Showing papers by "Dabing Zhang published in 2020"

PERSISTENT TAPETAL CELL2 Is Required for Normal Tapetal Programmed Cell Death and Pollen Wall Patterning.

Abstract: The timely programmed cell death (PCD) of the tapetum, the innermost somatic anther cell layer in flowering plants, is critical for pollen development, including the deposition and patterning of the pollen wall. Although several genes involved in tapetal PCD and pollen wall development have been characterized, the underlying regulatory mechanism remains elusive. Here we report that PERSISTENT TAPETAL CELL2 (PTC2), which encodes an AT-hook nuclear localized protein in rice (Oryza sativa), is required for normal tapetal PCD and pollen wall development. The mutant ptc2 showed persistent tapetal cells and abnormal pollen wall patterning including absent nexine, collapsed bacula, and disordered tectum. The defective tapetal PCD phenotype of ptc2 was similar to that of a PCD delayed mutant, ptc1, in rice, while the abnormal pollen wall patterning resembled that of a pollen wall defective mutant, Transposable Element Silencing Via AT-Hook, in Arabidopsis (Arabidopsis thaliana). Levels of anther cutin monomers in ptc2 anthers were significantly reduced, as was expression of a series of lipid biosynthetic genes. PTC2 transcript and protein were shown to be present in the anther after meiosis, consistent with the observed phenotype. Based on these data, we propose a model explaining how PTC2 affects anther and pollen development. The characterization of PTC2 in tapetal PCD and pollen wall patterning expands our understanding of the regulatory network of male reproductive development in rice and will aid future breeding approaches.

Defective Pollen Wall 3 (DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice

Abstract: In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

Genome-Wide Analysis of the GRAS Gene Family in Barley (Hordeum vulgare L.).

Abstract: The GRAS (named after first three identified proteins within this family, GAI, RGA, and SCR) family contains plant-specific genes encoding transcriptional regulators that play a key role in gibberellin (GA) signaling, which regulates plant growth and development. Even though GRAS genes have been characterized in some plant species, little research is known about the GRAS genes in barley (Hordeum vulgare L.). In this study, we observed 62 GRAS members from barley genome, which were grouped into 12 subgroups by using phylogenomic analysis together with the GRAS genes from Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa). Chromosome localization and gene structure analysis suggested that duplication events and abundant presence of intronless genes might account for the massive expansion of GRAS gene family in barley. The analysis of RNA-seq data indicates the expression pattern of GRAS genes in various tissues at different stages in barley. Noteworthy, our qRT-PCR analysis showed the expression of 18 candidate GRAS genes abundantly in the developing inflorescence, indicating their potential roles in the barley inflorescence development and reproduction. Collectively, our evolutionary and expression analysis of GRAS family are useful for future functional characterization of GA signaling in barley and agricultural improvement.

Rice pollen aperture formation is regulated by the interplay between OsINP1 and OsDAF1.

Abstract: The aperture on the pollen surface provides an exit for the emerging pollen tube. Apertures exhibit huge morphological variation across plant species-grasses, including rice, possess a complex aperture consisting of an annulus and an operculum-but little is known about how this species-specific cell-surface pattern forms. Here, we report a lectin receptor-like kinase in Oryza sativa, OsDAF1, which is essential for annulus formation and thus for fertility. OsDAF1 is evenly distributed in early microsporocytes but localizes to the distal pre-aperture site at the tetrad stage. We further reveal that the rice orthologue of a key aperture factor in Arabidopsis, OsINP1, has conserved and diversified roles in rice aperture formation. Disruption of OsINP1 prevents formation of the aperture, precluding pollen-tube germination. Furthermore, our results demonstrate that OsINP1 is required for polarization of OsDAF1 via direct protein interaction, suggesting that OsINP1 has an additional role in the formation of annulus that is absent in Arabidopsis. Our study reveals the importance of the aperture for rice grain yield and reveals mechanisms controlling pollen aperture development in cereal species.

