Cambrollé et al%2c 2014 Evaluating tolerance to calcareous soils in Vitis vinifera ssp.

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Evaluating tolerance to calcareous soils in Vitis vinifera ssp.
sylvestris
J. Cambrollé & J. L. García & R. Ocete &
M. E. Figueroa & M. Cantos
Received: 9 April 2015 /Accepted: 24 June 2015
# Springer International Publishing Switzerland 2015
Abstract
Aims We evaluate tolerance to soil lime in Vitis vinifera
ssp. sylvestris to explore the physiological mechanisms
involved in plant tolerance to calcareous soil conditions.
Methods The effects of soil CaCO3 content (0–60%) on
growth, photosynthetic performance and mineral nutri-
ent content were analyzed in Vitis vinifera ssp. sylvestris
from two populations, native to calcareous and non-
calcareous soils, respectively, and in the lime-tolerant
grapevine rootstock B41B^.
Results The reduction in relative growth rate of plants
exposed to 20 and 40 % CaCO3 was around 70 % in the
B41B^ rootstock, whereas the reduction inwild grapevine
plants from populations native to calcareous and non-
calcareous soils was around 30 and 40 %, respectively.
Wild grapevines showed a greater ability to maintain the
integrity of their photosynthetic apparatus despite the
nutritional disorders caused by lime-stress conditions in
comparison to grapevine rootstock B41B^. Plants from
the population found in highly calcareous soil were ca-
pable of maintaining Fe uptake and translocation to
leaves even under extremely high lime conditions
(40 % CaCO3) and were more efficient in controlling leaf
concentrations of the main macronutrients in comparison
to wild grapevines from the other studied population.
Conclusions Variation in the maintenance of essential
mineral nutrient status may be a crucial factor in plant
tolerance to calcareous soil conditions.
Keywords Calcareous soils . Grapevine .Mineral
nutrition . Photosynthesis
Abbreviations
A Net photosynthetic rate
Chl a Chlorophyll a
Chl b Chlorophyll b
Ci Intercellular CO2 concentration
Cx+c Carotenoids
F0 Minimal fluorescence level in the dark-adapted
state
Fm Maximal fluorescence level in the dark-
adapted state
Plant Soil
DOI 10.1007/s11104-015-2576-4
Responsible Editor: Hans Lambers.
J. Cambrollé : R. Ocete :M. E. Figueroa
Facultad de Biología, Universidad de Sevilla, P.O. Box 1095,
41080 Sevilla, Spain
R. Ocete
e-mail: ocete@us.es
M. E. Figueroa
e-mail: figueroa@us.es
J. L. García :M. Cantos
Instituto de Recursos Naturales y Agrobiología de Sevilla
(C.S.I.C.), P.O. Box 1052, 41080 Sevilla, Spain
J. L. García
e-mail: jlgarcia@irnase.csic.es
M. Cantos
e-mail: cantos@irnase.csic.es
J. Cambrollé (*)
Departamento Biología Vegetal y Ecología, Facultad de
Biología, Universidad de Sevilla, Av. Reina Mercedes 6,
41012 Seville, Spain
e-mail: cambrolle@us.es
Fs Steady state fluorescence yield
Fv Variable fluorescence level in the dark-adapted
state
Fv/Fm Maximum quantum efficiency of PSII
photochemistry
ΦPSII Quantum efficiency of PSII
Gs Stomatal conductance
RGR Relative growth rate
Introduction
Grapevine (Vitis vinifera L.) is the most economically
important deciduous fruit crop in the world. It is the
source for a variety of products in the food, wine and
pharmaceutical industries (Hvarleva et al. 2009; Gao
et al. 2010). Cultivated grapevine is an extremely het-
erogeneous species, with an estimated 10–20,000 culti-
vars in existence (Ambrosi et al. 1994). Commonly
cultivated varieties lack effective resistance to a range
of devastating diseases and to certain abiotic stresses,
such as soil salinity, water deficit or high soil lime
content, as a result of the biodiversity loss mainly caused
by human selection over a long period of time in order to
improve viticulture and oenological features.
Lime-induced chlorosis affects many annual crops
and perennial plants growing on calcareous soils and is
a major problem for grapevine and high value fruit trees,
especially in the Mediterranean region or in other semi-
arid areas (Bavaresco et al. 2006). Physiological stress
caused by calcareous soil conditions has a great impact
on fruit yield and quality in grapes (Tangolar et al.
2008). In viticulture, the most useful method to over-
come this stress is to graft the grape varieties on lime-
tolerant rootstocks (such as Fercal, 140 Ru or 41B).
Despite the many studies that have been carried out
(e.g., Bavaresco et al. 2000; Díaz et al. 2009; Nikolic
et al. 2000), the ideal rootstock has not yet been found
and the mechanisms involved in plant tolerance to cal-
careous soil conditions remain unclear.
Our group has recently demonstrated that plants of
Vitis vinifera ssp. sylvestris from a population growing
in calcareous soil exhibit high tolerance to lime stress
(Cambrollé et al. 2014), but the physiological mecha-
nisms that determine the higher tolerance to calcareous
soil conditions of this wild subspecies, compared to
other varieties, remain completely unknown.
Moreover, several key issues remain to be clarified.
