Tomato Fruit Development and Ripening Are Altered by the Silencing of LeEIN2 Gene

Loss-of-function ethylene insensitive 2 （EIN2） mutations showed ethylene insensitivity in Arabidopsis, which indicated an essential role of EIN2 in ethylene signaling. However, the function of EIN2 in fruit ripening has not been investigated. To gain a better understanding of EIN2, the temporal regulation of LeEIN2 expres- sion during tomato fruit development was analyzed. The expression of LeEIN2 was constant at different stages of fruit development, and was not regulated by ethylene. Moreover, LeEIN2-silenced tomato fruits were developed using a virus-induced gene silencing fruit system to study the role of LeEIN2 in tomato fruit ripening. Silenced fruits had a delay in fruit development and ripening, related to greatly descended expression of ethylene-related and ripening-related genes in comparison with those of control fruits. These results suggested LeEIN2 positively mediated ethylene signals during tomato development. In addition, there were fewer seeds and Iocules in the silenced fruit than those in the control fruit, like the phenotype of parthenocarpic tomato fruit. The content of auxin and the expression of auxin-regulated gene were declined in silenced fruit, which indicated that EIN2 might be important for crosstalk between ethylene and auxin hormones.     

Soaking Summer Squash Seeds in Low Concentrations of Cobalt Solution Before Sowing Increased Plant Growth, Femaleness, and Fruit Yield via Increasing Plant Ethylene Level

Soaking summer squash (Cucurbita pepo cv. Eskandarany) seeds in continuously aerated solutions of 0.25, 0.50, and 1.00 ppm Co²⁺ for 48 h before sowing strongly increased plant growth, femaleness, and fruit yield compared with those of water- (control) or 0.5 mm AOA (aminooxyacetic acid)-soaked seeds. Following the same pattern, plants of Co²⁺-soaked seeds produced significantly higher ethylene levels as early as the seedling stage (14 days after seed sowing) up to the onset of flower initiation (OFI) stage (30 days after seed sowing), with more pronounced levels of all measured parameters for plants of 1.00 ppm Co²⁺-soaked seeds. Plants of AOA-soaked seeds, however, behaved in a pattern similar to those of control in all measured parameters. The only exception was the significant ethylene inhibition noticed only at the plant seedling stage with AOA-seed soaking, which indicated the short term of AOA inhibition to ethylene when applied as early as the seed germination stage. When AOA was applied foliarly before and at the OFI stage, the increased plant femaleness obtained with Co²⁺ seed soaking was arrested. It is indicated, therefore, that summer squash plant femaleness is more responsive to plant ethylene-modulated levels before or at the OFI stage than earlier stages. Furthermore, all seed soaking treatments had no effect on plant leaf number or plant and fruit Co²⁺ content, which strongly indicated that the positive impact of Co²⁺ on summer squash plant growth and femaleness was mainly the result of the so-called ``low Co²⁺ level-induced ethylene.' The percentage of fruit yield increase reached about 26, 40, and 56% over the control by 0.25, 0.50, and 1.00 ppm Co²⁺ seed soaking, respectively, whereas AOA seed soaking resulted in only a 4.5% yield reduction. To ensure the permanent positive impact of Co²⁺ on ethylene production as well as the short period of AOA inhibition, seeds were resoaked in water or AOA for 8 h after being soaked in 1.00 ppm Co²⁺ solution for 40 h. The results obtained emphasized the previous findings because AOA inhibition was restricted on ethylene production only at the seedling stage. Meanwhile, all Co²⁺ positive effects were obtained 2 weeks later, even with AOA seed resoaking. These data strongly suggested that the positive impact of Co²⁺ on ethylene production is more permanent than the negative impact of AOA. Hence its application, in low concentrations, as early as the seed germination stage, strongly increased summer squash plant growth, femaleness, and fruit yield by increasing the plant ethylene level. Received July 21, 1997; accepted November 10, 1997     

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.     

