Effects of photoperiod and drought on the induction of crassulacean ...

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Effects of photoperiod and drought on the induction of crassulacean acid metabolism and the reproduction of plants of Talinum triangulare A. Herrera

Abstract: To examine the effects of day length on the induction of crassulacean acid metabolism (CAM) by drought in the tropical species, Talinum triangulare (Jacq.) Willd. (Portulacaceae), plants were subjected to drought under different photoperiods. Nocturnal acid accumulation was 52 µmol H+··g–1 fresh mass (FM) in plants grown under a 10 h light : 14 h dark photoperiod and 76 µmol H+·g–1 FM in plants grown under 13 h light : 11 h dark, whereas it was only 10 µmol H+·g–1 FM in plants grown under 18 h light : 6 h dark. Plants were subjected to drought under short days and under short days with a night interruption of 1.5 h white light, aiming to simulate a long day, while minimally affecting daily carbon balance. Only droughted plants under normal short days accumulated acids during the night. Absence of CAM could not be attributed to differences due to photoperiod in either biomass allocation, chlorophyll content, or leaf water content. Photoperiod did not significantly affect fecundity in watered plants, whereas drought markedly reduced fecundity in plants with night interruption relative to plants under normal short days. Reproductive effort, calculated as seeds per gram leaf, was significantly higher in droughted plants under normal short days and watered plants with and without night interruption than in droughted plants with night interruption. Key words: CAM, crassulacean acid metabolism, drought, fecundity, induction, photoperiod, reproductive effort, reproduction, Talinum triangulare Résumé : Afin d’examiner les effets de la longeur du jour sur l’induction du métabolisme acide crassulacéen par la sécheresse, chez l’espèce tropicale Talinum triangulare (Jacq.) Willd. (Portulacaceae), l’auteur à soumis des plantes à la sécheresse sous différentes photopériodes. L’accumulation nocturne d’acide est de 52 µmol H+·g–1 FM chez les plantes soumises à une photopériode de 10 h lumière (L) : 14 h ombrage (O) et de 76 µmol H+·g–1 FM chez les plantes soumises à 13 h L : 11 h O, alors qu’elle n’est que de 10 µmol avec 18 h L : 6 h O. Des plantes ont été soumises à la sécheresse sous des jours courts et des jours courts avec une interruption nocturne de 1,5 h de lumière blanche, dans le but de simuler un jour long tout en affectant au minimum la balance carbonée quotidienne. Seulement les plantes soumises à la sécheresse sous des jours courts normaux ont accumulé de l’acide pendant la nuit. L’absence de CAM ne peut pas être attribuée aux différences liées à la photopérode, que ce soit dans l’allocation de la biomasse, la teneur en chlorophylle ou le contenu en eau des feuilles. La photopériode n’affecte pas significativement la fécondité chez les plantes irriguées, alors que la sécheresse la réduit chez les plantes avec interruption nocturne, comparativement aux plantes soumises aux jours courts normaux. L’effort reproductif, calculé en termes de graines/g de feuille, est significativement plus élevé chez les plantes soumises à la sécheresse sous des jours courts normaux, et les plantes irriguées avec ou sans interruption nocturne, que chez les plantes asséchées avec interruption nocturne. Mots clés : CAM, métabolisme acide crassulacéen, sécheresse, fécondité, induction, photopériode, effort reproductif, reproduction, Talinum triangulare. [Traduit par la Rédaction]

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Introduction Crassulacean acid metabolism (CAM) is currently regarded as an adaptive response to water deficit (Kluge and Ting 1978). In inducible-CAM species, such as Mesembryanthemum crystallinum L. (Winter 1985) and Talinum triangulare (Jacq.) Willd. (Herrera et al. 1991), most of the carbon is fixed by the C3 pathway; therefore, the adaptive advantages of CAM to stress are not obvious. Inducible Received August 2, 1998. A. Herrera. Instituto de Biología Experimental, Centro de Botánica Tropical, Instituto de Biología Experimental, Universidad Central de Venezuela, Apartado 47577, Caracas 1041A, Venezuela. e-mail: [email protected] Can. J. Bot. 77: 404–409 (1999)

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CAM has been proposed as a mechanism for extending the life cycle into the dry season and providing additional resources for reproduction (Winter 1985). Relative fitness is the capacity of a genotype to survive and reproduce in relation to other genotypes (Futuyma 1986). So far, no study has been published in which the effect of the operation of CAM on relative fitness has been shown. To demonstrate that CAM is adaptive, a measure of fitness should be made in CAM plants in which this metabolism is not in operation and compared with plants performing CAM; it should thus be possible to infer the effects of the operation of CAM on one or both of the components of relative fitness (fecundity and survival; Pianka 1974). The importance of day length on the induction of CAM has been recognized in several species. Plants of the tropical © 1999 NRC Canada


