HISTOLOGICAL AND HISTOCHEMICAL CHANGES IN DEVELOPING AND RIPENING PEACHES III. CATECHOL TANNIN CONTENT PER CELL

Reeve , R. M. (Western Regional Res. Lab., U. S. Dept. of Agriculture, Albany, Calif.) Histological and histochemical changes in developing and ripening peaches. III. Catechol tannin content per cell. Amer. Jour. Bot, 46(9): 645–650. Illus. 1959.—Photometric measurements were made of the densities of catechol tannin test colors histochemically developed with a nitrous acid reaction in fresh sections of fruits. The measured values showed a linear relationship with both section thickness and tannin concentration. When average cell size, section thickness. and the volume of tissue photometrically illuminated are known, it is possible to calculate an amount of phenolic substance per cell. Such values calculated for the naturally occurring catechol tannins in developing and ripening peaches revealed very large changes in these substances per cell. Catechol tannins so estimated increased about 90-fold from the beginning of cell enlargement until the fruit was slightly over half its mature size, a period of about 16-fold increase in volume of the average cell. After a maximum tannin content had been reached, a decrease of about 8-fold per cell was found as the fruit matured and ripened. The early changes in tannin content, when expressed on a unit volume basis, were much less pronounced (about 5-fold increases) and agreed in general with published chemical data for increases in chlorogenic acid on a weight basis during a comparable growth period in a sweet cherry. The semiquantitative, percell expressions of these histochemical results show relation to established phenomena of growth.     

HISTOLOGICAL AND HISTOCHEMICAL CHANGES IN DEVELOPING AND RIPENING PEACHES. II. THE CELL WALLS AND PECTINS

Reeve , R. M. (U.S.D.A., Albany, California.) Histological and histochemical changes in developing and ripening peaches. II. The cell walls and pectins. Amer. Jour. Bot. 46(4): 241–247. Illus. 1959.—Histological and histochemical observations on developing and ripening clingstone and freestone peaches have revealed that, after cell divisions have ceased in the mesocarp, cell wall thickening and cell enlargement in the mesocarp parenchyma increase until the fruit is nearly full cell size. The cell walls then decrease in thickness as the fruit ripens and softens. Degree of methyl esterification of the pectic substances, as estimated histochemically, remains at about 75–80% in immature fruits during their cell-enlargement phase of growth. Percent of methyl esterification apparently is much lower, or amounts of esterified pectates are very low during the meristematic phases of fruit growth. Just prior to ripening, degree of esterification increases and approaches 100% in hard, ripe fruit at about the same time that the parenchyma cell walls exhibit their greatest thickness or degree of hydration. The degree of esterification of the pectic substances then rapidly decreases and the cell walls become appreciably thinner as the ripening fruit softens. Further relation of these changes in wall thickness, in degree of esterification of the pectins, and in other cell wall carbohydrates to the textural qualities of ripening fruits is discussed. Interpretations concerning cell wall plasticity, cell growth and relation between auxin and changes in pectins also are presented.     

山茱萸果实发育过程中单宁物质的分布与积累特征

选取不同发育时期的山茱萸果实作为研究对象,采用果实形态观察法、显微及超微技术、组织化学定位法以及紫外分光光度计法对山茱萸果实发育过程中单宁物质分布及积累特征进行观察分析,并以单因素ANOVA检验不同发育时期单宁含量的差异,以揭示单宁物质在山茱萸果实发育中的变化规律,为山茱萸果实涩味调控机制研究提供理论依据。结果表明:(1)山茱萸果实发育过程中果皮颜色和果实体积变化明显,可将其发育过程划分为幼果期、中果期、成熟期3个时期;单宁物质主要分布在山茱萸果实中果皮的单宁细胞中。(2)在山茱萸果实发育过程中单宁细胞数目呈先增后减的变化趋势,幼果期单宁细胞从无到有,随着果实发育单宁细胞数目不断增多,至中果期单宁细胞数目开始减少。(3)单宁含量的变化规律与单宁细胞数目的变化一致,单宁含量在花后120 d时达到最多,随后逐渐减少。(4)单宁物质首先在细胞质的小液泡中积累,中央大液泡形成后则为单宁物质积累的主要场所,其积累形态主要有颗粒状、不规则状和板块状3种;单宁细胞中线粒体数目较多,中果期后期及成熟期在中央大液泡液泡膜附近有电子致密物质积累。研究认为,山茱萸果实中中果皮薄壁细胞为单宁物质积累的专属细胞,即单宁细胞,单宁物质的合成运输与液泡、囊泡以及线粒体的作用密切相关;成熟期山茱萸果实总单宁含量降低,涩味降低,表明单宁物质积累的动态变化与植物对环境的适应性和果实涩味息息相关,可结合代谢组和转录组的方法对山茱萸果实中单宁物质的合成机制进行进一步研究。     

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.     

