[Show abstract][Hide abstract] ABSTRACT: Lodging has been a major roadblock to attaining increased crop productivity. In an attempt to understand the mechanism for culm strength in rice, we isolated an effective quantitative trait loci (QTL), STRONG CULM3 (SCM3), the causal gene of which is identical to rice TEOSINTE BRANCHED1 (OsTB1), a gene previously reported to positively control strigolactone (SL) signaling. A near-isogenic line (NIL) carrying SCM3 showed enhanced culm strength and increased spikelet number despite the expected decrease in tiller number, indicating that SL also has a positive role in enhancing culm strength and spikelet number. We produced a pyramiding line carrying SCM3 and SCM2, another QTL encoding APO1 involved in panicle development. The NIL-SCM2+SCM3 showed a much stronger culm than NIL-SCM2 and NIL-SCM3 and an increased spikelet number caused by the additive effect of these QTLs. We discuss the importance of utilizing suitable alleles of these STRONG CULM QTLs without inducing detrimental traits for breeding.
[Show abstract][Hide abstract] ABSTRACT: Some ferns possess the ability to control their sex ratio to maintain genetic variation in their colony with the aid of antheridiogen
pheromones, antheridium (male organ)–inducing compounds that are related to gibberellin. We determined that ferns have evolved
an antheridiogen-mediated communication system to produce males by modifying the gibberellin biosynthetic pathway, which is
split between two individuals of different developmental stages in the colony. Antheridiogen acts as a bridge between them
because it is more readily taken up by prothalli than bioactive gibberellin. The pathway initiates in early-maturing prothalli
(gametophytes) within a colony, which produce antheridiogens and secrete them into the environment. After the secreted antheridiogen
is absorbed by neighboring late-maturing prothalli, it is modified in to bioactive gibberellin to trigger male organ formation.
[Show abstract][Hide abstract] ABSTRACT: Although the introduction of semi-dwarf trait into rice has led to improved lodging resistance making it capable of supporting high grain yield, lodging still remains a concern when attempting to further increase the grain yield of rice. However, improving the lodging resistance in rice by depending on the semi-dwarf trait alone is possible only up to a certain limit, beyond which other traits may be needed for reinforcement. To search for alternative traits relating to high lodging resistance, we identified 9 rice mutant lines possessing improved culm strength. To evaluate whether such lines can be useful for breeding lodging resistant rice, small organ size1 (smos1) mutant having increased lodging resistance but low tiller number and low grain yield, was chosen as a representative for a breeding trial. smos1 was crossed with ST-4 (from the Stock rice collection of Nagoya University Togo field #4), a cultivar with high tiller number and high grain yield, and from their progeny, LRC1 (lodging resistance candidate-1) was selected. Although the low tiller number trait of smos1 was not fully reversed in LRC1, this was compensated by an increase in grain weight per panicle, thereby resulting in high grain yield per plant. This important attribute of LRC1 was further enhanced by the improved lodging resistance trait inherited from smos1. Such improved lodging resistance in LRC1 and smos1 was revealed to be mainly due to increased culm diameter and culm thickness, which led to a high section modulus (SM) value, a parameter defining the physical strength of the culm. Since smos1 possesses high breaking-type lodging resistance which is different from semi-dwarf plants with high bending-type lodging resistance, an alternative approach of using thick culm lines for the creation of rice with increased lodging resistance is hereby proposed.
PLoS ONE 07/2014; 9(7):e96009. DOI:10.1371/journal.pone.0096009 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of INDETERMINATE DOMAIN (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.