Firstly, a direct comparative evaluation of lime tolerance
between this wild subspecies and a commercial variety
of grapevine has never been performed under the same
experimental conditions. Moreover, wild grapevine
populations present considerable genetic polymorphism
and wide variability (McGovern et al. 1996) and it is not
known whether the higher degree of lime tolerance
reported by Cambrollé et al. (2014) could be explained
simply by inter-population differences. This knowledge
is essential for enhancing the adaptation of vines to
calcareous soil conditions, and could expand our knowl-
edge about the physiological mechanisms involved in
plant tolerance to calcareous soil conditions.
The objectives of this study were therefore: (1) to
evaluate differences in tolerance to calcareous soil con-
ditions between wild grapevine plants from two popu-
lations, native to calcareous and non-calcareous soils,
respectively, and a lime-tolerant commercial rootstock
of grapevine, through analysis of plant growth and
physiological response to a range of soil CaCO3 con-
tents from 0 to 60%; and (2) to comparatively determine
the physiological traits involved in lime tolerance in
wild grapevine by examining the extent to which
CaCO3 content determines plant performance in terms
of effects on photosynthetic apparatus (PSII photochem-
istry), gas exchange characteristics, photosynthetic pig-
ments and concentrations of Fe, N, P, S, K and Cu
within plant tissues.
Materials and methods
Plant material and calcium carbonate treatments
The wild subspecies ofVitis vinifera (V. vinifera (L.) ssp.
sylvestris (Gmelin) Hegi) is the only native Eurasian
subspecies and represents a valuable genetic resource
for cultivated grapevines (Negrul 1938). Two natural
populations from southern Spain were selected for
study; one growing in a hypercalcic calcisol soil (FAO
et al.1999) with 62–67 % calcium carbonate (B14/Rute/
1^ population), and the other growing in a humic fluvisol
(FAO et al. 1999) with 0 % calcium carbonate (B14/
Montoro/4^ population). Both populations were located
in the Subbetic mountain range of southern Spain (Ocete
et al. 2007). In addition, plants of the hybrid rootstock
B41B^ (Vitis vinifera L. cv. Chasselas x Vitis berlandieri
Planch.) were used for comparison with the two wild
grapevine populations. This rootstock is considered to
Plant Soil
be lime-tolerant (Bavaresco et al. 2003; Pavlousec 2013)
and is used by viticulturists on calcareous soils
worldwide.
Plants were obtained by micropropagation of axillary
buds from individuals of the three study plants described
above, according to López et al. (2004). The resulting
plants were adapted to outdoor conditions following
Cantos et al. (1993), transferred to individual plastic
pots (diameter 11 cm) filled with perlite and placed in
a glasshouse with minimum-maximum temperatures of
21–25 °C, relative humidity of 40–60 % and naturaldaylight (minimum and maximum light flux: 200 and
1000 μmol m−2 s−1, respectively). Pots were carefully
irrigated with 20 % Hoagland’s solution (Hoagland and
Arnon 1938) as required. When the plantlets reached
around 20 cm in height, they were transferred to four
different calcium carbonate soil treatments: 0, 20, 40
and 60 % CaCO3 (15 replicate pots per treatment for
each Vitis accession). The different soil treatments were
prepared by mixing sterilized fine siliceous sand with
finely divided CaCO3, following Cambrollé et al.
(2014).
Growth
From each treatment, three complete plants (roots and
shoots) were harvested at the beginning, and the remain-
ing 12 at the end of the experiment (i.e., following
30 days of treatment). These plants were dried at
80 °C for 48 h and then weighed.
Relative growth rate (RGR) of whole plants was
calculated using the formula:
RGR ¼ ln Bf – ln Bið Þ � D−1 g g−1month−1� �
where Bf = final dry mass, Bi = initial dry mass (average
of the three plants from each treatment dried at the
beginning of the experiment; i.e., 12 plants per acces-
sion) and D = duration of experiment (months).
Gas exchange
Gas exchange measurements were taken from randomly
selected, fully expanded leaves (for each study plant and
calcium carbonate treatment, n=20, i.e., one measure-
ment from each of the 12 replicate plants, plus eight
extra measurements taken randomly), following 30 days
of treatment, using an infrared gas analyzer in an open
system (LI-6400, LI-COR Inc., Neb., USA). Net
photosynthetic rate (A), intercellular CO2 concentration
(Ci) and stomatal conductance to CO2 (Gs) were deter-
mined at an ambient CO2 concentration of 400 μmol
mol−1 at 20 – 25 °C, 50±5 % relative humidity and a
photon flux density of 1600 μmol m−2 s−1. Values of the
parameters A, Ci and Gs were calculated using the
standard formulae of Von Caemmerer and Farquhar
(1981).
