A flavin-dependent monooxygenase produces nitrogenous tomato aroma volatiles using cysteine as a nitrogen source.

Tomato (Solanum lycopersicum) produces a wide range of volatile chemicals during fruit ripening, generating a distinct aroma and contributing to the overall flavor. Among these volatiles are several aromatic and aliphatic nitrogen-containing compounds for which the biosynthetic pathways are not known. While nitrogenous volatiles are abundant in tomato fruit, their content in fruits of the closely related species of the tomato clade is highly variable. For example, the green-fruited species Solanum pennellii are nearly devoid, while the red-fruited species S. lycopersicum and Solanum pimpinellifolium accumulate high amounts. Using an introgression population derived from S. pennellii, we identified a locus essential for the production of all the detectable nitrogenous volatiles in tomato fruit. Silencing of the underlying gene (SlTNH1;Solyc12g013690) in transgenic plants abolished production of aliphatic and aromatic nitrogenous volatiles in ripe fruit, and metabolomic analysis of these fruit revealed the accumulation of 2-isobutyl-tetrahydrothiazolidine-4-carboxylic acid, a known conjugate of cysteine and 3-methylbutanal. Biosynthetic incorporation of stable isotope-labeled precursors into 2-isobutylthiazole and 2-phenylacetonitrile confirmed that cysteine provides the nitrogen atom for all nitrogenous volatiles in tomato fruit. Nicotiana benthamiana plants expressing SlTNH1 readily transformed synthetic 2-substituted tetrahydrothiazolidine-4-carboxylic acid substrates into a mixture of the corresponding 2-substituted oxime, nitro, and nitrile volatiles. Distinct from other known flavin-dependent monooxygenase enzymes in plants, this tetrahydrothiazolidine-4-carboxylic acid N-hydroxylase catalyzes sequential hydroxylations. Elucidation of this pathway is a major step forward in understanding and ultimately improving tomato flavor quality.

Growth Response of Cuttings to Drought and Intermittent Flooding for Three Salix Species and Implications for Riverbank Soil Bioengineering.

Willows are used as cuttings or in fascines for riverbank soil bioengineering, to control erosion with their high resprouting ability and rapid growth. However, water availability is highly variable along riverbanks both in time and space and constitutes a major stress limiting willow establishment. A species-specific understanding of willow cutting response to water stress is critical to design successful riverbank soil bioengineering projects given exclusive use of local species is often recommended. In a three-month greenhouse experiment, we investigated the effects of three soil moisture treatments (drought-soil saturation-intermittent flooding) on survival, biomass production and root growth of cuttings of three willow species used for soil bioengineering along NE American streams (Salix discolor-S. eriocephala-S. interior). Cutting survival was high for all species and treatments (>89%). Biomass production and root volume only differed between species. S. eriocephala produced the highest biomass and root volume, and S. discolor invested more in belowground than aboveground biomass. Root length responded to soil moisture differently between species. Under intermittent flooding, S. eriocephala produced shorter roots, while S. interior produced longer roots. For riverbank soil bioengineering, S. eriocephala should be favored at medium elevation and S. interior at lower elevation.

Valorization of snow crab (Chionoecetes opilio) cooking effluents for food applications.

BACKGROUND: Seafood processing generates significant amounts of solid and liquid waste in the environment. Such waste represents a potential source of high-value biomolecules for food, pharmaceutic and cosmetic applications. There are very few studies on the valorization of wastewaters compared to solid by-products. However, cooking waters are characterized by a high organic polluting load, which could contain valuable molecules such as proteins, pigments and flavor compounds. Snow crab (Chionoecetes opilio) processing is included among the most important processes in Canadian fisheries, although its cooking effluent composition is not well characterized. RESULTS: The present study concentrated and valorized the biomass in snow crab cooking wastewaters for the development of products for food applications. A membrane process was designed and optimized to concentrate the effluents. The chemical composition of the concentrates was analyzed, including characterizing the flavor profile compounds. The extracts were mainly composed of proteins (592 g kg-1 ) and minerals (386 g kg-1 ) and contained desirable flavor compounds. Their functional properties (solubility, water-holding capacity, oil-holding capacity) and antioxidant activities were also assessed, and their safety was verified. CONCLUSION: The cooking effluents generated by snow crab processing facilities, usually considered as waste, can be concentrated and turned into a natural aroma for the food industry. © 2019 Society of Chemical Industry.

Sling-based Exercise for External Rotator Muscles: Effects on Shoulder Profile in Young Recreational Tennis Players

Context: Tennis playing generates specific adaptations, particularly at the dominant shoulder. It remains to be established whether shoulder strength balance can be restored by sling-based training for adolescent recreational tennis players. Objective: We added a sling-based exercise for shoulder external rotators to investigate its effects on external rotator muscle strength, on internal rotator muscle strength, on glenohumeral range of motion and on tennis serve performance. Design: Test-retest design. Setting: Tennis training sports facilities. Participants: Twelve adolescent male players volunteered to participate in this study (age: 13.3 ± 0.5 years; height: 1.64 ± 0.07 cm, mass: 51.7 ± 5.8 kg, International Tennis Number: 8). Intervention: The procedure spanned 10 weeks. For the first five weeks, players performed their regular training (RT) twice a week. For the last five weeks, a sling-based exercise (SE) for strengthening the shoulder external rotator muscles was added to their regular training. Main Outcome Measures: Maximal isometric strength of shoulder external and internal rotator muscles and glenohumeral range of motion in external and internal rotation were assessed in both shoulders. Serve performance was also evaluated by accuracy and post-impact ball velocity, using a radar gun. Results: No change was found in any measurement after the RT period. Significant increases in external (~+5%; p<0.001) and internal (~+2%; p<0.05) rotator muscle strength and in external/internal strength ratio (~+4%; p<0.001) were observed after the SE period. Serve velocity and accuracy were significantly improved after SE (~+2% and ~+24%, respectively; p<0.05 for both), while no clinically meaningful alterations in ranges of motion were observed. Conclusions: Prophylactic intervention through sling-based exercise for strengthening shoulder external rotator muscles appears effective in restoring strength balance at the dominant shoulder, and may prevent adolescent tennis players from sustaining degenerative shoulder problems which could later impair their performance of daily and work-related tasks.

Role of an esterase in flavor volatile variation within the tomato clade.

Tomato flavor is dependent upon a complex mixture of volatiles including multiple acetate esters. Red-fruited species of the tomato clade accumulate a relatively low content of acetate esters in comparison with the green-fruited species. We show that the difference in volatile ester content between the red- and green-fruited species is associated with insertion of a retrotransposon adjacent to the most enzymatically active member of a family of esterases. This insertion causes higher expression of the esterase, resulting in the reduced levels of multiple esters that are negatively correlated with human preferences for tomato. The insertion was evolutionarily fixed in the red-fruited species, suggesting that high expression of the esterase and consequent low ester content may provide an adaptive advantage in the ancestor of the red-fruited species. These results illustrate at a molecular level how closely related species exhibit major differences in volatile production by altering a volatile-associated catabolic activity.

Biochemical dynamics during postharvest: Highlighting the interplay of stress during storage and maturation of fresh produce.

The lifecycle of fresh produce involves a sequence of biochemical events during their ontology, and these events are particularly significant for climacteric fruits. A high demand during ripening is observed in these plant products, which is reflected in a high rate of respiration and ethylene production. Increased respiratory demand triggers the activation of secondary pathways such as alternate oxidase, which do not experience critical increases in energy consumption in non-climacteric fruit. In addition, biochemical events produced by external factors lead to compensatory responses in fresh produce to counteract the oxidative stress caused by the former. The dynamics of these responses are accompanied by signaling, where reactive oxygen species play a pivotal role in fresh product cell perception. This review aims to describe the protection mechanisms of fresh produce against environmental challenges and how controlled doses of abiotic stressors can be used to improve quality and prolong their shelf-life through the interaction of stress and defense mechanisms.

Diverse roles of strigolactone signaling in maize architecture and the uncoupling of a branching-specific subnetwork.

