Figure 1.
Flowering times of Arabidopsis wild-type (WT) and mutants of different flowering pathways under drought stress.
(A) Early flowering of WT (Col-0 and Ler-0) plants under drought stress and long-day conditions.
(B) Flowering times of mutants of the photoperiod (gi, co), autonomous (flc-3), and phytohormone (gai) pathways under drought stress and long-day conditions.
(C) Flowering times of WT (Col-0) plants under drought stress and short-day conditions.
(D) Counted flowering times (days) of plants with different genotypes under CK and DR conditions. * flowering significantly earlier under DR condition than under CK condition.
DR : Drought treatment began from 10days before flowering.
Figure 2.
Abundance of mRNAs of flowering-time and circadian-clock–regulated genes in Arabidopsis under long-day control (CK) and drought (DR) conditions.
The expressions of GI (A), FKF1 (B), CO (C), FT (D) were analyzed by real time-PCR in Ler-0 plants grown in LDs. For each gene, the first peak on the first day under CK conditions was standardized to a level of 1. Open and closed bars along the horizontal axis represent light and dark periods, respectively, measured in hours from dawn. Each experiment was done twice with similar results.
===/ /=== represents the 5-d recovery period with watering. * indicated a significant difference (P<0.05).
DR: Drought treatment began from the 10th day age and maintained for 10 days.
Figure 3.
Up-regulation of miRNA172E under drought conditions.
Each experiment was done triple with similar results.
(A) Change in pri-miRNA172 levels under drought conditions( Ler-0).
(B) Change in mature miRNA172 levels under drought conditions in wild-type plants. * P<0.05.
(C) RT-PCR analysis of Pri-miRNA172A and Pri-miRNA172E in the gi mutant under drought and control conditions.
(D) Changes in mature miRNA172A/B and miRNA172E levels under drought conditions in the gi mutant.
DR: Drought treatment began from the 14day age. For the mature miRNA assay, samples were collected at the 8th day of DR treatment.
Figure 4.
The gi mutant is sensitive to drought stress.
(A) The phenotypes of wild-type plants ( Ler-0) and gi mutants under drought stress. (B) Transpiration rates of wild type, gi and miRNA172A (A1-10) /D (D6-3) /E (E1-2, E38-6) over-expressing plants.
(C) Water loss in wild type, gi mutants, and plants over-expressing miRNA172A (A1-10) /D (D6-3) /E (E1-2, E38-6).
DR treatment began from10 day age and maintained for 10 days.
Figure 5.
Differential gene expression in wild type (WT) and gi mutants under drought conditions as measured by digital gene expression.
(A) Differential gene expression in WT( Ler-0) and gi mutants under drought conditions.
(B) Venn diagram of up- and downregulated genes in WT and gi mutants with and without drought treatment.
(C) Differential expression of WRKY genes in gi and WT under CK (standard) and DR(drought) conditions. Red: upregulated in gi compared with WT; green: down-regulated in gi compared with WT.
DR treatment began from10 day age and maintained for 10 days.
Figure 6.
Transcriptional levels of WRKY genes in wild type (Ler-0) and gi mutants under standard (CK, white rectangles) and drought (DR, black rectangles) conditions.
Results are averages of three biological replicates. *, significantly different (P<0.05) expression levels between gi mutants and wild-type plants under CK or DR. DR treatment began from10 day age and maintained for 10 days.
Figure 7.
Phylogenetic analysis of Arabidopsis WRKY genes used in this study and WRKY genes from Hordeum vulgare.
Data were analyzed by the neighbor joining method. Annotations indicate the regulation of Arabidopsis WRKY genes by GI. The number above each branch-point referred to the bootstrap value (maximum is 100), which implied the reliability of existing clades in the tree. The system has performed 1000 replicates to construct the phylogram. The number in each clade represented the percentages of success for constructing the existing clade. 0.1 means 10% substitution rate between two sequences.
Figure 8.
Yeast two-hybrid system analysis of WRKY and TOE1.
Using TOE1 as bait identified WRKY44 as a potential protein interactor. Selective plates lacking adenine, histidine, tryptophan, and leucine (–Ade, –His, –Trp, –Leu) and control plates lacking only tryptophan (–Trp) are shown. Empty vectors (BD) and expressed proteins (TOE1) are indicated. Plates were photographed after 4 d. Potential interactors exhibited positive galactosidase activity (blue).
Figure 9.
Transcriptional level of WRKY20, WRKY44, and WRKY51 in co and miRNA172–over-expressing plants (miRNA172-OX) under standard (CK, white rectangles) and drought (DR, black rectangles) conditions.
Controls for the co mutant and miRNA172-OX was Col-0, the wild type in their respective ecotype backgrounds. Results are averages of three biological repeats. * Significantly different (P<0.05) expression between miRNA172-OX and WT under both CK and DR conditions. E1-2 line was used as miRNA172-OX. DR treatment began from10 day age and maintained for 10 days.
Figure 10.
A schematic working model for the involvement of GI and WRKY in drought defense and drought escape in Arabidopsis.
SD, short day; LD, long day;→, up-regulated; ┥, down-regulated;<->, interact at protein level.