Changes in assimilation capacity during leaf development in broad ...

Journal of Horticultural Science & Biotechnology (2007) 82 (1) 69–78

Changes in assimilation capacity during leaf development in broadleaved Prunus persica and sclerophyllous Olea europaea By S. MARCHI1,2, D. GUIDOTTI2, L. SEBASTIANI1 and R. TOGNETTI3* 1 BioLabs, Polo Sant’Anna Valdera, Scuola Superiore Sant’Anna, Viale Rinaldo Piaggio, I-56025 Pontedera (PI), Italy 2 Aedit s.r.l., Viale Rinaldo Piaggio, I-56025 Pontedera (PI), Italy 3 Dipartimento di Scienze e Tecnologie per l’Ambiente e il Territorio (STAT), Università degli Studi del Molise, Contrada Fonte Lappone, I-86090 Pesche (IS), Italy (e-mail: [email protected]) (Accepted 13 October 2006) SUMMARY Net photosynthesis, dark respiration, chlorophyll and carbohydrate content, and leaf growth in plants of deciduous peach (Prunus persica (L.) Batsch.) and evergreen olive (Olea europaea L.) grown in controlled environmental conditions were measured to assess changes in carbon balance during leaf development at the 6th, 12th and 16th nodes (first flush) from the beginning of expansion to full maturity. A simulation model was written including growth traits, physiological measurements and meteorological data. In both species, gas exchange varied with the percentage of leaf expansion, regardless of the node examined. The relationships between net daily CO2 assimilation (24 h), chlorophyll content, and the ratio of leaf mass per area, across all nodes, differed between species. Photosynthetic capacity differed significantly between different degrees of leaf expansion in both species. The model predicted differences in leaf carbohydrate balance between peach and olive. The onset of carbohydrate export from individual leaves varied with species, being 18 – 19 d and 28 – 29 d from emergence in peach and olive, respectively.

S

easonal patterns in carbon accumulation are largely determined by the dynamics of shoot growth (Johnson and Lakso, 1986a). Since leaves are the main source of CO2 assimilation in plants, leaf area development is a major determinant of carbon accumulation. Carbohydrate metabolism in leaves changes during development (Turgeon, 1989). Young leaves depend, in part, on carbohydrates imported from other regions of the plant, while mature leaves produce an excess of photoassimilates and act as the major source of transport sugars. The timing of the transition from the import (sink) to the export (source) phase coincides with a net positive accumulation of carbon in the leaf. In spite of this, leaf area dynamics are often not adequately represented in many carbon-balance models for perennial plants. Many of these models focus on carbon inputs and do not include the dynamics of shoot growth (Buwalda, 1991; Johnson and Lakso, 1986b). The importance of including the dynamics of shoot growth to fully characterise seasonal patterns in carbon accumulation, was emphasised by Grossman and DeJong (1994). In particular, shoot node (leaf, layer) number seems to be an appropriate variable for modelling shoot leaf area expansion, as it represents the structural development of the shoot (Greer et al., 2004). Source-sink relationships, and the regulation of carbon allocation determine crop yield in plants. Modelling is essential to generate an accurate carbohydrate balance for leaves and other organs (Kappes and Flore, 1986). Information is needed on carbohydrate accumulation by leaves, and on the rates of carbohydrate fixation and loss *Author for correspondence.

by photosynthesis and respiration, respectively. Carbohydrate content measurements are destructive. To obtain a time-course of carbohydrate accumulation, it is therefore necessary to correlate carbohydrate content to parameters that can be measured non-destructively. The adaptive significance of evergreen vs. deciduous leaves has received wide attention in the literature (Chabot and Hicks, 1982). Models that consider the carbon associated with growing leaves with different life spans have been used to suggest reasons for the relative success of contrasting leaf phenologies at various scales (Kikuzawa, 1991). Significant differences in aboveground annual production in neighbouring deciduous and evergreen trees may not be clearly related to leaf longevity (Gower et al., 1993). In general, leaves of dicotyledonous plants stop importing and begin to export photo-assimilates when they are 30 – 60% fullyexpanded (Turgeon, 1989). Developing leaves continue to import photo-assimilates from source leaves for a period after they have begun to export their own photosynthetic products. However, changes in sinksource status have not been studied intensively for plants growing in Mediterranean-type agro-ecosystems, and comparisons between evergreen and deciduous species are rare (Miyazawa et al., 2003). The present work is based on companion studies on the carbon economies of Olea europaea L. and Prunus persica L. plantlets (Marchi et al., 2005a, b). In both species, shoot and leaves expanded in a sigmoid pattern, with differences among the sampled nodes (6th, 12th and 16th from the base; first flush). Photosynthesis varied with leaf development. Young leaves had low CO2 assimilation rates that were reflected in their low

