www.elsevier.com/locate/agee

Agriculture, Ecosystems and Environment 106 (2005) 189–198

Analysis of chlorophyll fluorescence transients in mandarin

leaves during a photo-oxidative cold shock and recovery

F. Pietrinia, D. Chaudhurib, A.P. Thapliyalc, A. Massaccia,*

aInstitute of Agro-Environment and Forest Biology of National Research Council,

Via Salaria Km 29,300, 00016 Monterotondo Scalo (Roma), ItalybRubber Research Institute of India, Regional Research Station, Rubber Board, Guwahati, AssamcRubber Research Institute of India, Regional Research Station, Rubber Board, Tura, Meghalaya

Accepted 30 June 2004

Abstract

In temperate climates, plants are more frequently exposed to sudden and strong decrease of temperature combined with high

light intensity that compromise the photosynthetic efficiency and, often, the development and their survival. To investigate

deeper the effects of such a photo-oxidative cold shock on photosynthesis of mandarin (Citrus reticulata Blanco) we used

transient fluorescence analysis and gas exchange techniques. Mandarin plants were acclimated at 13 8C or grown at 25 8C for 30

days, then single leaves were exposed for 6 h at 5 8C and 1200 mmol photons m�2 s�1 and again exposed for 72 h under growth

conditions. Acclimated leaves showed lower photosynthesis and higher total carotenoid contents than non-acclimated leaves.

The photo-oxidative cold shock inhibited photosystem II (PSII) efficiency, as suggested by the reduction of Fv/Fm ratios, in all

leaves. Carotenoid content was reduced in non-acclimated leaves. A time-dependent increase in the initial fluorescence level

(F0) and a faster decline in Fv/Fm ratio indicated a stronger PSII inhibition in non-acclimated leaves in comparison with

acclimated leaves. The chlorophyll a fluorescence kinetic showed in all leaves complex changes in the O–J, J–I and I–P phases.

The I and P steps of the fluorescence rise were particularly affected in non-acclimated leaves. Photosynthesis, fluorescence and

pigment contents, measured in leaves maintained for 2 h in the dark and then returned for 72 h to growth conditions, showed a

different pattern of recovery between leaves. Photosynthesis and fluorescence completely recovered in acclimated leaves after

72 h under growth conditions. On the contrary, non-acclimated leaves still showed very low values of photosynthesis and Fv/Fm,

suppression of the sigmoidal shape of fluorescent transients and further reduction of chlorophyll contents, indicating the

presence of irreversible damages to PSII and, probably, an anticipated senescence. It is suggested that carotenoids have a relevant

role in protecting leaves of mandarin from the effects of a photo-oxidative cold shock.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Chlorophyll fluorescence; Cold-acclimation; Photosystem II; Photo-oxidative cold shock; Recovery; Mandarin (Citrus reticulata

Blanco)

* Corresponding author. Tel.: +39 0690672537; fax: +39 069064492.

E-mail address: angelo.massacci@ibaf.cnr.it (A. Massacci).

0167-8809/$ – see front matter # 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.agee.2004.10.007

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198190

1. Introduction

The occurrence of unexpected frosts, numerous in

the last decades, causes serious damages to many

cultivated species (Zinoni et al., 2002). The higher

frequency of the phenomenon seems to be attributed to

the underway process of hearth warming (EPA

WBBsite, 2004). The higher temperatures, in fact,

anticipate the growth season so that the new leaves are

emitted when the risk of frost is elevated. Never-

theless, if frosts are associated with high light

damaging effects are even more severe (Long et al.,

1994). In these conditions, light energy arriving to

chloroplasts is not utilisable for photosynthesis and

this energy cannot be all convertible into heat. The

formation of singlet oxygen and other active oxygen

species becomes, thus, inevitable and, when the

capacity of the scavenging mechanisms is overloaded,

these species are damaging to pigments, thylakoid

membranes and various components of the photo-

chemical process (Asada and Takahashi, 1987). The

protective conversion of excess light energy into heat

is, then, an extremely important process. It is

considered as a safety valve for photosynthesis and

it is associated with a prompt regulatory reduction of

PSII activity in response to either a sudden or slow

build-up of high transthylakoid DpH (Niyogi, 2000).

When stress factors are removed, PSII function can,

however, recover with a half time depending on the

intensity of the perturbation: 7–10 min or, even,

several hours. In both cases, xanthophylls and

phosphatase inhibitors seem to be involved in a

mechanistically similar process that through confor-

mational changes can induce such rapid or sustained

energy dissipations (Gilmore and Ball, 2000; Xu et al.,

1999). Xanthophylls represent about 80% of total

carotenoids and are mostly associate with polypep-

tides of minor antenna such as CP24, CP26 and CP29

(Bassi et al., 1993) while b-carotene, the other major

component of total carotenoids, is associated with the

core reaction centres of PSII and PSI (Peter and

Thomber, 1991). Studies on an overwintering ever-

green (snow gum) suggested that the conversion

capacity of excess energy into heat increased when

leaves were acclimated to low temperatures (Ottander

et al., 1995). The lower request of energy absorption

and electron transport at PSII induced also in maize a

different organisation and pigment composition of the

photochemical apparatus (Haldimann et al., 1995).

Mandarin, a species growing in temperate climates,

seems to possess some chilling acclimation capacity

but it suffers particularly photo-oxidative cold shock

episodes in early spring. Newly emitted leaves are, in

fact, severely damaged under such a condition

(Yelenosky, 1982; Cakmak et al., 1995). Changes

induced by acclimation to winter temperatures could

involve all cell components particularly chloroplasts.

The capacity of energy dissipation and of other

protective mechanisms (e.g. antioxidants) in these

organelles, in fact, must be increased to prevent

irreversible damages to the photochemical process

that might trigger anticipated senescence. Chlorophyll

a fluorescence is considered as a proper technique to

study the extent of such damaging modifications in

acclimated or stressed leaves (Leipner et al., 1997).

Particularly, the Fv/Fm ratio obtained by the measured

basal F0 and maximum Fm fluorescence emission [Fv/

Fm = (Fm � F0)/Fm] is recognised as a good indicator

of photoinhibitory or photo-oxidative effects on PSII

(Maxwell and Johnson, 2000; Baker et al., 1994).

Further, other fluorescence parameters, derived by the

theory of fluxes, have been suggested to describe

changes of absorbed, dissipative, trapping, and

electron transport fluxes (Lazar, 1999; Strasser et

al., 2000; Force et al., 2003). The analysis of these

parameters, named JIP test, is performed on the typical

fluorescence induction from the basal emission F0 (0)

to a maximum emission level Fm (P) through two

intermediate steps called, according to a recent

terminology, J and I (Strasser and Govindjee, 1992;

Strasser et al., 1995). The fluorescence induction has a

typical sigmoidal shape evidencing three main phases:

O–J, J–I and I–P. Specifically, the first phase O–J is

determined by the charge separation reactions, the

photochemical event that leads to the reduction of the

primary quinone acceptor QA, and seems influenced

by the S-state of the oxygen evolving complex (OEC).

The following J–I phase is sensitive to temperature

and, as well as the I–P phase, reflects the accumulation

of reduced quinones Q�AQ�

B and of Q�AQ�2

B , respec-

tively (Neubauer and Schreiber, 1987; Schreiber and

Krieger, 1996). The heterogeneity of PSII could

account for the occurrence of two phases related to the

reduction of secondary quinones. The time needed to

reach the P step from time zero, when fluorescence

rises, is that needed to reduce the plastoquinone pool

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198 191

(PQ) and is limited by the slow events in the electron

transport at PSII which include the S-states of OEC

(Stirbet et al., 1998). Although a satisfactory inter-

pretation relating all phases to specific photochemical

events is not yet available the analysis of fluorescence

induction could be, however, considered as an

interesting complement to the information obtainable

by the various classical and well investigated

fluorescence parameters (e.g. F0, Fv, Fv/Fm, qE, qI,

and DF/Fm) (Govindjee, 1995; Force et al.,

2003).

In this study we exposed newly developed leaves at

13 and 25 8C to a photo-oxidative shock at 5 8C and

1200 mmol photons m�2 s�1 and to a recovery period

of 72 h under their growth conditions. The effects of

this treatment were analysed by measuring gas

exchange, fluorescence transients and pigment con-

tents. Our results show that changes induced by low

temperature acclimation allowed mandarin leaves to

tolerate the photo-oxidative shock and to recover

completely photosynthesis and photochemical activ-

ities. The increase of carotenoid contents is suggested

to play an important role in this tolerance.

2. Materials and methods

2.1. Plant material

One-year-old leaves of mandarin (Citrus reticulata

Blanco) were placed in climatic chambers under a

photoperiod of 14 h (600 mmol photons m�2 s�1) at

25 � 2 8C (non-acclimated) for 45 days. At the end of

this period, some leaves were moved to a second

chamber under the same conditions except that the

temperature was set at 13 � 2 8C. Approximately 30

days later, new leaves emitted at this temperature were

fully expanded.

2.2. Photo-oxidative cold shock and recovery

The central part of a last fully expanded leaf was

enclosed in a cuvette in the dark for 20 min at 25 or

13 8C and then for 40 min at 5 8C. After the dark

adaptation at 5 8C, the leaf was exposed at

1200 mmol photons m�2 s�1 for 6 h. Recovery from

this treatment was induced for 2 h in the dark and for

72 h under growth conditions (25 or 13 8C and

600 mmol photons m�2 s�1 for 14 h). This experiment

was replicated four times.

2.3. Fluorescence measurements

Fluorescence transients (OJIP) were measured at

room temperature by a Plant Efficiency Analyser

(Hansatech, King’s Lynn, Norfolk, UK) according to

Strasser et al. (1995). The transients were induced by a

light provided by an array of six light-emitting diodes

(peak 650 nm), which focused on the sample surface

to give homogenous illumination over the exposed

area (4 mm in diameter). The chlorophyll a fluores-

cence signals were recorded for 5 s with data

acquisition rate of 10 ms for the first 2 and 1 ms for

the rest of the time. The fluorescence signal at 50 ms,

the earliest measurement free of any artefacts related

to the electronics of the instrument (Haldimann and

Strasser, 1999), was considered as F0. The maximal

measured fluorescence Fm and the variable fluores-

cence, Fv (obtained by the difference between the

initial fluorescence, F0, and the maximal fluorescence,

Fm of dark adapted leaves), were used to calculate the

Fv/Fm ratios. The duration of dark adaptation required

to obtain the relaxation of the rapid energy-dependent

non-photochemical quenching was experimentally

established to 20 min. At least four repetitions were

done for each measurement.

2.4. Measurements of CO2 assimilation

Measurements of CO2 gas exchange were made

using a LI-6400 (LI-COR, Inc., Lincoln, NE, USA)

portable IRGA analyser. A central part of the leaf was

clamped into a cuvette and illuminated with red light

of 600 mmol m�2 s�1. Measurements were taken

under steady CO2 uptake maintained for 5 min, at

the constant relative humidity of 50% and at the

temperature of 25 8C. Four replicates were done for

this measurement.

2.5. Pigment analysis

Two square centimeters of the last full-expanded

leaf used for photosynthesis and fluorescence mea-

surements were ground under dim light in a mortar

containing liquid N2. When the leaf was reduced to a

fine powder, 2 ml of acetone–water (80%, v/v) were

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198192

added to extract the pigments. The samples were

centrifuged at 12 000 � g and 5 8C for 10 min, and the

supernatant was removed and used for pigment

determinations. Absorbance was measured at 470,

646.8 and 663.2 nm with a spectrophotometer (Perkin

Elmer, Norwalk, CT, USA). The extinction coeffi-

cients and the equations reported by Lichtenthaler

(1987) were used to calculate the chlorophyll a and b

and total carotenoid contents. Four replicates were

done for each measurement.

2.6. Statistical analysis

In any one experiment, all the measurements were

made on at least four replicates. To evaluate the

significance of photosynthesis activity, fluorescence

and contents of pigments data the Student–Newman–

Keuls test was performed.

3. Results

3.1. Effects of cold-acclimation

3.1.1. Chlorophyll a fluorescence

Cold-acclimation did not apparently induce

changes in Fv/Fm ratios (Fig. 1) and F0 (Fig. 2).

The fluorescence yield of cold-acclimated leaves

typically arose from the normalised initial fluores-

cence F0 (O) to the maximum Fm (P) with the

Fig. 1. Time course of Fv/Fm changes in dark adapted leaves of non-acc

symbols) mandarin leaves, obtained at 25 8C (under the left grey bar), at 5 8C5 8C and 1200 mmol photons m�2 s�1 (under the white bar), after the recove

following 72 h at 13 (under the bold hatched bar) or 25 8C (under the light

significant difference in respective values between the non-acclimated

mean � S.E. (n = 4).

expected intermediate steps J and I. However,

acclimated leaves showed in comparison with non-

acclimated leaves a slight reduction of the signal

magnitude and of the delay in the time at which the J,

P, and I steps occurred.

3.1.2. Gas exchange

Table 1 shows the results of gas exchange

measurements. These indicate that cold-acclimation

decreased the activity of CO2 uptake, while reduced

more strongly and similarly stomatal conductance and

water transpiration. Further, the A/Ci curves (not

shown) confirmed that there were no substantial

differences in the CO2 uptake between leaves when

this was measured at 25 8C, while it was higher in

acclimated than in non-acclimated leaves when

measured at 13 8C.

3.1.3. Photosynthetic pigments

Fig. 3 shows the increase of total carotenoid

contents and only a slight reduction of chlorophyll a

and b in acclimated leaves compared with non-

acclimated leaves.

3.2. Photo-oxidative cold shock treatment

3.2.1. Chlorophyll a fluorescence

The acclimation at 5 8C in the dark before the shock

treatment did not affect Fv/Fm (Fig. 1) and F0 (Fig. 2).

