Expression of the C3-C4 intermediate character in somatic hybrids between Brassica napus and the C3-C4 species Moricandia arvensis

The wild crucifer Moricandia arvensis is a potential source of alien genes for the genetic improvement of related Brassica crops. In particular M. arvensis has a C₃-C₄ intermediate photosynthetic mechanism which results in enhanced recapture of photorespired CO₂ and may increase plant water-use efficiency. In order to transfer this trait into Brassica napus, somatic hybridisations were made between leaf mesophyll protoplasts from cultured M. arvensis shoot tips and hypocotyl protoplasts from three Brassica napus cultivars, Ariana, Cobra and Westar. A total of 23 plants were recovered from fusion experiments and established in the greenhouse. A wide range of chromosome numbers were observed among the regenerated plants, including some apparent mixoploids. Thirteen of the regenerated plants were identified as nuclear hybrids between B. napus and M. arvensis on the basis of isozyme analysis. The phenotypes of these hybrids were typically rather B. napus-like, but much variability was observed, including variation in flower colour, leaf shape and colour, leaf waxiness, fertility and plant vigour. CO₂ compensation point measurements on the regenerated plants demonstrated that 3 of the hybrids express the M. arvensis C₃-C₄ intermediate character at the physiological level. Semi-thin sections through leaf tissues of these 3 plants revealed the presence of a Kranz-like leaf anatomy characteristic of M. arvensis but not found in B. napus. This is the first report of the expression of this potentially important agronomic trait, transferred from Moricandia, in M. arvensis x B. napus hybrids.     

Development of a strategy for transgenic studies and monitoring of transgene expression in two closely related Moricandia species possessing a C3 or C3–C4 intermediate photosynthetic phenotype

In order to establish a model system for comparative studies of C₃ and C₃–C₄ intermediate photosynthesis, the development of efficient transformation systems and the monitoring of transgene behaviour and stability were carried out in two closely related Moricandia species (Brassicaceae): the C₃–C₄ photosynthetic intermediate species M. arvensis and the C₃ species M. moricandioides. In this study the green fluorescent protein (gfp) reporter gene was used as a vital marker gene while the use of the β‐glucuronidase (gusA) gene was based on the highly sensitive detection of its activity. For Agrobacterium‐mediated transformation of leaf explants, a cauliflower mosaic virus 35S promoter‐driven, modified version of gfp, the mgfp5‐ER gene and the gusA gene, respectively, were introduced into the new dual binary transformation vector system pGreen/pSoup (Hellens et al. 2000, Plant Mol Bio 42: 819–832). GFP5 produced bright‐green fluorescence in transformed tissues that was distinctly detected 5–12 days following transformation in developing calli of the two species. Visual screening, combined with antibiotic selection, enabled early and easy identification of transformation events and contributed to improvements in the transformation strategies. Transgene integration studies demonstrated that mgfp5‐ER was inserted with low copy number in the M. arvensis plant lines and the transgene was transmitted in a Mendelian fashion to T₁ and T₂ progenies. GFP5 expression levels in a population of 100 independent primary transformed M. arvensis plant lines (T₀) showed great variation between transformation events (coefficient of variation of 108%). The mgfp5‐ER or gusA reporter genes were expressed in 90–95% of the kanamycin‐resistant M. arvensis plant lines and in up to 98% of the independent M. moricandioides plant lines.     

Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C₃-C₄ intermediate species Moricandia arvensis (L.) DC has been compared with that of the C₃ species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO₂ and hence a lower rate of photorespiration.Abbreviations kDa kilodalton - RuBP ribulose-1,5-bisphosphate     

Evidence from photosynthetic characteristics for the hybrid origin of Diplotaxis muralis from a C3-C4 intermediate and a C3 species

Artificial hybridization studies have been carried out between plants with different photosynthetic types to study the genetic mechanism of photosynthetic types. However, there are only few reports describing the possibility of natural hybridization between plants with different photosynthetic types. A previous cytological and morphological study suggested that a cruciferous allotetraploid species, Diplotaxis muralis (L.) DC. (2n = 42), originated from natural hybridization between D. tenuifolia (L.) DC. (2n = 22) and D. viminea (L.) DC. (2n = 20). These putative parents have recently been reported to be a C (3)-C (4) intermediate and a C (3) species, respectively. If this hybridization occurred, D. muralis should have characteristics intermediate between those of the C (3)-C (4) intermediate and C (3) types. We compared leaf structures and photosynthetic characteristics of the three species. The bundle sheath (BS) cells in D. tenuifolia included many centripetally located chloroplasts and mitochondria, but those of D. viminea had only a few organelles. The BS cells in D. muralis displayed intermediate features between the putative parents. Glycine decarboxylase P protein was confined to the BS mitochondria in D. tenuifolia, but accumulated mainly in the mesophyll mitochondria in D. viminea. In D. muralis, it accumulated in both the BS and the mesophyll mitochondria. Values of CO (2) compensation point and its response to changing light intensity were also intermediate between the putative parents. These data support the theory that D. muralis was created by natural hybridization between species with different photosynthetic types.     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

