Rice GLUCAN SYNTHASE-LIKE5 promotes anther callose deposition to maintain meiosis initiation and progression.

Callose is a plant cell wall polysaccharide whose deposition is spatiotemporally regulated in various developmental processes and environmental stress responses. The appearance of callose in premeiotic anthers is a prominent histological hallmark for the onset of meiosis in flowering plants; however, the biological role of callose in meiosis remains unknown. Here, we show that rice (Oryza sativa) GLUCAN SYNTHASE LIKE5 (OsGSL5), a callose synthase, localizes on the plasma membrane of pollen mother cells (PMCs) and is responsible for biogenesis of callose in anther locules through premeiotic and meiotic stages. In Osgsl5 mutant anthers mostly lacking callose deposition, aberrant PMCs accompanied by aggregated, unpaired, or multivalent chromosomes were frequently observed and, furthermore, a considerable number of mutant PMCs had untimely progress into meiosis compared to that of wild-type PMCs. Immunostaining of meiosis-specific protein HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS2 in premeiotic PMCs revealed precocious meiosis entry in Osgsl5 anthers. These findings provide insights into the function of callose in controlling the timing of male meiosis initiation and progression, in addition to roles in microsporogenesis, in flowering plants.

Comparison of constitutive promoter activities and development of maize ubiquitin promoter- and Gateway-based binary vectors for rice.

In transgenic experiments, we often face fundamental requirements such as overexpressing a certain gene, developing organelle markers, testing promoter activities, introducing large genomic fragments, and combinations of them. To fulfill these multiple requirements in rice, we developed simple binary vectors with or without maize ubiquitin (UBQ) promoter, Gateway cassette and fluorescent proteins. First, we compared stabilities of cauliflower mosaic virus 35S and maize UBQ promoters for constitutive gene expression in transgenic rice. We show that the 35S promoter was frequently silenced after shoot regeneration, whereas maize UBQ promoter achieved stable expression in various young tissues. Binary vectors with Gateway cassettes under the control of the UBQ promoter allowed us to develop stable organelle markers for nuclei, microtubules and P-bodies in rice. The maize UBQ promoter can be easily replaced with any promoters of interest as exemplified by reporters of mitotic cells and provascular bundles. Finally, by introducing two genomic fluorescent reporters, we showed utilities of the Gateway cassette and two selection markers in large DNA fragment transfer and sequential transformations, respectively. Thus, these binary vectors provide useful choices of transgenic experiments in rice.

The Thiamin-Requiring 3 Mutation of Arabidopsis 5-Deoxyxylulose-Phosphate Synthase 1 Highlights How the Thiamin Economy Impacts the Methylerythritol 4-Phosphate Pathway.

The thiamin-requiring mutants of Arabidopsis have a storied history as a foundational model for biochemical genetics in plants and have illuminated the central role of thiamin in metabolism. Recent integrative genetic and biochemical analyses of thiamin biosynthesis and utilization imply that leaf metabolism normally operates close to thiamin-limiting conditions. Thus, the mechanisms that allocate thiamin-diphosphate (ThDP) cofactor among the diverse thiamin-dependent enzymes localized in plastids, mitochondria, peroxisomes, and the cytosol comprise an intricate thiamin economy. Here, we show that the classical thiamin-requiring 3 (th3) mutant is a point mutation in plastid localized 5-deoxyxylulose synthase 1 (DXS1), a key regulated enzyme in the methylerythritol 4-phosphate (MEP) isoprene biosynthesis pathway. Substitution of a lysine for a highly conserved glutamate residue (E323) located at the subunit interface of the homodimeric enzyme conditions a hypomorphic phenotype that can be rescued by supplying low concentrations of thiamin in the medium. Analysis of leaf thiamin vitamers showed that supplementing the medium with thiamin increased total ThDP content in both wild type and th3 mutant plants, supporting a hypothesis that the mutant DXS1 enzyme has a reduced affinity for the ThDP cofactor. An unexpected upregulation of a suite of biotic-stress-response genes associated with accumulation of downstream MEP intermediate MEcPP suggests that th3 causes mis-regulation of DXS1 activity in thiamin-supplemented plants. Overall, these results highlight that the central role of ThDP availability in regulation of DXS1 activity and flux through the MEP pathway.

Regulation of the plastochron by three many-noded dwarf genes in barley.

