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Dynamics of non-structural carbohydrates in two Ficus species after transfer to deep shade
Environmental and Experimental Botany 54 (2005) 148–154
Dynamics of non-structural carbohydrates in two Ficus species after transfer to deep shade Erik J. Veneklaasa,b,∗ , Franka den Oudena b
a Plant Ecophysiology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
Accepted 25 June 2004
Abstract The present study aims at assessing the role of stored carbohydrates of F. benjamina L. and F. binnendijkii (Miq.) Miq., two tropical trees of horticultural significance, during acclimation to very low light intensities. Plants were grown at an average photosynthetic photon flux density (PPFD) of 90 mol m−2 s−1 , and then transferred to a low-light treatment, with a PPFD of 5 mol m−2 s−1 . Plants of both species showed negative growth rates for approximately 1 week, coinciding with a substantial decrease in the levels of total non-structural carbohydrates (TNC; starch and soluble sugars) in leaves and stems. TNC reached much lower levels in developing leaves than in fully grown leaves, and some of these developing leaves were shed in F. benjamina. In stems and leaves combined, TNC levels were 142 mg g−1 (plant) in F. benjamina and 160 mg g−1 (plant) in F. binnendijkii at the time of transfer to low light. Thirty days later, in fully acclimated plants, these levels had stabilised at 116 and 112 mg g−1 . The relatively minor damage to the plants and short duration of a negative carbon balance, despite the low mean PPFD 5 mol m−2 s−1 , illustrate the capacity of both F. benjamina and F. binnendijkii to tolerate deep shade. Stored carbohydrates are important in plants that grow in environments like forest understories or indoor environments, where carbon balance can be negative for several days. © 2004 Elsevier B.V. All rights reserved. Keywords: Carbon balance; Carbon storage; Growth rate; Acclimation; Ficus benjamina
1. Introduction
∗ Corresponding author at: School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 8 6488 3584; fax: +61 8 6488 1108. E-mail address: [email protected] (E.J. Veneklaas).
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.06.010
Seedlings and saplings of woody plants in tropical forests generally experience low light intensities, due to the shade cast by taller trees. Most species have therefore developed a tolerance to shade, ranging from highly tolerant understorey specialists to relatively intolerant treefall gap specialists (Whitmore, 1996).
E.J. Veneklaas, F. den Ouden / Environmental and Experimental Botany 54 (2005) 148–154
Light environments of all forest microsites vary, however, either in predictable ways (within a day, between seasons) or in unpredictable ways (due to weather or, often more drastically, due to forest canopy dynamics). Plants respond by adjusting their morphology (e.g. sun versus shade leaves) and/or physiology/biochemistry (e.g. concentrations of chlorophyll and photosynthetic enzymes, respiration rate). The present paper focuses on the adaptive response of two tropical woody species to a decrease in light intensity. Such a decrease happens in nature, for example, when canopies close, but is also highly relevant for ornamental plants that are transferred from greenhouses to indoor environments. In particular, we observed changes in reserve carbohydrates, since we expected them to be a crucial carbon source during the acclimation process, and useful indicators of changes in the plant’s carbon balance. At different levels of photosynthetic photon flux density (PPFD, mol m−2 s−1 ), plants gain different amounts of carbon through photosynthesis, and partition this carbon differently to the construction and maintenance of biomass and several metabolic functions (Veneklaas and Poorter, 1998). A continuous supply of assimilates at high PPFD allows high metabolic rates and high growth rates whilst a pool of reserve carbohydrates can be maintained. A sudden decrease of PPFD causes an immediate carbon balance problem as tissues with high metabolic activity, particularly growing organs, draw on scarce assimilates. In these circumstances, stored carbohydrates are an important, or even the only, source of energy. Once plants have acclimated to shade, they have much lower carbon requirements, due to low growth rates and reduced maintenance costs (Veneklaas and Poorter, 1998; Noguchi et al., 2001a). Carbon is stored in most species as insoluble (starch) and soluble (mainly sucrose) carbohydrates (Chapin et al., 1990). Both fractions are highly dynamic but turnover rates vary widely between species and growing conditions (Farrar, 1989; Noguchi et al., 2001a). Not all stored carbon is available to the plant, yet reserve storage is generally considered to be a buffer for situations when demand exceeds supply (Chapin et al., 1990). Kobe (1997) provides empirical data and a model showing the importance of stored carbohydrates for survival and growth in shade. In the experiment described here we analysed the response of two woody species to a sudden decrease in PPFD in terms of growth and reserve carbohydrates.
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Both species grow in tropical forests of Southeast Asia (Corner, 1965) but are also widely used as indoor ornamental foliage plants. In a previous experiment (E.J. Veneklaas and F. Den Ouden, unpublished), F. binnendijkii was found to have higher starch concentrations in its stems, and its leaf growth suffered less from a transfer to deep shade than was the case in F. benjamina. We therefore hypothesised that a greater amount of storage carbohydrates confers greater shade tolerance to F. binnendijkii. Throughout the paper we use the term shade tolerance to indicate the ability to respond adaptively to transfer into deep shade, minimising losses (of carbon, or plant parts, e.g. leaves), and adjusting rapidly in order to survive and grow.
