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Endodormancy (ED) and cold hardiness (CH) are two strategies utilized by grapevine (Vitis vinifera L.) buds to survive unfavorable winter conditions. Each phenomenon 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 structural 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 necessary for the further development of CH.
Relationship Between Endodormancy and Cold Hardiness
in Grapevine Buds
´n Rubio
´bora Dantas
Ricardo Bressan-Smith
Francisco J. Pe
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
(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
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
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
¨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
Laboratorio de Bioquı
´mica Vegetal, Facultad de Ciencias,
Universidad de Chile, Casilla 653, Santiago, Chile
Centro de Ciencias e Tecnologias Agropecuarias,
Universidade Estadual do Norte Fluminense, Av Alberto
Lamego 2000, Campos dos Goytacazes, RJ, Brazil
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
parameter which is an estimate of the mean time required
to reach 50 % bud break under forced conditions (Pe
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
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
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
from 200 to 280 °C at a rate of 5 °C min
. 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
´, Brazil (50120S) and exposed to different
J Plant Growth Regul
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
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
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)
(0.5 lg9lL
primer, 1 lL dNTP mix (10 mM), and Superscript
(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
required to reach 50 % bud break under forced conditions). Values of
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.
J Plant Growth Regul
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
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.
Seasonal Variations of Cold Hardiness
and Endodormancy in Grapevine Buds
The depth of dormancy measured as BR
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
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
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
3 weeks of exposure to cold, starch content was reduced by
50 mg GFW
, whereas in buds exposed to ambient tem-
peratures (14 °C) for the same period of time, it was
reduced only by 10 mg GFW
(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
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).
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
Overlapping BR
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
2.1 ±0.2
VvCSLE 1.1 ±0.1 1.0 ±0.2
1.5 ±0.2 1.1 ±0.2
VvCSLG 1.0 ±0.3 1.0 ±0.4 1.1 ±0.4
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
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
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
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
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
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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... Remarkably, perhaps because of the widespread acceptance of HC, surprisingly few studies have documented the natural dynamics of bud dormancy in field conditions, particularly in climates where chilling may be quantitatively lacking. The few studies that have documented bud dormancy have limited the seasonal window to autumn and winter (Kühn, Ormeño-Núñez, Jaque-Zamora, & Pérez, 2009;Rubio, Dantas, Bressan-Smith, & Pérez, 2016) or how chilling influences this transition (Perez, Rubio, & Ormeno-Nunez, 2007). ...
... This concurs with cv. Thompson Seedless in Santiago of Chile (34° S) which develops dormancy from mid-summer under critical photoperiod of about 13 h and at a mean daily temperature of 21°C reaching peak-dormancy by the end of autumn (Kühn et al., 2009;Parada, Noriega, Dantas, Bressan-Smith, & Pérez, 2016;Rubio et al., 2016). Dormancy onset in the Flame Seedless cultivar occurred at 12-12.50 h photoperiod. ...
... Thompson Seedless has a milder expression of dormancy in temperate climates in comparison to cv. Flame Seedless (Kühn et al., 2009;Parada et al., 2016;Rubio et al., 2016). ...
Full-text available
Abstract Grapevine (Vitis vinifera L.) is the most widely cultivated fruit crop worldwide, contributing substantially to rural economies. The production cycle and productivity depend on seasonal cues and can range from a strongly deciduous habit in cool‐temperate climates to evergreen in subtropical and tropical climates. The influence of the different seasonal conditions on the dynamics of the perennating bud, including the degree of growth and metabolic quiescence, cell cycle status and internal tissue oxygen status between different climatic zones is largely unknown. This knowledge is important for adapting to changing climate conditions and for crop expansion to wider regions. This study investigated the growth and metabolic physiology of the perennating bud of commercially grown cv. Flame Seedless table grapes from Mediterranean and subtropical climate in Western Australia, from summer until late winter. Climate data were obtained, showing differences in minimum (night) temperature between the two climates, reflected by differences in calculated chilling units. Bud dormancy increased during autumn of both climates;however, the onset and depth of dormancy of buds from the subtropical region were attenuated relative to the Mediterranean condition. Stark contrasts were also observed in metabolism. The respiration of subtropical‐grown buds increased over fivefold during late autumn and winter, while that of Mediterranean‐grown buds increased less than twofold. This was also reflected in less desiccation of the subtropical‐grown buds, and an apparently greater degree of hypoxia within the bud during late winter, prior to bud burst. Collectively, these data show pronounced differences in growth and metabolic physiology of commercially grown table grapes, which provide a foundation for investigating the influence of differing climate and seasonality on the growth and productivity of table grapes and how these may be managed through breeding and agronomy.
