Revision of the relationship between anther morphology and pollen sterility by cold stress at the booting stage in rice.

BACKGROUND AND AIMS: Cold stress in rice (Oryza sativa) plants at the reproductive stage prevents normal anther development and causes pollen sterility. Tapetum hypertrophy in anthers has been associated with pollen sterility in response to cold at the booting stage. Here, we re-examined whether the relationships between anther abnormality and pollen sterility caused by cold stress at the booting stage in rice can be explained by a monovalent factor such as tapetum hypertrophy. METHODS: After exposing plants to a 4-d cold treatment at the booting stage, we collected and processed anthers for transverse sectioning immediately and at the flowering stage. We anatomically evaluated the effect of cold treatment on anther internal morphologies, pollen fertilities and pollen numbers in the 13 cultivars with various cold sensitivities. KEY RESULTS: We observed four types of morphological anther abnormalities at each stage. Pollen sterility was positively correlated with the frequency of undeveloped locules, but not with tapetum hypertrophy as commonly believed. In cold-sensitive cultivars grown at low temperatures, pollen sterility was more frequent than anther morphological abnormalities, and some lines showed remarkably high pollen sterility without any anther morphological alterations. Most morphological anomalies occurred only in specific areas within large and small locules. Anther length tended to shorten in response to cold treatment and was positively correlated with pollen number. One cultivar showed a considerably reduced pollen number, but fertile pollen grains under cold stress. We propose three possible relationships to explain anther structure and pollen sterility and reduction due to cold stress. CONCLUSIONS: The pollen sterility caused by cold stress at the booting stage was correlated with the frequency of entire locule-related abnormalities, which might represent a phenotypic consequence, but not a direct cause of pollen abortion. Multivalent factors might underlie the complicated relationships between anther abnormality and pollen sterility in rice.

Diploid Male Gametes Circumvent Hybrid Sterility Between Asian and African Rice Species.

In F1 hybrids of Oryza sativa (Asian rice) and Oryza glaberrima (African rice), heterozygosity leads to a complete gamete abortion because of allelic conflict at each of the 13 hybrid sterility (HS) loci. We systematically produced 19 plants from the F1 hybrids of both the rice species by the anther culture (AC) method. Five of the 19 interspecific hybrid plants were partially fertile and able to produce seeds. Unlike ordinal doubled haploid plants resulting from AC, these regenerated plants showed various ploidy levels (diploid to pentaploid) and different zygosities (completely homozygous, completely heterozygous, and a combination). These properties were attributable to meiotic anomalies in the interspecific hybrid F1 plants. Examination of the genetic structures of the regenerated plants suggested meiotic non-reduction took place in the interspecific hybrid F1 plants. The centromeric regions in the regenerated plants revealed that the abnormal first and/or second divisions of meiosis, namely the first division restitution (FDR) and/or second division restitution (SDR), had occurred in the interspecific hybrid. Immunohistochemical observations also verified these phenomena. FDR and SDR occurrences at meiosis might strongly lead to the formation of diploid microspores. The results demonstrated that meiotic anomalies functioned as a reproductive barrier occurred before the HS genes acted in gamete of the interspecific hybrid. Although such meiotic anomalies are detrimental to pollen development, the early rescue of microspores carrying the diploid gamete resulted in the fertile regenerated plants. The five partially fertile plants carrying tetraploid genomes with heterozygous alleles of the HS loci produced fertile diploid pollens, implying that the diploid gametes circumvented the allelic conflicts at the HS loci. We also proposed how diploid male gametes avoid HS with the killer-protector model.

Genetic Properties Responsible for the Transgressive Segregation of Days to Heading in Rice.

