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ScienceDirect Shedding light on integrative GA signaling Hao Xu, Qian Liu, Tao Yao and Xiangdong Fu Gibberellic acid (GA) regulates a diversity of processes associated with plant growth and development. The DELLA proteins act as repressors of GA signaling, and are destabilized by GA. Although it is known that GA signaling integrates various endogenous and environmental signals, the molecular basis of their modulation of plant growth and development is only now beginning to be understood. The current suggestion is that the DELLA proteins act as one possible quantitative modulator of plant growth, achieved by integrating multiple environmental and hormonal signals via protein–protein interactions. This review discusses recent elaborations of the de-repression model proposed to describe the GA response, and focuses on integrative networks thought to regulate plant growth, development and the adaptation to a fluctuating environment. Addresses The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, PR China Corresponding author: Fu, Xiangdong ([email protected])
Current Opinion in Plant Biology 2014, 21:89–95 This review comes from a themed issue on Cell signalling and gene regulation 2014 Edited by Xiangdong Fu and Junko Kyozuka
http://dx.doi.org/10.1016/j.pbi.2014.06.010 1369-5266/# 2014 Elsevier Ltd. All rights reserved.
Introduction The phytohormone GA belongs to a large family of tetracyclic diterpenoids. Since its discovery in the 1950s, 136 structural variants have been identified in a variety of plants, fungi and bacteria (http://www.planthormones.info/ga1info.htm). Only a limited number of these, namely GA1, GA3, GA4, GA5, GA6 and GA7, are thought to have biological activity [1]. In most plant species, GA1 and GA4 are the primary forms associated with the modulation of growth and development; they are active throughout the plant’s life cycle, including seed germination, hypocotyl elongation, leaf expansion and floral induction [2]. Although exogenous GA supply and grafting experiments have both demonstrated that bioactive GA and some of its precursors can be translocated over long distances within the plant [3–5], the sites of GA production and action generally overlap. As an www.sciencedirect.com
example, two fluorescently labeled GA compounds (GA3Fl and GA4-Fl) have been shown to accumulate specifically in the endodermis of the root elongation zone [6]. The revelation that the endodermis must have a particular role in GA regulation is consistent with findings that the endodermis is the primary GA-responsive tissue in the Arabidopsis thaliana root [7]. GA promotes plant growth by promoting the degradation of the DELLA proteins, a family of nuclear growth repressors [2]. The GA signal is perceived by the soluble receptor GA-INSENSITIVE DWARF1 (GID1) [8–10]. The binding of bioactive GA to GID1 induces a conformational change which allows GA–GID1 to interact with the DELLA N-terminal domain [11,12], which in turn promotes GID1-GA–DELLA association with the SCFSLY1/GID2 E3 ubiquitin ligase F-box protein component (SLY1 in A. thaliana and GID2 in rice), thereby targeting the DELLA proteins for degradation via the 26S proteasome pathway [13,14]. It has been proposed that the DELLA proteins can function as transcriptional repressors by blocking the activity of bHLH transcription factors, and it has been experimentally shown that when they directly interact with PHYTOCHROME INTERACTING FACTORS (PIFs), the latter’s DNA binding ability to its promoter target is compromised, thereby affecting light-responsive gene expression and hypocotyl elongation [15,16]. Although the DELLA proteins themselves lack a DNA-binding domain, they are able to function indirectly as transcriptional activators either by interacting with the DNA binding INDETERMINATE DOMAIN (IDD) family proteins [17], or by inactivating the JASMONATE ZIM-domain (JAZ) transcriptional repressors [18]. The DELLA proteins have recently been shown to be involved in protein– protein interactions with certain key regulatory proteins belonging to various hormonal and environmental signaling pathways. This review discusses recent advances in the understanding of GA-mediated growth responses and focuses on the integrative mechanisms underlying the action of the DELLA proteins as a modulator of plant growth, development and adaptation to environmental changes.
GA-induced growth is dependent on the degradation of the DELLA proteins The A. thaliana DELLA proteins GA-INSENSITIVE (GAI), REPRESSOR-of-ga1-3 (RGA), RGA-LIKE1 (RGL1), RGL2 and RGL3, as well as the rice SLENDER RICE1 (SLR1) protein all repress GA-responsive growth [2]. GA stimulates growth by promoting the 26S proteasome-dependent degradation of the DELLA Current Opinion in Plant Biology 2014, 21:89–95
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proteins, thereby relieving the DELLA-imposed growth restraint [19–21]. The recent advances in our understanding of the de-repression model make it possible to explain the role of GA and the function of the DELLA proteins [8–14]. When the formation of the GID1–GA–DELLA complex promotes the interaction between the DELLA C-terminus and the F-box protein component of SCFSLY1/GID2 complex [20–23], leading to the E3 ubiquitin ligase-driven polyubiquitination of the DELLA proteins, and their subsequent targeting for degradation via the ubiquitin 26S proteasome pathway (Figure 1). The FLAG-SLY1 fusion protein co-immunoprecipitates with the HA-GID1b, consistent with the regulatory model which holds that SLY1 controls the stability of the DELLA proteins through its interaction with the GID1–GA–DELLA protein complex [24]. Thus, a key
event of GA-mediated growth stimulation is the degradation of the DELLA proteins. The post-translational modification of the DELLA proteins has suggested an alternative mechanism for controlling their stability and activity. O-GlcNAcylation catalyzed by the SPINDLY protein (similar to animal Olinked N-acetylglucosamine transferase) and phosphorylation catalyzed by a casein kinase have been proposed to increase DELLA protein activity [25,26]. The SUMOylation of the DELLA proteins represents an additional means of DELLA-dependent growth control. When the SUMOylated DELLA proteins interact with SUMOinteracting motif in GID1, GA access to GID1 may be blocked, and as a result, high levels of non-SUMOylated DELLA protein can accumulate; the beneficial effect of
Figure 1
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Schematic representation of the DELLA-mediated GA signaling de-repression regulatory model. The DELLA proteins repress growth, with their stability and activity being enhanced by O-GlcNAcylation, phosphorylation and SUMOylation (DELLA-M: modified DELLA proteins). The DELLA proteins restrain growth by sequestering transcription factors into inactive protein complexes. In addition, the DELLA–PFD interaction locks the PFD complex within the nucleus thereby affecting microtubule organization. Conversely, GA promotes the proteasome-dependent degradation of the DELLA proteins. The binding of bioactive GA to the GA receptor GID1 promotes the GID1–DELLA interaction. The formation of the GID1–GA–DELLA complex subsequently enhances the interaction between the DELLA C terminus and the E3 ubiquitin ligase SCFSLY1/GID2 F-box protein, resulting in the polyubiquitination of DELLA and its targeting for degradation via the 26S proteasome pathway. The GA-induced degradation of the DELLA proteins releases transcription factors, a process which either activates the expression of GA-responsive genes or allows the trafficking of the PFD complex into the cytoplasm, where it can promote GA-induced cell expansion. Current Opinion in Plant Biology 2014, 21:89–95
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Control of plant growth and GA signaling Xu et al. 91
this process is to constrain growth in the face of salinity stress [27].