70

Modelling sink-source transition in peach and olive

chlorophyll concentration. Net daily CO2 assimilation was negative in young expanding leaves. The sink-source transition, defined as the time when the increase in daily carbohydrate exchange rate exceeded the daily increase in leaf carbohydrate content, occurred before full leaf expansion. In olive leaves, the sink-source transition occurred between 10 – 30% expansion, depending on the node. Below 30 – 50% full expansion, peach leaves might not respond to assimilate requirements from sinks, being sinks themselves. The transition from import to export is a function of the development of the photosynthetic system during leaf ontogeny, with the achievement of a net positive carbon balance (Marchi, 2004). In this study, we report on changes in the assimilation capacity of peach and olive plantlets, with growth confined to a single axis with leaves, stem and roots, without the complications of flowers and fruits. The aim was to develop a framework for understanding speciesspecific relationships between development and physiology in the early phases of leaf growth. The specific focus was on the area expansion of individual leaves along the stem, firstly to take into account the effects of radiation, and secondly to model leaf area expansion. We developed a simple simulation model incorporating growth traits, physiological measurements and meteorological data. We hypothesised that young leaves would have a lower assimilation potential than mature leaves, and that differences in the sink-source transition between peach and olive would reflect the different leaf habits of mesophyte deciduous vs. sclerophyllous evergreen species.

MATERIALS AND METHODS Plant material and growth conditions One-year-old saplings of ‘Big Top’ peach on GF677 rootstock (P. persica P. amygdalus) were grown in 3.5 l plastic pots. Current year self-rooted cuttings (harvested from mother plants in August of the previous year), from ‘Leccino’ olive were grown in 1.5 l plastic pots at Pisa (43°43’ N; 10°23’ E). All plants were trained to a single shoot in a naturally illuminated greenhouse, covered with a nursery net to reduce incoming radiation. Maximum photosynthetically active radiation was approx. 600 µmol photons m–2 s–1. Pots containing a universal loam:perlite:field soil mix [1:1:1 (v:v:v) for peach], or sphagnum peat:pumice [1:1 (v:v) for olive] were watered regularly to field capacity. At the beginning of leaf area expansion (early March for both peach and olive), olive and peach plants were fertilised with 50 ml and 100 ml half-strength Hoagland solution, respectively. The treatment was repeated approx. every 20 d throughout the experiment. Pests were chemically controlled and pots were weeded manually. Growth analysis Leaf area was monitored on ten control plants per species, approx. every 5 d or 10 d for peach and olive, respectively, throughout the experimental period. Leaf growth was described by a logistic model (Marchi et al., 2005a, b). The asymptote was fixed at 100% (full single leaf expansion). The model was fitted to the data by the iterative process of the Marquardt-Levenberg algorithm (SigmaPlot; SPSS Inc., San Rafael, CA, USA). The

length (L) and the width (l) of each leaf lamina were measured and converted to leaf area (cm2) after calibration (Marchi et al., 2005a, b). The calibration curves were established from preliminary destructive leaf samplings and measurements of leaf areas using a planimeter (LI-3000; LI-COR Inc., Lincoln, NE, USA). The ages of the expanding leaves used for measurements were estimated from their length and width (area), using the logistic equation. Gas exchange and chlorophyll content Gas exchange measurements were made on attached leaves at different rates of surface expansion using a portable gas exchange system LI-6400 (LI-COR). Measurements took into account differences in phenology and growth patterns between species, from March to May for peach, and from March to September for olive. Measurements were done in duplicate during sunny days under natural photosynthetic photon flux density (PPFD) and air temperature conditions (relative humidity inside the cuvette was kept at 70 ± 2%). Mean daily temperatures during measurements were 20° – 24°C and 20° – 28°C for peach and olive, respectively. Photosynthetic rate (A; µmol CO2 m–2 s–1), dark respiration (R; µmol CO2 m–2 s–1, at the end of the dark period), stomatal conductance (gs; mol H2O m–2 s–1), and intercellular CO2 concentration (Ci; µmol CO2 mol–1 air) were measured on 20 plants at the 12th node from the base, and the Ci to ambient CO2 concentration (Ca) ratio (Ci/Ca; µmol µmol–1) was calculated. In the greenhouse, in the early morning, we first measured the rate of dark respiration (mitochondrial) after shading the cuvette with an aluminium cover, as the rate of dark respiration would increase with the accumulation of photosynthetates (Noguchi et al., 1996). Dark respiration was extrapolated at different temperatures using a Q10 relation as follows: R = RnQ10(T–Tn)/10

(Q10 = 2.2)

where Tn is the leaf temperature (°C) at which Rn was measured, and T the leaf temperature (°C) at which R was calculated. For Mediterranean climates, Q10 is expected to be around 2.2 (Larcher, 1983). The plants were then brought in to the open, avoiding direct sunlight, to measure the daily course of gas exchange between the leaf and the atmosphere. The daily CO2 exchange rate was obtained by subtracting the night-time CO2 loss (by respiration) from the daytime CO2 gain (by photosynthesis; derived from gas exchange measurements). To estimate the daytime CO2 assimilation gain by photosynthesis, we integrated diurnal photosynthetic values from dawn to dusk, for each peach and olive leaf measured. Photosynthetic rates at dawn and at dusk were considered equal to zero. To estimate night-time CO2 loss by respiration, we multiplied the dark respiration rate by the period in which the PPFD was zero, and assumed that the dark respiration rate was constant during the night. After gas exchange measurements, plants were harvested and the leaves were oven-dried at 65°C until constant weight, then weighed for subsequent analysis. The leaf dry mass per area (LMA; g m–2) was determined by dividing the dry mass of each leaf by the corresponding area.

