Arabidopsis Basic Helix-Loop-Helix 34 (bHLH34) Is Involved in Glucose Signaling through Binding to a GAGA Cis-Element

Abstract

The modulation of glucose (Glc) homeostasis and signaling is crucial for plant growth and development. Nevertheless, the molecular signaling mechanism by which a plant senses a cellular Glc level and coordinates the expression of Glc-responsive genes is still incompletely understood. Previous studies have shown that Arabidopsis thaliana plasma membrane Glc-responsive regulator (AtPGR) is a component of the Glc-responsive pathway. Here, we demonstrated that a transcription factor bHLH34 binds to 5'-GAGA-3' element of the promoter region of AtPGR in vitro, and activates beta-glucuronidase (GUS) activity upon Glc treatment in AtPGR promoter-GUS transgenic plants. Gain- and loss-of-function analyses suggested that the bHLH34 involved in the responses to not only Glc, but also abscisic acid (ABA) and salinity. These results suggest that bHLH34 functions as a transcription factor in the Glc-mediated stress responsive pathway as well as an activator of AtPGR transcription. Furthermore, genetic experiments revealed that in Glc response, the functions of bHLH34 are different from that of a bHLH104, a homolog of bHLH34. Collectively, our findings indicate that bHLH34 is a positive regulator of Glc, and may affect ABA or salinity response, whereas bHLH104 is a negative regulator and epistatic to bHLH34 in the Glc response.

Keywords: AtPGR; activator; bHLH34; glucose-responsive element; transcription factor.

FIGURE 1

Electrophoretic mobility shift assay (EMSA) analyses of bHLH34. (A) A schematic diagram of the four (P1, P2, P3, and P4) fragments derived from the promoter 5′ upstream region (P999) of the AtPGR gene. The numbers indicate the nucleotide positions relative to the translation start site, ATG (A as +1). (B) Identification of the DNA-binding promoter fragment of the bHLH34 protein. Experiments were performed three times and similar results were obtained. The ³²P-radiolabeled P1, P2, P3, and P4 DNA fragments incubated in the absence (–) or presence (+) of His-bHLH34 (arrow). 35S, 35S promoter without GAGA motif as a negative control. (C) Sequences of the six DNA binding elements (DBEs) in AtPGR promoter. GAGA motifs are shown in bold. (D) Identification of the DNA-binding promoter site in the bHLH34 protein. Experiments were carried out two times and similar results were obtained. Lane 1, the ³²P-radiolabeled DBE1-6 oligonucleotides incubated in the absence of His-bHLH34; lane 2, the ³²P-radiolabeled DBE1-6 oligonucleotides incubated in the presence of His-bHLH34 (arrow); and lane 3, the ³²P-radiolabeled DBE1 or the ³²P-radiolabeled DBE2 oligonucleotide incubated in the presence of MBP. (E) bHLH34 directly binds to the DBE1 oligonucleotide in the EMSA. The His-bHLH34 protein incubated with ³²P-radiolabeled DBE1. An unlabeled DBE1 probe was used as the competitor (10-, 25-, 50- or 100-fold excess) to show binding specificity. The arrow indicates the shifted band. (F) bHLH34 specifically binds to the GAGA element. Mutations in GAGA element abolish bHLH34 binding to DBE1. A list of DBE1 and m1 to m4 point-mutated or four-nucleotide-mutated m5 probes. Mutated nucleotides are boldfaced. The binding sequence of bHLH34 was determined using various mutated sequences. The arrow indicates the position of the DBE1 probe-His-bHLH34 complex. (G) bHLH34 binds to E-box (ccggcCACTTGtgcca) in an EMSA. Lane 1, the ³²P-radiolabeled- E-box (ccggcCACTTGtgcca) incubated in the absence of His-bHLH34; lane 2, the ³²P-radiolabeled- E-box (ccggcCACTTGtgcca) incubated with His-bHLH34.