C3-C4 intermediate photosynthetic characteristics of cassava (Manihot esculenta Crantz)

Cassava, bean and maize leaves were fed with¹⁴CO₂ in light and the primary products of photosynthesis identified 5 and 10 seconds after assimilation. In maize, approximately three quarters of the labelled carbon was incorporated in C₄ acids, in beans about two thirds in PGA, and in cassava approximately 40–60% in C₄ acids with 30–50% in PGA. These data indicate that cassava possesses the C₄ photosynthetic cycle, however due to the lack of typical Kranz anatomy appreciable carbon assimilation takes place directly through the Calvin-Benson-Bassham cycle.     

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     

Photorespiratory properties of protoplasts from C3-C4 intermediate species of moricandia

Protoplasts were isolated from leaves of the C₃-C₄ intermediate species, Moricandia arvensis (L.) DC. and Moricandia spinosa Pomel. Analysis by light and transmission electron microscopy indicated that these purified preparations contained both mesophyll protoplasts (MP) and bundle-sheath protoplasts (BSP). Conventional density gradient centrifugation procedures failed to yield separations of pure protoplasts from each cell-type. With these heterogeneous suspensions of MP and BSP, values measured for (i) the percentage inhibition of photosynthetic CO₂ fixation by O₂, (ii) the apparent K_m(CO)2 of photosynthesis, and (iii) dark/light ratios of the rate of ¹⁴CO₂ evolution during decarboxylation of exogenous [1-¹⁴C] glycine were not significantly different from those determined for protoplast preparations from related or representative C₃ plants, including M. foetida, Nicotiana tabacum, and Triticum aestivum. In contrast, previous comparisons with C₃ species, using intact leaf tissue from M. arvensis, have shown a reduced sensitivity of net photosynthesis to inhibition by O₂ [Holaday et al., Plant Sci. Lett., 27 (1982) 181] and an enhanced capacity for the photosynthetic refixation of CO₂ evolved during decarboxylation of exogenous photorespiratory substrates [Holbrook et al., Plant Physiol., 77 (1985) 578]. We conclude that these photosynthetic properties, associated with reduced photorespiration by M. arvensis and M. spinosa, are dependent upon the integrity of the anatomical and ultrastructural arrangement of bundle-sheath and mesophyll cells in these C₃-C₄ intermediate species.     

Cellular expression pattern of the glycine decarboxylase P protein in leaves of an intergeneric hybrid between the C3-C4 intermediate species Moricandia nitens and the C3 species Brassica napus

 An intergeneric hybrid plant was produced between the C₃-C₄ intermediate species Moricandia nitens and the C₃ species Brassica napus by sexual hybridization and in vitro embryo rescue. The hybrid nature of the plant was apparent in its morphology and flower pigmentation and was confirmed by leaf isozyme patterns. The overall plant morphology and the shape and thickness of leaves of the hybrid plant were intermediate between those of the parent species. However, the bundle-sheath cells of the hybrid resembled those of the C₃ parent and lacked the organelle development of the C₃-C₄ intermediate parent. Immunogold labelling for the presence of the P subunit of the mitochondrial glycine decarboxylase complex revealed a very similar labelling density on mitochondria in bundle-sheath and mesophyll cells in B. napus, while in  M. nitens the P subunit was only detectable in bundle sheath cells. In the hybrid the labelling density on mesophyll cell mitochondria was almost half of that on the bundle-sheath mitochondria. The CO₂ compensation point of the hybrid was significantly less than that of the C₃ parent but was not as low, nor as responsive to changes in light intensity, as for the C₃-C₄ parent. Received: 23 October 1997 / Accepted: 28 November 1997     

Phosphoenolpyruvate carboxylase reduces photorespiration in Panicum milioides, a C3-C4 intermediate species.

