Frost survival and gene expression in timothy (Phleum pratense L.) cultivars as affected by age and selection in diverse field environments

Abstract

The sustainable production of perennial grasses in Northern Norway is at risk due to the ongoing climate change. The predicted increase in temperatures and variable weather patterns are further expected to create challenges for winter survival of timothy (Phleum pratense L.). Knowledge about the molecular mechanisms underlying freezing tolerance is crucial for developing robust cultivars. The current study is aimed at identifying genes involved in freezing stress response of timothy and studying gene expression differentiation due to field selection in contrasting environments using RNAseq. Four timothy cultivars were field tested for three years in Tromsø and Vesterålen, in Northern Norway. The surviving material from the field tests, along with plants raised from the original seed lots, were subjected to freezing tests. LT50 values varied across cultivars and materials. Many genes coding for transcription factors and proteins known to play an important role in freezing tolerance, like dehydrins, c‐repeat binding factors, and late embryogenesis abundant proteins were upregulated with decreasing temperatures. Moreover, genes associated with glycolysis/gluconeogenesis, TCA cycle, glutathione metabolism, proteasome pathways and genes encoding autophagy‐related proteins, plasma membrane‐associated proteins, sugar and amino acid transporters had elevated expression in field survivors compared to plants raised from the original material. The lower freezing stress tolerance of field survivors despite the elevated expression of several stress‐responsive genes might be due to a combination of selection in the field and the age effect. Furthermore, differences in freezing stress response between northern and southern adapted cultivars and surviving material from two field trial locations are discussed.

Surviving under Ice: Insights into gene expression changes during ice encasement in timothy (Phleum pratenseL.)

“…However, results of the current study cannot explain the decline in ICET in Engmo and the relatively stable expression of ICET in Grindstad among field survivors when compared to their corresponding original material. A similar trend was observed in one of our previous studies investigating freezing stress responses in the same plant material (Pashapu et al, 2024). The differences in their stress tolerances and the contrasting ability to maintain stress tolerances throughout different stages of plant development make Engmo and Grindstad the ideal candidates to study the effect of plant age on stress responses.…”

“…Upregulation of genes controlling cell cycle and organ size, i.e., growth-regulating factor 3-like, cell number regulator, lateral organ boundary (LOB) domain-containing protein, and TOUSLED serine/threonine-protein kinase, and downregulation of many genes linked to cell wall modification and growth, i.e., expansins, cellulose synthase, xyloglucan endotransglucosylase/hydrolase, fasciclin-like arabinogalactan proteins, pectin esterase, and polygalacturonase support a LOQS response under ice encasement (Table 2). Moreover, the expression of DREB1/CBF TFs, dehydrins, LEA proteins, early-responsive to dehydration 15-like, heat shock TFs and heat shock proteins, core genes involved in freezing stress responses of timothy (Pashapu et al, 2024) were observed to be upregulated under ice encasement (Table 2), supporting our initial hypothesis. It would be interesting to compare the expression of these genes under freezing and ice encasement stress.…”

“…Differentially expressed genes under ice encasement A total of 12,142 genes were identified as differentially expressed at different contrasts under long-term ice encasement conditions in timothy; CA vs T1 (2,663 down; 5,100 up), CA vs T2 ( 4,526 down; 4,981 up) and T1 vs T2 (217 down; 49 up). As hypothesized, the DEGs identified under ice encasement stress (Table 2) overlap with some of the core freezing, and hypoxia responses in plants ( Bailey-Serres and Voesenek, 2010;Jethva et al, 2022;Pashapu et al, 2024). Some of the DEGs identified include ethylene-responsive transcription factors (ERFs), plant cysteine oxidase (PCOs), prolyl 4-hydroxylase, sucrose non-fermenting-1-related protein kinase (SnRK) (hypoxia signalling), alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH), pyruvate decarboxylase 2 (PDC2), sucrose synthase, pyruvate kinase, ATP-dependent 6phosphofructokinase (glycolysis), dehydration responsive element-binding 1 (DREB1/C-repeat binding factor (CBF)), late embryogenesis abundant (LEA) proteins, dehydrins, early-responsive to dehydration (ERD) (cold/freezing responses), respiratory burst oxidase homolog protein (RBOH), alternative oxidase (AOX), peroxidases, glutathione S-transferase, superoxide dismutase (SOD) (ROS signalling & homeostasis) (Figure 3, Figure S2, Table 2).…”

“…The plant material used in the current study is the same as described in Pashapu et al (2024). To briefly summarize, 4-year-old surviving plants of the timothy cultivars Engmo, Noreng, Grindstad, and Snorri were collected from field plots in Tromsø (69.4° N, 19.4° E) and Vesterålen (68.7° N, 15.4° E) in late August/early September 2020.…”