Proceedings of the National Academy of Sciences 05/2014; 111(21). DOI:10.1073/pnas.1321669111 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Traditional breeding for high-yielding rice has been dependent on the widespread use of fertilizers and the cultivation of gibberellin (GA)-deficient semi-dwarf varieties. The use of semi-dwarf plants facilitates high grain yield since these varieties possess high levels of lodging resistance, and thus could support the high grain weight. Although this approach has been successful in increasing grain yield, it is desirable to further improve grain production and also to breed for high biomass. In this study, we re-examined the effect of GA on rice lodging resistance and biomass yield using several GA-deficient mutants (e.g. having defects in the biosynthesis or perception of GA), and high-GA producing line or mutant. GA-deficient mutants displayed improved bending-type lodging resistance due to their short stature; however they showed reduced breaking-type lodging resistance and reduced total biomass. In plants producing high amounts of GA, the bending-type lodging resistance was inferior to the original cultivars. The breaking-type lodging resistance was improved due to increased lignin accumulation and/or larger culm diameters. Further, these lines had an increase in total biomass weight. These results show that the use of rice cultivars producing high levels of GA would be a novel approach to create higher lodging resistance and biomass.
PLoS ONE 02/2014; 9(2):e86870. DOI:10.1371/journal.pone.0086870 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: GRAS proteins belong to a plant specific protein family that participates in diverse and important functions in growth and development. GRAS proteins are typically composed of a variable N-terminal domain and highly conserved C-terminal GRAS domain. Despite the importance of the GRAS domain, little biochemical or structural analyses have been reported, mainly due to difficulties with purification of sufficient quality and quantity of protein. This study is focused on one of the most extensively studied GRAS proteins, the rice DELLA protein (SLR1), which is known to be involved in gibberellin (GA) signaling. Using a baculovirus-insect cell expression system we have achieved overproduction and purification of full-length SLR1. Limited proteolysis of the full-length SLR1 indicated that a region including the entire GRAS domain (SLR1(206-625)) is protease resistant. Based on those results, we have constructed an expression and purification system of the GRAS domain (SLR1(206-625)) in E. coli. Several physicochemical assays have indicated that the folded structure of the GRAS domain is rich in secondary structural elements and that alanine substitutions for six cysteine residues improves protein folding without impairing function. Furthermore, by NMR spectroscopy we have observed direct interaction between the purified GRAS domain and the GA receptor GID1. Taken together, our purified preparation of the GRAS domain of SLR1 is suitable for further structural and functional studies that will contribute to precise understanding of the plant regulation mechanism through DELLA and GRAS proteins.
Protein Expression and Purification 01/2014; 95. DOI:10.1016/j.pep.2014.01.006 · 1.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Through co-expression network analysis, we identified 123 rice transcription factors (TFs) as candidate rice secondary cell wall regulators (Hirano et al., the accompanying paper). To validate whether these TFs are associated with secondary cell wall formation, six TF genes belonging to either the MYB, NAC or homeodomain containing TF families were over-expressed or down-regulated in rice. With the exception of OsMYB58/63-RNAi plants, all transgenic plants showed phenotypes possibly related to secondary cell wall alteration, such as dwarfism, narrow and dark green leaves, and also altered rice cinnamyl alcohol dehydrogenase 2 (OsCAD2) gene expression and lignin content. These results suggest that many of the 123 candidate secondary cell wall regulating TFs are likely to function in rice secondary cell wall formation.Further analyses were performed for the OsMYB55/61 and OsBLH6 TFs, one representing TF in which the Arabidopsis ortholog is known to participate in lignin biosynthesis (AtMYB61) and one for which no previous involvement in cell wall formation has been reported even in Arabidopsis (BLH6). OsMYB55/61 and OsBLH6-GFP fusion proteins localized to the nucleus of onion epidermal cells. Moreover, expression of a reporter gene driven by the OsCAD2 promoter was enhanced in rice calli when OsMYB55/61 or OsBLH6 were transiently expressed, demonstrating that they function in secondary cell wall formation. These results show the validity of identifying potential rice secondary cell wall TFs through the rice co-expression network analysis.
