Homologs of APETALA1/FRUITFULL in Solanum plants

The MADS-box APETALA1 genes control plant transition to flowering and the floral morphogenesis proper. The experimental evidence of APETALA1 overexpression presumes that this class of genes can also directly affect time to flowering. We therefore cloned and compared homologs of APETALA1 class genes from potato (Solanum tuberosum cultivars adapted to long day conditions) and its wild relative Solanum demissum, a short-day subtropical species. The homologs isolated from these plants belong to the subclass FRUITFULL. The inconsiderable variations in the primary structure of these homologs cannot explain the diverse photoperiodic reactions of particular Solanum genotypes.     

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.     

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。     

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.     

Fleshy Fruit Expansion and Ripening Are Regulated by the Tomato SHATTERPROOF Gene TAGL1

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.     

The Ripening of Tomato Fruit as Affected by the Injection of Certain Chemicals

Some chemical agents known to uncouple oxidation from phosphorylationin biological systems were injected into mature green tomatofruit. Limited amounts of the substances accelerated the ripeningof fruit left on the plants but had no effect on picked fruit.L-Methionine, regarded as having a coupling action on oxidativephosphorylation, appeared to lengthen the ripening period. Aswell as speeding up ripening, DNP and CPA were also shown toincrease the activity of polygalacturonase, but not pectinesterase,in unpicked tomato fruit. It is concluded that even when subjectto the action of uncoupling substances, production of enzymesprobably necessary for the furtherance of the ripening processcontinues. The methods by which this process in tomato fruitcould be maintained are examined, and the possibility is discussedthat ‘loose’ coupling is the mechanism by whichan energy source is provided for the endergonic cell processestaking place during ripening.     

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.     

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。     

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.     

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.     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c—a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-¹⁴C]putrescine and [terminal labeled-³H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.     

Early Application of Ethrel Extends Tomato Fruit Cell Division and Increases Fruit Size and Yield with Ripening Delay

Flowers of tomato (Lycopersicon esculentum Mill.) plants cv. Castle Rock were sprayed with 100 ppm of ethrel, 0.5 mm aminooxyacetic acid (AOA), or water (control) 2 days after anthesis. The fruit period of cell division was extended up to 16–18 days after anthesis with the application of ethrel but reduced from 10–12 days (control) down to only 6–8 days with the application of AOA. In a trend opposite to AOA application, fruits that received ethrel treatment were of higher ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) levels than control. This was noticed not only during the first 2 weeks after anthesis but also during the fruit climacteric phase. Mesocarp cells of ethrel-treated fruits were greater in number/mm² but smaller in size than control; an opposite trend was obtained with the application of AOA. This was observed for a period of 18 days after anthesis, but by that time or at earlier ages, fruits of AOA treatment were larger in size and heavier in weight than control, and both were larger and heavier than ethrel-treated ones. At 5 weeks after anthesis and thereafter, the fruit response to all treatments was totally reversed because early ethrel-treated fruits became significantly larger in size and heavier in weight with a ripening delay of about 10 and 15 days compared with those of control and AOA-treated ones, respectively. When the same treatments were applied to the whole plant, similar results were obtained because the early application of ethrel increased the fruit yield by about 15% over control with a pronounced ripening delay; an opposite trend was obtained with the application of AOA. No significant differences were found among all treatments in terms of flower or fruit abscission or fruit number/plant. The data suggest that ethylene regulates tomato fruit transmission from cell division to cell enlargement. In addition, fruit cell division is terminated only when endogenous ethylene decreases to its basal level, allowing cell enlargement to dominate and proceed as in the case of the early application of AOA. The ripening delay of ethrel-treated fruits may be caused by the longer time required for the increased cell number to reach maturation. A low level of ethrel application at the tomato early fruiting stage may be used for increasing fruit yield by increasing fruit size and consequently its quality. Received June 1, 1998; accepted December 7, 1998     

Changes in Structural Features of Free N-Glycan and Endoglycosidase Activity during Tomato Fruit Ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man_8-9GlcNAc₁, became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-β-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of α-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.     

Relationship between Chlorophylls and Carotenoids of Ripening Tomato Fruit as Influenced by Potassium Nutrition

Studies of fruit pigmentation at various stages of ripeningshowed that the chlorophylls generally decreased as the totalcarotenoids increase during ripening. At any stage of ripening,the carotenoid content of K-deficient fruit was lower than thatof normal fruit. The relationship between the two pigment systemswas altered by the K. status of the fruit. When the rate ofcarotenogenesis was low (green and red stages), the chlorophyllswere higher in high K-fruit than in K-deficient fruit. Whenthe rate of carotenoid synthesis was rapid (breaker to lightred stages), the chlorophyll level of K-deficient fruit washigher than that of normal fruit.