Salsola arbusculiformis and S. laricifolia (Chenopodiaceae) in China

To examine diagnostic characters and test phylogenetic relationships between Salsola arbusculiformis and the closely similar S. laricifolia, characters were examined from previous reports and herbarium specimens, and sequences of nrDNA ITS and cpDNA psbB–psbH were compared for these species as well as other representative taxa of Salsoleae. Our results 1) confirm most of the morphological differences highlighted in previous studies, such as those of leaf size, bracts, bracteoles, style, fruiting perianth, and anther appendages; 2) show that S. arbusculiformis is a typical Irano–Turanian species, mainly distributed from Iran and Turkmenistan to westernmost China (Tacheng, Ili, and Bole regions, Xinjiang), while S. laricifolia is a central Asian floristic element mainly distributed from east Kazakhstan and Kyrgyzstan to northern China (north Xinjiang, inner Mongolia, Ningxia and Gansu provinces), and Mongolia; and 3) strongly support their sister relationship, with samples from five different populations clustering together for each of the two species.     

Occurrence of C3 and C4 photosynthesis in cotyledons and leaves of Salsola species (Chenopodiaceae)

Most species of the genus Salsola (Chenopodiaceae) that have been examined exhibit C₄ photosynthesis in leaves. Four Salsola species from Central Asia were investigated in this study to determine the structural and functional relationships in photosynthesis of cotyledons compared to leaves, using anatomical (Kranz versus non-Kranz anatomy, chloroplast ultrastructure) and biochemical (activities of photosynthetic enzymes of the C₃ and C₄ pathways, ¹⁴C labeling of primary photosynthesis products and ¹³C/¹²C carbon isotope fractionation) criteria. The species included S. paulsenii from section Salsola, S. richteri from section Coccosalsola, S. laricina from section Caroxylon, and S. gemmascens from section Malpigipila. The results show that all four species have a C₄ type of photosynthesis in leaves with a Salsoloid type Kranz anatomy, whereas both C₃ and C₄ types of photosynthesis were found in cotyledons. S. paulsenii and S. richteri have NADP- (NADP-ME) C₄ type biochemistry with Salsoloid Kranz anatomy in both leaves and cotyledons. In S. laricina, both cotyledons and leaves have NAD-malic enzyme (NAD-ME) C₄ type photosynthesis; however, while the leaves have Salsoloid type Kranz anatomy, cotyledons have Atriplicoid type Kranz anatomy. In S. gemmascens, cotyledons exhibit C₃ type photosynthesis, while leaves perform NAD-ME type photosynthesis. Since the four species studied belong to different Salsola sections, this suggests that differences in photosynthetic types of leaves and cotyledons may be used as a basis or studies of the origin and evolution of C₄ photosynthesis in the family Chenopodiaceae.This revised version was published online in October 2005 with corrections to the Cover Date.     

Photosynthesizing Tissue Development in C4 Cotyledons of Two Salsola Species (Chenopodiaceae)

The quantitative anatomy of developing cotyledons of NAD-malic enzyme species Salsola incanescens and NADP-malic enzyme species S. paulsenii (Chenopodiaceae) was studied. S. incanescens belongs to the group of species with foliar type of seedling development characterized by slowly growing cotyledons and a rosette form at juvenility. The rosette is the consequence of fast leaf formation, which was correlated with a low rate of leaf growth. S. paulsenii belongs to the group with the cotyledonous type of seedling development. A high growth rate of cotyledons, slow leaf formation, and absence of the rosette characterize this type. Slow leaf formation was correlated with a high rate of leaf growth. The Kranz–anatomy in cotyledons of S. incanescens (atriplicoid type) and S. paulsenii (salsoloid type) determines the duration of cotyledon development proceeding for 15 days after seed germination. The rate of growth changes during the developmental period was correlated with the type of seedling development. Cotyledons of a foliar species S. incanescens exhibit 2 to 5 times slower growth changes in cotyledon area, width, thickness, volume of mesophyll and bundle sheath cells, and number of chloroplasts per bundle sheath cell than the cotyledons of a cotyledonous species S. paulsenii. During cotyledon development in both species, the number of chloroplasts per mesophyll cell remained unchanged, and developmental changes in the bundle sheath occurred at higher rate than in mesophyll cells. Thus, these two indices seem to be independent of the type of Kranz–anatomy. The presence of atriplicoid type cotyledons in the species with salsoloid structure of true leaves might indicate a close genetic relationship between these two patterns of Kranz-anatomy.     