[Show abstract][Hide abstract] ABSTRACT: The plant secondary cell wall is the major source of lignocellulosic biomass, a renewable energy resource which can be used for bioethanol production. To comprehensively identify transcription factors (TFs), glycosyltransferase (GT), and glycosyl hydrolase (GH) involved in secondary cell wall formation in rice (Oryza sativa), co-expression network analysis was performed using 68 microarray data points for different rice tissues and stages. In addition to rice genes encoding orthologs of Arabidopsis thaliana TFs known to regulate secondary cell wall formation, the network analysis suggested many novel TF genes likely to be involved in cell wall formation. In the accompanying paper (Hirano et al.), several of these TFs are shown to be involved in rice secondary cell wall formation.Based on a comparison of the rice and Arabidopsis networks, TFs were classified as common to both species or specific to each plant species, suggesting that in addition to a common transcriptional regulatory mechanism of cell wall formation, the two plants may also use species-specific groups of TFs during secondary wall formation. Similarly, genes encoding GT and GH were also classified as genes showing species-common or species-specific expression patterns. In addition, genes for primary or secondary cell wall formation were also suggested.The list of rice TF, GT, and GH genes provides an opportunity to unveil the regulation of secondary cell wall formation in grasses, leading to optimization of the cell wall for biofuel production.
[Show abstract][Hide abstract] ABSTRACT: When the gibberellin (GA) receptor GIBBERELLIN INSENSITIVE DWARF 1 (GID1) binds to GA, GID1 interacts with DELLA proteins, repressors of GA signaling. This interaction inhibits the suppressive function of DELLA protein and thereby activates the GA response. However, how DELLA proteins exert their suppressive function and how GID1s inhibit suppressive function of DELLA proteins is unclear. By yeast one-hybrid experiments and transient expression of the N-terminal region of rice DELLA protein (SLR1) in rice callus, we established that the N-terminal DELLA/TVHYNP motif of SLR1 possesses transactivation activity. When SLR1 proteins with various deletions were over-expressed in rice, the severity of dwarfism correlated with the transactivation activity observed in yeast, indicating that SLR1 suppresses plant growth through transactivation activity. This activity was suppressed by the GA-dependent GID1-SLR1 interaction, which may explain why GA responses are induced in the presence of GA. The C-terminal GRAS domain of SLR1 also exhibits a suppressive function on plant growth, possibly by directly or indirectly interacting with the promoter region of target genes. Our results indicate that the N-terminal region of SLR1 has two roles in GA signaling: interaction with GID1 and transactivation activity.
The Plant Journal 03/2012; 71(3):443-53. DOI:10.1111/j.1365-313X.2012.05000.x · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gibberellins (GAs) are tetracyclic, diterpenoid plant hormones, essential for many developmental processes in higher plants. Plants perceive GA through a nuclear-localized GA receptor, GA INSENSITIVE DWARF1 (GID1). From sequence similarity, it is suggested that GID1 evolved from a hormone-sensitive lipase (HSL), and recent x-ray crystallography of the GA-GID1 complex has given insights into how GID1 recognizes GA. Analyses of the GA signaling pathway in several plant species further suggest that the GID1-mediated GA signaling pathway emerged in the vascular plant lineage and since then regulation of GA recognition specificity seems to have been fine tuned to strictly regulate the on-off GA signal.
Protein and Peptide Letters 09/2011; 19(2):180-5. DOI:10.2174/092986612799080248 · 1.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step of monolignol biosynthesis. The rice genome contains 12 CAD-like genes, and whereas the proteins encoded by OsCAD2 and OsCAD7 are known to function in monolignol biosynthesis, the degree to which these enzymes contribute to this process and the involvement of the enzymes encoded by the remaining ten genes is unclear. This paper investigates the role of OsCAD2 and the nine other OsCAD-like proteins in monolignol biosynthesis. Among the OsCAD genes analyzed, OsCAD2, an enzyme belonging to the bona fide CAD phylogenetic group, was the most abundantly expressed gene in the uppermost internode, and was expressed at levels that were more than seven times greater than those of the second most abundantly expressed gene, OsCAD1. Promoter-GUS analysis of OsCAD2 (pCAD::GUS) in the internode, sheath, and roots revealed that GUS expression was strong in tissues that accumulated high levels of lignin. Furthermore, expression always preceded lignin accumulation, showing the tight correlation between OsCAD2 expression and monolignol biosynthesis. Additionally, expression of pCAD::GUS was well synchronized with that of rice caffeic acid 3-O-methyltransferase (OsCOMT::GUS), suggesting that the two enzymes function cooperatively during monolignol biosynthesis. Co-expression network analysis of eight OsCAD genes further revealed that, among the OsCAD genes, expression of OsCAD2 was most tightly associated with the transcription of lignin biosynthesis-related genes. These results suggest that OsCAD2 is largely responsible for monolignol biosynthesis in rice, which is similar to that indicated for the predominant role of other plant bona fide CAD protein to monolignol biosynthesis.
