Structure and expression of an ethylene-related mRNA from tomato.

Messenger RNAs homologous to a cDNA clone (pTOM 13) derived from a ripe-tomato-specific cDNA library are expressed during tomato fruit ripening and after the wounding of leaf and green fruit material. Both responses involve the synthesis of the hormone ethylene. Accumulation of the pTOM 13--homologous RNA during ripening is rapid and sustained, and reaches its maximum level in orange fruit. Following mechanical wounding of tomato leaves a pTOM 13--homologous RNA shows rapid induction within 30 minutes, which occurs before maximal ethylene evolution (2-3 h). This RNA also accumulates following the wounding of green tomato fruit. Northern blot analysis of poly(A)+ RNA indicates that the length of the mRNA is about 1400 nucleotides. Nucleotide sequence analysis showed the cDNA insert to contain the complete coding region of the pTOM 13 protein (33.5 kD) and an unusual 5' structure of ten dT-nucleotides. Hybridisation of the pTOM 13 cDNA insert to Southern blots of tomato DNA indicates the presence of only a small number of homologous sequences in the tomato genome.     

Mechanical wounding and abscission in citrus

Fruit detachment force (FDF), ethylene evolution, fruit and leaf drop were determined in Citrus sinensis for periods up to 96 h after mechanical wounding. Injury by removing a thin section of mature fruit flavedo reduced FDF, increased ethylene evolution and promoted abscission. Injuring flavedo 1 cm below the calyx was more effective at reducing FDF than injuring flavedo at the equator or the blossom‐end of mature fruit. Injuring the calyx or peduncle of mature fruit, or injuring three leaves closest to the mature fruit did not reduce FDF. Immature fruitlets either did not abscise or underwent low rates of abscission in response to mechanical wounding, depending on age. Inhibiting ethylene binding in wounded mature fruit with 1‐methylcyclopropene (1‐MCP) increased ethylene evolution compared with wounded fruit alone, but the reduction in FDF was similar. When an ethylene biosynthesis inhibitor (aminoethoxyvinylglycine, AVG) was used, reduction in FDF of wounded mature fruit exposed to AVG was similar to that of wounded fruit alone but ethylene production was markedly reduced. Wounding mature leaf blades in the presence or absence of 1‐MCP resulted in elevated but equal ethylene evolution up to 48 h after wounding, however, no leaf drop occurred. Thereafter, ethylene evolution was higher in 1‐MCP‐treated wounded leaves. Removing up to 77% of the total mature leaf area did not cause leaf drop, nor did wounding tissue across the laminar or petiolar abscission zones. Leaflets of 5 mm length reached nearly 100% abscission after mechanical wounding, whereas wounding leaves 20 mm length resulted in 15% abscission. The data suggest that mechanical wounding of flavedo results in mature fruit abscission, and ethylene binding may not be mandatory to initiate abscission in citrus fruit. The differential response of fruit and leaves at different ages to wounding may be related to potential contribution to carbohydrate accumulation, and production and sensitivity of tissues to an abscission signal(s).     

The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, in peach fruit softening

Ethylene promotes fruit ripening, including softening. The fruit of melting-flesh peach (Prunus persica (L). Batsch) cultivar 'Akatsuki' produces increasing levels of ethylene, and the flesh firmness softens rapidly during the ripening stage. On the other hand, the fruit of stony hard peach cultivars 'Yumyeong', 'Odoroki', and 'Manami' does not soften and produces little ethylene during fruit ripening and storage. To clarify the mechanism of suppression of ethylene production in stony hard peaches, the expression patterns of four ethylene biosynthesis enzymes were examined: ACC synthases (Pp-ACS1, Pp-ACS2, and Pp-ACS3) and ACC oxidase (Pp-ACO1). In the melting-flesh cultivar 'Akatsuki', Pp-ACS1 mRNA was dramatically induced after harvesting, and a large amount of ethylene was produced. On the other hand, in stony hard peaches, Pp-ACS1 mRNA was not induced during the ripening stage, and ethylene production was inhibited. Since Pp-ACS1 mRNA was induced normally in senescing flowers, wounded leaves, and wounded immature fruit of 'Yumyeong', Pp-ACS1 was suppressed only at the ripening stage, and was not a defect in Pp-ACS1. These results indicate that the suppression of fruit softening in stony hard peach cultivars was caused by a low level of ethylene production, which depends on the suppressed expression of Pp-ACS1.     