山茱萸核果的解剖结构和组织化学定位

用解剖学和组织化学的方法研究了山茱萸 (MacrocarpiumofficinacleSieb .etZucc .)核果的解剖结构和皂甙、多糖的组织化学定位。结果表明 :山茱萸核果的外果皮革质 ,由一层被覆较厚角质膜的表皮细胞构成 ;中果皮肉质 ,由多列薄壁细胞构成 ,含色素细胞不均匀分布 ,靠近外果皮的薄壁细胞大多为含色素细胞 ,使果实呈现红色 ,向内的薄壁细胞体积渐渐增大 ,在较大的薄壁细胞以及维管束周围的薄壁细胞内常含有色素块。组织化学定位显示 :外中果皮的含色素细胞的色素块中含有丰富的皂甙和多糖 ,果实的中果皮的薄壁细胞在未成熟时就已经形成皂甙 ,并随着果实的成熟逐渐增加积累     

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.     

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.     

扁桃幼果发育的形态解剖学研究

以普通扁桃品种‘纸皮’为研究对象,观察测定了扁桃幼果生长发育动态,并采用石蜡切片法研究了扁桃幼果发育过程。结果表明,扁桃幼果鲜重、体积及果径增长均呈单“S”曲线。大体可分为3个生长时期,增长速率为:第Ⅱ期>第I期>第Ⅲ期。扁桃果实由单心皮上位子房发育而成,边缘胎座,横生胚珠。果皮由外果皮、中果皮和内果皮构成:外果皮是一种复合结构,由表皮毛和表皮细胞组成;中果皮主要是由薄壁细胞和分布其中的维管束组成;内果皮也有丰富的维管束组织分布,其木质化顺序由外向内。中果皮细胞分裂终止早于内果皮。     

A comparative study of melting and non-melting flesh peach cultivars reveals that during fruit ripening endo-polygalacturonase (endo-PG) is mainly involved in pericarp textural changes, not in firmness reduction

Peach softening is usually attributed to the dismantling of the cell wall in which endo-polygalacturonase (endo-PG)-catalysed depolymerization of pectins plays a central role. In this study, the hypothesis that the function of endo-PG is critical for achieving a melting flesh fruit texture but not for reducing fruit firmness was tested by comparing pericarp morphology and endo-PG expression and localization in melting (MF) and non-melting flesh (NMF) fruit at successive stages of ripening. MF Bolero, Springbelle, and Springcrest, and NMF Oro-A and Jonia cultivars were analysed. Both MF and NMF fruit were left to ripen on the tree and reached a firmness of <10 Newtons (N). The image analysis of pericarp tissues revealed that during softening the loss of cell turgidity was a process common to mesocarp cells of all MF and NMF fruit and was clearly visible in peaches with a firmness of less than ～20?N. In contrast, the loss of cell adhesion was a feature exclusively observed in ripe MF fruit pericarp. In this ripe fruit, large numbers of endo-PG isoforms were highly expressed and the enzyme localization corresponded to the middle lamella. As a consequence, wide apoplastic spaces characterized the pericarp of ripe MF peaches. In contrast, no loss of cell adhesion was observed in any NMF fruit or in unripe MF peaches. Accordingly, no endo-PG was detected in unripe NMF fruit, whereas few and poorly expressed enzyme isoforms were revealed in ripe NMF and in unripe MF peaches. In this fruit, the poorly expressed endo-PG localized mainly in vesicles within the cytoplasm and inner primary cell wall. On the whole the results suggested that endo-PG function was needed to achieve melting flesh texture, which was characterized by wide apoplastic spaces and partially deflated mesocarp cells. Conversely, endo-PG activity had no critical influence on the reduction of fruit firmness given the capacity of NMF peaches to soften, reaching values of 5-10?N. As in tomato, the change of symplast/apoplast water status seems to be the main process through which peach fruit regulates its firmness.     

