Journal of Plant Physiology 244 (2020) 153049

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Journal of Plant Physiology

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Downregulation of three novel candidate genes is important for freezing T

tolerance of field and laboratory cold acclimated barley
Anna Fiust*, Marcin Rapacz
Department of Plant Physiology, University of Agriculture, Podłużna 3, 30-239, Krakow, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: Diversity arrays technology (DArT) marker sequences for barley were used for identifying new potential can-
Freezing tolerance didate genes for freezing tolerance (FT). We used quantitative trait loci (QTL) genetic linkage maps for FT and
Candidate gene photosynthetic acclimation to cold for six- and two-row barley populations, and a set of 20 DArT markers
Barley obtained using the association mapping of parameters for photosynthetic acclimation to low temperatures in
barley for the bioinformatics analyses. Several nucleotide and amino acid sequence, annotation databases and
associated algorithms were used to identify the similarities of six of the marker sequences to potential genes
involved in plant low temperature response. Gene ontology (GO) annotations based on similarities to database
sequences were assigned to these marker sequences, and indicated potential involvement in signal transduction
pathways in response to stress factors and epigenetic processes, as well as auxin transport mechanisms.
Furthermore, relative gene expressions for three of six of new identified genes (Hv.ATPase, Hv.DDM1, and
Hv.BIG) were assessed within four barley genotypes of different FT. A physiological assessment of FT was
conducted based on plant survival rates in two field-laboratory and one laboratory experiments. The results
suggested that plant survival rate after freezing but not the degree of freezing-induced leaf damage between the
tested accessions can be correlated with the degree of low-temperature downregulation of the studied candidate
genes, which encoded proteins involved in the control of plant growth and development. Additionally, candidate
genes for qRT-PCR suitable for the analysis of cold acclimation response in barley were suggested after vali-
dation.

1. Introduction temperature during late autumn, called cold acclimation or hardening,

Freezing tolerance (FT), the major component of winter hardiness,
is a key trait with economic and agronomic importance that strongly
limits the geographic distribution of barley cultivation. FT can be de-
fined as the ability of a plant to survive freezing temperatures, prevent
damage to vegetative tissues, and minimize other negative effects of
freezing temperatures on future yield potential (Galiba et al., 2009).
Several component traits, i.e., cold acclimation processes, including the
hardening and intrinsic capacity of vegetative tissues to survive freeze-
induced desiccation, and to recover efficiently from accompanying
stress, determine the final FT of plants (Visioni et al., 2013). Plant
adaptive response triggered mostly by a gradual decrease in
leads to a series of biochemical changes that enhance the cold resistance
of sensitive tissues (Pecchioni et al., 2012). Cold acclimation is crucial
for induction of genetic mechanisms responsible for FT (Sandve et al.,
2011; van der Schoot and Rinne, 2011). From a genetic point of view,
FT is a polygenic trait affected by several genes with different chro-
mosome locations (Vágújfalvi et al., 2012).
Knowledge of the variable nature of winter injuries and plant re-
sponse mechanisms to freezing is extensive. Various documented en-
vironmental factors, e.g., weather pattern fluctuations (Rapacz et al.,
2014), as well as distinctions between the effects of low temperature
tolerance genes and the pleiotropic effects of developmental genes, may
strongly affect plant reactions. Moreover, freezing tolerance changes

Abbreviations: ADP, ADP ribosylation factor 1-like protein; CBF, C-repeat binding factor; CDD, Conserved Domain Database; COR, cold-regulated genes; DarT,
diversity arrays technology; DREB, dehydration responsive element binding; EL, electrolyte leakage; FLM, a field-laboratory method; FR, frost resistance locus; FT,
freezing tolerance; GO, gene ontology; HSP90, cytosolic heat shock protein 90; L- TUB, a-Tubulin; LEA, late embryogenesis abundant; MAS, marker-assisted selection;
PSII, photosystem II; qPCR, quantitative polymerase chain reaction; QTL, quantitative trait locus; RLK, receptor-like kinase; sAMD, homologue of S-adeno-
sylmethionine decarboxylase
⁎
Corresponding author.
E-mail addresses: bednarczyk.an@gmail.com (A. Fiust), rrrapacz@cyf-kr.edu.pl (M. Rapacz).

