Fig 1.
Activation-tagged locus and activated genes in the stc8 mutant.
(A) Integrated location point of T-DNA for activation, in chromosome 2. (B) Expression of At2g41130, encoding bHLH106 and (C) At2g41140 encoding CRK1. Total RNA was extracted from calli grown on CIM supplemented with 150 mM NaCl. Expression was determined by real-time RT-PCR and normalized using ACTIN2 (ACT2) and shown as ratios of transcript levels to those of the wild-type without salt. Error bars represent ±SEM from four experimental replicates. Here are “n.s.” for no significant difference and * for P < 0.05 in ANOVA.
Fig 2.
(A) Integrated locations of T-DNA for gene disruption at the bHLH106 locus in SALK_109295 and GABI_560F05, designated as bHLH106-KO1 and bHLH106-KO2, respectively. The pair of opposing arrowheads shows the primers employed for RT-PCR. (B) bHLH106 gene expression in KO plants. Expression was determined by real-time RT-PCR and normalized using ACTIN2 (ACT2). Error bars represent ±SEM from four experimental replicates.
Fig 3.
Phenotypes of bHLH106-KO line growing on culture medium containing NaCl, KCl, or LiCl.
(A) bHLH106-KO1 and control plants were germinated and grown on standard MS medium for 4 days, and then transferred to media containing different concentrations of NaCl, 110 mM KCl, or 10 mM LiCl. Plants were then kept vertical in rectangular culture plates for 2 weeks. SUF4-KO is a KO line of SUF4 (At1g30970) for suppressor of FRI4 as an unrelated gene, and was used as a control for KO lines generated by T-DNA at the other loci. (B) Statistic data of plant growth with (red column) or without (blue column) NaCl in the same experiments shown in panel A. Fresh weights of whole plants were measured with or without 125 mM NaCl. Fresh weights of shoots and roots were measured with or without 100 mM NaCl. Error bars represent ±SEM from six experimental replicates. The numbers above columns are ratios of fresh weight of each line with salt to that without salt. Here are “n.s.” for no significant difference and * for P < 0.05 in ANOVA of the ratios. (C) Statistic data of root length in the same experiments shown in panel A. Here are “n.s.” for no significant difference and * for P < 0.05 in ANOVA.
Fig 4.
Phenotypes of OX lines of bHLH106.
(A) Expression of bHLH106 in OX lines. bHLH106 was driven by the CaVM 35S promoter and F2 homozygous lines used for analysis. Total RNA was extracted from whole plants grown on standard MS. Expression was determined by real-time RT-PCR and normalized using ACTIN2 (ACT2). Error bars represent ±SEM from three experimental replicates. (B) Phenotypes of bHLH106-OX grown on culture medium containing different concentrations of NaCl. The culture and treatment of plants with NaCl was as described in the legend for Fig 3. (C) Statistic data of shoot weight of plants growth with (red column) or without (blue column) 100 mM NaCl in the same experiments shown in panel B. Error bars represent ±SEM from six experimental replicates. The numbers above columns are ratios of fresh weight of each line with salt to that without salt. Here is * for P < 0.05 in ANOVA.
Fig 5.
Expression of bHLH106 with regard to salt-response and organ-specificity.
(A) bHLH106 transcripts in response to salt. Plants were transferred to salt-containing medium 4 days after germination, and kept vertical for 2 weeks. Total RNA was extracted from whole plants and subjected to real-time RT-PCR with normalization using ACTIN2 (ACT2). Error bars represent ±SEM from three experimental replicates. All the P values are less than 0.05 between 0 mM NaCl and 100 mM or 125 mM NaCl. (B) bHLH106 transcripts in different organs. The wild-type plants were grown under standard conditions. RNA was extracted from mature plants. Expression was determined by real-time RT-PCR and normalized using ACTIN2 (ACT2). Error bars represent ±SEM from three experimental replicates.
Fig 6.
Response of bHLH106-KO and bHLH106-OX lines to ABA.
bHLH106-KO1 and bHLH106-OX3 lines were grown in medium containing different concentrations of ABA for 2 weeks. The percentages are surviving rates of employed lines in comparison with their survival on the standard MS medium without ABA.
Fig 7.
Intracellular localization of bHLH106.
The construct of bHLH106 fused with GFP was bombarded into onion epidermal cells. Transient expression of GFP was observed. (A) Bombarded with GFP alone construct, pTH2 [49, 64]. (B, and C) Bombarded with bHLH106 fused with GFP.
Fig 8.
Interaction of bHLH106 with a variety of G-box sequences.
Interactions were examined by electrophoresis mobility shift assay (EMSA). (A) Nucleotide sequences of regions containing a possible G-box were employed for EMSA as probes. (B) Thirteen kinds of 20-mer G-box sequences consisting of 5′-C/GA/GNNT/G/AG/C-3′ (M1 to M13) and arbitrary degenerate probes (C1 and C2). The “+” and “-”denote the presence or absence of bHLH106 protein in EMSA. (C) Competition experiments using the 5′-CACGTG-3′ sequences against the bHLH106 protein.
Fig 9.
Representation of bHLH106 integrating functions of multiple genes through their G-box to confer salt tolerance on dedifferentiated cells of Arabidopsis.
bHLH106 directly regulated 198 genes possessing G-box sequences positively, and 36 genes containing G-box ones negatively (S1 and S2 Tables). The up-regulated genes are classified into 12 different groups on the basis of functions (S3 Table). The down-regulated genes are categorized into abiotic stress-responsive ones and the others (S4 Table). The number at each gene group is that of members. The arrow shows positive regulation, and the arrow interrupted with a bar indicates negative regulation. Detailed functions of these genes are described in “S1 Discussion.”