Characterization of mutants in Arabidopsis showing increased sugar-specific gene expression, growth, and developmental responses

Abstract

Sugars such as sucrose serve dual functions as transported carbohydrates in vascular plants and as signal molecules that regulate gene expression and plant development. Sugar-mediated signals indicate carbohydrate availability and regulate metabolism by co-coordinating sugar production and mobilization with sugar usage and storage. Analysis of mutants with altered responses to sucrose and glucose has shown that signaling pathways mediated by sugars and abscisic acid interact to regulate seedling development and gene expression. Using a novel screen for sugar-response mutants based on the activity of a luciferase reporter gene under the control of the sugar-inducible promoter of the ApL3 gene, we have isolated high sugar-response (hsr) mutants that exhibit elevated luciferase activity and ApL3 expression in response to low sugar concentrations. Our characterization of these hsr mutants suggests that they affect the regulation of sugar-induced and sugar-repressed processes controlling gene expression, growth, and development in Arabidopsis. In contrast to some other sugar-response mutants, they do not exhibit altered responses to ethylene or abscisic acid, suggesting that the hsr mutants may have a specifically increased sensitivity to sugars. Further characterization of the hsr mutants will lead to greater understanding of regulatory pathways involved in metabolite signaling.

Figure 1.

Gene expression in the hsr mutants. A and B, Luciferase activity in the hsr mutants. Luciferase levels were measured in 10-d-old seedlings of the A3L3 parental line and hsr mutants grown on media containing different concentrations of Suc (A) and Glc (B). Seedlings were sprayed with luciferin and analyzed luminometrically. Data were calculated from at least 50 seedlings. At sugar concentrations of 0.2% (w/v) or higher, hsr seedlings exhibited significantly higher (P < 0.005) levels of luciferase activity compared with the A3L3 parental line. C, Reverse transcription (RT)-PCR analysis of transcript levels in the hsr mutants. RT-PCR was performed on first strand cDNA made from 10-d-old seedlings grown in constant light on medium containing 0% or 3% (w/v) Suc. cDNA was standardized by reference to an actin standard. The number of amplification cycles used for each set of gene specific primers were: actin, 22; ApL3, 26; Atβ-Amy, 26; VSP1, 26; and STP1, 24. C, Control parental line A3L3.

Figure 2.

Growth regulator responses of the hsr mutants. A, Root elongation in response to 5 μm ABA. Seedlings were grown on sugar-free half-strength Murashige and Skoog medium for 4 d before transfer to vertical plates containing 5 μm ABA. Root elongation was measured after 4 d and relative root elongation was calculated relative to root growth on ABA-free plates (n = 18). The ABA-insensitive mutant abi1 was included as a control. There were no significant differences in ABA sensitivity between the A3L3 parental line and the hsr mutants. B, Hypocotyl elongation in response to ACC treatment. Approximately 100 seedlings of the parental line A3L3, hsr1, hsr2-1, hsr3, hsr4, the ethylene-insensitive line ein2, and the ethylene-resistant line etr1 were stratified for 4 d on Murashige and Skoog medium supplemented with 1% (w/v) Suc in the presence and absence of 10 μm ACC. Hypocotyl length was measured 6 d after transfer to 20°C in the dark. There were no significant differences between the A3L3 parental line and the hsr mutants.

Figure 3.

Carbohydrate levels and uptake in the hsr mutants. A, Levels of Glc, Fru, Suc, and starch were measured in 10-d-old seedlings grown on medium containing 1% (w/v) Suc in continuous light. The hsr2-1 allele was used. Significant differences (Student's t test; P < 0.05) to A3L3 are indicated by an asterisk. B, Glc uptake in 7-d-old seedlings was measured using U-¹⁴C-Glc. Ws is the ecotype transformed with the 35S:STP1 transgene overexpressing the STP1 hexose transporter. sd of three replicates is given. Independent repeats of uptake assays gave similar results. The hsr2-1 allele was used.

Figure 4.

Phenotypes of the hsr mutants in response to sugars. A, Seedling establishment in response to different Suc concentrations was measured by plating approximately 100 seedlings on medium containing different concentrations of Suc and stratifying these for 4 d before transfer to constant light at 20°C. The percentage of seedlings exhibiting expanded green cotyledons after 5 d was measured. At ≥5% (w/v) Suc, all hsr mutants exhibited significantly reduced seedling establishment (P < 0.05) compared with the parental line A3L3. B, Seedling survival on medium containing 3.75 mm Man was tested by germinating seedlings on medium without Man for 5 d and subsequent transfer to Man-containing plates for a further 5 d. The hsr2-1 allele was used. Significant differences (Student's t test; P < 0.05) to A3L3 are indicated by an asterisk. C, The chlorophyll content of 5-d-old seedlings was measured after growth on medium containing increasing concentrations of Suc. The results were obtained from pools of 100 seedlings for each data point. At ≥3% (w/v) Suc, all hsr mutants exhibited significantly reduced chlorophyll content (P < 0.05) compared with the parental line A3L3. The experiment was repeated three times with consistent results. The hsr2-1 allele was used. D, The anthocyanin content of 5-d-old seedlings was measured after growth on medium containing increasing concentrations of Suc. The results were obtained from pools of 100 seedlings for each data point. At ≥5% (w/v) Suc, all hsr mutants exhibited significantly elevated anthocyanin levels (P < 0.05) compared with the parental line A3L3. The experiment was repeated three times with consistent results. The hsr2-1 allele was used.

Figure 5.

Dark development phenotypes of the hsr mutants. A, The different stages of dark development used for scoring responses to sugars are shown. Seedlings were grown on medium containing 0.1% (w/v) Suc (Stage 1), 0.25% (w/v) Suc (Stage 2), and 1.0% (w/v) Suc (Stages 3 and 4) on vertical plates in the dark at 20°C for 3 weeks. Similar results were obtained with Glc. B, The development of hsr mutants grown on medium containing 0.5% (w/v) Glc was scored after 3 weeks growth in the dark on vertical plates. The stages of development reached in samples of 200 seedlings were scored according Figure 5A. Ws is the parental line of the transgenic line 35S:STP1 that overexpresses the STP1 hexose transporter.