https://doi.org/10.1016/j.jplph.2019.153049
Received 7 March 2019; Received in revised form 25 July 2019; Accepted 30 July 2019
Available online 01 October 2019
0176-1617/ © 2019 Elsevier GmbH. All rights reserved.
A. Fiust and M. Rapacz
seasonally due to additional stimuli beyond low temperatures. In the
grass family, the main factor influencing an FT increase during the
hardening process is low temperature, but photoperiod and the redox
state of photosystem II (PSII) are also important (Rapacz et al., 2014;
Sandve et al., 2011)
However, currently no plant receptors responsible for receiving a
signal for low temperature in the hardening process have been identi-
fied. The standard model assumes that the induction of cold-regulated
(COR) genes is a consequence of changes in the physical properties of
membranes during cold stress (Miura and Furumoto, 2013). Moreover,
Ca2+ accumulation in plant tissues is connected with FT (Dinari et al.,
2013). Plant exposure to low temperatures in the hardening process
leads to a reprogramming of gene expression and cell metabolism, as
well as a remodeling of cell structures (Viswanathan and Zhu, 2002).
Vernalization genes are reported to be the primary group of genes
involved in cereal FT (Kosová et al., 2008). Genetic studies in barley
and wheat conclude that differences in the expression of the VRN1 and
VRN3 genes (and in coding regions of VRN2) affect the delay of flow-
ering until the end of the winter, and protect from freezing sensitive
floral primordia. A second group of genes identified to affect FT are
transcription C-repeat binding factors (CBFs) localized in the frost re-
sistance-2 (FR2) locus. CBFs are COR regulators, and are induced by
low temperatures to confer tolerance to subsequent freezing tempera-
tures (acclimation) (Galiba et al., 2009; Kosová et al., 2008; Knight and
Knight, 2012).
Other crucial FT pathways are regulated by transcription factors
associated with dehydration stress, and affect for example the expres-
sion of barley Hva1 gene, which encodes a member of the third group of
late embryogenesis abundant (LEA) proteins (Thomashow, 1998),
wheat WCOR410 or barley DHN5, which encode another proteins of the
dehydrin family (Miura and Furumoto, 2013). A key protein involved in
FT in barley is the dehydration responsive element binding-1 (DREB1)
CBF. DREB1 is a member of the A-2 subgroup of the DREB subfamily,
which belongs to the APETALA2 transcription factor family, and is
apparently involved in abiotic stress signaling pathways (Akhtar et al.,
2012).
Moreover, the involvement of epigenetic mechanisms, a modifica-
tion of DNA activity by methylation, histone modification or chromatin
remodeling without alteration of the nucleotide sequence, involved in
the response to environmental cues and abiotic stresses (i.e. cold) has
been documented (Luo et al., 2012; Jung and Park, 2013; Stockinger
et al., 2001; Vlachonasios et al., 2003; Pavangadkar et al., 2010; Zhu
et al., 2008). Gene expression and DNA recombination are strongly
affected by structural chromatin traits which is shaped by the activity of
enzymes (Visioni et al., 2013; Jerzmanowski, 2007). Chromatin un-
folding involves i.e. the action of ATP-dependent remodeling complexes
(Visioni et al., 2013; Pecchioni et al., 2012), covalent modifications and
deposition of histone proteins (Kouzarides, 2007; Pfluger and Wagner,
2007; Zhang et al., 2007; Draker and Cheung, 2009).
The knowledge regarding roles of auxins in cold stress responses of
plants is limited. Nevertheless, some reports suggest that cold stress
induced change in the plant growth and development is tightly linked
to the intracellular auxin gradient, which is regulated by the polar
deployment and intracellular trafficking of auxin carriers (Hannah
et al., 2005; Khurana and Jain, 2009). Moreover, it has been shown that
a significant number of auxin-regulated genes are additionally affected
by cold in Arabidopsis and rice (Hannah et al., 2005; Khurana and Jain,
2009) and that application of auxin analogues on Brassica napus during
cold hardening stimulated accumulation of freeze-protective metabo-
lites and soluble sugars (Gavelienė et al., 2013).
Chlorophyll fluorescence-based techniques are often used to study
the genetic control of FT and winter hardiness. Molecular markers as-
sociated with barley photosynthetic acclimation to cold and FT were
described (Rapacz et al., 2010; Tyrka et al., 2015), and the primary QTL
indicators of barley FT were identified on chromosome 5H (Francia
et al., 2004) and 2H (Tyrka et al., 2015; Pasquariello et al., 2014).
2
Journal of Plant Physiology 244 (2020) 153049
We hypothesized that further candidate genes for freezing tolerance
may be identified in barley with bioinformatics tools on the base of
DArT marker sequences. The DArT marker system has been extensively
used in various genetic applications, including genetic diversity ana-
lyses, the construction of genetic linkage maps, and quantitative trait
locus (QTL) linkage analyses, in several crops (Jaccoud, 2001; Gupta
et al., 2008), including barley (Wenzl et al., 2006).
For this purpose, sequences from a set of DArT markers dedicated
for barley FT were analyzed against a set of databases and associated
algorithms (including NCBI BLAST (Altschul et al., 1990), GenBank
(Benson et al., 2013) and IPK barley BLAST server of International
Barley Sequencing Consortium (IBSC, http://webblast.ipk-gatersleben.
de/barley/)). Thorough characterization of the proteins identified in
our study was conducted using several databases (UniProtKB/Swiss-
Prot (Magrane and Consortium, 2011) and InterPro (Hunter et al.,
2008) to gain insight into structure and potential function.
Many recent reports of plant response to freezing temperatures
focus on a simple analysis of changes in the expression of known reg-
ulatory genes under stress conditions (Akhtar et al. 2013; Agarwal
et al., 2006). Verification of changes in gene expression of three po-
tential candidate genes for barley FT identified in bioinformatics ana-
lysis in the study, was assessed using quantitative polymerase chain
reaction (qPCR) analyses within a set of four barley genotypes with the
different freezing tolerance level. The differences in FT of tested lines
were assessed within physiological experiments using realistic (field or
semi-field) conditions of cold acclimation.
2. Materials and methods
2.1. Plant material
Plant material consisted of four unrelated advanced breeding lines
with different FTs: POA_7333/06-1 and 51-1 (tolerant), G2056 and
POA_7183/06-6 (susceptible), derived by DANKO Plant Breeding
company (Choryń, Poland). The accessions were preliminary tested in
two years winter hardiness observations in two locations and freezing
tolerance assessment also performed during two years by the field-la-
boratory method (FLM) as described previously by Rapacz et al. (2015)
for wheat and triticale.
2.2. Molecular markers and bioinformatics analysis
Two QTL genetic linkage maps for FT and photosynthetic acclima-
tion to low temperature for two (six- and two-rowed) barley popula-
tions (Tyrka et al., 2015), and the set of 20 DArT markers obtained by
Cwynar (2011, unpublished) in the association mapping of parameters
for photosynthetic acclimation to low temperatures in barley (Table
A.1) were used for our bioinformatics analyses.
Marker set was selected on the base of association mapping analysis
of 94 unrelated winter barley accessions with different freezing toler-
ance rate. The physiological characterization of tested lines included
freezing tolerance tests assessed on the base of field-laboratory method
(FLM) and fluorescence measurements conducted as described above
for the selection of extreme accessions. The structure of tested popu-
lation, the association of DArT markers and the physiological para-
meters for photosynthetic acclimation to low temperatures as well as
barley FT was evaluated using dedicated statistical algorithms such as
Structure and Tassel (Pritchard et al., 2000; Bradbury et al., 2007). The
set of 20 DArT markers with the highest association with physiological
parameters (Table A.1) were selected for bioinformatics analysis.
Initially, those DArT markers homologous to noncoding sequences
were rejected (BLAST (Altschul et al., 1990), GeneBank (Benson et al.,
2013)). The remaining sequences were subjected to successive bioin-
formatics analyses.
The general characterization of the marker sequences and simila-
rities to potential FT genes was performed with BLAST (BLASTn and
A. Fiust and M. Rapacz
tBLASTx) [http://blast.ncbi.nlm.nih.gov; Altschul et al., 1990] against
GenBank sequences [https://www.ncbi.nlm.nih.gov/genbank; Benson
et al., 2013]. We physically located the markers on the barley genome
sequence using the IPK barley BLAST server [http://webblast.ipk-
gatersleben.de] against three barley variety genomes: Morex,
Bowman and Barke. The GeneID program (Blanco et al., 2002) was used
to predict potential gene boundaries and exon/intron borders. Subse-
quently, those marker sequences with the highest nucleotide identities
(more than 70%, BLAST) to documented FT genes in GenBank were
searched against the Swiss-Prot (SIB, Swiss Institute of Bioinformatics),
TrEMBL (EBI, European Bioinformatics Institute)and PIR (Protein In-
formation Resource) databases using BLASTx (Altschul et al., 1990).
Analyzed sequenced were searched against NCBI Conserved Domain
Database (CDD) (Marchler-Bauer et al., 2015). The predicted Gene
Ontology (GO) annotations of identified candidate genes were assigned
based on Blast2GO software (Götz et al., 2008). Nucleotide sequence
alignments of 1) Hv.ATPase, 2) Hv.BIG, 3) Hv.DDM1 gene and sequences
homologous for other species (Fig. A.5.1-3) were done using ClustalW
Multiple alignment tool with BioEdit software (http://www.mbio.ncsu.
edu/bioedit/bioedit.html). Nucleotide sequences of candidate genes for
other species were obtained from NCBI (www.ncbi.nlm.nih.gov).
2.3. Freezing tolerance assessment: field-laboratory method and electrolyte
leakage analysis
The FT of four barley genotypes dedicated for gene expression
analysis was evaluated with a field-laboratory method (FLM) (Koch and
Lehman, 1969) in three different experiments (field condition of two
winter seasons: 2014/15 – FT_1 and 2015/16 – FT_ 2 and laboratory
experiment in controlled conditions – FT_ 3). We used an FLM based on
the protocol described by Rapacz et al. (2008). Seeds of tested barley
genotypes were sown in boxes in the last week of September in five
replicates (rows in different boxes, 12 seeds of each replicate) for each
genotype. Relatively high temperatures during cold hardening in the
autumn of 2014/15 were followed by a significant decrease in tem-
perature (−11.2 °C) at the turn of December/January (Fig. A.2). Winter
2015/16 weather conditions were more beneficial for plant survival
because of a lower variance in the average daily temperatures pre-
ceding freezing events (−13.7 °C) in the field experiment (Fig. A.2).
After 2 weeks of hardening in the field (when the maximum daily
temperature did not exceed 4 °C) plants were transferred to a freezing
chamber, and subjected to the freeze-thaw cycles. Minimum freezing
