Dominant and pleiotropic effects of a GAI gene in wheat results from a lack of interaction between DELLA and GID1

Dominance, semidominance, and recessiveness are important modes of Mendelian inheritance. The phytohormone gibberellin (GA) regulates many plant growth and developmental processes. The previously cloned semidominant GA-insensitive (GAI) genes Reduced height1 (Rht1) and Rht2 in wheat (Triticum aestivum) were the basis of the Green Revolution. However, no completely dominant GAI gene has been cloned. Here, we report the molecular characterization of Rht-B1c, a dominant GAI allele in wheat that confers more extreme characteristics than its incompletely dominant alleles. Rht-B1c is caused by a terminal repeat retrotransposons in miniature insertion in the DELLA domain. Yeast two-hybrid assays showed that Rht-B1c protein fails to interact with GA-INSENSITIVE DWARF1 (GID1), thereby blocking GA responses and resulting in extreme dwarfism and pleiotropic effects. By contrast, Rht-B1b protein only reduces interaction with GID1. Furthermore, we analyzed its functions using near-isogenic lines and examined its molecular mechanisms in transgenic rice. These results indicated that the affinity between GID1 and DELLA proteins is key to regulation of the stability of DELLA proteins, and differential interactions determine dominant and semidominant gene responses to GA.     

Interaction of BTB-TAZ protein MdBT2 and DELLA protein MdRGL3a regulates nitrate-mediated plant growth

Nitrate acts as a vital signal molecule in the modulation of plant growth and development. The phytohormones gibberellin (GA) is also involved in this process. However, the exact molecular mechanism of how nitrate and GA signaling pathway work together in regulating plant growth remains poorly understood. In this study, we found that a nitrate-responsive BTB/TAZ protein MdBT2 participates in regulating nitrate-induced plant growth in apple (Malus × domestica). Yeast two-hybridization, protein pull-down, and bimolecular fluorescence complementation (BiFC) assays showed that MdBT2 interacts with a DELLA protein MdRGL3a, which is required for the ubiquitination and degradation of MdRGL3a proteins via a 26S proteasome-dependent pathway. Furthermore, heterologous expression of MdBT2 partially rescued growth inhibition caused by overexpression of MdRGL3a in Arabidopsis. Taken together, our findings indicate that MdBT2 promotes nitrate-induced plant growth partially through reducing the abundance of the DELLA protein MdRGL3a.

The BTB-TAZ protein interacts with and promotes ubiquitination and degradation of DELLA protein, thus regulating plant growth in response to nitrate.     

Overexpression of RoDELLA impacts the height, branching, and flowering behaviour of Pelargonium?×?domesticum transgenic plants

Key message

We reported the cloning of a rose DELLA gene. We obtained transgenic Pelargonium lines overexpressing this gene which presented several phenotypes in plant growth, root growth, flowering time and number of inflorescences.

Abstract

Control of development is an important issue for production of ornamental plant. The plant growth regulator, gibberellins (GAs), plays a pivotal role in regulating plant growth and development. DELLA proteins are nuclear negative regulator of GA signalling. Our objective was to study the role of GA in the plant architecture and in the blooming of ornamentals. We cloned a rose DELLA homologous gene, RoDELLA, and studied its function by genetic transformation of pelargonium. Several transgenic pelargonium (Pelargonium?×?domesticum ‘Autum Haze’) lines were produced that ectopically expressed RoDELLA under the control of the 35S promoter. These transgenic plants exhibited a range of phenotypes which could be related to the reduction in GA response. Most of transgenic plants showed reduced growth associated to an increase of the node and branch number. Moreover, overexpression of RoDELLA blocked or delayed flowering in transgenic pelargonium and exhibited defects in the root formation. We demonstrated that pelargonium could be used to validate ornamental gene as the rose DELLA gene. RoDELLA overexpression modified many aspects of plant developmental pathways, as the plant growth, the transition of vegetative to floral stage and the ability of rooting.     

Arabidopsis DELLA Protein Degradation Is Controlled by a Type-One Protein Phosphatase,TOPP4

Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway.     

Role of gibberellic acid in tomato defence against potato purple top phytoplasma infection

Infection of tomato by potato purple top (PPT) phytoplasma causes disruption of gibberellin (GA) homeostasis in the plant host. Such pathologically‐induced GA deficiency can be partially reversed by exogenous application of GA. This study was designed to explore the role of GA in tomato defence response against phytoplasmal disease, and to determine whether pretreatment with GA would protect healthy tomato seedlings from subsequent phytoplasmal infection and disease development. Our results revealed that, following exogenous GA application and subsequent PPT phytoplasma graft inoculation, there was an apparently coordinated down‐regulation of the gene encoding a key GA signalling component and growth repressor known as DELLA protein (GAI) and up‐regulation of genes involved in salicylic acid (SA) synthesis (ICS1), signalling (NIM1) and downstream defence responses (PRP‐1). Our results also indicated that differential regulation of the above genes was correlated with an increase in activities of defence‐related enzymes β‐1,3‐glucanase and chitinase. The data presented in this communication provide evidence to suggest that GA may act via DELLA and SA signalling pathways to modulate host defence in response to PPT phytoplasma infection. Although the GA pretreatment‐induced defence was not sufficient to prevent a systemic infection, it reduced phytoplasma titre and significantly attenuated disease symptoms. While the actual molecular mechanism underlying the GA‐induced plant defence remains elusive, findings from the current study open new opportunities for in‐depth studies of the functional role of the GA signalling network during defence response against phytoplasmal infection.     

