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Relationship Between Endodormancy and Cold Hardiness
in Grapevine Buds
Sebastia
´n Rubio
1
•De
´bora Dantas
2
•Ricardo Bressan-Smith
2
•Francisco J. Pe
´rez
1
Received: 14 April 2015 / Accepted: 16 June 2015
ÓSpringer Science+Business Media New York 2015
Abstract Endodormancy (ED) and cold hardiness (CH)
are two strategies utilized by grapevine (Vitis vinifera L.)
buds to survive unfavorable winter conditions. Each phe-
nomenon is triggered by different environmental cues—ED
by short-day (SD) photoperiod and cold hardiness (CH) by
low temperatures. In grapevine buds, CH occurs mainly via
the supercooling of intracellular water, a phenomenon
associated with the low temperature exotherm (LTE). The
seasonal dynamics of ED and CH were studied on
grapevines buds by determining the BR
50
(time required to
reach 50 % of bud break under forced conditions) and the
LTE, which measure the depth of ED and the level of CH,
respectively. Overlapping BR
50
and LTE curves revealed
that CH began to develop in late April, when buds were
fully endodormant and daily mean temperatures had started
to drop below 14 °C, suggesting that ED is a prerequisite
for the acquisition of full CH. Increase in starch content
and thickening of the cell wall (CW) of meristematic cells
which occurs in dormant buds could be involved in struc-
tural and metabolic changes that favor CH subsequent
acquisition. Interestingly, the thickening of the CW and the
synthesis of starch which are associated with ED were
induced by a SD-photoperiod, while the hydrolysis of
starch, the accumulation of soluble sugars, and the up-
regulation of dehydrin genes, which are associated with
CH, were induced by low temperatures. Overall, the results
indicate that structural, metabolic, and transcriptional
changes that occur during ED in grapevine buds are nec-
essary for the further development of CH.
Keywords Cold hardiness Cell wall Dormancy
Dehydrins Exotherms Grapevine buds Supercooling
Introduction
Grapevines (Vitis vinifera L.) develop axillary buds con-
taining embryonic shoots from which whole branches
develop after perceiving specific signals (Rohde and Bha-
lerao 2007). The decreasing photoperiod during late sum-
mer is the environmental signal that triggers the transition
of buds into endodormancy (ED) (Fennell and Hoover
1991;Ku
¨hn and others 2009; Grant and others 2013). ED is
a physiological state characterized by growth inhibition,
arrest of cell division, and reduced metabolic and respira-
tory activity. Operationally ED is characterized by a delay
in the bud-break response under forced conditions (Lang
1987; Dennis 2003). The development of ED is part of the
process by which buds adapt to unfavorable winter con-
ditions. One of the main functions of ED is to avoid bud
break in response to a transient warm spell during winter,
which could not only avoid further damage by frost (Jian
and others 1997), but also play an important role in
preparing plants for freezing temperatures (Sakai and
Larcher 1987). In tree species with photoperiodically-in-
duced dormancy such as birch (Betula pendula), the per-
ception of decreasing day-length results in growth
cessation, development of a terminal bud, and progression
Electronic supplementary material The online version of this
article (doi:10.1007/s00344-015-9531-8) contains supplementary
material, which is available to authorized users.
&Francisco J. Pe
´rez
frperez@uchile.cl
1
Laboratorio de Bioquı
´mica Vegetal, Facultad de Ciencias,
Universidad de Chile, Casilla 653, Santiago, Chile
2
Centro de Ciencias e Tecnologias Agropecuarias,
Universidade Estadual do Norte Fluminense, Av Alberto
Lamego 2000, Campos dos Goytacazes, RJ, Brazil
123
J Plant Growth Regul
DOI 10.1007/s00344-015-9531-8
to a dormant and more freezing-tolerant state (Rinne and
others 2001). In contrast, species of Vitis do not set a ter-
minal bud in response to SD-photoperiod and the shoot
apical area does not enter into ED nor cold acclimates;
however, upon reaching a critical day-length (CDL), other
hallmark phenotypes such as periderm development,
growth cessation, and latent bud dormancy are induced
(Fennell and Hoover 1991; Wake and Fennell 2000;
Sreekantan and others 2010; Grant and others 2013).
