Flaveria Trinervia Research Articles

Hepatoprotective activity of Tridecan-1-ol isolatedfrom Flaveria trinervia (Speng). C. Mohr

Background- Flaveria trinervia (Asteraceae), a medicinal herb has been used in Indian traditionl medicine to cure jaundice. Reports on phytochemical and pharmacological investigations are very scarce. A phytoconstiuent Tridecan-1-ol and screened for hepatoprotective activity. -To evaluate hepatoprotective effects of Tridecan-1-ol against CCl4 induced hepatic damage on Wister albino rats. Methods- Tridecan-1-ol was isolated from petroleum extract of Flaveria trinervia using silica gel column chromatography and the structure was confirmed by IR, MASS,and 1H NMR spectroscopic studies. The parameters employed to confirm the hepatoprotectivity were the estimation of liver function serum markers such as total bilirubin, total protein, alanine transaminase, aspartate transaminase, alkaline phosphatase activities and the histological profile of the liver tissue. Results-The LD50 of petroleum ether extract and the constituent Tridecan -1-ol was found to be 500 and 150 mg/kg body weight respectively. Tridecan -1-ol showed significant hepatoprotective activity than the crude extract and it was compared with the standard hepatoprotective drug silymarin. The histological profile of the liver tissue ofTridecan-1-ol administered rats showed the presence of normal hepatic cords, absence of necrosis and fatty infiltration as similar to the controls. Conclusion-Tridecan-1-ol afforded significant hepatoprotectivity against CCl4 induced liver damage and supported the traditional claim of Flaveria trinervia as a hepatoprotective herb.

Antioxidant and antipyretic studies on methanolic extraction of flaveria trinervia leaves

The aim of the present study was to perform antipyretic and antioxidant activity of Flaveria trinervia Methanolic leaves extract by different in-vivo and in-vitro models. The different pharmacognostical parameters were evaluated as per standard procedure. Finally, antipyretic (Brewer’s Yeast Pyrexia model) and antioxidant activity (Hydroxyl Radaical scavenging activity, determination of Reducing Power, Metal chelating activity, Carbon tetrachloride (ccl4) induced lipid peroxidation, Inhibitory Test on Protein Oxidative Modification, Lipid peroxidation assay, Catalase assay and Reduced glutathione assay) was evaluated by adopting different methods. The extracts showed a marked antipyretic effect by causing a reduction in yeast-induced fever. Methanolic extract (200 and 400 mg/kg) showed the effect to the same degree as paracetamol (50mg/kg, i.p.). The experimentally induced laboratory model was employed in evaluating the antipyretic activities of Methanolic extracts of Flaveria trinervia. The extract caused a better hypothermal activity against yeast-induced pyrexia in rats. Free radical scavenging activity of Flaveria trinervia. was found because polyphenolic compounds present in plant contribute significantly to the total antioxidant capacity of the leaves. Flavonoids play some important pharmacological roles against diseases, such as cardiovascular diseases, cancer, inflammation and allergy.

Molecular characterization of Alfalfa mosaic virus (AMV) isolates in alfalfa and other plant species in different regions in Saudi Arabia

In a survey conducted in 2012 and 2013, 1166 samples from alfalfa plants and 202 samples from symptomatic weeds and cultivated plants growing adjacent to alfalfa fields were collected. Using DAS-ELISA, Alfalfa mosaic virus (AMV) was detected in 58.4% of the alfalfa samples and in 63.9% of the weeds and cultivated plants samples. ELISA detection of AMV was confirmed by testing representative samples from all regions by RT-PCR using a pair of primers (AMV/F and AMV/R) specific to the AMV coat protein (CP) gene. The size of the major product obtained from AMV-infected plants was identical to the 700 bp size expected from the CP gene of AMV. The amino acids and the nucleotide sequences of 17 Saudi AMV isolates from alfalfa, and 16 from other cultivated plants and weeds detected in different regions in Saudi Arabia showed a percentage of similarity that ranged between 87.9%–100% among them and 86.2% - 100% when compared with sequences of 15 different AMV isolates reported in the GenBank. This is the first time AMV was detected in Vigna unguiculata and in the following weed plant species: Chenopodium quinoa, Convolvulus arvensis, Malva parviflora, Hibiscus spp., Hippuris vulgaris, Cichorium intybus, and Flaveria trinervia in Saudi Arabia.

