Arabidopsis Anther Development Research Articles

BRI1 EMS SUPPRESSOR1 genes regulate abiotic stress and anther development in wheat (Triticum aestivum L.).

BRI1 EMS SUPPRESSOR1 (BES1) family members are crucial downstream regulators that positively mediate brassinosteroid signaling, playing vital roles in the regulation of plant stress responses and anther development in Arabidopsis. Importantly, the expression profiles of wheat (Triticum aestivum L.) BES1 genes have not been analyzed comprehensively and systematically in response to abiotic stress or during anther development. In this study, we identified 23 BES1-like genes in common wheat, which were unevenly distributed on 17 out of 21 wheat chromosomes. Phylogenetic analysis clustered the BES1 genes into four major clades; moreover, TaBES1-3A2, TaBES1-3B2 and TaBES1-3D2 belonged to the same clade as Arabidopsis BES1/BZR1 HOMOLOG3 (BEH3) and BEH4, which participate in anther development. The expression levels of 23 wheat BES1 genes were assessed using real-time quantitative PCR under various abiotic stress conditions (drought, salt, heat, and cold), and we found that most TaBES1-like genes were downregulated under abiotic stress, particularly during drought stress. We therefore used drought-tolerant and drought-sensitive wheat cultivars to explore TaBES1 expression patterns under drought stress. TaBES1-3B2 and TaBES1-3D2 expression was high in drought-tolerant cultivars but substantially repressed in drought-sensitive cultivars, while TaBES1-6D presented an opposite pattern. Among genes preferentially expressed in anthers, TaBES1-3B2 and TaBES1-3D2 expression was substantially downregulated in thermosensitive genic male-sterile wheat lines compared to common wheat cultivar under sterile conditions, while we detected no obvious differences under fertile conditions. This result suggests that TaBES1-3B2 and TaBES1-3D2 might not only play roles in regulating drought tolerance, but also participate in low temperature-induced male sterility.

A single nucleotide polymorphism in an R2R3 MYB transcription factor gene triggers the male sterility in soybean ms6 (Ames1)

Key messageIdentification and functional analysis of the male sterile gene MS6 in Glycine max.Soybean (Glycine max (L.) Merr.) is an important crop providing vegetable oil and protein. The male sterility-based hybrid breeding is a promising method for improving soybean yield to meet the globally growing demand. In this research, we identified a soybean genic male sterile locus, MS6, by combining the bulked segregant analysis sequencing method and the map-based cloning technology. MS6, highly expressed in anther, encodes an R2R3 MYB transcription factor (GmTDF1-1) that is homologous to Tapetal Development and Function 1, a key factor for anther development in Arabidopsis and rice. In male sterile ms6 (Ames1), the mutant allele contains a missense mutation, leading to the 76th leucine substituted by histidine in the DNA binding domain of GmTDF1-1. The expression of soybean MS6 under the control of the AtTDF1 promoter could rescue the male sterility of attdf1 but ms6 could not. Additionally, ms6 overexpression in wild-type Arabidopsis did not affect anther development. These results evidence that GmTDF1-1 is a functional TDF1 homolog and L76H disrupts its function. Notably, GmTDF1-1 shows 92% sequence identity with another soybean protein termed as GmTDF1-2, whose active expression also restored the fertility of attdf1. However, GmTDF1-2 is constitutively expressed at a very low level in soybean, and therefore, not able to compensate for the MS6 deficiency. Analysis of the TDF1-involved anther development regulatory pathway showed that expressions of the genes downstream of TDF1 are significantly suppressed in ms6, unveiling that GmTDF1-1 is a core transcription factor regulating soybean anther development.

BZR1 Family Transcription Factors Function Redundantly and Indispensably in BR Signaling but Exhibit BRI1-Independent Function in Regulating Anther Development in Arabidopsis

BRASSINAZOLE-RESISTANT 1 family proteins (BZRs) are central transcription factors that govern brassinosteroid (BR)-regulated gene expression and plant growth. However, it is unclear whether there exists a BZR-independent pathway that mediates BR signaling. In this study, we found that disruption of all BZRs in Arabidopsis generated a hextuple mutant (bzr-h) displaying vegetative growth phenotypes that were almost identical to those of the null mutant of three BR receptors, bri1brl1brl3 (bri-t). By RNA sequencing, we found that global gene expression in bzr-h was unaffected by 2 h of BR treatment. The anthers of bzr-h plants were loculeless, but a similar phenotype was not observed in bri-t, suggesting that BZRs have a BR signaling-independent regulatory role in anther development. By real-time PCR and in situ hybridization, we found that the expression of SPOROCYTELESS (SPL), which encodes a transcription factor essential for anther locule development, was barely detectable in bzr-h, suggesting that BZRs regulate locule development by affecting SPL expression. Our findings reveal that BZRs are indispensable transcription factors required for both BR signaling and anther locule development, providing new insight into the molecular mechanisms underlying the microsporogenesis in Arabidopsis.

