Gibberellin Signaling: A Theme and Variations on DELLA Repression

GAs are a family of tetracyclic diterpenoid plant hormones that stimulate plant growth and developmental transitions. As sessile organisms, plants rely on developmental plasticity to respond to environmental challenges. Plant hormones regulate developmental responses to diverse environmental stimuli such as changes in light, temperature, moisture, animal feeding, and disease pressure. GAs stimulate seed germination in response to changes in light, temperature, and moisture (Koornneef and van der Veen, 1980; Yamauchi et al., 2004; Seo et al., 2009). GA also stimulates stem elongation and leaf expansion through cell expansion and cell division in response to light or dark (photomorphogenesis and skotomorphogenesis; Ogawa et al., 2003; Alabadí et al., 2008; Feng et al., 2008; de Lucas et al., 2008; Gallego-Bartolomé et al., 2011). GA stimulates developmental transitions from meristematic to shoot growth, from juvenile to adult leaf development, and from vegetative growth to flowering, and also stimulates aspects of flower development (Telfer et al., 1997; Yu et al., 2004; Galinha et al., 2009). The appropriate regulation of these events is essential to the survival of plant species and to successful crop production. GA stimulates many aspects of plant growth and development by lifting DELLA (Asp-Glu-Leu-Leu-Ala) protein repression of these events. This article will review multiple biochemical mechanisms for the regulation of and response to DELLA repression. Studies using plants with altered GA biosynthesis or catabolism have resulted in a wealth of knowledge of the diverse roles of GA in plant growth and development (for review, see Sun and Gubler, 2004; Yamaguchi, 2008). GA biosynthesis enzyme mutants of dicots and monocots are GA sensitive, showing defects in growth and development that are rescued by GA application. GA-sensitive mutants of rice (Oryza sativa) and barley (Hordeum vulgare) exhibit dwarfism, infertility, and failure to mobilize stored reserves during seed germination through α-amylase induction (Zwar and Chandler, 1995; Chandler and Robertson, 1999; Sakamoto et al., 2004). Similar phenotypes are seen in Arabidopsis (Arabidopsis thaliana) mutants affecting enzymes acting later in the biosynthesis pathway, GA 3-oxidase (GA3ox) and GA20ox (Hedden and Phillips, 2000; Plackett et al., 2012). Since GA3ox and GA20ox belong to multigene families, single mutants are fertile semidwarves. In Arabidopsis and tomato (Solanum lycopersicum), mutants affecting early GA biosynthesis enzymes such as ent-copalyl diphosphate synthase (Sun and Kamiya, 1994) cause failure in seed germination, growth as a dark green dwarf, failure to transition to flowering under short days, and partial to complete infertility (Koornneef and van der Veen, 1980; Karssen et al., 1989; Wilson et al., 1992). Overexpression of the gene encoding the GA catabolic enzyme GA2ox increases GA turnover, leading to reduced grain germination and α-amylase induction in wheat (Triticum aestivum; Appleford et al., 2007) and to failures in seed development and pollen tube growth in Arabidopsis (Singh et al., 2002). GA signaling is often controlled through direct regulation of hormone accumulation mediated by changes in GA20ox, GA3ox, and GA2ox expression in response to environmental or developmental stimuli. This is logical, as the hormone is the first step in a hormone signaling pathway. Stimulation of Arabidopsis seed germination by red light or cold imbibition and inhibition of germination by far-red light are associated with increased and decreased GA accumulation, respectively (for review, see Seo et al., 2009). Far-red light inhibits seed germination by inducing GA turnover through GA2ox2 and inhibiting the GA biosynthesis GA3ox genes, whereas red light or cold stimulates germination by inducing the biosynthesis genes GA3ox or GA20ox and inhibiting GA2ox expression (Penfield et al., 2006; Oh et al., 2007). The germination of seed imbibing in the cold is stimulated by increased GA levels, but cold acclimation of adult plants is associated with decreased GA. Induction of the C-repeat-binding factor genes by cold acclimation induces the