Chlorophyll fluorescence
Chlorophyll fluorescence was measured in randomly
selected, fully developed leaves (n=20) using a portable
modulated fluorimeter (FMS-2, Hansatech Instruments
Ltd., England) following 30 days of treatment. Light-
and dark-adapted fluorescence parameters were mea-
sured at dawn (stable, 50 μmol m−2 s−1 ambient light)
and midday (1600 μmol m−2 s−1) in order to investigate
the effect of soil CaCO3 content on the sensitivity of
study plants to photoinhibition. Values of variable fluo-
rescence (Fv=Fm - F0) and maximum quantum efficien-
cy of PSII photochemistry (Fv/Fm) were calculated from
F0 and Fm. Using fluorescence parameters determined in
both light- and dark-adapted states, the following were
calculated: quantum efficiency of PSII (ΦPSII=(Fm′ –
Fs)/ Fm′), which measures the proportion of light
absorbed by the chlorophyll associated with PSII that
is used in photochemistry (Maxwell and Johnson 2000);
and non-photochemical quenching (NPQ=(Fm – Fm′) /
Fm′), which is linearly related to heat dissipation
(Maxwell and Johnson 2000).
Photosynthetic pigments
At the end of the experimental period, photosynthetic
pigments were extracted from fully expanded leaves of
plants grown under each treatment (n=12), using the
methods described in Cambrollé et al. (2011a). Pigment
concentrations (μg g−1 fwt) were calculated following
the method of Lichtenthaler (1987).
Mineral analysis
At the end of the experimental period, leaf samples were
carefully washed with distilled water and then dried at
80 °C for 48 h and ground (Cambrollé et al. 2011b).
Samples of 0.5 g each were then digested by wet oxida-
tion with concentrated HNO3, under pressure in a mi-
crowave oven to obtain the extract. Concentrations of
Plant Soil
Fe, P, S, K and Cu in the extracts were determined by
optical spectroscopy inductively coupled plasma (ICP-
OES) (ARL-Fison 3410, USA). Total N concentration
was determined by Kjeldahl digestion using an elemen-
tal analyzer (Leco CHNS-932, Spain).
Statistical analysis
Statistical analysis was carried out using Statistica v. 6.0
(Statsoft Inc.). Pearson coefficients were calculated to
assess the correlation between different variables. Data
were analyzed using one- and two-way analyses of
variance (F-test). Data were tested for normality with
the Kolmogorov-Smirnov test and for homogeneity of
variance with the Brown-Forsythe test. Tukey tests were
applied to significant test results for identification of
important contrasts.Measured differences between fluo-
rescence at dawn and midday were compared using the
Student test (t-test).
Results
Growth
Relative growth rate (RGR) decreased significantly with
external CaCO3 content (r=−0.71, p<0.001; r=−0.73,
p<0.001; r=−0.68, p<0.001, for B14/Rute/1^, B41B^
and B14/Montoro/4^ plants, respectively). In the case of
the grapevine rootstock B41B^, RGR was drastically
affected at 20 % CaCO3 and showed no response to
further increases in external CaCO3 content. There were
no significant differences between RGR in the three
study plants at 60 % CaCO3 (ANOVA, Tukey test,
p>0.05). However, at 20 and 40 % CaCO3 content,
RGR was significantly lower in B41B^ plants than in
B14/Rute/1^ and B14/Montoro/4^ wild grapevine plants
(ANOVA, Tukey test, p<0.01, in both cases).
Furthermore, at 20 % and 40 % CaCO3, the B14/Rute/
1^ plants presented significantly higher values of RGR
than those of B14/Montoro/4^ plants (ANOVA, Tukey
test, p<0.001, in both cases; Fig. 1). Plants from the B14/
Rute/1^ and B14/Montoro/4^wild populations that were
treated with 60 % CaCO3 exhibited chlorosis from
around the second week of treatment; in the case of
the B41B^ plants, leaf chlorosis was detected early in
plants exposed to external CaCO3 content of 20 % and
above.
Gas exchange
Net photosynthesis rate (A) decreased significantly with
increasing external CaCO3 content (r=−0.87, p<0.001;
r=−0.83, p<0.001; r=−0.87, p<0.001, for B14/Rute/1^,
B41B^ and B14/Montoro/4^ plants, respectively). Under
exposure to 20 and 40 % CaCO3, values of A showed a
similar trend to those of RGR, with the highest values
found in B14/Rute/1^ wild grapevine plants and signif-
icantly lower values recorded in 41B rootstock com-
pared to both wild grapevine plants (ANOVA, Tukey
test, p<0.05, in both cases). Moreover, A was directly
correlated with RGR (r=0.68, p<0.01; r=0.85, p<0.01;
r=0.66, p<0.01, for B14/Rute/1^, B41B^ and B14/
Montoro/4^ plants, respectively). Relative to the con-
trol, mean reduction of A in the 20 and 40 % CaCO3
treatments was 33 % in B14/Rute/1^, 59 % in B14/
Montoro/4^, and 74 % in B41B^ plants (Fig. 2a).
Stomatal conductance (Gs) showed a decreasing
trend with CaCO3 content in B14/Rute/1^ and B14/
Montoro/4^ wild grapevine plants. In contrast, Gs in
41B plants was drastically affected at 20 % CaCO3 and
showed no response to further increases in external
CaCO3 content (Fig. 2b). In all three cases, intercellular
CO2 concentration (Ci) showed no significant variations
up to 40 % CaCO3 content (ANOVA, p>0.05, in all
cases), while a marked increase was recorded at the
highest external CaCO3 treatment (Fig. 2c).
