Gibberellin-GID1-DELLA: A Pivotal Regulatory Module for Plant Growth and Development

The diterpenoid hormone GA controls diverse developmental processes throughout the life cycle of a plant. Physiological and genetic studies show that active GA promotes seed germination and vegetative growth. In some species, GA also induces flowering and regulates flower, fruit, and seed development. This article highlights recent advances in our understanding of the molecular mechanisms of GA metabolism, transport, perception, and signaling, and the regulatory circuit between the GA pathway and other pathways to control plant growth and development in response to internal and external cues.

GA METABOLISM AND TRANSPORT

The biochemical pathway of GA biosynthesis and catabolism in plants is well defined, and genes encoding most enzymes in this pathway have been identified (Yamaguchi, 2008). Although many GA derivatives are present in a given plant, only very few GAs are biologically active. GA biosynthesis appears to be tightly linked to the site of GA responses as bioactive GAs have been found to be more abundant in rapidly growing tissues (Yamaguchi, 2008). This idea is also supported by recent studies on the expression of GA metabolism genes and phenotypic characterization of GA-deficient mutants (Sun, 2008; Yamaguchi, 2008). However, transport of GA intermediates or active GAs may also play an important role in modulating GA responses. For example, the cereal aleurone is well known for its response to embryo-produced GA (namely expression of hydrolytic enzyme genes in this tissue). In Arabidopsis (Arabidopsis thaliana), GA₄ (the major bioactive GA in this plant) can be transported from rosette leaves to the shoot apex to facilitate flower initiation in Arabidopsis (Eriksson et al., 2006). This was demonstrated by feeding labeled GA₄ to leaves and then measuring GA₄ levels in the shoot apical meristem. GA 3-oxidase (GA3ox) catalyzes the final step for bioactive GA production. Studies of GA3ox mutants suggest that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth in Arabidopsis (Hu et al., 2008). In developing siliques, active GAs are transported from the seed endosperm to the surrounding maternal tissues where they promote fruit growth. In germinating seeds, a subset of the GA-responsive genes are expressed in different cell types from those of GA3ox genes, also suggesting that GAs need to be transported between cells to regulate gene expression (Ogawa et al., 2003). It will be important to investigate the molecular mechanism of GA transport and how GA metabolism and transport are coordinated to modulate the local GA levels in response to the internal developmental program and environmental cues. Development of gene markers and/or fluorescent sensors to monitor bioactive GA molecules in planta will greatly facilitate these studies.

GA RECEPTOR AND EARLY EVENTS IN GA SIGNALING

Recent genetic, biochemical, and structural studies have elucidated the molecular mechanism of GA perception and initial steps in GA signaling in plants (Fig. 1; Ueguchi-Tanaka et al., 2007; Sun, 2008). The GA signal is perceived by the GA receptor GID1 (for GA INSENSITIVE DWARF1), which is a soluble protein that is localized to both cytoplasm and nucleus. DELLA proteins are nuclear transcriptional regulators, which repress GA signaling and restrict plant growth presumably by causing transcriptional reprogramming. Binding of GA to GID1 enhances the interaction between GID1 and DELLA, resulting in rapid degradation of DELLAs via the ubiquitin-proteasome pathway. A specific ubiquitin E3 ligase complex (SCF^SLY1/GID2) is required to recruit DELLA for polyubiquitination and subsequent degradation by the 26S proteasome. Recently, crystal structures of GA-GID1 and GA-GID1-DELLA complexes have been determined (Murase et al., 2008; Shimada et al., 2008). Without GA binding, the N-terminal extension (N-Ex) of GID1 has a flexible structure that is highly sensitive to protease treatment. Binding of GA to the C-terminal domain of GID1 induces a conformational switch of its N-Ex to cover the GA-binding pocket (like closing the lid), as well as creates hydrophobic surfaces for DELLA binding (Fig. 1). Although there is no direct contact between DELLA and GA, DELLA binding further stabilizes the GA-GID1-DELLA complex. These studies indicate that bioactive GA is an allosteric inducer of its receptor GID1. The current model also suggests that binding of GA-GID1 to DELLA enhances recognition of DELLA by the F-box protein of the ubiquitin E3 ligase SCF complex (SLY1 in Arabidopsis and GID2 in rice [Oryza sativa]; Fig. 1). This is different from the mechanism of auxin perception: Auxin functions as a molecular glue that brings the F-box protein (TIR1 and its homologs) and its substrate protein (IAA/AUX) together without altering the conformations of these proteins (Tan et al., 2007).

