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Low temperatures damage many temperate crops, including grapevine, which, when exposed to chilling, can be
affected by symptoms ranging from reduced yield up to
complete infertility. We have previously demonstrated
that Burkholderia phytofirmans PsJN, a plant growth-promoting rhizobacteria (PGPR) that colonizes grapevine, is
able to reduce chilling-induced damage. We hypothesized
that the induced tolerance may be explained at least
partly by the impact of bacteria on grapevine photosynthesis or carbohydrate metabolism during cold acclimation. To investigate this hypothesis, we monitored herein
the fluctuations of photosynthesis parameters (net photosynthesis [Pn], intercellular CO2 concentration, stomatal
conductances, PSII, and total chlorophyll concentration),
starch, soluble sugars (glucose, fructose, saccharose, mannose, raffinose, and maltose), and their precursors during
5 days of chilling exposure (4C) on grapevine plantlets.
Bacterization affects photosynthesis in a nonstomatal
dependent pattern and reduced long-term impact of chilling on Pn. Furthermore, all studied carbohydrates known
to be involved in cold stress tolerance accumulate in nonchilled bacterized plantlets, although some of them remained more concentrated in the latter after chilling
exposure. Overall, our results suggest that modification of
carbohydrate metabolism in bacterized grapevine plantlets may be one of the major effects by which this PGPR
reduces chilling-induced damage.
Abiotic stresses are responsible for 50% of crop loss worldwide (Boyer 1982; Bray et al. 2000; Nagarajan and Nagarajan
2010). Among them, exposure to low temperatures is one of
the most damaging environmental factors affecting plants
(Boyer 1982; Nagarajan and Nagarajan 2010). Upon chilling
(low temperature above 0C), common temperate crops such
as maize, potato, and grapevine can suffer severe damage (Ait
Barka et al. 2006; McKersie and Leshem 1994; Nagarajan and
Nagarajan 2010; Ruelland et al. 2009). Nevertheless, at chilling temperatures, plants may enter a process called acclimation, which improves tolerance and reduces damage due to
long-term chilling exposure (Ruelland et al. 2009). Cold acclimation involves several metabolic adjustments such as i) induction of cold-specific gene expression (Chinnusamy et al. 2007;
Nakashima and Yamaguchi-Shinozaki 2006; Thomashow 2010),
O. Fernandez and A. Theocharis have contributed equally to this work.
Corresponding author: E. A. Barka; E-mail: ea.barka@univ-reims.fr
496 / Molecular Plant-Microbe Interactions
Fig. 1. Photosynthesis parameters in bacterized (B) and nonbacterized (NB) plantlets during 5 days of chilling exposure. A, Net photosynthesis (Pn); B, internal
CO2 stomatal concentration (Ci); C, stomatal conductance (gs); D, effective PSII quantum yield (PSII). Values are means ( standard error) of two independent experiments. If noted, asterisks (*) show significant difference (P < 0.05) between NB and B (day 0) or NB chilled and B chilled (days 1, 2, and 5)
plantlets.
Vol. 25, No. 4, 2012 / 497
Fig. 3. A, Starch and B, total soluble sugars (TSS) in bacterized (B) and
nonbacterized (NB) plantlets during 5 days of chilling exposure. TSS concentration was calculated as the sum of glucose + fructose + maltose + sucrose + raffinose + mannose. Values are means ( standard error) of three
independent experiments. If noted, asterisks (*) show significant difference (P < 0.05) between NB and B (day 0) or NB chilled and B chilled
(days 1, 2, and 5) plantlets.
Glycolysis fluctuations.
To gain insight into the bacterization effect on glycolysis before or during chilling treatment, the levels of phosphoenolpyruvate (PEP) and pyruvate were monitored (Fig. 6). Addition-
Fig. 4. A, Sugars and galactinol and B, phosphated and nucleotined intermediates concentration in bacterized (B) and nonbacterized (NB) plantlets before
chilling exposure. Left axis indicates concentration for A, mannose, glucose, fructose, and sucrose and B, G1P, G6P, F6P, M6P, and UDPG; right axis indicates concentration for A, galactinol, raffinose, and maltose and B, S6P and ADPG. Values are means ( standard error) of three independent experiments. If
noted, asterisks (*) show significant difference (P < 0.05) between NB and B plantlets.