C3、C4和C3-C4中间型植物的进化

介绍了有关C3、C4和C3-C4中间型植物进化的形态学、生理学、分子生物学、遗传学等方面的证据;推断地球上首先出现C3植物,然后是C3-C4中间类型植物,最后出现C4植物.     

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.     

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)     

C3 and C4 leaf anatomy types in Camphorosmeae (Camphorosmoideae,Chenopodiaceae)

Complementary to our previous project on the molecular phylogeny of Camphorosmeae, the leaf anatomy of ca. 35 species including all non-Australian and selected Australian species was studied by use of light microscopy. Nine anatomical leaf types were described, compared to previous classifications, and discussed with regard to their putative evolution on the background of phylogenetic trees. Particular emphasis was given to the relationships between the C₃ and C₄ leaf types: Chenolea type (C₃), Eokochia type (C₃), Neokochia type (C₃), Sedobassia type (C₃/C₄ intermediate), Bassia prostrata type (C₄), B. muricata type (C₄), B. eriantha type, B. lasiantha type (C₄), Camphorosma type (C₄). The main results and conclusions were: (1) Two unusual new C₃ leaf types: Chenolea with microfenestrate chlorenchyma, Eokochia with unique complex vascular bundles; (2) Sedobassia interpreted as anatomically C₃/C₄ intermediate by kranz-like bundle sheath cells is the first C₃/C₄ intermediate in Camphorosmeae and found in a derived position; (3) Neokochia type detected as the likely starting point for all four C₄ leaf types and for the C₃/C₄ intermediate; (4) hypodermis of C₄ types originated from outermost chlorenchyma layer of C₃ types and lost multiple times during further evolution; (5) atriplicoid Bassia. lasiantha type without water storage tissue evolved from kochioid B. muricata type; (6) two independent gains of C₄ photosynthesis, one in Bassia and one in Camphorosma; (7) depending on the lineage, leaf architecture remains comparatively stable (Australian Camphorosmeae) or shows an unexpected plasticity (Bassia scoparia group).     

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.     

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)     

Reduced Apparent Photorespiration by the C(3)-C(4) Intermediate Species, Moricandia arvensis and Panicum milioides

The CO₂/O₂ specificity factor of sucrose gradient purified ribulose 1,5-bisphosphate carboxylase/oxygenase from the C₃-C₄ intermediate plants Moricandia arvensis (79 ± 1) and Panicum milioides (89 ± 2) was similar to the respective values of the enzyme from the closely related C₃ species, Moricandia foetida (80 ± 5) and Panicum laxum (86 ± 2). Thus, the kinetic properties of this bifunctional enzyme do not explain the reduced rates of photorespiration exhibited by either of these intermediate 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.

     

Origin and evolution of C4 photosynthesis in the tribe Salsoleae (Chenopodiaceae) based on anatomical and biochemical types in leaves and cotyledons

 The Chenopodiaceae genus Salsola contains a large number of species with C₄ photosynthesis. Along with derivative genera they have a prominent position among the desert vegetation of Asia and Africa. About 130 species from Asia and Africa were investigated to determine the occurrence of C₃ versus C₄ syndrome in leaves and cotyledons, and to study specific anatomical and biochemical features of photosynthesis in both photosynthetic organs. The species studied belong to all six previously identified sections of the tribe Salsoleae based on morphological characters. Types of photosynthesis were identified using carbon ¹³C/¹²C isotope fractionation. The representatives of all systematic groups were investigated for mesophyll anatomy and biochemical subtypes by determination of enzyme activity (RUBPC, PEPC, NAD- and NADP-ME and AAT) and primary photosynthetic products. Two photosynthetic types (C₃ and C₄) and two biochemical subtypes (NAD- and NADP-ME) were identified in both leaves and cotyledons. Both Kranz and non-Kranz type anatomy were found in leaves and cotyledons, but cotyledons had more diversity in anatomical structure. Strong relationships between anatomical types and biochemical subtypes in leaves and cotyledons were shown. We found convincing evidence for a similar pattern of structural and biochemical features of photosynthesis in leaves and cotyledons within systematic groups, and evaluated their relevance at the evolutionary level. We identified six groups in tribe Salsoleae with respect to photosynthetic types and mesophyll structure in leaves and cotyledons. Two separate lineages of biochemical and anatomical evolution within Salsoleae were demonstrated based on studies of leaves and cotyledons. The sections Caroxylon, Malpighipila, Cardiandra and Belanthera have no C₃ species and only the NAD-ME C₄ subtype has been found in leaves. We suggest the C₄ species in the NADP-ME lineage evolved in Coccosalsola and Salsola sections, and originated in the subsection Arbuscula. Coccosalsola contains many species with C₃ and/or C₃-C₄ intermediate photosynthesis. Within these main evolutionary lineages, species of different taxonomic groups (sections and subsections) had differences in anatomical or/and biochemical features in leaves and cotyledons. We conclude that structural and biochemical changes in the photosynthetic apparatus in species of the tribe Salsoleae were a key factor in their evolution and broad distribution in extreme desert environments. Received January 25, 2001 Accepted July 17, 2001     

