Planta

Planta (Berl.)128, 85--92 (1976)

9 by Springer-Verlag 1976

Hydrogen Isotope Discrimination in Higher Plants: Correlations with Photosynthetic Pathway and Environment H. Ziegler 1, C.B. Osmond 1' z, W. Stichler 3, and P. Trimborn 3 1 Botanisches Institut der Technischen Universit/it, Arcisstral3e 21, D-8000 Mfinchen 2 2 Department of Environmental Biology, Australian National University, Canberra City, ACT 2601 3 Institut ftir Radiohydrometrie der Gesellschaft ffir Strahlen- und Umweltforschung mbH, Miinchen

Summary. The ratio of deuterium to hydrogen (expressed as 6D) in hydrogen released as water during the combustion of dried plant material was examined. The 6D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C3 plants show mean 5D values of - 132O/oofor shoots and - 117O/oofor roots; C4 plants show mean 6D values of -91~ for shoots and -77~ for roots and CAM plants a 6D value of -75~ for roots and shoots. The difference between the 5D value of shoot material from C3 and C4 plants was confirmed in species growing under a range of glasshouse conditions. This difference in 6D value between C3 and C4 species does not appear to be due to differences in the 6D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C3 and C4 plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in 5D values are discussed. In CAM plants grown in the laboratory or collected from the field 6D values range from -75O/0o to +50O/oo and are correlated with ~ 3 C values. When deprived of water, the 6D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different 6D value of available water in areas of increasing aridity. Whatever the origin of the variable 5D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.

Introduction Natural abundance stable isotope studies With 13C have been particularly useful in the delineation of C3

and C4 plants (Bender, 1968; Smith and Brown, 1973) and to the understanding of the photosynthetic strategies of CAM plants under natural conditions (Lerman, 1975; Osmond, 1975; Osmond et aI., 1975). The ~13C values characteristic of these plant groups are determined first, by the isotope discrimination characteristics of the primary carboxylases (Whelan et al., 1973) and second, by the physical conditions in which the subsequent metabolic interconversion takes place (Lerman and Queiroz, 1974; Osmond etal., 1975). Relatively little is known of hydrogen isotope discrimination at natural abundance in living systems. Schiegl and Vogel (1970) reported 6D values for plant sap and for wood samples, in addition to data on the 6D value in water supplies, marine organisms, coal oil and natural gas. They showed that plant sap was enriched in deuterium, and that wood was depleted, when compared with deuterium in precipitation and ground water. Smith and Epstein (1970) reported 6D values for a variety of salt marsh plants and animals which used the same water source. These authors cited unpublished evidence that the 6D value of plant material collected along an altitudinal gradient reflected the changing cSD value of ground water with altitude. In all tissues examined, Smith and Epstein (1970) found the lipid fraction to be substantially depleted in deuterium. In this paper we report 6D values obtained from a variety of plants growing under natural, as well as controlled environmental conditions. These show correlation with respect to photosynthetic pathway, and indicate that ~D value of CAM plants reflects plant water relations. Factors contributing to the 6D value of plant materials are more complex than those determining the 613 C value, but potentially important factors are discussed. Materials and Methods Plant materials were collected in the field as described earlier (Osmond et al., 1975). In controlled growth experiments seeds or

86

H. Ziegler et al. : Hydrogen Isotope Discrimination in Higher Plants

fruits of C 3 and C 4 plants and plantlets of several C A M species were placed in the same tray of soil in a controlled environment cabinet (Heraeus Model K K B 600 L). The cabinet was maintained at 23 ~ day 17~ night (10 h day); the light intensity (metal-arc lamp) averaged 10,000 lux, and relative humidity was 59% during the day and 65% at night. The plants were irrigated with the same water. In some experiments with Kalanchoe daigremontiana plants were grown in single pots at 25 ~ in continuous light with light intensity (metal-arc lamp) 10,000 lux, supplemented by a 6 0 W tungsten lamp. The plants received the same irrigation water until the time they were transferred to another cabinet maintained with the same light intensity but a 12 h, 27 ~ day and 12 h, 15 ~ night. No further water was added during the subsequent 2 4 week period of the experiments. Plant materials were either dried at 85 ~ or were extracted in boiling water before drying to produce an insoluble fraction which was dried at 85 ~ and a soluble fraction which was lyophilised. These samples were combusted to CO, and water as described previously (Schiegl and Vogel, 1970; Osmond et al., 1975). The 613C value of CO2 was determined on a specially equipped mass spectrometer and is presented as permille deviation from the PDBstrandard (Craig, 1975; Smith and Epstein, 1970) as follows: ~13C=[ 13C/12C s a m p l e 13C/12 C standard

l] x i03 [O/0o]

The ~D value was determined on H2 evolved by reducing the water of combustion with heated zink (Schiegl and Vogel, 1970) and is reported as permille deviation from "standard mean ocean water" (SMOW) (Craig, 1961 ; Smith and Epstein, 1970): riD=

[

D/Hsample D/H standard

-1

1

x 103 [~

These data are referred to as 6D value (metabolic hydrogen) to distinguish them from determination based on water samples obtained from different tissue fractions, referred to as 6D value (tissue water). 6D values were obtained for water used to irrigate the plants, and for water transpired from the leaves. Transpired water was collected by absorption of the water vapor in a column filled with molecular sieve (A 4). The 6D value (tissue water) of different tissues was obtained by analysis of the water collected by vacuum distillation o f freshly sampled plant parts. Errors in determinations of 6 ~a c value were less than + 0.3% and in determination of 6D value (metabolic hydrogen), 12 assays of 3 samples of spinach leaf gave errors of less than +2o/00.

Table 1. The 3D (tissue water) value in different parts o f plants grown under controlled conditions. (Irrigation water -78.2% 0; transpiration water in the case of Spinacia - 152. t ~ Species

Spinacia oleracea (C3) Pisurn sativurn (C3) Amaranthus edulis (C4) Sedum praealtum (CAM)

Mean

6D value (tissue water) [~ root

shoot

- 71.0 - 75.0 - 78.0

- 33 - 41 - 42 - 32

- 73.0

- 37

- 68.0

The data are similar to those obtained by Schiegl and Vogel (1970) for plant sap samples which were collected from plants under field conditions, but which showed greater deuterium enrichment than observed here. These data together indicate that a significant fractionation occurs during evaporation of water from the leaf. Presumably this arises from the difference in vapour pressure of D 2 0 or D H O and H 2 0 which results in the enrichment of deuterium in leaf tissue water and the depletion of deuterium in the water vapour. There are no evident differences in the degree of enrichment of deuterium in leaf water for the leaves of the C3, C4 or CAM plants under our conditions (Table l) and in each case root tissue water appears to be in equilibrium with the irrigation water, rather than with leaf water: there in no discrimination during the uptake of water by the root. These data suggest that it may be important to relate the 6D value (metabolic hydrogen) to the 6D value (tissue water) for each tissue, rather than to that of the water supplied. Whichever 6D value is appropriate, the data in Table 1 show that physical factors are unlikely to account for the differences in 6D value (metabolic hydrogen) obtained for plants with different metabolic pathways when grown under controlled conditions.

Results and Discussion

6D Value (Tissue Water) in the Plant System The physical pathway of water movement into plants is more complex than that for CO2 and, as water exchange greatly exceeds CO2 exchange, it is important to assess the extent of hydrogen isotope fractionation due to physical factors. Table 1 shows that transpired water obtained from spinach plants is substantially depleted in deuterium (-152.1%o) in comparison with the irrigation water (-78.2%o). The fractionation during transpiration is, however, in the same range expected to result from evaporation of water from soil without a plant. In 4 species, water in shoot tissue, but not root tissue, is somewhat enriched in deuterium.

6D Value (Metabolic Hydrogen) and Its Correlation with Pathway of Photosynthetic Carbon Metabolism Comparisons of c~13C value, and 6D value (metabolic hydrogen) in roots and shoots of C3, C4 and CAM species grown under identical environmental conditions are given in Table 2. The mean 613C values of the C3 plants (-30.8O/oo) and of the C4 plants (mean, - 12.4~ are consistent with those reported previously and the values for CAM plants (mean, - 2 6 . 9 % o ) indicate that dark CO2 fixation makes a relatively minor contribution to net CO/assimilation in these well watered plants (Osmond, 1975; Osmond et al., 1975). In C3 plants the mean 6D values (recta-

H. Ziegler

et al. : H y d r o g e n

Isotope Discrimination

T a b l e 2. T h e 6 D v a l u e ( m e t a b o l i c h y d r o g e n ) of shoots and roots of plants grown under conditions (6D irrigation water - 78.2%o) Species

Shoot 6D

in Higher Plants

a n d t h e 313 C v a l u e identical, controlled

Root 613C

(incor-

613C

6D (incor-

porated)

porated)

(~

(~

(%0)

(~

- 131 -135

- 32.5 -26.2

-129

-27.6

-141

-33.1

-136

-33.2

Pisumsativum

-120

-31.0

-

-29.3

Helianthusannuus

-131

-31.5

-123

-32.2

Mean

-132

-30.8

-117

-30.6

- 89 -101

-13.1 -13.1

-

82

-12.8

C3 S p e c i e s

Spinacia oleracea

81

C4 Species

Zeamays Pennisetumtyphoides

-

83

--10.6

-

69

-11.8

Amaranthusedulis

-

91

-12.6

-

81

-11.5

Mean

-

91

-12.4

-

77

-12.0

CAM

Species

Kalanchoe daigremontiana

-

69

-28.4

-

-

67

-23.8

-

Bryophyllurn tubiflorum

-

78

-26.9

-

80

-24.9

Sedumpraealtum

-

82

-28.4

-

72

-27.1

Mean

-

74

-26.9

-

76

-26.0

87

Several C3, C4 and CAM plants grown under different conditions in Munich and Canberra were also examined (Table 3). The conditions of growth ranged from outdoor soil culture to glasshouse solution culture and the 6D value of the irrigation water was not monitored in any of the experiments. The range and averages of the 6D value (metabolic hydrogen) for C3 plants and Ca plants are closely comparable with those found under strictly controlled conditions (Table 2). The two groups are distinguished by an average difference of about -40o/oo in the 6D value under these highly variable conditions. These data suggest that the riD value (metabolic hydrogen) may be at least as characteristic of the photosynthetic pathway as is the 6~3C value. It is interesting to note that the direction of isotope discrimination is the same for both 3C and D, i.e. C3 species show substantial discrimination against a3C and D, whereas C4 plants do not. If the values are referred to the 6 ~3C value of CO2 in air (approximately -7~ and the 6D value of the irrigation water (-78~ then C4 plants and C3 plants show a reasonable proportionality in their discrimination characteristics. Thus, with respect to CO2 and H20, C4 plants discriminate about - 5 % 0 for 3C and about - 100/o0 for D, whereas C3 plants discriminate about -20O/oo for 13C and -50o/oo for D.

-

bolic hydrogen) of roots and shoots are comparable (the lower root value is due to a single sample) and there is little indication that these values reflect the different 6D value (tissue water) in these tissues noted in Table 1. The C3 plants are depleted about - 5 5 % o in deuterium relative to the irrigation water, comparable with that found by marsh plants (Smith and Epstein, 1970). However, if the 6D values are related to the appropriate 6D value for tissue water in roots and shoots (Table 1) then the similarity between shoot and root disappears. (Corrected values are -95O/oo for shoots and -44O/oo for roots.) It is quite significant to note that the 6D of the root dry matter from plants of each photosynthetic type closely resembles that of the shoot dry matter: It is reasonable to assume that the biosynthetic incorporation of H or D proceeds exclusively or at least predominantly in the shoot, and that the root is supplied with assimilates from the shoot and uses them without further discrimination.

Changes in (~13C Value and 6D Value (Metabolic Hydrogen) in C A M Plants Subject to Water Stress

Several studies have shown that the 613C value of CAM plants changes when plants are exposed to water stress, and suggest that water availability may be an important factor in determining the ~ I3C value of these plants under natural conditions (Osmond et al., 1973; Osmond et al., 1975; Mooney, Troughton and Berry, 1974). These data have been correlated with the extent to which dark CO/fixation contributes carbon (with a 613 C value of about - 1 1 ~ to the total carbon assimilated by the plant (Osmond, 1975 ; Nalborczyk, LaCroix and Hill, 1975). In Tables 2 and 3 there is a suggestion that differences in the ~13C value of CAM plants may be correlated with the cSD value (metabolic hydrogen). Well irrigated CAM plants grown in controlled environment cabinets show 613C values of -26O/o0 (Table 2) and mature plants of the same species grown in the glasshouse show ~ 13C values of - 16~ (Table 3). Corresponding 6D values (metabolic hydrogen) were - 74O/oo and - 30O/oo. The change in ~ 3 C value and cSD value is in the same direction, and the proportionality of the discrimination against the isotopes is similar to that noted between Ca and C4 plants above.

H. Ziegler et al. : Hydrogen Isotope Discrimination in Higher Plants

88

Table 3. The 313 C and 6D value (metabolic hydrogen) of shoots of C3, C4 and CAM plants grown in different conditions Species and pathway

Growth conditions

613C value

6D value

(~

(~

Spinach1 oleracea (C3)

Controlled environment Cabinet, soil, Munich

-22.1 (7) a - 2 1 . 5 (3)

- 134 (7) - 1 2 9 (3)

Beta vulgaris (C3)

Outdoors, soil, Munich

-27.4

-117

Triticum sp. (C3)

Glasshouse, soil, Canberra

-26.8

-

Atriplex hastata (C3)

Glasshouse, solution, Canberra

-26.9

-141

-24.9

- 122

Glasshouse, soil, Munich

- 1 3 . 0 (3) - 1 3 . 4 (2)

-- 84 - 70

Mean C3

Zea mays (C4)

88

Glasshouse, soil, Canberra

-12.4

-

69

Sorghum bicolor (C4)

Glasshouse, soil, Canberra

-11.8

-

77

Amaranthus edulis (C4)

Outdoors, soil, Munich

- 12.3

-

87

A triplex spongiosa (C4)

Glasshouse, solution, Canberra

--12.8

-

98

--12.6

-

81

Mean C4

(2) (2)

Kalanchoe daigr emontiana (CAM)

Glasshouse, soil, Munich

-16.2

-

37.4

Bryophyllum tubiflorum (CAM)

Glasshouse, soil, Munich

-16.5

-

22.0

Numbers in parenthesis refer to number of replicate experiments. J

ff/ '

i

O

-10-

-20-

/

/

-30-

-40-

f J / f

-50-

7o

16o 9 "~

days

Fig. 1.6D-values in the soluble and insoluble fraction of Kalanchoe daigremontiana leaves after different periods of cultivation in continuous light and water supply (o insoluble, ~ soluble) and with short day treatment without irrigation ( o insoluble, 9 soluble). Details in the text

The influences of water stress on the 613C and 6D value (metabolic hydrogen) in Kalanchoe daigremontiana was examined under controlled conditions. Plants grown for 73 days in continuous light (25 ~) were transferred to a cabinet with short days (6 h,

25 ~ and cold nights (18 h, 15 ~ and were deprived of water. The 613 C and 6D value (metabolic hydrogen) were determined on the dried soluble extract and on the insoluble residue of the sixth leaf pair at intervals during the following 26 days. Changes in 613C were small, but in the same direction as previously observed. The 6D value (metabolic hydrogen) in both soluble and insoluble fractions became substantially less negative throughout the experiments (Fig. 1) confirming the correlation noted in Tables 2, 3. The largest changes were observed in the soluble fraction (cf. 6 a3C value, in which larger changes were measured in the insoluble fraction). When integrated with the insoluble fraction, by correcting for weight of both fractions, the 6D value (metabolic hydrogen) of the whole leaf declined from - 4 4 % 0 to - 14% o in the 23 day experiment. These data suggest that water stress, which increased the dependence of CAM plants on dark CO2 fixation (Kluge and Fischer, 1967), results in considerable enrichment of deuterium in the metabolic hydrogen of these plants. We do not have data on the effect of water stress on the 6D value (tissue water) in CAM

plants. Correlations between 613C Value and 6D Value (Metabolic Hydrogen) in CAM Plants Under Natural Conditions The range of 6D values (metabolic hydrogen) measured in CAM plants growing under natural conditions

H. Ziegler et al. : Hydrogen Isotope Discrimination in Higher Plants

89

greatly exceeds the values recorded in Tables 2, 3 and Fig. 1. As shown in Fig. 2, species of Opuntia spp collected in Eastern Australia are substantially enriched in deuterium. In the genus Sempervivum, samples collected in the European Alps show a correlation between 6 ~3C value and 6D value (metabolic hydrogen) similar to that observed in Kalanchoe daigremontiana grown in laboratory (Fig. 2). Sedum spp, which do not display marked dark CO2 fixation under natural conditions (Osmond et al., 1975), show 6D values similar to those of well irrigated CAM plants in the laboratory (Fig. 2). In these data it is extraordinarily difficult to account for variation in the 6D value which may be due to different 6D values in ground water, particularly in arid sites (Elhalt etal., 1963; Schiegl and Vogel, 1970), to changes which have occurred during the drought stress on the plants (cf. Fig. 1) and to discrimination during CO2 fixation by different metabolic pathways. There is some evidence that the 6D value of ground water may influence the 6D value (metabolic hydrogen) in plant materials. Schiegl and Vogel (1970) noted that mean 6D values of ground water and wood were about -80~ and - 115O/oorespectively in Germany; about -45% o and -75~ respectively in the Netherlands and -23~ and -49~ respectively in South Africa. We have noticed a tendency for the 6D value (metabolic hydrogen) of Sedum spp. (CAM plants which evidently do not display dark CO2 fixation under natural conditions in the European alps) to be correlated with altitude. Samples of Sedum album and

9 \

-$ I000

~p

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Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment.

The ratio of deuterium to hydrogen (expressed as δD) in hydrogen released as water during the combustion of dried plant material was examined. The δD ...
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