JXB Advance Access originally published online on July 12, 2006
Journal of Experimental Botany 2006 57(11):2687-2695; doi:10.1093/jxb/erl040
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RESEARCH PAPER |
Relationships between leaf conductance to CO2 diffusion and photosynthesis in micropropagated grapevine plants, before and after ex vitro acclimatization
1Consiglio per la Ricerca e Sperimentazione in Agricoltura–Istituto Sperimentale per le Colture Industriali, via di Corticella 133, I-40128 Bologna, Italy
2Potsdam Institute for Climate Impact Research (PIK), PO Box 601203, D-14412 Potsdam, Germany
3Laboratoire d'Ecologie, Systématique et Evolution (ESE), CNRS-UMR 8079, Bât. 362, UFR scientifique d'Orsay, Université de Paris-XI, F-95401 Orsay Cedex, France
*To whom correspondence should be addressed. E-mail: gianni.fila{at}entecra.it
Received 7 April 2006; Accepted 20 April 2006
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Abstract |
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In vitro-cultured plants typically show a low photosynthetic activity, which is considered detrimental to subsequent ex vitro acclimatization. Studies conducted so far have approached this problem by analysing the biochemical and photochemical aspects of photosynthesis, while very little attention has been paid to the role of leaf conductance to CO2 diffusion, which often represents an important constraint to CO2 assimilation in naturally grown plants. Mesophyll conductance, in particular, has never been determined in in vitro plants, and no information exists as to whether it represents a limitation to carbon assimilation during in vitro growth and subsequent ex vitro acclimatization. In this study, by means of simultaneous gas exchange and chlorophyll fluorescence measurements, the stomatal and mesophyll conductance to CO2 diffusion were assessed in in vitro-cultured plants of the grapevine rootstock ‘41B’ (Vitis vinifera ‘Chasselas’xVitis berlandieri), prior to and after ex vitro acclimatization. Their impact on electron transport rate partitioning and on limitation of potential net assimilation rate was analysed. In vitro plants had a high stomatal conductance, 155 versus 50 mmol m–2 s–1 in acclimatized plants, which ensured a higher CO2 concentration in the chloroplasts, and a 7% higher electron flow to the carbon reduction pathway. The high stomatal conductance was counterbalanced by a low mesophyll conductance, 43 versus 285 mmol m–2 s–1, which accounted for a 14.5% estimated relative limitation to photosynthesis against 2.1% estimated in acclimatized plants. It was concluded that mesophyll conductance represents an important limitation for in vitro plant photosynthesis, and that in acclimatization studies the correct comparison of photosynthetic activity between in vitro and acclimatized plants must take into account the contribution of both stomatal and mesophyll conductance.
Key words: Acclimatization, chlorophyll fluorescence, cuticular conductance, gas exchange, grapevine, in vitro culture, mesophyll conductance, photosynthesis, stomatal conductance
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Introduction |
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In vitro-cultured plants are frequently reported to be very sensitive to the abrupt environmental changes that they experience when they are removed from culture containers and placed under ex vitro conditions (Pospí
ilová et al., 1999). For this reason, an acclimatization period is normally required after ex vitro transplantation, during which gradual transfer to greenhouse or outdoor climatic conditions (light and relative humidity above all) are applied in order to reduce stress, and to acclimatize in vitro plants to the new growing conditions progressively. The success of this process is highly dependent on the extent of the morphological and/or physiological abnormalities acquired during in vitro growth, which may become incompatible with external life. One of the earliest identified detrimental factors for ex vitro acclimatization is the poor photosynthetic capacity of in vitro plants (Donnelly and Vidaver, 1984; Grout, 1988). This limitation arises from the low light and the restricted supply of CO2 imposed by the confined microenvironment (Solárová, 1989; Kozai, 1991; Chaves, 1994; Arigita et al., 2002), and the down-regulation brought about by the addition of exogenous sugars to the nutrient media (Capellades et al., 1991; Lees et al., 1991; Hdider and Desjardins, 1994; Serret et al., 1997).
From a survey of the literature on this subject, it was noticed that the approach followed by most studies on ex-vitro acclimatization is to compare the effect of various culture conditions [e.g. variation of photosynthetic photon flow density (PPFD), CO2, sugars in the nutrient media], before and/or after ex vitro transplantations. Comparison of photosynthetic capacity is typically made on a net assimilation basis, most often neglecting the fact that carbon assimilation also depends on the availability of CO2 at the carboxylation sites, which is restricted by the resistances to diffusion along the pathway from the atmosphere to the chloroplasts. Photosynthesis tends to be considered essentially as a biochemical or photochemical issue, even when treatments known to affect leaf conductances, such as CO2 enrichment (Singsaas et al., 2003), are applied. In fact, a well-established notion in ecophysiological research on naturally grown plants is that overlooking the contribution of leaf conductance leads to over- or underestimation of the real carbon assimilating capacity, with the inevitable misinterpretations of results.
The objective of this study was 2-fold: (i) to assess the leaf conductances to CO2 diffusion and their contribution to photosynthesis in in vitro-cultured plants; and (ii) to check whether changes in CO2 diffusion are part of the acclimatization to ex vitro conditions.
While information exists about stomatal conductance in in vitro plants, which is usually reported to be very high, nothing is known about the limitation to CO2 diffusion inside the leaf.
Special attention has therefore been reserved for this latter one, for which a series of simultaneous fluorescence and gas exchange measurements were undertaken on in vitro and greenhouse-acclimatized plants of the grapevine rootstock ‘41B’ (Vitis vinifera ‘Chasselas’xVitis berlandieri). Beyond the usual gas exchange parameters (net assimilation rate and stomatal conductance), the combined measurements allowed estimation of electron transport rate partitioning and the assessment of mesophyll conductance to CO2.
The results are expected to fill a knowledge gap in the photosynthetic physiology in in vitro-cultured plants, and to support a more consistent interpretation of photosynthesis measurements in acclimatization studies.
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Materials and methods |
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General experimental set-up
All experiments were conducted on two sets of plants: (i) in vitro-cultured plants, before ex vitro acclimatization (henceforth referred to as ‘in vitro plants’); and (ii) in vitro-cultured plants after ex vitro acclimatization (henceforth referred to as ‘acclimatized plants’).
Simultaneous gas exchange and chlorophyll fluorescence measurements were applied in order to observe net assimilation rate and to estimate stomatal and mesophyll conductances. According to the methodology used (Epron et al., 1995), determination of mesophyll conductance required the previous estimation of electron transport rate partitioning, which was achieved by means of the method described by Genty et al. (1989). This method utilizes the close correspondence between the actual quantum yield of photosystem II (PSII) photochemistry, 
PSII, and the actual quantum yield of carbon assimilation, CO2, as a relative measurement of electron transport rate. A dedicated set of experiments was therefore run in order to ‘calibrate’ this relationship on the present plant material.
Two other complementary sets of experiments were run to measure the optical properties of the leaves and the cuticular conductance to CO2. One leaf was sampled per individual plant. Five replicates for each treatment were used in the measurements of leaf optical properties and in the gas exchange/fluorescence experiments, while three replicates were used for the assessment of cuticular conductance.
Plant material and culture conditions
All experiments were conducted on the grapevine rootstock, ‘41B’ (Vitis vinifera ‘Chasselas’xVitis berlandieri). Nodal cuttings were cultured for 30 d in 25x250 mm tubes, covered with a loose metallic cap, and containing 20 ml of solidified nutrient medium (Charles, 1992) with 25 g l–1 sucrose. Growth room conditions were constant temperature, 27 ±1 °C, with a 12 h photoperiod at a PPFD of 60 µmol m–2 s–1 provided by cool-white fluorescent lamps (Osram, Munich, Germany). Acclimatization to ex vitro conditions was conducted as follows: 4-week-old in-vitro plants were transferred to the greenhouse in pots filled with a peat and sand mixture 1:1 (v/v), and kept in a PVC [poly(vinyl chloride)] box with a semi-transparent cover to avoid desiccation. After 1 week, the cover was removed, and the plants were grown in a temperature-controlled greenhouse (max. PPFD: 600 µmol m–2 s–1; day/night temperature: 26/12 °C).
Measurements on in vitro plants were carried out after 4 weeks from the last subculture, when there were four to six fully expanded leaves and a developed root system, whereas on acclimatized plants measurements were carried out 2 months after ex vitro transplantation.
Optical properties of the leaves and chlorophyll determination
Leaf absorption spectra were measured in the visible (400–700 nm) light range, using an integrating sphere (LI-1800-12S, Li-Cor, Inc., Lincoln, NE, USA) coupled to a spectroradiometer SE590 (Spectron Engineering, Denver, CO, USA). The concentration of chlorophyll pigments was determined on the same leaf samples after extraction in N,N-dimethylformamide according to Moran (1982).
Gas exchange and chlorophyll fluorescence measurements
Measurements were carried out on the youngest fully developed leaves. Since in vitro plant leaves typically show a rapid wilting when they are taken out of the tubes, their stem was cut under water, just above the root insertion zone, and then kept in a tube filled with distilled water during the measurements. This procedure allowed water to flow directly into the stem, and was effective in preserving leaves from dehydration during the experiments (Fila et al., 1998).
Coupled gas exchange and chlorophyll fluorescence emission measurements were carried out in an open system as described by Ghashghaie and Cornic (1994). This consisted of a thermostated leaf assimilation chamber with a section area of 1.7 cm2, which was entirely covered by leaves of both types of plants, and a gas volume of 6.4 ml. In- and outcoming air passed separately through a one-channel infrared gas analyser (Binos; Leybold Heraeus, Hanau, Germany) and a dew-point hygrometer (Elcowa electronique, Mulhouse France; system 1100 DP) for CO2 and H2O partial pressure measurements. Air flow rate was set at 20 l h–1. Actinic light was provided by a slide projector, and light intensity was modulated through neutral density filters. The actual PPFD was measured with a Li-190 Li-Cor quantum sensor.
In vivo chlorophyll fluorescence emission from the upper leaf surface was measured with a chlorophyll pulse-amplitude modulation fluorometer (PAM-101, H. Walz, D-8521 Effeltrich, Germany), connected to the assimilation chamber window through a branched fibre-optic system, based on the one described by Dietz et al. (1985). The frequency of modulated light was 1.6 kHz, and saturating flash pulses (1 s) of white light (12 000 µmol m–2 s–1) were provided by a KL 1500 Schott light source (Schott, Wiesbaden, Germany).
Three fluorescence levels were measured: F0, Fs, and Fm (F'm under illumination; Maxwell and Johnson, 2000). From these measurements the actual quantum yield of PSII photochemistry, PSII=(F'm–F's)/F'm was calculated (Genty et al., 1989).
Light and CO2 responses
A light response curve was established to assess net assimilation rate and leaf conductances to CO2, which were recorded at light saturation, and the electron transport rate partitioning.
After measuring dark respiration, the light was switched on, and light-response curves were recorded in the PPFD range between 0 and 600 µmol m–2 s–1, at either 0.21 mol O2 mol–1 air (i.e. 21% O2) and 350 µmol CO2 mol–1 air (normal air) or 0.01 mol O2 mol–1 air (i.e. 1% O2) and 600 µmol CO2 mol–1 air (non-photorespiratory conditions).
An intercellular CO2 (Ci)-response curve was established to determine S*, the apparent S, for the estimation of Cc (CO2 concentration in the chloroplasts) and gm (mesophyll conductance) according to the model of Epron et al. (1995). CO2 was varied in the range where Rubisco activity is limiting for photosynthesis, i.e. below 500 µmol CO2 mol–1, air at a PPFD of 200 µmol m–2 s–1 and where the relationship against Ci is linear. The chosen PPFD intensity was at the limit or below the saturated region of the light-response curve of in vitro plants and acclimatized plants, respectively. CO2 partial pressure was varied by mixing different ratios of CO2-free air with compressed air containing 10% CO2.
Measurements of cuticular conductance
Substomatal CO2 concentration, Ci, was calculated according to the model of von Caemmerer and Farquhar (1981). This model ignores the contribution of cuticular transpiration to the leaf gas exchange, thus allowing a simplified calculation of Ci. This assumption proved to be actually valid in most situations, but it is questionable for in vitro plants, where the effectiveness of cuticle on transpiration control has been debated (Wetzstein and Sommer, 1982, Sutter, 1988). In order to check the consistency of the Ci simplified calculation, especially for in vitro plants, an additional series of measurements was carried out to assess the value of cuticular conductance (gc).
Grapevine leaves are hypostomatous, hence transpiration from the upper surface occurs only through the cuticle. Using the same gas exchange apparatus, the conductance from the upper leaf surface was measured by isolating the lower surface with a self-adhesive transparent plastic ribbon. Measurements were conducted at a PPFD of 200 µmol m–2 s–1 and 350 µmol mol–1 CO2, at a vapour pressure deficit varying between 0.5 and 2.5 kPa (in all the other light- and CO2-response measurements the vapour pressure deficit was always between 0.7 and 1.0 kPa). The measured conductance was taken as the value of gc, although this implied the further assumption that the gas exchange properties of the cuticles are identical for both epidermises. Since the cuticle discriminates against CO2, gc for CO2 was finally estimated as 15% of gc for water vapour (Boyer et al., 1997).
Non-cyclic electron flow analysis
Simultaneous measurement of fluorescence yield and CO2 exchange under non-photorespiratory conditions made it possible to estimate the rate of photosynthetic electron flow JT according to Genty et al. (1989), who found a linear relationship between the quantum yield of PSII, PSII, and the quantum yield of CO2 assimilation (CO2) under non-photorespiratory conditions. This relationship allows PSII to be utilized as a relative measure of the whole chain photosynthetic electron transfer. A good linear correlation was obtained for both in vitro and acclimatized plants (Fig. 1). The two relationships were found to be statistically different with the ANCOVA test (P=0.05), which rejected the homogeneity of regression slope assumption. Both equations [PSII=11.55CO2–0.02 (acclimatized plants) and PSII=9.24CO2+0.01 (in vitro plants)] were therefore used.
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The total electron transport rate JT was then calculated (Cornic and Briantais, 1991):
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PSII and CO2. The whole expression is multiplied by 4, which accounts for the number of electrons, which must be transported to reduce one molecule of CO2. By adopting the model of Epron et al. (1995), it was possible to estimate the partitioning of the total electron flow, JT, into two fractions: Jc, the electron flow associated with CO2 reduction, and Jo, the flow funnelled into collective O2-dependent dissipative processes, such as photorespiration and the Mehler reaction. The calculation required the knowledge of the day respiration (Rd), which is usually estimated by the respiration rate measured in the dark, R. This represents a simplification, since it has been well established that mitochondrial respiration is partly inhibited in the light (Brooks and Farquhar, 1985; Atkin et al., 2000). It was therefore assumed that day respiration is 50% of the respiration measured in the dark. This value was chosen because it was intermediate to the range of variation reported in the literature (between 16% and 77%, as reported by Atkin et al., 2000).
Mesophyll conductance determination
The estimation of mesophyll conductance was performed following the approach of Epron et al. (1995). These authors determined in vivo an apparent specificity factor of Rubisco (S*), as the slope of the linear regression between Jc/Jo and the ratio between Ci and atmospheric O2:
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From the comparison of S* to S, measured in vitro on the purified enzyme, it is possible to compute the actual CO2 concentration in the chloroplast (Cc) by means of the following relationship:
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After Cc calculation, the mesophyll conductance (gm) was computed at light saturation and ambient CO2 (350 µmol mol–1) with the relationship:
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Sensitivity analysis
The estimation of Cc and gm was made ignoring gc, and using postulated values for Rd and S. A sensitivity analysis was therefore undertaken to assess to what extent these estimates were sensitive to the uncertainty associated with these assumptions.
Estimates of Cc and gm were recalculated from a wide sample of combinations of gc, Rd, and S, where each factor was allowed to vary across the entire range of its variation interval. The variability thus generated represented a scenario of ‘total uncertainty’ against which the relative weight of each factor was assessed. This was done by eliminating in turn the variability associated with each factor, by extracting a subset of combinations containing its postulated value (gc=0, Rd=50% of R, S=2700) and observing how the entire variability was affected.
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Chlorophyll content and optical properties
Total chlorophyll content, expressed on a leaf area basis, was not statistically different between in vitro and acclimatized plants at a 5% error level (Student's t-test, n=5). The ratios between Chl a and Chl b were similar and lower than 2, characteristic of shade plants (Table 1). Light absorption spectra were identical in both cases (not shown). The coefficient of light absorption in the photosynthetically active radiation range (400–700 nm) was 0.780 for both types of plants.
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Net leaf CO2 exchange
Leaves of acclimatized plants had a higher net CO2 assimilation rate measured under saturating light. In an atmosphere containing 0.21 mol O2 mol–1 air (normal air) the recorded values were 4.1±0.20 against 6.1±0.67 µmol m–2 s–1 in in vitro and acclimatized plants, respectively. Under non-photorespiratory conditions the respective values were 6.5±0.48 against 11.6±0.73 µmol m–2 s–1 (Table 2), and the maximum quantum yield of net CO2 uptake by leaves (the slope of the A/PPFD curve) was almost identical at 0.072 and 0.070 mol CO2 mol absorbed photons–1 (not shown), which is very close to the mean value found in a wide range of C3 plants by Long et al. (1993).
