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.

BcMF27 , a Pectin Methylesterase Gene, Regulates Pollen Development And Pollen Tube Growth in Brassica Campestris

Functional pollen grains are an essential ingredient of successful reproduction in flowering plants and are protected by outer walls. Pectin methylesterases (PMEs) modify pectin, a structural component of pollen intine. However, there are few studies on PMEs. Artificial microRNA (amiRNA) and overexpression technology was performed to investigate the function of pollen-specific PME gene, BcMF27, in pollen development. Knockdown of BcMF27 led to pollen wall collapse, 20% of which unknown material adhered to. Wall-collapsed pollen had abnormally thick intine outside of the germinal furrows. A portion of the cytoplasm was degraded in the remaining pollen with unknown material on the wall, in addition to a thick intine. Overexpression of BcMF27 resulted in 66.67% pollen wall disruption, causing an abnormally thick intine. In addition, functional interruption of BcMF27 gave rise to pollen tubes twisted in vitro. Taken together, BcMF27 contributes to the intine morphogenesis during pollen development and stabilizes pollen tube elongation. This research can promote knowledge of PMEs function and the molecular mechanism in pollen wall construction.

Tetraketide α-pyrone reductases in sporopollenin synthesis pathway in Gerbera hybrida: diversification of the minor function

The structurally robust biopolymer sporopollenin is the major constituent of the exine layer of pollen wall and plays a vital role in plant reproductive success. The sporopollenin precursors are synthesized through an ancient polyketide biosynthetic pathway consisting of a series of anther-specific enzymes that are widely present in all land plant lineages. Tetraketide α-pyrone reductase 1 (TKPR1) and TKPR2 are two reductases catalyzing the final reduction of the carbonyl group of the polyketide synthase-synthesized tetraketide intermediates to hydroxylated α-pyrone compounds, important precursors of sporopollenin. In contrast to the functional conservation of many sporopollenin biosynthesis associated genes confirmed in diverse plant species, TKPR2’s role has been addressed only in Arabidopsis, where it plays a minor role in sporopollenin biosynthesis. We identified in gerbera two non-anther-specific orthologues of AtTKPR2, Gerbera reductase 1 (GRED1) and GRED2. Their dramatically expanded expression pattern implies involvement in pathways outside of the sporopollenin pathway. In this study, we show that GRED1 and GRED2 are still involved in sporopollenin biosynthesis with a similar secondary role as AtTKPR2 in Arabidopsis. We further show that this secondary role does not relate to the promoter of the gene, AtTKPR2 cannot rescue pollen development in Arabidopsis even when controlled by the AtTKPR1 promoter. We also identified the gerbera orthologue of AtTKPR1, GTKPR1, and characterized its crucial role in gerbera pollen development. GTKPR1 is the predominant TKPR in gerbera pollen wall formation, in contrast to the minor roles GRED1 and GRED2. GTKPR1 is in fact an excellent target for engineering male-sterile gerbera cultivars in horticultural plant breeding.

Overexpression of Sesame Polyketide Synthase A Leads to Abnormal Pollen Development in Arabidopsis

Background: Sesame is a great reservoir of bioactive constituents and unique antioxidant components and is widely used for its nutritional and medicinal value. The expanding demands for sesame seeds are putting pressure on sesame breeders to develop reliable high-yielding varieties. Heterosis utilization is an efficient way to increase sesame yield. Polyketide synthases (PKSs) are critical enzymes in the biosynthesis of sporopollenin, a primary component of pollen exine. Their in planta functions are being investigated for application in crop breeding.Results: In this study, we cloned the sesame POLYKETIDE SYNTHASE A (SiPKSA) and examined its function in male sterility. SiPKSA was specifically expressed in sesame flower buds, and its expression was significantly higher in sterile sesame anthers than in fertile anthers at the tetrad and microspore development stage. Further overexpression of SiPKSA in Arabidopsis caused transgenic plants male sterile. Ultrastructural observation showed that the pollen grains of SiPKSA-overexpressing plants contained few cytoplasmic inclusions and exhibited an abnormal pollen wall structure, with a thicker exine layer compared with wild type. In agreement with it, the expression of a set of sporopollenin biosynthesis-related genes and the contents of fatty acids and phenolics were significantly altered in anthers of SiPKSA-overexpressing plants compared with wild type during anther development. Conclusion: These findings highlighted that overexpression of SiPKSA in Arabidopsis might cause excessive sporopollenin biosynthesis to influence pollen and pollen wall development, leading to male sterile, suggesting that its manipulation might improve hybrid breeding in sesame and other crop species.

BcMF27, A Pectin Methylesterase Gene, Regulates Pollen Development and Pollen Tube Growth in Brassica Campestris

Functional pollen grains are an essential ingredient of successful reproduction in flowering plants and are protected by outer walls. Pectin methylesterases (PMEs) modify pectin, a structural component of pollen intine. However, there are few studies on PMEs. Artificial microRNA (amiRNA) and overexpression technology was performed to investigate the function of pollen-specific PME gene, BcMF27, in pollen development. Knockdown of BcMF27 led to pollen wall collapse, 20% of which unknown material adhered to. Wall-collapsed pollen had abnormally thick intine outside of the germinal furrows. A portion of the cytoplasm was degraded in the remaining pollen with unknown material on the wall, in addition to a thick intine. Overexpression of BcMF27 resulted in 66.67% pollen wall disruption, causing an abnormally thick intine. In addition, functional interruption of BcMF27 gave rise to pollen tubes twisted in vitro. Taken together, BcMF27 contributes to the intine morphogenesis during pollen development and stabilizes pollen tube elongation. This research can promote knowledge of PMEs function and the molecular mechanism in pollen wall construction.

OsMS188 Is a Key Regulator of Tapetum Development and Sporopollenin Synthesis in Rice

Background During anther development, the tapetum provides essential nutrients and materials for pollen development. In rice, multiple transcription factors and enzymes essential for tapetum development and pollen wall formation have been cloned from male-sterile lines. Results In this study, we obtained several lines in which the MYB transcription factor OsMS188 was knocked out through the CRISPR-Cas9 approach. The osms188 lines exhibited a male-sterile phenotype with aberrant development and degeneration of tapetal cells, absence of the sexine layer and defective anther cuticles. CYP703A3, CYP704B2, OsPKS1, OsPKS2, DPW and ABCG15 are sporopollenin synthesis and transport-related genes in rice. Plants with mutations in these genes are male sterile, with a defective sexine layer and anther cuticle. Further biochemical assays demonstrated that OsMS188 binds directly to the promoters of these genes to regulate their expression. UDT1, OsTDF1, TDR, bHLH142 and EAT1 are upstream regulators of rice tapetum development. Electrophoretic mobility shift assays (EMSAs) and activation assays revealed that TDR directly regulates OsMS188 expression. Additionally, protein interaction assays indicated that TDR interacts with OsMS188 to regulate downstream gene expression. Conclusion Overall, OsMS188 is a key regulator of tapetum development and pollen wall formation. The gene regulatory network established in this work may facilitate future investigations of fertility regulation in rice and in other crop species.