Plant Physiol. Biochem., 1999, 37 (7/8), 587-595 I 0 Elsevier, Paris

Effects of CO, and sugars on photosynthesis and composition of avocado leaves grown in vitro

Gloria de la Viiiaa,b*, Fernando Pliego-Alfaro”, Simon P. Driscollb, Valerie J. Mitchellb, Martin A. Parryb, David W. Lawlorb

a Departamento de Biologia Vegetal (Fisiologia Vegetal), Facultad de Ciencias, Universidad de Mglaga, Campus de Teatinos s/n, 2907 l- MBlaga, Spain

h Department of Biochemistry and Physiology, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK

* Author to whom correspondence should be addressed (fax +44 01582 760981; e-mail gloria.delavina@bbsrc.ac.uk)

(Received February 26, 1999; accepted May 11, 1999)

Abstract - The effects of micropropagation conditions on avocado (Per-sea americana Mill.) have been measured in leaves and plants cultured in vitro. The consequences of the type and concentration of sugar in the medium and of carbon dioxide concentration in the atmosphere on the rates of photosynthesis and amounts of ribulose 1,5-biphosphate carboxylase-oxygenase (EC 4.1.1.39; Rubisco) and total soluble protein (TSP) were measured. At the highest sucrose supply (87.6 mM), Rubisco content was substantially decreased in leaves, and even more when elevated CO, (1 000 pmol.mol-‘) was supplied. Maximum photosynthetic rate (P,,,) was significantly decreased when plants developed in high sucrose and elevated CO,. However, Rubisco concentration was significantly greater when glucose was supplied at the same molar concentration or when the concentration of sucrose was small (14.6 mM), and no differences were observed due to the CO, concentration in the air in these treatments. The ratio of Rubisco to total soluble protein (Rubisco/TSP) was dramatically decreased in plants grown in the highest concentration of sucrose and with elevated CO,. Leaf area and ratio of leaf fresh weight/(stem + root) fresh weight, were greater in plants grown with CO2 enriched air. However, upon transplanting, survival was poorer in plants grown on low sucrose/high CO, compared to those grown on high sucrose/high CO,. 0 Elsevier, Paris

Elevated CO2 / in vitro acclimation / micropropagated plants / Persea amerkana / photosynthesis / Rubisco / sucrose

ANOVA, analyses of variance / PAR, photosynthetic active radiation I PFD, photon flux density I P,, net photosynthesis rate / P,(600), net photosynthesis rate at 600 pmol.m%’ / Rubisco, ribulose l&biphosphate carboxylase-oxygenase (EC 4.1.1.39) I Rubisco/TSP, percentage of Rubisco to total soluble protein

1. INTRODUCTION

Micropropagation is an established technique for rapid propagation of uniform plants, including tree species of economic importance, such as avocado (Persea americana Mill.). After multiplication of the plants in vitro, microshoots or rooted plantlets are transferred to pots in the greenhouse, where they must adapt to very different growth conditions. Often the transplants of woody species survive only poorly: the reasons are not well understood. It is likely that it is caused by inadequate rates of photosynthesis in the micropropagated plants. Their photosynthetic rate is usually one order of magnitude lower than those grown in normal conditions, and frequently so low that their carbon balance is negative over a wide range of

Plant Physiol. Biochem., 0981-9428/99/7-8/O Elsevier, Paris

irradiances. Moreover, leaves developed in vitro may never develop photosynthetic competence, so plants must produce new leaves when transferred to a new environment to be able to photosynthesize, grow and survive [34]. These effects of micropropagation may be responsible for the poor performance upon trans- planting.

During growth in vitro, photosynthetic tissue may acclimate to the environment. Acclimation refers to the biochemical, physiological and morphological changes of plants in response to environmental condi- tions. In vitro, micropropagated plants usually grow on large concentration of sugar from the culture medium, under poor irradiance and with limited CO, in the gas phase. It is likely that these factors act in combination,

588 G. de la Vifia et al.

resulting in a poor photosynthetic performance [ 12, 16, 17, 371 although this is not always observed [23. 28,331. Decreasing sugar concentration and increasing irradiance and CO, in the culture vessels may be a way of increasing maximum photosynthetic rate (P,,,) in vitro [ 11, 22, 421. In some cases, replacing sucrose by glucose during the last phases of in vitro culture, which is usually called the in vitro acclimation (or acclimatization) phase [42], increases P,,, and im- proves survival upon transplanting [29]. Decreased Rubisco content is often correlated with decreased photosynthetic rate and with increased concentration of sugars in cell cultures [32]. This correlation is also observed in other experimental systems in which sugars accumulate in leaves [24]. The increased P,,, in vitro, observed when sucrose is decreased, suggests that the exogenous carbohydrate supply inhibits devel- opment of the photosynthetic system and photosyn- thetic rate [7, 15, 20, 291, specifically by decreasing Rubisco activity [ 181.

The aim of the work described here was to examine how culture conditions, e.g. type and concentration of sugar and CO, concentration in the air, affect photo- synthetic rate and amount of Rubisco and total soluble protein in leaves of rooted avocado microshoots, as well as their growth in vitro. The effects are related to subsequent establishment of plants in the glass house.

2. RESULTS

2.1. Gas exchange

After 8 weeks of growth, the P,,, of leaves from micropropagated avocado plants was lower in the elevated sucrose plus high CO, treatment than in the other treatments (table 1, Jigure 1). There were no differences between the other treatments. The dark respiration rate was not affected by treatments. When 1 000 umol.mol-’ COZ in air was supplied during gas exchange measurements under non-limiting irra- diance (600 umol.m-*.s-‘), P, was greater than in 350 pm01 CO,.m-‘.s-’ (table r) for plants grown in all treatments except for leaves of plants developed under elevated CO, and with high sucrose concentration in the culture medium, where the increases were not significant.

Net photosynthesis increased with increasing pho- ton flux @gure I), saturating at about 600 pmol photonsm-‘.ss’ . The maximum rate of photosynthesis was similar in all treatments except for plants grown in high sucrose concentration and elevated CO, which was smaller than all other treatments. Light compen- sation point was between 9.4 and 10.4 pmol photons.m-2s’ in sucrose treatments. In glucose treatments, the LCP was higher (17.3 pmol photons.m-2s1) when ambient CO, was supplied than

Table I. Maximum rate of net photosynthesis, P,,, (pmol CO,,m-‘s’), and the rate at non-limiting irradiance (600 umol.m~‘.s~‘), P,(600) (urnof CO,.m-‘,s-‘), measured under normal (350 pmol.mol-‘) and elevated (1 000 pmol.mol-‘) CO,, dark respiration rate, R, (pmol CO,.m-*.s-‘) and light compensation point, LCP (umol photonsm-‘,s-‘), measured under ambient CO, (350 pmol.molF’), chlorophyll (a+b) content, (mgg’ dry weight) total soluble protein concentration (g,m-*), ribulose l,%bisphosphate carboxylase-oxygenase concentration (g.m-‘). Significant differences due to growth conditions are indicated by small letters and those due to CO, concentration used during P, measurement are shown by capital letters, (LSD P = 0.05). In the treatment indicated by -, it was not possible to measure P, at elevated CO,. Values are means +_ SE obtained from four independent samples.

- ~____-.. ~~~~~~. .--~- ._____~~ Sucrose Glucose

14.6 mM 87.6 mM 87.6 mM ~. ___.-

co, co* co, __-

350 umol~mol-’ 1 000 pmol~mol-’ 350 pmol~mol--’ 1 000 pmol~mol-’ 350 pmol~mol~’ 1 000 pmol~molF’ -.

P ,nax 2.2 f 0.6 a 2.2 10.2 a 2.3 2~ 0.5 a 0.8 + 0.1 b 1.8+0.2a 1.8 + 0.2 a PJ600) normal CO2 2.1 + 0.3 aA 1.8 kc 0.2 aA 1.9 5 0.2 a 0.9 + 0. I bA I .8 f 0. I aA 1.7+0.1 aA P,(600) high CO, 3.6 f 0.5 bB 2.7 f 0.2 aB - I.1 f0.1 aA 2.5 kO.1 aB 2.3 +O.l aB Rd 0.3 * 0.0 a 0.2 * 0.0 a 0.2 + 0.0 a 0.2 * 0.0 a 0.3 f0.1 a 0.2 + 0.0 a LCP 9.6 9.4 10.4 9.7 17.3 4.3 Total protein 0.37f0.12bc 0.59+O.lOab 0.91 f0.13 a 0.15i0.04c 0.63 f 0.17 ab 0.62 f 0.08 ab Rubisco 0.21 + 0.08 a 0.32 f 0.1 I a 0.05 + 0.01 b 0.01 + 0.00 c 0.25 + 0.02 a 0.45 f 0.16 a Chl (a+b) in old leaves 34.6 f 2.7 a 35.9 + 2.9 a 35.053.1 a 31.8f2.1 b 38.4f 1.7 a 39.9rt l.Oc. Chl (a+b) in leaves young 28.8 +_ 1.4 b 35.3 * 1 .o a 30.6 I!I 1.4 a 35.0 +_ 2.9 a 31.7 * 1.4 a 40.7 +_ 2.1 c Total Chl (a+b) 30.8 f 1.7 a 35.6 4 I .6 a 31.8f0.8a 33.7 zk 2.2 a 35.2 f 0.8 a 40.5 + 1.5 b _____ ____-.. ____.-.- ___--- -

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Photosynthesis acclimation in micropropagated avocado 589

-0. I I I I I I I I I I I I 0 400 800 1200 0 400 800 1200

PFD (pmol.me2.s“)

Figure 1. Photosynthesis rate (PJ versus photon flux (PF) for leaves of micropropagated avocado plants under different growth conditions: A, 87.6 mM sucrose and 350 pmol,mol-’ CO,; B, 87.6 mM sucrose and 1 000 pmobmol-’ CO,; C, 14.6 mM sucrose and 350 pmol.mol-’ CO,; D, 14.6 mM sucrose and 1 000 pmol.mol-’ CO,; E, 87.6 mM glucose and 350 pmol,mol-’ CO,; F, 87.6 mM glucose and 1 000 pmol~mol-’ CO,.

when the atmosphere was enriched with 1 000 pmol.mol-’ CO, (4.3 pmol photonsm+s-‘) (table r). The apparent quantum yield (the slope of the net photosynthetic rate at limiting irradiance) calcu- lated from jgure I was smaller in high sucrose treat- ments (0.012 pmol CO,.pmol-’ photons) than in the low sucrose (0.027 pmol CO,+mol-’ photons) and glucose (0.022 pmol CO,+mol-’ photons) treatments. Differences in apparent quantum yield between CO, treatments were negligible. No statistical assessment of differences in LCP and’apparent quantum yield is possible.

2.2. Rubisco, total soluble protein and chlorophyll contents

Amounts of Rubisco and total soluble protein per unit leaf area of micropropagated avocado varied with treatment (table I>. The higher concentration of su- crose decreased Rubisco content of leaves compared

to the lower sucrose concentration and the glucose treatment, which were not statistically different. A CO, concentration of 1 000 pmolmoll’ compared to the ambient CO, concentration significantly decreased the amount of Rubisco in the leaves of avocado plants grown with the high sucrose concentration (table r>. However, the elevated CO, did not significantly in- crease the amount of Rubisco in leaves from the treatment with the lower sucrose concentration or the glucose treatment, which was equimolar to it. The TSP behaved differently than Rubisco (table r). It was largest in the high sucrose and ambient CO, treatment and decreased most with elevated CO, at the highest sucrose concentration. The amount of TSP per unit area was smaller, at ambient CO,, in the lower sucrose concentration than in the glucose treatment, but was the same at elevated CO,. Equimolar concentrations of sucrose and glucose did not affect Rubisco concentra- tion and TSP in the same way, e.g. the amount of Rubisco was slightly higher in leaves grown with glucose in the medium than with sucrose. Carbon dioxide had a much greater impact on Rubisco and TSP amounts in the leaves when combined with concentrated sucrose than with the other carbohydrate treatments. The ratio Rubisco/TSP was extremely low in the high sucrose treatments (8 % in high CO, and 4.3 % in ambient CO,), compared with that observed in low sucrose (53.5 % in high CO, and 57.6 % in ambient COJ or glucose (73.1 % in high CO, and 36.0 % in ambient CO,).

Although decreased concentration of sucrose in the medium correlated with a significant increase in Rubisco, a parallel increase in P,,, was observed only under high CO, (/Figure 1). In contrast, elevated CO, in combination with abundant sucrose produced the op- posite effect, decreasing Rubisco concentration and P max significantly. Plants grown in these conditions were the only ones that did not increase their P, when saturating light intensity and elevated CO, were used during photosynthetic measurements (PJ600)) (tu- ble I).

The chlorophyll content was not affected by the treatments (table r> despite the large effects of carbo- hydrate and CO, treatments on TSP and Rubisco.

2.3. Growth measurements The whole plant fresh weights, when grown under

different environmental conditions, were not signifi- cantly different. However, the ratio of leaf to stem plus root fresh weight was always greater in plants grown at elevated CO*, reflecting smaller root development (table II).

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590 G. de la Viiia et al.

Table II. Growth parameters of avocado plants cultured under different growth conditions. Sucrose concentrations (14.6 and 87.6 mM) and glucose (87.6 mM), at two CO, concentrations (350 and 1 000 pmol.mol-‘). Significant differences based on LSD (P = 0.05). Values are means f SE obtained from four independent samples.

Sucrose Glucose

14.6 mM 87.6 mM 87.6 mM

co, co, co1

350 ~rnol~mol~’ 1 000 ~mol.mol-’ 350 pmol~mol-’ 1 000 ymol.mol-’ 350 ~mol~mol~’ 1 000 ~.mol~mol- ’

Total leaf area (cm*) 30.3 ?L- 3.2 a 33.8 f 3.3 a Young leaf area (cm’) 21.4 _+ 2.7 a 21.1 k2.5 a Root fresh weight (g) 0.4 f 0.0 a 0.3fO.l b Leaves/(Stem + Root) 0.9fO.l a 1.3kO.l b Whole plant fresh weight (g) 0.7 f0.1 a 0.8 f 0.0 a Whole plant length (cm) 2.6 f 0.3 a 3.9 f 0.4 b

_________

Survival rate of micropropagated plants trans- planted to sand and grown in the glass house, was lower for those grown in the smaller sucrose concen- tration than in the large one (both at elevated CO,). After 2 months, 100 % of the plants from the higher sucrose concentration had survived in comparison to only 70 % of those from the lower sucrose concentra- tion. After 6 months, survival rates were 90 % and 70 %, respectively. However, those plants from the lower sucrose treatment which survived transplanting, grew better than those from the higher sucrose con- centration: after 4 months, the differences in leaf area were statistically significant (84.0 and 47.2 cm2, re- spectively). Stems grew longer and more leaves were produced on plants from the low compared to the high sucrose concentration (length Il. 1 and 7.6 cm and leaf number 13.5 and 11 .O, respectively).

3. DISCUSSION

This study was on the effects of sugars in the culture medium and CO, in the atmosphere on the total soluble and Rubisco protein and on the photosynthetic rate of micropropagated avocado. A specific inhibitory effect of sucrose compared with equimolar glucose on Rubisco concentration and the ratio of Rubisco to total soluble protein was observed. Total soluble protein content was very large when avocado plants were grown with concentrated sucrose under ambient CO,, but Rubisco content was much reduced. Such, condi- tions promote heterotrophic metabolism in tissue cul- ture [22] and this appears to be the case in avocado leaves grown in culture, hence the effects on Rubisco.

Sucrose in the medium also decreased P,,, when the cultures were grown in CO,-enriched air. The rate

Plant Physiol. Biochem.

27.6fl.6a 18.4k2.4a

0.4 f 0.0 a 0.8 f 0.1 a 0.9 fr 0.1 a 2.8kO.l a

32.9 f 2.5 a 19.3i 1.9a

0.3 k 0.1 b 1.5fO.l b

0.8 + 0.0 a 4.8 + 0.9 b

27.9 -t 3.5 a 15.5 5 3.0 b 0.4 -t 0.0 a 0.8 + 0.0 a 0.9 kO.1 a 3.0fO.l a

28.8 k 4.0 a 20.2 f 2.7 a

0.3 + 0.0 b 1.1 kO.1 b 0.8 i 0. I a 4.0 f 0.9 b

of photosynthesis was greatest for plants grown with the smaller concentration of sucrose in the medium and CO, in the atmosphere. Light compensation points were low in all treatments as expected in plants grown at low light intensities. The apparent quantum yield was smallest with high sucrose, suggesting that su- crose decreases photosynthetic efficiency. No decrease in the maximum rate of photosynthesis was observed at very large photon flux, indicating that photoinhibi- tion was not affecting the measurements, which were made for only short periods to avoid such an effect.