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species, Kalanchoë blossfeldiana Poelln., perform CAM only during short days (Brulfert et al. 1988). In Portulacaria afra (L.) Jacq. (subtropical), long days are more inductive of CAM in watered plants, whereas droughted plants have a typical CAM pattern regardless of photoperiod (Guralnick et al. 1984). A nocturnal interruption with a few minutes of red light inhibits CAM in K. blossfeldiana (Queiroz and Brulfert 1982). Plants that grow in low-latitude ecosystems are not expected to exhibit morphogenetic responses to photoperiod. Only a few studies have shown an effect of day length on flowering of tropical species (Clements 1968; Njoku 1959). In growth-cabinet, greenhouse, and field studies on the induction of CAM in T. triangulare (Portulacaceae), a pantropical, perennial, deciduous herb, day length varied between 11 h 45 min and 12 h 30 min. Under controlled conditions a change in day length between 10 and 18 h does not affect flowering in T. triangulare (Njoku 1958), but the effect of photoperiod on CAM in this, or any tropical inducible-CAM species, has not been reported. The induction of CAM and of flowering and fruiting in M. crystallinum and Mesembryanthemum nodiflorum L. in their natural habitat occurs toward the middle to the end of summer (Winter 1985; Sayed and Hegazy 1994) under photoperiods of 13–14 h light : 10–11 h dark. In plants of M. nodiflorum the relationship between CAM induction, seen as an increase in nocturnal acid accumulation, and reproductive output during summer was interpreted as a strategy to delay resource allocation to reproductive biomass and to maximize reproductive yield before the end of the lifespan (Sayed and Hegazy 1994). Greenhouse-grown plants of T. triangulare produce more seeds under drought than under watering, and age-dependent fecundity increases with drought, with age-dependent survival being the same for both treatments; a significant and positive correlation between nocturnal acid accumulation and reproductive effort was also found (Taisma and Herrera 1998). The hypothesis of the present work is that changes in photoperiod affect the induction of CAM by drought in plants of T. triangulare and that the occurrence of CAM increases fecundity, relative to plants under drought without CAM. Changes in nocturnal acid accumulation (∆H+), fecundity, and reproductive effort of plants of T. triangulare subjected to water deficit were followed under different photoperiods.

Materials and methods Experiment I Plants of T. triangulare (Portulacaceae), grown from seeds collected in Venezuela, were maintained from April to June in the greenhouse of the Botanic Gardens of the University of Cambridge, Cambridge, U.K. (photoperiod 15–16 h light : 8–9 h dark) in 1.5-L pots filled with organic soil. Fourteen days before the beginning of the water-deficit treatment, a group of plants was placed in a controlled-environment growth room with a light period of 13 h (SD, short day), and a second group in a similar growth room with a light period of 10 h (VSD, very short day); a third group was left in the greenhouse under a photoperiod of 18 h (LD, long day), since only two growth rooms were available. Illumination in the growth rooms was provided by one mercury vapour lamp (HQI-E400W/DV, Urban Enviroscape Ltd., UK) providing light with a spectrum similar to daylight (red : far red ratio - 1.4) placed

405 50 cm above the plants. Illumination in the greenhouse was supplemented with sodium halide lamps (Wotan, Phillips, the Netherlands) during the last hours of the day. Photosynthetic photon flux density (PPFD) in the different photoperiodic regimes was 600 ± 80 µmol·m–2·s–1 in the VSD and SD treatments (average of the illuminated area), and 300–1500 µmol·m–2·s–1 in the LD treatment. Ten plants in each photoperiod were watered daily (control); another 10 were subjected to drought.

Experiment II Young plants collected in Venezuela were planted in 8-L pots filled with organic soil and grown in the gardens of the Instituto de Biología Experimental in Caracas for 3 months with commercial fertilizer (N–P–K 15:15:15) and daily watering. When plants reached an average height of 50 cm, they were placed in two adjacent greenhouses in Caracas with natural illumination and similar microclimatic conditions. In one of the greenhouses, seven watered controls and seven droughted plants were illuminated from 00:00 to 01:30 with a PPFD of 200 µmol·m–2·s–1 provided by 100-W incandescent and fluorescent lamps (General Electric, Caracas, Venezuela) to guarantee a red : far red ratio similar to daylight (Downs 1979; McCree 1979). This treatment with a night interruption will be designated as NI. Another seven control and seven droughted plants were placed in the other greenhouse without night interruption (SD). Air temperature (T) was measured with thermistors connected to a model YSI 46 telethermometer (Yellow Springs Instruments Co., Yellow Springs, Ohio). PPFD was measured with an LI-190S quantum sensor connected to an LI-185 meter (LI-COR Instruments, Lincoln, Nebr.). Relative humidity (RH) was measured with built-in hygrothermographs in the growth rooms and the greenhouse (experiment I) or with a hygrothermograph (Weather Measure Corp., Sacramento, Calif.) in the greenhouses (experiment II).

