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.     

Photosynthesis of F(1) Hybrids between C(4) and C(3)-C(4) Species of Flaveria

Photosynthetic characteristics were studied in several F₁ hybrids between C₄ and C₃-C₄ species of Flaveria. Stable carbon isotope ratios, O₂ inhibition of apparent photosynthesis, and phosphoenolpyruvate carboxylase activities in the hybrids were similar to the means for the parents. Values of CO₂ compensation concentrations were nearer to those of the C₄ parent and apparent photosynthesis was below that of both parents, being only 60 and 74% of that of the lowest (C₃-C₄) parent in two experiments. Reductions of CO₂ compensation concentration and O₂ inhibition of apparent photosynthesis as well as increases in carbon isotope ratios and phosphoenolpyruvate carboxylase activities compared to values in C₃-C₄ species suggest transfer of a limited degree of C₄ photosynthesis to the F₁ hybrids. However, the lower apparent photosynthesis of the hybrids suggests that transfer of C₄ characteristics to non-C₄ species is detrimental unless characteristics associated with C₄ photosynthesis are fully developed. There was a highly significant negative correlation (r = −0.90) between CO₂ compensation concentration and the logarithm of phosphoenolpyruvate carboxylase activity in the parents and hybrids, suggesting involvement of this enzyme in controlling the CO₂ compensation concentration. Although bundle-sheath cells were more developed in leaves of hybrids than in C₃-C₄ parents, they appeared to contain lower quantities of organelles than those of the C₄ parent. Reduced quantities of organelles in bundle-sheath cells could indicate incomplete compartmentation of partial pathways of the C₄ cycle in the hybrids. This may mean that the reduction of CO₂ compensation and O₂ inhibition of apparent photosynthesis relative to the C₃-C₄ parents is less dependent on fully developed Kranz anatomy than is increased apparent photosynthesis.     

Photosynthetic Characteristics of C3-C4 Intermediate Flaveria Species II. Kinetic Properties of Phosphoenolpyruvate Carboxylase from C3, C4 and C3-C4 Intermediate Species

The kinetic properties of phosphoenolpyruvate (PEP) carboxylasehave been studied among several Flaveria species: the C₃ speciesF. cronquistii, the C₃–C₄ species F. pubescens and F.linearis, and the C₄ species F. trinervia. At either pH 7 or8, the maximum activities (in µmol.mg Chl^–1.h^–1)for F. pubescens and linearis (187–513) were intermediateto those of the C₃ species (12–19) and the C₄ species(2,182–2,627). The response curves of velocity versusPEP concentration were hyperbolic for the C₃ and C₃–C₄species at either pH 7 or 8 while they were sigmoidal for theC₄ species at pH 7 and hyperbolic at pH 8. The K_m values forPEP determined from reciprocal plots were lowest in the C₃ species,and of intermediate value in the C₃–C₄ species comparedto the K' values of the C₄ species determined from Hill plotsat either pH 7 or 8. Glucose-6-phosphate (G6P) decreased theK_m values for PEP at both pH 7 and 8 in the C₃ and C₃–C₄species. In the C₄ species, G6P decreased the K' values at pH8 but increased the K' values at pH 7. In all cases, G6P hadits effect by influencing the activity at limiting PEP concentrationswith little or no effect on the maximum activity. At pH 8 andlimiting concentrations of PEP the degree of stimulation ofthe activity by G6P was greatest in the C₄ species, intermediatein F. linearis, a C₃–C₄ species, and lowest in the C₃species. In several respects, the PEP carboxylases of the C₃–C₄Flaveria species have properties intermediate to those of theC₃ and C₄ species. (Received April 30, 1983; Accepted August 22, 1983)     

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.     