Freezing tolerance or cold hardiness (CH) also develops in
grapevine buds in response to low non-freezing tempera-
tures, a phenomenon known as cold acclimation (Thoma-
show 1999; Mills and others 2006; Ferguson and others
2011,2014). It has been reported that the dormant buds of
Vitis riparia (Pierquet and others 1977) exhibit deep
supercooling of intracellular water, suggesting that the
depression of the freezing point is the mechanism through
which grapevine buds adapt to subfreezing temperatures.
Differential thermal analysis (DTA) has been widely used
to measure the exotherms of deep supercooled buds, two
exotherms are generally observed in cold-acclimated buds,
a high-temperature exotherm (HTE) and a low-temperature
exotherm (LTE), which correspond to the heat released
during the freezing of extracellular and intracellular water,
respectively (Burke and others 1976). Lethal tissue damage
takes place in buds at temperatures below LTE, indicating
that LTE can serve as a measure of CH (Pierquet and
Stushnoff 1980; Mills and others 2006; Ferguson and
others 2011). Several factors can influence the decrease in
the supercooling of water; however, the properties of a cell,
tissue, or organ that allow it to undergo deep supercooling
remain enigmatic, despite the prevalence of this ability in
many plant species (Gusta and Wisniewski 2013).The role
of sugars in the development of CH has been well docu-
mented in grapevines; total soluble sugars increase during
the initial stage of CH, and it is speculated that raffinose
plays an important role in cold acclimation in grapevine
buds (Grant and others 2013; Hamman and others 1996).
Dehydrins, a class of hydrophilic, thermostable stress
proteins that belong to the late embryogenesis abundant
(LEA) family, are expressed in response to drought,
salinity, cold, and osmotic stress (Nylander and others
2001). In buds of Vitis labruscana L. cv. Concord, a heat
stable 27 KD protein that accumulates in response to cold,
was identified as immunologically related to dehydrins by a
strong reaction with the antidehydrin antibody (Salzman
and others 1996). Recently, four dehydrin genes were
identified in V. vinifera and V. yeshanensis, and their
responsiveness to various forms of abiotic and biotic
stresses was studied (Yang and others 2012). This study
examined the relationship between ED and CH in grape-
vine buds, and characterized partially the structural,
metabolic, and transcriptional changes that occur during
ED which are necessary for the further acquisition of CH at
low temperatures.
Materials and Methods
Seasonal Variations on the Depth of Dormancy
in Buds of V. vinifera cv. Thompson Seedless
The bud-break response of single-bud cuttings under forced
conditions is a common indicator used to describe the
depth of dormancy in grapevines (Koussa and others 1994;
Dennis 2003). This system makes it possible to work with a
large number of buds, providing a proper representation of
the dormancy status of a given bud population at a specific
point in time during the dormancy cycle. Canes were col-
lected every 2–3 weeks, between 11 December and mid-
August 2012, from 8-year-old V. vinifera L cv. Thompson
seedless growing at the experimental station of the Chilean
National Institute of Agriculture Research (INIA), located
in Santiago (33°340S latitude). Detached canes each car-
rying ten buds in positions 5–14 were transferred to the
laboratory, and cut into single-bud cuttings. Forty of these
cuttings (10–12 cm length) were mounted on a
polypropylene sheet and floated in tap water in a plastic
container on each collection date. The cuttings were then
transferred to a growth chamber set at 23 ±2°C with a
16 h photoperiod (forcing conditions). Every 5 days, water
was replaced in the container and bud break was assayed
for a period of 30 days. The appearance of visible green
tissue at the tip of the bud was indicative of bud break. The
depth of bud dormancy was determined using BR
50,
a
parameter which is an estimate of the mean time required
to reach 50 % bud break under forced conditions (Pe
´rez
and others 2007). The depth of dormancy has been previ-
ously determined in buds of V. vinifera cv. Thompson
seedless in the same place and with the same methodology,
obtaining similar results (Pe
´rez and others 2007; Vergara
and Pe
´rez 2010).
Temperature Measurements
Temperature data were collected every hour from the
weather station of the National Institute of Agricultural
Research (INIA, La Platina) located at 33°340S latitude
70°400W longitude, 100 m from the vineyard.
