Introduction:
Acclimation to cool temperatures in winter annuals has two main components: (i) activation of survival traits, such as enhanced freezing tolerance, that permit survival during periods of subfreezing temperatures (Kang et al., 2013; Oakley, Ågren, Atchison, & Schemske, 2014; Thomashow, 1999; Zhen & Ungerer, 2008), and (ii) activation of growth-maintenance traits, such as photosynthetic upregulation, that facilitate continued growth and productivity on cool days (Anderson, Chow, & Park, 1995; Bode, Ivanov, & Hüner, 2016; Hüner et al., 2012; Huner, Öquist, & Sarhan, 1998). The mechanisms behind photosynthetic upregulation under winter conditions include synthesis of greater numbers of proteins involved in photosynthesis (Huner et al., 1993; Stitt & Hurry, 2002; Strand et al., 1999) as well as a greater capacity for sugar export from leaves (Adams, Cohu, Muller, & Demmig-Adams, 2013; Dumlao et al., 2012; Leonardos, Savitch, Huner, Öquist, & Grodzinski, 2003), which compensates for reduced enzyme activity under cool temperature. In addition, leaves of winter annuals grown in cool versus warm temperatures are thicker and contain more chloroplast-rich mesophyll cells per unit area (Adams, Stewart, Cohu, Muller, & Demmig-Adams, 2016; Cohu, Muller, Adams, & Demmig-Adams, 2014; Gorsuch, Pandey, & Atkin, 2010). By virtue of this enhancement of biochemical and structural features for photosynthesis and sugar export/transport, overwintering herbaceous plants are able to maintain high sugar production and transport to underground storage, while minimizing exposure of above-ground portions to freezing events by reducing leaf surface area (Eremina, Rozhon, & Poppenberger, 2016). Notably, a similar upregulation of photosynthetic capacity and leaf thickness takes place in many species during acclimation to high growth-light intensity (Boardman, 1977; Gauhl, 1976; Munekage, Inoue, Yoneda, & Yokota, 2015; Yano & Terashima, 2004), including Arabidopsis thaliana(Hoshino, Yoshida, & Tsukaya, 2019; Stewart, Polutchko, Adams, & Demmig-Adams, 2017 ). Common regulatory networks may thus be involved in both cold and high-light acclimation, including the level of excitation pressure sensed by the chloroplast (Anderson et al., 1995; Hüner et al., 2012; Hüner, Dahal, Bode, Kurepin, & Ivanov, 2016).
It has been proposed that the transcription factor family of C-repeat-Binding Factors (CBFs) may link photosynthetic upregulation in response to growth under cool temperatures and/or high light intensity to enhanced freezing tolerance (Hüner et al., 2014, 2016). A. thaliana contains three tandemly duplicated CBF paralogs (CBF1 , CBF2 , and CBF3 ; abbreviated toCBF1–3 in this text) that are strongly induced by cold treatment and together direct many of the transcriptional and physiological changes necessary for enhanced freezing tolerance (Knight & Knight, 2012; Shi, Ding, & Yang, 2018; Thomashow, 1999). Laboratory studies have 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 for strongly reduced induction of freezing-tolerance genes and freezing tolerance itself (Gilmour, Fowler, & Thomashow, 2004; Jia et al., 2016; Park, Gilmour, Grumet, & Thomashow, 2018; Zhao et al., 2016). CBFover-expressing lines exhibited higher freezing tolerance as well as greater leaf thickness, chlorophyll levels, and photosynthetic rates per unit area even after growth under low light and warm conditions (Gilmour et al., 2004; Savitch et al., 2005). Thus, CBF overexpression is sufficient to induce 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 the Swedish ecotype exhibited much greater foliar phenotypic plasticity in response to both growth light intensity and temperature compared to the Italian ecotype (Adams, Cohu, Amiard, & Demmig-Adams, 2014; Adams et al., 2016; Adams, Stewart, Polutchko, & Demmig-Adams, 2018; Cohu, Muller, Demmig-Adams, & Adams, 2013a; Cohu, Muller, Stewart, Demmig-Adams, & Adams, 2013b; Stewart et al., 2015, 2016, 2017). The Swedish ecotype was also shown to have a greater tolerance to deep-freezing events relative to the Italian ecotype (Oakley et al., 2014; Park et al., 2018). The CBF s were identified as a QTL for fitness (Ågren et al., 2013) and freezing tolerance (Oakley et al., 2014), and subsequent work revealed that IT possesses an 8-bp deletion in its CBF2 gene that renders the CBF2 transcription factor nonfunctional (Gehan et al., 2015). While IT exhibited greater freezing tolerance when transformed with the SWCBF2 allele (Gehan et al., 2015), the loss of functional CBF2 in IT is not sufficient to account for its lower freezing tolerance relative to SW (Park et al., 2018).
In the present study, IT and SW were grown under several differing conditions using a factorial design of light intensity and temperature regimes. Transcriptome data from fully expanded leaves were generated to compare expression patterns of genes associated with freezing tolerance and photosynthesis, and chloroplast redox state (redox 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 parental ecotypes were subsequently grown alongside their corresponding mutant lines that encode nonfunctional CBF1–3 proteins, it:cbf123 and sw:cbf123 , respectively, and a mutant line of SW that encodes a nonfunctional CBF2, sw:cbf2 (Park et al., 2018). Fully expanded leaves of these plants were assayed for freezing tolerance, morphological and photosynthetic characteristics, and expression of genes associated with these phenotypic traits.
iv. a. Materials and Methods: