Amaranthus Edulis Research Articles

Characterisation of seeds of a C4 phosphoenolpyruvate carboxylase‐deficient mutant of Amaranthus edulis

Seeds from the C(4) plant Amaranthus edulis were studied as part of the characterisation of a mutant (designated LaC(4) 2.16), which contains reduced amounts (5% of wild type) of the photosynthetic leaf form of phosphoenolpyruvate carboxylase (PEPC). On a per seed basis, the amount of PEPC activity was not significantly altered, while the weight and protein content of the mutant seeds were 34% lower than that of the wild type. Western gel blot analysis detected two PEPC polypeptides with molecular masses of 105 kDa (minor) and 100 kDa (major). The determination of in vitro phosphorylation in reconstituted assays revealed the presence of both calcium-dependent and calcium-independent PEPC-kinase activities in protein extracts of wild-type and mutant seeds. However, PEPC proteins were phosphorylated in dry seeds, and PEPC phosphorylation did not occur in vivo during seed imbibition in the presence of (32) P-phosphate. In contrast, (32) P-phosphate was incorporated into a range of proteins in wild-type seeds, but not in mutant seeds. In addition, ATP content was much reduced in germinating mutant seeds and this did not increase following the supply of phosphate. Collectively, these data suggest that the deficiency in C(4) PEPC in mutant A. edulis leaves has no effect on C(3) -type PEPC content and phosphorylation state in seeds, but causes impairment of energy production, thereby accounting for the reduced germination of the mutant.

Co-regulation of dark and light reactions in three biochemical subtypes of C4 species

Regulation of light harvesting in response to changes in light intensity, CO(2) and O(2) concentration was studied in C(4) species representing three different metabolic subtypes: Sorghum bicolor (NADP-malic enzyme), Amaranthus edulis (NAD-malic enzyme), and Panicum texanum (PEP-carboxykinase). Several photosynthetic parameters were measured on the intact leaf level including CO(2) assimilation rates, O(2) evolution, photosystem II activities, thylakoid proton circuit and dissipation of excitation energy. Gross rates of O(2) evolution (J(O)₂'), measured by analysis of chlorophyll fluorescence), net rates of O(2) evolution and CO(2) assimilation responded in parallel to changes in light and CO(2) levels. The C(4) subtypes had similar energy requirements for photosynthesis since there were no significant differences in maximal quantum efficiencies for gross rates of O(2) evolution (average value = 0.072 O(2)/quanta absorbed, approximately 14 quanta per O(2) evolved). At saturating actinic light intensities, when photosynthesis was suppressed by decreasing CO(2), ATP synthase proton conductivity (g (H) (+)) responded strongly to changes in electron flow, decreasing linearly with J(O)₂', which was previously observed in C(3) plants. It is proposed that g (H) (+) is controlled at the substrate level by inorganic phosphate availability. The results suggest development of nonphotochemical quenching in C(4) plants is controlled by a decrease in g (H) (+), which causes an increase in proton motive force by restricting proton efflux from the lumen, rather than by cyclic or pseudocyclic electron flow.

The biochemistry of Rubisco in Flaveria

C(4) plants have been reported to have Rubiscos with higher maximum carboxylation rates (kcat(CO(2))) and Michaelis-Menten constants (K(m)) for CO(2) (K(c)) than the enzyme from C(3) species, but variation in other kinetic parameters between the two photosynthetic pathways has not been extensively examined. The CO(2)/O(2) specificity (S(C/O)), kcat(CO(2)), K(c), and the K(m) for O(2) (K(o)) and RuBP (K(m-RuBP)), were measured at 25 degrees C, in Rubisco purified from 16 species of Flaveria (Asteraceae). Our analysis included two C(3) species of Flaveria, four C(4) species, and ten C(3)-C(4) or C(4)-like species, in addition to other C(4) (Zea mays and Amaranthus edulis) and C(3) (Spinacea oleracea and Chenopodium album) plants. The S(C/O) of the C(4) Flaveria species was about 77 mol mol(-1), which was approximately 5% lower than the corresponding value in the C(3) species. For Rubisco from the C(4) Flaverias kcat(CO(2)) and K(c) were 23% and 45% higher, respectively, than for Rubisco from the C(3) plants. Interestingly, it was found that the K(o) for Rubisco from the C(4) species F. bidentis and F. trinervia were similar to the C(3) Flaveria Rubiscos (approximately 650 microM) while the K(o) for Rubisco in the C(4) species F. kochiana, F. australasica, Z. mays, and A. edulis was reduced more than 2-fold. There were no pathway-related differences in K(m-RuBP). In the C(3)-C(4) species kcat(CO(2)) and K(c) were generally similar to the C(3) Rubiscos, but the K(o) values were more variable. The typical negative relationships were observed between S(C/O) and both kcat(CO(2)) and K(c), and a strongly positive relationship was observed between kcat(CO(2)) and Kc. However, the statistical significance of these relationships was influenced by the phylogenetic relatedness of the species.

