Changbin Chen

Regulation of Arabidopsis tapetum development and function by DYSFUNCTIONAL TAPETUM1 (DYT1) encoding a putative bHLH transcription factor

In flowering plants, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes that regulate anther cell differentiation and function. Here, we report the analysis of a new Arabidopsis male sterile mutant, dysfunctional tapetum1 (dyt1). The dyt1 mutant exhibits abnormal anther morphology beginning at anther stage 4, with tapetal cells that have excess and/or enlarged vacuoles and lack the densely stained cytoplasm typical of normal tapetal cells. The mutant meiocytes are able to complete meiosis I, but they do not have a thick callose wall; they often fail to complete meiotic cytokinesis and eventually collapse. DYT1 encodes a putative bHLH transcription factor and is strongly expressed in the tapetum from late anther stage 5 to early stage 6, and at a lower level in meiocytes. In addition, the level of DYT1 mRNA is reduced in the sporocyteless/nozzle (spl/nzz) and excess microsporocytes1/extra sporogenous cell (ems1/exs) mutants; together with the mutant phenotypes, this suggests that DYT1 acts downstream of SPL/NZZ and EMS1/EXS. RT-PCR results showed that the expression levels of many tapetum-preferential genes are reduced significantly in the dyt1 mutant, indicating that DYT1 is important for the expression of tapetum genes. Our results support the hypothesis that DYT1 is a crucial component of a genetic network that controls anther development and function.

A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.)

In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately 85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing a rice gene.

The BAM1/BAM2 Receptor-Like Kinases Are Important Regulators of Arabidopsis Early Anther Development

Anther development involves the formation of several adjacent cell types required for normal male fertility. Only a few genes are known to be involved in early anther development, particularly in the establishment of these different cell layers. Arabidopsis thaliana BAM1 (for BARELY ANY MERISTEM) and BAM2 encode CLAVATA1-related Leu-rich repeat receptor-like kinases that appear to have redundant or overlapping functions. We characterized anther development in the bam1 bam2 flowers and found that bam1 bam2 anthers appear to be abnormal at a very early stage and lack the endothecium, middle, and tapetum layers. Analyses using molecular markers and cytological techniques of bam1 bam2 anthers revealed that cells interior to the epidermis acquire some characteristics of pollen mother cells (PMCs), suggesting defects in cell fate specification. The pollen mother-like cells degenerate before the completion of meiosis, suggesting that these cells are defective. In addition, the BAM1 and BAM2 expression pattern supports both an early role in promoting somatic cell fates and a subsequent function in the PMCs. Therefore, analysis of BAM1 and BAM2 revealed a cell–cell communication process important for early anther development, including aspects of cell division and differentiation. This finding may have implications for the evolution of multiple signaling pathways in specifying cell types for microsporogenesis.

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes.

BackgroundMeiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.ResultsA high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.ConclusionThese findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

LEUNIG has multiple functions in gynoecium development in Arabidopsis

Summary: The Arabidopsis gene LEUNIG was previously found to regulate floral organ identity. In this work we describe gynoecial phenotypes of newly isolated strong leunig alleles, leunig‐101, leunig‐102, and leunig‐103. Gynoecia of these strong leunig mutants are united only at the basal part, leaving four unfused parts at the apex. Among them two medial ones are styles capped with stigmas, and two lateral ones are protrusions from valves. The gynoecium with unfused apex in leunig arises as a unit from a basal meristematic zone, suggesting that LEUNIG is required for normal congenital gynoecium fusion. The epidermal cells on growing inner surfaces of leunig gynoecium failed to fuse after they contact each other, indicating that LEUNIG is essential for the proper postgenital fusion. The epidermal cells at the very distal portion of protruded valves mimic those on wild‐type styles, and those valves occasionally also have stigma‐like tissues, indicating that LEUNIG function is required for the valve identity determination. We have also analyzed clavata1‐4 leunig‐101, clavata2‐1 lug‐101, fruitfull‐1 leunig‐101, and pinoid‐1 leunig‐101 double mutants. clavata1‐4 leunig‐101 and clavata2‐1 leunig‐101 exhibited additive phenotypes of single mutants, suggesting that LEUNIG and CLAVATA genes function in different pathways. In contrast, FRUITFULL and PINOID genes interact with LEUNIG to regulate gynoecium development. genesis 26:42–54, 2000.

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana.

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.

Microarray Analysis of Gene Expression Involved in Anther Development in rice (Oryza sativa L.)

