Journal of Plant Physiology 229 (2018) 122–131

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Journal of Plant Physiology

journal homepage: www.elsevier.com/locate/jplph

Gene expression in two contrasting hybrid clones of Eucalyptus camaldulensis T

x Eucalyptus urophylla grown under water deficit conditions
⁎
Guilherme Silva Martins , Natália Chagas Freitas, Wesley Pires Flausino Máximo,
Luciano Vilela Paiva
Laboratório Central de Biologia Molecular, Departamento de Química, Universidade Federal de Lavras, 37200-000, Lavras, MG, Brazil

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

Keywords: The physiological and molecular responses to water stress are mediated by a range of mechanisms, many of
Eucalyptus which involve abscisic acid (ABA)-dependent signaling pathways. In addition, plants contain drought response
Gene expression genes that can be induced by ABA-independent routes, mediated by secondary messengers such as Ca2+, or
Abiotic stress regulated by epigenetic modifications. The complex processes involved in the response to water stress can be
RT-qPCR
investigated using molecular techniques to evaluate the expression patterns of genes of interest and to infer the
behavior of different genotypes and species. In the present study, we first analyzed the stability of a set of
reference genes for normalization of the gene expression with real-time quantitative polymerase chain reaction
(RT-qPCR), since there were no results related to the genotype used in this study. We verified that although there
were some variations between algorithms used, the three most stable reference genes were SAND, PP2A-3 and
EF-1α. The expressions of genes encoding for proteins associated with drought-tolerance responses, namely 9-cis-
epoxycarotenoid dioxygenase 3 (EgrNCED3), pyrabactin resistance 1 (EgrPYR1), dehydration-responsive ele-
ment-binding 2.5 (EgrDREB2.5) transcription factors, calcium-dependent protein kinase 26 (EgrCDPK26), me-
thyl transferase 1 (EgrMET1) and deficient in DNA methylation 1 (EgrDDM1) protein, were determined by RT-
qPCR in leaf samples from drought sensitive (VM05) and drought tolerant (VM01) clones of the hybrid
Eucalyptus camaldulensis x Eucalyptus urophylla grown under water stress and irrigation conditions. When the two
clones were maintained under conditions of water deficiency, VM01 exhibited higher expression levels of
EgrNCED3 and EgrPYR1 genes than VM05 at all sampling times, implying that ABA biosynthesis and subsequent
induction of the ABA-dependent cascade mediated by the PYR1-ABA receptor complex were enhanced in the
tolerant clone. Under water-stress conditions, this clone also presented increased expression of the EgrDREB2.5
gene, representative of an ABA-independent cascade, and of the EgrCPK26 gene, related to stomatal opening and
closure. On the other hand, the expression levels of EgrMET1 and EgrDDM1 genes in the sensitive clone were
higher than in the tolerant clone under all conditions, showing a putative impact of epigenetic modifications on
tolerance to water deficiency. The results obtained indicate that the superior ability of the VM01-tolerant clone
to perceive water deficiency and activate drought-resistance genes is associated with the high expression levels
of EgrNCED3, EgrPYR1 and EgrDREB2.5 under water-stress conditions. These findings will facilitate future re-
search on the functional characterization of stress-related response genes, the identification of molecular mar-
kers, the evaluation of drought tolerance and genetic transformation in tree species.

Abbreviations: ABA, abscisic acid; BLAST, Basic Local Alignment Search Tool; CDPK, calcium-dependent protein kinase; CTAB, cetyltrimethylammonium bromide;
DDM1, deficient in DNA methylation 1; DREB, dehydration-responsive element-binding; EF1α, elongation factor 1α; ERE, ethylene responsive element; IDH, iso-
citrate dehydrogenase; LEA, late embryogenesis abundant; MET1, methyl transferase 1; NCED3, 9-cis-epoxycarotenoiddioxygenase 3; PP2A, protein phosphatase 2A;
PP2C, protein phosphatase 2C; PYRI/PYL/RCAR, pyrabactin resistance 1/pyrabactin resistance 1-like/regulatory components of ABA receptors; RT-qPCR, real-time
quantitative polymerase chain reaction; SAND, SAND domain-containing proteins; SLAC1, slow anion channel 1; SNF1, sucrose non-fermenting 1; SnRK2, SNF1-
related protein kinase 2; TUB, β-tubulin; UBQ, ubiquitin
⁎
Corresponding author.
E-mail addresses: guilhermesm91@hotmail.com (G.S. Martins), natalia_biotec@hotmail.com (N.C. Freitas), wesleypfm@hotmail.com (W.P.F. Máximo),
luciano@dqi.ufla.br (L.V. Paiva).

https://doi.org/10.1016/j.jplph.2018.07.007
Received 19 February 2018; Received in revised form 21 July 2018; Accepted 25 July 2018
Available online 26 July 2018
0176-1617/ © 2018 Elsevier GmbH. All rights reserved.
G.S. Martins et al.
1. Introduction
Water deficit is one of the conditions that most threatens production
in Eucalyptus plantations because it affects vital processes such as
photosynthesis, respiration, carbohydrate metabolism and ion absorp-
tion. In order to reduce or even avoid the impact of water deficit, plants
frequently undergo biochemical and morphological changes that enable
them to acclimatize to stressful conditions (Chaves et al., 2009).
Therefore, understand the complex mechanisms underlying physiolo-
gical and molecular responses to water stress is crucial for the gen-
eration and selection of tolerant genotypes (Ahuja et al., 2010). In
South America, one of the hybrids with the greater economic im-
portance is Eucalytpus camaldulensis vs Eucalyptus urophylla mainly be-
cause of its drought tolerance, wood quality, disease resistance and
shoot development. However, regardless its better capacity to tolerate
drought conditions compared to its counterparts, the productivity is
still affected by this abiotic stress.
A diverse array of strategies may be involved in the adaptation of a
plant to water stress, including stomatal closure, induction of stress
responsive proteins and accumulation of cell protective metabolites
mediated by the phytohormone abscisic acid (ABA) (Umezawa et al.,
2010). The enzyme that catalyzes the rate-limiting step in the ABA
biosynthesis is 9-cis-epoxycarotenoid dioxygenase (NCED3), and many
reports have confirmed that increases in NCED3 gene expression are
associated with higher production of ABA and ABA-induced responses
to water stress (Iuchi et al., 2001; Daszkowska-Gollec and Szarejko,
2013) in some species as, for example, Arabidopsis thaliana (Woo et al.,
2011), Zea mays (Su et al., 2011) and Nicotiana tabacum (Zhang et al.,
2009).
Findings from studies involving A. thaliana led to the concept of an
ABA perception/signaling model that elicits drought tolerance and in-
volves three main components, namely the Pyrabactin resistance 1/
Pyrabactin resistance 1-like/Regulatory components of ABA receptors
(PYR1/PYL/RCAR) and two phosphatase/kinase enzyme pairs with
opposite functions, i.e. protein phosphatase 2C (PP2C) and sucrose non-
fermenting (SNF1)-related protein kinase 2 (SnRK2) (Mehrotra et al.,
2014). The conformational changes that occur when intracellular ABA
binds to the PYR1/PYL/RCAR ABA receptor induce the inactivation of
PP2C proteins and the activation of SnRK2 proteins culminating in the
expression of genes and ABA-dependent transcription factors and, ul-
timately the triggering of the downstream pathway (Gonzalez-Guzman
et al., 2012; Santiago et al., 2009; Yoshida et al., 2015).
Drought-tolerance responses can also be induced by ABA-in-
dependent routes (Kuromori et al., 2014) involving, for example, the
attachment of Dehydration-responsive element-binding (DREB) tran-
scription factors to drought-responsive genes through recognition of A/
GCCGAC sequences in promoter regions (Agarwal and Jha, 2010;
Matsukura et al., 2010). Genes encoding DREB transcription factors
have been described and evaluated in A. thaliana (Liu et al., 1998,
2000), Glycine max (Chen et al., 2007; Mizoi et al., 2012), Oryza sativa
(Matsukura et al., 2010), Triticum aestivum (Shen et al., 2003) and Z.
mays (Qin et al., 2007).
Additionally, drought-resistance responses can be mediated by
secondary messengers such as Ca2+, the mobilization of which from
intracellular compartments generates increased concentration in the
cytoplasm resulting in conformational changes and activation of cal-
cium-dependent protein kinases (CDPKs) (Boudsocq and Sheen, 2013).
Members of the CDPK multigene family participate in the activation
and inhibition of a range of enzymes, ion channels and transcription
factors. For example, the AtCPK6 gene of Arabidopsis plays a role in
stomatal closure through activation of guard-cell slow anion channel 1
(SLAC1) and calcium channels and the scavenging of reactive oxygen
species (Geiger et al., 2010), while some studies have demonstrated
that the over-expression of AtCPK6 can induce drought tolerance in
various species by regulating the production of osmolytes such as
proline (Xu et al., 2010; Schulz et al., 2013).
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Journal of Plant Physiology 229 (2018) 122–131
Furthermore, it is known that the epigenome strongly influences the
expression or repression of genes at times of stress, and that epigenetic
regulatory mechanisms enable plants to survive and reproduce suc-
cessfully in unfavorable environments. The epigenetic changes in re-
sponse to water stress that have received most research attention are
DNA methylation, histone modifications and chromatin remodeling
(Han and Wagner, 2013). Although the mechanisms have not been fully
elucidated, it has been shown that DNA methylation and decomposition
of chromatin in plants grown under drought conditions are mediated by
DNA methyltransferases such as MET1 and chromatin remodeling fac-
tors such as deficient in DNA methylation 1 (DDM1) protein (Karan
et al., 2012; Kim et al., 2014).
Gene expression analysis is commonly employed in plant research
for identifying molecular markers, performing genetic manipulation
and evaluating the tolerance to biotic and abiotic stresses. Alterations in
the expression of specific target genes in relation to endogenous control
genes (reference genes) can be detected using real-time quantitative
polymerase chain reaction (RT-qPCR), the advantages of which include
reliability, swiftness, sensitivity and specificity (Pfaffl et al., 2004).
With the aim of identifying different strategies for drought-resistance in
Eucalyptus spp. we have: (i) evaluated the expression of six genes en-
coding for proteins associated with drought-tolerance responses,
namely EgrNCED3, EgrPYR1, EgrDREB2.5, EgrCDPK26, EgrMET1 and
EgrDDM1, in leaf samples from drought sensitive (VM05) and drought
tolerant (VM01) clones of the hybrid Eucalyptus camaldulensis x Eu-
calyptus urophylla using RT-qPCR, and (ii) correlated the levels of ex-
pression with the physiological and morphological characteristics of
these genotypes grown under water stress and irrigation conditions.
2. Materials and methods
2.1. Plant material, growth conditions and sampling
3 months-old seedlings (∼40 cm length) of two clones (VM01-tol-
erant and VM05-sensitive) of the hybrid E. camaldulensis Dehnh. x E.
urophylla S.T.Blake were used in this study. They were planted in pots
containing 1500 mL of CSC® commercial substrate (Carolina Soil do
Brasil, Santa Cruz do Sul, RS, Brazil) composed of sphagnum moss,
expanded vermiculite, dolomitic limestone, agricultural gypsum and
traces of NPK. Prior to the experiment, seedlings of both clones were
subjected to constant irrigation for 10 days in a greenhouse with con-
trolled temperature and humidity, and subsequently submitted to either
water stress (no irrigation) or constant irrigation conditions. The ex-
periment was carried out in biological triplicate for each sampling time
(5, 10 and 15 days of water stress or irrigation conditions), such that a
total of 18 seedlings were assessed for each clone. For each plant, 4
leaves per seedling, localized in the middle node of the plant were
harvested for the subsequent experiments.
2.2. Analysis of water potential
A Scholander pressure pump was used to measure the internal water
potential in plants before dawn (typically between 2 and 4 h a.m) on the
sampling day. The tests were performed on totally expanded mature
leaves located at the middle third portion of each seedling.
2.3. Extraction of total RNA and cDNA synthesis
Total RNA was extracted from leaf tissues using the method de-
scribed by Chang et al. (1993) with some modifications. Briefly, ce-
tyltrimethylammonium bromide (CTAB) extraction buffer (containing
2% w/v CTAB, 2% w/v polyvinylpyrrolidone-40, 2 M NaCl, 100 mM
Tris-HCl pH 8.0, 25 mM ethylenediaminetetraacetic acid pH 8.0 and
0.5 g L−1 spermidine) was preheated to 65 °C and added, along with β-
mercaptoethanol (0.2%). The mixture was homogenized for 2 min and
extracted twice with chloroform : isoamyl alcohol (24:1; v/v). Each
G.S. Martins et al.
extraction was followed by centrifugation at 9000 g for 10 min at 4 °C A
quarter volume of 10 M LiCl was added to the combined supernatants
and the resulting mixture was allowed to stand for 6 h at 4 °C and
subsequently centrifuged at 9000 g for 30 min at 4 °C. The precipitate
was resuspended in preheated (37 °C) SSTE buffer (containing 1 M
NaCl, 0.5% sodium dodecyl sulfate, 10 mM Tris-HCl pH 8.0 and 1 mM
ethylenediaminetetraacetic acid pH 8.0), and two volumes of iso-
propanol added to the suspension. The mixture was allowed to stand for
1 h at −20 °C and subsequently centrifuged at 10,000 g for 20 min at
4 °C. The final precipitate was washed with 75% ethanol and re-
suspended in diethylpyrocarbonate-treated water.
In order to remove any traces of contaminating DNA, the RNA ex-
tracts were treated with Ambion® Turbo DNA-free kit reagents (Life
Technologies, Carlsbad, CA, USA) according to the manufacturer's in-
structions. The quantity and purity of total RNA samples were de-
termined from 280/260 and 260/230 nm absorbance ratios measured
using a NanoDrop® 1000 spectrophotometer (Life Technologies). Ratios
of 1.8 and 2.3, respectively, were accepted as pure RNA and electro-
phoresis with agarose gel 2% was carried out to observe RNA integrity.
cDNA synthesis were performed with 1000 ng aliquots of RNA using
Applied Biosystems™ High-Capacity cDNA Reverse Transcription kits
(Life Technologies) according to the manufacturer's recommendations.
2.4. Design of primers
Basic Local Alignment Search Tool (BLAST) searches were per-
formed to compare reference and target protein sequences, available on
Phytozome (http://phytozome.jgi.doe.gov) database where the genome
of E. grandis was previously uploaded (Myburg et al., 2014). The
alignment was done using A. thaliana L. orthologous genes in the E.
grandis sequence. The protein sequences acquired using the BLAST were
used to predict the nucleotide sequences of the specific genes, and ap-
propriate primers were designed using the Invitrogen® OligoPerfect
Designer (Life Technologies) tool.
The genes evaluated as reference candidates for normalization of
RT-qPCR data included those encoding SAND domain-containing pro-
teins (SAND), Elongation factor 1α (EF1α), Protein phosphatases 2 A
(PP2 A-1 and PP2 A-3), Ubiquitin (UBQ), NADP+-dependent Isocitrate
dehydrogenase (IDH) and β-tubulin (TUB). The target drought-toler-
ance genes evaluated in the present study were EgrCPK26, EgrDREB2,
EgrNCED3, EgrPYR1, EgrMET1 and EgrDDM1. The BLAST information of
all the genes used in this study (E. grandis genome ID) as well as the
primers sequences can be found in Supplementary Tables 1 and 2.
2.5. RT-qPCR analysis
PCR analyses were performed using the Applied Biosystems 7500
Real-Time PCR thermal cycler (Life Technologies Corporation). In all
assays, the samples analyzed comprised biological and technical tri-
plicates, including an inter-assay (pool genomic DNA from both clones)
sample to ensure reproducibility between runs. The specificity of each
primer pair was verified from the dissociation (melt) curves acquired
after 40 cycles. PCR amplification efficiency (E) and regression coeffi-
cient (R2) were determined for the validation of primers according to
the standard curve method after using a set of all cDNA samples with 5-
fold serial dilution (Supplementary Tables 1 and 2).
2.6. Screening of reference genes
Expression levels of the reference gene candidates were assessed in
five cDNA pools from the E. camaldulensis x E. urophylla hybrid leaves
described before. The five sets comprise: (i) a cDNA pool of all samples
(All the Samples); (ii) a cDNA pool of all samples from the VM01-tol-
erant clone; (iii) a cDNA pool of all samples from the VM05-sensitive
clone; (iv) a cDNA pool of all samples from seedlings grown under
water stress conditions (both clones); and (v) a cDNA pool of all
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Journal of Plant Physiology 229 (2018) 122–131
samples from seedlings grown under irrigation conditions (both clones).
The relative stability of candidate genes was analyzed using the sta-
tistical algorithms geNorm, with the stability being expressed as M
value (Vandesompele et al., 2002), NormFinder, expressed as stability
value (Andersen et al., 2004), Bestkeeper, expressed as coefficient of
variation and standard deviation (Pfaffl et al., 2004) and Delta-Ct, ex-
pressed as quantification cycle values (Silver et al., 2006). The results
obtained using these algorithms were submitted to the web-based Re-
fFinder tool which compares and classifies individual reference genes
by assigning a weight to each one, based on its ranking by the four
algorithms, and then calculates the geometric mean of the weights to
yield a final overall ranking (Xie et al., 2012)
2.7. Relative expression of target genes
The data acquired in the RT-qPCR analyses were normalized using
the most stable reference genes tested in this study. Relative expression
evaluation was performed as described previously (Pfaffl, 2001). The
different calibrators for each drought-related gene were chosen as the
samples with the lowest expression: EgrNCED3 (irrigated VM05 clone;
day-5), EgrPYR1 (irrigated VM01 clone; day-5), EgrDREB2.5 (irrigated
VM05- clone; day-5), EgrCPK26 (irrigated VM05 clone; day-10),
EgrMET1 (irrigated VM01 clone; day-5), and EgrDDM1 (water stressed
VM01 clone; day-15). For the statistic, the software GraphPrism 7 was
used. Data analysis were performed by two-way ANOVA followed by
the multiple comparisons Tukey's test at significance superior to 5%.
3. Results and discussion
3.1. Stability of selected reference genes
The efficiency of the RT-qPCR technique depends on the selection of
appropriate reference genes in order to normalize expression data and
minimize inter-sample variation during the early experimental steps
(Yang et al., 2014; Zhu et al., 2013). Ideal reference genes should be
constitutively expressed at relatively constant levels under the experi-
mental conditions tested. Since there is no universal reference gene that
can be used in all situations, it is necessary to identify reference genes
more suitable for each specific experimental situation (Czechowski
et al., 2005; Imai et al., 2014). Although RT-qPCR has been commonly
employed in gene expression analysis in plants under water stress
(Wang et al., 2011; Okamoto et al., 2013; Kidokoro et al., 2015), it is
still necessary to select suitable reference genes for the specific geno-
type, tissue and severity of stress assessed (Imai et al., 2014). In this
context, few studies have evaluated reference genes suitable for studies
involving Eucalyptus species grown under conditions of water stress
(Boava et al., 2010; Fernández et al., 2010; Moura et al., 2012).
In the present study, seven reference genes were assessed as possible
reference gene candidates, and the characteristics of the corresponding
primer pairs are presented in Supplementary Table 1. The gene candi-
dates SAND and PP2 A-3 showed the most stable expression according
to the geNorm (Fig. 1), NormFinder (Table 1) and Delta-Ct (Table 2)
algorithms, although some variation was observed in the VM05-sensi-
tive clone samples. However, there was divergence among the algo-
rithms regarding the third most stable gene and such incongruence
could be observed in all the sample sets tested. According to previous
validation studies, it is essential to normalize RT-qPCR data with at
least three reference genes (Fernández et al., 2010; Chan et al., 2014;
Da Silva et al., 2015), hence the selection of a third reference gene was
of particular importance. The variation in expression levels of these
genes in five sample sets of the two hybrid clones (VM01-tolerant and
VM05- sensitive) can be found in Supplementary Fig. 1.
The mathematical approach used by the BestKeeper algorithm dif-
fers substantially from that employed by the other algorithms (Pfaffl
et al., 2004; Zhu et al., 2013), and the rankings proposed by this
method (Table 3) diverged substantially from those of the other three.
G.S. Martins et al. Journal of Plant Physiology 229 (2018) 122–131