S. MARCHI, D. GUIDOTTI, L. SEBASTIANI and R. TOGNETTI

71

Total chlorophyll was estimated with a portable chlorophyll meter (SPAD-502; Minolta Camera Co., Osaka, Japan), during the course of experiment. Data were converted into total concentrations of chlorophyll per unit leaf area (µmol m–2) using a calibration curve (Marchi et al., 2005a, b). These regressions were established from preliminary destructive leaf sampling, chlorophyll extraction in N,N-dimethylformamide, and spectrophotometric determinations (Lambda 6 UV-VIS; Perkin-Elmer, Boston, MA, USA) according to Moran (1982). Chlorophyll contents were monitored during shoot development on the same plants used for gas exchange measurements at the 6th, 12th and 16th nodes from the base.

temperature dependence of the model parameters, were calculated using the approach of Harley et al. (1992) and Wullschleger (1993). The CO2 compensation point (c; in Pa) was calculated as the point that the model function crossed the x-axis (A = 0). Relative stomatal (i.e., gasphase) limitation to A (lg; %) was calculated using the empirical function describing A as a rectangular hyperbolic function of Ci, from which stomatal supply vs. mesophyll demand of CO2 can be extrapolated (Farquhar and Sharkey, 1982). The lg was then estimated relative to the photosynthetic rate with all stomatal limitation removed (Jones 1985):

Photosynthetic response curves The CO2 response curves (A/Ci) were measured at the 12th node at two different rates of surface expansion (50% and 100%) on four replicate plants per species. Measurements were done in late-April (peach) or midJune (olive) using the LI-6400, considering differences between species for maximum gas exchange. The leaf chamber temperature and humidity were adjusted to maintain a leaf-to-air vapour pressure difference of approx. 1.0 kPa and 1.2 kPa for peach and olive, respectively (air temperature 21° – 26°C, average maximum daily temperature of each period of measurement), taking into account seasonal variations between species-specific measurements. The A/Ci curves were obtained by changing the CO2 concentration entering the cuvette from 50 – 800 µmol mol–1 by means of an external CO2 cartridge mounted on the LI-6400 console, automatically controlled by a CO2 injector. The CO2 assimilation rate was first measured by setting the reference CO2 concentration near ambient (390 µmol mol–1), then at 300, 200, 100, 50, 400, 400, 600, 800 µmol mol–1. Gas exchange was determined at each step after maintaining the leaf at the new CO2 concentration and recording the data automatically once the total coefficient of variation was less than 1%. Light intensity (LED source, red-blue 6400-02B) was maintained at 800 µmol photons m–2 s–1 (above the photosynthetic light saturation for these plants developed in controlled conditions). The response of leaf A to Ci was analysed according to the mechanistic model of CO2 assimilation proposed by Farquhar et al. (1980), and subsequently modified by Sharkey (1985) and Harley and Sharkey (1991). The biochemical model describing A calculates three parameters potentially limiting photosynthesis: Vcmax [maximum carboxylation rate of ribulose-1,5bisphosphate carboxylase-oxygenase (Rubisco) in µmol CO2 m–2 s–1]; Jmax (ribulose-1,5-bisphosphate; RuBP, regeneration capacity mediated by electron transport rate; in µmol electrons m–2 s–1); and TPU (rate of triose phosphate utilisation for sucrose and starch synthesis; in µmol CO2 m–2 s–1). Day respiration rate (mitochondrial; in µmol CO2 m–2 s–1) was designated Rday and resulted from processes other than photorespiration. The model was parameterised and fitted to the experimental data using Photosyn Assistant (Dundee Scientific, Dundee, UK). The Michaelis-Menten constants for carboxylation and oxygenation, the specificity factor values of Rubisco for CO2/O2, the efficiency of light energy conversion, and

where A is the mean actual assimilation rate and A0 the assimilation rate that would occur if resistances to CO2 diffusion were zero (A at Ci equal to the growth Ca). The light response curves (A/Q) were measured on the same plants, leaves and times as for the A/Ci curves, using the LI-6400. The light response curves were obtained by varying the light intensity from 2,000 – 0 µmol photons m–2 s–1 using the light source provided with the equipment (LED source, red-blue 6,400-02B) and mounted on the LI-6400 cuvette, which enabled automatic changes of photon flux density (2,000, 1,500, 1,000, 500, 200, 100, 50, 20 and 0 µmol photons m–2 s–1) with 200 s intervals. The CO2 flux entering the cuvette was adapted by means of CO2 cartridges to maintain an internal chamber CO2 concentration of 400 µmol mol–1. Leaf chamber temperature and humidity were adjusted to maintain a leaf-to-air vapour pressure difference of approx. 1.2 kPa and 1.3 kPa for peach and olive, respectively (22° – 26°C), taking into account seasonal variations between species-specific measurements. The A/Q curves, when started at low light levels, resulted in a limitation of photosynthesis at high light due to insufficient stomatal opening. The response of leaf A to Q was modelled by a non-rectangular hyperbola, where the initial slope was the apparent quantum efficiency (ø; in µmol CO2 µmol–1 photons). The light compensation point (l; in µmol photons m–2 s–1) and apparent dark respiration (Rd; in µmol CO2 m–2 s–1) were estimated from axis intercepts. The light saturated maximum (Asat; in µmol CO2 m–2 s–1) was the upper asymptote (Prioul and Chartier, 1977). All these parameters were determined by fitting data to the quadratic model function using Photosyn Assistant (Dundee Scientific). The light compensation point was calculated as the point that the model function crossed the x-axis (A = 0).

