Jasmonate Zim-Domain Protein 9 Interacts With Slender Rice 1 to Mediate the Antagonistic Interaction Between Jasmonic and Gibberellic Acid Signals in Rice.
ABSTRACT: The jasmonic acid (JA) and gibberellic acid (GA) signaling pathways interact to coordinate stress responses and developmental processes. This coordination affects plant growth and yield, and is mediated by interactions between the repressors of each pathway, the JASMONATE ZIM-DOMAIN PROTEIN (JAZ) and DELLA proteins. In this study we attempted to identify rice (Oryza sativa) JAZs that interact with rice DELLAs such as SLENDER RICE 1 (SLR1). Analysis of protein-protein interactions showed that OsJAZ8 and OsJAZ9 interact with SLR1; OsJAZ9 also interacted with the SLR1-LIKE (SLRL) protein SLRL2. Based on this broader interaction, we explored the function of OsJAZ9 in JA and GA responses by analyzing transcript levels of the JA-responsive gene OsbHLH148 and the GA-responsive gene OsPIL14 in OsJAZ9-overexpressing (OsJAZ9-Ox) and osjaz9 mutant plants. OsbHLH148 and OsPIL14 encode key transcription factors controlling JA and GA responses, respectively, and JA and GA antagonistically regulate their expression. In OsJAZ9-Ox, the expression of OsbHLH148 was downregulated and the expression of OsPIL14 was upregulated. By contrast, in osjaz9 mutants, the expression of OsbHLH148 was upregulated and the expression of OsPIL14 was downregulated. These observations indicated that OsJAZ9 regulates both JA and GA responses in rice, and this finding was supported by the opposite expression patterns of OsDREB1s, downstream targets of OsbHLH148 and OsPIL14, in the OsJAZ9-Ox and osjaz9 plants. Together, these findings indicate that OsJAZ9 suppresses JA responses and promotes GA responses in rice, and the protein-protein interaction between OsJAZ9 and SLR1 is involved in the antagonistic interplay between JA and GA.
Project description:The rice SLR1 gene encodes the DELLA protein, and a loss-of-function mutation is dwarfed by inhibiting plant growth. We generate slr1-d mutants with a semi-dominant dwarf phenotype to target mutations of the DELLA/TVHYNP domain using CRISPR/Cas9 genome editing in rice. Sixteen genetic edited lines out of 31 transgenic plants were generated. Deep sequencing results showed that the mutants had six different mutation types at the target site of the TVHYNP domain of the SLR1 gene. The homo-edited plants selected individuals without DNA (T-DNA) transcribed by segregation in the T1 generation. The slr1-d7 and slr1-d8 plants caused a gibberellin (GA)-insensitive dwarf phenotype with shrunken leaves and shortened internodes. A genome-wide gene expression analysis by RNA-seq indicated that the expression levels of two GA-related genes, GA20OX2 (Gibberellin oxidase) and GA3OX2, were increased in the edited mutant plants, suggesting that GA20OX2 acts as a convert of GA12 signaling. These mutant plants are required by altering GA responses, at least partially by a defect in the phytohormone signaling system process and prevented cell elongation. The new mutants, namely, the slr1-d7 and slr1-d8 lines, are valuable semi-dominant dwarf alleles with potential application value for molecule breeding using the CRISPR/Cas9 system in rice.
Project description:Submergence-tolerant rice maintains viability during complete submergence by limiting underwater elongation until floodwaters recede. Acclimation responses to submergence are coordinated by the submergence-inducible Sub1A, which encodes an ethylene-responsive factor-type transcription factor (ERF). Sub1A is limited to tolerant genotypes and sufficient to confer submergence tolerance to intolerant accessions. Here we evaluated the role of Sub1A in the integration of ethylene, abscisic acid (ABA), and gibberellin (GA) signaling during submergence. The submergence-stimulated decrease in ABA content was Sub1A-independent, whereas GA-mediated underwater elongation was significantly restricted by Sub1A. Transgenics that ectopically express Sub1A displayed classical GA-insensitive phenotypes, leading to the hypothesis that Sub1A limits the response to GA. Notably Sub1A increased the accumulation of the GA signaling repressors Slender Rice-1 (SLR1) and SLR1 Like-1 (SLRL1) and concomitantly diminished GA-inducible gene expression under submerged conditions. In the Sub1A overexpression line, SLR1 protein levels declined under prolonged submergence but were accompanied by an increase in accumulation of SLRL1, which lacks the DELLA domain. In the presence of Sub1A, the increase in these GA signaling repressors and decrease in GA responsiveness were stimulated by ethylene, which promotes Sub1A expression. Conversely, ethylene promoted GA responsiveness and shoot elongation in submergence-intolerant lines. Together, these results demonstrate that Sub1A limits ethylene-promoted GA responsiveness during submergence by augmenting accumulation of the GA signaling repressors SLR1 and SLRL1.
Project description:The plant hormone gibberellin (GA) is crucial for multiple aspects of plant growth and development. To study the relevant regulatory mechanisms, we isolated a rice mutant earlier flowering1, el1, which is deficient in a casein kinase I that has critical roles in both plants and animals. el1 had an enhanced GA response, consistent with the suppression of EL1 expression by exogenous GA(3). Biochemical characterization showed that EL1 specifically phosphorylates the rice DELLA protein SLR1, proving a direct evidence for SLR1 phosphorylation. Overexpression of SLR1 in wild-type plants caused a severe dwarf phenotype, which was significantly suppressed by EL1 deficiency, indicating the negative effect of SLR1 on GA signalling requires the EL1 function. Further studies showed that the phosphorylation of SLR1 is important for maintaining its activity and stability, and mutation of the candidate phosphorylation site of SLR1 results in the altered GA signalling. This study shows EL1 a novel and key regulator of the GA response and provided important clues on casein kinase I activities in GA signalling and plant development.
