A pepper MSRB2 gene confer drought tolerance in rice through the protection of chloroplast-targeted genes

Background: The perturbation of the steady state of reactive oxygen species (ROS) due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 (responsive to ABA protein 21) promoter with/without a selection marker, the bar gene. Results: A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol (DTT). An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Conclusions: Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice.OAIID:oai:osos.snu.ac.kr:snu2014-01/102/0000005113/8SEQ:8PERF_CD:SNU2014-01EVAL_ITEM_CD:102USER_ID:0000005113ADJUST_YN:NEMP_ID:A077085DEPT_CD:517CITE_RATE:3.73FILENAME:msrb-김연기.pdfDEPT_NM:식물생산과학부EMAIL:[email protected]_YN:YCONFIRM:

RapaNet: A Web Tool for the Co-Expression Analysis of Brassica rapa Genes

Accumulated microarray data are used for assessing gene function by providing statistical values for co-expressed genes; however, only a limited number of Web tools are available for analyzing the co-expression of genes of Brassica rapa . We have developed a Web tool called RapaNet ( http://bioinfo.mju.ac.kr/arraynet/brassica300k/query/ ), which is based on a data set of 143 B rapa microarrays compiled from various organs and at different developmental stages during exposure to biotic or abiotic stress. RapaNet visualizes correlated gene expression information via correlational networks and phylogenetic trees using Pearson correlation coefficient ( r ). In addition, RapaNet provides hierarchical clustering diagrams, scatterplots of log ratio intensities, related pathway maps, and cis -element lists of promoter regions. To ascertain the functionality of RapaNet, the correlated genes encoding ribosomal protein (L7Ae), photosystem II protein D1 (psbA), and cytochrome P450 monooxygenase in glucosinolate biosynthesis (CYP79F1) were retrieved from RapaNet and compared with their Arabidopsis homologues. An analysis of the co-expressed genes revealed their shared and unique features

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

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

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

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