[Show abstract][Hide abstract] ABSTRACT: The DELLA protein SLENDER RICE1 (SLR1) is a repressor of gibberellin (GA) signaling in rice (Oryza sativa), and most of the GA-associated responses are induced upon SLR1 degradation. It is assumed that interaction between GIBBERELLIN INSENSITIVE DWARF1 (GID1) and the N-terminal DELLA/TVHYNP motif of SLR1 triggers F-box protein GID2-mediated SLR1 degradation. We identified a semidominant dwarf mutant, Slr1-d4, which contains a mutation in the region encoding the C-terminal GRAS domain of SLR1 (SLR1(G576V)). The GA-dependent degradation of SLR1(G576V) was reduced in Slr1-d4, and compared with SLR1, SLR1(G576V) showed reduced interaction with GID1 and almost none with GID2 when tested in yeast cells. Surface plasmon resonance of GID1-SLR1 and GID1-SLR1(G576V) interactions revealed that the GRAS domain of SLR1 functions to stabilize the GID1-SLR1 interaction by reducing its dissociation rate and that the G576V substitution in SLR1 diminishes this stability. These results suggest that the stable interaction of GID1-SLR1 through the GRAS domain is essential for the recognition of SLR1 by GID2. We propose that when the DELLA/TVHYNP motif of SLR1 binds with GID1, it enables the GRAS domain of SLR1 to interact with GID1 and that the stable GID1-SLR1 complex is efficiently recognized by GID2.
The Plant Cell 08/2010; 22(8):2680-96. DOI:10.1105/tpc.110.075549 · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: sd1 is known as the 'green revolution' gene in rice because its application in rice breeding has dramatically increased rice yield. Since the 'green revolution,' sd1 has been extensively used to produce modern semi-dwarf varieties. The extensive use of limited dwarfing sources may, however, cause a bottleneck effect in the genetic background of rice varieties. To circumvent this problem, novel and useful sources of dwarf genes must be identified. In this study, we identified three semi-dominant dwarf mutants. These mutants were categorized as dn-type dwarf mutants according to the elongation pattern of internodes. Gibberellin (GA) response tests showed that the mutants were still responsive to GA, although at a reduced rate. Map-based cloning revealed that the dwarf phenotype in these mutants was caused by gain-of-function mutations in the N-terminal region of SLR1. Degradation of the SLR1 protein in these mutants occurred later than in the wild type. Reduced interaction abilities of the SLR1 protein in these mutants with GID1 were also observed using the yeast two-hybrid system. Crossing experiments indicated that with the use of an appropriate genetic background, the semi-dominant dwarf alleles identified in this study could be used to alleviate the deficiency of dwarfing genes for breeding applications.
[Show abstract][Hide abstract] ABSTRACT: The rice (Oryza sativa) DELLA protein SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GID1 causes SLR1 protein degradation involving the F-box protein GID2; this triggers GA-associated responses such as shoot elongation and seed germination. In GA-insensitive and GA biosynthesis mutants, SLENDER RICE1 (SLR1) accumulates to high levels, and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F-box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. The level of SLR1 protein in gid2 was decreased by loss of GID1 function or treatment with a GA biosynthesis inhibitor, and dwarfism was enhanced. Conversely, overproduction of GID1 or treatment with GA(3) increased the SLR1 level in gid2 and reduced dwarfism. These results indicate that derepression of SLR1 repressive activity can be accomplished by GA and GID1 alone and does not require F-box (GID2) function. Evidence for GA signaling without GID2 was also provided by the expression behavior of GA-regulated genes such as GA-20oxidase1, GID1, and SLR1 in the gid2 mutant. Based on these observations, we propose a model for the release of GA suppression that does not require DELLA protein degradation.
