Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : I. Leaf Anatomy, Photosynthetic Responses to O(2) and CO(2), and Activities of Key Enzymes in the C(3) and C(4) Pathways

Four species of the genus Flaveria, namely F. anomala, F. linearis, F. pubescens, and F. ramosissima, were identified as intermediate C₃-C₄ plants based on leaf anatomy, photosynthetic CO₂ compensation point, O₂ inhibition of photosynthesis, and activities of C₄ enzymes. F. anomala and F. ramosissima exhibit a distinct Kranz-like leaf anatomy, similar to that of the C₄ species F. trinervia, while the other C₃-C₄ intermediate Flaveria species possess a less differentiated Kranz-like leaf anatomy. Photosynthetic CO₂ compensation points of these intermediates at 30°C were very low relative to those of C₃ plants, ranging from 7 to 14 microliters per liter. In contrast to C₃ plants, net photosynthesis by the intermediates was not sensitive to O₂ concentrations below 5% and decreased relatively slowly with increasing O₂ concentration. Under similar conditions, the percentage inhibition of photosynthesis by 21% O₂ varied from 20% to 25% in the intermediates compared with 28% in Lycopersicon esculentum, a typical C₃ species. The inhibition of carboxylation efficiency by 21% O₂ varied from 17% for F. ramosissima to 46% for F. anomala and were intermediate between the C₄ (2% for F. trinervia) and C₃ (53% for L. esculentum) values. The intermediate Flaveria species, especially F. ramosissima, have substantial activities of the C₄ enzymes, phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, NADP-malic enzyme, and NADP-malate dehydrogenase, indicating potential for C₄ photosynthesis. It appears that these Flaveria species may be true biochemical C₃-C₄ intermediates.     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

The Influence of Leaf Age on C(4) Photosynthesis and the Accumulation of Inorganic Carbon in Flaveria trinervia, a C(4) Dicot

Characteristics of C₄ photosynthesis were examined in young, mid-age, and mature leaves of Flaveria trinervia (an NADP-malic enzyme-type C₄ dicot). The turnover of [4-¹⁴C] (malate plus aspartate) following a pulse with ¹⁴CO₂ was similar in leaves of different ages (apparent half-time of 18-25 seconds). However, the rate of ¹⁴CO₂ incorporation in mid-age leaves was about 1.5-fold higher than in young leaves, and about 2.5-fold higher than in mature leaves. The rate of ¹⁴CO₂ fixation was proportional to the total active pool of malate plus aspartate but was not correlated with the total photosynthetically derived inorganic carbon pool. The leaf's ability to concentrate inorganic carbon photosynthetically declined during leaf expansion, from 29 down to 7 nanomoles per milligram chlorophyll. Similarly, the active aspartate pool also declined during leaf expansion, from about 123 down to 20 nanomoles per milligram chlorophyll. Enhanced metabolism of aspartate to CO₂ and pyruvate in young leaves is suggested to facilitate the maintenance of high CO₂ levels in bundle sheath cells which are thought to have a higher conductance to CO₂.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : II. Inhibition of Apparent Photosynthesis by a Phosphoenolpyruvate Carboxylase Inhibitor