Transgressive segregation produces hybrid progeny phenotypes that exceed the parental phenotypes. Unlike heterosis, extreme phenotypes caused by transgressive segregation are heritably stable. We examined transgressive phenotypes of flowering time in rice, and revealed transgressive segregation in F2 populations derived from a cross between parents with similar (proximal) days to heading (DTH). The DTH phenotypes of the A58 × Kitaake F2 progenies were frequently more extreme than those of either parent. These transgressive phenotypes were maintained in the F3 and F4 populations. Both A58 and Kitaake are japonica rice cultivars adapted to Hokkaido, Japan, which is a high-latitude region, and have a short DTH. Among the four known loci required for a short DTH, three loci had common alleles in A58 and Kitaake, implying there is a similar genetic basis for DTH between the two varieties. A genome-wide single nucleotide polymorphism (SNP) analysis based on the F4 population identified five new quantitative trait loci (QTL) associated with transgressive DTH phenotypes. Each of these QTL had different degrees of additive effects on DTH, and two QTL had an epistatic effect on each other. Thus, a genome-wide SNP analysis facilitated the detection of genetic loci associated with extreme DTH phenotypes, and revealed that the transgressive phenotypes were produced by exchanging the complementary alleles of a few minor QTL in the similar parental phenotypes.

Identification of quantitative trait loci for rice grain quality and yield-related traits in two closely related Oryza sativa L. subsp. japonica cultivars grown near the northernmost limit for rice paddy cultivation.

Quantitative trait loci (QTLs) associated with eating quality, grain appearance quality and yield-related traits were mapped in recombinant inbred lines (RILs) derived from closely related rice (Oryza sativa L. subsp. japonica) cultivars, Yukihikari (good eating quality) and Joiku462 (superior eating quality and high grain appearance quality). Apparent amylose content (AAC), protein content (PC), brown grain length (BGL), brown grain width (BGWI), brown grain thickness (BGT), brown grain weight per plant (BGW) and nine yield-related traits were evaluated in 133 RILs grown in four different environments in Hokkaido, near the northernmost limit for rice paddy cultivation. Using 178 molecular markers, a total of 72 QTLs were detected, including three for AAC, eight for PC, two for BGL, four for BGWI, seven for BGT, and six for BGW, on chromosomes 1, 2, 3, 4, 6, 7, 8, 9, 11 and 12. Fifteen intervals were found to harbor multiple QTLs affecting these different traits, with most of these QTL clusters located on chromosomes 4, 6, 8, 9 and 12. These QTL findings should facilitate gene isolation and breeding application for improvement of eating quality, grain appearance quality and yield of rice cultivars.

Ethylene-dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize.

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen-deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene-dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1-aminocyclopropane-1-carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen-deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.

Development of genome-wide PCR-based markers from insertion, deletion and single nucleotide polymorphisms for closely related Japanese rice cultivars and identification of QTLs for the appearance of cooked rice and polished rice.

Appearance of rice grain is an important property, affecting its acceptance by consumers. Moreover, appearance is a complex characteristic involving many components, including glossiness and whiteness. The genetic bases for the glossiness of cooked rice and the whiteness of polished rice (WPR) were determined using 133 recombinant inbred lines (RILs) derived from a cross between two closely related cultivars from Hokkaido, Joiku462, with high glossiness and whiteness, and Yukihikari, an ancestor of Joiku462 with low glossiness and whiteness. Analyses identified 167 genome-wide InDel markers, five cleaved amplified polymorphic sequences (CAPS) and eight derived CAPS markers differentiating the parental lines. The glossiness area (GLA) and glossiness strength (GLS) of cooked rice and WPR were determined for RILs in two locations, Pippu and Sapporo, Hokkaido. Four QTLs were detected. qGLA10 and qGLS9 were detected on chromosomes 10 and 9, respectively, with both being significant at both geographic locations. qWPR1 on chromosome 1 was significant at Pippu, and qWPR4 on chromosome 4 was significant at Sapporo. The Joiku462 alleles at all QTLs increased each trait. The PCR-based markers flanking these four QTLs may be useful for improvement of GLA, GLS and WPR.

Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure.

Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots.

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.

The rice RCN11 gene encodes ß1,2-xylosyltransferase and is required for plant responses to abiotic stresses and phytohormones.