lg = 100 (A0 – A) / A0

Modelling the sink-source transition A simple model was developed to simulate single-leaf development as a function of hourly solar radiation. Climatic data were obtained from a standard meteorological station (ARSIA, Regione Toscana, Italy). The model is object-oriented (leaf) and uses meteorological data as inputs. The model integrates the experimental data of leaf development and gas exchange to obtain a model environment, thus simulating the import and export of carbohydrates (CH2O), estimating the sink-source balance of growing leaves. The model was open, and based on resource availability (input), which may be integrated with experimental data (fruit


72

development, crown structure, light interception, water and nutrient balance, etc.) to increase the quality of the simulation (output). The development of individual leaves was simulated from day-0, with the minimum surface area fixed at 0.1 cm2, and the maxima at 50 cm2 and 8 cm2 for peach and olive, respectively (values close to experimental observations). For each day, the model computed carbon balance and leaf development. The percentage of leaf development was considered to increase following a sigmoid pattern as a function of the Julian day of the year, from the beginning of simulation: a area(%) = —————— days b 1 + —— x

( ) 0

in which a (the asymptote) was fixed at 100, b (the width of the gradual transition from low to high values or curve shape, per day) equalled –2.41 and –2.2635, and x0 (the value at which the function was 50% of its width or curve displacement along the x-axis, in d) equals 13.6 and 21.1057 for peach and olive, respectively. The percentage of leaf development was used as input variable when estimating photosynthesis and respiration. The effective surface area (in cm2) was then determined according to the percentage of expansion and maximum surface area at full development. The dark respiration rate (R; µmol CO2 m–2 s–1) as a function of leaf area expansion (%) was computed, fitting a logarithmic model to the experimental data (see Figure 1 for parameters). The light response curve of photosynthesis was modelled by the following equation: øQ + Amax(gross) – (øQ + Amax(gross))2 – 4øQsAmax(gross) A = ————————————————————— –R 2s in which A (µmol CO2 m–2 s–1) is the net assimilation rate, Q is the incident radiation (µmol photons m–2 s–1), ø is the apparent quantum efficiency (experimental values from A/Q curves averaged 0.1400 and 0.1135 µmol CO2 µmol–1 photons for peach and olive, respectively), s is an interpolation a-dimensional parameter describing the convexity of the curve (0.275 and 0.700 for peach and olive, respectively), and R is the dark respiration estimated through the logarithmic equation (and assumed constant during the night). Values of daily maximum photosynthesis (Amax), measured experimentally, were interpolated fitting a linear (peach) or logarithmic (olive) function to leaf area expansion (%) (see Figure 2 for parameters) and, implementing R equations, the maximum gross photosynthesis Amax(gross) was estimated. Over a 24-h period, A and R were multiplied by the leaf area (LA), then transformed to total CH2O,

considering the atomic weights of carbon, hydrogen and oxygen: net µmol CO2 m–2 s–1 = [LA (A + R)] / 104 net CH2O (g) = (net µmol CO2 m–2 s–1) 30 / 106 For each mole of CO2, the synthesis of a “basic mole of carbohydrate” with a molecular weight of 30 was assumed. The leaf was considered to be a sink for CH2O when assimilation was less than the increment of CH2O needed for growth. Such an increase was estimated from the linear regression between the percentage of leaf development and leaf dry mass (DM) minus ash concentration (determined after combustion of the sample in a muffle furnace for 6 h at 550°C). The gravimetric difference between the DM and the corresponding ash concentration was considered to be equivalent to total leaf CH2O. The organic nitrate content, volatilised during combustion, was considered negligible for our purposes. The origin of the regression was set to zero: total CH2O (g) = b area (%) The coefficient b represents the amount of CH2O required per unit increase in percentage leaf area expansion (Table I). The analysis of percentile intervals (10% and 90%) indicated extreme values, representing the minimum and maximum CH2O accumulation rates (i.e., levels of assimilated CH2O accumulation at a certain percentage of leaf area development). In the model, minimum and maximum CH2O accumulation rates were used to simulate input and output translocation of leaf assimilates. If the simulation generated an accumulation of CH2O into leaves lower than 10%, the difference between total CH2O formed and the amount of CH2O considered as the minimum was ascribed to translocation from the plant to the leaf. The estimated value of 10% was considered to be the minimum for CH2O accumulated into leaves. If the simulation generated an accumulation of CH2O into leaves higher than 90%, the difference between total CH2O formed and the amount of CH2O considered as the maximum was ascribed to translocation from the leaf to the plant. The estimated value of 90% was considered to be the maximum for CH2O accumulated into leaves. Statistical analysis Plants were assembled in a completely randomised design. All data were averaged on a plant basis and individual means used for statistical analysis. One-way analysis of variance (ANOVA) for foliage parameters of each species was performed. Statistical analysis was conducted using the Statistica statistical package (StatSoft Inc., Tulsa, OK, USA). Separation of means was performed using an LSD-test, at the 0.05 significance

TABLE I Descriptive statistics of modelled carbohydrate (CH2O) accumulation rates (Gaussian distribution of b coefficients for total analysed leaves) for peach and olive CH2O accumulation rate (g d–1)

Confidence limit

Percentile interval

Species

N

Min.