temperature was −12 °C and the freezing protocol was detailed else-
where (Rapacz et al., 2008).
Plants were grown in a greenhouse in the laboratory-controlled
experiment (day/night: temperature 16/25 °C; length – 8 h/16 h). After
4 weeks, the plants were transferred for 3 weeks to hardening chambers
with controlled hardening conditions (day/night: temperature 4/3 °C;
length – 8 h/16 h, light intensity 300 μmol (quanta) m−2 s-1. Control
samples for the laboratory experiments were plants assessed before
transfer to hardening chambers. Experimental sample plants were ex-
posed to freezing-thawing cycles as described for the field-laboratory
study. After the hardening period, plants in both the field-laboratory
and laboratory experiments were transferred to an unheated glasshouse
and left in a temperature of ∼10–15 °C for 3 weeks. FT was expressed
as the percentage of plant survival.
Samples for tEL50 analysis were collected before transfer to the
freezing chamber. Cell membrane integrity was assessed using elec-
trolyte leakage analysis (Flint et al., 1967) as previously described by
Rapacz et al. (2001). The percentage of electrolyte leakage was de-
termined according to Flint et al. (1967) based on the equation EL =
(EL1/EL2) × 100%. A five-point freezing temperature profile (−4 °C,
−6 °C, −8 °C, −10 °C, and −12 °C) was used for the control plants.
The tEL50 (temperature of EL = 50%) was calculated with a multiple
regression model in Statistica 13.1 (Dell, Round Rock, TX, USA).
3
Journal of Plant Physiology 244 (2020) 153049
2.4. Validation of reference genes and gene expression analysis
Samples for gene expression analysis from all three experiments
(FT_1, FT_2, and FT_3) (Fig. A.2) were collected from control plants and
the experimental plants after hardening, simultaneously with samples
for tEL50 analysis. Up to 90–100 mg of barley plant tissue, in three
biological replicates for each treatment and genotype, were frozen in
liquid nitrogen and used for RNA isolation. Total RNA was purified
from second leaf tissues using an RNeasy Plant Mini Kit (Qiagen, Venlo,
Netherlands). cDNA synthesis and genomic DNA elimination were
performed using a QuantiTect Reverse Transcription Kit (Qiagen,
Venlo, Netherlands). Qiagen kits were used as per manufacturer’s
suggested protocols. The final concentration and quality of cDNA were
determined spectrophotometrically (Nanodrop 2000c, Thermo
Scientific, Wilmington, DE, USA).
We used those predicted homology regions (BLASTn; Altschul et al.,
1990) of DArT and potential candidate gene to design primers for qPCR
analysis. The primers, both for the reference sequences (Table A.3-1)
and the target genes (Table A.3-2) were designed using Primer Express
Software v. 3.0.1 (Applied Biosystems by Life Technologies, Carlsbad,
CA, USA). qPCR runs were performed in 96-well plates in a reaction
volume of 25 μl with Power SYBR™ Green PCR Master Mix (Applied
Biosystems by Life Technologies, Carlsbad, CA, USA) (Table A.3). The
final concentration of qPCR ingredients (Table A.4-1) and temperature
profiles (Table A.4-2) are given in Table A.4. Each qPCR run was re-
peated three times for a final replication number of nine (three biolo-
gical samples × three technical instrumental replicates). The expres-
sion of target genes was calculated using Pfaffl’s mathematical model
for relative quantification (Pfaffl, 2001). Genes with stable expression
levels for qPCR were used as references and validated in a set of 16
randomly selected samples from the conducted experiments. The ex-
pression stability of the nine potential reference genes was evaluated on
the base of Ct (threshold value) using qBasePLUS (Hellemans et al.,
2007) and BestKeeper (Pfaffl et al., 2004).
3. Results
3.1. Bioinformatics identification of candidate freezing tolerance genes
Six of our DArT barley FT marker sequences correlate with relevant
gene annotations (Table 1). These particular markers all reliably map to
specific contigs of the barley genome (Table 1).
We identified a putative protein kinase (UniProt accession number:
N1R5V0) in the bPb-3722 and bPb-7975 marker sequences (Table 1).
GO annotations associated with bPb-3722 corroborate kinase activity
for the encoded protein, and localize it as an integral component of the
plant cell wall (Table 1). Additionally, the localization of the bPb-3722
marker sequence in bin 6H_06.2 on chromosome 6H on the integrated
barley linkage map described by Marcel (Hv-Integrated 2009; Grain-
Genes) indicates that the position of the sequence co-localizes with
several QTLs for nonhost barley resistance to heterologous rust fungi:
Vada(2)_6.3, Vada(2)_3.2, Vada(2)_5.7, Rec(4)_4.2, and Vada(2)_11.2
(Jafary et al., 2008). Presumably homologous CBL-interacting protein
kinase activity was also identified in the bPb-7975 sequence (Table 1).
The highest similarity of this marker is to a Triticum protein (26% of
query cover with 98% of sequences identity, UniProt accession number:
A0A287H6Z1) with predicted protein phosphorylation and signal
transduction biological functions (GO:0006468, GO:0007165, Table 1).
Moreover, the bPb-7975 marker sequence location maps to bin 05.1_k5
of chromosome 2H, which co-localizes with the Rphq18 locus for barley
rust resistance (Jafary et al., 2008), and the Rbgq7 locus for barley re-
sistance to powdery mildew (Aghnoum et al., 2009).
We identified a presumed homology of the bPb-3642 marker se-
quence to a probable chromatin-remodeling complex ATPase chain.
Various grasses encode the highest level of regional similarity for this
function (Table 1). The identification of the cl26465 protein domains
 A. Fiust and M. Rapacz