PRX9 and PRX40 Are Extensin Peroxidases Essential for Maintaining Tapetum and Microspore Cell Wall Integrity during Arabidopsis Anther Development.

Pollen and microspore development are essential steps in the life cycle of all land plants that generate male gametes. Within flowering plants, pollen development occurs inside of the anther. Here, we report the identification of two class III peroxidase-encoding genes, PEROXIDASE9 (PRX9) and PRX40, that are genetically redundant and essential for proper anther and pollen development in Arabidopsis (Arabidopsis thaliana). Arabidopsis double mutants devoid of functional PRX9 and PRX40 are male sterile. The mutant anthers display swollen, hypertrophic tapetal cells and pollen grains, suggesting disrupted cell wall integrity. These phenotypes lead to nearly 100%-penetrant pollen degeneration upon anther maturation. Using immunochemical and biochemical approaches, we show that PRX9 and PRX40 likely cross-link extensins to contribute to tapetal cell wall integrity during anther development. This work suggests that PRX9 and PRX40 encode Arabidopsis extensin peroxidases and highlights the importance of extensin cross-linking during pollen development.

CIK Receptor Kinases Determine Cell Fate Specification during Early Anther Development in Arabidopsis.

Appropriate cell division and differentiation ensure normal anther development in angiosperms. BARELY ANY MERISTEM 1/2 (BAM1/2) and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), two groups of leucine-rich repeat receptor-like protein kinases, are required for early anther cell specification. However, little is known about the molecular mechanisms underlying these two RLK-mediated signaling pathways. Here, we show that CLAVATA3 INSENSITIVE RECEPTOR KINASEs (CIKs), a group of novel coreceptor protein kinase-controlling stem cell homeostasis, play essential roles in BAM1/2- and RPK2-regulated early anther development in Arabidopsis thaliana The archesporial cells of cik1/2/3 triple and cik1/2/3/4 quadruple mutant anthers perform anticlinal division instead of periclinal division. Defective cell division and specification of the primary and inner secondary parietal cells occur in these mutant anthers. The disordered divisions and specifications of anther wall cells finally result in excess microsporocytes and a lack of one to three parietal cell layers in mutant anthers, resembling rpk2 or bam1/2 mutant anthers. Genetic and biochemical analyses indicate that CIKs function as coreceptors of BAM1/2 and RPK2 to regulate archesporial cell division and determine the specification of anther parietal cells.

Phosphorylation of SPOROCYTELESS/NOZZLE by the MPK3/6 Kinase Is Required for Anther Development.

Germ cells are indispensable carriers of genetic information from one generation to the next. In contrast to the well-understood process in animals, information on the mechanism of germ cell initiation in plants is very limited. SPOROCYTELESS/NOZZLE was previously identified as an essential regulator of diploid germ cell (archesporial cell) differentiation in the stamens and ovules of Arabidopsis (Arabidopsis thaliana). Although SPOROCYTELESS (SPL) transcription is activated by the floral organ identity regulator AGAMOUS and epigenetically regulated by SET DOMAIN GROUP2, little is known about the regulation of the SPL protein. Here, we report that the protein kinases MPK3 and MPK6 can both interact with SPL in vitro and in vivo and can phosphorylate the SPL protein in vitro. In addition, phosphorylation of the SPL protein by MPK3/6 is required for SPL function in the Arabidopsis anther, as measured by its effect on archesporial cell differentiation. We further demonstrate that phosphorylation enhances SPL protein stability. This work not only uncovers the importance of SPL phosphorylation for its regulatory role in Arabidopsis anther development, but also supports the hypothesis that the regulation of precise spatiotemporal patterning of germ cell initiation and that differentiation is achieved progressively through multiple levels of regulation, including transcriptional and posttranslational modification.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination.

Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

Abundant protein phosphorylation potentially regulates Arabidopsis anther development.

As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development.

SPL8 Acts Together with the Brassinosteroid-Signaling Component BIM1 in Controlling Arabidopsis thaliana Male Fertility.

