Three genes that affect sugar sensing (abscisic acid insensitive 4, abscisic acid insensitive 5, and constitutive triple response 1) are differentially regulated by glucose in Arabidopsis

Abstract

Mutant characterization has demonstrated that ABI4 (Abscisic Acid [ABA] Insensitive 4), ABI5 (ABA Insensitive 5), and CTR1 (Constitutive Triple Response 1) genes play an important role in the sugar signaling response in plants. The present study shows that the transcripts of these three genes are modulated by glucose (Glc) independently of the developmental arrest caused by high Glc concentrations. ABI4 and ABI5 transcripts accumulate in response to sugars, whereas the CTR1 transcript is transiently reduced followed by a rapid recovery. The results of our kinetic studies on gene expression indicate that ABI4, ABI5, and CTR1 are regulated by multiple signals including Glc, osmotic stress, and ABA. However, the differential expression profiles caused by these treatments suggest that distinct signaling pathways are used for each signal. ABI4 and ABI5 response to the Glc analog 2-deoxy-Glc supports this conclusion. Glc regulation of ABI4 and CTR1 transcripts is dependent on the developmental stage. Finally, the Glc-mediated regulation of ABI4 and ABI5 is affected in mutants displaying Glc-insensitive phenotypes such as gins, abas, abi4, abi5, and ctr1 but not in abi1-1, abi2-1, and abi3-1, which do not show a Glc-insensitive phenotype. The capacity of transcription factors, like the ones analyzed in this work, to be regulated by a variety of signals might contribute to the ability of plants to respond in a flexible and integral way to continuous changes in the internal and external environment.

Figure 1.

Glc induction of the ABI4 transcript and spatial expression pattern. A, Glc regulation of the ABI4 gene. Northern blot from Columbia-0 (Col-0) plants grown for 2 d on standard liquid media and exposed to Murashige and Skoog (MS) 7% (w/v) Glc for 0, 6, and 12 h. Each lane contains 8 μg of total RNA. The complete ABI4 cDNA was used as a probe. B, β-Glucuronidase (GUS)-specific activity was measured in dry seeds (DS), 1-h-imbibed seeds (IS), 3-d cold-treated seeds (CTS), or germinating seedlings in standard media (SM) or Murashige and Skoog 7% (w/v) Glc for 1, 3, and 10 d. Each point represents the mean of four independent lines and four biologically independent experiments, expressed as nanomoles of methylumbelliferone per microgram of total protein per minute. C, GUS activity of Glc-treated seedlings relative to the GUS level of their corresponding standard media grown plants, normalized to 1 in each case. Bars = sd (sometimes smaller than the scale). GUS staining of ABI4::GUS seedlings grown on standard media for 3 (D) or 10 (E) d. GUS staining of ABI4::GUS seedlings grown on MS 4% (w/v) Glc for 3 (F) or 10 (G) d. GUS staining of ABI4::GUS seedlings grown on MS 7% (w/v) Glc for 3 (H) or 10 (I) d. The value of a representative experiment of the GUS-specific activity of transgenic lines grown in each condition is shown. J, Glc regulation of the GUS transcript level. Transgenic plants carrying the ABI4::GUS expression cassette were grown in standard liquid media for 2 d followed by the exposure to Murashige and Skoog 7% (w/v) Glc. Samples were taken at 0, 6, and 12 h. Five micrograms of total RNA from each sample were loaded in each lane and hybridized with a 600-bp fragment of the GUS gene.

Figure 2.

ABI4, ABI5, and CTR1 transcript accumulation is regulated by Glc. RT-PCR analysis of total RNA from 3-d-old wild-type (Wassilewskija [Ws]) seedlings grown in standard media and transferred to Murashige and Skoog 7% (w/v) Glc for 1, 3, 6, 24, and 72 h. The PCR product of APT1 was used as a cDNA loading control. Specific primers were used to amplify ABI4, ABI5, CTR1, and APT1 gene transcripts. The lengths of the PCR products are 974, 183, 353, and 478 bp for ABI4, ABI5, CTR1, and APT1, respectively. The linear phase of the exponential PCR reaction was corroborated for each gene (data not shown). A representative experiment from three biologically independent experiments is shown, including only the 24-h transference (T24) control for simplification. The means ± se of all three experiments and their corresponding transfer controls are included in Figure 4, D to F.

Figure 3.

ABI4 response to osmotic stress. Northern blots from wild-type (Col-0) and ABI4::GUS transgenic 2-d-old seedlings grown on standard liquid media and exposed to Murashige and Skoog 7% (w/v) mannitol for 0, 6, and 12 h or Murashige and Skoog 7% (w/v) Glc for 12 h (G). Each lane contains 8 μg of total RNA. The complete ABI4 cDNA and a 600-bp GUS fragment were used as probes. rRNAs were used as loading control.

Figure 4.

Glc regulation of ABI4, ABI5, and CTR1 genes is not due to the osmotic effect of Glc. Three-day-old WT (Ws) seedlings grown in standard media were transferred to standard media plus either 7% (w/v) mannitol (A), 100 μm ABA (B), or 0.5 mm 2DG (C) for 1, 3, 6, and 24 h. For simplification, only the 24-h transfer (T24) control was included. RT-PCR was performed as described in Figure 2. The rd29A gene was used as control of ABA and osmotic treatments. Comparative histograms of the densitometric quantification of the PCR products from biologically independent experiments are shown for ABI4 (D), ABI5 (E), and CTR1 (F) in response to 7% (w/v) Glc, 0.5 mm 2DG, 7% (w/v) mannitol, and 100 μm ABA and including the transfer controls (T control) for each time point. In the case of ABI4, where no detectable transcript levels were found, such as 0 h and most of the T control times, no bar is observed (D). The APT1 RT-PCR product was used as internal control in each reaction, and the value of each condition and the relative units of the amplification signals obtained by the densitometric analysis of each gene are normalized to the amplification signal of the control APT1 gene (arbitrary units). The data on the graphic correspond to the mean ± se of three separate experiments.

Figure 5.

Glc regulation of ABI4 and CTR1 transcript accumulation is affected by developmental signals. Six-day-old WT (Ws) seedlings grown in standard media and transferred to Murashige and Skoog 7% (w/v) Glc (A), standard media plus 0.5 mm 2DG (B), or 100 μm ABA (C) for the indicated times. For simplification, the 24-h transfer (T24) control is included, but similar levels to this control were observed in the rest of the control times (data not shown). The PCR product of APT1 was used as a cDNA loading control. Samples were collected and used for RT-PCR analysis as described. This picture is representative of three biologically independent experiments.

Figure 6.

ABI4 and ABI5 Glc regulation is affected in ABA-deficient, -insensitive, and ethylene signaling mutants. A, Indicated genotypes were grown in standard media for 3 d (T0) and transferred to the same media (-) or Murashige and Skoog 7% (w/v) Glc (+) for 6 h (T6). gin (B), aba and ctr1 (C), and abi (D) plants were grown on standard media (-), Murashige and Skoog 7% (w/v) Glc (+), or Murashige and Skoog 7% (w/v) Glc plus 100 nm ABA (*) during 18 d. Col-0 ecotype (aba2-1 and ctr1-1 genetic background) has exactly the same response as the Landsberg erecta (Ler) ecotype under these conditions. For simplification, the Col-0 ecotype PCR is omitted in this figure. Samples were harvested and used for RT-PCR analysis as described. The PCR product of APT1 was used as a cDNA loading control. A representative experiment from three biologically independent experiments is shown.