Accumulation of carbohydrates in plants frequently, but not consistently, correlates with decreased photo- synthetic rates and smaller content of Rubisco both in micropropagated and normally grown plants. Carbo- hydrate accumulation in normally grown plants is promoted by elevated CO, concentration which in- creases the size of the assimilate source [5, 40, 411 or by decreasing sink-strength so that the demand for carbohydrates is decreased [3]. Such a situation occurs with nutrient deficiencies, e.g. deficiency of suitable source of nitrogen [27]. The plants in our study were supplied with sufficient nutrient to allow growth. It is unlikely that the decrease in Rubisco was due to inadequate nitrogen, for example, as the TSP concen- tration was large when Rubisco was very small (e.g. in high sucrose and ambient CO,). Thus, sucrose has a more specific effect.

Micropropagated plants are supplied with very large concentration of sugar in the rooting medium and if CO, concentration is also increased then total carbo- hydrate supply should be large. Capellades et al. [7, 81 observed greater starch accumulation and smaller P,,, in leaves of micropropagated Rosa sp. grown in media with a high sucrose concentration than in media containing low sucrose. Dependence of P,,, and

Photosynthesis acclimation in micropropagated avocado 591

Rubisco activity on the sugar of the culture media has been reported in micropropagated plants of several species [7, 18, 201. The type of sugar is also impor- tant [36]. The avocado P,,, from tissue culture is inhibited more by sucrose than glucose in our study. This indicates a specific effect of sucrose. However, sucrose may be hydrolysed, in the rooting medium, to glucose and fructose by invertase excreted from the plant. Complete hydrolysis would double the molar concentration of glucose plus fructose from the su- crose and thus increase the osmotic potential of the medium. This, possibly, could account for the decrease in Rubisco. However, this is unlikely as an osmotic effect should be a general effect on protein synthesis, but total soluble protein was not affected to the same extent as Rubisco. As the total molar concentration of glucose would be the same in the hydrolysed 87.6 mM sucrose and in 87.6 mM glucose, the effects of the treatments would be the same. However, they differed greatly. Therefore, we conclude that the decrease in Rubisco and photosynthesis is a specific effect of large sucrose concentration in the medium and not caused by the breakdown of sucrose. A similar specific inhibi- tory effects of sucrose, compared with glucose, oc- curred on the P,,, of tissue cultures of Clematis [29].

In cultures of Rosa sp. the observed decrease in P max due to sucrose was thought to be due to the accumulation of phosphorylated compounds and depletion of Pi in the chloroplast stroma, thus inhibit- ing ATP synthesis [7]. However, other physiological processes could produce such an effect. As we have shown for avocado, micropropagated plants grown with concentrated exogenous sugars for long periods adapt by decreasing the amount of proteins related to assimilatory functions, as shown by Desjardins [lo]. This long-term effect has also been reported in straw- berry tissue culture [ 191, leaf cell suspension cul- tures [35] and in plants growing under conditions that promote sugar accumulation [14, 24, 391. The effect may be even more evident in woody than in herba- ceous species, as they usually have lower growth rates and, in consequence, lower sink-strength.

Although the Rubisco concentration in leaves was increased by decreasing sucrose concentration in the culture medium, P,,, was only increased by this treatment in CO,-enriched air. However, the values of P, measured were still low, showing that other factors may be limiting photosynthesis, e.g. acclimation to low light during growth. This is supported by the very small concentrations of soluble protein and Rubisco and Pm,, which were ten times smaller in our micro- propagated avocado than usual in shade plants grow-

ing in the field [26]. Small rates of P,,, and small concentrations of Rubisco and total soluble proteins in leaves of tissue-cultured avocado plants, could be a result of acclimation to very small irradiance. How- ever, it is also possible, in long-term culture condi- tions, that factors which promote accumulation of sugars in leaves, e.g. concentrated sucrose in the culture medium and elevated CO,, are responsible for the effect, although as we did not measure the concen- trations of different sugars in the leaves, we cannot relate the changes observed to changes in concentra- tion of a specific sugar. We conclude that the small photosynthetic rate of micropropagated avocado is a consequence of long-term effects of large concentra- tions of sucrose in the culture media and the decreased Rubisco content of the leaves.

