Physio-anatomical modifications and element allocation pattern in Alternanthera tenella Colla. associated with phytoextraction of chromium.

Intensive industrial activities have elevated chromium (Cr) concentrations in the environment, particularly in soil and water, posing a significant threat due to its cytotoxic and carcinogenic properties. Phytoremediation has emerged as a sustainable and economical alternative for detoxifying pollutants. In this context, an attempt has been made to assess the efficacy of Cr remediation by the invasive plant Alternanthera tenella Colla. The study investigated morphological, anatomical, and physiological adaptations in plant tissues in response to 240 µM of K2Cr2O7, considering elemental distribution patterns and bioaccumulation potential. Growth parameter assessments revealed a notable 50% reduction in root elongation and biomass content; however, the plant exhibited a comparatively higher tolerance index (47%) under Cr stress. Chromium significantly influenced macro and micro-elemental distribution in plant tissues, particularly in roots and leaves. Structural modifications, including changes in the thickness and diameter of xylem walls in the root, stem, and leaf tissues of Cr-treated A. tenella, were observed. Distinct cell structural distortions and Cr deposit inclusions in the xylem wall and inner parenchyma cells were distinct. Under Cr stress, there was a reduction in pigment content and metabolites such as proteins and soluble sugars, while proline, phenol, and malondialdehyde showed a twofold increase. The concentration of Cr was higher in the shoots of A. tenella (185.7 mg/kg DW) than in the roots (179.625 mg/kg DW). With a high BCFroot value (16.23) and TF > 1, coupled with effective mechanisms to cope with metal stress, A. tenella emerges as an ideal candidate for chromium phytoextraction.

Biosynthesis of Betalains Elicited by Methyl Jasmonate in Two Species of Alternanthera Genus: Antagonistic Regulations Result in Increase of Pigments.

Natural pigments are components very important in the dye industry. The betalains are pigments found in plants from Caryophyllales order and are relevant in the food manufacturing. The main source of betalains is beetroot, which has unfavorable aftertaste. Therefore, the demand for alternative species producing betalains has increased. Elicitor molecules such as methyl jasmonate (MeJA) induce metabolic reprogramming acting in the biosynthesis of specialized metabolites and can enhance pigment concentrations. Here, we used this strategy to identify if treatment with MeJA at 100 µM can promote the accumulation of betalains and other bioactive compounds in Alternanthera philoxeroides and Alternanthera sessilis. We performed the gene expression, concentration of betalains, phenols, flavonoids, amino acids (phenylalanine and tyrosine), and antioxidant activity. The results showed that MeJA treatment increased betalains and other bioactive compounds in the two Alternanthera species but A. sessilis had a better performance. One key factor in this pathway is related to the phenylalanine and tyrosine concentration. However, the species have distinct metabolic regulation: in A. philoxeroides, high concentrations of betalain pigments increase the tyrosine concentration and gene expression (include ADH) under MeJA and in A. sessilis, high concentrations of betalain pigments reduce the gene expression and tyrosine concentration after 2 days under MeJA. This study brings new questions about betalain biosynthesis and sheds light on the evolution of this pathway in Caryophyllales.

A biochemical and lipidomic approach to perceive Halimione portulacoides (L.) response to mercury: An environmental perspective.

The impact of hazardous materials, such as Hg, on life is far from being understood and due to the high number of polluted sites it has generated great concern. A biochemical and lipidomic approach was used to assess the effects of Hg on the saltmarsh halophyte Halimione portulacoides. Plants were collected at two sites of a Hg contaminated saltmarsh. Hg accumulation and distribution in the plant, biochemical parameters (antioxidant and metabolic) and lipid profiles were determined and compared between plant organs and sites (s1 and s2). Hg did not induce antioxidant enzyme activity. Lipid profiles changed under Hg exposure, especially in leaves, decreasing the unsaturation level, the membrane fluidity and stability, and evidencing that membrane lipid remodeling influences plant tolerance to Hg. This knowledge can help select the most appropriate methodologies for the restoration of Hg polluted hotspots, curtailing a serious environmental problem threatening saltmarshes.

Protoplast Isolation and Transfection in the Single-Cell C4 Species Bienertia sinuspersici.

