Up-regulation of <i>miRNA172E</i> under drought conditions.

<div><p>Each experiment was done triple with similar results.</p> <p>(<b>A</b>) Change in <i>pri-miRNA172</i> levels under drought conditions( Ler-0). </p> <p>(<b>B</b>) Change in mature <i>miRNA172</i> levels under drought conditions in wild-type plants. * <i>P</i><0.05. </p> <p>(<b>C</b>) RT-PCR analysis of <i>Pri-miRNA172A</i> and <i>Pri-miRNA172E</i> in the <i>gi</i> mutant under drought and control conditions. </p> <p>(<b>D</b>) Changes in mature <i>miRNA172A/B</i> and <i>miRNA172E</i> levels under drought conditions in the <i>gi</i> mutant.</p> <p>DR: Drought treatment began from the 14day age. For the mature miRNA assay, samples were collected at the 8^th day of DR treatment.</p></div

Transcriptional levels of <i>WRKY</i> genes in wild type (Ler-0) and <i>gi</i> mutants under standard (CK, white rectangles) and drought (DR, black rectangles) conditions.

<p>Results are averages of three biological replicates. *, significantly different (<i>P</i><0.05) expression levels between <i>gi</i> mutants and wild-type plants under CK or DR. DR treatment began from10 day age and maintained for 10 days. </p

The <i>gi</i> mutant is sensitive to drought stress.

<div><p>(<b>A</b>) The phenotypes of wild-type plants ( Ler-0) and <i>gi</i> mutants under drought stress. (<b>B</b>) Transpiration rates of wild type, <i>gi</i> and <i>miRNA172A</i> (A1-10) <i>/D</i> (D6-3) <i>/E</i> (E1-2, E38-6) over-expressing plants. </p> <p>(<b>C</b>) Water loss in wild type, <i>gi</i> mutants, and plants over-expressing <i>miRNA172A</i> (A1-10) <i>/D</i> (D6-3) <i>/E</i> (E1-2, E38-6).</p> <p>DR treatment began from10 day age and maintained for 10 days.</p></div

Yeast two-hybrid system analysis of WRKY and TOE1.

<p>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).</p

Abundance of mRNAs of flowering-time and circadian-clock–regulated genes in <i>Arabidopsis</i> under long-day control (CK) and drought (DR) conditions.

<div><p>The expressions of <i>GI</i> (<b>A</b>), <i>FKF1</i> (<b>B</b>), <i>CO</i> (<b>C</b>), <i>FT</i> (<b>D</b>) 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.</p> <p>===/ /=== represents the 5-d recovery period with watering. * indicated a significant difference (P<0.05).</p> <p>DR: Drought treatment began from the 10^th day age and maintained for 10 days.</p></div

Phylogenetic analysis of <i>Arabidopsis</i><i>WRKY</i> genes used in this study and <i>WRKY</i> genes from <i>Hordeum vulgare</i>.

<p>Data were analyzed by the neighbor joining method. Annotations indicate the regulation of <i>Arabidopsis </i><i>WRKY</i> genes by <i>GI</i>. 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. </p

Transcriptional level of <i>WRKY20</i>, <i>WRKY44</i>, and <i>WRKY51</i> in <i>co</i> and <i>miRNA172</i>–over-expressing plants (miRNA172-OX) under standard (CK, white rectangles) and drought (DR, black rectangles) conditions.

<p>Controls for the co mutant and miRNA172-OX was <i>Col-</i>0, the wild type in their respective ecotype backgrounds. Results are averages of three biological repeats. * Significantly different (<i>P</i><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. </p

Single Probe for Imaging and Biosensing of pH, Cu²⁺ Ions, and pH/Cu²⁺ in Live Cells with Ratiometric Fluorescence Signals

It is very essential to disentangle the complicated inter-relationship between pH and Cu in the signal transduction and homeostasis. To this end, reporters that can display distinct signals to pH and Cu are highly valuable. Unfortunately, there is still no report on the development of biosensors that can simultaneously respond to pH and Cu²⁺, to the best of our knowledge. In this work, we developed a single fluorescent probe, AuNC@FITC@DEAC (AuNC, gold cluster; FITC, fluorescein isothiocyanate; DEAC, 7-diethylaminocoumarin-3-carboxylic acid), for biosensing of pH, Cu²⁺, and pH/Cu²⁺ with different ratiometric fluorescent signals. First, 2,2′,2″-(2,2′,2″-nitrilotris­(ethane-2,1-diyl)­tris­((pyridin-2-yl-methyl)­azanediyl))­triethanethiol (TPAASH) was designed for specific recognition of Cu²⁺, as well as for organic ligand to synthesize fluorescent AuNCs. Then, pH-sensitive molecule, FITC emitting at 518 nm, and inner reference molecule, DEAC with emission peak at 472 nm, were simultaneously conjugated on the surface of AuNCs emitting at 722 nm, thus, constructing a single fluorescent probe, AuNC@FITC@DEAC, to sensing pH, Cu²⁺, and pH/Cu²⁺ excited by 405 nm light. The developed probe exhibited high selectivity and accuracy for independent determination of pH and Cu²⁺ against reactive oxygen species (ROS), other metal ions, amino acids, and even copper-containing proteins. The AuNC-based inorganic–organic probe with good cell-permeability and high biocompatibility was eventually applied in monitoring both pH and Cu²⁺ and in understanding the interplaying roles of Cu²⁺ and pH in live cells by ratiometric multicolor fluorescent imaging

Differential gene expression in wild type (WT) and <i>gi</i> mutants under drought conditions as measured by digital gene expression.

<div><p>(<b>A</b>) Differential gene expression in WT( Ler-0) and <i>gi</i> mutants under drought conditions. </p> <p>(<b>B</b>) Venn diagram of up- and downregulated genes in WT and <i>gi</i> mutants with and without drought treatment. </p> <p>(<b>C</b>) Differential expression of <i>WRKY</i> genes in <i>gi</i> and WT under CK (standard) and DR(drought) conditions. Red: upregulated in <i>gi</i> compared with WT; green: down-regulated in <i>gi</i> compared with WT. </p> <p>DR treatment began from10 day age and maintained for 10 days. </p></div

Flowering times of <i>Arabidopsis</i> wild-type (WT) and mutants of different flowering pathways under drought stress.

<div><p>(<b>A</b>) Early flowering of WT (Col-0 and Ler-0) plants under drought stress and long-day conditions. </p> <p>(<b>B</b>) Flowering times of mutants of the photoperiod (<i>gi</i>, <i>co</i>), autonomous (flc-3), and phytohormone (gai) pathways under drought stress and long-day conditions. </p> <p>(<b>C</b>) Flowering times of WT (Col-0) plants under drought stress and short-day conditions. </p> <p>(<b>D</b>) Counted flowering times (days) of plants with different genotypes under CK and DR conditions. * flowering significantly earlier under DR condition than under CK condition.</p> <p>DR : Drought treatment began from 10days before flowering.</p></div