... Photoperiod (Fennell and Hoover, 1991;Wake and Fennell, 2000) and physical maturation processes (Pouget, 1963) influence the subsequent transition into dormancy sensu stricto; however, the transition is diffuse (Lavee and May, 1997). Data of several grapevine varieties from 26° to 34° latitude suggest that the depth of dormancy increases to a maximum prior to, or during, early winter (Lavee and May, 1997;Rubio et al., 2016;Parada et al., 2016;Zheng et al., 2018a). Several studies show that cold hardiness and responses to chilling correlate well with bud phenology and bud burst in autumn and winter (Ferguson et al., 2011(Ferguson et al., , 2014Kovaleski et al., 2018;Kovaleski and Londo, 2019). ...
... Carignan (43°36ʹN) (Pouget, 1963;Nigond, 1967), and reasonably well with cv. Thompson Seedless (33°34ʹS) (Rubio et al., 2016(Rubio et al., , 2019Parada et al., 2016), cv. Chardonnay, and cv. ...
... However, according to more binary definitions, as related to the capacity to resume growth, the physiology of buds in late summer is quite unexplained. In contrast, the growth physiology of buds collected from late autumn onwards, following the commencement of chilling, are congruent with the contemporary literature on grapevine (Rubio et al., 2016;Zheng et al., 2018a). The question of why this pronounced disconnect during late summer was not observed by other studies cited earlier may reflect genotypic differences which remain to be explored. ...
Full-text available
Grapevine (Vitis vinifera L.) displays wide plasticity to climate, however the physiology of dormancy along a seasonal continuum is poorly understood. Here we investigated the apparent disconnect between dormancy and the underlying respiratory physiology and transcriptome of grapevine buds, from bud set in summer to bud burst in spring. The establishment of dormancy in summer was pronounced and reproducible, however this was coupled with little or no change in physiology, indicated by respiration, hydration and tissue oxygen tension. The release of dormancy was biphasic; the depth of dormancy declined substantially by mid-autumn, while the subsequent decline towards spring was moderate. Observed changes in physiology failed to explain the first phase of dormancy decline, in particular. Transcriptome data contrasting development from summer through to spring also indicated that dormancy was poorly reflected by metabolic quiescence during summer and autumn. Gene ontology and enrichment data revealed the prevailing influence of abscisic acid (ABA)-related gene expression during the transition from summer to autumn, and promoter motif analysis suggested photoperiod may play an important role in regulating ABA functions during the establishment of dormancy. Transcriptomic data from later transitions reinforced the importance of oxidation and hypoxia as physiological cues to regulate the maintenance of quiescence and resumption of growth. Collectively these data reveal a novel disconnect between growth and metabolic quiescence in grapevine following bud set, which requires further experimentation to explain the phenology and dormancy relationships.
... However, the buds of grapevine, as well as the buds of other deciduous fruit tree species, in addition to enter into ED acclimate to the cold during the fall-winter season and develop cold hardiness or freezing tolerance. This cold acclimation (CA) occurs only when the buds are in the endodormant state [10,3,11]. Although, ED and cold hardiness are two strategies utilized by grapevine buds to survive unfavorable winter conditions, their relationships is poorly understood. To investigate which molecular processes and physiological pathways are activated or repressed by ED and cold hardiness, and which are shared by both stimuli, we used RNA-sequencing (RNA-seq) technology, which allows analysis of massive amounts of expressed genes [12], and has been successfully applied in the investigation of dormancy and cold hardiness in grapevine buds [13]. ...
... ED and cold hardiness are two phenomena experienced by the buds of most deciduous fruit trees, and are crucial for tree survival during the winter season, and for their sprouting during spring. In grapevine buds, both phenomena are regulated by different environmental cues, ED is triggered by decreasing photoperiod [1,2,3], while cold hardiness is triggered by LT [10,11]. Here, we compared the transcriptomic data of grapevine buds exposed to LT and SD by means of RNA-seq technology to investigate the role of LT in ED, cold hardiness and budbreak in grapevine. ...