The Plasticity of Root Systems in Response to External Phosphate

Abstract: Phosphate is an essential macro-element for plant growth accumulated in the topsoil. The improvement of phosphate uptake efficiency via manually manipulating root system architecture is of vital agronomic importance. This review discusses the molecular mechanisms of root patterning in response to external phosphate availability, which could be applied on the alleviation of phosphate-starvation stress. During the long time evolution, plants have formed sophisticated mechanisms to adapt to environmental phosphate conditions. In terms of root systems, plants would adjust their root system architecture via the regulation of the length of primary root, the length/density of lateral root and root hair and crown root growth angle to cope with different phosphate conditions. Finally, plants develop shallow or deep root system in low or high phosphate conditions, respectively. The plasticity of root system architecture responds to the local phosphate concentrations and this response was regulated by actin filaments, post-translational modification and phytohormones such as auxin, ethylene and cytokinin. This review summarizes the recent progress of adaptive response to external phosphate with focus on integrated physiological, cellular and molecular signaling transduction in rice and Arabidopsis.

Grass-Specific EPAD1 Is Essential for Pollen Exine Patterning in Rice

Abstract: The highly variable and species-specific pollen surface patterns are formed by sporopollenin accumulation. The template for sporopollenin deposition and polymerization is the primexine that appears on the tetrad surface, but the mechanism(s) by which primexine guides exine patterning remain elusive. Here, we report that the Poaceae-specific EXINE PATTERN DESIGNER 1 (EPAD1), which encodes a nonspecific lipid transfer protein, is required for primexine integrity and pollen exine patterning in rice (Oryza sativa). Disruption of EPAD1 leads to abnormal exine pattern and complete male sterility, although sporopollenin biosynthesis is unaffected. EPAD1 is specifically expressed in male meiocytes, indicating that reproductive cells exert genetic control over exine patterning. EPAD1 possesses an N-terminal signal peptide and three redundant glycosylphosphatidylinositol (GPI)-anchor sites at its C terminus, segments required for its function and localization to the microspore plasma membrane. In vitro assays indicate that EPAD1 can bind phospholipids. We propose that plasma membrane lipids bound by EPAD1 may be involved in recruiting and arranging regulatory proteins in the primexine to drive correct exine deposition. Our results demonstrate that EPAD1 is a meiocyte-derived determinant that controls primexine patterning in rice, and its orthologs may play a conserved role in the formation of grass-specific exine pattern elements.

Transcriptome profiling reveals phase-specific gene expression in the developing barley inflorescence

Abstract: The shape of an inflorescence varies among cereals, ranging from a highly branched panicle in rice to a much more compact spike in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). However, little is known about the molecular basis of cereal inflorescence architecture. We profiled transcriptomes at three developmental stages of the barley main shoot apex — spikelet initiation, floral organ differentiation, and floral organ growth — and compared them with those from vegetative seedling tissue. Transcript analyses identified 3688 genes differentially transcribed between the three meristem stages, with a further 1394 genes preferentially expressed in reproductive compared with vegetative tissue. Co-expression assembly and Gene Ontology analysis classified these 4888 genes into 28 clusters, revealing distinct patterns for genes such as transcription factors, histone modification, and cell-cycle progression specific for each stage of inflorescence development. We also compared expression patterns of VRS (SIX-ROWED SPIKE) genes and auxin-, gibberellic acid- and cytokinin-associated genes between two-rowed and six-rowed barley to describe regulators of lateral spikelet fertility. Our findings reveal barley inflorescence phase-specific gene expression, identify new candidate genes that regulate barley meristem activities and flower development, and provide a new genetic resource for further dissection of the molecular mechanisms of spike development.

Genome-wide analysis of RopGEF gene family to identify genes contributing to pollen tube growth in rice (Oryza sativa).

