Specific Changes of Exocarp and Mesocarp Occurring during Softening Differently Affect Firmness in Melting (MF) and Non Melting Flesh (NMF) Fruits

Melting (MF) and non melting flesh (NMF) peaches differ in their final texture and firmness. Their specific characteristics are achieved by softening process and directly dictate fruit shelf life and quality. Softening is influenced by various mechanisms including cell wall reorganization and water loss. In this work, the biomechanical properties of MF Spring Crest’s and NMF Oro A’s exocarp and mesocarp along with the amount and localization of hydroxycinnamic acids and flavonoids were investigated during fruit ripening and post-harvest. The objective was to better understand the role played by water loss and cell wall reorganization in peach softening. Results showed that in ripe Spring Crest, where both cell turgor loss and cell wall dismantling occurred, mesocarp had a little role in the fruit reaction to compression and probe penetration response was almost exclusively ascribed to the epidermis which functioned as a mechanical support to the pulp. In ripe Oro A’s fruit, where cell wall disassembly did not occur and the loss of cell turgor was observed only in mesocarp, the contribution of exocarp to fruit firmness was consistent but relatively lower than that of mesocarp, suggesting that in addition to cell turgor, the integrity of cell wall played a key role in maintaining NMF fruit firmness. The analysis of phenols suggested that permeability and firmness of epidermis were associated with the presence of flavonoids and hydroxycinnamic acids.     

European, Chinese and Japanese pear fruits exhibit differential softening characteristics during ripening

Softening characteristics were investigated in three types of pear fruit, namely, European pear 'La France', Chinese pear 'Yali', and Japanese pear 'Nijisseiki'. 'La France' fruit softened dramatically and developed a melting texture during ripening, while 'Yali' fruit with and without propylene treatment showed no change in flesh firmness and texture during ripening. Non-treated 'Nijisseiki' did not show a detectable decrease in flesh firmness, whereas continuous propylene treatment caused a gradual decrease in firmness resulting in a mealy texture. In 'La France', the analysis of cell wall polysaccharides revealed distinct solubilization and depolymerization of pectin and hemicellulose during fruit softening. In 'Nijisseiki', propylene treatment led to the solubilization and depolymerization of pectic polysaccharides to a limited extent, but not of hemicellulose. In 'Yali', hemicellulose polysaccharides were depolymerized during ripening, but there was hardly any change in pectic polysaccharides except in the water-soluble fraction. PC-PG1 and PC-PG2, two polygalacturonase (PG) genes, were expressed in 'La France' fruit during ripening, while only PC-PG2 was expressed in 'Nijisseiki' and neither PC-PG1 or PC-PG2 was expressed in 'Yali'. The expression pattern of PC-XET1 was constitutive during ripening in all three pear types. PG activity measured by the reducing sugar assay increased in all three pears during ripening. However, viscometric measurements showed that the levels of endo-PG activity were high in 'La France', low in 'Nijisseiki', and undetectable in 'Yali' fruits. These results suggest that, in pears, cell wall degradation is correlated with a decrease in firmness during ripening and the modification of both pectin and hemicellulose are essential for the development of a melting texture. Furthermore, the data suggest that different softening behaviours during ripening among the three pear fruits may be caused by different endo-PG activity and different expression of PG genes.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

Development of Ty1-copia retrotransposon-based SSAP molecular markers for the study of genetic diversity in peach

Sequence-specific amplified polymorphism (SSAP) technology is a novel, anchored PCR approach derived from AFLP, which amplifies the region between a transposon insertion and an adjacent restriction site and have higher levels of polymorphism. In the current study, we developed 16 SSAP markers based on the long terminal repeat (LTR) sequences of Ty1-copia retrotransposons in the peach and used them for DNA profiling of 52 individual peaches: 44 peach cultivars and 8 ornamental peaches. These primer combinations produced a total of 1,553 fragments and 1,517 polymorphic bands with a polymorphism percentage of 97.7%. Furthermore, the Shannon's information index of each primer combination ranged from 0.1593 to 0.4456. Neighbor-joining analyses revealed two main genetic clusters, corresponding to the fruit flesh types: (A-1) MF (melting flesh) with clingstone and ornamental peaches; (A-2) MF with freestone and NMF (non-melting flesh) with clingstone. Finally, cluster analyses revealed that all ornamental peaches are closely related to the MF with clingstone peach cultivars. The application of these primer combinations identified using SSAP will facilitate future cultivar identification and germplasm management in peaches.     

