Sunflower Cotyledons Research Articles

Expression of Recombinant Human Glucocerebrosidase Protein in Sunflowers

Molecular farming has become one of the most significant implementations of modern biotechnology to generate modified plant crops to produce medicinal proteins. Agrobacterium is one plant genetic engineering tool that integrates genes of interest inside a host plant. In recent years, the need to produce recombinant proteins as therapeutics has growing rapidly, and human glucocerebrosidase is one of the proteins that is need to treat disease. In this study, specific primers were designed to amplify Hu-GBA1 gene from constructed pGEM-GBA plasmid which was cloned into the plant expression vector pCAMBIA1304. The generated recombinant pCAMBIA1304-GBA plasmid was used to transform A. tumefaciens LBA4404 and applied for transformation of sunflower cotyledon explants. Colony PCR technique was used to confirm the presence of Hu-GBA1 gene in transformed A. tumefaciens. Agrobacterium containing pCAMBIA1304-GBA was suspended in Infection Medium (IM) supplement with 200 mM acetosyringone. A bacterial suspension was used to transform sunflower cotyledons. After infection, cotyledons were co-cultivated in Co-cultivation medium (CCM), supplied with 200 mM acetosyringone without antibiotics. The cotyledons were then transferred to selection media containing 7.5 mg/L Hygromycin and 250 mg/L Cefotaxime and grown for additional 14 days at 25℃ in photoperiod of 16h L/8h D. The transformed sunflower cotyledons were successfully generated complete plant with used 6-Benzylaminopurine and Naphthalene acetic acid as growth hormones. The presence of the Hu-GBA1 gene in the genomic DNA of transgenic sunflower plant was proven by PCR as a band of 1561bp size. The GBA mRNA expression in modified sunflowers was detected by qRT-PCR compared with control GBA mRNA. Enzyme Linked Immunoassay was done on crude recombinant protein that extracted from transformed sunflower using Human Glucosylceramide ELISA Kit, the Elisa test results confirmed the production of recombinant glucocerebrosidase and the concentration of crude recombinant enzyme extracted from transformed sunflower with GBA1 gene was 0.45 ng/µl

Expression of Recombinant Human Glucocerebrosidase Protein in Sunflowers

Molecular farming has become one of the most significant implementations of modern biotechnology to generate modified plant crops to produce medicinal proteins. Agrobacterium is one plant genetic engineering tool that integrates genes of interest inside a host plant. In recent years, the need to produce recombinant proteins as therapeutics has growing rapidly, and human glucocerebrosidase is one of the proteins that is need to treat disease. In this study, specific primers were designed to amplify Hu-GBA1 gene from constructed pGEM-GBA plasmid which was cloned into the plant expression vector pCAMBIA1304. The generated recombinant pCAMBIA1304-GBA plasmid was used to transform A. tumefaciens LBA4404 and applied for transformation of sunflower cotyledon explants. Colony PCR technique was used to confirm the presence of Hu-GBA1 gene in transformed A. tumefaciens. Agrobacterium containing pCAMBIA1304-GBA was suspended in Infection Medium (IM) supplement with 200 mM acetosyringone. A bacterial suspension was used to transform sunflower cotyledons. After infection, cotyledons were co-cultivated in Co-cultivation medium (CCM), supplied with 200 mM acetosyringone without antibiotics. The cotyledons were then transferred to selection media containing 7.5 mg/L Hygromycin and 250 mg/L Cefotaxime and grown for additional 14 days at 25℃ in photoperiod of 16h L/8h D. The transformed sunflower cotyledons were successfully generated complete plant with used 6-Benzylaminopurine and Naphthalene acetic acid as growth hormones. The presence of the Hu-GBA1 gene in the genomic DNA of transgenic sunflower plant was proven by PCR as a band of 1561bp size. The GBA mRNA expression in modified sunflowers was detected by qRT-PCR compared with control GBA mRNA. Enzyme Linked Immunoassay was done on crude recombinant protein that extracted from transformed sunflower using Human Glucosylceramide ELISA Kit, the Elisa test results confirmed the production of recombinant glucocerebrosidase and the concentration of crude recombinant enzyme extracted from transformed sunflower with GBA1 gene was 0.45 ng/µl

Expression and properties of the mitochondrial and cytosolic forms of fumarase in sunflower cotyledons

Fumarase (EC 4.2.1.2) is encoded in sunflower (Helianthus annuus L.) by two genes (FUM1 and FUM2) expressing correspondingly the mitochondrial and the cytosolic form. Both forms have been purified from sunflower cotyledons and characterized. Three quarters of fumarase activity is located in the mitochondrial and one quarter in the cytosolic fraction. The cytosolic form has lower pH optimum than the mitochondrial form, it possesses higher affinity to malate, activated by Mn2+ and less efficiently by Mg2+ while the mitochondrial form is activated only by Mg2+. It is proposed that the mitochondrial form is involved in the respiratory processes linked to the tricarboxylic acid cycle and the cytosolic form participates in the utilization of succinate produced in the glyoxylate cycle providing the flux to gluconeogenesis in germinating sunflower seeds.

