Phylogenetic and amino acid comparisons of the MSRBs.

<p>(A) Phylogenetic tree of the MSRBs from <i>Arabidopsis thaliana</i> (AtMSRB1: NP_564640, AtMSRB2: NP_567639, AtMSRB3: NP_567271, AtMSRB4: NP_192390, AtMSRB5: NP_192392, AtMSRB6: NP_192393, AtMSRB7: NP_567637, AtMSRB8: NP_193915, AtMSRB9: NP_567638), Oryza sativa (OsMSRB1.1: AK063588, OsMSRB1.2: AK111486, OsMSRB3: AK071730, OsMSRB5: AK068764), and <i>Capsicum annuum</i> (CaMSRB2: EF144171, CaMSRB1: EF144174, CaMSRB3: EF144173). The phylogenetic tree was constructed by ClustalW and Phylip package (Neighbor-joining method) with full-length sequences including transit peptides and visualized using TreeView. (B) The amino acid sequence alignment of the conserved SelR domain of the MSRBs from <i>Arabidopsis thaliana, Oryza sativa</i>, and <i>Capsicum annuum</i> was constructed using ClustalW (<a href="http://www.ebi.ac.uk/Tools/msa/clustalo/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalo/</a>). Four putative MSRB signature motifs are boxed, and two conserved Cys residues are indicated with arrows.</p

Detection of recombinant proteins containing methionine sulfoxide (MetSO) residues by western blotting.

<p>Rubi (Ribulose-bisphosphate carboxylase activase, Os11g0707000), PBGD (Porphobilinogen deaminase, Os02g0168800), and Cys (Cysteine synthase, Os12g0625000) were cloned into the pET-DsRed vector, expressed in <i>E. coli</i>, purified, and treated with 0.3% H₂O₂. These recombinant proteins containing MetSO were detected by western blotting with the methionine sulfoxide polyclonal antibody (Cayman). kDa, molecular mass indicators (in kDa The experiments were representative of three independent experiments.</p

Changes in chlorophyll fluorescence during drought stress.

<p>The leaves were detached from four-week-old plants of the transgenic and WT lines and were air-dried at 25°C under continuous light (100 µmol protons m⁻² S⁻¹). The <i>Fv/Fm</i> value was measured by mini PAM according to the time course. The results shown are the mean ± SD, n = 5 replicates for each group. The experiments were representative of three independent experiments.</p

MetSO/Met ratio in individual methionine residues of PBGD.

<p>The gray and black bars represent the Met and MetSO residues, respectively. The MetSO content is expressed as the percent of MetSO in relation to the each Met residues (Met + MetSO). The figures that shown are in the bar graph represent the absolute numbers of Met or MetSO. The results shown are the mean ± SD, n = 3 replicates for each group (*<i>P</i><0.05 compared to PBGD + H₂O₂ + MSRB2 value).</p

Subcellular Localization of CaMSRB2 by transient expression.

<p>The expression of CaMSRB2 fused in-frame to DsRed was driven by the <i>CaMV</i> 35S promoter in rice protoplasts and examined under a confocal microscope. GFP with an N-terminal chloroplast transit peptide was constructed under the control of the <i>RbcS</i> promoter as a chloroplast marker. White scale bar represents 5 um.</p

Statistical analysis of up- and down-regulated genes in MapMan BINs.

<p>A bin containing a significantly higher number of up- or down-regulated genes are prited in bold.</p

Expression of nine genes in the WT and CaMSRB2-transformed rice as detected by real-time PCR.

<p>The y-axis shows the relative expression level normalized to that of the WT plants that were grown under drought conditions for 2 days. Os04g0414700 (photosystem I PsaO domain-containing protein), Os03g0778100 (photosystem-1 F subunit), Os08g0502700 (pyridoxal phosphate-dependent transferase), Os08g0248800 (aspartate carbamoyltransferase 3), Os04g0644600 (esterases and lipases epoxide hydrolase family protein), Os07g0693800 (fatty acid desaturase), Os07g0412100 (granule-bound starch synthase Ib, chloroplast precursor), Os02g0596000 (rhodanese-like domain-containing protein), Os03g0736400 (methylase putative domain-containing protein), and Os04g0465500 (GCN5-related N-acetyltransferase domain-containing protein). The results shown are the mean ± SD, n = 3 replicates for each group (^***<i>P</i><0.001; **<i>P</i><0.01; and *<i>P</i><0.05 compared to WT). The experiments were representative of two independent experiments.</p

Venn diagrams of differentially expressed genes.

<p>Blue, yellow, and green represent the genes that were down- (A) or up-regulated (B) more than 2-fold in WT and transgenic plants after drought treatment compared to WT plants under normal conditions, respectively. A total of 1,981 and 1,722 genes were commonly down- or up-regulated in both WT and transgenic plants under drought stress, respectively. A total of 947 and 481 genes were only down- or up-regulated in the WT plants under drought stress, respectively.</p

Response of CaMSRB2-transformed rice to drought stress.

<p>(A) Phenotypes of transgenic plants and WT under drought stress. Four-week-old plants were treated under drought stress conditions for 3 days. The pictures were taken after re-watering for 5 days. The experiments were representative of four independent experiments. (B) The drought tolerance was evaluated by the survival rate. The survival rates were calculated from 18 plants for each genotype. (C) The chlorophyll index from ten plants was measured using a chlorophyll meter (SPAD502). (B), (C) WT and transgenic plants were re-watered for two weeks. The results shown are the mean ± SD, n = 4 replicates for each group (***P<0.001, **P<0.01, and *P<0.05 compared to WT).</p