The non-miR156 targeted SBP-box gene SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8 (SPL8), plays an important role in Arabidopsis anther development, where its loss-of-function results in a semi-sterile phenotype. Fully male-sterile plants are obtained when a spl8 loss-of-function mutation is introduced into a 35S:MIR156 genetic background, thereby revealing functional redundancy between SPL8 and miR156-targeted SBP-box genes. Here, we show that BIM1, a gene encoding a bHLH protein involved in brassinosteroid signaling and embryonic patterning, functions redundantly with SPL8 in its requirement for male fertility. Although bim1 single mutants displayed a mild fertility problem due to shortened filaments in some flowers, mutation of BIM1 significantly enhanced the semi-sterile phenotype of the spl8 mutant. Expression of both SPL8 and BIM1 was detected in overlapping expression domains during early anther developmental stages. Our data suggest that in regulating anther development, SPL8 and BIM1 function cooperatively in a common complex or in synergistic pathways. Phylogenetic analysis supports the idea of an evolutionary conserved function for both genes in angiosperm anther development.

SPL8 and miR156‐targeted SPL genes redundantly regulate Arabidopsis gynoecium differential patterning

SPL8 and miR156-targeted SPL genes are known to play an essential role in Arabidopsis anther development. Here we show that these SPL genes are also expressed within the developing gynoecium, where they redundantly control development of the female reproductive tract. Whereas the gynoecium morphology in the spl8 single mutant is largely normal, additional down-regulation of miR156-targeted SPL genes results in a shortened style and an apically swollen ovary narrowing onto an elongated gynophore. In particular, the septum does not form properly and lacks a transmitting tract. Loss of SPL8 function enhances the mutant phenotypes of ett, crc and spt, indicating a functional overlap between SPL8 and these genes in regulating gynoecium development. Furthermore, gynoecium development of 35S:MIR156b spl8-1 double mutants shows enhanced sensitivity to a polar auxin transport inhibitor, and the expression pattern of the auxin biosynthesis gene YUCCA4 is altered compared to wild-type. Our observations imply that SPL8 and miR156-targeted SPL genes control gynoecium patterning through interference with auxin homeostasis and signalling.

Regulation of the Arabidopsis anther transcriptome by DYT1 for pollen development

Several genes encoding transcription factors have been shown to be essential for male fertility in plants, suggesting that transcriptional regulation is a major mechanism controlling anther development in Arabidopsis. DYSFUNCTIONAL TAPETUM 1 (DYT1), a putative bHLH transcription factor, plays a critical role in regulating tapetum function and pollen development. Here, we compare the transcriptomes of young anthers of wild-type and the dyt1 mutant, demonstrating that DYT1 is upstream of at least 22 genes encoding transcription factors and regulates the expression of a large number of genes, including genes involved in specific metabolic pathways. We also show that DYT1 can bind to DNA in a sequence-specific manner in vitro, and induction of DYT1 activity in vivo activated the expression of the downstream transcription factor genes MYB35 and MS1. We generated DYT1-SRDX transgenic plants whose fertility was dramatically reduced, implying that DYT1 probably acts as a transcriptional activator. Furthermore, we used yeast two-hybrid assays to show that DYT1 forms homodimers and heterodimers with other bHLH transcription factors. Our results demonstrate the important role of DYT1 in regulating anther transcriptome and function, and supporting normal pollen development.

