OsPhyA modulates rice flowering time mainly through OsGI under short days and Ghd7 under long days in the absence of phytochrome B.

Phytochromes recognize light signals and control diverse developmental processes. In rice, all three phytochrome genes-OsphyA, OsphyB, and OsphyC-are involved in regulating flowering time. We investigated the role of OsPhyA by comparing the osphyA osphyB double mutant to an osphyB single mutant. Plants of the double mutant flowered later than the single under short days (SD) but bolted earlier under long days (LD). Under SD, this delayed-flowering phenotype was primarily due to the decreased expression of Oryza sativa GIGANTEA (OsGI), which controls three flowering activators: Heading date 1 (Hd1), OsMADS51, and Oryza sativa Indeterminate 1 (OsId1). Under LD, although the expression of several repressors, e.g., Hd1, Oryza sativa CONSTANS-like 4 (OsCOL4), and AP2 genes, was affected in the double mutant, that of Grain number, plant height and heading date 7 (Ghd7) was the most significantly reduced. These results indicated that OsPhyA influences flowering time mainly by affecting the expression of OsGI under SD and Ghd7 under LD when phytochrome B is absent. We also demonstrated that far-red light delays flowering time via both OsPhyA and OsPhyB.

OsMPK6 plays a critical role in cell differentiation during early embryogenesis in Oryza sativa.

The formation of body axes is the basis of morphogenesis during plant embryogenesis. We identified embryo-lethal mutants of rice (Oryza sativa) in which T-DNAs were inserted in OsMPK6 Embryonic organs were absent because their development was arrested at the globular stage. Similar to observations made with gle4, shootless, and organless, the osmpk6 mutations affected the initial step of cell differentiation. Expression of an apical-basal axis marker gene, OSH1, was reduced in the mutant embryos while that of the radial axes marker genes OsSCR and OsPNH1 was not detected. The signal for ROC1, a protodermal cell marker, was weak at the globular stage and gradually disappeared. Transcript levels of auxin and gibberellin biosynthesis genes were diminished in osmpk6 embryos. In addition, phytoalexin biosynthesis genes were down-regulated in osmpk6 and a major diterpene phytoalexin, momilactone A, did not accumulate in the mutant embryos. These results indicate that OsMPK6 begins to play a critical role during early embryogenesis, especially when the L1 radial axis is being formed.

Defective Tapetum Cell Death 1 (DTC1) Regulates ROS Levels by Binding to Metallothionein during Tapetum Degeneration.

After meiosis, tapetal cells in the innermost anther wall layer undergo program cell death (PCD)-triggered degradation. This step is essential for microspore development and pollen wall maturation. We identified a key gene, Defective Tapetum Cell Death 1 (DTC1), that controls this degeneration by modulating the dynamics of reactive oxygen species (ROS) during rice male reproduction. Mutants defective in DTC1 exhibit phenotypes of an enlarged tapetum and middle layer with delayed degeneration, causing male sterility. The gene is preferentially expressed in the tapetal cells during early anther development. In dtc1 anthers, expression of genes encoding secretory proteases or lipid transporters is significantly reduced, while transcripts of PCD regulatory genes, e.g. UDT1, TDR1, and EAT1/DTD, are not altered. Moreover, levels of DTC1 transcripts are diminished in udt1, tdr, and eat1 anthers. These results suggest that DTC1 functions downstream of those transcription factor genes and upstream of the genes encoding secretory proteins. DTC1 protein interacts with OsMT2b, a ROS scavenger. Whereas wild-type plants accumulate large amounts of ROS in their anthers at Stage 9 of development, those levels remain low during all stages of development in dtc1 anthers. These findings indicate that DTC1 is a key regulator for tapetum PCD by inhibiting ROS-scavenging activity.

OsMLO12, encoding seven transmembrane proteins, is involved with pollen hydration in rice.

Hydration is the first step in pollen germination. However, the process is not well understood. OsMLO12 is highly expressed in mature pollen grains; plants containing alleles caused by transfer DNA insertions do not produce homozygous progeny. Reciprocal crosses between wild-type and OsMLO12/osmlo12 plants showed that the mutant alleles were not transmitted through the male gametophyte. Microscopic observations revealed that, although mutant grains became mature pollen with three nuclei, they did not germinate in vitro or in vivo due to a failure in hydration. The OsMLO12 protein has seven transmembrane motifs, with an N-terminal extracellular region and a C-terminal cytosolic region. We demonstrated that the C-terminal region mediates a calcium-dependent interaction with calmodulin. Our findings suggest that pollen hydration is regulated by MLO12, possibly through an interaction with calmodulin in the cytosol.

Rice glycosyltransferase1 encodes a glycosyltransferase essential for pollen wall formation.

The pollen wall consists of an exine and an intine. The mechanism underlying its formation is not well understood. Glycosyltransferases catalyze the modification of biological molecules by attaching a single or multiple sugars and play key roles in a wide range of biological processes. We examined the role of GLYCOSYLTRANSFERASE1 (OsGT1) in pollen wall development in rice (Oryza sativa). This gene is highly expressed in mature pollen, and plants containing alleles caused by transfer DNA insertion do not produce homozygous progeny. Reciprocal crosses between OsGT1/osgt1 and the wild type indicated that the mutation leads to a male gametophyte defect. Microscopic analyses revealed that osgt1 pollen developed normally to the pollen mitosis stage but failed to produce mature grains. In osgt1 pollen, intine structure was disrupted. In addition, starch and protein levels were much lower in the mutant grains. Recombinant OsGT1 transferred glucose from UDP-glucose to the third and seventh positions of quercetin, a universal substrate of glycosyltransferases. Consistent with the role of OsGT1, an OsGT1-green fluorescent protein fusion protein was localized to the Golgi apparatus. Taken together, our results suggest that OsGT1 is a Golgi-localized glycosyltransferase essential for intine construction and pollen maturation, providing new insight into male reproductive development.