Chlorophyll fluorescence
Values of maximum quantum efficiency of PSII (Fv/Fm)
were lower at midday than at dawn (t-test, p<0.01, in all
cases). In B41B^ and B14/Montoro/4^ plants, Fv/Fm
measured at midday significantly decreased with in-
creasing soil lime content (r =−0.76, p<0.001;
r=−0.82, p<0.001, for B41B^ and B14/Montoro/4^
plants, respectively). Midday Fv/Fm values of B14/
Rute/1^ plants showed a slight decreasing trend with
increasing soil lime content up to the 40 % CaCO3
treatment, with a sharp decline observed at the highest
lime content, which reached significantly lower values
than those of the control (ANOVA, Tukey test,
p<0.005; Fig. 3a). Values of Fv/Fm measured at dawn
also differed in the three study plants: in the B41B^
plants, dawn Fv/Fm showed a significant decreasing
trend withincreasing external lime content (r=−0.72,
p<0.001), reaching its lowest value at 60 % CaCO3. In
contrast, in both the B14/Rute/1^ and B14/Montoro/4^
Plant Soil
wild grapevine plants, dawn Fv/Fm decreased slightly up
to the 40 % CaCO3 treatment, maintaining values
around 0.80, and then decreased substantially on expo-
sure to the highest CaCO3 level, reaching values of
around 0.50. At 20 and 40 % CaCO3 content, dawn
Fv/Fm values in B41B^ plants ranged around 0.6, and
were significantly lower than those recorded in the B14/
Rute/1^ and^14/Montoro/4^ wild grapevine plants
(ANOVA, Tukey test, p<0.05, in both cases). There
were no significant differences between the dawn Fv/
Fm values of the B14/Rute/1^ and B14/Montoro/4^
plants (two-way ANOVA, p>0.05; Fig. 3b).
Quantum efficiency of PSII (ΦPSII) was significant-
ly lower at midday than at dawn (t-test, p<0.001, in all
cases). In all three study plants, midday ΦPSII signifi-
cantly declined at 20 % CaCO3 and did not respond to
further increases in external lime content (Fig. 3c). At
dawn, ΦPSII showed a similar pattern to that of Fv/Fm,
with minimum values at 60 % CaCO3 in all three cases
and a more pronounced decline in ΦPSII values in the
B41B^ plants compared to the wild grapevine plants
(Fig. 3d).
In 41B plants, non-photochemical quenching (NPQ)
measured at midday increased significantly on exposure
to 20 % CaCO3 (ANOVA, Tukey test, p<0.005) but
showed no clear response to further increases in external
CaCO3 content. In both the B14/Rute/1^ and B14/
Montoro/4^ wild grapevine plants, NPQ at midday
showed little variation until the 40 % CaCO3 treatment,
and then increased substantially on exposure to the
highest CaCO3 level (Fig. 3e). In contrast, dawn NPQ
values of 41B plants showed an increasing trend with
CaCO3 level, whereas in both the B14/Rute/1^ and B14/
Montoro/4^ wild grapevine plants NPQ did not show a
clear relationship with increasing external lime content
(Fig. 3f).
Photosynthetic pigments
In all three study plants, pigment concentrations signif-
icantly decreased on exposure to increasing external
CaCO3 content (Chl a: r=−0.81, p<0.001; r=−0.84,
p<0.001; r =−0.82, p<0.001. Chl b: r =−0.77,
p<0.001; r=−0.81, p<0.001; r=−0.76, p<0.001, for
B14/Rute/1^, B41B^ and B14/Montoro/4^ plants, re-
spectively). In the 20 and 40 % CaCO3 treatments, both
Chl a and Chl b were significantly lower in B41B^
plants than in B14/Rute/1^ and B14/Montoro/4^ wild
grapevine plants (ANOVA, Tukey test, p<0.05, in all
cases). There were no significant differences between
the pigment concentrations of the B14/Rute/1^ and B14/
Montoro/4^ plants (two-way ANOVA, p>0.05, in both
cases; Fig. 4a and b).
Chemical analysis of plant samples
Leaf iron, nitrogen, phosphorus, potassium and copper
concentrations were similar in B41B^ rootstock and B14/
Montoro/4^ wild grapevine plants (two-way ANOVA,
p>0.05, in all cases). At 20 % CaCO3, mean values of
all the analyzed nutrients were higher in B14/Rute/1^
plants than in B41B^ and B14/Montoro/4^ plants, with
Fig. 1 Relative growth rate in
plants of V. vinifera x
V. berlandieri B41B^ (○),
V. vinifera ssp. sylvestris from the
B14/Rute/1^ population (●) and
V. vinifera ssp. sylvestris from the
B14/Montoro/4^ population (∇),
in response to treatment with a
range of external CaCO3 contents
for 30 days. Values represent the
mean±standard error, n=12
Plant Soil
these differences significant for the N, P, S, K and Cu
concentrations (ANOVA, Tukey test, p<0.05, in all
cases). Moreover, at 40 % CaCO3, leaf concentrations
of Fe, N, P and S were also significantly higher in B14/
Rute/1^ wild grapevine plants (ANOVA, Tukey test,
p<0.005, in all cases) (Fig. 5).
