Showing papers by "Dabing Zhang published in 2014"

Jasmonic acid regulates spikelet development in rice

Abstract: The spikelet is the basal unit of inflorescence in grasses, and its formation is crucial for reproductive success and cereal yield. Here, we report a previously unknown role of the plant hormone jasmonic acid (JA) in determining rice (Oryza sativa) spikelet morphogenesis. The extra glume 1 (eg1) and eg2 mutants exhibit altered spikelet morphology with changed floral organ identity and number, as well as defective floral meristem determinacy. We show that EG1 is a plastid-targeted lipase that participates in JA biosynthesis, and EG2/OsJAZ1 is a JA signalling repressor that interacts with a putative JA receptor, OsCOI1b, to trigger OsJAZ1's degradation during spikelet development. OsJAZ1 also interacts with OsMYC2, a transcription factor in the JA signalling pathway, and represses OsMYC2's role in activating OsMADS1, an E-class gene crucial to the spikelet development. This work discovers a key regulatory mechanism of grass spikelet development and suggests that the role of JA in reproduction has diversified during the flowering plant evolution.

ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis

Abstract: Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning.

Molecular Control of Grass Inflorescence Development

Abstract: The grass family is one of the largest families in angiosperms and has evolved a characteristic inflorescence morphology, with complex branches and specialized spikelets. The origin and development of the highly divergent inflorescence architecture in grasses have recently received much attention. Increasing evidence has revealed that numerous factors, such as transcription factors and plant hormones, play key roles in determining reproductive meristem fate and inflorescence patterning in grasses. Moreover, some molecular switches that have been implicated in specifying inflorescence shapes contribute significantly to grain yields in cereals. Here, we review key genetic and molecular switches recently identified from two model grass species, rice (Oryza sativa) and maize (Zea mays), that regulate inflorescence morphology specification, including meristem identity, meristem size and maintenance, initiation and outgrowth of axillary meristems, and organogenesis. Furthermore, we summarize emerging networks of genes and pathways in grass inflorescence morphogenesis and emphasize their evolutionary divergence in comparison with the model eudicot Arabidopsis thaliana. We also discuss the agricultural application of genes controlling grass inflorescence development.

The Rice Basic Helix-Loop-Helix Transcription Factor TDR INTERACTING PROTEIN2 Is a Central Switch in Early Anther Development

Abstract: In male reproductive development in plants, meristemoid precursor cells possessing transient, stem cell–like features undergo cell divisions and differentiation to produce the anther, the male reproductive organ. The anther contains centrally positioned microsporocytes surrounded by four distinct layers of wall: the epidermis, endothecium, middle layer, and tapetum. Here, we report that the rice (Oryza sativa) basic helix-loop-helix (bHLH) protein TDR INTERACTING PROTEIN2 (TIP2) functions as a crucial switch in the meristemoid transition and differentiation during early anther development. The tip2 mutants display undifferentiated inner three anther wall layers and abort tapetal programmed cell death, causing complete male sterility. TIP2 has two paralogs in rice, TDR and EAT1, which are key regulators of tapetal programmed cell death. We revealed that TIP2 acts upstream of TDR and EAT1 and directly regulates the expression of TDR and EAT1. In addition, TIP2 can interact with TDR, indicating a role of TIP2 in later anther development. Our findings suggest that the bHLH proteins TIP2, TDR, and EAT1 play a central role in regulating differentiation, morphogenesis, and degradation of anther somatic cell layers, highlighting the role of paralogous bHLH proteins in regulating distinct steps of plant cell–type determination.

Rice CYP703A3, a cytochrome P450 hydroxylase, is essential for development of anther cuticle and pollen exine.