Max.

Mean

–95%

+95%

10%

90%

SD

P. persica O. europaea

63 71

0.00088 0.00011

0.00349 0.00086

0.00194 0.00055

0.00182 0.00051

0.00206 0.00059

0.00147 0.00035

0.00242 0.00077

0.00046 0.00017

The number of samples (N) and the standard deviation (SD) of the model are also reported (see text for details).

73

S. MARCHI, D. GUIDOTTI, L. SEBASTIANI and R. TOGNETTI -1

Daily maximum photosynthetic rate (µmol m s )

0

-2

-2

y = -4.182 + 0.547lnx 2 R = 0.18 P = 0.0005

-1

Dark respiration (µmol m s )

A -1

-2

-3

th

6 node 12th node 16th node -4 0

20

40

60

80

100

Leaf area expansion (%)

16

A 14 12

y = 0.675 + 0.120x 2 R = 0.76 P < 0.0001

10 8 6 4

th

6 node 12th node 16th node

2 0 0

20

40

60

80

100

Leaf area expansion (%) -1

Daily maximum photosynthetic rate (µmol m s )

0

-2

-2

-1

Dark respiration (µmol m s )

B

-1

-2

y = -4.248 + 0.820lnx R2 = 0.62 P < 0.0001

-3

th

6 node 12th node th 16 node

-4 0

20

40

60

80

100


16

B y = 2.291 - 1.563lnx R2 = 0.33 P < 0.0001

14 12 10 8 6 4

6th node 12th node 16th node

2 0 0

20

40

60

80

100


FIG. 1 Changes in dark respiration rate (R) during leaf development (% leaf area expansion) measured on leaves at the 6th, 12th and 16th nodes from the base, in Prunus persica (Panel A) and Olea europaea (Panel B). Regression equations, squared correlation coefficients (R2) and significance levels (P) are also reported.

level. The slope and elevation of regression lines were tested for homogeneity of error, and coefficients were compared for significance by the Student’s t-test.

RESULTS In both species, gas exchange varied with the percentage of leaf expansion, regardless of the node examined (Figure 1; Figure 2; Figure 3). In peach and olive, R measured at the end of the dark period decreased with increasing leaf expansion, following a

FIG. 2 Changes in daily maximum photosynthetic rate (Amax) during leaf development (% leaf area expansion) measured on leaves at the 6th, 12th and 16th nodes from the base, in Prunus persica (Panel A) and Olea europaea (Panel B). Regression equations, squared correlation coefficients (R2) and significance levels (P) are also reported.

logarithmic pattern. In peach, maximum daily A and the corresponding gs increased linearly with leaf development. In olive, the increase in maximum daily A, and the corresponding gs, with leaf development was not linear and followed a logarithmic pattern. The relationship between maximum daily A and the corresponding gs resulted in generally low values of linear regression coefficients (R2 = 0.38 – 0.39). In both species, daily courses of gas exchange showed a general tendency for A to vary with leaf expansion more sharply than the corresponding gs (data not shown). The values

TABLE II Gas exchange parameters estimated from response curves of net photosynthesis (A) to intercellular CO2 concentration (Ci) measured on leaves of the 12th node at two different degrees of full leaf surface expansion (50% and 100%) in peach and olive Species P. persica O. europaea

Degree of leaf expansion 100% 50% P-level 100% 50% P-level

Vcmax

Jmax

TPU

35.2 (3.4) 26.6 (1.5) 0.0616 68.8 (6.0) 52.9 (3.5) 0.0641

107.6 (3.9) 92.3 (4.1) 0.0366 120.2 (5.4) 93.9 (2.3) 0.0042

6.81 (0.40) 6.56 (0.55) 0.7327 4.89 (0.30) 4.27 (0.13) 0.0995

Rday 2.23 (0.28) 2.55 (0.16) 0.3532 1.39 (0.09) 1.98 (0.19) 0.0294

c

lg

5.46 (0.12) 6.84 (0.28) 0.0036 6.00 (0.18) 7.06 (0.32) 0.0266

25.2 (0.9) 29.3 (1.2) 0.2579 29.5 (2.3) 44.3 (3.4) 0.0111

Values are the means (± SE) for each degree of leaf expansion and species (n = 4 individuals). Cuvette conditions, parameters and equations are defined in the text. Units are µmol CO2 m–2 s–1 for Vcmax, TPU, and Rday; µmol electrons m–2 s–1 for Jmax; Pa for c; and lg values are percentages. The level of significance (ANOVA; P-level) for differences between the two degrees of leaf expansion are reported.