DELLA proteins restrain growth by controlling both cell division and expansion The growth of plant organs reflects a combination of cell proliferation and cell expansion. The short root phenotype of the A. thaliana GA-deficient ga1-3 mutant results from a reduction in the size of the root meristem, but since this characteristic can be reversed by deleting GAI and RGA [28,29], suggesting that the DELLA proteins regulate root growth and development via an effects on cell division. The product of STUNTED acts downstream of the DELLA proteins and promotes cell division by repressing cyclin-dependent kinase inhibitor genes [30]. It has long been known that the effect of GA on axial cell expansion is related to the transverse alignment of the cortical microtubule (CMT) array [31]. The CMTs in the epidermal cells of the Lemna minor root become disorganized following exposure to uniconazole-P, but recover when the root is provided with GA3 [32]. However, little is known regarding the molecular basis of GA-mediated microtubule orientation. A recent study has revealed that GA regulates microtubule organization via an interaction between the DELLA proteins and the prefoldin complex (PFD), a cochaperone required for tubulin folding [33]. In the absence of GA, the DELLA–PFD interaction locks the complex within the nucleus, so compromises a/b-tubulin heterodimer availability in the cytoplasm. Conversely, GA induces the degradation of the DELLA proteins and thus allows PFD to be trafficked into the cytoplasm, thereby increasing the amount of active tubulin subunits and consequently promoting cell expansion (Figure 1). This finding has provided a novel mechanistic link between the GA–DELLA regulatory module and CMT organization.
GA signal transduction requires DELLAmediated protein–protein interactions The DELLA proteins belong to the family of plantspecific GRAS proteins thought to act as transcriptional regulators. Because the DELLA proteins lack their own DNA-binding domain, other transcription factors are required as intermediate components. The in vivo interaction with the bHLH transcription factors PIF3 and PIF4 has been shown to affect the DNA binding activity of the PIFs, resulting in an inhibition of PIF-mediated gene expression and hypocotyl elongation [15,16]. The DELLA proteins also inhibit the activity of ALCATRAZ (ALC), a protein required for fruit development [34]. In addition, the DELLA proteins can interact with BRASSINAZOLE-RESISTANT1 (BZR1), inhibiting its DNA binding both in vitro and in vivo; the supply of GA releases this inhibition [35,36]. The co-activation of PIF proteins by BZR1 highlights a further role in brassinosteroid (BR) signal integration, and reveals that the formation of the www.sciencedirect.com
PIF4-DELLA-BZR1 complex can dynamically regulate plant growth and development in response to GA, BR and light [35,37]. The DELLA proteins can interact with the DNA-binding domains of EIN3/EIL1 to repress EIN3/ EIL1-regulated HOOKLESS1 (HLS1) expression and apical hook development [38]. The interaction between the DELLA proteins and miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors interferes with SPL transcriptional activity, thereby delaying floral induction through inactivating the expression of miR172 and MADS-box genes [39]. Taken together, the DELLA proteins act as transcriptional repressors by preventing the binding of DELLA-associated transcription factors to their promoter target (Figure 2). The DELLA proteins as transcriptional activator can provide an alternative mechanism for the integration of environmental and endogenous signals to modulate plant development and defense responses. They interact directly with JAZ proteins, the key repressors of jasmonate (JA) signaling, and consequently enhance the transcriptional activation potential of MYC2 [18]. JA is proposed to promote the degradation of JAZ proteins, thereby releasing MYC2 and subsequently activating JAresponsive genes. The DELLA proteins compete with MYC2 for binding to JAZ proteins; the JAZ-DELLA complex inhibits the binding of JAZ to MYC2, which in turn enhances the activity of MYC2 and consequently up-regulates target gene expression [18]. The formation of the JAZ-DELLA complex also inhibits interaction between DELLA and PIF3 proteins [40], and modulates JA-mediated defense in A. thaliana (Figure 2). The inactivation of the DELLA proteins can result from a competitive protein–protein interaction. The loss-offunction mutant scl3 (SCARECROW-LIKE 3) displays a phenotype which mimics GA deficiency and shows a higher than wild type expression level of GA synthesis genes [41]. The interaction between SCL3 and DELLA proteins, in conjunction with the SHR/SCR pathway, indicating that SCL3 is a positive regulator of GAmediated root growth and development [41,42]. The DELLA proteins can interact with certain RING domain proteins (previously known as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3) [43]. The boi quadruple mutant displays an increased GA sensitivity, whereas the over-expression of BOI reduces GA sensitivity. Both BOI and DELLA proteins target the same promoters of various GA-responsive genes, and repress their expression by interacting with each other [43]. SWI3C, one of the core non-catalytic subunits of the SWI/SNF-type chromatin-remodeling complexs (CRCs), interacts with both DELLA and SPINDLY [44], a finding which suggests that DELLA-mediated GA signaling requires the function of SWI3C-containing SWI/ Current Opinion in Plant Biology 2014, 21:89–95
