The evolution of photosynthetic capacity and the antioxidant
enzymatic system during acclimatization of micropropagated
Calathea
plants
J.M. Van Huylenbroeck
a,*, A. Piqueras
b, P.C. Debergh
caDepartment of plant genetics and breeding (D6P),CLO Gent,Ministry of Small Enterprises,Traders and Agriculture,Caritasstraat 21,
9090 Melle, Belgium
bDepartamento de Nutricion y Fisiologia Vegetal,CEBAS (CSIC),PO BOX 4195,30080 Murcia, Spain
cFaculty of Agricultural and Applied Biological Sciences,Department of Plant Production,Horticulture,Uni6ersity Gent,Coupure links 653,
B-9000 Gent, Belgium
Received 18 October 1999; received in revised form 8 December 1999; accepted 6 January 2000
Abstract
The effects of an increased PPFD on photosynthesis, the functioning of the photosynthetic apparatus and the response of the antioxidant enzymatic system were studied during the ex vitro establishment of micropropagatedCalathea‘Maui Queen’ plantlets. Measured chlorophyll and carotenoids contents in ex vitro formed leaves were almost three times higher compared to the in vitro formed ones. At the end of the acclimatization, an inverse relation between PPFD and the chlorophyll (a+b)/carotenoids ratio was observed. During the first days after transplantationCalatheaplants are not photosynthetically active, as is illustrated by the photosynthetic light response curves. With the appearance of new leaves, higher photosynthetic capacities were observed and light saturation point increased (days 17 and 25). Also the maximal photosynthetic efficiency enlarged as shown by the increased initial slope of the curves.Fv/Fmdecreased directly after transplantation of the micropropagated plantlets, afterwards a recovery was observed, but highest Fv/Fm values were observed in low light (LL) plants. The photochemical quenching coefficient increased gradually during the first two weeks of the acclimatization. In high light (HL) plants,qPdecreased directly after transfer, while this was not observed in LL and medium light (ML). During the acclimatization period to increasing light intensities significant changes in the activity of the antioxidant enzymatic system were observed. A decrease in superoxide dismutase (SOD) activity was measured during the first half of the acclimatization period followed by a recovery in ML and HL plants by day 35. Dehydroascorbate reductase (DHAR) activity decreased during acclimatization. At the end of the experimental period the lowest levels were measured in ML plants. Catalase (CAT) activity increased significantly during the first two weeks after transfer, a clear inverse relationship to PPFD was detected. The relation between the adquisition of full photosynthetic capacity and the activation of the enzymatic antioxidant system in the leaves of calathea plants during ex vitro acclimatization is discussed. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Acclimatization ex vitro; Antioxidant enzymes; Photosynthesis;Calathea; Micropropagation
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1. Introduction
In vitro culture is an efficient vegetative propa-gation system for many high-value cultivars. Nev-ertheless, micropropagation is in some cases
restricted due to considerable losses during the ex vitro acclimatization (in greenhouse or field). Therefore improvement in survival and a better knowledge of the physiology of in vitro cultured plants are of considerable interest.
Directly after transfer to ex vitro conditions, micropropagated plants are very susceptible to various stresses, because they have not yet devel-oped adequate patterns of resource allocation and
* Corresponding author. Tel.: +32-9-2722862; fax: + 32-9-2722901.
E-mail address:[email protected] (J.M. Van Huylen-broeck)
morphological and physiological features required by the new environment [1]. Low photosynthesis rates [2,3] and the malfunctioning of the water housekeeping system [4] are two of the major constrains of tissue cultured plants. A switch to autotrophy and changes in stomatal functioning and cuticula composition are observed during ac-climatization [5 – 7]. Plantlets are exposed to low photosynthetic photon flux densities (PPFD). Once in the greenhouse, PPFD is much higher and plants become very susceptible to light stress. Ex-cessive radiation during acclimatization can even lead to chronic photoinhibition [8,9]. Besides pho-toinhibition, plants also suffer due to the differ-ence between in vitro and ex vitro relative humidity [10]. Both phenomena, photoinhibition and water stress, can generate toxic O2 species [11 – 13]. As protection against the latter, plant cells have developed several antioxidant and enzy-matic scavenging systems as superoxide dismutase (SOD), catalase (CAT), peroxidase, dehydroascor-bate redutase (DHAR) and gluthathione reductase (GR) [14]. Accordingly, there are many examples relating SOD and enzymes of the H2O2scavenging pathway to environmental stresses in plants [15]. However, limited information on the possible role of these protective enzymatic systems during the acclimatization process and their relation with photoinhibition and malfunctioning in the transpi-ration system of micropropagated plants is avail-able. Recently, it was demonstrated that micropropagated plants develop antioxidant mechanisms during acclimatization [16].
