SUGAR-INSENSITIVE3, a RING E3 ligase, is a new player in plant sugar response

Abstract

Sugars, such as sucrose and glucose, have been implicated in the regulation of diverse developmental events in plants and other organisms. We isolated an Arabidopsis (Arabidopsis thaliana) mutant, sugar-insensitive3 (sis3), that is resistant to the inhibitory effects of high concentrations of exogenous glucose and sucrose on early seedling development. In contrast to wild-type plants, sis3 mutants develop green, expanded cotyledons and true leaves when sown on medium containing high concentrations (e.g. 270 mm) of sucrose. Unlike some other sugar response mutants, sis3 exhibits wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol, a gibberellic acid biosynthesis inhibitor, on seed germination. Map-based cloning revealed that SIS3 encodes a RING finger protein. Complementation of the sis3-2 mutant with a genomic SIS3 clone restored sugar sensitivity of sis3-2, confirming the identity of the SIS3 gene. Biochemical analyses demonstrated that SIS3 is functional in an in vitro ubiquitination assay and that the RING motif is sufficient for its activity. Our results indicate that SIS3 encodes a ubiquitin E3 ligase that is a positive regulator of sugar signaling during early seedling development.

Figure 1.

Suc-insensitive phenotype of sis3-1. Seeds of sis3-1 and Ws-2 wild type were sown on minimal medium containing the indicated sugars and grown under continuous light at 22°C for 2 weeks. The percentages of seeds that germinated and developed into seedlings with green expanded cotyledons and true leaves were scored. Data represent the means of three independent assays. Error bars represent sd. Asterisks indicate where the phenotype of the sis3-1 mutant differed from that of the wild type by P < 0.05, according to Student's t test. Sorbitol-containing medium was included as an osmotic control. This experiment was repeated with similar results.

Figure 2.

Map-based cloning of SIS3. A, Map of chromosome 3 surrounding SIS3. The number of recombinants (rec) found in 1,200 DNA samples is shown. SIS3 is located between 17,701 and 17,774 kb on bacterial artificial chromosome (BAC) T17F15. B, Structure of the SIS3 genomic sequence displaying positions of T-DNA insertions relative to the beginning of the start codon. Boxes represent exons and lines represent introns. Black boxes denote coding regions. C, Quantitative real-time RT-PCR analysis of relative SIS3 transcript levels in mutant and wild-type (Col-0) plants. Binding locations of primer pairs A and B are denoted by the short arrows in B. Error bars indicate sd.

Figure 3.

SIS3 mediates sugar response during early seedling development. Seedlings homozygous for the sis3-2 and sis3-3 mutations are more resistant to 300 mm Suc and Glc than are wild-type Col-0 seedlings. Complementation of sis3-2 with a genomic clone of At3g47990 (sis3 com) restores the mutant to a wild-type level of sugar sensitivity. Sorbitol-containing medium was included as an osmotic control. Mutant, transgenic, and wild-type seedlings exhibit similar phenotypes on medium containing 300 mm sorbitol. Data represent the means of three independent assays ± sd. Asterisks indicate results where the phenotypes of sis3-2 and sis3-3 differ from that of the wild type by P < 0.05, according to Student's t test. The results of the sis3-2 line transformed with a genomic clone of At3g47990 (sis3 com) did not differ significantly from those of the wild type. The image at bottom shows the growth phenotypes of sis3-2, sis3-3, sis3 com, and Col-0 wild type on 300 mm Suc after 12 d.

Figure 4.

