Glutamine-synthetase isoforms appearing in sunflower cotyledons during germination

Ion-exchange chromatography has been used to separate the isoforms of glutamine synthetase (GS; EC 6.3.1.2) appearing in sunflower (Helianthus annuus L. cv. Peredovic) cotyledons during seedling growth under different light and nitrogen conditions. Both in dry and imbibed seeds, only a single form of GS (GS_s) was detected. Upon seed germination, the GS_s isoform was gradually replaced by cytosolic (GS₁) and plastidic (GS₂) isoforms. Light and nitrate decreased the levels of GS₁. In contrast, the appearance of GS₂ was greatly stimulated by light. Nitrate also had a positive effect, particularly in the light. Light and nitrate acted synergistically on the appearance of GS₂. The GS₂:GS₁ ratio in cotyledons of 9-d-old seedlings ranged from about 2, in darkness and nitrate-deprivation conditions, to 16 under light and nitrate application. The possible physiological roles of the distinct GS isoforms appearing in the epigeal cotyledons of sunflower during germination, and their differential regulation by light and nitrate, are discussed.Abbreviations GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic GS - GS_s GS from seeds This work was supported by a grant from Dirección General de Investigatión Científica y Técnica (PB90-0777) and Plan Andaluz de Investigación (3261), Spain. P.C. gratefully acknowledges receipt of a scholarship from Junta de Andalucía. The valuable technical assistance of Mrs. G. Alcalá is greatly appreciated. We are also grateful to Eurosemillas (Córdoba) for supplying us with sunflower seeds.     

Responsive regions for direct organogenesis in sunflower cotyledons

Regeneration efficiency from three different regions of cotyledonary explants was examined in six sunflower inbred lines. Proximal, middle and distal regions from seedling cotyledons were cultured on regeneration medium supplemented with growth regulators. Plant regeneration by direct organogenesis was observed after four weeks. Significant differences among inbred lines were found for regeneration percentage and average number of shoots per total explants. Also a decreasing regeneration capacity was observed from proximal to distal sections for all inbred lines. Regeneration ability from cotyledonary explants in this species is strongly influenced by the genotype and by the region from which the explant was obtained. The distance to the cotyledonary node plays a preponderant role in the expression of shoot forming capacity. Shoot differentiation via seedling cotyledons depends upon the presence of the proximal region of cotyledon regardless of the genotype.     

Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower (Helianthus annuus L.)

Lack of a genotype-independent transformation protocol for sunflower is a major bottleneck in improving this important oilseed crop. An efficient Agrobacterium-mediated transformation protocol is described, which was adaptable across a broad range of sunflower genotypes. The improved transformation approach used cotyledons from mature seeds vertically split through the embryo axis. The LBA 4404 Agrobacterium strain was used, which carried pCAMBIA 2301 plasmid containing UidA as the reporter gene and nptII as the selectable marker for transformants on kanamycin. Bacterial titer, cotyledon type, acetosyringone concentration and vacuum application enhanced the transformation efficiency. Wounding, enzyme pretreatment and sonication significantly reduced the transformation frequency. Putative transgenic shoots were obtained through both axillary proliferation and adventitious shoot regeneration. Following two and three cycles of selection on kanamycin for axillary and adventitious shoots, respectively, putative transformed shoots were obtained at an average frequency of 3.0?%. Reporter gene histochemical assay and molecular characterization of primary and T₁ transgenic plants revealed stable transgene integration, expression and monogenic inheritance. The standardized procedure was tested on 28 genotypes comprising sets of inbred, maintainer, restorer and hybrid lines. Transformation was successful in all genotypes albeit with variable frequency in all except the hybrid lines wherein it was stable around 4.0?%. The procedure opens possibilities of directly improving any commercial genotypes of sunflower.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Improved shoot development and rooting from mature cotyledons of sunflower

