Proteomic analysis of endoplasmic reticulum stress responses in rice seeds

The defects in storage proteins secretion in the endosperm of transgenic rice seeds often leads to endoplasmic reticulum (ER) stress, which produces floury and shrunken seeds, but the mechanism of this response remains unclear. We used an iTRAQ-based proteomics analysis of ER-stressed rice seeds due to the endosperm-specific suppression of OsSar1 to identify changes in the protein levels in response to ER stress. ER stress changed the expression of 405 proteins in rice seed by > 2.0-fold compared with the wild-type control. Of these proteins, 140 were upregulated and 265 were downregulated. The upregulated proteins were mainly involved in protein modification, transport and degradation, and the downregulated proteins were mainly involved in metabolism and stress/defense responses. A KOBAS analysis revealed that protein-processing in the ER and degradation-related proteasome were the predominant upregulated pathways in the rice endosperm in response to ER stress. Trans-Golgi protein transport was also involved in the ER stress response. Combined with bioinformatic and molecular biology analyses, our proteomic data will facilitate our understanding of the systemic responses to ER stress in rice seeds

Proteomic analysis of endoplasmic reticulum stress responses in rice seeds

The defects in storage proteins secretion in the endosperm of transgenic rice seeds often leads to endoplasmic reticulum (ER) stress, which produces floury and shrunken seeds, but the mechanism of this response remains unclear. We used an iTRAQ-based proteomics analysis of ER-stressed rice seeds due to the endosperm-specific suppression of OsSar1 to identify changes in the protein levels in response to ER stress. ER stress changed the expression of 405 proteins in rice seed by >2.0- fold compared with the wild-type control. Of these proteins, 140 were upregulated and 265 were downregulated. The upregulated proteins were mainly involved in protein modification, transport and degradation, and the downregulated proteins were mainly involved in metabolism and stress/defense responses. A KOBAS analysis revealed that protein-processing in the ER and degradation-related proteasome were the predominant upregulated pathways in the rice endosperm in response to ER stress. Trans-Golgi protein transport was also involved in the ER stress response. Combined with bioinformatic and molecular biology analyses, our proteomic data will facilitate our understanding of the systemic responses to ER stress in rice seeds

CFLAP1 and CFLAP2 Are Two bHLH Transcription Factors Participating in Synergistic Regulation of AtCFL1-Mediated Cuticle Development in Arabidopsis

The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1) and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1

35S:<i>CFLAP1SRDX</i> plants had opposite phenotypes to 35S:<i>CFLAP1</i> plants.

<p>(A) The schematic diagram of 35S:<i>CFLAP1SRDX</i> construct. CaMV35S, CFLAP1 CDS, EAR motif and Nos represent the 35S promoter, <i>CFLAP1</i> coding sequence, the repression domain of 12 amino acid residues, and the <i>NOS</i> terminator sequence, respectively. (B) and (C) Transient effector-reporter expression assay in tobacco leaves, with the <i>CO</i> promoter driving a luciferase gene as a reporter, and the 35S:<i>CFLAP1</i> and 35S:<i>CFLAP1SRDX</i> as effectors. Luminescence imaging is shown 48 hours after co-infiltration with the constructs indicated at left. (D) The result of RT-PCR. From right to left, wild type, transgenic lines 35S:<i>CFLAP1SRDX-1</i>, 35S:<i>CFLAP1SRDX-41</i>, 35S:<i>CFLAP1SRDX-43</i>, and 35S:<i>CFLAP1SRDX-48</i>, respectively.<i>TUB2</i> was used as an internal control. (E) Epicuticular wax components of 35S: <i>CFLAP1SRDX-48</i> and wild-type stems. Numbers indicate the main chain length of each constituent. Each value is the mean + SD of three biological replicates. At least 5 individual stems were used for each replicate. (F) Epicuticular wax components of 35S: <i>CFLAP1SRDX-48</i> and wild-type rosette leaves. Numbers indicate the main chain length of each constituent. Each value is the mean + SD of three biological replicates. At least 7 individual rosette leaves were used for each replicate. Level of significance obtained with a Student’s <i>t</i> test is marked by the following: *, p<0.05; ***, p<0.01.</p

Lipid-related pathways were oppositely regulated in 35S:<i>CFLAP1</i> plants and 35S:<i>CFLAP1SRDX</i> plants.

<p>(A) The orange columns indicate the numbers of the up-regulated genes mapped to the enriched pathways. The green columns indicate the numbers of the down-regulated genes mapped to the enriched pathways. Detailed information was further shown in Tables <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005744#pgen.1005744.t002" target="_blank">2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005744#pgen.1005744.t003" target="_blank">3</a>. Arrows highlight similar pathways with opposite expression trends in 35S:<i>CFLAP1</i> plants and 35S:<i>CFLAP1SRDX</i> plants. (B) Relative expression levels of <i>FDH</i>, <i>BDG</i>, <i>KCS8</i> and <i>DEWAX</i> in wild type, 35S:<i>CFLAP1</i> and 35S:<i>CFLAP1-SRDX</i> plants. The expression level in wild type is set to 1.0. The error bars represent the SD of three biological replicates.</p

Epicuticular wax components analysis in 35S:<i>CFLAP1</i> and wild-type plants.

<p>(A) Epicuticular wax components in 35S:<i>CFLAP1</i> and wild-type stems. Numbers indicate the main chain length of each constituent. Each value is the mean + SD of three biological replicates. At least 5 individual stems were used for each replicate. (B) Epicuticular wax components of 35S:<i>CFLAP1</i> and wild-type rosette leaves. Numbers indicate the main chain length of each constituent. Each value is the mean + SD of three biological replicates. At least 7 individual rosette leaves were used for each replicate. Level of significance obtained with a Student’s <i>t</i> test is marked by the following: *, p<0.05; ***, p<0.01.</p

The expression patterns of <i>CFLAP1</i> and <i>CFLAP2</i>.

<p>(A) Analysis of CFLAP1/2 expression levels in different organs of wild-type <i>Arabidopsis</i> by qRT-PCR. The expression level of seedling is set to 1.0, and error bars represent the SD of three biological replicates. (B) to (F) The expression pattern of CFLAP1 in <i>Arabidopsis</i>. (B) GUS-stained one-week-old seedling. (C) and (D) Higher magnification of boxed regions in (B). (E) GUS-stained two-week-old seedling. (F) Higher magnification of boxed region in (E). (G) to (L) The expression pattern of CFLAP2 in <i>Arabidopsis</i>. (G) GUS-stained one-week-old seedling. (H) GUS-stained two-week-old seedling. (I) GUS-stained trichomes in true leaves of two-week-old seedling. (J) GUS-stained inflorescence stem with buds, flowers, and siliques. (K) GUS-stained young silique. (L) GUS-stained the abscission zone of mature silique. Bars = 2 mm in (B) and (H), 1 mm in (C), (E), (G), (J), (K) and (L), 500 μm in (F) and (I), 100 μm in (D).</p