2. Materials and methods Plants of F. benjamina L. ‘Exotica’ and F. binnendijkii (Miq.) Miq. ‘Amstel Queen’ were obtained from a commercial grower. After gently removing the potting mix from the root systems, single plants were placed in 5.5 l PVC tubes filled with finegrade perlite. After two weeks of acclimation to the growing conditions, mean plant dry mass was 0.5 g for both species. From that point, plants grew for another 4 weeks until commencement of the experiment. A 2 cm surface layer of gravel was used to minimise drying and disturbance of the perlite. Irrigations were carried out three times a week with 250 ml of nutrient solution containing 2.4 mM KNO3 , 1.8 mM Ca(NO3 )2 , 0.8 mM MgSO4 , 0.6 mM KH2 PO4 and micronutrients. The experiment was carried out in a climatised greenhouse, with temperature set at 23/20 ◦ C (day/night) and air humidity at 70%. Photosynthetic photon flux density averaged 90 mol m−2 s−1 , using natural illumination and supplementary light (photoperiod 17 h). In the low-light treatment, which started after 6 weeks, PPFD was reduced to 5.5% (5 mol m−2 s−1 ) of that of the high-light treatment, using black shade cloth. Growth, biomass allocation and non-structural carbohydrate concentrations were assessed before and after transfer to the low-light environment, as well as on control plants. Destructive samplings were carried out at the time of transfer and 3, 6, 15 and 30 days after transfer, in all cases during morning hours. Shoots of all sample plants were cut and kept at 4 ◦ C until processing. Leaves and stems were separated, and leaf
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area determined (Licor LI-3000, Lincoln, Nebraska). They were then immediately freeze-dried. Roots were washed and then also freeze-dried. When plants were transferred to low light, the number and position of their leaves was determined to allow separation of leaves that would grow in low light from those that had grown in high light. Leaves that had attained less than half their final size on the day of transfer were considered as low-light-grown leaves. Destructive sampling and leaf demographical observations involved twelve replicate plants for the lowlight treatment, and six replicate plants for the highlight treatment. Relative growth rates (RGRm , rate of dry mass increase per unit dry weight present; RGRa , rate of leaf area increase per unit leaf area present) and their standard errors were calculated following Venus and Causton (1979). Soluble sugars and starch were determined for the stem and leaf fractions. Equal parts of finely ground freeze-dried tissue of six replicate plants were mixed to obtain one lumped sample for each plant part and species. The tissues were extracted twice with 80% ethanol (30 min at 30 ◦ C) to obtain soluble sugars. The residue was treated for 3 h with 3% HCl (125 ◦ C) to hydrolyse starch. Both fractions were analysed spectrophotometrically using the anthrone method (Hewitt, 1958), and results expressed as glucose equivalents. The sum of soluble sugars and starch is referred to as total non-structural carbohydrates (TNC).
3. Results 3.1. Growth Plants of F. benjamina and F. binnendijkii had attained dry masses of 2.7 and 1.7 g, respectively, at the time of transfer to low light. There were small differences in biomass allocation, with the leaf fraction representing 53 and 62%, the stem fraction 18 and 14%, and the root fraction 29 and 24% of plant mass for F. benjamina and F. binnendijkii, respectively. Mean leaf size for the two species was 10.1 and 8.2 cm2 , respectively. Transfer from the high-light to the low-light environment caused an immediate drop in growth rates (Fig. 1). Patterns in F. benjamina and F. binnendijkii were not significantly different judging from the large standard errors associated with the means (Fig. 1),
Fig. 1. Total plant mass-based (a) and leaf area-based (b) relative growth rates (with standard errors) of two Ficus species in response to a decrease in photosynthetic flux density from 90 to 5 mol m−2 s−1 on day 0. Growth rates at day 0 are averages for the intervals just before and just after that day, for plants grown at 90 mol m−2 s−1 .
but both species showed negative relative growth rates (RGRm ) for several days after transfer, when the plants that stayed in high light presented RGRs of 59 and 54 mg g−1 d−1 , respectively. It can be estimated through interpolation that it took approximately 13 and 11 days for F. benjamina and F. binnendijkii, respectively, to return to their original mass. Only then did plants invest mass in new leaves (Fig. 2). The leaf mass per area (LMA) of these new leaves was almost twice as low as that of high-light-grown leaves: it decreased from 32 to 18 g m−2 in F. benjamina, and from 57 to 30 g m−2 in F. binnendijkii. Relative growth rates of leaf area also fell sharply (Fig. 1b) but neither of the two species showed a net loss of leaf area compared to the values at the time of
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resenting a depletion of 19 and 30%, respectively. Approximately 70–80% of the TNC in the above-ground parts of the plant were located in the leaves, which is a reflection of the fraction of plant biomass they represent.