Abstract: Anther cuticle and pollen exine act as protective envelopes for the male gametophyte or pollen grain, but the mechanism underlying the synthesis of these lipidic polymers remains unclear. Previously, a tapetum-expressed CYP703A3, a putative cytochrome P450 fatty acid hydroxylase, was shown to be essential for male fertility in rice (Oryza sativa L.). However, the biochemical and biological roles of CYP703A3 has not been characterized. Here, we observed that cyp703a3-2 caused by one base insertion in CYP703A3 displays defective pollen exine and anther epicuticular layer, which differs from Arabidopsis cyp703a2 in which only defective pollen exine occurs. Consistently, chemical composition assay showed that levels of cutin monomers and wax components were dramatically reduced in cyp703a3-2 anthers. Unlike the wide range of substrates of Arabidopsis CYP703A2, CYP703A3 functions as an in-chain hydroxylase only for a specific substrate, lauric acid, preferably generating 7-hydroxylated lauric acid. Moreover, chromatin immunoprecipitation and expression analyses revealed that the expression of CYP703A3 is directly regulated by Tapetum Degeneration Retardation, a known regulator of tapetum PCD and pollen exine formation. Collectively, our results suggest that CYP703A3 represents a conserved and diversified biochemical pathway for in-chain hydroxylation of lauric acid required for the development of male organ in higher plants.

Rice actin-binding protein RMD is a key link in the auxin–actin regulatory loop that controls cell growth

Abstract: The plant hormone auxin plays a central role in plant growth and development. Auxin transport and signaling depend on actin organization. Despite its functional importance, the mechanistic link between actin filaments (F-actin) and auxin intracellular signaling remains unclear. Here, we report that the actin-organizing protein Rice Morphology Determinant (RMD), a type II formin from rice (Oryza sativa), provides a key link. Mutants lacking RMD display abnormal cell growth and altered configuration of F-actin array direction. The rmd mutants also exhibit an inhibition of auxin-mediated cell elongation, decreased polar auxin transport, altered auxin distribution gradients in root tips, and suppression of plasma membrane localization of auxin transporters O. sativa PIN-FORMED 1b (OsPIN1b) and OsPIN2 in root cells. We demonstrate that RMD is required for endocytosis, exocytosis, and auxin-mediated OsPIN2 recycling to the plasma membrane. Moreover, RMD expression is directly regulated by heterodimerized O. sativa auxin response factor 23 (OsARF23) and OsARF24, providing evidence that auxin modulates the orientation of F-actin arrays through RMD. In support of this regulatory loop, osarf23 and lines with reduced expression of both OsARF23 and OsARF24 display reduced RMD expression, disrupted F-actin organization and cell growth, less sensitivity to auxin response, and altered auxin distribution and OsPIN localization. Our findings establish RMD as a crucial component of the auxin–actin self-organizing regulatory loop from the nucleus to cytoplasm that controls rice cell growth and morphogenesis.

Origins and Evolution of WUSCHEL-Related Homeobox Protein Family in Plant Kingdom

Abstract: WUSCHEL-related homeobox (WOX) is a large group of transcription factors specifically found in plants. WOX members contain the conserved homeodomain essential for plant development by regulating cell division and differentiation. However, the evolutionary relationship of WOX members in plant kingdom remains to be elucidated. In this study, we searched 350 WOX members from 50 species in plant kingdom. Linkage analysis of WOX protein sequences demonstrated that amino acid residues 141–145 and 153–160 located in the homeodomain are possibly associated with the function of WOXs during the evolution. These 350 members were grouped into 3 clades: the first clade represents the conservative WOXs from the lower plant algae to higher plants; the second clade has the members from vascular plant species; the third clade has the members only from spermatophyte species. Furthermore, among the members of Arabidopsis thaliana and Oryza sativa, we observed ubiquitous expression of genes in the first clade and the diversified expression pattern of WOX genes in distinct organs in the second clade and the third clade. This work provides insight into the origin and evolutionary process of WOXs, facilitating their functional investigations in the future.

Tuberculate fruit gene Tu encodes a C2 H2 zinc finger protein that is required for the warty fruit phenotype in cucumber (Cucumis sativus L.).

Abstract: Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.

Seed metabolomic study reveals significant metabolite variations and correlations among different soybean cultivars

Abstract: Soybean [Glycine max (L.) Merr.] is one of the world's major crops, and soybean seeds are a rich and important resource for proteins and oils. While "omics" studies, such as genomics, transcriptomics, and proteomics, have been widely applied in soybean molecular research, fewer metabolomic studies have been conducted for large-scale detection of low molecular weight metabolites, especially in soybean seeds. In this study, we investigated the seed metabolomes of 29 common soybean cultivars through combined gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry. One hundred sixty-nine named metabolites were identified and subsequently used to construct a metabolic network of mature soybean seed. Among the 169 detected metabolites, 104 were found to be significantly variable in their levels across tested cultivars. Metabolite markers that could be used to distinguish genetically related soybean cultivars were also identified, and metabolite-metabolite correlation analysis revealed some significant associations within the same or among different metabolite groups. Findings from this work may potentially provide the basis for further studies on both soybean seed metabolism and metabolic engineering to improve soybean seed quality and yield.