The role of phosphoenolpyruvate carboxylase during C4 photosynthetic isotope exchange and stomatal conductance.

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) plays a key role during C(4) photosynthesis and is involved in anaplerotic metabolism, pH regulation, and stomatal opening. Heterozygous (Pp) and homozygous (pp) forms of a PEPC-deficient mutant of the C(4) dicot Amaranthus edulis were used to study the effect of reduced PEPC activity on CO(2) assimilation rates, stomatal conductance, and (13)CO(2) (Delta(13)C) and C(18)OO (Delta(18)O) isotope discrimination during leaf gas exchange. PEPC activity was reduced to 42% and 3% and the rates of CO(2) assimilation in air dropped to 78% and 10% of the wild-type values in the Pp and pp mutants, respectively. Stomatal conductance in air (531 mubar CO(2)) was similar in the wild-type and Pp mutant but the pp mutant had only 41% of the wild-type steady-state conductance under white light and the stomata opened more slowly in response to increased light or reduced CO(2) partial pressure, suggesting that the C(4) PEPC isoform plays an essential role in stomatal opening. There was little difference in Delta(13)C between the Pp mutant (3.0 per thousand +/- 0.4 per thousand) and wild type (3.3 per thousand +/- 0.4 per thousand), indicating that leakiness (), the ratio of CO(2) leak rate out of the bundle sheath to the rate of CO(2) supply by the C(4) cycle, a measure of the coordination of C(4) photosynthesis, was not affected by a 60% reduction in PEPC activity. In the pp mutant Delta(13)C was 16 per thousand +/- 3.2 per thousand, indicative of direct CO(2) fixation by Rubisco in the bundle sheath at ambient CO(2) partial pressure. Delta(18)O measurements indicated that the extent of isotopic equilibrium between leaf water and the CO(2) at the site of oxygen exchange () was low (0.6) in the wild-type and Pp mutant but increased to 0.9 in the pp mutant. We conclude that in vitro carbonic anhydrase activity overestimated as compared to values determined from Delta(18)O in wild-type plants.

CO2 diffusion resistance of bundle sheath cells in Amaranthus edulis, as revealed from the study of a PEP carboxylase deficient mutant

A mutant of the NAD-ME type C4 plant Amaranthus edulis, that lacks PEP carboxylase in the mesophyll cells was studied. Analysis of CO2 response curves of the mutant in the absence of a functional C4 cycle, provided a direct means of determining the liquid phase diffusive resistance of atmospheric CO2 to the sites of RuBP carboxylation inside the bundle sheath (BS) chloroplasts (rbs) within intact plants. The magnitude of rbs has been difficult to determine and previous estimates using different methods have ranged from 15 -1400 m2 s mol-1. The values of rbs in A. edulis were 70-180 m2 s mol-1 and they increased as the leaf matured. This is about 70-fold higher than the liquid phase resistance or diffusion of CO2 to Rubisco in the mesophyll cells of C3 plants. The values of rbs in A. edulis are sufficient for C4 photosynthesis to elevate the CO2 concentration inside the BS cells and thus reduce photorespiration. High reassimilation of photorespired CO2 was demonstrated in both mutant and wild type plants at limiting CO2 concentrations, which can be explained by high rbs. Based on O2 sensitivity analysis of wild type plants, the calculated CO2 concentration in BS cells under maximum rates of CO2 fixation in young leaves (lower rbs) and in expanded leaves (higher rbs) was about the same (1000-1200 m bar), which is sufficient to minimise photorespiration. This indicates that overcycling of the pathway and the rate of leakage of CO2 from the BS cells was higher in the younger leaves.

Control of C4 photosynthesis: effects of reduced activities of phosphoenolpyruvate carboxylase on CO2 assimilation in Amaranthus edulis L.

Heterozygous mutants of Amaranthus edulis deficient in PEP carboxylase (PEPC) have been used to study the control of photosynthetic carbon assimilation. A reduction in PEPC activity led to a decrease in the initial slope of the relationship between the CO2 assimilation rate and the intercellular CO2 concentration and to a decrease in photosynthesis at high light intensities, consistent with a decrease in the capacity of the C4 cycle in high light. PEPC exerted appreciable control on photosynthetic flux in the wild-type, with a relatively high flux control coefficient of 0.35 in saturating light and ambient CO2. The flux control coefficient was decreased in low light or increased in low CO2 or in plants containing lower PEPC activity. However, the rate of CO2 assimilation decreased down to about 55% PEPC, followed by an up-turn in the light-saturated photosynthetic rate as PEPC was further reduced, suggesting the existence of a mechanism that compensates for the loss of PEPC activity. The amounts of photosynthetic metabolites, including glycine and serine, also showed a biphasic response to decreasing PEPC. There was a linear relationship between the activity of PEPC and the activation state of the enzyme. A possible mechanism of compensation involving photorespiratory intermediates is discussed.

Utilization of O2 in the metabolic optimization of C4 photosynthesis

ABSTRACTThe combined effects of O2 on net rates of photosynthesis, photosystem II activity, steady‐state pool size of key metabolites of photosynthetic metabolism in the C4 pathway, C3 pathway and C2 photorespiratory cycle and on growth were evaluated in the C4 species Amaranthus edulis and the C3 species Flaveria pringlei. Increasing O2 reduced net CO2 assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O2 up to 5–10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O2 dependent photorespiration, O2 is required for maximizing C4 cycle activity to concentrate CO2 in bundle sheath cells. Therefore, the response of net photosynthesis to O2 in C4 plants may result from the balance of these two opposing effects. Under 21 versus 5% O2, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.

Photorespiratory metabolism of glyoxylate and formate in glycine-accumulating mutants of barley and Amaranthus edulis

Glycine-accumulating mutants of barley (Hordeum vulgare L.) and Amaranthus edulis (Speg.), which lack the ability to decarboxylate glycine by glycine decarboxylase (GDC; EC 2.1.2.10), were used to study the significance of an alternative photorespiratory pathway of serine formation. In the normal photorespiratory pathway, 5,10-methylenetetrahydrofolate is formed in the reaction catalysed by GDC and transferred to serine by serine hydroxymethyltransferase. In an alternative pathway, glyoxylate could be decarboxylated to formate and formate could be converted into 5,10-methylenetetrahydrofolate in the C1-tetrahydrofolate synthase pathway. In contrast to wild-type plants, the mutants showed a light-dependent accumulation of glyoxylate and formate, which was suppressed by elevated (0.7%) CO2 concentrations. After growth in air, the activity and amount of 10-formyltetrahydrofolate synthetase (FTHF synthetase; EC 6.3.4.4), the first enzyme of the conversion of formate into 5,10-methylenetetrahydrofolate, were increased in the mutants compared to the wild types. A similar increase in FTHF synthetase could be induced by incubating leaves of wild-type plants with glycine under illumination, but not in the dark. Experiments with 14C showed that the barley mutants incorporated [14C]formate and [2-14C]glycollate into serine. Together, the accumulation of glyoxylate and formate under photorespiratory conditions, the increase in FTHF synthetase and the ability to utilise formate and glycollate for the formation of serine indicate that the mutants are able partially to compensate for the lack of GDC activity by bypassing the normal photorespiratory pathway.

The isolation and characterisation of a mutant of the C 4 plant Amaranthus edulis deficient in NAD-malic enzyme activity

A mutant of Amaranthus edulis (Speg.) lacking activity of the C4 leaf form of NAD-malic enzyme (ME; EC 1.1.1.39) has been isolated. Homozygous mutant (5% wild-type ME activity) and heterozygous (50% wild-type ME activity) F2 plants were shown to contain both the α and β NAD-ME subunits in similar amounts to those detected in the wild-type leaves. The rate of photosynthetic CO2 assimilation was reduced in the homozygous mutant to 5% of that observed for the wild-type leaves. Other C4 enzymes were not down-regulated in the mutant plants. There was little difference in photosynthetic rate of the heterozygous plants compared to the wild-type, suggesting that NAD-ME exerts little control over the rate of C4 photosynthesis, and that in the wild-type the enzyme has a very low control coefficient. The activity loss in the heterozygote may therefore be compensated by regulatory mechanisms that increase the activity of the enzyme in vivo. Data for bundle-sheath strands indicated that although the homozygous mutants were able to oxidise malate via the Krebs cycle, they were unable to convert malate to pyruvate and alanine via NAD-ME.