Table 1
Gene ontology annotations for six DArT markers of frost tolerance in winter barley. * – marker position according to Tyrka et al. [18], H – chromosome position. MA – association mapping, QA – barley QTL genetic
linkage maps for freezing tolerance and photosynthetic acclimation to cold. The highest similarity [%] of the marker sequence is presented in brackets. GO – gene ontology of predicted protein assessed by Blast2GO
software, “-”– no prediction.
Marker H* Position Contig Source BLASTx/BLASTn Accession code Species (query cover [%]/ UniProt GO term prediction
(cM)* (NCBI) similarity [%])

bPb-7975 2H 47,4 CAJW010244014 MA CBL-interacting protein KJ561800.1 Triticum aestivum (26% / A0A287H6Z1 Biological Process: GO:0006468 protein
kinase 98%) Triticum urartu Non-specific serine/threonine phosphorylation, GO:0007165 signal transduction
protein kinase (Hordeum vulgare Molecular Function: GO:0004672 protein kinase
subsp. Vulgare) 98,2% activity, GO:0005524 ATP binding
Cellular Component: -
bPb-5262 2H 11 CAJW010062577 QA Predicted auxin transport XM_020321677.1 Aegilops tauschii subsp. A0A287R7A1 Uncharacterized Biological Process: -
protein [BIG] Tauschii (100% / 97%) protein (Hordeum vulgare subsp. Molecular Function: GO:0005488 binding,
Vulgare) 100% GO:0008270 zinc ion binding
Cellular Component: -
bPb-3642 3H 35,9 CAJW010135263 MA Probable chromatin- XM_020298382.1 Aegilops tauschii subsp. A0A287K4P7 Chromatin- Biological Process: GO:0006338 chromatin
remodeling complex ATPase Tauschii (57% / 98%), remodeling complex ATPase remodeling, GO:0043044 ATP-dependent chromatin
chain Brachypodium distachyon (Hordeum vulgare subsp. Vulgare) remodeling