Leaf Fe and N concentrations of both B41B^ root-
stock and B14/Montoro/4^ wild grapevine plants de-
creased significantly with external CaCO3 level (Fe:
r=−0.87, p<0.01; r=−0.92, p<0.001. N: r=−0.70,
p<0.05; r=−0.93, p<0.001, for B41B^ and B14/
Montoro/4^ plants, respectively), whereas in the case
of the B14/Rute/1^ plants, Fe and N concentrations
showed little variation until the 40 % CaCO3 treatment,
and presented their lowest value at 60% CaCO3 (Fig. 5a
and b). In the B41B^ and B14/Montoro/4^ plants, leaf P
decreased significantly on exposure to 20 % CaCO3
(ANOVA, Tukey test, p<0.005, in both cases) but
showed no clear response to further increases in external
CaCO3 content; in the case of B14/Rute/1^ plants, leaf P
concentration did not show a clear relationship with
increasing lime content, with a marked increase occur-
ring at 20 % CaCO3 (Fig. 5c).
Leaf S concentration showed a different response to
CaCO3 level in the three study plants: In B14/Rute/1^
plants, leaf S showed no clear trend in relation to CaCO3
content until the 40 % CaCO3 treatment, and then de-
creased, reaching its lowest value at the highest CaCO3
level. Leaf S concentration in B14/Montoro/4^ wild
grapevine plants significantly decreased with external
CaCO3 content (r=−0.93, p<0.001). In contrast, in the
case of B41B^ rootstock plants, leaf S decreased at 20 %
CaCO3 and did not respond to further increases in
external CaCO3 level (Fig. 5d).
Leaf K concentration slightly decreased under expo-
sure to 40 and 60 % external CaCO3 in B14/Rute/1^
plants; however, in all three study plants, there were no
significant differences in leaf K between plants exposed
to CaCO3 and control plants (ANOVA, Tukey test
p>0.05, in all cases; Fig. 5e). There was a significant
decreasing trend in leaf Cu concentration with increas-
ing external CaCO3 level in B41B^ and B14/Montoro/4^
plants, (r=−0.80, p<0.005; r=−0.84, p<0.005, for
B41B^ and B14/Montoro/4^ plants, respectively); in
contrast, in B14/Rute/1^ wild grapevine plants, leaf Cu
showed no clear relationship with external CaCO3 con-
tent (Fig. 5f).
Discussion
The hybrid rootstock B41B^, which is used by viticul-
turists on calcareous soils worldwide, proved to be less
tolerant to lime stress than Vitis vinifera ssp. sylvestris
from the two studied populations. In our study, the
reduction in relative growth rate of plants exposed to
20 and 40 % CaCO3 was around 70 % in the B41B^
rootstock, whereas the reduction in B14/Montoro/4^ and
B14/Rute/1^ wild grapevine plants was around 40 and
Fig. 2 Net photosynthetic rate, A (a), stomatal conductance, Gs
(b), and intercellular CO2 concentration, Ci (c) in randomly select-
ed, fully developed leaves of plants of V. vinifera x V. berlandieri
rootstock B41B^ (○), V. vinifera ssp. sylvestris from the B14/Rute/
1^ population (●) and V. vinifera ssp. sylvestris from the B14/
Montoro/4^ population (∇), in response to treatment with a range
of external CaCO3 contents for 30 days. Note scale differences.
Values represent the mean±standard error, n=20
Plant Soil
30 %, respectively. The highest external CaCO3 treat-
ment caused a similar growth reduction in the three
study plants (around 60–70 % relative to the non-
calcareous control). The reduced growth recorded in
plants exposed to soil lime is likely to be attributable
to the reduction in photosynthetic carbon assimilation.
In all B41B^ rootstock plants and wild grapevines from
both populations, increasing external CaCO3 induced
considerable effects on net photosynthesis rate (A) and
stomatal conductance (Gs), with no direct relationship
between both parameters since there was no reduction in
intercellular CO2 concentration (Ci). It should be em-
phasized that the deleterious effects of 20 and 40 %
external lime on gas exchange parameters were consid-
erably more pronounced in B41B^ rootstock plants.
Moreover, at these soil CaCO3 contents, wild grapevine
plants from B14/Rute/1^ population showed consider-
ably higher values of A and Gs than the B14/Montoro/4^
plants. Three-year-old plants of V. vinifera L. cv BPinot
Blanc^ vines, grafted onto the lime-susceptible root-
stock B3309C^, experienced a reduction of around
50 % in net photosynthesis rate under exposure to
16 % active lime (Bavaresco et al. 2006).
A recent study by Covarrubias and Rombolà (2013)
showed that the presence of bicarbonate in the nutrient
so lu t ion caused a subs t an t i a l dec rease in
Fig. 3 Maximum quantum efficiency of PSII photochemistry, Fv/
Fm, quantum efficiency of PSII, ΦPSII, and non-photochemical
quenching, NPQ, at midday (a, c, e) and at dawn (b, d, f), in
randomly selected, fully developed leaves of V. vinifera x
V. berlandieri rootstock B41B^ (○), V. vinifera ssp. sylvestris from
the B14/Rute/1^ population (●) and V. vinifera ssp. sylvestris from
the B14/Montoro/4^ population (∇), in response to treatment with a
range of external CaCO3 contents for 30 days. Note scale differ-
ences. Values represent the mean±standard error, n=20
Plant Soil
phosphoenolpyruvate carboxylase (PEPC) activity in
the Fe-chlorosis tolerant B140 Ruggeri^ grapevine root-
stock. PEPC activity is considered a physiological
marker of Fe deficiency (Covarrubias et al. 2014). In
this way, the effects of increasing soil lime on photo-
synthetic function detected in our study could be partly
related to a decrease in the activity of certain enzymes
implied in photosynthesis. On the other hand, our fluo-
rescence analysis showed that the reduction in photo-
synthetic activity could be partially due to the effects of
external lime content on the photosynthetic apparatus:
Maximum quantum efficiency of PSII (Fv/Fm) and
quantum efficiency of PSII (ΦPSII) were both affected
by external CaCO3 content, suggesting that lime stress
enhances the photoinhibition induced by light stress.