Fig. 1. Stability of reference gene candidates, according to the geNorm algorithm (M values), in five sample sets from two clones (VM01-tolerant and VM05-sensitive)
of the hybrid E. camaldulensis x E. urophylla seedlings grown under water stress and irrigation conditions. Sample sets comprised: pool of all samples (all the samples);
VM01-tolerant samples (VM01 (Tolerant)); VM05-sensitive samples (VM05 (Sensitive)); samples from seedlings grown under irrigation conditions (Water treatment);
and samples from seedlings grown under water stress conditions (Drought treatment). The stability of a reference gene is inversely proportional to its M value.

Moreover, the VM01-tolerant clone samples yielded lower CV and SD
values compared with the other sample sets. Nevertheless, according to
BestKeeper, the most stable reference genes were EF1α and SAND. Ac-
cording to the overall ranking provided by RefFinder (Table 4), the
most stable reference genes in three of the five sample sets were SAND,
PP2 A-3 and EF1α, and these were selected for the purposes of this
study.
3.2. VM01-tolerant clone demonstrates higher water potential and ABA-
related genes expression in drought treatment
Levels of the phytohormone ABA increase in response to stress
conditions, leading to the induction of various defense genes including
those coding for late embryogenesis abundant (LEA) proteins, tran-
scription factors, protein kinases, protein channels, transport proteins
and enzymes associated with the synthesis of osmoprotective com-
pounds, phospholipid signaling and stomatal closure (Urano et al.,
2009; Cutler et al., 2010; Fujita et al., 2011; Lim et al., 2015). In at-
tempting to investigate the ABA-dependent response in drought stress,
previous studies used genes related to ABA biosynthesis and signaling,
such as NCED3 and PYR1, although few data is available concerning the
molecular footprint of trees species under such abiotic stress. Interest-
ingly, seedlings of the VM01-tolerant clone grown under conditions of
water stress exhibited higher levels of EgrNCED3 expression compared
with the watered VM01 and VM05 clone at days 10 and 15 (Fig. 2A;
Supplementary Fig. 3A). In VM05-sensitive seedlings grown under
water stress conditions, higher expression of EgrNCED3 comparing to
control conditions was observed only at day 5, but the level of ex-
pression decreased over time (Fig. 2A; Supplementary Fig. 3B). Under
constant irrigation, the water potentials of both clones showed little