Leaf Epiphytic Bacteria of Plants Colonizing Mine Residues: Possible Exploitation for Remediation of Air Pollutants.

Plant surfaces are known as an important sink for various air pollutants, including particulate matter and its associated potentially toxic elements (PTE). Moreover, leaves surface or phylloplane is a habitat that harbors diverse bacterial communities (epiphytic). However, little is known about their possible functions during phytoremediation of air pollutants like PTE. The study of leaf epiphytic bacteria of plants colonizing mine residues (MR) containing PTE is thus a key to understand and exploit plant–epiphytic bacteria interactions for air phytoremediation purposes. In this research, we aimed (i) to characterize the functions of epiphytic bacteria isolated from the phylloplane of Brickellia veronicifolia, Flaveria trinervia, Gnaphalium sp., and Allionia choisyi growing spontaneously on multi-PTE contaminated MR and (ii) to compare these against the same plant species in a non-polluted control site (NC). Concentrations (mg kg-1) of PTE on MR leaf surfaces of A. choisyi reached up to 232 for Pb, 13 for Cd, 2,728 for As, 52 for Sb, 123 for Cu in F. trinervia, and 269 for Zn in Gnaphalium sp. In the four plant species, the amount of colony-forming units per cm2 was superior in MR leaves than in NC ones, being A. choisyi the plant species with the highest value. Moreover, the proportion of isolates tolerant to PTE (Zn, Cu, Cd, and Sb), UV light, and drought was higher in MR leaves than in those in NC. Strain BA15, isolated from MR B. veronicifolia, tolerated 150 mg Zn L-1, 30 mg Sb L-1, 25 mg Cu L-1; 80 mg Pb L-1, and was able to grow after 12 h of continuous exposition to UV light and 8 weeks of drought. Plant growth promotion related traits [N fixation, indole acetic acid (IAA) production, and phosphate solubilization] of bacterial isolates varied among plant species isolates and between MR and NC sampling condition. The studied epiphytic isolates possess functions interesting for phytoremediation of air pollutants. The results of this research may contribute to the development of novel and more efficient inoculants for microbe-assisted phytoremediation applied to improve air quality in areas exposed to the dispersion of metal mine tailings.

Detection of new viruses in alfalfa, weeds and cultivated plants growing adjacent to alfalfa fields in Saudi Arabia.

A total of 1368 symptomatic plant samples showing different virus-like symptoms such as mottling, chlorosis, mosaic, yellow mosaic, vein clearing and stunting were collected from alfalfa, weed and cultivated plant species growing in vicinity of alfalfa fields in five principal regions of alfalfa production in Saudi Arabia. DAS-ELISA test indicated occurrence of 11 different viruses in these samples, 10 of which were detected for the first time in Saudi Arabia. Eighty percent of the alfalfa samples and 97.5% of the weed and cultivated plants samples were found to be infected with one or more of these viruses. Nine weed plant species were found to harbor these viruses namely, Sonchus oleraceus, Chenopodium spp., Hibiscus spp., Cichorium intybus, Convolvulus arvensis, Malva parviflora, Rubus fruticosus, Hippuris vulgaris, and Flaveria trinervia. These viruses were also detected in seven cultivated crop plants growing adjacent to the alfalfa fields including Vigna unguiculata, Solanum tuberosum, Solanum melongena, Phaseolus vulgaris, Cucurbita maxima, Capsicum annuum, and Vicia faba. The newly reported viruses together with their respective percent of detection in alfalfa, and in both weeds and cultivated crop plant species together were as follows: Bean leaf roll virus (BLRV) {12.5 and 4.5%}, Lucerne transient streak virus (LTSV) {2.9 and 3.5%}, Bean yellow mosaic virus (BYMV) {1.4 and 4.5%}, Bean common mosaic virus (BCMV) {1.2 and 4.5%}, Red clover vein mosaic virus (RCVMV) {1.2 and 4%}, White clover mosaic virus (WCIMV) {1.0 and 5%}, Cucumber mosaic virus (CMV) {0.8 and 3%}, Pea streak virus (PeSV) {0.4 and 4.5%} and Tobacco streak virus (TSV) {0.3 and 2.5%}. Alfalfa mosaic virus (AMV), the previously reported virus in alfalfa, had the highest percentage of detection in alfalfa accounting for 58.4% and 62.8% in the weeds and cultivated plants. Peanut stunt virus (PSV) was also detected for the first time in Saudi Arabia with a 66.7% of infection in 90 alfalfa samples collected from the surveyed regions during the last visit that tested negative to all the previously detected viruses.

