SLR1 Protein Research Articles

The JA-OsJAZ6-DELLA module controls the tillering and drought stress response in rice

Jasmonic acid (JA) plays crucial functions during plant growth and stress response, but its roles and regulatory mechanism in plant branching remain largely unknown. Rice basal branching (tillering) is an essential agronomic trait that affects crop production. Here, we report that OsJAZ6, the repressor of JA signaling, negatively modulates rice tillering and drought stress tolerance. Loss-of-function mutants of OsJAZ6 exhibit a significant increase in tiller number, while OsJAZ6ΔJas-overexpression lines produce fewer tillers than wild-type plants. Further investigations show that function loss of OsJAZ6 promotes the tiller bud growth rather than formation. Mechanistic studies show that OsJAZ6 interacts with rice DELLA/SLR1 (SLENDER RICE 1), a transcription repressor of gibberellin (GA) signaling, and the interaction promotes SLR1 degradation, which further facilitates the degradation of rice tillering regulator MOC1 (MONOCULM 1), thereby inhibiting the tiller bud growth. In agreement, the slr1 mutant exhibits fewer tillers than wild type. Consistently, application of JA promotes the growth of tiller bud and thus increases the tiller number, while GA treatment results in opposite result. Meanwhile, osjaz6 mutants display enhanced drought tolerance, coupled with increased JA sensitivity, while the slr1 mutant shows the reverse behavior. Collectively, our data demonstrate that OsJAZ6 negatively modulates rice tillering as well as drought stress tolerance by destabilizing SLR1 protein. Our data shed light on the regulatory mechanism of controlling the tiller development and drought stress response in rice by the JA-OsJAZ6-SLR1 module.

OsCSN1 Regulates the Growth and Development of Rice Seedlings through the Degradation of SLR1 in the GA Signaling Pathway

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is involved in various regulations during plant development. The CSN is a highly conserved protein complex with nine subunits, and CSN1 acts in a network of signaling pathways critical for plant development. Although CSN1 has been widely studied in Arabidopsis thaliana, there have been few investigations on CSN1 in rice. In this paper, using the CRISPR/Cas9 system, CSN1 was edited from Oryza sativa subsp. japonica (rice). After screening out the OsCSN1 knockout mutant and OsCSN1 reduce mutant, the phenotype and protein expression were identified under different light conditions. Experiments showed that in OsCSN1 knockout mutant and OsCSN1 reduce mutant, the SLR1 protein was rapidly degraded at the rice seedling. In this study, the OsCSN1 acted as a negative regulator to affect seedling growth and development through CUL4-based E3 ligase, which is involved in the degradation of SLR1 in the GA signaling pathway. However, its direct target and mechanism of action are not clear.

The rice YABBY4 gene regulates plant growth and development through modulating the gibberellin pathway.

YABBY genes encode seed plant-specific transcription factors that play pivotal roles in diverse aspects of leaf, shoot, and flower development. Members of the YABBY gene family are primarily expressed in lateral organs in a polar manner and function to specify abaxial cell fate in dicotyledons, but this polar expression is not conserved in monocotyledons. The function of YABBY genes is therefore not well understood in monocotyledons. Here we show that overexpression of the rice (Oryza sativa L.) YABBY4 gene (OsYABBY4) leads to a semi-dwarf phenotype, abnormal development in the uppermost internode, an increased number of floral organs, and insensitivity to gibberellin (GA) treatment. We report on an important role for OsYABBY4 in negative control of the expression of a GA biosynthetic gene by binding to the promoter region of the gibberellin 20-oxidase 2 gene (GA20ox2), which is a direct target of SLR1 (the sole DELLA protein negatively controlling GA responses in rice). OsYABBY4 also suppresses the expression level of SLR1 and interacts with SLR1 protein. The interaction inhibits GA-dependent degradation of SLR1 and therefore leads to GA insensitivity. These data together suggest that OsYABBY4 serves as a DNA-binding intermediate protein for SLR1 and is associated with the GA signaling pathway regulating gene expression during plant growth and development.

Phosphorylation of d-allose by hexokinase involved in regulation of OsABF1 expression for growth inhibition in Oryza sativa L.