Seed germination rates and plant development and growth under abiotic stress are important aspects of crop productivity. Here, our characterization of the rice (Oryza sativa L.) mutant reduced culm number11 (rcn11) showed that RCN11 controls growth of plants exposed to abnormal temperature, salinity and drought conditions. RCN11 also mediates root aerenchyma formation under oxygen-deficient conditions and ABA sensitivity during seed germination. Molecular studies showed that the rcn11 mutation resulted from a 966-bp deletion that caused loss of function of ß1,2-xylosyltransferase (OsXylT). This enzyme is located in the Golgi apparatus where it catalyzes the transfer of xylose from UDP-xylose to the core ß-linked mannose of N-glycans. RCN11/OsXylT promoter activity was observed in the basal part of the shoot containing the shoot and axillary meristems and in the base of crown roots. The level of RCN11/OsXylT expression was regulated by multiple phytohormones and various abiotic stresses suggesting that plant specific N-glycosylation is regulated by multiple signals in rice plants. The present study is the first to demonstrate that rice ß1,2-linked xylose residues on N-glycans are critical for seed germination and plant development and growth under conditions of abiotic stress.

RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

Rice RCN1/OsABCG5 mutation alters accumulation of essential and nonessential minerals and causes a high Na/K ratio, resulting in a salt-sensitive phenotype.

Mineral balance and salt stress are major factors affecting plant growth and yield. Here, we characterized the effects of rice (Oryza sativa L.) reduced culm number1 (rcn1), encoding a G subfamily ABC transporter (OsABCG5) involved in accumulation of essential and nonessential minerals, the Na/K ratio, and salt tolerance. Reduced potassium and elevated sodium in field-grown plants were evident in rcn1 compared to original line 'Shiokari' and four independent rcn mutants, rcn2, rcn4, rcn5 and rcn6. A high Na/K ratio was evident in the shoots and roots of rcn1 under K starvation and salt stress in hydroponically cultured plants. Downregulation of SKC1/OsHKT1;5 in rcn1 shoots under salt stress demonstrated that normal function of RCN1/OsABCG5 is essential for upregulation of SKC1/OsHKT1;5 under salt stress. The accumulation of various minerals in shoots and roots was also altered in the rcn1 mutant compared to 'Shiokari' under control conditions, potassium starvation, and salt and d-sorbitol treatments. The rcn1 mutation resulted in a salt-sensitive phenotype. We concluded that RCN1/OsABCG5 is a salt tolerance factor that acts via Na/K homeostasis, at least partly by regulation of SKC1/OsHKT1;5 in shoots.

Strigolactone and cytokinin act antagonistically in regulating rice mesocotyl elongation in darkness.

Strigolactones (SLs) are a group of phytohormones that control plant growth and development including shoot branching. Previous studies of the phenotypes of SL-related rice (Oryza sativa) dwarf (d) mutants demonstrated that SLs inhibit mesocotyl elongation by controlling cell division. Here, we found that the expression of cytokinin (CK)-responsive type-A RESPONSE REGULATOR (RR) genes was higher in d10-1 and d14-1 mutants than in the wild type. However, CK levels in mesocotyls of the d mutants were not very different from those in the wild type. On the other hand, application of a synthetic CK (kinetin) enhanced mesocotyl elongation in the d mutants and the wild type. d10-1 and d14-1 mesocotyls were more sensitive to CK than wild-type mesocotyls, suggesting that the up-regulation of the CK-responsive type-A RR genes and the higher elongation of mesocotyls in the d mutants are mainly due to the increased sensitivity of the d mutants to CK. Co-treatment with kinetin and a synthetic SL (GR24) confirmed the antagonistic functions of SL and CK on mesocotyl elongation. OsTCP5, which encodes a transcription factor belonging to the cell division-regulating TCP family, was also regulated by SL and CK and its expression was negatively correlated with mesocotyl length. These findings suggest that OsTCP5 contributes to the SL- and CK-controlled mesocotyl elongation in darkness.

The rice REDUCED CULM NUMBER11 gene controls vegetative growth under low-temperature conditions in paddy fields independent of RCN1/OsABCG5.