Seasonal Variations on LTE in Buds of V. vinifera
cv. Thompson Seedless
Canes collected weekly from field-grown V. vinifera cv.
Thompson seedless between 22 April and 27 August 2012
J Plant Growth Regul
123
were cut into single buds. Exotherms were determined in
single buds by differential thermal analysis (DTA).
Kryoscan, a freezing and data acquisition device that uses
Peltier elements (PE) for the cooling and detection modules
(Badulescu and Ernst 2006) was employed for DTA. The
temperature of the cooling block was pre-chilled at 10 °C
using a water bath and further chilled by a pyramidal
configuration of PE connected to a temperature controller
(PXR-4, Fuji electronic system, Japan). The temperature
controller regulates the passing voltage through the PE
according to a temperature sensor connected to the cooling
block, and by the programmed temperature ramp. Minia-
ture PE (Peltier modules series Opto Tec, Laird Tech-
nologies-Engineered Thermal Solutions, USA) instead of
thermocouples were used to record exotherms, because
they yield a relatively high voltage difference which is less
susceptible to electrical noise, and no external zero refer-
ences are needed (Badulescu and Ernst 2006). These sen-
sitive PE detect temperature gradients generated by the
exotherms and convert the thermal signal to voltage out-
puts. The data acquisition system (Measurement Comput-
ing USB 120BLS, USA) and DasyLab software were used
to measure and collect the output voltage and the temper-
ature. Signals were recorded every 2 s, and a decrease in
temperature of 4 °C per h starting at 10 °C and ending at
-30 °C was programmed (Mills and others 2006). Gen-
erally, two peaks were observed, one corresponded to HTE
that was assigned to the freezing point of extracellular
(apoplast) water, which is non-lethal (Burke and others
1976) and the other corresponded to LTE that was assigned
to the freezing point of intracellular water, which is lethal
(Burke and others 1976). Because lethal tissue damage in
grapevine buds occurs at temperatures below the LTE, this
value can serve as a measure of CH (Pierquet and Stush-
noff 1980; Wolf and Cook 1994; Mills and others 2006).
Values for each date correspond to the average of 12 bio-
logical replicates of single buds (Fig. 1). LTE measure-
ments were repeated in the same location and with the
same variety during 2013.
Effect of Temperature on LTE of Dormant and Non-
dormant Buds of V. vinifera cv. Thompson
Seedless
To analyze the effects of temperature on exotherms of
dormant and non-dormant buds, single-bud cuttings of V.
vinifera cv. Thompson seedless collected on 27 December
(non-dormant) and 10 June 2012 (dormant) were exposed to
low (5 °C, cooled) and room (14 °C, non-cooled) temper-
atures, and exotherms were measured in single buds over
time (12 buds at each collection time). Dormant buds prior
to harvesting were exposed in the field to approximately
200 chilling hours, and therefore, were partially cold
acclimated (LTE =-15 °C) before the experiments.
Effect of Dormancy and Low Temperatures
on the Starch and Soluble Sugar Content of Buds
of V. vinifera cv. Thompson Seedless
To study the effect of dormancy on the levels of starch in
grapevine buds, starch levels were determined in buds of V.
vinifera cv. Thompson seedless collected on 27 December
(non-dormant) and 10 June 2012 (dormant). To study the
effect of low temperature on the starch and soluble sugar
content in grapevine buds, buds of V. vinifera cv.
