4.1 ⎪ Response of plant function and gene expression to growth under HLC conditions
Neither cool temperature alone (maximum daytime leaf temperature of 16°C) nor high light alone strongly induced expression ofCBF1–3, but combined high light and cool temperature (HLC) acted synergistically to induce CBF1–3 . Increased excitation pressure in the chloroplast serves as one of the signals that induce elevatedCBF1–3 expression under HLC conditions (Hüner et al.2012, 2016) and is integrated with additional photosynthetic retrograde signals (Lee & Thomashow 2012; Noren et al. 2016). Overwintering herbaceous plants, experiencing cold temperatures and the associated high excitation pressure in the chloroplast, enact the suite of acclimatory responses demonstrated here, including upregulation of photosynthetic capacity and leaf thickness (with more mass and chlorophyll per area), reduced leaf expansion, and enhanced freezing tolerance (see also Cohu et al. 2013b, 2014; Muller et al.2014; Sanderson et al. 2020). These changes can allow overwintering species to achieve full acclimation, defined as a new homeostasis where internal stress (with signs of oxidative stress) is minimized or absent.
The pronounced acclimation of plant form and function in SW and IT plants grown in HLC conditions was associated with sweeping changes in gene expression, with approximately 5.2% of total leaf transcriptome upregulated and 4.9% downregulated in HLC relative to growth in low light and warm temperature (LLW). The most strongly enriched gene ontology categories in HLC were related to starch metabolism, sucrose biosynthesis, and cold acclimation, which is consistent with the upregulation of photosynthetic capacity and of freezing tolerance. Continued photosynthetic productivity under cool temperatures in the absence of significant growth generates carbohydrate that can be stored in sink tissues (Demmig-Adams et al. 2017; Adams et al.2018a) and also contribute to the accumulation of compatible solutes and freezing point depression (Wanner & Junttila 1999; Reyes-Diaz et al. 2006; Cao, Song & Su 2007).
Pathways repressed in HLC in both SW and IT included those associated with growth hormones. Reduction of rosette expansion under winter conditions, involving decreased rates of cell elongation during leaf development (Yano & Terashima 2004; Hoshino et al. 2019) helps to minimize foliar freezing damage. Pathways repressed in HLC in both SW and IT included not only those associated with growth hormones (e.g., brassinosteroids and auxin polar transport) but also with water transport. In fact, vascular tissue is one of the targets of growth hormones (Fàbregas et al. 2015; Etchells, Peter Etchells & Turner 2017) and acclimation to cool temperature is associated with adjustments of vascular anatomy (Cohu et al. 2013a; Adamset al. 2016; Stewart et al. 2016). Thus, effects of freezing on the plant vasculature may be involved in the pronounced vulnerability of it:cbf123 plants. Further research is warranted into the possibility that the much greater impairment of freezing tolerance of whole plants by CBF1–3- deficiency in the IT compared to the SW background may be due to the well-characterized phenomenon of freeze-thaw-induced water conduit embolism and its dependence on xylem anatomy (Lens et al. 2013). Further research should also address plant ontology and age given that the electrolyte leakage assays in this study were performed on excised leaves of plants acclimated for multiple weeks, whereas, to achieve the necessary sample size, whole plants of a younger age/smaller size were used for survival assays.