Effects of fruit set sequence and defoliation on cell number,cell size and hormone levels of tomato fruits (Lycopersicon esculentum Mill.) within a truss

Fruit size within a tomato (Lycopersicon esculentum Mill.) truss depends on both fruit position in the truss and the time of pollination among fruits. In the natural pollination sequence a difference of 5 days in the pollination of proximal and distal flowers results in significant final size differences between proximal and distal fruits. These final size differences were eliminated when all flowers were pollinated simultaneously. At anthesis proximal ovaries have higher cell numbers than distal ovaries but the cell division activity and cell enlargement in both positions was similar in the first 10 days of fruit growth. Simultaneous pollination resulted in lower cell numbers in proximal but higher cell numbers in distal fruits compared to control fruits.Hormone levels in different sized fruits were measured using radioimmunoassays. Cytokinin concentration during the cell division period indicated a possible role in the regulation of cell division. With other hormones no obvious correlations were found. The results are discussed in relation to factors determining final fruit size in tomato.     

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.     

Endogenous Levels of Phenolics in Tomato Fruit during Growth and Maturation

Changes in the metabolism of several types of phenolics in the pulp and pericarp of tomato (Lycopersicon esculentum) fruit var. Ailsa Craig and Pik-Red were related to the stage of development. The highest levels of chlorogenic acid were found in the pulp and pericarp at the earliest stage of fruit development, and quantities declined rapidly during fruit ripening. Levels of rutin, found only in the pericarp, followed a similar pattern of change. The p-coumaric acid conjugate of rutin was found in low levels through fruit growth and ripening. High levels of p-coumaric acid glucoside were detected in the pulp only as the fruit matured with no rapid decline in levels during ripening. The decline of chlorogenic acid and rutin levels during fruit ripening paralleled the decline in indole-3-acetic acid levels measured previously in the pericarp tissues of these two varieties of tomato fruit during maturation. These phenolics are among those that have been suggested as regulants of auxin metabolism. Received April 30, 1996; accepted December 26, 1996     

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。     

Hormonal Regulation of Tomato Fruit Development: A Molecular Perspective

Fruit development is a complex yet tightly regulated process. The developing fruit undergoes phases of cell division and expansion followed by numerous metabolic changes leading to ripening. Plant hormones are known to affect many aspects of fruit growth and development. In addition to the five classic hormones (auxins, gibberellins, cytokinins, abscisic acid and ethylene) a few other growth regulators that play roles in fruit development are now gaining recognition. Exogenous application of various hormones to different stages of developing fruits and endogenous quantifications have highlighted their importance during fruit development. Information acquired through biochemical, genetic and molecular studies is now beginning to reveal the possible mode of hormonal regulation of fruit development at molecular levels. In the present article, we have reviewed studies revealing hormonal control of fruit development using tomato as a model system with emphasis on molecular genetics.     

通过表达ACC脱氨酶基因控制番茄果实的成熟

乙烯在跃变型果实的成熟过程中起着触发呼吸跃变和促进果实成熟的作用。细菌来源的1-氨基环丙烷-1-羧酸(ACC)脱氨酶能降解乙烯的直接前体ACC,从而抑制植物体内乙烯的合成。我们用PCR方法从假单孢杆菌中克隆到ACC脱氨酶基因并通过农杆菌介导的方法将其转入番茄(Lycopersicun esculentum)中。再生植株经Southern blot检测证明,ACC脱氨酶基因已整合到番茄基因组中并稳定表达。转基因番茄果实成熟期的推迟时间与体内乙烯的抑制程度有相关性。转基因番茄植株乙烯的合成降低80%左右,果实在离体条件下可保鲜75d左右。研究ACC脱氢酶基因在植物体内的作用可阐明高等植物体内乙烯的作用机理并为培育耐贮藏果蔬品种打下基础。     

A model for an early stage of tomato fruit development: cell multiplication and cessation of the cell proliferative activity

Changes in cell number during the early period of tomato fruit development were analysed by means of a deterministic model of cell multiplication. The period commenced at the seed stage with one theoretical cell undergoing intensive cell division, and ended when the cell number became nearly constant. The model takes into consideration the proliferative activity of the fruit cell population which, a few days before flower anthesis, begins to decrease progressively after each mitotic cycle. Model parameters, namely the time at which proliferative activity diminishes, its rate of decrease and the length of the cell cycle, were estimated by fitting the model to observed cell population dynamics in tomato fruits growing in three different positions on the truss. It is hypothesized that the molecular mechanism responsible for the cessation of mitosis in growing fruits is associated with shortening of telomeric ends of nuclear DNA, as suggested previously for other growing cell populations.     