GA turnover GA2ox genes (Achard et al., 2008). Decreased GA levels enhance cold tolerance and suppress plant growth in the cold. GA stimulates the transition from meristematic growth to shoot differentiation. KNOX genes maintain the meristem by repressing the GA biosynthesis GA20ox enzymes and activating the transcript accumulation of the GA turnover GA2ox enzymes (for review, see Galinha et al., 2009). GA20ox expression, and presumably GA accumulation, is high in new shoots but depleted in the meristem. The GA signal is perceived by a soluble receptor protein, GA-INSENSITIVE DWARF1 (GID1). The mechanisms of GA perception are conserved, showing agreement in Arabidopsis and rice, where the signaling pathway has been studied in the greatest detail (Table I). The GID1 gene was identified through map-based cloning of a GA-insensitive mutant in rice, where there is a single copy of the gene (Ueguchi-Tanaka et al., 2005). GA-insensitive GID1 mutants have defined a single barley homolog, GSE1 (Gubler et al., 2002; Chandler et al., 2008), and three Arabidopsis homologs, GID1a, GID1b, and GID1c (Griffiths et al., 2006; Nakajima et al., 2006; Iuchi et al., 2007; Willige et al., 2007). Mutations in the GA receptor result in phenotypes similar to those resulting from severe GA biosynthesis mutations, but they are not rescued by GA application. GID1 protein localizes mainly to the nucleus but also appears to localize to the cytoplasm (Ueguchi-Tanaka et al., 2005; Willige et al., 2007). GID1 encodes a homolog of mammalian hormone-sensitive lipase (Ueguchi-Tanaka et al., 2005). X-ray crystallography demonstrated two key features of the GID1 protein (Murase et al., 2008; Shimada et al., 2008). First, the hormone-sensitive lipase catalytic domain that normally binds a lipid has evolved to bind GAs. Second, the N-terminal “lid” domain of GID1 interacts hydrophobically with the γ-lactone ring of GA4 and upon GA binding folds over the GA-binding pocket (Fig. 1A). This GA-dependent conformational change causes the GID1 N-terminal helical lid domain to behave like “molecular glue” to interact with DELLA repressor target proteins (Ueguchi-Tanaka et al., 2007; Murase et al., 2008). The theme where a small hormone molecule enables a protein-protein interaction is common in plants. For example, the hormones auxin and jasmonate stimulate the interaction of their hormone receptors with the downstream target proteins through direct interactions with both receptor and target proteins (Tan et al., 2007; Sheard et al., 2010). The GID1-GA-DELLA complex binds GA4 with a higher affinity than GID1 alone, indicating that DELLA binding stabilizes the receptor-hormone interaction (Nakajima et al., 2006; Ueguchi-Tanaka et al., 2007). The GID1-GA complex stimulates plant growth and development by down-regulating DELLA repressors through direct protein-protein interaction. DELLA targets in boldface have been biochemically verified through coimmunoprecipitation or pull-down assay. DELLA targets in boldface have been biochemically verified through coimmunoprecipitation or pull-down assay. Proteolysis-dependent and -independent GA signaling models. A, The canonical GA signaling model illustrating GA-dependent GID1-DELLA complex formation resulting in DELLA recognition and ubiquitylation by the SCFSLY1 E3. Polyubiquitylation leads to DELLA proteolysis by the 26S proteasome, thereby lifting DELLA repression of GA responses. B, Proteolysis-independent GA signaling in sly1 mutants occurs when GID1-GA-DELLA complex formation blocks DELLA repression of GA responses without DELLA destruction. C, EL1-mediated phosphorylation of DELLA activates DELLA as a repressor of GA responses. DELLA genes are defined as repressors of GA signaling because gain-of-function mutations in DELLA genes lead to reduced GA signaling (i.e. dwarfism), whereas loss of function leads to increased GA signaling (i.e. tall or slender phenotype). GID1 lifts DELLA repression through direct protein-protein interaction. All DELLA repressors have an N-terminal DELLA regulatory domain containing the conserved amino acid sequence Asp-Glu-Leu-Leu-Ala (DELLA) and a C-terminal GRAS (for GAI, RGA, and SCARECROW) functional domain (Fig. 2; Peng et al., 