Moreover, the decrease in Fv/Fm and ΦPSII was follow-
ed by an increase in NPQ, thus indicating that a part of
the excitation energy that was not utilised for
photochemistry was dissipated in the form of heat. In
B41B^ plants, midday values of Fv/Fm at all CaCO3
treatments did not recover at dawn and in fact remained
lower than the control parameters for unstressed plants
(Björkman and Demmig 1987), indicating the occur-
rence of chronic photoinhibition or photodamage. In
all probability, this decline in Fv/Fm was due to the
decrease in the concentration of chlorophyll recorded
in all CaCO3 treatments.
It is interesting to note that the deleterious effects of
CaCO3 on photosynthetic function were considerably
less marked in the wild grapevine plants than in the
B41B^ lime-tolerant rootstock: At 20 and 40 %
CaCO3, dawn Fv/Fm values in wild grapevine plants
remained around the optimal values for unstressed
plants and the reduction in chlorophyll concentration
was considerably lower than that of the B41B^ plants.
Integration of our results suggests that the higher pho-
tosynthetic rates recorded in wild grapevines compared
to B41B^ plants may be related to a greater ability to
maintain the integrity of the photosynthetic apparatus
under lime-stress conditions. This ability during stress is
of particular significance and is a characteristic of stress
resistance because it allows plants to recover and fully
utilize available resources upon relief from stress (Liu
and Dickmann 1993).
Mineral nutrients play primary roles in photosynthet-
ic CO2 reduction, synthesis and partitioning of photo-
synthates (Mengel and Kirkby 1982). Calcareous soil
conditions strongly impair the bioavailability of iron for
plant requirements and may often interfere with essen-
tial nutrient uptake and transport (Bert et al. 2013;
Zancan et al. 2008). In our experiment, the B41B^
grapevine rootstock and B14/Montoro/4^ wild grape-
vine plants suffered a considerable reduction in leaf Fe
concentration at external CaCO3 contents from 20 %
upwards. These results agree with those of Bavaresco
et al. (2003), who reported a reduction in leaf Fe con-
centration of around 23 % in plants of V. vinifera L. cv.
BPinot blanc^ grafted onto the lime-tolerant B41B^ root-
stock under exposure to 19.3 % active lime. In contrast,
plants from the B14/Rute/1^ wild grapevine population
were found to be capable of maintaining leaf Fe concen-
tration up to the 40 % CaCO3 treatment. Moreover, in
our study, leaf concentrations of all the analyzed nutri-
ents were virtually unaffected by 20 and 40 % CaCO3
soil contents in B14/Rute/1^ plants, thus demonstrating a
more efficient control of the nutritional status under
CaCO3 stress than that of the B41B^ rootstock and wild
Fig. 4 Chlorophyll a (chl a) (a) and Chlorophyll b (chl b) (b) in
randomly selected, fully developed leaves of V. vinifera x
V. berlandieri rootstock B41B^ (○), V. vinifera ssp. sylvestris from
the B14/Rute/1^ population (●) and V. vinifera ssp. sylvestris from
the B14/Montoro/4^ population (∇), in response to treatment with a
range of external CaCO3 contents for 30 days. Note scale differ-
ences. Values represent the mean±standard error, n=12
Plant Soil
grapevines from B14/Montoro/4^ population, which suf-
fered considerable reductions in leaf concentration of
nitrogen, phosphorus, sulphur and copper at external
CaCO3 contents from 20 % upwards. In our study, the
reduction in leaf N recorded in B41B^ and B14/Montoro/
4Bplants at 20 % CaCO3 was similar to that obtained by
Bavaresco et al. (2003) in V. vinifera L. cv. BPinot blanc^
grafted onto B41B^ rootstock grown in 19.3 % active
lime (around 50 %, relative to the non-calcareous
control).