MACRO: A Combined Microchip-PCR and Microarray System for High-Throughput Monitoring of Genetically Modified Organisms

Abstract: The monitoring of genetically modified organisms (GMOs) is a primary step of GMO regulation. However, there is presently a lack of effective and high-throughput methodologies for specifically and sensitively monitoring most of the commercialized GMOs. Herein, we developed a multiplex amplification on a chip with readout on an oligo microarray (MACRO) system specifically for convenient GMO monitoring. This system is composed of a microchip for multiplex amplification and an oligo microarray for the readout of multiple amplicons, containing a total of 91 targets (18 universal elements, 20 exogenous genes, 45 events, and 8 endogenous reference genes) that covers 97.1% of all GM events that have been commercialized up to 2012. We demonstrate that the specificity of MACRO is ∼100%, with a limit of detection (LOD) that is suitable for real-world applications. Moreover, the results obtained of simulated complex samples and blind samples with MACRO were 100% consistent with expectations and the results of indepen...

Metabolic map of mature maize kernels

Abstract: Metabolites in maize kernels are associated not only with nutritional value but also physiological properties such as maturation, desiccation, and germination. However, comprehensive information concerning the metabolome of maize kernels is limited. In this study, we identified 210 metabolites in mature kernels of 14 representative maize lines using a non-targeted metabolomic profiling approach. Further statistical analysis revealed that 75 metabolites were significantly variable among those tested lines, and certain metabolites out of the detected 210 metabolites played critical roles in distinguishing one line from another. Additionally, metabolite–metabolite correlation analysis dissected key regulatory elements or pathways involved in metabolism of lipids, amino acids and carbohydrates. Furthermore, an integrated metabolic map constructed with transcriptomic, proteomic and metabolic data uncovered characteristic regulatory mechanisms of maize kernel metabolism. Altogether, this work provides new insights into the maize kernel metabolome that would be useful for metabolic engineering and/or molecular breeding to improve maize kernel quality and yield.

Dwarf Tiller1, a Wuschel-related homeobox transcription factor, is required for tiller growth in rice

Abstract: Unlike many wild grasses, domesticated rice cultivars have uniform culm height and panicle size among tillers and the main shoot, which is an important trait for grain yield. However, the genetic basis of this trait remains unknown. Here, we report that DWARF TILLER1 (DWT1) controls the developmental uniformity of the main shoot and tillers in rice (Oryza sativa). Most dwt1 mutant plants develop main shoots with normal height and larger panicles, but dwarf tillers bearing smaller panicles compared with those of the wild type. In addition, dwt1 tillers have shorter internodes with fewer and un-elongated cells compared with the wild type, indicating that DWT1 affects cell division and cell elongation. Map-based cloning revealed that DWT1 encodes a WUSCHEL-related homeobox (WOX) transcription factor homologous to the Arabidopsis WOX8 and WOX9. The DWT1 gene is highly expressed in young panicles, but undetectable in the internodes, suggesting that DWT1 expression is spatially or temporally separated from its effect on the internode growth. Transcriptomic analysis revealed altered expression of genes involved in cell division and cell elongation, cytokinin/gibberellin homeostasis and signaling in dwt1 shorter internodes. Moreover, the non-elongating internodes of dwt1 are insensitive to exogenous gibberellin (GA) treatment, and some of the slender rice1 (slr1) dwt1 double mutant exhibits defective internodes similar to the dwt1 single mutant, suggesting that the DWT1 activity in the internode elongation is directly or indirectly associated with GA signaling. This study reveals a genetic pathway synchronizing the development of tillers and the main shoot, and a new function of WOX genes in balancing branch growth in rice.

GMO quantification: valuable experience and insights for the future

Abstract: Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.

