Chitosan Augments Tetramycin against Soft Rot in Kiwifruit and Enhances Its Improvement for Kiwifruit Growth, Quality and Aroma.

In this study, the co-application of chitosan and tetramycin against kiwifruit soft rot and its effects on the disease resistance, growth, quality and aroma of kiwifruit were investigated. The results show that chitosan could effectively enhance tetramycin against soft rot of kiwifruit with the field control efficacy of 85.33% for spraying chitosan 100 time + 0.3% tetramycin AS 5000-time dilution liquid, which was higher than 80.99% for 0.3% tetramycin AS 5000-time dilution liquid and significantly (p < 0.01) higher than 40.66% for chitosan 100-time dilution liquid. Chitosan could significantly (p < 0.05) improve the promoting effects of tetramycin on total phenolics, total flavonoids, SOD activity of kiwifruit compared to tetramycin during storage for 0-28 days and enhance the disease resistance of kiwifruit. Moreover, the co-application of chitosan and tetramycin was more effective than tetramycin or chitosan alone in enhancing fruit growth, improving fruit quality and increasing fruit aroma. This study highlights that chitosan can be used as an adjuvant to enhance tetramycin against soft rot of kiwifruit and promote tetramycin's improvement for the single fruit volume and weight, vitamin C, soluble sugar, soluble solid, dry matter, soluble protein, titratable acidity and aroma of kiwifruit.

The kiwifruit enzyme actinidin enhances the hydrolysis of gluten proteins during simulated gastrointestinal digestion.

This study investigated the effect of actinidin, a cysteine protease in kiwifruit, on the hydrolysis of gluten proteins and digestion-resistant gluten peptides (synthetic 33-mer peptide and pentapeptide epitopes) under static simulated gastrointestinal conditions. Actinidin efficacy in hydrolysing gliadin was compared with that of other gluten-degrading enzymes. Actinidin hydrolysed usually resistant peptide bonds adjacent to proline residues in the 33-mer peptide. The gastric degree of hydrolysis of gluten proteins was influenced by an interaction between pH and actinidin concentration (P < 0.05), whereas the pentapeptide epitopes hydrolysis was influenced only by the actinidin concentration (P < 0.05). The rate of gastric degree of hydrolysis of gliadin was greater (P < 0.05) by actinidin (0.8%/min) when compared to papain, bromelain, and one commercial enzyme (on average 0.4%/min), while all exogenous enzymes were able to hydrolyse the pentapeptide epitopes effectively. Actinidin is able to hydrolyse gluten proteins under simulated gastric conditions.

Phenolic Profiles, Antioxidant Capacities, and Inhibitory Effects on Digestive Enzymes of Different Kiwifruits.

In order to obtain high-quality kiwifruits with health-promoting characteristics, physicochemical properties, phenolic profiles, antioxidant capacities, and inhibitory effects on digestive enzymes (pancreatic lipase and α-glucosidase), of fourteen different types of kiwifruit obtained from China were systematically investigated and compared. Noticeable variations in the fruits' physicochemical properties and phenolic profiles were observed among them. The total phenolic content of Actinidia chinensis cv. Hongshi, A. chinensis cv. Jinshi, and A. chinensis cv. Jinlong were 16.52 ± 0.26 mg GAE/g DW (dry weight), 13.38 ± 0.20 mg GAE/g DW, and 11.02 ± 0.05 mg GAE/g DW, respectively, which were much higher than those of the other tested kiwifruits. According to high performance liquid chromatography (HPLC) analysis, phenolic compounds, including procyanidin B1, procyanidin B2, (-)-epicatechin, chlorogenic acid, gallic acid, and quercetin-3-rhamnoside, were found to be the major compounds in kiwifruits, while procyanidin B1, procyanidin B2, and chlorogenic acid were the most abundant phenolic compounds. Furthermore, all the tested kiwifruits exerted remarkable antioxidant capacities and inhibitory effects on pancreatic lipase and α-glucosidase. Indeed, A. chinensis cv. Hongshi, Actinidia chinensis cv. Jinshi, and Actinidia chinensis cv. Jinlong exhibited much better antioxidant capacities and inhibitory effects on digestive enzymes than those of the other tested kiwifruits. Particularly, A. polygama showed the highest inhibitory activity on α-glucosidase. Therefore, Actinidia chinensis cv. Hongshi, Actinidia chinensis cv. Jinshi, and Actinidia chinensis cv. Jinlong, as well as A. polygama could be important dietary sources of natural antioxidants and natural inhibitors against pancreatic lipase and α-glucosidase, which is helpful for meeting the growing demand for high-quality kiwifruits with health-promoting characteristics in China.