4
71,8% Molecular Function: GO:0003676 nucleic acid
binding, GO:0003677 DNA binding, GO:0005524
ATP binding, GO:0016818 hydrolase activity, acting
on acid anhydrides, in phosphorus-containing
anhydrides, GO:0016887 ATPase activity,
GO:0031491 nucleosome binding
Cellular Component: GO:0005634 nucleus,
GO:0016589 NURF complex
bPb-2778 3H 190 CAJW010041750 QA ATP-dependent DNA helicase XM_020301224.1 Aegilops tauschii subsp. (67% M8AIE8 ATP-dependent DNA Biological Process: -
[DDM1] / 86%) helicase DDM1 (Triticum urartu) Molecular Function: GO:0005524 ATP binding
84% Cellular Component: -
bPb-3241 5H 117,2 CAJW010008158 QA blt101 promotor sequence AB370200.1 Hordeum vulgare (61% / – Biological Process: -
88%) Molecular Function: -
Cellular Component: GO:0016021 integral
component of membrane
bPb-3722 6H 68,5 CAJW010276189 MA Predicted protein: G-type XM_010229404.3 Brachypodium distachyon N1R5V0 Putative receptor protein Biological Process: GO:0006468 protein
lectin S-receptor-like serine/ (52% / 71%), Aegilops kinase ZmPK1 (Aegilops tauschii) phosphorylation
threonine-protein kinase tauschii Oryza sativa 83,5% Molecular Function: GO:0004672 protein kinase
SD2-5 activity, GO:0005524 ATP binding
Cellular Component: GO:0005886 plasma
membrane
Journal of Plant Physiology 244 (2020) 153049
A. Fiust and M. Rapacz Journal of Plant Physiology 244 (2020) 153049

(NCBI Conserved Domain Database (CDD), https://www.ncbi.nlm.nih. Table 3

gov/Structure/cdd/cdd.shtml (Marchler-Bauer et al., 2015) in the bPb-
3642 marker sequence suggests potential activity in ATP-dependent
chromatin remodeling processes as well as helicase activity (Table 1).
The bPb-2778 marker maps to chromosome 3H on the genetic
linkage map of two-row winter barley, co-localized with the
QFT.P44.3H.2 and QFTF.P44.3H.2 loci for FT (Tyrka et al., 2015). bPb-
2778 is most similar to DDM1 gene sequences (Table 1). DDM1 encodes
an ATP-dependent DNA helicase with chromatin remodeling activity. A
conserved domain characteristic of helicase-related ATPases (SNF2)
family (NCBI CDD: cl26465) was identified within the marker sequence
translation. GO annotation association corroborates regulation of DNA
methylation and chromatin condensation activity (Table 1).
The bPb-5262 marker localizes within the QFT.P44.2H locus for
barley cold acclimation potential (Tyrka et al., 2015). It encodes a
potential BIG-like predicted auxin transport protein (Table 1). General
functions of the encoded protein associated to GO annotation are polar
auxin transport, and activation of auxin-induced signaling pathways
(GO:0009734, GO:0009926).
The Pb-3241 marker sequence is most similar to a barley promoter
sequence encoded by the blt101 gene associated with low-temperature
induction (Table 1). The physical localization of the marker in bin
5H_10.5 co-localizes it with Rbgq15 (Aghnoum et al., 2009).
The three DArT marker sequences (Hv.ATPase, Hv.BIG, Hv.DDM1)
with the highest query cover rate and similarities (Table 1; Fig. 2) to
potential FT genes were used for further molecular analyses.
3.2. Freezing tolerance
Effects of autumn weather conditions on barley FT within 2014/15
(FT_1) and 2015/16 (FT_2) were observed (Table 2). FT_2 plants were
characterized by higher survival rates, very similar to those observed
after controlled cold acclimation (FT_3). The barley accession FT
rankings were however the same. The most freezing tolerant was
POA_7333/06-1, and G2056 was characterized with the lowest survival
rate. Different results were obtained in the tEL50 assessments. In FT_1
the results of the test were unreliable, probably due to frost damage of
the leaves in the field prior to sampling causing very low repeatability
of the single EL values (Table 2). In FT_2 the plants were characterized
with lower tEL50 values than in FT_3, but the FT ranking was similar
with the highest leaf freezing damage rate in the case of POA_7333/06-
1 (the best survival rate), and a very similar FT in the remainder of the
studied accessions (Table 2).
3.3. Candidate gene expression
Three reference genes (ADP, SV = 0.694, sAMD, SV = 0.921 and L-
TUB, SV = 0.968) were selected by BestKeeper as the most stably ex-
pressed under our experimental conditions (Table 3). Whereas, qBa-
sePLUS analysis indicates ADP (M = 0.698) and HSP90 (M = 0.731) to
be the most appropriate references for gene expression analysis. A re-
latively low level of gene expression variation was also observed for
Ranking of candidate reference genes and expression stability values calculated
by BestKeeper and qBasePLUS for 22 samples of barley genotypes under different
experimental conditions. M– qBasePLUS measure of gene expression stability, SV
– standard deviation values of BestKeeper.
gene Best Keeper [SV] qBase PLUS (M)
ADP 0.694 0.698
SAMD 0.921 0.779
L-TUB 0.968 1.033
HSP70 1.035 1.117
GAPDH 1.155 0.857
ACT 1.175 0.852
UBI 1.29 1.087
EF1 1.477 0.894
HSP90 1.483 0.731
sAMD (M = 0.779) (Table 3). The geometric means of the expression of
the two proposed reference sequences, the ADP and sAMD genes, were
then used as a proper reference for further genetic analyses of potential
barley FT genes in our study.
Differences were observed in the expression of the three target gene
candidates for barley winter hardiness genes within the set of four
tested barley accessions (Fig. 1). All three candidate genes, Hv.ATPase,
Hv.DDM1, and Hv.BIG, are involved in the regulation of growth and
development, and are, in general, more downregulated, or less upre-
gulated during cold acclimation of tolerant plants. Hardening condi-
tions result in significant changes in the gene expression of Hv.ATPase
(Fig. 1). The largest decrease in the relative number of mRNA Hv.AT-
Pase transcripts was predominantly observed with the POA_7333/06-1
and 51-1 accessions (Fig. 1a), that is, those with higher FT rates
(Table 2). A downregulation of cold-induced Hv.DDM1 and Hv.BIG
genes was also observed (Fig. 1b, c). A decrease in the expression of
Hv.DDM1 and Hv.BIG occurred more often in tolerant than in suscep-
tible barley accessions (Fig. 1b, c). The laboratory conditions of cold
hardening proved to be crucial to assess changes in the gene expression
of Hv.ATPase, Hv.DDM1, and Hv.BIG in plants affected by cold tem-
peratures in comparison to non-stressed barley genotypes.
4. Discussion
In this study we aimed to confirm the hypothesis that new genes
candidates for FT genes in barley may be identified with bioinformatics
tools on the base of DArT marker sequences associated with barley
freezing tolerance indices. Molecular markers associated with genes
and QTLs affecting important traits are frequently used as indirect se-
lection criteria for improving breeding efficiency via marker-assisted
selection (MAS) (Ashraf and Foolad, 2013). However, the identification
of such associations based on the linkage of molecular markers and
unique genomic regions can be complicated by recombination events
(Ott et al., 2015). DArT is commonly used in genotype polymorphism
studies by providing access to multiple polymorphic loci spread over a
genome without any previous sequence information (Jaccoud, 2001;

