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.