Abstract: In plants, the key roles played by RopGEF-mediated ROP signaling in diverse processes, including polar tip growth, have been identified. Despite their important roles in reproduction, a comprehensive analysis of RopGEF members has not yet been performed in rice (Oryza sativa). To determine whether RopGEF regulators are involved in rice pollen tube growth, we performed genome-wide analysis of this family in rice. Phylogenomic and meta-expression analysis of eleven RopGEFs in rice showed that four genes were preferentially expressed in mature pollen. These four genes contain the plant-specific Rop nucleotide exchanger (PRONE) domain and possible phosphorylated residues, suggesting a conserved role in polar tip growth with Arabidopsis thaliana. In subcellular localization analysis of the four RopGEFs through tobacco (Nicotiana benthamiana) infiltration, four proteins were predominantly identified in plasma membrane. Moreover, double mutants of RopGEF2/8 exhibited reduced pollen germination, causing partial male sterility. These genes possess unique cis-acting elements in their promoters compared with the other RopGEF genes. In this study, four RopGEF genes were identified as pollen-specific gene in eleven members of rice, and the expression pattern, promoter analysis, and evolutionary relationship of the RopGEF family were studied compared with Arabidopsis. Our study indicated that four RopGEF genes might function during pollen germination in distinct subcellular localization. Our study could provide valuable information on the functional study of RopGEF in rice.

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis

Abstract: Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid (Aux/IAA) gene family in barley (Hordeum vulgare L.).

Abstract: Aux/IAA genes are early auxin-responsive genes and essential for auxin signaling transduction There is little information about Aux/IAAs in the agriculturally important cereal, barley Using in silico method, we identified and subsequently characterized 36 Aux/IAAs from the barley genome Based on their genomic sequences and the phylogenic relationship with Arabidopsis and rice Aux/IAA, the 36 HvIAAs were categorized into two major groups and 14 subgroups The indication of the presence or absence of these domains for the biological functions and acting mechanisms was discussed The cis-element distributions in HvIAA promoters suggests that the HvIAAs expressions may not only regulated by auxin (the presence of AuxREs and TGA-element) but also by other hormones and developmental and environmental cues We then studied the HvIAAs expression in response to NAA (1-Naphthaleneacetic acid) using quantitative real-time PCR (qRT-PCR) Like the promoter analysis, only 14 HvIAAs were upregulated by NAA over two-fold at 4 h HvIAAs were clustered into three groups based on the spatiotemporal expression data We confirmed by qRT-PCR that most HvIAAs, especially HvIAA3, HvIAA7, HvIAA8, HvIAA18, HvIAA24 and HvIAA34, are expressed in the developing barley spike compared within seedling, suggesting their roles in regulating spike development Taken together, our data provide a foundation for further revealing the biological function of these HvIAAs

Molecular and genetic pathways for optimizing spikelet development and grain yield

Abstract: The spikelet is a unique structure of inflorescence in grasses that generates one to many flowers depending on its determinate or indeterminate meristem activity. The growth patterns and number of spikelets, furthermore, define inflorescence architecture and yield. Therefore, understanding the molecular mechanisms underlying spikelet development and evolution are attractive to both biologists and breeders. Based on the progress in rice and maize, along with increasing numbers of genetic mutants and genome sequences from other grass families, the regulatory networks underpinning spikelet development are becoming clearer. This is particularly evident for domesticated traits in agriculture. This review focuses on recent progress on spikelet initiation, and spikelet and floret fertility, by comparing results from Arabidopsis with that of rice, sorghum, maize, barley, wheat, Brachypodium distachyon, and Setaria viridis. This progress may benefit genetic engineering and molecular breeding to enhance grain yield.

Dissection of flag leaf metabolic shifts and their relationship with those occurring simultaneously in developing seed by application of non-targeted metabolomics

Abstract: Rice flag leaves are major source organs providing more than half of the nutrition needed for rice seed development. The dynamic metabolic changes in rice flag leaves and the detailed metabolic relationship between source and sink organs in rice, however, remain largely unknown. In this study, the metabolic changes of flag leaves in two japonica and two indica rice cultivars were investigated using non-targeted metabolomics approach. Principal component analysis (PCA) revealed that flag leaf metabolomes varied significantly depending on both species and developmental stage. Only a few of the metabolites in flag leaves displayed the same change pattern across the four tested cultivars along the process of seed development. Further association analysis found that levels of 45 metabolites in seeds that are associated with human nutrition and health correlated significantly with their levels in flag leaves. Comparison of metabolomics of flag leaves and seeds revealed that some flavonoids were specific or much higher in flag leaves while some lipid metabolites such as phospholipids were much higher in seeds. This reflected not only the function of the tissue specific metabolism but also the different physiological properties and metabolic adaptive features of these two tissues.

