Effect of LiCl on phosphoenolpyruvate carboxylase kinase and the phosphorylation of phosphoenolpyruvate carboxylase in leaf disks and leaves of <Emphasis Type="Italic">Sorghum vulgare</Emphasis>

In the present work, the effect of LiCl on phosphoenolpyruvate carboxylase kinase (PEPCase-k), C₄ phosphoenolpyruvate carboxylase (PEPCase: EC 4.1.1.31) and its phosphorylation process has been investigated in illuminated leaf disks and leaves of the C₄ plant Sorghum vulgare. Although this salt induced severe damages to older leaves, it did not significantly alter the physiological parameters (photosynthesis, transpiration rate, intercellular CO₂ concentration) of young leaves. An immunological approach was used to demonstrate that the PEPCase-k protein accumulated rapidly in illuminated leaf tissues, consistent with the increase in its catalytic activity. In vivo, LiCl was shown to strongly enhance the light effect on PEPCase-k protein content, this process being dependent on protein synthesis. In marked contrast, the salt was found to inhibit the PEPCase-k activity in reconstituted assays and to decrease the C₄ PEPCase content and phosphorylation state in LiCl treated plants. Short-term (15 min) LiCl treatment increased IP₃ levels, PPCK gene expression, and PEPCase-k accumulation. Extending the treatment (1 h) markedly decreased IP₃ and PPCK gene expression, while PEPCase-k activity was kept high. The cytosolic protein synthesis inhibitor cycloheximide (CHX), which blocked the light-dependent up-regulation of the kinase in control plants, was found not to be active on this process in preilluminated, LiCl-treated leaves. This suggested that the salt causes the kinase turnover to be altered, presumably by decreasing degradation of the corresponding polypeptide. Taken together, these results establish PEPCase-k and PEPCase phosphorylation as lithium targets in higher plants and that this salt can provide a means to investigate further the organization and functioning of the cascade controlling the activity of both enzymes.     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

Changes in phenotype of transgenic amaranth <Emphasis Type="Italic">Amaranthus retroflexus</Emphasis> L., overexpressing <Emphasis Type="Italic">ARGOS-LIKE</Emphasis> gene

Amaranth is a new and promising crop for the Russian climate, notable for its well-balanced amino acid composition. Yield increase using the methods of genetic engineering is a challenging task. We generated transgenic plants of amaranth with expression of the Arabidopsis thaliana ARGOS-LIKE gene under the control of the dahlia mosaic virus promoter. We achieved 1.4% transformation effectiveness. In comparison with wild-type amaranth, we observed a 21% increase in stem length, 79% increase in leaf length, and 190% increase in fresh weight of transgenic plants. It was shown that ARGOS-LIKE gene of A. thaliana along with the dahlia mosaic virus promoter can be used to increase primarily the green weight of shoot and leaf size of amaranth.     

Evolution of the C<Subscript>4</Subscript> phosphoenolpyruvate carboxylase promoter of the C<Subscript>4</Subscript> species <Emphasis Type="Italic">Flaveria trinervia</Emphasis>: the role of the proximal promoter region

Background  

The key enzymes of photosynthetic carbon assimilation in C₄ plants have evolved independently several times from C₃ isoforms that were present in the C₃ ancestral species. The C₄ isoform of phosphoenolpyruvate carboxylase (PEPC), the primary CO₂-fixing enzyme of the C₄ cycle, is specifically expressed at high levels in mesophyll cells of the leaves of C₄ species. We are interested in understanding the molecular changes that are responsible for the evolution of this C₄-characteristic PEPC expression pattern, and we are using the genus Flaveria (Asteraceae) as a model system. It is known that cis-regulatory sequences for mesophyll-specific expression of the ppcA1 gene of F. trinervia (C₄) are located within a distal promoter region (DR).     

Effects of the maize C<Subscript>4</Subscript> phosphoenolpyruvate carboxylase (<Emphasis Type="Italic">ZmPEPC</Emphasis>) gene on nitrogen assimilation in transgenic wheat

Nitrogen (N) is the primary limiting factor for crop growth, development, and productivity. Transgenic technology is a straightforward strategy for improving N assimilation in crops. The present study assessed the effects of maize C₄ phosphoenolpyruvate carboxylase (ZmPEPC) gene overexpression on N assimilation in three independent transgenic lines and wild-type (WT) wheat (Triticum aestivum L.). The transgenic wheat lines depicted ZmPEPC overexpression and higher PEPC enzyme activity relative to that in the WT. The leaves of the transgenic wheat lines subjected to low N treatment showed an increase in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) expression, content, and carboxylase activity. The transgenic wheat lines also depicted an upregulation of genes associated with the anaplerotic pathway for the TCA cycle, suggesting that more carbon (C) skeleton material is being allocated for N assimilation under low N conditions. Furthermore, ZmPEPC expression in transgenic wheat lines induced the upregulated of genes associated primary N metabolism, including TaNR, TaGS2, TaGOGAT, TaAspAT, and TaASN1. The average total free amino acid content in the transgenic wheat lines was 48.18% higher than that in the WT, and asparagine (Asn), glutamine (Gln), aspartic acid (Asp), and serine (Ser) were also markedly enhanced. In addition, elementary analysis showed that N and C content, and the biomass of the transgenic wheat lines increased with low N treatment. Yield trait analysis indicated that ZmPEPC overexpression improved grain yield by increasing 1000-grain weight. In conclusion, ZmPEPC overexpression in wheat could modulate C metabolism, significantly improve N assimilation, enhances growth, and improves yield under low N conditions.     

