SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant

Abstract

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.

Figure 1

Analysis of SPY::GUS1 expression. The expression of the SPY gene during plant development was examined using the SPY::GUS1 reporter gene, which expresses GUS under the control of the SPY promoter. SPY5′ is SPY genomic sequence from an HindIII site 2,361 bp upstream of the 5′ end of exon 1, all of exon 1 (which is not translated), intron 1, and the first 16 nucleotides of exon 2 just before the SPY start codon. An asterisk represents the stop codon. For A through D, I, and J, the GUS staining reaction was allowed to proceed at 25°C, whereas other images were stained at 37°C to increase the intensity of the staining (see “Materials and Methods”). The numbers in A and B indicate seedling age in days. The plant in H is 3 weeks old. The plants in I and J were treated with either 2 μL of ethanol or 2 μL of ethanol containing 20 μg of GA₃, respectively, and stained 24 h after treatment.

Figure 2

SPY-GFP is present predominantly in the nucleus. Localization of SPY-GFP in the roots of SPY::SPY-GFP plants. An asterisk represents the stop codon. A, SPY-GFP is expressed in roots. B, GFP is localized predominantly to the nucleus of cells in the zone of elongation.

Figure 3

SPY is present predominantly in the nucleus. A, Anti-SPY antibody was used to detect SPY in protein extracts from Arabidopsis seedlings (lane 1) and from cauliflower (Brassica oleracea var. botrytis) heads (lane 2). Each lane contained 50 μg of protein. SPY was detected using two different antisera (see “Materials and Methods”) with similar results. B, Identical blots, each containing nuclear and crude cytosolic protein preparations from cauliflower inflorescence, were probed with anti-SPY, antihistone, and antitubulin antibodies. Each lane contained approximately 35 μg of protein.

Figure 4

spy-4 roots grow abnormally on slanted plates. WT and spy-4 seedlings were germinated on slanted plates on 1% (w/v) agar as described in “Materials and Methods” to compare root growth and development. A, WT Ws seedlings. B, spy-4 (Ws background) seedlings. C, Enlarged view of WT roots showing uniform twisting pattern. D, Enlarged view of spy-4 roots showing abnormal twisting pattern.

Figure 5

SPY is required for normal “root waving.” Seedlings were grown on slanted plates as for Figure 4, and the angle of the root tip from the vertical (relative to the highest part of the plate) determined as shown in the inset. The data show the root angle observed from the front of the plates. The frequency value represents the number of seedlings in each class.