Auxin regulates Arabidopsis anther dehiscence, pollen maturation, and filament elongation

We provide evidence on the localization, synthesis, transport, and effects of auxin on the processes occurring late in Arabidopsis thaliana stamen development: anther dehiscence, pollen maturation, and preanthesis filament elongation. Expression of auxin-sensitive reporter constructs suggests that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 suggest that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. We found that in tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes. Our results suggest that auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, while auxin transport contributes to the independent regulation of preanthesis filament elongation.     

The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development

In flowering plants, tapetum degeneration is proposed to be triggered by a programmed cell death (PCD) process during late stages of pollen development; the PCD is thought to provide cellular contents supporting pollen wall formation and to allow the subsequent pollen release. However, the molecular basis regulating tapetum PCD in plants remains poorly understood. We report the isolation and characterization of a rice (Oryza sativa) male sterile mutant tapetum degeneration retardation (tdr), which exhibits degeneration retardation of the tapetum and middle layer as well as collapse of microspores. The TDR gene is preferentially expressed in the tapetum and encodes a putative basic helix-loop-helix protein, which is likely localized to the nucleus. More importantly, two genes, Os CP1 and Os c6, encoding a Cys protease and a protease inhibitor, respectively, were shown to be the likely direct targets of TDR through chromatin immunoprecipitation analyses and the electrophoretic mobility shift assay. These results indicate that TDR is a key component of the molecular network regulating rice tapetum development and degeneration.     

Programmed cell death progressively models the development of anther sporophytic tissues from the tapetum and is triggered in pollen grains during maturation

To characterize the spatial and temporal occurrence of programmed cell death (PCD) in Lilium anther tissues, we used both microscopical and molecular markers of apoptosis for developmental stages from meiosis to pollen release. The first hallmarks of PCD include cell condensation and shrinkage of the cytoplasm, separation of chromatin into delineated masses, and DNA fragmentation in the tapetum as early as the premeiosis stage. PCD then extended to other anther sporophytic tissues, leading to anther dehiscence. Although the PCD clearly affected the endothecium and the epidermis, these two cell layers remained alive until anther dehiscence. In pollen, no sign of PCD was found until pollen mitosis I, after what apoptotic features developed progressively in the vegetative cell. In addition, DNA ladders were detected in all sporophytic tissues and cell types throughout pollen development, whereas in the male gametophyte DNA ladders were only detected during pollen maturation. Our data suggest that PCD is a progressive and active process affecting all the anther tissues, first being triggered in the tapetum.     

New views of tapetum ultrastructure and pollen exine development in Arabidopsis thaliana

Background and Aims

The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy.

Methods

In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat.

Key Results

Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively.

Conclusions

This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed.     

The loculus content and tapetum during pollen development in Lilium

 The ratio of loculus volume to the volume of the entire anther began to increase from the microspore mother cell stage and reached 32.3% at anthesis. The content of the loculus was examined in Lilium during pollen development and two waves could be distinguished. From the premeiotic stage until the vacuolated microspore stage, the loculus consisted of neutral polysaccharides, pectins and proteins. These substances originated from tapetal activity from the premeiotic stage until the young microspore stage. Dictyosomes and rough endoplasmic reticulum seemed to be involved in tapetal secretion, although, in some mitochondria, vesicles progressively developed as early as premeiosis and increased until the young microspore stage, which could reveal their involvement in the secretion process. At this stage, numerous cytoplasmic vesticles containing material similar to the locular material fused with the plasma membrane of the tapetum so that vesicle content was in contact with the loculus. It seems that tapetal and callose wall degradation at the late tetrad stage may also have contributed to the production of material in the loculus. From pollen mitosis to anthesis, the anther loculus contained mainly the pollenkitt which was synthesized in the tapetum between the young microspore stage and the vacuolated microspore stage. At the young microspore stage, proplastids divided and developed into elaioplasts and smooth endoplasmic reticulum (SER) increased dramatically. Pollenkitt had a double origin: some droplets were extruded directly from the plastid stroma through the plastid envelopes; the others were unsaturated lipid globules, which presumably derived from the interaction between SER saccules and plastids. Received: 2 September 1997 / Revision accepted: 12 March 1998     

WUSCHEL regulates cell differentiation during anther development

During anther development a series of cell specification events establishes the male gametophyte and the surrounding sporophytic structure. Here we show that the homeobox gene WUSCHEL, originally identified as a central regulator of stem cell maintenance, plays an important role in cell type specification during male organogenesis. WUS expression is initiated very early during anther development in the precursor cells of the stomium and terminates just before the stomium cells enter terminal differentiation. At this stage the stomium cells and the neighboring septum cells that separate the pollen sacs undergo typical cell wall thickening and degenerate which leads to rupture of the anther and pollen release. In wus mutants, neither stomium cells nor septum cells differentiate or undergo cell death and degenerate. As a consequence, the anther stays intact and pollen is not released. CLAVATA3 which is activated by WUS in stem cell maintenance, is not activated in anthers indicating a novel pathway regulated by WUS. Comparing WUS function in stem cell maintenance and sexual organ development suggests that WUS expressing cells represent a conserved signaling module that regulates behavior and communication of undifferentiated cells.     

Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development

F-box proteins, components of the Skp1-Cullin1-F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins interact with Skp1 through the F-box motif and with ubiquitination substrates through C-terminal protein interaction domains. F-box proteins regulate plant development, various hormonal signal transduction processes, circadian rhythm, and cell cycle control. We isolated an F-box protein gene from wheat spikes at the onset of flowering. The Triticum aestivum cyclin F-box domain (TaCFBD) gene showed elevated expression levels during early inflorescence development and under cold stress treatment. TaCFBD green fluorescent protein signals were localized in the cytoplasm and plasma membrane. We used yeast two-hybrid screening to identify proteins that potentially interact with TaCFBD. Fructose bisphosphate aldolase, aspartic protease, VHS, glycine-rich RNA-binding protein, and the 26S proteasome non-ATPase regulatory subunit were positive candidate proteins. The bimolecular fluorescence complementation assay revealed the interaction of TaCFBD with partner proteins in the plasma membranes of tobacco cells. Our results suggest that the TaCFBD protein acts as an adaptor between target substrates and the SCF complex and provides substrate specificity to the SCF of ubiquitin ligase complexes.     

An investigation of the role of the anther tapetum during microspore development using genetic cell ablation

The effects on anther development of a fusion of the Arabidopsis anther-specific apg gene promoter to a ribonuclease (barnase) in transgenic tobacco plants were examined. Contrary to expectations, viable pollen grains were produced by these plants despite the demonstration that ribonuclease expression in the microspores and tapetum caused targeted cell ablation. Transformed plants were reduced in male fertility due to ablation of a proportion of pollen dependent on apg-barnase locus number. Plants were otherwise phenotypically normal and fully female fertile, confirming the anther-specific nature of the apg promoter. In microspores inheriting an apg-barnase locus following meiosis, loss of cell viability, as judged by fluorescein diacetate staining, occurred during mid to late microspore development. Microspores not inheriting a transgene went on to mature into viable pollen grains. Premature degeneration of the tapetum was also observed as a result of apg-barnase expression, but this did not appear to disrupt the subsequent microspore and pollen developmental programmes. This was substantiated by observations of microspore development in plants in which the tapetum was rescued from ablation by crossing in a second transgene encoding a tapetum-specific inhibitor of the ribonuclease. It was determined that tapetum cell disruption occurs at the early to mid uninucleate microspore stage in apg-barnase transformants. The data presented show that after this point in microspore development the tapetum is no longer essential for the production of viable pollen in tobacco.     

A novel F-box protein represses endothecial secondary wall thickening for anther dehiscence in Arabidopsis thaliana

In plants, the regulation of protein turnover by the ubiquitin proteasome system (UPS) is a key posttranslational mechanism underlying diverse cellular processes. However, the participation of the UPS in cellular processes involved in anther dehiscence, especially endothecial secondary wall (ESW) thickening, has not been characterized. Here, we report that a novel F-box protein in arabidopsis, designated SAF1 (Secondary wall thickening-Associated F-box 1), negatively regulates ESW thickening in the anther. SAF1 is predominantly expressed in flower tissues and interacts with Arabidopsis-Skp1-like 19 (ASK19). SAF1-overexpressed (Ox) lines showed reduced fertility due to a lack or loss of ESW thickening in the anther and inhibition of the expression of relevant genes, such as IRREGURAR XYLEMs (IRXs) in flowers. These findings suggest that the novel Skp/Cul/F-box (SCF) complex consisting of SAF1 as an F-box protein and ASK19 as a Skp functions in secondary wall thickening of the anther endothecium.     

ORE9, an F-box protein that regulates leaf senescence in Arabidopsis

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.     

Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation

Among the >200 members of the leucine-rich repeat receptor kinase family in Arabidopsis thaliana, only a few have been functionally characterized. Here, we report a critical function in anther development for the SOMATIC EMBRYOGENESIS RECEPTOR KINASE1 (SERK1) and SERK2 genes. Both SERK1 and SERK2 are expressed widely in locules until stage 6 anthers and are more concentrated in the tapetal cell layer later. Whereas serk1 and serk2 single insertion mutants did not show developmental phenotypes, serk1 serk2 double mutants were not able to produce seeds because of a lack of pollen development in mutant anthers. In young buds, double mutant anthers developed normally, but serk1 serk2 microsporangia produced more sporogenous cells that were unable to develop beyond meiosis. Furthermore, serk1 serk2 double mutants developed only three cell layers surrounding the sporogenous cell mass, whereas wild-type anthers developed four cell layers. Further confocal microscopic and molecular analyses showed that serk1 serk2 double mutant anthers lack development of the tapetal cell layer, which accounts for the microspore abortion and male sterility. Taken together, these findings demonstrate that the SERK1 and SERK2 receptor kinases function redundantly as an important control point for sporophytic development controlling male gametophyte production.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S2, encoded by the S2 allele, with the expected features of the Sp gene was identified. AhSLF-S2 is located 9 kb downstream of S2 RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S2 are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S2 is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.