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The responses of photosynthetic capacity, chlorophyll fluorescence and chlorophyll content of

nectarine (Prunus persica var. Nectarina Maxim) to greenhouse and field grown conditions

Abstract

Three nectarine (Prunus persica var. Nectarina Maxim.) cultivars grown under solar-heated
greenhouse and open-field in northwest China, were tested to evaluate their photosynthetic
and chlorophyll fluorescence response to both growth conditions, and whether nectarine
plants acclimate to the solar-heated greenhouse growth condition. Comparisons of light-
saturated photosynthetic capacity (Amax) and CO2-saturated photosynthetic capacity
(RuBPmax) indicated that each cultivar (Z, Zao-Hongzhu; H, Hua-Guang; Y, Yan-Guang)
maintained similar rates of light-saturated and CO2-saturated carbon assimilation when grown
in both conditions. The curve of diurnal variation of net photosynthetic (PN) rate showed
double peaks in open-field but single when grown in greenhouse. Compared with open-field-
grown plants, a significant increase of daily average PN was found in Z but decreased in Y in
greenhouse. The diurnal variation of Fv/Fm indicate that plants grown in greenhouse
experience less photoinhibition than in open-field condition. A reduction in chlorophyll (chl)
a/b ratio in leaves of greenhouse grown plants with significant increase in chlorophyll (chl) b
content were obtained. The results suggest that some nectarine cultivars have the ability to
acclimate to the solar-heated greenhouse growth condition. # 2007 Elsevier B.V. All rights
reserved.

Introduction

1. Introduction Nectarine has become one of the most important fruit trees in China. In
the greenhouse, nectarine fruits mature at least 60 days earlier than in open-field
conditions. As demand for yeararound fresh fruit has increased, the price of nectarine
fruits produced in greenhouse was six times higher than in open-field. Now, the
production of nectarine fruits in solar-heated (heated only by the sun), plastic-covered
greenhouse has markedly increased to 110,000 ha in China. Yield of nectarine is a
function of the production of assimilates by photosynthesis and translocation of
assimilates to reproductive sinks, the alteration of the photosynthetic rate (PN) can
influence also plant growth. Climate, soil nutrients and water have long been
recognized as the main factors influencing agricultural productivity (Boyer, 1982;
Fischer and Turner, 1978; Novoa and Loomis, 1981). Water and nutrients could be
managed easily through fertilization and irrigation. Nevertheless, in production, it was
difficult to control other micro-climate factors (light intensity, temperature, CO2
concentration, Fig. 1) not only in open-field, but also in solar-heated greenhouse.
Despite the necessity of light for autotrophic organisms, no plant is capable of using
100% of maximum solar irradiation for photosynthesis (Demmig-Adams et al., 1997).
Most plants have the ability to acclimate to a specific light environment. Chlorophyll
concentration, Calvin cycle intermediates, chloroplast density and the activity and
expression of antioxidant enzymes have all been reported to fluctuate during the
acclimation process (Demmig-Adams et al., 1997; Herna´ndez et al., 2004a,b; Karpinski
et al., 1997; Yoshimura et al., 2000). Nectarine develops well under long day and high
light intensity conditions. Under these climates, the plant often acclimates to light, and
this acclimation promotes plant growth and fruit development. Similar observations
have been reported in other crops such as cucumber and pepper (Wang et al., 1999).
However, in open-field, most plants receive more sunlight than they can actually use
for photosynthesis, as a result, photosynthesis produces more NADPH and ATP than
necessary. This accumulation of redox and energy equivalents will decrease the
plastoquinone pool and/or inhibit the watersplitting complex, inevitably leading to PSII
inactivation, the so-called photoinhibition (Anderson et al., 1997; Karpinski et al., 1997,
1999). Under these circumstances, besides adjusting light absorption, plants have ways
of getting rid of excess light energy that has already been absorbed. This update will
focus on protective non-photochemical mechanisms that quench singlet-excited
chlorophylls and harmlessly dissipate excess excitation energy as heat. These
nonphotochemical quenching (NPQ) processes occur in almost all photosynthetic
eukaryotes, and they help to regulate and protect photosynthesis in environments in
which light energy absorption exceeds the capacity for light utilization (Patricia et al.,
2001).
For the plants grown in greenhouse and open-field conditions, the growth periods and
micro-climates were significantly different. In this experiment, the growth period of
nectarine trees in greenhouse was from 24 January to 28 October, whereas, in open-
field, it was from 15 April to 22 October. The growth period was prolonged about 90
days in greenhouse grown condition (Fig. 2). Meanwhile, the production of nectarine
in greenhouses during winter/spring frequently encounters long period of unfavorable
weather conditions such as fluctuant CO2 concentration and temperature in
combination with low light (Fig. 1). In recent years, photosynthesis of fruit tree has
been studied in banana (Thomas and Turner, 2001), mango (Shivashankara and
Mathai, 2000), grape (Cohen et al., 2005; Patakas et al., 2003), apple (Mierowska et al.,
2002) and jujube (Su and Liu, 2005), etc. However, little information is available on the
responses of photosynthesis to solar-heated greenhouse conditions in woody plant
including nectarine. We hypothesized that the greenhouse growth condition could be
regarded as a kind of environment stress compared to the original open-field growth
condition. Therefore, the objective of this research was to determine: (1) whether
differences in the photosynthetic response to different growth conditions
(photosynthetic photon flux density and CO2 concentration, especially) and different
nectarine genotypes exist and (2) whether the studied cultivars show acclimation to
the new greenhouse growth conditions.