Photosynthetic Characteristics of C(3)-C(4) Intermediate Flaveria Species : III. Reduction of Photorespiration by a Limited C(4) Pathway of Photosynthesis in Flaveria ramosissima

The initial products of photosynthesis by the C₃ species Flaveria cronquistii, the C₄ species F. trinervia, and the C₃-C₄ intermediate species F. ramosissima were determined using a pulse-chase technique with ¹⁴CO₂-¹²CO₂. The intermediate species F. ramosissima incorporated at least 42% of the total soluble ¹⁴C fixed into malate and aspartate after 10 seconds of photosynthesis in ¹⁴CO₂, as compared with 90% for the C₄ species F. trinervia and 5% for the C₃ species F. cronquistii. In both F. ramosissima and F. trinervia, turnover of labeled malate and aspartate occurred during a chase period in ¹²CO₂, although the rate of turnover was slower in the intermediate species. Relative to F. cronquistii, F. ramosissima showed a reduced incorporation of radioactivity into serine and glycine during the pulse period. These results indicate that a functional C₄ pathway of photosynthesis is operating in F. ramosissima which can account for its reduced level of photorespiration, and that this species is a true biochemical intermediate between C₃ and C₄ plants.     

Photosynthetic Plasticity in Flaveria brownii: Growth Irradiance and the Expression of C(4) Photosynthesis

Photosynthesis was examined in leaves of Flaveria brownii A. M. Powell, grown under either 14% or 100% full sunlight. In leaves of high light grown plants, the CO₂ compensation point and the inhibition of photosynthesis by 21% O₂ were significantly lower, while activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and various C₄ cycle enzymes were considerably higher than those in leaves grown in low light. Both the CO₂ compensation point and the degree of O₂ inhibition of apparent photosynthesis were relatively insensitive to the light intensity used during measurements with plants from either growth conditions. Partitioning of atmospheric CO₂ between Rubisco of the C₃ pathway and phosphoenolpyruvate carboxylase of the C₄ cycle was determined by exposing leaves to ¹⁴CO₂ for 3 to 16 seconds, and extrapolating the labeling curves of initial products to zero time. Results indicated that ~94% of the CO₂ was fixed by the C₄ cycle in high light grown plants, versus ~78% in low light grown plants. Thus, growth of F. brownii in high light increased the expressed level of C₄ photosynthesis. Consistent with the carbon partitioning patterns, photosynthetic enzyme activities (on a chlorophyll basis) in protoplasts from leaves of high light grown plants showed a more C₄-like pattern of compartmentation. Pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase were more enriched in the mesophyll cells, while NADP-malic enzyme and ribulose 1,5-bisphosphate carboxylase/oxygenase were relatively more abundant in the bundle sheath cells of high light than of low light grown plants. Thus, these results indicate that F. brownii has plasticity in its utilization of different pathways of carbon assimilation, depending on the light conditions during growth.     

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.     

Assimilatory sulfate reduction in C(3), C(3)-C(4), and C(4) species of Flaveria

The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5'-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C(4) monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C(4) photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C(3), C(3)-C(4), C(4)-like, and C(4) species of the dicot genus Flaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C(4)-like and C(4) species than in C(3) and C(3)-C(4) species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C(4) species Flaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C(4) plants and therefore is neither a prerequisite nor a consequence of C(4) photosynthesis.     

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.     

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.     

In vitro plant regeneration from leaf explants of C3 and C3-C4-intermediate Flaveria species

Summary A procedure is described for the invitro regeneration of whole plants of Flaveria cronquistii (C₃ species) F. pubescens and F. chloraefolia (both C₃-C₄ intermediate species) using different concentrations of 6-benzylaminopurine and alpha-napnthalenic acid.Abbreviations BAP 6-benzylaminopurine - NAA alpha-naphthalenic acid - MS medium Murashige-Skoog-medium     

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.     

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     

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.     

Activities of Principal Photosynthetic and Photorespiratory Enzymes in Leaf Mesophyll and Bundle Sheath Protoplasts from the C3-C4 Intermediate Flaveria ramosissima

Mesophyll and bundle sheath protoplasts were differentiallyisolated for the first time from leaves of a C₃-C₄ intermediate,Flaveria ramosissima. Protoplasts were partially purified fromleaf digests following differential centrifugation and flotationon dextran step-gradients. Two mesophyll and one bundle sheathfraction were obtained, with relative purities of the preparationsdetermined visually as >95% for mesophyll and >80% forbundle sheath. Representative C₃ and C₄ photosynthetic enzymes had substantialactivities, on a chlorophyll basis, in all three protoplastpreparations. The activity of phosphoenolpyruvate carboxylasewas highest in the lower density mesophyll fraction and lowestin the bundle sheath fraction. Conversely, the activity of NADP-malicenzyme was highest in the bundle sheath, and lowest in the lightermesophyll preparation. Ribulose 1,5-bisphosphate carboxylase/oxygenasehad similar activity in all three preparations, as did glycolateoxidase. However, glycine decarboxylase was about 3-fold enrichedin the bundle sheath fraction. The data indicate that the partialcompartmentation of photorespiratory metabolism may contributealong with limited C₄ photosynthesis to reducing photorespirationin this intermediate species. (Received April 27, 1988; Accepted June 17, 1988)     