Focusing on a comparison of the physiological re-
sponse of wild grapevine plants from both studied pop-
ulations, integration of our results indicates that, com-
pared to the B14/Montoro/4^ plants, the higher
photosynthetic and growth rates of plants from the
B14/Rute/1^ population under exposure to 20 and
40 % CaCO3 could be related to a greater physiological
capacity for controlling their mineral composition under
lime-stress conditions. Iron is an essential nutrient for
plant growth and plays a crucial role in several metabol-
ic pathways, including hormone synthesis and other
fundamental redox reactions (Briat et al. 1995; Briat
and Lobréaux 1997). Nitrogen is also critical for plant
growth and development, since it is needed to synthe-
size amino acids, which are the building elements of
proteins, nucleotides and numerous other metabolites
and cellular components (Nunes-Nesi et al. 2010). In
our experiment, efficient control of nutrient status in
Fig. 5 Total iron (a), nitrogen (b), phosphorus (c), sulphur (d),
potassium (e) and copper (F) concentrations in the leaves of plants
of V. vinifera x V. berlandieri B41B^ (○), V. vinifera ssp. sylvestris
from the B14/Rute/1^ population (●) and V. vinifera ssp. sylvestris
from the B14/Montoro/4^ population (∇), in response to treatment
with a range of external CaCO3 contents for 30 days. Note scale
differences. Values represent the mean±standard error, n=3
Plant Soil
B14/Rute/1^ wild grapevine plants under lime stress
may have contributed to the maintenance of higher
growth rates than those recorded in wild grapevines
from the B14/Montoro/4^ population: this may be
achieved both directly, through the effects of nutrients
on plant metabolism and development, and indirectly,
for example, by the effects of certain nutrients in the
regulation of enzymes involved in the photosynthetic
process.
Summarizing our results, in all B41B^ rootstock
and wild grapevines from both studied populations,
the highest soil CaCO3 content (60 %) drastically
inhibited photosynthetic function and induced con-
siderable nutrient imbalances, which probably
caused a reduction in carbon gain and the observed
drastic reduction in growth. Although 20 and 40 %
soil CaCO3 similarly affected the leaf nutrient con-
tent of B41B^ rootstock and B14/Montoro/4^ plants,
wild grapevines from this population showed a
greater ability to maintain the integrity of their pho-
tosynthetic apparatus despitethe nutritional disor-
ders caused by lime-stress conditions, which could
explain the higher photosynthetic and growth rates
recorded in these plants, relative to the B41B^ root-
stock. Wild grapevine can be considered a highly
lime-tolerant subspecies of Vitis vinifera. Plants
from the population grown in hypercalcic calcisol
soil (B14/Rute/1^) present a higher degree of lime-
tolerance and, compared to other wild grapevine
populations, could constitute an elite gene pool for
the development of new lime stress-tolerant varieties
of grapevine. These plants are capable of maintain-
ing Fe uptake and translocation to leaves even under
extremely high lime conditions (40 % CaCO3) and
proved to be more efficient in controlling leaf con-
centrations of the main macronutrients (N, P and S)
in comparison to wild grapevines from the other
studied population. Our study suggests that variation
in the maintenance of essential mineral nutrient sta-
tus may be a crucial factor in plant tolerance to
calcareous soil conditions.
Acknowledgements We thank the Consejo Superior de
Investigaciones Científicas (CSIC) for financial support (project
201140E122) and the Seville University Glasshouse General Ser-
vice for their collaboration. J. Cambrollé thanks the University of
Seville for a research contract (IV Plan Propio de Investigación,
research projects ref. 5/2012). The authors are also grateful to María
del Mar Parra for technical assistance and to Mr. K. MacMillan for
revision of the English version of the manuscript.
References
Ambrosi H, Dettweiler E, Rühl EH, Schmid J, Schumann F (1994)
Farbatlas Rebsorten. Ulmer Verlag, Stuttgart
Bavaresco L, Cantù E, Trevisan M (2000) Chlorosis occurrence,
natural arbuscular-mycorrhizal infection and stilbene root
concentration of ungrafted grapevine rootstocks growing on
calcareous soil. J Plant Nutr 23:1685–1697
Bavaresco L, Giachino E, Pezzutto S (2003) Grapevine rootstock
effects on lime-induced chlorosis, nutrient uptake, and
source-sink relationships. J Plant Nutr 26:1451–1465
Bavaresco L, Bertamini M, Iacono F (2006) Lime-induced chlo-
rosis and physiological responses in grapevine (Vitis vinifera
L. cv. Pinot blanc) leaves. Vitis 45:45–46
Bert P-F, Bordenave L, Donnart M, Hévin C, Ollat N, Decroocq S
(2013) Mapping genetic loci for tolerance to lime-induced
iron deficiency chlorosis in grapevine rootstocks (Vitis sp.).
Theor Appl Genet 126:451–473
Björkman O, Demmig B (1987) Photon yield of O2 evolution and
chlorophyll fluorescence characteristics at 77 K among vas-
cular plants of diverse origins. Planta 170:489–504
Briat J-F, Lobréaux S (1997) Iron transport and storage in plants.
Trends Plant Sci 2:187–193
Briat J-F, Fobis-Loisy I, Grignon N, Lobréaux S, Pascal N, Savino
G et al (1995) Cellular and molecular aspects of iron metab-
olism in plants. Biol Cell 84:69–81
Cambrollé J, Redondo-Gómez S, Mateos-Naranjo E, Luque T,
Figueroa ME (2011a) Physiological responses to salinity in
the yellow-horned poppy, Glaucium flavum. Plant Physiol
Biochem 49:186–194
Cambrollé J, Mateos-Naranjo E, Redondo-Gómez S, Luque T,
Figueroa ME (2011b) The role of two Spartina species in
phytostabilization and bioaccumulation of Co, Cr, and Ni in
the Tinto-Odiel estuary (SW Spain). Hydrobiologia 671:95–
103
Cambrollé J, García JL, Figueroa ME, Cantos M (2014)
Physiological response to soil lime in wild grapevine.