2. Materials and methods


2.1. Plant material and experimental site description
Two sets of experiments were conducted in Gansu province, northwest China. The
climate is continental monsoon type with severe and dry winter, and high temperature
and concentrated rainfall in the summer. Annual sunshine is around 2650 h, annual
average available accumulated temperature (10 8C) is 3242 8C, and there are 195
frost-free days in a normal year.
For field experiments, the plant materials were planted in April 2001 with a designed
plant population of 825 plants ha1 . Row space was 4 m. In 2004, plants received a
total of 230 kg N ha1 and 68 kg P ha1 . Conventional farming techniques were used.
For greenhouse experiments, 3-year-old seedlings were transplanted into 15-L
containers filled with a substrate of three soil: 1 organic fertilizer: 0.5 sand (by volume)
on 2 April 2003. On 9 November 2003, containers were placed into the greenhouse at
a depth of 40 cm and a designed plant population of 6660 plants ha1 , seven times
higher than that used in the open-field experiment. Plants received a total of 300 kg N
ha1 and 100 kg P ha1 . Of the N and P nutrients, 70% was applied at seedling and the
remainder was used during the whole growing season. Special techniques for
nectarine grown in greenhouse were used.Three cultivarsZao-Hongzhu (Z), Hua-Guang
(H) and Yan-Guang (Y) plants were used for greenhouse and open-field experiments,
30 days after blossom in 2004 (Fig. 2), respectively.

2.2. Gas exchange measurements


PPFD response curves were conducted on sunny days between 09:00 and 13:00 by
using a CIRAS-1 (PP Systems, Hansentech,UK) infrared gas analyzer modified with a
temperature and light controlled cuvette. A 12 V mobile battery was connected to the
system to supply the necessary power requirements. Preliminary measurements
indicated that before 09:00 and after 13:00, stomatal conductance was limited and
carbon assimilation was unusually low. All plants were exposed to 15 min of full
sunlight to allow for stomatal opening before taking measurements. The air cuvette
temperature and the air CO2 concentration was maintained at 25 8C and 400 mL L1 ,
respectively. PPFD was decreased from 1600 to 20 mmol m2 s 1 (1600, 1400, 1200,
1000, 800, 600, 400, 300, 200, 150, 100, 80, 40 and 20 mmol m2 s 1 ). Assimilation was
recorded at each light level following a 10 min acclimation period. Measurements
were repeated to obtain at least three stable readings for each of the marked leaves.
Diurnal variation of PN in open field and greenhouse were taken at 08:00, 10:00,
12:00, 14:00, 16:00 and 18:00. PN–Ci curves were generated to investigate specific
characteristics of photosynthesis and to estimate potential photosynthetic capacity.
Marked leaf was placed in the cuvette at 25 8C, and 1000 mmol m2 s 1 PPFD, the CO2
concentration was increased from 20 mL L1 to 2000 mL L1 (20, 40, 80, 100, 150, 200,
400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000 mL L1 ). The air within the cuvette
was maintained at