Hybrids have been made between species of Flaveria exhibiting varying levels of C₄ photosynthesis. The degree of C₄ photosynthesis expressed in four interspecific hybrids (Flaveria trinervia [C₄] × F. linearis [C₃-C₄], F. brownii [C₄-like] × F. linearis, and two three-species hybrids from F. trinervia × [F. brownii × F. linearis]) was estimated by inhibiting phosphoenolpyruvate carboxylase in vivo with 3,3-dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate (DCDP). The inhibitor was fed to detached leaves at a concentration of 4 mm, and apparent photosynthesis was measured at atmospheric levels of CO₂ and at 20 and 210 mL L⁻¹ of O₂. Photosynthesis at 210 mL L⁻¹ of O₂ was inhibited 32% by DCDP in F. linearis, by 60% in F. brownii, and by 87% in F. trinervia. Inhibition in the hybrids ranged from 38 to 52%. The inhibition of photosynthesis by 210 mL L⁻¹ of O₂ was increased when DCDP was used, except in the C₄ species, F. trinervia, in which photosynthesis was insensitive to O₂. Except for F. trinervia, control plants with less O₂ sensitivity (more C₄-like) exhibited a progressively greater change in O₂ inhibition of photosynthesis when treated with DCDP. This increased O₂ inhibition probably resulted from decreased CO₂ concentrations in bundle sheath cells due to inhibition of phosphoenolpyruvate carboxylase. The inhibition of photosynthesis by DCDP is concluded to underestimate the degree of C₄ photosynthesis in the interspecific hybrids because increased direct assimilation of atmospheric CO₂ by ribulose bisphosphate carboxylase may compensate for inhibition of phosphoenolpyruvate carboxylase.     

Photosynthesis in Flaveria brownii A.M. Powell : A C(4)-Like C(3)-C(4) Intermediate

Leaves of Flaveria brownii exhibited slightly higher amounts of oxygen inhibition of photosynthesis than the C₄ species, Flaveria trinervia, but considerably less than the C₃ species, Flaveria cronquistii. The photosynthetic responses to intercellular CO₂, light and leaf temperature were much more C₄-like than C₃-like, although 21% oxygen inhibited the photosynthetic rate, depending on conditions, up to 17% of the photosynthesis rate observed in 2% O₂. The quantum yield for CO₂ uptake in F. brownii was slightly higher than that for the C₄ species F. trinervia in 2% O₂, but not significantly different in 21% O₂. The quantum yield was inhibited 10% in the presence of 21% O₂ in F. brownii, yet no significant inhibition was observed in F. trinervia. An inhibition of 27% was observed for the quantum yield of F. cronquistii in the presence of 21% O₂. The photosynthetic response to very low intercellular CO₂ partial pressures exhibited a unique pattern in F. brownii, with a break in the linear slope observed at intercellular CO₂ partial pressure values between 15 and 20 μbar when analyzed in 21% O₂. No significant break was observed when analyzed in 2% O₂. When taken collectively, the gas-exchange results reported here are consistent with previous biochemical studies that report incomplete intercellular compartmentation of the C₃ and C₄ enzymes in this species, and suggest that F. brownii is an advanced, C₄-like C₃-C₄ intermediate.     

Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis

The capability to reassimilate CO₂ originating from intracellular decarboxylating processes connected with the photorespiratory glycolate pathway and-or decarboxylation of C₄ acids during C₄ photosynthesis has been investigated with four species of the genus Flaveria (Asteraceae). The C₃-C₄ intermediate species F. pubescens and F. anomala reassimilated CO₂ much more efficiently than the C₃ species F. cronquistii and, with respect to this feature, behaved similarly to the C₄ species F. trinervia. Therefore, under atmospheric conditions the intermediate species photorespired with rates only between 10–20% of that measured with F. cronquistii. At low oxygen concentrations (1,5%) the reassimilation potential of F. anomala approached that of F. trinervia and was distinct from that found with F. pubescens. The data are discussed with respect to a possible sequence of events during evolution of C₄ photosynthesis. If compared with related data for C₃-C₄ intermediate species from other genera they support the hypothesis that, during evolution of C₄ photosynthesis, an efficient capacity for CO₂ reassimilation evolved prior to a CO₂-concentrating mechanism.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - D reassimilation coefficient - R _n , R _t net, total CO₂ evolution as measured with 0.03 and 3% CO₂, respectively - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: I. Initial Products of CO(2) Assimilation and Levels of Whole Leaf C(4) Metabolites

Labeling patterns from ¹⁴CO₂ pulses to leaves and whole leaf metabolite contents were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. During the first one to two minutes of illumination, malate was the primary initial product of ¹⁴CO₂ assimiltion (about 77% of total ¹⁴C incorporated). After about 5 minutes of illumination, the proportion of initial label to aspartate increased from 16 to 66%, and then gradually declined during the following 7 to 10 minutes of illumination. Nutrition experiments showed that the increase in ¹⁴CO₂ partitioning to aspartate was delayed about 2.5 minutes in plants grown with limiting N, and was highly dampened in plants previously treated 10 to 12 days with ammonia as the sole N source. Measurements of C₄ leaf metabolites revealed several transients in metabolite pools during the first few minutes of illumination, and subsequently, more gradual adjustments in pool sizes. These include a large initial decrease in malate (about 1.6 micromoles per milligram chlorophyll) and a small initial decrease in pyruvate. There was a transient increase in alanine levels after 1 minute of illumination, which was followed by a gradual, prolonged decrease during the remainder of the induction period. Total leaf aspartate decreased initially, but temporarily doubled in amount between 5 and 10 minutes of illumination (after its surge as a primary product). These results are discussed in terms of a plausible sequence of metabolic events which lead to the formation of the intercellular metabolite gradients required in C₄ photosynthesis.     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: II. Metabolism of Products of CO(2) Fixation after Different Illumination Times

The metabolism of fixed ¹⁴CO₂ and the utilization of the C-4 carboxyl of malate and aspartate were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. Pulse/chase experiments indicated that both malate and aspartate appeared to function directly in the C₄ cycle at all times during the induction period (examined after 30 seconds, 5 minutes and 20 minutes illumination). However, the rate of loss of ¹⁴C-label from the C-4 position of malate plus aspartate was relatively slow after 30 seconds of illumination, compared to treatments after 5 or 20 minutes of illumination. Similarly, the appearance of label in other photosynthetic products (e.g. 3-phosphoglycerate, sugar phosphates, alanine) during the chase periods was generally slower after only 30 seconds of leaf illumination, compared to that after 5 of 20 minutes illumination. This may be due to the lower rate of photosynthesis after 30 seconds illumination. The appearance of label in carbons 1→3 of each C₄ acid during the chase periods was relatively slow after either 30 seconds or 5 minutes illumination, while there was a relatively rapid accumulation of label in carbons 1→3 of both C₄ acids after 20 minutes illumination. Thus, while the turnover rate of the ¹⁴C-4 label in both C₄ acids increased only during the first 5 minutes of the induction period, only later during induction is there an increased rate of appearance of label in other carbon atoms of the C₄ acids. The implied source of ¹⁴C for labeling of the 1→3 positions of the C₄ acids is an apparent carbon flux from 3-phosphoglycerate of the reductive pentose phosphate pathway to phosphoenolpyruvate of the C₄ cycle.     

Transfer of C(4) Photosynthetic Characters through Hybridization of Flaveria Species

Transfer of C₄ photosynthetic traits was studied through hybridization of Flaveria trinervia (Spreng.) Mohr (C₄) and Flaveria brownii A.M. Powell (C₄-like) with Flaveria linearis Lag. (C₃-C₄) and the C₃ species Flaveria pringlei Gandoger (C₃). Fertility was low, based on irregular chromosome pairing and low pollen stainability, except in F. brownii × F. linearis which had bivalent pairing and 76% stainable pollen. Hybrids had apparent photosynthesis values of 71 to 148% of the midparental means, while the CO₂ compensation concentration was similar to the C₄ or C₄-like parent, except in hybrids having the C₃ species F. pringlei as a parent. Inhibition of apparent photosynthesis by O₂, and phosphoenolpyruvate carboxylase and NADP-malic enzyme activities and subunit levels in the hybrids were closer to the C₃ or C₃-C₄ parent. The species F. brownii and F. trinervia were equal in their capacity to transfer reduced O₂ inhibition of AP and CO₂ compensation concentration values to hybrids with F. linearis (C₃-C₄), although hybrids with F. trinervia had higher PEPC activity. The O₂ inhibition of AP was correlated with the logarithm of activities of phosphoenolpyruvate carboxylase (r = −0.95) and NADP-malic enzyme (r = −0.87). These results confirm that C₄ traits can be transferred by hybridization of C₃-C₄ and C₄ or C₄-like species, with a higher degree of C₄ photosynthesis than exists in C₃-C₄ species, and at least in F. brownii × F. linearis, fertile progeny are obtained.     