We have developed an optimized protocol for isolating protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. The isolated protoplasts maintained the integrity of the unique single-cell C4 intracellular compartmentation of organelles as observed in chlorenchyma cells after cell wall digestion. Approximately over 80% of isolated protoplasts expressed the fusion reporter gene following the polyethylene glycol-mediated transfection procedures. Overall, fluorescent protein fusion tagged with various intraorganellular sorting signals validated the potential use of the transient gene expression system in subcellular localization and organelle dynamics studies.

Physiological responses of Agriophyllum squarrosum and Setaria viridis to drought and re-watering.

Drought resistance of psammophyte determines survival and growth, but their responses to drought are not well understood. We conducted a pot experiment to study how physiological characteristics respond to drought and rehydration. We found that watering to 60-65% of field capacity (the control) provided more water than was required by Agriophyllum squarrosum and its leaves became yellow and slightly wilted. The total chlorophyll content and Fm (maximum fluorescence after dark adaptation) in control were lower than in the drought treatment, and both decreased after rehydration. With increasing drought duration and intensity, the relative water content (RWC), chlorophyll content, Fm, and the quantum efficiency of photosystem II (Fv/Fm) of Setaria viridis decreased, but malondialdehyde and membrane permeability increased. During the late drought, the activities of three antioxidant enzymes in A. squarrosum increased to prevent membrane lipid peroxidation; for S. viridis, only peroxidase and superoxide dismutase activities increased. After rehydration, RWC of both species increased, but Fv/Fm of A. squarrosum and Fm of S. viridis did not recover under severe drought. Our research illustrated that A. squarrosum is better adapted to arid environment than S. viridis, but the high soil moisture content is not conducive to normal growth of A. squarrosum.

Separation of betacyanins from Iresine herbstii Hook. ex Lindl. leaves by high-speed countercurrent chromatography in a polar solvent system.

Betacyanins, natural plant pigments, from Iresine herbstii Hook. ex Lindl. leaf extract were separated for the first time by high-speed countercurrent chromatography (HSCCC) in a highly polar solvent system composed of PrOH-ACN-(NH4)2SO4satd.soln-H2O (1.0:0.5:1.2:1.0; v/v/v/v) in the tail-to-head mode. The flow rate of the mobile phase was 2.0 ml/min and the column rotation speed was 860 rpm. The retention of the stationary phase was 81.0%. For the identification of separated betacyanins in the crude extract and in the HSCCC fractions, as well as for the molecular formulas and multi-step fragmentation pattern elucidation, liquid chromatography with tandem mass spectrometry and high-resolution ion-trap time-of-flight mass spectrometry were performed. The innovative application of the HSCCC system for fractionation of the betacyanins present in I. herbstii leaves enabled effective separation as well as preconcentration of the pigments for further low- and high-resolution LC-MS/MS analysis. HSCCC separation enabled identification of 22 betacyanins, of which 18 had not been detected previously in the leaves of I. herbstii, and four of these betacyanins (sinapoyl-gomphrenin and coumaroyl-gomphrenin as well as their epimers) were identified for the first time in the Iresine genus.

Betacyanins from Gomphrena globosa L. flowers: Incorporation in cookies as natural colouring agents.

A betacyanin rich extract was obtained from the flowers of Gomphrena globosa L. by ultrasound-assisted extraction and dried either by lyophilization or spray-drying, was tested as a natural colourant in cookies and compared to a commercial colourant. The extracts were characterized in terms of betacyanin content and antioxidant potential. The effects of the colourants incorporation in the cookies were assessed through proximate composition, soluble sugars, fatty acids, color, texture and microbial load, over a shelf life of 30 days. Considering all the assays and analyzing the results through a 2-way analysis of variance, the cookies incorporated with spray-dried colourant showed the most intense pink coloration while cookies incorporated with lyophilized extract lost less color intensity over time. Thus, betacyanin extracts have potential as pink natural alternatives to synthetic colourants in the food industry.

Plants' genetic variation approach applied to zinc contamination: secondary metabolites and enzymes of the antioxidant system in Pfaffia glomerata accessions.

