Evidence for the probable oil body association of a thiol-protease, leading to oleosin degradation in sunflower seedling cotyledons

The activity of a 65 kDa, cytosolic protease from sunflower seedling cotyledons coincides with the degradation of oleosins during seed germination. Further investigations carried out in this laboratory have demonstrated the probable association of a thiol-protease with oil bodies, leading to gradual degradation of oleosins during seedling growth. Evidence to this effect have been brought out through zymographic detection of protease activity from oil bodies, degradation of oleosins by electrophoretically eluted protease from the seedling cotyledons and inhibition of protease activity by thiol-protease inhibitor, such as N-ethylmaleimide (NEM). In addition to these biochemical evidence, visualization of thiol-protease activity has also been achieved by a novel fluorescence microscopic method and confocal imaging. It involves the uptake and binding of a fluorogenic thiol-protease inhibitor (fluorescein mercuric acetate, FMA) at the intracellular thiol-protease activity sites in protoplasts, leading to fluorescence emission at 523 nm following excitation at 499 nm. Maximum protease activity is observed in 4-d-old seedling cotyledons, coinciding with the phase of active triacylglycerol (TAGs) hydrolysis. All these observations provide evidence for the expression of the said thiol-protease activity on the oil body surface, leading to gradual proteolysis of oleosins during seed germination.     

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.     

Glycophyte salt resistance

By perfusion of entire sunflower stems with NaCl solutions of various concentrations, we studied the phenomenon of sodium decrement, i.e., sodium retaining in the stem and leaf petioles. Such retaining could comprise up to 50–80% of initial sodium concentration. It depended on the rate of perfusion, the length of xylem vessels, and NaCl concentration. When perfusion with 100–500 mM NaCl concentrations (high for glycophytes) lasted for 10–12 days, we did not observe any decrease in the degree of sodium decrement. Simultaneously with sodium decrement, other ions (K⁺ and Ca²⁺) were secreted into the perfusate, thus providing for physiological equilibrating the monosalt solution supplied to the stem base. The high salt concentration in the perfusate induced a decrease in the hydraulic conductance of the vessels. The conclusion is that stressful NaCl solutions attain the shoot meristem and reproductive organs as an “equilibrated” salt solution and at a declined rate of xylem flow. The mechanisms of observed phenomenon of glycophyte salt resistance are discussed, the main of them being related to osmosis-dependent responses of stem living cells and the processes of ion exchange between the cells and xylem vessel content.     

Maintenance and constructive respiration in various organs ofHelianthus annuus L. andZinnia elegans L.

Maintenance and constructive respiration were examined in leaves, stems and roots of sunflower seedlings and in detached leaves of zinnia. Maintenance and constructive respiratory coefficients in sunflower leaves were smaller than those in stems and roots, and zinnia leaves. It was suggested that matter accumulation influenced maintenance respiratory coefficient.     

Microsampling: an alternative approach using sesquiterpene lactones for systematics

The localization of sesquiterpene lactones in glandular trichomes of numerous Asteraceae makes it possible to use a microtechnique to investigate their occurrence. General aspects of the utility and value of the method in comparison with standard procedures are discussed with special focus on the advantage of a quick, inexpensive and reproducible analytical technique in systematic and biochemical studies. Using analytical HPLC, it was possible to establish the sesquiterpene lactone profiles of all 64 known taxa of the genus Helianthus. Intraspecific variation of sesquiterpene lactones was found to be low in comparison to morphological characters. Hybrids were shown to possess the additive sesquiterpene lactone patterns of their parents. This suggests that the chemistry of these compounds could be a useful tool in taxonomy.     

Genomic alterations in the interspecific hybrid Helianthus annuus×Helianthus tuberosus

 The genome of a Helianthus annuus (2n=34) ×Helianthus tuberosus (2n=102) hybrid was studied at cytological, biochemical and molecular levels and compared to those of the parental species. Cytophotometric analyses showed that the hybrid has a 4C DNA content higher than expected and with a larger variability than in the parents. This high variability is probably not related to chromosome-number variations since the hybrid always had 2n=68 chromosomes. Moreover, hybrid interphase nuclei showed lower heterochromatin condensation than the parental ones. Thermal denaturation of genomic DNAs indicated that quantitative variation of some DNA families occurred in the hybrids compared to parents. Finally, molecular analyses of DNAs restricted with different enzymes, after Southern blotting and hybridization with HR probes, showed restriction patterns in the hybrid different from those observed in parents. These results indicate that interspecific hybridization between H. annuus and H. tuberosus may determine quantitative variation of some DNA families and differential DNA methylations that probably modify the nuclear structure. These phenomena are probable responses to a “genomic shock” following the interspecific cross. Received: 22 May 1998 / Accepted: 4 June 1998     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh ₁ and Adh ₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh ₂ isozymes were more than twice as active as those of Adh ₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh ₁ ^F /Adh ₁ ^F , Adh ₂ ^S /Adh ₂ ^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh ₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Ultrastructure and autoradiography of dormant and activated parenchyma ofHelianthus tuberosus

Summary Parenchyma cells of dormant tubers ofHelianthus tuberosus L. cv. OB1 (Jerusalem artichoke) contain a very low amount of hormones, therefore they respond to 2,4-D or IAA treatment by dividing and synthesizing RNA, DNA, and polyamines.In particular the activation of the dormant tissues induces an early synthesis of DNA, which reaches the maximum at 3 hours, much before the beginning of the S phase (12 hours). By supplying [6-³H] thymidine and carrying out electron microscopic autoradiography, we were able to determine that plastids and mitochondria were the organelles responsible for this early synthesis while the DNA in the nucleus first appeared labeled at 15 hours.In addition, ultrastructural observations carried out to compare the dormant cells with activated ones, showed an increase in the nucleolar volume, a different organization of the tubular complex of the plastids and several other ultrastructural changes which indicate that at 3 hours some fundamental metabolic processes are already active; they become even more evident later on.The implications of these results in the physiology of the tuber cells during activation are discussed.     

Generation of white mold disease-resistant sunflower plants expressing human lysozyme gene

Sunflower plants were transformed via co-cultivation of previously bombarded hypocotyl explants with Agrobacterium tumefaciens harboring the plasmid pNGL that contains the human lysozyme gene. The transformed shoots were selected using kanamycin and regenerated plants were analyzed using histochemical β-glucuronidase assay. Southern, Western and Northern blot analyses indicated the transfer, expression and stable integration of the foreign DNA into the sunflower genome. Resistance against the phytopathogenic fungus Sclerotinia sclerotiorum, which causes white mold disease, was confirmed using a phytopathogenic test and microscopic observation of the infection process.