Humberto Prieto

Browning in Annona cherimola fruit: Role of polyphenol oxidase and characterization of a coding sequence of the enzyme

Cherimoya (Annona cherimola Mill.) fruit is an attractive candidate for food processing applications as fresh cut. However, along with its desirable delicate taste, cherimoya shows a marked susceptibility to browning. This condition is mainly attributed to polyphenol oxidase activity (PPO). A general lack of knowledge regarding PPO and its role in the oxidative loss of quality in processed cherimoya fruit requires a better understanding of the mechanisms involved. The work carried out included the cloning of a full-length cDNA, an analysis of its properties in the deduced amino sequence, and linkage of its mRNA levels with enzyme activity in mature and ripe fruits after wounding. The results showed one gene different at the nucleotide level when compared with previously reported genes, but a well-conserved protein, either in functional and in structural terms. Cherimoya PPO gene (Ac-ppo, GenBank DQ990911) showed to be present apparently in one copy of the genome, and its transcripts could be significantly detected in leaves and less abundantly in flowers and fruits. Analysis of wounded matured and ripened fruits revealed an inductive behavior for mRNA levels in the flesh of mature cherimoya after 16 h. Although the highest enzymatic activity was observed on rind, a consistent PPO activity was detected on flesh samples. A lack of correlation between PPO mRNA level and PPO activity was observed, especially in flesh tissue. This is probably due to the presence of monophenolic substrates inducing a lag period, enzyme inhibitors and/or diphenolic substrates causing suicide inactivation, and proenzyme or latent isoforms of PPO. To our knowledge this is the first report of a complete PPO sequence in cherimoya. Furthermore, the gene is highly divergent from known nucleotide sequences but shows a well conserved protein in terms of its function, deduced structure, and physiological role.

The Atmospheric Monitoring System of the JEM-EUSO space mission

An Atmospheric Monitoring System (AMS) is a mandatory and key device of a space-based mission which aims to detect Ultra-High Energy Cosmic Rays (UHECR) and Extremely-High Energy Cosmic Rays (EHECR) from Space. JEM-EUSO has a dedicated atmospheric monitoring system that plays a fundamental role in our understanding of the atmospheric conditions in the Field of View (FoV) of the telescope. Our AMS consists of a very challenging space infrared camera and a LIDAR device, that are being fully designed with space qualification to fulfil the scientific requirements of this space mission. The AMS will provide information of the cloud cover in the FoV of JEM-EUSO, as well as measurements of the cloud top altitudes with an accuracy of 500 m and the optical depth profile of the atmosphere transmittance in the direction of each air shower with an accuracy of 0.15 degree and a resolution of 500 m. This will ensure that the energy of the primary UHECR and the depth of maximum development of the EAS ( Extensive Air Shower) are measured with an accuracy better than 30% primary energy and 120 g=cm 2 depth of maximum development for EAS occurring either in clear sky or with the EAS depth of maximum development above optically thick cloud layers. Moreover a very novel radiometric retrieval technique considering the LIDAR shots as calibration points, that seems to be the most promising retrieval algorithm is under development to infer the Cloud Top Height (CTH) of all kind of clouds, thick and thin clouds in the FoV of the JEM-EUSO space telescope.

Molecular characterization of the chalcone isomerase gene family in Deschampsia antarctica

Deschampsia antarctica (Poaceae) is one of the two vascular plants known to have colonized the Antarctic region. Studies examining the biosynthesis of flavonoids, compounds which plants use, for example, for protection against overexposure to UV light or as antioxidants that scavenge free radicals and other oxidative species, in D. antarctica may provide clues to its success in that extreme environment. We characterized the family of genes encoding chalcone isomerase (CHI EC 5.5.1.6), an important enzyme involved in flavonoid biosynthesis, in D. antarctica. Sequence analysis of the three family members revealed differences in numbers of introns and lengths of coding regions among the three and suggest that DaCHI3 is likely a pseudogene (ψDaChi2). Salinity stress resulted in differential mRNA expression of the DaCHI genes with ψDaCHI2 exhibiting the earliest response (3-h post-treatment), induced by as much as sevenfold, while DaCHI1 and DaCHI2 mRNAs accumulated later (3d and 5d post-treatment, respectively) and, in the case of DaCHI2, with a response of nearly sixfold. We discuss how differences in the proposed gene structures, deduced protein characteristics, and mRNA expression patterns suggest that the members of this gene family may have unique functions in the phenylpropanoid pathway in D. antarctica.

