Regulation of flowering time in Arabidopsis

 

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Flowering pathway

Gibberellin pathway

References

 

The Gibberellin transduction pathway

 

RGA (Repressor of the ga1-3 mutant) (also: GRS)

 

GRS (GAI-Related Sequence) was cloned and sequenced by Peng et al. (1997), and later re-named RGA (Silverstone et al., 1998). RGA encodes a regulatory protein of the VHIID family, with a highly conserved C-terminal GRAS domain,  (Lee et al., 2002. See also: Wen and Chang, 2002) and an N-terminal DELLA domain. It contains Leucine repeats that may be involved in protein-protein interaction. It also contains a putative nuclear localization signal (Silverstone et al., 1998).

The sequence contains no introns (Tyler et al., 2004) and encodes a predicted 587 amino-acids protein of 64 kDa (Peng et al., 1997; Dill et al., 2004).

The RGA sequence shows high similarities with GAI. See here for sequence and function similarities between  the DELLA protein genes

RGA  is a low-abundance, nuclear protein :

The RGA protein could not be detected in immunoblot analysis, which indicates low protein levels (Dill et al., 2004). In the WT, the protein is mostly present in the rosette leaves (Tyler et al., 2004).

According to transient expression assays performed on onion epidermal cells, the RGA protein accumulates in the nucleus (Silverstone et al., 1998). These results were later confirmed in stable transformation assays (Silverstone et al., 2001).

RGA expression:

In the WT, the gene is expressed in all the tissues studied (Silverstone et al., 1998; Tyler et al., 2004). The expression level of RGA is higher in the stem, a bit lower in the rosette and roots, and lower in siliques and flowers (Silverstone et al., 1998).

However, the expression profile of RGA does not necessarily reflect the RGA protein activity, and over-expression of RGA (under the control of the 35S promoter) does not confer any obvious phenotype (Dill et al., 2001).

rga loss-of-function mutants:

The first rga mutants (alleles rga-1 to rga-17) were identified through a ga1-3 suppressor screen (Silverstone et al., 1997(1)). A second series of mutagenesis (fast neutrons) led to the isolation of the mutant alleles rga-18 to rga-27 (Silverstone et al., 1998).

The rga loss-of-function mutations partially complements the ga1-3 phenotype (dwarf and late-flowering). As the loss of the RGA function partially de-represses the GA signaling pathway, this means that RGA is a negative regulator of the GA response (Silverstone et al., 1997(1); King et al., 2001).

However, in the WT background, the rga loss-of-function mutants have no obvious phenotype: the plants are a bit paler than the WT but have overall the same flowering time (Silverstone et al., 1997(1); Silverstone et al., 1998). This suggests that another gene exists, with a redundant function (likely GAI).

All the rga loss-of-function mutants are recessive.

  • In rga-1, a point mutation causes a premature stop codon. The mutant protein lacks 67 amino acids in the C-terminal domain (including the GRAS domain) (Silverstone et al., 1998).  Unlike the native RGA, the mutant protein  is resistant to GA-mediated degradation, and is present in large amounts in the rga-1 mutants (Dill et al., 2004).

  • rga-2 is a mis-sense point mutation, which does not seem to affect the gene expression (Silverstone et al., 1998).

  • rga-20, rga-24 and rga-26 have deletions in the coding sequence, spanning the entire coding sequence in the case of rga-24 and rga-20 (obviously, these are null mutations) (Silverstone et al., 1998). The rga-24 mutant is a commonly used loss-of-function mutant.

  • In rga-22, a single amino-acid deletion occurs in the GRAS domain. The resulting protein is still sensitive to GA-mediated degradation (Dill et al., 2004).

  • rga-28 contains a T-DNA insertion in the RGA coding sequence that makes the protein non-functional (Tyler et al., 2004).

rga-D17 gain-of-function allele:

rga-D17 is a mutant allele constructed in such a way that its DELLA domain carries the same alteration as the gai-1 gain-of-function mutant. The rga-D17 allele lacks 17 amino-acids in the DELLA domain of the N-terminus.

Unlike the rga loss-of-function mutants, the rga-D17 mutant is dwarfed and does not respond to GA treatment (Dill et al., 2001). The rga-D17 mutant phenotype is semi-dominant.

  • The DELLA domain is not necessary for nucleus localization: Stable GFP fusion experiments show that the rga-D17 mutant protein is still localized in the nucleus (Dill et al., 2001).

  • The DELLA domain is necessary for GA-induced degradation: The RGA protein levels are low in the WT, higher in the ga1-3 mutants and very high in the ga1-3 rga-D17 mutants, and not affected by GA treatment. This means that the rga-D17 mutant protein is not sensitive to GA-mediated degradation any more (Dill et al., 2001). The  DELLA domain is therefore necessary for the GA-dependent proteolysis of RGA. See also here ( interaction between the GA signal and SLY1 and DELLA proteins), and here (Interaction between GAs and RGA).

 

GA-induced degradation of RGA protein by SLY1

Other GA effects on RGA

Redundancy between DELLA protein genes

RGA down-regulates miR159

SPY up-regulates RGA