An ethylene-related cDNA from ripening apples

We report the isolation of a ripening-related apple cDNA which is complementary to a mRNA which may be involved in ethylene production. Poly(A)⁺ RNA was extracted from cortical tissue of ripe apple fruit (Malus domestica Borkh cv. Golden Delicious) and a cDNA library constructed in the plasmid vector pSPORT. The library was screened with pTOM13, a tomato cDNA clone thought to code for ACC oxidase in that fruit. An apple cDNA clone (pAP4) was isolated and sequenced. The 1182 bp cDNA insert includes an open reading frame of 942 bp, and shows strong homology with reported tomato and avocado sequences, both at the nucleic acid and amino acid levels. The polypeptide has a calculated molecular mass of 35.4 kDa and a calculated pI of 5.15. In apple cortical tissue, expression of pAP4-complementary RNA increased with ethylene production by the fruit during ripening. Expression was also enhanced in both ethylene-treated and wounded fruit.     

The pch2Δ Mutation in Baker's Yeast Alters Meiotic Crossover Levels and Confers a Defect in Crossover Interference

Pch2 is a widely conserved protein that is required in baker''s yeast for the organization of meiotic chromosome axes into specific domains. We provide four lines of evidence suggesting that it regulates the formation and distribution of crossover events required to promote chromosome segregation at Meiosis I. First, pch2Δ mutants display wild-type crossover levels on a small (III) chromosome, but increased levels on larger (VII, VIII, XV) chromosomes. Second, pch2Δ mutants show defects in crossover interference. Third, crossovers observed in pch2Δ require both Msh4-Msh5 and Mms4-Mus81 functions. Lastly, the pch2Δ mutation decreases spore viability and disrupts crossover interference in spo11 hypomorph strains that have reduced levels of meiosis-induced double-strand breaks. Based on these and previous observations, we propose a model in which Pch2 functions at an early step in crossover control to ensure that every homolog pair receives an obligate crossover.     

Organisation and expression of a wound/ripening-related small multigene family from tomato

cDNA clones derived from a ripe tomato fruit cDNA library were used to investigate changes in the abundance of specific mRNAs in ripening fruit and wounded leaves. mRNAs related to one cDNA clone (pTOM 13) were expressed in both situations. This clone was used to identify homologous sequences in a tomato genomic library. Three groups of related clones that hybridised to the pTOM 13 cDNA insert were identified and subcloned into plasmid vectors. Genomic Southern analysis of tomato DNA using gene-specific DNA fragments isolated from the subcloned DNAs indicated that all pTOM 13 closely related genes had been isolated. RNA dot blot analysis with these DNA fragments as probes indicated differential expression of this small multigene family in leaves and fruit.     

The pch2Δ Mutation in Baker's Yeast Alters Meiotic Crossover Levels and Confers a Defect in Crossover Interference

Pch2 is a widely conserved protein that is required in baker's yeast for the organization of meiotic chromosome axes into specific domains. We provide four lines of evidence suggesting that it regulates the formation and distribution of crossover events required to promote chromosome segregation at Meiosis I. First, pch2Δ mutants display wild-type crossover levels on a small (III) chromosome, but increased levels on larger (VII, VIII, XV) chromosomes. Second, pch2Δ mutants show defects in crossover interference. Third, crossovers observed in pch2Δ require both Msh4-Msh5 and Mms4-Mus81 functions. Lastly, the pch2Δ mutation decreases spore viability and disrupts crossover interference in spo11 hypomorph strains that have reduced levels of meiosis-induced double-strand breaks. Based on these and previous observations, we propose a model in which Pch2 functions at an early step in crossover control to ensure that every homolog pair receives an obligate crossover.     

Postharvest response of avocado fruits of different maturity to delayed ethylene treatments

The effect of 10, 100, 1000, 10,000 ppm of ethylene, applied for 3, 6, 12, and 24 hours, on the ripening rate of “Hass” avocado (Persea americana Mill.) fruits at three stages of maturity was investigated. Ethylene treatments were started either immediately after picking or 2 days later. A sharp peak of ethylene production was found to precede full softening by about 2 days and the occurrence of this peak was used to determine ripening rate. Hastening of fruit ripening was much more marked following ethylene treatment which began 2 days after harvesting than immediately after. The difference in response diminished gradually as the fruit became more mature.     

