Pretransition and progressive softening of bovine carbonic anhydrase II as probed by single molecule atomic force microscopy

To develop a simple method for probing the physical state of surface adsorbed proteins, we adopted the force curve mode of an atomic force microscope (AFM) to extract information on the mechanical properties of surface immobilized bovine carbonic anhydrase II under native conditions and in the course of guanidinium chloride-induced denaturation. A progressive increase in the population of individually softened molecules was probed under mildly to fully denaturing conditions. The use of the approach regime of force curves gave information regarding the height and rigidity of the molecule under compressive stress, whereas use of the retracting regime of the curves gave information about the tensile characteristics of the protein. The results showed that protein molecules at the beginning of the transition region possessed slightly more flattened and significantly more softened conformations compared with that of native molecules, but were still not fully denatured, in agreement with results based on solution studies. Thus the force curve mode of an AFM was shown to be sensitive enough to provide information concerning the different physical states of single molecules of globular proteins.     

Structural and functional analysis of tomato β‐galactosidase 4: insight into the substrate specificity of the fruit softening‐related enzyme

Plant β‐galactosidases hydrolyze cell wall β‐(1,4)‐galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato β‐galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of β‐(1,4)‐galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1–7 that belongs to the β‐galactosidase/exo‐β‐(1,4)‐galactanase subfamily. The enzyme can hydrolyze a wide range of plant‐derived (1,4)‐ or 4‐linked polysaccharides, and shows a strong ability to attack β‐(1,4)‐galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with β‐d ‐galactose. TBG4 comprises a catalytic TIM barrel domain followed by three β‐sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 β‐galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards β‐(1,6)‐galactobiose, and ~0.6‐fold activity towards β‐(1,4)‐galactobiose, compared with wild‐type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant β‐galactosidases towards β‐1,4 and β‐1,6 linkages.     

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

Background

One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening.

Scope

In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected.     

Mango - Postharvest Biology and Biotechnology

Mango is one of the choicest fruits in the world and popular due to its delicate taste, pleasant aroma and nutritional value. Mango is indigenous to north-east India and north Burma, but now grown in over 90 countries. In the past two decades, mango production has increased appreciably with international trade jumping approximately four-fold valued close to US$ 950 million. Mango belongs to the category of climacteric fruits and its ripening is initiated and proceeded by a burst in ethylene production and a dramatic rise in the rate of respiration. Although there are a few hundred mango cultivars grown in the Indian subcontinent and other parts of the world, the most popular cultivars are generally highly perishable and ripen within 7 to 9 days of harvest at ambient temperature. Currently, the export potential and international trade of mango is limited due to several factors such as its perishable nature, disease and pest infestation, and susceptibility of certain premium cultivars to chilling injury when stored at low temperatures. Efforts are ongoing to develop technologies for improved storage and packaging, and overcome limitations encountered during storage and transit. Controlled atmosphere (CA) and hypobaric storage of mango are powerful means to overcome its perishable nature. The composition of CA varies among cultivars to ensure its original taste, flavor and aroma. Edible coating on the fruit skin may further cut down the rate of deterioration. Recently, significant advances have been made in understanding ripening characteristics of mango at the molecular level. Candidate genes related to ethylene biosynthesis and signalling, cell wall modification, aroma production and stress response have been cloned and characterized for future use in mango improvement. Efforts are also being made to establish a suitable transformation and plant regeneration system so that transgenic mango with added value and increased shelf life for long distance transportation could be developed.     

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls

Background and AimsThe programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by β-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested.MethodsWe developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action.Key ResultsIn model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide (‘ΔUA–GalA’), taken as diagnostic of PL action. ΔUA–GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA–GalA from higher homologues. The ΔUA–GalA was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA–GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA–GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA–GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol^–1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action.ConclusionsThe results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.     

Ethephon application induces symptoms of fruit tissue degeneration in watermelon

Vegetable Research Division, NHRI, 475 Imok-dong, Jangan-gu, Suwon, Gyeonggi-do, 440-706, Republic of Korea Flesh tissue degeneration (called blood-black heart; BBH) of watermelon (Citrullus vulgaris S.) is occasionally observed in Korea. Fruits with BBH have lower quality, a dark-red flesh, and reduced firmness of the rind, often producing an unpleasant flavor. Although causal factors are thought to be undesirable soil moisture conditions, e.g., drought or water-logging, temperature extremes, or virus infection, the mechanism for this physiological disorder is not clearly understood. It is possible that ethylene gas (C₂H₄) is involved in degrading the cell walls. To determine such an implication for ethylene in the occurrence of BBH, we foliar-applied various concentrations of ethephon (100, 200, 400, 800, or 1000 mg L^-1) to watermelon plants at 5 or 10 d prior to harvesting, then monitored the development of this disorder in their fruits. At 400 mg L^-1 or higher, quality was diminished and the fruit had softer rind tissues. About 25% of the fruits harvested at that level exhibited BBH versus 100% of the fruits exposed to 800 or 1000 mg L^-1 ethephon. Concomitant with the onset of BBH, those affected fruits produced elevated amounts of ethylene gas during the 4-d measurement period. Therefore, a high incidence of BBH may well be related to this increased ethylene production, which can be triggered by both unfavorable environmental conditions and inappropriate cultural practices.     

Antioxidant Systems in Ripening Tomato Fruits

Two cultivars of tomato (Lycopersicon esculentum Mill.), Selection-7 (shelf life 7–8 d) and ARTH-3 (shelf life 14–15 d) were analyzed for oxidative stress and the antioxidant enzyme system at different stages of fruit ripening. The results presented here suggest that during the early stages of fruit ripening, efficient antioxidant system protects the tomato fruits against the damaging effect of progressive oxidative stress. At later stages, however, oxidative damage occurs due to decreased activities of the ROS scavenging enzymes.     

热处理对脱涩后火柿生理生化变化的影响

以CO2脱涩后的火柿为试材,研究了脱涩后火柿在5℃条件下贮藏过程中主要生理生化变化及35℃热处理对其软化生理的影响。结果表明,柿果可溶性单宁含量的继续下降是柿果软化的一个原因;乙烯在脱涩柿果的软化中起着主导作用,呼吸速率及PE、PG和纤维素酶活性的变化与乙烯存在着紧密的关系。35℃热处理抑制了脱涩火柿可溶性单宁含量的下降速率,使乙烯生成量降低和乙烯峰出现时间推迟,刺激了早期的呼吸速率,抑制了后期的呼吸速率,降低了PE、PG和纤维素酶的活性,从而达到了延缓脱涩柿果轮化的目的。     

Polygalacturonase: a candidate gene for the soft flesh and deciduous fruit mutation in Capsicum

The soft flesh and deciduous fruit of pepper (Capsicum spp.) originated from the wild C. frutescens BG 2816 accession is a complete dominant trait controlled by the S gene. We constructed an F₂ population from a cross of BG 2816 (SS) and the bell-type C. annuum cultivar Maor (ss) and determined that S cosegregated with the tomato fruit-specific endo-polygalacturonase (PG) gene. The soft flesh and deciduous fruit phenotypes were observed together in all F₂ individuals, indicating a pleiotropic effect of PG on the two traits. We mapped S to pepper chromosome 10 in the region corresponding to that in which PG was previously mapped in tomato. Northern, RT-PCR and western analyses and enzyme activity assays, collectively, indicated that PG is not detected in green, breaker or red fruits of Maor, nor in green fruits of BG 2816. Accumulation of PG mRNA and protein was detected in the fruits of BG 2816, and it increased during ripening from breaker to red stages. The sequence analysis of partial PG cDNA isolated from BG 2816 revealed high homology (87% identity) with the tomato PG. The resemblance of the soft flesh and deciduous fruit phenotypes to PG-associated phenotypes in other fruit crops, the complete linkage between Sand PG, and the greater expression of PG in the fruits of BG 2816 than in those of Maor, all strongly indicate that PG is a candidate gene for S.