Observation of ultra-high energy cosmic rays in cloudy conditions by the space-based JEM-EUSO Observatory

JEM-EUSO is a space observatory that will be located on-board the Japanese Experiment Module at the International Space Station. It will observe Extensive Air Showers (EAS) induced by ultra-high energy cosmic rays using the Earths atmosphere as detector. In addition to clear sky observations, EAS are also observable in cloudy conditions if a sufficiently large part of the EAS development occurs above the cloud. The atmospheric monitoring system plays a fundamental role in our understanding of the atmospheric conditions in the field of view of the telescope.

Antioxidant responses of in vitro shoots of Deschampsia antarctica to polyethylene glycol treatment

Abstract To understand the adaptability to environmental stresses by Deschampsia antarctica, one of the two vascular plants growing in Antarctica, we analysed the activity of several antioxidant enzymes, including peroxidase (POD, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6)) and glutathion reductase (GR, EC 1.6.4.2), in shoots subjected to drought stress (PEG-8000, -0.3 MPa). Additionally, levels of total phenolic compounds, flavonoids and ascorbate, were determined. The content of malondialdehyde (MDA), chlorophyll and hydrogen peroxide did not change as a result of PEG-8000 treatment. In addition, treated plants showed higher enzymatic activity of CAT, POD and GR in shoots than control plants. In addition, a high capacity to scavenge free radicals was also detected in stressed plants. These results seem to indicate that in D. antarctica tolerance of drought stress is associated with enhanced activity of antioxidant enzymes and free radical scavenging capacity.

Genetically engineered Thompson Seedless grapevine plants designed for fungal tolerance: selection and characterization of the best performing individuals in a field trial

Abstract The fungi Botrytis cinerea and Erysiphe necator are responsible for gray mold and powdery mildew diseases, respectively, which are among the most devastating diseases of grapes. Two endochitinase (ech42 and ech33) genes and one N-acetyl-β-d-hexosaminidase (nag70) gene from biocontrol agents related to Trichoderma spp. were used to develop a set of 103 genetically modified (GM) ‘Thompson Seedless’ lines (568 plants) that were established in open field in 2004 and evaluated for fungal tolerance starting in 2006. Statistical analyses were carried out considering transgene, explant origin, and plant response to both fungi in the field and in detached leaf assays. The results allowed for the selection of the 19 consistently most tolerant lines through two consecutive years (2007–2008 and 2008–2009 seasons). Plants from these lines were grafted onto the rootstock Harmony and established in the field in 2009 for further characterization. Transgene status was shown in most of these lines by Southern blot, real-time PCR, ELISA, and immunostrips; the most tolerant candidates expressed the ech42–nag70 double gene construct and the ech33 gene from a local Hypocrea virens isolate. B. cinerea growth assays in Petri dishes supplemented with berry juices extracted from the most tolerant individuals of the selected population was inhibited. These results demonstrate that improved fungal tolerance can be attributed to transgene expression and support the iterative molecular and physiological phenotyping in order to define selected individuals from a population of GM grapevines.

Observation of Ultra-High Energy Cosmic Rays in cloudy conditions by the JEM-EUSO Space Observatory

Source of Ultra-high Energy Cosmic Rays (several times 10 19 eV) are still unidentified. Overcoming their extremely small fluxes, a detector with huge observation are as is needed to investigate the energy and arrival direction distribution of EECRs. JEM-EUSO is a unique experiment that will be located in the International Space Station to observe extensive air showers (EAS) by monitoring night part of Earth atmosphere. In addition to clear sky condition, the extensive air showers in cloudy condition are also observable by taking advantage of the certain fraction of EAS develop above the cloud. In the preset work, using Monte Carlo simultions for test clouds, the cloud impact to the trigger efficiency was estimated taking into account the statistics of cloud property.

Comparative Study of Two Table Grape Varieties with Contrasting Texture during Cold Storage

Postharvest softening of grape berries is one of the main problems affecting grape quality during export. Cell wall disassembly, especially of pectin polysaccharides, has been commonly related to fruit softening, but its influence has been poorly studied in grapes during postharvest life. In order to better understand this process, the Thompson seedless (TS) variety, which has significantly decreased berry texture after prolonged cold storage, was compared to NN107, a new table grape variety with higher berry firmness. Biochemical analysis revealed a greater amount of calcium in the cell wall of the NN107 variety and less reduction of uronic acids than TS during cold storage. In addition, the activity of polygalacturonase was higher in TS than NN107 berries; meanwhile pectin methylesterase activity was similar in both varieties. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) suggests a differential pectin metabolism during prolonged cold storage. Results revealed lower pectin fragments in TS after 60 days of cold storage and shelf life (SL) compared to 30 days of cold storage and 30 + SL, while NN107 maintained the same fragment profile across all time points evaluated. Our results suggest that these important differences in cell wall metabolism during cold storage could be related to the differential berry firmness observed between these contrasting table grape varieties.

Evaluation of the Resistance of Transgenic C5 Plum (Prunus domestica L.) against Four Chilean Plum Pox Virus Isolates through Micro-Grafting

The transgenic plum (Prunus domestica L.) C5, in which the coat protein (CP) gene of the Plum pox virus (PPV) is inserted, represents a unique example of the use of genetic engineering for fruit crop improvement in Prunus spp. Field trials in Poland, Romania, and Spain have demonstrated resistance of C5 to several D and M strain PPV isolates. In Chile, the quarantine regulations for PPV and for genetically modified (GM) plants require that the testing of C5 for resistance to Chilean PPV isolates be done under controlled isolated conditions. To carry out these tests C5 shoots were multiplied in vitro and micro-grafted onto four Adesoto101 (Prunus insititia L.) rootstock populations that had been previously infected each with one of four Chilean PPV-Ds. Tests were carried out under controlled conditions in a biosafety greenhouse. Symptoms appearance, virus detection, and viral mRNA levels for the cylindrical inclusion (CI) and CP genes were determined during three consecutive growing seasons. Complete resistance to all PPV isolates was demonstrated during the first 2 yr in all of the C5 plants. In the third season, four of 10 C5 plants showed mild symptoms on leaves close to the graft union and low but detectable CI mRNA levels in the C5 scions. These results support the effectiveness of using of micro-grafting on P. insititia for PPV resistance studies, especially in the limited space of a quarantine facility; whereas resistance levels in C5 after 3 yr indicate the importance of long term and field scale evaluations.

Candidate nematicidal proteins in a new Pseudomonas veronii isolate identified by its antagonistic properties against Xiphinema index.

The nematode Xiphinema index affects grape vines and transmits important viruses associated with fanleaf degeneration. Pseudomonas spp. are an extensive bacterial group in which important biodegradation and/or biocontrol properties can occur for several strains in the group. The aim of this study was to identify new Pseudomonas isolates with antagonist activity against X. index. Forty bacterial isolates were obtained from soil and root samples from Chilean vineyards. Thirteen new fluorescent pseudomonads were found and assessed for their antagonistic capability. The nematicide Pseudomonas protegens CHA0 was used as a control. Challenges of nematode individuals in Kings B semi-solid agar Petri dishes facilitated the identification of the Pseudomonas veronii isolate R4, as determined by a 16S rRNA sequence comparison. This isolate was as effective as CHA0 as an antagonist of X. index, although it had a different lethality kinetic. Milk-induced R4 cultures exhibited protease and lipase activities in cell supernatants using both gelatin/tributyrin Petri dish assays and zymograms. Three proteins with these activities were isolated and subjected to mass spectrometry. Amino acid partial sequences enabled the identification of a 49-kDa protease similar to metalloprotease AprA and two lipases of 50 kDa and 69 kDa similar to LipA and ExoU, respectively. Electron microscopy analyses of challenged nematodes revealed degraded cuticle after R4 supernatant treatment. These results represent a new and unexplored property in this species associated with the presence of secretable lipases and protease, similar to characterized enzymes present in biocontrol pseudomonads.