Article pubs.acs.org/jpr
Proteome Analysis of Cold Response in Spring and Winter Wheat (Triticum aestivum) Crowns Reveals Similarities in Stress Adaptation and Differences in Regulatory Processes between the Growth Habits Klára Kosová,*,† Pavel Vítámvás,† Sébastien Planchon,‡ Jenny Renaut,‡ Radomíra Vanková,§ and Ilja Tom Prásǐ l† †
Department of Genetics and Plant Breeding, Crop Research Institute, Drnovská 507, 16106 Prague 6, The Czech Republic Centre de Recherche Public, Gabriel Lippmann, 41 Rue du Brill, 4422 Belvaux, Luxembourg § Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojová 263, 16502 Prague 6, The Czech Republic ‡
S Supporting Information *
ABSTRACT: A proteomic response to cold treatment (4 °C) has been studied in crowns of a frost-tolerant winter wheat cultivar Samanta and a frost-sensitive spring wheat cultivar Sandra after short-term (3 days) and long-term (21 days) cold treatments. Densitometric analysis of 2-D differential in gel electrophoresis (2D-DIGE) gels has resulted in the detection of 386 differentially abundant protein spots, which reveal at least a two-fold change between experimental variants. Of these, 58 representative protein spots have been selected for MALDI-TOF/TOF identification, and 36 proteins have been identified. The identified proteins with an increased relative abundance upon cold in both growth habits include proteins involved in carbohydrate catabolism (glycolysis enzymes), redox metabolism (thioredoxin-dependent peroxidase), chaperones, as well as defense-related proteins (protein revealing similarity to thaumatin). Proteins exhibiting a cold-induced increase in the winter cultivar include proteins involved in regulation of stress response and development (germin E, lectin VER2), while proteins showing a cold-induced increase in the spring cultivar include proteins involved in restoration of cell division and plant growth (eIF5A2, glycine-rich RNA-binding protein, adenine phosphoribosyltransferase). These results provide new insights into cold acclimation in spring and winter wheat at the proteome level and enrich our previous work aimed at phytohormone dynamics in the same plant material. KEYWORDS: 2D-DIGE analysis, cold stress, spring and winter growth habit, wheat crown, cold acclimation, development response
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INTRODUCTION Cold is an important abiotic stress, limiting agricultural productivity in temperate climates. Cold is similar to other abiotic stresses in that it induces an active stress response that aims to allow continued plant survival during exposure. The plant stress response is dynamic and involves several distinct phases including an alarm phase, an acclimation phase, a resistance phase, and an exhaustion phase.1−4 The plant stress response has been characterized in a number of studies by a profound reorganization of the transcriptome, proteome, and metabolome. Proteins play a crucial role in plant stress response because they are directly involved in both structural and metabolic changes. The effects of cold on the proteome in © XXXX American Chemical Society
several plant species exposed to low temperatures have been summarized in Kosová et al.3 Physiological studies reveal that cold poses a significant stress on plants, leading to a profound reorganization of the whole cellular metabolism and resulting in a shift from an active growth and development to an active stress acclimation. This shift can be observed through the induction of several stress-responsive proteins such as chaperones, redox metabolism enzymes, and defense-related Special Issue: Agricultural and Environmental Proteomics Received: June 24, 2013
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dx.doi.org/10.1021/pr400600g | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
out on the same plant material, the same growth stage (a threeleaf stage), and the same growth conditions, and thus they provide a complex view on winter and spring wheat response to cold treatment, enabling us to identify similarities as well as differences between the spring and winter wheat growth habits.