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The GA–DELLA regulatory module affects many aspects of plant growth and development. The degradation of the DELLA proteins is modulated by the endogenous level of GA in a dose-dependent manner. The accumulation of the DELLA proteins is also regulated by auxin, ethylene and abscisic acid, as well as by the plant’s nutritional status, ambient light and temperature conditions and the presence of abiotic stress. The DELLA proteins function as transcriptional repressors by physically interacting with PIFs, BZR1, SPLs, EIN3/EIL1 and ALC transcription factors in a way which reduces their abilities to bind with their target promoter. DELLA proteins also function as transcriptional activators by acting through the DNA binding INDETERMINATE DOMAIN (IDD) family proteins. The in vivo binding of the DELLA-IDD complex with the GA regulator SCARECROW-LIKE 3 (SCL3) promoter activates SCL3 expression. In addition, the DELLA proteins interact with JAZs and consequently interfere with the JAZ-MYC2 interaction, thereby enhancing the activity of MYC2.
SNF CRCs. The recent observation that DWARF14 (D14, a probable candidate strigolactone receptor) can physically interact with SLR1 suggests that the DELLA proteins may mediate cross-talk between strigolactone and GA signaling [45]. However, experimental evidence identifying the function of a D14-DELLA interaction has not yet been forthcoming.
DELLA proteins may act as a quantitative modulator of plant growth in response to environmental changes Comparative studies have suggested that the functional GID1–GA–DELLA regulatory module is highly conserved among vascular plants [46]. The common central thread is the GA-induced degradation of the DELLA proteins, which is controlled by the level of endogenous GA in a dose-dependent manner. The relative abundance of the DELLA proteins is also regulated by auxin, ethylene and Current Opinion in Plant Biology 2014, 21:89–95
abscisic acid, as well as by certain environmental factors, such as the nutritional status of the plant, the ambient temperature and the presence of salinity stress [47–51] (Figure 2). As sessile organisms, plants have evolved to survive adverse environmental conditions by adapting their pattern of growth and development to their immediate environment. Small changes in ambient temperature can affect both the plant’s growth rate and its flowering time [52]. GA is required for temperature-mediated hypocotyl elongation, and the lack of the DELLA protein SLN1 in barley largely overcomes the inhibition of growth imposed by low temperature [19], suggesting that the DELLA proteins can be an important component of the plant response to ambient temperature. PIF4 activates FT expression in a temperature-dependent manner [52], and the loss-of-function pif4-101 mutant is unable to elongate its hypocotyl at high temperature [53], indicating that PIF4 is a central hub mediating the warm temperature www.sciencedirect.com
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response of A. thaliana. Due to the DELLA proteins modulate PIF4 activity through the PIF4-DELLA interaction, a possible mechanism whereby changes in DELLA protein abundance could quantitatively influence regulation of temperature-responsive hypocotyl elongation and flowering time [52]. Controlling the abundance of the PIF4-BZR1-DELLA complex via the dosage-dependent regulation of the GA-induced destabilization of the DELLA proteins could provide an efficient means for the plant to respond to ambient environmental changes [35–37]. It has been shown that an important function of the DELLA proteins relates to their ability to enhance the plant’s level of both salinity tolerance and resistance against necrotrophs via the potentiation of JA signaling [18,50]. The WD-repeat/bHLH/MYB complex has recently been implicated as a direct target of the DELLA proteins. Both DELLA and JAZ proteins interact with, and attenuate the WD-repeat/bHLH/MYB complex to inactivate the expression of downstream genes; one effect is the repression of trichome formation [54]. Therefore, the GA-mediated and JA-mediated degradation of, respectively, DELLA and JAZ proteins represents a mechanism to enable the coordinated activation of the WD-repeat/bHLH/MYB complex, with consequent effects on growth, development and the defense response (Figure 2).
