Calcium in plant senescence and fruit ripening

Abstract. Calcium has long been associated with regulation of the ripening process of fruit and post-harvest storage life. Specifically, maintenance of relatively high calcium concentrations in fruit tissue results in a slower rate of ripening, as seen in lower respiration rates, reduced ethylene production, and slower softening of fruit flesh. There are also some specific fruit disorders such as bitter pit, which can be prevented if sufficient calcium is present. Senescence of other plant tissues such as leaves and flowers has also been shown to be retarded by the application of calcium.
Work leading to the above information is reviewed and discussed in the context of what is currently known of cellular regulation of calcium in plants. The major sites for the action of calcium in senescence and ripening are suggested to be in membrane structure and function, and in cell wall structure. Although high external concentrations of calcium are an advantage in reducing the rate of senescence or ripening, it is emphasized that normal cell function requires the maintenance of low concentrations of free calcium in the cell cytosol. It is suggested that one possible feature of senescence is a breakdown in such cellular regulation.     

Indole-3-acetic acid, ethylene, and abscisic acid metabolism in developing muskmelon (Cucumis melo L.) fruit

Hormonal metabolism associated with fruit development in muskmelon was investigated by measuring IAA, ABA, and ACC levels in several tissues at various stages of development. In addition, levels of conjugated IAA and ABA were determined in the same tissues. Ethylene production, which is believed to signal the ripening and senescence of mature fruit, was also measured. Ethylene production was highest in the outer tissue near the rind and gradually declined during maturation, except for a dramatic increase in all fruit tissues at the climacteric. In contrast to ethylene production, ACC levels increased during maturation and remained equal throughout the fruit until the climacteric, when levels in the outer tissues increased nearly 5-fold over levels in the inner tissues. The consistent presence of ACC indicates that ACC oxidase rather than the availability of ACC regulates ethylene production in developing fruits. ABA and ABA esters generally declined during maturation, however an increase in ABA esters associated with the outer mesocarp tissue was observed in fully mature, climacteric fruit. IAA and IAA conjugates were only found in the outer tissue near the rind, and their levels remained low until the fruit was fully mature and entering the climacteric. At that time, increased levels of conjugates were detected. The late burst of hormonal metabolism in the outer mesocarp tissue appeared to signal its degeneration and the deterioration that typically occurs in ripening fruit. The tissue-specific conjugation of IAA and ABA, in addition to the production of climacteric ethylene, may represent part of the signaling mechanism initiating ripening and eventual deterioration of tissues in muskmelon fruits.Abbreviations ABA abscisic acid - ACC 1-aminocylopropane-1-carboxylic acid - DAP days after pollination - IAA indole-3-acetic acid     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

果实成熟的基因调控

果实的成熟过程是由一系列生理生化变化过程组成,这些变化过程受到外界环境条件、植物激素和基因的调控。随着近年来有关果实成熟衰老的基因的分离、定性及反义基因技术在控制果实成熟上的成功应用,对揭示果实成熟衰老的分子机理起到了重要作用。本文就近年来果实成熟基因调控研究进展作一简要评述 。     

Ultrastructure of the Mesocarp of Mature Avocado Fruit and Changes Associated with Ripening

The mesocarp tissue of ripening avocado fruits was studied byfreeze fracture, thin section and scanning electron microscopy.Carbon dioxide and ethylene production by individual fruit weremonitored, and samples were analysed at several stages of theripening process. The tissue is composed primarily of large, isodiametric, lipid-containingparenchyma cells. At maturity these cells contain the normalcomplement of cell organelles, and all membranes appear intact.When ripening begins, several changes in the ultrastructureoccur. The most obvious changes are a loosening and eventualbreakdown of the cell wall, and swelling and vesiculation ofthe rough endoplasmic reticulum. In freeze fracture replicasa significant increase in the number of intramembranous particlesin the EF face of the plasmamembrane was observed at the climactericpeak. In post-climacteric, soft fruit the particle density of theEF face of the plasmamembrane decreased to the density observedin the membrane of pre-climacteric cells. All of the organellesand membranes appear whole and intact whether examined by thinsection, freeze fracture or scanning electron microscopy. However,the cell walls in post-climacteric fruit have almost completelydisappeared. These results indicated that the ripening process per se inavocados does not involve a complete loss of compartmentationnor a breakdown of organelle and membrane integrity. It may,however, lead to these or similar senescence changes as a resultof the loss of the cell walls. The variations in particle densityof the plasmamembrane during ripening may reflect one or moreof several structural, compositional, or functional membranephenomena, and this aspect of ripening warrants further study. Persea americana Mill., avocado pear, freeze fracture, fruit ripening, scanning electron microscopy, senescence, ultrastructure     