Characterization of Mutant Sunflower Acetoacetyl CoA Thiolase

Sunflower (Helianthus annus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism and other residues have been implicated in the stabilization of the enolate intermediate in the synthetic reaction. Site‐directed mutagenesis of the various amino acid residues was done and the effect of these mutations on the catalytic activity was measured.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Temperature-dependent chlorophyll accumulation and photosystem II assembly during etioplast to chloroplast transition in sunflower cotyledons

Abstract Despite numerous data dealing with the biogenesis of photosynthetic membranes connected with specific functional alterations in higher plants this is still an insufficiently understood topic and is one of the most promising areas of research in plant biochemistry. The main goal of our study was to detect the impact of different temperatures on chlorophyll biosynthesis and the maximum quantum yield of PSII (Fv/Fm). Therefore, we investigated the greening processes in etiolated sunflower cotyledons (Helianthus annuus L.) grown at different temperatures (10, 20 and 30 °C) during 24 h. The dynamics of chlorophyll a and b (Chl a and Chl b) accumulation as well as photosystem II (PSII) effi ciency were observed. We also evaluated combined effects of different temperatures (20 and 30 °C) and short-term application of increased irradiation (800 μmol m-2 s-1) on effective quantum yield of PSII (ΔF/F’m) and non photochemical quenching (NPQ) in cotyledons with fully developed PSII. Our results showed reduced chlorophyll accumulation and the arrest of PSII assembly at 10 °C in comparison with 20 and 30 °C. Further, the increased irradiance induced equal down regulation of effective quantum yield of PSII at 20 and 30 °C, with significantly higher capability of heat dissipation at 30 °C.

Photomodulation of fatty acid composition and lipoxygenase activity in sunflower seedling cotyledons

The dynamics of fatty acid composition is an important metabolic index of seed germination and seedling development process. Present investigations indicate a preferential mobilization of fatty acids, particularly linoleic acid, out of the oil bodies in sunflower seedling cotyledons. A lack of selectivity in the utilization of saturated fatty acids during seed germination is also evident. Lipoxygenase (LOX) may play a role in the reconstitution of membrane lipid composition as cells expand during seed germination. LOX activity is absent in sunflower seeds and appears during germination. Photo modulation of LOX activity is evident by its enhanced expression in light-grown seedling cotyledons. Since sunflower LOX activity is easily solubilized by mild detergent, such as 2 % Triton X-100, the association of LOX with the membrane does not seem to be very strong unlike other membrane proteins, such as oleosins, which can be extracted only by stringent procedures. Sunflower LOX has only one pH optimum with linoleic and linolenic acids as substrates, indicating the presence of single isoform in the total soluble protein (TSP) and oil body (OB) extracts. OB-associated LOX exhibits substrate-dependent variation in its pH optima. Specific activity of TSP and OB-associated LOX increases as seedling growth progresses. Sunflower TSP-LOX activity is inhibited by some hydrophobic thiols. TSP as well as OB-associated LOX activity in sunflower cotyledons is modulated by Ca2+ availability in a negative way. A critical role of LOX is, thus, evident during seed germination in sunflower.

The effect of epibrassinolide on senescence in horizontal sunflower (Helianthus annuus L.) seedlings

This study examined the effect of epibrassinolide (eBL) on senescence occurring in cotyledons of sunflower ( Helianthus annuus L.) seedlings, which were kept in vertical and horizontal positions. 10 -11 M and 10 -9 M eBL and 2,3,5-triiodobenzoic acid (TIBA), an inhibitor of auxin transport, were sprayed to the 14 days old seedlings. From the 15 th day on, some of the seedlings from the control and the experimental groups were kept in a horizontal position, and senescence process of the cotyledons of these seedlings was observed. Applications of eBL (especially 10 -9 M) were found to have induced senescence both in horizontally and vertically positioned plants. When it was applied with TIBA, a marked delay of senescence was noted in the lower cotyledons of the horizontally positioned plants. Total chlorophyll and protein amounts and peroxidase activity were determined. In case brassinosteroids are applied with TIBA, they do not affect senescence, implying that they accelerate senescence in the presence of auxin. Since auxin is distributed asymmetrically, eBL and auxin accumulated in the lower cotyledons may lead to accelerated senescence. This study showed that eBL and auxin may act as a senescence signal in the vertically and horizontally positioned seedlings, and earlier death of the lower cotyledons than the upper ones may be a result of asymmetrical auxin distribution. In this study, it was revealed that promoting effect of eBL on senescence occured only in the presence of auxin. Keywords: eBL – Epibrassinolide, TIBA - 2,3,5-triiodobensoic acid, Auxin, Senescence, Sunflower ( Helianthus annuus L.). *Corresponding Author: Serap Saglam-Cag (e-mail:sercag@istanbul.edu.tr) (Received: 13.09.2012 Accepted: 04.03.2013)

Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds

Genetic improvement of sunflower (Helianthus annuus L.) through the use of biotechnological tools requires a reliable in vitro shoot regeneration system. Tissue culture protocols reported to date for sunflower suffer from low efficiency, poor reproducibility, genotype dependence and a tendency for flowering in vitro. The present study describes an efficient protocol system for shoot regeneration via direct adventitious shoot organogenesis from cotyledons of mature seeds of sunflower. About 169 media combinations comprising 12 different growth regulator combinations in various concentrations were assessed for induction of shoots from cotyledons derived from mature seeds and also from seedling tissues of 2–20-day-old seedlings. Appearance of shoots from seedling tissues was sporadic and the frequency of shoot regeneration was low. Cotyledon explants from mature seeds were consistent with regard to frequency of adventitious shoot regeneration and number of shoots per explant. A high frequency (93.86 %) of adventitious shoot regeneration was obtained within 2 weeks of culture initiation on Murashige and Skoog (MS) medium supplemented with 9.84 μM 2-isopentenyladenine (2-iP), 2.85 μM indole-3-acetic acid (IAA) and 0.45 μM thidiazuron (TDZ). Use of 2-iP in the shoot induction and elongation media prevented precocious flowering. Statistical analysis revealed significant effects of explant orientation, age of seedlings, and genotype on adventitious organogenesis. Maximum shoot regeneration was obtained when cotyledons from 0 and 1-day-old seedlings were placed with their adaxial surface in contact with the medium surface. The protocol developed was tested on 42 genotypes and found to be applicable to a wide range of genotypes. Histological studies indicated that the shoots originated predominantly through adventive organogenesis from the sub-epidermal and cortical regions.

Expression and Purification of Sunflower Thiolase Mutants

Sunflower (Helianthus annus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of mutant forms of these two thiolases from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and constructs were transformed into BL‐21 E. coli that were induced for production of the thiolases with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble thiolases were purified and were used to characterize their enzymatic properties.

PH modulation differs during sunflower cotyledon colonization by the two closely related necrotrophic fungi Botrytis cinerea and Sclerotinia sclerotiorum

During pathogenesis on sunflower cotyledons, Botrytis cinerea and Sclerotinia sclerotiorum show a striking resemblance in symptom development. Based on pH change profiles, the colonization process of both fungi can be divided into two stages. The first stage is associated with a pH decrease, resulting from an accumulation of citric and succinic acids. The second stage is correlated with a pH increase, resulting from an accumulation of ammonia. In this article, we also report that oxalic acid is produced at the late stage of the colonization process and that ammonia accumulation is concomitant with a decrease in free amino acids in decaying tissues. Sclerotinia sclerotiorum produces eight-fold more oxalic acid and two-fold less ammonia than B. cinerea. Consequently, during sunflower cotyledon colonization by B. cinerea, pH dynamics differ significantly from those of S. sclerotiorum. In vitro assays support the in planta results and show that decreases in pH are linked to glucose consumption. At different stages of the colonization process, expression profiles of genes encoding secreted proteases were investigated. This analysis highlights that the expression levels of the B. cinerea protease genes are higher than those of S. sclerotiorum. This work suggests that the overt similarities of S. sclerotiorum and B. cinerea symptom development have probably masked our recognition of the dynamic and potentially different metabolic pathways active during host colonization by these two necrotrophic fungi.