Thompson seedless collected on 10 June 2012 (dormant)
were used. The buds (0.2 g approx.) were ground with a
mortar and pestle in liquid nitrogen and extracted 39with
3 ml of cold acetone and 19with a mixture of chloroform
and isoamyl alcohol (24:1). The suspension was cen-
trifuged at 13,000 rpm for 3 min, and the pellet was dried
and extracted with 2 ml 80 % (v/v) ethanol for 30 min in a
water bath heated to 60 °C. This extraction was repeated 3
times, and the supernatants were collected, pooled, and
dried. The starch content of the pellet was determined after
ethanol extraction of the soluble sugars by acid extraction
using the anthrone reagent (Hansen and Moller 1975). The
dried supernatant obtained from ethanol extraction was
dissolved in 100 ll of pyridine and an aliquot of 15 ll was
derivatized by adding 5 ll BSTFA (Sigma-Aldrich, USA);
the mixture was then heated at 90 °C for 30 min. The
chromatographic analyses of the derivatized samples were
performed using a Shimadzu GC 2014 gas chromatograph
(Shimadzu Corporation, Kyoto, Japan) equipped with a
CBP1 capillary column and an FID detector. The operating
conditions were as follows: injector and detector temper-
atures were 180 and 300 °C, respectively; carrier gas flow
(helium) at 1.0 ml/min; injection volume of 1 ll with a
flow splitter at a ratio of 50:5. The oven was programmed
to temperatures of 60–200 °C at a rate of 30 °C min
-1
and
from 200 to 280 °C at a rate of 5 °C min
-1
. Standard
curves were constructed for the determination of the
sucrose, glucose, fructose, and starch concentrations.
Influence of Dormancy and SD-Photoperiod on CW
Thickness in Meristematic Cells
The thickness of the cell wall (CW) of meristematic cells
of non-dormant buds (collected 27 December 20012) and
dormant buds (collected 10 June 2012) of V. vinifera cv.
Thompson seedless grown in Santiago, Chile (33°340S
70°400W) was examined by transmission electron micro-
scopy (TEM). The thickness of the CW of meristematic
cells of buds of V. vinifera cv. Italia melhorada grown in
Messoro
´, Brazil (50120S) and exposed to different
J Plant Growth Regul
123
photoperiods was also analyzed by TEM. Sections were
taken from the middle of the buds and were fixed in 2 %
formaldehyde, post-fixed for 2 h in 0.1 mg 9ml
-1
osmium tetra-oxide, dehydrated in a graded series of
ethanol (25, 50, 70, 90, and 3 9100 % for at least 1 h for
each), and embedded in Spurr’s resin. Thin sections were
cut with a diamond knife, stained in uranyl acetate and lead
citrate, and examined with a Jeol 100 SX electron micro-
scope. Three buds per treatment were analyzed to deter-
mine the thickness of the CW.
Photoperiod Treatments on V. vinifera cv. Italia
Melhorada
In a collaborative project with colleagues from Brazil, the
effect of different photoperiod regimes on the thickness of
the cell wall (CW) and on the expression of cellulose
synthase, lacasse, and dehydrin genes was performed.
Photoperiod experiments were carried out in Messoro
´,
Brazil due to small variations in photoperiod and temper-
ature in the area, making it easier to conduct this type of
experiment. Cuttings of V. vinifera cv. Italia melhorada on
rootstock IAC 572 grown at the Federal University of
Rural Semi-Arid (UFERSA), located in Messoro
´, Brazil
(5°1201600S), where the natural photoperiod during the
whole year is (12/12 h day/night) and temperature fluctu-
ates between 29 and 31 °C, were used as plant material for
photoperiod experiments (3 replicates per treatment).
Rooted cuttings (15 per treatment) were planted into mix
1:1:1 (v: v: v) soil, sand, and muck in 5 l pots. As growth
commenced, one shoot was allowed to develop on each
cutting. Cuttings having uniform growth with 12–16 leaves
were selected and randomly assigned to each photoperiod
treatment for 8 weeks. Photoperiod experiments were
conducted in a greenhouse under LD (14/10 h day/night)
and SD-photoperiod (10/14 h day/night), because the crit-
ical day-length (CDL) for dormancy transition in V. vini-
fera is about 13 h (Ku
¨hn and others 2009). Supplemental
light was provided automatically in the afternoon at 17:
30 h using a 100 W fluorescent tube; light restriction was
imposed with a black plastic sheet in the early morning at
4:30 h. After the treatments, buds were lyophilized for
gene expression analysis, and fixed in 2 % formaldehyde
for TEM analysis.
Photoperiod and Low-Temperature Effect
on the Expression of Dehydrin Genes
For photoperiod experiments, total RNA was isolated from
lyophilized buds (0.05–0.1 g) of V. vinifera cv. Italia
melhorada. For low-temperatures experiments, total RNA
was isolated from buds (0.5–0.7 g) of V. vinifera cv.