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Table 1
Classification of reference gene candidates according to stability values determined using the NormFinder algorithm in five sample sets from two clones (VM01-
tolerant and VM05-sensitive) of the hybrid E. camaldulensis x E. urophylla seedlings grown under water stress and irrigation conditions.
Rank Sample pool VM01-tolerant VM05-sensitive Water stress conditions Irrigation conditions

Gene Stability value Gene Stability value Gene Stability value Gene Stability value Gene Stability value

1 SANDa 0.5433 SAND 0.4196 PP2 A-3 0.5371 PP2 A-3 0.5433 SAND 0.3057
2 PP2 A-3b 0.8528 PP2 A-3 0.8365 SAND 0.5773 SAND 0.8528 PP2 A-3 0.782
3 EF1ac 0.976 IDH 0.844 TUB 0.7223 TUB 0.976 EF1a 0.9634
4 TUBd 1.196 EF1a 1.1454 UBQ 0.8005 UBQ 1.196 TUB 1.4756
5 UBQe 1.3761 PP2 A-1 1.3531 EF1a 0.9621 EF1a 1.3761 UBQ 1.5321
6 PP2 A-1b 1.7669 TUB 1.6235 IDH 1.0545 IDH 1.7669 PP2 A-1 1.8796
7 IDHf 2.3691 UBQ 1.7485 PP2 A-1 1.2907 PP2 A-1 2.3691 IDH 2.3413

a
SAND domain-containing proteins.
b
protein phosphatase 2 A (1/3).
c
elongation factor 1α (EF1α).
d
β-tubulin.
e
ubiquitin.
f
NADP+-dependent isocitrate dehydrogenase).

variation throughout the experimental period, and ranged from -0.3 to
-0.6 MPa (Fig. 3). In agreement with the RT-qPCR data, under water
stress conditions, the water potential of the VM01-tolerant clone was
still unaffected at day 10, while that of the VM05-sensitive clone de-
creased considerably reaching a potential of −0.7 MPa within this
period. Following 15 days of water stress, the signs of wilting of VM01-
tolerant seedlings were less severe than those of their sensitive coun-
terparts and the water potential of the tolerant clone was substantial
higher (−0.8 versus −1.75 Mpa, respectively), thus confirming the
phenotypic differences between the two clones (Supplementary Fig. 2).
Previous investigations involving contrasting genotypes with other
species grown under water stress conditions have shown that tolerant
genotypes exhibit higher water potentials and lower transpiration rates.
On the other hand, because of increased water loss, sensitive genotypes
exhibit oxidative stress, higher leaf temperature, lower chlorophyll
content, damaged photosynthetic apparatus and leaf senescence
(Damatta et al., 2003; Costa e Silva et al., 2009; Yang and Miao, 2010;
Anjum et al., 2011). In addition to the previous results, the expression
of EgrPYR1, a component of ABA biosynthesis and signaling, in samples
from the VM01-tolerant clone grown under drought conditions also
showed an increase after 5 days of drought treatment, as the previous
gene (Fig. 2B; Supplementary Fig. 3C). When this gene was evaluated in
the sensitive clone, it was observed a significant higher expression
during drought condition than in the irrigated treatment (control) at 5
days. However, similar to EgrNCED3, EgrPYR1 did not show any sig-
nificant difference between drought and control conditions at 10 and 15
days in the sensitive clone (Fig. 2B; Supplementary Fig. 3D).
NCED gene products are responsible for the oxidative cleavage of
carotenoid precursors in the plastids to yield xanthoxin, which is
transported to the cytosol and ultimately converted into ABA
(Daszkowska-Gollec and Szarejko, 2013). It has been shown that ABA
concentrations in A. thaliana plants submitted to water stress were more
highly correlated with expression levels of NCED3 than with those of
other genes of the same family (Endo et al., 2008; Hao et al., 2009;
Jensen et al., 2013; Sussmilch et al., 2017; Urano et al., 2017). In ad-
dition, high levels of ABA production correlate with the overexpression
of some members of the NCED gene family in leaf and root tissues to
tolerate water stress conditions (Zhang et al., 2008; Kim and Maik,
2010; Yoshida et al., 2014, 2015). In Eucalyptus spp., despite the lack of
molecular biology and drought-related genes analysis, several studies
have demonstrated the correlation between the ABA concentration,
stomata conductance, proline accumulation, photosynthetic capacity
and drought tolerance (Gibson et al., 1991; Woodward and Bennett,
2005; Sixto et al., 2016; Nolan et al., 2017). Moreover, Eucalyptus tol-
erant varieties have been shown to accumulate ABA at higher amounts
compared to susceptible ones (Li and Wang, 2003; Valdés et al., 2013).
Several other studies have shown the importance of NCED3 genes in
ABA biosynthesis and, consequently, in drought response in different
species (Zhang et al., 2015; Pedrosa et al., 2017; Huang et al., 2018).
Regarding the higher expression of EgrNCED3 in VM01, we suggest that
the higher performance of this clone under drought may be partially
due to the higher biosynthesis of ABA and consequently activation of