Strigolactones (SLs) control lateral branching in diverse species by regulating transcription factors orthologous to Teosinte branched1 (Tb1). In maize (Zea mays), however, selection for a strong central stalk during domestication is attributed primarily to the Tb1 locus, leaving the architectural roles of SLs unclear. To determine how this signaling network is altered in maize, we first examined effects of a knockout mutation in an essential SL biosynthetic gene that encodes CAROTENOID CLEAVAGE DIOXYGENASE8 (CCD8), then tested interactions between SL signaling and Tb1. Comparative genome analysis revealed that maize depends on a single CCD8 gene (ZmCCD8), unlike other panicoid grasses that have multiple CCD8 paralogs. Function of ZmCCD8 was confirmed by transgenic complementation of Arabidopsis (Arabidopsis thaliana) max4 (ccd8) and by phenotypic rescue of the maize mutant (zmccd8::Ds) using a synthetic SL (GR24). Analysis of the zmccd8 mutant revealed a modest increase in branching that contrasted with prominent pleiotropic changes that include (1) marked reduction in stem diameter, (2) reduced elongation of internodes (independent of carbon supply), and (3) a pronounced delay in development of the centrally important, nodal system of adventitious roots. Analysis of the tb1 zmccd8 double mutant revealed that Tb1 functions in an SL-independent subnetwork that is not required for the other diverse roles of SL in development. Our findings indicate that in maize, uncoupling of the Tb1 subnetwork from SL signaling has profoundly altered the balance between conserved roles of SLs in branching and diverse aspects of plant architecture.

Beneficial 'unintended effects' of a cereal cystatin in transgenic lines of potato, Solanum tuberosum.

BACKGROUND: Studies reported unintended pleiotropic effects for a number of pesticidal proteins ectopically expressed in transgenic crops, but the nature and significance of such effects in planta remain poorly understood. Here we assessed the effects of corn cystatin II (CCII), a potent inhibitor of C1A cysteine (Cys) proteases considered for insect and pathogen control, on the leaf proteome and pathogen resistance status of potato lines constitutively expressing this protein. RESULTS: The leaf proteome of lines accumulating CCII at different levels was resolved by 2-dimensional gel electrophoresis and compared with the leaf proteome of a control (parental) line. Out of ca. 700 proteins monitored on 2-D gels, 23 were significantly up- or downregulated in CCII-expressing leaves, including 14 proteins detected de novo or up-regulated by more than five-fold compared to the control. Most up-regulated proteins were abiotic or biotic stress-responsive proteins, including different secretory peroxidases, wound inducible protease inhibitors and pathogenesis-related proteins. Accordingly, infection of leaf tissues by the fungal necrotroph Botryris cinerea was prevented in CCII-expressing plants, despite a null impact of CCII on growth of this pathogen and the absence of extracellular Cys protease targets for the inhibitor. CONCLUSIONS: These data point to the onset of pleiotropic effects altering the leaf proteome in transgenic plants expressing recombinant protease inhibitors. They also show the potential of these proteins as ectopic modulators of stress responses in planta, useful to engineer biotic or abiotic stress tolerance in crop plants of economic significance.

A protease activity-depleted environment for heterologous proteins migrating towards the leaf cell apoplast.

Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1- and S1-type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ∼45% and improving the accumulation of a murine diagnostic antibody, C5-1, co-secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5-1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.

Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases.

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, generally involving the site-directed mutagenesis of functionally relevant amino acids or the selection of improved inhibitor variants by phage display approaches. Here, we propose a novel approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including arthropod proteases, to appreciate the extent of functional variability among the plant cystatin family. The most, and less, potent of these cystatins were then used as 'donors' of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model 'recipient' inhibitor. In brief, inhibitory activities against Cys proteases strongly differed from one plant cystatin to another, with Ki (papain) values diverging by more than 30-fold and inhibitory rates against arthropod proteases varying by up to 50-fold depending on the enzymes assessed. In line with theoretical assumptions from docking models generated for different Cys protease-cystatin combinations, structural element substitutions had a strong impact on the activity of recipient cystatin SlCYS8, positive or negative depending on the basic inhibitory potency of the donor cystatin. Our data confirm the wide variety of cystatin inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Impacts of pH and Base Substitution during Deaerator Treatments of Herring Milt Hydrolysate on the Odorous Content and the Antioxidant Activity.

Despite the biological interest in herring milt hydrolysate (HMH), its valorization is limited by its unpleasant odor resulting from the presence of mainly amine and carbonyl compounds. Recently, a deaerator was demonstrated as an interesting avenue to reduce the odorous content of HMH. However, the removal rate of amine and carbonyl compounds was highly dependent on the operating conditions, and the impact of such a process on the biological potential of HMH was not considered. Therefore, this study aimed to optimize the deaerator process by assessing the impacts of the combination of deaerator treatments at neutral and basic pH, the increase in pH from 10 to 11, and the substitution of NaOH by KOH on the odorous content and the antioxidant activity of HMH. Results showed that the highest deodorization rate of HMH was obtained when a deaerator treatment at neutral pH was combined with another one at basic pH using KOH for alkalization. This condition resulted in a decrease in the dimethylamine and trimethylamine contents by 70%, while certain compounds such as 2,3-pentanedione, methional, (E,E)-2,4-heptadienal, or (E,Z)-2,6-nonadienal were almost completely removed. Removal mechanisms of the targeted compounds were totally identified, and the performance of the developed process was confirmed by sensory analysis. Lastly, it was shown that the antioxidant potential of HMH was not affected by the deodorization process. These results demonstrated the feasibility of deodorizing a complex matrix without affecting its biological potential.

SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato.

The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch-inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C(13) cyclohexenone and C(14) mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source-sink interactions and production of arbuscular mycorrhiza-induced apocarotenoids.

Effect of two thermoresistant non-starter lactic acid bacteria strains on volatilome profile during Cheddar ripening simulation.

Dairy farm management practices can modify milk microbiota and therefore modulate non-starter lactic acid bacteria (NSLAB) found in cheese. These NSLAB can cause organoleptic defects. This study aimed to investigate the impact of two potential NSLAB in Cheddar cheesemaking: Lactiplantibacillus plantarum RKG 2-212 a strain isolated both in corn silage and raw milk, and Lactobacillus delbrueckii RKG R10, a strain isolated after pasteurisation of milk from a farm using grass and legume silage, and corn silage. The whole genome of these two lactobacilli was first sequenced. Then, the thermoresistance was evaluated after treatment at 60 °C for 5 min and compared to reference strains. Both lactobacilli were highly thermoresistant compared to other three lactic acid bacteria which are Lactococcus lactis subsp. cremoris ATCC 19257 and SK11, and L. plantarum ATCC 14917 (P < 0.0001). They lost less than 1 log cfu/mL (Δlog) and their genome contained a great number of copy number of genes coding for heat shock protein. During a Pearce test activity simulating Cheddar cheesemaking, the two lactobacilli did not show interaction with the starter Lcc. lactis subsp. cremoris SK11, and their population remained stable. During a ripening simulation, L. delbrueckii RKG R10 had a slight loss in viability in cheese slurry samples incubated at 30 °C for 12 d. However, L. plantarum RKG 2-212 had considerable growth, from 6.51 to 8.3 log cfu/g. This growth was associated with the acidification of the slurries (P < 0.0001). The presence of the lactobacilli modified the profile of volatile compounds evaluated by gas chromatography-mass spectrometry, accounting for 10.7% of the variation. The strain L. plantarum RKG 2-212 produced volatile compounds in greater quantity that could be associated with organoleptic defects such as acetic acid and 2-methylbutyraldehyde. Therefore, silage can be a vector of thermoresistant lactic acid bacteria for milk which can lead to flavor defects in cheese.

Development of a New Deodorization Method of Herring Milt Hydrolysate: Impacts of pH, Stirring with Nitrogen and Deaerator Treatment on the Odorous Content.

Herring milt hydrolysate (HMH) presents the disadvantage of being associated with an unpleasant smell limiting its use. Thus, to develop a new effective and easy-to-use deodorization method, this research aimed to deepen the knowledge regarding the impacts of pH (pH 7 vs. pH 10), overnight stirring with nitrogen (+N vs. -N) and deaerator treatment (+D vs. -D) on the odorous content of HMH. This latter included dimethylamine (DMA), trimethylamine (TMA), trimethylamine oxide (TMAO) and the most potent odor-active compounds of HMH. Results showed that pH had a huge impact on the targeted compounds resulting in higher detected concentrations of DMA, TMA and TMAO at pH 10 than at pH 7 (p < 0.05) while the opposite trend was observed for the most potent odor-active compounds of HMH (p < 0.05). Moreover, independently of the pH condition, the overnight stirring with or without nitrogen had no impact (p > 0.05). Finally, the deaerator treatment was more effective to remove TMA and DMA at pH 10 than at pH 7 (p < 0.05) while the opposite trend was observed for the most potent odor-active compounds (p < 0.05). Sensory analysis confirmed that the application of pH 10 -N +D and pH 7 -N +D + alkalization pH 10 conditions led to the least odorous products (p < 0.05).

2-DE proteome maps for the leaf apoplast of Nicotiana benthamiana.

We provide 2-D gel reference maps for the apoplastic proteome of Nicotiana benthamiana leaves infiltrated or not with the bacterial gene vector Agrobacterium tumefaciens. About 90 proteins were analyzed by LC-MS/MS for identification and function assignment. We show, overall, an effective response of the plant to agroinfiltration involving a specific, cell wall maintenance-independent up-regulation of defense protein secretion. The proteome maps described should be a useful tool for systemic studies on plant-pathogen interactions or cell wall metabolism. They also should prove useful for the monitoring of secreted recombinant proteins and their possible pleiotropic effects along the cell secretory pathway of N. benthamiana leaves used as an expression platform for clinically useful proteins.

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls.

Recombinant protease inhibitors represent useful tools for the development of insect-resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad-spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI-expressing and control (comparator) lines, followed by a two-dimensional gel electrophoresis (2-DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI-expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2-DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2-DE showed unaltered deposition patterns in SlCDI-expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI-expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel-generation transgenic plants.

A companion protease inhibitor for the protection of cytosol-targeted recombinant proteins in plants.

We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in-built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359-368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI-expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%-40% depending on the light regime. Out of approximately 700 proteins detected on 2-D gels, only 20 exhibited a significantly altered level on a protein-specific basis, whereas most proteins were up-regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase-PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two- to threefold in SlCDI-expressing lines. These observations, along with the unrelated biological functions assigned to MS-identified proteins up-regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI-expressing lines, suggest a general, proteasome-independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human alpha(1)-antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by approximately 2.5-fold in SlCDI-expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein-stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.

Assessment of the Performance of Electrodialysis in the Removal of the Most Potent Odor-Active Compounds of Herring Milt Hydrolysate: Focus on Ion-Exchange Membrane Fouling and Water Dissociation as Limiting Process Conditions.

Herring milt hydrolysate (HMH), like many fish products, presents the drawback to be associated with off-flavors. As odor is an important criterion, an effective deodorization method targeting the volatile compounds responsible for off-flavors needs to be developed. The potential of electrodialysis (ED) to remove the 15 volatile compounds identified, in the first part of this work, for their main contribution to the odor of HMH, as well as trimethylamine, dimethylamine and trimethylamine oxide, was assessed by testing the impact of both hydrolysate pH (4 and 7) and current conditions (no current vs. current applied). The ED performance was compared with that of a deaerator by assessing three hydrolysate pH values (4, 7 and 10). The initial pH of HMH had a huge impact on the targeted compounds, while ED had no effect. The fouling formation, resulting from electrostatic and hydrophobic interactions between HMH constituents and ion-exchange membranes (IEM); the occurrence of water dissociation on IEM interfaces, due to the reaching of the limiting current density; and the presence of water dissociation catalyzers were considered as the major limiting process conditions. The deaerator treatment on hydrolysate at pH 7 and its alkalization until pH 10 led to the best removal of odorant compounds.

Functional divergence of principal alcohol o-acyltransferase for biosynthesis of volatile acetate esters among tomato wild species (Solanum Sect. Lycopersicon).

Volatile esters are the chemicals that have multiple physiological functions including plant defense responses and reproduction. From a human perspective, the esters largely contribute to the fruity aroma of freshy fruits. Composition of volatile esters show a significant diversity among the wild tomato species (Solanum sect. Lycopersicon). To address the basis for this divergence, here we conducted functional analysis of a gene encoding major alcohol o-acyltransferase (AAT1) that catalyzes volatile ester formation. Although AAT1 transcripts were highly expressed in the ripe fruits of all the wild species examined, their enzymatic properties significantly differed due to amino acid sequence variations. Notably, AAT1s from S. pennellii showed the highest ability to produce acetate esters whereas AAT1s from S. neorickii, S. chmielewskii and S. habrochaites had the lowest activities. Further, screenings using domain-swapped or point-mutated AAT1s allowed us to identify Met/Thr352 as one of the critical residues related to the transferase activity with acetyl-CoA. This finding is potentially applied to aroma engineering in which a site-directed mutagenesis at this position in alcohol o-acyltransferases could enable to manipulate volatile ester levels in ripe fruits.

A SELDI-TOF MS procedure for the detection, quantitation, and preliminary characterization of low-molecular-weight recombinant proteins expressed in transgenic plants.

We describe a SELDI-TOF MS procedure for the rapid detection and quantitation of low-molecular-weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens-mediated transformation, and then used as test material for the analyses. Real-time RT-PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI-TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.