Thompson seedless. In both cases, total RNA was extracted
and purified using a modification of the method of Chang
and others (1993), as described in Noriega and others
(2007). DNA was removed by treatment with RNAase-free
DNAase (1 U/lg) (Invitrogen, CA, USA) at 37 °C for
30 min. First-strand cDNA was synthesized from 5 lgof
purified RNA with 1 lL oligo(dT)
12–18
(0.5 lg9lL
-1
)as
primer, 1 lL dNTP mix (10 mM), and Superscript
Ò
II RT
(Invitrogen, USA). Gene expression analysis was per-
formed by quantitative real-time PCR, and carried out in an
Eco Real-Time PCR system (illumina, Inc. SD, USA),
using KAPA SYBR FAST mix (KK 4602) and KAPA Taq
Fig. 1 Comparison of daily mean temperature, endodormancy (ED),
and cold hardiness (CH) in grapevine buds. aEndodormancy (ED)
and cold hardiness (CH) development in V. vinifera cv. Thompson
seedless. bDaily mean temperatures in Santiago, Chile (33°340S)
during the year 2012. The depth of ED was determined by BR
50
(time
required to reach 50 % bud break under forced conditions). Values of
BR
50
for each collection date were determined by Probit Analysis
(Minitab statistical software) and bars represent s.d. (n=40). CH
was determined by measuring the LTE by differential thermal
analysis (DTA) using Peltier modules (TEM). Bars represent s.d.
(n=16)
J Plant Growth Regul
123
DNA Polymerase (Kapa Biosystem, USA). Primers suit-
able for the amplification of 100–150 bp products for each
gene under study were designed using the PRIMER3
software (Table 1 supplement) (Rozen and Skaletsky
2000). The amplification of cDNA was performed under
the following conditions: denaturation at 94 °C for 2 min
and 40 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C
for 45 s. Three biological replicates with three technical
repetitions were performed for each treatment. Transcript
levels were calculated by the DDCq method (Livak and
Schmittgen 2001) using VvUBIQUITIN as the reference
gene. VvUBIQUITIN was selected as a reference gene
because the transcript level was stable across treatments
(Rubio and others 2014).
Statistical Analysis
The depth of bud dormancy was estimated by BR
50
and the
corresponding averages and standard deviations (s.d.) were
calculated by mean of the Probit analysis (Minitab 13.31
Minitab Inc, USA). For pairwise comparison, Student’s
ttest a=0.05 was used.
Results
Seasonal Variations of Cold Hardiness
and Endodormancy in Grapevine Buds
The depth of dormancy measured as BR
50
and the level of
CH measured as LTE were determined in buds of V.
vinifera cv. Thompson seedless throughout the year 2012
(Fig. 1a). The vines were grown in Santiago, Chile
(33°340S) and daily mean temperatures of the location are
shown in Fig. 1b.
Temperature Affects LTE Values Only in Dormant
Buds
Significant differences in the values of LTE were detected
in cooled (exposed to 5 °C) and non-cooled (exposed to
14 °C) dormant buds. After 3 weeks of exposure to low
temperatures, the value of LTE decreased from -15 to
-16.5 °C, whereas in buds exposed to ambient tempera-
ture the value of LTE increased from -15 to -13 °C.
Moreover, the difference among buds exposed to both
treatments, increased with the progress of time, and after
6 weeks of treatment, the difference increased from 3 to
Fig. 2 Cold acclimation and deacclimation of dormant grapevine
buds exposed to low (5 °C) and ambient (14 °C) temperatures.
aDormant buds with partial cold acclimation were collected on 10
June after being exposed to 200 chilling hours in the field. Cold
acclimation was determined by measuring the low-temperature
exotherm (LTE) by DTA in single buds over time. bLow (5 °C)
and ambient (14 °C) temperature effects on the high-temperature
exotherm (HTE) in non-dormant buds. Canes were collected on
December 27. Values correspond to the average of 12 single and bars
represent s.d. and asterisk indicates significant differences (Student’s
ttest a=0.05)
J Plant Growth Regul
123
6°C (Fig. 2a). In contrast, in non-dormant buds, a broad
large peak was detected at -7±1°C, which did not vary
with the temperatures (Fig. 2b). This peak was interpreted
as the result of an overlap between HTE and LTE.