果实成熟过程中组织超微结构的变化

依据电镜下观察的果实成熟期间果肉组织结构的变化状态,综合论述了果实成熟过程中,果肉细胞、细胞壁构造、亚细胞结构及细胞间隙的变化,揭示了果实构造的变化与成熟衰老的密切关系。     

Ultrastructure of tomato fruit ripening and the role of polygalacturonase isoenzymes in cell wall degradation

Ultrastructural changes in the pericarp of tomato (Lycopersicon esculentum Mill) fruit were followed during ripening. Ethylene production was monitored by gas chromatography and samples analyzed at successive stages of the ripening process.

Changes in the cytoplasmic ultrastructure were not consistent with the suggestion that ripening is a `senescence' phenomenon. A large degree of ultrastructural organization, especially of the mitochondria, chromoplasts, and rough endoplasmic reticulum, was retained by ripe fruit.

Striking changes in the structure of the cell wall were noted, beginning with dissolution of the middle lamella and eventual disruption of the primary cell wall. These changes were correlated with appearance of polygalacturonase (EC 3.2.1.15) isoenzymes. Application of purified tomato polygalacturonase isoenzymes to mature green fruit tissue duplicated the changes in the cell wall noted during normal ripening. Possible roles of the polygalacturonase isoenzymes in cell wall disorganization are discussed.

     

果实成熟和衰老的分子调控机制

成熟和衰老是果实生命周期中的两个重要阶段, 直接影响果实的品质保持及采后寿命。果实的成熟、衰老是一个复杂的生理过程, 受诸多内源因子的调控和外源因素的影响。该文重点综述近年来果实成熟、衰老分子机制方面的研究进展, 以及外源信号分子对果实成熟和衰老的调控作用。     

Evidences for Chlorogenic Acid — A Major Endogenous Polyphenol Involved in Regulation of Ripening and Senescence of Apple Fruit

To learn how the endogenous polyphenols may play a role in fruit ripening and senescence, apple pulp discs were used as a model to study the influences of chlorogenic acid (CHA, a major polyphenol in apple pulp) on fruit ripening and senescence. Apple (‘Golden Delicious’) pulp discs prepared from pre-climacteric fruit were treated with 50 mg L^-1 CHA and incubated in flasks with 10 mM MES buffer (pH 6.0, 11% sorbitol). Compared to the control samples, treatment with CHA significantly reduced ethylene production and respiration rate, and enhanced levels of firmness and soluble solids content of the pulp discs during incubation at 25°C. These results suggested that CHA could retard senescence of the apple pulp discs. Proteomics analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry (MALDI-TOF/TOF) revealed that the expressions of several key proteins correlated to fruit ripening and senescence were affected by the treatment with CHA. Further study showed that treating the pulp discs with CHA remarkably reduced levels of lipoxygenase, β-galactosidase, NADP-malic enzyme, and enzymatic activities of lipoxygenase and UDP-glucose pyrophosphorylase, all of which are known as promoters of fruit ripening and senescence. These results could provide new insights into the functions of endogenous phenolic compounds in fruit ripening and senescence.     

Reduction in Pectin Methylesterase Activity Modifies Tissue Integrity and Cation Levels in Ripening Tomato (Lycopersicon esculentum Mill.) Fruits

Pectin methylesterase (PME, EC 3.1.1.11) is an ubiquitous enzyme in the plant kingdom; however, its role in plant growth and development is not yet understood. Using transgenic tomato (Lycopersicon esculentum Mill.) fruits that show more than 10-fold reduction in PME activity because of expression of an antisense PME gene, we have investigated the role of PME in tomato fruit ripening. Our results show that reduced PME activity causes an almost complete loss of tissue integrity during fruit senescence but shows little effect on fruit firmness during ripening. Low PME activity in the transgenic fruit pericarp modified both accumulation and partitioning of cations between soluble and bound forms and selectively impaired accumulation of Mg2+ over other major cations. Decreased PME activity was associated with a 30 to 70% decrease in bound Ca2+ and Mg2+ in transgenic pericarp. Levels of soluble Ca2+ increase 10 to 60%, whereas levels of soluble Mg2+ and Na+ are reduced by 20 to 60% in transgenic pericarp. Changes in cation levels associated with lowered PME activity do not affect the rate of respiration or membrane integrity of fruit during ripening. Overall, these results suggest that PME plays a role in determining tissue integrity during fruit senescence, perhaps by regulating cation binding to the cell wall.     

PG在番茄果实成熟中的作用及二价金属离子与乙烯对PG活性的影响

对采后番茄果实的电镜观察表明:当果实成熟衰老时,叶绿体数量减少,多数基粒结构丧失;成熟果实胞壁中胶层水解成中空的电子透明区,初生壁的纤丝也发生一定程度的水解,相邻细胞分离;外源 PG(多聚半乳糖醛酸酶)提取物处理绿熟期果实组织,也可引起胞壁结构和叶绿体发生与正常衰老相同的变化。Ca~(2+)、Mg~(2+)、Co~(2+)二价金属离子处理果实,可明显降低番茄红素含量和 PG 活性,延缓果实软化。外源乙烯处理果实,可促进番茄红素的形成,提高 PG活性,并能解除钙对 PG 活性的抑制。本文也对 PG 在乙烯和 Ca~(2+)调节果实成熟中的作用进行了讨论。     

果实成熟衰老过程中蛋白质组学研究进展

蛋白质组学已开始应用于果实成熟衰老研究,以明确蛋白差异表达与成熟衰老的关系和深入揭示果实成熟衰老过程的分子机制。本文综述了蛋白质组学在果实成熟衰老研究中的重要性、果实样品蛋白的提取制备方法,重点介绍了蛋白质组学在果实成熟衰老机制、果实抗病性机制、冷害机制以及采后处理对果实成熟调控研究中的应用,分析了蛋白质组学在果实成熟衰老研究中存在的不足,提出了今后研究的方向。     

The Role of Polygalacturonase (PG) in Tomato Fruit Ripening and Effects of Divalent Metal Ions and Ethylene on PG Activity

It has been reported that PG is a key enzyme related to the tomato fruit ripening. In this study tomato fruits were harvested at the mature-green stage and stored at room temperature. The cell ultrastructure of pericarp tissue was observed at different ripening stages, and the effects of treatments with ethylene and calcium on PG activity and fruit ripening were examined. The object of this study is to elucidate the role of PG in regulation of tomato fruit ripening by ethylene and calcium. PG activity, was undetectable at mature-green stage, but it rose rapidly as fruif ripening. The rise in PG activity was coincided with the dechnmg of fruit firmness during ripening of tomato fruits. The observation of cell ultrastructure showed that the most of grana in chloroplast were lost and the mitochondrial cristae decreased as fruit ripening. Striking changes of cell wall structure was most noted, beginning with dissolution of the middle lamella and eventual disruption of primary cell wall. A similar pattern of changes of cell wall and chloroplast have been observed in pericarp tissue treated with PG extract. In fruits treated with calcium and other divalent metal ions atmature-green stage, the lycopene content and PG activity decreased dramatically. Ethylene application enhanced the formation of lycopene and PG activity. The inhibition of Ca2+ on PG ac ivity was removed by ethylene. Based on the above results, it was demonstrated that PG played a major role in ripening of tomato fruits, and suggested that the regulation of fruit ripening by ethylene and Ca2+ was all mediated by PG. PG induced the hydrolysis of cell wall and released the other hydrolytic enzymes, then effected the ripening processes follow up.     

Transient increase in locular pressure and occlusion of endocarpic apertures in ripening tomato fruit

Locular pressure was monitored during ripening of tomato (Lycopersicon esculentum Mill.) fruit and the anatomy of the endocarp surface examined using scanning electron microscopy. The manometric pressure of the locule tissue increased from 0 in mature-green fruit to 10 to 50 Pa at the turning or pink stages, and then subsided in ripe fruit. Nonclimacteric fruit containing the ripening inhibitor (rin) mutation showed a similar pattern of internal pressure accumulation during senescence. Build-up of locular tissue pressure occurred in fruit ripening, on or off the plant, as well as in fruit with different susceptibility to cuticle cracking. Apertures ranging from 18-31 μm in width and 33-41 μm in length, with densities ranging from 6.7 to 47.9 apertures · mm⁻² were observed in the endocarp of mature-green fruit. These apertures were progressively occluded during early ripening and were absent in late ripening fruit. Aperture occlusion might result in reduced gas exchange between the locule and external fruit atmosphere, resulting in modification of the locular gas composition.     