Antioxidant activity of Flaveria trinervia (Sprengel) C. Mohr

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in plant extracts, the aim of the present study was to investigate the antioxidant capacity of Flaveria trinervia. Leaves of F. trinervia were subjected to cold extration with petroleum ether, chloroform, methanol and ethanol solvents, and chloroform extraction showed effective antioxidant property. The results showed potent antioxidant properties against nitric oxide radical with IC50 values of 640±1.2 µg/ml and diphenylpicrylhydrazyl (DPPH) radical with IC50 values of 125±4.4 µg/ml. Key words: Chloroform extract, diphenylpicrylhydrazyl (DPPH), nitric oxide, free radical antioxidant activity.

Erratum: A plastidial sodium-dependent pyruvate transporter

Nature 476, 472–475 (2011) In this Letter, the DNA Data Bank of Japan accession code for Flaveria trinervia BASS4 should be AB522103. This has been corrected online.

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 C 3, C 4, and intermediate species growing under natural conditions in south-central Mexico. We found that Flaveria pringlei (C 3) was the most widely distributed species with multiple growth habits. Numerous populations of Flaveria kochiana (C 4), a recently described species with a previously unknown distribution, were located in the Mixtec region of Oaxaca. Flaveria cronquistii (C 3) and Flaveria ramosissima (C 3-C 4) were only located in the Tehuacán Valley region while Flaveria trinervia (C 4) was widely distributed. Only one population of Flaveria angustifolia (C 3-C 4) and Flaveria vaginata (C 4-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 C 4 cycle characteristics and physiological performance in the Flaveria populations examined. Furthermore, C 3 species were not found at higher elevation than C 4 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.

Evolution and function of a cis-regulatory module for mesophyll-specific gene expression in the C4 dicot Flaveria trinervia.

C(4) photosynthesis presents a sophisticated integration of two complementary cell types, mesophyll and bundle sheath cells. It relies on the differential expression of the genes encoding the component enzymes and transporters of this pathway. The entry enzyme of C(4) photosynthesis, phosphoenolpyruvate carboxylase (PEPC), is found exclusively in mesophyll cells, and the expression of the corresponding gene is regulated at the transcriptional level. In the C(4) dicot Flaveria trinervia, the mesophyll-specific expression of the C(4) PEPC gene (ppcA) depends on a 41-bp segment in the distal promoter region referred to as MEM1 (for mesophyll expression module1). Here, we show that a MEM1 sequence found in the orthologous ppcA gene from the C(3) species Flaveria pringlei is not able to direct mesophyll-specific gene expression. The two orthologous MEM1 sequences of F. pringlei and F. trinervia differ at two positions, a G-to-A exchange and the insertion of the tetranucleotide CACT. Changes at these two positions in the C(3) MEM1 sequence were necessary and sufficient to create a mesophyll-specificity element during C(4) evolution. The MEM1 of F. trinervia enhances mesophyll expression and concomitantly represses expression in bundle sheath cells and vascular bundles.

Ethylene modulates transcription of the phosphoenolpyruvate carboxykinase gene in grapefruit flavedo

