Membrane-associated DELLA degradation modulates growth under carbon/nitrogen imbalance.

Regulation of Seed Germination: The Involvement of Multiple Forces Exerted via Gibberellic Acid Signaling

Gibberellins (GA) are known to influence phase change in Arabidopsis (Arabidopsis thaliana) as well as the development of trichomes, which are faithful epidermal markers of shoot maturation. They modulate these developmental programs in part by antagonizing DELLA repressors of growth, GIBBERELLIC ACID INSENSITIVE (GAI) and REPRESSOR OF ga1-3 (RGA). In this study, we have probed the relative roles played by RGA, GAI, and two homologs, RGA-LIKE1 (RGL1) and RGL2, in these processes and investigated molecular mechanisms through which they influence epidermal differentiation. We found that the DELLAs act collectively to regulate trichome initiation on all aerial organs and that the onset of their activity is accompanied by the repression of most genes known to regulate trichome production. These effects are consistent with the results of genetic analysis, which conclusively place theses genes downstream of the DELLAs. We find that repression of trichome regulatory genes is rapid, but involves an indirect, rather than a direct, molecular mechanism, which requires de novo protein synthesis. DELLA activity also influences postinitiation events and we show that GAI is a major repressor of trichome branching, a role in which it is antagonized by RGL1 and RGL2. Finally, we report that, in contrast to most other effects, the repression by GA applications of flower trichome initiation is not dependent on RGA, GAI, RGL1, or RGL2. In summary, our data show that DELLA proteins are central to trichome development in Arabidopsis and that their effect can be largely explained by their transcriptional influence on trichome initiation activators.

DELLA proteins restrain germination and elongation growth in Arabidopsis thaliana COP9 signalosome mutants

We investigated the physiological function of three Arabidopsis thaliana homologs of the gibberellin (GA) receptor GIBBERELLIN-INSENSITIVE DWARF1 (GID1) by determining the developmental consequences of GID1 inactivation in insertion mutants. Although single mutants developed normally, gid1a gid1c and gid1a gid1b displayed reduced stem height and lower male fertility, respectively, indicating some functional specificity. The triple mutant displayed a dwarf phenotype more severe than that of the extreme GA-deficient mutant ga1-3. Flower formation occurred in long days but was delayed, with severe defects in floral organ development. The triple mutant did not respond to applied GA. All three GID1 homologs were expressed in most tissues throughout development but differed in expression level. GA treatment reduced transcript abundance for all three GID1 genes, suggesting feedback regulation. The DELLA protein REPRESSOR OF ga1-3 (RGA) accumulated in the triple mutant, whose phenotype could be partially rescued by loss of RGA function. Yeast two-hybrid and in vitro pull-down assays confirmed that GA enhances the interaction between GID1 and DELLA proteins. In addition, the N-terminal sequence containing the DELLA domain is necessary for GID1 binding. Furthermore, yeast three-hybrid assays showed that the GA-GID1 complex promotes the interaction between RGA and the F-box protein SLY1, a component of the SCF(SLY1) E3 ubiquitin ligase that targets the DELLA protein for degradation.

Fruit Development: New Directions for an Old Pathway

Origin, evolution, and molecular function of DELLA proteins in plants

Gibberellins (GAs) modulate diverse developmental processes throughout the plant life cycle. However, the interaction between GAs and the circadian rhythm remains unclear. Here, we report that MUT9p-LIKE KINASE1 (MLK1) and MLK2 mediate the interaction between GAs and the circadian clock to regulate hypocotyl elongation in Arabidopsis thaliana DELLA proteins function as master growth repressors that integrate phytohormone signaling and environmental pathways in plant development. MLK1 and MLK2 interact with the DELLA protein REPRESSOR OF ga1-3 (RGA). Loss of MLK1 and MLK2 function results in plants with short hypocotyls and hyposensitivity to GAs. MLK1/2 and RGA directly interact with CIRCADIAN CLOCK ASSOCIATED1 (CCA1), which targets the promoter of DWARF4 (DWF4) to regulate its roles in cell expansion. MLK1/2 antagonize the ability of RGA to bind CCA1, and these factors coordinately regulate the expression of DWF4 RGA suppressed the ability of CCA1 to activate expression from the DWF4 promoter, but MLK1/2 reversed this suppression. Genetically, MLK1/2 act in the same pathway as RGA and CCA1 in hypocotyl elongation. Together, our results provide insight into the mechanism by which MLK1 and MLK2 antagonize the function of RGA in hypocotyl elongation and suggest that MLK1/2 coordinately mediate the regulation of plant development by GAs and the circadian rhythm in Arabidopsis.