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Measurements of the A/Ci response provided evidence that the carboxylation efficiency, the slope of the linear part of the curve, was higher for leaves of greenhouse-acclimatized plants, which also showed a lower CO2 compensation point (Fig. 2A).
Cuticular conductance
Leaf conductance to water vapour diffusion of the upper leaf surface was 20.6 for in vitro plants and 4.5 mmol m–2 s–1 for acclimatized plants, while the sums of cuticular and stomatal conductances, measured on the lower surface, were 142.4 and 46.4 mmol m–2 s–1 in in vitro and acclimatized plants, respectively. That means that in acclimatized plants, cuticular transfer conductance to water vapour was 9.6% of leaf conductance, while in in vitro plants it was 14.5%. On the hypothesis that the ratio of gas permeabilities of the cuticle is identical to the ratio of the diffusivities of CO2 and H2O in air, gc(CO2) would be 12.9 and 2.8 mmol m–2 s–1 in in vitro plants and acclimatized plants, respectively (Table 3). Introducing these gc values to the estimation of Ci, and considering a 15% value of the ratio gc(CO2)/gc(H2O), due to the cuticle discrimination effect against CO2 (Boyer et al., 1997), the deviation from the simplified Ci estimation was –1.15% in acclimatized plants and –0.65% in in vitro plants. For this reason, the simplified estimates were maintained in all subsequent calculations.
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Electron transport rate partitioning
In acclimatized plants JT saturated at a PPFD of 350 µmol m–2 s–1 at a maximum value of 55.3 µmol m–2 s–1, where 67.6% of the total electron flow was funnelled towards the CO2 reduction pathway. In in vitro plants, JT was inferior to that recorded on acclimatized plants: at light-saturation; above 200 µmol m–2 s–1 PPFD, it was 29.9 µmol m–2 s–1. The proportion diverted to Jc was 74.8% of JT (Table 2).
Mesophyll conductance
The apparent Rubisco specificity factor S* was 1801 (S*/S=0.67) in in vitro plants and 2411 (S*/S=0.89) in acclimatized plants (not shown). At the ambient external CO2 concentration of 350 µmol mol–1 and at light saturation, Cc was 195.3±1.15 µmol mol–1 in in vitro plants, i.e. 55.8% of Ca, and 66.7% of Ci. In acclimatized plants Cc was 168.2±2.71 µmol mol–1, 89.3% of Ci, and 48.0% of Ca (Table 3).
The maximum carboxylation efficiency, determined on the initial slope of the A/Cc curve (Fig. 2B), was +73.8% higher (0.0168 versus 0.0292 mol m–2 s–1) than the value determined on a Ci basis in in vitro plants, while in acclimatized plants the change was +12.1% (0.0371 versus 0.0416 mol m–2 s–1; Fig. 2).
Mesophyll conductance was 42.8 ±1.38 and 285.4±11.31 mmol m–2 s–1 for in vitro and acclimatized plants, respectively (Table 3).
The relative photosynthesis limitation (Li) due to gm led to a 14.5% decrease with respect to the potential net assimilation rate in in vitro plants (Fig. 2B). For acclimatized plants the calculated Li was 2.1%.
Sensitivity analysis
The variability associated to gc, Rd, or S was compared with the total uncertainty variability by means of cumulative frequency distributions of Cc and gm obtained. When the full range of uncertainty for gc, S, and Rd was considered, 95% of Cc estimates varied between 162 and 274 µmol mol–1 in in vitro plants, and between 147 and 193 µmol mol–1 in acclimatized plants (Fig. 3). In both treatments, the Cc distribution was almost the same in the subsets with fixed Rd or gc, while in the fixed S-subset the variability was reduced to the interval 177–212 µmol mol–1 in in vitro plants. In acclimatized plants no particular effect of constant S on the frequency distribution was noticeable.