80% relative humidity to minimize stomatal conductance. Assimilation was recorded at


each CO2 concentration level following a 10 min acclimation period. Measurements
were repeated to obtain at least three stable readings for each of the marked leaves.
2.3. Chlorophyll fluorescence measurements
The diurnal course maximal photosystem (PS) II photochemical efficiency (Fv/Fm) were
measured with a Plant Efficiency Analyzer (PEA, Hansatech FMS2, Hansatech
Instruments Ltd, Kings Lynn, UK) after the leaves were darkadapted for 40 min using
clips. The maximal fluorescence (Fm) with all PSII reaction centers closed was
determined by a 0.8 s saturating pulse at 8000 mmol m2 s 1 in dark-adapted leaves,
sufficient for Fm to be reached in all cultivars investigated.
2.4. Chlorophyll content Following the final measurements as above mentioned, leaves
were collected in early morning, and transported in ice box to the laboratory for later
determinations of contents of chlorophyll (chl) content. Chlorophyll was extracted by
placing three discs (2.36 cm2 ) in 8.0 mL N,N0 -dimethylformamide (DMF) in the dark
at room temperature on a rotary shaker for 48 h. Chlorophyll (chl) a and b were
determined spectrophotometrically as described by Porra et al. (1989).
2.5. Experimental design and statistical analysis Ten individual uniform sized plants of
each cultivar were selected randomly, two fully expanded, disease-free, fully sunlit
leaves were measured on each plant. Namely, 20 leaves were measured totally for
each treatment. Three leaves which had the maximal and similar photosynthetic
capacity among these 20 leaves on different plants were chosen as three replications
per treatment. These three leaves were tagged and used for the following
measurements. The tagging process was completed 1–2 days before the formal
measurements. Standard error (S.E.) was calculated and differences in mean values for
each treatment were tested at p < 0.05 according to least significant difference test
(LSD).
3. Results
3.1. Diurnal variation and average PN
Growing conditions had significant effects on photosynthetic patterns. In open-field
conditions, the curve of diurnal variation of PN appeared to be a marked double-peak
in a sunny day. However, in greenhouse, a single-peak curve was obtained (Fig. 3).
Normally, the daily highest PN values were achieved at 14:00–15:00 h in greenhouse
and at 10:00 in open-field (Fig. 3). The average PN were 9.97, 8.43, 7.40 mmol m2 s 1
in greenhouse and 8.25, 8.37, 8.09 mmol m2 s 1 in open-field for cultivars Z, H and Y,
respectively. For the cultivar Z and H, the daily average PN increased in greenhouse
conditions, conversely, the cultivar Y had significant lower daily average PN in
greenhouse than in open-field conditions (Fig. 3, Table 1). On the other hand, in open-
field growth conditions, the daily average PN among the three cultivars did not differ
significantly, nevertheless, in greenhouse, it was significantly different from each other
(Table 1).
3.2. PN–PPFD and PN–Ci curves
We observed a similar response pattern of PN to PPFD and Ci for all the three cultivars
(Fig. 4). Under greenhouse condition, all the three cultivars had a higher RuBPmax and
lower LSP, CCP and CSP than grown in open-field. Meanwhile, the LCP and Amax
values of cultivars Z and H increased, however, for cultivar Y, these two values
decreased when they were grown in greenhouse (Table 1). The RuBPmax and Amax
values did not change significantly between the two grown conditions for the all three
cultivars. In the both grown conditions, the significant differences existed in almost all
these parameters among three cultivars. Cultivar Y had the highest Amax value (20.47
mmol m2 s 1 ) in open-field, however, when grown in greenhouse, it had the lowest
Amax value (18.7 mmol m2 s 1 ) among the three cultivars (Table 1).
3.3. Chlorophyll fluorescence and chlorophyll content
Diurnal variation of photochemical efficiencies of PSII (Fv/ Fm) showed significant
differences between the two grown conditions (Fig. 5). For each cultivars, the Fv/Fm
were similar in the morning or evening, and the values were close to 0.85; moreover,
the lowest Fv/Fm (0.64, 0.62, 0.58 for Z, H and Y, respectively) was found around 15:00
h under open-field grown conditions (Fig. 5). However, diurnal courses of Fv/Fm values
of those three cultivars changed a little in greenhouse, they presented weak valleys
and varied between 0.78 and 0.85 (Fig. 5). For all the three cultivars, total chlorophyll
content values did not show significant difference between the both growth
conditions, although chl b content was significantly higher in leaves from plants grown
in greenhouse than in open-field. Therefore, chl a/b ratios were significantly lower in
the leaves from plants grown in greenhouse than in open-field.