The rice gene DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1) is required for early tapetum development and meiosis.

Tapetum development and meiosis play crucial roles in anther development. Here we identified a rice gene, DEFECTIVE TAPETUM AND MEIOCYTES 1 (DTM1), which controls the early stages of that development. This gene encodes for an endoplasmic reticulum (ER) membrane protein that is present only in cereals. Our T-DNA insertion mutations gave rise to abnormal tapetal formation. Cellular organelles, especially the ER, were underdeveloped, which led to hampered differentiation and degeneration of the tapetum. In addition, the development of pollen mother cells was arrested at the early stages of meiotic prophase I. RNA in-situ hybridization analyses showed that DTM1 transcripts were most abundant in tapetal cells at stages 6 and 7, and moderately in the pollen mother cells and meiocytes. Transcripts of UDT1, which functions in tapetum development during early meiosis, were reduced in dtm1 anthers, as were those of PAIR1, which is involved in chromosome pairing and synapsis during meiosis. However, expression of MSP1 and MEL1, which function in anther wall specification and germ cell division, respectively, was not altered in the dtm1 mutant. Moreover, transcripts of DTM1 were reduced in msp1 mutant anthers, but not in udt1 and pair1 mutants. These results, together with their mutant phenotypes, suggest that DTM1 plays important roles in the ER membrane during early tapetum development, functioning after MSP1 and before UDT1, and also in meiocyte development, after MEL1 and before PAIR1.

OsPUB15, an E3 ubiquitin ligase, functions to reduce cellular oxidative stress during seedling establishment.

The plant U-box (PUB) protein functions as an E3 ligase to poly-ubiquitinate a target protein for its degradation or post-translational modification. Here, we report functional roles for OsPUB15, which encodes a cytosolic U-box protein in the class-II PUB family. Self-ubiquitination assays showed that bacterially expressed MBP-OsPUB15 protein has E3 ubiquitin ligase activity. A T-DNA insertional mutation in OsPUB15 caused severe growth retardation and a seedling-lethal phenotype. Mutant seeds did not produce primary roots, and their shoot development was significantly delayed. Transgenic plants expressing the OsPUB15 antisense transcript phenocopied these mutant characters. The abnormal phenotypes were partially rescued by two antioxidants, catechin and ascorbic acid. Germinating seeds in the dark also recovered the rootless defect. Levels of H2O2 and oxidized proteins were higher in the knock-out mutant compared with the wild type. OsPUB15 transcript levels were increased upon H2O2, salt and drought stresses; plants overexpressing the gene grew better than the wild type under high salinity. These results indicate that PUB15 is a regulator that reduces reactive oxygen species (ROS) stress and cell death.

OsCOL4 is a constitutive flowering repressor upstream of Ehd1 and downstream of OsphyB.

Plants recognize environmental factors to determine flowering time. CONSTANS (CO) plays a central role in the photoperiod flowering pathway of Arabidopsis, and CO protein stability is modulated by photoreceptors. In rice, Hd1, an ortholog of CO, acts as a flowering promoter, and phytochromes repress Hd1 expression. Here, we investigated the functioning of OsCOL4, a member of the CONSTANS-like (COL) family in rice. OsCOL4 null mutants flowered early under short or long days. In contrast, OsCOL4 activation-tagging mutants (OsCOL4-D) flowered late in either environment. Transcripts of Ehd1, Hd3a, and RFT1 were increased in the oscol4 mutants, but reduced in the OsCOL4-D mutants. This finding indicates that OsCOL4 is a constitutive repressor functioning upstream of Ehd1. By comparison, levels of Hd1, OsID1, OsMADS50, OsMADS51, and OsMADS56 transcripts were not significantly changed in oscol4 or OsCOL4-D, suggesting that OsCOL4 functions independently from previously reported flowering pathways. In osphyB mutants, OsCOL4 expression was decreased and osphyB oscol4 double mutants flowered at the same time as the osphyB single mutants, indicating OsCOL4 functions downstream of OsphyB. We also present evidence for two independent pathways through which OsPhyB controls flowering time. These pathways are: (i) night break-sensitive, which does not need OsCOL4; and (ii) night break-insensitive, in which OsCOL4 functions between OsphyB and Ehd1.

OsMADS50 and OsMADS56 function antagonistically in regulating long day (LD)-dependent flowering in rice.

In much of the tropics and subtropics, rice (Oryza sativa L.) is grown under long days (LDs). Therefore, LD must play a major role in inducing flowering signal in rice. However, little is known on LD-dependent flowering signal in the species. We previously reported that OsMADS50, which is highly homologous to Arabidopsis SOC1, functions as a positive regulator for flowering. However, its detailed photoperiodic mechanism was not yet elucidated. Here, we report the functional analysis of OsMADS50 and its closely related gene OsMADS56. Knock-out of OsMADS50 caused a late-flowering phenotype only under LD conditions. Overexpression of OsMADS56 (56OX) also resulted in delayed flowering under LD. In the osmads50 mutants and 56OX transgenic plants, transcripts of Ehd1, Hd3a and RFT1 were reduced, although that of OsLFL1 increased. On the other hand, mRNA levels of OsGI, Hd1, OsId1, OsDof12, Ghd7, Hd6 and SE5 were unchanged. These observations imply that OsMADS50 and OsMADS56 function antagonistically through OsLFL1-Ehd1 in regulating LD-dependent flowering. Yeast two-hybrid and co-immunoprecipitation analyses indicated an interaction between those two proteins as well as their formation of homodimers. These results suggest that OsMADS50 and OsMADS56 may form a complex that regulates downstream target genes.