Procera is a putative DELLA mutant in tomato (Solanum lycopersicum): effects on the seed and vegetative plant

The procera (pro) mutant of tomato exhibits a well-characterizedconstitutive gibberellic acid (GA) response phenotype. The tomatoDELLA gene LeGAI in the pro mutant background contains a pointmutation that results in an amino acid change in the conservedVHVID putative DNA-binding domain in LeGAI to VHEID. This samepoint mutation is in four different genetic backgrounds exhibitingthe pro phenotype, suggesting that this mutation co-segregateswith the pro phenotype. Complementation of the mutant with aconstitutively expressed wild-type LeGAI gene sequence was notconclusive due to the infertility of transgenic plants. Thepro mutation alters tomato branching architecture through differentialsuppression of axillary bud development, indicating a role forDELLA proteins in the regulation of plant structure. Isolatedgib-1 pro double mutant embryo axes, which are unable to synthesizeGA, germinate faster than their wild-type counterparts, andexert greater embryo growth potential. The pro mutation is thereforeregulating GA responses within the tomato embryo. Transientexpression of a LeGAI–GFP (green fluorescent protein)fusion protein in onion epidermis results in its location tothe nucleus, and this protein is rapidly degraded by the proteasomein the presence of GA. Key words: Branching pattern, DELLA, embryo growth potential, tomato seed germination Received 12 October 2007; Revised 27 November 2007 Accepted 28 November 2007     

Biochemical Insights on Degradation of Arabidopsis DELLA Proteins Gained From a Cell-Free Assay System

The phytohormone gibberellic acid (GA) regulates diverse aspects of plant growth and development. GA responses are triggered by the degradation of DELLA proteins, which function as repressors in GA signaling pathways. Recent studies in Arabidopsis thaliana and rice (Oryza sativa) have implied that the degradation of DELLA proteins occurred via the ubiquitin-proteasome system. Here, we developed an Arabidopsis cell-free system to recapitulate DELLA protein degradation in vitro. Using this cell-free system, we documented that Lys-29 of ubiquitin is the major site for ubiquitin chain formation to mediate DELLA protein degradation. We also confirmed the specific roles of GA receptors and multisubunit E3 ligase components in regulating DELLA protein degradation. In addition, blocking DELLA degradation with a PP1/PP2A phosphatase inhibitor in our cell-free assay suggested that degradation of DELLA proteins required protein Ser/Thr dephosphorylation activity. Furthermore, our data revealed that the LZ domain of Arabidopsis DELLA proteins is essential for both their stability and activity. Thus, our in vitro degradation system provides biochemical insights into the regulation of DELLA protein degradation. This in vitro assay system could be widely adapted for dissecting cellular signaling pathways in which regulated proteolysis is a key recurrent theme.     

Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation,to promote viral infection and symptom development in N. benthamiana

The phytohormone gibberellin (GA) is a vital plant signaling molecule that regulates plant growth and defense against abiotic and biotic stresses. To date, the molecular mechanism of the plant responses to viral infection mediated by GA is still undetermined. DELLA is a repressor of GA signaling and is recognized by the F-box protein, a component of the SCF^SLY1/GID2 complex. The recognized DELLA is degraded by the ubiquitin-26S proteasome, leading to the activation of GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptoms and abnormal flower development. The infection by ALCScV significantly altered the expression of GA pathway-related genes and decreased the content of endogenous GA in N. benthamiana. Furthermore, ALCScV-encoded C4 protein interacts with the DELLA protein NbGAI and interferes with the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI, leading to inhibition of the GA signaling pathway. Silencing of NbGAI or exogenous GA₃ treatment significantly reduces viral accumulation and disease symptoms in N. benthamiana plants. The same results were obtained from experiments with the C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism by which geminivirus C4 proteins control viral infection and disease symptom development by interfering with the GA signaling pathway.     

Release of the repressive activity of rice DELLA protein SLR1 by gibberellin does not require SLR1 degradation in the gid2 mutant

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.     

