Barley Ethylene Insensitive Like1 (BEIL1), and Barley Growth Regulating Factor1 (BGRF1), were characterized and their in vitro DNA-binding capacities verified. Given the homology of BERF1 and BEIL1 to ethylene signaling proteins, we investigated if these factors might play a dual role in intron-mediated regulation and ethylene response. In transgenic rice (Oryza sativa), constitutive expression of the corresponding genes produced phenotypic alterations consistent with perturbations in ethylene levels and variations in the expression of a key gene of ethylene biosynthesis. In barley, ethylene treatment results in partial suppression of the Kap phenotype, accompanied by up-regulation of BERF1 and BEIL1 expression, followed by down-regulation of Bkn3 mRNA levels. In rice protoplasts, BEIL1 activates the expression of a reporter gene driven by the 305-bp intron element, while BERF1 can counteract this activation. Thus, BEIL1 and BERF1, likely in association with other Kap intron-binding proteins, should mediate the fine-tuning of Bkn3 expression by ethylene. We propose a hypothesis for the cross talk between the KNOX and ethylene pathways.
[Show abstract][Hide abstract] ABSTRACT: Transposons are effective mutagens alternative to T-DNA for the generation of insertional mutants in many plant species including those whose transformation is inefficient. The current strategies of transposon tagging are usually slow and labor-intensive and yield low frequency of tagged lines. We have constructed a series of transposon tagging vectors based on three approaches: (i) AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain (GVG) chemical-inducible expression system; (ii) deletion of AcTPase via Cre-lox site-specific recombination that was initially triggered by Ds excision; and (iii) suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element (HPT-Ds). We tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful resources for functional genomics of rice and other crop plants. The vectors are freely available for the community.
[Show abstract][Hide abstract] ABSTRACT: Plant responses mediated by phytochrome A display a first phase saturated by transient light signals and a second phase requiring sustained excitation with far-red light (FR). These discrete outcomes, respectively so-called very-low-fluence response (VLFR) and high-irradiance response (HIR), are appropriate in different environmental and developmental contexts but the mechanisms that regulate the switch remain unexplored. Promoter analysis of a light-responsive target gene revealed a motif necessary for HIR but not for VLFR. This motif is required for binding of the Bell-like homeodomain 1 (BLH1) to the promoter in in vitro and in yeast 1-hybrid experiments. Promoter substitutions that increased BLH1 binding also enhanced HIR. blh1 mutants showed reduced responses to continuous FR and to deep canopy shadelight, but they retained normal responses to pulsed FR or red light and unfiltered sunlight. BLH1 enhanced BLH1 expression and its promotion by FR. We conclude that BLH1 specifically regulates HIR and not VLFR of phytochrome A.
Proceedings of the National Academy of Sciences 08/2009; 106(32):13624-9. DOI:10.1073/pnas.0906598106 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction The homeodomain leucine zipper (HD-Zip) genes have been shown to encode transcription factors that have diverse functions in plant development and were implicated in stress responses(Ref.1, 2, 3). Rice gene Oshox4 (HOMEOBOX4) belongs to HD-Zip family I genes and its expression is drought-repressed (Ref 1 and 2). In this study, rice Oshox4 gene under the control of CaMV35s promoter was over expressed in IR64 mega variety through Agrobacterium-mediated transformation to explore the role of this transcription factor for drought tolerance and rice morphogenesis . Single-copy transgenic events were selected for evaluation under drought stress. Figure 3. Longitudinal section of 2nd internodes showing decreased cell expansion in Oshox4 transgenic plant which was reversed after GA spraying. Figure 5. Correlation of tiller and plant height of Oshox4 transgenic rice. Materials and methods Oshox4 under the control of CaMV35s promoter was introduced and transformed into IR64 immature embryos via Agrobacterium-mediated transformation. More than 50 events were obtained. Two independent single-copy transgenic events screened by Southern DNA analysis were selected for phenotypic evaluation in a soil dry-down experiment under controlled conditions using the Fraction of Transpirable Soil Water (FTSW) as stress co-variable (Ref. 4). Two treatments were applied, severe drought stress reaching FTSW of 0.2 and flooded control. Each treatment consist of 5 replicates. An amount of 30uM gibberellin acid was sprayed on 35s::Oshox4 transgenic plants at heading stage. Free hand section, microscopy and reverse transcription PCR was done to check the stem cell expansion and gene expression in flag leaf. Results 1. When transgenic plants were sprayed with 30uM gibberellin (GA) at heading stage, the semi-dwarf phenotype induced by the over-expression of Oshox4 was reversed, implying that Oshox4 gene may regulate plant response to soil water deficit by its involvement in GA metabolism (Figure1, 2, and 3). 2. The results of the dry-down showed that the leaf relative water content (RWC) of the Oshox4 transgenic plants was 2-fold higher than the control (null) (P-value is 0.000149 and 0.000867 respectively.) at FTSW 0.2 (Figure 4). This resulted in higher plant water status and better growth under drought. 3. There is no significant difference in biomass content between the Oshox4 transgenic events and the wild-type. The semi-dwarf transgenics have more number of tillers. Moreover, there is a correlation between tiller and plant height of Oshox4 transgenic rice (Figure 5). 4. RT-PCR results showed that the expression of OsGA2ox3 and OsGA2ox5 was up-regulated in Oshox4 transgenic plants. Exogenous GA increased the expression of OsGA2ox5 but decreased the expression of OsGA2ox3 (Figure 6 and 7).