The nuclear DELLA proteins are highly conserved repressors of hormone gibberellin (GA) signaling in plants. In Arabidopsis thaliana, GA derepresses its signaling pathway by inducing proteolysis of the DELLA protein REPRESSOR OF ga1-3 (RGA). SLEEPY1 (SLY1) encodes an F-box-containing protein, and the loss-of-function sly1 mutant has a GA-insensitive dwarf phenotype and accumulates a high level of RGA. These findings suggested that SLY1 recruits RGA to the SCFSLY1 E3 ligase complex for ubiquitination and subsequent degradation by the 26S proteasome. In this report, we provide new insight into the molecular mechanism of how SLY1 interacts with the DELLA proteins for controlling GA response. By yeast two-hybrid and in vitro pull-down assays, we demonstrated that SLY1 interacts directly with RGA and GA INSENSITIVE (GAI, a closely related DELLA protein) via their C-terminal GRAS domain. The rga and gai null mutations additively suppressed the recessive sly1 mutant phenotype, further supporting the model that SCFSLY1 targets both RGA and GAI for degradation. The N-terminal DELLA domain of RGA previously was shown to be essential for GA-induced degradation. However, we found that this DELLA domain is not required for protein-protein interaction with SLY1 in yeast (Saccharomyces cerevisiae), suggesting that its role is in a GA-triggered conformational change of the DELLA proteins. We also identified a novel gain-of-function sly1-d mutation that increased GA signaling by reducing the levels of the DELLA protein in plants. This effect of sly1-d appears to be caused by an enhanced interaction between sly1-d and the DELLA proteins.

Plant–pathogen interactions involve intricate signaling networks that coordinate the plant immune response. Recognition of pathogens through pattern recognition receptors (PRRs) triggers activation of mitogen-activated protein kinase (MAPK) pathways, initiating a cascade of defense mechanisms. Central to these responses is the synthesis of phytohormones such as salicylic acid (SA), auxins–indole-3-acetic acid (IAA), and gibberellins–gibberellic acid (GA), pivotal for immune activation. This review explores the multifaceted roles of these phytohormones in plant immunity, drawing on recent findings from Arabidopsis thaliana and Gossypium hirsutum studies. The review discusses MAPK-mediated activation of TGA1/4 (TGACG sequence-specific binding protein 1/4) transcription factors enhancing SA biosynthesis via isochorismate synthase (ICS). Increased SA levels activate NPR1, promoting gene expression in immune-related pathways including systemic acquired resistance (SAR). Concurrently, pathogen-induced IAA synthesis activates auxin-responsive genes crucial for immune responses. Elevated biosynthesis of IAA from L-tryptophan activates these genes by degrading repressor molecules. IAA acts antagonistically to SA, conserving energy during pathogen infection. Additionally, GA is vital for plant growth and development, operating DELLA (Asp–Glu–Leu–Leu–Ala) protein degradation with the formation of a complex with gibberellin insensitive dwarf 1 (GID1). Once DELLA prevents releasing GA-related response reactions, it is extremelly crucial for GA actions. In general, the review explores the intricate interplay between SA, IAA, and GA, highlighting SA's antagonistic regulation of GA signaling and the synergistic effects of auxin and GA. Understanding these hormone–mediated pathways is crucial for elucidating precise mechanisms underlying plant immunity. Insights gained could inform strategies to enhance plant resistance against pathogens, contributing to sustainable agriculture and global food security efforts.