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Background: With respect to grapevine buds, short days (SDs) induces endodormancy (ED), while low temperature (LT) induces cold hardiness. However, the relationship between both of these environmental cues has been explored little. In this study, transcriptomic data based on an RNA-sequencing (RNA-seq) analysis of grapevine buds exposed to LT and SDs were compared. Results: A total of 6121 differentially expressed genes (DEGs) were identified in the comparison between grapevine buds subjected to LT and control buds, while 1336 were identified in the comparison between grapevine buds subjected to SDs and long days (LDs). Cluster analysis showed that most gene differentially expressed in response to SDs were downregulated, while most genes differentially expressed in response to LT were upregulated. A small number of the DEGs were simultaneously upregulated or downregulated in response to LT and the SDs, while conversely, a large number of them were downregulated in response to SDs but upregulated in response to LT. Gen Ontology (GO) enrichment analysis of the DEGs downregulated in response to SDs and upregulated in response to LT indicated that most of these DEGs were related to the cell cycle. These results were somewhat surprising, since although cell cycle genes are downregulated during ED of grapevine buds as a consequence of SD conditions, their upregulation caused by LT was unexpected, since in other species, these genes are downregulated in response to LT. Conclusion: Repression of transcriptome and cell cycle genes in grapevine buds in response to SD, and massive upregulation of transcriptome and cell cycle genes in response to LT support the idea that SD induces the ED, and that the LT induces the release of the buds from the ED.
... Only DHN1, DHN2, and DHN3 were upregulated in V. vinifera cv. Thompson seedless dormant buds following exposure to cold temperature and downregulated when exposed to shorter photoperiod (Rubio et al. 2016). A dehydrin with a predicted weight of 13.3 kDa, likely DHN1, was upregulated during seed development following salt and drought stress, as well as ABA application (Hanana et al. 2014). ...
Full-text available
Winter survival of Vitis vinifera Linnaeus in cool climate viticultural areas can be jeopardized due to inadequate cold hardiness. Dehydrins are a family of proteins commonly found in plant tissue in response to dehydration stress and cold exposure. To determine their presence and relationship to cold hardiness in overwintering grapevines, compound buds of V. vinifera cv. Sauvignon blanc were sampled from a commercial vineyard every two to three weeks throughout the 2016-2017 winter. Proteins were extracted and separated by SDS-PAGE, and potential dehydrins were immunoblotted with a commercial antibody raised against the dehydrin K-segment consensus sequence. Six protein bands were identified in four Sauvignon blanc clones at 23, 26, 35, 41, 48 and 90 kDa, showing a serological relation to dehydrins due to their reaction with the K-segment antibody. The bands at 23, 41, 48, and 90 kDa were confirmed as dehydrins following trypsin digestion and LC-MS/MS with Mascot analysis. Their fluctuations throughout the dormant season were quantified by immunoblotting and three patterns emerged: the 23, 26 and 35 kDa proteins peaked immediately prior to deacclimation; the 41 and 48 kDa proteins peaked during maximum hardiness and decreased towards deacclimation while the 90 kDa plateaued during the same period. Maximum hardiness and relative dehydrin band intensity were positively correlated (p < 0.050) for all but the 23 kDa protein. The variation in accumulation patterns and relationships to cold hardiness indicates that these dehydrin proteins are likely regulated by different molecular processes and could play different roles in cryo-protection throughout dormancy.
... The changes in water and starch content in the vine tissues responded slowly to the variation of temperatures (either low or high), occurring during ecodormancy or prior to budbreak. Although transition to endodormancy is primarily driven by short-day photoperiod, a significant role in the subsequent acquisition of cold hardiness depends on the temperature exposition (Kovaleski et al., 2018;Rubio et al., 2016). In our study, autumn conditions were consistent with climate change increasing temperatures (mainly higher at night, data not shown) and the presence of plastic cover in the vineyard could have prevented a regular initiation of chilling accumulation. ...
