Comparative water-use efficiencies of three species of Peperomia (piperaceae) having different photosynthetic pathways

j. Plant PhyswL VOL 150. pp. 259-263 (1997)

Comparative Water-Use Efficiencies of Three Species of Peperomia (Piperaceae) Having Different Photosynthetic Pathways BRENT

R.

HELLIKER

1

and CRAIG E.

MARTIN

2

Department of Botany, University of kansas, Lawrence, Kansas 66045-2106, USA Received March 7, 1996 . Accepted May 15,1996

Summary

Water-use efficiencies (WUE) were compared among three species of P~eromia (Piperaceae) selected for their diversity in photosynthetic pathways. P~eromia obtusifolia is a C 3 plant that exhibits a small degree of CAM-cycling; P. camptotricha is a C 3-CAM intermediate, having relatively high rates of CO 2 uptake during both the day and the night; and P. scandens is a CAM plant. All three congeners have succulent leaves and share a similar ecological niche; they typically grow as epiphytes (occasionally as terrestrials) in tropical and subtropical forests. Thus, these species of Peperomia provide an ideal set of truly comparable plants to test the long-held, but recently challenged, generalization that the WUE of CAM plants is substantially greater than that of non-CAM species. Although daytime values of WUE were lower in P. scandens than in P. obtusifolia and P. camptotricha, values of nighttime WUE and 24-hour WUE for the CAM species greatly exceeded corresponding values for the other two Peperomia species. Thus, the results of this study support past generalizations that a high WUE is a consequence of the CAM photosynthetic pathway.

Key words: P~eromia camptotricha, P~eromia obtusifolia, P~eromia scandens, C3 -CAM, CO2 exchange, Crassulacean acid metabolism, transpiration, water-use efficiency. Abbreviations: CAM : : : Crassulacean acid metabolism; PPF : : : photosynthetic photon flux; vpd : : : vapor pressure deficit; WUE : : : water-use efficiency. Introduction

As a result of daytime stomatal closure and the restriction of stomatal opening and consequent gas exchange to the nighttime, water losses due to transpiration are often substantially less in plants with Crassulacean acid metabolism (CAM) than in their C 3 and C 4 counterparts (Kluge and Ting, 1978; Winter, 1985). This savings in water loss is typically so great that it offsets the low CO 2 uptake rates characteristic of CAM plants, such that their water-use efficiencies (WOE, the amount of carbon gained per amount of water I Current address: Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA. 2 Correspondence. @

1997 by Gwtav Fischer Verlag, Jena

lost) greatly exceed those of non-CAM plants. Thus, this high WUE is considered to be the chief advantage of CAM for plants growing in arid regions. In fact, it is generally considered that water conservation is the raison d'hrt for the evolution of CAM in higher plants (Kluge and Ting, 1978; Osmond, 1978). Although there can be no doubt that the WUE of most CAM plants greatly exceeds WUE values for most C 3 and C4 plants (Salisbury and Ross, 1992; Lareber, 1995), the nature of such comparisons has recently been challenged by results of several studies. In most reviews of CAM (e.g., Kluge and Ting, 1978; Winter, 1985), values of WUE represent ranges of values obtained from numerous species, with an emphasis