Regeneration and development of shoots from sunflower cotyledons were improved by optimizing explant age, plant growth regulator concentrations, and duration of exposure to plant growth regulators during shoot initiation, development and rooting. Shoot initiation required only a brief exposure to auxin and cytokinin, and minimizing the duration of exposure to high levels of plant growth regulators improved shoot development. Rooting was improved in terms of both the number of shoots that rooted and the time required for rooting by incorporating activated charcoal in the lower layer of a 2-layer rooting medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glyoxysomal acetoacetyl-CoA thiolase and 3-oxoacyl-CoA thiolase from sunflower cotyledons

Following chromatography on hydroxyapatite, the elution profile of the thiolase activity of the glyoxysomal fraction from sunflower (Helianthus annuus L.) cotyledons exhibited two peaks when the enzyme activity was assayed with acetoacetyl-CoA as substrate. Only one of these two activity peaks was detectable when a long-chain thiolase substrate was used in the activity assay. The proteins (thiolase I and thiolase II) underlying the two activity peaks detected with acetoacetyl-CoA were of glyoxysomal origin. They were purified using glyoxysomal matrices as starting material, and biochemically characterized. Thiolase I is an acetoacetyl-CoA thiolase (EC 2.3.1.9) exhibiting activity only towards acetoacetyl-CoA (Km = 11 microM). Its contribution to the total glyoxysomal thiolytic activity towards acetoacetyl-CoA amounted to about 15%. Thiolase II is a 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The activity of the enzyme towards 3-oxoacyl-CoAs increased with increasing chain length of the substrate. Thiolase II exhibited a Km value of 27 microM with acetoacetyl-CoA as substrate. and Km values between 3 and 7 microM with substrates having a carbon chain length from 6 to 16 carbon atoms. The thiolase activity of the glyoxysomes towards acetoacetyl-CoA and 3-oxopalmitoyl-CoA exceeded the glyoxysomal butyryl-CoA and palmitoyl-CoA beta-oxidation rates, respectively, by about 10-fold at all substrate concentrations employed (1-15 microM).     

Activity and hematin content of catalase from greening sunflower cotyledons

The specific activity of catalase purified from the peroxisomes of sunflower cotyledons declines in parallel with the total cotyledonary catalase activity during the transition of peroxisomes from glyoxysomal to leaf peroxisomal function. The hematin content of the purified catalase however, remains constant at 4 hematin groups per catalase molecule. The absorbance coefficients of catalase at 404 and 280 nm were determined to be 372 and 540/mM/cm, respectively.     

High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis

Summary A plant regeneration methodvia somatic embryogenesis of severalHelianthus annuus L. genotypes was developed. Starting from cotyledonary explants high frequency embryo induction was obtained following several subcultures on defined media. An appropriate cotyledon developmental stage was identified. Etiolated explants and darkness treatment were necessary to obtain somatic embryos in all tested genotypes. After 20–25 days on somatic induction medium containing an auxin:cytokinin ratio of 1:1, the germination of embryos was induced by a reduction of the hormonal ratio (1:2). Shoots were excised from callus and transferred onto a medium containing various vitamins. The range of embryogenesis frequency was 33–72%, depending on the genotype. High frequency of rooting (49–82%) was obtained using a medium supplemented with 0.5 mg/L of ancymidol and by a reduction of photoperiod. A large percentage of somatic embryos developed into normal regenerated plants producing viable seeds.Abbreviations MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - BAP benzylaminopurine - GA₃ gibberellic acid - EIM embryo induction medium - GM germination medium - VM vitamins medium - RA2 ancymidol rooting medium - EtOH ethanol     

Organization of lipid reserves in cotyledons of primed and aged sunflower seeds

Imbibing sunflower (Helianthus annuus L., cv. Briosol) seeds at water potentials between –2 MPa and –5 MPa leads to faster (priming) or slower (accelerated ageing) germination depending on the temperature and duration of treatment. Mobilization of food reserves may be associated with the changes in seed vigor. To study this, morphological, biochemical and phase properties of lipid, the major food reserve in sunflower, were compared in freshly harvested (i.e., control), primed and aged sunflower cotyledons using electron microscopy, biochemical analyses and differential scanning calorimetry, respectively. Lipid bodies became smaller and more dispersed throughout the cytoplasm during priming and ageing. Despite ultrastructural changes, there were few measured changes in biochemistry of the neutral lipid component; lipid content, proportion of saturated and unsaturated fatty acids and level of free fatty acids were unchanged in primed and slightly aged seeds, with only severely aged seeds showing a net decrease in polyunsaturated fatty acids and an increase in free fatty acids. Subtle changes in the calorimetric behavior of lipids within sunflower cotyledons were observed. Sunflower lipids exhibited polymorphic crystalline and amorphous solid phases when cooled to <–100°C, but priming decreased the rate of crystallization in vivo and ageing increased the rate of crystallization, but decreased percentage crystallinity. The observed changes in thermal behavior in vivo are consistent with losses and gains, respectively, of interacting non-lipid moieties in the triacylglycerol matrix.     

Turnover of catalase heme and apoprotein moieties in cotyledons of sunflower seedlings

The turnover of catalase apoprotein and catalase heme was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings by density labeling of apoprotein and radioactive labeling of heme moieties. The heavy isotope (50% ²H₂O) and the radioactive isotope ([¹⁴C]5-aminolevulinic acid) were applied either during growth in the dark (day 0-2.5) or in the light (day 2.5 and 5). Following isopycnic centrifugation of catalase purified from cotyledons of 5-day-old seedlings, superimposition curve fitting was used to determine the amounts of radioactive heme moieties in native and density-labeled catalase. Data from these determinations indicated that turnover of catalase heme and apoprotein essentially was coordinate. Only small amounts of heme groups were recycled into newly synthesized apoprotein during growth in the light, and no evidence was found for an exchange of heme groups in apoprotein moieties. It followed from these observations that degradation of catalase apoprotein was slightly faster than that of catalase heme. A degradation constant for catalase apoprotein of 0.263 per day was determined from the data on heme recycling and the degradation constant of catalase heme determined previously to be 0.205 per day (R Eising, B Gerhardt [1987] Plant Physiol 84: 225-232).     

Genetic control of organogenesis in cotyledons of sunflower (Helianthus annuus)

Genetic variability for regeneration ability was evaluated by studying direct organogenesis from cotyledons of thirteen genotypes including three cytoplasmic male sterile, three maintenor, three restorer inbred lines, and four F1 hybrids obtained by crosses between some of these inbred lines. The experimental design was a complete randomized block with three replications. A high genetic variability for organogenesis parameters between genotypes was observed in this study. Evidence of cytoplasmic effect and nucleo-cytoplasmic interaction for some of regeneration parameters was observed. The data also showed the importance of additive genetic control for organogenesis parameters in most genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Changes in lipid status and glass properties in cotyledons of developing sunflower seeds

Biochemical events involved in the acquisition of germinability and storability during orthodox seed development are well documented; however, the roles played by the physical organization of lipids and water are poorly characterized. The aim of this work was to determine, using a thermodynamic approach, whether changes in thermal properties of lipid reserves, and intracellular glasses might play a role in sunflower (Helianthus annuus L.) seed development. Triacyglycerols (TAGs) accumulated in cotyledons until the end of seed filling, which occurred 42 days after anthesis (DAA). Further seed development, leading to mature seed at 58 DAA, was mainly associated with an enlargement of lipid bodies without significant changes either in the lipid content or in their composition. When cooled to -100 degrees C, lipid reserves from cotyledons of mature seeds displayed alpha and beta' polymorphic crystalline structures; however, the ability to form alpha crystals, which was an indicator of lipid purity, progressively appeared during seed development. Characteristics of lipid melting confirmed that seed maturation drying was associated with changes in TAG physical organization. Cotyledon development was associated with an increase in the temperature of glass to rubber transition (Tg), thus suggesting a decrease in molecular mobility during maturation drying. This phenomenon was concomitant with an increase in raffinose content. Our results demonstrate that physical characteristics of lipid reserves and glasses of sunflower cotyledons are developmentally regulated and might play a role in acquisition of seed germinability and storability.     