In the present study we investigated the effects of an increased PPFD during the ex vitro estab-lishment of micropropagated Calathea ‘Maui Queen’ on photosynthesis, the functioning of the photosynthetic apparatus and the enzymatic activ-ities in the scavenging systems of activated oxygen.
2. Materials and methods
2.1. Plant material and light treatments
Calathea louisae Gagnep. ‘Maui Queen’ plantlets were regularly micropropagated accord-ing to Dunston and Sutter [17], at three months intervals, on agar-solidified medium containing 3% sucrose and 2% glucose. Cultures were maintained at 2291°C under cool-white fluorescent lamps
(PPFD at plant level 30 mmol m−2s−1and a 16 h photoperiod). For acclimatization, rooted shoots were transplanted to a peat substrate and placed in a growth room (2191°C, RH 80 – 90%, CO2 -concentration 350 ppm, day – night regime 16 h light and 8 h darkness). Three different PPFDs were applied (40 mmol m−2 s−1, low light, LL; 120 mmol m−2s−1, medium light, ML; 360 mmol m−2 s−1, high light, HL).
Leaf samples were taken at transplanting (day 0) and after 4, 7, 11, 16, 26, 42 days and stored in liquid nitrogen. Photosynthesis and chlorophyll a fluorescence parameters were measured at day 0, 3, 6, 10, 17, 25 and 40.
2.2. Photosynthesis measurements
Net CO2 gas exchange rates were measured as described in Van Huylenbroeck et al. [8], using a portable ADC-LCA3 photosynthesis measuring system and a temperature and light controlled modified Parkinson Leaf Chamber (PP-Systems, Hertfordshire, United Kingdom). Dark respiration rates (mmol CO2 m−2s−1) as well as CO2 uptake rates (mmol CO2 m−2 s−1) at a PPFD of 40, 120 and 360 mmol m−2 s−1 were measured. During measurements leaf temperature was controlled at 20°C and CO2 concentration was 350 ml l−1.
2.3. Chlorophyll fluorescence measurements
Chlorophyll fluorescence on the adaxial side of the last fully developed leaf was measured with a pulse-amplitude-modulation fluorometer (Model PAM-2000, Walz, Effeltrich, Germany) as de-scribed earlier in detail [8]. Minimal fluorescence (F0) was determined after 15 min dark adaptation, while maximal fluorescence (Fm) was measured after a saturation pulse. The fluorescence ratio
Fv/Fm, with Fv=Fm−F0 being the variable fluorescence, was then calculated. Photochemical quenching (qp) at three different light intensities was measured, using the saturation pulse method. Hereto plants were subsequently illuminated dur-ing 4 min with continuous actinic light of 40, 120 or 360 mmol m−2 s−1. At each light intensity, saturation pulses were given at 20 s intervals dur-ing the last 2 min of illumination. Afterwards a 4 s long far red illumination was given to determine
2.4. Pigment determination
Pigments were extracted in darkness using the methanol – chloroform (1:3 v/v) procedure of Quail et al. [19]. Chlorophyll a and b as well as total carotenoids in the chloroform layer were calcu-lated according to Wellburn [20].
2.5. Enzyme extraction and assays
For enzyme determination approximately 1 g fresh weight of plant tissue was homogenized in 3 ml pre-cooled extraction buffer consisting of 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 2.5 mM PMSF, 8% (w/v) insoluble PVP and 0.2% Triton X-100. The homogenate was filtered through cheesecloth (Miracloth) and cen-trifuged three times during 5 min at 10 000×g at 4°C. The supernatant was used for enzyme assay. The protein concentration was determined accord-ing to Lowry et al. [21] usaccord-ing bovine serum albu-min as a standard. All enzymes were assayed spectrophotometrically at 25°C.
