In a preliminary physiological study conducted by (Maffei et al. 1988) on C. citratus grown in humid temperate climates, some of the PEP-carboxylase kinetic characteristics, and Rubisco and glycolate oxidase activities were found to be comparable to those of C4 plants.
The C4 mechanism was also confirmed by the 13C/12C stable isotope ratio analyses (513C = -13.0). These results are in accordance with 513C values measured on other species of Cymbopogon (Rajendrudu and Das 1981) in which 513C value of -11.0 for C. flexuosus, -9.7 for C. martinii, -11.6 for C. nardus, -10.3 for C. pendulus, and -11.3 for C. winterianus, respectively, were recorded.
From a biochemical point of view, the three types of the basic C4 pathway differ mainly in the C4 acid transported into the bundle sheath cells (malate and aspartate) and in the way in which it is decarboxylated; they are named (based on the enzymes that catalyse their decarboxylation) NADP-dependent malic enzyme (NADP-ME) found in the chloroplasts, NAD-dependent malic enzyme (NAD-ME) found in mitochondria, and phosphoenolpyruvate (PEP) carboxykinase (PCK), found the cytosol of the bundle sheath cells (Edwards and Walker 1983; Ghannoum et al. 2001; Hatch et al. 1975; Jenkins et al. 1989; Huang et al. 2001). Furthermore, a characteristic leaf anatomy, biochemistry, and physiology are associated with each of the C4 types (Dengler and Nelson 1999; Hattersley and Watson 1976). A clear indication of the C4 photosynthetic pathway of C. citratus and the variant to which it belongs was obtained by estimating the activities of NADP-ME (EC 184.108.40.206), NADP-MDH (EC 220.127.116.11), PPDK (EC 18.104.22.168), NAD-ME (EC 22.214.171.124), and PCK (EC 126.96.36.199) as well as some kinetic characteristics of NADP-ME and NADP-MDH. Adaptation to a particular environment is a complex process involving a number of physiological, morphological, and ecological factors (Ghannoum et al. 2001; Huang et al. 2001).
Therefore, enzyme activities were recorded at the low and high temperatures typical of humid-temperate climates, in order to evaluate the adaptability of C. citratus. In order to estimate increases or decreases in the reaction rate due to changes in the protonation state, groups involved in the catalysis and/or binding of substrates as a consequence of pH fluctuations, activities were also recorded at different pH values.
1.3.1 Characterization of the Photosynthetic Variant
Further studies dealing with the characterization of the C4 variant indicated an NADP-dependent malic enzyme photosynthetic pathway in C. citratus.
The biochemical subtype was established through the estimation of the highest activities of NADP-dependent malic enzyme (NADP-ME, EC 188.8.131.52), NADP-dependent malate dehydrogenase (NADP-MDH, E.C. 184.108.40.206), pyruvate, orthophosphate dikinase (PPDK, E.C. 220.127.116.11), NAD-dependent malic enzyme (NAD-ME, E.C. 18.104.22.168), and phosphoenolpyruvate carboxykinase (PCK, E.C. 22.214.171.124) and some kinetic, along with some chemical-physical parameters of NADP-ME and NADP-MDH.
Extraction and partial purification sequentially involved precipitation with crystalline ammonium sulfate, dialysis, and anion exchange (DEAE-Sephacell). Both, extraction and assays were conducted according to Ashton (1990). The low activity values of PPDK (90.28 nKat mg-1 prot), PCK (<1 nKat mg-1 prot), and NAD-ME (52.51 nKat mg-1 prot) in C. citratus leaf extracts did not allow to determine the kinetic characteristics, such as Km and Vmax, and/or other chemical-physical parameters of these enzymes. NADP-MDH and NADP-ME presented relatively high activity values (15.93 mKat mg-1 and 12.56 mKat mg-1 prot, respectively). NADP-ME activity was 239-fold greater than NAD-ME activity. The kinetics of NADP-MDH and NADP-ME were therefore measured to gain a clearer picture of the photosynthetic pathway. The low activity of PPDK found in plant extracts of C. citratus agrees with the literature data for C4 plants (Ashton 1990) and with data on species of the same photosynthetic subtype (Ashton 1990; Bertea et al. 2001). The low levels of NAD-ME and PCK activities found in our extracts clearly indicated the absence of a C4 variant utilizing these two pathways. The relatively high activities found for NADP-MDH allowed us to determine some of its kinetic characteristics (Km and Vmax), which were comparable to those of plants belonging to the NADP-ME photosynthetic variant (Ashton 1990; Bertea et al. 2003).
Km values obtained for OAA and NADPH (NADP-MDH) were 29.0 (±0.014) mM and 31,67 (±0.09) mM, respectively. With regard to NADP-ME, the apparent Km values for NADP+ and malate were 19.40 (±0.08) and 242.0 (±0.008) mM, respectively. In the case of NADP-MDH, Vmax values for OAA and NADPH were 12.52 (±0.021) and 14.97 (±0.012) mKat mg-1 prot, respectively.
NADP-ME Vmax values for malate and NADP+ were 8.63 (±0.507) and 18.60 (±0.007) mKat mg-1 prot, respectively. In general, relatively high activities of NADP-MDH and NADP-ME allowed a partial characterization of these enzymes and provided evidence for an NADP-ME subtype for C. citratus. The apparent kinetic properties of both enzymes were comparable to those of plants belonging to this subtype (Ashton 1990; Hatch et al. 2007; Hatch et al. 1975), and were consistent with a high photosynthetic activity, even when the plant was cultivated in a temperate climate.
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