Enzymic and Photosynthetic Characteristics of Reciprocal F(1) Hybrids of Flaveria pringlei (C(3)) and Flaveria brownii (C(4)-Like Species)

The activities of key C₄ enzymes in gel-filtered, whole-leaf extracts and the photosynthetic characteristics for reciprocal F₁ hybrids of Flaveria pringlei (C₃) and F. brownii (C₄-like species) were measured to determine whether any inherited C₄-photosynthetic traits are responsible for their reduced CO₂ compensation concentration values (AS Holaday, S Talkmitt, ME Doohan Plant Sci 41: 31-39). The activities of phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and NADP-malic enzyme (ME) for the reciprocal hybrids are only about 7 to 17% of those for F. brownii, but are three- to fivefold greater than the activities for F. pringlei. The low activities of these enzymes in the hybrids appear to be the result of a partial dominance of F. pringlei genes over certain F. brownii genes. However, no such dominance occurs with respect to the expression of genes for NADP-malate dehydrogenase, which is as active in the hybrids as in F. brownii. In contrast to the situation with the enzymes above, cytoplasmic factors appear to determine the inheritance of NAD-ME. The NAD-ME activity in each hybrid is comparable to that in the respective maternal parent. Pulse-chase ¹⁴CO₂ incorporation analyses at ambient CO₂ levels indicate that the hybrids initially assimilate 7 to 9% of the total assimilated CO₂ into C₄ acids as compared to 3.5% for F. pringlei. In the hybrids, the percentage of ¹⁴C in malate decreases from an average of 6.5 to 2.1% after a 60-second chase in ¹²CO₂/air. However, this apparent C₄-cycle activity is too limited or inefficient to substantially alter CO₂ exchange from that in F. pringlei, since the values of net photosynthesis and O₂ inhibition of photosynthesis are similar for the hybrids and F. pringlei. Also, the ratio of the internal to the external CO₂ concentration and the initial slopes of the plot of CO₂ concentration versus net photosynthesis are essentially the same for the hybrids and F. pringlei. At 45 micromoles CO₂ per mole and 0.21 mole O₂ per mole, the hybrids assimilate nearly fivefold more CO₂ into C₄ acids than does F. pringlei. Some turnover of the malate pool occurs in the hybrids, but the labelling of the photorespiratory metabolites, glycine and serine, is the same in these plants as it is in F. pringlei. Thus, although limited C₄-acid metabolism may operate in the hybrids, we conclude that it is not effective in altering O₂ inhibition of CO₂ assimilation. The ability of the hybrids to assimilate more CO₂ via phosphoenolpyruvate carboxylase at low levels of CO₂ than does F. pringlei may result in an increased rate of reassimilation of photorespiratory CO₂ and CO₂ compensation concentrations below that of their C₃ parent. If the hybrids do possess a limited C₄ cycle, it must operate intracellularly. They are not likely to have inherited an intercellular compartmentation of C₄ enzymes, since F. brownii has incomplete compartmentation of key C₃ and C₄ enzymes.     

Degree of C(4) Photosynthesis in C(4) and C(3)-C(4)Flaveria Species and Their Hybrids : I. CO(2) Assimilation and Metabolism and Activities of Phosphoenolpyruvate Carboxylase and NADP-Malic Enzyme

The degree of C₄ photosynthesis was assessed in four hybrids among C₄, C₄-like, and C₃-C₄ species in the genus Flaveria using ¹⁴C labeling, CO₂ exchange, ¹³C discrimination, and C₄ enzyme activities. The hybrids incorporated from 57 to 88% of the ¹⁴C assimilated in a 10-s exposure into C₄ acids compared with 26% for the C₃-C₄ species Flaveria linearis, 91% for the C₄ species Flaveria trinervia, and 87% for the C₄-like Flaveria brownii. Those plants with high percentages of ¹⁴C initially fixed into C₄ acids also metabolized the C₄ acids quickly, and the percentage of ¹⁴C in 3-phosphoglyceric acid plus sugar phosphates increased for at least a 30-s exposure to ¹²CO₂. This indicated a high degree of coordination between the carbon accumulation and reduction phases of the C₄ and C₃ cycles. Synthesis and metabolism of C₄ acids by the species and their hybrids were highly and linearly correlated with discrimination against ¹³C. The relationship of ¹³C discrimination or ¹⁴C metabolism to O₂ inhibition of photosynthesis was curvilinear, changing more rapidly at C₄-like values of ¹⁴C metabolism and ¹³C discrimination. Incorporation of initial ¹⁴C into C₄ acids showed a biphasic increase with increased activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme (steep at low activities), but turnover of C₄ acids was linearly related to NADP-malic enzyme activity. Several other traits were closely related to the in vitro activity of NADP-malic enzyme but not phosphoenolpyruvate carboxylase. The data indicate that the hybrids have variable degrees of C₄ photosynthesis but that the carbon accumulation and reduction portions of the C₄ and C₃ cycles are well coordinated.     

Flaveria pringlei (C3) andFlaveria trinervia (C4) under NaCl stress

The C₄ speciesFlaveria trinervia is obviously better adapted to saline environments than the C₃ speciesF. pringlei. Treatment with 100 mM NaCl diminished crop growth rate inF. pringlei by 38% but not inF. trinervia. Under saline conditions, more assimilates were invested in leaf growth inF. trinervia but not inF. pringlei. Electrolyte concentration inF. trinervia in control and salt treated plants is lower than inF. pringlei. Fluorescence data do not indicate a damage of PS 2 charge separation in both species. Whether the C₄ photosynthetic pathway inF. trinervia is responsible for the improved salt tolerance compared toF. pringlei remains an open question.     

Interspecific variation in assimilation of 14CO2 into C4 acids by leaves of C3, C4 and C3−C4 intermediate Flaveria species near the CO2 compensation concentration

The assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of C₃, C₄ and C₃–C₄ intermediate Flaveria species was investigated near the CO₂ compensation concentration ^* in order to determine the potential role of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) in reducing photorespiration in the intermediates. Relative to air concentrations of CO₂, the proportion of CO₂ fixed by PEP carboxylase at ^* increased in all six C₃–C₄ intermediate species examined. However, F. floridana J.R. Johnston and F. ramosissima Klatt were shown to be markedly less responsive to reduced external CO₂, with only about a 1.6-fold enhancement of CO₂ assimilation by PEP carboxylase, as compared to a 3.0- to 3.7-fold increase for the other C₃–C₄ species examined, namely, F. linearis Lag., F. anomala B.L. Robinson, F. chloraefolia A. Gray and F. pubescens Rydb. The C₃ species F. pringlei Gandoger and F. cronquistii A.M. Powell exhibited a 1.5- and 2.9-fold increase in labeled malate and aspartate, respectively, at ^*. Assimilation of CO₂ by PEP carboxylase in the C₄ species F. trinervia (Spreng.) C. Mohr, F. australasica Hook., and the C₄-like species F. brownii A.M. Powell was relatively insensitive to subatmospheric levels of CO₂. The interspecific variation among the intermediate Flaverias may signify that F. floridana and F. ramosissima possess a more C₄-like compartmentation of PEP carboxylase and ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) between the mesophyll and bundle-sheath cells. Chasing recently labeled malate and aspartate with ¹²CO₂ for 5 min at ^* resulted in an apparent turnover of 25% and 30% of the radiocarbon in these C₄ acids for F. ramosissima and F. floridana, respectively. No substantial turnover was detected for F. linearis, F. anomala, F. chloraefolia or F. pubescens. With the exception of F. floridana and F. ramosissima, it is unlikely that enhanced CO₂ fixation by PEP carboxylase at the CO₂ compensation concentration is a major mechanism for reducing photorespiration in the intermediate Flaveria species. Moreover, these findings support previous related ¹⁴CO₂-labeling studies at air-levels of CO₂ which indicated that F. floridana and F. ramosissima were more C₄-like intermediate species. This is further substantiated by the demonstration that F. floridana PEP carboxylase, like the enzyme in C₄ plants, undergoes a substantial activation (2.2-fold) upon illuminating dark-adapted green leaves. In contrast, light activation was not observed for the enzyme in F. linearis or F. chloraefolia.Abbreviations and symbols PEP phosphoenolpyruvate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - CO₂ compensation concentration - ^* a subatmospheric level of CO₂ approximating Published as Paper No. 8832, Journal Series, Nebraska Agricultural Research Division     