Low temperature tolerance during vegetative growth is an important objective in rice (Oryza sativa L.) breeding programs. We isolated a novel reduced culm number mutant, designated reduced culm number11 (rcn11), by screening under low-temperature condition in a paddy fields. Since the shoot architecture of the rcn11 was very similar to that of the rcn1, we examined whether RCN11 is involved in RCN1/OsABCG5-associated vegetative growth control. The rcn11 mutant has no mutation in the RCN1/OsABCG5 gene and rcn11 has no effect on RCN1/OsABCG5 gene expression. In the rcn1 mutant, RCN1/OsABCG5 was upregulated showing that RCN1/OsABCG5 is controlled by negative feedback regulation. Absence of an effect of rcn11 on RCN1/OsABCG5 feedback regulation supported that RCN11 is not involved in the RCN1/OsABCG5-associated transport system. A genetic allelism test and molecular mapping study showed that rcn11 is independent of rcn1 on rice chromosome 3 and located on chromosome 8. The rcn1 rcn11 phenotype suggests that RCN11 acts on vegetative growth independent of RCN1/OsABCG5. A root development comparison between rcn1 and rcn11 in young seedlings represented that rcn11 reduced crown root number and elongation, whereas rcn1 reduced lateral root density and elongation. Thus, rcn11 will shed new light on vegetative growth control under low temperature.

Multiple and independent origins of short seeded alleles of GS3 in rice.

GRAIN SIZE 3 (GS3) is a cloned gene that is related to seed length. Here we report the discovery of new deletion alleles at the GS3 locus, each of which confer short seed. We selected ten short seeded cultivars from a collection of 282 diverse cultivars. Sequence analysis across the GS3 gene in these ten cultivars identified three novel alleles and a known allele that contain several independent deletion(s) in the fifth exon of GS. These independent deletion variants each resulted in a frameshift mutation that caused a premature stop codon, and they were functionally similar to one another. Each coded for a truncated gene product that behaved as an incomplete dominant allele and conferred a short seeded phenotype. Haplotype analysis of these sequence variants indicated that two of the variants were of japonica origin, and two were from indica. Transformation experiments demonstrated that one of the deletion alleles of GS3 decrease the cell number in the upper epidermis of the glume, resulting in a significant reduction in seed length. The multiple and independent origins of these short seeded alleles indicate that farmers and early breeders imposed artificial selection favoring short seeds.

Genome-wide analysis and expression profiling of half-size ABC protein subgroup G in rice in response to abiotic stress and phytohormone treatments.

The roles of the proteins encoded by half-size adenosine triphosphate-binding cassette transporter subgroup G (ABCG) genes in abiotic stress responses are starting to be established in the dicot model Arabidopsis thaliana. In the monocot model rice, the functions of most half-size ABCG proteins in abiotic stress responses are unknown. Rcn1/OsABCG5 is an essential transporter for growth and development under abiotic stress, but its molecular function remains largely unclear. Here, we present a comprehensive overview of all 30 half-size ABCG genes in rice, including their gene structures, phylogeny, chromosome locations, and conserved motifs. Phylogenetic analysis revealed that the half-size OsABCG proteins were divided to four classes. All seven rice intronless genes, including Rcn1/OsABCG5, were in Class III, like the 12 intronless ABCG genes of Arabidopsis. The EST and FL-cDNA databases provided expression information for 25 OsABCG genes. Semi-quantitative and quantitative RT-PCR analyses demonstrated that seven OsABCG genes were up-regulated in seedlings, shoots or roots following treatments with abiotic stresses (6, 17, 42 °C, NaCl, or mannitol) and abscisic acid. Another 15 OsABCG genes were up-regulated under at least one of the abiotic stress conditions and other phytohormones besides abscisic acid. Hierarchical clustering analysis of gene expression profiles showed that expression of the OsABCG genes could be classified into four clusters. The Rcn1/OsABCG5 cluster was up-regulated by abscisic acid and included OsABCG2, 3, 13, and 27. The present study will provide a useful reference for further functional analysis of the ABCGs in monocots.

Strigolactones negatively regulate mesocotyl elongation in rice during germination and growth in darkness.

