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.     

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.     

CO(2) Exchange, Cytogenetics, and Leaf Anatomy of Hybrids between Photosynthetically Distinct Flaveria Species

Hybrids between the C₄-like species, Flaveria brownii, A. M. Powell and the C₃-C₄ intermediate species Flaveria linearis Lag., Flaveria floridana Johnston, and Flaveria oppositifolia (DC.) Rydb. exhibited bivalent chromosome pairing during meiosis and stainability of pollen was high, ranging from 51 to 95%. An F₂ population produced from an F. brownii × F. linearis F₁ hybrid, exhibited bivalent chromosome pairing and high pollen stainability indicating a high degree of fertility in the hybrid. Oxygen inhibition of apparent photosynthesis averaged 6.8% for F. brownii and 22.2% for the C₃-C₄ species (in two experiments), and F₁ hybrids exhibited inhibitions which were intermediate to their parents. Values of carbon dioxide compensation concentration determined at low irradiance were 4.0, 34.0, and 6.5 microliters per liter for F. brownii, F. linearis and their F₁ hybrid, respectively. The mean value at low irradiance for 33 F₁ plants was 6.8 microliters per liter, and individual values ranged only from 3.7 to 11.7 microliters per liter. Anatomical characteristics for the F₁ hybrid leaves were intermediate to those of the parents, and there was considerable variation among F₂ plants derived from F. brownii × F. linearis. In the F₂ population δ¹³C values ranged from −27‰ to −20‰. The expression of more C₄-like characteristics by the F₁ hybrids in this study and their apparent high fertility make them promising specimens for producing segregating populations for use in C₄ inheritance studies.     

Cold stability of pyruvate,orthophosphate dikinase of Flaveria brownii

The nucleotide sequences of the complementary DNA of pyruvate, P_i dikinase (PPDK) from Flaveria bidentis, a C₄ plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a C₄-like plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.     

AM真菌影响入侵植物黄顶菊与本土物种狗尾草竞争生长的机理研究

该研究以入侵植物黄顶菊[Flaveria bidentis(L.)Kunt]和本土伴生植物狗尾草为材料,通过筛选出黄顶菊单一优势群落AM真菌,于温室盆栽条件下,采用2物种单播、混播以及接种AM真菌和不接种共6个处理,分析AM真菌对黄顶菊和狗尾草的根系侵染率、相对竞争强度、植株氮磷钾光合利用率、以及丙二醛含量和保护酶活性的影响,探讨AM真菌对黄顶菊与狗尾草竞争生长的机理。结果显示:(1)黄顶菊根际土壤AM真菌共包括4属10种,其中优势种为Glomus constrictum、Glomus perpusillum、Glomus reticulatum;盆栽接种AM真菌后,黄顶菊的根系侵染率显著高于本土伴生植物狗尾草,但接种AM真菌后黄顶菊相对竞争强度显著降低了29.57%,却对狗尾草相对竞争强度无显著影响。(2)接种AM真菌使黄顶菊植株光合氮、磷、钾利用率显著升高,但对伴生植物狗尾草的光合氮、磷、钾利用率均无显著影响。(3)接种AM真菌对黄顶菊植株POD和CAT活性以及MDA含量无显著影响,但显著增加了SOD和APX活性,而伴对生植物狗尾草的POD、CAT和APX活性均显著降低,MDA含量显著提高。研究表明,AM真菌对黄顶菊和狗尾草具有不同的选择性,AM真菌的定植促进了黄顶菊的竞争生长,增加了植株N、P含量、光合养分利用率以及抗氧化酶活性;但显著降低了本土伴生植物狗尾草的N、P吸收以及抗氧化酶活性。因此,AM真菌在竞争生长中对黄顶菊产生了偏利反馈,有助于黄顶菊的入侵。     

Water use efficiency inFlaveria andMoricandia species

The water use efficiency (WUE) of the C₃?C₄ intermediate speciesFlaveria anomala andF. pubescens was similar to that found inF. cronquistii (C₃). Compared to this values, the value inF. brownii (C₄-like) was significantly increased and was doubled inF. trinervia (C₄).Moricandia arvensis, a species with an enhanced CO₂ reassimilation potential has a very similar water use efficiency asM. moricandioides (C₃ but a lower transpiration rate.     

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.     