Photosynthetic Characteristics of the C(3)-C(4) Intermediate Parthenium hysterophorus

The weedy species Parthenium hysterophorus (Asteraceae) possesses a Kranz-like leaf anatomy. The bundle sheath cells are thick-walled and contain numerous granal chloroplasts, prominent mitochondria, and peroxisomes, all largely arranged in a centripetal position. Both mesophyll and bundle sheath chloroplasts accumulate starch. P. hysterophorus exhibits reduced photorespiration as indicated by a moderately low CO₂ compensation concentration (20-25 microliters per liter at 30°C and 21% O₂) and by a reduced sensitivity of net photosynthesis to 21% O₂. In contrast, the related C₃ species P. incanum and P. argentatum (guayule) lack Kranz anatomy, have higher CO₂ compensation concentrations (about 55 microliters per liter), and show a greater inhibition of photosynthesis by 21% O₂. Furthermore, in P. hysterophorus the CO₂ compensation concentration is relatively less sensitive to changes in O₂ concentrations and shows a biphasic response to changing O₂, with a transition point at about 11% O₂. Based on these results, P. hysterophorus is classified as a C₃-C₄ intermediate. The activities of diagnostic enzymes of C₄ photosynthesis in P. hysterophorus were very low, comparable to those observed in the C₃ species P. incanum (e.g. phosphoenolpyruvate carboxylase activity of 10-29 micromoles per milligram of chlorophyll per hour). Exposures of leaves of each species to ¹⁴CO₂ (for 8 seconds) in the light resulted in 3-phosphoglycerate and sugar phosphates being the predominant initial ¹⁴C products (77-84%), with ≤4% of the ¹⁴C-label in malate plus aspartate. These results indicate that in the C₃-C₄ intermediate P. hysterophorus, the reduction in leaf photorespiration cannot be attributed to C₄ photosynthesis.     

Photosynthesis in Flaveria brownii, a C(4)-Like Species: Leaf Anatomy, Characteristics of CO(2) Exchange, Compartmentation of Photosynthetic Enzymes, and Metabolism of CO(2)

Light microscopic examination of leaf cross-sections showed that Flaveria brownii A. M. Powell exhibits Kranz anatomy, in which distinct, chloroplast-containing bundle sheath cells are surrounded by two types of mesophyll cells. Smaller mesophyll cells containing many chloroplasts are arranged around the bundle sheath cells. Larger, spongy mesophyll cells, having fewer chloroplasts, are located between the smaller mesophyll cells and the epidermis. F. brownii has very low CO₂ compensation points at different O₂ levels, which is typical of C₄ plants, yet it does show about 4% inhibition of net photosynthesis by 21% O₂ at 30°C. Protoplasts of the three photosynthetic leaf cell types were isolated according to relative differences in their buoyant densities. On a chlorophyll basis, the activities of phosphoenolpyruvate carboxylase and pyruvate, Pi dikinase (carboxylation phase of C₄ pathway) were highest in the larger mesophyll protoplasts, intermediate in the smaller mesophyll protoplasts, and lowest, but still present, in the bundle sheath protoplasts. In contrast, activities of ribulose 1,5-bisphosphate carboxylase, other C₃ cycle enzymes, and NADP-malic enzyme showed a reverse gradation, although there were significant activities of these enzymes in mesophyll cells. As indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the banding pattern of certain polypeptides of the total soluble proteins from the three cell types also supported the distribution pattern obtained by activity assays of these enzymes. Analysis of initial ¹⁴C products in whole leaves and extrapolation of pulse-labeling curves to zero time indicated that about 80% of the CO₂ is fixed into C₄ acids (malate and aspartate), whereas about 20% of the CO₂ directly enters the C₃ cycle. This is consistent with the high activity of enzymes for CO₂ fixation by the C₄ pathway and the substantial activity of enzymes of the C₃ cycle in the mesophyll cells. Therefore, F. brownii appears to have some capacity for C₃ photosynthesis in the mesophyll cells and should be considered a C₄-like species.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Expression of C4-like photosynthesis in several species of Flaveria

