1 ⎪ INTRODUCTION
Functional acclimation to cool temperatures in winter annuals has two essential components. These are activation of traits that (i) permit survival during periods of subfreezing temperatures (e.g., enhanced freezing tolerance; Thomashow 1999; Zhen & Ungerer 2008; Kang et al. 2013; Oakley, Ågren, Atchison & Schemske 2014) and (ii) support for continued productivity on cool days via upregulation of photosynthetic capacity, which compensates for cold-dependent inhibition of enzymatic activity (Berry & Björkman 1980; Hüner, Öquist & Sarhan 1998; Savitch et al. 2002; Cohu, Muller, Adams & Demmig-Adams 2014). Photosynthetic capacity is enhanced by synthesis of greater numbers of photosynthetic proteins (Hüner et al. 1993; Strandet al. 1999; Stitt & Hurry 2002; Demmig-Adams, Stewart & Adams 2017; Adams, Stewart & Demmig-Adams 2018a) as well as augmentation of related features, such as the infrastructure for photosynthate export from leaves (Leonardos, Savitch, Huner, Öquist & Grodzinski 2003; Dumlao et al. 2012; Adams, Cohu, Muller & Demmig-Adams 2013; Adams, Stewart, Cohu, Muller & Demmig-Adams 2016). Leaves of winter annuals grown in cool versus warm temperatures are also thicker and contain more chloroplast-rich mesophyll cells per unit area (Gorsuch, Pandey & Atkin 2010; Cohu et al. 2014; Adams et al.2016). By virtue of upregulation of photosynthetic capacity in leaves that develop under cool temperatures (Cohu, Muller, Stewart, Demmig-Adams & Adams 2013b), plants are able to maintain sugar production and transport for underground storage while limiting above-ground growth and exposure of leaves to freezing temperature (Eremina, Rozhon & Poppenberger 2016). This enhancement of photosynthesis-related traits illustrates how acclimatory adjustment leads to a new homeostasis that minimizes internal stress despite a challenging environment (Anderson, Chow & Park 1995). Notably, a similar upregulation of photosynthesis-related features takes place during acclimation to high growth-light intensity (Gauhl 1976; Boardman 1977; Munekage, Inoue, Yoneda & Yokota 2015) in many species, includingArabidopsis thaliana (Stewart et al. 2017b; Stewart, Polutchko, Adams & Demmig-Adams 2017a; Hoshino, Yoshida & Tsukaya 2019). Common regulatory networks may thus be involved in both cold and high light acclimation, such as signaling networks that respond to the level of excitation pressure in the chloroplast (Anderson et al.1995; Hüner et al. 2012; Hüner, Dahal, Bode, Kurepin & Ivanov 2016).
The transcription factor family of C-repeat-Binding Factors (CBFs) has been proposed as a regulatory network that may orchestrate photosynthetic upregulation and enhance freezing tolerance in response to growth under cool temperatures and/or high light intensities (Savitchet al. 2005; Hüner et al. 2016). Arabidopsis thaliana contains three tandemly duplicated CBF paralogs (CBF1 , CBF2 , and CBF3 ; abbreviated toCBF1–3 in this text) that are strongly induced by cold temperature and orchestrate transcriptional and physiological changes necessary for enhanced freezing tolerance (Thomashow 1999; Knight & Knight 2012; Shi, Ding & Yang 2018). Laboratory studies revealed largely overlapping functions for the CBF1–3 transcription factors as well as a requirement for combined loss-of-function mutations in all three genes to strongly reduce induction of freezing-tolerance genes and freezing tolerance itself (Gilmour, Fowler & Thomashow 2004; Zhaoet al. 2016; Jia et al. 2016). CBF over-expressing lines exhibited higher freezing tolerance as well as greater leaf thickness, chlorophyll levels, and photosynthetic rates per unit area even when grown under low light and warm temperature (Gilmour et al. 2004; Savitch et al. 2005). Thus, CBF overexpression induced both the survival trait of enhanced freezing tolerance and the productivity-maintenance trait of photosynthetic upregulation.
Following a five-year, reciprocal transplant investigation of twoA. thaliana ecotypes (Ågren & Schemske 2012), Rodasen-47 from Sweden (SW) and Castelnuovo-12 from Italy (IT), numerous studies provided insight into the ecophysiology and genetics underlying local adaptation in this model organism. Anatomical and physiological studies revealed that SW exhibited considerably greater foliar phenotypic plasticity in response to both growth light intensity and temperature compared to IT (Cohu et al. 2013b; Adams, Cohu, Amiard & Demmig-Adams 2014; Adams, Stewart, Polutchko & Demmig-Adams 2018b; Stewart et al. 2015, 2016, 2017b). While possessing a similar constitutive freezing tolerance, in warm-grown plants, SW also induced greater freezing tolerance relative to IT when grown under controlled cold conditions (Gehan et al. 2015; Park, Gilmour, Grumet & Thomashow 2018; Sanderson et al. 2020). Under field growth conditions, the CBF1–3 region was identified as a QTL for fitness (Ågren, Oakley, McKay, Lovell & Schemske 2013) as well as freezing tolerance (Oakley et al. 2014). In fact, IT possesses a naturally occurring 8-bp deletion in its CBF2 gene that renders the CBF2 transcription factor nonfunctional (Gehan et al. 2015). Nevertheless, CBF2-deficient lines of SW still maintained greater cold-induced freezing tolerance than IT (Park et al. 2018; Sanderson et al. 2020). Likewise, a CBF1–3- deficient line created in SW maintained greater cold induced freezing tolerance than a CBF1–3- deficient line created in IT (Park et al. 2018).
In the present study, IT and SW were grown under a factorial design of different light intensity and temperature regimes. Transcriptome data from fully expanded leaves were generated to compare expression patterns of genes associated with the functional traits of freezing tolerance and photosynthesis, and chloroplast redox state (reduction state of the primary electron acceptor of photosystem II, QA) was assessed to address the relationship between chloroplast excitation pressure and CBF1–3 expression levels. Under the two most different growth conditions, the wild-type ecotypes, IT and SW, were grown alongside the corresponding CBF1–3-deficient mutant lines it:cbf123 and sw:cbf123 (Park et al. 2018). Fully expanded leaves of these plants that had developed under the respective growth conditions were assayed for freezing tolerance, morphological and photosynthetic characteristics, and expression of genes associated with the latter phenotypic traits.