500

-1

0.25

A 0.20

A

-2

-2

Net daily CO2 assimilation (mmol m d )


-1

Daily maximum stomatal conductance (mol m s )

74

y = 0.034 + 1.157x R2 = 0.33 P < 0.0001

0.15

0.10

th


0.05

400 6th node 12th node 16th node

300

y = -1261.2 + 248.7lnx 2 R = 0.44 P < 0.0001

200

100

0

-100 0

100

200

300

400

500

0.00 0

20

40

60

80

100

-2

Total chlorophyll content (µmol m ) 500

-1


0.55

B

-2

-2

-1

Daily maximum stomatal conductance (mol m s )


B

0.50

th

6 node th 12 node th 16 node

0.45 y = 0.006 + 0.002x 2 R = 0.02 P = 0.2199

0.40 0.35 0.30 0.25 0.20 0.15 0.10

400 th


300

200

100 y = -396.6 + 106.5lnx 2 R = 0.33 P < 0.0001

0

-100 0

0.05

100

200

300

400

500

Total chlorophyll content (µmol m-2)

0.00 0

20

40

60

80

100


FIG. 3 Changes in daily maximum stomatal conductance (gs) during leaf development (% leaf area expansion) measured on leaves at the 6th, 12th and 16th nodes from the base, in Prunus persica (Panel A) and Olea europaea (Panel B). Regression equations, squared correlation coefficients (R2) and significance levels (P) are also reported.

of Ci/Ca, averaged across all stages of leaf development, were 0.675 ± 0.047 and 0.696 ± 0.029 in peach and olive, respectively. Values of Ci commonly decreased with increasing leaf expansion. The increase in net daily CO2 assimilation (24 h) was associated with increased chlorophyll content and higher LMA, across all nodes in peach (Figure 4). In olive, the relationship between net daily CO2 assimilation and chlorophyll content was still positive, although this was not the case for the correlation with LMA (Figure 5). Photosynthetic capacity differed between degrees of leaf expansion in both species (Table II). Lower Vcmax

FIG. 4 Relationships between net daily CO2 assimilation and total chlorophyll content measured on leaves at the 6th, 12th and 16th nodes from the base, in Prunus persica (Panel A) and Olea europaea (Panel B). Regression equations, squared correlation coefficients (R2) and significance levels (P) are also reported.

and Jmax values were observed in leaves at 50% full surface expansion. The value of TPU remained constant; while, with increasing degrees of leaf expansion, Rday and lg decreased significantly only in olive, and c declined in both species. Photosynthetic activity increased significantly from 50% to 100% leaf expansion in both species (Table III), with Asat constantly higher in more expanded leaves, while ø was unaffected by leaf size. Other parameters differed between degrees of leaf expansion only in olive, Rd and l decreasing significantly with leaf size. To assess the importance of photosynthetic capacity, respiration rates, and the carbon economy of developing

TABLE III Gas exchange parameters estimated from response curves of net photosynthesis (A) to incident light intensity (Q) measured on leaves of the 12th node at two different degrees of full leaf surface expansion (50% and 100%) in peach and olive Species

Degree of expansion

P. persica

100% 50% P-level 100% 50% P-level

O. europaea

Asat 13.15 (1.40) 9.64 (0.26) 0.0491 13.95 (0.91) 8.67 (1.01) 0.0082

ø 0.162 (0.025) 0.136 (0.017) 0.4168 0.118 (0.005) 0.109 (0.003) 0.1632

Rd –2.68 (0.24) –2.65 (0.16) 0.9204 –0.27 (0.08) –0.48 (0.04) 0.0442

Values are the means (± SE) for each leaf degree of expansion and species (n = 4 individuals). Cuvette conditions, parameters and equations are defined in the text. Units are µmol CO2 m–2 s–1 for Asat and Rd; µmol CO2 µmol–1 photons for ø; and µmol photons m–2 s–1 for l. The level of significance (ANOVA; P-level) for differences between the two degrees of leaf expansion are reported.

l 17.43 (2.47) 20.13 (1.80) 0.4115 2.22 (0.62) 4.39 (0.23) 0.0166

75


500

-1


S. MARCHI, D. GUIDOTTI, L. SEBASTIANI and R. TOGNETTI

A

-2

y = -821.2 + 256.1lnx R2 = 0.31 P < 0.0001

400

0

20

40

60

80

100

0.02

th


300

A 0.00

Total CH2O (g)

200

100

0

-0.02

-0.04 sink source

-100 0

10

20

30

40

50

60

70

80

-0.06

Leaf dry mass per area ratio (g m-2) 0

-1


-0.08 500

10

20

B

30

40

50

60

-2

Leaf age (d)

400 th


300


200

0

20

40

60

80

100

0.02

100

B

y = 681.6 - 117.7lnx R2 = 0.05 P = 0.0625

0.00

-100 0

10

20

30

40

50

60

70

80

90

100 110 120 130

Leaf dry mass per area ratio (g m-2)

FIG. 5 Relationships between net daily CO2 assimilation and leaf dry mass per area ratio (LMA) measured on leaves at the 6th, 12th and 16th nodes from the base, in Prunus persica (Panel A) and Olea europaea (Panel B). Regression equations, squared correlation coefficients (R2) and significance levels (P) are also reported.