On the contrary, the photo-oxidative cold shock

limated (25 8C, open symbols) and cold-acclimated (13 8C, closed

in the dark (under the black bar), during the cold-shock treatment at

ry at 25 8C for 2 h in the dark (under the right grey bar), and after the

hatched bar) and 600 mmol photons m�2 s�1. An asterisk indicates a

and cold-acclimated plants (P < 0.05). Each point represents the

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198 193

Fig. 2. Time course of initial, F0, fluorescence changes in dark adapted leaves of non-acclimated (open symbols) (25 8C) and cold-acclimated

(closed symbols) (13 8C) mandarin plants, at 25 8C (under the left grey bar) and 5 8C in the dark (under the black bar), during the cold shock

treatment at 5 8C and 1200 mmol m�2 s�1 (under the white bar), after recovery at 25 8C for 2 h in the dark (under the right grey bar) and after the

following 72 h at 13 (under the bold hatched bar) or 25 8C (under the light hatched bar) and 600 mmol photons m�2 s�1. An asterisk indicates a

significant difference in respective values between the non-acclimated and cold-acclimated plants (P < 0.05). Each point represents the

mean � S.E. (n = 4).

strongly and similarly reduced Fv/Fm in all leaves after

6 h. F0 decreased in acclimated leaves and increased

in non-acclimated leaves: differences at the end of the

photo-oxidative cold shock treatment reached about

30%. It can be observed a different time-dependence

of the reduction, with the acclimated leaves slower in

reducing Fv/Fm than non-acclimated leaves (Fig. 1).

The cold shock treatment reduced also drastically the

fluorescence yield from F0 to Fm (Fig. 4a and b) and

the sigmoid shape of O–J, J–I and I–P phases was

partially lost. Again, these changes were slower in

cold-acclimated than non-acclimated leaves. Further,

Fig. 4a shows that, after 1 h from the cold shock

application, the I step disappeared in acclimated

leaves, while it was still present in non-acclimated

leaves. Inserts in Fig. 4a and b show fluorescence

transients in a different scale of the vertical axis. This

Table 1

Changes in net photosynthesis (A), stomatal conductance (gs) and transpi

(13 8C) mandarin plants before cold shock treatment and after 72 h of re

Growth conditions A (mmol CO2 m�2 s�1)

25 8C (before treatment) 5.42 � 0.4 a

13 8C (before treatment) 3.89 � 0.5 b

25 8C (after recovery) 0.62 � 0.1 c

13 8C (after recovery) 3.81 � 0.5 b

All measurements were made at 25 8C and at a light intensity of 600 mmol

letter are not significantly different (P < 0.05) according to Student–New

permits to observe that, in non-acclimated leaves, the I

step disappeared after 2 h, while P step was

completely suppressed and O–J was drastically

reduced after 5 h of shock treatment.

3.2.2. Gas exchange

During and at the end of the cold shock treatment

the gas exchange activity was not detectable.

3.2.3. Photosynthetic pigments

Fig. 3 shows that no large differences were found in

chlorophyll a and b of all leaves between values taken

before and after the cold shock treatment. On the

contrary, a reduction of total carotenoids was

measured in non-acclimated leaves as an effect of

the cold shock.

ration (E) in leaves of non-acclimated (25 8C) and cold-acclimated

covery at their growth conditions

gs (mol H2O m�2 s�1) E (mol H2O m�2 s�1)

0.0814 � 0.003 a 2.02 � 0.04 a

0.0377 � 0.002 b 0.98 � 0.05 b

0.0035 � 0.002 c 0.50 � 0.03 c

0.0377 � 0.004 b 0.96 � 0.05 b

photons m�2 s�1. Means in the same column followed by the same

man–Keuls test. Values are mean � S.E. of four replicates.

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198194

Fig. 3. Changes in the chlorophyll a (black bar), b (grey bar) and

total carotenoid (white bar) contents in leaves of non-acclimated

(25 8C) and cold-acclimated (13 8C) mandarin plants, before, at the

end of the cold shock treatment at 5 8C and 1200 mmol m�2 s�1, and

after 72 h of recovery at 25 or 13 8C and 600 mmol m�2 s�1 (growth

conditions). Different letters above bars with the same colour

indicate significant differences (P < 0.05). Values are means of

four replicates. Bars indicate S.E.

Fig. 4. Traces of the chlorophyll a fluorescence transients showing

the rise from the initial fluorescence level, F0 (O), through the

intermediate steps, J and I, to fluorescence maximum, Fm (P), in dark

adapted leaves of non-acclimated (25 8C, a) and cold-acclimated

(13 8C, b) mandarin plants during the cold shock treatment at 5 8Cand 1200 mmol photons m�2 s�1. Inserts show the same transients

on a different fluorescence scale. The measurements were taken at

various time intervals of the treatment as indicated by the numbers

adjacent to traces. For details see Section 2.