Water-use efficiency and nitrogen-use efficiency of C(3) -C(4) intermediate species of Flaveria Juss. (Asteraceae)

Plants using the C(4) pathway of carbon metabolism are marked by greater photosynthetic water and nitrogen-use efficiencies (PWUE and PNUE, respectively) than C(3) species, but it is unclear to what extent this is the case in C(3) -C(4) intermediate species. In this study, we examined the PWUE and PNUE of 14 species of Flaveria Juss. (Asteraceae), including two C(3) , three C(4) and nine C(3) -C(4) species, the latter containing a gradient of C(4) -cycle activities (as determined by initial fixation of (14) C into C-4 acids). We found that PWUE, PNUE, leaf ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) content and intercellular CO(2) concentration in air (C(i) ) do not change gradually with C(4) -cycle activity. These traits were not significantly different between C(3) species and C(3) -C(4) species with less than 50% C(4) -cycle activity. C(4) -like intermediates with greater than 65% C(4) -cycle activity were not significantly different from plants with fully expressed C(4) photosynthesis. These results indicate that a gradual increase in C(4) -cycle activity has not resulted in a gradual change in PWUE, PNUE, intercellular CO(2) concentration and leaf Rubisco content towards C(4) levels in the intermediate species. Rather, these traits arose in a stepwise manner during the evolutionary transition to the C(4) -like intermediates, which are contained in two different clades within Flaveria.     

Quantity and Kinetic Properties of Ribulose 1,5-Bisphosphate Carboxylase in C3, C4, and C3-C4 Intermediate Species of Flaveria (Asteraceae)

The kinetic properties of ribulose 1,5-bisphosphate carboxylase(RuBPC) appear to have been modified during evolution of photosynthesisto adjust to changes in substrate availability. C₄ plants areconsidered to have a higher concentration of CO₂ available toRuBPC than C₃plants. In this study, the K_m(CO₂ and catalyticcapacity (k_cat) of RuBPC and the ratio of RuBPC protein to totalsoluble protein from several Flaveria species, including C₃,C₃-C₄ intermediate, and C₄ species, were determined. The C₃and intermediate species had similar K_m(CO₂) values while theC₄ species on average had higher K_m(CO₂) values. The mean ratioof K_cat/K_m for species of each group was similar, supportingthe hypothesis that changes in K_m and K_cat, are linked. Theallocation of total soluble protein to RuBPC was lowest in theC₄ Flaveria species, intermediate in the C₃-C₄ species, andhighest in the C₃ species. The results suggest that during evolutionof C₄ photosynthesis adjustments may occur in the quantity ofRuBPC prior to changes in its kinetic properties. (Received January 4, 1989; Accepted April 11, 1989)     

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.     

Flaveria属C_4种和C_3-C_4中间型种杂交一代的CO_2交换特性

用Flaveria属不同的C_4种和C_3-C_4中间型种杂交,鉴定了杂种F_1 C_4光合CO_2的交换特性。结果表明:F.brownii(C_4)和F.floridana(C_3-C_4)正反交F_1表观光合强度在两亲值之间,有的植株偏向C_4亲本,有的偏向C_3-C_4亲本。利用这种偏向分布可从F_1中选出高光合能力的材料。从F.palmeri(C_4)和F.pubescens(C_3-C_4);F.brownii(C_4)和F.floridana(C_3-C_4);F.palmeri(C_4)和F.floridana(C_3-C_4);F.trineroia(C_4)和F.anomala(C_3-C_4)的正反交,可以看出:用C_4作父本,杂种F_1具有类似C_4种的耐高温的光合特性。从O_2抑光合反应看,F_1接近中亲值,稍偏向C_3-C_4亲本。CO_2补偿点却与C_4亲本相似,不因正反交而发生偏向分布。通过遗传操作,有可能将C_4光合特性传递给C_2植物。     

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.