The 24-nucleotides (nt) phased secondary small interfering RNA (phasiRNA) is a unique class of plant small RNAs abundantly expressed in monocot anthers at early meiosis. Previously, 44 intergenic regions were identified as the loci for longer precursor RNAs of 24-nt phasiRNAs (24-PHASs) in the rice genome. However, the regulatory mechanism that determines spatiotemporal expression of these RNAs has remained elusive. ETERNAL TAPETUM1 (EAT1) is a basic-helix-loop-helix (bHLH) transcription factor indispensable for induction of programmed cell death (PCD) in postmeiotic anther tapetum, the somatic nursery for pollen production. In this study, EAT1-dependent non-cell-autonomous regulation of male meiosis was evidenced from microscopic observation of the eat1 mutant, in which meiosis with aberrantly decondensed chromosomes was retarded but accomplished somehow, eventually resulting in abortive microspores due to an aberrant tapetal PCD. EAT1 protein accumulated in tapetal-cell nuclei at early meiosis and postmeiotic microspore stages. Meiotic EAT1 promoted transcription of 24-PHAS RNAs at 101 loci, and importantly, also activated DICER-LIKE5 (DCL5, previous DCL3b in rice) mRNA transcription that is required for processing of double-stranded 24-PHASs into 24-nt lengths. From the results of the chromatin-immunoprecipitation and transient expression analyses, another tapetum-expressing bHLH protein, TDR INTERACTING PROTEIN2 (TIP2), was suggested to be involved in meiotic small-RNA biogenesis. The transient assay also demonstrated that UNDEVELOPED TAPETUM1 (UDT1)/bHLH164 is a potential interacting partner of both EAT1 and TIP2 during early meiosis. This study indicates that EAT1 is one of key regulators triggering meiotic phasiRNA biogenesis in anther tapetum, and that other bHLH proteins, TIP2 and UDT1, also play some important roles in this process. Spatiotemporal expression control of these bHLH proteins is a clue to orchestrate precise meiosis progression and subsequent pollen production non-cell-autonomously.

Autophagy plays crucial roles in the recycling of metabolites, and is involved in many developmental processes. Rice mutants defective in autophagy are male sterile due to immature pollens, indicating its critical role in pollen development. However, physiological roles of autophagy during seed maturation had remained unknown. We here found that seeds of the rice autophagy-deficient mutant Osatg7-1, that produces seeds at a very low frequency in paddy fields, are smaller and show chalky appearance and lower starch content in the endosperm at the mature stage under normal growth condition. We comprehensively analyzed the effects of disruption of autophagy on biochemical properties, proteome and seed quality, and found an abnormal activation of starch degradation pathways including accumulation of α-amylases in the endosperm during seed maturation in Osatg7-1. These results indicate critical involvement of autophagy in metabolic regulation in the endosperm of rice, and provide insights into novel autophagy-mediated regulation of starch metabolism during seed maturation.

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T 0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T 1 generation. These results show that many Oryza species can be transformed by using modified immatureembryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

Autophagy has recently been shown to be required for tapetal programmed cell death (PCD) and pollen maturation in rice. A transcriptional regulatory network is also known to play a key role in the progression of tapetal PCD. However, the relationship between the gene regulatory network and autophagy in rice anther development is mostly unknown. Here, we comprehensively analyzed the effect of autophagy disruption on gene expression profile during the tapetal PCD in rice anther development using high-throughput RNA sequencing. Expression of thousands of genes, including specific transcription factors and several proteases required for tapetal degradation, fluctuated synchronously at specific stages during tapetal PCD progression in the wild-type anthers, while this fluctuation showed significant delay in the autophagy-deficient mutant Osatg7-1. Moreover, gene ontology enrichment analysis in combination with self-organizing map clustering as well as pathway analysis revealed that the expression patterns of a variety of organelle-related genes as well as genes involved in carbohydrate/lipid metabolism were affected in the Osatg7-1 mutant during pollen maturation. These results suggest that autophagy is required for proper regulation of gene expression and quality control of organelles and timely progression of tapetal PCD during rice pollen development.

We have previously shown that autophagy is required for post meiotic anther development including programmed cell death-mediated degradation of the tapetum and pollen maturation in rice. However, the spatiotemporal dynamics of autophagy in the tapetum remain poorly understood. We here established an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum cells by expressing green fluorescent protein-tagged AtATG8, a marker for autophagosomes. 3D-imaging analysis revealed that the number of autophagosomes/autophagy-related structures is extremely low at the tetrad stage (stage 8), and autophagy is dramatically induced at the uninucleate stages (stage 9-10) throughout the tapetal cells during anther development. The present monitoring system for autophagy offers a powerful tool to analyze the regulation of autophagy in rice tapetal cells during pollen maturation.

The Endosperm Balance Number (EBN) assigned to each species can foresee success or failure of a given interspecific cross in potatoes, although the underlying molecular basis is poorly studied. We found that the cross of Solanum demissum as female with a breeding line Saikai 35 constantly produced larger seeds (0.94 mg) than those from the reciprocal cross (0.39 mg), suggesting a slightly lower EBN in S. demissum. Crossing behaviors, measured by berry-setting rates, seeds/berry and seed size, in the reciprocal F 1 and BC 1 progenies suggest at least three genetic factors involved in normal seed development: 1) a cytoplasmic factor, and nuclear genome-encoded factors functioned 2) in female gametophyte and 3) in pollen. Thus, these materials are useful in exploring the molecular mechanism of EBN, because the degree of imbalanced EBN could be measured as quantitative traits.Resumen El número de balance endospérmico (EBN) asignado a cada especie puede anticipar éxito o fracaso en una cruza dada interespecífica en papa, aunque la base molecular en la que se respalda es estudiada pobremente. Encontramos que la cruza de Solanum demissum como hembra con una línea de mejoramiento Saikai 35 produjo constantemente semillas más grandes (0.94 mg) que las de la cruza recíproca (0.39 mg), sugiriendo un EBN ligeramente más bajo en S. demissum. Los comportamientos de las cruzas, medidos por los niveles de formación de frutos, semillas/fruto y tamaño de semilla, en las progenies de F1 recíproca y BC1, sugieren por lo menos tres factores genéticos involucrados en el desarrollo normal de semillas: 1) un factor citoplásmico, y funcionamiento de factores citoplásmicos codificados por el genomio nuclear 2) en gametofitos de la hembra y 3) en polen. De aquí que estos materiales son útiles en la exploración del mecanismo molecular de EBN, porque el grado de desbalanceamiento de EBN pudiera medirse como caracteres cuantitativos.

No abstract

A Mexican hexaploid wild species Solanum demissum has been extensively used by backcrossing to tetraploid cultivars in potato breeding. Because of practical difficulty in chromosome counting of the aneuploid progeny, selection in backcross generations has been limited to agronomic performances. We employed flow cytometry (FC), which could distinguish plants with the chromosome number difference less than 1.4 with 95% probability. Thus, FC is useful in selecting near-tetraploid level plants in a short period and at earlier generations.Resumen Una especie Silvestre Mexicana hexaploide, Solanum demissum, ha sido utilizada extensivamente mediante retrocruzas con variedades tetraploides en el mejoramiento de la papa. Debido a dificultades prácticas en el conteo cromosómico de la progenie aneuploide , la selección de las generaciones en las retrocruzas se ha limitado a los comportamientos agronómicos. Nosotros empleamos citometría de flujo (FC), que puede distinguir plantas con diferencia en el número cromosómico menor a 1.4 con un 95% de probabilidad. Así, FC es útil en la selección de plantas con un nivel cercano al tetraploide en un período corto y en generaciones más tempranas.