3.3. Recovery from the photo-oxidative cold shock

3.3.1. Fluorescence

Fig. 1 shows that recovery of the initial Fv/Fm ratio

in non-acclimated leaves was about 25% after 72 h

under growth conditions. On the contrary, recovery

was close to 100% in acclimated leaves. Fig. 5a and b

reports the effects of recovery on traces of fluores-

cence transients from F0 to Fm. Non-acclimated leaves

did not clearly show, even after 72 h, the typical OJIP

polyphasic transients (Fig. 5a). On the contrary, traces

obtained from acclimated leaves were showing

again these transients and the fluorescence yield

was close to that observed before the cold-shock

treatment (Fig. 5b). On another hand, F0 of non-

acclimated leaves remained higher even after recov-

ery, while returned similar to the initial value in

acclimated leaves.

3.3.2. Gas exchange

After 72 h of recovery, CO2 net assimilation,

stomatal conductance and water transpiration of

acclimated leaves were similar to those measured

before the shock treatment (Table 1). On the contrary,

non-acclimated leaves still showed very low values of

these gas exchange parameters.

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198 195

Fig. 5. Traces of the chlorophyll a fluorescence transients showing

the rise from the initial fluorescence level, F0 (O), through the

intermediate steps, J and I, to fluorescence maximum, Fm (P), in

20 min dark adapted leaves of non-acclimated (25 8C, a) and

cold-acclimated (13 8C, c) mandarin plants during the recovery at

25 or 13 8C and 600 mmol m�2 s�1 (growth conditions). Measure-

ments were taken 24, 48 and 72 h after the treatment. Grey

lines represents the traces of the chlorophyll a fluorescence tran-

sients showing the rise of non-acclimated (panel a) and cold-

acclimated (panel b) leaves before the treatment. For details see

Section 2.

3.3.3. Photosynthetic pigments

After 72 h of recovery, chlorophyll contents in

acclimated leaves did not change while in non-

acclimated leaves they decreased strongly with respect

to contents measured before and after the cold shock.

Carotenoid content, also, did not change in acclimated

leaves and did not recover in non-acclimated leaves

after the strong reduction caused by the shock

treatment (Fig. 3).

4. Discussion

A sudden decrease of 20 8C (cold shock) in a non-

acclimated leaf exposed to a constant photon flux

density of 1200 mmol m�2 s�1and to 25 8C lowers

theoretically to about one fourth the total electrons

needed for photosynthesis (assuming Q10 as 2) and

consequently increases by 75% the amount of photons

that are absorbed but not converted into photochemi-

cal activity. It is evident that under such a condition

only a great capacity and efficiency of photon

dissipation could avoid that some electrons are,

however, channelled from water through the two

photosystems, reduce oxygen instead of carbon and

produce an irreversible dysfunction of the photo-

chemical process (Huner et al., 1993). Certainly,

antioxidants can scavenge little fluxes of reduced

oxygen species at the sites where they are formed but

their activity can be considered as very low and easily

overloaded (Asada and Takahashi, 1987). This

situation raises questions about possible mechanisms,

including protective dissipation or antioxidants, which

are involved in a successful response of mandarin to a

sudden photo-oxidative cold shock. The first possi-

bility to consider, assuming that leaves grown at 25 8Cwould be incapable to withstand such a stressful

condition, is whether the mechanisms induced or

enhanced by an acclimation at chilling temperatures

are involved in the resistance to cold shock. To this

scope, we acclimated some leaves at 13 8C and

exposed them to cold shock and to a recovery period in

parallel with leaves grown at 25 8C. Rapid techniques

based on fluorescence and gas exchange associated

with pigment analysis were expected to provide

general answers to the above questions by investigat-

ing the performance of photosynthesis processes

affected by these treatments.

4.1. Effects of cold-acclimation

Pigment analysis showed that cold-acclimation

increased total carotenoids (Fig. 3) and did not

significantly affect chlorophyll contents. It is known

that carotenoids are involved in the safe heat

dissipation of excess absorbed energy and singlet

oxygen (Asada and Takahashi, 1987), this last formed

when an excess of photons are loaded to the light

harvesting antenna. Thus, it is reasonable to assume

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198196

Fig. 6. Traces of the chlorophyll a fluorescence transients showing

the rise from F0 (O), the initial fluorescence level, through the

intermediate J and I steps and up to Fm (P), the maximum fluor-

escence, in 20 min dark adapted leaves of non-acclimated (25 8C,

grey line) and cold-acclimated (13 8C, black line) mandarin plants.