A DELLA-independent pathway is involved in the regulation of GA responses According to the de-repression model, the DELLA proteins are key repressors of GA signaling. However, several recent evidences suggest that a DELLA-independent pathway may be involved in the GA response. As mentioned above, SPINDLY negatively regulates GA signaling. In the loss-of-function A. thaliana spy mutant, both the gai phenotype and the cytokinin response are suppressed [21,55]. The cytosol-localized SPY protein promotes cytokinin responses and suppresses GA signaling, but a nuclear-localized SPY-GFP fusion protein fails to complement the impaired spy phenotype. Conversely, the quadruple-DELLA mutant (lacking GAI, RGA, RGL1 and RGL2) displays a cytokinin-sensitive phenotype. The conclusion is that GA can regulate the cytokinin responses through a DELLA-independent pathway [55]. A recent study has revealed that the GA–DELLA regulatory system regulates fruit development in A. thaliana [56]. In fact, parthenocarpic fruit development is also induced by GA in both the global (lacking GAI, RGA, RGL1, RGL2 and RGL3) and ga1 global mutants, indicating the existence of a DELLA-independent GA signaling pathway affecting fruit growth [56]. The exogenous application of GA is unable to restore normal fruit elongation in the gid1a-1 gid1b-1 gid1c-1 triple mutant, confirming that DELLA-independent GA signaling www.sciencedirect.com
requires GID1-mediated GA perception. As compared with quadruple-DELLA and global mutants, GA treatment does not induce fruit elongation in the quadruple-DELLA spt-2 mutant. The implication is that SPATULA (SPT) acts as a repressor in the regulation of GA-induced fruit development [56], although experimental evidence for the participation of SPT in DELLA-independent GA signaling has yet to be provided.
Conclusion GA is an important regulator of plant growth and development. The recent knowledge regarding the GID1mediated perception of GA and degradation of the DELLA proteins have advanced our understanding of the molecular basis of GA signaling. It is of considerable interest that the DELLA proteins modulate a range of hormonal and environmental signaling activities through protein–protein interactions. Several questions still remain to be resolved. Although it has long been known that a plasma membrane-localized receptor may be required for GA signaling in the aleurone layer, the GID1– GA–DELLA protein complex is localized to the nucleus. GID1 itself is present in the cytoplasm, which raises the possibility that its presence there may be required for a GID1-dependent but DELLA-independent GA signaling pathway. Although the DELLA proteins interact with a diversity of regulatory proteins that function in various signaling pathways, it is unclear how the DELLA proteins are successfully assembled into specific complexes in response to cues emanating from the plant’s developmental status and/or from the external environment. Therefore, a combined biochemical and systems biology approach will be necessary to elucidate how the GA–DELLA module integrates the various relevant endogenous signals and environmental cues to ensure that the plant copes with a fluctuating environment.
Acknowledgements Due to space limitations we apologize to participants whose excellent work could not be mentioned here. This work was supported by National Natural Science Foundation of China (91117015 and 91335207).
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impinge on a common transcription module in Arabidopsis. Nat Cell Biol 2012, 14:810-817. 36. Gallego-Bartolome´ J, Minguet EG, Grau-Enguix F, Abbas M, Locascio A, Thomas SG, Alabadı´ D, Bla´zquez MA: Molecular mechanism for the interaction between gibberellin and brassinosteroid signaling pathways in Arabidopsis. Proc Natl Acad Sci USA 2012, 109:13446-13451. 37. Oh E, Zhu JY, Wang ZY: Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses. Nat Cell Biol 2012, 14:802-809. 38. An F, Zhang X, Zhu Z, Ji Y, He W, Jiang Z, Li M, Guo H: Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings. Cell Res 2012, 22:915-927. 39. Yu S, Galva˜o VC, Zhang YC, Horrer D, Zhang TQ, Hao YH, Feng YQ, Wang S, Schmid M, Wang JW: Gibberellin regulates the Arabidopsis floral transition through miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE transcription factors. Plant Cell 2012, 24:3320-3332. 40. Yang DL, Yao J, Mei CS, Tong XH, Zeng LJ, Li Q, Xiao LT, Sun TP, Li J, Deng XW et al.: Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade. Proc Natl Acad Sci USA 2012, 109:1192-1200. 41. Heo JO, Chang KS, Kim IA, Lee MH, Lee SA, Song SK, Lee MM, Lim J: Funneling of gibberellin signaling by the GRAS transcription regulator SCARECROW-LIKE 3 in the Arabidopsis root. Proc Natl Acad Sci USA 2011, 108:2166-2171. 42. Zhang ZL, Ogawa M, Fleet CM, Zentella R, Hu J, Heo JO, Lim J, Kamiya Y, Yamaguchi S, Sun TP: SCARECROW-LIKE 3 promotes gibberellin signaling by antagonizing master growth repressor DELLA in Arabidopsis. Proc Natl Acad Sci USA 2011, 108:2160-2165. 43. Park J, Nguyen KT, Park E, Jeon J-S, Choi G: DELLA proteins and their interacting RING finger proteins repress gibberellin responses by binding to the promoters of a subset of gibberellin-responsive genes in Arabidopsis. Plant Cell 2013, 25:927-943. 