Zinc (Zn) is a micronutrient, but its excessive concentration can impair plant growth and development. Fertilizers, liming materials, pesticides and fungicides containing Zn have contributed to increase its concentration in agricultural soils. The aim of the present study is to evaluate the effect of Zn excess on the non-enzymatic (anthocyanin and ß-ecdysone) and enzymatic (superoxide dismutase-SOD and guaiacol peroxidase-GPX) antioxidant system of two P. glomerata accessions (JB and GD) grown in hydroponic system and soil, under short- and long-term exposure times. Three Zn levels (2, 100 and 200 µM) and two short-term exposure times (7 and 14 d) were tested in the hydroponic experiment. Three Zn levels (2, 100 and 200 mg kg-1) and two long-term exposure times (34 and 74 d) were tested in the soil experiment. The effects of Zn excess on P. glomerata accessions depended on the growth system and exposure time. Zinc excess in both tested growth systems resulted in significant change in the tissue oxidative process (MDA concentration) in both accessions, as well as broadened the antioxidant system response, which was based on antioxidant enzymes (SOD and GPX) and secondary metabolites (anthocyanins and ß-ecdysone). The highest anthocyanin concentration was observed in accession JB, which was grown in hydroponics, but tissue anthocyanin concentration increased in both accessions, regardless of growth medium and exposure time. The ß-ecdysone concentration in the roots increased in both accessions, but accession GD was more responsive to Zn excess. There was significant physiological variation in P.glomerata accessions in response to Zn excess.

Blue and red light affects morphogenesis and 20-hydroxyecdisone content of in vitro Pfaffia glomerata accessions.

The combination of different colors from light-emitting diodes (LEDs) may influence growth and production of secondary metabolites in plants. In the present study, the effect of light quality on morphophysiology and content of 20-hydroxyecdysone (20E), a phytoecdysteroid, was evaluated in accessions of an endangered medicinal species, Pfaffia glomerata, grown in vitro. Two accessions (Ac22 and Ac43) were cultured in vitro under three different ratios of red (R) and blue (B) LEDs: (i) 1R:1B, (ii) 1R:3B, and (iii) 3R:1B. An equal ratio of red and blue light (1R:1B) increased biomass accumulation, anthocyanin content, and 20E production (by 30-40%). Moreover, 1R:1B treatment increased the size of vascular bundles and vessel elements, as well as strengthened xylem lignification and thickening of the cell wall of shoots. The 1R:3B treatment induced the highest photosynthetic and electron transport rates and enhanced the activity of oxidative stress-related enzymes. Total Chl content, Chl/Car ratio, and NPQ varied more by accession type than by light source. Spectral quality affected primary metabolism differently in each accession. Specifically, in Ac22 plants, fructose content was higher under 1R:1B and 1R:3B treatments, whereas starch accumulation was higher under 1R:3B, and sucrose under 3R:1B. In Ac43 plants, sugars were not influenced by light spectral quality, but starch content was higher under 3R:1B conditions. In conclusion, red and blue LEDs enhance biomass and 20E production in P. glomerata grown in vitro.

Endophytic association of bioactive and halotolerant Humicola fuscoatra with halophytic plants, and its capability of producing anthraquinone and anthranol derivatives.

Halophytic plants growing in harsh desert environments are rich reservoirs of unique endophytic microorganisms. Here, healthy fresh plants of the families Tamaricaceae and Amarantaceae at three saline locations in Iran were investigated for their bioactive endophytic fungi. Among a vast number of isolates, eight isolates were identified as Humicola fuscoatra (Sordariomycetes, Pezizomycotina, Ascomycota) by microscopy and representative DNA sequences of the 5.8S rDNA (ITS) and partial ß-tubulin (TUB2). Those isolates were halotolerant, and highly bioactive, so that their intra- and extra-cellular metabolites possessed in vitro antifungal, antibacterial and antiproliferative activities, against a number of fungal and bacterial plant pathogens including the fungi Arthrobotrys conoides, Pyrenophora graminea, Pyricularia grisea and the bacteria Agrobacterium tumefaciens, Pseudomonas syringae and Xanthomonas oryzae. Chemical analyses of metabolites from the endophytes using HNMR, CNMR, NOESY, COSY, HMBC, HSQC, DEPT, TOCSY and EI MASS techniques identified 3,8-dihydroxy-1-methyl-9,10-anthracenedione (aloesaponarin II; an anthraquinone derivative), 1,8,9-anthracenetriol structure (chrysarobin; an anthranol derivative) and 2,4-di-tert-butylthiophenol in fungal extracts. To the best of our knowledge, this is the first report of endophytic association of halotolerant H. fuscoatra isolates with Tamaricaceae and Amarantaceae, and their bioactivity against plant pathogens. Also, the capability of chrysarobin and aloesaponarin II production is new to the fungal kingdom. These findings may find application in agriculture, pharmacology, and biotechnology.