Project description:Gibberellins (GAs) are phytohormones that regulate various developmental processes in plants. The initial GA signalling events involve the binding of a GA to the soluble GA receptor protein GID1, followed by the binding of the complex to the negative transcriptional regulator of GA signaling, the DELLA protein. Although X-ray structures for certain Arabidopsis GID1/GA/DELLA protein complexes have previously been determined, examination of these complexes did not fully clarify how a DELLA protein recognizes and binds to a GID1/GA complex. Herein, we present a study aimed at physically defining, via a combination of gel chromatography, isothermal titration calorimetry (ITC), small-angle X-ray scattering experiments (SAXS), NMR spectroscopy and mutagenesis, how the rice DELLA protein (SLR1) binds to the rice GID1/GA complex. We have identified the shortest SLR1 sequence (M28-A112) that binds the rice GID/GA complex tightly. The binding constant for the ternary complex that includes SLR1(M28-A112) is 2.9?×?107?M-1; the binding is enthalpically driven and does not depend on the chemical nature of the bound GA. Furthermore, the results of SAXS, ITC, and gel filtration experiments indicate that when free in solution, SLR1(M28-A112) is a natively unfolded protein. The NMR experiments expand this observation to show that the unfolded mutant also contains a small amount of marginally stable secondary structure. Conversely, the protein has a highly ordered structure when bound one-to-one to GID1/GA.
Project description:The widespread introduction of semi-dwarf1 (sd1), also known as the 'Green Revolution' gene, has dramatically increased rice yield. However, the extensive use of limited sources of dwarf genes may cause 'bottleneck' effects in breeding new rice varieties. Alternative dwarf germplasms are quite urgent for rice breeding. Here, we characterized a new allele of the rice Slr1-d mutant, Slr1-d6, which reduced plant height by 37%, a much milder allele for dwarfism. Slr-d6 was still responsive to gibberellin (GA) to a reduced extent. The mutation site in Slr1-d6 was less conserved in the TVHYNP domain, leading to the specific semi-dominant dwarf phenotype. Expression of SLR1 and five key GA biosynthetic genes was disturbed in Slr1-d6, and the interaction between Slr1-d6 and GID1 was decreased. In the genetic background of cultivar 9311 with sd1 eliminated, Slr1-d6 homozygous plants were ~70 cm tall. Moreover, Slr1-d6 heterozygous plants were equivalent in height to the standard sd1 semi-dwarf 9311, but with a 25% yield increase, showing its potential application in hybrid rice breeding.
Project description: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.
Project description:The breeding of cereals with altered gibberellin (GA) signaling propelled the 'Green Revolution' by generating semidwarf plants with increased tiller number. The mechanism by which GAs promote shoot height has been studied extensively, but it is not known what causes the inverse relationship between plant height and tiller number. Here we show that rice tiller number regulator MONOCULM 1 (MOC1) is protected from degradation by binding to the DELLA protein SLENDER RICE 1 (SLR1). GAs trigger the degradation of SLR1, leading to stem elongation and also to the degradation of MOC1, and hence a decrease in tiller number. This discovery provides a molecular explanation for the coordinated control of plant height and tiller number in rice by GAs, SLR1 and MOC1.
Project description:Many years after the discovery of GID1 as the GA receptor, the belief in the existence of GA-binding protein (GBP) in aleurone cells still persists. Actually, some recent reviews and textbooks still contain information about the possibility of an alternative GA perception mechanism mediated by GBPs. Thus, to finally clarify this issue, we examined the expression of GA-regulated genes using rice embryoless half-seeds of four kinds of GA signaling mutants, each one independently carrying mutated GID1, DELLA, and GA-related F-box, which are all encoded by single genes in rice, allowing for convenient study of the GA perception system. The comprehensive microarray analysis using these rice mutants revealed that all genes tested to be responsive to GA in the wild-type (WT) aleurone cells consistently did not respond to GA in the gid1 or slr1 mutant. All microarray experiments were performed according to the manufacturerM-bM-^@M-^Ys manual. The Feature Extraction software (Agilent Technologies) was used to delineate and measure the Cy3 and Cy5 signal intensities of each spot in the array. The resulting data were normalized using the Variance-stabilizing normalization (VSN) algorithm.
Project description:Plant-microbe mutualisms can improve plant defense, but the impact of root endophytes on below-ground herbivore interactions remains unknown. We investigated the effects of the root endophyte Piriformospora indica on interactions between rice (Oryza sativa) plants and its root herbivore rice water weevil (RWW; Lissorhoptrus oryzophilus), and how plant jasmonic acid (JA) and GA regulate this tripartite interaction. Glasshouse experiments with wild-type rice and coi1-18 and Eui1-OX mutants combined with nutrient, jasmonate and gene expression analyses were used to test: whether RWW adult herbivory above ground influences subsequent damage caused by larval herbivory below ground; whether P. indica protects plants against RWW; and whether GA and JA signaling mediate these interactions. The endophyte induced plant tolerance to root herbivory. RWW adults and larvae acted synergistically via JA signaling to reduce root growth, while endophyte-elicited GA biosynthesis suppressed the herbivore-induced JA in roots and recovered plant growth. Our study shows for the first time the impact of a root endophyte on plant defense against below-ground herbivores, adds to growing evidence that induced tolerance may be an important root defense, and implicates GA as a signal component of inducible plant tolerance against biotic stress.
Project description: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.