Gibberellin metabolism and signaling.

Gibberellins (GAs) are plant hormones with a tetracyclic diterpenoid structure that are involved in various important developmental processes. Two GA-deficient mutants were isolated: a semidwarf mutant "sd1", which was found to have a defective GA20ox2 gene and was introduced to the world in a green revolution cultivar, and a severe dwarf allele of "d18", with a defective GA3ox2 gene. Based on the phenotypic similarity of d18, rice dwarf mutants were screened, further classifying them into GA-sensitive and GA-insensitive by applying exogenous GA3. Finally, GA-deficient rice mutants at 6 different loci and 3 GA signaling mutants (gid1, gid2, and slr1) were isolated. The GID1 gene encodes a GA nuclear receptor, and the GID1-DELLA (SLR1) system for GA perception is widely used in vascular plants. The structural characteristics of GID1 and GA metabolic enzymes have also been reviewed.

Evolutionary alterations in gene expression and enzymatic activities of gibberellin 3-oxidase 1 in Oryza.

Proper anther and pollen development are important for plant reproduction. The plant hormone gibberellin is important for anther development in rice, but its gametophytic functions remain largely unknown. Here, we report the functional and evolutionary analyses of rice gibberellin 3-oxidase 1 (OsGA3ox1), a gibberellin synthetic enzyme specifically expressed in the late developmental stages of anthers. Enzymatic and X-ray crystallography analyses reveal that OsGA3ox1 has a higher GA7 synthesis ratio than OsGA3ox2. In addition, we generate an osga3ox1 knockout mutant by genome editing and demonstrate the bioactive gibberellic acid synthesis by the OsGA3ox1 action during starch accumulation in pollen via invertase regulation. Furthermore, we analyze the evolution of Oryza GA3ox1s and reveal that their enzyme activity and gene expression have evolved in a way that is characteristic of the Oryza genus and contribute to their male reproduction ability.

The Dual Function of OsSWEET3a as a Gibberellin and Glucose Transporter Is Important for Young Shoot Development in Rice.

Translocation and long-distance transport of phytohormones are considered important processes for phytohormone responses, as well as their synthesis and signaling. Here, we report on the dual function of OsSWEET3a, a bidirectional sugar transporter from clade I of the rice SWEET family of proteins, as both a gibberellin (GA) and a glucose transporter. OsSWEET3a efficiently transports GAs in the C13-hydroxylation pathway of GA biosynthesis. Both knockout and overexpression lines of OsSWEET3a showed defects in germination and early shoot development, which were partially restored by GA, especially GA20. Quantitative reverse transcription PCR, GUS staining and in situ hybridization revealed that OsSWEET3a was expressed in vascular bundles in basal parts of the seedlings. OsSWEET3a expression was co-localized with OsGA20ox1 expression in the vascular bundles but not with OsGA3ox2, whose expression was restricted to leaf primordia and young leaves. These results suggest that OsSWEET3a is expressed in the vascular tissue of basal parts of seedlings and is involved in the transport of both GA20 and glucose to young leaves, where GA20 is possibly converted to the bioactive GA1 form by OsGA3ox2, during early plant development. We also indicated that such GA transport activities of SWEET proteins have sporadically appeared in the evolution of plants: GA transporters in Arabidopsis have evolved from sucrose transporters, while those in rice and sorghum have evolved from glucose transporters.

Diverse panicle architecture results from various combinations of Prl5/GA20ox4 and Pbl6/APO1 alleles.

Panicle architecture directly affects crop productivity and is a key target of high-yield rice breeding. Panicle length strongly affects panicle architecture, but the underlying regulatory mechanisms are largely unknown. Here, we show that two quantitative trait loci (QTLs), PANICLE RACHIS LENGTH5 (Prl5) and PRIMARY BRANCH LENGTH6 (Pbl6), independently regulate panicle length in rice. Prl5 encodes a gibberellin biosynthesis enzyme, OsGA20ox4. The expression of Prl5 was higher in young panicles resulting in panicle rachis elongation. Pbl6 is identical to ABERRANT PANICLE ORGANIZATION 1 (APO1), encoding an F-box-containing protein. We found a novel function that higher expression of Pbl6 is responsible for primary branch elongation. RNA-seq analysis revealed that these two genes independently regulate panicle length at the level of gene expression. QTL pyramiding of both genes increased panicle length and productivity. By combining these two genes in various combinations, we designed numerous panicle architecture without trade-off relationship.

A common allosteric mechanism regulates homeostatic inactivation of auxin and gibberellin.

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

GWAS with principal component analysis identifies a gene comprehensively controlling rice architecture.

Elucidation of the genetic control of rice architecture is crucial due to the global demand for high crop yields. Rice architecture is a complex trait affected by plant height, tillering, and panicle morphology. In this study, principal component analysis (PCA) on 8 typical traits related to plant architecture revealed that the first principal component (PC), PC1, provided the most information on traits that determine rice architecture. A genome-wide association study (GWAS) using PC1 as a dependent variable was used to isolate a gene encoding rice, SPINDLY (OsSPY), that activates the gibberellin (GA) signal suppression protein SLR1. The effect of GA signaling on the regulation of rice architecture was confirmed in 9 types of isogenic plant having different levels of GA responsiveness. Further population genetics analysis demonstrated that the functional allele of OsSPY associated with semidwarfism and small panicles was selected in the process of rice breeding. In summary, the use of PCA in GWAS will aid in uncovering genes involved in traits with complex characteristics.

