Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species

Brzi test za ispitivanje otpornosti suncokreta na tribenuron metil

A rapid laboratory test for detection of tribenuron-methyl resistant sunflower genotypes was developed. Four homozygous and four heterozygous tribenuron-methyl resistant hybrids, as well as a susceptible one were grown on MS medium supplemented with different concentrations of herbicide (2.0 µM, 2.5 µM, 3.0 µM, 3.5 µM and 4.0 µM) and with pH either 7 or 8. The effect of medium pH and herbicide concentration on above-ground part and root mass of sunflower seedlings was observed. The test enabled visual discrimination between resistant and susceptible genotypes in 5 days, and discrimination between homozygous and heterozygous resistant genotypes in 12 days. All tested herbicide concentrations and morphological parameters were found to be suitable for discrimination of tribenuron-methyl susceptible genotypes. The best pH and herbicide concentration combination and morphological parameter for discrimination between homozygous and heterozygous resistant sunflower genotypes were 3.0 µM at pH 7 and root mass, respectively.Razvijen je brzi laboratorijski test za detekciju genotipova suncokreta otpornih na tribenuron metil. Po četiri hibrida suncokreta homozigotno i heterozigotno otporna na tribenuron metil, kao i jedan neotporan hibrid su gajeni na MS podlozi sa pH vrednošću ili 7 ili 8 i sa različitim koncentracijama herbicida Express 50X, čija je aktivna materija tribenuron-metil (2.0 µM, 2.5 µM, 3.0 µM, 3.5 µM i 4.0 µM). Ispitivan je uticaj pH i koncentracije herbicida u podlozi na masu nadzemnog dela i korena klijanaca testiranih hibrida. Vizuelne razlike između otpornih i neotpornog hibrida su uočene nakon 5 dana kulture, dok su se razlike između homozigotnih i heterozigotnih otpornih hibrida pojavile nakon 12 dana kulture. Sve testirane koncentracije herbicida i testirani morfološki parametri su se pokazali kao dobri za identifikaciju neotpornih genotipova. Najbolja kombinacija pH i koncentracije herbicida za razlikovanje homozigotnih od heterozigotnih otpornih genotipova je bila 3.0 µM na pH 7, a morfološki parametar masa korena

Augmentation de la résistance à la gale commune chez des variétés de pommes de terre produites au Québec

Abstract: Common scab is a globally important disease of potato (Solanum tuberosum L.) that reduces the quality of tubers and decreases their market value. This disease is caused by the bacterium Streptomyces scabies (syn. scabiei) which induce the formation of superficial, pitted or raised lesions on the tuber surface. S. scabies synthesizes a toxin, thaxtomin A, which is essential for the development of disease symptoms. The best approach for common scab management is the development of resistant potato varieties. However, little is known about the factors that contribute to common scab resistance. Increasing resistance to thaxtomin A may be a way to increase resistance to the disease. The use of thaxtomin A as an agent for direct somatic cell selection had allowed the production of potato varieties with increased tolerance to common scab. The strategy used in this project was based on a progressive adaptation of potato calli to thaxtomin A before regeneration of plantlets from somatic embryos. We successfully adapted several potato varieties widely used or newly selected in Canada: Kennebec, Envol, Chieftain, Rubiconde and Belle d’Août. A total of 92 somaclonal variants adapted to thaxtomin A were produced after adjusting regeneration conditions. Randomly selected somaclones were tested for scab resistance. For an initial study of scab resistance, we developed a new method based on the infection of mini potato tubers developing from leaf buds. The increased resistance of the somaclones selected using this test was confirmed with S. scabies infection test in planta. The adaptation of potato variety Russet Burbank to thaxtomin A (0.5 µM) had resulted in the production of a somaclonal variant, RB9, which had increased resistance to common scab. Here, LS-MS/MS proteomic analysis of soluble tuber proteins showed that improved RB9 resistance to common scab was associated with changes in the tuber proteome. Changes included increased accumulation of major tuber proteins, such as patatins, lipoxygenases and serine protease inhibitors (Kunitz-type). The abundance of certain proteins associated with oxidative stress was also altered. We found S. scabies infection to cause changes in the abundance of patatins, lipoxygenases, and Kunitz-type protease inhibitors. These proteins tended to accumulate in the initial stages of infection in both Russet Burbank and RB9 tubers. The RB9 tubers showed a constant accumulation of lipoxygenases in response to S. scabies during the whole period of infection, while in tubers of the original variety, infection caused the accumulation of lipoxygenases only in the first days of infection. Moreover, young RB9 potato tubers had a reinforced periderm, with more periderm layers and an altered cell morphology compared to the Russet Burbank tubers. Thus, the improvement in scab resistance caused by adaptation to thaxtomin A was associated with both proteomic and morphological changes. Thaxtomin A is an essential factor for the development of common scab symptoms. When applied to tuber slices, it causes browning of the tuber flesh. Toluidine blue O staining of tuber flesh sections treated with thaxtomin A here resulted in a blue coloration which is characteristic of phenolic compounds. The extent of the browning did not reflect the common scab resistance of Russet Burbank and Yukon Gold varieties but it corresponded instead to the level of phenols accumulation. Treatment with thaxtomin A caused the death of tuber parenchyma cells of both varieties. However, the degree of cell death, determined by staining with Evans blue, did not correlate with the extent of browning. The application of 2-aminoindan-2-phosphonic acid, a competitive inhibitor of phenylalanine-ammonia lyase, simultaneously with the thaxtomin A reduced the development of browning on potato slices. Thus, the darkening of the tuber slices caused by thaxtomin A treatment was caused at least in part by the accumulation of phenolic compounds in the tuber flesh cells. Results obtained in the course of this project allow a better understanding of the interaction between potato tubers and pathogenic S. scabies. They also reveal the occurrence of protein factors that could be involved in potato resistance to common scab.La gale commune est une maladie de la pomme de terre (Solanum tuberosum L.) d'importance mondiale qui réduit la qualité des tubercules et diminue leur valeur marchande. Cette maladie caractérisée par la formation de lésions superficielles, profondes ou surélevées sur la surface du tubercule est causée par la bactérie Streptomyces scabies (syn. scabiei). S. scabies synthétise une toxine, la thaxtomine A, qui est essentielle pour le développement des symptômes de la maladie. La meilleure approche pour la gestion de la gale commune est le développement de variétés de pomme de terre résistantes. Cependant, les facteurs qui contribuent à la résistance à la gale commune ne sont pas bien connus. L'augmentation de la résistance à la thaxtomine A peut être un moyen d'augmenter la résistance à la maladie. L’utilisation de la thaxtomine A comme agent de sélection directe des cellules somatiques a permis la production de variétés de pomme de terre ayant une plus grande résistance à la gale commune. Dans ce projet, nous avons utilisé une stratégie basée sur l’adaptation progressive des cals de pomme de terre à la thaxtomine A suivie de la régénération de plantules à partir d’embryons somatiques. Nous avons ainsi adapté à la thaxtomine A plusieurs variétés de pommes de terre cultivées ou nouvellement sélectionnées au Canada tel que Kennebec, Envol, Chieftain, Rubiconde et Belle d’Août. Au total, 92 variants somaclonaux adaptés à la thaxtomine A ont été produits après l’ajustement des conditions de régénération. Des somaclones sélectionnés au hasard ont été testés pour leur résistance à la gale. Pour une première étude de la résistance à la gale commune, nous avons développé une nouvelle méthode basée sur l'infection de mini tubercules de pomme de terre se développant à partir de bourgeons foliaires. L'augmentation de la résistance des somaclones sélectionnés à l'aide de ce test a été confirmée par l'infection de plantes en pots. En second lieu, l'adaptation de la varieté de pomme de terre Russet Burbank à la thaxtomine A (0,5 µM) a produit le variant somaclonal RB9, qui possède une résistance augmentée à la gale commune. L’analyse protéomique a montré qu'une amélioration de la résistance de RB9 à la gale commune est associée à des modifications du protéome du tubercule. Les changements comprenaient une accumulation accrue des protéines principales du tubercule, telles que les patatines, les lipoxygénases et les inhibiteurs des protéases (type Kunitz). L'abondance de certaines protéines associées au stress oxydatif a également été modifiée. Nous avons constaté que l'infection par S. scabies provoque des changements dans l'abondance des patatines, des lipoxygénases et des inhibiteurs de protéase de type Kunitz. Ces protéines ont tendance à s'accumuler pendant les premiers stades de l'infection dans les tubercules de Russet Burbank et de RB9. Les tubercules de RB9 ont montré une accumulation constante des lipoxygénases en réponse à S. scabies durant toute la période d'infection, alors que dans les tubercules de la variété d'origine, l'infection a provoqué l'accumulation des lipoxygénases uniquement dans les premiers jours de l'infection. De plus, les jeunes tubercules de RB9 avaient un périderme renforcé, avec plus de couches des cellules suberisées et une morphologie cellulaire modifiée par rapport aux tubercules de la variété d'origine. Ainsi, l'amélioration de la résistance à la gale commune provoquée par l'adaptation à la thaxtomine A a été associée à des changements protéomiques et morphologiques. La thaxtomine A est un facteur essentiel pour le développement des symptômes de la gale commune. Lorsqu'elle est appliquée sur des tranches de tubercules, elle provoque un brunissement de la chair des tubercules. Une coloration au Bleu de toluidine O des tranches de chair de tubercule traitées à la thaxtomine A a ici permis d’observer une coloration bleue typique des composés phénoliques. Par contre, la superficie du brunissement ne reflètait pas la résistance des variétés Russet Burbank et Yukon Gold à la gale commune, mais correspondait plutôt au niveau d'accumulation des phénols. Le traitement avec la thaxtomine A a provoqué la mort des cellules du parenchyme des tubercules des deux variétés. Cependant, le degré de mort cellulaire, déterminé par une coloration au bleu d'Evans, n'était pas corrélée avec la superficie du brunissement. L'application d'acide 2-aminoindane-2-phosphonique, un inhibiteur compétitif de la phénylalanine-ammonia lyase, simultanément avec la thaxtomine A a réduit le développement du brunissement sur les tranches de pomme de terre. Ainsi, le brunissement des tranches de tubercule induit par le traitement à la thaxtomine A était provoqué, du moins en partie, par l'accumulation de composés phénoliques dans les cellules de la chair du tubercule. Les résultats obtenus dans le cadre de ce projet permettent de mieux comprendre l'interaction entre des tubercules de pomme de terre et S. scabies. Ils mettent en évidence des facteurs protéiques qui pourraient être impliqués dans la résistance de la pomme de terre à la gale commune

