Methyl jasmonate deficiency alters cellular metabolome, including the aminome of tomato (Solanum lycopersicum L.) fruit

Exogenous treatment with jasmonates (JA) has been shown to reduce the levels of polyamines in many plants. But the role of endogenous JA on polyamine biosynthesis or other cellular metabolites has thus far remained uninvestigated. We developed transgenic tomato (Solanum lycopersicum L.) having severely reduced methyl JA levels by silencing a fruit ripening-associated lipoxygenase (LOX), SlLoxB, using a truncated LOX gene under the control of the constitutive CaMV35S promoter. The LOX suppressed and MeJA-deficient fruits had lowered polyamine levels. Thus, these transgenic fruits were used as a plant model to evaluate the effects of reduced endogenous MeJA on cellular metabolites in ripening tomato fruits using NMR spectroscopy. During on-shelf ripening, transgenic fruits were significantly reduced in the content of 19 out of 30 metabolites examined, including Ile, Val, Ala, Thr, Asn Tyr, Glu, Gln, His, Phe, Trp, GABA, citrate, succinate, myo-inositol, unidentified compound B, nucleic acid compound Nucl1, choline, and trigonelline as compared to the wild-type azygous counterparts. A significant increase in β-glucose levels in transgenic fruits was observed at the pink stage. The transgenic fruits were equivalent to the wild type in lycopene level and chlorophyll degradation rates. Taken together, these results show that intracellular MeJA significantly regulates overall primary metabolism, especially aminome (amino acids and polyamines) of ripening fruits.     

Uronic acid-containing oligosaccharins: Their biosynthesis,degradation and signalling roles in non-diseased plant tissues

Pectic oligosaccharides, produced by microbial enzymes, are well-known oligosaccharins, eliciting defence responses in diseased plants. Regulatory roles in non-diseased plants have also been proposed: e.g. oligosaccharides, generated in vitro by hydrolysis of pectins, promote ripening in tomato (Lycopersicon esculentum L.) and other fruits. Endo-polygalacturonase (endo-PG; EC 3.2.1.15) occurs in many fruits and theoretically could generate such oligosaccharides. However, the mere occurrence of an enzyme does not prove that it acts; the proposed products (oligosaccharides) must be sought in vivo. Recent evidence indicates that fruit tissues do naturally produce pectic oligosaccharides, some of which promote ripening when added to unripe fruits. Such studies, complementing earlier work on in-vitro generated oligosaccharides, provide a means of discovering novel oligosaccharins. Unlike fruits, cell cultures of rose (Rosa sp.) produce no pectic oligosaccharides. Their `absence' is not due to excessively rapid turnover: when [<sup>14</sup>C]-oligogalacturonides are added, they undergo only slow hydrolysis. The hydrolysis is by exo-polygalacturonases (exo-PG; EC 3.2.1.67), yielding free galacturonic acid (GalA). If no oligogalacturonides are added, no GalA accumulates in the medium; therefore, exo-PG does not normally operate in healthy rose cells. Exo-PG is presumably `in reserve', available to trim oligogalacturonides made by phytopathogens. We conclude that the in-vivo action of a wall enzyme is best studied at the level of carbohydrate metabolism in vivo rather than by assay of extracted enzymes in vitro. The major uronic acid-containing oligosaccharide produced by rose cell cultures is α-D-mannopyranosyl-(1→4)-α-D-glucuronopyranosyl-(1→2)-myo-inositol, which may be a novel phytoglycolipid-derived oligosaccharin related to the inositolphosphoglycans that mediate insulin action.     

Ethylene: role in fruit abscission and dehiscence processes

Two peaks of ethylene production occur during the development of cotton fruitz (Gossypium hirsutum L.). These periods precede the occurrence of young fruit shedding and mature fruit dehiscence, both of which are abscission phenomena and the latter is generally assumed to be part of the total ripening process. Detailed study of the dehiscence process revealed that ethylene production of individual, attached cotton fruits goes through a rising, cyclic pattern which reaches a maximum prior to dehiscence. With detached pecan fruits (Carya illinoensis [Wang.] K. Koch), ethylene production measured on alternate days rose above 1 microliter per kilogram fresh weight per hour before dehiscence began and reached a peak several days prior to complete dehiscence. Ethylene production by cotton and pecan fruits was measured just prior to dehiscence and then the internal concentration of the gas near the center of the fruit was determined. From these data a ratio of production rate to internal concentration was determined which allowed calculation of the approximate ethylene concentration in the intact fruit prior to dehiscence and selection of appropriate levels to apply to fruits. Ethylene at 10 microliters per liter of air appears to saturate dehiscence of cotton, pecan, and okra (Hibiscus esculentus L.) fruits and the process is completed in 3 to 4 days. In all cases some hastening of dehiscence was observed with as little as 0.1 microliter of exogenous ethylene per liter of air. The time required for response to different levels of ethylene was determined and compared to the time course of ethylene production and dehiscence. We concluded that internal levels of ethylene rose to dehiscence-stimulating levels a sufficience time before dehiscence for the gas to have initiated the process. Since our data and calculations indicate that enough ethylene is made a sufficient time before dehiscence, to account for the process, we propose that ethylene is one of the regulators of natural fruit dehiscence, an important component of ripening in some fruits. Our data also suggest a possible involvement of ethylene in young fruit abscission.     

