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: