¹³C‐Kinetic Isotope Effects in Photosynthetic Carboxylation Reactions and δ¹³C‐Values of Plant Material

Abstract

AblItraet. The influenceof different parameters on B 13 C-values in plants in vivo and on the 13C-isotope effect of the carboxylation reactions in vitro were examined using isotope ratio mass spectrometry.The primary fixation of carbon in photosynthesis occurs in so-called Cs-plants via the ribulosebisphosphate carboxylase (RuBP-C) reaction, and in Cs-plants via the phosphoenolpyruvate carboxylase (PEP-C) reaction. The observed difference in 813C-values" of the organic material of these two groups of plants is a consequence of the different isotope discrimination by the two carboxylation reactions. I Changes of ecological conditions during growth are accompanied by variations of the 8 13 C-values within these groups or even for a single plant species.r"In particular, rise of temperature during growth generally leads to more negative 8 I'C-values; therefore the temperature coefficient of the discrimination effect seems to be positive. On the other hand, from corresponding in vitro experiments on isotope discrimination by the carboxylation reactions of RuBP-C and PEP-C, rather contradictory results were reported.v'" Whelan et aI. 1 observed a strong decrease in isotope discrimination by RuBP-C from sorghum with increasing temperature, whereas Christeller et al.6 found temperature independence for the isotope effect of the RuBP-C-reaction (enzyme from soy beans). As methods and enzymes used in these experiments differed from each other, and as from general experience in enzyme kinetics a negative temperature coefficient of the reaction was to be expected, we investigated the influence of different parameters on 8 13C-values of plants in vivo and, in parallel experiments, on the I'C-isotope effect of the carboxylation reactions in vitro. EXPERIMENTALSamples of plant material (5 mg) were converted to CO 2 in a commercial micro-combustion apparatus in a He/02 gas flow. The CO2 was isolated in a liquid nitrogen trap and then expanded into the sample reservoir of an isotope ratio mass spectrometer with double inlet system (MM 903, VG Micromass). The B 13 C-values were determined relative to a suitable standard. The in vitro experiments with enzymes were performed on incubation mixtures with an excessof carbon dioxide. After defined partial reaction, controlled spectrophotometrically, the remaining CO 2 was transferred by a He carrier gas flowinto a liquid nitrogen trap and assayed for 13C as mentioned above; the kinetic isotope effect was determined from the change of the B13C-value of the CO 2 before and after incubation. RESULTS AND DISCUSSIONThe isotope effect of the PEP-C-reaction was klJk l, = 1.002 ± 0.0003 (25°C; pH 8.0) with a temperature coefficient of -0.OOOO3/"C; no difference was found between enzymes from wheat and maize, representing C,-and Cz-plants respectively. The low value observed for k I2/k 13 is in line with the synchronous mechanism for the carboxylation step proposed by Utter.' The dependence of the kinetic isotope effect of the PEP-C-reaction on the pH-value could be mainly correlated with the th...

Molecular and isotopic stratigraphy in an ombrotrophic mire for paleoclimate reconstruction

“…However, a negative temperature coefficient of Ϫ0.27‰°C Ϫ1 for mean December temperatures has been reported in tree rings of Juniperus (Leavitt and Long, 1983), while a temperature coefficient of Ϫ0.7‰°C Ϫ1 was recorded for the rings of naturally grown Elm trees in Massachusetts (Farmer, 1979). In several growth chamber experiments, negative carbon isotope temperature coefficients have also been recorded in whole plants and leaves (Smith et al, 1973;Troughton and Gard, 1975;Smith et al, 1976;Schmidt et al, 1978). However, the mechanism leading to these negative coefficients remains elusive, although in an investigation of fractionation by CO 2 -fixing enzymes isolated from soybean leaves revealed a negative carbon isotope temperature coefficient (Ϫ0.22‰°C Ϫ1 ) for RuBP carboxylase (initial carboxylating enzyme in C 3 plants; Christeller et al, 1976).…”

Physical and chemical basis of carbon isotope fractionation in plants

“…Rubisco catalyses the first step in CO2 fixation in C^, plants: I HC-OH I HC-OH I CH2-OPO3 CO-HC-OH +2 H+ (11) It was recognized early that isotope fractionation by this enzyme was probably a source of the large isotope fractionation seen in C3 plants (Park & Epstein 1960, 1961. A variety of investigators have measured this fractionation by combustion analysis (Christeller, Laing & Troughton 1976: Estep, Tabita & van Baalen 1978Wong, Benedict & Kohel 1979), by specific analysis of individual carbon atoms in substrate and product (Roeske & O'Leary 1984), and by analysis of remaining CO2 during the carboxylation (Schmidt et al 1978: Winkler et al 1982Guy ct al. 1987).…”

Prediction of atmospheric δ13CO2 using fossil plant tissues

“…The value a = 4.4‰ was calculated from diffusivity comparisons of 13 CO 2 versus 12 CO 2 in air [ Craig , 1953, 1954] and is generally accepted. Depending upon the role envisioned for the primary enzyme that facilitates carboxylation, published estimates of b range from 27.0 to 29.0‰ [ Christeller et al , 1976; Farquhar , 1979; Schmidt et al , 1978; Wong et al , 1979]. Published estimates of ( c i / c a ) range from 0.38 to 0.89 [ Arens et al , 2000] and are meant to reflect a time‐averaged value resulting from moment‐to‐moment stomatal regulation of gas exchange.…”