Ethylene: Response of Fruit Dehiscence to CO(2) and Reduced Pressure

These studies were conducted to determine whether ethylene serves as a natural regulator of fruit wall dehiscence, a major visible feature of ripening in some fruits. We employed treatments to inhibit ethylene action or remove ethylene and observed their effect on fruit dehiscence. CO₂ (13%), a competitive inhibitor of ethylene action in many systems, readily delayed dehiscence of detached fruits of cotton (Gossypium hirsutum L.), pecan (Carya illinoensis [Wang.] K. Koch), and okra (Hibiscus esculentus L.). The CO₂ effect was duplicated by placing fruits under reduced pressure (200 millimeters mercury), to promote the escape of ethylene from the tissue. Dehiscence of detached fruits of these species as well as attached cotton fruits was delayed. The delay of dehiscence of cotton and okra by both treatments was achieved with fruit harvested at intervals from shortly after anthesis until shortly before natural dehiscence. Pecan fruits would not dehisce until approximately 1 month before natural dehiscence, and during that time, CO₂ and reduced pressure delayed dehiscence. CO₂ and ethylene were competitive in their effects on cotton fruit dehiscence. All of the results are compatible with a hypothetical role of ethylene as a natural regulator of dehiscence, a dominant aspect of ripening of cotton, pecan, and some other fruits.     

An Antisense Gene Stimulates Ethylene Hormone Production during Tomato Fruit Ripening

The ripening of many fruits is controlled by an increase in ethylene hormone concentration. E8 is a fruit ripening protein that is related to the enzyme that catalyzes the last step in the ethylene biosynthesis pathway, 1-aminocyclopropane-1-carboxylic (ACC) oxidase. To determine the function of E8, we have transformed tomato plants with an E8 antisense gene. We show here that the antisense gene inhibits the accumulation of E8 protein during ripening. Whereas others have shown that reduction of ACC oxidase results in reduced levels of ethylene biosynthesis, we find that reduction of the related E8 protein produces the opposite effect, an increase in ethylene evolution specifically during the ripening of detached fruit. Thus, E8 has a negative effect on ethylene production in fruit.     

Glucose Inhibits ACC Oxidase Activity and Ethylene Biosynthesis in Ripening Tomato Fruit

Experiments were carried out to evaluate the effect of glucose on ripening and ethylene biosynthesis in tomato fruit (Lycopersicon esculentum Mill.). Fruit at the light-red stage were vacuum infiltrated with glucose solutions post-harvest and changes in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC, ACC oxidase, and ethylene production monitored over time. ACC oxidase activity was also measured in pericarp discs from the same fruits that were treated either with glucose, fructose, mannose, or galactose. While control fruit displayed a typical peak of ethylene production, fruit treated with glucose did not. Glucose appeared to exert its effect on ethylene biosynthesis by suppressing ACC oxidase activity. Fructose, mannose, and galactose did not inhibit ACC oxidase activity in tomato pericarp discs. Glucose treatment inhibited ripening-associated colour development in whole fruit. The extent of inhibition of colour development was dependent upon the concentration of glucose. These results indicate that glucose may play an important role in ethylene-associated regulation of fruit ripening.     

Shortening the breeding cycle of papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.) by culturing embryos treated with ethrel

To shorten the breeding cycle of papaya by embryo culture, we attempted to improve the embryo quality (dimensions, germination ability, and/or seedling vigor) by treating fruits (attached to plants) with ethrel solution. Forty-five-, 60-, and 75-day-old fruits (DOF) were treated with 100 μl l⁻¹ ethrel for 10 or 20 days. The fruits treated with ethrel tended to be lighter in weight compared to those treated with water (the control). Seed coat coloration in 85 DOF (75 DOF plus 10 days of ethrel treatment [DET]) and 95 DOF (75 DOF plus 20 DET) was clearly promoted by the ethrel treatment. Embryo development was enhanced by ethrel treatments in 55 DOF (10 DET), i.e., the ethrel-treated embryos progressed to the mature cotyledon stage earlier than those in the control. The embryo size (length and width) was significantly larger in 55 DOF treated with ethrel than those of the control. In 55 and 65 DOF (20 DET), the embryo germination rates were higher in the ethrel-treated group than those in the control. The seedlings in the ethrel group grew more vigorously than those in the control. Therefore, we found that the embryo quality was improved by the ethrel treatment in 65–95 DOF. The breeding cycle of papaya could be shortened by embryo culture using embryos qualitatively improved by ethrel treatment, i.e., the period (generally 6–9 months) from pollination to seedling establishment could be shortened by approximately 3 months using these breeding techniques.