Table 2
Classification of reference gene candidates according to quantification cycle (Cq) values determined using the Delta-Ct algorithm in five sample sets from two clones
(VM01-tolerant and VM05-sensitive) of the hybrid E. camaldulensis x E. urophylla seedlings grown under water stress and irrigation conditions.
Rank Sample pool VM01-tolerant VM05-sensitive Water stress conditions Irrigation conditions

Gene Cq value Gene Cq value Gene Cq value Gene Cq value Gene Cq value

1 SANDa 1.55 SAND 1.36 SAND 0.570 SAND 1.78 SAND 1.56
2 PP2 A-3b 1.65 PP2 A-3 1.48 PP2 A-3 0.636 PP2 A-3 1.91 PP2 A-3 1.65
3 EF1ac 1.73 IDH 1.52 TUB 0.712 EF1a 2.10 EF1a 1.78
4 TUBd 1.88 EF1a 1.66 UBQ 0.854 TUB 2.26 TUB 2.09
5 UBQe 1.98 PP2 A-1 1.81 EF1a 0.928 UBQ 2.38 UBQ 2.10
6 PP2 A-1b 2.19 TUB 1.96 IDH 1.053 PP2 A-1 2.70 PP2 A-1 2.30
7 IDHf 2.65 UBQ 2.08 PP2 A-1 1.325 IDH 2.84 IDH 2.64

a
SAND domain-containing proteins.
b
protein phosphatase 2 A (1/3).
c
elongation factor 1α (EF1α).
d
β-tubulin.
e
ubiquitin.
f
NADP+-dependent isocitrate dehydrogenase).

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Table 3
Classification of reference gene candidates according to coefficient of variation (CV) and standard deviation (SD) values determined using the BestKeeper algorithm
in five sample sets from two clones (VM01-tolerant and VM05-sensitive) of the hybrid E. camaldulensis x E. urophylla seedlings grown under water stress and irrigation
conditions.
Rank Sample pool VM01-tolerant VM05-sensitive Water stress conditions Irrigation conditions

Gene CV SD Gene CV SD Gene CV SD Gene CV SD Gene CV SD

b
1 EF1a 6.23 1.67 IDH 3.68 0.92 SAND 6.72 1.64 IDH 6.2 1.48 EF1a 6,12 1,62
2 SANDa 6.7 1.6 PP2 A-1 4.51 1.10 IDH 7.72 1.66 EF1a 6.73 1.83 SAND 6,53 1,56
3 TUBc 6.79 1.74 EF1a 4.49 1.19 UBQ 7.54 1.71 TUB 6.84 1.8 PP2 A-1 6,85 1,73
4 UBQd 7.61 1.75 TUB 5.18 1.32 EF1a 7.84 2.12 SAND 7.6 1.87 TUB 7,24 1,84
5 IDHe 8.36 1.94 SAND 5.63 1.32 TUB 8.38 2.17 UBQ 8.22 1.95 PP2 A3 9,45 2,08
6 PP2A-1f 8.05 2.05 PP2 A-3 7.2 1.55 PP2 A-3 10.78 2.45 PP2 A-1 9.5 2.49 UBQ 9,67 2,34
7 PP2 A-3f 9.58 2.12 UBQ 8.05 1.88 PP2 A-1 10.55 2.81 PP2 A-3 11.01 11.01 IDH 10,35 2,27

a
SAND domain-containing proteins.
b
elongation factor 1α (EF1α).
c
β-tubulin.
d
ubiquitin.
e
NADP+-dependent isocitrate dehydrogenase.
f
protein phosphatase 2 A (1/3).

Table 4
Final overall ranking estimated by the RefFinder tool in five sample sets from two clones (VM01-tolerant and VM05-sensitive) of hybrid E. camaldulensis x E. urophylla
seedlings grown under water stress and irrigation conditions.
Rank Sample pool VM01-tolerant VM05-sensitive Drought conditions Irrigation conditions

Gene Geometric mean of Gene Geometric mean of Gene Geometric mean of Gene Geometric mean of Gene Geometric mean of
the weights the weights the weights the weights the weights