Table 2
Freezing tolerance of barley accessions measured in three independent field-laboratory experiments as survival rate after freezing at −12 °C ± SE (standard error)
and temperature after freezing at which 50% of the electrolytes leak out of the leaves: tEL50 [°C] ± confidence intervals for p = 0.05.
Accession Experiment

FT_1 FT_2 FT_3

survival rate [%] tEL50 [°C] survival rate [%] tEL50 [°C] survival rate [%] tEL50 [°C]

POA_7333/06-1 41.42 ± 5.08 −4.64 ± 0.21 80.00 ± 12.25 −11.39 ± 0.68 78.09 ± 13.93 −9.78 ± 0.54
51-1 28.33 ± 4.25 −4.50 ± 0.18 54.09 ± 8.24 −12.92 ± 0.43 62.5 ± 6.71 −10.67 ± 0.29
G2056 8.97 ± 0.31 −7.25 ± 1.11 8.48 ± 4.12 −13.41 ± 0.51 8.72 ± 2.23 −11.51 ± 0.36
POA_7183/06-6 16.48 ± 1.99 −7.74 ± 1.02 36.93 ± 6.04 −12.39 ± 0.35 43.61 ± 5.51 −11.14 ± 0.38

5
A. Fiust and M. Rapacz
Fig. 1. Relative gene expression of Hv.ATPase (a), Hv.DDM1 (b), and Hv.BIG (c)
genes in four barley genotypes in three independent experiments. FT_1, FT_2 –
gene expression measured before the first freezing event respectively during
2014/15 and 2015/16 winter season, FT_3 – gene expression measured after
cold acclimation in controlled conditions. The value 1 indicates the gene ex-
pression in control plants. Normalization with two reference genes: ADP and
sAMD.
Wenzl et al., 2004). In the present study, information derived by
bioinformatics analyses of DArT marker sequences was used to describe
new suggested FT candidate genes in barley.
Bioinformatics analyses indicate potential homologies of six barley
DArT marker sequences to several potential FT genes described in other
6
Journal of Plant Physiology 244 (2020) 153049
plants. bPb-7975, localized on chromosome 2H, is 94%–98.2% similar
to wheat CBL- interacting and Ca2+-dependent protein kinases. GO
annotation associated with the sequences include involvement in signal
transduction pathways activated by osmotic, drought, or low tem-
perature stresses, and stress related gene expression regulation (Cheong
et al., 2003; Albrecht et al., 2003). The encoded protein, CBL1, is a cold
activated negative regulator of signal transduction in Arabidopsis
thaliana (Cheong et al., 2003; Albrecht et al., 2003).
Another similarity to a stress response element was observed for
bPb-3722. A sequence similar (72%) to bPb-3722 encodes a serine/
threonine kinase receptor (G-type) in Brachypodium distachyon. Despite
relatively little knowledge of receptor-like kinase (RLK) roles, studies in
rice and Arabidopsis confirm the variable expression of these genes
under stress by salinity, drought, and temperature change, and after
injury (Vaid et al., 2012). These studies suggest a potential role of the
RLK transmembrane protein family responsible for stimuli perception in
plant response to low temperatures (Vaid et al., 2012).
Two different markers, bPb-3642 (Hv.ATPase) and bPb-2778, both
localized on chromosome 3H, are quite similar to genes involved in
epigenetic regulation. Such modifications of DNA activity occur
through post-translational mechanisms, methylation, acetylation,
phosphorylation, and/or ubiquitination, and result in changes in the
amount and/or activity of histone proteins and chromatin remodeling
without any changes of nucleotide sequence (Luo et al., 2012). Epige-
netic modification plays an important role in plant response to abiotic
stress. The effects of DNA methylation on the transcription of stress-
resistance genes have been investigated by the hypomethylation of the
NtGPDL coding sequence induced by cold and salt stress in Nicotiana
tabacum (Wada et al., 2004). The best-known mechanism of chromatin
change is histone modification. Owing to the reversible character of the
processes conducted by acetyltransferase (HAT) and deacetylase
(HDAC), histone modification is thought to be a flexible way to regulate
the activity of genes in response to environmental stress. The effect of
HAT and HDAC in the regulation of FT gene activity is described in
many Arabidopsis thaliana studies (Vlachonasios et al., 2003;
Pavangadkar et al., 2010; Zhu et al., 2008) Moreover, other proteins,
GCN5 and ADA2b, not involved in the cold-activated histone acetyla-
tion of promoter regions of COR genes, are involved in nucleosome
modifications at low temperatures (Vlachonasios et al., 2003;
Pavangadkar et al., 2010).
The bPb-2778 marker sequence is similar (84%–90%) to decreased
DNA methylation-1 (DDM1) genes; associated GO annotations indicate
it encodes a protein belonging to a small and well-conserved family
involved in DNA methylation in plants. The relation of DDM1 activity to
cytosine methylation, which decreases within repetitive DNA regions,
as well as the potential effect of DDM1 on chromatin remodeling was
investigated in Arabidopsis thaliana (Jeddeloh et al., 1999). The key role
of the BIG protein, most similar (91%–100%) to the bPb-5262 marker
sequence translation, is attendance in the polar transport of auxins (Gil
et al., 2001). It is involved in response pathways for the concentration
of gibberellins and phosphates (Desgagné-Penix et al., 2005; López-
Bucio et al., 2005). Auxins are tremendously important to plant growth
and development, and are a fundamental link in adaptation to en-
vironmental changes. Intracellular auxin distribution under optimal
growth conditions affects the adaptability of plants in embryo forma-
tion, organogenesis, and meristematic induction, as well as tropism
control (Vieten et al., 2007). Despite the important function of auxins in
the regulation of many key aspects of plant growth and development,
the role of auxins in response to low temperature stress is not yet un-
derstood. Nevertheless, studies by Shibasaki et al. (2009), confirmed in
Arabidopsis the low-temperature induced inhibition of intracellular
transport by a number of proteins, specifically auxin transporters, and
as a consequence the disruption of auxin transport.
DArT sequence analysis effectively identifies functional annotation
associations. The potential structural and functional information ob-
tained can contribute meaning to unknown genomic regions of barley.
A. Fiust and M. Rapacz
However, the predictive methods of bioinformatics, and interspecies
variability, may result in the generation of false positive results, so in
vivo experimental verification is suggested. In our study, verification of
the expression of three of six identified DArT sequences homologous to
potential FT genes (Hv.ATPase, Hv.DDM1, and Hv.BIG) was conducted
in plant material with different FT assessed within field-laboratory
experiments. The gene expression analysis were compared with phy-
siological assessments results of tested accessions.
In our study, the estimation of plant survival rates after freezing
gave somewhat different results than measurements of leaf tissue
freezing damage as assayed by an electrolyte leakage test. Extremely
different results were obtained for POA_7333/06-1, which had the best
survival rates in all the experiments, and was characterized with a
slightly higher electrolyte leakage rate after freezing than the rest of the
studied accessions. These results do not confirm the assumption of
potential relationships between these indicators of barley FT shown
before (Rapacz, 2002, 2011; Ahmad et al., 2010). It may be that in the
barley accessions studied in our experiment, all characterized by quite
similar leaf FT levels, factors other than leaf damages determine the
ability to regenerate after freezing. In our case, during the cold accli-
mation of the better surviving plants, we observed a lower expression of
three genes (Hv.ATPase, Hv.BIG, and Hv.DDM1), which we assume are
involved in the activation of plant growth.
Although reference genes for qPCR studies should be validated for
exact experimental design (Kozera and Rapacz, 2013; Rapacz et al.,
2012), genes validated under similar experiment or in the set of dif-
ferent experiments are often used (Faccioli et al., 2007; Janská et al.,
2013). In the case of our study, where experimental conditions were
very variable in multiple field experiment, no single gene was char-
acterized with the expression stable enough and the use of two genes
(ADP and sAMD) was suggested. This indicates that the proper valida-
tion of reference gene expression stability is particularly important in
the case of field experiments.
According to many studies, the cessation of growth is a prerequisite
of cold acclimation efficiency, and particularly protects crowns and