Rapid plant regeneration of a C4 dicot species: Amaranthus edulis

The effects of TDZ (N-phenyl-N′-1,2,3-thidiazol-5-ylurea) and CPPU (N-(2-chloro-4-pyridil)-N′-phenylurea) on bud initiation of Amaranthus edulis thin cell layers (tTCLs) explants were investigated. tTCLs (0.2–0.4 mm thick) excised from 1 week old seedlings were cultured on Murashige and Skoog (MS) basal salts medium supplemented with varying concentrations of TDZ or CPPU ranging from 1 to 20 μM and 1 week after culture, buds were initiated. The optimal concentrations were 2 μM for TDZ and 10 μM for CPPU. Buds developed into normal plants after transfer onto a MS basal salts medium containing BA to obtain normal internode elongation and onto a MS basal salts medium containing IBA or TDZ for rooting.

Oxygen Requirement and Inhibition of C4 Photosynthesis. An analysis of c4 plants deficient in the c3 and c4 cycles An Analysis of C4 Plants Deficient in the C3 and C4 Cycles

The basis for O2 sensitivity of C4 photosynthesis was evaluated using a C4-cycle-limited mutant of Amaranthus edulis (a phosphoenolpyruvate carboxylase-deficient mutant), and a C3-cycle-limited transformant of Flaveria bidentis (an antisense ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] small subunit transformant). Data obtained with the C4-cycle-limited mutant showed that atmospheric levels of O2 (20 kPa) caused increased inhibition of photosynthesis as a result of higher levels of photorespiration. The optimal O2 partial pressure for photosynthesis was reduced from approximately 5 kPa O2 to 1 to 2 kPa O2, becoming similar to that of C3 plants. Therefore, the higher O2 requirement for optimal C4 photosynthesis is specifically associated with the C4 function. With the Rubisco-limited F. bidentis, there was less inhibition of photosynthesis by supraoptimal levels of O2 than in the wild type. When CO2 fixation by Rubisco is limited, an increase in the CO2 concentration in bundle-sheath cells via the C4 cycle may further reduce the oxygenase activity of Rubisco and decrease the inhibition of photosynthesis by high partial pressures of O2 while increasing CO2 leakage and overcycling of the C4 pathway. These results indicate that in C4 plants the investment in the C3 and C4 cycles must be balanced for maximum efficiency.

CO2 AND O2 DEPENDENCE OF PS II ACTIVITY IN C4 PLANTS HAVING GENETICALLY PRODUCED DEFICIENCIES IN THE C3 OR C4 CYCLE

The CO2 dependence of rates of CO2 fixation (A) and photochemistry of PS II at 5, 15 and 30% O2 were analyzed in the C4 plant Amaranthus edulis having a C4 cycle deficiency [phosphoenolpyruvate carboxylase (PEPC) mutants], and in the C4 plant Flaveria bidentis having a C3 cycle deficiency [Rubisco small subunit antisense (αSSU)]. In the wild type (WT) A. edulis and its heterozygous mutant having less than 50% WT PEPC activity there was a similar dependence of A and PS II photochemistry on varying CO2, although the CO2 saturated rates were 25% lower in heterozygous plants. The homozygous plants having less than 2% PEPC of the WT had significant levels of photorespiration at ambient levels of CO2 and required about 30 times ambient levels for maximum rates of A. Despite variation in the capacity of the C4 cycle, more than 91% of PS II activity was linearly associated with A under varying CO2 at 5, 15 and 30% O2. However, the WT plant had a higher PS II activity per CO2 fixed under saturating CO2 than the homozygous mutant, which is suggested to be due to elimination of the C4 cycle and its associated requirement for ATP from a Mehler reaction. In the αSSU F. bidentis plants, a decreased rate of A (35%) and PS II activity (33%) accompanied a decrease in Rubisco capacity. There was some increase in alternative electron sinks at high CO2 when the C3 cycle was constrained, which may be due to increased flux through the C4 cycle via an ATP generating Mehler reaction. Nevertheless, even with constraints on the function of the C4 or C3 cycle by genetic modifications, analyses of CO2 response curves under varying levels of O2 indicate that CO2 assimilation is the main determinant of PS II activity in C4 plants.

