Protein-protein interactions

DELLAs were known to interact in a GA-dependent manner, with the GA receptor GID1, recruiting a cognate F-box protein (GID2 in rice and SLY1 in A. thaliana), to form a tripartite interaction (Willige et al., 2007, Griffiths et al., 2006, Hirano et al., 2010). F-box protein binding targets DELLA proteins for proteasome-mediated degradation. In the absence of degradation, the tripartite interaction blocks and inactivates the C-terminal domain of DELLA proteins, disrupting their regulatory roles.

DELLA proteins likely do not directly bind DNA. They regulate transcription indirectly, by binding transcription factors. DELLA proteins, in the absence of GA, bind PIF3 (PHYTOCHROME INTERACTING FACTOR 3) protein, a basic helix-loop-helix (bHLH)-type transcription factor, and prevent it from binding its target promoters. PIF3 binds DNA to regulate other transcription factors, which in turn activate genes involved in hypocotyl expansion and

skotomorphogenesis. The result of RGA-PIF3 interaction is impaired hypocotyl elongation. PIFs also interact with the light-activated form of the photoreceptor phytochrome B (phyB), and are degraded through the 26S proteasome system (Feng et al., 2008). Bimolecular

fluorescence complementation (BiFC) imaging visualising direct RGA-PIF3 interaction in vivo in the nucleus suggests that the DELLA interaction with PIF3 does not affect PIF3 interaction with phyB, but rather affects PIF3’s DNA-binding transcription regulation activity. In support of this,

in vitro pre-binding of PIF3 to a G-box-containing DNA oligonucleotide inhibits RGA-PIF3

interaction. The authors confirmed that the RGA protein itself does not bind DNA using Chromatin ImmunoPrecipitation (ChIP) experiments with 38 GA-responsive promoters, all giving a negative result.

PIF4 (PHYTOCHROME INTERACTING FACTOR 4) is a bHLH transcription factor homologous to

PIF3 (de Lucas et al., 2008). PIF4,like PIF3, promotes cell elongation in the dark. It is negatively

regulated by light through phyB -dependent proteasome-mediated protein destabilisation and by interaction with DELLA proteins, which bind to PIF4 DNA-binding surface and block its

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interaction with DNA. RGA, GAI, RGL1 and RGL3 all interact with PIF3 and PIF4. RGA has a higher affinity for PIF4 than for PIF3. Given that DELLAs bind to the conserved DNA-binding domain of PIF3 and PIF4, it is possible, but not yet proven, that DELLAs may bind all members of the PIF bHLH subfamily of transcription factors.

Besides PIF3 and PIF4, GAI and RGL2 interact with other bHLH transcription factors of subfamily 15, SPATULA (SPT), PHYTOCHROME INTERACTING FACTOR-LIKE 2 and 5 (PIL2, PIL5) (Gallego-Bartolome et al., 2010). SPT and PIL5 inhibit GA biosynthesis at GA3ox (1&2) in cold (and to a lesser extent in the dark) in germination and seedling growth. However, independent investigation found that the regulation of SPT is independent of GA, despite its dependence on DELLA proteins (Sidaway-Lee et al., 2010). These contradictory findings indicate another complex and yet unexplored regulatory loop, or suggest that DELLAs bound to these bHLH proteins are insensitive to GA.

ALCATRAZ (ALC) is a bHLH transcription factor in the same subfamily as PIF3 and -4.ALC is

required for the specification of cells in the separation layer of the silique. This layer secretes enzymes into the replum (valve margin) to break it down and separate the two valves of the silique for seed dispersal. DELLAs (RGA and possibly GAI and RGL2) directly interact with ALC

and stop its function (Arnaud et al., 2010). Overall fruit patterning in A. thaliana is controlled by GA; interaction of GA signalling with the DELLA-mediated inhibition of ALC has not been investigated.

JA ZIM-DOMAIN 1 (JAZ1), a repressor of Jasmonic Acid (JA) signalling, interacts with DELLA

proteins. Binding to DELLA protein prevents JAZ1from blocking MYC2, the repressor of JA signalling. From analysis of truncated of RGA and JAZ mutants, DELLA motif and the N-terminal of the two leucine zippers are the RGA domains that interact with the NT and Jaz (or tify) domains of JAZ1 (Hou et al., 2010). GA signalling leads to degradation of DELLA proteins, thereby releasing JAZ1 from inhibition. JAZ1 is then free to inhibit MYC2 and repress JA signalling. JA signalling is involved in necrotrophic response to pathogens, mediating necrosis; GA signalling is therefore repressing this type of response. GA also modulates the salicylic acid (SA) signalling pathway-mediated biotrophic response to pathogens. JAZ1 has also found to interact with MYB21 and MYB24 (R2R3-MYB transcription factors) that mediate the JA- regulated development of stamens in Arabidopsis (Song et al., 2011).

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SCARECROW-LIKE 3 (SCL3), a GRAS superfamily protein that has a C-terminal domain

conserved with DELLA proteins, interacts with some DELLA proteins. Interaction of SCL3 with RGA and possibly GAI and RGL1 attenuates DELLA activity during germination and seedling growth in the root and hypocotyl (Heo et al., 2011, Zhang et al., 2011). SCL3 is a positive regulator of GA synthesis, up regulating GA20ox and GA3ox expression. SCL3 is a DNA binding protein; it is (auto) down regulating its own expression by binding to the SCL3 promoters. DELLA promotes SCL3 expression and this may be by direct promoter binding of DELLA-SCL3 complex. The root meristem and ground tissue specification is controlled by SCARECROW- SHORTROOT (SCR-SHR) GRAS protein heterodimer activity which promotes SCL3 expression. Based on these findings it was proposed that SCR-SHRand SCL3-SCL3and SCL3-DELLA

interactions are balanced to integrate the GA signal into control of root meristematic size and activity through differentiation and elongation. SCL3 promotes GA-induced root elongation by reducing DELLA activity in the endodermal cells. SCL3 is also involved in plant development in tissues and organs other than the root, but this may be mostly DELLA-independent.