DCET1 Controls Male Sterility Through Callose Regulation, Exine Formation, and Tapetal Programmed Cell Death in Rice

In angiosperms, anther development comprises of various complex and interrelated biological processes, critically needed for pollen viability. The transitory callose layer serves to separate the meiocytes. It helps in primexine formation, while the timely degradation of tapetal cells is essential for the timely callose wall dissolution and pollen wall formation by providing nutrients for pollen growth. In rice, many genes have been reported and functionally characterized that are involved in callose regulation and pollen wall patterning, including timely programmed cell death (PCD) of the tapetum, but the mechanism of pollen development largely remains ambiguous. We identified and functionally characterized a rice mutant dcet1, having a complete male-sterile phenotype caused by defects in anther callose wall, exine patterning, and tapetal PCD. DCET1 belongs to the RNA recognition motif (RRM)-containing family also called as the ribonucleoprotein (RNP) domain or RNA-binding domain (RBD) protein, having single-nucleotide polymorphism (SNP) substitution from G (threonine-192) to A (isoleucine-192) located at the fifth exon of LOC_Os08g02330, was responsible for the male sterile phenotype in mutant dcet1. Our cytological analysis suggested that DCET1 regulates callose biosynthesis and degradation, pollen exine formation by affecting exine wall patterning, including abnormal nexine, collapsed bacula, and irregular tectum, and timely PCD by delaying the tapetal cell degeneration. As a result, the microspore of dcet1 was swollen and abnormally bursted and even collapsed within the anther locule characterizing complete male sterility. GUS and qRT-PCR analysis indicated that DCET1 is specifically expressed in the anther till the developmental stage 9, consistent with the observed phenotype. The characterization of DCET1 in callose regulation, pollen wall patterning, and tapetal cell PCD strengthens our knowledge for knowing the regulatory pathways involved in rice male reproductive development and has future prospects in hybrid rice breeding.

βVPE is involved in tapetal degradation and pollen development by activating proprotease maturation in Arabidopsis thaliana

Vacuolar processing enzyme (VPE) is responsible for the maturation and activation of vacuolar proteins in plants. We found that βVPE was involved in tapetal degradation and pollen development by transforming proproteases into mature protease in Arabidopsis thaliana. βVPE was expressed specifically in the tapetum from stages 5 to 8 of anther development. The βVPE protein first appeared as a proenzyme and was transformed into the mature enzyme before stages 7–8. The recombinant βVPE protein self-cleaved and transformed into a 27 kDa mature protein at pH 5.2. The mature βVPE protein could induce the maturation of CEP1 in vitro. βvpe mutants exhibited delayed vacuolar degradation and decreased pollen fertility. The maturation of CEP1, RD19A, and RD19C was seriously inhibited in βvpe mutants. Our results indicate that βVPE is a crucial processing enzyme that directly participates in the maturation of cysteine proteases before vacuolar degradation, and is indirectly involved in pollen development and tapetal cell degradation.

βVPE is involved in tapetal degradation and pollen development by activating proprotease maturation in Arabidopsis thaliana

Vacuolar processing enzyme (VPE) is responsible for the maturation and activation of vacuolar proteins in plants. We found that βVPE was involved in tapetal degradation and pollen development by transforming proproteases into mature protease in Arabidopsis thaliana. βVPE was expressed specifically in the tapetum from stages 5–8 of anther development. The βVPE protein first appeared as a proenzyme and transformed into the mature enzyme before stages 7–8. The recombinant βVPE protein self-cleaved and transformed to a 27-kD mature protein at pH 5.2. The mature βVPE protein could induce the maturation of CEP1 in vitro. βvpe mutants exhibited delayed vacuolar degradation and decreased pollen fertility. The maturation of CEP1, RD19A, and RD19C were seriously inhibited in βvpe mutants. Our results indicate that βVPE is a crucial processing enzyme that directly participates in the maturation of cysteine proteases before vacuolar degradation, and is indirectly involved in pollen development and tapetal cell degradation.

Comparative anther and pollen tetrad development in functionally monoecious Pseuduvaria trimera (Annonaceae), and evolutionary implications for anther indehiscence

The multiple evolutionary origins and diverse morphologies of unisexual flowers in angiosperms indicate that many different developmental mechanisms [sporophytic and (or) gametophytic tissues] underlie patterns of sex differentiation, yet, these mechanisms leading to unisexuality remain largely unresolved. In Pseuduvaria trimera (W.G. Craib) Y.C.F. Su & R.M.K. Saunders, morphologically hermaphroditic flowers are functionally female due to indehiscent anthers, but the developmental and anatomical mechanisms preventing their dehiscence are still unknown. Anther and pollen development were compared in both male and functionally female flowers using histological observations to test whether anther indehiscence results from a sporophytic and (or) gametophytic default. The epidermis, endothecium, middle layers, and pollen development were identical in the two floral morphs, but variations occurred in the tapetum and stomium regions. In male flowers, concurrently with the binucleate tapetal cell degeneration, the appearance of intercellular spaces and lysis of the stomium region cells lead to anther dehiscence. Conversely, in the functionally female flowers, trinucleate tapetum appears with delayed degradation, and the persistent cells with a highly vacuolated cytoplasm and stomium region remain intact at maturity. Sporophytic tissues with tapetum abnormalities and stomium integrity are, thus, responsible for anther indehiscence. Lack of microspore rotation in P. trimera might indicate a different evolutionary origin of pollen tetrad formation in this family.

PRX9 and PRX40 are extensin peroxidases essential for maintaining tapetum and microspore cell wall integrity during Arabidopsis anther development

Pollen and microspore development is an essential step 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, PRX9 and PRX40, that are genetically redundant and essential for proper anther and pollen development in 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 ultimately lead to nearly 100%-penetrant pollen degeneration upon anther maturation. Using immunochemical and biochemical approaches, we show that PRX9 and PRX40 are likely extensin peroxidases that contribute to the establishment of tapetal cell wall integrity during anther development. This work identifies PRX9 and PRX40 as the first extensin peroxidases to be described in Arabidopsis and highlights the importance of extensin cross-linking during plant development.

Escherichia colimazEF Toxin-Antitoxin System as a Tool to Target Cell Ablation in Plants

<b><i>Background/Aims:</i></b> The <i>Escherichia coli</i> MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the <i>mazEF</i> toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation. <b><i>Methods:</i></b> A double transformation strategy, combining expression of the <i>mazE</i> antitoxin gene under the control of the <i>CaMV 35S</i> promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the <i>mazF </i>gene under the control of the <i>TA29</i> tapetum-specific promoter in transgenic tobacco, was applied. <b><i>Results:</i></b> No transgenic <i>TA29-mazF</i> line could be regenerated, suggesting that the <i>TA29</i> promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated<i> 35S-mazE</i>/<i>TA29-mazF</i> double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen. <b><i>Conclusion:</i></b> These results show that the <i>E. coli</i><i>mazEF</i> system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation.

The function of the tapetal tissue during microsporogenesis in Lilium

The main functional activity -of the tapetum in the <em>Lilium</em> anther is the synthesis of reserve lipids and carotenoid pigments. The fusion of these substances during tapetum desintegration results in the formation of pollenkitt Pollernkitt participates in the formation both of the exine and of sporopollenin-containing structures of the tapetal cell (orbicules, tapetal and peritapetal membranes) during the last steps of anther development.