Micropropagated avocado plants accumulated more fresh mass in the leaves than in stems plus roots when grown in CO,-enriched air, compared to plants grown under ambient CO,. Root fresh weight was clearly smaller. However, stem length was greater when plants were grown in elevated CO, compared to ambient CO*. Thus, CO, affects plant development, accentuat- ing the capacity for formation of shoots and photosyn- thetic organs and their components. Similar results have been reported by Kozai [22] in several species growing with CO,-enriched air. However, despite the beneficial effects of low sucrose and elevated CO, concentrations on the last phase of growth in vitro, the survival of plants after transfer to the glass house was not as good as that of plants grown with large sucrose and elevated CO, concentrations [9]. Capellades et al. [7] observed that the survival of Rosa sp. trans- plants was improved by very high sucrose concentra- tion in vitro (although this treatment decreased the P,,,), probably due to a large accumulation of starch which is the substrate for growth during acclimation to glass house conditions [7]. Our data suggest that the benefits of improved photosynthesis and growth in vitro are not sufficient to confer good survival. Possi- bly this could be due to relatively small carbohydrate reserves and large surface area of plants grown with low sucrose, which may result in plants suffering greater water stress, photoinhibition, etc., than those grown with high sucrose.

In conclusion, high sucrose concentration in the culture medium has specific effects on micropropa- gated avocado, decreasing the amount of Rubisco and the ratio Rubisco/TSP, and giving rise to low rates of photosynthesis correlated with a small amount of total soluble protein and Rubisco per unit area of leaves. This could be explained as a long-term acclimation

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592 G. de la Vifia et al.

process in the very extreme environment of micro- propagated plants,. with large sugar concentrations, weak irradiance and depletion of the CO, in culture vessels. Thus, decreasing sucrose in the culture me- dium in combination with increasing irradiance and CO, concentrations, increased the capacity for photo- synthesis and improve the growth of the. micropropa- gated avocado plants. However, these changes did not improve survival of plants following transplanting and acclimation to glass house conditions.

4.1.3. Treatments applied __ After rooting, irradiance was increased to

8.5 ltmol.m-‘s’ and two partial pressures of CO, were applied only during the photoperiod: ambient co (350 ymol~mol-‘) and elevated CO, (1 0’00 ymol.mol-’ in air). Each rooting group (87.6 or 14.6 mM sucrose and 87.6 mM glucose) was divided between ambient and 1 000 lrmol~mol-’ CO, treat- ments, and grown in vitro 8 weeks further.

4. METHODS

4.1. Plant growth conditions

4.1.1. Multiplication phase

An in vitro seedling of the avocado (Persea ameri- cana Mill.) rootstock G.A.-13 [21] was used as a source of plant material in this study. A stock of proliferating shoots was established following the procedure of Barcelo-Mufioz et al. [4] with subcultur- ing at g-week intervals. The multiplication medium was essentially MS medium [3 I] with the N,,K macro-elements formulation [30] supplemented with 4.4 uM benzyladenine (BA) and 8 g.L-’ agar (Difco). Ten shoots were cultured in glass vessels (300 mL capacity, 7.5 x 8.5 cm) containing 50 mL solid me- dium. Sucrose concentration was 87.6 mM and pH 5.7. All chemicals were supplied by Sigma Chemi- cal Co. (Poole, Dorset, UK) unless otherwise stated. Plants were maintained under a daylength of 16 h, at 35 ymol.m-2s’ of PAR from Grolux (Sylvania) fluo- rescent tubes and at a constant temperature of 25 IL I “C.

4.1.2. Rooting phase

Microshoots were rooted in 150 x 25 mm diameter glass test tubes, with the COZ concentration of the growth room (350 pmol~mol-‘) and a low PAR flux (35 pmol.m-2.ss’) as described by Barcelo-MuAoz et al. [4] proceeding in two phases. In the first stage, shoots were incubated in the presence of 4.9 pM indol-3-butyric acid for 3 d. In the second stage, the auxin was omitted and 1 g.L-’ activated charcoal added to the medium. The culture medium used was MS [31] with macro-elements at 0.3x. Carbohydrate concentrations during rooting were 87.6 or 14.6 mM sucrose or 87.6 mM glucose. After 4 weeks, 80 % of the plants in each sugar treatment had rooted and these rooted plantlets were used in this study.