Figure 1.

Model of GA signaling in plants. Bioactive GA binding induces a conformational switch in the N-Ex of GID1 for DELLA binding, which in turn promotes a conformational transition in the GRAS domain of the DELLA protein for SCF^SLY1/GID2 recognition. DELLA protein will then be polyubiquitinated and degraded via the ubiquitin-proteasome pathway. SPY (an OGT) may activate DELLA by GlcNAc-modification, whereas EL1 (a casein kinase in rice) may phosphorylate and activate DELLA.

In addition to GA-dependent proteolysis, recent studies also suggest that DELLA activity may be modulated by other mechanisms. Overexpression of GID1 could inactivate DELLA by direct interaction without protein degradation (Ariizumi et al., 2008). Posttranslational modifications such as glycosylation and phosphorylation may also affect DELLA activity. SPINDLY (SPY), an O-linked GlcNAc (O-GlcNAc) transferase (OGT), is a GA signaling repressor (Olszewski et al., 2002). In animal systems, O-GlcNAc modification of Ser or Thr residues of target proteins by OGT often interferes with phosphorylation by protein kinases. Epistasis analysis between spy and della and two-dimensional gel-blot analysis of DELLA modifications suggest that SPY activates DELLA by GlcNAc modification, whereas phosphorylation of DELLA by an unknown GA-activated protein kinase may compete with SPY and inactivate DELLA (Shimada et al., 2006; Silverstone et al., 2007). However, EARLIER FLOWERING1 (EL1), encoding a casein kinase in rice, has been shown recently to function as a repressor of GA signaling (Dai and Xue, 2010). The results of this study also suggested that phosphorylation of DELLA by EL1 is required for DELLA activity and stability. This recent finding seems to challenge the current working model described above, although it is possible that phosphorylations at distinct sites by two different protein kinases may have opposite effects on DELLA activity. Direct evidence for O-GlcNAc modification of DELLA by SPY and the effects of GlcNAc modification and phosphorylation on DELLA function will require further investigation.

MOLECULAR MECHANISM OF DELLA-REGULATED GROWTH

DELLA proteins belong to the GRAS family of plant-specific nuclear proteins, which do not contain any canonical DNA-binding domain and therefore, are likely to regulate expression of their target genes by interacting with other transcription factors. To investigate the mechanism of DELLA-mediated growth repression, several putative DELLA direct targets in Arabidopsis were identified by expression microarrays, and DELLA was shown to be associated with several promoters of its target genes by chromatin immunoprecipitation-quantitative PCR analysis (Zentella et al., 2007). Interestingly, DELLA induces expression of upstream GA biosynthetic genes and GA receptor genes, suggesting that DELLA functions in maintaining GA homeostasis via a feedback mechanism (Fig. 2). Other DELLA-induced target genes encode transcription factors/regulators and RING-type ubiquitin E3 ligases. One of the RING ubiquitin E3 ligases, XERICO, is important for abscisic acid (ABA) accumulation. Thus, DELLA inhibits GA-mediated responses (e.g. seed germination) in part by up-regulating ABA levels through XERICO. Sequence comparison and chromatin immunoprecipitation-quantitative PCR using promoters of DELLA targets did not uncover any conserved DELLA-responsive cis-elements, suggesting that DELLA interacts with different transcription factors to regulate expression of target genes.

Figure 2.

Interaction circuit between GA-GID1-DELLA signaling module and other internal and external cues. The GA-GID1-DELLA regulatory module is highlighted in orange. Signals that promote bioactive GA accumulation are labeled in green, whereas signals that reduce GA levels are highlighted in purple. Activation or inhibition could be via different modes of action. PD, Protein degradation; PPI, protein-protein interaction; TC, transcription; SAM, shoot apical meristem; PHY, PHYTOCHROME; SOM, SOMNUS.

