Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis

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.

Figure 1.

The protein defects caused by 19 spy alleles. A, Schematic representation of the SPY protein with the nature and location of 19 spy mutations indicated. The numbered boxes represent the TPRs. CDI and CDII are conserved domains found in OGTs (Roos and Hanover, 2000). The dotted line indicates the portion of SPY encoded by the PstI fragment affected in spy-18. The defects in spy-1 to spy-5 were determined previously (Jacobsen et al., 1996). B, Locations of the mutations affecting TPRs 6, 8, and 9. The underlined amino acids of TPR 8 and 9 are predicted to be deleted in spy-1, spy-2, and spy-8. spy-7 causes replacement of the bolded amino acids with Phe. C, C-terminal alignment of SPY (Ath) and OGTs from C. elegans (Cel) and rat (Rat). Consensus residues (Con) represent conserved amino acids among the three sequences. The amino acid changes caused by the spy alleles are indicated below the consensus. The portion of SPY encoded by the PstI fragment that was affected in spy-18 is indicated by the dotted line above the SPY sequence. The spy-20 mutation deleted one nucleotide of codon 879 (Table I), which affects the amino acids after Ala-879 (underlined). The new amino acids encoded by the frame-shifted region are shown below the consensus. Due to a single nucleotide polymorphism, position 879, A, is an Ala in Ler and Ser in Col-0.

Figure 2.

The spy alleles flowered earlier than wild type in SD. A, Number of days from sowing until floral buds are clearly visible. B, Number of rosette and cauline leaves produced by the primary inflorescence stem after bolting. The values plotted are the means ± se of 10 plants. Some error bars are too small to be seen. The spy alleles are organized in the order of the mutations in the SPY genomic sequence. P, Promoter; CT, C terminus.

Figure 3.

SD conditions partially restored the fertility of the spy mutants. Fertility was measured as the average number of seeds per silique in the first 15 siliques on the primary inflorescence stems of 10 plants that were grown in LD or SD conditions. The values plotted are the means ± se of 10 plants. The spy alleles are organized in the order of the mutations in the SPY genomic sequence. *, Sterile; §, not determined.

Figure 4.

Abnormal trichome branching and inflorescence phyllotaxis in the spy mutants. A, Most of the trichomes on wild-type Ler leaves have three branches, whereas spy-16, -18, and -20 produce higher percentages of four branched trichomes. Examples of three- (wild type), four- (spy-16 and -18), and five-branch trichomes are shown. B, spy-2 and spy-18 showed abnormal inflorescence phyllotaxy in comparison to wild-type Ler. [See online article for color version of this figure.]

Figure 5.

spy-8 partially suppresses rga-Δ17 but does not restore GA responsiveness. Plants were grown on soil for 36 d with 100 μm GA₃ (+) or water (−) treatment (starting at day 18). All plants are homozygous for the genotype(s) indicated, except rga-Δ17; homo, plants are homozygous for rga-Δ17; hemi, plants are hemizygous for rga-Δ17. Representative plants are shown. 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.]

Figure 6.

The levels of RGA and rga-Δ17 proteins, but not mRNA, are elevated in the spy mutant backgrounds. A to C, The blots contain 25 μg (in A and B) or 50 μg (in C) total protein from 8-d-old seedlings after 2-h treatment with water (−) or 100 μm GA₃ (+) as labeled. Affinity-purified rabbit anti-RGA antibodies were used to detect the RGA (64 kD) and rga-Δ17 (62 kD) proteins (form I). The extra band (form II) may be a modified rga-Δ17. The images in B were developed after a shorter exposure on film than A and C. An image of the Ponceau-stained blot is shown below the immunoblot in C to confirm equal loading. D, Relative RGA mRNA levels determined by quantitative RT-PCR. Total RNA was isolated from wild type and various mutants after 100 μm GA₃ (+) or water (−) treatment for 2 h. The relative RGA mRNA levels were determined by running three quantitative RT-PCR reactions for each sample and normalized using the housekeeping gene GAPC. Bars = means ± se. The value of water-treated wild type was arbitrarily set to 1.0. Proteins or mRNA in A to D were extracted from homozygous lines as labeled, except that the sample for the rga-Δ17 line was extracted from a mixture of hemi- and homozygous plants. E, GFP fluorescence in root tips of transgenic lines expressing GFP-RGA in wild-type or spy-8 backgrounds. The images were captured by confocal laser microscopy under an identical setting.