Comparative metabolomic analysis of wild type and mads3 mutant rice anthers

Abstract: Rice (Oryza sativa L.) MADS3 transcription factor regulates the homeostasis of reactive oxygen species (ROS) during late anther development, and one MADS3 mutant, mads3-4, has defective anther walls, aborted microspores and complete male sterility. Here, we report the untargeted metabolomic analysis of both wild type and mads3-4 mature anthers. Mutation of MADS3 led to an unbalanced redox status and caused oxidative stress that damages lipid, protein, and DNA. To cope with oxidative stress in mads3-4 anthers, soluble sugars were mobilized and carbohydrate metabolism was shifted to amino acid and nucleic acid metabolism to provide substrates for the biosynthesis of antioxidant proteins and the repair of DNA. Mutation of MADS3 also affected other aspects of rice anther development such as secondary metabolites associated with cuticle, cell wall, and auxin metabolism. Many of the discovered metabolic changes in mads3-4 anthers were corroborated with changes of expression levels of corresponding metabolic pathway genes. Altogether, this comparative metabolomic analysis indicated that MADS3 gene affects rice anther development far beyond the ROS homeostasis regulation.

Exine Export in Pollen

Abstract: Pollen as a sperm cell carrier is mainly protected by outer pollen wall (called exine) from physical and biological stresses. The major composition of exine is the highly resistant biopolymer sporopollenin, which mainly consists of hydrophobic lipids, phenylpropanoids, and aromatic compounds. The biosynthesis of these constituents has been shown to be catalyzed by enzymes preferentially expressed in the sporophytic tapetal layer, a nutritive tissue supporting pollen development. How the synthesized sporopollenin precursors are exported from tapetal cells onto the surface of microspore for pollen exine formation remains largely unknown. Here, we review the structure of tapetal cella and pollen exine in the model monocot rice (Oryza sativa) and the model dicot Arabidopsis thaliana. In addition, we highlight the update understanding on the role of ATP-binding cassette (ABC), lipid transfer protein (LTP), and multidrug and toxic efflux (MATE) transporters in trafficking of sporopollenin precursors across tapetal cells for exine development in rice and Arabidopsis. We also discuss the future research focus on the transport of sporopollenin precursors for exine synthesis.

Crystallization and preliminary crystallographic analysis of defective pollen wall (DPW) protein from Oryza sativa.

Abstract: The defective pollen wall (dpw) gene of Oryza sativa encodes a fatty acid reductase (DPW) which plays important roles in primary fatty alcohol synthesis. DPW catalyzes the synthesis of 1-hexadecanol. The enzyme shows a higher specificity for palmitoyl-ACP than for palmitoyl-CoA as the substrate, and can only use NADPH as the cofactor. To gain an understanding of the molecular mechanism underlying the reaction catalyzed by DPW, the gene encoding DPW without the N-terminal 80 amino acids (DPWΔ80) was cloned into pET-28a vector and was overexpressed in Escherichia coli. DPWΔ80 was purified to homogeneity and screened for crystallization. DPWΔ80 in complex with NADPH produced crystals that diffracted X-rays to a resolution of 3.4 A. The crystals belonged to space group P61 or P65, with unit-cell parameters a = b = 222.8, c = 114.0 A, α = β = 90, γ = 120°.