Measurements The magnitude of CAM was assessed through the determination of nocturnal acid accumulation (∆H+) in expanded leaves from the third and fourth nodes of different plants. The leaves (n = 6) were harvested at the end and at the beginning of the light period and frozen at –20°C until processing. Leaves were ground with mortar and pestle with deionized, distilled water; the macerate was boiled for 1 min and titrated with 1 mM KOH to pH 7.0 using a pH meter (model PHM61, Radiometer, Copenhagen, Denmark) after Nobel (1988). Chlorophyll content was measured in a segment of the leaf taken at lights-off for the determination of ∆H+; the segment was immersed in 3 mL of dimethyl sulphoxide and placed at 55°C for approximately 8 h; absorbance measurements were taken in the solution (Hiscox and Israelstam 1979). Leaf water content (LWC) was determined in segments of the same leaf collected at lights-off for the determination of ∆H+. Fresh and dry (60°C for 3 days) masses were determined. Leaf total and segment area were calculated with empirical equations determined for length and width in watered plants at the beginning of each experiment. Previous determinations (unpublished) showed that expanded leaf area does not change with drought. Carbon isotope discrimination (δ13C) was determined in triplicate in ground dried leaves after Ehleringer and Osmond (1989). Leaf CO2 exchange (A) was measured in one leaf from three different plants using an LCA2 infrared gas analyzer connected to a PLC(B) assimilation chamber and an ASU(MF) air pump (The Analytical Development Co., Ltd., Hoddesdon, U.K.). Mass of plant organs is expressed as a percentage of total dry mass (60°C for a week). Mean fecundity (F) was calculated as F = (mean seed number/fruit) × (fruit number/plant) with values of number of fruits per plant (four plants per treatment) and seeds per fruit (10 fruits from each 4 plants) counted at the end of experi© 1999 NRC Canada



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Can. J. Bot. Vol. 77, 1999 Table 1. Values of microclimatic parameters measured in the photoperiodic treatments of experiments I and II. Air T (°C)

RH (%)

Photoperiod

Day length (h)

Maximum

Minimum

Minimum

Maximum

VSD SD (I) LD SD (II) NI

10 13 18 12 12

29.2±0.2 31.0±0.0 31.3±0.7 31.9±1.5 32.7±1.2

23.2±0.2 23.0±0.0 24.5±0.3 18.7±0.4 18.7±0.4

50.0±4.5 43.0±0.5 39.0±3.0 41.0±4.2 41.0±4.2

69.0±2.4 83.0±1.5 73.0±0.7 84.0±3.5 84.0±3.5

Note: Values are mean ± SE (n ≥ 3) of daily courses.

ment II (24 days). Reproductive effort (RE), considered a satisfactory surrogate measure of relative fitness (Travis 1992), was calculated as total number of seeds per gram leaf dry mass. Results, presented as means ± SE, were analyzed through oneway ANOVA. Differences were considered significant when p ≤ 0.05.

Fig. 1. Changes with length of drought in nocturnal acid accumulation of watered (open symbols) and droughted (solid symbols) plants grown under the photoperiods of experiment I. VSD, very short days; SD, short days; LD, long days. Values are means ± SE (n = 6).