proteins as well as alterations in energy metabolism and a downregulation of several development-related proteins. Common wheat (Triticum aestivum) is a major cereal crop grown in temperate climate zones. The wheat gene pool can generally be divided into two major growth habits − a winter growth habit possessing a recessive vernalization gene Vrn1 and a dominant vernalization gene Vrn2 and revealing a vernalization requirement and a spring growth habit possessing a dominant vernalization gene Vrn1 whose expression cannot be inhibited by a Vrn2 gene product.5 The winter and spring growth habits differ in the dynamics of cold response as well as development. The winter growth habit has a vernalization requirement that functions as an evolutionary adaptation preventing the plants from a premature transition into a coldsensitive reproductive phase under adverse environmental conditions during winter. In contrast, spring growth habit does not have vernalization requirement, and it is characterized by a relatively faster plant development.5,6 An induction of several genes (transcription factors and genes involved in signaling) has been observed in both spring and winter cultivars of common wheat upon cold. However, the activation of cold acclimation-related genes in the spring cultivar was only transient, and it was overriden by another regulatory program associated with a restoration of an active growth and development.7 Proteins are directly involved in an acquisition of an enhanced cold tolerance. Recently, high-throughput proteomic approaches have helped the researchers to unravel the complex regulatory network underlying the plants dynamic response to cold. Proteomic studies have contributed to the identification and characterization of several novel proteins involved in the plant cold response. In cereals, there have already been several proteomic works published on cold response in both winter8−13 as well as spring14 wheat and barley cultivars. The papers published on the winter cultivars have been focused on a shortterm cold acclimation as well as a long-term vernalization responses. The effect of subzero temperatures (−5 °C) simulating a spring freeze stress on wheat proteome was studied by Han et al.15 The paper of Rinalducci et al.14 published on the spring wheat cultivar was focused on a longterm cold response until transition to flowering. However, to our best knowledge, no comparison of proteomic response during the initial phases of cold treatment in a spring and a winter cultivar within a single experiment has been published. The present study is focused on a comparison of the plant cold response in a frost-tolerant winter wheat cultivar Samanta and a frost-sensitive spring cultivar Sandra at the proteome level. It complements our previous work4 aimed at determining the dynamics of plant frost tolerance (lethal temperature of 50% of sample (LT50) values, respectively), dehydrin WCS120 accumulation, and a complex analysis of phytohormones (abscisic acid (ABA) and its derivatives, cytokinins (CKs), gibberellins (GAs), indolyl acetic acid (IAA), jasmonic acid (JA), salicylic acid (SA), and aminocyclopropane carboxylic acid (ACC)) during a 21-day cold treatment. The present study has been focused on proteome analysis of crown tissues in both cultivars grown under control conditions or subjected to 3 or 21 days of cold treatment. The detection, identification, and functional annotation of proteins showing differential abundance between control and cold treatments as well as between the growth habits can contribute to our knowledge on cold acclimation mechanisms in wheat growth habits with contrasting frost tolerance. Both studies have been carried
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MATERIALS AND METHODS
Plant Material and Sample Preparation
A frost-tolerant winter wheat cultivar Samanta (W) and a frostsensitive spring wheat cultivar Sandra (S) were used for the proteomic experiment. Data on acquired frost tolerance determined as lethal temperature of 50% of the sample (LT50), WCS120 protein relative accumulation as well as selected phytohormone levels in both cultivars during cold treatment were published in Kosová et al.4 The seeds were germinated under controlled conditions in the dark (20 °C). The germinated seeds were transferred to pots filled with soil and grown in a growth chamber under controlled conditions (12 h photoperiod, 350 μmol m−2 s−1). Plants were grown for the first 3 weeks at 20 °C; then, the temperature was decreased to 4 °C. Crown samples for proteome analysis were taken at 0 (control), 3 (early acclimation), and 21 (full acclimation) days of cold. In both experiments, cold treatment was applied on 21day old plants that were in a three-leaf stage. Six experimental variants (spring cultivar at 0 days of cold (S 0 d), spring cultivar at 3 days of cold (S 3 d), spring cultivar at 21 days of cold (S 21 d), winter cultivar at 0 days of cold (W 0 d), winter cultivar at 3 days of cold (W 3 d), winter cultivar at 21 days of cold (W 21 d)) and four biological replicates per each variant were investigated. Samples were immediately frozen in liquid nitrogen and stored at −80 °C until further use. For total protein extraction, trichloroacetic acid (TCA)/acetone method followed by protein extraction into phenol and subsequent protein precipitation using ammonium acetate was used.16 Proteomic Analysis
Dry protein pellets were resolved in lysis buffer prepared according to GE Healthcare manual for 2D-DIGE analysis, pH of the solution was adjusted to 8.5, and protein concentration was determined by 2D Quant kit (GE Healthcare). The protein samples (30 μg) were labeled with CyDye minimal dyes (GE Healthcare) according to manufacturer’s instructions. (For details, see Supplementary Table S1 and Supplementary Figure S1 in the Supporting Information.) The samples were loaded onto 24 cm IPG linear strip with a pI range 5−8 (Bio-Rad). The isoelectric focusing was carried out in IEF Cell (Bio-Rad) using a rapid voltage slope to reach the maximum of 10 000 V and the total of 72 000 Vh. The second dimension was carried out as SDS-PAGE using 12.5% (v/v) resolving gel (EttanDALTsix, GE Healthcare). Scanning of the 2D-DIGE gels was carried out using PharosFX fluorescence imager (BioRad) with the following emission wavelengths for the individual CyDyes: Cy2 506 nm, Cy3 572 nm, and Cy5 669 nm. Densitometric analysis of scanned images was carried out using PDQuest Advanced 8.0.1 (Bio-Rad) multichannel application (for 2D-DIGE analysis). Protein spot normalization was carried out using local regression model, and spot manual editing was carried out using group consensus tool. The statistical criteria applied in the search for differentially abundant protein spots (at least a 2 fold change; p ≤ 0.05) are described in the Statistical Analysis section. Out of the differentially abundant protein spots, the protein spots present in at least 80% of gels and revealing