Overexpression of a novel cytochrome P450 monooxygenase gene, CYP704B1, from Panax ginseng increase biomass of reproductive tissues in transgenic Arabidopsis.

Abstract: Cytochrome P450 monooxygenase 704B (CYP704B), a member of the CYP86 clan, was found to be needed in Arabidopsis and rice to biosynthesize precursors of sporopollenin through oxidizing fatty acids. In the present study, we cloned and characterized a CYP704B gene in Panax ginseng, named PgCYP704B1. It shared high sequence identity (98-99%) with CYP704 of Arabidopsis, Theobroma cacao, and Morus notabilis. The phylogenetic comparison of ginseng and higher plants between the members of CYP86 clan revealed that ginseng CYP704 was categorized as a group of CYP704B with dicot plants. The expression of PgCYP704B1 is low in the stem, leaf, and fruit, and high in flower buds, particularly detected in the young gametic cell and tapetum layer of the developing anther. Arabidopsis plants overexpressing PgCYP704B1 improved plant biomass such as plant height, siliques and seed number and size. A cytological observation by transverse and longitudinal semi-thin sections of the siliques cuticles revealed that the cell length increased. Furthermore a chemical analysis showed that PgCYP704B1ox lines increased their cutin monomers contents in the siliques. Our results suggest that PgCYP704B1 has a conserved role during male reproduction for fatty acid biosynthesis and its overexpression increases cutin monomers in siliques that eventually could be used for seed production.

NERD1 is required for primexine formation and plasma membrane undulation during microsporogenesis in Arabidopsis thaliana

Abstract: The primexine formation and plasma membrane undulation are the crucial steps of pollen wall formation in many angiosperms. However, the molecular mechanism underlining these processes is largely unknown. In Arabidopsis, NEW ENHANCER OF ROOT DWARFISM1 (NERD1), a transmembrane protein, was reported to play pleiotropic roles in plant development including male fertility control; while, how NERD1 disruption impacts male reproduction is yet unclear. Here, we revealed that the male sterility of nerd1 mutants is attributed to defects in early steps of pollen wall formation. We found that nerd1-2 is void of primexine formation and microspore plasma membrane undulation, defective in callose deposition. Consequently, sporopollenin precursors are unable to deposit and assemble on the microspore surface, but instead accumulated in the anther locule and tapetal cells, and ultimately leading to microspore abortion. NERD1 is localized in the Golgi and is expressed in both vegetative and reproductive organs, with the highest expression in reproductive tissues, including the tapetum, male meiocytes, tetrads and mature pollen grains. Our results suggest that NERD1 is required for the primexine deposition and microspore plasma membrane undulation, thus essential for sporopollenin assembly and pollen exine formation.

Effective identification of CRISPR/Cas9-induced and naturally occurred mutations in rice using a multiplex ligation-dependent probe amplification-based method

Abstract: A multiplex ligation-dependent probe amplification (MLPA)-based method was developed and successfully utilized to efficiently detect both CRISPR/Cas9-induced and naturally occurred mutations in rice. The site-specific nuclease-based CRISPR/Cas9 system has emerged as one of the most efficient genome editing tools to modify multiple genomic targets simultaneously in various organisms, including plants for both fundamental and applied researches. Screening for both on-target and off-target mutations in CRISPR/Cas9-generated mutants at the early stages is an indispensable step for functional analysis and subsequent application. Various methods have been developed to detect CRISPR/Cas9-induced mutations in plants. Still, very few have focused on the detection of both on- and off-targets simultaneously, let alone the detection of natural mutations. Here, we report a multiplex capable method that allows to detect CRISPR/Cas9 induced on- and off-target mutations as well as naturally occurred mutation based on a multiplex ligation-dependent probe amplification (MLPA) method. We demonstrated that unlike other methods, the modified target-specific MLPA method can accurately identify any INDELs generated naturally or by the CRISPR/Cas9 system and that it can detect natural variation and zygosity of the CRISPR/Cas9-generated mutants in rice as well. Furthermore, its high sensitivity allowed to define INDELs down to 1 bp and substitutions to a single nucleotide. Therefore, this sensitive, reliable, and cheap method would further accelerate functional analysis and marker-assisted breeding in plants, including rice.