FIGURE 2

bHLH34 activates AtPGR expressions. (A) Expression levels of bHLH34 in P999-GUS and two independent P999-GUS/bHLH34-overexpressing (OX1-1/P999-GUS, OX2-5/P999-GUS) transgenic plants were confirmed by reverse transcription (RT)-PCR using RNA extracted from 14-day-old seedlings. Actin 1 served as an internal RT-PCR control. (B) The activities of the 999-bp regulatory promoter region (P999) of the AtPGR gene were examined by means of GUS as a reporter in transgenic plants (P999-GUS). These transgenic plants overexpressing bHLH34 (OX1-1/P999-GUS, OX2-5/P999-GUS) were analyzed histochemically by treatment with H₂O or 6% glucose for 12 h. 35S pro-GUS served as a positive control for analysis of GUS activity. Scale bars = 5 mm. (C,D) P999-GUS, OX1-1/P999-GUS, OX2-5/P999-GUS (C), and 35S pro-GUS (D) seedlings grown on the MS medium for 12 days were carefully taken out and treated with H₂O or 6% Glc for 12 h. Subsequently, seedlings were subjected to GUS staining, and GUS activity was measured. The values of GUS activities are averages of three independent enzymatic assays. Each assay was performed with extracts obtained from three individual seedlings of each transgenic plant. Error bars indicate standard deviations, and different letters above bars indicate a statistical difference (ANOVA, P < 0.05).

FIGURE 3

Expression of bHLH34 in Arabidopsis. (A,B) Expression profiles for AtPGR gene in various organs and at different developmental stages. Results are relative to the bHLH34 expression and represent the average of three independent biological replicates (n = 5–25, mean values ± SD, ANOVA, P < 0.05). Different letters above bars indicate a statistical difference. (A) RNA levels were confirmed by qPCR using total RNA isolated from roots (Rt), stems (St), leaf (Lf), and flowers (Fl). (B) RNA levels were determined by qPCR using total RNA isolated at the indicated plant sampling stages. (C–F) Expression of the bHLH34 in Arabidopsis under glucose, ABA, NaCl, and mannitol stress conditions. qPCR analyses of the expression of bHLH34 involved in glucose (C), ABA (D), NaCl (E), or mannitol (F) responses. Total RNA samples were obtained from 14-day-old seedlings treated with 6% glucose, 100 μM ABA, 150 mM NaCl, or 400 mM mannitol at the indicated time points. Error bars indicate standard deviations of three independent experiments, and different letters above bars indicate a statistical difference (ANOVA, P < 0.05). Each experiment was performed with total RNA of each sample obtained from 20 seedlings. Arabidopsis Actin 1 was used as the internal control. HXK1 (C), RAB18 (D), RD29A (E), or RAB18 (F) gene served as a control for the glucose, ABA, salt, or mannitol stress treatment, respectively.

FIGURE 4

The influence of bHLH34 transgenic plants on glucose (Glc), ABA, and salt stress insensitivity. (A,B) Effects of Glc treatment on cotyledon greening. Seeds of the samples were sown on MS agar plates supplement with 5% Glc and allowed to grow for 10 days. The photograph shows that bHLH34-overexpressing lines (OX2-1 and OX3-5) were better development and greener than the WT and bhlh34 RNAi (ri2-2 and ri5-1) plants under Glc stress condition (A). Seedlings with green cotyledons were counted (triplicates, n = 50 each). Error bars indicate standard deviations, and different letters above bars indicate a statistical difference (ANOVA, P < 0.05) (B). (C) Sensitivity of germination to ABA. Seeds were sown on MS agar plates supplement with 1 μM ABA and allowed to grow for indicated days, and germination was counted (triplicates, n = 50 each). Error bars indicate standard deviations for three independent experiments, and different letters above bars indicate a statistical difference (ANOVA, P < 0.05). (D) Seeds of the samples were sown on the MS medium containing 1 μM ABA and permitted to grow for 14 days. The photograph shows that bHLH34-overexpressing lines were greener than the WT and bhlh34 RNAi plants under ABA condition. (E) Sensitivity of cotyledon greening to ABA. Seeds were sown on the MS medium containing 1 μM ABA and permitted to grow for 14 days, and seedlings with green cotyledons were counted (triplicates, n = 50 each). Error bars indicate standard deviations for three independent experiments, and different letters above bars indicate a statistical difference (ANOVA, P < 0.05). (F) Effects of salt stress on plant growth. Seeds were sown on MS medium containing 150 mM NaCl and permitted to grow for 21 days. The photograph shows that bHLH34-overexpressing lines were better development than the WT and bhlh34 RNAi plants under salt stress condition. (G) Effects of salt treatment on cotyledon greening. Seeds were sown on MS medium supplement with 150 mM NaCl and allowed to grow for 21 days, and surviving plants was counted (triplicates, n = 35 each). Error bars indicate standard deviations, and different letters above bars indicate a statistical difference (ANOVA, P < 0.05).

