Characterization of chilling-shock responses in four genotypes of Miscanthus reveals the superior tolerance of M. x giganteus compared with M. sinensis and M. sacchariflorus

Abstract

Background and aims: The bioenergy grass Miscanthus is native to eastern Asia. As Miscanthus uses C4 photosynthesis, the cooler temperatures experienced in much of northern Europe are expected to limit productivity. Identification of genetic diversity in chilling tolerance will enable breeders to generate more productive varieties for these cooler regions. Characterizing the temporal relationships between photosynthesis, carbohydrate and molecular expression of relevant genes is key to understanding genotypic differences in tolerance or sensitivity.

Methods: To characterize chilling responses in four Miscanthus genotypes, plants were exposed to a sudden reduction in temperature. The genotypes studied comprised of two M. sinensis, one M. sacchariflorus and one inter-species hybrid, M. × giganteus. Changes in photosynthesis (Asat), carbohydrate composition and the expression of target transcripts were observed following chilling-shock. After 4 d the decline in leaf elongation rate (LER) in the different genotypes was measured.

Results: Following chilling-shock the greatest decline in Asat was observed in M. sacchariflorus and one M. sinensis genotype. Carbohydrate concentrations increased in all genotypes following chilling but to a lesser extent in M. sacchariflorus. Two stress inducible genes were most highly expressed in the genotypes that experienced the greatest declines in Asat and LER. Miscanthus × giganteus retained the highest Asat and was unique in exhibiting no decline in LER following transfer to 12 °C.

Conclusions: Miscanthus × giganteus exhibits a superior tolerance to chilling shock than other genotypes of Miscanthus. The absence of sucrose accumulation in M. sacchariflorus during chilling-shock suggests an impairment in enzyme function. A candidate transcription factor, MsCBF3, is most highly expressed in the most sensitive genotypes and may be a suitable molecular marker for predicting chilling sensitivity.

Fig. 1.

(A) Light saturated photosynthesis (A_sat) at 28 °C (Warm) and after transfer to 12 °C (Chilled) in the four genotypes. (B) Percentage reduction in leaf CO₂ assimilation following transfer. Values are means ± s.e., n = 3.

Fig. 2.

Soluble sugar contents of the four genotypes at 28 °C (Warm) and after transfer to 12 °C (Chilled): sucrose (A), glucose (B) and fructose (C). Plants were transferred to 12 °C at 2 h. Values are means ± s.e., n = 3.

Fig. 3.

Starch contents at 28 °C (Warm) and at 12 °C (Chilled) in the four genotypes. Values are means ± s.e., n = 3.

Fig. 4.

Fold change in expression between cold/warm conditions of (A) MsPGM1, (B) MsBAM3, (C) MsCBF3 and (D) MsRD19a. Expression levels at 28 °C and 12 °C were calculated relative to an internal control gene (MsYLS8). Fold change is the relative expression at 12 °C/relative expression at 28 °C. Values are means ± s.e., n = 3. * Significant differences to 28 °C (Tukey test, P < 0·05). Primer sequences are listed in Supplementary Data Table S2.

Fig. 5.

Pearson's correlation coefficient for fold changes in (A) MsPGM1 and (B) MsBAM3 against sucrose content after transfer to 12 °C; n = 3. Time-points correspond to T0, 2, 4 and 8 h after transfer to 12 °C. Fold change in relative gene expression is as described in Fig. 4.

Fig. 6.

Average leaf elongation rate of the four genotypes at 28 °C for 4 d (Warm), 12 °C chilling-shock for 24 h before a return to 28 °C for 3 d (Mixed), 12 °C chilling-shock maintained for 4 d (Cold).Values are means + s.e., n = 4–6. Different letters show significant differences between treatments for the same genotype (Student's two-tailed t-test, P < 0·05).