Genetic analysis of the slow-melting flesh character in peach

The slow-melting flesh (SMF) trait in peach [Prunus persica (L.) Batsch] defines a slower process of postharvest fruit-softening than the prevalent melting flesh (MF) types. This gives a longer shelf life and a delayed harvest-time resulting in better fruit quality. Unlike other known fruit texture traits, SMF is difficult to measure and has a complex inheritance. We examined this character over 2 years in the offspring of two crosses, both with “Big Top,” an SMF nectarine, as the female parent, and with a melting flesh (MF) nectarine as the male parent (“Armking” and “Nectaross”). Following harvest, a texturometer was used to provide a textural profile analysis, and fruit firmness evolution was measured with a penetrometer over a period of 5 days’ storage at 20 °C. Linkage maps were constructed with a high-density SNP chip, and a phenotype-genotype analysis allowed the detection of three independent genomic regions where most QTLs (quantitative trait loci) were located. Two of these, on linkage groups 4 and 5, explained the variability for two characters—maturity date and firmness loss—that is, the QTL on linkage group 4 found in the MF parents and that on linkage group 5 in Big Top. A third region on linkage group 6, which identified a QTL for maturity date only in Armking, has no apparent association to the softening process. The relationship between maturity date and fruit-firmness loss and a hypothesis on the inheritance of the SMF character are discussed.     

Latency of Infection in Unripe Avocados by Colletotrichum gloeosporioides is Not Due to Inadequate Enzyme Potential

Colletotrichum gloeosporioides produced exo-pectin lyase and protease in a) liquid cultures with incorporated washed cell wall material from unripe or ripe avocado and b) autoclaved immature fruit. The activity of exo-pectin lyase and protease produced in liquid cultures incorporating washed cell walls from immature fruits was almost the same as when washed cell walls from ripe fruits were incorporated. Ripe fruit tissue rotted by the fungus contained exo-pectin lyase, endo-polygalacturonase (endo-PG) and protease. The endo-PG was found to be endogenous to avocado fruit, and had a pH optimum of 5.5. The pH optima of exo-pectin lyase and protease were 8.5 and 7.5 respectively in all three enzyme preparations. All these enzyme preparations completely macerated avocado fruit tissue discs in vitro in less than 3 h of incubation but not potato tuber discs. Neither immature nor ripe fruit contained substances, proteinaceous or otherwise, which could inhibit the exo-pectin lyase or protease activity of these preparations. The results indicated that C. gloeosporioides possesses sufficient enzyme potential to invade cell walls of unripe fruit and that the fruit tissue does not have a mechanism to inactivate such enzymes.     

柿果实采后软化过程中细胞壁组分代谢和超微结构的变化

柿果实采后果胶酯酶活性迅速上升,其活性与果实硬度的下降呈明显的负相关。多聚半乳糖醛酸酶活性增加缓慢,但其活性与果实硬度的下降无明显相关性。β-半乳糖苷酶活性迅速增加,其活性与果实硬度的下降呈明显的负相关。纤维素酶活性呈逐渐上升趋势,与果实硬度的下降也呈明显的负相关。伴随着细胞壁水解酶活性的增加,果实原果胶和纤维素含量迅速下降,而水溶性果胶含量则迅速上升。柿果刚采收时细胞壁结构完整,3d后细胞壁中胶层基本被溶解,甚至初生壁也局部发生降解。     

Water-soaked symptom of 'Andesu' netted melon fruit does not develop under anaerobic nitrogen atmospheres during ripening

'Andesu' netted melon fruits (Cucumismelo. L.) were ventilated with pure nitrogen or air for 12 days from35 to 47 DAA or 6 days from 47 to 53 DAA (ripe and over-ripe stages,respectively). Exposure to anaerobic nitrogen atmosphere resulted in higheracetaldehyde and ethanol concentrations, while lower sucrose concentration wasrecorded in the mesocarp compared with air-ventilated fruit. However,water-soaked symptom was not developed under anaerobic nitrogen atmospheres,suggesting that the formation of water-soaking mesocarp tissue does not resultfrom increased alcoholic fermentation and/or decreased soluble sugaraccumulation in the flesh. Anaerobic nitrogen atmospheres also resulted in lowethylene production, high flesh firmness, and inhibition of depolymerisation ofpolyuronides and non-cellulosic neutral sugars in the cell walls. Theimportanceof increased intercellular spaces and membrane permeability on the developmentof water-soaked symptom was suggested.     

Two forms of exopolygalacturonase increase as peach fruits ripen

Abstract. Freestone peach cultivars are distinguished from clingstone cultivars by a more extensive softening of the mesocarp tissue, and by the separation of mesocarp and endocarp during ripening. Cultivars of both types have been reported to develop exopolygalacturonase activity during ripening, but the enzyme has not been characterized in any detail. During development of freestone peaches ( Prunus persica L. var Coronet), two exopolygalacturonase enzymes were detected 42, 65 and 85 d after full bloom and in ripe fruit. During ripening one enzyme (exoPG 1) increased 36-fold and the other (exoPG 2) 90-fold but exoPG 2 accounted for a 73% of the total activity in ripe fruit. ExoPG 1 was purified 24-fold and exoPG 2 540-fold. ExoPG 2 is a slightly acidic glycoprotein. ExoPG 1 and exoPG 2 differ slightly in their pH optima and in their responses to calcium: each produces monogalacturonic acid as a reaction product. Similar enzymes were found in Flavorerest, a semi-freestone peach.     