FIGURE 5

Expression of abiotic stress-regulated genes in bHLH34 transgenic plants. (A–G) mRNA levels of AtPGR (A), GIN6 (B), AtAPR2 (C), ABI1 (D), ABO3 (E), RD29B (F), or AtOZF2 (G) were measured by qPCR using total RNA from 14-day-old WT, two independent bhlh34 RNAi (ri2-2, ri5-1), and two independent bHLH34-overexpressing (OX2-1, OX3-5) seedlings, which were treated with 6% Glc, 100 μM ABA, or 150 mM NaCl with gentle shaking for the indicated period. The mean value of three technical replicates was normalized to the level of Actin 1 mRNA, an internal control. Error bars indicate standard deviations, and different letters above bars indicate a statistical difference (n = 20 each, ANOVA, P < 0.05).

FIGURE 6

Genetic analysis of bhlh104, bhlh34 RNAi (ri2-2) and bhlh34/bhlh104 double mutants (D2-1 and D3-1) under normal and glucose (Glc) conditions. (A,B) bHLH34 (A) and bHLH104 (B) expression levels in WT, bhlh104, bhlh34, and two independent bhlh34/bhlh104 mutant lines (D2-1 and D3-1) were confirmed by qPCR using RNA extracted from 10-day-old seedlings. The mean value of three technical replicates was normalized to the level of Actin 1 mRNA, an internal control. Error bars indicate standard deviations, and different letters above bars indicate a statistical difference (n = 20 each, ANOVA, P < 0.05). (C) Seed germination assays in the normal condition. Seeds of WT, bhlh104, bhlh34 RNAi (ri2-2) and bhlh34/bhlh104 mutants (D2-1 and D3-1) were sown on the sterile MS medium and permitted to grow for indicated days, germination was scored (triplicates, n = 50 each). Error bars indicate standard deviations for three independent experiments, and different letters above bars indicate a statistical difference (50 seeds per point, ANOVA, P < 0.05). (D) Seeds of the samples were sown on the MS medium and permitted to grow for 6 days. The photograph indicates that bhlh104 showed development and green cotyledons similar to those of WT and bhlh34, while bhlh34/bhlh104 lines had paler green leaves. (E) Cotyledon greening assays in the normal condition. Seeds were sown on the MS medium and permitted to grow for indicated days, green cotyledons were counted (triplicates, n = 50 each). Error bars indicate standard deviations for three independent experiments, and different letters above bars indicate a statistical difference (50 seeds per point, ANOVA, P < 0.05). (F) Effects of Glc treatment on seed germination. Seeds were sown on the MS medium containing 6% Glc and permitted to grow for indicated days, germination was scored (triplicates, n = 50 each). Error bars indicate standard deviations for three independent experiments, and different letters above bars indicate a statistical difference (50 seeds per point, ANOVA, P < 0.05). (G) Seeds of the samples were sown on the MS medium and permitted to grow for 14 days. The photograph shows that bhlh104 and bhlh34/bhlh104 mutant lines show better development and are greener than the WT and bhlh34 at high Glc concentration. (H) Effects of Glc treatment on cotyledon greening. Seeds were sown on the MS medium and permitted to grow for 14 days, green cotyledons were counted (triplicates, n = 50 each). Error bars indicate standard deviations for three independent experiments, and different letters above bars indicate a statistical difference (ANOVA P < 0.05).