4. Discussion
The extent of the photosynthetic response of mature leaves to an increased PPFD
depends on complex interactions between photoinhibitory damage, repair,
photoprotection and acclimation of the photosynthetic machinery (Pearcy and Sims,
1994; Turnbull et al., 1993). Double-peak curves of diurnal variation of PN in sunny
days in open-field were more likely the result of photoinhibition caused by high PPFD
of 1000–1300 mmol m2 s 1 (Matos et al., 1998; Figs. 1 and 3). In greenhouse, because
of the isolation and the respiration of plants inside, CO2 concentration was higher than
open-field in the early morning. Before the air vents were open, CO2 concentration
decreased sharply accompanied with the beginning of photosynthesis, although no
photoinhibition occurred and the PPFD was usually lower than 1000 mmol m2 s 1 , the
decline/few increase of photosynthetic rates was found in all the three cultivars at
10:30 correlated with the decrease in CO2 concentration (Figs. 1 and 3). Furthermore,
it is worthy to note that daily average PN of each cultivar was very similar in openfield;
however, significant differences were found among three cultivars’ average PN when
the plants were grown in greenhouse (Table 1, Fig. 3). Perhaps, cultivars Z and H
adapted to greenhouse conditions better than cultivar Y. Nectarine is a light-
acclimated plant. According to Kitao et al. (2000), when nectarine plants grow under
open-field condition, they have the ability to increase Amax as well as other energy
dissipating mechanisms. They typically have a higher Amax when exposed to high light
intensity. Likewise, Amax of nectarines grown in greenhouse can be reduced.
Surprisingly, in this test, statistical analyses showed that there were no significant
differences in Amax between the two growing conditions for these three cultivars
(Table 1).
It was also reported that high light acclimated plants had increased respiration and LCP
(Lambers et al., 1998), another characteristic of plants that have acclimated to a high
light environment. But, in this test, Z and H cultivars were able to acclimate and
increase Amax and LCP in the greenhouse environment. These results also differ from
those reported by Wang et al. (2000), who found a reduction in Amax and LCP in ‘Shu-
Guang’, another nectarine cultivar used in greenhouse production in China. The whole
micro-ecosystem is an important factor in photosynthetic ability of plants and it may
have been a confusing factor when comparing with previous reports where different
protecting environments were used. In the current study, plants were grown in solar-
heated, plastic-covered greenhouse, which may have overcome the adverse effects
associated with photosynthetic restriction. Chlorophyll (chl) fluorescence continues to
be a mainstay in studies of photosynthetic regulation and plant responses to the
environment due to its sensitivity, convenience, and nonintrusive quality (Rascher et
al., 2000; Schreiber et al., 1995). It is frequently used to determine the state of energy
distribution in the thylakoid membrane, the quantum efficiency of PSII, and the extent
of photoinhibition (Critchley, 1998; Maxwell and Johnson, 2000). Under intense
sunlight, photochemical damage was reflected in either the increase in original
fluorescence (Fo), or in the decrease in maximum fluorescence (Fm), or in the ratio of
variable (Fv = Fm Fo) to maximal fluorescence (Fv/Fm) (Bjo¨rkman and Demmig, 1987;
Demmig-Adams and Adams, 1992; Thomas and Turner, 2001).
