Hypolignification: a decisive factor in the development of hyperhydricity

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH

3':5'-Cyclic AMP-dependent 3'

Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 × 10^8 cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 × 10-8 M final concentration) increased cGMP levels from 1 pmol per 10^7 cells to 7 pmol per 10^7 cells in 10 sec and basal levels were recovered in 20-25 sec. cGMP accumulation did not occur when cells were in the cAMP-insensitive stage. cAMP-sensitive cells responded with increased cGMP levels when triggered by 5 × 10-8 M 5'-CH2-cAMP or 10-5 M adenosine-5'-methylmonophosphate (5'-AMPMe) but not after addition of 5 × 10-8 M 3':5'-cyclic IMP (cIMP) or 5 × 10-8 M 5'-AMP. As agonists of cAMP, 5'-CH2-cAMP and 5'-AMPMe have, respectively, more than 10% and 1% the chemotactic activity of cAMP, while cIMP has 0.01% the activity of cAMP and 5-AMP is inactive up to a concentration of 10-3 M. cAMP-mediated cGMP formation was dependent upon cAMP concentration, with a half-maximal cAMP concentration of about 10-8 M. This cAMP concentration agrees closely with that necessary for half-maximal receptor occupation. cAMP-mediated cGMP formation was independent of the presence of extracellular Ca2+; cell aggregation and chemotaxis were also independent of the presence of external Ca2+. Therefore, cAMP action does not depend on stimulation of the Ca2+ influx. cAMP was found to mediate desensitization of cAMP-dependent cGMP formation. Additon of 5 × 10-8 M cAMP to sensitive cells induced a desensitization period that lasted 1-5 min. Desensitization was dependent on the cAMP concentration. Finally, we propose that the translation of a chemotactic signal from the cell surface to pseudopod formation in Dictyostelium involves changes in the levels of cGMP

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable

Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples

Apple scab resistance genes, HcrVf1 and HcrVf2, were isolated including their native promoter, coding and terminator sequences. Two fragment lengths (short and long) of the native gene promoters and the strong apple rubisco gene promoter (PMdRbc) were used for both HcrVf genes to test their effect on expression and phenotype. The scab susceptible cultivar ‘Gala’ was used for plant transformations and after selection of transformants, they were micrografted onto apple seedling rootstocks for scab disease tests. Apple transformants were also tested for HcrVf expression by quantitative RT-PCR (qRT-PCR). For HcrVf1 the long native promoter gave significantly higher expression that the short one; in case of HcrVf2 the difference between the two was not significant. The apple rubisco gene promoter proved to give the highest expression of both HcrVf1 and HcrVf2. The top four expanding leaves were used initially for inoculation with monoconidial isolate EU-B05 which belongs to race 1 of V. inaequalis. Later six other V. inaequalis isolates were used to study the resistance spectra of the individual HcrVf genes. The scab disease assays showed that HcrVf1 did not give resistance against any of the isolates tested regardless of the expression level. The HcrVf2 gene appeared to be the only functional gene for resistance against Vf avirulent isolates of V. inaequalis. HcrVf2 did not provide any resistance to Vf virulent strains, even not in case of overexpression. In conclusion, transformants carrying the apple-derived HcrVf2 gene in a cisgenic as well as in an intragenic configuration were able to reach scab resistance levels comparable to the Vf resistant control cultivar obtained by classical breeding, cv. ‘Santana’

Physiological and morpho-anatomical analyses of hyperhydric Arabidopsis thaliana influenced by media components

Hyperhydricity is a physiological anomaly that significantly affects the growth and proliferation rate of crops cultivated by tissue culture techniques. To better understand the mechanisms that govern hyperhydricity incidence, we examined the effects of several media components, particularly cytokinin and gelling agents. These elements were found to be influential in both in vitro propagation and the development of hyperhydricity. Our study revealed that Arabidopsis thaliana seedlings had a greater manifestation of hyperhydricity symptoms when exposed to high cytokinin concentrations compared with the control. The presence of gelrite led to the manifestation of hyperhydric symptoms by elevated water build-up in the apoplast. The phenomenon of stomata closure was observed in the hyperhydric leaves, resulting in an increased ability to retain water and a decrease in the transpiration rates when compared to their respective control leaves. Additionally, histological examinations of the cross sections of hyperhydric leaves revealed an irregular cellular arrangement and large intercellular spaces. Furthermore, hyperhydric seedlings displayed impaired cuticular development in comparison to their normal seedlings