The sis3 mutant lines display wild-type or near wild-type responses to ABA and GAs during seed germination. A, Seeds were sown on minimal medium (Kranz and Kirchheim, 1987) supplemented with 0, 0.5, or 1 μm ABA and incubated at 22°C under continuous light for 9 d prior to scoring. Germination is defined as the emergence of the radicle from the seed coat. Seeds homozygous for the abi4-103 mutation, which has previously been shown to confer an ABA-resistant phenotype (Laby et al., 2000), were used as a positive control. Data represent the means of three independent assays ± sd. The asterisk indicates where mutant and wild-type (Col-0) results differed at P < 0.05 according to Student's t test. B, Seeds were sown as above on medium containing 0 or 30 μm paclobutrazol. Seeds homozygous for the spy3 mutation, which has previously been shown to confer a paclobutrazol-resistant phenotype (Jacobsen and Olszewski, 1993), were used as a positive control. Data represent the means of three independent assays ± sd. The asterisk indicates where mutant and wild-type (Col-0) results differed at P < 0.05 according to Student's t test.

Figure 5.

Root growth phenotypes of wild-type, sis3-2, and sis3-3 plants. Sterilized seeds were suspended in a 0.15% agar solution and incubated at 4°C in the dark for 3 d. Seeds were then sown on solid MS medium and placed in the light at 22°C for 3 d. Germinated seeds were next transferred to solid MS medium supplemented with the indicated concentrations of ABA and grown vertically in continuous light at 22°C for 5 d before measuring primary root lengths. The sis3-2 and sis3-3 seedlings had slightly longer roots than wild-type seedlings on medium containing 0 μm ABA, although the mutant and wild-type results did not differ at P < 0.05, according to Student's t test. To account for the slightly longer mutant root lengths on medium lacking ABA, the root lengths of seedlings grown on medium containing ABA were divided by the average root length of the same plant line on medium lacking ABA to obtain a length ratio. Data represent means ± sd (n = 30). The length ratios of the mutants did not differ from those of the wild type at P < 0.05 for any of the media tested.

Figure 6.

Gene expression analyses. A, Organ-specific expression of the SIS3 gene. Total RNA was extracted from aerial parts of the indicated organs of 32-d-old wild-type Col-0 plants grown on soil under continuous light. SIS3 mRNA levels were measured using quantitative real-time RT-PCR. Data were obtained from two biologically independent experiments, and relative mRNA levels were determined using ACTIN2 as a reference. The expression level of SIS3 in roots was set to 100. Data represent means ± sd. B, Quantitative real-time RT-PCR analysis of SIS3 transcript levels in wild-type Col-0 germinating seeds treated with 300 mm Glc or sorbitol (sorb). The Glc-repressed CAB4 gene was used as a positive control for Glc treatment. Data were obtained from two biologically independent experiments, and relative mRNA levels were determined using ACTIN2 as a reference. The SIS3 mRNA level in sorbitol-treated Col-0 was set to 100. Data represent means ± sd. C, Quantitative real-time RT-PCR analysis of sugar-regulated gene expression in sis3-2, sis3-3, and wild-type Col-0 germinating seeds treated with 100 mm Glc or sorbitol. Relative mRNA levels were determined using ACTIN2 as a reference. The mRNA levels in sorbitol-treated wild-type Col-0 were set to 100. Data represent means ± sd. D, Quantitative real-time RT-PCR analysis of genes postulated to be involved in sugar response pathways. Wild-type Col-0 and sis3-3 germinating seeds were treated with 100 mm Suc or sorbitol. Relative mRNA levels were determined using ACTIN2 as a reference. The mRNA levels in sorbitol-treated wild-type Col-0 were set to 100. Data represent means ± sd.

Figure 7.

SIS3 has ubiquitin E3 ligase activity. A, Schematic depiction (not to scale) of putative SIS3 protein domains predicted by different software programs (see text for more details). The prediction of a signal peptide or signal anchor is inconsistent between different software programs and is thus depicted with a question mark. B, E3 ubiquitin ligase activity of SIS3 in vitro. GST-SIS3 fusion proteins containing the full-length SIS3 protein (FL) or only the RING domain of SIS3 (RING) were incubated with E1, E2 (UBC8), and ubiquitin (UBQ). Polyubiquitin chains were visualized with anti-ubiquitin antibodies. Omission of E1, E2, or ubiquitin resulted in a loss of ubiquitination. GST by itself served as a negative control. Numbers on the left indicate the molecular masses of marker proteins in kD.