Panicum milioides represents the first well-documented example of a higher plant species with reduced photorespiration and O₂ inhibition of photosynthesis. We have investigated the biochemical mechanism(s) involved in reducing O₂ sensitivity of photosynthesis in this species by parallel enzyme inhibitor experiments with thin leaf slices of P. milioides and C₃ and C₄Panicum species. The reduced O₂ sensitivity of net photosynthesis in P. milioides gradually increased with increasing concentrations of the phosphoenolpyruvate carboxylase (EC 4.1.1.31) inhibitors, maleate and malonate. At saturating levels of inhibitor, photosynthesis in 2% O₂ was decreased by about 18%, and the inhibitory effects of both 21% O₂ and 49% O₂ were identical to those observed with a C₃Panicum species in the absence or presence of inhibitor. A significant potential for C₄ photosynthesis in P. milioides, compared to its complete absence in a C₃Panicum species, was demonstrated on the basis of: (a) a coupling of leaf slice CO₂ fixation by phosphoenolpyruvate carboxylase with the C₃ cycle; (b) NAD-malic enzyme (EC 1.1.1.39)-dependent aspartate and malate decarboxylation in leaf slices; (c) a full complement of C₄ cycle enzymes in leaf extracts, including pyruvate, P_i dikinase (EC 2.7.9.1) and NAD-malic enzyme; and (d) Kranz-like leaf anatomy with numerous plasmodesmata traversing the mesophyll-bundle sheath interfacial cell wall. These data indicate that the reduced photorespiration and O₂ inhibition of photosynthesis in P. milioides is due to phosphoenolpyruvate carboxylase participation, possibly by creating a limited C₄-like CO₂ pump, rather than an altered ribulose 1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39).     

Effect of varying environments on photosynthetic parameters of C3, C3-C4 and C4 species of Panicum

Summary CO₂ exchange characteristics and the activity of the carboxylating enzymes phosphoenolpyruvate carboxylase (PEP-C, E.C. 4.1.1.31) and ribulose 1,5-bisphosphate carboxylase (RuBP-C, E.C. 4.1.1.39) during one year in the greenhouse and at two levels of light and temperature in growth chambers were determined in the C₃-C₄ intermediate species P. milioides Nees ex. Trin. These results were compared with those of P. bisulcatum Thumb. (C₃) and P. maximum Jacq. (C₄). Under all tested conditions, and even when the influence of leaf surface temperature on photosynthetic rates and CO₂ compensation points were measured, the biochemical and physiological behaviour of the C₃-C₄ intermediate was more similar to that of the C₃ plant than the C₄ species. The C₄ plant P. maximum, however, responded positively, mainly in terms of PEP-C activity and photosynthetic rate, to the regime of high light and temperature. The results presented indicate that in the C₃-C₄ Panicum grown in high light and temperature no direct relationships between a low CO₂ compesation point and superior growth are evident. It has still to be clarified why in nature a photosynthetic-photorespiratory pathway leading to an intermediate CO₂ compensation value has evolved in P. milioides.     

Photosynthetic,hydraulic and biomass properties in closely related C3 and C4 species

In plants, most water is absorbed by roots and transported through vascular conduits of xylem which evaporate from leaves during photosynthesis. As photosynthesis and transport processes are interconnected, it was hypothesized that any variation in water transport demand influencing water use efficiency (WUE), such as the evolution of C₄ photosynthesis, should affect xylem structure and function. Several studies have provided evidence for this hypothesis, but none has comprehensively compared photosynthetic, hydraulic and biomass allocation properties between C₃ and C₄ species. In this study, photosynthetic, hydraulic and biomass properties in a closely related C₃ Tarenaya hassleriana and a C₄ Cleome gynandra are compared. Light response curves, measured at 30°C, showed that the C₄ C. gynandra had almost twice greater net assimilation rates than the C₃ T. hassleriana under each increasing irradiation level. On the contrary, transpiration rates and stomatal conductance were around twice as high in the C₃, leading to approximately 3.5 times higher WUE in the C₄ compared with the C₃ species. The C₃ showed about 3.3 times higher hydraulic conductivity, 4.3 times greater specific conductivity and 2.6 times higher leaf‐specific conductivity than the C₄ species. The C₃ produced more vessels per xylem area and larger vessels. All of these differences resulted in different biomass properties, where the C₄ produced more biomass in general and had less root to shoot ratio than the C₃ species. These results are in support of our previous findings that WUE, and any changes that affect WUE, contribute to xylem evolution in plants.     