SummaryThe role of ethylene in citrus fruit physiology is poorly understood. Gene expression studies with ethylene and ethylene inhibitors suggest a regulatory role for the low basal levels of endogenous ethylene in citrus fruits; however, only a few ethylene-modulated genes have been reported. 1-Methylcyclopropene (1-MCP), an ethylene antagonist that inhibits ethylene signal transduction by preventing ethylene from binding to its receptors, is a useful tool to further identify genes associated with the regulatory roles of ethylene. We determined the effects of ethylene and 1-MCP on gene expression in grapefruit flavedo. Total RNA from fruit treated with air, 5 µl l–l ethylene, or 0.3 µl l–l 1-MCP for 48 h was used in differential-display reverse transcription-polymerase chain reaction (DD-RT-PCR) analysis. Our hypothesis was that genes whose expression is induced by ethylene would be up-regulated in response to ethylene, whereas genes whose expression is inhibited by ethylene would be up-regulated in response to 1-MCP. A 440 bp cDNA, induced by ethylene, was used as a probe to screen a cDNA library of flavedo, and obtain a clone with a deduced amino acid sequence 88% similar to the enzyme phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.49) from both Arabidopsis and Flaveria trinervia. We designated this gene CpPEPCK (Citrus paradisi phosphoenolpyruvate carboxykinase). Northern blot and RT-PCR analyses revealed increased expression of CpPEPCK in grapefruit flavedo after exposure to ethylene, and with increased exposure time. In contrast, treatment with 1-MCP inhibited the accumulation of CpPEPCK transcripts in the same tissue. Although CpPEPCK is expressed in leaves, roots, and petals, higher transcript abundance was found in the flavedo of ethylene-treated fruit than in other tissues. This study provides additional evidence for a role for ethylene in citrus fruit physiology.

Abundant Expression in Vascular Tissue of Plant TAF10, an Orthologous Gene for TATA Box-binding Protein-associated Factor 10, in Flaveria trinervia and Abnormal Morphology of Arabidopsis thaliana Transformants on its Overexpression

TAF10 is one of the TATA box-binding protein-associated factors (TAFs), which constitute the TFIID complex. We isolated a plant TAF10 ortholog from a Flaveria trinervia cDNA library, and named it ftTAF10. The ftTAF10 polypeptide contains a histone-fold motif, which is highly conserved among the TAF10s of other organisms. A transiently expressed green fluorescent protein (GFP) fusion protein was translocated into the nuclei of onion epidermal cells, suggesting that the ftTAF10 functions in nuclei. The transcript level was higher in stems and roots than in leaves, and in situ hybridization of F. trinervia seedlings revealed that the ftTAF10 transcript is accumulated abundantly in vascular tissues of hypocotyls, in the central cylinder of roots, and slightly in bundle sheath cells of leaves. Overexpression of ftTAF10 in Arabidopsis under the cauliflower mosaic virus 35S promoter caused two kinds of abnormal morphology, limitation of the indeterminate inflorescence and production of deformed leaves. These results indicate the possibility that ftTAF10 is a plant 'selective TAF' involved in the expression of a subset of vascular abundant genes, and that its appropriate gene expression is necessary for normal development.

Screening of Alternanthera repens., Boerhavia coccinea., Flaveria trinervia., Tournefortia densiflora., and Vitex mollis. Extracts to Evaluate their Antibacterial Activity and Effect on Smooth Muscle. I

Organic extracts of five medicinal plants (Alternanthera repens. (L) Kuntze, Boerhavia coccinea. Mill, Flaveria trinervia. (Spreng) C. Mohr, Tournefortia densiflora. M. et G., and Vitex mollis. Kunth), widely used in traditional Mexican medicine for the treatment of diarrhea and dysentery, were evaluated for antibacterial and intestinal antispasmodic activity. The antibacterial properties were determined in vitro. by the agar dilution method against multi-resistant enteric bacteria obtained from clinical isolates of Escherichia coli. R166 and R170, Salmonella typhi. R1234 and R 1330, and Enterobacter cloacae. R819, and against ATCC strains Shigella sonnei. 11060, Escherichia coli. 25922, Proteus mirabilis. 43071, and Salmonella typhimurium. 14028. In order to evaluate the effect of the extracts on the contraction induced in vitro., preparations of intestine from guinea-pigs were used. The integral extracts of B. coccinea. and T. densiflora. were the most active species with a MIC of <2 mg/ml. The highest antispasmodic activity was obtained with V. mollis. extract, 37% motility inhibition. These findings could be useful in the search for new therapeutic agents.

Cis-Regulatory elements for mesophyll-specific gene expression in the C4 plant Flaveria trinervia, the promoter of the C4 phosphoenolpyruvate carboxylase gene.

