Natural Variation in OsPRR37 Regulates Heading Date and Contributes to Rice Cultivation at a Wide Range of Latitudes

Heading date and photoperiod sensitivity are fundamental traits that determine rice adaptation to a wide range of geographic environments. By quantitative trait locus （QTL） mapping and candidate gene analysis using whole- genome re-sequencing, we found that Oryza sativa Pseudo-Response Regulator37 （OsPRR37; hereafter PRR37） is respon- sible for the Early heading7-2 （EH7-2）/Heading date2 （Hd2） QTL which was identified from a cross of late-heading rice ＇Milyang23 （M23）＇ and early-heading rice ＇H143＇. H143 contains a missense mutation of an invariantly conserved amino acid in the CCT （CONSTANS, CO-like, and TOC1） domain of PRR37 protein. In the world rice collection, different types of nonfunctional PRR37 alleles were found in many European and Asian rice cultivars. Notably, the japonica varieties harboring nonfunctional alleles of both Ghd7/Hd4 and PRR37/Hd2 flower extremely early under natural long-day condi- tions, and are adapted to the northernmost regions of rice cultivation, up to 53~ N latitude. Genetic analysis revealed that the effects of PRR37 and Ghd7 alleles on heading date are additive, and PRR37 down-regulates Hd3a expression to suppress flowering under long-day conditions. Our results demonstrate that natural variations in PRR37/Hd2 and GhdT/ Hd4 have contributed to the expansion of rice cultivation to temperate and cooler regions.

Rice Transcription Factor OsDOFll Modulates Sugar Transport by Promoting Expression of Sucrose Transporter and SWEET Genes

Sucrose is produced in mesophyll cells and transferred into phloem cells before it is delivered long- distance to sink tissues. However, little is known about how sucrose transport is regulated in plants. Here, we identified a T-DNA insertional mutant of Oryza sativa DNA BINDING WITH ONE FINGER 11 （OsDOF11）, which is expressed in the vascular cells of photosynthetic organs and in various sink tissues. The osdofll mutant plants are semi-dwarf and have fewer tillers and smaller panicles as compared with wild-type （WT） plants. Although sucrose enhanced root elongation in young WT seedlings, this enhance- ment did not occur in osdof11 seedlings due to reduced sucrose uptake. Sugar transport rate analyses revealed that less sugar was transported in osdofll plants than in the WT. Expression of four Sucrose Transporter （SUT） genes-OsSUT1, OsSUT3, OsSUT4, and OsSUT5-as well as two Sugars Will Eventually be Exported Transporters （SWEET） genes, OsSWEETll and OsSWEET14, was altered in various organs of the mutant, including the leaves. Chromatin immunoprecipitation assays showed that OsDOFll directly binds the promoter regions of SUT1, OsSWEETll, and OsSWEET14, indicating that the expression of these transporters responsible for sucrose transport via apopiastic loading is coordinately controlled by OsDOFll. We also observed that osdofll mutant plants were less susceptible to infection byXanthomonas oryzae pathovar oryzae, suggesting that OsDOFll participates in sugar distribution during pathogenic in- vasion. Collectively, these results suggest that OsDOFll modulates sugar transport by regulating the expression of both SUT and SWEET genes in rice.

Roles of lignin biosynthesis and regulatory genes in plant development

Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing Iignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non-lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant develop- ment. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism.

