Caiyun Yang

The ABORTED MICROSPORES Regulatory Network Is Required for Postmeiotic Male Reproductive Development in Arabidopsis thaliana

This study identifies targets and interacting factors of an Arabidopsis basic helix-loop-helix protein, ABORTED MICROSPORES (AMS), which is known to be required for pollen development. AMS is found to regulate the expression of several genes involved in metabolism and pollen wall deposition. The Arabidopsis thaliana ABORTED MICROSPORES (AMS) gene encodes a basic helix-loop-helix (bHLH) transcription factor that is required for tapetal cell development and postmeiotic microspore formation. However, the regulatory role of AMS in anther and pollen development has not been fully defined. Here, we show by microarray analysis that the expression of 549 anther-expressed genes was altered in ams buds and that these genes are associated with tapetal function and pollen wall formation. We demonstrate that AMS has the ability to bind in vitro to DNA containing a 6-bp consensus motif, CANNTG. Moreover, 13 genes involved in transportation of lipids, oligopeptides, and ions, fatty acid synthesis and metabolism, flavonol accumulation, substrate oxidation, methyl-modification, and pectin dynamics were identified as direct targets of AMS by chromatin immunoprecipitation. The functional importance of the AMS regulatory pathway was further demonstrated by analysis of an insertional mutant of one of these downstream AMS targets, an ABC transporter, White-Brown Complex homolog, which fails to undergo pollen development and is male sterile. Yeast two-hybrid screens and pull-down assays revealed that AMS has the ability to interact with two bHLH proteins (AtbHLH089 and AtbHLH091) and the ATA20 protein. These results provide insight into the regulatory role of the AMS network during anther development.

MALE STERILITY1 Is Required for Tapetal Development and Pollen Wall Biosynthesis

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene is critical for viable pollen formation and has homology to the PHD-finger class of transcription factors; however, its role in pollen development has not been fully defined. We show that MS1 transcription appears to be autoregulated by the wild-type MS1 transcript or protein. Using a functional green fluorescent protein (GFP) fusion to analyze the temporal and spatial expression of MS1, we demonstrate that the MS1:GFP protein is nuclear localized within the tapetum and is expressed in a developmentally regulated manner between late tetraspore and microspore release, then rapidly breaks down, probably by ubiquitin-dependent proteolysis. Absence of MS1 expression results in changes in tapetal secretion and exine structure. Microarray analysis has shown that 260 (228 downregulated and 32 upreglated) genes have altered expression in young ms1 buds. These genes are primarily associated with pollen wall and coat formation; however, a number of transcription factors and Cys proteases have also been identified as the putative primary regulatory targets of MS1. Ectopic expression of MS1 alters transcriptional regulation of vegetative gene expression, resulting in stunted plants with increased levels of branching, partially fertile flowers and an apparent increase in wall material on mature pollen. MS1 therefore plays a critical role in the induction of pollen wall and pollen coat materials in the tapetum and, ultimately, the production of viable pollen.

Arabidopsis MYB26/MALE STERILE35 Regulates Secondary Thickening in the Endothecium and Is Essential for Anther Dehiscence

The Arabidopsis thaliana MYB26/MALE STERILE35 (MS35) gene is critical for the development of secondary thickening in the anther endothecium and subsequent dehiscence. MYB26 is localized to the nucleus and regulates endothecial development and secondary thickening in a cell-specific manner in the anther. MYB26 expression is seen in anthers and also in the style and nectaries, although there is no effect on female fertility in the ms35 mutant. MYB26 expression in anthers occurs early during endothecial development, with maximal expression during pollen mitosis I and bicellular stages, indicating a regulatory role in specifying early endothecial cell development. Overexpression of MYB26 results in ectopic secondary thickening in both Arabidopsis and tobacco (Nicotiana tabacum) plants, predominantly within the epidermal tissues. MYB26 regulates a number of genes linked to secondary thickening, including IRREGULAR XYLEM1 (IRX1), IRX3, IRX8, and IRX12. Changes in expression were also detected in two NAC domain genes, NAC SECONDARY WALL–PROMOTING FACTOR1 (NST1) and NST2, which have been linked to secondary thickening in the anther endothecium. These data indicate that MYB26 regulates NST1 and NST2 expression and in turn controls the process of secondary thickening. Therefore, MYB26 appears to function in a regulatory role involved in determining endothecial cell development within the anther and acts upstream of the lignin biosynthesis pathway.

PERSISTENT TAPETAL CELL1 Encodes a PHD-Finger Protein That Is Required for Tapetal Cell Death and Pollen Development in Rice

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.

The final split: the regulation of anther dehiscence

Controlling male fertility is an important goal for plant reproduction and selective breeding. Hybrid vigour results in superior growth rates and increased yields of hybrids compared with inbred lines; however, hybrid generation is costly and time consuming. A better understanding of anther development and pollen release will provide effective mechanisms for the control of male fertility and for hybrid generation. Male sterility is associated not only with the lack of viable pollen, but also with the failure of pollen release. In such instances a failure of anther dehiscence has the advantage that viable pollen is produced, which can be used for subsequent rescue of fertility. Anther dehiscence is a multistage process involving localized cellular differentiation and degeneration, combined with changes to the structure and water status of the anther to facilitate complete opening and pollen release. After microspore release the anther endothecium undergoes expansion and deposition of ligno-cellulosic secondary thickening. The septum separating the two locules is then enzymatically lysed and undergoes a programmed cell death-like breakdown. The stomium subsequently splits as a consequence of the stresses associated with pollen swelling and anther dehydration. The physical constraints imposed by the thickening in the endothecium limit expansion, placing additional stress on the anther, so as it dehydrates it opens and the pollen is released. Jasmonic acid has been shown to be a critical signal for dehiscence, although other hormones, particularly auxin, are also involved. The key regulators and physical constraints of anther dehiscence are discussed.