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A different situation was observed in the gm dataset, where the highest variability for the total uncertainty scenario was observed in acclimatized plants, with 95% of combinations ranging between 120 and 2595 mmol m–2 s–1, and between 33 and 368 mmol m–2 s–1 in in vitro plants. Even in this case, taking a fixed S value remarkably reduced the variability into the interval 38–56 mmol m–2 s–1 in in vitro plants, while in acclimatized plants none of the factors when taken individually could attenuate the overall variability.
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Discussion |
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The objective of this study was to determine to what extent photosynthesis in in vitro-cultured plants is limited by constraints to CO2 diffusion in the leaves, and in particular by mesophyll conductance, which had never been determined on in vitro plants up to then. The analysis utilized methodologies well established for naturally grown plants, but which were never tested on in vitro-cultured plants. It was therefore necessary to dedicate part of the work to verify the consistency of the principal assumptions involved in the procedures. The aspects that needed particular attention were the incidence of cuticular conductance, the relationships between chlorophyll fluorescence and the electron transport rate (Genty's plot), and the postulated values of S and Rd.
Cuticular conductance
A higher gc in in vitro plants was hypothesized on the ground of microscopic observations reporting thin or absent cuticle for in vitro plants (Wetzstein and Sommer, 1982; Sutter, 1988). However, direct measurements of gc were similar to that recorded on outdoor grown plants (Fuchigami et al., 1981; Shackel et al., 1990; Santamaria and Kerstiens, 1994). This is not in contradiction with the microscopic results, since it has been established that cuticular permeability to gases is not related to its thickness (Kerstiens, 1996). It was found that gc to H2O was 14% of total leaf conductance (gs+gc) for in vitro plants and below 10% for acclimatized plants. Considering that gc to CO2 is much inferior to that for H2O—well below at 13% according to Boyer et al. (1997)—it was found that the effect of gc on Ci and subsequent gm estimation, was negligible.
Genty's relationship
Estimation of electron transport rate was based on the relationship between PSII and CO2, which was linear for both in vitro and acclimatized plants, but was characterized by a different regression equation. An effect of leaf optical properties can be excluded, as the light absorption spectra were perfectly coincident in the visible region (data not shown), and the chlorophyll content was not different either (Table 1).
The difference in the regression could be related to the different thickness of the leaf. It is known that the fluorescence signal is detected only from the superficial layers of cells, while all the layers across the whole leaf thickness contribute to the gas exchange measured (Maxwell and Johnson, 2000; Tsuyama et al., 2003). For a given PSII, a lower CO2 would be expected from a thicker leaf, where gradients in light distribution and CO2 assimilation activity may establish, and this is precisely what was observed (Fig. 1).
Sensitivity against Rd and S assumptions
The consistency of the postulated values of Rd and S was verified through a sensitivity analysis. In in vitro plants, S was the parameter with the highest effect in reducing the uncertainty of Cc and gm estimates while, in acclimatized plants, none of the factors, individually taken, was able to ameliorate the accuracy of the estimates. In any case, even under full uncertainty conditions, the variability of the estimates for in vitro plants was narrower than that for acclimatized plants (Fig. 3). This behaviour could be explained as follows: the function yielding gm is hyperbolic since it has (Ci–Cc) as the denominator, which was calculated for a range of Rd values (Fig. 4). This means that the same relative variation of (Ci–Cc) results in different relative variations of gm, whether this falls in the horizontal or in the vertical asymptotic region of the function. In acclimatized plants, due to lower Ci values, the (Ci–Cc) difference tends to be closer to 0. Under these conditions, small variations of Rd (which affects S*, then Cc) or small experimental inaccuracies in gas exchange measurement are sufficient to induce very large variations in gm (Fig. 4). This intrinsic instability of the estimations explains why taking one fixed factor at a time is not sufficient to reduce the overall variability. However, precise knowledge of gm when this is very high is of little practical importance, since in this case it is more convenient to approximate Ci=Cc and ignore gm.
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All this considered, it was concluded that in vitro plants showed a weak gm, reasonably contained in the interval 38–56 mmol m–2 s–1, which proved to be an appreciable limitation for CO2 diffusion in in vitro leaves and for photosynthesis (Fig. 2B). After acclimatization, gm increases, and its limitation effect is reduced.
Relationship between gs–gm and photosynthetic activity
Over the past decades, studies on in vitro plants often presented large discrepancies in characterizing their photosynthetic ability and its impact on ex vitro acclimatization. Desjardins et al. (1995) pointed out that many of these discrepancies, previously attributed to intrinsic species-specific photosynthetic characteristics (Grout, 1988), were more explainable by the high variability in the culture conditions and measuring techniques adopted, which made it difficult to compare and generalize results.