4. Discussion
Fig. 2. Cumulative mass of new leaves of two Ficus species after a decrease in photosynthetic photon flux density from 90 to 5 mol m−2 s−1 on day 0.
transfer to low light. Thus, some leaf expansion occurred during the period of net loss of plant mass. 3.2. Non-structural carbohydrates Trends in TNC in response to the transfer to a lowlight environment were similar for the two species, both in leaves and stems. Concentrations of both starch and soluble sugars decreased in low light and remained well below the values of high-light plants (Fig. 3a–h). TNC levels in low light were much lower in expanding leaves than in fully grown leaves. Eight days after transfer, however, this difference had disappeared (Fig. 3a–d). Stems, in comparison with leaves, had somewhat lower concentrations of soluble sugars, but considerably higher concentrations of starch (particularly in F. binnendijkii). The dynamics of sugar and starch concentrations were generally similar. In high-light plants, the ratio of soluble sugars to TNC was 0.50 and 0.45 in leaves, and 0.28 and 0.25 in stems of F. benjamina and F. binnendijkii, respectively. These ratios decreased upon transfer to low light, to about 0.30 in leaves (F. benjamina only) and 0.18 in stems (both species) 3 days after transfer. Thereafter, the sugar to TNC ratio gradually returned to its original value in both leaves and stems, although this occurred at a slower pace for F. benjamina. In stems and leaves combined, TNC levels were 142 mg g−1 (plant) in F. benjamina and 160 mg g−1 (plant) in F. binnendijkii before transfer to low light. Thirty days later, in fully acclimated plants, these levels had stabilised at 116 and 112 mg g−1 (Fig. 4), rep-
Both Ficus species were greatly affected by the sudden decrease in PPFD to which they were exposed, but in the end, adjusted well and did not suffer major damage. Initially, growth rates decreased dramatically, and there was even a net loss of mass. While it took close to 2 weeks to recover this lost mass, positive growth rates were achieved approximately 1 week after transfer to low light (Figs. 1 and 2). This period roughly coincided with the time it took for TNC to stabilise in leaf and stem tissues (Fig. 3). Based on these observations, we hypothesise that the likely course of the process of physiological acclimation to low light was: (1) the carbon balance became negative as the light intensity dropped below the photosynthetic light compensation point and respiration continued at high rates typical for high-light environments; (2) requirements for carbon (mainly maintenance, as there is little growth) were largely met by accessing stored carbon; (3) respiration rates decreased and acclimated net photosynthetic rates increased through metabolic adjustments, resulting in a low but positive carbon balance; (4) new, lower TNC levels and growth rates were established. Confirmation of this model will require new experiments that specifically address rates of photosynthesis and respiration. The course of the described process is highly relevant for the health and survival of leaves and the whole plant, and thus for the ability of plants to cope with a sudden decrease in light intensity. The adjustments in carbon metabolism enabled both F. benjamina and F. binnendijkii to retain almost all of their foliage, and these adjustments were very similar in the two species. Leaf shedding was limited to leaves that were still developing at the time of transfer to low light. In these leaves, TNC levels were extremely low (Fig. 3). On the basis of these results we cannot conclude whether abscission was due to the low TNC levels per se or the lack of import of carbohydrates. The effect of low light on plant mass (negative RGRm ) tended to be slightly greater in F. benjamina
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Fig. 3. Concentrations of soluble sugars and starch in leaves and stems of two Ficus species in response to a decrease in photosynthetic photon flux density from 90 to 5 mol m−2 s−1 on day 0; (a)–(d), leaves: (a) and (b), soluble sugars in F. benjamina and F. binnendijkii; (c) and (d), starch in F. benjamina and F. binnendijkii. H: high-light plants; L: low-light plants. Ln depicts leaves that had not fully developed on day 0. (e)–(h), stems: (e) and (f), soluble sugars in F. benjamina and F. binnendijkii; (g) and (h), starch in F. benjamina and F. binnendijkii. H: high-light plants; L: low-light plants.
compared to F. binnendijkii in the first week after transfer, and the effect on leaf area (RGRa ) appeared slightly slower but lasted longer (Fig. 1). Loss of mass was caused by decreasing TNC levels during the first 3 days
after transfer, when the depletion of TNC accounted for all of the decrease in stem and leaf mass. In the subsequent 5 days, TNC depletion had slowed down yet loss of dry mass continued. A limited amount of leaf
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Fig. 4. Total non-structural carbohydrate concentrations in shoots of F. benjamina and F. binnendijkii in response to a decrease in photosynthetic flux density from 90 to 5 mol m−2 s−1 on day 0.
shedding was probably partly responsible for this. Although we did not monitor leaf demography, we did observe loss of some of the developing leaves of F. benjamina after transfer, while this was hardly the case in F. binnendijkii. These were low numbers of small leaves, but may explain some of the plant’s mass loss between days 3 and 8. This explanation is consistent with Fig. 1b, showing the lowest RGRa in F. benjamina around day 6. The fact that TNC levels were extremely low in developing leaves (Fig. 3) and that there was no shedding of fully grown leaves, suggests that low TNC was responsible rather than a systemic signal for abscission. The relatively minor damage to the plants and short duration of a negative carbon balance, despite the low mean PPFD 5 mol m−2 s−1 , illustrates the capacity of both F. benjamina and F. binnendijkii to adjust to and survive in deep shade. The shade tolerance of these species is known from tests of indoor plant quality, however post-harvest quality trials for indoor foliage plants are often carried out at PPFDs of 10–20 mol m−2 s−1 (Reyes et al., 1996a,b). The capacity of our plants to tolerate a PPFD as low as 5 mol m−2 s−1 may be due to the relatively low PPFD at which our plants were grown (90 mol m−2 s−1 ). It has been experimentally shown that plants acclimated to moderate PPFD suffer fewer leaf losses upon storage in the dark or low light than plants that were grown at high PPFD (Conover and Poole, 1984; Ben-Jaacov et al., 1985; Collins and Blessington, 1985). Interestingly, Bulle and De Jongh, (2001) show that an acclimation period that is too short (e.g. 3 days for F. benjamina) actually increases leaf losses.