The Plant Cell 10/2008; 20(9):2437-46. DOI:10.1105/tpc.108.061648 · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the involvement of phytohormones during rice microspore/pollen (MS/POL) development, endogenous levels of IAA, gibberellins (GAs), cytokinins (CKs) and abscisic acid (ABA) in the mature anther were analyzed. We also analyzed the global expression profiles of genes related to seven phytohormones, namely auxin, GAs, CKs, brassinosteroids, ethylene, ABA and jasmonic acids, in MS/POL and tapetum (TAP) using a 44K microarray combined with a laser microdissection technique (LM-array analysis). IAA and GA(4) accumulated in a much higher amount in the mature anther compared with the other tissues, while CKs and ABA did not. LM-array analysis revealed that sets of genes required for IAA and GA synthesis were coordinately expressed during the later stages of MS/POL development, suggesting that these genes are responsible for the massive accumulation of IAA and GA(4) in the mature anther. In contrast, genes for GA signaling were preferentially expressed during the early developmental stages of MS/POL and throughout TAP development, while their expression was down-regulated at the later stages of MS/POL development. In the case of auxin signaling genes, such mirror-imaged expression observed in GA synthesis and signaling genes was not observed. IAA receptor genes were mostly expressed during the late stages of MS/POL development, and various sets of AUX/IAA and ARF genes were expressed during the different stages of MS/POL or TAP development. Such cell type-specific expression profiles of phytohormone biosynthesis and signaling genes demonstrate the validity and importance of analyzing the expression of phytohormone-related genes in individual cell types independently of other cells/tissues.
[Show abstract][Hide abstract] ABSTRACT: Gibberellin (GA) perception is mediated by GID1 (GA-INSENSITIVE DWARF1), a receptor that shows similarity to hormone-sensitive lipases. A key event in GA signaling is the degradation of DELLA proteins, which are negative regulators of GA response that interact with GID1 in a GA-dependent manner. This GID1-DELLA GA-perception system is conserved among vascular plants but is not found in the moss Physcomitrella patens. The identification of factors in GA signaling downstream of DELLA and the development of a new concept of DELLA function beyond its role as a repressor of GA signaling are important advances. DELLA proteins appear to have at least two other distinct roles: maintaining GA homeostasis and regulating cross-talk between GA and other plant hormones.
[Show abstract][Hide abstract] ABSTRACT: In rice (Oryza sativa) and Arabidopsis thaliana, gibberellin (GA) signaling is mediated by GIBBERELLIN-INSENSITIVE DWARF1 (GID1) and DELLA proteins in collaboration with a GA-specific F-box protein. To explore when plants evolved the ability to perceive GA by the GID1/DELLA pathway, we examined these GA signaling components in the lycophyte Selaginella moellendorffii and the bryophyte Physcomitrella patens. An in silico search identified several homologs of GID1, DELLA, and GID2, a GA-specific F-box protein in rice, in both species. Sm GID1a and Sm GID1b, GID1 proteins from S. moellendorffii, showed GA binding activity in vitro and interacted with DELLA proteins from S. moellendorffii in a GA-dependent manner in yeast. Introduction of constitutively expressed Sm GID1a, Sm G1D1b, and Sm GID2a transgenes rescued the dwarf phenotype of rice gid1 and gid2 mutants. Furthermore, treatment with GA(4), a major GA in S. moellendorffii, caused downregulation of Sm GID1b, Sm GA20 oxidase, and Sm GA3 oxidase and degradation of the Sm DELLA1 protein. These results demonstrate that the homologs of GID1, DELLA, and GID2 work in a similar manner in S. moellendorffii and in flowering plants. Biochemical studies revealed that Sm GID1s have different GA binding properties from GID1s in flowering plants. No evidence was found for the functional conservation of these genes in P. patens, indicating that GID1/DELLA-mediated GA signaling, if present, differs from that in vascular plants. Our results suggest that GID1/DELLA-mediated GA signaling appeared after the divergence of vascular plants from the moss lineage.
The Plant Cell 11/2007; 19(10):3058-79. DOI:10.1105/tpc.107.051524 · 9.58 Impact Factor