We previously reported that a rare sugar D-allose, which is the D-glucose epimer at C3, inhibits the gibberellin-dependent responses such as elongation of the second leaf sheath and induction of α-amylase in embryo-less half seeds in rice (Fukumoto et al. 2011). D-Allose suppresses expressions of gibberellin-responsive genes downstream of SLR1 protein in the gibberellin-signaling through hexokinase (HXK)-dependent pathway. In this study, we discovered that D-allose induced expression of ABA-related genes including OsNCED1-3 and OsABA8ox1-3 in rice. Interestingly, D-allose also up-regulated expression of OsABF1, encoding a conserved bZIP transcription factor in ABA signaling, in rice. The D-allose-induced expression of OsABF1 was diminished by a hexokinase inhibitor, D-mannoheptulose (MNH). Consistently, D-allose also inhibited Arabidopsis growth, but failed to trigger growth retardation in the glucose-insensitive2 (gin2) mutant, which is a loss-of-function mutant of the glucose sensor AtHXK1. D-Allose activated AtABI5 expression in transgenic gin2 over-expressing wild-type AtHXK1 but not in gin2 over-expressing the catalytic mutant AtHXK1(S177A), indicating that the D-allose phosphorylation by HXK to D-allose 6-phosphate (A6P) is the first step for the up-regulation of AtABI5 gene expression as well as D-allose-induced growth inhibition. Moreover, overexpression of OsABF1 showed increased sensitivity to D-allose in rice. These findings indicated that the phosphorylation of D-allose at C6 by hexokinase is essential and OsABF1 is involved in the signal transduction for D-allose-induced growth inhibition.

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.

Isolation and characterization of dominant dwarf mutants, Slr1-d, in rice

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.

Specificity and Similarity of Functions of the Aux/IAA Genes in Auxin Signaling of Arabidopsis Revealed by Promoter-Exchange Experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14

As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis (Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2::axr2-1::GFP and pMSG2::slr-1::GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2::msg2-1::GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1::GFP or slr-1::GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2::msg2-1::GFP and pAXR2::slr-1::GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.

EUI1, Encoding a Putative Cytochrome P450 Monooxygenase, Regulates Internode Elongation by Modulating Gibberellin Responses in Rice

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control, and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellin was elevated in the mutant, whereas its growth in response to gibberellin is similar to that of the wild type, suggesting that the higher level accumulation of gibberellin in the eui1 mutant causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes which encode gibberellin biosynthesis enzymes were altered. We cloned the EUI1 gene, which encodes a putative cytochrome P450 monooxygenase, by map-based cloning and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the gibberellin-deficient-like phenotypes, which could be partially reversed by supplementation with gibberellin. Furthermore, apart from the alteration of expression levels of the gibberellin biosynthesis genes, accumulation of SLR1 protein was found in the overexpressing transgenic plants, indicating that the expression level of EUI1 is implicated in both gibberellin-mediated SLR1 destruction and a feedback regulation in gibberellin biosynthesis. Therefore, we proposed that EUI1 plays a negative role in gibberellin-mediated regulation of cell elongation in the uppermost internode of rice.

Gibberellin Signal Transduction in Rice

The rice SLR1 gene is an ortholog of ArabidopsisGA1 and RGA, which function as negative regulators of gibberellin (GA) signal transduction. Genetic, physiological and biochemical analyses have revealed that the SLR1 protein, which is localized in the nucleus, is degraded by an upstream GA signal to induce the downstream GA actions. In other words, SLR1 suppresses GA activity and the GA-dependent degradation of the SLR1 protein releases SLR1-suppressed GA action. We identified the link between SLR1 degradation and GA signaling when we were analyzing the rice GA-insensitivedwarf (GID2) mutant. The GID2 gene encodes a putative F-box protein that is a component of an SCF complex associated with protein degradation via the 26S proteasome. Biochemical analyses of SLR1 and GID2 suggest that the proteasome-dependent degradation of SLR1 is initiated by phosphorylatian of SLR1 and mediated by an SCF complex involving GID2. In this review, we propose a model for GA signal transduction in rice.

Pollen-stigma adhesion in Brassica spp involves SLG and SLR1 glycoproteins.