Our results show that a large fraction of stem and leaf TNC was used in the first week of low light, when photosynthetic carbon gain must have been very low and respiratory carbon loss high. This raises the question if high TNC confers shade tolerance. Although differences between the species were small, F. binnendijkii, in comparison with F. benjamina, had somewhat higher TNC levels at the start of the experiment (Fig. 4), was less affected by low light in its RGR (Fig. 1), and shed fewer developing leaves. The use of TNC as an indicator of shade tolerance is, however, complicated by the fact that plants acclimated to very high light intensity suffer considerably from transfer to low light despite their high TNC content (Milks et al., 1979). This is presumably due to the greater contrast between a plant’s optimal physiological state in very high light intensity compared to shade, one possible factor being the higher carbohydrate requirements of high-light-adapted plants (cf. Noguchi et al., 2001b). In one contrasting report, Fails et al. (1982) placed high-light grown (full sun) and low-light grown (75% shade) F. benjamina at a PPFD of 20 mol m−2 s−1 for twelve weeks, and found greater leaf growth in highlight grown plants, apparently at the expense of root mass, while there was no net growth. However, reexamination of the data shows that leaf growth was not actually greater in these plants when expressed on a total plant or leaf mass basis, and the decrease in root mass may well have been due to a combination of root turnover and local use of TNC. Remobilisation of carbohydrates from roots to leaves seems unlikely, particularly when reasonable amounts of carbohydrates are present in shoots.
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TNC levels in low-light acclimated plants may correlate well with tolerance of deep shade. Reyes et al. (1996a,b) found much higher TNC in the shadetolerant palm Chamaedora elegans than in the shadeintolerant palm Chrysalidocarpus lutescens growing at 20 mol m−2 s−1 . The two species of the present study did not differ in that respect (Fig. 4), which would be in accordance with the observation of similar shade tolerance. Our results show that stored carbohydrates are very important in plants that suffer a temporary carbon crisis. The level of PPFD that our plants experienced is not uncommon in forest understories or indoor environments, where additional abiotic stresses (drought, scarcity of nutrients) may further reduce carbon gain by photosynthesis. Moreover, plants of greater size are likely to suffer more and adjust less readily to low PPFD: with increasing plant size, biomass partitioning shifts to plant parts that are not photosynthetically active, and self-shading increases (Veneklaas and Poorter, 1998). Further, insights in the dynamics of stored carbohydrates and the rate of metabolic and morphological adjustments are required to predict the impact of a sudden decrease in light intensity. Acknowledgements We would like to thank Hans Lambers (Utrecht and Perth) for his support to our project before, during, and long after the experiment; Kees Berg (Bergen) for information on Ficus taxonomy; and Ficus grower De Amstel B.V. (De Kwakel, The Netherlands) for plant material. This study was supported by a grant from the Dutch Technology Foundation (STW). References Ben-Jaacov, J., Ziv, D., Steinitz, B., 1985. Clonal variability in response to light intensity during growth and to subsequent dark storage of Ficus benjamina and Ficus retusa. HortScience 20, 934–936. Bulle, A., De Jongh, M., 2001. Effects of growing conditions on the shelf life of Ficus benjamina. Acta Hortic. 543, 113–117. Chapin III, F.S., Schulze, E.-D., Mooney, H.A., 1990. The ecology and economics of storage in plants. Annu. Rev. Ecol. Syst. 21, 423–447.
Collins, P.C., Blessington, T.M., 1985. Keeping quality of Ficus benjamina as affected by production light levels and postproduction light quality and level. HortScience 20, 390–391. Conover, C.A., Poole, R.T., 1984. Acclimatization of indoor foliage plants. Hortic. Rev. 6, 119–154. Corner, E.J.H., 1965. Check-list of Ficus in Asia and Australasia. The Gardens’ Bulletin, Singapore 21, pp. 1–186. Fails, B.S., Lewis, A.J., Barden, J.A., 1982. Light acclimatization potential of Ficus benjamina. J. Am. Soc. Hortic. Sci. 107, 762– 766. Farrar, J.F., 1989. The carbon balance of fast-growing and slowgrowing species. In: Lambers, H., Cambridge, M.L., Konings, H., Pons, T.L. (Eds.), Causes and Consequences of Variation in Growth Rate and Productivity of Higher Plants. SPB Academic Publishing BV, The Hague, pp. 241–256. Hewitt, B.R., 1958. Spectrophotometric determination of total carbohydrate. Nature 182, 246–247. Kobe, R.K., 1997. Carbohydrate allocation to storage as a basis of interspecific variation in sapling survivorship and growth. Oikos 80, 226–233. Milks, R.R., Joiner, J.N., Garard, L.A., Conover, C.A., Tjia, B., 1979. Influence of acclimatization on carbohydrate production and translocation of Ficus benjamina L. J. Am. Soc. Hortic. Sci. 104, 410–413. Noguchi, K., Go, C.-S., Miyazawa, S.-I., Terashima, I., Ueda, S., Yoshinari, T., 2001a. Costs of protein turnover and carbohydrate export in leaves of sun and shade species. Aust. J. Plant Physiol. 28, 37–47. Noguchi, K., Nakajima, N., Terashima, I., 2001b. Acclimation of leaf respiratory properties in Alocasia odora following reciprocal transfers of plants between high- and low-light environments. Plant Cell Environ. 