1997; Silverstone et al., 1998; Pysh et al., 1999; Itoh et al., 2002). The N-terminal DELLA regulatory domain is an intrinsically disordered domain that folds and becomes structured upon GID1 protein binding (Sun et al., 2010). Mutations in the DELLA and TVHYNP regions of the regulatory domain interfere with the ability to bind the GID1 receptor, leading to a semidominant GA-insensitive dwarf phenotype due to the inability to down-regulate the DELLA repressor (Peng et al., 1997; Dill and Sun, 2001; Itoh et al., 2002; Silverstone et al., 2007; Asano et al., 2009). Similar phenotypes have been detected in the PFYRE and SAW regions of the GRAS domain, which form secondary interactions with GID1 (Hirano et al., 2010). The DELLA regulatory domain also contains a Ser/Thr/Val-rich domain believed to be involved in the regulation of DELLA function by phosphorylation, since deletion of both the TVHYNP and Ser/Thr/Val domains blocks DELLA phosphorylation (Itoh et al., 2005a; Silverstone et al., 2007; Dai and Xue, 2010). The DELLA gene of barley is called SLENDER1 (SLN1) and the DELLA gene of rice is called SLENDER RICE1 (SLR1), in reference to the “tall” phenotypes resulting from loss-of-function mutations in the GRAS functional domain (Table I; Chandler et al., 2002; Itoh et al., 2002). The roles of the five Arabidopsis DELLA repressor genes have been determined based on the ability of loss-of-function alleles to rescue phenotypes of the ga1-3 GA biosynthesis mutant. Mutations in REPRESSOR OF GA1-3 (RGA), GA-INSENSITIVE (GAI), and RGA-LIKE (RGL1) rescue plant height; mutations RGA, RGL2, and RGL1 rescue flowering; while mutations in RGL2, RGA, GAI, and RGL3 rescue seed germination (Dill and Sun, 2001; King et al., 2001; Lee et al., 2002; Cheng et al., 2004, Tyler et al., 2004; Cao et al., 2005; Piskurewicz and Lopez-Molina, 2009). DELLA RGL2 has the strongest effect on seed germination, whereas DELLA RGA has the strongest effect on plant height. Promoter-swap experiments have shown that the partially specialized functions of these two Arabidopsis DELLA proteins appear to result mainly from tissue-specific gene expression (Gallego-Bartolomé et al., 2010). DELLA protein organization illustrating the conserved domains and subdomains involved in GID1 binding (purple), target binding (blue), nuclear localization (gray), and SLY1/GID2 binding (green). Also depicted are the invariant Arg (R) and Tyr residues (Y) conserved among the STAT-like GRAS protein family and specific Ser residues (Ser-196 and Ser-510) that are sites of EL1-directed phosphorylation. GA lifts DELLA repression of GA responses by targeting DELLA for destruction via the ubiquitin-proteasome pathway. This model was originally based on the observation that rescue of dwarfism due to GA deficiency by GA treatment was associated with DELLA protein disappearance (Silverstone et al., 2001; Itoh et al., 2002). The ubiquitin-proteasome pathway was implicated when it was that mutations in the genes of Arabidopsis and the rice homolog resulted in a GA-insensitive phenotype associated with an inability to target DELLA for destruction et al., 2003; et al., SLY1/GID2 is the of an (for and that the of DELLA protein et al., 2003; et al., 2003; Dill et al., 2004; et al., 2004; et al., 2004; et al., 2005; et al., and 2011). The of an complex through a C-terminal protein-protein interaction domain and interacts with the of the complex via the N-terminal domain and 2004). The SLY1/GID2 the C-terminal and domains to bind the DELLA protein and domains (Fig. 2; Dill et al., 2004; et al., 2004; et al., et al., 2011). bind the Arabidopsis protein in and a complex with that the domain in et al., 2004; et al., 2011). on a SCFSLY1 the formation of to DELLA through than through et al., 2009). GA treatment of the ga1-3 GA biosynthesis mutant in DELLA destruction through ubiquitylation in or in et al., 2004; and 2007; et al., 2011). This disappearance be by 26S et al., 2002; et al., 2005). of DELLA proteins targets for destruction via the 26S The formation of the GID1-GA-DELLA complex the protein-protein interaction DELLA and the protein demonstrated that binding to DELLA is stimulated by GID1-GA-DELLA complex formation (Griffiths et al., 2006; et