Flowering Research in Taiwan

Flowering is an important and highly regulated process. Taiwanese researchers have contributed significantly to understanding flowering biology, including cell biology, cytogenetics and functional genomics, in rice, lily and especially orchids. In recognition of this effort, the PCP Editorial Board is publishing a special issue on flowering research in Taiwan. This issue contains seven articles on cutting-edge topics or reviews of flowering research carried out in Taiwan, focusing on research into orchids and lily. An anther consists of sporophytic tissues of three outer cell layers and an innermost layer, the tapetum. The tapetum has attracted the most attention. Tapetum cells are important in nurturing the maturing microspores, and their ablation leads to microspore death and thus male sterility (Hesse et al. 1993). In a review paper, M.-D. Huang et al. (pp. 1459–1466) provide information about current studies on the transcriptome of the anther sporophyte, including microarray and next-generation sequencing data. The authors illustrate a proposed gene regulation network of anther development in Arabidopsis using the most updated references. In addition, they point out potential studies worthwhile pursuing, including those of ‘secretomes’, antisense transcripts and small RNAs. Finally, they propose a model for exine precursor biosynthesis in tapetum cells and transport of these precursors from the tapetum cell to the microspore surface. With an estimated >25,000 species, orchids are the most species-rich of all angiosperm families (Leitch et al. 2009). They show a wide diversity of epiphytic and terrestrial growth forms and have successfully colonized almost every habitat on earth. The expansion of the orchid family probably took place in a comparatively short period as compared with that of most flowering plant families, which suggests exceptionally high speciation rates (Gill 1989). The production of orchids in the world has increased 12–18% every year since 2000. Taiwan is one of the world-renowned countries exporting orchids, because the climate is congenial to the growth of orchids and because of the rich resource of native species and advances in breeding techniques to produce more beautiful and elegant new varieties. Taiwan’s orchid industry has been recognized by the international community as prosperous in terms of sales volume, orchid plantation, orchid-growing technologies and financial resources. The most popular potted flowering plants are Phalaenopsis hybrids, and Oncidium are the top-traded cut flowers. Most, if not all, orchid flowers on the market are hybrids, such as Phalaenopsis spp., although most researchers use inbred lines such as Phalaenopsis equestris as materials for detailed studies. Fig. 1 illustrates several examples of orchid varieties, their mutants, disease and insect infection, as well as orchid production. In the second review paper of this issue, Hsiao et al. (pp. 1467–1486) provide an overview of the research activities in orchid biology and biotechnology, including the status of genomics, transformation technology, flowering regulation, molecular regulatory mechanisms of floral development, scent production and color presentation. The authors also point out the pros and cons of using orchids for genome research. Oncidium is a valuable floral genus of orchid. The yellow color is most often seen in the flower market. The largest flower structure is the labellum, also known as the lip (fig. 1E, p. 1533). Chang et al. (pp. 1532–1545) used 454 sequencing to perform deep sequencing of the Oncidium transcriptome using six organs. A value-added expressed sequence tag (EST) database, OncidiumOrchidGenomeBase (http://predictor.nchu. edu.tw/oogb/), was constructed and provides a foundation for molecular genetics and functional genomics for modifying the flowering quality and desirable agronomic traits of Oncidium. The authors also identified flowering time-associated genes, including photoreceptors and floral pathway integrators. Su et al. (pp. 1501–1514) performed transcriptome analysis of mixed vegetative and reproductive tissues of the moth orchid Phalaenopsis aphrodite and constructed the Orchidstra genome database (http://orchidstra.abrc.sinica.edu.tw) for searching and linking to corresponding Pfam, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Fu et al. (2011) prepared an EST database from 11 tissues collected from three orchid species, i.e. P. equestris, P. aphrodite and P. bellina, using both traditional and next-generation sequencing. This OrchidBase is freely available at http://lab.fhes.tn.edu.tw/est. Altogether, these three transcriptome databases consist of about 100,000 contigs from four orchid species and thus provide tremendous resources for orchid functional genomics studies as well as breeding application. Although the floral ABCDE model is generally thought to be ubiquitous in the core eudicots, studies of basal angiosperms,

AtMYB103 is a crucial regulator of several pathways affecting Arabidopsis anther development

Previous reports indicated that AtMYB103 has an important role in tapetum development, callose dissolution, and exine formation in A. thaliana anthers. Here, we further characterized its function in anther development by expression pattern analysis, transmission electron microscopy observation of the knockout mutant, and microarray analysis of downstream genes. A total of 818 genes differentially expressed between ms188 and the wild-type were identified by global expression profiling analysis. Functional classification showed that loss-of-function of AtMYB103 impairs cell wall modification, lipid metabolic pathways, and signal transduction throughout anther development. RNA in situ hybridization confirmed that transcription factors acting downstream of AtMYB103 (At1g06280 and At1g02040) were expressed in the tapetum and microspores at later stages, suggesting that they might have important roles in microsporogenesis. These results indicated that AtMYB103 is a crucial regulator of Arabidopsis anther development.

The ASH1 HOMOLOG 2 (ASHH2) Histone H3 Methyltransferase Is Required for Ovule and Anther Development in Arabidopsis

BackgroundSET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction.Methodology/Principal FindingsA slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to ∼90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2.Conclusions/SignificanceThe severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.

Prediction of anther-expressed gene regulation in Arabidopsis

Anther development in Arabidopsis, a popular model plant for plant biology and genetics, is controlled by a complex gene network. Despite the extensive use of this genus for genetic research, little is known about its regulatory network. In this paper, the direct transcriptional regulatory relationships between genes expressed in Arabidopsis anther development were predicted with an integrated bioinformatic method that combines mining of microarray data with promoter analysis. A total of 7710 transcription factor-gene pairs were obtained. The 80 direct regulatory relationships demonstrating the highest confidence were screened from the initial 7710 pairs; three of the 80 were validated by previous experiments. The results indicate that our predicted results were reliable. The regulatory relationships revealed by this research and described in this paper may facilitate further investigation of the molecular mechanisms of anther development. The bioinformatic method used in this work can also be applied to the prediction of gene regulatory relationships in other organisms.

Receptor‐like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.