young leaves from freeze damage (Østrem et al., 2018, 2015; Hüner
et al., 2012; Huner et al., 1993). Leaf and shoot regeneration in
monocotyledonous plants occurs from tillering nodes, and even full
damage to leaves under unfavorable environmental conditions does not
indicate a plant’s potential to restore the assimilation surface after
stress (Kosová et al., 2013). It has been suggested that a key aspect of
winter barley plant recovery after freezing is the ability of meristematic
tissue to survive, which depends directly on the level of mechanical
damage, as well as cold acclimation of tillering nodes (Tanino and
McKersie, 1985).
The differences in the Hv.ATPase and Hv.DDM1 gene expression
within frost-susceptible and -tolerant barley plants may suggest the
importance of the sequences we identified in the regulation of the other
genes involved in cold responsiveness of plants as well as growth ces-
sation during cold acclimation.
Both the Hv.ATPase and Hv.DDM1 genes are suggested to be in-
volved in the ATP-dependent chromatin remodeling process caused by
multi-protein complexes belonging to the SNF2 superfamily, all with a
highly conserved, central ATPase catalytic subunit (Flaus et al. 2006).
Epigenetic changes resulting from ATPase activities determine dis-
orders in hormonal signaling pathways (Han et al. 2012; Archacki et al.
2013), as well as modifications of transcriptional activity (activation/
repression) of a number of genes (Sudarsanam and Winston, 2000).
Although, the complex network of chromatin regulators such as
Hv.ATPase is still waiting to be fully elucidated. However, the central
role that SWI/SNF complexes have in regulation of plant growth and
development is clear. It has been confirmed that, the Arabidopsis SNF2
mutants showed the deficiencies in growth and development (Wagner
and Meyerowitz, 2002; Hurtado et al., 2006; Gentry and Hennig, 2014).
Observed alterations in the gene expression pattern between barley
genotypes with the different freezing tolerance may indicate a low-
7
Journal of Plant Physiology 244 (2020) 153049
temperature effect on chromatin structure resulting from the different
enzyme activity (Mlynárová et al., 2007). It may be suggested that the
occurred changes caused by remodeling complexes activate the cascade
of interaction on the basal transcriptional machinery and/or with gene-
specific DNA-binding factors and regulate the expression of i.e. devel-
opment genes (Vignali et al., 2000; Mlynárová et al., 2007).
The involvement of the DDM1 protein as a key factor connecting
chromatin remodeling processes with the introduction/maintenance of
appropriate DNA methylation levels has been confirmed in Arabidopsis.
Moreover, a high specificity of DDM1 for transposons was also reported
(Teixeira and Colot, 2009). A significant decrease in the expression
level of mRNA for the Hv.DDM1 coding sequence in our experiments
potentially resulting in the increase of the methylation level may in-
dicate a significant influence on a reduction in epigenetic control at low
temperatures. The similar differences between tolerant genotypes with
the higher DNA methylation level and susceptible genotypes char-
acterized by the evident demethylation was observed in Ribes germ-
plasm (Johnston et al., 2009). Results indicating the involvement of
DNA methylation in molecular regulation of the cold responsiveness of
plants were also conducted in rice (Hua et al., 2005; Chakrabarti et al.,
2011). The DNA methylation was additionally shown to play an im-
portant role in the vernalization process promoting early flowering in
Arabidopsis (Finnegan et al., 1998) or inactivation of transposable ele-
ments (Miura et al., 2001; Singer et al., 2001).
Different expression profiles within sensitive and tolerant barley
plants observed for the Hv.BIG gene may indicate the decreased effi-
ciency of auxin transport to the leaves during cold acclimation in the
case of genotypes characterized by a better recovery rate after freezing.
It may be suggested that observed decrease of auxin transport by auxin
carrier protein in leaves may enhance the gradient auxins accumulation
in the crowns and roots (Korver et al., 2018; Zažímalová et al., 2010)
and finally improve the plant regeneration ability after freezing. The
impact of the higher endogenous auxin concentration in roots on the
enhancement of drought tolerance was observed in Arabidopsis (Im Kim
et al., 2013; Shi et al., 2014). The studies conducted on Arabidopsis
confirmed that endogenous auxins might participate in the positive
regulation of drought tolerance, through regulation of root archi-
tecture, ROS metabolism, ABA-responsive genes expression, and me-
tabolic homeostasis (Shi et al., 2014). All of these aspects of auxin ac-
tion may be important during freezing recovery as it was shown that the
lack of root development limited the ability of plants to survive freezing
without effect on electrolyte leakage in leaves (Chen et al., 1983).
The importance of auxin transport in the adaptation to progressive
environmental changes has previously been suggested (Vieten et al.,
2007). Moreover, it has been confirmed that, the combination of both
local and polar auxin transports control many processes, such as flower
and vascular development, lateral root growth, embryogenesis, coty-
ledon development, root initiation development in plants (Chandler,
2009). Moreover, the complex hormonal network integrating auxins
and reactive oxygen species (ROS) regulates the steady-state balance of
plants and control the diverse aspects of plant growth and development
such as cell cycle (Fehér et al., 2008), cell wall plasticity (Teale et al.,
2006) as well as abiotic stress adaptation in general (De Tullio et al.,
2010; Tognetti et al., 2010).
Differences in the FT of barley plants evaluated by three in-
dependent field-laboratory and laboratory tests confirmed the effect of
weather conditions before winter on the ability of plants to survive. Our
experimental results corroborate that the effectiveness of cold accli-
mation is modified in the field by many complex factors, including
temperature, photoperiod, irradiance, PSII redox state, soil-water po-
tential, and nutrient status (Rapacz et al., 2014). Environmental effects
were visible in the differences in the expression levels of our candidate
genes. Differences in the expression of Hv.ATPase, Hv.BIG, and
Hv.DDM1 novel gene candidates for FT in barley were confirmed within
a set of four tested genotypes. The variation in gene expression profiles
for the tested genes in field experiments (FT_1 and FT_2) suggests the
A. Fiust and M. Rapacz
influence of complex environmental factors in the molecular back-
ground of cold acclimation in barley.
Our results demonstrate the possibility of the effective use of DArT
markers for the identification of FT gene candidates in barley.
Bioinformatics analysis of DArT marker sequences can be exploited in
the identification of hypothetical and functional connections with po-
tential coding sequences and corresponding proteins involved in stress
response pathways. We suggest, for the first time, that the down-
regulation of some genes (Hv.ATPase, Hv.DDM1, and Hv.BIG) during
cold acclimation may contribute to the observed differences in FT be-
tween barley accessions. Our choice of plant materials was based on
long-term observations of field winter survival differences, as well as
the gene expression profiles measured in the field. Thus, these results
may be applicable also to winter survival rates and usefull for breeding
purposes.
Authorship of the paper
Corresponding author Anna Fiust have made a significant con-
tribution to the conception, design, execution of studies presented in
this paper as well as writing original draft of this article. AF was project
leader with a significant contribution in the acquisition of the financial
support for the project leading to this publication.
Marcin Rapacz have made a significant contribution to the con-
ception, design and supervising all aspects of conducted studies. MR
was a project supervisor and contributed in writing of the final version
and approved the manuscript.
Declaration of Competing Interest
On behalf of all authors, the corresponding author states that there
is no conflict of interest.
Acknowledgement
This work was supported by the National Science Centre, Poland
[UMO-2013/09/N/NZ9/01588].
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jplph.2019.153049.
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