leaves, the daily CH2O balance of peach and olive leaves was modelled for the beginning of growing season in 2 consecutive years (Figure 6; Figure 7). The simulation exploited measured solar radiation during Spring 2002 and 2003, considering that leaves started developing when mean daily temperatures were above 10°C for at least 3 d (early March). Peach leaves were sinks of CH2O until 11 – 12 d from emergence (37.5% and 42.5% expansion in 2002 and 2003, respectively) and became sources of CH2O 18 – 19 d after emergence (66.3% and 69.1% expansion in 2002 and 2003, respectively). Olive leaves were sinks of CH2O until 13 – 14 d from emergence (25% and 28.3% expansion in 2002 and 2003, respectively) and became sources of CH2O 29 – 28 d from emergence (67.2% and 65.5% expansion in 2002 and 2003, respectively). The time interval during which leaves were acting simultaneously as importers and exporters of CH2O did not vary with year or with species. The two periods examined provided similar results, regardless of species; although, in 2002, the sink-source transition occurred slightly earlier (both in terms of leaf age and percentage development). When the whole Spring season (March – June) was considered, the total CH2O formed by a peach leaf was 5.14 g and 6.46 g in 2002 and 2003, respectively. In olive, the values were consistently lower, at 0.65 g in 2002 and 0.82 g in 2003.

Total CH2O (g)

0

-0.02

-0.04 sink source

-0.06

-0.08 0

10

20

30

40

50

60

Leaf age (d) FIG. 6 Modelled daily carbohydrate (CH2O) balance in peach leaves for the beginning of the growing season in two consecutive years, 2002 (Panel A) and 2003 (Panel B). The simulation made use of measured solar radiation during the Spring, and considered that leaves start developing when mean daily temperatures are > 10°C for at least 3 d (early March).

DISCUSSION In peach, the daily maximum A increased linearly with leaf development, and the corresponding gs showed similar trends. This behaviour is common to other deciduous fruit trees, such as apple (Kenney and Johnson, 1981). In olive, a plateau in diurnal gas exchange was observed at about 30% of final area expansion. Values of R, generally higher in peach than in olive, decreased with increasing leaf development in both species. During the early stages of leaf growth, the synthesis of chlorophyll, proteins and structural compounds were high, resulting in high catabolic rates to support energy needs. As the photosynthetic system matures, the requirement for respiratory energy decreases rapidly (Kozlowski, 1992). Olive leaves apparently reached photosynthetic maturity (i.e., chloroplast development) earlier than peach leaves. Stomatal development was also more gradual in peach


76

Leaf area expansion (%) 0

20

40

60

80

100

0.002

A

Total CH2O (g)

0.000

-0.002

-0.004

-0.006

sink source

-0.008

-0.010 0

10

20

30

40

50

60

70

Leaf age (d)

Leaf area expansion (%) 0

20

40

60

80

100

0.002

B

Total CH2O (g)

0.000

-0.002

-0.004

-0.006

sink source

-0.008

-0.010 0

10

20

30

40

50

60

70

Leaf age (d) FIG. 7 Modelled daily carbohydrate (CH2O) balance in olive leaves for the beginning of the growing season in two consecutive years, 2002 (Panel A) and 2003 (Panel B). The simulation made use of measured solar radiation during the Spring, and considered that leaves start developing when mean daily temperatures are > 10°C for at least 3 d (early March).

than in olive, but was less effective in influencing leaf CO2 assimilation at all nodes examined. In expanding leaves of olive, photosynthesis was noticeably limited by high respiration, while this was not the case in peach leaves. In olive, foliar tissues were still immature and the initial energy required for their construction probably originated, for the most part, from respiration. In peach, leaves had slower growth at the beginning of development, when photosynthesis was also low. Expanding leaves of both species were able to optimise the allocation of N in order to maintain an equilibrium between their enzymatic (Rubisco) and light-harvesting (chlorophyll) capacities (Thompson et al., 1992). Mean values of Jmax and Vcmax for fullyexpanded leaves of both peach and olive resembled those reported by Wullschleger (1993) for many C3 species at 134 µmol electrons m–2 s–1 and 64 µmol CO2 m–2 s–1, respectively. In peach, Smith and Neales (1977) found