Wound ethylene and 1-aminocyclopropane-1-carboxylate synthase in ripening tomato fruit

Ethylene production, 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ACC-synthase activity were compared in intact and wounded tomato fruits (Lycopersicon esculentum Mill.) at different ripening stages. Freshly cut and wounded pericarp discs produced relatively little ethylene and had low levels of ACC and of ACC-synthase activity. The rate of ethylene synthesis, the level of ACC and the activity of ACC synthase all increased manyfold within 2 h after wounding. The rate of wound-ethylene formation and the activity of wound-induced ACC synthase were positively correlated with the rate of ethylene production in the intact fruit. When pericarp discs were incubated overnight, wound ethylene synthesis subsided, but the activity of ACC synthase remained high, and ACC accumulated, especially in discs from ripe fruits. In freshly harvested tomato fruits, the level of ACC and the activity of ACC synthase were higher in the inside parts of the fruit than in the pericarp. When wounded pericarp tissue of green tomato fruits was treated with cycloheximide, the activity of ACC synthase declined with an apparent half life of 30–40 in. The activity of ACC synthase in cycloheximide-treated, wounded pericarp of ripening tomatoes declined more slowly.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Inhibition of Biosynthesis of Polyisoprenol Sugars in Chick Embryo Microsomes by Tunicamycin

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-¹³C] and [2-¹³C]cinnamic acids, and [2-¹³C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4′-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.     

Changes in the content and biosynthesis of phytoalexins in banana fruit

Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1-2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-(13)C] and [2-(13)C]cinnamic acids, and [2-(13)C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4'-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.     

香蕉果实XET cDNA的克隆及其在成熟软化的果实果皮和果肉中的表达分析

木葡聚糖内糖基转移酶(Xyloglucan endotransglycosylase,XET)通过分解细胞壁半纤维素多糖的主要成分--木葡聚糖而参与果实软化.为了阐明香蕉(Musa acuminata.Colla cv.GrandNain)果实成熟过程中的软化与细胞壁代谢酶XET基因表达模式的关系,采用RT-PCR和RACE-PCR方法,首次从成熟香蕉果实果肉中分离了编码XT基因的全长cDNA(MA-XET1,全长1 095 bp).序列分析表明,MA-XET1的5'端和3'端的非翻译区分别为66 bp和1 89bp,该片段含有一个完整的开放读码框,编码280个氨基酸,推导的MA-XET1蛋白质中存在XET蛋白的催化活性部位DEIDFEFL.Southern杂交表明,MA-XET1在香蕉基因组中由多拷贝基因编码.Northern分析显示,跃变前期的果肉中,不能检测MA-XET1基因的表达,跃变期的果实果肉中MA-XET1表达增加,跃变后期该基因表达略有减弱;在跃变前期的果实果皮中,MA-XET1的积累较低,跃变期的果实果皮中积累大幅增加,而后迅速下降.Propylene(丙烯,乙烯的类似物)处理降低香蕉果实果皮和果肉的硬度,而且propylene促进MA-XET1在果皮和果肉中的积累.这些结果表明,MA-XET1参与香蕉果实成熟过程中的果皮和果肉软化,并且,MA-XET1的表达在转录水平上受乙烯调控.     

Ultrastructural changes in the cell walls of ripening apple and pear fruit

Ultrastructural changes in the cell walls of “Calville de San Sauveur” apples (Malus sylvestris Mill) and “Spadona” pear (Pyrus communis L.) fruit were followed during ripening. In apple, structural alterations in cell walls became apparent at advanced stages of softening and showed predominantly dissolution of the middle lamella. In pears softening was also associated with the dissolution of the middle lamella, and in addition a gradual disintegration of fibrillar material throughout the cell wall. In fully ripe fruit almost all of the fibrillar arrangement in the cell wall was lost. Application of enzyme solutions containing polygalacturonase and cellulase to tissue discs from firm pear fruit led to ultrastructural changes observed in naturally ripening pears. In apple polygalacturonase alone was sufficient to dissolve the middle lamella region of the cell walls, as was also found to occur in naturally ripening fruit. In both apple and pear the cell wall areas containing plasmodesmata maintained their structural integrity throughout the ripening process. At advanced stages of ripening vesicles appeared in the vicinity of plasmodesmata.