Environ Exp Bot 105:25–31
Cantos M, Liñán J, Pérez-Camacho F, Troncoso A (1993)
Obtención de plantas selectas de vid, variedad Zalema, libres
de la virosis Bentrenudo corto^. Acta Horticult II:705–709
Covarrubias JI, Rombolà AD (2013) Physiological and biochem-
ical responses of the iron chlorosis tolerant grapevine root-
stock 140 Ruggeri to iron deficiency and bicarbonate. Plant
Soil 370:305–315
Covarrubias JI, Pisi A, Rombolà AD (2014) Evaluation of sus-
tainable management techniques for preventing iron chloro-
sis in the grapevine. Aust J Grape Wine Res 20:149–159
Díaz I, del Campillo MC, Cantos M, Torrent J (2009) Iron defi-
ciency symptoms in grapevine as affected by the iron oxide
and carbonate contents of model substrates. Plant Soil 322:
293–302
FAO, SICS, ISRIC (1999) Base Referencial Mundial del Recurso
Suelo. In: Informes sobre recursos mundiales de suelo, n.°
84. Fao, Roma
Gao F, Shu X, Ali MB, Howard S, Li N, Winterhagen P, Qiu W,
Gassmann W (2010) A functional EDS1 ortholog is differ-
entially regulated in powdery mildew-resistant and -
susceptible grapevines and complements an Arabidopsis
eds1 mutant. Planta 231:1037–1047
Plant Soil
Hoagland D, Arnon DI (1938) The water culture method for
growing plants without soil. Calif AES Bull 347:1–39
Hvarleva T, Bakalova A, Rusanov K, Diakova G, Ilieva I,
Atanassov A, Atanassov I (2009) Toward marker assisted
selection for fungal disease resistance in grapevine.
Biotechnol Biotechnol Equip 23:1431–1435
Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments
of photosynthetic biomembranes. Methods Enzymol 148:
350–382
Liu Z, Dickmann DI (1993) Responses of two hybrid Populus
clones to flooding, drought, and nitrogen availability. II. Gas
exchange and water relations. Can J Bot 71:927–938
LópezMA, Cantos M, Ocete R, Gómez I, Gallardo A, Troncoso A
(2004) Ecological aspects and conservation of wild grapevine
populations in the s.w. of the Iberian Peininsula. Acta
Horticult 652:81–85
Maxwell K, Johnson GN (2000) Chorophyll fluorescence- a prac-
tical guide. J Exp Bot 51:659–668
McGovern PE, Fleming SJ, Katz SH (1996) The origins and
ancient history of wine. Overseas Publishers Association,
Amsterdam
Mengel K, Kirkby A (1982) Principles of plant nutrition.
International Potash Institute, Bern
Negrul AM (1938) Evolution of cultivated forms of grapes. C R
Acad USSR N S 18:585–588
Nikolic M, Römheld V, Merkt N (2000) Effect of bicarbonate on
uptake and translocation of 59Fe in two grapevine rootstocks
differing in their resistance to Fe deficiency chlorosis. Vitis
39:145–149
Nunes-Nesi A, Fernie AR, Stitt M (2010) Metabolic and signaling
aspects underpinning the regulation of plant carbon nitrogen
interactions. Mol Plant 3:973–996
Ocete R, Cantos M, López MA, Gallardo A, Pérez MA, Troncoso
A, Lara M, Failla O, Ferragut FJ, Liñán J (2007)
Caracterización y conservación del recurso fitogenético vid
silvestre en Andalucía. Consejería deMedio Ambiente, Junta
de Andalucía, Sevilla
Pavlousec P (2013) Tolerance to Lime-Induced Chlorosis and
Drought in Grapevine Rootstocks. In: Vahdati, Leslie (eds)
Abiotic stress – Plant Responses and Applications in
Agriculture. In Tech, 277–306
Tangolar SG, ÜnlüG, Tangolar S, Daşgan Y, Yilmaz N (2008)Use
of in vitro method to evaluate some grapevine varieties for
tolerance and susceptibility to sodium bicarbonate-induced
clorosis. In Vitro Cell Dev Biol Plant 44:233–237
Von Caemmerer S, Farquhar GD (1981) Some relationships be-
tween the biochemistry of photosynthesis and the gas ex-
change of leaves. Planta 153:377–387
Zancan S, Sugliaa I, Roccab LN, Ghisia R (2008) Effects of UV-B
radiation on antioxidant parameters of iron-deficient barley
plants. Environ Exp Bot 63:71–79
Plant Soil
	Evaluating tolerance to calcareous soils in Vitis vinifera ssp. sylvestris
	Abstract
	Abstract
	Abstract
	Abstract
	Abstract
	Introduction
	Materials and methods
	Plant material and calcium carbonate treatments
	Growth
	Gas exchange
	Chlorophyll fluorescence
	Photosynthetic pigments
	Mineral analysis
	Statistical analysis
	Results
	Growth
	Gas exchange
	Chlorophyll fluorescence
	Photosynthetic pigments
	Chemical analysis of plant samples
	Discussion
	References