average values of 148 µmol electrons m–2 s–1 and 53 µmol CO2 m–2 s–1 for Jmax and Vcmax, respectively. In olive, Loreto and Sharkey (1990) found average values of 63 µmol electrons m–2 s–1 and 16 µmol CO2 m–2 s–1; while Centritto et al. (2003) reported values of 110 – 120 µmol electrons m–2 s–1 and 68 – 76 µmol CO2 m–2 s–1 for Jmax and Vcmax, respectively. Photosynthetic capacity increased with leaf expansion, particularly in terms of Jmax (and consequently maximum A). The less marked increase in Vcmax might indicate a comparatively lower consumption of RuBP, with respect to the regeneration capacity of RuBP itself, in agreement with the model of Farquhar et al. (1980). Olive leaves used a relatively lower proportion of their N for light harvesting processes, and a higher fraction for carboxylation activities than peach leaves. Indeed, the Jmax to Vcmax ratio averaged 3.05 and 3.47 in peach, and 1.75 and 1.77 in olive, for leaves at 50% or 100% expansion, respectively. Wullschleger (1993) found an average TPU value, for 16 C3 species, of 10.1 µmol CO2 m–2 s–1, which is comparable to our observations. Net CO2 assimilation during leaf development was only marginally affected by TPU, suggesting no feedback between CH2O synthesis (growth) and assimilation of CO2 in the short term. In rapidly expanding olive leaves, photosynthetic capacity was somewhat limited by high Rday, as energy to construct immature tissues would be provided mainly by respiration (Lambers et al., 1983). During the early stages of leaf growth, the synthesis of chlorophyll, proteins and structural compounds was high in olive, resulting in high catabolic rates to support energy needs (Marchi, 2004). As the photosynthetic system matured, the requirement for respiratory energy decreased rapidly. This was not true in more slowly expanding peach leaves. Values of c decreased consistently with an increasing degree of leaf expansion, and similarly in both species, suggesting a general reduction in photorespiration. The effect of lg on A potentially decreased as olive leaves expanded fully, despite gs increasing slightly with leaf development compared to photosynthetic capacity. Conversely, such an increase was more pronounced and linear with leaf development in peach. Stomatal dysfunction was, however, unlikely because there was a sufficiently good degree of coupling between A and gs in both species, albeit with a low correlation coefficient. Younger leaves of both species had low Asat values that increased progressively during leaf development, presumably because of improved metabolic capacity (Greer and Halligan, 2001). However, stomata showed a relatively higher sensitivity to varying light intensity as leaf development progressed. The increase in ø with leaf development, overall, was not significant, indicating a similar photosynthetic capacity to maximise utilisation of light across all leaf ages. Younger developing leaves showed higher Rd values in olive; while fully-developed leaves had high Rd values in peach, suggesting that the construction process was still active (Miyazawa et al., 1998). In olive, values of l decreased with the progress of leaf development, representing a decrease in light demand, whereas this was not the case in peach, which showed insignificant variations in l. The simulation model was based on the concept that every plant grew as a collection of semi-autonomous,

S. MARCHI, D. GUIDOTTI, L. SEBASTIANI and R. TOGNETTI although interactive organs (DeJong, 1999). The availability of CH2O was strictly dependent on, and connected to the growth and development of each plant organ (Farrar, 1993; Gifford and Evans, 1981; Marcelis, 1994). At any specific time, the partitioning of photoassimilates was determined by resource availability, growth capacity and the maintenance requirement of each organ, with environmental conditions acting as modulators. In this study, we have modelled the production and partitioning of photo-assimilates in two important fruit tree species, starting from single-leaf development in relation to the growth environment. Although pruning and planting systems may alter the growth and form of a fruit tree, generally these practices do not directly affect the behaviour of single leaves. In peach and olive leaves, the sink-source transition did not apparently differ over the two periods considered for comparison. However, the sink-source transition, estimated through the simulation based on measured solar radiation, differed from the experimental results gathered from plants growing in controlled environments. In the case of the growth chamber, such a change of conditions occurred at a much earlier leaf age (and stage of development) compared to those obtained through the model. It is therefore important to implement experimental data with climatic parameters to obtain reliable estimates. The model allowed us to estimate the total amount of CH2O produced by single leaves, and the quantity of assimilates that can be exported to other organs at a particular time. This simple approach may be useful for further analysis of CH2O partitioning at the whole plant level, and to compare shoots bearing fruits with branches without reproductive organs. To increase the reliability of the model, further analyses at the single leaf level and/or on the whole canopy, are warranted in order to limit the scattering of the data. The start of the gross export of CH2O is a first step in leaf development towards autotrophy (i.e., the onset of the net export of assimilates; Turgeon, 1989). The time period during which leaves were acting simultaneously as sinks and sources of CH2O did not vary with year or

77

species. However, variability in final leaf size and the time of initiation of CH2O export between plants and years may be high; thus, over a range of absolute leaf sizes, some leaves operate as exporters while others do not. Larger differences in temperature and light intensity than in our analysis might be expected, causing significant variations in growth between different seasons. In sour cherry, Kappes and Flore (1989) found that gross CH2O export from the 7th and 10th leaves was initiated when the area of the 7th leaf reached 8.5 cm2 (27% expansion) and when that of the 10th leaf reached 14 – 21 cm2 (48 – 72% expansion) in the first year of study. After 2 years, gross CH2O export was delayed when the leaf area was greater at full expansion [e.g., for the 7th leaf, 26 cm2 (47% expansion) and for the 10th leaf 36 cm2 (78% expansion)]. These differences might explain the differences reported for other woody plants, such as grapevine, that started gross CH2O export at 50% full leaf expansion in California (Hale and Weaver 1962), while only at 30% full leaf expansion in Switzerland (Koblet, 1969). Small increases in leaf growth rate due to nutrition or temperature, or decreases in photosynthetic rate due to light conditions, may cause a delay in the start of gross CH2O export in peach and olive, which is influenced by CH2O supply and demand. This simple modelling exercise, incorporating growth, physiological and meteorological data, and aimed at developing a tool to study CH2O partitioning in single leaves based on sink-source relationships, should stimulate further systematic analyses on whole plants. Further studies are needed at the canopy level, and on older leaves, to determine age-dependent differences in CO2 assimilation between peach and olive. The refinement of the present modelling environment could also help to reveal critical periods that limit the photosynthetic productivity of peach and olive trees throughout the growing season in Mediterranean-type agro-ecosystems, particularly under changing climatic conditions. We thank ARSIA (Extension Service in Agriculture and Forestry), Regione Toscana, Italy, for providing the meteorological data.

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