260

BRENT R HELLIKER and CRAIG E. MARTIN

on well-studied taxa. As a result, plants of widely dissimilar phylogenies, morphologies, and ecological niches are compared (see, for example, Larcher, 1995). Although the difference in photosynthetic pathway will undoubtedly influence rates of water loss and, hence, values ofWUE, morphological and ecological differences can also have substantial impacts on rates of gas exchange. For example, most terrestrial CAM plants are succulent (Kluge and Ting, 1978), whereas most C 3 and C4 taxa are not. Furthermore, the majority of terrestrial CAM plants grow in extremely arid environments and exhibit numerous adaptations that minimize water loss. Many of the non-CAM species included in comparisons ofWUE values are crop plants that do not grow in arid environments and, therefore, lack many morphological adaptations that reduce rates of water loss. Thus, it is possible, at least in some cases, that the higher WUE normally attributed to the restriction of stomatal opening to the night in CAM plants might also reflect radical differences in morphology and adaptation to habitat. There have been surprisingly few comparisons of water conservation in CAM and non-CAM plants that utilize phylogenetically related and morphologically similar plants, as well as plants that grow in comparable environments. Surveys of the water relations and gas exchange characteristics of CAM and C 3 epiphytic bromeliads in situ in Trinidad revealed higher values of WUE in the CAM plants relative to the C 3 taxa, although differences were small and not entirely consistent (Griffiths et al., 1986; Smith et al., 1986). On the other hand, the C 3 species tended to grow in moister habitats, the C 3 and CAM species were in different genera, and there were often morphological differences between the epiphytes with different photosynthetic pathways. In a study of terrestrial plants with the same leaf-succulent morphology and in the same genus in the Crassulaceae, Gravatt and Martin (1992) found only slight differences in WUE among species of Sedum with CAM, CAM-cycling (see Ting, 1985 and Martin, 1996), and C 3 photosynthesis. One problem with this comparison, however, is the disparate nature of the habitats from which the different species were collected, and, presumably, to which they were adapted. For example, the C 3 taxa were collected from moist, shady or cool habitats whereas those species with CAM-cycling grew on seasonally arid and warm rock outcrops, and the CAM species was collected from a warm, arid desert (Gravatt and Martin, 1992). Perhaps the best comparisons of water use by succulent plants with and without CAM have been made by von Willert and colleagues (see von Willert et al., 1992). In several comparisons of stem and leaf succulents growing side-by-side in the extremely arid Namib Desert in southern Mrica, water-use efficiencies of the CAM species were not higher than those of the C 3 species. Such comparisons, however, were usually made between unrelated taxa. A genus of plants that appears ideal for comparisons of the putatively superior water-conservative nature of CAM, relative to C 3 photosynthesis, is Peperomia in the tropical family Piperaceae. Modes of carbon metabolism in this semi-epiphytic genus vary from C 3 to CAM, with several variations of C 3-CAM intermediacy (Virzo De Santo et al., 1978, 1983; Ting et al., 1985; Holthe et al., 1992). Many species in the genus have succulent leaves and grow on the ground or, more

commonly, as epiphytes in moist tropical forests (Tebbs, 1993). Therefore, in order to test the commonly assumed generalization that the CAM pathway is primarily responsible for the high WUE of CAM plants, gas exchange characteristics, including WUE, were compared among three species of Peperomia selected for their morphological and ecological similarity, but having dissimilar photosynthetic pathways.

Materials and Methods Cuttings of Peperomia obtusifolia (L.) A. Dietr., P. camptotricha Miq., and P. scandem Ruiz & Pav6n were obtained from plants in the greenhouses at the University of California at Riverside. It is likely that all plants of each species used in the current study were genetically identical. The cuttings were rooted and grown in a standard greenhouse soil mix [7: 2 : 1 : 1 (v/v) mixture of clay loam, peat moss, Perlite, and vermiculite) under the following approximate environmental conditions in the greenhouse at the University of Kansas: 1000 Ilmol. m- 2 • s-I maximum photosynthetic photon flux (PPF; natural photoperiod), day/night air temperature ranges of 2736/15-26 ·C, and day/night vapor pressure deficit (vpd) ranges of 1.3-3.0/0.7-1.6kPa. Plants were always kept well-watered and fertilized weekly (stock material: 18 % total N, 18 % available P20 5, 18 % soluble K20, and trace elements). Gas exchange of the plants was measured after a year of vigorous growth. A week prior to gas exchange measurements, all immature leaves were removed &om the shoots to be measured. This was done to avoid complications in interpretation of the data because young leaves of at least two of the species examined exhibit different metabolic characteristics than the older leaves (Sipes and Ting, 1985; Holthe et al., 1987). The shoots were then sealed in gas-exchange cuvettes with the following internal environmental conditions: 900 Ilmol· m -2. S-I PPF (12 h photoperiod), 30/20·C day/night air temperatures, and 2.4/0.5 kP,,; day/night vpd. Rates of net CO 2 and H 20 exchange of the shoots were monitored continuously for three days with an open-flow gas exchange system incorporating a differential in&ared gas analyzer and dew point meters (for further information on this system, see Gravatt and Martin, 1992). The potted soil with roots outside the gas exchange cuvette was kept wellwatered throughout the measurements. Rates of gas exchange were calculated using equations &om Sestak et al. (1971) and from Farquhar and Sharkey (1982). Data presented in this study were from the second 24 h period of each experiment (the first day and night allowed plants to acclimate to the cuvette conditions). All WUE values were calculated on a molar basis based on integration of the areas under the gas exchange curves using the so&ware program InPlot (GraphPad So&ware, San Diego, CA). At the end of the second day, and again at the end of the second night, a leaf was removed from a shoot inside the cuvette and stored at -65·C until analyzed for malic acid concentration. Gas exchange rates were adjusted for this loss of leaf material. Upon thawing, the leaf tissue was sliced and its liquid removed by centrifugation as in Smith and Liittge (1985). The malic acid concentration of this extract was determined by comparison with results from known concentrations of malic acid using the enzymatic/spectrophotometric method of Gutmann and Wahlefeld (1974). Dry mass of the tissue was measured after drying one week at 65 .c. Because sample sizes were small, and the data were o&en not normally distributed nor homoscedastic, all data were rank-transformed before the application of parametric statistics (see Conover and Iman, 1981; and Potvin and Roff. 1993). Morning and evening means of leaf malic acid concentrations were compared using a ttest, whereas WUE values of the three species were compared using an analysis-of-variance, followed by the T'-method multiple-com-