Abstract Photosynthetic metabolism was investigated in leaves of five species of Flaveria (Asteraceac), all previously considered to be C₄ plants. Leaves were exposed to ¹⁴CO₂ for different intervals up to 16s. Extrapolation of ¹⁴C-product curves to zero time indicated that only F. trinervia and F.bidentis assimilated atmospheric CO₂ exclusively through phosphoenolpyruvate carboxylase. The proportion of direct fixation of ¹⁴CO₂ by ribulose-I, 5-bisphosphate carboxylase/oxygenase (Rubisco) ranged from 5 to 10% in leaves of F. australasica. F. palmeri and F. vaginata. Protoplasts of leaf mesophyll and bundle sheath cells were utilized to examine the intercellular compartmentation of principal photosynthetic enzymes. Leaves of F. australasica, F. palmeri and F. vaginata contained 5 to 7% of the leaf's Rubisco activity in the mesophyll cells, while leaves of F. trinervia and F. bidentis contained at most 0.2 to 0.8% of such activity in their mesophyll cells. Thus, F. trinervia and F. bidentis have the complete C₄ syndrome, while F. australasica, F. palmeri and F. vaginata are less advanced, C₄-like species.     

Photosynthetic/Photorespiratory Carbon Metabolism in the C(3)-C(4) Intermediate Species, Moricandia arvensis and Panicum milioides

The distribution of ¹⁴C in photosynthetic metabolites of two naturally occurring higher plants with reduced photorespiration, Moricandia arvensis and Panicum milioides, in pulse and pulse-chase ¹⁴CO₂ incorporation experiments was similar to that for the C₃ species, M. foetida and Glycine max. After 6 seconds of ¹⁴CO₂ incorporation, only about 6% of the total ¹⁴C fixed was in malate and aspartate in both M. arvensis and P. milioides. The apparent turnover of the C₄ acids was very slow, and malate accumulated during the day in M. arvensis. Thus, C₄ acid metabolism by M. arvensis and P. milioides had no significant role in photosynthetic carbon assimilation under the conditions of our experiments (310 microliters CO₂ per liter, 21% O₂, 1100 or 1900 micromoles photon per square meter per second, 27°C).

After a 36-second chase period in air containing 270 microliters CO₂ per liter, about 20% of the total ¹⁴C fixed was in glycine with M. arvensis, as compared to 15% with M. foetida, 14% with P. milioides, and 9% with G. max. After a 36-second chase period in 100 microliters CO₂ per liter, the percentage in glycine was about twice that at 270 microliters CO₂ per liter in the C₃ species and P. milioides, but only 20% more ¹⁴C was in glycine in M. arvensis. These data suggest that either the photorespiratory glycine pool in M. arvensis is larger than in the other species examined or the apparent turnover rate of glycine and the flow of carbon into glycine during photorespiration are less in M. arvensis. An unusual glycine metabolism in M. arvensis may be linked to the mechanism of photorespiratory reduction in this crucifer.

