Male-sterility of thermosensitive genic male-sterile rice is associated with premature programmed cell death of the tapetum

The tapetum plays a crucial role in pollen development. This secretory tissue produces numerous nutritive proteins necessary for pollen maturation. The tapetum, whose cells undergo programmed cell death (PCD), is completely diminished by the time the pollen is fully mature. Our previous studies on a thermosensitive genic male-sterile (TGMS) rice (Oryza sativa L.) suggested that male-sterility was due to failure in pollen development. In this paper we describe how further analysis of the TGMS rice revealed that male-sterility is associated with premature PCD of the tapetum. Cytological observations of TGMS rice anthers at various developmental stages indicated that PCD initiates at an early stage of pollen development and continues until the tapetal cells are completely degraded, resulting in pollen collapse. Transmission electron microscopy showed the morphologically distinct hallmarks of apoptosis, including cytoplasmic shrinkage, membrane blebbing, and vacuolation. Identification of DNA fragmentation using the TUNEL assay supports the hypothesis that premature PCD is associated with male-sterility in the rice. The tissue-specific feature of the thermosensitive genic male-sterile phenotype is discussed with regard to PCD during anther development.     

Abolition of the tapetum suicide program ruins microsporogenesis

Microsporogenesis in angiosperms takes places within the anther. Microspores are surrounded by a layer of cells, the tapetum, which degenerates during the later stages of pollen development with cytological features characteristic of programmed cell death (PCD). We report herein that the expression of AtBI-1, which suppresses Bax-induced cell death, in the tapetum at the tetrad stage inhibits tapetum degeneration and subsequently results in pollen abortion, while activation of AtBI-1 at the later stage does not. Our results demonstrate that the PCD signal commences at the tetrad stage and that the proper timing of PCD in the tapetum is essential for normal microsporogenesis.     

A VPE-like protease NtTPE8 exclusively expresses in the integumentary tapetum and is involved in seed development

Programmed cell death(PCD) is an essential process for development, and shows conserved cytological features in both plants and animals. Caspases are well-known critical components of the PCD machinery in animals. However, currently few typical counterparts have been identified in plants and only several caspase-like proteases are known to be involved in plant PCD, indicating the existence of great challenge for confirming new caspase-like proteases and elucidating the mechanisms regulating plant PCD. Here, we report a novel cysteine protease, NtTPE8, which was extracted from tobacco seeds and confirmed as a new caspase-like protease.Recombinant NtTPE8 exhibited legumain and caspase-like proteolytic activities, both of which could be inhibited by the pan-caspase inhibitor(Z-VAD-FMK). Notably, NtTPE8 possessed several caspase activities and the capacity to cleave the cathepsin H substrate FVR, indicating a unique character of NtTPE8. NtTPE8 was exclusively expressed in the integumentary tapetum and thus, is the first specific molecular marker reported to date for this cell type. Downregulation of NtTPE8 caused seed abortion, via disturbing early embryogenesis, indicating its critical role in embryogenesis and seed development. In conclusion, we identified a novel caspase-like cysteine protease, NtTPE8,exclusively expressed in the integumentary tapetum that is involved in seed development.     

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1(OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator(GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of Os GPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. Os GPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that At GPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of Os GPT1 in the tapetum degeneration and pollen development. The divergent functions of Os GPT1 and At GPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.     

Premature Tapetum Degeneration: a Major Cause of Abortive Pollen Development in Photoperiod Sensitive Genic Male Sterility in Rice

Photoperiod-sensitive genic male-sterile (PSGMS) rice ( Oryza sativa L.), a natural mutant found in the rice cultivar Nongken 58, is very useful for the development of hybrid rice cultivars. Despite its widespread use in breeding programs, the initial stage of the abortive development of PSGMS rice and the possible cytological mechanisms of pollen abortion have not been determined. In the present study, a systematic cytological comparison of the anther development of PSGMS rice with its normal fertile counterpart is conducted. The results show that pollen abortion in PSGMS rice first occurs before the pollen mother cell (PMC) stage, and continues during the entire process of pollen development until pollen degradation. The abortive process was closely associated with the abnormal behavior of the tapetum. Although tapetum degeneration in PSGMS rice initiates already at the PMC stage, it proceeds slowly and does not complete until the breakdown of the pollen. Such cytological observations were supported by the results of the TUNEL (TdT-mediated dUTP Nick End Labeling) assay, which detects DNA fragmentation resulting from programmed cell death (PCD), indicating that the premature tapetum degeneration is in the process of PCD.     

An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development

The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.     

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.     

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.     

Positional cloning of the rice male sterility gene ms-IR36, widely used in the inter-crossing phase of recurrent selection schemes

The monogenetic recessive male-sterile gene ms-IR36 is widely used to facilitate the inter-crossing phase of recurrent selection in rice (Oryza sativa), but its segregation within the progeny disturbs other breeding phases. Marker-assisted early identification of msms and Msms seedlings would help overcome this drawback. Using successively bulked segregant analysis and large F2 populations, we mapped the ms-IR36 gene to a 33-kb region on the short arm of chromosome 2 that includes 10 candidate genes. Sequencing of these candidates together with checking rice genome annotations and expression databases allowed the target to be narrowed down to one candidate gene already isolated and characterized as the tapetum degeneration retardation (TDR) gene and reported to be involved in tapetal programmed cell death. Comparison of the sequence of the TDR gene between male-sterile (MS) and male-fertile (MF) IR36 plants detected one non-synonymous nucleotide substitution affecting the active domain of the encoded protein. Perfect co-segregation was observed between polymorphism at this nucleotide (SNP) and the MS/MF phenotype of 946 F2 plants. Spatial modelling of the active domain of the candidate protein reinforced the candidate status of the only SNP identified. Histological characterization of anther development in MS IR36 revealed defects identical to the ones observed in mutants used for the isolation and characterization of the TDR gene: delayed/non-degradation of tapetum tissue and collapse of the haploid microspores. We concluded that ms-IR36 corresponded to the TDR gene with a different mutation from the earlier one described in the same gene. No significant linkage drag was associated with ms-IR36. A SNP-based marker that enables simple early identification of MS plants and MF plants with the Msms genotype was designed.     

Ultrastructural aspects and programmed cell death in the tapetal cells of Lathyrus undulatus Boiss

Programmed cell death (PCD) in the tapetum of Lathyrus undulatus L. was analyzed based on light, fluorescence and electron microscopy to characterize its spatial and temporal occurrence. Development and processes of PCD in secretory tapetal cells of Lathyrus undulatus L. were correlated with the sporogenous cells and pollen grains. At early stages of development the tapetal cells appeared similar to pollen mother cells, structurally. Concurrent with meiosis, tapetum expanded both tangentially and radially as vacuoles increased in size. Tapetal cells most fully developed at young microspore stage. However, tapetum underwent substantial changes in cell organization including nucleus morphology monitored by DAPI. The TUNEL staining confirmed the occurrence of intra-nucleosomal DNA cleavage. In addition to nuclear degeneration which is the first hallmark of PCD other diagnostic features were observed at vacuolated microspore stage intensely; such as chromatin condensation at the periphery of the nucleus, nuclear membrane degeneration, chromatin release to the cytoplasm, vacuole collapse according to tonoplast rupture, shrinkage of the cytoplasm, the increase and enlargement of the endoplasmic reticulum cisternae and disruption of the plasma membrane. After vacuole collapse due to possible release of hydrolytic enzymes the cell components degraded. Tapetal cells completely degenerated at bicellular pollen stage.