Characterization of a calcium/calmodulin-regulated SR/CAMTA gene family during tomato fruit development and ripening

Background  

Fruit ripening is a complicated development process affected by a variety of external and internal cues. It is well established that calcium treatment delays fruit ripening and senescence. However, the underlying molecular mechanisms remain unclear.     

Identification of cDNA clones for tomato (Lycopersicon esculentum Mill.) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene

Changes in gene expression during foliar senescence and fruit ripening in tomato (Lycopersicon esculentum Mill.) were examined using in-vitro translation of isolated RNA and hybridization against cDNA clones.During the period of chlorophyll loss in leaves, changes occurred in mRNA in-vitro translation products, with some being reduced in prevalence, whilst others increased. Some of the translation products which changed in abundance had similar molecular weights to those known to increase during tomato fruit ripening. By testing RNA from senescing leaves against a tomato fruit ripening-related cDNA library, seven cDNA clones were identified for mRNAs whose prevalence increased during both ripening and leaf senescence. Using dot hybridization, the pattern of expression of the mRNAs corresponding to the seven clones was examined. Maximal expression of the majority of the mRNAs coincided with the time of greatest ethylene production, in both leaves and fruit. Treatment of mature green leaves or unripe fruit with the ethylene antagonist silver thiosulphate prevented the onset of senescence or ripening, and the expression of five of the seven ripening- and senescence-related genes.The results indicate that senescence and ripening in tomato involve the expression of related genes, and that ethylene may be an important factor in controlling their expression.Abbreviations cDNA copy-DNA - MW molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

番茄果实成熟过程中钙调素含量变化及其与乙烯生成的关系

应用酶联免疫吸附法(ELISA)测定番茄(Lycopersicon esculentum Mill大红品种)果实成熟过程中钙调素(CaM)含量的变化。果实开始成熟(发白期),CaM含量随着呼吸跃变上升,成熟时(粉红期)达到最大,过熟衰老时则下降。果实内部乙烯浓度、ACC含量及其合成酶活性也随跃变而增加,随过熟衰老而降低。GaM含量在果实不同部位中的分布有明显差异,跃变上升期以子房腔组织含量最高,并由中心向外逐渐降低,外周果皮含量最低。此时用外源乙烯催熟处理促进各部位CaM增加。成熟衰老时子房腔组织首先衰老,CaM含量大为降低,但在中柱和果皮中却高于跃变上升期。外源乙烯促进衰老使CaM下降。Ca~(2+)促进番茄圆片CaM含量增高和乙烯产生,CaM抑制剂CPZ,TFP在降低CaM含量的同时也抑制乙烯的产生。     

Ripening of fleshy fruit: Molecular insight and the role of ethylene

Development and ripening in fruit is a unique phase in the life cycle of higher plants which encompasses several stages progressively such as fruit development, its maturation, ripening and finally senescence. During ripening phase, several physiological and biochemical changes take place through differential expression of various genes that are developmentally regulated. Expression and/or suppression of these genes contribute to various changes in the fruit that make it visually attractive and edible. However, in fleshy fruit massive losses accrue during post harvest handling of the fruit which may run into billions of dollars worldwide. This encouraged scientists to look for various ways to save these losses. Genetic engineering appears to be the most promising and cost effective means to prevent these losses. Most fleshy fruit ripen in the presence of ethylene and once ripening has been initiated proceeds uncontrollably. Ethylene evokes several responses during ripening through a signaling cascade and thousands of genes participate which not only sets in ripening but also responsible for its spoilage. Slowing down post ripening process in fleshy fruit has been the major focus of ripening-related research. In this review article, various developments that have taken place in the last decade with respect to identifying and altering the function of ripening-related genes have been described. Role of ethylene and ethylene-responsive genes in ripening of fleshy fruit is also included. Taking clues from the studies in tomato as a model fruit, few case studies are reviewed.