Deep Super-SAGE transcriptomic analysis of cold acclimation in lentil (Lens culinaris Medik.)

Abstract

Background: Frost is one of the main abiotic stresses limiting plant distribution and crop production. To cope with the stress, plants evolved adaptations known as cold acclimation or chilling tolerance to maximize frost tolerance. Cold acclimation is a progressive acquisition of freezing tolerance by plants subjected to low non-freezing temperatures which subsequently allows them to survive exposure to frost. Lentil is a cool season grain legume that is challenged by winter frost in some areas of its cultivation.

Results: To better understand the genetic base of frost tolerance differential gene expression in response to cold acclimation was investigated. Recombinant inbred lines (RILs) from the cross Precoz x WA8649041 were first classified as cold tolerant or cold susceptible according to their response to temperatures between -3 to -15 °C. Then, RILs from both extremes of the response curve were cold acclimated and the leaf transcriptomes of two bulks each of eight frost tolerant and seven cold susceptible RILs were investigated by Deep Super-SAGE transcriptome profiling. Thus, four RNA bulks were analysed: the acclimated susceptible, the acclimated tolerant and the respective controls (non-acclimated susceptible and non-acclimated tolerant). Approximately 16.5 million 26 nucleotide long Super-SAGE tags were sequenced in the four sets (between ~3 and 5.4 millions). In total, 133,077 different unitags, each representing a particular transcript isoform, were identified in these four sets. Tags which showed a significantly different abundance in any of the bulks (fold change ≥4.0 and a significant p-value <0.001) were selected and used to identify the corresponding lentil gene sequence. Three hundred of such lentil sequences were identified. Most of their known homologs coded for glycine-rich, cold and drought-regulated proteins, dormancy-associated proteins, proline-rich proteins (PRPs) and other membrane proteins. These were generally but not exclusively over-expressed in the acclimated tolerant lines.

Conclusions: This set of candidate genes implicated in the response to frost in lentil represents an useful base for deeper and more detailed investigations into this important agronomic trait in future.

Keywords: Cold acclimation; Deep Super-SAGE; Frost; Gene expression; Lentil; Stress.

Fig. 1

Scheme of the cold treatments to determine the level of frost response of the Precoz x WA8649041 RILs

Fig. 2

Number of Precoz x WA8649041 RILs in each of the 12 levels of response to frost. Arrows indicate the level shown by the two parents. Double-headed red arrows indicate the pairs of levels considered as susceptible or tolerant for the Deep Super-SAGE experiment. Two set of bulked RNA were obtained, one from the eight tolerant RILs and another one from the seven susceptible RILs

Fig. 3

Number of unitags found in each of the lentil RNA bulk sets or common to two or more sets