Verticillium dahliae's VdNEP acts both as a plant defence elicitor and a pathogenicity factor in the interaction with Helianthus annuus

The soil-borne fungus Verticillium dahliae Kleb. causes a serious wilt disease in many economic crops worldwide, including sunflower. We investigated the role of V. dahliae's necrosis and ethylene-inducing proteins (VdNEP) in the interaction with sunflower. We inoculated two highly and two weakly aggressive V. dahliae isolates onto moderately resistant and susceptible sunflower hybrids. We also synthesized VdNEP protein in vitro and infiltrated it into sunflower control plants to compare its effects with those of the fungus. VdNEP induced wilting symptoms, i.e. chlorosis, necrosis and vascular discoloration but also triggered host defence responses, i.e. hypersensitive response in Nicotiana benthamiana leaves and sunflower cotyledons. It activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves, as well as pathogenesis-related genes (Ha-PR-3 and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and those encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO. The latter suggested that more than one signalling pathway may be involved in the V. dahliae-sunflower interaction. Two other genes (Ha-PAL and Ha-NML1), related to the salicylic acid pathway, were slightly downregulated by VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defences. The phenotypic changes induced by VdNEP indicate that this protein acts both as a defence elicitor and a pathogenicity factor.

Sunflower cotyledons cope with copper stress by inducing catalase subunits less sensitive to oxidation

Copper is an essential trace element for living organisms, in excess, can be toxic to the cell because of its capacity to generate reactive oxygen species (ROS). Catalase (CAT) catalyzes the dismutation of hydrogen peroxide into water and dioxygen and in plants it is located in peroxisomes and glyoxysomes. Different metals can induce changes in CAT activity, but the mechanism underlying its changes is unclear. After 4 h of treatment with 5 and 10 μM CuCl 2 a decrease in the specific CAT activity was detected in sunflower cotyledons of post-germinative heterotrophic seedlings. At 8 h of treatment, 5 μM Cu 2+ produced an induction of CAT activity while only a complete recovery to control values was observed for 10 μM Cu 2+ treated seedlings. These activity variations were not related to the level of CAT protein expression, but they did correlate with the oxidative state of the CAT protein. This indicates that the mechanism of CAT inactivation by Cu 2+ involves oxidation of the protein structure. The level of the mRNA of CATA3 and CATA4 increased with the presence of the metal after 4 h of exposure. These CAT genes code for the synthesis of CAT subunits less sensitive to oxidation, which would prevent the copper-induced oxidative inactivation of CAT.

The interaction of light irradiance with auxin in regulating growth of Helianthus annuus shoots

Plants growing under canopy shade or in near-neighboring proximity of taller vegetation are the receivers of shade light conditions. The effect of light irradiance (photosynthetically active radiation [PAR]), one of the main components of shade light, on the growth of various tissues of sunflower seedlings and the possible role of auxin were investigated. Gradual reductions in PAR irradiance level from near-normal to low and very low result in significant and gradual increases in sunflower hypocotyl growth and endogenous auxin content. Similar reductions in PAR level resulted in significant and gradual decreases in sunflower cotyledon and leaf growth, and endogenous auxin content. Exogenously applied auxin increased hypocotyl elongation under near-normal PAR, where IAA levels are below optimum, but decreased elongation under very low PAR, where IAA levels are already at optimum. These results suggests that auxin acts as positive growth regulator of sunflower hypocotyls subjected to low light irradiance stress. This is further supported by the transfer experiments where seedlings transferred, for example, from near-normal PAR to very low PAR showed increased elongation associated with increased IAA levels. Therefore, it is reasonable to conclude that light irradiance-mediated changes in hypocotyl elongation of young sunflower seedlings are regulated by endogenous auxin levels.

Localization of lipoxygenase activity on the oil bodies and in protoplasts using a novel fluorescence imaging method

Lipoxygenase (linoleate:oxygen oxidoreductase; EC 1.13.11.12; LOX) catalyzes oxygenation of polyenoic fatty acids, which precedes the degradation of storage lipids during seed germination in sunflower. In the present work, it has been confirmed that 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) produces fluorescence in presence of lipid hydroperoxides (LOX reaction products). This work provides new information on spatial localization of transiently enhanced LOX activity in protoplasts from 5d old seedling cotyledons of sunflower (Helianthus annuus L. cv. Morden) by exploiting H(2)DCFDA as a probe for fluorescence detection from LOX activity sites. Use of LOX inhibitors [nordihydroguaiaretic acid (NDGA) and propyl gallate (PG)] confirms oil bodies as LOX activity sites. Oil body surface has been shown to possess LOX activity in 5d old seedling cotyledons.

Expression and Purification of Sunflower Cotyledon Acetoacetyl CoA Thiolase Mutants

Sunflower (Helianthus annuus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of mutant forms of Thiolase II from an E. coli bacterial expression system. Mutants generated by site‐directed mutagenesis were cloned into the pET151 expression vector, and this construct was transformed into BL‐21 E. coli that were induced for production of the Thiolase II with IPTG (Isopropyl β‐D‐1‐thiogalactopyranoside). The soluble Thiolase II was purified to apparent homogeneity using Ni‐NTA agarose affinity chromatography as indicated by a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.

Amino acid changes during sunflower infection by the necrotrophic fungus B. cinerea

Metabolic changes that occur in host tissues during a necrotrophic plant/fungal interaction have been poorly investigated. Whereas carbon metabolism reprogramming and photosynthesis disturbances have been studied,1 data on plant amino acids stores during infection are scarce. Here we report an analysis of sunflower cotyledon amino acid content during infection with the necrotrophic fungus Botrytis cinerea, by using 13C-NMR spectroscopy. A rapid disappearance of plant amino acids was observed, most probably due to fungal assimilation. In order to explore amino acid changes due to host reaction, we investigated the amino acid content in healthy and invaded region of infected leaves. During the course of infection, glutamate and total amino acid stores were affected at distance in the non invaded region. Glutamate depletion was correlated to an enhanced sunflower glutamate dehydrogenase (GDH) transcription level in the area invaded by pathogen. Our data suggest that glutamate could be transferred to the invaded region to supply nitrogen. Such a strategy could delay cell death, and consequently disturb fungal progression in plant tissues.

Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic fungus Botrytis cinerea: from plant hexoses to mannitol

The main steps for carbon acquisition and conversion by Botrytis cinerea during pathogenesis of sunflower cotyledon were investigated here. A sequential view of soluble carbon metabolites detected by NMR spectroscopy during infection is presented. Disappearance of plant hexoses and their conversion to fungal metabolites were investigated by expression analysis of an extended gene family of hexose transporters (Bchxts) and of the mannitol pathway, using quantitative PCR. In order to analyse the main fungal metabolic routes used by B. cinerea in real time, we performed, for the first time, in vivo NMR analyses during plant infection. During infection, B. cinerea converts plant hexoses into mannitol. Expression analysis of the sugar porter gene family suggested predominance for transcription induced upon low glucose conditions and regulated according to the developmental phase. Allocation of plant hexoses by the pathogen revealed a conversion to mannitol, trehalose and glycogen for glucose and a preponderant transformation of fructose to mannitol by a more efficient metabolic pathway. Uptake of plant hexoses by B. cinerea is based on a multigenic flexible hexose uptake system. Their conversion into mannitol, enabled by two simultaneously expressed pathways, generates a dynamic intracellular carbon pool.

EFFECT OF GROWTH REGULATORS, SALINITY AND HEAVY METAL STRESSES ON ACID PHOSPHATASE ACTIVITY OF SUNFLOWER COTYLEDONS

Acid phosphatase (EC 3. 1. 3. 2) activity was measured in sunflower (Helianthus annus L.) cotyledons. The present results indicate that the tested growth regulators indoleacetic acid (IAA), naphthylacetic acid (NAA) and indolephenoxyacetic acid (IPA) induced acid phosphatase activity at concentration range 100-400 µM. The enzyme was inhibited by CdCl2 and PbCl2 at 20-100 µM. Cd2+ and Pb2+ enhanced lipid peroxidation and H2O2 content. Malondialdehyde (MDA) content was reduced in cotyledons pretreated with GA3. Salinity induced lipid peroxidation and increased H2O2 content. NaCl at low concentrations (50 and 100 mM) induced the enzyme activity whereas the higher concentrations (150-300 mM) reduced the activity. Coronatine (COR) at 100 µM alleviated partially the toxic effect of salinity.

Optimization of expression and purification of acetoacetyl CoA thiolase from sunflower cotyledon

Sunflower (Helianthus annus L) cotyledons have two thiolase activities which have been identified previously. Thiolase I (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase II (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of a Thiolase I from an E. coli expression system. The sunflower thiolase was cloned into the pBAD‐HisB expression vector, and this construct was transformed into BL‐21 E. coli which were induced for production of the Thiolase I with 0.2% arabinose. The soluble Thiolase 1 was purified using Ni‐NTA agarose affinity chromatography with a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.

Investigation of amino acid residues involved in the sunflower thiolase reaction mechanism

Acetoacetyl CoA thiolase is one of two thiolase activities found in sunflower cotyledons. This thiolase activity is involved in the oxidation of the short‐chain substrate acetoacetyl CoA to produce two acetyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism. The purpose of this research is to do site‐directed mutagenesis of the corresponding cysteine and histidine residues in the sunflower acetoacetyl CoA thiolase