In flowering plants, anthers bear male gametophytes whose development is regulated by the elaborate coordination of many genes. In addition, both gibberellic acid (GA3) and jasmonic acid (JA) play important roles in anther development and pollen fertility. To facilitate the analysis of anther development genes and how GA3 and JA regulate anther development, we performed microarray experiments using a 10-K cDNA microarray with probes derived from seedlings, meiotic anthers, mature anthers and GA3- or JA-treated suspension cells of rice. The expression level change of 2155 genes was significantly (by 2-fold or greater) detected in anthers compared with seedlings. Forty-seven genes, representing genes with potential function in cell cycle and cell structure regulation, hormone response, photosynthesis, stress resistance and metabolism, were differentially expressed in meiotic and mature anthers. Moreover, 314 genes responded to either GA3 or JA treatment, and 24 GA3- and 82 JA-responsive genes showed significant changes in expression between meiosis and the mature anther stages. RT-PCR demonstrated that gene y656d05 was not only highly expressed in meiotic anthers but also induced by GA3. Strong RNA signals of y656d05 were detected in pollen mother cells and tapetum in in situ hybridization. Further characterization of these candidate genes can contribute to the understanding of the molecular mechanism of anther development and the involvement of JA and GA3 signals in the control of anther development in rice.

Novel Meiotic miRNAs and Indications for a Role of PhasiRNAs in Meiosis

Small RNAs (sRNA) add additional layers to the regulation of gene expression, with siRNAs directing gene silencing at the DNA level by RdDM (RNA-directed DNA methylation), and micro RNAs (miRNAs) directing post-transcriptional regulation of specific target genes, mostly by mRNA cleavage. We used manually isolated male meiocytes from maize (Zea mays) to investigate sRNA and DNA methylation landscapes during zygotene, an early stage of meiosis during which steps of meiotic recombination and synapsis of paired homologous chromosomes take place. We discovered two novel miRNAs from meiocytes, zma-MIR11969 and zma-MIR11970, and identified putative target genes. Furthermore, we detected abundant phasiRNAs of 21 and 24 nt length. PhasiRNAs are phased small RNAs which occur in 21 or 24 nt intervals, at a few hundred loci, specifically in male reproductive tissues in grasses. So far, the function of phasiRNAs remained elusive. Data from isolated meiocytes now revealed elevated DNA methylation at phasiRNA loci, especially in the CHH context, suggesting a role for phasiRNAs in cis DNA methylation. In addition, we consider a role of these phasiRNAs in chromatin remodeling/dynamics during meiosis. However, this is not well supported yet and will need more additional data. Here, we only lay out the idea due to other relevant literature and our additional observation of a peculiar GC content pattern at phasiRNA loci. Chromatin remodeling is also indicated by the discovery that histone genes were enriched for sRNA of 22 nt length. Taken together, we gained clues that lead us to hypothesize sRNA-driven DNA methylation and possibly chromatin remodeling during male meiosis in the monocot maize which is in line with and extends previous knowledge.

The transcriptome landscape of early maize meiosis

BackgroundA major step in the higher plant life cycle is the decision to leave the mitotic cell cycle and begin the progression through the meiotic cell cycle that leads to the formation of gametes. The molecular mechanisms that regulate this transition and early meiosis remain largely unknown. To gain insight into gene expression features during the initiation of meiotic recombination, we profiled early prophase I meiocytes from maize (Zea mays) using capillary collection to isolate meiocytes, followed by RNA-seq.ResultsWe detected ~2,000 genes as preferentially expressed during early meiotic prophase, most of them uncharacterized. Functional analysis uncovered the importance of several cellular processes in early meiosis. Processes significantly enriched in isolated meiocytes included proteolysis, protein targeting, chromatin modification and the regulation of redox homeostasis. The most significantly up-regulated processes in meiocytes were processes involved in carbohydrate metabolism. Consistent with this, many mitochondrial genes were up-regulated in meiocytes, including nuclear- and mitochondrial-encoded genes. The data were validated with real-time PCR and in situ hybridization and also used to generate a candidate maize homologue list of known meiotic genes from Arabidopsis.ConclusionsTaken together, we present a high-resolution analysis of the transcriptome landscape in early meiosis of an important crop plant, providing support for choosing genes for detailed characterization of recombination initiation and regulation of early meiosis. Our data also reveal an important connection between meiotic processes and altered/increased energy production.

Comparative Transcriptomics of Early Meiosis in Arabidopsis and Maize

Though sexually reproductive plants share the same principle and most processes in meiosis, there are distinct features detectable. To address the similarities and differences of early meiosis transcriptomes from the dicot model system Arabidopsis and monocot model system maize, we performed comparative analyses of RNA-seq data of isolated meiocytes, anthers and seedlings from both species separately and via orthologous genes. Overall gene expression showed similarities, such as an increased number of reads mapping to unannotated features, and differences, such as the amount of differentially expressed genes. We detected major similarities and differences in functional annotations of genes up-regulated in meiocytes, which point to conserved features as well as unique features. Transcriptional regulation seems to be quite similar in Arabidopsis and maize, and we could reveal known and novel transcription factors and cis-regulatory elements acting in early meiosis. Taken together, meiosis between Arabidopsis and maize is conserved in many ways, but displays key distinctions that lie in the patterns of gene expression.