灵武长枣果实发育过程中阿拉伯半乳糖蛋白组织化学分布

为揭示灵武长枣（Ziziphus jujuba ‘Lingwu Changzao’）果实发育过程中阿拉伯半乳糖蛋白（AGPs）的分布规律,以膨大前期、快速膨大期、着色期和完熟期灵武长枣果实为试验材料,通过组织化学和免疫荧光定位的方法,研究了不同发育时期果实AGPs的分布特征。结果显示：βGlcY-AGPs形成的棕红色沉淀和MAC204抗体识别的抗原在各时期果实的外果皮及相邻的内部数层排列紧密的中果皮小细胞的细胞壁和细胞内部均有分布。中果皮大型卵圆形薄壁细胞的细胞壁和细胞内在膨大前期均有βGlcY-AGPs形成的棕红色沉淀和MAC204抗体识别的抗原分布,而在快速膨大期、着色期和完熟期均主要分布于薄壁细胞的细胞壁上,大部分细胞内部无分布;随着果实发育成熟,中果皮薄壁细胞间隙拉大排列更加松散,出现细胞破裂,βGlcY-AGPs形成的棕红色沉淀和MAC204抗体识别的抗原分布逐渐减少。各时期果实维管束中维管束鞘、木质部、韧皮部、形成层的所有细胞的细胞壁和细胞内部都分布有βGlcY-AGPs形成的棕红色沉淀和MAC204抗体识别的抗原,维管束数量和大小随果实发育及体积的进一步增大逐渐减少,βG...     

核桃果皮的发育解剖学研究

核桃果皮的发育过程可分为３个阶段,发育时期：中、内三层果皮的界线不清,维管束处于发育初期;发育中期：随着中果皮最外侧两层石细胞的出现和薄壁组织细胞体积的迅速扩大以及维管束轮数的增加,使三层果皮具较明显的界面,发育后期：中果皮的维管束递增到４－５轮,     

Comparison of ripening processes in intact tomato fruit and excised pericarp discs

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L^*a^*b^* color space, changing from green (a^* < −5) to red (a^* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO₂ production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a^* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a^* = −5), appeared as discs turned yellow-red (a^* = 5), and increased another eight-fold as discs became full red (a^* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a^* = 0) to red.     

The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches

Ethylene has long been regarded as the main regulator of ripening in climacteric fruits. The characterization of a few tomato mutants, unable to produce climacteric ethylene and to ripen their fruits even following treatments with exogenous ethylene, has shown that other factors also play an important role in the control of climacteric fruit ripening. In climacteric peach and tomato fruits it has been shown that, concomitant with ethylene production, increases in the amount of auxin can also be measured. In this work a genomic approach has been used in order to understand if such an auxin increase is functional to an independent role played by the hormone during ripening of the climacteric peach fruits. Besides the already known indirect activity on ripening due to its up-regulation of climacteric ethylene synthesis, it has been possible to show that auxin plays a role of its own during ripening of peaches. In fact, the hormone has shown the ability to regulate the expression of a number of different genes. Moreover, many genes involved in biosynthesis and transport and, in particular, the signalling (receptors, Auxin Response Factors and Aux/IAA) of auxin had increased expression in the mesocarp during ripening, thus strengthening the idea that this hormone is actively involved in the ripening of peaches. This study has also demonstrated the existence of an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene and genes in the ethylene domain regulated by auxin.     

Cellulase, Fruit Softening and Abscission in Red RaspberryRubus idaeusL. cv Glen Clova

The ripening of raspberry fruit (Rubus ideausL. cv Glen Clova)is associated with a climacteric rise in ethylene production.As the fruit pigments change from green to red there is a progressivesoftening, loss of skin strength and a breakdown of cell wallsin the mesocarp. An increase in cellulase (endo-1,4-ß-D-glucanase)in both drupelets and receptacles accompanies these changes.The localization of cellulase in the regions of the fruit associatedwith abscission zones suggest the enzyme may be involved infruit separation as well as softening. Rubus idaeusL; raspberry; fruit ripening; ethylene; abscission; cell wall breakdown; cellulase; endo-1,4-ß-D-glucanase     