Rice-Specific Mitochondrial Iron-Regulated Gene （MIR） Plays an Important Role in Iron Homeostasis

Mitochondria utilize iron （Fe）, but the proteins involved in mitochondrial Fe regulation are not characterized in plants. We cloned and characterized a mitochondrial iron-regulated （MIR） gene in rice involved in Fe homeostasis. MIR, when expressed in tobacco BY-2 cells, was localized to the mitochondria. MIR transcripts were greatly increased in response to Fe deficiency in roots and shoot tissue. MIR is not homologous to any known protein, as homologs were not found in the rice or Arabidopsis genome databases, or in the EST database for other organisms. Growth in the MIR T-DNA knockout rice mutant （mir） was significantly impaired compared to wild-type （WT） plants when grown under Fe-deficient or -sufficient conditions. Furthermore, mir plants accumulated more than twice the amount of Fe in shoot and root tissue compared to WT plants when grown under either Fe-sufficient or -deficient conditions. Despite the high accumulation of Fe in roots and shoots, mir plants triggered the expression of Fe-deficiency-inducible genes, indicating that mir may not be able to utilize Fe for physiological functions. These results clearly suggest that MIR is a rice-specific mitochondrial protein, recently evolved, and plays a significant role in Fe homeostasis.

Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism

We characterized the function of the rice cytosolic hexokinase OsHXK7 （Qryza sativa HexokindseZ）, which is highly upregulated when seeds germinate under O2- deficient conditions. According to transient expression assays that used the promoter：luciferase fusion construct, OsHXK7 enhanced the glucose （Glc）-dependent repression of a rice n-amylase gene （RAmy3D） in the mesophyll protoplasts of maize, but its catalytically inactive mutant alleles did not. Consistently, the expression of OsHXK7, but not its catalytically inactive alleles, complemented the Arabidopsis glucose insensitive2-1 （gin2-1） mutant, thereby resulting in the wild type characteristics of Glc-dependent repression, seedling development, and plant growth. Interestingly, OsHXK7-mediated Glc-dependent repression was abolished in the O2-deficient mesophyll protoplasts of maize. This result provides compelling evidence that OsHXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions, but its signaling role is suppressed when O2 is deficient. The germination of two null OsHXK7 mutants, oshxk7-1 and oshxk7-2, was affected by O2 deficiency, but overexpression enhanced germination in rice. This result suggests the distinct role that OsHXK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O2-deficient rice.

A rice tubulin tyrosine ligase-like 12 protein affects the dynamic and orientation of microtubules

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin.Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase(TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein(Os TTLL12)andgeneratedoverexpression Os TTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this Os TTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of Os TTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.

Genome-Wide Identification and Functional Analysis of Genes Expressed Ubiquitously in Rice

Genes that are expressed ubiquitously throughout all developmental stages are thought to be necessary for basic biological or cellular functions. Therefore, determining their biological roles is a great challenge. We identified 4034 of these genes in rice after studying the results of Agilent 44K and Affymetrix meta- anatomical expression profiles. Among 105 genes that were characterized by loss-of-function analysis, 79 were classified as members of gene families, the majority of which were predominantly expressed. Using T-DNA insertional mutants, we examined 43 genes and found that loss of expression of six genes caused developing seedor seedling-defective phenotypes. Of these, three are singletons without similar family members and defective phenotypes are expected from mutations. Phylogenomic analyses integrating genome-wide transcriptome data revealed the functional dominance of three ubiquitously expressed fam- ily genes. Among them, we investigated the function of OsO3g19890, which is involved in ATP generation within the mitochondria during endosperm development. We also created and evaluated functional net- works associated with this gene to understand the molecular mechanism. Our study provides a useful strategy for pheonome analysis of ubiquitously expressed genes in rice.

Mutation of Oryza sativa CORONATINE INSENSITIVE 1b(OsCOI1b) delays leaf senescence

Jasmonic acid （JA） functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA‐responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice （Oryza sativa） genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant func-tions in development. Here, we use the oscoi1b‐1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence‐promoting conditions. oscoi1b‐1 mutants stayed green during dark‐induced and natural senescence, with substantial retention of chlorophylls and photosyn-thetic capacity. Furthermore, several senescence‐associated genes were downregulated in oscoi1b‐1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1‐1 plants containing 35S：OsCOI1a or 35S：OsCOI1b rescued the delayed leaf senescence during dark incubation, sug-gesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b‐1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1‐mediated JA signaling affects spikelet fertility and grain filling.