The plastochron, the time interval between the formation of two successive leaves, is an important determinant of plant architecture. We genetically and phenotypically investigated many-noded dwarf (mnd) mutants in barley. The mnd mutants exhibited a shortened plastochron and a decreased leaf blade length, and resembled previously reported plastochron1 (pla1), pla2, and pla3 mutants in rice. In addition, the maturation of mnd leaves was accelerated, similar to pla mutants in rice. Several barley mnd alleles were derived from three genes-MND1, MND4, and MND8. Although MND4 coincided with a cytochrome P450 family gene that is a homolog of rice PLA1, we clarified that MND1 and MND8 encode an N-acetyltransferase-like protein and a MATE transporter-family protein, which are respectively orthologs of rice GW6a and maize BIGE1 and unrelated to PLA2 or PLA3. Expression analyses of the three MND genes revealed that MND1 and MND4 were expressed in limited regions of the shoot apical meristem and leaf primordia, but MND8 did not exhibit a specific expression pattern around the shoot apex. In addition, the expression levels of the three genes were interdependent among the various mutant backgrounds. Genetic analyses using the double mutants mnd4mnd8 and mnd1mnd8 indicated that MND1 and MND4 regulate the plastochron independently of MND8, suggesting that the plastochron in barley is controlled by multiple genetic pathways involving MND1, MND4, and MND8. Correlation analysis between leaf number and leaf blade length indicated that both traits exhibited a strong negative association among different genetic backgrounds but not in the same genetic background. We propose that MND genes function in the regulation of the plastochron and leaf growth and revealed conserved and diverse aspects of plastochron regulation via comparative analysis of barley and rice.

Construction and applications of a B vitamin genetic resource for investigation of vitamin-dependent metabolism in maize.

The B vitamins provide essential co-factors for central metabolism in all organisms. In plants, B vitamins have surprising emerging roles in development, stress tolerance and pathogen resistance. Hence, there is a paramount interest in understanding the regulation of vitamin biosynthesis as well as the consequences of vitamin deficiency in crop species. To facilitate genetic analysis of B vitamin biosynthesis and functions in maize, we have mined the UniformMu transposon resource to identify insertional mutations in vitamin pathway genes. A screen of 190 insertion lines for seed and seedling phenotypes identified mutations in biotin, pyridoxine and niacin biosynthetic pathways. Importantly, isolation of independent insertion alleles enabled genetic confirmation of genotype-to-phenotype associations. Because B vitamins are essential for survival, null mutations often have embryo lethal phenotypes that prevent elucidation of subtle, but physiologically important, metabolic consequences of sub-optimal (functional) vitamin status. To circumvent this barrier, we demonstrate a strategy for refined genetic manipulation of vitamin status based on construction of heterozygotes that combine strong and hypomorphic mutant alleles. Dosage analysis of pdx2 alleles in endosperm revealed that endosperm supplies pyridoxine to the developing embryo. Similarly, a hypomorphic bio1 allele enabled analysis of transcriptome and metabolome responses to incipient biotin deficiency in seedling leaves. We show that systemic pipecolic acid accumulation is an early metabolic response to sub-optimal biotin status highlighting an intriguing connection between biotin, lysine metabolism and systemic disease resistance signaling. Seed-stocks carrying insertions for vitamin pathway genes are available for free, public distribution via the Maize Genetics Cooperation Stock Center.

Autonomous and non-autonomous functions of the maize Shohai1 gene, encoding a RWP-RK putative transcription factor, in regulation of embryo and endosperm development.

In plants, establishment of the basic body plan during embryogenesis involves complex processes of axis formation, cell fate specification and organ differentiation. While molecular mechanisms of embryogenesis have been well studied in the eudicot Arabidopsis, only a small number of genes regulating embryogenesis has been identified in grass species. Here, we show that a RKD-type RWP-RK transcription factor encoded by Shohai1 (Shai1) is indispensable for embryo and endosperm development in maize. Loss of Shai1 function causes variable morphological defects in the embryo including small scutellum, shoot axis bifurcation and arrest during early organogenesis. Analysis of molecular markers in mutant embryos reveals disturbed patterning of gene expression and altered polar auxin transport. In contrast with typical embryo-defective (emb) mutants that expose a vacant embryo pocket in the endosperm, the endosperm of shai1 kernels conforms to the varied size and shape of the embryo. Furthermore, genetic analysis confirms that Shai1 is required for autonomous formation of the embryo pocket in endosperm of emb mutants. Analyses of genetic mosaic kernels generated by B-A translocation revealed that expression of Shai1 in the endosperm could partially rescue a shai1 mutant embryo and suggested that Shai1 is involved in non-cell autonomous signaling from endosperm that supports normal embryo growth. Taken together, we propose that the Shai1 gene functions in regulating embryonic patterning during grass embryogenesis partly by endosperm-to-embryo interaction.

Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.

To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms.