Dynamic characteristics of sugar accumulation and related enzyme activities in sweet and non-sweet watermelon fruits

Sugar content largely determines watermelon fruit quality. We compared changes in sugar accumulation and activities of carbohydrate enzymes in the flesh (central portion) and mesocarp of elite sweet watermelon line 97103 (Citrullus lanatus subsp. vulgaris) and exotic non-sweet line PI296341-FR (C. lanatus subsp. lanatus) to elucidate the physiological and biochemical mechanisms of sugar accumulation in watermelon fruit. The major translocated sugars, raffinose and stachyose, were more unloaded into sweet watermelon fruit than non-sweet fruit. During the fruit development, acid α-galactosidase activity was much higher in flesh of 97103 than in mesocarp of 97103, in flesh and mesocarp of PI296341-FR fruit. Insoluble acid invertase activity was higher in 97103 flesh than in 97103 mesocarp, PI296341-FR flesh or mesocarp from 18 days after pollination (DAP) to 34 DAP. Changes in soluble acid invertase activity in 97103 flesh were similar to those in PI296341-FR flesh and mesocarp from 18 DAP to full ripening. Sucrose synthase and sucrose phosphate synthase activities in 97103 flesh were significantly higher than those in 97103 mesocarp and PI296341-FR fruits from 18 to 34 DAP. Only insoluble acid invertase and sucrose phosphate synthase activities were significantly positively correlated with sucrose content in 97103 flesh. Therefore, phloem loading, distribution and metabolism of major translocated sugars, which are controlled by key sugar metabolism enzymes, determine fruit sugar accumulation in sweet and non-sweet watermelon and reflect the distribution diversity of translocated sugars between subspecies.     

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.     

山茱萸果皮的解剖学研究

本文报导了山茱萸果皮的解剖学研究结果.外果皮由一层表皮细胞构成.中果皮外方为3-4(5)层厚角组织细胞,这些细胞通常含有单宁;其内方包含薄壁细胞、单宁细胞和8束维管束.单宁细胞成团或零星分布于薄璧细胞中,前者的体积明显大于后者.单宁细胞的单宁与多糖结合在一起.维管束含有环纹、螺纹、梯纹、网纹及孔纹管胞,还有少数散生的纤维.内果皮高度木质化,主要由多种形状的石细胞组成,其间分布了肉眼可见、排列成环状的异细胞,偶见少数生活石细胞及薄壁细胞.内果皮有3束维管束.     

The molecular interaction of human salivary histatins with polyphenolic compounds.

Dietary tannins are polyphenols that are effectively precipitated by salivary histatins (Hsts), a novel family of tannin binding proteins. Epigallocatechin gallate (EGCG), a flavan-3-ol ester related to condensed tannins (polymerized products of flavan-3-ols), and pentagalloyl glucose (PGG), a hydrolyzable tannin, were used to evaluate the molecular nature of Hst-polyphenol interaction. NMR demonstrated that Hst5, a representative Hst, bound to EGCG in a hydrophobic manner via basic and aromatic residues. In contrast, proline plays a dominant role in polyphenol binding to other tannin precipitating proteins. The role of basic and aromatic amino acids in EGCG binding was investigated using a series of modified Hsts in each of which one type of amino acid was substituted by Ala. EGCG bound to all modified Hsts, but the binding was diminished. Optimal EGCG binding also depended on the primary structure, as a polypeptide with randomised Hst5 sequence showed significantly diminished interaction with EGCG. Soluble EGCG/Hst5 complexes containing up to seven molecules of EGCG per mol of Hst5 had a 1-mM dissociation constant. In contrast to EGCG, PGG formed small soluble complexes with Hst5 consisting of only one molecule each of PGG and Hst5, as demonstrated by analytical ultracentrifugation. These complexes became insoluble upon binding of additional molecules of PGG. Diminished PGG binding was seen to a peptide with a Hst5 randomized sequence showing the importance of the primary structure. Hsts may serve to form insoluble complexes with tannins thereby preventing their absorption from the intestines and potentially harmful biological effects. In contrast the much weaker interaction with EGCG may allow its uptake into the organism and exploitation of its antioxidant effect.