The adhesion of pollen grains to the stigma is the first step of pollination in flowering plants. During this step, stigmas discriminate between pollen grains that can and cannot be permitted to effect fertilization. This selection is operated by various constituents of the cell walls of both partners. Several genes structurally related to the self-incompatibility system that prevents self-pollination in Brassica spp are known to target their products into the stigma cell wall. We proposed previously that one of these genes, the one encoding the S locus glycoprotein (SLG)-like receptor 1 (SLR1), which is coexpressed with that encoding SLG, may participate in pollen-stigma adhesion. Here, we exploit a biomechanical assay to measure the pollen adhesion force and show that it is reduced both by transgenic suppression of SLR1 expression and by pretreatment of wild-type stigmas with anti-SLR1 antibodies, anti-SLG antibodies, or pollen coat-protein extracts. Our results indicate a common adhesive function for the SLR1 and SLG proteins in the pollination process.

Pollen-Stigma Adhesion in Kale Is Not Dependent on the Self-(In)Compatibility Genotype.

The adhesion of pollen on the stigmas of flowering plants is a critical step for the success of reproduction in angiosperms, long considered to present some specificity in terms of self-incompatibility. We carried out quantitative measurements of the pollen-stigma adhesion (expressed in Newtons) in kale (Brassica oleracea), using the flotation force of Archimedes exerted by dense sucrose solutions (50%, w/v) to release pollen grains fixed on the surface of stigmas. We demonstrate that pollen adhesion varies with the genotypes of the plants used as partners, but increases with time in all cases for about 30 to 60 min after pollination. There is no correlation with the self- or cross-status of the pollinations, nor with the self-compatible or -incompatible genotypes of the parents. Only late events of pollination, after the germination or arrest of the pollen tube, depend on compatibility type. Biochemical and physiological dissection of pollen-stigma adhesion points to major components of this interaction: among male components, the pollen coating, eliminated by delipidation (or modified by mutation in the case of the cer mutants of the related species Arabidopsis thaliana), plays a major role in adhesion; the genetic background of the pollen parent is also of some importance. On the female side, the developmental stage of the stigma and the protein constituents of the stigmatic pellicle are critical for pollen capture. The SLG and SLR1 proteins are not involved in the initial stages of pollen adhesion on the stigma but one or both may be involved in the later stages.

SLR1 function is dispensable for both self-incompatible rejection and self-compatible pollination processes inBrassica

TheSLR1 gene inBrassica is related both in DNA sequence and in pattern of expression to theS-locus glycoprotein (SLG) gene involved in the self-incompatibility mechanism which recognises and arrests the germination of self pollen. However,SLR1 shows minimal allelic variation and is expressed in both self-incompatible and compatibleBrassica lines and in related, self-compatible cruciferous plants. The function of the SLR1 protein is unknown. TheSLR1 gene was specifically ablated in self-incompatible and self-compatibleBrassica plants byAgrobacterium-mediated transformation with an antisense construct. Primary transformants and homozygous T2 progeny of both self-incompatibleB. oleracea and self-compatibleB. napus recipients were found to exhibit normal pollination responses despite having no detectable SLR1 glycoprotein. This shows that the high, wild-type level of SLR1 protein is not required to sustain the self-incompatibility reaction, nor is it necessary for successful intra-specific cross-pollination between compatible lines.

Immunodetection of Protein Glycoforms Encoded by Two Independent Genes of the Self-Incompatibility Multigene Family of Brassica

Glycoprotein products of two highly homologous Brassica S gene family members were studied: SLSG (S locus-specific glycoprotein), product of an SLG gene at the S locus, and SLR1 (S locus-related) protein, product of the SLR1 gene, a gene unlinked to the S locus. A polyclonal antibody directed against a trpE-SLR1 fusion protein facilitated study of the SLR1 protein. SLR1 protein was detected in a number of crucifer species. No variation in the level of this protein was found between self-compatible and self-incompatible plants. Both SLSG and SLR1 protein occurred as glycoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Each glycoform had several charge forms, indicated by elution patterns from a high performance liquid chromatography cation exchange column and behavior on two-dimensional gels. Deglycosylation of both SLSG and SLR1 protein caused loss of the glycoforms, which apparently arose from differences in glycosylation. Consistent with their apparent similar post-translational processing, immunolocalization showed that SLR1 protein, like SLSG, accumulated in the stigma papillae cell walls. Thus, both SLSG and SLR1 protein are present at the site of pollen-stigma interaction.