Dormancy and SD-Photoperiod Increase
the Thickness of the CW in Meristematic Cells
of Grapevine Buds
Significant differences between the thickness of the CW of
meristematic cells of dormant (1.4 ±0.3 lM) and non-
dormant buds (0.28 ±0.1 lM) of V. vinifera cv Thompson
seedless were observed (Fig. 3c, d). Because SD-pho-
toperiod induces ED in grapevine buds (Fennell and
Hoover 1991;Ku
¨hn and others 2009;Pe
´rez and others
2009; Grant and others 2013), the effect of photoperiod on
the thickening of the CW of meristematic cells of buds of
V. vinifera cv. Italia melhorada was examined by TEM.
After 8 weeks of treatment, significant differences between
the thickness of the CW of meristematic cells exposed to
SD (0.75 ±0.1 lM) and LD-photoperiods (0.3 ±
0.05 lM) were observed (Fig. 3a, b).
Photoperiod Regulation of ‘‘Cellulose synthase
and Laccase’’ Genes of Grape Buds
All cellulose synthase genes of grapevine buds analyzed
were down-regulated by SD-photoperiod. After 8 weeks of
treatment, the expression of VvCSA3 and VvCSLG was
down-regulated by SD-photoperiod, whereas the expres-
sion of VvCSLE was down-regulated only after 2 weeks
(Table 1). Conversely, the expression of VvLAC14, a gene
involved in laccase synthesis, was up-regulated by SD-
photoperiod, whereas the expression of the other laccase
genes analyzed was not affected by photoperiod (Table 1).
Dormancy Increases Starch Accumulation and Low
Temperatures Increase Starch Breakdown
and Soluble Sugar Content in Dormant Buds
Starch accumulates during the development of ED in
grapevine buds, and the levels of starch in non-dormant
buds were significantly lower than those found in dormant
buds (Fig. 4a). On the other hand, low temperature (5 °C)
reduced the content of starch in dormant buds. After
Fig. 3 Electron
photomicrographs that illustrate
the effect of photoperiod and
endodormancy (ED) on the
thickening of the cell wall (CW)
of meristematic cells in buds of
V. vinifera L cv. Italia
melhorada and cv. Thompson
seedless. Photoperiod studies
were performed in V. vinifera
cv. Italia melhorada exposed to
aLD-photoperiod and bSD-
photoperiod for 8 weeks.
Endodormancy studies were
performed in buds of V. vinifera
cv. Thompson seedless
collected on c27 December
(non-dormant), and d10 June
2012 (dormant). Scale
bars =2lM
J Plant Growth Regul
123
3 weeks of exposure to cold, starch content was reduced by
50 mg GFW
-1
, whereas in buds exposed to ambient tem-
peratures (14 °C) for the same period of time, it was
reduced only by 10 mg GFW
-1
(Fig. 4b). Accordingly low
temperatures increased the content of soluble sugars,
sucrose (Suc), glucose (Glc), and fructose (Fru) in dormant
buds (Fig. 5).
Effect of Photoperiod and Low Temperatures
on the Expression of Dehydrin Genes in Grapevine
Buds
The expression of VvDHN1,VvDHN2, and VvDHN3, but
not of VvDHN4 was strongly up-regulated by low tem-
peratures in buds of V. vinifera cv. Thompson seedless.
Analysis was performed by RT-qPCR after 2 weeks of
treatment (Fig. 6a). Conversely, the expression of
VvDHN1,VvDHN2, and VvDHN3 was down-regulated by
SD-photoperiod, whereas the expression of VvDHN4 was
up-regulated in buds of V. vinifera cv. Italia melhorada
(Fig. 6b).
Discussion
The present study shows that the development of dormancy
in grapevine buds is a prerequisite for the acquisition of full
CH; whereas bud dormancy is characterized by the thick-
ening of the CW of meristematic cells and starch accu-
mulation, CH is characterized by starch breakdown,
soluble sugar accumulation, and up-regulation of dehydrin
genes.