Dehydroascorbate reductase (DHAR) (EC 1.8.5.1) was measured according to Foyer and Haliwell [22]. The reaction mixture contained 50 mM phosphate buffer (pH 6.8) and 2.5 mM glu-tathione. The reaction was started with dehy-droascorbic acid (0.5 mM). The absorption coefficient for ascorbate at 265 nm is 14 mM
cm−1. Control rates in the absence of enzyme extract were subtracted.
Glutathione reductase (GR) (EC 1.6.4.2) activ-ity was determined according to Foyer and Halli-well [23]. The assay contained 50 mM Tris buffer (pH 7.8), 1.0 mM EDTA and 0.12 mM NADPH (absorption coefficient at 340 nm: 6.22 mM cm−1). The reaction was started with 0.8 mM GSSG. Control rates obtained in the absence of GSSG and of enzyme extract were subtracted.
Ascorbate peroxidase (1.11.1.7) activity was de-termined and extracted as described by Amako et al. [24]. The reaction mixture contained 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 0.1 mM hydrogen peroxide and 0.5 mM ascorbate. The hydrogen peroxide dependent oxidation of ascorbate was followed by a decrease in the ab-sorbance at 290 nm (absorption coefficient 2.8 mM cm−1). Guaiacol peroxidase activity was measured according to Chance and Maehly [25] using a reaction mixture with 50 mM phosphate buffer (pH 7) and 5 mM guaiacol (absorption coefficient at 436 nm: 25.5 mM cm−1). The reac-tion was started with 20 mM hydrogen peroxide. Catalase (CAT) (EC 1.11.1.6) activity was mea-sured as described by Aebi [26]. The decomposi-tion of H2O2 was monitored by the decrease in absorbance at 240 nm. For the assay a 50 mM phosphate buffer (pH 7.8) and 10 mM H2O2were used.
Superoxide dismutase (SOD) (EC 1.15.11) activ-ity was determined by the cytochrome c method using xanthine – xanthine oxidase as the source of superoxide radicals, and a unit of activity was defined according to McCord and Fridovich [27].
2.6. Statistical analysis
A two factor analysis of variance (ANOVA) was conducted. When significant differences occur, mean separation was performed using the LSD (P=0.5) method. Data are presented9standard errors.
3. Results
3.1. Pigments
No relation between total chlorophyll or carotenoid content and PPFD was observed in
Fig. 1. Changes in total chlorophyll content (A), carotenoids (B), chlorophyll a to b (C) and chlorophyll to carotenoids ratio (D) in leaves of micropropagated Calathea ‘Maui Queen’ acclimatized at a PPFD of 40 ( ), 120 () or 360
Fig. 2. Evolution of light response curves as measured at different moments during the acclimatization of micropropagated
Calathea‘Maui Queen’ plantlets grown at a PPFD of 40 ( ), 120 () or 360 ()mmol m−2s−1. Values are means9S.E. (n=6) (if not visible, S.E. smaller than data label).
leaves of Calathea (Fig. 1(A) and (B)). Pigment concentration increased significantly about three weeks after transplantation when new leaves ap-peared. Measured chlorophyll and carotenoids contents in those new leaves were almost three times higher compared to the in vitro formed ones (Fig. 1(A) and (B)). Chlorophyll a to b ratio increased from 1.95 to 2.290.1 (Fig. 1(C)). The maximal ratio was reached faster in ML and HL plants. The chlorophyll (a+b)/carotenoids ratio of LL plants increased slightly during acclimatiza-tion (Fig. 1(D)). On contrary, a decrease in this ratio was found during the first days for ML and HL plants. At the end of the acclimatization, an inverse relation between PPFD and the chloro-phyll (a+b)/carotenoids ratio was observed (Fig. 1(D)).
3.2. Photosynthesis and fluorescence measurements
During the first days after transplantation Ca
-latheaplants are practically not photosynthetically active as is illustrated by the photosynthetic light response curves (Fig. 2). From day 6 on, positive values were measured, although saturation was already reached at very low PPFD. With the ap-pearance of new leaves, higher photosynthetic ca-pacities were observed and light saturation point increased (day 17 and 25). Also the maximal pho-tosynthetic efficiency enlarged as was shown by the increased initial slope of the curves. At the end of the experimental period (day 40) an inhibition
of photosynthesis was observed for ML and HL plants, while net photosynthesis for LL plants continued to increase.