Fig. 5. Concentration ratio of A, sugars and galactinol and B, phosphated and nucleotined intermediates in bacterized (B) and nonbacterized (NB) plantlets
during 5 days of chilling exposure (4C). At day 0, ratio is calculated as concentration of sugars in NB versus B plantlets and at days 1, 2, and 5 as concentration of sugars in NB chilled versus B chilled plantlets. Black line indicates ratio of 100%.
Vol. 25, No. 4, 2012 / 499
chlorophyll concentrations but not the quantum yield of photosystem II (Figs. 1D and 2). Effects on photosynthesis parameters
have been described in the literature for other beneficial plant
microbe interactions. In contrast with our results, long-term ex-
DISCUSSION
In a previous study, we showed that B. phytofirmans was
able to enhance tolerance of grapevine to low temperatures by
cold acclimation (Ait Barka et al. 2006). To explore the mechanisms underlying bacterial-induced cold tolerance, we investigated the fluctuations of both photosynthesis and carbohydrate
metabolism during acclimation.
Photosynthesis parameters are modified
in grapevine plantlets by both chilling and bacterization.
Both Pn and PSII were markedly reduced in NBC and BC
plantlets without disruption of Ci and gs. This is not surprising
because a decrease in Pn and PSII is a classical physiological
response of plants to chilling stress (Bertamini et al. 2005;
Hendrickson et al. 2004). The cessation of growth resulting
from cold stress reduces the capacity for energy utilization
which, in turn, probably results in feedback inhibition of photosynthesis (Ruelland and Zachowski 2010).
B. phytofirmans PsJN reduced photosynthesis in a nonstomatal-dependent pattern. This is demonstrated by the fact that
B plantlets displayed a lower Pn but similar Ci and gs relative to
NB plantlets (Fig. 1A to C). Furthermore, bacterization affected
Table 1. Analysis of sugars, galactinol, and phosphated and nucleotined intermediates in bacterized and nonbacterized plantlets during 5 days of chilling
exposurea
Concentration (mol g1 FW)
NBC (duration of cold exposure)
Metabolites
Mannose
Glucose
Fructose
Sucrose
Galactinol
Raffinose
Maltose
G1P
G6P
F6P
M6P
S6P
ADPG
UDPG
a
NB
1 day
2 days
5 days
1 day
2 days
5 days
3.29 0.15
5.75 1.06
4.49 0.95
5.46 0.46
0.11 0.02
0.11 0.05
0.01 0.002
139 5
277 14
69 5
85 4
2 0.1
0.2 0.02
77 3
3.52 0.61
11.53a 1.23
10.26 0.86
12.84 0.63
0.23a 0.06
0.09a 0.04
1.43 0.28
337b 35
847b 70
183b 16
267b 27
17.2b 1.1
4.8b 1.2
80b 4
3.48a 0.18
19.01a 1.27
18.32 0.78
13.82 0.17
0.4a 0.08
0.22a 0.07
4.26 0.55
362b 19
809b 45
182b 10
276b 23
20.6b 0.4
7.3b 1.3
96b 3
3.69 0.14
31.99 0.26
31.30 0.44
18.39 0.56
0.69 0.11
0.57a 0.12
7.90b 0.59
352 42
839 50
201 11
266b 36
22.8 1.8
12.8 2.8
108 1
5.53 0.36
12.75 1.45
7.44 0.61
9.46 0.65
0.21 0.04
0.42 0.07
0.05 0.01
86 3
280 11
72 3
57 4
2.2 0.1
0.1 0.02
48 3
5.56 0.52
19.84b 1.02
12.91 1.01
11.80 0.98
0.47b 0.04
0.57b 0.13
1.01 0.18
109a 19
391a 60
89a 13
93a 16
8.5a 0.5
1.3a 0.2
41a 7
5.23b 0.34
26.82b 0.71
20.52 1.4
14.53 0.52
0.99b 0.07
0.99b 0.15
3.10 0.07
170a 43
507a 76
118a 19
129a 18
14.1 1.7
2.6a 0.9
51a 12
4.9 0.73
32.72 0.55
29.73 0.66
16.41 1.14
0.88 0.16
1.27b 0.10
5.87a 0.57
269 69
699 127
163 28
202a 53
17.9 1.9
9.3 2.7
91 17
NB = nonbacterized plantlets (26C), B = bacterized plantlets (26C), NBC = NB chilled plantlets (4C), and BC = B chilled plantlets (4C). Values are
means ( standard error) of three independent experiments. If noted, letters (a or b) indicate different means (P < 0.05) between BC and NBC plantlets.