261

Water-use efficiency in Peperomia

An

parison-of-means test. statistical tests were performed according Sokal and Rohlf (1981); significant differences were inferred when

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As determined previously by Ting and colleagues (Sipes and Ting, 1985; Ting et aI., 1985; Holthe et aI., 1987, 1992), the CO 2 exchange and malic acid data indicate that Peperomia obtusifolia is a C 3 plant with a small degree of CAM-cycling, P. camptotricha is a C 3-CAM intermediate having substantial amounts of net CO 2 uptake during both the day and the night, and P. scandens is a CAM plant (Figs. 1-3). All three species exhibited significant increases in leaf. malic acid concentrations overnight (Fig. 4), although the amount and fluctuation of malic acid in P. obtusifolia were very small. Thus, although the latter species should technically be considered as having CAM-cycling, the low amounts of malic acid, coupled with a net negative CO 2 uptake at night (Table 1; Fig. 1), led to its designation as a C 3 plant in this study. Comparisons of the CO 2 exchange and the malic acid data (Table 1) indicate that, in spite of the large amounts of nocturnal CO 2 uptake in P. camptotricha and P. scandens, a substantial amount of malic acid presumably results from the recycling of respiratory CO 2• This has been reponed previously

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Results and Discussion

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for all three species (Sipes and Ting, 1985; Ting et aI., 1985; Holthe et aI., 1987, 1992).

262

BRENT R. HELLIKER and CRAIG E. MARTIN

Molar water-use efficiencies were calculated from CO 2 and H 20 vapor exchange data integrated throughout the day, the night, and a 24 h period (Fig. 5). Only time periods (12 or 24 h) when net CO 2 exchange was positive, indicating net CO 2 uptake, were used. Thus, night WUE values were obtained for only three individuals of P. obtusifolia; likewise, two individuals of P. scandens did not exhibit a positive value for net CO 2 exchange throughout the day. Nighttime WUE of P. scandens exceeded values of the other two species threeto ten-fold (Fig. 5). Furthermore, although daytime WUE of P. scandens was substantially lower than corresponding values

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CO2 exchange, mmol kg-I daytime uptake 241.5 (149.3) 0(0) daytime loss daytime net 241.5 (149.3) nighttime uptake 18.2 (10.0) -28.2 (36.0) nighttime loss nighttime net -9.9 (42.5) 24hnet 231.6 (109.7) H 20 loss, mol kg-I daytime nighttime 24h Degree of COz-recycling

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for P. obtusifolia and P. camptotricha, the conservation of water at night in this CAM species more than compensated for the low daytime value, resulting in a 24 h WUE for P. scandens that was more than double the values for the C 3 and the C 3-CAM species of Peperomia (Fig. 5). Given the similarity in gross morphology of these species of Peperomia, coupled with the fact that all three grow in moist tropical forests, it is likely that the higher WUE observed in P. scandens, relative to the other tWo species, is solely a consequence of the CAM pattern of gas exchange whereby gas exchange is mosdy restricted to the nighttime when the evaporative demand of the atmosphere is low. This study confirms past generalizations (see, for example, Kluge and Ting, 1978; Osmond, 1978; Winter, 1985) that a high WUE is associated with the CAM pathway and not with succulence or leaf morphology alone. Previous reports to the contrary (e.g., Gravatt and Martin, 1992; von Willert et al., 1993) may indicate that factors other than photosynthetic pathway and gross morphology, e.g., adaptations to dissimilar environments or microhabitats, can dramatically influence the efficiency of water-use by plants, regardless of their photosynthetic pathway.

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Fig. 5: Integrated day (bars with slashes), night (solid bars), and 24h (cross-hatched bars) values of molar water-use efficiency (WUE) for Peperomia obtusifolia (C3), P. camptotricha (C3-CAM), and P. scandens (CAM). Bars represent means (lines projecting from the bars are standard deviations) of 6 plants (representative diurnal gas exchange data for one plant of each species are given in Figs. 1-3, and means of integrated values are given in Table 1), except N = 3 for P. obtusifolia night WUE and N = 4 for P. scandens day WUE. For each respective time period, only the CAM means are significantly different (*, P < 0.05) from values of both of the other species. Data are expressed on a dry-mass basis.

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Acknowledgments

Many thanks to Irwin Ting for generously providing the plant material, Katie Nus for expert assistance in the greenhouse, and Eddie Nowak and Anthony Kraybill for assistance in the laboratory.

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