Physiology and proteomic analysis reveals root, stem and leaf responses to potassium deficiency stress in alligator weed.

Alligator weed is reported to have a strong ability to adapt to potassium deficiency stress. Proteomic changes in response to this stress are largely unknown in alligator weed seedlings. In this study, we performed physiological and comparative proteomics of alligator weed seedlings between normal growth (CK) and potassium deficiency (LK) stress using 2-DE techniques, including root, stem and leaf tissues. Seedling height, soluble sugar content, PGK activity and H2O2 contents were significantly altered after 15 d of LK treatment. A total of 206 differentially expressed proteins (DEPs) were identified. There were 72 DEPs in the root, 79 in the stem, and 55 in the leaves. The proteomic results were verified using western blot and qRT-PCR assays. The most represented KEGG pathway was "Carbohydrate and energy metabolism" in the three samples. The "Protein degradation" pathway only existed in the stem and root, and the "Cell cycle" pathway only existed in the root. Protein-protein interaction analysis demonstrated that the interacting proteins detected were the most common in the stem, with 18 proteins. Our study highlights protein changes in alligator weed seedling under LK stress and provides new information on the comprehensive analysis of the protein network in plant potassium nutrition.

Maintenance of photosynthesis and the antioxidant defence systems have key roles for survival of Halopeplis perfoliata (Amaranthaceae) in a saline environment.

Coastal salt marsh plants employ various combinations of morphological and physiological adaptations to survive under saline conditions. Little information is available on salinity tolerance mechanisms of Halopeplis perfoliata, a C3 stem succulent halophyte. We investigated the growth, photosynthesis and antioxidant defence mechanisms of H. perfoliata under saline conditions (0, 150, 300 and 600 mM NaCl) in an open greenhouse. Optimal shoot succulence, projected shoot area and relative growth rate were obtained in the low (150 mm NaCl) salinity treatment, while growth was inhibited at the highest salinity (600 mm NaCl). The CO2 compensation point and carbon isotope composition of biomass confirmed C3 photosynthesis. Increases in salinity did not affect the photosynthetic pigment content or maximum quantum efficiency of PSII of H. perfoliata. Assimilation of CO2 (A) also remained unaffected by salinity. A modest effect on some gas exchange and photochemistry parameters was observed at 600 mm NaCl. With increasing salinity, there was a continual increase in respiration, suggesting utilisation of energy to cope with saline conditions. Under 300 and 600 mm NaCl, there was an increase in H2 O2 and MDA with a concomitant rise in AsA, GR content and CAT activity. Hence, H. perfoliata appears to be an obligate halophyte that can grow up to seawater salinities by modulating photosynthetic gas exchange, photochemistry and the antioxidant defence systems.

Effects of parental light environment on growth and morphological responses of clonal offspring.

Environments experienced by parent ramets of clonal plants can potentially influence fitness of clonal offspring ramets. Such clonal parental effects may result from heritable epigenetic changes, such as DNA methylation, which can be removed by application of DNA de-methylation agents such as 5-azacytidine. To test whether parental shading effects occur via clonal generation and whether DNA methylation plays a role in such effects, parent plants of the clonal herb Alternanthera philoxeroides were first subjected to two levels of light intensity (high versus low) crossed with two levels of DNA de-methylation (no or with de-methylation by application of 5-azacytidine), and then clonal offspring taken from each of these four types of parent plant were subjected to the same two light levels. Parental shading effects transmitted via clonal generation decreased growth and modified morphology of clonal offspring. Offspring responses were also influenced by DNA methylation level of parent plants. For clonal offspring growing under low light, parental shading effects on growth and morphology were always negative, irrespective of the parental de-methylation treatment. For clonal offspring growing under high light, parental shading effects on offspring growth and morphology were negative when the parents were not treated with 5-azacytidine, but neutral when they were treated with 5-azacytidine. Overall, parental shading effects on clonal offspring performance of A. philoxeroides were found, and DNA methylation is likely to be involved in such effects. However, parental shading effects contributed little to the tolerance of clonal offspring to shading.

Characterization of modified Alternanthera philoxeroides by diethylenetriamine and its application in the adsorption of copper(II) ions in aqueous solution.