Overlapping BR
50
and LTE curves of buds of V. vinifera
cv. Thompson seedless grown in Santiago, Chile revealed
that CH began to develop in late April when buds were
fully endodormant. However, these results do not assure
whether a relationship exists between CH and ED, because
the drop in temperatures, and therefore, the initiation of
chilling accumulation could coincide with the stage of ED
in this region. To get more insight into the existence of a
relationship between dormancy and CH in grapevine buds,
Table 1 Effect of photoperiod on the expression of cellulose syn-
thase VvCSA3,VvCSLE,VvCSLG and laccase VvLAC7,VvLAC9,
VvLAC14 genes in buds of Vitis vinı´fera cv
Genes SD-photoperiod LD-photoperiod
2 weeks 8 weeks 2 weeks 8 weeks
VvCSA3 1.1 ±0.3 1.0 ±0.1 1.0 ±0.3
a
2.1 ±0.2
VvCSLE 1.1 ±0.1 1.0 ±0.2
a
1.5 ±0.2 1.1 ±0.2
VvCSLG 1.0 ±0.3 1.0 ±0.4 1.1 ±0.4
a
1.9 ±0.2
VvLAC7 1.1 ±0.6 1.0 ±0.1 0.8 ±0.2 1.5 ±0.6
VvLAC9 1.0 ±0.1 1.1 ±0.6 0.9 ±0.1 1.4 ±0.8
VvLAC14 1.0 ±0.2 1.1 ±0.1 1.0 ±0.3
a
0.4 ±0.1
Samples exposed to SD-photoperiod for 2 weeks serve as controls.
Values are the average of three biological replicates with three
technical repetitions. Italia melhorada after 2 and 8 weeks of
treatments
a
Significant differences Student’s ttest a=0.05 and ±correspond
to s.d
Fig. 4 Effect of dormancy and low temperature on the content of
starch in buds of V. vinifera cv. Thompson seedless. aStarch content
was determined in dormant buds (collected on 10 June) and in non-
dormant buds (collected on 27 December). bSingle dormant bud
cuttings exposed to ambient (14 °C) (non-cooled) and low temper-
atures (5 °C) (cooled) were weekly analyzed for starch content for a
period of 3 weeks. Values correspond to the average of three
biological replicates bars represent s.d. and asterisk indicates
significant differences (Student’s ttest a=0.05)
J Plant Growth Regul
123
temperature effects on LTE values were studied in dormant
and non-dormant buds of V. vinifera cv. Thompson seed-
less. Although the experiments were carried out in single-
bud cuttings in the dark, the results indicate that dormant
buds can be cold acclimated or deacclimated depending on
whether they were exposed to low or ambient temperatures.
Conversely, non-dormant buds were not cold acclimated
when they were exposed to low temperatures. Interestingly,
these results are consistent with reports indicating that the
buds of woody perennials cannot cold acclimate when the
development of ED is prevented by over-expressing PHYA
and FT genes (Olsen and others 1997; Tra
¨nker and others
2010).
Although SD-photoperiod induces ED in grapevine buds
(Fennell and Hoover 1991;Ku
¨hn and others 2009; Grant
and others 2013), its effect on LTE is very low (Grant and
others 2013), indicating that CH is mainly induced by low
temperatures in grapevine buds. As the thickening of the
CW and starch synthesis is associated with ED, and only
dormant buds are cold acclimated by low temperatures, it
seems likely that CW thickening and starch accumulation
that occurs during ED could play a significant role in the
subsequent development of CH in dormant buds. The
thickening of the CW that is triggered by SD-photoperiod
does not involve an increase in the expression of cellulose
synthase genes, suggesting that the synthesis of cellulose
during ED is not regulated transcriptionally. However, the
SD-photoperiod up-regulation of VvLAC14 suggests that
the potential increase in lignin synthesis during ED could
be transcriptionally regulated.