Cell separation in kiwifruit without development of a specialised detachment zone.

BACKGROUND: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The 'good-peeling' genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The 'poor-peeling' genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. RESULTS: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. CONCLUSIONS: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.

Effect of Putrescine Treatment on Chilling Injury, Fatty Acid Composition and Antioxidant System in Kiwifruit.

We investigated the effects of different concentrations (0, 1, 2 and 4 mM) of putrescine on chilling injury, fruit quality, ethylene production rate, fatty acid composition and the antioxidant system of cold-stored kiwifruit (Actinidia chinensis Planch. var. chinensis 'Hongyang'). We achieved a significant decrease in ethylene production, maintained fruit quality and alleviated chilling injury during storage via treatment with 2 mM putrescine. Furthermore, putrescine treatment inhibited increases in superoxide anion production rate and H2O2 concentration, while maintaining higher membrane lipid unsaturation as well as increased activities of superoxide dismutase and catalase. In addition, putrescine treatment enhanced the activities of antioxidant enzymes related to the ascorbate-glutathione cycle while causing higher levels of ascorbic acid and reduced glutathione. Our results suggest that induced tolerance against chilling injury via putrescine treatment in cold-stored kiwifruit may be due to enhanced antioxidant activity, increased unsaturation of membrane lipids, and inhibited ethylene production.

Comparative analysis on the distribution of protease activities among fruits and vegetable resources.

In this study, a comparative analysis on the distribution of protease activities among 90 plant resources, including fruits and vegetables, has been performed. Protease activities of plant extracts were assayed at different pH values (pH 3.0, pH 7.5 and pH 10.5) using casein as a substrate. Ten fruits and thirteen vegetables show protease activities above 10U/g. Pineapple, fig and papaya, which are used for commercial protease production, exhibited high protease activities. Additionally, high protease activities were detected in kiwifruit (28.8U/g), broccoli (16.9U/g), ginger (16.6U/g), leek (32.7U/g) and red pepper (15.8U/g) at different pH values. SDS-PAGE and zymograms confirmed that various types of proteases existed in the five plant extracts and might be explored. Furthermore, five plant extracts were treated by different protease inhibitors. These results show that there are still many plant resources unexplored, which may be promising candidates for plant-derived protease production.

Dietary actinidin from kiwifruit (Actinidia deliciosa cv. Hayward) increases gastric digestion and the gastric emptying rate of several dietary proteins in growing rats.

Dietary actinidin influences the extent to which some dietary proteins are digested in the stomach, and it is hypothesized that the latter modulation will in turn affect their gastric emptying rate (GE). In this study, the effect of dietary actinidin on GE and gastric digestion of 6 dietary protein sources was determined in growing rats. Each dietary protein source [beef muscle, gelatin, gluten, soy protein isolate (SPI), whey protein isolate, and zein] was included in 2 semisynthetic diets as the sole nitrogen source. For each protein source, 1 of the 2 diets contained actinidin [76.5 U/g dry matter (DM)] in the form of ground freeze-dried green kiwifruit (Actinidia deliciosa cv. Hayward), whereas the other diet contained freeze-dried gold kiwifruit (Actinidia chinensis cv. Hort16A), which is devoid of actinidin (3.4 U/g DM). For both diets, dietary kiwifruit represented 20% of the diet on a DM basis. The real-time GE was determined in rats gavaged with a single dose of the diets using magnetic resonance spectroscopy over 150 min (n = 8 per diet). Gastric protein digestion was determined based on the free amino groups in the stomach chyme collected from rats fed the diets (n = 8 per diet) that were later killed. GE differed across the protein sources [e.g., the half gastric emptying time (T(½)) ranged from 157 min for gluten to 266 min for zein] (P < 0.05). Dietary actinidin increased the gastric digestion of beef muscle (0.6-fold), gluten (3.2-fold), and SPI (0.6-fold) and increased the GE of the diets containing beef muscle (43% T(½)) and zein (23% T(½); P < 0.05). There was an inverse correlation between gastric protein digestion and DM retained in the stomach (r = -0.67; P < 0.05). In conclusion, dietary actinidin increased gastric protein digestion and accelerated the GE for several dietary protein sources. GE may be influenced by gastric protein digestion, and dietary actinidin can be used to modulate GE and protein digestion in the stomach of some dietary protein sources but not others.

Isolation and developmental expression analysis of L-myo-inositol-1-phosphate synthase in four Actinidia species.

Myo-inositol (MI) is an important polyol involved in cellular signal transduction, auxin storage, osmotic regulation, and membrane formation. It also serves as a precursor for the production of pinitol, ascorbic acid, and members of the raffinose family. The first committed step for MI formation is catalyzed by L-myo-inositol-1-phosphate synthase (MIPS). We isolated MIPS cDNA sequences from Actinidia eriantha, Actinidia rufa, and Actinidia arguta and compared them with that of Actinidia deliciosa. Each comprised 1533 bp, encoding 510 amino acids with a predicted molecular weight of 56.5 KDa. The MIPS protein was highly conserved in Actinidia, sharing 98.94% identity among species. The MIPS gene was expressed in the flowers, leaves, petioles, and carpopodia. Similarly high levels of expression were detected in the young fruit of all four species. Overall activity of the enzyme was also maximal in young fruit, indicating that this developmental stage is the key point for MI synthesis in Actinidia. Among the four species, A. arguta had the greatest concentration of MI as well as the highest ratios of MI:sucrose and MI:glucose+fructose. This suggests that conversion to MI from carbohydrates was most efficient in A. arguta during early fruit development.

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase.

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two-dimensional zymography and direct identification of zymographic spots by mass spectrometry.

BACKGROUND: Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow-up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes. RESULTS: Kiwi crude extracts were analysed by two-dimensional zymography on gelatin-containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI-ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. CONCLUSIONS: The innovative achievements of the present study are the: (1) two-dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI-ToF MS analysis of the zymographic spots.

Nematicidal effects of cysteine proteinases against sedentary plant parasitic nematodes.

Cysteine proteinases from the fruit and latex of plants, such as papaya, pineapple and fig, have previously been shown to have substantial anthelmintic efficacy, in vitro and in vivo, against a range of animal parasitic nematodes. In this paper, we describe the in vitro effects of these plant extracts against 2 sedentary plant parasitic nematodes of the genera Meloidogyne and Globodera. All the plant extracts examined caused digestion of the cuticle and decreased the activity of the tested nematodes. The specific inhibitor of cysteine proteinases, E-64, blocked this activity completely, indicating that it was essentially mediated by cysteine proteinases. In vitro, plant cysteine proteinases are active against second-stage juveniles of M. incognita and M. javanica, and some cysteine proteinases also affect the second-stage juveniles of Globodera rostochiensis. It is not known yet whether these plant extracts will interfere with, or prevent invasion of, host plants.

In vitro and in vivo anthelmintic efficacy of plant cysteine proteinases against the rodent gastrointestinal nematode, Trichuris muris.

Extracts of plants, such as papaya, pineapple and fig, are known to be effective at killing intestinal nematodes that inhabit anterior sites in the small intestine, such as Heligmosomoides polygyrus. In this paper, we demonstrate that similar in vitro efficacy also occurs against a rodent nematode of the large intestine, Trichuris muris, and confirm that the cysteine proteinases present in the plant extracts are the active principles. The mechanism of action of these enzymes involved an attack on the structural proteins of the nematode cuticle, which was similar to that observed with H. polygyrus. However, not all plant cysteine proteinases were equally efficacious because actinidain, from the juice of kiwi fruit, had no detrimental effect on either the motility of the worms or the nematode cuticle. Papaya latex was also shown to significantly reduce both worm burden and egg output of mice infected with adult T. muris, demonstrating that enzyme activity survived passage to the caecum and was not completely inactivated by the acidity of the host's stomach or destroyed by the gastric or pancreatic proteinases. Thus, the cysteine proteinases from plants may be a much-needed alternative to currently available anthelmintic drugs due to their efficacy and novel mode of action against different gastrointestinal nematode species.

Assessment of the anthelmintic effect of natural plant cysteine proteinases against the gastrointestinal nematode, Heligmosomoides polygyrus, in vitro.

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.