By a simple and convenient method of using epichlorohydrin as linkages, a novel Alternanthera philoxeroides (AP) derivative modified with diethylenetriamine (DAP) was synthesized, which can remove copper(II) ions (Cu(II)) in the water environment efficiently. The adsorption capacity of DAP for Cu(II) under various factors was measured using ultraviolet spectrophotometer. The adsorption capacity and removal ratio were 19.33 mg/g and 95.57% at pH 5.5 and 298 K. The kinetic and equilibrium study shows that pseudo-second-order kinetic (R2 = 0.9964) and Langmuir isotherm models (R2 > 0.982) could properly describe DAP adsorption behaviors, and thermodynamic parameters indicate a spontaneous endothermic process (ΔG = - 3.6636 kJ/mol). The combined results of SEM, XRD, FTIR, and XPS analyses reveal that the dominant contribution for enhancement in Cu(II) adsorption is made by the formation of an amino group. And the adsorption mechanism is mainly the complexation reaction. The adsorption efficiency of DAP remained above 72.06% after 6 cycles of adsorption-desorption, which indicated that DAP has good regenerability and stability. All the results suggest that DAP could serve as promising adsorbents for Cu(II) pollution minimization.

Reactive green dye remediation by Alternanthera philoxeroides in association with plant growth promoting Klebsiella sp. VITAJ23: A pot culture study.

Contamination of soil by textile effluent is a major threat found worldwide. These pollutants have diverse range of negative effects on the ecosystem, therefore restoration through cost effective biological strategy is the need of the hour. The aim of the current study was to enhance the decolourization of reactive green dye (RGD) using phytoremediation coupled with augmentation of effective bacteria to the rhizosphere. The isolate Klebsiella sp. VITAJ23 was isolated from textile effluent polluted soil and was assessed for its plant growth promoting traits (PGP) and the PGP functional genes were amplified. The soil was artificially polluted with RGD concentration ranging from 1000 to 3000 mg kg-1 and Alternanthera philoxeroides plantlets were planted in phyto and rhizoremediation treatments, the setup was maintained upto 60 d. The isolate VITAJ23 was augmented in the rhizoremediation setup and the morphological parameters were assessed at regular interval. There was a significant increase in the chlorophyll content as well as root and shoot length of the plant when treated with the bacterial suspension. Decolourization study revealed 79% removal of reactive green dye with an enhanced oxido-reductase enzyme activity in the setup bioaugmented with bacteria. The biodegraded metabolites were identified as 2-allylnapthalene, l-alanine, n-acetyl-and propenoic acid by GC-MS analysis and a plant-bacteria degradation pathway was predicted using computational tools. Inoculation of PGP-Klebsiella sp. VITAJ23 enhanced the rate of plant growth and dye degradation.

Nutrient enhancement of allelopathic effects of exotic invasive on native plant species.

Many ecosystems may suffer from both nutrient enrichment and exotic plant invasions simultaneously. Much has been known that nutrient inputs can promote growth and expansion of exotic invasive plants in wetlands, and that allelopathic effects of the exotic invasive plants can inhibit the growth of coexisting native plants, contributing to their invasion success. Thus, we hypothesized that allelopathic effects of exotics on natives in invaded ecosystems can be enhanced by nutrient enrichment. To test this hypothesis, we conducted two greenhouse hydroponic experiments. One is the monoculture experiment in which a widespread exotic invasive perennial Alternanthera philoxeroides and a native perennial Ludwigia peploides subsp. stipulacea in monoculture were subjected to five levels of nutrient supply. The other is the mixture experiment in which the two species in mixture were subjected to five levels of nutrient supply, each with and without activated carbon addition. Both A. philoxeroides and L. peploides grew better under higher level of nutrient availability in monoculture experiment. In the mixture experiment, A. philoxeroides formed less total and root biomass while L. peploides formed more in response to activated carbon addition and all of the responses had larger degree at higher level of nutrient availability, indicating A. philoxeroides had significant allelopathic effects on L. peploides and the effects was significantly enhanced by nutrient enrichment. Such results support our hypothesis and reveal a novel mechanism for exotic plant invasion in eutrophicated and invaded wetlands, i.e. nutrient enhancement of allelopathic effects of exotics on natives.

Cadmium associates with oxalate in calcium oxalate crystals and competes with calcium for translocation to stems in the cadmium bioindicator Gomphrena claussenii.

