Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

[Global proteomic and phosphoproteomic analysis of the premature maize anther].

Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.

The Arabidopsis CALLOSE DEFECTIVE MICROSPORE1 gene is required for male fertility through regulating callose metabolism during microsporogenesis.

During angiosperm microsporogenesis, callose serves as a temporary wall to separate microsporocytes and newly formed microspores in the tetrad. Abnormal callose deposition and dissolution can lead to degeneration of developing microspores. However, genes and their regulation in callose metabolism during microsporogenesis still remain largely unclear. Here, we demonstrated that the Arabidopsis (Arabidopsis thaliana) CALLOSE DEFECTIVE MICROSPORE1 (CDM1) gene, encoding a tandem CCCH-type zinc finger protein, plays an important role in regulation of callose metabolism in male meiocytes and in integrity of newly formed microspores. First, quantitative reverse transcription PCR and in situ hybridization analyses showed that the CDM1 gene was highly expressed in meiocytes and the tapetum from anther stages 4 to 7. In addition, a transfer DNA insertional cdm1 mutant was completely male sterile. Moreover, light microscopy of anther sections revealed that microspores in the mutant anther were initiated, and then degenerated soon afterward with callose deposition defects, eventually leading to male sterility. Furthermore, transmission electron microscopy demonstrated that pollen exine formation was severely affected in the cdm1 mutant. Finally, we found that the cdm1 mutation affected the expression of callose synthesis genes (CALLOSE SYNTHASE5 and CALLOSE SYNTHASE12) and potential callase-related genes (A6 and MYB80), as well as three other putative ß-1,3-glucanase genes. Therefore, we propose that the CDM1 gene regulates callose metabolism during microsporogenesis, thereby promoting Arabidopsis male fertility.