Cold acclimation proteome analysis reveals close link between the up-regulation of low-temperature associated proteins and vernalization fulfillment

Low-temperature (LT) stress is one of the major limiting factors in cereal production in cold high-altitude mountainous areas of Iran where cereals are exposed to variable periods of temperatures in the vernalization range during the autumn season. Cereals regulate their development through adaptive mechanisms that are responsive to low but nonfreezing temperatures. We exploited a proteomic approach to determine the interrelationship between vernalization fulfillment and expression of low-temperature (LT)-induced protein in most hardy Norstar and semi-hardy Azar2 wheat (Triticum aestivum L. em. Thell). These cultivars were subjected to 12 h of cold acclimating temperature (2 °C) over a period of 0-89 days. LT tolerance, as measured by LT50, and vernalization fulfillment, as estimated from final leaf number (FLN), was determined at intervals throughout the acclimation period. A significant decrease in FLN associated with LT treatment indicated that Norstar and Azar2 had vernalization responses. Azar2 achieved its vernalization fulfillment and maximum LT tolerance (∼ -8 °C) by 28 days of acclimation. However, Norstar had a longer vernalization requirement (between 35 and 42 days) and reached vernalization fulfillment and maximum LT tolerance (∼ -18.7 °C) about the same time as vernalization fulfillment. We applied a two-dimensional electrophoresis-based proteomics approach to analyze changes in the leaf proteome of two genotypes, Norstar and Azar2, during cold acclimation. Using MALDI-TOF/TOF mass spectrometry, 66 LT-associated proteins could significantly be identified. These proteins were categorized into cold-regulated proteins, antifreezing proteins, oxidative stress defense, photosynthesis, chloroplast post-transcriptional regulation, metabolisms, and protein synthesis. A close association between the vernalization fulfillment and the start of a decline in the protein accumulation of hardy Norstar with a long vernalization requirement and semi-hardy Azar2 with a short vernalization requirement was observed. This finding supported the hypothesis that developmental trait which was regulated by vernalization had a regulatory influence over LT proteome response and highlight a close link between the up-regulation of LT-associated proteins and vernalization fulfillment at the molecular level in wheat.