Meristem transformation of sunflower via Agrobacterium

Summary For transformation of sunflower (Helianthus annuus L. cv. Zebulon), shoot apical meristems were dissected from seeds and cocultivated with a disarmed Agrobacterium tumefaciens strain harboring a binary vector carrying genes encoding GUS- and NPTII-activity. The influence of the media conditions, the time of cocultivation and the stage of the developing seed on shoot development and meristem transformation was analysed. Transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by assays for GUS and NPTII. GUS-positive shoots were rooted on rockwool and transferred to soil. Transformation of shoot meristem cells occurred at low frequencies. Chimaeric expression of the two genes was observed in transformed plants. Integration of the foreign DNA in the sunflower genome was confirmed with the polymerase chain reaction.Abbreviations GUS ß-Glucuronidase - NPTII Neomycin phosphotransferase II     

Kinetin action on the development of microbody enzymes in sunflower cotyledons in the dark

Summary Removal of the roots from etiolated sunflower seedlings (Helianthus annuus L.) at various stages of development resulted in a premature or enhanced decline of the activities of catalase (E.C. 1.11.1.6) and isocitrate lyase (E.C. 4.1.3.1) (glyoxysomes), and hydroxypyruvate reductase (E.C. 1.1.1.26) and glycolate oxidase (E.C. 1.1.3.1) (leaf peroxisomes) in the cotyledons. Treatment of the cuttings with kinetin in the dark inhibited the loss of glyoxysomal enzyme activities and, at the same time, induced a three to fivefold increase in leaf-peroxisomal enzyme activities. The decline of glyoxysomal enzyme activities was also suppressed after application of both kinetin and cycloheximide (50 g/ml). The kinetin-mediated rise of leaf-peroxisomal enzyme activities was strongly curtailed in the presence of cycloheximide. The dose effect curves of kinetin action on the development of glyoxysomal and of leaf-peroxisomal enzyme activities were different, supporting the hypothesis that the mechanisms of kinetin action on the two microbody enzyme systems are different. Nitrogen nutrition of intact seedling effected the development of microbody enzyme activities in a pattern closely resembling that of kinetin action. Presumably endogenous cytokinins produced by the roots are involved in the regulation of microbody enzyme activities in cotyledons of dark-grown sunflower seedlings.     

Effect of benzyladenine on some enzymes of mitochondria and microbodies in excised sunflower cotyledons

Benzyladenine (BA) increases the rate of expansion of dark-grown sunflower (Helianthus annuus L.) cotyledons. The hormone slightly enhances the development of the two glyoxysomal enzymes, isocitrate lyase and malate synthetase, during the first 3 days of germination and greatly accelerates their decay in the 2 following days. The levels of the peroxisomal enzymes, glycolate oxidase and glyoxylate reductase, are enhanced by BA more than those of the two glyoxysomal enzymes. These effects of BA on microbody enzymes are very similar to those of white light. Mitochondrial enzyme activities are increased to a varying extent by BA: the increase is minimal for fumarase, and maximal for cytochrome oxidase. The level of cytochrome oxidase is enhanced 346% at the 5th day of germination. Also, the rate of O₂ consumption is increased by BA, but the time course of this increased O₂ consumption does not match with that of cytochrome oxidase. Fusicoccin, a fungal toxin, mimics the effect of BA on cotyledon expansion, but fails to duplicate its action on microbody enzymes. This suggests that the effect of BA on microbody enzymes is not closely linked with the mechanism of growth promotion.     

Transglutaminases and their substrates in kinetin-stimulated etioplast-to-chloroplast transformation in cucumber cotyledons

In the light of our previous work, we know that there is a relationship between bound polyamines and the chloroplast differentiation process. This relationship may represent an important component of the process and be part of the mechanism of kinetin action, which stimulates chloroplast differentiation. To clarify the nature of the binding of polyamines to chloroplast structures, the possible involvement of transglutaminases in kinetin-stimulated chloroplast photodevelopment was investigated. Immunodetection of transglutaminases revealed bands at 77, 50 and 30 kDa both in etioplasts and chloroplasts. The data indicated a positive correlation between enzyme level and activity. It also demonstrated the regulation of transglutaminase protein expression by kinetin. The suborganellar location of transglutaminases by electron microscopy showed that the enzyme is peculiarly localised, mainly in pro-thylakoids and appressed grana thylakoids. The data corroborated that spermidine post-translational modification of certain plastid proteins of 58, 29, 26 and 12 kDa occurred. The results we obtained suggest that transglutaminases take part in the formation of the chloroplast structure via a mechanism whereby polyamines bind to their protein substrates. These findings about the effect of kinetin on conjugation provide a new contribution to the understanding of the mechanism of kinetin action on etioplast-to chloroplast transformation.     

Cloning, expression, and purification of glyoxysomal 3-oxoacyl-CoA thiolase from sunflower cotyledons

The glyoxysomal beta-oxidation system in sunflower (Helianthus annuus L.) cotyledons is distinguished by the coexistence of two different thiolase isoforms, thiolase I and II. So far, this phenomenon has only been described for glyoxysomes from sunflower cotyledons. Thiolase I (acetoacetyl-CoA thiolase, EC 2.3.1.9) recognizes acetoacetyl-CoA only, while thiolase II (3-oxoacyl-CoA thiolase, EC 2.3.1.16) exhibits a more broad substrate specificity towards 3-oxoacyl-CoA esters of different chain length. Here, we report on the cloning of thiolase II from sunflower cotyledons. The known DNA sequence of Cucumis sativus 3-oxoacyl-CoA thiolase was used to generate primers for cloning the corresponding thiolase from sunflower cotyledons. RT-PCR was then used to generate an internal fragment of the sunflower thiolase gene and the termini were isolated using 5'- and 3'-RACE. Full-length cDNA was generated using RT-PCR with sunflower thiolase-specific primers flanking the coding region. The resultant gene encodes a thiolase sharing at least 80% identity with other plant thiolases at the amino acid level. The recombinant sunflower thiolase II was expressed in a bacterial system in an active form and purified to apparent homogeneity in a single step using Ni-NTA agarose chromatography. The enzyme was purified 53.4-fold and had a specific activity of 235 nkat/mg protein. Pooled fractions from the Ni-NTA column resulted in an 83% yield of active enzyme to be used for further characterization.     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

The biosynthesis of triacylglycerols in microsomal preparations of developing cotyledons of sunflower (Helianthus annuus L.)

The synthesis of triacylglycerols was investigated in microsomes (microsomal fractions) prepared from the developing cotyledons of sunflower (Helianthus annuus). Particular emphasis was placed on the mechanisms involved in controlling the C18- unsaturated-fatty-acid content of the oils. We have demonstrated that the microsomes were capable of: the transfer of oleate from acyl-CoA to position 2 of sn-phosphatidylcholine for its subsequent desaturation and the return of the polyunsaturated products to the acyl-CoA pool by further acyl exchange; the acylation of sn-glycerol 3-phosphate with acyl-CoA to yield phosphatidic acid, which was further utilized in diacyl- and tri-acylglycerol synthesis; and (3) the equilibrium of a diacylglycerol pool with phosphatidylcholine. The acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine coupled to the equilibration of diacylglycerol and phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C18 polyunsaturated fatty acids for triacylglycerol production. Similar reactions were found to operate in another oilseed plant, safflower (Carthamus tinctorius L.). On the other hand, the microsomes of avocado (Persea americana) mesocarp, which synthesize triacylglycerol via the Kennedy [(1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940] pathway, were deficient in acyl exchange and the diacylglycerol in equilibrium phosphatidylcholine interconversion. The results provide a working model that helps to explain the relationship between C18- unsaturated-fatty-acid synthesis and triacylglycerol production in oilseeds.