1 SANDa 1.00 SAND 1.50 SAND 1.19 SAND 1.41 SAND 1.19
2 PP2 A-3b 2.30 PP2 A-3 2.21 UBQ 2.00 PP2 A-3 2.30 PP2 A-3 2.11
3 EF1ac 2.71 IDH 2.45 PP2 A-3 3.13 EF1a 3.00 EF1a 2.28
4 TUBd 3.94 EF1a 3.46 TUB 3.41 TUB 3.56 TUB 4.23
5 UBQe 4.47 PP2 A-1 2.98 IDH 4.82 IDH 4.30 UBQ 4.95
6 PP2 A-1b 6.00 TUB 5.42 EF1a 4.95 UBQ 4.73 PP2 A-1 5.05
7 IDHf 6.44 UBQ 7.00 PP2 A-1 7.00 PP2 A-1 6.00 IDH 7.00

a
SAND domain-containing proteins.
b
protein phosphatase 2 A (1/3).
c
elongation factor 1α.
d
β-tubulin.
e
ubiquitin.
f
NADP+-dependent isocitrate dehydrogenase).

downstream response.
ABA-dependent events leading to drought tolerance may be trig-
gered by the interaction of PYR1/PYL/RCAR, PP2C and SNF1 down-
stream of ABA synthesis (Santiago et al., 2009; Gonzalez-Guzman et al.,
2012; Yoshida et al., 2015). The correlation previously reported be-
tween ABA production and ABA receptor activities of the PYR1 proteins
in the PYR1 /PYL/RCAR complex would explain the similarity of ex-
pression patterns of EgrNCED3 and EgrPYR1 in clone VM01 and VM05,
although both clones seem to induce the expression of these two genes
differently. Besides its role in ABA signaling, PYR1 proteins can interact
with a member of the PP2C superfamily, such as the Arabidopsis Ser/Thr
phosphatase of type 2C (AP2C1). This protein has been shown to affect

Fig. 2. Relative quantitative expression (RQE) of two target drought resistance genes in samples from two clones of hybrid Eucalyptus camaldulensis x Eucalyptus
urophylla seedlings grown under water stress conditions for 5, 10 and 15 days. The reference genes used for normalization were SAND, PP2 A-3 and EF1α. Panels show
the expression of: (A) EgrNCED3 (calibrator - irrigated VM05 clone at day 5); (B) EgrPYR1 (calibrator - irrigated VM01 clone at day 5). Data analysis were performed
by two-way ANOVA followed by the multiple comparisons Tukey's test at significance superior to 5%. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;
ns = non-significant). Error bars represent ± s.d. (n = 3).