Photosynthetic efficiency is also evaluated by chlorophyll fluorescence measurements (Figs. 3 and 4). Fv/Fm decreased directly after transplanta-tion of the micropropagated plantlets. This de-crease was significantly stronger with increasing PPFD, and was mainly due to a reduction in Fm (Fig. 3). Afterwards a recovery was observed, but highest Fv/Fm values were observed in LL plants.
Fig. 4. Photochemical quenching parameter (qp), measured at 360 (A), 120 (B) and 40 (C) (mmol m−2 s−1), for fully expanded leaves of micropropagatedCalathea ‘Maui Queen’ acclimatized at a PPFD of 40 ( ), 120 () or 360 ()mmol
m−2s−1. Values are means9S.E. (n=6) (if not visible, S.E. smaller than data label).
were measured for ML and HL plants at day 14 and 35 (Fig. 5(B)). DHAR activity decreased dur-ing acclimatization. At the end of the experimental period lowest levels were measured in ML plants (Fig. 5(C)). CAT activity increased significantly during the first two weeks after transfer (Fig. 5(D)). A clear inverse relationship to PPFD (highest activities measured in LL plants) was observed. As observed for CAT, also GR activity doubled during the acclimatization of micropropa-gated Calatheaplantlets (Fig. 5(E)). However, for GR no correlation with PPFD was found. Guaia-col peroxidase activities remained unchanged for LL plants, while a significant increase for ML and HL plants was observed at the end of the acclima-tization (Fig. 5(F)).
4. Discussion
Directly after transplantation, CO2 fixation by in vitro leaves of Calathea was very low (Fig. 2). Downregulation of photosynthesis in vitro is fre-quently observed during micropropagation, due to depletion of CO2 in the culture vessels [28] and
Fig. 5. Changes of SOD (A), ascorbate peroxidase (B), DHAR (C), CAT (D), GR (E) and guaiacol peroxidase (F) activity during acclimatization of micropropagated Calathea
‘Maui Queen’ at a PPFD of 40 (hatched, upwards), 120 (grey bar) or 360 (hatched, downwards) mmol m−2s−1. Activities measured at transplantation (day 0) are presented as black bars. Values are means9S.E. (n=6).
The photochemical quenching coefficient was low for in vitro plantlets (day 0), and increased gradu-ally during the first 2 weeks of the acclimatization (Fig. 4). In HL plants, qP decreased directly after transfer, while this was not observed in LL and ML.
3.3. Enzymatic acti6ities
feedback inhibition of the Calvin cycle by exoge-nous sugar supply resulting in deactivation of Rubisco [29]. Also Grout and Aston [2] measured a negative CO2balance in cauliflower and this was the case until two weeks after plants had been transferred to soil. During this period plants are very susceptible to additional stresses. Also in this study, an increase in light intensities had strong photoinhibitory effects, as was reflected by a fast decrease of the variable over the maximal fluores-cence ratio (Fig. 3). A similar process was ob-served during acclimatization of micropropagated
Spathiphyllum plants [7]. The low qp values found during the same period (Fig. 4) indicates that a high proportion of the primary quinone electron acceptor of PSII is in a reduced state and that a low percentage of the captured light energy is directed through the electron transport chain [9]. Starting from day 10, a switch from hetero-trophic to autohetero-trophic metabolism is observed and functional photosynthetic apparatus are devel-oped, as is reflected by the increased light satura-tion point and photosynthetic efficiency (Fig. 2). In addition to this metabolic switch, the photoin-hibitory effects of increased light intensities dimin-ished, as evidenced by the recovery of the Fv/Fm ratio (Fig. 3). In this period new ex vitro formed leaves and functional roots were fully developed and plants acquired complete autotrophic metabolism. Previous results have already demon-strated that the in vitro formed leaves of Calathea
never become fully autotrophic [7]. However, pro-longed exposure of the ex vitro formed leaves to ML and HL conditions had still an inhibitory effect on photosynthetic activity (lower Fv/Fm de-creasing net photosynthesis at day 40). These re-sults are in agreement with observations in micropropagated Liquidambar [30].