On the base of the present results, it is proposed that neglecting leaf conductances to CO2 diffusion could have represented an additional source of ambiguity in interpreting photosynthesis measurements in in vitro-cultured plants. In the first place, whenever stomatal conductance is very high, which is a frequent occurrence in in vitro plants, any gas exchange measurement carried out under normal air should take into account that the assimilation rate is affected by partial photorespiration suppression. This effect could explain why in vitro plants were sometimes found to have a higher photosynthesis than ex vitro plants, as in the study by Lee et al. (1985) who reported that in vitro plants of Liquidambar styraciflua had a higher net carbon assimilation than seedlings of the same species grown under the same light conditions. De et al. (1992) also recorded a superior photosynthesis rate in in vitro Asparagus plants compared with acclimatized ones grown at higher irradiances. As far as is known, only these latter authors, together with Pospíilová et al. (1998), recognized that carbon assimilation in in vitro plants was favoured by a high stomatal conductance, but they did not provide a quantitative estimate of its contribution.
The effect of gs should be analysed together with gm, which is not predictable and is much more difficult to measure. A special caution is especially necessary when comparing photosynthetic characteristics between treatments potentially affecting gm. An example of these treatments is represented by CO2 enrichment (Singsaas et al., 2003), which is one of the most frequently applied in in vitro culture research. Also sugar treatments, which have an impact on leaf morphology, might well affect leaf conductance beyond biochemical effects.
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Conclusions |
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At the time of ex vitro transplanting, in vitro plants showed a low photosynthetic activity, as expected from the culture conditions, which showed a peculiar dependency on leaf conductances to CO2 diffusion. A high gs in in vitro plants favoured the carboxylation activity of Rubisco, which was, however, attenuated by a low gm, which limited the potential assimilation rate by 14.5%. During acclimatization, the decrease in gs was compensated by an increase in gm that limited the potential assimilation by only 2.1%.
It is concluded that the characterization of photosynthetic competence in in vitro plants could gain accuracy by considering some variables, which are not usually taken into account in the analysis of these systems, such as the diffusion process of CO2. The results proved to be important for analysing photosynthesis results, and they could also suggest an alternative target for studies aimed at improving acclimatization protocols. So far, the culture objective was to raise photochemical/biochemical efficiency. It could, instead, be worthwhile exploring the conditions leading to ameliorated leaf CO2 diffusion efficiency.
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Acknowledgements |
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The authors are grateful to Professor Gabriel Cornic for his advice during the experiments and for helpful discussions. Thanks are also due to Dr Michael Hodges for critically reading the manuscript, to Dr Massimo Gardiman for his kind assistance in in vitro culture techniques, and to Dr Gianni Bellocchi who provided extensive help during statistical analysis. A special thank you goes to JXB editor-in-chief Professor Bill Davies and to the anonymous reviewers for their valuable comments. In vitro cultures of grapevine rootstock ‘41B’ were kindly provided by the company Moët-Hennessy Recherche, Epernay (France).
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Abbreviations |
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A, net CO2 assimilation rate; Ap, net potential CO2 assimilation rate at infinite gm; Chl, chlorophyll; Ca, ambient CO2 concentration; Ci, intercellular CO2 concentration; Cc, CO2 concentration in the chloroplasts; gc, cuticular conductance to H2O or CO2; gm, mesophyll conductance; gs, stomatal conductance; Fm and F'm, maximum yields of Chl fluorescence after dark and light exposure; F0 and F'0, yields of intrinsic Chl fluorescence emitted by photosystem II reaction centres in the ‘open’ state, after dark and light exposure, respectively; Fs, steady-state fluorescence yield under light; Fv and F'v, yields of variable fluorescence after dark and light exposure; JT, rate of total linear electron flow; Jc, rate of linear electron flow associated with photosynthetic carbon reduction; Jo, rate of linear electron flow associated with photorespiratory carbon oxidation; Li, relative limitation to potential net CO2 assimilation; PPFD, photosynthetic photon flow density; PSII, photosystem II; R, dark respiration; Rd, day respiration; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; S, Rubisco specificity factor; S*, apparent Rubisco specificity factor;
CO2, quantum yield of carbon assimilation; PSII, quantum yield of PSII. |
References |
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