Abstract
Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold de-acclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we performed comparative transcriptomic and proteomic analyses during CA and DA. As revealed by shotgun proteomics, we identified 3987 peptides originating from 1569 unique proteins and the corresponding mRNAs were analyzed. Among the 1569 genes, 658 genes were specifically induced at the transcriptional level during the process of cold acclimation. In order to investigate the relationship between mRNA and the corresponding protein expression pattern, a Pearson correlation was analyzed. Interestingly, 199 genes showed a positive correlation of mRNA and protein expression pattern, indicating that both their transcription and translation occurred during CA. However, 226 genes showed a negative correlation of mRNA and protein expression pattern, indicating that their mRNAs were transcribed during CA and were stored for the subsequent DA step. Under this scenario, those proteins were specifically increased during DA without additional transcription of mRNA. In order to confirm the negative correlation of mRNA and protein expression patterns, qRT-PCR and western blot analyses were performed. Mitochondrial malate dehydrogenase 1 (mMDH1) exhibited a negative correlation of mRNA and protein levels, which was characterized by CA-specific mRNA induction and protein accumulation specifically during DA. These data indicate that the expression of specific mRNAs and subsequent accumulation of corresponding proteins are not always in accordance under low temperature stress conditions in plants.
Highlights
From the ‡Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science (CSRS), Yokohama, Kanagawa, 230-0045, Japan; §Plant Proteomics Research Unit, RIKEN CSRS, Yokohama, Kanagawa, 230-0045, Japan; ¶Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, Morioka, Iwate, 0208550, Japan; ʈPlant Immunity Research Group, RIKEN CSRS, Yokohama, Kanagawa, 230-0045, Japan; **Kihara Institute for Biological Research, Yokohama City University, Yokohama, Kanagawa, 2440813, Japan; ‡‡Core Research for Evolutionary Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
We demonstrated that many cold-inducible genes were transcribed and translated, a similar number of corresponding coldinducible genes were not translated during the cold acclimation (CA) stage and were translated at a later stage such as de-acclimation
According to a correlation analysis of mRNA and protein expression patterns, over 200 genes showed negative correlation when their mRNA and protein expression patterns were compared during CA and DA (Fig. 2 and supplemental Table S3)
Summary
15N isotope-labeled standard proteins were prepared in order to normalize each sample. The peak heights of the identified peptides (Target) and the corresponding 15N isotope-labeled peptide (Standard) pairs in all samples were determined using Mass Navigator v1.2. In order to quantify protein expression levels, unique peptides were selected under the conditions where the corresponding peaks were detected in more than 36/45 experiments (three biological and three technical replicates with five time points of samples) after the “Quantitation score” (Mass Navigator) was cut off at 0.7. QRT-PCR and Western Blot Analysis—Total RNAs were prepared from CA and DA-treated samples (three biological replicates) using the Plant RNA Reagent (Invitrogen) and cDNAs were synthesized using Quantitech cDNA synthesis kit (Qiagen, Venlo, Netherlands). After centrifugation for 90 min at 4 °C at 40,000 rpm (275,000 ϫ g) using a SW41Ti rotor (Beckman Coulter, Brea, CA), the gradient was loaded into Monitor UV-900 (GE Healthcare) for scanning at 254 nm using a perista pump (ATTO, Tokyo, Japan)
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