Stock-Scion Interactions of Normal and Fruit Ripening Mutants rin and nor in Tomato

Scions of the non-ripening rin and nor tomato strains (Lycopersicum esculentum Mill.) were grafted on normal understock plants (cv. Rutgers) in an effort to study the influence of roots and vegetative tissue on the ripening behavior of the tomato fruit. Receiprocal grafts of ‘Rutgers’ scions on rin and nor understocks as well as grafted and ungrafted controls were also established. No alteration in the ethylene, and CO₂ evolution and color development of either mutant fruits on normal understock or of normal fruits on mutant understock occurred. We suggest that the inability of rin and nor mutant fruits to ripen normally stems either from the presence in mutant fruit of a non-translocatable ripening inhibitor, or from the absence of a non-translocatable ripening factor.     

Water Permeability during Tomato Fruit Development in Normal and rin Nonripening Mutant

This work tested one aspect of the relations between membrane permeability and fruit ripening. Membrane permeability was measured as [³H]water efflux rate from preloaded fruit pericarp disks. Different stages of fruit development were compared between two tomato (Lycopersicon esculentum Mill) strains: the normal Rutgers and the isogenic nonripening rin strain. The first significant increase in permeability was measured in Rutgers tissue at 110% of development, after fruit ripening had already begun as indicated by ethylene and CO₂ evolution and lycopene synthesis. The rin did not show any increase in tissue permeability during fruit development or maturation.     

番茄果实成熟突变体的研究进展

番茄是一类比较重要的经济作物,具有很高的营养价值,长期以来番茄一直是研究肉质果实生长和成熟的模式生物。近年来,许多番茄果实成熟突变体的发现为研究果实成熟机制提供了重要的生物材料,综述了番茄果实成熟的影响因素、番茄果实成熟相关的突变体及基于突变体对果实成熟的相关研究,为今后突变体及果实成熟机制的研究提供参考。     

Photosynthetic Activity of Ripening Tomato Fruit

Gas exchanges, chlorophyll (Chl) a fluorescence and carboxylation activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and phosphoenolpyruvate carboxylase (PEPC) were determined in tomato (Lycopersicon esculentum Mill.) fruits picked at different developmental stages (immature, red-turning, mature, and over-ripe). The fruits did not show signs of CO₂ fixation. However, photochemical activity was detectable and an effective electron transport was observed, the values of Chl fluorescence parameters in green fruits being similar to those determined in the leaves. The RuBPCO activity, which was similar to those recorded in the leaves at the immature stage of the fruit, decreased as the fruit ripened. PEPC activity was always higher than RuBPCO activity.     

PG与番茄果实成熟的关系

系统比较了转多聚半乳糖醛酸酶（PG）反义基因和对照番茄果实成熟过程中绿熟、转色、粉顶、粉红、全红5个时期的PG活性和与其相关的生理、生化组分的动态变化。实验表明,转基因果与对照果相比,PG活性始终处于较低水平,PG活性强烈被抑制是在全红期;果实的硬度、贮藏寿命指数都高于对照果;番茄红素合成积累进程被延缓;可溶性果胶含量、电解质外渗百分率均低于对照果。外源乙烯刺激引起对照果PG活性剧增,而转基因果表现钝化。讨论了PG活性与果实成熟、耐贮性及软化的关系,及对外源乙烯刺激的敏感性。首次明确提出PG活性在对照果中极大地表达,在转基因果中强烈被抑制是在全红期 ,而不是在整个成熟期;PG活性在果实软化中起直接和重要作用;PG活性的高低与番茄红素的合成与积累有关。     

Effect of Heat Treatment on the Development of Polygalacturonase Activity in Tomato Fruit during Ripening

When mature green tomato fruits are stored at 22?C for 30 days,they ripen normally and soften, but if they are kept at 33?Cfor 15 days (heat treatment), then stored at 22?C they do notsoften. The effect of heat treatment on the development of polygalacturonase(PG, EC 3.2.1.15 [EC] ) activities in tomato fruits during storagetherefore was studied. When mature green tomato fruits werestored at 22?C, PG activity, which had not been detectable inthe fruits, appeared as the color changed and increased dramaticallythereafter. PG activity, however, did not appear during heattreatment. When heat-treated fruits were transferred to 22?C,PG activity appeared after a 6-day lag period and increasedduring the next 30 days at 22?C to about 15% of the value detectedin ripe tomato fruits. The PG in ripe tomato fruits was composed of two isoenzymesthat had different mol wts. A high molecular form (PG-1, molwt 100K) appeared during the early stage of ripening and a lowmolecular form (PG-2, mol wt 44K) a little later. PG-2 increasedvigorously during ripening and eventually accounted for mostof the enzyme activity in the ripe fruits. Only a single isoenzyme(Y-PG, mol wt 100K), however, was detected in heat-treated tomatofruits stored at 22?C for 30 days. PG-1 and Y-PG gave the samemol wt on Sephacryl S-200 gel nitration, but could be separatedby Toyopearl HW-55 F gel filtration. (Received October 31, 1983; Accepted February 20, 1984)     

PG与番茄果实成熟的关系

系统比较了转多聚半乳糖醛酸酶(PG)反义基因和对照番茄果实成熟过程中绿熟、转色、粉顶、粉红、全红5个时期的PG活性和与其相关的生理、生化组分的动态变化.实验表明,转基因果与对照果相比,PG活性始终处于较低水平,PG活性强烈被抑制是在全红期;果实的硬度、贮藏寿命指数都高于对照果;番茄红素合成积累进程被延缓;可溶性果胶含量、电解质外渗百分率均低于对照果.外源乙烯刺激引起对照果PG活性剧增,而转基因果表现钝化.讨论了PG活性与果实成熟、耐贮性及软化的关系,及对外源乙烯刺激的敏感性.首次明确提出PG活性在对照果中极大地表达,在转基因果中强烈被抑制是在全红期,而不是在整个成熟期;PG活性在果实软化中起直接和重要作用;PG活性的高低与番茄红素的合成与积累有关.     

Association between Elemental Content and Fruit Ripening in rin and Normal Tomatoes

Analysis of Ca and other inorganic ions in the pericarp of rin, a nonripening mutant, and normal tomato (Lycopersicon esculentum Mill) fruits revealed significant differences in their accumulations at advanced stages of fruit development. During early stages of fruit development, soluble Ca was higher in Rutgers and there were no detectable changes in the accumulation patterns of the other inorganic ions. In the mutant rin, bound Ca continued to increase with age and it was twice as high as compared to earlier stages. In the normal tomato, bound Ca decreased about 3-fold at later stages of development. Mg and Mn also showed some changes similar to Ca. K continued to increase with age and the mutant rin had lower levels than Rutgers throughout development. Other ions such as P, Zn, Cu, and Co were similar in the mutant and normal fruits. These results are interpreted as indicating that high levels of bound divalent cations in the mutant rin may be associated with an altered membrane and cell wall and play a role in fruit ripening.     

Reversible Inhibition of Tomato Fruit Gene Expression at High Temperature (Effects on Tomato Fruit Ripening)

The reversible inhibition of three ripening-related processes by high-temperature treatment (38[deg]C) was examined in tomato (Lycopersicon esculentum L. cv Daniella) fruit. Ethylene production, color development, and softening were inhibited during heating and recovered afterward, whether recovery took place at 20[deg]C or fruit were first held at chilling temperature (2[deg]C) after heating and then placed at 20[deg]C. Ethylene production and color development proceeded normally in heated fruit after 14 d of chilling, whereas the unheated fruit had delayed ethylene production and uneven color development. Levels of mRNA for 1-aminocyclopropane-1-carboxylic acid oxidase, phytoene synthase, and polygalacturonase decreased dramatically during the heat treatment but recovered afterward, whereas the mRNA for HSP17 increased during the high-temperature treatment and then decreased when fruit were removed from heat. As monitored by western blots, the HSP17 protein disappeared from fruit tissue after 3 d at 20[deg]C but remained when fruit were held at 2[deg]C. The persistence of heat-shock proteins at low temperature may be relevant to the protection against chilling injury provided by the heat treatment. Protein levels of 1-aminocyclopropane-1-carboxylic acid oxidase and polygalacturonase also did not closely follow the changes in their respective mRNAs. This implied both differences in relative stability and turnover rates of mRNA compared to protein and nontranslation of the message that accumulated in low temperature. The results suggest that high temperature inhibits ripening by inhibiting the accumulation of ripening-related mRNAs. Ripening processes that depend on continuous protein synthesis including ethylene production, lycopene accumulation, and cell-wall dissolution are thereby diminished.     

Polygalacturonase Isozymes and Pectin Depolymerization in Transgenic rin Tomato Fruit

We have previously described the construction and expression of a chimeric gene that allows developmentally regulated expression of tomato (Lycopersicon esculentum) polygalacturonase in ripening-impaired, mutant (rin) tomato fruit (JJ Giovannoni, D DellaPenna, AB Bennett, RL Fischer [1989] The Plant Cell 1: 53-63). We now show that expression of the chimeric polygalacturonase gene in rin tomato fruit resulted in the accumulation of all three polygalacturonase isozymes (PG1, PG2A, and PG2B). Polyuronide solubilization and polyuronide depolymerization both reached their maximal levels in transgenic rin fruit prior to the appearance of PG2 isozymes. These results demonstrate that PG1, PG2A, and PG2B all arise by differential processing of a single gene product and further suggest that the PG1 isozyme is sufficient to carry out both polyuronide solubilization and depolymerization in vivo.     