44. Sarnowska EA, Rolicka AT, Bucior E, Cwiek P, Tohge T, Fernie AR, Jikumaru Y, Kamiya Y, Franzen R, Schmelzer E et al.: DELLAinteracting SWI3C core subunit of SWI/SNF chromatin remodeling complex modulates gibberellin responses and hormonal crosstalk in Arabidopsis. Plant Physiol 2013, 163:305317. This study reveals a link between GA signaling and the function of SWI3Ccontaining SWI/SNF chromatin-remodeling complexes (CRCs). SWI3C is shown to interact with both DELLA and SPINDLY, suggesting that an alteration in chromatin structure is required to induce DELLA-mediated GA signaling. 45. Nakamura H, Xue YL, Miyakawa T, Hou F, Qin HM, Fukui K, Shi X, Ito E, Ito S, Park SH et al.: Molecular mechanism of strigolactone perception by DWARF14. Nat Commun 2013, 4:2613. The authors show that the interaction between the probable strigolactone (SL) receptor D14 and SLR1 protein, which provides new insight into crosstalk between GA and SL signaling pathways. 46. Yasumura Y, Crumpton-Taylor M, Fuentes S, Harberd NP: Step-by-step acquisition of the gibberellin-DELLA
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growth-regulatory mechanism during land-plant evolution. Curr Biol 2007, 17:1225-1230. 47. Achard P, Vriezen WH, Van Der Straeten D, Harberd NP: Ethylene regulates Arabidopsis development via the modulation of DELLA protein growth repressor function. Plant Cell 2003, 15:2816-2825. 48. Fu X, Harberd NP: Auxin promotes Arabidopsis root growth by modulating gibberellin response. Nature 2003, 421:740-743. 49. Jiang C, Gao X, Liao L, Harberd NP, Fu X: Phosphate starvation root architecture and anthocyanin accumulation responses are modulated by the gibberellin-DELLA signaling pathway in Arabidopsis. Plant Physiol 2007, 145:1460-1470. 50. Djakovic-Petrovic T, Wit MD, Voesenek LACJ, Pierik R: DELLA protein function in growth responses to canopy signals. Plant J 2007, 51:117-126. 51. Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H, Moritz T, Van Der Straeten D, Peng J, Harberd NP: Integration of plant responses to environmentally activated phytohormonal signals. Science 2006, 311:91-94. 52. Kumar SV, Lucyshyn D, Jaeger KE, Alos E, Alvey E, Harberd NP, Wigge PA: Transcription factor PIF4 controls the thermosensory activation of flowering. Nature 2012, 484: 242-245. This work shows that PIF4 is both necessary and sufficient to trigger flowering in response to ambient temperature changes. Given that the DELLA–PIF4 interaction represses the activity of PIF4, the authors propose a possible mechanism by which changes in GA level (or signaling) could influence temperature-regulated flowering. 53. Koini MA, Alvey L, Allen T, Tilley CA, Harberd NP, Whitelam GC, Franklin KA: High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4. Curr Biol 2009, 19:408-413. 54. Qi T, Huang H, Wu D, Yan J, Qi Y, Song S, Xie D: Arabidopsis DELLA and JAZ proteins bind the WD-Repeat/bHLH/MYB complex to modulate gibberellin and jasmonate signaling synergy. Plant Cell 2014 http://dx.doi.org/10.1105/ tpc.113.121731. The paper describes how both DELLA and JAZ proteins can directly interact with and attenuate the WD-repeat/bHLH/MYB complex to inactivate the expression of target downstream genes, for example repressing trichome formation. The experiments provide a number of insights into the molecular framework involved in the integration of GA and JA signaling, which either in parallel, antagonistically or cooperatively regulate the growth and development of A. thaliana. 55. Maymon I, Greenboim-Wainberg Y, Sagiv S, Kieber JJ, Moshelion M, Olszewski N, Weiss D: Cytosolic activity of SPINDLY implies the existence of a DELLA-independent gibberellin-response pathway. Plant J 2009, 58:979-988. 56. Fuentes S, Ljung K, Sorefan K, Alvey E, Harberd NP, Østergaard L: Fruit growth in Arabidopsis occurs via DELLA-dependent and DELLA-independent gibberellin responses. Plant Cell 2012, 24:3982-3996. This genetic study provides the first evidence for the existence of a GID1dependent but DELLA-independent GA signaling pathway involved in the regulation of fruit growth in A. thaliana. The authors also show that SPATULA is a component of the regulation of DELLA-independent GA signaling and fruit development.
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ScienceDirect Shedding light on integrative GA signaling Hao Xu, Qian Liu, Tao Yao and Xiangdong Fu Gibberellic acid (GA) regulates a diversity of processes associated with plant growth and development. The DELLA proteins act as repressors of GA signaling, and are destabilized by GA. Although it is known that GA signaling integrates various endogenous and environmental signals, the molecular basis of their modulation of plant growth and development is only now beginning to be understood. The current suggestion is that the DELLA proteins act as one possible quantitative modulator of plant growth, achieved by integrating multiple environmental and hormonal signals via protein–protein interactions. This review discusses recent elaborations of the de-repression model proposed to describe the GA response, and focuses on integrative networks thought to regulate plant growth, development and the adaptation to a fluctuating environment. Addresses The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, PR China Corresponding author: Fu, Xiangdong ([email protected])
Current Opinion in Plant Biology 2014, 21:89–95 This review comes from a themed issue on Cell signalling and gene regulation 2014 Edited by Xiangdong Fu and Junko Kyozuka
http://dx.doi.org/10.1016/j.pbi.2014.06.010 1369-5266/# 2014 Elsevier Ltd. All rights reserved.