Functional analysis of cotton orthologs of GA signal transduction factors GID1 and SLR1

Gibberellic acid (GA) is both necessary and sufficient to promote fiber elongation in cultured fertilized ovules of the upland cotton variety Coker 312. This is likely due to the temporal and spatial regulation of GA biosynthesis, perception, and subsequent signal transduction that leads to alterations in gene expression and morphology. Our results indicate that the initiation of fiber elongation by the application of GA to cultured ovules corresponds with increased expression of genes that encode xyloglucan endotransglycosylase/hydrolase (XTH) and expansin (EXP) that are involved in promoting cell elongation. To gain a better understanding of the GA signaling components in cotton, that lead to such changes in gene expression, two GA receptor genes (GhGID1a and GhGID1b) and two DELLA protein genes (GhSLR1a and GhSLR1b) that are orthologous to the rice GA receptor (GID1) and the rice DELLA gene (SLR1), respectively, were characterized. Similar to the GA biosynthetic genes, expression of GhGID1a and GhGID1b is under the negative regulation by GA while GA positively regulates GhSLR1a. Recombinant GST-GhGID1s showed GA-binding activity in vitro that was augmented in the presence of GhSLR1a, GhSLR1b, or rice SLR1, indicating complex formation between the receptors and repressor proteins. This was further supported by the GA-dependent interaction of these proteins in yeast cells. Ectopic expression of the GhGID1a in the rice gid1-3 mutant plants rescued the GA-insensitive dwarf phenotype, which demonstrates that it is a functional GA receptor. Furthermore, ectopic expression of GhSLR1b in wild type Arabidopsis led to reduced growth and upregulated expression of DELLA-responsive genes.     

Transgenic tomato plants expressing strawberry <Emphasis Type="ItalicSmallCaps">d</Emphasis>-<Emphasis Type="Italic">galacturonic acid reductase</Emphasis> gene display enhanced tolerance to abiotic stresses

After analyzing tomato plants transformed with GalUR gene for their ascorbic acid contents, it was found that some transgenic lines contained higher levels of ascorbic acid compared to control plants. In the present study, callus induction rate was 50.2 % in the explant and shoot regeneration rate was 51.5 % from the callus with transformation efficiency of 3.0 %. Based on PCR and Southern blot analysis, three independent transformants containing the insert gene were selected. Phenotypic traits of these transgenic progeny were similar to those of control tomatoes. Tomatoes (H15) with high fruit ascorbic acid contents were selected for next generation (GalUR T₃) analysis. Transgenic tomatoes with increased ascorbic acid contents were found to be more tolerant to abiotic stresses induced by viologen, NaCl, or mannitol than non-transformed plants. In leaf disc senescence assay, the tolerance of these transgenic plants was better than control plants because they could retain higher chlorophyll contents. Under salt stress of less than 200 mM NaCl, these transgenic plants survived. However, control plants were unable to survive such high salt stress. Ascorbic acid contents in the transgenic plants were inversely correlated with MDA contents, especially under salt stress conditions. The GalUR gene was expressed in H15 tomatoes, but not in control plants. Higher expression levels of antioxidant genes (APX and CAT) were also found in these transgenic plants compared to that in the control plants. However, no detectable difference in SOD expression was found between transgenic plants and control plants. Results from this study suggest that the increase in ascorbic acid contents in plants could up-regulate the antioxidant system to enhance the tolerance of transgenic tomato plants to various abiotic stresses.     

Characterisation of the DELLA subfamily in apple (Malus x domestica Borkh.)

The hormone gibberellic acid (GA) regulates growth and development throughout the plant life cycle. DELLA proteins are key components of the GA signalling pathway and act to repress GA responses. The “DELLA” amino acid motif is highly conserved among diverse species and is essential for GA-induced destruction of DELLA proteins, which relieves repression. Six genes encoding the DELLA motif were identified within an apple expressed sequence tag (EST) database. Full-length cDNA clones were obtained by RACE and these were designated MdRGL1a/b, MdRGL2a/b, and MdRGL3a/b. Sequence alignment of the predicted proteins indicates that the MdDELLAs are 37–93% homologous to one another and 44–65% to the Arabidopsis DELLAs. The MdDELLAs cluster into three pairs, which reflect the presumed allopolyploid origins of the Maloideae. Expression analysis using quantitative real-time PCR indicates that all three pairs of MdDELLA mRNAs are expressed at the highest levels in summer arrested shoot tips and in autumn vegetative buds. Transgenic Arabidopsis expressing MdRGL2a have smaller leaves and shorter stems, take longer to flower in short days, and exhibit a reduced response to exogenous GA₃, indicating significant conservation of gene function between DELLA proteins from apple and Arabidopsis. Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings

Germination followed by seedling growth constitutes two essential steps in the initiation of a new life cycle in plants, and in cereals, completion of these steps is regulated by sugar starvation and the hormone gibberellin. A calcium-dependent protein kinase 1 gene (OsCDPK1) was identified by differential screening of a cDNA library derived from sucrose-starved rice suspension cells. The expression of OsCDPK1 was found to be specifically activated by sucrose starvation among several stress conditions tested as well as activated transiently during post-germination seedling growth. In gain- and loss-of-function studies performed with transgenic rice overexpressing a constitutively active or RNA interference gene knockdown construct, respectively, OsCDPK1 was found to negatively regulate the expression of enzymes essential for GA biosynthesis. In contrast, OsCDPK1 activated the expression of a 14-3-3 protein, GF14c. Overexpression of either constitutively active OsCDPK1 or GF14c enhanced drought tolerance in transgenic rice seedlings. Hence, our studies demonstrated that OsCDPK1 transduces the post-germination Ca²⁺ signal derived from sugar starvation and GA, refines the endogenous GA concentration and prevents drought stress injury, all essential functions to seedling development at the beginning of the life cycle in rice.