Plant metabolomics and metabolic biology

Abstract: Plants produce a large array of specializedmetabolites (natural products) that not only are essential for growth and development, but also play important roles in adaptation to the variable stressful environments. Also, many plant metabolites are essential nutrient elements for humans and serve as natural drugs. However, study of the biosynthesis of natural products is still in its infancy, because more than 90% of plant metabolites are unknown and only a small number of genes/ enzymes involved in metabolism are identified in the model plants Arabidopsis and rice. In the post‐genomics era, metabolomics – defined as the analysis of the total population of metabolites in a given sample, cell or tissue – and the integration of the data in the context of functional genomics, is attractive for analysis of global metabolic changes in plant development and responds to different environmental stresses. Metabolomic analysis would greatly accelerate our processes in deciphering the function of an individual gene in a metabolic pathway, building metabolic networks, identifying metabolic regulators, and more importantly establishing causal relations between metabolic pathways and biologic functions in plants. In this special issue on metabolomics and metabolic biology, we present one invited review and 10 research articles. Both gas chromatography mass spectrometry (GC‐ MS) and liquid chromatography mass spectrometry (LC‐MS) are widely used in plant metabolomic analysis. LC‐MS has good potential in separation of polar chemicals which constitute more than 80% of plant metabolites. However, currently, there is no consensus on which LC‐MS method should be used in plant metabolomic studies. In the invited review by Liu and Rochfort (2014), the most promising methods were reviewed, providing plant scientists with up‐to‐date and simplified information regarding the current status of polar metabolite analysis. In the research articles by Lin et al. (2014) and Agarrwal et al. (2014), metabolomic analysis was applied in studies of soybean cultivars, and the interaction between rice and the Asian rice gall midge (Orseolia oryzae Wood‐Mason), respectively. In both cases, metabolite markers (biomarkers) for distinguishing genetically related soybean cultivars and rice‐insect interaction were successfully identified. Untargeted metabolomic analysis ofmads3‐4 (a mutant in a gene encoding MADS3 transcription factor) and wild‐type mature anthers by Qu et al. (2014) indicated that the MADS3 gene affects rice anther development far beyond the reactive oxygen species homeostatic regulation. In two research articles by Schwahn et al. (2014) and Dong et al. (2014), sub‐metabolomic analysis was applied in focus on particular types of metabolites, for example, steroidal glycoalkaloids and flavonoids. Comparative analysis of flavonoid contents in various tissues from the two subspecies, Indica and Japonica, revealed tissue‐specific accumulation of most flavonoids and clear differential accumulation of different types of flavonoids. Based on the analysis of a large number of samples from four types of tissues of 169 accessions from tomato and its wild relatives, Schwahn et al. (2014) integrated species and tissue specificity information into the annotation and classification of steroidal glycoalkaloid peaks, allowing refinement of steroidal glycoalkaloid biosynthesis and detection of putative novel branch points. The cytochrome P450 (CYP) superfamily belonging to monooxygenase, has greatly expanded in the plant genomes. It is believed that members in this family play critical roles in biosynthesis of diversified natural products. In this special issue, the paper by Ma et al. (2014) annotates a total of 174 P450 genes from the mulberry (Morus notabilis C.K. Schn.) genome. The article by Yang et al. (2014) demonstrates that a cytochrome P450‐type carotene hydroxylase (PuCHY1) catalyzes the conversion of b‐carotene into zeaxanthin in the red alga Porphyra umbilicalis. Anthocyanin, a well‐studied plant specialized metabolite, is often accumulated to higher levels under stress, for example, cold, drought, and photo‐oxidative stress. Research by Gan et al. (2014) on the thf1I (thylakoid formation1) mutant in Arabidopsis revealed that the elevated anthocyanin content increases levels of methyl jasmonate, which is served as signals in coordination of plant metabolism and growth in photo‐oxidative stress. On the other hand, a study by Li et al. (2014) focusing on the glandular trichomes of Colquhounia seguinii identifies three new clerodane diterpenoids. Interestingly, all identified compounds showed antifeedant activity against the insect Spodoptera exigua. Finally, this special issue presents the work by Florian et al. (2014), which studies the relationship between transcripts and metabolite levels when plants grown under normal air conditions were transferred to different CO2 and O2 concentrations. An interesting conclusion is that the regulation of the photosynthesis pathways is not mediated at the level of transcription.

Characterization and phylogenetic analysis of allergenic Tryp_alpha_amyl protein family in plants.

Abstract: Most known allergenic proteins in rice (Oryza sativa) seed belong to the Tryp_alpha_amyl family (PF00234), but the sequence characterization and the evolution of the allergenic Tryp_alpha_amyl family members in plants have not been fully investigated. In this study, two specific motifs were found besides the common alpha-amylase inhibitors (AAI) domain from the allergenic Tryp_alpha_amyl family members in rice seeds (trRSAs). To understand the evolution and functional importance of the Tryp_alpha_amy1 family and the specific motifs for the allergenic one, a BLAST search identified 75 homologous proteins of trRSAs (trHAs) from 22 plant species including main crops such as rice, maize (Zea mays), wheat (Triticum aestivum), and sorghum (Sorghum bicolor) from all available sequences in the public databases. Statistical analysis showed that the allergenicity of trHAs is closely associated with these two motifs with high number of cysteine residues (p value = 0.00026), and the trHAs with and without the two mot...