Strigolactones (SLs) are newly discovered plant hormones that regulate plant growth and development including shoot branching. They also stimulate symbiosis with arbuscular mycorrhizal fungi. Rice has at least three genes that are involved in SL synthesis (D10, D17/HTD1 and D27) and at least two genes that are involved in SL signaling (D3) and SL signaling or downstream metabolism (D14/D88/HTD2). We observed that mesocotyl elongation in darkness was greater in rice mutants defective in these genes than in the wild type. Exogenous application of a synthetic SL analog, GR24, rescued the phenotype of mesocotyl elongation in the SL-deficient mutants, d10-1, d17-1 and d27-1, in a dose-dependent manner, but did not affect mesocotyl lengths of the SL-insensitive mutants, d3-1 and d14-1. No significant differences in cell length were found between the d mutants and the wild type, except for some cells on the lower half of the d3-1 mesocotyl that were shortened. On the other hand, the number of cells in the mesocotyls was 3- to 6-fold greater in the d mutants than in the wild type. Treatment with GR24 reduced the number of cells in the d10-1 mesocotyl to the wild-type level, but did not affect the number of cells in the d3-1 and d14-1 mesocotyls. These findings indicate that SLs negatively regulate cell division, but not cell elongation, in the mesocotyl during germination and growth of rice in darkness.

Rice shoot branching requires an ATP-binding cassette subfamily G protein.

* Shoot branching is important for the establishment of plant architecture and productivity. * Here, characterization of rice (Oryza sativa) reduced culm number 1 (rcn1) mutants revealed that Rcn1 positively controls shoot branching by promoting the outgrowth of lateral shoots. Molecular studies revealed that Rcn1 encodes a novel member of ATP-binding cassette protein subfamily G (ABCG subfamily), also known as the white-brown complex (WBC) subfamily, and is designated OsABCG5. * Rcn1 is expressed in leaf primordia of main and axillary shoots, and in the vascular cells and leaf epidermis of older leaves. In addition, Rcn1 is expressed in the crown root primordia, endodermis, pericycle and stele in the root. No effect on Rcn1 expression in shoots or roots was seen when the roots were treated with auxins. Phenotypic analyses of rcn1 and tillering dwarf 3 (d3) double mutants at the seedling stage clarified that Rcn1 works independently of D3 in the branching inhibitor pathway. * Rcn1 is the first functionally defined plant ABCG protein gene that controls shoot branching and could thus be significant in future breeding for high-yielding rice.

Rice tillering dwarf mutant dwarf3 has increased leaf longevity during darkness-induced senescence or hydrogen peroxide-induced cell death.

Senescence or cell death in plant leaves is known to be inducible by darkness or H(2)O(2). When the Arabidopsis gene MAX2/ORE9 is disrupted, leaf senescence or cell death in response to the above stimuli is delayed. Because the rice (Oryza sativa L.) gene DWARF3 (D3) is orthologous to MAX2/ORE9, we wished to know whether disruption of D3 also results in increased longevity in leaves. We found that darkness-induced senescence or H(2)O(2)-induced cell death in the third leaf [as measured by chlorophyll degradation, membrane ion leakage and expression of senescence-associated genes (SAGs)] in a d3 rice mutant was delayed by 1-3 d compared to that in its reference line Shiokari. Moreover, the mRNA levels of D3, HTD1 and D10, which are orthologs of Arabidopsis MAX2/ORE9, MAX3 and MAX4, respectively, increased during cell death. These results suggest that D3 protein in rice, like MAX2/ORE9 in Arabidopsis, is involved in leaf senescence or cell death.

Genetic interaction between 2 tillering genes, reduced culm number 1 (rcn1) and tillering dwarf gene d3, in rice.

Mutant genes, reduced culm number 1 (rcn1) and bunketsuwaito tillering dwarf (d3), affect tiller number in rice (Oryza sativa L.) in opposite directions. The d3 mutant was reported to increase tiller number and reduce plant stature. Our objective was to compare the phenotype of the d3rcn1 double mutant with each single mutant and parental rice cultivar "Shiokari" and to clarify whether the Rcn1 gene interacted with the D3 gene. We recovered a new rcn1 mutant from Shiokari and developed d3rcn1 double mutant with Shiokari genetic background. A new rcn1 mutant, designated as "S-97-61" exhibited a reduction in tiller number and plant stature to about the same level as the previously reported original rcn1 mutant. Three near-isogenic lines, rcn1 mutant, d3 mutant, and d3rcn1 double mutant, were grown together with the parental Shiokari. The reduction in tillering by the rcn1 mutation was independent of the d3 genotype, and tillering number of d3rcn1 double mutant was between those of the d3 and rcn1 mutants. These results demonstrated that the Rcn1 gene was not involved in the D3-associated pathway in tillering control.

The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp.

Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus.