Abstract
The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.
Highlights
Background spy6 spy-7 spy-8 spy-9 spy-10 spy-11 spy-12 spy-13 spy-14 spy-15 spy-16 spy-17 spy-18 spy-19 spy-20G (1823) / A ATACTTGC (2902–2909) / TT G (2778) / A; skipping of exon 8 (2710–2788)b G (1822) / A G (1823) / A G (1823) / A G (3893) / A C (3899) / T C (3899) / T G (3883) / A C (5076) / T C (5076) / T Deletion of 3# endc n.i.d T (5270) deletedGly-268 / Glu Ile-Leu-Ala (390–392) / Phe Partial deletion of TPR8 /TPR9 (354–376) Gly-268 / Arg Gly-268 / Glu Gly-268 / Glu Gly-570 / Asp Thr-572 / Met Thr-572 / Met Glu-567 / Lys Arg-815 / Trp Arg-815 / Trp Leu-782 to Ser-914 deleted n.i
The inflorescence stems of the spy-8 plants grew slower than wild type, but the final height is similar to wild type (Table II). [See online article for color version of this figure.]
Phenotypic characterization of 12 spy alleles revealed that mutations in TPRs 6, 8, and 9 or the catalytic region affect GA signaling in floral induction and fertility
Summary
The spy mutants exhibit pleiotropic phenotypes, some of which are related to elevated GA response. When comparing phenotypes of the homozygous rga-D17 line and the double homozygous spy-8 rga-D17 plant (Fig. 5; Table IV), we found that spy-8 partially rescued all the phenotypes of rga-D17 but did not restore its GA responsiveness These results are consistent with the hypothesis that SPY may activate the DELLA proteins by GlcNAc. Values for height and silique number are means 6 SE. To examine the effect of spy on the subcellular localization of RGA, we generated a spy-8 line that contains the PRGA:green fluorescent protein (GFP)-RGA transgene by crosses between spy-8 and a previously characterized transgenic Ler/PRGA:GFP-RGA line (Silverstone et al, 2001) This PRGA:GFP-RGA fusion gene was able to rescue the phenotype of a null rga allele, and the GFP-RGA fusion is localized to the nucleus in transgenic Arabidopsis (Silverstone et al, 2001; Fig. 6E). The less active form of RGA and rga-D17 in the spy-8 background may accumulate to a higher level
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