     

An examination of the advantages of C3-C4 intermediate photosynthesis in warm environments

Abstract. The photosynthetic responses to temperature in C₃, C₃-C₄ intermediate, and C₄ species in the genus Flaveria were examined in an effort to identify whether the reduced photorespiration rates characteristic of C₃-C₄ intermediate photosynthesis result in adaptive advantages at warm leaf temperatures. Reduced photorespiration rates were reflected in lower CO₂ compensation points at all temperatures examined in the C₃-C₄ intermediate, Flaveria floridana, compared to the C₃ species, F. cronquistii. The C₃-C₄ intermediate, F. floridana, exhibited a C₃-like photosynthetic temperature dependence, except for relatively higher photosynthesis rates at warm leaf temperatures compared to the C₃ species, F. cronquistii. Using models of C₃ and C₃-C₄ intermediate photosynthesis, it was predicted that by recycling photorespired CO₂ in bundle-sheath cells, as occurs in many C₃-C₄ intermediates, photosynthesis rates at 35°C could be increased by 28%, compared to a C₃ plant. Without recycling photorespired CO₂, it was calculated that in order to improve photosynthesis rates at 35°C by this amount in C₃ plants, (1) intercellular CO₂ partial pressures would have to be increased from 25 to 31 Pa, resulting in a 57% decrease in water-use efficiency, or (2) the activity of RuBP carboxylase would have to be increased by 32%, resulting in a 22% decrease in nitrogen-use efficiency. In addition to the recycling of photorespired CO₂, leaves of F. floridana appear to effectively concentrate CO₂ at the active site of RuBP carboxylase, increasing the apparent carboxylation efficiency per unit of in vitro RuBP carboxylase activity. The CO₂-concentrating activity also appears to reduce the temperature sensitivity of the carboxylation efficiency in F. floridana compared to F. cronquistii. The carboxylation efficiency per unit of RuBP carboxylase activity decreased by only 38% in F. floridana, compared to 50% in F. cronquistii, as leaf temperature was raised from 25 to 35°C. The C₃-C₄ intermediate, F. ramosissima, exhibited a photosynthetic temperature temperature response curve that was more similar to the C₄ species, F. trinervia, than the C₃ species, F. cronquistii. The C₄-like pattern is probably related to the advanced nature of C₄-like biochemical traits in F. ramosissima The results demonstrate that reductions in photorespiration rates in C₃-C₄ intermediate plants create photosynthetic advantages at warm leaf temperatures that in C₃ plants could only be achieved through substantial costs to water-use efficiency and/or nitrogen-use efficiency.     

Environmental Effects on Photorespiration of C(3)-C(4) Species : I. Influence of CO(2) and O(2) during Growth on Photorespiratory Characteristics and Leaf Anatomy

The possibility of altering CO₂ exchange of C₃-C₄ species by growing them under various CO₂ and O₂ concentrations was examined. Growth under CO₂ concentrations of 100, 350, and 750 micromoles per mole had no significant effect on CO₂ exchange characteristics or leaf anatomy of Flaveria pringlei (C₃), Flaveria floridana (C₃-C₄), or Flaveria trinervia (C₄). Carboxylation efficiency and CO₂ compensation concentrations in leaves of F. floridana developed under the different CO₂ concentrations were intermediate to F. pringlei and F. trinervia. When grown for 12 days at an O₂ concentration of 20 millimoles per mole, apparent photosynthesis was strongly inhibited in Panicum milioides (C₃-C₄) and to a lesser degree in Panicum laxum (C₃). In P. milioides, acute starch buildup was observed microscopically in both mesophyll and bundle sheath cells. Even after only 4 days exposure to 20 millimoles per mole O₂, the presence of starch was more pronounced in leaf cross-sections of P. milioides compared to those at 100 and 210 millimoles per mole. Even though this observation suggests that P. milioides has a different response to low O₂ with respect to translocation of photosynthate or sink activity than C₃ species, the concentration of total available carbohydrate increased in shoots of all species by 33% or more when grown at low O₂. This accumulation occurred even though relative growth rates of Festuca arundinacea (C₃) and P. milioides grown for 4 days at 210 millimoles per mole O₂, were inhibited 83 and 37%, respectively, when compared to plants grown at 20 millimoles per mole O₂.