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     

Glycolate synthesis in a C3 C4 and intermediate photosynthetic plant type

Excised leaves of a C₃-photosynthetic type, Hordeum vulgare,a C₄-type, Panicum miliaceum, and an intermediate-type, Panicummilioides, were allowed to take up through their cut ends a1 mM solution of butyl hydroxybutynoate (BHB), an irreversibleinactivator of glycolate oxidase. After 30 to 60 min in BHB,extractable glycolate oxidase activity could not be detectedin the distal quarter of the leaf blades. Following this pretreatment,recovery of ¹⁴C-glycolate from ¹⁴CO₂ incorporated in a 10 minperiod was nearly maximal for each of the three plant types.Labeled glycolate was 51% of the total ¹⁴CO₂ incorporated forthe C₃-species, 36% for the intermediate-species, and 27% forthe C₄-species Increased labeling of glycolate was compensatedfor primarily by decreased labeling of the neutral and basicfractions for the C₃ and intermediate-type species. In the C₄-type,label decreased primarily in the neutral and insoluble fractions,but increased in the basic fraction. A lower rate of glycolatesynthesis is indicative of a lower rate of photorespirationand consistent with a lower O₂/CO₂ ratio present in the bundle-sheathcells of C₄-plants. We conclude that both decreased glycolatesynthesis and the refixation of photorespiratory-released CO₂are important in maintaining a lower rate of photorespirationin C₄-plants compared to C₃ plants. Intermediate glycolate synthesisin Panicum milioldes is consistent with its intermediate levelof O₂ inhibition of photosynthesis and intermediate rate ofphotorespiration. (Received May 6, 1978; )     

Novel characteristics of cassava,Manihot esculenta Crantz,a reputed C3-C4 intermediate photosynthesis species

The cassava plant, Manihot esculenta, grows exceptionally well in low fertility and drought prone environments, but the mechanisms that allow this growth are unknown. Earlier, and sometimes contradictory, work speculated about the presence of a C₄-type photosynthesis in cassava leaves. In the present work we found no evidence for a C₄ metabolism in mature attached cassava leaves as indicated i) by the low, 2 to 8%, incorporation of ¹⁴CO₂ into C₄ organic acids in short time periods, 10 s, and the lack of ¹⁴C transfer from C₄ acids to other compounds in ¹²CO₂, ii) by the lack of C₄ enzyme activity changes during leaf development and the inability to detect C₄ acid decarboxylases, and iii) by leaf CO₂ compensation values between 49 and 65 l of CO₂ 1^–1 and by other infrared gas exchange photosynthetic measurements. It is concluded that the leaf biochemistry of cassava follows the C₃ pathway of photosynthesis with no indication of a C₃-C₄ mechanism.However, cassava leaves exhibit several novel characteristics. Attached leaves have the ability to effectively partition carbon into sucrose with nearly 45% of the label in sucrose in about one min of ¹⁴CO₂ photosynthesis, contrasting with 34% in soybean (C₃) and 25% in pigweed (C₄). Cassava leaves displayed a strong preference for the synthesis of sucrose versus starch. Field grown cassava leaves exhibited high rates of photosynthesis and curvilinear responses to increasing sunlight irradiances with a tendency to saturate only at high irradiances, above 1500 mol m^–2 s^–1. Morphologically, the cassava leaf has papillose epidermal cells on its lower mesophyll surface that form fence-like arrangements encircling guard cells. It is proposed that the active synthesis of sugars has osmotic functions in the cassava plant and that the papillose epidermal cells function to maintain a healthy leaf water status in various environments.Abbreviations ADP adenosine diphosphate - Asp aspartate - BSA bovine serum albumin - CoA coenzyme A - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - FBP fructose-1,6-biphosphate - Gly glycine - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - Mal malate - NAD nicotinamide adenine dinucleotide (oxidized form) - NADH nicotinamide adenine dinucleotide (reduced form) - NADP nicotinamide adenine dinucleotide phosphate (oxidized form) - PAR photosynthetic active radiation (400–700 nm) - PEP phosphenolpyruvate carboxylase - p-FBPase plastid fructose-1,6-biphosphatase - PGA 3-phosphoglyceric acid - PMSF phenylmethylsulfonyl fluoride - PVP polyvinylpyrrolidone - Rubisco ribulose-1,5-biphosphate carboxylase/oxygenase - RuBP ribulose-1,5-biphosphate - Ser serine - sugar-P sugar-phosphates     

Photosynthetic carbon metabolism in Panicum milioides, a C3-C4 intermediate species: evidence for a limited C4 dicarboxylic acid pathway of photosynthesis