Kinetic properties of phosphoenolpyruvate carboxylase from c(3), c(4), and c(3)-c(4) intermediate species of flaveria (asteraceae)

Flaveria cronquistii (C₃), F. chloraefolia (C₃-C₄), F. floridana (C₃-C₄), F. pubescens (C₃-C₄), F. anomala (C₃-C₄), F. linearis (C₃-C₄), F. brownii (C₄), F. palmeri (C₄), F. trinervia (C₄) and F. australasica (C₄), comprising 10 out of the 21 known species of the genus Flaveria (Asteraceae), were included in a comparative study of the kinetic and regulatory properties of green leaf phosphoenolpyruvate (PEP) carboxylase. At least three kinetically distinct enzyme-forms were identified on the basis of their affinities for PEP and the degree of allosterism with respect to this substrate. The kinetic properties of PEP carboxylase of most of the species seemingly were modified in vivo depending on the growth conditions of the plants. K_m(PEP_free)-values of the enzyme from the five C₃-C₄ intermediate species ranged from 6 micromolar (F. chloraefolia, low light-grown) to 38 micromolar (F. pubescens, high light-grown). In contrast, the K_m for PEP of PEP carboxylase from the C₃ species F. cronquistii (13 micromolar) apparently was not influenced by growth conditions. The response of the enzyme from the C₃ and C₃-C₄ species was hyperbolic in all cases. A second isoform with a lower affinity for PEP (88-100 micromolar), but also hyperbolic kinetics was found in the C₄ species F. brownii, whereas in the three other C₄ species examined a PEP carboxylase with a still lower affinity for PEP (187-221 micromolar) and sigmoidal kinetics was present. These isozyme-related kinetic data were supported by analyses of the elution behavior of the enzyme during anion-exchange chromatography on DEAE-Trisacryl M. The results are discussed with respect to the evolution of C₄ photosynthesis in the Flaveria genus.     

Expression of a C3 plant Rubisco SSU gene in regenerated C4 Flaveria plants

We report the successful transformation, via Agrobacterium tumefaciens infection, and regeneration of two species of the genus Flaveria: F. brownii and F. palmeri. We document the expression of a C₃ plant gene, an abundantly expressed ribulose 1,5-bisphosphate carboxylase/oxygenase small subunit gene isolated from petunia, in these C₄ plants. The organ-specific expression of this petunia gene in Flaveria brownii is qualitatively identical to its endogenous pattern of expression.     

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.     

Exogenous Nitric Oxide (as Sodium Nitroprusside) Ameliorates Polyethylene Glycol-Induced Osmotic Stress in Hydroponically Grown Maize Roots

The present study was designed to examine whether exogenous sodium nitroprusside (SNP) supplementation has any ameliorating action against PEG-induced osmotic stress in Zea mays cv. FRB-73 roots. Twenty percent or 40 % polyethylene glycol (PEG6000; ?0.5 MPa and ?1.76 MPa, respectively) treatment alone or in combination with 150 and 300 μM SNP was applied to hydroponically grown maize roots for 72 h. Although only catalase (CAT) activity increased when maize roots were exposed to PEG-induced osmotic stress, induction of this antioxidant enzyme was inadequate to detoxify the extreme levels of reactive oxygen species, as evidenced by growth, water content, superoxide anion radical (O ₂ ^?? ), hydroxyl radical (OH^?) scavenging activity, and TBARS content. However, supplementation of PEG-exposed specimens with SNP significantly alleviated stress-induced damage through effective water management and enhancement of antioxidant defense markers including the enzymatic/non-enzymatic systems. Exogenously applied SNP under stress resulted in the up-regulation of glutathione peroxidase (GPX), glutathione S-transferase (GST), ascorbate peroxidase (APX), glutathione reductase (GR), total ascorbate, and glutathione contents involved in ascorbate–glutathione cycle. On the other hand, growth rate, osmotic potential, CAT, APX, GR, and GPX increased in maize roots exposed to both concentrations of SNP alone, but activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase decreased. Based on the above results, an exogenous supply of both 150 and 300 μM SNP to maize roots was protective for PEG-induced toxicity. The present study provides new insights into the mechanisms of SNP (NO donor) amelioration of PEG-induced osmotic stress damages in hydroponically grown maize roots.     