al., 2010). GA stimulates coimmunoprecipitation with that GID1-DELLA complex formation also stimulates interaction in et al., 2011). DELLA proteins higher levels in GA biosynthesis and indicating that the GID1 receptor, and of the complex are for DELLA proteolysis via the ubiquitin-proteasome pathway et al., 2003; et al., 2003; et al., 2003; Dill et al., 2004; et al., 2004; et al., 2004; et al., 2005). pull-down and coimmunoprecipitation have shown DELLA in complex with SLY1/GID2 in et al., 2004; et al., 2011). and are to Arabidopsis in a et al., 2009). is a homolog of SLY1/GID2 both in Arabidopsis and rice called or et al., 2003; Itoh et al., 2003; et al., 2004; et al., 2004; and 2009). function in GA is the of DELLA protein accumulation, that mutations have a small effect in sly1 mutants et al., and 2011). of GA signaling through DELLA the of DELLA protein accumulation with the of GA-insensitive This is not the in sly1 and which DELLA protein but severe GA-insensitive phenotypes than GA biosynthesis or GA receptor mutants et al., 2003; Willige et al., 2007; Ueguchi-Tanaka et al., 2008). appears that DELLA be by a such that not of the DELLA protein that in mutants is functional as a repressor of GA signaling and 2007; et al., 2008; Ueguchi-Tanaka et al., 2008). model in which DELLA repression is by GID1-GA-DELLA complex formation was based on the phenotypes are rescued by GID1 gene without a in DELLA protein and GA signaling in of the for GID1-GA-DELLA complex GA hormone the GID1 and the DELLA domain for protein interaction. In light of it appears that GID1 causes GA signaling in sly1 mutants by binding DELLA protein than by DELLA proteolysis (Fig. Arabidopsis and the protein binds GA with a higher affinity and ability to interact with DELLA in the of GA. This GA-insensitive sly1 mutant phenotypes et al., 2008; et al., 2010). This binding a of GA signaling in the of GA. Arabidopsis to have expression levels, that of accumulation be essential to GA responses (Griffiths et al., 2006; Willige et al., 2007). DELLA be in the a for GA signaling mechanisms that DELLA destruction or in where DELLA destruction is by signaling or of auxin signaling (Achard et al., 2003; and the of DELLA to GA signaling without using a the ability to DELLA protein function as as are not since phenotype is the of events over and be by multiple signaling will to a direct for DELLA function or as also that DELLA be by phosphorylation and (for Studies that DELLA is but the of in DELLA or protein is DELLA phosphorylation was first in rice and in it was that DELLA phosphorylation DELLA affinity for the protein since target protein phosphorylation often stimulates protein target binding in et al., 2003; et al., 2004; and 2004). it that rice DELLA and Arabidopsis DELLA protein binding when et al., 2004; et al., 2004). of the protein-protein interaction demonstrated in affinity for or DELLA (Itoh et al., appear to the of barley DELLA and Arabidopsis RGL2 and RGA, that DELLA is to et al., 2002; et al., 2005; et al., 2009). that the protein by rice DELLA through direct protein phosphorylation and Xue, 2010). as a of GA signaling and a of since loss of function leads to increased GA associated with early flowering and increased α-amylase expression during seed The phenotypes are associated with a in the of DELLA protein GA with experiments that DELLA is of that DELLA by phosphorylation. First, protein DELLA in Second, mutations in DELLA and believed to phosphorylation, DELLA of gene mutations and to increased accumulation of This that DELLA be through phosphorylation these two the DELLA regulatory domain and the GRAS functional domain (Fig. will to EL1-directed DELLA phosphorylation these sites in and the effect of phosphorylation on DELLA encodes an that GA signaling in rice, and barley et al., 1998; et al., 2001; Shimada et al., 2006; et al., 2009). mutations in identified in for to the GA biosynthesis and for of ga1-3 and Silverstone et al., In the gain-of-function DELLA is associated not with DELLA disappearance but with DELLA phosphorylation (Silverstone et al., 2007). also the rice mutant without DELLA proteolysis and resulted in increased DELLA protein phosphorylation et al., Since also rice and