The traces were normalised at F0. For details see Section 2.

that the increase of total carotenoids is induced as a

photoprotective response by the cold-acclimation. In

other studies conducted in maize, a species sensitive to

cold photo-oxidation, acclimation at 14 8C modified

total carotenoid composition with the further pre-

dominance of xanthophylls (from 80 to 90%) over b-

carotene (from 20 to 11%), with respect to non-

acclimated leaves, but it did not increase the total

carotenoid content (Haldimann et al., 1995). It is very

likely that cold-acclimation induces similar (i.e. a

relative increase of xanthophylls) modifications of

carotenoid composition in mandarin to enhance the

heat dissipation capacity. On another hand, analysis of

fluorescence emission showed that cold-acclimation

did not affect Fv/Fm or F0 (Fig. 1). This is not

surprising since leaves of mandarin were emitted and

developed at 13 8C. A similar effect was observed on

leaves emitted and developed in presence of toxic and

oxidative concentration of Cd (Pietrini et al., 2003).

The interpretation of this effect requires more specific

and detailed experiments. Some changes were,

instead, induced in the polyphasic induction of

fluorescence (Fig. 6): the partial loosing of the typical

sigmoidal shape of O–J phase, the timing delay of J, I

and P steps normalised on F0, and the slight reduction

of fluorescence yield. It is clear that these effects are

attributable to some differences in the composition

and the organisation of PSII antenna and reaction

centres induced by acclimation; they likely reflect

changes in PSII grouping (Strasser and Stirbet, 2001).

A clear effect of cold-acclimation is the reduction of

the CO2 assimilation rate measured at 25 8C (Table 1).

The perfect overlapping of the CO2 assimilation rate

as a function of the internal concentration of CO2 (not

shown) for acclimated and non-acclimated leaves

indicated that the photosynthesis capacity was not

reduced and that the reduction of the CO2 assimilation

could be mostly related to reduced supply of CO2 to

the carboxylation sites (Massacci and Loreto, 2001).

4.2. Photo-oxidative cold shock treatment

On another hand, much larger differences in the

fluorescence emission were recorded between accli-

mated and non-acclimated leaves when they were

exposed to a photo-oxidative cold shock. First, the

basal fluorescence F0 decreased (Fig. 2) during the

shock treatment in cold-acclimated leaves and

increased after 2 h in non-acclimated leaves. Accord-

ing to Gilmore et al. (1996), F0 increases when the

photochemical apparatus is damaged or, more

specifically, when the number of functional chlor-

ophylls not connected to the reaction centres of PSII

increases. The decrease of F0 is, on the contrary, an

indication of a high-energy dissipation in the minor

antenna. The capacity of energy dissipation is

saturated after 2 h in non-acclimated leaves and

thereafter damages to the photochemical apparatus

occur. Further, as expected, Fv/Fm decreased strongly

at the end of the shock treatment in both acclimated

and non-acclimated leaves because of the reduction of

PSII quantum yield. The association of the F0

measurements suggest that in non-acclimated leaves

there is a damage component in this reduction. The

analysis of fluorescence induction performed during

the treatment showed some important differences

between changes induced in acclimated leaves

compared with non-acclimated leaves (Fig. 4a and

b). Specifically, the non-acclimated leaves showed an

almost complete suppression of the sigmoidal shape,

the almost complete disappearance of the P step and of

the J–I and I–P phases (Fig. 4b and insert). A slower

electron donation to PSII has been shown to be

associated to the disappearance of the P level

(Srivastava et al., 1997) together with the higher

unbalance between a stable PSI activity and a

damaged PSII (Stirbet et al., 1998). An important

observation on the results of Fig. 4a is that changes of

the J–I and I–P phases induced by cold-acclimation

are mostly similar to that observed in non-acclimated

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198 197

leaves but are time-dependent, they appears with a

delay of about 3 h. This suggests that acclimation

enhances the photoprotection but it cannot completely

remove some causes of the progressive damage to the

photochemical apparatus. An interesting aspect of this

cold shock treatment is the decrease of carotenoid

contents in non-acclimated leaves. Very likely the

energy load to the photochemical apparatus is so high

that singlet oxygen and other oxygen active species are

formed in large amount causing also destruction of b-

carotenes and of xantophylls. In maize, a species

sensitive to chilling, the exposure to 14 8C induced a

decrease of b-carotenes compensated by a similar

increase of xantophylls (Haldimann et al., 1995).

4.3. Recovery from the photo-oxidative cold shock

The return to growth conditions evidenced that all

effects of the cold treatment on the photosynthesis

process were reversible in cold-acclimated leaves and

irreversible in non-acclimated leaves (Table 1 and Fig.