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Previous studies have indicated that the rate of photorespiration in C4 plants is low or negligible. In this study, wild‐type and mutant leaves of the C4 plant Amaranthus edulis were treated with the glutamine synthetase inhibitor, phosphinothricin and the glycine decarboxylase inhibitor, aminoacetonitrile, at different concentrations of CO2. The time course of ammonia accumulation in leaves of the wild type was compared with a mutant lacking phosphoenolpyruvate carboxylase activity (EC 4.1.1.31), and with three different mutants that accumulated glycine. The increase in the concentration of ammonia in the leaves, stimulated by the treatments was used as a measurement of the rate of photorespiration in C4 plants.The application of glutamine and glycine maintained the rate of photorespiratory ammonia production for a longer period in the wild type, and increased the rate in a mutant lacking phosphoenolpyruvate carboxylase suggesting that there was a lack of amino donors in these plants.The calculated rate of photorespiration in Amaranthus edulis wild‐type leaves when the supply of amino donors was enough to maintain the photorespiratory nitrogen flow, accounted for approximately 6% of the total net photosynthetic CO2 assimilation rate. In a mutant lacking phosphoenolpyruvate carboxylase, however, this rate increased to 48%, when glutamine was fed to the leaf, a value higher than that found in some C3 plants. In mutants of Amaranthus edulis that accumulated glycine, the rate of photorespiration was reduced to 3% of the total net CO2 assimilation rate. The rate of ammonia produced during photorespiration was 60% of the total produced by all metabolic reactions in the leaves. The data suggests that photorespiration is an active process in C4 plants, which can play an important role in photosynthetic metabolism in these plants.

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

A study of photorespiratory ammonia production in the C4 plant Amaranthus edulis, using mutants with altered photosynthetic capacities

Control of Photosynthesis in Amaranthus edulis Mutants with Reduced Amounts of PEP Carboxylase

Mutants of the NAD-ME plant have been created using sodium azide. These contain reduced activities of PEP carboxylase ranging from 5 to 100% of wild-type. Those with greater than 50% of the wild-type PEPC content showed a lower maximum rate of photosynthesis and reduced carboxylation efficiency compared to the wild-type plants. The PEPC from these heterozygotes was less sensitive to malate inhibition than the PEPC of the wild-type plants and also had an increased phosphorylation state. Mutants containing 45 and 49% of wild-type PEPC exhibited a greater Amax than was observed for the 55, 66 and 70% mutants. We postulate that there is a compensatory mechanism that activates PEPC when the PEPC protein is less than 55% of the wild-type. Control coefficients were measured for PEP carboxylase from wild-type and heterozygous (55%) plants. Results suggest that activation of PEPC by phosphorylation and metabolites may be more an ‘on-off’ switch than a means of fine adjustment of PEPC activity in response to varying factors such as PEPC content, CO2 or temperature changes.

Estimation of diffusive resistance of bundle sheath cells to CO2 from modeling of C4 photosynthesis

Bundle sheath resistance to diffusion of CO2 (rc) is a critical component of C4 photosynthesis which allows accumulation of inorganic carbon in bundle sheath cells of C4 plants. Several analyses were made to evaluate the magnitude of rc in C4 plants. Experimental data on the O2 inhibition of photosynthesis (Dai et al. (1993) Plant Physiol 103: 83-90; (1995) Plant Physiol 107: 815-825) and rates of photorespiration (de Veau and Burris (1989) Plant Physiol 90: 500-511) in Z. mays at different stages of development were analyzed using mathematical models of C4 photosynthesis. In young and senescing leaves modeled values of rc and the CO2 partial pressure in bundle sheath cells (Cbs) were lower and fractional leakage of CO2 from bundle sheath cells (fL) was higher than in mature leaves. Diffusive resistance of bundle sheath cells of C4 plants was also evaluated by analyzing the response of photosynthetic rates to varying CO2 in Amaranthus edulis in which the C4 cycle was dysfunctional by chemical mutagenesis (Dever et al. (1995) J Exp Bot 46: 1363-1376) and in Sorghum bicolor, Panicum maximum and Panicum miliaceum in which the C4 cycle was chemically inhibited (Brown and Byrd (1993) Plant Physiol 103: 1183-1188). These analyses indicate that in mature leaves of C4 plants the values of rc are substantially lower (ca. 50-200 m(2) s mol(-1)) than previous suggested (ca. 500-1500 m(2) s mol(-1)) for C4 photosynthesis and that there is considerable leakage of CO2 from bundle sheath cells. Nevertheless, rc and Cbs values are sufficiently high in mature leaves to minimize photorespiration in C4 plants under normal levels of CO2.