A number of roles have been proposed for sugars in
freezing tolerance, including osmotic effects (Sakai and
Larcher 1987), decrease of ice nucleation point in super-
cooled liquid (Gunnink 1989), cryoprotection of proteins
and membranes (Ashworth and others 1993), and promo-
tion of glass formation (Levine and Slade 1998). Therefore,
it is possible sugars act in several capacities to affect
Fig. 5 Effect of low temperature on the accumulation of soluble
sugars in buds of V. vinifera cv. Thompson seedless. Dormant buds
were exposed to low (5 °C) (cooled) and ambient (14 °C) (non-
cooled) temperatures for 3 weeks. Soluble sugars were extracted and
measured by gas chromatography. Values are the average of three
biological replicates and bars correspond to s.d. and asterisk indicates
significant differences (Student’s ttest a=0.05)
Fig. 6 Effect of low temperature and photoperiod on the expression
of dehydrin genes (VvDHNs) in grapevine buds. aSingle-bud cuttings
of V. vinifera cv. Thompson seedless grapevines exposed to ambient
(14 °C) (non-cooled) and low temperatures (5 °C) (cooled) for
2 weeks. bV. vinifera cv. Italia melhorada exposed to LD and SD-
photoperiod for 8 weeks. Transcript levels were determined by RT-
qPCR, normalized against VvUBIQUITIN. Samples maintained at
ambient temperature 14 °C (non-cooled) serve as controls in low-
temperature experiments, and vines exposed to SD-photoperiod serve
as controls in the photoperiod experiment. Values are the average of
three biological replicates each with three technical repetitions, bars
represent s.d. and asterisk indicates significant differences (Student’s
ttest a=0.05)
J Plant Growth Regul
123
freezing tolerance depending on the tissue or conditions.
An increase of total soluble sugars and a decrease in starch
content have been observed coincidentally with an increase
in freezing tolerance in many plant species (Levitt 1980).
In this study, starch accumulation in grape buds was
associated with dormancy and starch breakdown and the
subsequent increase in sugar content with CH. Interest-
ingly, it has been reported that in V. amurensis, a wild
grapevine species with remarkable cold tolerance, the
expression of genes coding for starch-degrading enzymes
such as a-amylases was up-regulated by cold stress (Xin
and others 2013). This result was confirmed in V. vinifera
by Rubio and others (2014), who demonstrated that diverse
isogenes coding for a-amylases was up-regulated in dor-
mant buds exposed to low temperatures.
Recently, several studies have shown that the accumu-
lation of dehydrins (DHNs) and other stress proteins plays
an important role in the acclimation of woody plants to
unfavorable temperatures (Kosova and others 2007). DHNs
are a class of hydrophilic, thermostable stress proteins with
a high number of charged amino acids that belong to the
group II Late Embryogenesis Abundant (LEA) family.
Genes that encode these proteins are expressed during late
embryogenesis, as well as in vegetative tissue subjected to
drought, low-temperature and high-salt conditions (Ny-
lander and others 2001). Four dehydrin genes were iden-
tified in the genome of V. vinifera (VvDHNs), two
belonging to YnSKn type VvDHNs (VvDHN1,VvDHN4),
and two to SKn type VvDHNs (VvDHN2,VvDHN3), and
their expression pattern and stress response varied between
them (Yang and others 2012). In this study, low tempera-
tures up-regulated, whereas SD-photoperiod down- regu-
lated the expression of VvDHN1, VvDHN2, and VvDHN3.
Because, SD-photoperiod induces ED and low tempera-
tures induce CH in grapevine buds, it is likely that these
cold-induced VvDHNs are associated with the acquisition
of CH. Interestingly, it has been reported that V. riparia
(VrDHN1) protects lactate dehydrogenase (LDH) from
freeze–thaw damage more effectively than bovine serum
albumin (BSA), a protein with a known cryoprotective
function (Hughes and Graether 2011; Hughes and others
2013). In other woody perennials, DHN expression has
been associated with both ED and CH. In blueberry, DHN
proteins accumulate during cold acclimation and a rela-
tionship between the abundance of DHNs and CH has been
established (Arora and others 1997). In birch (Betula
pubescens), SD-photoperiod and low temperature induce
the expression of BPuDHN1, whereas BPuDHN2 was
exclusively induced by low temperatures (Welling and
others 2004). The potential significance of DHNs in the
acquisition of CH lies in the fact that plant cells undergo
dehydration during freezing stress due to the presence of
ice in extracellular spaces (Levitt 1980).
Acknowledgments The financial support of FONDECYT Project
1140318 is gratefully acknowledged.
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