Panicum milioides, a naturally occurring species with C4-like Kranz leaf anatomy, is intermediate between C3 and C4 plants with respect to photo-respiration and the associated oxygen inhibition of photosynthesis. This paper presents direct evidence for a limited degree of C4 photosynthesis in this C3-C4 intermediate species based on: (a) the appearance of 24% of the total 14C fixed following 4 s photosynthesis in 14CO2-air by excised leaves in malate and aspartate and the complete transfer of label from the C4 acids to Calvin cycle intermediates within a 15 s chase in 12CO2-air; (b) pyruvate- or alanine-enhanced light-dependent CO2 fixation and pyruvate stimulation ote- or alanine-enhanced light-dependent CO2 fixation and pyruvate stimulation of oxaloacetate- or 3-phosphoglycerate-dependent O2 evolution by illuminated mesophyll protoplasts, but not bundle sheath strands; and (c) NAD-malic enzyme-dependent decarboxylation of C4 acids at the C-4 carboxyl position, C4 acid-dependent O2 evolution, and 14CO2 donation from (4-14C)C4 acids to Calvin cycle intermediates during photosynthesis by bundle sheath strands, but not mesophyll protoplasts. However, P. milloides differs from C4 plants in that the activity of the C4 cycle enzymes is only 15 to 30% of a C4 Panicum species and the Calvin cycle and phosphoenolpyruvate carboxylase are present in both cell types. From these and related studies (Rathnam, C.K.M. and Chollet, R. (1979) Arch. Biochem. Biophys. 193, 346-354; (1978) Biochem. Biophys. Res. Commun. 85, 801-808) we conclude that reduced photorespiration in P. milioides is due to a limited degree of NAD-malic enzyme-type C4 photosynthesis permitting an increase in pCO2 at the site of bundle sheath, but not mesophyll, ribulose-bisphosphate carboxylase-oxygenase.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Comparisons of rbcL genes for the large subunit of ribulose-bisphosphate carboxylase from closely related C3 and C4 plant species

Ribulosebisphosphate carboxylase/oxygenase from C4 plants exhibits higher turnover rates and lower affinities for CO2 than the enzyme from C3 plants or C3-C4 intermediate species. This property is shown to be inherited maternally in reciprocal interspecific crosses between two Flaveria species, and thus must be specified by the chloroplast-encoded large subunits. To investigate the amino acid changes responsible, the chloroplast rbcL genes from three pairs of C3 and C4 species from three genera (Flaveria, Atriplex, and Neurachne) were cloned and sequenced. Comparisons of the predicted amino acid sequences from species of the same genus revealed a limited number of changes within each pair, ranging from three to six, of which only one (309Met (C3) to Ile (C4] was consistently observed. This residue occurs in the loop connecting the carboxyl end of beta strand 5 with the amino end of alpha helix 5 in the alpha/beta barrel of the large subunit, and is close to the active site in a region which makes interdomain and intersubunit contacts. However, it is unlikely that a change of this residue alone is responsible for the alteration of kinetic properties. Nucleotide sequence comparisons of the rbcL genes showed no significant or consistent changes in the promoter and transcribed but nontranslated regions to suggest why rbcL is not expressed in C4 leaf mesophyll cells. It is concluded that mutations in rbcL have led to an alteration of the kinetics but not the expression of ribulose-bisphosphate carboxylase.     

C4 isoform of NADP-malate dehydrogenase. cDNA cloning and expression in leaves of C4, C3, and C3-C4 intermediate species of Flaveria.

In C4 plants of the NADP-malic enzyme type, an abundant, mesophyll cell-localized NADP-malate dehydrogenase (MDH) acts to convert oxaloacetate, the initial product of carbon fixation, to malate before it is shuttled to the bundle sheath. Since NADP-MDH has different but important roles in leaves of C3 and C4 plants, we have cloned and characterized a nearly full-length cDNA encoding NADP-MDH from Flaveria trinervia (C4) to permit comparative structure/expression studies within the genus flaveria. The dicot genus Flaveria includes C3-C4 intermediate species, as well as C3 and C4 species. We show that the previously noted differences in NADP-MDH activity levels among C3, C4, and C3-C4 Flaveria species are in part due to interspecific differences in mRNA accumulation. We also show that the NADP-MDH gene appears to be present as a single copy among different Flaveria species, suggesting that a pre-existing gene has been reregulated during the evolution from C3 to C4 plants to accommodate the abundance and localization requirements of the C4 cycle.