Introduction The phytohormone GA belongs to a large family of tetracyclic diterpenoids. Since its discovery in the 1950s, 136 structural variants have been identified in a variety of plants, fungi and bacteria (http://www.planthormones.info/ga1info.htm). Only a limited number of these, namely GA1, GA3, GA4, GA5, GA6 and GA7, are thought to have biological activity [1]. In most plant species, GA1 and GA4 are the primary forms associated with the modulation of growth and development; they are active throughout the plant’s life cycle, including seed germination, hypocotyl elongation, leaf expansion and floral induction [2]. Although exogenous GA supply and grafting experiments have both demonstrated that bioactive GA and some of its precursors can be translocated over long distances within the plant [3–5], the sites of GA production and action generally overlap. As an www.sciencedirect.com
example, two fluorescently labeled GA compounds (GA3Fl and GA4-Fl) have been shown to accumulate specifically in the endodermis of the root elongation zone [6]. The revelation that the endodermis must have a particular role in GA regulation is consistent with findings that the endodermis is the primary GA-responsive tissue in the Arabidopsis thaliana root [7]. GA promotes plant growth by promoting the degradation of the DELLA proteins, a family of nuclear growth repressors [2]. The GA signal is perceived by the soluble receptor GA-INSENSITIVE DWARF1 (GID1) [8–10]. The binding of bioactive GA to GID1 induces a conformational change which allows GA–GID1 to interact with the DELLA N-terminal domain [11,12], which in turn promotes GID1-GA–DELLA association with the SCFSLY1/GID2 E3 ubiquitin ligase F-box protein component (SLY1 in A. thaliana and GID2 in rice), thereby targeting the DELLA proteins for degradation via the 26S proteasome pathway [13,14]. It has been proposed that the DELLA proteins can function as transcriptional repressors by blocking the activity of bHLH transcription factors, and it has been experimentally shown that when they directly interact with PHYTOCHROME INTERACTING FACTORS (PIFs), the latter’s DNA binding ability to its promoter target is compromised, thereby affecting light-responsive gene expression and hypocotyl elongation [15,16]. Although the DELLA proteins themselves lack a DNA-binding domain, they are able to function indirectly as transcriptional activators either by interacting with the DNA binding INDETERMINATE DOMAIN (IDD) family proteins [17], or by inactivating the JASMONATE ZIM-domain (JAZ) transcriptional repressors [18]. The DELLA proteins have recently been shown to be involved in protein– protein interactions with certain key regulatory proteins belonging to various hormonal and environmental signaling pathways. This review discusses recent advances in the understanding of GA-mediated growth responses and focuses on the integrative mechanisms underlying the action of the DELLA proteins as a modulator of plant growth, development and adaptation to environmental changes.
GA-induced growth is dependent on the degradation of the DELLA proteins The A. thaliana DELLA proteins GA-INSENSITIVE (GAI), REPRESSOR-of-ga1-3 (RGA), RGA-LIKE1 (RGL1), RGL2 and RGL3, as well as the rice SLENDER RICE1 (SLR1) protein all repress GA-responsive growth [2]. GA stimulates growth by promoting the 26S proteasome-dependent degradation of the DELLA Current Opinion in Plant Biology 2014, 21:89–95
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proteins, thereby relieving the DELLA-imposed growth restraint [19–21]. The recent advances in our understanding of the de-repression model make it possible to explain the role of GA and the function of the DELLA proteins [8–14]. When the formation of the GID1–GA–DELLA complex promotes the interaction between the DELLA C-terminus and the F-box protein component of SCFSLY1/GID2 complex [20–23], leading to the E3 ubiquitin ligase-driven polyubiquitination of the DELLA proteins, and their subsequent targeting for degradation via the ubiquitin 26S proteasome pathway (Figure 1). The FLAG-SLY1 fusion protein co-immunoprecipitates with the HA-GID1b, consistent with the regulatory model which holds that SLY1 controls the stability of the DELLA proteins through its interaction with the GID1–GA–DELLA protein complex [24]. Thus, a key
event of GA-mediated growth stimulation is the degradation of the DELLA proteins. The post-translational modification of the DELLA proteins has suggested an alternative mechanism for controlling their stability and activity. O-GlcNAcylation catalyzed by the SPINDLY protein (similar to animal Olinked N-acetylglucosamine transferase) and phosphorylation catalyzed by a casein kinase have been proposed to increase DELLA protein activity [25,26]. The SUMOylation of the DELLA proteins represents an additional means of DELLA-dependent growth control. When the SUMOylated DELLA proteins interact with SUMOinteracting motif in GID1, GA access to GID1 may be blocked, and as a result, high levels of non-SUMOylated DELLA protein can accumulate; the beneficial effect of
Figure 1
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Schematic representation of the DELLA-mediated GA signaling de-repression regulatory model. The DELLA proteins repress growth, with their stability and activity being enhanced by O-GlcNAcylation, phosphorylation and SUMOylation (DELLA-M: modified DELLA proteins). The DELLA proteins restrain growth by sequestering transcription factors into inactive protein complexes. In addition, the DELLA–PFD interaction locks the PFD complex within the nucleus thereby affecting microtubule organization. Conversely, GA promotes the proteasome-dependent degradation of the DELLA proteins. The binding of bioactive GA to the GA receptor GID1 promotes the GID1–DELLA interaction. The formation of the GID1–GA–DELLA complex subsequently enhances the interaction between the DELLA C terminus and the E3 ubiquitin ligase SCFSLY1/GID2 F-box protein, resulting in the polyubiquitination of DELLA and its targeting for degradation via the 26S proteasome pathway. The GA-induced degradation of the DELLA proteins releases transcription factors, a process which either activates the expression of GA-responsive genes or allows the trafficking of the PFD complex into the cytoplasm, where it can promote GA-induced cell expansion. Current Opinion in Plant Biology 2014, 21:89–95
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this process is to constrain growth in the face of salinity stress [27].