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.     

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.     

Anatomical types of leaves and assimilating shoots and carbon C/C isotope fractionation in Chinese representatives of Salsoleae s.l. (Chenopodiaceae)

To examine the anatomical types in Salsoleae s.l., and evaluate carbon isotope fractionation values for identifying the respective photosynthetic pathway, a total of 34 species representing 12 genera of Salsoleae s.l. in China were examined using light microscopy and carbon ¹³C/¹²C isotope fractionation. There are nine leaf anatomical types, namely, Sympegmoid (Sympegmoid type, Sympegmoid type II), Salsoloid with hypodermis (Salsola soda type, Salsola soda type II, Nanophyton type II), Salsoloid without hypodermis (Salsola kali type, Salsola kali type II, Nanophyton type, Climacoptera type II). Salsola soda type and Salsola soda type II are found in the assimilating shoots. Two new subtypes, Salsola soda type II and Nanophyton type II are reported. Anabasis brevifolia, A. eriopoda, A. elatior, A. truncata and A. salsa are of the Salsola soda type II, with a distinctive two-layered epidermis cells. Horaninowia ulicina is of the Nanophyton type II with hypodermis which distinguished from Nanophyton type; Both, Salsola kali type and Climacoptera type II exist in Climacoptera. The Climacoptera type II is distinguished from Climacoptera type by an adaxially interrupted Kranz layer. Salsola collina, S. zaidamica, S. praecox, S. pellucida and S. ruthenica in Salsola sect. Salsola have the Salsola kali type II. The Salsola kali type differs from Salsola kali type II having the palisade and Kranz cells interrupted by longitudinal collenchymatic ridges. Although carbon isotope fractionation data alone are already useful tools to identify photosynthesis, their determination in combination with other approaches, such as anatomical studies are necessary in order to render a structuring of all possibilities evolved among C₄ type Chenopodiaceae.     

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.     

Long-chain (C19-C29) 1-chloro-n-alkanes in leaf waxes of halophytes of the Chenopodiaceae

The hydrocarbon fraction of leaf waxes of three halophytes of the Chenopodiaceae common to Mediterranean salt marshes (Suaeda vera, Sarcocornia fruticosa and Halimione portulacoides) revealed the presence of a minor series of odd and even chains 1-chloro-n-alkanes ranging from C(19) to C(29). The identification of these new chlorinated plant constituents was based on a combination of mass spectrometry data with selective chlorine detection (CPG-AED) and was confirmed by comparison with authentic standards. The qualitative and quantitative distributions of these 1-chloro-n-alkanes varied inter-specifically. Homologues with an odd carbon-chain were predominant in all species but maximised at C(25) and C(27) in S. vera and S. fruticosa, and at C(27) and C(29) in H. portulacoides. Remarkably, 1-chloro-nonacosane was an abundant homologue only in this latter species. Leaves of S. vera contained 4 to 7 times more of total chloroalkanes than leaves of the other two species. These compounds accounted for 10, 4 and 1% of the hydrocarbon fraction of leaf waxes of S. vera, S. fruticosa and H. portulacoides, respectively. Attempts to link the occurrence of these chloroalkanes with other classes of leaf waxes (n-alkenes, n-aldehydes and n-alcohols) did not allowed a clear precursor-product relationship to be established. The biological functions as well as the mode of synthesis of alkylchlorides in (halophyte) plants remain unknown but undoubtedly deserve further attention.     

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.