Results When mean daily courses of microclimatic parameters were compared between growth conditions, significant differences were found in minimum and maximum RH among all treatments and in minimum night air T between photoperiods of experiments I and II (Table 1). It is important to note that no significant differences were found in either maximum and minimum RH or air T between greenhouses in which experiments SD (II) and NI were done. Differences in ∆ H+ during drought between watered and droughted plants and among photoperiods were found in the course of experiment I (Fig. 1). Nocturnal acid accumulation increased with drought on days 11 and 6 in VSD and SD plants, respectively, but not in LD plants. Watered plants in the three photoperiods showed no ∆ H+. Under LD, ∆ H+ of droughted plants did not differ significantly from watered plants at any time of the dry cycle. Although not quantified, differences in reproduction were not observed among photoperiods, with plants from all three treatments producing flowers and fruits during the experiment. In experiment II, droughted SD plants had a higher ∆H+ than droughted NI, watered NI, and watered SD plants, in all of which ∆ H+ was not statistically different from zero (Fig. 2). Since significant differences among growth conditions may cause significant differences in growth parameters, such as biomass allocation patterns, chlorophyll content, and LWC, comparisons of growth parameters among photoperiods and the relationship between changes in parameters and the maximum value of ∆ H+ were done. Significant changes in proportional biomass allocation to organs of watered plants were found among photoperiods. NI plants allocated more biomass to stems and less to roots than plants of the other photoperiods, and SD (I and II) plants had lower percent biomass allocated to roots than LD plants; no significant differences were detected in percent biomass of leaves among photoperiods (Fig. 3A; VSD plants were not harvested). Total chlorophyll content did not differ significantly among photoperiods in watered plants; it decreased significantly with drought only in VSD plants (Fig. 3B). Differences in LWC among photoperiods were found ( p = 0.022), and LWC decreased significantly with drought relative to

watering in all photoperiods except for SD II (Fig. 3C). Changes in ∆ H+ did not apparently follow changes in either the pattern of biomass allocation, chlorophyll content of watered or droughted plants, or LWC of watered or droughted plants. No significant differences between values of δ13C in droughted plants occurred; values were: SD (experiment I), –26.22 ± 0.36‰, and LD, –25.57 ± 0.19‰ ( p = 0.120). Measurements of CO2 exchange rates in experiment II made between 00:00 and 01:30 of day 20 of drought showed no assimilation during that period in any of the treatments; daily carbon balance was similar in SD and NI watered © 1999 NRC Canada



Herrera Fig. 2. Changes with length of drought in nocturnal acid accumulation of watered (open symbols) and droughted (solid symbols) plants under NI (night interruption, circles) and SD (short days, squares) photoperiods of experiment II. Values are means ± SE (n = 3).

407 Fig. 3. Correlation of nocturnal acid accumulation with (A) proportional biomass allocation to leaves (solid circles), stems (open circles), and roots (open triangles) of watered plants harvested at the end of the experiments; (B) chlorophyll content; and (C) leaf water content of droughted plants (solid symbols). The corresponding control values (open symbols) are included for the time when each ∆H+ was measured in droughted plants. Photoperiods under which each value of ∆H+ plotted was measured are indicated on the top abscissa. Values are means ± SE (n = 6).

plants (Fig. 4). No dark CO2 fixation was detected in droughted plants of either photoperiod since plants had been under drought for a period longer than the necessary to observe it before plants enter idling (i.e., approximately 9 days; Herrera et al. 1991). In experiment II no statistically significant differences in F of watered plants were found among photoperiods ( p = 0.073), whereas F was significantly reduced in droughted NI plants relative to droughted SD plants ( p = 0.019) (Fig. 5A). Reproductive effort was not significantly different between watered SD and NI plants ( p = 0.275), whereas it increased significantly in droughted SD plants relative to droughted NI plants ( p = 0.003); it was higher in droughted SD plants than in watered SD, in watered NI, or in droughted NI plants (Fig. 5B). Germination tests carried out with seeds obtained from fruits collected at the final harvest showed that those with a black testa were not dormant, whereas those with a brown-red testa did not germinate. The number of black seeds per plant was significantly higher in droughted SD than in droughted NI plants ( p = 0.0259; Fig. 5C).

Discussion In the present study, evidence for an effect of day length on the induction of CAM by drought in a tropical inducible species is presented. Under short days, watered plants of T. triangulare did not exhibit nocturnal accumulation of H+, whereas plants subjected to drought exhibited a peak in H+ accumulation a few days after the beginning of drought and a low value after prolonged drought; these results are in agreement with those reported previously (Herrera et al. 1991). In contrast, CAM was not induced by drought in plants maintained under long days for the same length of time. Simulating a long day by illuminating SD plants in the middle of the night effectively inhibited nocturnal acid accumulation in the absence of CO2 assimilation during the night

interruption, thus giving a similar carbon balance in NI and SD plants. Even though growth conditions differed in microclimatic parameters, day length was apparently the sole effector of changes in CAM induction by drought. Effects of photoperiod on either biomass allocation, chlorophyll content (as a measure of assimilatory capacity), or LWC did not allow any relationship with CAM induction to be established. Changes in ∆H+ could not be attributed to changes in thermoperiod among photoperiods. Differences in night RH and in PPFD among photoperiods are not likely causes for differences in the induction of CAM, since high values of © 1999 NRC Canada



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Can. J. Bot. Vol. 77, 1999

Fig. 4. Changes in net CO2 exchange after 20 days of treatment in watered (open symbols) and droughted (solid symbols) plants under NI (circles) and SD (squares) photoperiods of experiment II. The solid bar on the abscissa indicates the length of the dark period. The inserted open empty bar shows the length of the night interruption. Values are means ± SE (n = 3).