The best-elucidated molecular mechanism of DELLA-mediated growth regulation came from studies on the interaction between light and GA pathways. In etiolated Arabidopsis seedlings, PHYTOCHROME-INTERACTING FACTORs (PIFs) belonging to the subfamily 15 of bHLH transcription factors promote hypocotyl elongation. During deetiolation, phytochromes inhibit hypocotyl elongation by causing PIF degradation and also inhibiting GA accumulation and in turn increasing DELLA protein levels (Achard and Genschik, 2009). Two recent studies (de Lucas et al., 2008; Feng et al., 2008) reveal that DELLA inhibits hypocotyl elongation by binding directly to PIF3 and PIF4, and preventing expression of PIF3/PIF4 target genes. Therefore, in addition to light regulation of GA metabolism, cross talk between light and GA signaling pathways occurs through protein-protein interaction between PIF and DELLA (Fig. 2). Three additional bHLH subfamily-15 members PIF1 (PIL5), SPT, and PIL2 also interact with DELLA in yeast two-hybrid assays (Gallego-Bartolome et al., 2010). It will be important to determine whether DELLA regulates the functions of other members in this bHLH subfamily by direct protein-protein interactions. DELLA may also modulate gene expression via interaction with other classes of transcription factors and/or chromatin modification complexes. Future identification of additional DELLA interacting proteins by yeast two-hybrid assays and/or proteomic approaches will provide more complete understanding of the molecular mechanisms of DELLA function.

Interaction between light and GA also occurs during seed germination. In contrast to their antagonistic effects on hypocotyl elongation, both light and GA promote germination (Fig. 2). In the dark, PIL5 (PIF1) inhibits germination, in part, by binding to and activating transcription of promoters of two AtDELLA genes (RGA and GAI; Oh et al., 2007). PIL5 also reduces GA accumulation and increases ABA levels via SOMNUS (a nuclear zinc-finger protein; Kim et al., 2008). Interestingly, ABA was shown to promote transcription of another AtDELLA gene (RGL2; Piskurewicz et al., 2008). In the light, phytochromes mediate light-induced germination by causing PIL5 degradation and GA accumulation, the combined effects of which allow down-regulation of DELLA activity (by reducing transcription and increasing protein turnover).

In addition to mediating the cross talk between GA and light signaling pathways, DELLA plays a major role in modulating plant growth in response to internal cues (other hormone signals) and external biotic and abiotic stresses (Fig. 2; Achard and Genschik, 2009; Bari and Jones, 2009; Harberd et al., 2009). In most cases, DELLA stability is indirectly affected by other pathways through alteration of GA metabolism and bioactive GA levels. For example, auxin induces root and stem elongation, at least in part, by up-regulating GA biosynthetic genes (GA3ox) and down-regulating GA catabolism genes (GA2ox). During cold and salt stresses, AP2 transcription factors CBF1 and DDF1, respectively, induce expression of GA2ox genes. Similarly, stabilization of DELLA by ABA treatment is achieved by reduction of GA accumulation. In a GA biosynthesis mutant background, ABA pretreatment failed to inhibit GA-induced DELLA degradation (Zentella et al., 2007).

In conclusion, the GA-GID1-DELLA signaling module is regulated at multiple levels to achieve proper growth: (1) The amounts of bioactive GAs are affected via altered expression of GA metabolic genes by internal and external cues. GA transport may also play a role, although the regulatory mechanism is unknown; (2) elevated GA signals will activate GID1 to induce rapid proteolysis of DELLA; (3) in addition to protein degradation, DELLA activity is also regulated by transcription, posttranslational modifications, and direct protein-protein interactions; and (4) DELLA may play a role in maintaining GA homeostasis by feedback regulation of expression of GA biosynthesis and receptor genes. Further elucidation of how DELLA coordinates GA and other signaling activities will come from functional studies of DELLA modifications, DELLA target genes, and identification and functional characterization of additional DELLA interacting proteins.

SPATIAL AND TEMPORAL CONTROL OF THE GA-GID1-DELLA SIGNALING MODULE

To understand how the GA-GID1-DELLA module regulates plant growth and development, it will be necessary to monitor the sites and timing of the actions of this module. In the root, expression of a GA-resistant (gain-of-function) DELLA mutant protein in the endodermis (but not other cell types) appears to inhibit root elongation, suggesting that the primary site of GA-induced DELLA degradation for primary root elongation is in the endodermis (Ubeda-Tomas et al., 2008). Interestingly, recent studies showed that different phytohormones seem to act in distinct and not completely overlapping cell types (Jaillais and Chory, 2010). This is not too surprising because defects in different hormone pathways cause distinct phenotypes. With the available knowledge of the metabolic pathway and early signaling pathway for individual phytohormone, systems biology approach will help to visualize the complex regulatory webs (both transcriptional networks and protein interactomes) among these pathways in specific cell types to modulate different developmental events.