GA biosynthesis it appears that increased GA signaling in DELLA proteolysis GID1-DELLA complex The model as in with phosphorylation for of the residues and that loss of DELLA in decreased DELLA function as a of phosphorylation et al., 2004; Shimada et al., 2006; Silverstone et al., 2007). be in these since it has not been demonstrated that DELLA is is an since DELLA phosphorylation in mutants is to cause increased GA whereas EL1-directed DELLA phosphorylation is believed to cause decreased GA is that DELLA is by phosphorylation as a to GA signaling due to the or that phosphorylation amino acid residues have on DELLA these that DELLA be by will to DELLA GID1-DELLA binding affinity or is by the GID1 DELLA is to multiple environmental to plant growth and development by with multiple targets that gene The that DELLA proteins regulate was originally based on nuclear localization and protein on DELLA gene through protein interaction with specific factor DELLA proteins are of signal and of 1997; Peng et al., 1997; Silverstone et al., 1998; et al., 2000; et al., The GRAS functional domain of in that it contains two domains and also called or and a domain (Fig. 2; et al., GRAS subdomains PFYRE and SAW to the DELLA that of the is DELLA in and et al., 2006; et al., 2007; Gallego-Bartolomé et al., 2011). experiments that the Arabidopsis DELLA RGA localizes to the regions of genes et al., 2007). Since of these genes GA and DELLA it was that DELLA functions as a of repressors of GA such as the of acid et al., 2007). the DELLA domain to in a is also for function as a of stem elongation in rice in of (Hirano et al., 2012). Since DELLA than early it was that DELLA through interaction with proteins et al., 2007). DELLA protein was to interact with (for (for jasmonate and (for in and is believed to interact with (for (for and based on Lucas et al., 2008; Feng et al., 2008; et al., Gallego-Bartolomé et al., et al., et al., et al., 2011). is that DELLA protein interaction with and the domain, since mutations in the domain result in a gain-of-function GA-insensitive phenotype (Itoh et al., 2002; de Lucas et al., 2008; et al., et al., et al., 2010). In where a protein interaction has been it appears that DELLA with the ability of a protein to or a target (Fig. This not the model that DELLA is a of repressors of GA will to DELLA as a repressor or as a of gene on specific target and DELLA is to interact with many target that the N-terminal of is an intrinsically disordered it is that the DELLA intrinsically disordered multiple protein interactions (Sun et al., 2010). disordered regions have specific but interactions with multiple proteins (for review, see Sun et al., 2012). the of protein interactions in the of plant growth and development is with an on biochemically verified target proteins (Table I). A, DELLA targets through protein-protein interactions with B, model for DELLA of in response to light and the In the GA and GID1 levels are repressing ability to regulate levels during the leading to growth a in GID1 levels in DELLA and repression of leading to and In the red light which leading to destruction via the ubiquitin-proteasome pathway. of blocks DELLA RGA protein the GRAS family factor is a of GA since loss of function in GA-insensitive reduced seed germination, shoot and elongation et al., et al., 2011). and DELLA appear to in to based on the that DELLA RGA binds to protein, thereby repression of and the genes and et al., 2011). was originally to be a of GA because and that the is a direct DELLA target showing GA expression et al., 2007). a because the GA expression is a is that DELLA functions through interaction with the GRAS protein, that rice DELLA interact with in and that GRAS genes DELLA function in rice (Itoh et al., will to DELLA interacts with GRAS DELLA the response to through direct protein interaction with a repressor of jasmonate that Arabidopsis DELLA genes a in because the appears to plant growth by DELLA protein and because are to gene expression in response to et al., 2008). This that DELLA is involved in and disease responses is in light of the that DELLA genes and interacts with protein et al., 2010). repression of genes is when binding to the receptor and