In the context of climate change, in which some extreme weather and climate events have increased in frequency and intensity because of global warming, adaptive techniques in viticulture have become necessary to reduce the resulting negative impacts. This study on four table grape cultivars has evaluated the effects of different winter pruning treatments (time of pruning) on phenology, fruit composition and starch content in canes and ≥two-year-old wood. By shifting the pruning time from leaf fall up to budbreak (BBCH 08), a neutral response on yield and berry quality parameters (TSS, TA and pH) was observed for the 4 cultivars. Late pruning treatments resulted in shorter shoot lengths and delayed phenological stages for the early ripening cultivars. The partitioning of starch between canes and older wood was almost similar, although lower in canes, with average values of 13% and a significant reduction at budbreak in order to release soluble sugars for the initial vegetative growth. Starch was mainly located in newly formed xylem, i.e., in the parenchymatic rays where amyloplasts are located, whereas a smaller amount of starch was visible in the other tissues (phloem, cortex). The possibility of a late pruning until over budbreak can be considered a practice to avoid some late spring frost risks, often occurring in viticultural areas. Thus, a ‘precise’ application of winter pruning into a global warming viticulture context could have potential benefits, performing a cost-effective tool management without negative (or very limited for early cultivars) effects on yield and quality of table grapes.
... Change in cold hardiness in peach is closely related to the soluble sugar content and the accumulation of a 60 kDa dehydrin protein [49]. Our results indicate that cold hardiness is highly dependent on the depth of dormancy and was improved by ethephon applications (Figure 2B), which is generally consistent with other reports on grapevine [50], peach [6], and magnolia (Magnolia wufengensis) [51]. Peach buds acquire cold hardiness primarily via the supercooling of intracellular water, and it was shown that ethephon enhances supercooling in peach buds through increasing soluble sugar content [52]. ...
Full-text available
Spring frosts exacerbated by global climate change have become a constant threat to temperate fruit production. Delaying the bloom date by plant growth regulators (PGRs) has been proposed as a practical frost avoidance strategy. Ethephon is an ethylene-releasing PGR found to delay bloom in several fruit species, yet its use is often coupled with harmful effects, limiting its applicability in commercial tree fruit production. Little information is available regarding the mechanisms by which ethephon influences blooming and bud dormancy. This study investigated the effects of fall-applied ethephon on bud phenology, cold hardiness, and hormonal balance throughout the bud dormancy cycle in peach. Our findings concluded that ethephon could alter several significant aspects of peach bud physiology, including accelerated leaf fall, extended chilling accumulation period, increased heat requirements, improved cold hardiness, and delayed bloom date. Ethephon effects on these traits were primarily dependent on its concentration and application timing, with a high concentration (500 ppm) and an early application timing (10% leaf fall) being the most effective. Endogenous ethylene levels were induced significantly in the buds when ethephon was applied at 10% versus 90% leaf fall, indicating that leaves are essential for ethephon uptake. The hormonal analysis of buds at regular intervals of chilling hours (CH) and growing degree hours (GDH) also indicated that ethephon might exert its effects through an abscisic acid (ABA)-independent way in dormant buds. Instead, our data signifies the role of jasmonic acid (JA) in mediating budburst and bloom in peach, which also appears to be influenced by ethephon treatment. Overall, this research presents a new perspective in interpreting horticultural traits in the light of biochemical and molecular data and sheds light on the potential role of JA in bud dormancy, which deserves further attention in future studies that aim at mitigating spring frosts.
... The time required for each sample to reach budbreak, including right-censored observations of the buds that did not break during the treatment, was adjusted to the survival distribution function by the nonparametric Kaplan-Meier method [23]. The first sample of the year collected in early January before the onset of ED [24,25] was used as a reference of the behaviour of budbreak, when growth was not restricted within the buds (paradormant buds). A log-rank test was performed to compare the estimated survival distributions of the reference sample with samples collected at other dates. ...
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Changes in the level of hydrogen peroxide (H2O2) is a good indicator to monitor fluctuations in cellular metabolism and in the stress responses. In this study, the changes in H2O2 content during bud endodormancy (ED) and budbreak were analysed in grapevine (Vitis vinifera L.). The results showed a gradual increase in the H2O2 content during the development of bud ED, which was mainly due to an increase in the activity of peroxidases (PODs). The maximum H2O2 content reached in the grapevine buds coincided with the maximum depth of bud ED. In contrast, during budbreak, the H2O2 content decreased. As the plant hormones cytokinin (CK) and auxin play an important role in budbreak and growth resumption in grapevine, the effect of exogenous applications of H2O2 on the expression of genes involved in CK and auxin metabolism was analysed. The results showed that H2O2 represses the expression of the CK biosynthesis genes VvIPT3a and VvLOG1 and induces the expression of the CK-inactivating gene VvCKX3, thus reducing potentially the CK content in the grapevine bud. On the other hand, H2O2 induced the expression of the auxin biosynthesis genes VvAMI1 and VvYUC3 and of the auxin transporter gene VvPIN3, thus increasing potentially the auxin content and auxin transport in grapevine buds. In general, the results suggest that H2O2 in grapevine buds is associated with the depth of ED and negatively regulates its budbreak.
At the end of the summer season, grapevine buds (Vitis vinifera L) grown in temperate climates enter a state of winter recess or endodormancy (ED), which is induced by the shortening of the photoperiod, and during this period, the buds accumulate sucrose. In this study, we investigated whether the shortening of the photoperiod regulates the accumulation of sucrose in the buds in the same way as it regulates its entry into the ED. Because sucrose accumulation is regulated by genes that control its transport and degradation, the effect of the SD photoperiod and the transition of buds from paradormancy (PD) to ED on the expression of sucrose transporter (VvSUTs) and invertase genes (VvINVs) was studied. To analyze the possible role of sucrose during ED development, its effect on bud swelling and sprouting was studied on dormant and nondormant buds under forced growth conditions. The results showed that the SD photoperiod upregulates the expression of the VvSUT genes and downregulates that of the VvINV genes in grapevine buds. Additionally, during the transition of buds from PD to ED, the sucrose content increased, the expression of the VvINV genes decreased, and the expression of the VvSUT genes did not change significantly. Sucrose delayed bud swelling and sprouting when applied to dormant buds but had no effect when applied to nondormant buds. Therefore, we concluded that ED development and sucrose accumulation were synchronized events induced by the SD photoperiod and that a sucrose peak marks the end of ED development in grapevine buds.
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The effects of global warming on plants are not limited to the exacerbation of summer stresses; they could also induce dormancy dysfunctions. In January 2020, a bud break was observed in an old poly-varietal vineyard. Meteorological data elaboration of the 1951–2020 period confirmed the general climatic warming of the area and highlighted the particular high temperatures of the last winter. Phenological records appeared to be significantly correlated to wood hydration and starch reserve consumption, demonstrating a systemic response of the plant to the warm conditions. The eight cultivars, identified by single-nucleotide polymorphism (SNP) profiles and ampelographic description, grown in this vineyard showed different behaviors. Among them, the neglected Sprino, Baresana, Bianco Palmento, and Uva Gerusalemme, as well as the interspecific hybrid Seyve Villard 12.375, appeared to be the most interesting. Among the adaptation strategies to climate changes, the cultivar selection should be considered a priority, as it reduces the inputs required for the plant management over the entire life cycle of the vineyard. Hot Mediterranean areas, such as Salento, are a battlefront against the climate change impacts, and, thus, they represent a precious source of biodiversity for viticulture.
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In recent years, new vineyards have been established in southwestern Ontario. The open water of Lake Erie provides some winter protection for Vitis hybrids and winter-hardy Vitis vinifera L. cultivars in this area. However, winter damage is possible when vines are grown distant from the open water or when lakes are frozen. To better understand the risks to winter survival, the dormancy and chilling phenology were studied over three winters from 2013-2016. Ten dormant canes of two V. vinifera cultivars, ‘Chardonnay’ and ‘Riesling’, were collected weekly from September 1 until March 30 from the mature vines in a commercial vineyard located at St. Williams (Ontario). The canes defoliated in early October, and the endodormancy was completed by the end of December. The cumulative chilling hours (0-7.2 °C) from defoliation until the completion of endodormancy were averaged 606 hours for ‘Chardonnay’ and 665 hours for ‘Riesling’. ‘Chardonnay’ buds were slightly less hardy than ‘Riesling’ to cold temperatures, with a threshold of about -24 °C for ‘Chardonnay’ and -25 °C for ‘Riesling’. Most primary buds of both cultivars died after February 16, 2015, and more than half died after February 12, 2014, due to severe low temperatures of -33.1 and -26 °C, respectively.
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Attempts to discuss the various aspects of plant dormancy can be bewildering due to the excessive number of nonphysiological, independent terms that have arisen over the years. In the context of field observations and orchard management, this terminology has often been adequate. However, in the complex realm of scientific description of the processes that constitute dormancy, the terminology has not been able to keep pace with physiological investigation. In 1985, a set of alternative terms, endodormancy , ectodormancy , and ecodormancy , were suggested to improve the situation (14). During the past 2 years, R. Darnell, J. Early, G. Martin, and I have reviewed the dormancy literature to evaluate the strengths and weaknesses of new and previous terms. At various times, N. Arroyave, R. Biasi, R. Femandez-Escobar, G. Stutte, and others from around the world have contributed greatly to discussion and critical analysis of the requirements for a physiological nomenclature. In 1986, ectodormancy was replaced by paradormancy (16) due to the former’s spoken and written similarities to ecodormancy . This paper summarizes the communicative burden presented by the current terminology, the evolution of the new terms, the universal classification system in which the terms are used, and the implications for future dormancy research. These topics are presented in greater detail elsewhere (15).
