Comparative analysis of the cold acclimation and freezing tolerance capacities of seven diploid Brachypodium distachyon accessions

Abstract

Background and aims: Cold is a major constraint for cereal cultivation under temperate climates. Winter-hardy plants interpret seasonal changes and can acquire the ability to resist sub-zero temperatures. This cold acclimation process is associated with physiological, biochemical and molecular alterations in cereals. Brachypodium distachyon is considered a powerful model system to study the response of temperate cereals to adverse environmental conditions. To date, little is known about the cold acclimation and freezing tolerance capacities of Brachypodium. The main objective of this study was to evaluate the cold hardiness of seven diploid Brachypodium accessions.

Methods: An integrated approach, involving monitoring of phenological indicators along with expression profiling of the major vernalization regulator VRN1 orthologue, was followed. In parallel, soluble sugars and proline contents were determined along with expression profiles of two COR genes in plants exposed to low temperatures. Finally, whole-plant freezing tests were performed to evaluate the freezing tolerance capacity of Brachypodium.

Key results: Cold treatment accelerated the transition from the vegetative to the reproductive phase in all diploid Brachypodium accessions tested. In addition, low temperature exposure triggered the gradual accumulation of BradiVRN1 transcripts in all accessions tested. These accessions exhibited a clear cold acclimation response by progressively accumulating proline, sugars and COR gene transcripts. However, whole-plant freezing tests revealed that these seven diploid accessions only have a limited capacity to develop freezing tolerance when compared with winter varieties of temperate cereals such as wheat and barley. Furthermore, little difference in terms of survival was observed among the accessions tested despite their previous classification as either spring or winter genotypes.

Conclusions: This study is the first to characterize the freezing tolerance capacities of B. distachyon and provides strong evidence that some diploid accessions such as Bd21 have a facultative growth habit.

Keywords: Brachypodium distachyon; COR413; VRN1; cold acclimation; flowering; freezing tolerance; fructans; ice recrystallization inhibition; phenological development; proline; vernalization; winter hardiness.

Fig. 1.

Final leaf number of seven diploid Brachypodium distachyon accessions in response to cold exposure. Final leaf number was compiled for Brachypodium accessions previously shown to have spring (left panel) or winter (right panel) growth habits when cold acclimated under (A and B) SD and (C and D) LD conditions. Time points where plants did not flower during the course of the experiment are indicated by an asterisk. Statistically significant differences (P < 0·05) in final leaf number were observed until 28 d for Bd3-1, Bd21 and Bd30-1, until 35 d for Bd2-3, until 49 d for Bd29-1 and until 56 d for accessions Bd1-1 and Bd18-1.

Fig. 2.

Apical development of seven diploid Brachypodium distachyon accessions exposed to cold (4 °C) for the indicated time. Double-ridge formation was compiled for Brachypodium accessions previously shown to have (A) spring or (B) winter growth habits. Time points where plants never formed the double-ridge structure during the course of the experiment are indicated by an asterisk. Statistically significant differences (P < 0·05) in days to double-ridge values were observed until 28 d for Bd21 and Bd 30-1, until 42 d for Bd2-3 and Bd 3-1 and until 49 d of treatment for Bd1-1, Bd18-1 and Bd29-1.

Fig. 3.

Expression analysis of BradiVRN1 in response to low temperatures. (A–C) Relative transcript accumulation of BradiVRN1 in Brachypodium accessions Bd2-3, Bd3-1 and Bd21. (D and E) Relative transcript accumulation of BradiVRN1 in winter Brachypodium accessions Bd1-1 and Bd18-1. (F) Relative transcript accumulation of BradiVRN1 in Bd21 and Bd18-1 plants not exposed to 4 °C. Letters above bars represent statistical significance (P < 0·05); different letters indicate statistically different fold expression.

Fig. 4.

Accumulation of COR gene transcripts during cold acclimation in Brachypodium distachyon. Relative transcript accumulation of (A, B) BradiIRI and (C, D) BradiCOR413 in Bd2-3 and Bd18-1 plants exposed to LT for up to 42 d. Relative transcript accumulation in above-ground tissues was measured by quantitative real-time PCR and normalized to 18S rRNA transcript levels. Letters above bars represent statistical significance (P < 0·05); different letters indicate statistically different fold expression.

Fig. 5.

Freezing temperature tolerance (LT₅₀) of seven diploid Brachypodium distachyon accessions. (A) Plants at the three-leaf stage were either kept under normal conditions (non-acclimated) for 5 d (NA5) or cold acclimated for 28 d (CA28) before being subjected to WPFTs. After 2 weeks in normal growth conditions, survival counts were taken and the LT₅₀ was calculated. The experiment was repeated four times, and error bars indicate the standard deviation of the mean. (B and C) Relative transcript abundance of BradiIRI (B) and BradiCOR413 (C) in NA5 and CA28 plants used for WPFTs of seven diploid Brachypodium accessions. Letters above bars represent statistical significance (P < 0·05); different letters indicate statistically different fold expression.

Fig. 6.

Water-soluble sugar and proline concentration in leaves of seven diploid Brachypodium distachyon accessions exposed to cold. (A and C) WSS and (B and D) proline concentration of non-acclimated plants (NA0 and NA5), cold-acclimated plants (CA7, CA14, CA21 and CA28) and de-acclimated plants (DA1) of accessions previously shown to have a spring growth habit (left) or winter growth habit (right). The error bars indicate standard deviation of the mean.