Fig. 5. Changes with photoperiod in plants subjected during 24 days to watering (open bars) or drought (solid bars) in experiment II in (A) mean fecundity (B) reproductive effort, and (C) number of black seeds per plant. Values are means ± SE (n = 4). An asterisk on solid bars indicates significant differences in droughted plants between photoperiods.

∆ H+ have been measured in T. triangulare within a wide range of RH under field, greenhouse, and growth-chamber conditions and under full sun exposure as well as shade (Herrera et al. 1991). These results suggest that day length alone, rather than other enviromental factors, governs CAM induction by drought in T. triangulare and allow comparisons of ∆ H+ between growth regimes of different photoperiods and different microclimatic conditions to be made. The absence of differences in δ13C between LD and SD plants suggests that the contribution of dark CO2 uptake to carbon balance in leaves of this species is very small. In plants grown under short days and subjected to drought, dark CO2 fixation at the peak of CAM activity represents only 7% of the net carbon gain of plants in the C3 mode (calculated from Herrera et al. 1991). Since the night interruption did not inhibit either flowering or fruit production by plants of T. triangulare, it was possible to compare the fecundity of plants under drought with and without CAM. CAM induced by drought and short days was related to an increase in fecundity, since both F and RE were higher in droughted SD than in droughted NI plants, with no effect of photoperiod per se in watered plants. Besides, droughted SD plants produced more black (nondormant) seeds than droughted NI plants. Relative fitness of individuals would therefore increase because of an increase in fecundity. This would suggest that CAM constitutes an adaptation to drought in T. triangulare. Plants of M. crystallinum with CAM induced and exposed to air with CO2 during the night produce more flowers and seeds than those exposed to CO2-free air during the night, which have lower nocturnal malate accumulation (Winter and Ziegler 1992), suggesting that CAM increases the reproductive component of fitness in this species. Therefore, droughtinducible CAM in T. triangulare may be considered an adaptation to water stress.

The cost–benefit relationship of this adaptation in T. triangulare is probably low. The cost could be low since (i) the induction of the CAM by drought in T. triangulare is rapid, and thus, a large portion of the enzyme complement can already be present in plants in the C3 mode; (ii) the additional cost of performing CAM (approximately 10%; Winter and Smith 1996) is small relative to the cost of C3 fixation, already low in droughted plants; and (iii) leaves of T. triangulare have a high specific leaf area and turnover rate and may have a comparatively low cost of construction (Medina 1981). This may be particularly relevant in a perennial deciduous species. Thus, the advantages of CAM would not be as evident in carbon gain, as in its side effects on fecundity. Benefits of this adaptation are significant increases in F and RE (more seeds for same leaf mass). Germination of seeds from droughted plants was higher than in seeds of watered plants, which apparently contain an inhibitor of germination that must be leached to reach the same percent germination as in droughted plants (Taisma and Herrera 1998). A higher F, RE, and germination could imply an adaptive advantage for recruitment in CAM plants at the beginning of the rainy season. © 1999 NRC Canada



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Abscisic acid (ABA), the concentration of which increases with water deficit (Mansfield and McAinsh 1995), may affect both CAM induction (Chu et al. 1990) and seed development before maturation (Bewley 1997). However, ABA also increases with drought in non-CAM plants, and therefore, ABA concentration may not be the causal link between CAM and seed production. Fruits of M. crystallinum have a higher δ13C value than leaves, indicating that their mass increases mainly by carbon fixed through CAM (Winter 1985). These results, together with those from the present work, demonstrate the occurrence of a more favourable carbon balance for seed production during CAM operation.

Acknowledgements This work was financed by project Nos. CDCH 03.005.92, CDCH 03.10.3173.94, and CONICIT S1-1754 (Venezuela). The Department of Botany of the University of Cambridge provided growth facilities, technical assistance, and equipment for experiment I. P. Freeman and A.L. Friend cooperated at several stages. The British Council in Caracas partly financed a sabbatical leave for the author at the University of Cambridge. Determinations of δ13C were kindly made by M. Morecroft and M.A. Hall, of the University of Cambridge, with funds provided by F.I. Woodward. I thank E. Montes and A. Mendoza for their help during sampling. M.D. Fernández helped continually with critical observations, suggestions, and practical work. Thanks are due to R. Urich, L. Bulla, W. Tezara, and three anonymous referees for their critical reading of the manuscript.

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