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Cold hardiness and endogenous levels of soluble sugars were monitored during the dormant season for Chardonnay and Riesling (Vitis vinifera L) dormant buds and stem cortical tissues. Endogenous levels of glucose, fructose, raffinose, and stachyose were strongly associated with cold hardening, increasing from the onset of cold acclimation in August to maximum cold hardiness in December and January. During dehardening in March and April, endogenous levels of these sugars dropped as temperature increased. A high ratio of glucose and fructose to sucrose coincided with maximum cold hardiness, and a low ratio was associated with the dehardened condition in fall and spring. Sucrose levels, however, were not associated with cold hardiness in either cultivar. Neither cold hardiness nor soluble sugars of grape tissues were influenced by a late harvest compared to harvest at normal fruit maturity.
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Thermal analysis (TA) was used to evaluate dormant bud cold hardiness of nine Vitis cultivars weekly during the 1993–94 dormant period. TA hardiness estimates were expressed as either mean low-temperature exotherm temperature (MLTE) or temperatures lethal to 10% (LT 10 ), 50% (LT 50 ), or 90% (LT 90 ) of dormant bud sample. A destructive freeze on 19 Jan. 1994 presented an opportunity to compare dormant bud field survival with laboratory estimates of bud hardiness that had been derived from TA. Vineyard air temperatures of –24C caused primary bud kill that ranged from a mean of 15% with `Concord' to 100% with `Viognier'. With the exception of `Viognier' and one of two `Cabernet Sauvignon' clones, field mortality levels were accurately bracketed by TA estimates of LT 10 , MLTE, and LT 90 values, which had been obtained in the week preceding the freeze. `Viognier' bud hardiness was overestimated by ≈1.5C, and the hardiness of `Cabernet Sauvignon clone UCD#6' was underestimated by <1C. The discrepancy with `Viognier' may have been related to prior destruction of primary buds by bud necrosis and the misinterpretation of secondary bud exotherms as due to primary buds.
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The technique of differential thermal analysis was used to examine midwinter hardiness of bud and stem tissues of V. riparia. Low temperature freezing points (exotherms) were found to occur in both stem and deacclimated bud tissues. Exotherms were not exhibited by fully acclimated buds. The temperature at which the stem tissue exotherm occurred was independent of cooling rate and duration of a thawing pretreatment of 6-48 h. The initiation temperature of the bud exotherms varied with cooling rate and duration of a thawing pretreatment of 6-48 h, an increase in either caused the bud exotherm to occur at a higher temperature than that of the control. It is postulated that deep supercooling of the intracellular water in stem and bud tissues is the characteristic which limits the northern distribution of V. riparia.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1, 2, 3, 4.
Nonstructural carbohydrates (NSC), including water soluble carbohydrates (WSC) are thought to serve an important role in freezing tolerance of many plants. Raffinose family oligosaccharides (RFOs) are α-galactosyl derivatives of sucrose. The most common RFOs are the trisaccharide raffinose, the tetrasaccharide stachyose, and the pentasaccharide verbascose. RFOs are nearly ubiquitous in the plant kingdom and are found in a large variety of seeds from many different families. Severely cold winter temperatures can significantly impact grapevine productivity through tissue and organ destruction caused by freeze injury. Crop loss and the need to retrain vines after bud, cane, and trunk injury mean financial loss, often for one or more years. Buds of grapevine (Vitis vinifera L.), grown at the vineyard of the Institute of Fruit Science, Vegetable Science and Viticulture, University of Hohenheim, Germany, were sampled during winter and analyzed for their concentration of soluble sugars (i.e. glucose, fructose, sucrose, raffinose and stachyose) and thermal analysis was performed to determine their freezing points. Freezing of extracellular water was recorded from -5 to -16 °C with a minimum at the beginning of January; freezing of intra-cellular water was recorded from -11 to -24 °C. Apical buds are very important organs as they determine further growth and development of tree species. Bud physiological state, including saccharide metabolism, determines their growth activity. The concentration of soluble sugars was highest by the end of December. Sugar concentrations in basal buds were significant higher than in buds from intermediate and apical shoot sections. A significant correlation could be proofed between sugar concentrations (i.e. raffinose and stachyose) and air temperature before sampling. But there was no correlation between freezing temperature of extra-cellular or intra-cellular water and soluble sugars in bud tissues.