香蕉果实成熟软化过程中β-D-木聚糖苷酶活性变化

β-D-木聚糖苷酶是细胞壁半纤维素中阿拉伯木聚糖和木聚糖残基降解的主要酶,对香蕉贮藏过程中果皮、果肉中β-D-木聚糖苷酶活性以及果实硬度、呼吸强度和乙烯释放量的变化进行测定分析。结果显示:β-D-木聚糖苷酶活性在果实贮藏初期的变化很小,到果实硬度开始急剧下降时迅速增加,其增加量在果皮和果肉中分别为12和22倍以上,且果肉中的酶活性大于果皮中;乙烯吸收剂处理延缓了香蕉果实呼吸和乙烯的高峰出现以及果实硬度、果肉和果皮中β-D-木聚糖苷酶活性变化的速度和幅度,但并不改变其活性的变化趋势。结果证明,β-D-木聚糖苷酶能诱导香蕉果实成熟,在果实软化中起着十分重要的作用,且其活性受乙烯的调节。     

Pectin esterase, polygalacturonase and gel formation in peach pectin fractions

Peaches (Prunus persica cv. Hermoza) were stored at 0C in regular air (RA) or in controlled atmosphere (CA 10% CO2, 3% O2) for 4 weeks and then ripened for 4 days at 20 degrees C. Woolliness developed in the regular air stored fruit while the controlled atmosphere stored fruit ripened normally. In the woolly fruit symptoms of the disorder were greater in the inner mesocarp than in the outer. Polygalacturonase (PG) and pectin esterase (PE) activities differed in the outer and inner mesocarp of the affected fruit. PG activity was low and PE activity was high in the inner mesocarp of the woolly fruit during ripening relative to the outer mesocarp, while in the healthy fruit, activities were similar in both areas. Cell wall fractions of water-soluble, CDTA-soluble and carbonate-soluble pectins were prepared from freshly harvested peaches and incubated with PE and PG from ripe peaches at different ratios. Only the CDTA-soluble fraction formed a gel with peach enzymes, and the rate of gelation increased with increasing amounts of PE relative to PG. Both water-soluble and CDTA-soluble pectin fractions formed gels with commercial PE (extracted from orange peel). The PE extracted from peaches was stable when stored at 0 degrees C for 9 days, while PG activity was stable only for 1 day. We suggest that PE, acting on pectins in the cell wall in vivo may cause gel formation and that the CDTA-soluble polymers have the capacity to bind apoplastic water and create the dry appearance observed in woolly fruit.     

皱皮木瓜果实发育后期品质变化及其成熟阶段的划分初探

以湖北长阳产皱皮木瓜为材料,测定果实发育后期果实鲜质量、果长、果径、果色、果实硬度以及果肉干物质量、可溶性糖含量、总酸含量和总黄酮含量等品质指标的动态变化,划分不同成熟阶段,为判断果实适宜采收期、实现优质生产提供理论参考。结果表明:(1)皱皮木瓜果实发育后期果实鲜质量、果长、果径、果肉干物质量和可溶性糖含量均呈现上升趋势;果色由绿色、黄绿色渐变为淡黄色到黄色;果实硬度、果肉总酸和总黄酮含量呈先上升后下降趋势。(2)各品质指标快速变化的时间区域存在差异,果实鲜质量在花后105~150d增加较快,果色在150d后逐渐变黄,果实硬度在花后135~165d快速下降,果肉总酸、总黄酮含量则在花后105~120d快速增加至峰值。(3)根据主成分分析结果和各品质指标的变化特点,可初步将皱皮木瓜果实发育后期划分为未成熟(花后105d之前)、早期成熟(花后120~150d)和成熟(花后165~180d)3个阶段。研究表明,随着果实成熟度的提高,皱皮木瓜果实鲜质量、果色、果肉干物质量、可溶性糖含量等指标不断升高,果实硬度逐渐下降,其食用加工品质不断提升,而在早期成熟阶段(花后120~150d)果实的药用品质则相对较高。     

The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, in peach fruit softening