1). Cold-acclimation seems to induce physiological

and biochemical changes that confer mandarin the

capacity to tolerate a sudden and intense photo-

oxidative event. The increase of total carotenoids has

certainly some positive role in this acquired tolerance

as indicated by many works on xantophylls, the major

components of total carotenoids (Leipner et al., 1997;

Niyogi et al., 1997). On another hand, the high

susceptibility of mandarin to cold shocks is clearly

evidenced in the measurements of non-acclimated

leaves. In fact, chlorophyll contents were further

reduced during 72 h recovery. Photosynthesis

appeared irreversibly inhibited with damages to the

photochemical apparatus as indicated by the low Fv/Fm

(Fig. 1). F0 continued decreasing (Fig. 2). Dysfunctions

at donor and acceptor sides of PSII were maintained and

reflected in the shapeless fluorescence transients (Fig.

5a). Some studies have already related reduction of

photosynthesis with degradation of rubisco and chlor-

ophylls by active oxygen species as symptoms of

senescence (Pietrini et al., 1999; Desimone et al., 1998).

5. Conclusions

These results seem to indicate that the mechanisms

enhanced by cold-acclimation conferred mandarin the

capacity to tolerate a sudden and intense photo-

oxidative shock to leaves. The increase of total

carotenoids after cold-acclimation and their decrease

in non-acclimated leaves during the cold shock

indicates that these molecules might have an

important role in this capacity.

References

Asada, K., Takahashi, M., 1987. Production and scavenging of

active oxygen in photosynthesis. In: Kyle, D.J., Osmond,

C.B., Arntzen, C.D. (Eds.), Photoinhibition. Topics in Photo-

synthesis, vol. 9. Elsevier, Amsterdam, pp. 227–287.

Baker, N.R., Farage, P.K., Stirling, C.M., Long, S.P., 1994. Photo-

inhibition of crop photosynthesis in the field at low temperatures.

In: Baker, N.R., Bowyer, J.R. (Eds.), Photoinhibition of Photo-

synthesis from Molecular Mechanisms to the Field. bios Scien-

tific Publisher, Oxford, pp. 349–364.

Bassi, R., Pineau, B., Danese, P., Marquardt, J., 1993. Carotenoid-

binding proteins of photosystem II. Eur. J. Biochem. 212, 297–

303.

Cakmak, I., Atli, M., Kaya, R., Evliya, H., Marschner, H., 1995.

Association of high light and zinc deficiency in cold-induced

leaf chlorosis in grapefruit and mandarin trees. J. Plant Physiol.

146, 355–360.

Desimone, M., Wagner, E., Joanningmeier, U., 1998. Degradation of

active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/

oxygenase by chloroplastic proteases requires ATP-hydrolysis.

Planta 205, 459–466.

EPA WBBsite, 2004. http://www.epa.gov/globalwarming/.

Force, L., Critcley, C., van Rensen, J.J.S., 2003. New fluorescence

parameters for monitoring photosynthesis in leaves. Photosynth.

Res. 78, 17–33.

Gilmore, A.M., Ball, M.C., 2000. Protection and storage of chlor-

ophyll in over-wintering evergreens. Proc. Natl. Acad. Sci.

U.S.A. 97, 11098–11101.

Gilmore, A.M., Hazlett, T.L., Debrunner, P.G., Govindjee, 1996.

Comparative time-resolved photosystem II chlorophyll a fluor-

escence analyses reveal distinctive differences between photo-

inhibitory reaction center damage and xanthophyll cycle-

dependent energy dissipation. Photochem. Photobiol. 64,

552–563.

Govindjee, 1995. Sixty-three years since Kautsky: chlorophyll a

fluorescence. Aust. J. Plant Physiol. 22, 131–160.

Haldimann, P., Fracheboud, Y., Stamp, P., 1995. Carotenoid com-

position in Zea mays developed at sub-optimal temperature and

different light intensities. Physiol. Planta. 95, 409–414.

Haldimann, P., Strasser, R.J., 1999. Effects of anaerobiosis as probed

by the polyphasic chlorophyll a fluorescence rise kinetic in pea

(Pisum sativum L.). Photosynth. Res. 62, 67–83.

Huner, N.P.A., Oquist, G., Hurry, V.M., Krol, M., Falk, S., Griffith,

M., 1993. Photosynthesis, photoinhibition and low temperature

acclimation in cold tolerant leaves. Photosynth. Res. 37,

19–39.

F. Pietrini et al. / Agriculture, Ecosystems and Environment 106 (2005) 189–198198

Lazar, D., 1999. Chlorophyll a fluorescence induction. Biochim.

Biophys. Acta 1412, 1–28.

Leipner, J., Fracheboud, Y., Stamp, P., 1997. Acclimation by sub-

optimal growth temperature diminishes photo-oxidative damage

in maize leaves. Plant Cell Environ. 20, 366–372.