4.2. Gas exchange measurements

Rates of net photosynthesis (P,) were measured in the laboratory on whole plants. Test tubes in which plants were grown were connected to an open-circuit gas exchange system, using them as gas exchange cuvettes. Air was supplied from pressurized cylinders and flowed through each test tube at 7 * 0. I mL.s-’ Carbon dioxide concentration was controlled by a gas blender (GD 600; ADC Hoddesdon, UK) and mea- sured using an infra-red gas analyzer (225-Mk3; ADC Hoddesdon, UK). Net photosynthesis was measured at different quantum fluxes of PAR from a quartz halogen lamp (5OW Osram m37), by placing neutral density wire mesh screens between the lamp and the test tube. Photon flux at the position of leaves inside the test tubes was measured by a small sensor, calibrated against a quantum sensor (Li-Cor Corporation, NE. USA). Air temperature was measured with a fine-wire thermocouple in the test tube in a thermostatically controlled waterbath. Air was humidified by bubbling through water before entering the plant cuvette. Gas exchange was measured in darkness, to calculate dark respiration rate (Rd), and at 60. 220, 600 and I 200 umol.m-2.sp’ PAR at 350 pmol~mol-’ CO1 in air, to plot a net photosynthesis versus photon flux density (P,/PFD) curve. Carbon dioxide was then increased to 1 000 pmol.moll’ and irradiance de- creased to 600 umol.m-‘+-I to calculate P,, under this conditions (P,(600), table 1) and then to total darkness, to calculate R,. After measurements, which lasted for approximately 30 min, shoots were cut, the aerial parts of the plants removed and CO, exchange of the root system in the culture medium was measured at the two CO, concentrations. Net photosynthesis (umol CO,.m-‘.s-‘) was calculated by: P,, = (C x J/A) x (44/22.4) x (273/(273 + T,)) x (l/44), where C is the concentration of CO, (ltmol~moll’) in the air, J the flux of air entering the cuvette (mL.s ‘). A the whole plant leaf area (rn-‘) measured from drawings of the leaves, T, chamber air temperature (“C) and 44/22.4 the CO, molecular mass/volume ratio which is corrected for

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Photosynthesis acclimation in micropropagated avocado 593

temperature by 273/(273 + T,). The factor l/44 trans- formed pg of CO, to pmol of CO, from the molecular mass. When CO, was released from the plants by respiration (below the light compensation point), P, was calculated by substracting CO, released by the roots in the culture medium from the CO, released by the whole plant plus culture medium. When CO, was absorbed (above the light compensation point), P, was calculated by adding CO, released by the root system plus culture medium to the total CO, absorbed. The dark respiration rate of the leaves (Rd) was calculated by subtracting the CO2 released or gained for the root system plus culture medium to the CO, released by the whole plant plus culture medium. The magnitude of the CO, released or gained by the root system plus culture medium differed when measured in high or low CO,, and that was considered when calculating R,. h-radiance response curves in 340 pmol.moll’ CO, were done for all culture conditions. The maximum rate of photosynthesis (P,,,) and light compensation point (LCP) were determined from the Edwards and Walker [ 131 equation. Dark respiration rate (Rd) and P, in 600 pmol quanta.m-*.s-’ in ambient (350 umol.mol-‘) and elevated (1 000 umol.moll’) CO, were also calculated. Four to six plants per treatment were analysed.

4.3. Measurements of soluble protein and Rubisco content

After gas exchange, all leaves were cut from every plant, weighed, traced on graph paper to obtain the area and their estimated chlorophyll content. Leaves were them frozen in liquid nitrogen and kept for biochemical analyses. Four to six samples from each treatment were analysed. Soluble protein was ex- tracted by grinding the leaves in chilled pestle and mortar with 1 % (w/v) insoluble, HCl-washed PVP and 2.0 mL buffer containing 20 mM Hepes, 10 mM MgCl,, 1 mM Na,EDTA, 10 mM NaHCO, and 100 mM @mercaptoethanol plus 1 % (v/v) Tween 80 and 100 1tL 40 mM phenylmethyl-sulphonyl fluoride (PMSF), pH 7.6. Homogenates were centrifuged at 10 000 x g for 3 min and the supernatants assayed immediately. Total soluble protein was determined by the Bradford [6] method (after dilution of the crude extract with water), using bovine serum albumin as standard. Rubisco protein (EC 4.11.39) in the extracts was determined by the Laemmli [25] method. Proteins were separated by vertical denaturing SDS- polyacrylamide gel electrophoresis (PAGE) at room temperature using a discontinuous buffer system. A Mini-Protean II system (Bio-Rad) and commercial