Bright Fluorescent Vacuolar Marker Lines Allow Vacuolar Tracing Across Multiple Tissues and Stress Conditions in Rice.

Abstract: The vacuole is indispensable for cells to maintain their water potential and to respond to environmental changes. Nevertheless, investigations of vacuole morphology and its functions have been limited to Arabidopsis thaliana with few studies in the model crop rice (Oryza sativa). Here, we report the establishment of bright rice vacuole fluorescent reporter systems using OsTIP1;1, a tonoplast water channel protein, fused to either an enhanced green fluorescent protein or an mCherry red fluorescent protein. We used the corresponding transgenic rice lines to trace the vacuole morphology in roots, leaves, anthers, and pollen grains. Notably, we observed dynamic changes in vacuole morphologies in pollen and root epidermis that corresponded to their developmental states as well as vacuole shape alterations in response to abiotic stresses. Our results indicate that the application of our vacuole markers may aid in understanding rice vacuole function and structure across different tissues and environmental conditions in rice.

A high-quality genome assembly and annotation of the humpback grouper Cromileptes altivelas

Abstract: Cromileptes altivelas that belongs to Serranidae in the order Perciformes, is widely distributed throughout the tropical waters of the Indo-West Pacific regions Due to their excellent food quality and abundant nutrients, it has become a popular marine food fish with high market values Here, we reported a chromosome-level genome assembly and annotation of the humpback grouper genome using more than 103X PacBio long-reads and high-throughput chromosome conformation capture (Hi-C) technologies The N50 contig length of the assembly is as large as 414 Mbp, the final assembly is 107 Gb with N50 of scaffold 4478 Mb, and 9924% of the scaffold sequences were anchored into 24 chromosomes The high-quality genome assembly also showed high gene completeness with 27,067 protein coding genes and 3,710 ncRNAs This high accurate genome assembly and annotation will not only provide an essential genome resource for C altivelas breeding and restocking, but will also serve as a key resource for studying fish genomics and genetics

Transcriptome analysis of Fenneropenaeus chinensis (Decapoda: Penaeidae) after low-salinity exposure identifies differentially expressed genes in pathways potentially related to osmoregulation

Abstract: Ability to tolerate low salinity is a key factor affecting the distribution of the Chinese shrimp (Fenneropenaeus chinensis). Although previous studies have investigated the mechanisms underlying adaptations to low salinity in some crustaceans, little is known about low-salinity adaptations in F. chinensis, particularly at the molecular level. Here, to identify genes potentially associated with the molecular response of F. chinensis to low-salinity exposure, we compared the transcriptomes of F. chinensis in low-salinity (5 ppt) and normal-salinity (20 ppt) environments. In total, 45,297,936 and 44,685,728 clean reads were acquired from the low-salinity and control groups, respectively. De novo assembly of the clean reads yielded 159,130 unigenes, with an average length of 662.82 bp. Of these unigenes, only a small fraction (10.5% on average) were successfully annotated against six databases. We identified 3,658 differentially expressed genes (DEGs) between the low-salinity group and the control group: 1,755 DEGs were downregulated in the low-salinity group as compared to the control, and 1,903 were upregulated. Of these DEGs, 282 were significantly overrepresented in 38 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. Notably, several DEGs were associated with pathways important for osmoregulation, including the mineral absorption pathway (ATP1A, Sodium/potassium-transporting ATPase subunit alpha; CLCN2, Chloride channel 2; HMOX2, Heme oxygenase 2; SLC40A1/FPN1, Solute carrier family 40 iron-regulated transporter, member 1), the vasopressin-regulated water reabsorption pathway (AQP4, Aquaporin-4; VAMP2, Vesicle-associated membrane protein 2; RAB5, Ras-related protein Rab-5) and the ribosome pathway. Our results help to clarify the molecular basis of low-salinity adaptations in F. chinensis.