Lichtenthaler, H.K., 1987. Chlorophylls and carotenoids: pigments

of photosynthetic biomembranes. Methods in Enzymology, vol.

148, Academic Press, London, pp. 350–382.

Long, S.P., Humphries, S., Falkowski, P.G., 1994. Photoinhibition of

photosynthesis in nature. Annu. Rev. Plant Physiol. Plant Mol.

Biol. 45, 633–662.

Massacci, A., Loreto, F., 2001. Diffusive resistances to CO2 entry in

the leaves and their limitations to photosynthesis. In: Pessarakli,

M. (Ed.), Handbook of Plant and Crop Physiology. Marcel

Dekker, New York, pp. 327–336.

Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence—a

practical guide. J. Exp. Bot. 51, 659–668.

Neubauer, C., Schreiber, U., 1987. The polyphasic rise of chlor-

ophyll fluorescence upon onset of strong continuous illumina-

tion. I. Saturation characteristics and partial control by the

photosystem Il acceptor side. Z. Naturforsch. 42c, 1246–1254.

Niyogi, K.K., 2000. Safety valves for photosynthesis. Curr. Opin.

Plant Biol. 3, 455–460.

Niyogi, K.K., Bjorkman, O., Grossman, A.R., 1997. The roles of

specific xanthophylls in photoprotection. Proc. Natl. Acad. Sci.

U.S.A. 94, 14162–14167.

Ottander, C., Campbell, D., Oquist, G., 1995. Seasonal changes in

photosystem II organization and pigment composition in Pinus

sylvestris. Planta 197, 176–183.

Peter, G.F., Thomber, J.P., 1991. Biochemical composition and

organization pf higher plant photosystem light-harvesting pig-

ment-protein. J. Biol. Chem. 266, 16745–16754.

Pietrini, F., Iannelli, M.A., Battistelli, A., Moscatello, S., Loreto, F.,

Massacci, A., 1999. Effects on photosynthesis, carbohydrate

accumulation, and re-growth induced by temperature increase in

maize genotypes with different sensitivity to low temperature.

Aust. J. Plant Physiol. 26, 367–373.

Pietrini, F., Iannelli, A.M., Pasqualini, S., Massacci, A., 2003.

Interaction of cadmium with glutathione and photosynthesis

in developing leaves and chloroplasts of Phragmites australis

Trin. ex Steudel. Plant Physiol. 133 (2), 829–837.

Schreiber, U., Krieger, A., 1996. Two fundamentally different

variable chlorophyll fluorescence in vivo. FEBS Lett. 397,

131–135.

Srivastava, A., Guisse, B., Greppin, H., Strasser, R.J., 1997. Reg-

ulation of antenna structure and electron transport in photosys-

tem II of Pisum sativum under elevated temperature probed by

the fast polyphasic chlorophyll a fluorescence transient: OKJIP.

Biochim. Biophys. Acta 1320, 95–106.

Stirbet, A., Govindjee, Strasser, B.J., Strasser, R.J., 1998. Chlor-

ophyll a fluorescence induction in higher leaves: modelling and

numerical simulation. J. Theor. Biol. 193, 131–151.

Strasser, R.J., Govindjee, 1992. On the O–J–I–P fluorescent tran-

sient in leaves and D1 mutants of Chlamydomonas reinhardtii.

In: Murata, N. (Ed.), Research in Photosynthesis, vol. 4.

Kluwer Academic Publishers, Dordrecht, The Netherlands,

pp. 29–32.

Strasser, R.J., Srivastava, A., Govindjee, 1995. Polyphasic chlor-

ophyll a fluorescence transient in plant and cyanobacteria.

Photochem. Photobiol. 61, 32–42.

Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2000. The

fluorescence transient as a tool to characterize and screen

photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty,

P. (Eds.), Probing Photosynthesis: Mechanisms, Regulation

and Adaptation. Taylor and Francis, London, pp. 445–

483.

Strasser, R.J., Stirbet, A., 2001. Estimation of the energetic con-

nectivity of PSII centres in leaves using the fluorescence rise O–

J–I–P. Fitting of experimental data to three different PSII

models. Math. Comput. Simulat. 56, 451–461.

Xu, C.C., Jeon, Y.A., Hwang, H.J., Lee, C.H., 1999. Suppression of

zeaxanthin epoxidation by chloroplast phosphatase inhibitors in

rice leaves. Plant Sci. 146, 27–34.

Yelenosky, G., 1982. Chilling injury in leaves of citrus leaves at

1.7 8C. Hort. Sci. 17, 385–387.

Zinoni, F., Antolini, G., Campisi, T., Marletto, V., Rossi, F., 2002.

Characterization of Emilia Romagna in relation with late frost

risk. Chem. Phys. Earth 27, 1091–1102.