24, 831–839. Ottosen, C.O., Høyer, L., 1988. Keeping quality of various genotypes of Ficus benjamina after simulated dark shipping and storage indoors. HortScience 23, 586–587. Reyes, T., Nell, T.A., Barrett, J.E., Conover, C.A., 1996a. Irradiance level and fertilizer rate affect acclimatization of Chamaedora elegans Mart. HortScience 31, 839–842. Reyes, T., Nell, T.A., Barrett, J.E., Conover, C.A., 1996b. Testing the light acclimatization of Chrysalidocarpus lutescens Wendl. HortScience 31, 1203–1206. Veneklaas, E.J., Poorter, L., 1998. Growth and carbon partitioning of tropical tree seedlings in contrasting light environments. In: Lambers, H., Poorter, H., Van Vuuren, M.M.I. (Eds.), Inherent Variation in Plant Growth; Physiological Mechanisms and Ecological Consequences. Backhuys Publishers, Leiden, pp. 337– 361. Venus, J.C., Causton, D.R., 1979. Plant growth analysis: a reexamination of the methods of calculation of relative growth and net assimilation rates without using fitted functions. Ann. Bot. 43, 633–638. Whitmore, T.C., 1996. A review of some aspects of tropical rain forest seedling ecology with suggestions for further enquiry. In: Swaine, M.D. (Ed.), The Ecology of Tropical Forest Tree Seedlings, 17. Man and the Biosphere Series, Unesco, Paris, pp. 3–39.
Dynamics of non-structural carbohydrates in two Ficus species after transfer to deep shade Erik J. Veneklaasa,b,∗ , Franka den Oudena b
a Plant Ecophysiology, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
Accepted 25 June 2004
Abstract The present study aims at assessing the role of stored carbohydrates of F. benjamina L. and F. binnendijkii (Miq.) Miq., two tropical trees of horticultural significance, during acclimation to very low light intensities. Plants were grown at an average photosynthetic photon flux density (PPFD) of 90 mol m−2 s−1 , and then transferred to a low-light treatment, with a PPFD of 5 mol m−2 s−1 . Plants of both species showed negative growth rates for approximately 1 week, coinciding with a substantial decrease in the levels of total non-structural carbohydrates (TNC; starch and soluble sugars) in leaves and stems. TNC reached much lower levels in developing leaves than in fully grown leaves, and some of these developing leaves were shed in F. benjamina. In stems and leaves combined, TNC levels were 142 mg g−1 (plant) in F. benjamina and 160 mg g−1 (plant) in F. binnendijkii at the time of transfer to low light. Thirty days later, in fully acclimated plants, these levels had stabilised at 116 and 112 mg g−1 . The relatively minor damage to the plants and short duration of a negative carbon balance, despite the low mean PPFD 5 mol m−2 s−1 , illustrate the capacity of both F. benjamina and F. binnendijkii to tolerate deep shade. Stored carbohydrates are important in plants that grow in environments like forest understories or indoor environments, where carbon balance can be negative for several days. © 2004 Elsevier B.V. All rights reserved. Keywords: Carbon balance; Carbon storage; Growth rate; Acclimation; Ficus benjamina
1. Introduction
∗ Corresponding author at: School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. Tel.: +61 8 6488 3584; fax: +61 8 6488 1108. E-mail address: [email protected] (E.J. Veneklaas).
0098-8472/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2004.06.010
Seedlings and saplings of woody plants in tropical forests generally experience low light intensities, due to the shade cast by taller trees. Most species have therefore developed a tolerance to shade, ranging from highly tolerant understorey specialists to relatively intolerant treefall gap specialists (Whitmore, 1996).
E.J. Veneklaas, F. den Ouden / Environmental and Experimental Botany 54 (2005) 148–154
Light environments of all forest microsites vary, however, either in predictable ways (within a day, between seasons) or in unpredictable ways (due to weather or, often more drastically, due to forest canopy dynamics). Plants respond by adjusting their morphology (e.g. sun versus shade leaves) and/or physiology/biochemistry (e.g. concentrations of chlorophyll and photosynthetic enzymes, respiration rate). The present paper focuses on the adaptive response of two tropical woody species to a decrease in light intensity. Such a decrease happens in nature, for example, when canopies close, but is also highly relevant for ornamental plants that are transferred from greenhouses to indoor environments. In particular, we observed changes in reserve carbohydrates, since we expected them to be a crucial carbon source during the acclimation process, and useful indicators of changes in the plant’s carbon balance. At different levels of photosynthetic photon flux density (PPFD, mol m−2 s−1 ), plants gain different amounts of carbon through photosynthesis, and partition this carbon differently to the construction and maintenance of biomass and several metabolic functions (Veneklaas and Poorter, 1998). A continuous supply of assimilates at high PPFD allows high metabolic rates and high growth rates whilst a pool of reserve carbohydrates can be maintained. A sudden decrease of PPFD causes an immediate carbon balance problem as tissues with high metabolic activity, particularly growing organs, draw on scarce assimilates. In these circumstances, stored carbohydrates are an important, or even the only, source of energy. Once plants have acclimated to shade, they have much lower carbon requirements, due to low growth rates and reduced maintenance costs (Veneklaas and Poorter, 1998; Noguchi et al., 2001a). Carbon is stored in most species as insoluble (starch) and soluble (mainly sucrose) carbohydrates (Chapin et al., 1990). Both fractions are highly dynamic but turnover rates vary widely between species and growing conditions (Farrar, 1989; Noguchi et al., 2001a). Not all stored carbon is available to the plant, yet reserve storage is generally considered to be a buffer for situations when demand exceeds supply (Chapin et al., 1990). Kobe (1997) provides empirical data and a model showing the importance of stored carbohydrates for survival and growth in shade. In the experiment described here we analysed the response of two woody species to a sudden decrease in PPFD in terms of growth and reserve carbohydrates.