C(4) photosynthesis depends on the strict compartmentalization of CO(2) assimilatory enzymes. cis-regulatory mechanisms are described that ensure mesophyll-specific expression of the gene encoding the C(4) isoform of phosphoenolpyruvate carboxylase (ppcA1) of the C(4) dicot Flaveria trinervia. To elucidate and understand the anatomy of the C(4) ppcA1 promoter, detailed promoter/reporter gene studies were performed in the closely related C(4) species F. bidentis, revealing that the C(4) promoter contains two regions, a proximal segment up to -570 and a distal part from -1566 to -2141, which are necessary but also sufficient for high mesophyll-specific expression of the beta-glucuronidase reporter gene. The distal region behaves as an enhancer-like expression module that can direct mesophyll-specific expression when inserted into the ppcA1 promoter of the C(3) plant F. pringlei. Mesophyll expression determinants were restricted to a 41-bp segment, referred to as mesophyll expression module 1 (Mem1). Evolutionary and functional studies identified the tetranucleotide sequence CACT as a key component of Mem1.

Screening of Mexican Medicinal Plants for Antiprotozoal Activity – Part II

The activity of the crude extracts from eight medicinal plants used in Mexico to treat gastrointestinal infections were assayed against Entamoeba histolytica and Giardia lamblia axenic trophozoites. Alternanthera repens, Boerhavia coccinea, Flaveria trinervia, Leucaena esculenta, Tournefortia densiflora and Vitex mollis were found to have antiprotozoal activity, with extracts of Tournefortia densiflora being the most potent against both tested organisms.

Differential Regulation of Transcripts Encoding Cytosolic NADP-Malic Enzyme in C3 and C4 FlaveriaSpecies

A cytosolic NADP-malic enzyme (CYTME) has been described previously in several plants, all C3 species. CYTME is distinct from the chloroplastic NADP-malic enzyme (CHLME) that is highly active in C4 species. We show that at least one CytMe gene is present in all Flaveria spp., including C3, C4, and C3-C4 intermediate types. Based on the CytMe expression patterns in Flaveria pringlei (C3) and Flaveria trinervia (C4), we suggest CYTME has several distinct roles, including the supplying of NADPH for cytosolic metabolism, the supporting of wound response or repair, and the balancing of cellular pH in illuminated leaves. These three roles are likely correlated with CytMe mRNAs of apparent sizes 2.0, 2.2, and 2.4 kb, respectively, which differ in the length of the 5' untranslated regions. Various regulatory mechanisms involving RNA processing and translational efficiency are discussed.

Differential Regulation of Transcripts Encoding Cytosolic NADP-Malic Enzyme in C3 and C4Flaveria Species

A cytosolic NADP-malic enzyme (CYTME) has been described previously in several plants, all C3 species. CYTME is distinct from the chloroplastic NADP-malic enzyme (CHLME) that is highly active in C4 species. We show that at least oneCytMe gene is present in all Flaveriaspp., including C3, C4, and C3-C4 intermediate types. Based on theCytMe expression patterns in Flaveria pringlei (C3) and Flaveria trinervia(C4), we suggest CYTME has several distinct roles, including the supplying of NADPH for cytosolic metabolism, the supporting of wound response or repair, and the balancing of cellular pH in illuminated leaves. These three roles are likely correlated withCytMe mRNAs of apparent sizes 2.0, 2.2, and 2.4 kb, respectively, which differ in the length of the 5′ untranslated regions. Various regulatory mechanisms involving RNA processing and translational efficiency are discussed.

Phospho enolpyruvate carboxylase kinase involved in C 4 photosynthesis in Flaveria trinervia: cDNA cloning and characterization

In C 4 plants, phospho enolpyruvate carboxylase (PEPC; EC 4.1.1.31), a key enzyme in C 4 photosynthesis, is controlled by reversible phosphorylation of a conserved Ser residue near the N-terminus. We now report the first cloning of a cDNA from a C 4 plant, Flaveria trinervia, which encodes the specific protein kinase (FtPEPC-PK) involved in the phosphorylation of C 4-form PEPC. Several lines of supportive evidence are: strict substrate specificity of the recombinant enzyme, prominent light/dark response of the transcript level and abundant expression in leaves of C 4 plant ( F. trinervia) but very low expression in a C 3 plant of the same genus ( Flaveria pringlei). We also discuss the possibility that the FtPEPC-PK gene has co-evolved with the PEPC gene to participate in C 4 photosynthesis.