A biomechanical model of anther opening reveals the roles of dehydration and secondary thickening.

Summary Understanding the processes that underlie pollen release is a prime target for controlling fertility to enable selective breeding and the efficient production of hybrid crops. Pollen release requires anther opening, which involves changes in the biomechanical properties of the anther wall. In this research, we develop and use a mathematical model to understand how these biomechanical processes lead to anther opening. Our mathematical model describing the biomechanics of anther opening incorporates the bilayer structure of the mature anther wall, which comprises the outer epidermal cell layer, whose turgor pressure is related to its hydration, and the endothecial layer, whose walls contain helical secondary thickening, which resists stretching and bending. The model describes how epidermal dehydration, in association with the thickened endothecial layer, creates forces within the anther wall causing it to bend outwards, resulting in anther opening and pollen release. The model demonstrates that epidermal dehydration can drive anther opening, and suggests why endothecial secondary thickening is essential for this process (explaining the phenotypes presented in the myb26 and nst1nst2 mutants). The research hypothesizes and demonstrates a biomechanical mechanism for anther opening, which appears to be conserved in many other biological situations where tissue movement occurs.

Biphasic regulation of the transcription factor ABORTED MICROSPORES (AMS) is essential for tapetum and pollen development in Arabidopsis

Summary Viable pollen is essential for plant reproduction and crop yield. Its production requires coordinated expression at specific stages during anther development, involving early meiosis‐associated events and late pollen wall formation. The ABORTED MICROSPORES (AMS) transcription factor is a master regulator of sporopollenin biosynthesis, secretion and pollen wall formation in Arabidopsis. Here we show that it has complex regulation and additional essential roles earlier in pollen formation. An inducible‐AMS reporter was created for functional rescue, protein expression pattern analysis, and to distinguish between direct and indirect targets. Mathematical modelling was used to create regulatory networks based on wild‐type RNA and protein expression. Dual activity of AMS was defined by biphasic protein expression in anther tapetal cells, with an initial peak around pollen meiosis and then later during pollen wall development. Direct AMS‐regulated targets exhibit temporal regulation, indicating that additional factors are associated with their regulation. We demonstrate that AMS biphasic expression is essential for pollen development, and defines distinct functional activities during early and late pollen development. Mathematical modelling suggests that AMS may competitively form a protein complex with other tapetum‐expressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation.

ECHIDNA Protein Impacts on Male Fertility in Arabidopsis by Mediating trans-Golgi Network Secretory Trafficking during Anther and Pollen Development

ECHIDNA-mediated trans-Golgi network trafficking is needed for full male fertility in Arabidopsis and is required for anther and pollen formation, filament elongation, and anther dehiscence in addition to pollen tube formation. The trans-Golgi network (TGN) plays a central role in cellular secretion and has been implicated in sorting cargo destined for the plasma membrane. Previously, the Arabidopsis (Arabidopsis thaliana) echidna (ech) mutant was shown to exhibit a dwarf phenotype due to impaired cell expansion. However, ech also has a previously uncharacterized phenotype of reduced male fertility. This semisterility is due to decreased anther size and reduced amounts of pollen but also to decreased pollen viability, impaired anther opening, and pollen tube growth. An ECH translational fusion (ECHPro:ECH-YELLOW FLUORESCENT PROTEIN) revealed developmentally regulated tissue-specific expression, with expression in the tapetum during early anther development and microspore release and subsequent expression in the pollen, pollen tube, and stylar tissues. Pollen viability and production, along with germination and pollen tube growth, were all impaired. The ech anther endothecium secondary wall thickening also appeared reduced and disorganized, resulting in incomplete anther opening. This did not appear to be due to anther secondary thickening regulatory genes but perhaps to altered secretion of wall materials through the TGN as a consequence of the absence of the ECH protein. ECH expression is critical for a variety of aspects of male reproduction, including the production of functional pollen grains, their effective release, germination, and tube formation. These stages of pollen development are fundamentally influenced by TGN trafficking of hormones and wall components. Overall, this suggests that the fertility defect is multifaceted, with the TGN trafficking playing a significant role in the process of both pollen formation and subsequent fertilization.

Transcription factor MYB26 is key to spatial specificity in anther secondary thickening formation

MYB26 regulates anther secondary thickening via NST1 and NST2 specifically in the endothecium through a series of regulatory controls. Successful fertilization relies on the production and effective release of viable pollen. Failure of anther opening (dehiscence), results in male sterility, although the pollen may be fully functional. MYB26 regulates the formation of secondary thickening in the anther endothecium, which is critical for anther dehiscence and fertility. Here, we show that although the MYB26 transcript shows expression in multiple floral organs, the MYB26 protein is localized specifically to the anther endothecium nuclei and that it directly regulates two NAC domain genes, NST1 and NST2, which are critical for the induction of secondary thickening biosynthesis genes. However, there is a complex relationship of regulation between these genes and MYB26. Using DEX-inducible MYB26 lines and overexpression in the various mutant backgrounds, we have shown that MYB26 up-regulates both NST1 and NST2 expression. Surprisingly normal thickening and fertility rescue does not occur in the absence of MYB26, even with constitutively induced NST1 and NST2, suggesting an additional essential role for MYB26 in this regulation. Combined overexpression of NST1 and NST2 in myb26 facilitates limited ectopic thickening in the anther epidermis, but not in the endothecium, and thus fails to rescue dehiscence. Therefore, by a series of regulatory controls through MYB26, NST1, NST2, secondary thickening is formed specifically within the endothecium; this specificity is essential for anther opening.