Comparison of Propylene-induced Responses of Immature Fruit of Normal and rin Mutant Tomatoes

Continuous application of propylene to 40 to 80% mature fruits of normal tomato strains (Lycopersicon esculentum Mill.) advanced ripening in fruits of all ages by at least 50%. Although preclimacteric respiration was stimulated by propylene treatment, there was no concomitant increase in ethylene production. Once ripening commenced, the rates of endogenous ethylene production were similar in both propylene-treated and untreated fruits. Continuous exposure to propylene also stimulated respiration in immature fruits of rin, a nonripening mutant. Although respiration reached rates similar to those during the climacteric of comparable normal fruits there was no change in endogenous ethylene production which remained at a low level. Internal ethylene concentrations in attached 45 to 75% mature fruits of rin and a normal strain were similar. It is suggested that the onset of ripening in normal tomato fruit is not controlled by endogenous ethylene, although increased ethylene production is probably an integral part of the ripening processes.     

Evidence for changes in messenger RNA content related to tomato fruit ripening

Poly(A)-containing mRNA was purified from tomato fruits and translated in a wheat germ in vitro protein-synthesizing system. Comparison of the protein products produced in response to mRNA samples from unripe and ripening fruits provides evidence for changes in the amounts of mRNA coding for specific proteins during ripening.     

Partial Characterization of C(x) Cellulase and Cellobiase from Ripening Tomato Fruits

Cellulolytic enzymes were studied in extracts from the locular contents of ripening fruits of Lycopersicon esculentum var. KC-146. When acting on carboxymethyl cellulose, the enzyme preparations were capable of decreasing the viscosity of the reaction mixture and generating reducing groups, oligosaccharides, and glucose. Cellobiose cellotriose, cellotetrose, and cellopentose also served as substrates for glucose production.     

Physiological and molecular analyses of early and late Coffea arabica cultivars at different stages of fruit ripening

Coffee quality is strongly influenced by a great number of factors, among which the fruit ripening stage at harvest time has a major influence on this feature. Studies comprising ethylene production and the regulation of ethylene biosynthesis genes during the ripening process indicate that ethylene plays an important role on coffee fruit ripening. Coffee early cultivars usually show a more uniform ripening process although little is known about the genetic factors that promote the earliness of ripening. Thus, in order to better understand the physiological and genetic factors involved in the regulation of ripening time, and consequently ripening uniformity, this study aimed to analyze ethylene and respiration patterns during coffee ripening, as well as to analyze ACC oxidase, an ethylene biosynthesis enzyme, gene expression, in fruits of early (Catucaí 785-15) and late (Acauã) coffee cultivars. Coffee fruits were harvested monthly from 124 days after flowering (end of February), until complete maturation (end of June). Dry matter, moisture content, color, respiratory rate and ethylene production analysis were performed. In silico analysis identified a coffee ACC oxidase gene (CaACO-like) and its expression was analyzed by real-time PCR. Dry matter and relative water content constantly increased and gradually decreased, respectively, during fruit ripening, and the color analysis enabled the observation of the earliness in the ripening process displayed by Catucaí 785-15 and its higher fruit ripening uniformity. The results obtained from the CaACO-like expression analysis and respiration and ethylene analysis suggest that the differences in ripening behavior between the two coffee cultivars analyzed in this study may be related to the differences in their capacity to produce ethylene, with fruits of Catucaí 785-15 and Acauã showing a typical and an attenuated climacteric phase, respectively, which may have lead to differences in their ripening time and uniformity.     

Effect of prolonged root hypoxia on the antioxidant content of tomato fruit

Tomato fruit quality depends on its metabolite content, which in turn is determined by numerous metabolic changes occurring during fruit development and ripening. The aim of this work was to investigate whether flooding affects the nutritional quality of tomato fruit, focusing on compounds essential to human health: carotenoids and ascorbate. To this end, tomato plants (Solanum lycopersicum L. cv Micro-Tom) were submitted to prolonged root hypoxia (1–2% O₂) at first flower anthesis. Fruits were harvested at five stages of the ripening process and analysed for their carotenoid and ascorbate contents. Our results showed that the ripening of fruits that developed on hypoxia treated plants was not inhibited. However, root hypoxia significantly limits carotenoid and ascorbate accumulation in pericarp during fruit ripening, the strongest effects being observed at late stages of ripening. Limitation of both carotenoids and ascorbate accumulation seems to be primarily mediated by the reduced level of expression of genes of the corresponding metabolic pathway.     

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。     

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.     

Transplantation Studies with Immature Fruit of Normal, and rin and nor Mutant Tomatoes

The aim of the work reported herein was to determine whether the lack of normal ripening in fruits of rin and nor tomato mutants is due to the presence of ripening inhibitors or to the lack of ripening factors in the fruit. A fruit tissue transplantation technique was developed for this purpose.