Effect of age, period and birth-cohort on the frequency of glucose-6-phosphate dehydrogenase deficiency in Sardinian adults.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an inherited disorder common in Sardinia. In this study, the frequency variation of G6PD-deficiency across age groups and birth cohorts was investigated using Age-Period-Cohort analysis.Data were collected from the clinical records of 11,252 patients (6975 women, age range 17-94 years) who underwent endoscopy between 2000 and 2016 at a teaching hospital (University of Sassari), Italy. G6PD status was assessed by enzymatic assay based on G6PD/6GPD ratio. A Poisson log-linear regression model was used to identify age and time trend in G6PD deficiency.Enzyme deficiency was detected in 11.4% of the entire cohort (men: 7.9%; women: 13.6%). Age-Period-Cohort analysis showed no inflection points across age groups, especially after age 80. The effects of time period and birth cohorts on G6PD deficiency were negligible (frequencies before and after 1950 were 11.0% and 11.8%, respectively).These findings indicate that the frequency of G6PD deficiency does not vary significantly in oldest subjects. The lack of evidence for selection across the malaria eradication time may be explained by other factors, including somatic cell selection or misclassification of heterozygotes women as G6PD normal in the older birth cohorts. Additional molecular studies may help clarify these issues. Key message The frequency of glucose-6-phosphate dehydrogenase deficiency is stable across age groups and does not vary in generations born before or after malaria eradication

Functional properties of two mutants of human glucose 6-phosphate dehydrogenase, R393G and R393H, corresponding to the clinical variants G6PD Wisconsin and Nashville

AbstractTwo severe Class I human glucose-6-phosphate dehydrogenase (G6PD, EC1.1.1.49) mutations, G6PDWisconsin (nt1177 C→G, R393G) and G6PDNashville (nt1178 G→A, R393H), affect the same codon, altering a residue in the dimer interface close to the “structural” NADP+ site. These mutations are predicted to influence interaction with the bound “structural” NADP+, long supposed to be crucial for enzyme stability. Recombinant proteins corresponding to these mutants have been constructed, expressed and purified to homogeneity. Steady-state kinetic parameters of the mutant enzymes were comparable to those of normal human G6PD, indicating that the mutations do not alter catalytic efficiency drastically. However, investigations of thermostability, urea denaturation, protease digestion, and hydrophobic exposure demonstrated that G6PD R393H is less stable than normal G6PD or R393G, and stability was more NADP+-dependent. Apoenzymes were prepared by removal of “structural” NADP+. Again the G6PDNashville protein was markedly less stable, and its dissociation constant for “structural” NADP+ is ∼500 nM, about 10 times higher than values for R393G (53 nM) and normal G6PD (37 nM). These results, together with structural information, suggest that the instability of the R393H protein, enhanced by the weakened binding of “structural” NADP+, is the likely cause of the severe clinical manifestation observed for G6PDNashville. They do not, however, explain the basis of disease in the case of G6PDWisconsin

Is Diabetes a Model for Gene-environment Interaction in Premature Senescence?

A vast majority of human diseases or disorders are invariably associated with the complex regulations and functions of genes. These also involved in the integration of environmental signals for cells to modulate the functional output of the genome. The ageing process reflects the overall presentation of an organism and the interaction of its genes with the environment. Ageing results from a plethora of processes, which potentially include factors such as diabetes, immune system decline, oxidative damage, rates of apoptosis, and telomere shortening. Persons detected with shorter telomere lengths usually present with accelerated biological ageing, especially those with diabetes mellitus, glucose intolerance, and hypertension. Adverse social factors may be contributory to a worse prognosis for subjects with diabetes mellitus as regards early ageing.The deduction is that diabetes is inextricably linked with "premature ageing". There is appreciable variation in the rate of progression of ageing in humans. A simple biological model may not be sufficient to observe both the clinical and theoretical modalities to understand the mechanisms of gene-environment interaction for diabetes mellitus and premature ageing. The potential clinical and gerontological implications of this study are to configure the  population at risk for developing premature ageing among diabetic patients in order to develop early therapeutic strategies and curtail financial costs to prevent untoward sequelae. Keywords: Diabetes; model; gene; environment; premature ageing; senescence, therapy, costs

G6PD Deficiency at Sumba in Eastern Indonesia Is Prevalent, Diverse and Severe: Implications for Primaquine Therapy against Relapsing Vivax Malaria

Safe treatment of Plasmodium vivax requires diagnosis of both the infection and status of erythrocytic glucose-6-phosphate dehydrogenase (G6PD) activity because hypnozoitocidal therapy against relapse requires primaquine, which causes a mild to severe acute hemolytic anemia in G6PD deficient patients. Many national malaria control programs recommend primaquine therapy without G6PD screening but with monitoring due to a broad lack of G6PD deficiency screening capacity. The degree of risk in doing so hinges upon the level of residual G6PD activity among the variants present in any given area. We conducted studies on Sumba Island in eastern Indonesia in order to assess the potential threat posed by primaquine therapy without G6PD screening. We sampled 2,033 residents of three separate districts in western Sumba for quantitative G6PD activity and 104 (5.1%) were phenotypically deficient (\u3c4.6U/gHb; median normal 10U/gHb). The villages were in two distinct ecosystems, coastal and inland. A positive correlation occurred between the prevalence of malaria and G6PD deficiency: 5.9% coastal versus inland 0.2% for malaria (P\u3c0.001), and 6.7% and 3.1% for G6PD deficiency (P\u3c0.001) at coastal and inland sites, respectively. The dominant genotypes of G6PD deficiency were Vanua Lava, Viangchan, and Chatham, accounting for 98.5%of the 70 samples genotyped. Subjects expressing the dominant genotypes all had less than 10% of normal enzyme activities and were thus considered severe variants. Blind administration of anti-relapse primaquine therapy at Sumba would likely impose risk of serious harm