CDNA cloning and characterization of a C4 PEPC kinase from Flaveria trinervia

PEPC activity in C4 plants is regulated by reversible phosphorylation mainly in response to light. The cDNA for this specific protein kinase (PEPC-PK) has not yet been cloned, though several cDNAs for PEPC-PKs from C3 and CAM plants have been isolated recently. A complete cDNA (~1.4 kb) encoding PEPC-PK was isolated from Flaveria trinervia, a dicot C4 plant, by screening cDNA library with a F. trinervia PEPC-PK fragment prepared by PCR with degenerate primers. The cloned PEPC-PK contained an ORF for a protein of 281 amino acid residues, and it was composed of the kinase domain only. The recombinant PEPC-PK expressed in E. coli phosphorylated a specific serine residue of maize C4-form PEPC near the N-terminus, whereas it did not phosphorylate a mutant PEPC in which the serine residue of the phosphorylation-site have been substituted for aspartate (S15D). The activity of the PEPC-PK was Ca2+-independent. The interesting enzymatic properties will also be presented. Northern blot analysis showed that the transcripts accumulated abundantly in leaves in daytime, but not at night. The transcripts were not detected in roots and stems. Genomic Southern blot analysis under the high-stringency conditions indicated that the F. trinervia genome contained more than two copies. These results suggested that this PEPC-PK plays a major role in the regulatory phosphorylation of PEPC for C4 photosynthesis.

Phosphoenolpyruvate carboxylase isoforms from an aquatic monocot: structure/function relationships

Growth at low [CO2] induces a change from C3 to C4 photosynthesis in the aquatic angiosperm, Hydrilla verticillata. A unique feature of this system is that Kranz anatomy is lacking, and both the C4 and Calvin cycles operate within the same cell. Phosphoenolpyruvate carboxylase (PEPC) is the initial carboxylase in the C4 system, and it also has anapleurotic functions in both C3 and C4 plants. Three full-length PEPC cDNA sequences were isolated from Hydrilla, two from leaf tissue and one from root, which showed 95% to 99% homology among the three isoforms. The objective of this study was to identify candidate residues in the isoforms that potentially define characteristic C3 and C4 kinetic and regulatory behavior. Three-dimensional models of the deduced amino acids were constructed using co-ordinates of the E. coli model (Kai et al 1999). Comparative analyses between these and that of the C4 PEPC from Flaveria trinervia were undertaken. None of the three Hydrilla isoforms had the C4 signature Ser, although differential regulation of expression studies indicated that one, Hvpepc4, was the photosynthetic isoform. Most of the substitutions and deletions found in this isoform were outside the known conserved regions, but they still might contribute towards functional variation. Identification of these residues will provide information for site directed mutagenesis experiments to elucidate the structure/function relationship of Hydrilla PEPCs in C3 and C4 photosynthesis.

The C4 specific properties of Flaveria trinervia phosphoenolpyruvate carboxylase is divided in two regions that synergetically interact

The evolution of the C4 photosynthetic pathway was facilitated by the reqruitment of genes already present in ancestral C3 plants. In order to function in the C4 cycle not only the level and pattern of expression of these genes had to be changed, but also the enzymatic properties had to be optimised. The C4 phosphoenolpyruvate carboxylase gene (ppcA) of the C4 species F. trinervia and its orthologous counterpart of the C3 plant F. pringlei were chosen to identify the C4-specific property determinants in the enzyme sequence. By constructing a series of C3/C4 chimerical enzymes it was shown that the serine residue at position 774 is crucial for the C4 enzyme. The results also suggested that region 2 (amino acids 296 to 437) contains additional determinant(s) for C4 properties in Flaveria phosphoenolpyruvate carboxylases. In the present work we investigated the influence of region 2 alone and in combination with site-directed C3/C4 mutagenesis of position 774. The results show that region 2 contains determinants for C4 properties. However, in order to transform a C3 enzyme into C4 type both region 2 and position 774 must be of C4 sequences. In addition, we will present results that show that a synergistic interaction exists between the two C4 specific determinants.