Modeling can be useful in predicting budbreak, especially in the subtropics, where special treatments may be required to supplement natural chilling. It has limited practical use in the cooler temperate regions, as chilling is always adequate. The Utah Model appears to be reliable in the cooler regions, whereas the dynamic model may be more useful in warmer zones. Regardless of the model used, attention must be paid to species and cultivar, and appropriate adjustments made. To standardize methods for determining the stage of rest, researchers should first decide upon the portion of the plant to be used as the experimental unit (e.g., single-node cutting, entire cutting, rooted cutting, whole plant). This will vary, of course, with the purpose of the research. Single-node cuttings are of little use for determining response of whole trees, as bud development in the latter will be affected by correlative inhibition, including apical dominance. Cuttings bearing many buds will be the better choice for researchers wishing to predict the field response. The larger the cutting, the better the expected response. The source of cuttings is also important, especially for theoretical studies; previous year's shoots are normally used, but their vigor could affect response. Therefore, selected shoots should be similar in length and taken from similar positions on the plant. The environmental conditions to be used, especially temperature, must be established for each species and cultivar used. A temperature of 15-20°C should be adequate. Light should be supplied unless it can be shown to have no effect. The criteria to be used for evaluation of response, including observation time and stage of bud development, must also be agreed upon. These conditions will undoubtedly differ among species, but some agreement should be possible within species. Greening of the bud scales alone appears to be too limited a response to predict behavior under orchard conditions. On the other hand, full bloom may be difficult to obtain with small cuttings. Recording time to reach a given stage of development ("speed of budbreak") appears to be superior to recording the percentage budbreak during a specific time interval. The problem with the method is selecting a stage that will be reached by partially chilled buds so that comparisons can be made throughout the period of dormancy. Otherwise, response can be quantified only during the later stages of dormancy. Despite all precautions, differences in results will occur; e.g., contrast the results of several workers in using single-node cuttings of apple (see above). Such differences may reflect differences in choice of plant material, or in environmental conditions during the previous growing season. Some of these problems might be solved by using a cooperative approach. For example, experimental protocols could be established and tested by researchers at several locations, using the same cultivars, and/or cuttings could be exchanged, so that the responses of cuttings from the same trees could be compared at different locations.
The purpose of this study was to identify morphological, physiological, and biochemical changes in Vitis genotypes in response to photoperiod regimes. Experiments were conducted under greenhouse conditions using cold-sensitive Cabernet franc (Vitis vinifera) and cold-tolerant Couderc 3309 (3309C, V. riparia x V. rupestris) and Concord (V. labruscana). Potted vines were exposed to short day (SD) (8 hr) or long day (LD) (16 hr) for 4, 6, and 8 weeks. Shoot growth, periderm formation, dormancy, freezing tolerance (lethal temperature that kills 50% of primary buds: LT50), and soluble sugar concentrations in leaf and bud tissues were examined. Shoot growth slowed in all cultivars under SD accompanied with increased periderm formation and dormancy depth. Concord initiated these changes first, followed by 3309C, then Cabernet franc. The three cultivars did not show differences in freezing tolerance under LD, with LT50 ranging between -6.1 and -8.1 degrees C. However, freezing tolerance increased by 0.7, 2.0, and 2.7 degrees C after 4, 6, and 8 weeks under SD, respectively. Freezing tolerance of Concord increased after 4 weeks of SD treatment, whereas that of 3309C and Cabernet franc did not increase until after 6 weeks of SD treatment. Among all sugars, raffinose had distinctive responses associated with photoperiod, remaining low and similar (0.5 to 2.3 mg/g dry weight) under LD. Under SD, raffinose concentration was generally higher, ranging from 2.2 to 5.7 mg/g dry weight in leaves and 1.6 to 3.7 mg/g dry weight in buds, with cold-tolerant 3309C and Concord accumulating higher concentrations compared to cold-sensitive Cabernet franc. These results suggest that raffinose accumulation might be an early step in response to photoperiod coinciding with slowed shoot growth, the induction of endodormancy, and the initial acquisition of freezing tolerance.