protein leading to proteolysis via the ubiquitin-proteasome pathway. In the of binds the thereby repressing gene expression involved in and responses as as the repression of In the of DELLA binds thereby ability to bind and (Fig. in leads to and the expression of and DELLA as a of response by repressor through protein-protein interaction. GA signaling growth responses to through the DELLA In the through with an and to whereas in light resulting in short and green GA signaling developmental responses to dark The gene in the light, and the and genes in the GA signaling appears to regulate protein in the presumably through increased et al., 2008). is an that targets the protein for destruction. In red light, the and interact with the form of which in leads to their destruction through the ubiquitin-proteasome pathway. In the and that stimulates growth and and 2002; et al., 2004). GA and DELLA growth in the dark through the protein-protein interaction (Fig. de Lucas et al., 2008; Feng et al., 2008). In the of DELLA binds to and their ability to the of genes involved in that DELLA interfere with the ability of to bind target Lucas et al., 2008). GA stimulates growth in the dark by targeting DELLA for thereby protein DELLA and also appear to have on genes involved in and et al., 2011). that in the protein binds the and the expression of a gene involved in that DELLA proteins the ability of to in the The of DELLA in gene expression involved in is in light of the that and are by the et al., 2007; et al., 2008; et al., 2011). and levels are whereas DELLA protein levels are as GID1 levels and are in the light, when gene expression is et al., 2011). and protein levels are the of the when their growth et al., 2007). a on the model by Feng et in which DELLA inhibits function the transition from to (Fig. is in growth and GID1 levels leading to increased DELLA protein levels and repression of the of red to far-red light increases to destruction through interaction with the form of DELLA also appears to in development through interaction with protein, in Arabidopsis et al., 2010). GA and both stimulate of the in the The that the DELLA proteins RGA, GAI, and RGL2 interact with the protein in and that DELLA the of involved in by direct protein interaction. to GA stimulates development in by lifting DELLA repression of GA stimulates and DELLA plant growth both through on cell expansion and GA stimulates the expression of enzymes involved in cell and genes cell division and also stimulates associated with cell expansion et al., 2003; et al., et al., 2012). GA appears to stimulate cell expansion in through on downstream auxin and auxin gene expression is by the gene which in is by the factor et al., 2010). DELLA RGA activates transcript accumulation, DELLA a repressor of GA stimulates expression through DELLA destruction. with Willige et that GA biosynthesis and signaling are for auxin and appropriate accumulation of and auxin cell expansion downstream of GA signaling is by showing that genes both and gene (Gallego-Bartolomé et al., et al., 2011). GA stimulates cell division in by lifting the DELLA repression of a that stimulates cell by repressing genes et al., 2012). GA cell division and expansion through multiple for on GA signaling have since it was first that GA functions by targeting DELLA repressors for acting as a of a have demonstrated that GA stimulates GID1-DELLA protein GA signaling mechanisms that are of DELLA DELLA and identified multiple downstream DELLA target proteins of diverse multiple for a functional interaction DELLA and to be determined DELLA is and is for DELLA repression DELLA repression is stimulated by DELLA phosphorylation, but it is not phosphorylation residues has on will to the ability of DELLA to bind GID1 or downstream protein targets and is not the GID1 protein DELLA of downstream protein-protein DELLA plant development through diverse the domain protein and of the and GRAS The mechanisms and such diverse protein interactions The mechanisms by which DELLA with target protein DELLA behave as a of gene but of DELLA function as a of a is The of and mechanisms for lifting DELLA repression to be to was not due to of the for and of the signal and of jasmonate