127
G.S. Martins et al.
Fig. 3. Leaf water potential (determined using a Scholander pressure pump) as
a function of time in two clones of the hybrid Eucalyptus camaldulensis x
Eucalyptus urophylla. Assessment was performed in seedlings of VM01-tolerant
clone grown under irrigation (—●—) and water stress (—■—) conditions, and
in seedlings of VM05-sensitive clone grown under irrigation (—▼—) and water
stress (—▲—) conditions. (*p < 0.05, **p < 0.01, ****p < 0.0001). Error bars
represent ± s.d. (n = 3).
activity in the voltage-gated potassium ion channel KAT1 of A. thaliana
that mediates potassium influx into guard cells and may regulate sto-
matal opening (Brock et al., 2010; Jones et al., 2014). Although we also
see a higher expression of EgrNCED3 and EgrPYR1 in VM05 at 5 days,
we believe that this clone’s response to drought vary from the tolerant
one where it might induce ABA in the beginning of the drought stress or
other response related to the two genes previously described. However,
this strategy may not be as effective as the one from the VM01 clone
3.3. VM01-tolerant clone exhibits higher expression of the ABA-
independent gene EgrDREB2.5, but not EgrCPK26
The protein product of EgrDREB2.5, a member of the family of
ethylene responsive element (ERE) binding factors, can attach to the
promoter regions of genes related to water deficiency response. In
drought tolerant and transgenic genotypes, high expression of DREB2
was correlated with the activation of genes coding for enzymes asso-
ciated with cell homeostasis and repair, such as LEA and dehydrin
proteins, osmolyte biosynthesis and antioxidant enzymes (Chen et al.,
2007; Khan, 2011). E. grandis genome encodes 6 DREB2 genes which all
showed to be responsive to drought conditions. Here, we evaluated the
difference in EgrDREB2.5 expression between the two clones. This
DREB2 protein possessed the highest expression pattern between the
others five in drought treatment (Nguyen et al., 2016; Cao et al., 2015).
As shown in Fig. 4A EgrDREB2.5 expression increased progressively
with respect to time in VM01-tolerant seedlings grown under water
Fig. 4. Relative quantitative expression (RQE) of two target drought resistance genes in samples from two clones of hybrid Eucalyptus camaldulensis x Eucalyptus
urophylla seedlings grown under water stress conditions for 5, 10 and 15 days. The reference genes used for normalization were SAND, PP2 A-3 and EF1α. Panels show
the expression of: (A) EgrDREB2.5 (calibrator - irrigated VM05 clone at day 5) and (B) EgrCPK26 (calibrator - irrigated VM05 clone at day 10). Data analysis were
performed by two-way ANOVA followed by the multiple comparisons Tukey's test at significance superior to 5%. (*p < 0.05, **p < 0.01,****p < 0.0001, ns = non-
significant). Error bars represent ± s.d. (n = 3).
128
Journal of Plant Physiology 229 (2018) 122–131
stress conditions and, after 10 days of treatment, was significantly
higher than that observed in the VM05 seedlings grown under drought
conditions. In contrast, no alterations in EgrDREB2.5 expression were
observed in samples from the VM05-sensitive clone grown under water
stress or irrigation conditions (Fig. 4A; Supplemantary Fig. 4D). These
data correlate with the superiority of VM01 clone in stress conditions
since DREB2 transcription factors are essential for ABA-independent
response during drought being a good candidate for drought-tolerance
molecular markers. Regulation of Ca2+ levels forms another important
plant strategy against water deficiency. The changes induced in cyto-
plasmic Ca2+ concentrations following the perception of stress are re-
cognized by calcium-binding proteins that, in turn, convey the in-
formation downstream to initiate a phosphorylation cascade leading to
regulation of gene expression (Kudla et al., 2010). In this regard, genes
of the CDPK family perform an array of functions in plants including
environmental stress signaling following the perception of abiotic stress
stimuli. However, in the present study, we did not see a significant
difference of the EgrCPK26 expression between all the treated and non-
treated seedlings (Fig. 4B; Supplementary Fig. 4A and B). According to
these results, we suggest less relevance of CDPK candidate on both
clones’ tolerance compared to the other genes previously described.
3.4. Expression of epigenetically-regulated drought-resistance genes
(EgrMET1 and EgrDDM1)
DNA methylation is carried out by DNA methyltransferases, of
which plants have at least three classes that differ in protein structure
and function. The MET1 enzymes, for example, add methyl groups to
CG sequences in a specific manner (Law and Jacobsen, 2010). It has
been shown that the methylation status of promoter and coding regions
of genes in some species are altered as result of abiotic stresses, and that
such methylation may be associated with both gene activation and re-
pression (Sahu et al., 2013). According to Song et al. (2012), salinity
stress in G. max led to an increase in MET1 activity that, in turn, af-
fected the methylation status and expression levels of four salt-re-
sponsive transcription factors. Moreover, abiotic-stresses (aluminum,
low temperature and salt stresses) induced demethylation and tran-
scriptional activation of a gene encoding a glycerophosphodiesterase-
like protein in tobacco (Choi and Sano, 2007).
With regard to the alteration of chromatin structure, it has been
suggested that DDM1 protein, a member of the broad SWI2/SNF2
protein family, acts as a chromatin-remodeling factor. DDM1 is a he-
licase-like protein that directs energy derived from ATP hydrolysis into
the mechanical modification of chromatin structure. The protein moves
along the DNA helix unwinding the nucleosomes and allowing access of
transcription factors and methyltransferases to DNA bases (Ryan and
Owen-Hughes, 2011). In A. thaliana, high expression of the DDM1 gene
G.S. Martins et al.
Fig. 5. Relative quantitative expression (RQE) of two target genes related to epigenetic regulation in samples from two clones of hybrid Eucalyptus camaldulensis x
Eucalyptus urophylla seedlings grown under different conditions. The reference genes used for normalization were SAND, PP2 A-3 and EF1α. Panels show the
expression of: (A) EgrMET1 (calibrator - irrigated VM01 clone at day 5); and (B) EgrDDM1 (calibrator - water stressed VM01 clone at day 15). Data analysis were
performed by two-way ANOVA followed by the multiple comparisons Tukey's test at significance superior to 5%. (*p < 0.05, **p < 0.01, ***p < 0.001
****p < 0.0001, ns = non-significant). Error bars represent ± s.d. (n = 3).
was found to be directly correlated with elevated levels of hetero-
chromatin, the densely packed DNA that is less accessible to binding
proteins (Zemach et al., 2013). However, the results obtained by these
authors indicate that DDM1 enables DNA methyltransferases to access
H1-containing heterochromatin, thereby modifying the methylome of
the plant.
In the present study, the expression levels of EgrMET1 and EgrDDM1
were generally higher in samples from VM05-sensitive seedlings grown
under water stress and irrigation condition compared to profiles ob-
served in the tolerant clone, which presented only basal levels of these
genes regardless the growth conditions (Fig. 5A and B). It is likely that
alterations in the transcriptome profile of the VM05-sensitive clone are
due to the degree of chromatin compaction, requiring greater DDM1
protein activity to facilitate the access of transcription machinery.
Surprisingly, EgrDDM1 expression was not as consistent as EgrMET1. At
day 10 and 15, the expression of the former decreases drastically in
VM05-sensitive clone under water stress and irrigated conditions, re-
spectively, while the latter gene does not show significant difference
(Supplementary Fig. 5 A, B,C and D). Further studies are required in
order to clarify the mechanisms of epigenetic regulation towards
drought response of these two clones.
The phenotypic characteristics of VM01-tolerant and VM05-sensi-
tive clones grown under water stress and irrigation conditions verify the
greater drought tolerance of the former. An association between the
expression of the target genes EgrNCED3, EgrPYR1 and EgrDREB2 and
the superior response of the VM01-tolerant clone to water deficiency
bring the hypothesis that the VM01 strategy might include increase
DREB-dependent response during time and ABA biosynthesis and re-
sponse during all period of stress. To the best of our knowledge, there
are few literature reports that relate drought tolerance to the expression
of stress-responsive genes in Eucalyptus spp. Our findings will facilitate
future research on the functional characterization of stress-related re-
sponse genes, the identification of molecular markers, the evaluation of
drought tolerance and genetic transformation in other Eucalyptus spe-
cies and hybrids.
Acknowledgments
This work was supported by National Council for Scientific and
Technological Development (CNPq), Minas Gerais State Agency for
Research and Development (FAPEMIG) and Coordination for the
Improvement of Higher Education Personnel (CAPES). We also ac-
knowledge Vallourec Inc. to give us the plant material and making
possible this study.
129
Journal of Plant Physiology 229 (2018) 122–131
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.2018.07.007.
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