[Show abstract][Hide abstract] ABSTRACT: Transgenic methods offer a complementary approach for classical breeding to improve the tolerance of plants toward biotic and abiotic stresses. The objective of our study at RC Biotechnology LIPI in Indonesia, in collaboration with the Institute of Biology of Leiden University (The Netherlands), is to explore the use of HD-Zip transcription factors in improving the performance of rice under dry conditions. Enhancing drought resistance entails transgenic expression of HD-Zip transcription factors involved in drought response of rice. Earlier, seven HD-Zip I and II genes were identified in rice that were analyzed for drought-responsiveness. One of the two dehydration-repressed HD-Zip genes was induced by 4 hours of flooding, as a treatment opposite to drought. This Oshox4 gene from the HD-Zip gene family was selected for study in transgenic plants. Transgenic Nipponbare lines overexpressing this particular gene developed smaller leaves and exhibited a reduction in senescence compared with the controls. Transformation of Indonesian rice with Oshox4 and characterization of transgenic lines, including drought phenotyping, are now being carried out. Experiments carried out in Arabidopsis showed that overexpression of this particular HD-Zip gene can confer resistance to drought. Our second approach aims at identifying novel drought-resistance genes and is based on insertional mutagenesis in rice using gene trap and activation tag constructs.
Drought frontiers in rice - Crop improvement for increased rainfed production, Edited by Serraj R, Bennett J, Hardy B, 01/2009: pages pp 351-364; World Scientific Publishing Co., Singapore., ISBN: 978-981-4280-00-6
[Show abstract][Hide abstract] ABSTRACT: Monosaccharides transporters play important roles in assimilate supply for sink tissue development. In this study, a new monosaccharide transporter gene OsMST6 was identified from rice (Oryza sativa L.). The predicted OsMST6 protein shows typical features of sugar transporters and shares 79.6% identity with the rice monosaccharide transporter OsMST3. Heterologous expression in yeast (Saccharomyces cerevisiae) demonstrated that OsMST6 is a broad-spectrum monosaccharide transporter, with a K
m of 266.1 μΜ for glucose. OsMST6-green fluorescent protein fusion protein is localized to the plasma membrane in plant. Semi-quantitative RT-PCR analysis exhibited that OsMST6 is expressed in all tested organs/tissues. In developing seeds, OsMST6 expression level is high at the early and middle grain filling stages and gradually declines later. Further analysis detected its expression in both maternal and filial tissues. RNA in situ hybridization analysis indicated that OsMST6 is predominantly expressed in the vascular parenchyma of the chalazal vein, cross-cells, nucellar tissue and endosperm of young seeds, in mesophyll cells of source leaf blades, and in pollens and the connective vein of anthers. In addition, OsMST6 expression is up-regulated by salt stress and sugars. The physiological role of OsMST6 for seed development and its roles in other sink and source tissues are discussed.
[Show abstract][Hide abstract] ABSTRACT: The homeodomain leucine zipper (HD-Zip) genes encode transcription factors that have diverse functions in plant development and have often been implicated in stress adaptation. The HD-Zip genes are the most abundant group of homeobox (HB) genes in plants and do not occur in other eukaryotes. This paper describes the complete annotation of the HD-Zip families I, II and III from rice and compares these gene families with Arabidopsis in a phylogeny reconstruction. Orthologous pairs of rice and Arabidopsis HD-Zip genes were predicted based on neighbour joining and maximum parsimony (MP) trees with support of conserved intron-exon organization. Additionally, a number of HD-Zip genes appeared to be unique to rice. Searching of EST and cDNA databases and expression analysis using RT-PCR showed that 30 out of 31 predicted rice HD-Zip genes are expressed. Most HD-Zip genes were broadly expressed in mature plants and seedlings, but others showed more organ specific patterns. Like in Arabidopsis and other dicots, a subset of the rice HD-Zip I and II genes was found to be regulated by drought stress. We identified both drought-induced and drought-repressed HD-Zip genes and demonstrate that these genes are differentially regulated in drought-sensitive versus drought-tolerant rice cultivars. The drought-repressed HD-Zip family I gene, Oshox4, was selected for promoter-GUS analysis, showing that drought-responsiveness of Oshox4 is controlled by the promoter and that Oshox4 expression is predominantly vascular-specific. Loss-of-function analysis of Oshox4 revealed no specific phenotype, but overexpression analysis suggested a role for Oshox4 in elongation and maturation processes.