In the present study, it should be noted that in response to increasing PPFD, Fv/Fm
decreased as the PPFD increased in both conditions, however, the decrease in open-
field was more severe (Fig. 5). These results indicate some degree of photoinhibition
under high light around 15:00 h in a sunny day, and confirmed that the double-peak
curves of diurnal variation of PN in the sunny day in open-field was the result of
photoinhibition caused by high PPFD (1000–1300 mmol m2 s 1 ) (Figs. 1, 3 and 5).
Additionally, photoinhibition is typically associated with, but not necessarily
accompanied by chlorophyll degradation and a reduction of Amax (Critchley, 1998;
Demmig-Adams et al., 1998). After the mid-day, rapid recoveries of Fv/Fm were found
in all the three cultivars, this confirmed a photoprotective mechanism which might aid
photosynthesis recovery when favorable conditions are restored. Chl b is usually the
main component of light harvesting chlorophyll protein (LHCP), so that the amount of
this pigment is important for the capacity of the leaf to accommodate to the shade
condition (Kura-Hotta et al., 1987; Lei et al., 1996), whereas chl a is concentrated
around PSII. To capture as much light as possible, shade-grown plants typically have
more light-harvesting complexes per unit area than sun-grown plants (Jason et al.,
2004). In this study, reduction in chl a/b ratio in leaves of greenhouse grown plants
with significant increase in chl b content (Table 2) appeared most likely due to changes
in the organization of both lightharvesting and electron transport components
(Schiefthaler et al., 1997). On the other hand, lower chl b content could be an
indication of chlorophyll destruction by excess irradiance in open-field conditions
(Jason et al., 2004). Our results are supporting the idea that higher chlorophyll a to
chlorophyll b ratio, without significant change of total chlorophylls content, it should
be considered as a protecting mechanism and not just as the consequence of the
open-field condition.
Total chl a + b content were not significantly different between both growing
conditions for three cultivars (Table 2). It may be speculated that the level of total chl a
+ b was high enough to enable the high non-photochemical quenching (NPQ) values in
both growing conditions and facilitated efficient photoprotection of the
photosynthetic apparatus against excess irradiation. This was confirmed by the
photosynthetic recovery in the afternoon. Data from the present study indicate that in
the three cultivars investigated, H and Z have the ability to acclimate to the
greenhouse grown condition (Fig. 1, 2) as reported in other plants (Anderson et al.,
1995), the strategy of acclimation – involving adjustments in reaction centre
stoichiometry (Bailey et al., 2001; Chow et al., 1990; Walters and Horton, 1994),
altered antenna size (Bailey et al., 2001; Leong and Anderson, 1984) and altered levels
of Rubisco (Bailey et al., 2001; Seemann et al., 1987; Walters and Horton, 1994). Such
acclimation is readily monitored through the consequent changes in the ratio of
chlorophyll a to chlorophyll b (chl a/ b), and in the light saturated rate of
photosynthesis (Amax), these changes were also found in our experiments.
Acknowledgements The authors thank to Dr. Lorenzo Burgos and Jose A. Hernandez—
their valuable suggestions and grammar corrections on this article. Thanks to Minghui
Ban and Junqiang Niu for technical assistance.