Geographic distributions and physiological characteristics of co-existing Flaveria species in south-central Mexico

The genus Flaveria consists of 23 species with significant variation in photosynthetic physiologies. We tested whether photosynthetic pathway variation in seven co-existing Flaveria species corresponds to geographic distributions or physiological performance in C₃, C₄, and intermediate species growing under natural conditions in south-central Mexico. We found that Flaveria pringlei (C₃) was the most widely distributed species with multiple growth habits. Numerous populations of Flaveria kochiana (C₄), a recently described species with a previously unknown distribution, were located in the Mixtec region of Oaxaca. Flaveria cronquistii (C₃) and Flaveria ramosissima (C₃-C₄) were only located in the Tehuacán Valley region while Flaveria trinervia (C₄) was widely distributed. Only one population of Flaveria angustifolia (C₃-C₄) and Flaveria vaginata (C₄-like) were located near Izúcar de Matamoros. Midday leaf water potential differed significantly between Flaveria species, but did not vary according to growth habit or photosynthetic pathway. The quantum yield of photosystem II did not vary between species, despite large differences in leaf nitrogen content, leaf shape, plant size and life histories. We did not find a direct relationship between increasing C₄ cycle characteristics and physiological performance in the Flaveria populations examined. Furthermore, C₃ species were not found at higher elevation than C₄ species as expected. Our observations indicate that life history traits and disturbance regime may be the primary controllers of Flaveria distributions in south-central Mexico.     

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.     

Getting the most out of natural variation in C4 photosynthesis

C₄ photosynthesis is a complex trait that has a high degree of natural variation, involving anatomical and biochemical changes relative to the ancestral C₃ state. It has evolved at least 66 times across a variety of lineages and the evolutionary route from C₃ to C₄ is likely conserved but not necessarily genetically identical. As such, a variety of C₄ species are needed to identify what is fundamental to the C₄ evolutionary process in a global context. In order to identify the genetic components of C₄ form and function, a number of species are used as genetic models. These include Zea mays (maize), Sorghum bicolor (sorghum), Setaria viridis (Setaria), Flaveria bidentis, and Cleome gynandra. Each of these species has different benefits and challenges associated with its use as a model organism. Here, we propose that RNA profiling of a large sampling of C₄, C₃–C₄, and C₃ species, from as many lineages as possible, will allow identification of candidate genes necessary and sufficient to confer C₄ anatomy and/or biochemistry. Furthermore, C₄ model species will play a critical role in the functional characterization of these candidate genes and identification of their regulatory elements, by providing a platform for transformation and through the use of gene expression profiles in mesophyll and bundle sheath cells and along the leaf developmental gradient. Efforts should be made to sequence the genomes of F. bidentis and C. gynandra and to develop congeneric C₃ species as genetic models for comparative studies. In combination, such resources would facilitate discovery of common and unique C₄ regulatory mechanisms across genera.     

Utilization of O2 in the metabolic optimization of C4 photosynthesis

The 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%.     

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.     

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.     

Anatomical and enzymic studies of leaves of a C3 × C4Flaveria F1 hybrid exhibiting reduced photorespiration

Flaveria pringlei exhibits C₃ CO₂ compensation concentration (Г) values averaging 53 μl CO₂/l at 21% (v/v) O₂ and 25 ± 2°C. When this species is hybridized with the C₄ species, F. brownii (male) ($\text{Г = 6 μ}\text{l CO}{}_2\text{/}\text{l}$), the F₁ hybrid plants exhibit an average Г value of 31 μl CO₂/l at 21% O₂.Although light micrographs of leaf cross-sections show that the leaves of the hybrid plants possess the mesophyll arrangement characteristic of F. pringlei leaves, the hybrid plants have some bundle-sheath chloroplasts. However, the numbers of these organelles do not appear to be intermediate with respect to the numbers in the parents and are closest to the small number present in the bundle-sheath cells of F. pringlei leaves. The activities of key C₄ enzymes (in μmol · mg Chl^?1 · h^?1) are: phosphoenolpyruvate (PEP) carboxylase, 121; pyruvate, orthophosphate (P_i) dikinase, 26; NADP-malate dehydrogenase, 2529; and NADP-malic enzyme, 82. All of these activities are substantially higher than in F. pringlei, but are only 7–10% of those in F. brownii (with the exception of the NADP-malate dehydrogenase activity). These data suggest that a C₄ cycle might be operating to a limited extent in the hybrid plants resulting in reduced photorespiration.Whether or not C₄ photosynthesis occurs in these hybrid plants, they represent the first reported C₃ × C₄ F₁ hybrids to exhibit reduced Γ-values. This cross and its reciprocal should be useful models for studying the anatomical and biochemical factors determining the development of limited C₄ photosynthesis in C₃ species.