Jasmonate regulates juvenile-to-adult phase transition in rice.

Juvenile-to-adult phase transition is an important shift for the acquisition of adult vegetative characteristics and subsequent reproductive competence. We identified a recessive precocious (pre) mutant exhibiting a long leaf phenotype in rice. The long leaf phenotype is conspicuous in the second to the fourth leaves, which are juvenile and juvenile-to-adult transition leaves. We found that morphological and physiological traits, such as midrib formation, shoot meristem size, photosynthetic rate and plastochron, in juvenile and juvenile-to-adult transition stages of the pre mutant have precociously acquired adult characteristics. In agreement with these results, expression patterns of miR156 and miR172, which are microRNAs regulating phase change, support the accelerated juvenile-to-adult phase change in the pre mutant. The mutated gene encodes an allene oxide synthase (OsAOS1), which is a key enzyme for the biosynthesis of jasmonic acid (JA). The pre mutant showed a low level of JA and enhanced sensitivity to gibberellic acid, which promotes the phase change in some plant species. We also show that prolonged plastochron in the pre mutant is caused by accelerated PLASTOCHRON1 (PLA1) function. The present study reveals a substantial role of JA as a negative regulator of vegetative phase change.

Genetic interaction between rice PLASTOCHRON genes and the gibberellin pathway in leaf development.

BACKGROUND: The rice PLASTOCHRON (PLA) genes PLA1 and PLA2 regulate leaf maturation and the temporal pattern of leaf initiation. Although the function of PLA genes in the leaf initiation process has been analyzed, little is known about how they affect leaf growth. Previously, we suggested that PLA1 and PLA2 function downstream of the gibberellin (GA) signal transduction pathway. In the present study, we examined the phenotype of a double mutant of pla and slender rice 1 (slr1), which is a constitutive GA response mutant. By analyzing these double mutants, we discuss the relationship between PLA-related and GA-dependent pathways and the possible function of PLA genes in leaf growth. FINDINGS: Single slr1 and pla mutants exhibited elongated and dwarf phenotypes in the vegetative stage, respectively. The stature and leaf size of the pla1/slr1 and pla2/slr1 double mutants were intermediate between those of the pla and slr1 single mutants. However, the effects of slr1 on leaf elongation were markedly suppressed in the pla1 and pla2 mutant backgrounds. On the other hand, the change in cell length in the double mutants was almost the same as that in the single mutants. An expression analysis of genes involved in GA biosynthesis and catabolism indicated that feedback regulation functioned normally in the pla/slr1 double mutants. CONCLUSIONS: Our genetic results confirm that PLA genes regulate leaf growth downstream of the GA pathway. Our findings also suggest that PLA1 and PLA2 are partly required for GA-dependent leaf elongation, mainly by affecting cellular proliferation.

Rice PLASTOCHRON genes regulate leaf maturation downstream of the gibberellin signal transduction pathway.

Rice PLASTOCHRON 1 (PLA1) and PLA2 genes regulate leaf maturation and plastochron, and their loss-of-function mutants exhibit small organs and rapid leaf emergence. They encode a cytochrome P450 protein CYP78A11 and an RNA-binding protein, respectively. Their homologs in Arabidopsis and maize are also associated with plant development/organ size. Despite the importance of PLA genes in plant development, their molecular functions remain unknown. Here, we investigated how PLA1 and PLA2 genes are related to phytohormones. We found that gibberellin (GA) is the major phytohormone that promotes PLA1 and PLA2 expression. GA induced PLA1 and PLA2 expression, and conversely the GA-inhibitor uniconazole suppressed PLA1 and PLA2 expression. In pla1-4 and pla2-1 seedlings, expression levels of GA biosynthesis genes and the signal transduction gene were similar to those in wild-type seedlings. GA treatment slightly down-regulated the GA biosynthesis gene GA20ox2 and up-regulated the GA-catabolizing gene GA2ox4, whereas the GA biosynthesis inhibitor uniconazole up-regulated GA20ox2 and down-regulated GA2ox4 both in wild-type and pla mutants, suggesting that the GA feedback mechanism is not impaired in pla1 and pla2. To reveal how GA signal transduction affects the expression of PLA1 and PLA2, PLA expression in GA-signaling mutants was examined. In GA-insensitive mutant, gid1 and less-sensitive mutant, Slr1-d1, PLA1 and PLA2 expression was down-regulated. On the other hand, the expression levels of PLA1 and PLA2 were highly enhanced in a GA-constitutive-active mutant, slr1-1, causing ectopic overexpression. These results indicate that both PLA1 and PLA2 act downstream of the GA signal transduction pathway to regulate leaf development.