149
Both species grow in tropical forests of Southeast Asia (Corner, 1965) but are also widely used as indoor ornamental foliage plants. In a previous experiment (E.J. Veneklaas and F. Den Ouden, unpublished), F. binnendijkii was found to have higher starch concentrations in its stems, and its leaf growth suffered less from a transfer to deep shade than was the case in F. benjamina. We therefore hypothesised that a greater amount of storage carbohydrates confers greater shade tolerance to F. binnendijkii. Throughout the paper we use the term shade tolerance to indicate the ability to respond adaptively to transfer into deep shade, minimising losses (of carbon, or plant parts, e.g. leaves), and adjusting rapidly in order to survive and grow.
2. Materials and methods Plants of F. benjamina L. ‘Exotica’ and F. binnendijkii (Miq.) Miq. ‘Amstel Queen’ were obtained from a commercial grower. After gently removing the potting mix from the root systems, single plants were placed in 5.5 l PVC tubes filled with finegrade perlite. After two weeks of acclimation to the growing conditions, mean plant dry mass was 0.5 g for both species. From that point, plants grew for another 4 weeks until commencement of the experiment. A 2 cm surface layer of gravel was used to minimise drying and disturbance of the perlite. Irrigations were carried out three times a week with 250 ml of nutrient solution containing 2.4 mM KNO3 , 1.8 mM Ca(NO3 )2 , 0.8 mM MgSO4 , 0.6 mM KH2 PO4 and micronutrients. The experiment was carried out in a climatised greenhouse, with temperature set at 23/20 ◦ C (day/night) and air humidity at 70%. Photosynthetic photon flux density averaged 90 mol m−2 s−1 , using natural illumination and supplementary light (photoperiod 17 h). In the low-light treatment, which started after 6 weeks, PPFD was reduced to 5.5% (5 mol m−2 s−1 ) of that of the high-light treatment, using black shade cloth. Growth, biomass allocation and non-structural carbohydrate concentrations were assessed before and after transfer to the low-light environment, as well as on control plants. Destructive samplings were carried out at the time of transfer and 3, 6, 15 and 30 days after transfer, in all cases during morning hours. Shoots of all sample plants were cut and kept at 4 ◦ C until processing. Leaves and stems were separated, and leaf
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area determined (Licor LI-3000, Lincoln, Nebraska). They were then immediately freeze-dried. Roots were washed and then also freeze-dried. When plants were transferred to low light, the number and position of their leaves was determined to allow separation of leaves that would grow in low light from those that had grown in high light. Leaves that had attained less than half their final size on the day of transfer were considered as low-light-grown leaves. Destructive sampling and leaf demographical observations involved twelve replicate plants for the lowlight treatment, and six replicate plants for the highlight treatment. Relative growth rates (RGRm , rate of dry mass increase per unit dry weight present; RGRa , rate of leaf area increase per unit leaf area present) and their standard errors were calculated following Venus and Causton (1979). Soluble sugars and starch were determined for the stem and leaf fractions. Equal parts of finely ground freeze-dried tissue of six replicate plants were mixed to obtain one lumped sample for each plant part and species. The tissues were extracted twice with 80% ethanol (30 min at 30 ◦ C) to obtain soluble sugars. The residue was treated for 3 h with 3% HCl (125 ◦ C) to hydrolyse starch. Both fractions were analysed spectrophotometrically using the anthrone method (Hewitt, 1958), and results expressed as glucose equivalents. The sum of soluble sugars and starch is referred to as total non-structural carbohydrates (TNC).
3. Results 3.1. Growth Plants of F. benjamina and F. binnendijkii had attained dry masses of 2.7 and 1.7 g, respectively, at the time of transfer to low light. There were small differences in biomass allocation, with the leaf fraction representing 53 and 62%, the stem fraction 18 and 14%, and the root fraction 29 and 24% of plant mass for F. benjamina and F. binnendijkii, respectively. Mean leaf size for the two species was 10.1 and 8.2 cm2 , respectively. Transfer from the high-light to the low-light environment caused an immediate drop in growth rates (Fig. 1). Patterns in F. benjamina and F. binnendijkii were not significantly different judging from the large standard errors associated with the means (Fig. 1),
Fig. 1. Total plant mass-based (a) and leaf area-based (b) relative growth rates (with standard errors) of two Ficus species in response to a decrease in photosynthetic flux density from 90 to 5 mol m−2 s−1 on day 0. Growth rates at day 0 are averages for the intervals just before and just after that day, for plants grown at 90 mol m−2 s−1 .