DELLA proteins restrain growth by controlling both cell division and expansion The growth of plant organs reflects a combination of cell proliferation and cell expansion. The short root phenotype of the A. thaliana GA-deficient ga1-3 mutant results from a reduction in the size of the root meristem, but since this characteristic can be reversed by deleting GAI and RGA [28,29], suggesting that the DELLA proteins regulate root growth and development via an effects on cell division. The product of STUNTED acts downstream of the DELLA proteins and promotes cell division by repressing cyclin-dependent kinase inhibitor genes [30]. It has long been known that the effect of GA on axial cell expansion is related to the transverse alignment of the cortical microtubule (CMT) array [31]. The CMTs in the epidermal cells of the Lemna minor root become disorganized following exposure to uniconazole-P, but recover when the root is provided with GA3 [32]. However, little is known regarding the molecular basis of GA-mediated microtubule orientation. A recent study has revealed that GA regulates microtubule organization via an interaction between the DELLA proteins and the prefoldin complex (PFD), a cochaperone required for tubulin folding [33]. In the absence of GA, the DELLA–PFD interaction locks the complex within the nucleus, so compromises a/b-tubulin heterodimer availability in the cytoplasm. Conversely, GA induces the degradation of the DELLA proteins and thus allows PFD to be trafficked into the cytoplasm, thereby increasing the amount of active tubulin subunits and consequently promoting cell expansion (Figure 1). This finding has provided a novel mechanistic link between the GA–DELLA regulatory module and CMT organization.
GA signal transduction requires DELLAmediated protein–protein interactions The DELLA proteins belong to the family of plantspecific GRAS proteins thought to act as transcriptional regulators. Because the DELLA proteins lack their own DNA-binding domain, other transcription factors are required as intermediate components. The in vivo interaction with the bHLH transcription factors PIF3 and PIF4 has been shown to affect the DNA binding activity of the PIFs, resulting in an inhibition of PIF-mediated gene expression and hypocotyl elongation [15,16]. The DELLA proteins also inhibit the activity of ALCATRAZ (ALC), a protein required for fruit development [34]. In addition, the DELLA proteins can interact with BRASSINAZOLE-RESISTANT1 (BZR1), inhibiting its DNA binding both in vitro and in vivo; the supply of GA releases this inhibition [35,36]. The co-activation of PIF proteins by BZR1 highlights a further role in brassinosteroid (BR) signal integration, and reveals that the formation of the www.sciencedirect.com
PIF4-DELLA-BZR1 complex can dynamically regulate plant growth and development in response to GA, BR and light [35,37]. The DELLA proteins can interact with the DNA-binding domains of EIN3/EIL1 to repress EIN3/ EIL1-regulated HOOKLESS1 (HLS1) expression and apical hook development [38]. The interaction between the DELLA proteins and miR156-targeted SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors interferes with SPL transcriptional activity, thereby delaying floral induction through inactivating the expression of miR172 and MADS-box genes [39]. Taken together, the DELLA proteins act as transcriptional repressors by preventing the binding of DELLA-associated transcription factors to their promoter target (Figure 2). The DELLA proteins as transcriptional activator can provide an alternative mechanism for the integration of environmental and endogenous signals to modulate plant development and defense responses. They interact directly with JAZ proteins, the key repressors of jasmonate (JA) signaling, and consequently enhance the transcriptional activation potential of MYC2 [18]. JA is proposed to promote the degradation of JAZ proteins, thereby releasing MYC2 and subsequently activating JAresponsive genes. The DELLA proteins compete with MYC2 for binding to JAZ proteins; the JAZ-DELLA complex inhibits the binding of JAZ to MYC2, which in turn enhances the activity of MYC2 and consequently up-regulates target gene expression [18]. The formation of the JAZ-DELLA complex also inhibits interaction between DELLA and PIF3 proteins [40], and modulates JA-mediated defense in A. thaliana (Figure 2). The inactivation of the DELLA proteins can result from a competitive protein–protein interaction. The loss-offunction mutant scl3 (SCARECROW-LIKE 3) displays a phenotype which mimics GA deficiency and shows a higher than wild type expression level of GA synthesis genes [41]. The interaction between SCL3 and DELLA proteins, in conjunction with the SHR/SCR pathway, indicating that SCL3 is a positive regulator of GAmediated root growth and development [41,42]. The DELLA proteins can interact with certain RING domain proteins (previously known as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3) [43]. The boi quadruple mutant displays an increased GA sensitivity, whereas the over-expression of BOI reduces GA sensitivity. Both BOI and DELLA proteins target the same promoters of various GA-responsive genes, and repress their expression by interacting with each other [43]. SWI3C, one of the core non-catalytic subunits of the SWI/SNF-type chromatin-remodeling complexs (CRCs), interacts with both DELLA and SPINDLY [44], a finding which suggests that DELLA-mediated GA signaling requires the function of SWI3C-containing SWI/ Current Opinion in Plant Biology 2014, 21:89–95
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Figure 2
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The GA–DELLA regulatory module affects many aspects of plant growth and development. The degradation of the DELLA proteins is modulated by the endogenous level of GA in a dose-dependent manner. The accumulation of the DELLA proteins is also regulated by auxin, ethylene and abscisic acid, as well as by the plant’s nutritional status, ambient light and temperature conditions and the presence of abiotic stress. The DELLA proteins function as transcriptional repressors by physically interacting with PIFs, BZR1, SPLs, EIN3/EIL1 and ALC transcription factors in a way which reduces their abilities to bind with their target promoter. DELLA proteins also function as transcriptional activators by acting through the DNA binding INDETERMINATE DOMAIN (IDD) family proteins. The in vivo binding of the DELLA-IDD complex with the GA regulator SCARECROW-LIKE 3 (SCL3) promoter activates SCL3 expression. In addition, the DELLA proteins interact with JAZs and consequently interfere with the JAZ-MYC2 interaction, thereby enhancing the activity of MYC2.
SNF CRCs. The recent observation that DWARF14 (D14, a probable candidate strigolactone receptor) can physically interact with SLR1 suggests that the DELLA proteins may mediate cross-talk between strigolactone and GA signaling [45]. However, experimental evidence identifying the function of a D14-DELLA interaction has not yet been forthcoming.