The perception of the phytohormone gibberellin (GA) by its nuclear receptor GIBBERELLIN INSENSITIVE DWARF1 (GID1) triggers polyubiquitination and proteasomal degradation of master growth regulators-DELLA proteins-mediated by the SCFSLY1/GID2 E3 ubiquitin ligase complex. DELLA-encoding genes are known as 'Green Revolution' genes, as their dominant mutations lead to semidwarf cereal varieties with significantly higher yields due to reduced GA response. DELLAs function as central signaling hubs, coordinating diverse physiological responses by interacting with key transcription factors across multiple cellular pathways. While the DELLA domain mediates GA-GID1 binding, the mechanism of SCFSLY1/GID2 recruitment remained unknown. Additionally, GA-GID1 binding can inhibit DELLA protein activity independently of its proteolysis, although the underlying mechanism was unclear. Here, we present the cryo-EM structures of GA3-GID1A complexed with a full-length DELLA protein in Arabidopsis, RGA (REPRESSOR OF ga1-3), and the GA3-GID1A-RGA-SLY1-ASK1 complex. We show that the DELLA domain of RGA functions as a molecular bridge to enhance its GRAS domain binding to GID1A through direct interactions with both the GRAS domain and GID1A. Disrupting either intramolecular (DELLA-GRAS) or intermolecular (GRAS-GID1A) interactions weakens RGA-GID1 binding. Contrary to prior models, SLY1 binds the GRAS domain's concave surface without inducing conformational changes. Combining AlphaFold modeling and yeast three-hybrid assays, we demonstrate that GID1 binding to the RGA GRAS domain blocks its interactions with INDETERMINATE DOMAIN (IDD) transcription factors, explaining how GA-GID1 relieves growth suppression independently of DELLA degradation.

Our results provide further insight into the regulation of DELLA proteins in Arabidopsis . We clarified that phosphorylation modification of the six conserved sites is important for RGA functions and stability. The DELLA proteins, important plant growth and development repressors mediate the gibberellin (GA) signaling pathway. Although these proteins exhibit phosphorylation and de-phosphorylation states at the molecular level, little is known regarding the effects of different modifications of DELLA proteins on the regulation of their bioactivity and stability at the genetic level. In this study, six conserved serine (Ser)/threonine (Thr) sites of REPRESSOR OF ga1-3 (RGA) were substituted with alanine (RGA6A) or aspartic acid (RGA6D) to mimic the states of constitutive de-phosphorylation and phosphorylation, respectively. We found that the overexpression of de-phosphomimic RGA in Col-0 plants caused GA-overdose phenotypes, which were similar to DELLA-deficient mutant. These phenotypes were probably attributed to de-phosphomimic RGA, which retained its transcriptional activation activity that induces GA biosynthetic genes, but lost the transcription repressor function that inhibits GA-responsive genes. Further, de-phosphomimic RGA was unstable and easily degradable unlike the wild-type RGA, suggesting that the de-phosphorylated form is necessary for its degradation. In contrast, phosphomimic RGA overexpression caused GA-deficient phenotypes with non-degradable RGA. These phenotypes were probably due to phosphomimic RGA, which represses GA-responsive gene expression instead of inducing GA biosynthetic genes. In addition, phosphomimic RGA was stable and hardly degradable, which aggravated the RGA-inhibiting function in GA signaling. In conclusion, we show that the six conserved Ser/Thr sites are important for the different bioactivities of the RGA protein that regulate the GA response, and also for RGA stability via the mimicking of phosphorylation/de-phosphorylation.

Functional identification of mango MiGID1A and MiGID1B genes confers early flowering and stress tolerance.

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 isozyme composition and kinetics of production of a-amylase were studied in three samples of barley. Acid-dehusked caryopses and endosperm halves from a sample of Klages and from two samples of Clipper that differed in their ability to produce a-amylase in the absence of added gibberellic acid were incubated on moist sand at two temperatures in the presence or absence of gibberellic acid. The isozyme composition of the extracts was analysed on isoelectric-focusing-polyacrylamide gels and chromatofocusing columns. The endosperm halves of Klages and Clipper (type A) produced very low levels of enzyme in the absence of gibberellic acid while similar amounts of enzyme activity were found in both (+) and (-) gibberellic acid-treated incubates of Clipper (type B) endosperm halves. In these latter incubates, the initial kinetics of production, the proportion of a-amylase 1 to total enzyme activity and the effect of incubation temperature on the kinetics of production in both acid-dehusked caryopses and endosperm halves were similar, and were comparable to those observed in gibberellic acid-treated incubates of Klages and Clipper (type A). Since Clipper (type B) endosperm halves can produce a-amylase in the absence of added gibberellic acid, its role in this process should be reexamined.

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.