Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in <i>Arabidopsis thaliana</i> with Tiling Microarray

<div><p>Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix <i>Arabidopsis</i> tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.</p></div

Flowchart of the algorithm used for detecting stress-regulated genes and alternative splicing (see text).

<p>Flowchart of the algorithm used for detecting stress-regulated genes and alternative splicing (see text).</p

Example of EST or cDNA evidence from TAIR genome browser supporting predicted alternative splicing type.

<p>A red rectangle marks the EST or cDNA sequence supporting the predicted alternative splicing of the sample genes <i>AT1G47530</i>, <i>AT3G06620</i> and <i>AT3G47630</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066511#pone-0066511-t001" target="_blank">Table 1</a>).</p

Defining splicing type according to probes expression level.

<p>The common forms of alternative splicing represented here are; exon skipping, intron retention, alternative 3' acceptor site and alternative 5' donor site. Boxes joined by lines represent the exons and introns, respectively, of immature transcripts; diagonal lines indicate splicing patterns. Highly expressed probes are depicted as short thick bars while probes with low expression are depicted as short thin bars below the transcript. The splicing variant (right side) is defined as intron retention when all probes in an intron are significantly highly expressed, i.e., have near exon level expression. All other cases of altered intron probes expression are defined as unknown, as the splicing variant can include exon skipping, intron retention or alternative 5' or 3'.</p

A sample list of detected transcripts with putative stress-regulated alternative splicing.

<p>The listed genes were used for validation of predicted cold-regulated alternative splicing events by RT-PCR. All genes, except AT1G47530, were not defined as cold responsive based on showing less than 2 and more than -2 fold change (FDR <0.05).</p>a<p>Example of cold responsive gene.</p>b<p>Alternative splicing types are intron retention (r) or unknown (u, possible exon skipping, alternative 5'/3' or intron retention).</p>c<p>The alternatively spliced intron is not completely excised in either cold treatment or control (ctrl).</p>d<p>Predictions that were confirmed by RT-PCR.</p

Clustergram representing correlation between transcriptomes from cold treatment conditions and stress treated plants.

<p>The transcriptomes of the WGA (2h, 10h, and 24h) experiments are clustered according to their similarity to each stress index, which are shown on the horizontal axis. The indexes for drought, osmotic, salt, cold and heat stress are from Kilian <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066511#pone.0066511-Kilian1" target="_blank">[4]</a>. The transcriptomes screened by vector-based analysis are shown on the vertical axis. Correlation values are color-coded from blue (negative correlation) to red (positive correlation). Neutral correlation values are white. The data for 2h and 10h were obtained from Matsui <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066511#pone.0066511-Matsui1" target="_blank">[11]</a></p

The expression pattern of genes in control and cold-treated plants as examined by RT-PCR.

<p>Primers flanking the retained intron were used for amplification. <i>RD29A</i> (<i>AT5G52310</i>), a gene induced by cold, was used for verification of the effectiveness of the cold-treatment. Amplification of <i>Cyclophilin</i> (<i>AT4G38740</i>) was used to demonstrate an equal quantity of template in each PCR reaction. Asterisks indicate cold regulated splice variants. Crosses indicate genes with transcripts predicted to trigger NMD (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066511#pone.0066511.s003" target="_blank">Figure S3</a>).</p

Chilling Stress Upregulates α‑Linolenic Acid-Oxidation Pathway and Induces Volatiles of C₆ and C₉ Aldehydes in Mango Fruit

Mango-fruit storage period and shelf life are prolonged by cold storage. However, chilling temperature induces physiological and molecular changes, compromising fruit quality. In our previous transcriptomic study of mango fruit, cold storage at suboptimal temperature (5 °C) activated the <i>α-</i>linolenic acid metabolic pathway. To evaluate changes in fruit quality during chilling, we analyzed mango “Keitt” fruit peel volatiles. GC–MS analysis revealed significant modulations in fruit volatiles during storage at suboptimal temperature. Fewer changes were seen in response to the time of storage. The mango volatiles related to aroma, such as <i>δ</i>-3-carene, <i>(Z)-β-</i>ocimene, and terpinolene, were downregulated during the storage at suboptimal temperature. In contrast, C₆ and C₉ aldehydes and alcoholsα-linolenic acid derivatives 1-hexanal, <i>(Z)</i>-3-hexenal, <i>(Z)</i>-3-hexenol, <i>(E)-</i>2-hexenal, and nonanalwere elevated during suboptimal-temperature storage, before chilling-injury symptoms appeared. Detection of those molecules before chilling symptoms could lead to a new agro-technology to avoid chilling injuries and maintain fruit quality during cold storage at the lowest possible temperature

Evaluation of physiological parameters of ‘Ettinger’ fruit during cold-quarantine treatments.

<p>Physiological parameters of treated [modified atmosphere (MA), methyl jasmonate (MJ), low-temperature conditioning (LTC)] or non-treated ‘Ettinger’ after cold storage (black column) and further shelf storage (white column). (A) Overall decay displayed in percentage. (B) Firmness displayed in Newton. (C) Blossom-end rot displayed in percentage. Data are mean ± SE.</p

Chilling symptoms of ‘Ettinger’ after cold-quarantine treatments.

<p>Evaluation of chilling-injury (CI) symptoms in treated [modified atmosphere (MA), methyl jasmonate (MJ), low-temperature conditioning (LTC)] and non-treated ‘Ettinger’ after cold-quarantine treatments (15 days at 5°C or at 1°C, then storage at 5°C for the rest of the overall 3 weeks in cold storage), followed by 7 days of shelf storage. (A) Representative pictures of ‘Ettinger’ fruit after 3 weeks of cold storage followed by shelf storage. (B) CI severity ranked from 1–3 after cold storage (black column) and further shelf storage (white column). Data are mean ± SE. Different letters (lowercase letters and uppercase letters refer to after cold storage and shelf life, respectively) indicate significant differences at <i>P</i> < 0.05 by one-way ANOVA and Duncan's multiple range test.</p