Plant Mol Biol (2015) 87:1–15
Global changes in gene expression, assayed by microarray
hybridization and quantitative RT-PCR, during acclimation
of three Arabidopsis thaliana accessions to sub-zero temperatures
after cold acclimation
Mai Q. Le · Majken Pagter · Dirk K. Hincha
Received: 2 April 2014 / Accepted: 7 October 2014 / Published online: 14 October 2014
© Springer Science+Business Media Dordrecht 2014
Particularly photosynthesis-related genes are down-regulated
and genes belonging to the functional classes of cell wall
biosynthesis, hormone metabolism and RNA regulation of
transcription are up-regulated. Collectively, these data pro-
vide the ﬁrst global analysis of gene expression during sub-
zero acclimation and allow the identiﬁcation of candidate
genes for forward and reverse genetic studies into the molec-
ular mechanisms of sub-zero acclimation.
Keywords Arabidospis thaliana · CBF regulon · Cold
acclimation · Freezing tolerance · Transcription factor ·
Together with drought, cold is considered the most impor-
tant factor limiting the distribution of plants on Earth.
Freezing damage to native vegetation and crop plants is
additionally a ubiquitous problem of major economic sig-
niﬁcance (Steponkus 1984). The freezing tolerance of most
temperate plant species increases upon exposure to low,
non-freezing temperatures over a period of days to weeks.
This adaptive process is termed cold acclimation (Thom-
ashow 1999; Xin and Browse 2000). Freezing tolerance
beyond that resulting from exposure to low, non-freezing
temperatures is conferred on cold acclimated plants by
exposure to temperatures slightly below freezing but before
freezing injury occurs. This is commonly referred to as
sub-zero acclimation and has been described in a number
of plant species, such as Medicago sativa (Castonguay et
al. 1993; Monroy et al. 1993), Triticum aestivum (Herman
et al. 2006) and other cereals (Livingston 1996), Agrostis
(Espevig et al. 2011) and Arabidopsis thaliana (Le et al.
2008; Livingston et al. 2007).
Abstract During cold acclimation plants increase in freez-
ing tolerance in response to low non-freezing temperatures.
This is accompanied by many physiological, biochemical
and molecular changes that have been extensively investi-
gated. In addition, plants of many species, including Arabi-
dopsis thaliana, become more freezing tolerant during expo-
sure to mild, non-damaging sub-zero temperatures after
cold acclimation. There is hardly any information available
about the molecular basis of this adaptation. Here, we have
used microarrays and a qRT-PCR primer platform covering
1,880 genes encoding transcription factors (TFs) to moni-
tor changes in gene expression in the Arabidopsis acces-
sions Columbia-0, Rschew and Tenela during the ﬁrst 3 days
of sub-zero acclimation at −3 °C. The results indicate that
gene expression during sub-zero acclimation follows a tighly
controlled time-course. Especially AP2/EREBP and WRKY
TFs may be important regulators of sub-zero acclimation,
although the CBF signal transduction pathway seems to be
less important during sub-zero than during cold acclimation.
Globally, we estimate that approximately 5 % of all Arabi-
dopsis genes are regulated during sub-zero acclimation.
Mai Q. Le and Majken Pagter have contributed equally to this
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-014-0256-z) contains supplementary
material, which is available to authorized users.
M. Q. Le · M. Pagter · D. K. Hincha (*)
Max-Planck-Institut für Molekulare Pﬂanzenphysiologie, Am
Mühlenberg 1, 14476 Potsdam, Germany
M. Q. Le
Hanoi University of Sciences, 334 Nguyen Trai, Thanh Xuan,