With the formation of new leaves, a strong increase in chlorophyll and carotenoids amounts are seen (Fig. 1). Donelly and Vidaver [31] also found higher pigment contents in ex vitro formed leaves of Rubus. The decrease of chlorophyll con-tent at day 40 for ML and HL plants, proved the photodamaging effect of a prolonged exposure to higher light intensities. The chlorophyll a to b ratio increased during acclimatization, as was re-ported by Donelly and Vidaver [31] forRubus. On the other hand, a reverse relationship between light intensity and chlorophyll (a+b)/carotenoids ratio was observed (Fig. 1). This relative increase
in carotenoids observed in ML and HL plants reflects a flexible functional response of the photo-synthetic apparatus to the different light environ-ments, since the photoprotective role of carotenoids against photooxidative damage is well documented [32,33].
The changes observed in the antioxidant enzy-matic activities during acclimatization of Calathea
indicate a progressive activation of this enzymatic system against oxidative stress. During the ac-climatizaton of micropropagated plants, two situa-tions of environmental stress able to generate activated oxygen species may occur: photoinhibi-tion [8] and moderated water stress [34,35]. Some of the changes observed in our experiment for SOD, CAT, GR and ascorbate peroxidase activi-ties during the acclimatization period resemble those found in plants exposed to mild water stress [36,35]. Enhancements in the activity of activated oxygen-scavenging enzymes during water or other environmental stress situations probably occurs in the chloroplasts, because in photosynthetic tissues, ascorbic acid-dependent antioxidants as ascorbate peroxidase and GR are predominantly localised in the chloroplasts, which are the major sites of H2O2 production in leaves [37]. The activity of antioxi-dant enzymes may also be important for the scav-enging of toxic O2 species in other cell compartments. There is increasing evidence indi-cating the importance of the cytosolic isoforms of antioxidative enzymes in plants grown under vari-ous environmental stress conditions [38,39]. In re-sponse to changes in light intensity SOD, CAT, ascorbate peroxidase, GR and guaiacol peroxidase activities showed clear changes for each acclima-tization date. The increase of SOD, ascorbate peroxidase and GR activities under high light conditions reveal a protection against photooxida-tive stress linked to photoinhibition, as it has been previously reported by Logan et al. [39] in Cucur
species via the Mehler-peroxidase pathway as sink for reducing equivalents and/or a mechanism to increment the transthylakoid membrane pH gradi-ent [41,42]. In the case of GR, by the end of the acclimatization period this activity was inhibited at higher light intensities. GR utilises NADPH to re-reduce oxidized glutathione. Since this com-pound is the reductive substrate for the enzyme DHAR that reduces the oxidation product of ascorbate, chloroplastic GR could be considered as part of the Mehler-peroxidase pathway. Our results showed that the acclimatory pattern in relation to light intensity for DHAR is correlated to some extend to GR although it differed from those of SOD and ascorbate peroxidase by the end of the acclimatization period. There could be sev-eral possibilities to explain this. In first place, chloroplasts have two pathways to reduce MDA (monodehydroascorbate): direct photoreduction as well as an enzymatic reduction via MDA reduc-tase using GR [43]. In addition GR activity may be influenced by the need for reduced glutathione for cellular processes not associated with the Meh-ler-peroxidase pathway as for example sulphur metabolism and maintenance of protein redox bal-ance [44]. From all the enzymes studied in this work only CAT activity exhibited an inverse rela-tion to light intensity. This result is not surprising since it is well documented that catalase synthesis and degradation is light sensitive [45] and high light intensities accelerate this process in Calathea
leaves. The observed pattern of activity for CAT in relation to light intensity strengthens the signifi-cance of the acclimatory changes observed for the predominantly chlorplastic ascorbate peroxidase. It is possible that the pattern observed for CAT is related to its role in photorespiration and/or detoxification of H2O2formed as a result of mito-chondrial electron transport [14].
Guaiacol peroxidases are involved in hardening the cell walls during development [46]. This could explain the increase in peroxidase activity ob-served in micropropagated Calathea plants at the end of the acclimatization, when new leaves are fully developed.
Our work suggests that tissue-cultured plants develop a functional photosynthetic apparatus and antioxidant enzymatic protective system during ac-climatization. A good climatic control during the critical days of the acclimatization process will lead to better results.
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