Table 2. Pyruvate kinase (PK) and phosphofructo-kinase (PFK) ratios in bacterized (B) and nonbacterized (NB) plantlets during 5 days of chilling exposurea
NBC (duration of cold exposure)
Ratios
PK
PFK
a
NB
0.45
1,655
1 day
2 days
5 days
1.33
3,189
1.62
1,283
3.04
261
1 day
2 days
0.95
3,363
1.27
1,887
5 days
3.7
631
NB and B plantlets at 26C, NBC = NB chilled plantlets (4C) and BC: B chilled plantlets (4C). PK and PFK ratios were calculated as
pyruvate/phosphoenolpyruvate and F6P/fructose 1,6-bisphosphate, respectively, in order to represent an estimate of PK and PFK enzyme activities.
perature tolerance. Therefore, this phenomenon may partly explain how the B. phytofirmans strain improves tolerance to
chilling.
MATERIALS AND METHODS
Plant material and in vitro growth conditions.
Plantlets of Vitis vinifera Chardonnay were micropropagated by nodal explants grown on 15 ml of agar medium in 25
mm-culture tubes as described earlier (Ait Barka et al. 2006).
Cultures were performed in a growth chamber at constant temperature of 26C under white fluorescent light (200 mol m2
s1) with 16 h of light per day.
Bacterial inoculum and plant bacterization.
The bacterial inoculum was produced by transferring two
loops of B. phytofirmans PsJN tagged with green fluorescent
protein (Sessitsch et al. 2005) to 100 ml of Kings B liquid
medium in a 250-ml Erlenmeyer flask incubated at 20C at
150 rpm for 48 h. Bacteria were collected by centrifugation
(3,000 g for 15 min) and washed twice with phosphatebuffer saline (PBS; 10 mM, pH 6.5). The pellet was resuspended in PBS and used as inoculum. The bacterial concentration was estimated by spectrophotometry (600 nm) and adjusted
to 3 108 CFU ml1 with PBS (Pillay and Nowak 1997). Roots
of 2-week-old plantlets were immersed in bacterial inoculum
(3 108 CFU ml1) or PBS (control) for 10 s. After inoculation, plantlets were grown as described above for 4 weeks before chilling treatment.
Chilling treatment.
Half of B and NB plantlets were transferred to a cold growth
chamber maintained at 4C under 16 h of light (white fluorescent light, 200 mol m2 s1) and 8 h of darkness, whereas the
control plantlets were maintained at 26C. Each treatment was
replicated at least three times and each replicate consisted of
six plantlets.
Leaves from NB and B plantlets were sampled at 0, 1, 2,
and 5 days after chilling exposure. The four conditions for
plantlet treatments were abbreviated as follows: NB plantlets
at 26C (NB), B plantlets at 26C (B), NB plantlets exposed to
4C (NBC), and B plantlets exposed to 4C (BC).
Leaf gas exchanges.
The Pn, gs, and Ci were measured with an open gas exchange
system (LI-6400; Li-Cor, Lincoln, NE, U.S.A.) using equations
developed by Caemmerer and Farquhar (1981). The infrared
gas analysis system was equipped with a clamp-on leaf cuvette
that exposed 6 cm2 of leaf area. Air temperature and humidity
were maintained at 25C and 30%, respectively. The level of
photosynthetically active radiations provided by a red and blue
light-emitting diode (Li-6400-02, Li-Cor) was fixed to 1,500
mol m2 s1. CO2 concentration was maintained at a constant
level of 400 mol liter1 using a CO2 injector with a highpressure liquid CO2 cartridge source (LI-6400-01; Li-Cor).
Gas exchange measurements were performed on six plantlets
per condition and on three leaves per plantlet.
Chlorophyll fluorescence.
Chlorophyll fluorescence reflects the functionality of the
photosynthetic apparatus because it results from absorbed
light. Chlorophyll fluorescence was evaluated simultaneously
with gas exchange measurement at ambient CO2 concentration
and temperature. Leaf chlorophyll fluorescence was quantified
using a pulse-modulated fluorometer (FMS2; Hansatech,
Kings Lynn, U.K.). The measuring system applies an array of
blue light-emitting diodes, adjusted to 10 Hz (peak wavelength
502 / Molecular Plant-Microbe Interactions
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