but both species showed negative relative growth rates (RGRm ) for several days after transfer, when the plants that stayed in high light presented RGRs of 59 and 54 mg g−1 d−1 , respectively. It can be estimated through interpolation that it took approximately 13 and 11 days for F. benjamina and F. binnendijkii, respectively, to return to their original mass. Only then did plants invest mass in new leaves (Fig. 2). The leaf mass per area (LMA) of these new leaves was almost twice as low as that of high-light-grown leaves: it decreased from 32 to 18 g m−2 in F. benjamina, and from 57 to 30 g m−2 in F. binnendijkii. Relative growth rates of leaf area also fell sharply (Fig. 1b) but neither of the two species showed a net loss of leaf area compared to the values at the time of
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resenting a depletion of 19 and 30%, respectively. Approximately 70–80% of the TNC in the above-ground parts of the plant were located in the leaves, which is a reflection of the fraction of plant biomass they represent.
4. Discussion
Fig. 2. Cumulative mass of new leaves of two Ficus species after a decrease in photosynthetic photon flux density from 90 to 5 mol m−2 s−1 on day 0.
transfer to low light. Thus, some leaf expansion occurred during the period of net loss of plant mass. 3.2. Non-structural carbohydrates Trends in TNC in response to the transfer to a lowlight environment were similar for the two species, both in leaves and stems. Concentrations of both starch and soluble sugars decreased in low light and remained well below the values of high-light plants (Fig. 3a–h). TNC levels in low light were much lower in expanding leaves than in fully grown leaves. Eight days after transfer, however, this difference had disappeared (Fig. 3a–d). Stems, in comparison with leaves, had somewhat lower concentrations of soluble sugars, but considerably higher concentrations of starch (particularly in F. binnendijkii). The dynamics of sugar and starch concentrations were generally similar. In high-light plants, the ratio of soluble sugars to TNC was 0.50 and 0.45 in leaves, and 0.28 and 0.25 in stems of F. benjamina and F. binnendijkii, respectively. These ratios decreased upon transfer to low light, to about 0.30 in leaves (F. benjamina only) and 0.18 in stems (both species) 3 days after transfer. Thereafter, the sugar to TNC ratio gradually returned to its original value in both leaves and stems, although this occurred at a slower pace for F. benjamina. In stems and leaves combined, TNC levels were 142 mg g−1 (plant) in F. benjamina and 160 mg g−1 (plant) in F. binnendijkii before transfer to low light. Thirty days later, in fully acclimated plants, these levels had stabilised at 116 and 112 mg g−1 (Fig. 4), rep-
Both Ficus species were greatly affected by the sudden decrease in PPFD to which they were exposed, but in the end, adjusted well and did not suffer major damage. Initially, growth rates decreased dramatically, and there was even a net loss of mass. While it took close to 2 weeks to recover this lost mass, positive growth rates were achieved approximately 1 week after transfer to low light (Figs. 1 and 2). This period roughly coincided with the time it took for TNC to stabilise in leaf and stem tissues (Fig. 3). Based on these observations, we hypothesise that the likely course of the process of physiological acclimation to low light was: (1) the carbon balance became negative as the light intensity dropped below the photosynthetic light compensation point and respiration continued at high rates typical for high-light environments; (2) requirements for carbon (mainly maintenance, as there is little growth) were largely met by accessing stored carbon; (3) respiration rates decreased and acclimated net photosynthetic rates increased through metabolic adjustments, resulting in a low but positive carbon balance; (4) new, lower TNC levels and growth rates were established. Confirmation of this model will require new experiments that specifically address rates of photosynthesis and respiration. The course of the described process is highly relevant for the health and survival of leaves and the whole plant, and thus for the ability of plants to cope with a sudden decrease in light intensity. The adjustments in carbon metabolism enabled both F. benjamina and F. binnendijkii to retain almost all of their foliage, and these adjustments were very similar in the two species. Leaf shedding was limited to leaves that were still developing at the time of transfer to low light. In these leaves, TNC levels were extremely low (Fig. 3). On the basis of these results we cannot conclude whether abscission was due to the low TNC levels per se or the lack of import of carbohydrates. The effect of low light on plant mass (negative RGRm ) tended to be slightly greater in F. benjamina
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Fig. 3. Concentrations of soluble sugars and starch in leaves and stems of two Ficus species in response to a decrease in photosynthetic photon flux density from 90 to 5 mol m−2 s−1 on day 0; (a)–(d), leaves: (a) and (b), soluble sugars in F. benjamina and F. binnendijkii; (c) and (d), starch in F. benjamina and F. binnendijkii. H: high-light plants; L: low-light plants. Ln depicts leaves that had not fully developed on day 0. (e)–(h), stems: (e) and (f), soluble sugars in F. benjamina and F. binnendijkii; (g) and (h), starch in F. benjamina and F. binnendijkii. H: high-light plants; L: low-light plants.