Primary partitioning and storage of photosynthate in sucrose and starch in leaves of C4 plants

A procedure involving pulse labelling of leaves with 14CO2 was developed to measure the primary (initial) partitioning of photosynthate between sucrose and starch. Partitioning of photosynthate into sucrose and starch was determined in leaves of C4 plants and compared with the patterns of storage of carbon in these products during the light period. The ratio of primary partitioning into sucrose and starch varied from about 0.5 in those species that accumulated mostly starch in the leaves (Amaranthus edulis L., Atriplex spongiosa F. Muell. and Flaveria trinervia (Spreng.) C. Mohr) to about 8 in Eleusine indica (L.) Gaertn., which accumulated mostly sucrose. No label was detected in free glucose or fructose. Generally there was a reasonable link between the primary partitioning of photosynthate and the type of carbohydrate stored in the leaf during the day. However, the ratio of carbon initially partitioned into sucrose versus starch was about 3 to 4 times higher in leaves of NADP-malic enzyme-type monocotyledonous species compared with phosphoenolpyruvate carboxykinase-type species, although the ratio of sucrose to starch accumulated in leaves during the day was very similar in the two groups. Sucrose and starch were the principal carbohydrates accumulated in leaves during the day. None of the species examined contained significant amounts of fructan and only one species, Atriplex spongiosa, contained substantial amounts of hexose sugars. In most of the species studied, the proportion of photosynthate partitioned into starch was greater at the end of the day than at the beginning. With the exception of Flaveria trinervia, the rate of CO2 assimilation did not decline during the day, showing that, under our conditions, accumulation of carbohydrate in the leaves did not lead to feedback inhibition of photosynthesis in these C4 species.

The isolation and characterization of mutants of the C4 photosynthetic pathway

Abstract Three novel classes of the C4 plant Amaranthus edulis are described. (1) A mutant that contains less than 10% of the normal activity and protein of phosphoenol-pyruvate carboxylase. (2) A mutant that is deficient in NAD-malic enzyme activity, but contains the normal two subunits of the enzyme protein. (3) Four mutant plants that accumulate high concentrations of glycine following exposure to air. A common characteristic of all the mutant plants is that they grow poorly in air and exhibit very low rates of photosynthetic CO2 assimilation. The characteristics of the mutant lines are discussed with reference to our current knowledge of the regulation of the C4 photosynthesis pathway. The possibility that C4 plants undergo photorespiratory nitrogen and carbon metabolism when grown in air is considered in detail.

Effect of Carbonic Anhydrase Inhibition on Photosynthesis by Leaf Pieces of C3 and C4 Plants

The role of carbonic anhydrase (CA) in the photosynthesis of C3 and C4 plants was investigated by use of the effects of the CA inhibitor ethoxyzolamide on peeled and vacuum infiltrated leaf pieces. Two C4(Zea mays and Amaranthus edulis) and three C3 (Spinacea oleracea, Nicotiana rustica and Hordeum vulgare) species were studied. The results clearly showed that photosynthesis at limiting inorganic carbon was more severely inhibited in the C4 species compared with the C3 species. However, the degree of inhibition in the C4 species was less than might have been expected based on the rate limitation which the conversion of CO2 to HCO3- would impose on the provision of substrate to PEP carboxylase. The reasons for this lower than expected inhibition is discussed in terms of the lack of penetration of ethoxyzolamide, the diffusion of HCO3- through plasmodesmata and the possible existence of a plasma membrane CA activity.

C3–C4 intermediate photosynthesis: linking physiology to gene expression

C₃–C₄ intermediate photosynthesis: linking physiology to gene expression