pH and CO<Subscript>2</Subscript> effects on <Emphasis Type="Italic">Coelastrella</Emphasis> (<Emphasis Type="Italic">Scotiellopsis</Emphasis>) <Emphasis Type="Italic">rubescens</Emphasis> growth and metabolism

We studied effects of рН and СО₂ enrichment on the physiological condition and biochemical composition of a carotenogenic microalga Coelastrella (Scotiellopsis Vinatzer) rubescens Kaufnerová et Eliás (Scenedesmaceae, Sphaeropleales, Chlorophyceae), a promising source of natural astaxanthin. The microalga was grown at a constant pH (5, 6, 7 or 8) maintained by direct СО₂ injection. The air-sparged culture served as the control. Cell division rate and size, dry biomass productivity, the rates of nitrogen and phosphorus uptake as well as photosynthetic pigment and total lipid content and fatty acid composition were followed. С. rubescens possessed a narrow-range рН tolerance (the optimum рН 6–7). Under these conditions, the highest values of the maximum (1.0–1.1 1/day) and average (0.3–0.35 1/day) specific growth rate, chlorophyll а (4.8–4.9%) and total carotenoid dry weight percentages (1.7–1.8%) were recorded. Cell lipid fatty acid unsaturation index (1.851) and polyunsaturated fatty acid percentage (36–39%) and С18:3 ω3/С18:1 ω9 ratio (3.8–4.5) were also the highest under these conditions. A decline of рН to 5 brought about severe stress manifesting itself as a cell division cessation, photosynthetic apparatus reduction, two-fold increase in cell volume, accumulation of dry weight and lipids and a considerable decline in fatty acid unsaturation. Cultivation of С. rubescens without СО₂ enrichment resulted in a rapid alkalization of the medium to рН 9.5–10.5 impairing the physiological condition of the cells. Reasons of the deteriorative effects of suboptimal pH values on the physiological condition of C. rubescens are discussed.     

Drought tolerance and proteomics studies of transgenic wheat containing the maize C<Subscript>4</Subscript> phosphoenolpyruvate carboxylase (<Emphasis Type="Italic">PEPC</Emphasis>) gene

Enhancing drought tolerance of crops has been a great challenge in crop improvement. Here, we report the maize phosphoenolpyruvate carboxylase (PEPC) gene was able to confer drought tolerance and increase grain yield in transgenic wheat (Triticum aestivum L.) plants. The improved of drought tolerance was associated with higher levels of proline, soluble sugar, soluble protein, and higher water use efficiency. The transgenic wheat plants had also a more extensive root system as well as increased photosynthetic capacity during stress treatments. The increased grain yield of the transgenic wheat was contributed by improved biomass, larger spike and grain numbers, and heavier 1000-grain weight under drought-stress conditions. Under non-stressed conditions, there were no significant increases in these of the measured traits except for photosynthetic rate when compared with parental wheat. Proteomic research showed that the expression levels of some proteins, including chlorophyll A-B binding protein and pyruvate, phosphate dikinase, which are related to photosynthesis, PAP fibrillin, which is involved in cytoskeleton synthesis, S-adenosylmethionine synthetase, which catalyzes methionine synthesis, were induced in the transgenic wheat under drought stress. Additionally, the expression of glutamine synthetase, which is involved in ammonia assimilation, was induced by drought stress in the wheat. Our study shows that PEPC can improve both stress tolerance and grain yield in wheat, demonstrating the efficacy of PEPC in crop improvement.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Purification and characterization of cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript> from <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

Cytochrome c ₆ , (cyt c ₆) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E _m) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Cry1C,Cry2A</Emphasis> and <Emphasis Type="Italic">Cry9C</Emphasis> genes into <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and plant regeneration

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

Electrostatic attraction between cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> and cytochrome <Emphasis Type="Italic">c</Emphasis> affects kinetics of cytochrome <Emphasis Type="Italic">c</Emphasis> reduction

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.     

Superoxide dismutase activity in C<Subscript>3</Subscript> and C<Subscript>3</Subscript>/CAM intermediate species of <Emphasis Type="Italic">Clusia</Emphasis>

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 μmol m⁻² s⁻¹ for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.