Cadmium (Cd) was shown to co-localise with calcium (Ca) in oxalate crystals in the stems and leaves of Cd tolerant Gomphrena claussenii, but Cd binding remained unresolved. Using synchrotron radiation X-ray absorption near edge spectroscopy we demonstrate that in oxalate crystals of hydroponically grown G. claussenii the vast majority of Cd is bound to oxygen ligands in oxalate crystals (>88%; Cd-O-C coordination) and the remaining Cd is bound to sulphur ligands (Cd-S-C coordination). Cadmium binding to oxalate does not depend on the amount of Ca supplied or from which organs the crystals originate (stems and mature leaves). By contrast, roots contain no oxalate crystals and therein Cd is bound predominantly by S ligands. The potential to remove Cd by extraction of Cd-rich oxalate crystals from plant material should be tested in phytoextraction or phytomining strategies.

Physiological and quantitative proteomic analyses unraveling potassium deficiency stress response in alligator weed (Alternanthera philoxeroides L.) root.

KEY MESSAGE: Physiological and iTRAQ based proteomic analysis provided new insights into potassium deficiency stress response in alligator weed root. Alligator weed (Alternanthera philoxeroides) has a strong ability to adapt to potassium deficiency (LK) stress. Proteomic changes in response to this stress are largely unknown in alligator weed. In this study, we investigated physiological and molecular mechanisms under LK using isobaric tags for relative and absolute quantitation to characterize proteome-level changes in this plant. First, root physiology, 2, 3, 5-Triphenyl-trazolium chloride (TTC) assay and peroxidase activity were significantly altered after 10 and 15 days of LK treatment. The comparative proteomic analysis suggested a total of 375 proteins were differential abundance proteins. The proteomic results were verified by western blot assays and quantitative real-time PCR. Correlation analysis of transcription and proteomics suggested protein processing in the endoplasmic reticulum, endocytosis, and spliceosome pathways were significantly enriched. The protein responsible for energy metabolism, signal sensing and transduction and protein degradation played crucial roles in this stress. Twelve ubiquitin pathway related proteins were identified in our study, among them 11 proteins were up-regulated. All protein ubiquitination of lysine using pan antibodies were also increased after LK treatment. Our study provide a valuable insights of molecular mechanism underlying LK stress response in alligator weed roots and afford a vital basis to further study potassium nutrition molecular breeding of other plant species.

Responses in shoot elongation, carbohydrate utilization and growth recovery of an invasive species to submergence at different water temperatures.

Widely distributed amphibious exotic plant species may respond plastically to water temperatures when submerged. Alternanthera philoxeroides, a highly flood-tolerant species, originates from tropical regions and has successfully invaded temperate regions. The wide distribution of this species suggests it can respond to flooding at different water temperatures. In this study, the plastic responses of A. philoxeroides plants to submergence at water temperatures of 10 °C, 20 °C and 30 °C were investigated. The A. philoxeroides plants had large pools of non-structural carbohydrates, which were readily mobilized upon submergence. Submergence hindered biomass accumulation and decreased the carbohydrate content level and respiration rate (P < 0.05). Water temperature had remarkable effects on shoot elongation, carbohydrate utilization and recovery growth. With decreasing water temperature, the respiration rate was lower and carbohydrate content decreased more slowly, but the post-submergence biomass accumulation was faster (P < 0.05), indicating a beneficial effect of low water temperature for recovery. However, high water temperatures accelerated shoot elongation (P < 0.05), which benefitted the submerged plants more if contact with air was restored. These results suggest that the species can respond to different water temperatures plastically, which may provide hints for its invasion success in regions with diverse climates.

Changes in redox regulation during transition from C3 to single cell C4 photosynthesis in Bienertia sinuspersici.

Bienertia sinuspersici performs single cell C4 photosynthesis without Kranz anatomy. Peripheral and central cytoplasmic compartments in a single chlorenchyma cell act as mesophyll cells and bundle sheath cells. Development of this specialized mechanism is gradual during plant development. Young leaves perform C3 photosynthesis, while mature leaves have complete C4 cycle. The aim of this work was to investigate changes in redox regulation and antioxidant defence during transition from C3 to single cell C4 photosynthesis in B. sinuspersici leaves. First, we confirmed gradual development of C4 with protein blot and qRT-PCR analysis of C4 enzymes. After this activities and isoenzymes of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and H2O2 and TBARS and glutathione pool and redox status (GSH/GSSG) were determined in young, developing and mature leaves during transition from C3 to single cell C4 photosynthesis. Activities of SOD, APX and POX decrease, while GR and DHAR were increased. However, most striking results were the changes in isoenzyme patterns of SOD, CAT and GR which were gradual through transition to C4 photosynthesis.