compared to F. binnendijkii in the first week after transfer, and the effect on leaf area (RGRa ) appeared slightly slower but lasted longer (Fig. 1). Loss of mass was caused by decreasing TNC levels during the first 3 days
after transfer, when the depletion of TNC accounted for all of the decrease in stem and leaf mass. In the subsequent 5 days, TNC depletion had slowed down yet loss of dry mass continued. A limited amount of leaf
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Fig. 4. Total non-structural carbohydrate concentrations in shoots of F. benjamina and F. binnendijkii in response to a decrease in photosynthetic flux density from 90 to 5 mol m−2 s−1 on day 0.
shedding was probably partly responsible for this. Although we did not monitor leaf demography, we did observe loss of some of the developing leaves of F. benjamina after transfer, while this was hardly the case in F. binnendijkii. These were low numbers of small leaves, but may explain some of the plant’s mass loss between days 3 and 8. This explanation is consistent with Fig. 1b, showing the lowest RGRa in F. benjamina around day 6. The fact that TNC levels were extremely low in developing leaves (Fig. 3) and that there was no shedding of fully grown leaves, suggests that low TNC was responsible rather than a systemic signal for abscission. The relatively minor damage to the plants and short duration of a negative carbon balance, despite the low mean PPFD 5 mol m−2 s−1 , illustrates the capacity of both F. benjamina and F. binnendijkii to adjust to and survive in deep shade. The shade tolerance of these species is known from tests of indoor plant quality, however post-harvest quality trials for indoor foliage plants are often carried out at PPFDs of 10–20 mol m−2 s−1 (Reyes et al., 1996a,b). The capacity of our plants to tolerate a PPFD as low as 5 mol m−2 s−1 may be due to the relatively low PPFD at which our plants were grown (90 mol m−2 s−1 ). It has been experimentally shown that plants acclimated to moderate PPFD suffer fewer leaf losses upon storage in the dark or low light than plants that were grown at high PPFD (Conover and Poole, 1984; Ben-Jaacov et al., 1985; Collins and Blessington, 1985). Interestingly, Bulle and De Jongh, (2001) show that an acclimation period that is too short (e.g. 3 days for F. benjamina) actually increases leaf losses.
Our results show that a large fraction of stem and leaf TNC was used in the first week of low light, when photosynthetic carbon gain must have been very low and respiratory carbon loss high. This raises the question if high TNC confers shade tolerance. Although differences between the species were small, F. binnendijkii, in comparison with F. benjamina, had somewhat higher TNC levels at the start of the experiment (Fig. 4), was less affected by low light in its RGR (Fig. 1), and shed fewer developing leaves. The use of TNC as an indicator of shade tolerance is, however, complicated by the fact that plants acclimated to very high light intensity suffer considerably from transfer to low light despite their high TNC content (Milks et al., 1979). This is presumably due to the greater contrast between a plant’s optimal physiological state in very high light intensity compared to shade, one possible factor being the higher carbohydrate requirements of high-light-adapted plants (cf. Noguchi et al., 2001b). In one contrasting report, Fails et al. (1982) placed high-light grown (full sun) and low-light grown (75% shade) F. benjamina at a PPFD of 20 mol m−2 s−1 for twelve weeks, and found greater leaf growth in highlight grown plants, apparently at the expense of root mass, while there was no net growth. However, reexamination of the data shows that leaf growth was not actually greater in these plants when expressed on a total plant or leaf mass basis, and the decrease in root mass may well have been due to a combination of root turnover and local use of TNC. Remobilisation of carbohydrates from roots to leaves seems unlikely, particularly when reasonable amounts of carbohydrates are present in shoots.
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TNC levels in low-light acclimated plants may correlate well with tolerance of deep shade. Reyes et al. (1996a,b) found much higher TNC in the shadetolerant palm Chamaedora elegans than in the shadeintolerant palm Chrysalidocarpus lutescens growing at 20 mol m−2 s−1 . The two species of the present study did not differ in that respect (Fig. 4), which would be in accordance with the observation of similar shade tolerance. Our results show that stored carbohydrates are very important in plants that suffer a temporary carbon crisis. The level of PPFD that our plants experienced is not uncommon in forest understories or indoor environments, where additional abiotic stresses (drought, scarcity of nutrients) may further reduce carbon gain by photosynthesis. Moreover, plants of greater size are likely to suffer more and adjust less readily to low PPFD: with increasing plant size, biomass partitioning shifts to plant parts that are not photosynthetically active, and self-shading increases (Veneklaas and Poorter, 1998). Further, insights in the dynamics of stored carbohydrates and the rate of metabolic and morphological adjustments are required to predict the impact of a sudden decrease in light intensity. Acknowledgements We would like to thank Hans Lambers (Utrecht and Perth) for his support to our project before, during, and long after the experiment; Kees Berg (Bergen) for information on Ficus taxonomy; and Ficus grower De Amstel B.V. (De Kwakel, The Netherlands) for plant material. This study was supported by a grant from the Dutch Technology Foundation (STW). References Ben-Jaacov, J., Ziv, D., Steinitz, B., 1985. Clonal variability in response to light intensity during growth and to subsequent dark storage of Ficus benjamina and Ficus retusa. HortScience 20, 934–936. Bulle, A., De Jongh, M., 2001. Effects of growing conditions on the shelf life of Ficus benjamina. Acta Hortic. 543, 113–117. Chapin III, F.S., Schulze, E.-D., Mooney, H.A., 1990. The ecology and economics of storage in plants. Annu. Rev. Ecol. Syst. 21, 423–447.
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