Effect of Antisense Suppression of Endopolygalacturonase Activity on Polyuronide Molecular Weight in Ripening Tomato Fruit and in Fruit Homogenates

Fruit of tomato (Lycopersicon esculentum Mill.) in which endopolygalacturonase (PG) activity had been suppressed to <1% of wild-type levels were slightly firmer than nontransgenic controls later in ripening. Enzymically inactive cell walls were prepared from these ripening fruit using Tris-buffered phenol. When extracted with chelator followed by Na2CO3, the amounts of pectin solubilized from cell walls of nontransgenic control or from transgenic antisense PG fruit were similar. Size-exclusion chromatography analysis showed that, relative to controls, in antisense PG fruit polyuronide depolymerization was delayed in the chelator-soluble fraction throughout ripening and reduced in the Na2CO3-soluble fraction at the overripe stage. Reduced pectin depolymerization rather than altered extractability thus may have contributed to enhanced fruit firmness. Substantially larger effects of suppressed PG activity were detected in tomato fruit homogenates processed to paste. In control paste the majority of the polyuronide was readily soluble in water and was very highly depolymerized. In antisense PG paste the proportion of polyuronide solubilized by water was reduced, and polyuronides retained a high degree of polymerization. The suppression of fruit PG activity thus has a small effect on polyuronide depolymerization in the fruit but a much larger effect in paste derived from these fruit. This indicates that in the cell wall PG-mediated degradation of polyuronide is normally restricted but that in tissue homogenates or in isolated cell walls this restriction is removed and extensive pectin disassembly results unless PG is inactivated.     

Degradation of Cell Wall Polysaccharides during Tomato Fruit Ripening

Changes in neutral sugar, uronic acid, and protein content of tomato (Lycopersicon esculentum Mill) cell walls during ripening were characterized. The only components to decline in amount were galactose, arabinose, and galacturonic acid. Isolated cell walls of ripening fruit contained a water-soluble polyuronide, possibly a product of in vivo polygalacturonase action. This polyuronide and the one obtained by incubating walls from mature green fruit with tomato polygalacturonase contained relatively much less neutral sugar than did intact cell walls. The ripening-related decline in galactose and arabinose content appeared to be separate from polyuronide solubilization. In the rin mutant, the postharvest loss of these neutral sugars occurred in the absence of polygalacturonase and polyuronide solubilization. The enzyme(s) responsible for the removal of galactose and arabinose was not identified; a tomato cell wall polysaccharide containing galactose and arabinose (6:1) was not hydrolyzed by tomato β-galactosidase.     

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.     

Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant.     

Differential Expression of the Two Subunits of Tomato Polygalacturonase Isoenzyme 1 in Wild-Type and rin Tomato Fruit

The [beta] subunit of tomato (Lycopersicon esculentum Mill.) fruit polygalacturonase 1 is a cell wall glycoprotein that binds to and apparently regulates the catalytic PG2 polypeptide in vivo. [beta] Subunit and polygalacturonase 2 (PG2) expression have been investigated in both wild-type and ripening inhibitor (rin) mutant fruit. During fruit development and ripening, [beta] subunit expression was unrelated to expression of the catalytic PG2 protein. In wild-type fruit, [beta] subunit mRNA and protein were first detected early in development and increased to maximal levels before PG2 mRNA and protein were detected. At the onset of ripening [beta] subunit mRNA decreased dramatically, but [beta] subunit protein levels remained stable. In rin fruit, which fail to ripen, [beta] subunit expression was similar to that in wild type, although PG2 mRNA and protein were not detected. These data suggest that [beta] subunit expression is ethylene independent and regulated primarily by developmental cues. This conclusion is supported by results from ethylene-treated immature (20 days after pollination) wild-type and rin fruit in which no significant differences were observed in [beta] subunit expression patterns in response to ethylene treatment. Surprisingly, RNA blot analysis indicated that catalytic PG2 mRNA was induced in immature rin fruit after 3 d of exogenous ethylene treatment. In addition, [beta] subunit mRNA and protein were also detected at lower levels in root, leaf, and flower tissues of both genotypes, suggesting a broader functional role for the protein.     

CNR转录因子在番茄果实成熟过程中的功能

SPL转录因子在植物中广泛存在并参与植物生长、发育和成熟过程,CNR是SPL转录因子家族中的一个成员,其作用机制尚不清楚.通过RNA-seq、qRT-PCR和染色质免疫共沉淀技术(ChIP)确定转录因子CNR直接作用的新的靶基因,旨在揭示番茄果实成熟过程中CNR的转录调控网络.通过RNA-seq筛选出野生型AC和突变体...     

Carboxypeptidase Activity of Tomato Fruit during the Ripening Process and Some Enzymatic Properties

Carboxypeptidase activity of tomato fruit reached a maximum at an early period of ripening. During storage of the fruit at 25°C, the enzyme activity decreased, accompanied by a fall of the pH value of the sap.

The enzyme was apparently localized in the soluble fraction, as determined by differential centrifugation.

The enzyme was optimally active at pH 5.0 ~ 5.5, was most stable at pH 4.5 ~ 6.5, and was strongly inhibited by DFP and HgCl₂, but not by EDTA and 1,10-phenanthroIine. Z-dipeptides containing arginine, proline and several neutral amino acids were hydrolyzed by the enzyme.

The similarity of the enzymatic properties of the present enzyme to those of other plant carboxypeptidases and pig kidney cathepsin A is also discussed.