Ethylene promotes fruit ripening, including softening. The fruit of melting-flesh peach (Prunus persica (L). Batsch) cultivar 'Akatsuki' produces increasing levels of ethylene, and the flesh firmness softens rapidly during the ripening stage. On the other hand, the fruit of stony hard peach cultivars 'Yumyeong', 'Odoroki', and 'Manami' does not soften and produces little ethylene during fruit ripening and storage. To clarify the mechanism of suppression of ethylene production in stony hard peaches, the expression patterns of four ethylene biosynthesis enzymes were examined: ACC synthases (Pp-ACS1, Pp-ACS2, and Pp-ACS3) and ACC oxidase (Pp-ACO1). In the melting-flesh cultivar 'Akatsuki', Pp-ACS1 mRNA was dramatically induced after harvesting, and a large amount of ethylene was produced. On the other hand, in stony hard peaches, Pp-ACS1 mRNA was not induced during the ripening stage, and ethylene production was inhibited. Since Pp-ACS1 mRNA was induced normally in senescing flowers, wounded leaves, and wounded immature fruit of 'Yumyeong', Pp-ACS1 was suppressed only at the ripening stage, and was not a defect in Pp-ACS1. These results indicate that the suppression of fruit softening in stony hard peach cultivars was caused by a low level of ethylene production, which depends on the suppressed expression of Pp-ACS1.     

YFT1基因突变影响番茄果色和硬度

番茄（Solanum lycopersicum）是全球第一大经济作物,其品质倍受科学家和消费者关注,果色和硬度是决定番茄品质的重要经济性状。为了解析YFT1（YELLOW FRUIT TOMATO1）调控番茄果色发育和硬度形成机制,本文对野生型cv.M82和突变体yft1的果色、硬度和果皮显微结构进行了比较分析。结果显示,在35 dpa（days post anthesis）,yft1和cv.M82番茄果色、硬度、果皮细胞大小和形状无明显差异。随着果实发育cv.M82番茄由绿色转为橙色/浅红（47 dpa）和红色（54 dpa）;并由硬变软,54 dpa的果实硬度（13.68±1.78N）仅相当于35 dpa的（35.51±1.09N）1/3。同时,从转色期（47 dpa）果皮细胞由内向外逐渐变大;内果皮薄壁细胞由圆形（35 dpa）依次变成卵形（47 dpa）和不规则形（54 dpa）,并观察到了细胞内陷和胞间空隙消失（54 dpa）。与cv.M82不同,yft1番茄的果色、硬度和果皮细胞显微结构随果实发育（35~54 dpa）变化不明显;而其果实硬度（47~54 dpa）显著高于cv.M82。这些结果均暗示YFT1突变使番茄果实发育延迟,并影响了果色发育和硬度的形成。该研究将为番茄果色发育和果色形成机制的揭示提供重要的表型证据。     

β-Galactosidase, polygalacturonase and pectinesterase in differential softening and cell wall modification during papaya fruit ripening

Papaya ( Carica papaya L. cv. Eksotika) fruit softens differentially in relation to position of the tissue. The inner mesocarp tissue is softer, and its firmness decreases more rapidly during ripening than that of the outer mesocarp tissue. As the fruit ripens, pectin solubility and depolymerisation increase. Hemicellulose, too, appears to be depolymerised but, unlike pectins, this apparent degradation of hemicellulose is associated with an increase rather than a decrease in its level. Pectin and hemicellulose depolymerisation began in the inner mesocarp tissue at about the same time as β-galactosidase (EC 3.2,1.23) activity started to increase and tissue firmness began to decrease more rapidly. In contrast, pectin solubilisation in both outer and inner mesocarp tissues occurred steadily throughout ripening at a comparable rate and paralleled closely the increase of polygalacturonase (PG; EC 3.2.1.67) and pectinesterase (EC 3.1.1.11). In general, irrespective of enzyme distribution, tissue softening during ripening was more closely related to changes in β-galactosidase activity than to PG or pectinesterase activity. Papaya, β-galactosidase appears to be an important wall degrading enzyme and may contribute significantly to differential softening, perhaps by complementing the action of polygalacturonase. Polygalacturonase activity increased with increasing depth of the mesocarp tissue, as did softening of the fruit.     

采前喷施草酸对芒果果实细胞钙含量和分布的影响

利用焦锑酸钾沉淀-透射电子显微镜观察的方法, 观测了采前喷施草酸或钙后采收期芒果(Mangifera indica)果实细胞钙含量和分布情况, 探讨钙含量和分布变化对果实成熟和衰老的影响。结果表明, 与对照相比, 采前经草酸或钙处理的果实果皮和果肉细胞排列较规则且致密, 淀粉粒分布较多。采前草酸或钙处理均能显著提高芒果果皮和外果肉组织的钙含量;疏松结合态钙均匀分布在果皮和果肉细胞的细胞壁、细胞膜、液泡膜和质体中, 并在液泡内堆积; 而对照果实的液泡膜模糊, 钙颗粒较少。实验证明采前喷施草酸或钙能维持果实细胞的形态, 提高果实细胞的钙含量, 影响钙的分布, 有利于保持果实的硬度并可增加果实营养。