DELLA proteins may act as a quantitative modulator of plant growth in response to environmental changes Comparative studies have suggested that the functional GID1–GA–DELLA regulatory module is highly conserved among vascular plants [46]. The common central thread is the GA-induced degradation of the DELLA proteins, which is controlled by the level of endogenous GA in a dose-dependent manner. The relative abundance of the DELLA proteins is also regulated by auxin, ethylene and Current Opinion in Plant Biology 2014, 21:89–95
abscisic acid, as well as by certain environmental factors, such as the nutritional status of the plant, the ambient temperature and the presence of salinity stress [47–51] (Figure 2). As sessile organisms, plants have evolved to survive adverse environmental conditions by adapting their pattern of growth and development to their immediate environment. Small changes in ambient temperature can affect both the plant’s growth rate and its flowering time [52]. GA is required for temperature-mediated hypocotyl elongation, and the lack of the DELLA protein SLN1 in barley largely overcomes the inhibition of growth imposed by low temperature [19], suggesting that the DELLA proteins can be an important component of the plant response to ambient temperature. PIF4 activates FT expression in a temperature-dependent manner [52], and the loss-of-function pif4-101 mutant is unable to elongate its hypocotyl at high temperature [53], indicating that PIF4 is a central hub mediating the warm temperature www.sciencedirect.com
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response of A. thaliana. Due to the DELLA proteins modulate PIF4 activity through the PIF4-DELLA interaction, a possible mechanism whereby changes in DELLA protein abundance could quantitatively influence regulation of temperature-responsive hypocotyl elongation and flowering time [52]. Controlling the abundance of the PIF4-BZR1-DELLA complex via the dosage-dependent regulation of the GA-induced destabilization of the DELLA proteins could provide an efficient means for the plant to respond to ambient environmental changes [35–37]. It has been shown that an important function of the DELLA proteins relates to their ability to enhance the plant’s level of both salinity tolerance and resistance against necrotrophs via the potentiation of JA signaling [18,50]. The WD-repeat/bHLH/MYB complex has recently been implicated as a direct target of the DELLA proteins. Both DELLA and JAZ proteins interact with, and attenuate the WD-repeat/bHLH/MYB complex to inactivate the expression of downstream genes; one effect is the repression of trichome formation [54]. Therefore, the GA-mediated and JA-mediated degradation of, respectively, DELLA and JAZ proteins represents a mechanism to enable the coordinated activation of the WD-repeat/bHLH/MYB complex, with consequent effects on growth, development and the defense response (Figure 2).
A DELLA-independent pathway is involved in the regulation of GA responses According to the de-repression model, the DELLA proteins are key repressors of GA signaling. However, several recent evidences suggest that a DELLA-independent pathway may be involved in the GA response. As mentioned above, SPINDLY negatively regulates GA signaling. In the loss-of-function A. thaliana spy mutant, both the gai phenotype and the cytokinin response are suppressed [21,55]. The cytosol-localized SPY protein promotes cytokinin responses and suppresses GA signaling, but a nuclear-localized SPY-GFP fusion protein fails to complement the impaired spy phenotype. Conversely, the quadruple-DELLA mutant (lacking GAI, RGA, RGL1 and RGL2) displays a cytokinin-sensitive phenotype. The conclusion is that GA can regulate the cytokinin responses through a DELLA-independent pathway [55]. A recent study has revealed that the GA–DELLA regulatory system regulates fruit development in A. thaliana [56]. In fact, parthenocarpic fruit development is also induced by GA in both the global (lacking GAI, RGA, RGL1, RGL2 and RGL3) and ga1 global mutants, indicating the existence of a DELLA-independent GA signaling pathway affecting fruit growth [56]. The exogenous application of GA is unable to restore normal fruit elongation in the gid1a-1 gid1b-1 gid1c-1 triple mutant, confirming that DELLA-independent GA signaling www.sciencedirect.com
requires GID1-mediated GA perception. As compared with quadruple-DELLA and global mutants, GA treatment does not induce fruit elongation in the quadruple-DELLA spt-2 mutant. The implication is that SPATULA (SPT) acts as a repressor in the regulation of GA-induced fruit development [56], although experimental evidence for the participation of SPT in DELLA-independent GA signaling has yet to be provided.
Conclusion GA is an important regulator of plant growth and development. The recent knowledge regarding the GID1mediated perception of GA and degradation of the DELLA proteins have advanced our understanding of the molecular basis of GA signaling. It is of considerable interest that the DELLA proteins modulate a range of hormonal and environmental signaling activities through protein–protein interactions. Several questions still remain to be resolved. Although it has long been known that a plasma membrane-localized receptor may be required for GA signaling in the aleurone layer, the GID1– GA–DELLA protein complex is localized to the nucleus. GID1 itself is present in the cytoplasm, which raises the possibility that its presence there may be required for a GID1-dependent but DELLA-independent GA signaling pathway. Although the DELLA proteins interact with a diversity of regulatory proteins that function in various signaling pathways, it is unclear how the DELLA proteins are successfully assembled into specific complexes in response to cues emanating from the plant’s developmental status and/or from the external environment. Therefore, a combined biochemical and systems biology approach will be necessary to elucidate how the GA–DELLA module integrates the various relevant endogenous signals and environmental cues to ensure that the plant copes with a fluctuating environment.
Acknowledgements Due to space limitations we apologize to participants whose excellent work could not be mentioned here. This work was supported by National Natural Science Foundation of China (91117015 and 91335207).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Current Opinion in Plant Biology 2014, 21:89–95