Evolution and diversification of the plant gibberellin receptor GID1.

The plant gibberellin (GA) receptor GID1 shows sequence similarity to carboxylesterase (CXE). Here, we report the molecular evolution of GID1 from establishment to functionally diverse forms in eudicots. By introducing 18 mutagenized rice GID1s into a rice gid1 null mutant, we identified the amino acids crucial for GID1 activity in planta. We focused on two amino acids facing the C2/C3 positions of ent-gibberellane, not shared by lycophytes and euphyllophytes, and found that adjustment of these residues resulted in increased GID1 affinity toward GA4, new acceptance of GA1 and GA3 carrying C13-OH as bioactive ligands, and elimination of inactive GAs. These residues rendered the GA perception system more sophisticated. We conducted phylogenetic analysis of 169 GID1s from 66 plant species and found that, unlike other taxa, nearly all eudicots contain two types of GID1, named A- and B-type. Certain B-type GID1s showed a unique evolutionary characteristic of significantly higher nonsynonymous-to-synonymous divergence in the region determining GA4 affinity. Furthermore, these B-type GID1s were preferentially expressed in the roots of Arabidopsis, soybean, and lettuce and might be involved in root elongation without shoot elongation for adaptive growth under low-temperature stress. Based on these observations, we discuss the establishment and adaption of GID1s during plant evolution.

OsIDD2, a zinc finger and INDETERMINATE DOMAIN protein, regulates secondary cell wall formation.

Previously, we found 123 transcription factors (TFs) as candidate regulators of secondary cell wall (SCW) formation in rice by using phylogenetic and co-expression network analyses. Among them, we examined in this work the role of OsIDD2, a zinc finger and indeterminate domain (IDD) family TF. Its overexpressors showed dwarfism, fragile leaves, and decreased lignin content, which are typical phenotypes of plants defective in SCW formation, whereas its knockout plants showed slightly increased lignin content. The RNA-seq and quantitative reverse transcription polymerase chain reaction analyses confirmed that some lignin biosynthetic genes were downregulated in the OsIDD2-overexpressing plants, and revealed the same case for other genes involved in cellulose synthesis and sucrose metabolism. The transient expression assay using rice protoplasts revealed that OsIDD2 negatively regulates the transcription of genes involved in lignin biosynthesis, cinnamyl alcohol dehydrogenase 2 and 3 (CAD2 and 3), and sucrose metabolism, sucrose synthase 5 (SUS5), whereas an AlphaScreen assay, which can detect the interaction between TFs and their target DNA sequences, directly confirmed the interaction between OsIDD2 and the target sequences located in the promoter regions of CAD2 and CAD3. Based on these observations, we conclude that OsIDD2 is negatively involved in SCW formation and other biological events by downregulating its target genes.

Regulation of Strigolactone Biosynthesis by Gibberellin Signaling.

Strigolactones (SLs) are a class of plant hormones that regulate diverse physiological processes, including shoot branching and root development. They also act as rhizosphere signaling molecules to stimulate the germination of root parasitic weeds and the branching of arbuscular mycorrhizal fungi. Although various types of cross talk between SLs and other hormones have been reported in physiological analyses, the cross talk between gibberellin (GA) and SLs is poorly understood. We screened for chemicals that regulate the level of SLs in rice (Oryza sativa) and identified GA as, to our knowledge, a novel SL-regulating molecule. The regulation of SL biosynthesis by GA is dependent on the GA receptor GID1 and F-box protein GID2. GA treatment also reduced the infection of rice plants by the parasitic plant witchers weed (Striga hermonthica). These data not only demonstrate, to our knowledge, the novel plant hormone cross talk between SL and GA, but also suggest that GA can be used to control parasitic weed infections.

SMALL ORGAN SIZE 1 and SMALL ORGAN SIZE 2/DWARF AND LOW-TILLERING Form a Complex to Integrate Auxin and Brassinosteroid Signaling in Rice.

Although auxin and brassinosteroid (BR) synergistically control various plant responses, the molecular mechanism underlying the auxin-BR crosstalk is not well understood. We previously identified SMOS1, an auxin-regulated APETALA2-type transcription factor, as the causal gene of the small organ size 1 (smos1) mutant that is characterized by a decreased final size of various organs in rice. In this study, we identified another smos mutant, smos2, which shows the phenotype indistinguishable from smos1. SMOS2 was identical to the previously reported DWARF AND LOW-TILLERING (DLT), which encodes a GRAS protein involved in BR signaling. SMOS1 and SMOS2/DLT physically interact to cooperatively enhance transcriptional transactivation activity in yeast and in rice nuclei. Consistently, the expression of OsPHI-1, a direct target of SMOS1, is upregulated only when SMOS1 and SMOS2/DLT proteins are both present in rice cells. Taken together, our results suggest that SMOS1 and SMOS2/DLT form a keystone complex on auxin-BR signaling crosstalk in rice.

Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells.

Antheridiogen determines sex in ferns via a spatiotemporally split gibberellin synthesis pathway.

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.

DELLA protein functions as a transcriptional activator through the DNA binding of the indeterminate domain family proteins.

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.

New approach to increasing rice lodging resistance and biomass yield through the use of high gibberellin producing varieties.

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.

A novel AP2-type transcription factor, SMALL ORGAN SIZE1, controls organ size downstream of an auxin signaling pathway.

The organ size of flowering plants is determined by two post-embryonic developmental events: cell proliferation and cell expansion. In this study, we identified a new rice loss-of-function mutant, small organ size1 (smos1), that decreases the final size of various organs due to decreased cell size and abnormal microtubule orientation. SMOS1 encodes an unusual APETALA2 (AP2)-type transcription factor with an imperfect AP2 domain, and its product belongs to the basal AINTEGUMENTA (ANT) lineage, including WRINKLED1 (WRI1) and ADAP. SMOS1 expression was induced by exogenous auxin treatment, and the auxin response element (AuxRE) of the SMOS1 promoter acts as a cis-motif through interaction with auxin response factor (ARF). Furthermore, a functional fluorophore-tagged SMOS1 was localized to the nucleus, supporting the role of SMOS1 as a transcriptional regulator for organ size control. Microarray analysis showed that the smos1 mutation represses expression of several genes involved in microtubule-based movement and DNA replication. Among the down-regulated genes, we demonstrated by gel-shift and chromatin immunoprecipitation (ChIP) experiments that OsPHI-1, which is involved in cell expansion, is a target of SMOS1. SMOS1 homologs in early-diverged land plants partially rescued the smos1 phenotype of rice. We propose that SMOS1 acts as an auxin-dependent regulator for cell expansion during organ size control, and that its function is conserved among land plants.

Expression and purification of a GRAS domain of SLR1, the rice DELLA protein.

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 Escherichia 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.

DELLA and SCL3 balance gibberellin feedback regulation by utilizing INDETERMINATE DOMAIN proteins as transcriptional scaffolds.

DELLA proteins are key negative regulators in the phytohormone gibberellin's (GA) signaling. In addition to this role, the DELLA proteins upregulate the gene expression levels of the positive regulators in GA signaling, such as GA 20-oxidase, GA receptor, and a transcriptional regulator, SCARECROW-LIKE3 (SCL3), which enables the regulation of GA feedback. Since DELLAs lack a known DNA binding domain, other transcription factor(s) that recruit DELLAs to DNA are essential for this regulation. Recently, we showed that the INDETERMINATE DOMAIN family proteins serve as transcriptional scaffolds to exert the transactivation activity of DELLAs. This finding and further analyses regarding the function of SCL3 indicate that the balance of the DELLAs and SCL3 protein levels (both are GRAS proteins) regulates downstream gene expression through IDDs binding to DNA. Here, we review the regulatory system in plants similar to ours and also discuss the interactive network between GRAS and IDD proteins.

Regulatory Networks Acted Upon by the GID1-DELLA System After Perceiving Gibberellin.

Recent studies have revealed that DELLA proteins (DELLAs) interact with various kinds of transcription factors (TFs) and other kinds of proteins to regulate GA signaling. Here, we enumerate some of these DELLA interactors to show the multiple functions of DELLAs in the GA signaling pathway. Through interaction with TFs, DELLAs regulate the expression of many genes in an inhibitory or enhancing manner under various biological events including the crosstalk between GA and other phytohormones, and the development of organs and tissues. DELLA-interacting TFs can be categorized into two types in terms of the effect of DELLA on their transacting activity. The first group includes those that are inhibited by DELLAs in terms of their DNA-binding activity, which includes the phytochrome interacting factor family of proteins involved in hypocotyl elongation, chlorophyll biosynthesis, fruit patterning, and cotyledon expansion; squamosa promoter binding-like proteins involved in floral transition; ethylene insensitive 3 and EIN3-like 1 proteins involved in GA-ethylene crosstalk; brassinazole-resistant 1 involved in GA-brassinosteroid crosstalk; and jasmonate ZIM domain and MYC2 proteins involved in GA-jasmonate crosstalk. The second group includes those TFs that affected in terms of their transcriptional activity but not DNA-binding activity upon interaction with DELLA, which includes the ABA-insensitive 3 and ABI5 involved in GA-abscisic acid crosstalk, indeterminate domain involved in feedback regulation of GA signaling, and Botrytis-susceptible interactor proteins involved in DELLAs transrepression activity. We also mentioned that interaction of DELLAs with proteins besides TFs regulates the crosstalk between GA and strigolactone, and tubulin folding. The interaction of all of these various TFs and proteins with DELLAs strongly demonstrates that DELLA functions as a hub protein linking GA signaling to a myriad of biological events.

The suppressive function of the rice DELLA protein SLR1 is dependent on its transcriptional activation activity.

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.