gels (Mini-Protean II No. 161-0902 Bio-Rad, 4 % W/V

polyacrylamide content in the stacking gel, 15 % polyacrylamide content in the separating gel, 0.375 mM Tris-HCl, pH 8.8) were used. The electro- phoresis buffer was 25 mM Tris/192 mM glycine/ 0.1 % SDS (Bio-Rad 161-0732). Electrophoresis was carried out at 120 V. Purified Rubisco protein from wheat was used as standard. The leaves of four different plantlets were analysed in each treatment and each replicate was assayed twice. Gels were stained overnight with 0.1 % w/v Coomasie Brilliant Blue R in water/methanol/acetic acid (50/40/10, v/v/v) and destained in water/methanol/acetic acid (50/40/10, v/v/v) and then in water/ethanol/acetic acid (X5/1 O/5, v/v/v). Bands migrating to the same position as the Rubisco standard were cut from the destained gels and extracted overnight at 37 “C in glass vials containing 1.5 or 2.0 mL 1 % (w/v) SDS solution. The concen- tration of Rubisco protein in leaf extracts was calcu- lated from measurements of standards and unknowns at 585 nm in a spectrophotometer (Cecil Instruments, UK). Chlorophyll was measured, using a non- destructive chlorophyll meter (SPAD-502, Minolta Japan). Ten measurements were taken every 0.5 cm on both sides of the central vein of each leaf, to obtain an average chlorophyll content per unit leaf area, which was used to determine average chlorophyll content of the whole leaf area of the plant. Chlorophyll content of old leaves (the leaves plants had at the start of the rooting phase) and in young leaves, developed during the rooting and in vitro acclimation phase, were determined separately. The chlorophyll meter was calibrated by comparison with chlorophyll content of leaves measured by Arnon’s method [I] using the leaves of two different plantlets.

4.4. Growth measurements

Young leaf and total leaf areas were calculated by the methods described above. Fresh weight of the whole plant and of leaves, stems and roots was measured and the ratio of leaf/(stem + root) was calculated. Plant height was also measured.

4.5. Growth ex vitro

An experiment was done with ten plantlets from the high and from the low sucrose treatment, both grown with elevated CO,. Plantlets were transplanted into sterile sand and inoculated with mycorrhizal inoculum of Glomus intrurradice (MICROBIO, Herts., UK) as describe by Azcon-Aguilar et al. [2] and grown in a greenhouse for 6 months. They were kept under con-

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594 G. de la Vifia et al.

stant 100 % relative humidity and 25 _+ 3 “C for the first month; afterwards, they were grown under more variable relative humidity and temperature (80 + 10 % and 25 + 5 “C, respectively) for the same period. After 2 months growing ex vitro, they were transferred to open benches with relative humidity 6q f 10 % and temperature 25 + 10 “C. After the first month, ex vitro plants were irrigated twice a month with a nutrient solution made with Hakaphos Verde (BASF), (N/P/K/Mg 15/10/15/2) at a concentration of 0.1 g.L-’ (first 4 weeks) or 1 g.L-’ (after the first 4 weeks). Sequestrene (Ciba Geigy) at 0.8. lop2 g.L-’ was added to the nutrient solution monthly. Plants were watered with sterile distilled water. Survival of the plants and growth in height, leaf number and area were measured over the growing period.

4.6. Statistical analyses

ANOVA and Kruskal-Wallis tests were used to analyse the data and assess significance. Differences between means were detected by LSD and the U-Mann Whitney rank sum test [38].

Acknowledgments

This work was funded by the ‘Consejeria de Edu- cation y Ciencia’ (FPIyD scholarship, Junta de An- dalucia, Spain) and CYCIT (project AGF 95-0588- CO2-01). Support was also provided by a European Science Foundation grant from the Whole Plant Physi- ology Network and by The British Council (Cultural, Educational and Technical Cooperation Programme). We thank Dr Mathew Paul for critical reading of the manuscript. IACR-Rothamsted is a grant-aided Insti- tute of the Biotechnology and Biological Sciences Research Council.

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