Effect of low oxygen and high carbon dioxide on the levels of ethylene and 1-aminocyclopropane-1-carboxylic acid in ripening apple fruits

The association of the level of ACC and the ethylene concentration in ripening apple fruit (Malus sylvestris Mill, var. Ben Davis) was studied. Preclimacteric apple contained small amounts of ACC and ethylene. With the onset of the climacteric and a concomitant decrease in flesh firmness, the level of ACC and ethylene concentration both increased markedly. During the postclimacteric period, ethylene concentration started to decline, but the level of ACC continued to increase. Ethylene production and loss of flesh firmness of fruits during ripening were greatly suppressed by treatments with low O₂ (O₂ 1–3%, CO₂ O%) or high CO₂ (CO₂ 20–30%, O₂ 15–20%) at the preclimacteric stage. However, after 4 weeks an accumulation of ACC was observed in treated fruits when control fruit was at the postclimacteric stage. Treatment of fruit with either low O₂ or high CO₂ at the climacteric stage resulted in a decrease of ethylene production. However, the ACC level in fruit treated with low O₂ was much higher than both control and high CO₂ treated fruit; it appears that low O₂ inhibits only the conversion of ACC to ethylene, resulting in an accumulation of ACC. Since CO₂ inhibits ethylene production but does not result in an accumulation of ACC, it appears that high CO₂ inhibits both the conversion of ACC to ethylene and the formation of ACC.     

Inheritance of the<Emphasis Type="Italic"> Md-ACS1</Emphasis> gene and its relationship to fruit softening in apple (<Emphasis Type="Italic">Malus</Emphasis> ×<Emphasis Type="Italic"> domestica</Emphasis> Borkh.)

The 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene is a member of the ACS gene family that is involved in apple (Malus × domestica Borkh.) fruit ripening. Presence of an allele (Md-ACS1-2) of this gene is associated with low internal ethylene concentration in some apple cultivars. In this study, inheritance of Md-ACS1 was determined for 50 apple cultivars/advanced selections and 101 F₁ seedlings from five populations. Following this, the softening pattern of apples stored at 20°C for up to 40 days was examined using 35 fruiting cultivars/selections of defined Md-ACS1 status. Md-ACS1 is inherited in a Mendelian fashion and was found to be linked to fruit softening. Maturity season of genotypes also significantly affected fruit softening. Late-season genotypes in the Md-ACS1-2/2 class had the slowest rate of softening, while early-season Md-ACS1-1/1 genotypes had the most rapid softening rate. The implications of these results are discussed in relation to parental selection and breeding for storage ability in apple.Communicated by H. Nybom     

Md-ACS1 and Md-ACO1 genotyping of apple (<Emphasis Type="Italic">Malus</Emphasis> x <Emphasis Type="Italic">domestica</Emphasis> Borkh.) breeding parents and suitability for marker-assisted selection

Fruit ethylene production genotypes for Md-ACS1 and Md-ACO1 were determined for 60 apple cultivars and 35 advanced breeding selections. Two alleles for each gene are commonly found in cultivated apple. Earlier studies showed that genotypes homozygous for the ACS1-2 allele produce less ethylene and have firmer fruit than ACS1-1/2 and ACS1-1/1 genotypes. ACO1 plays a minor role compared to ACS1, with homozygous ACO1-1 having lower ethylene production. In this study, ACS1-2 and ACO1-1 homozygotes had firmer fruit at harvest and after 60 days of 0–1°C cold storage compared to other genotypes. These genotypes, ACS1-2/2 and ACO1-1/1, were observed for the following 8 of 95 cultivars/selections: “Delblush”, “Fuji”, “Pacific Beauty”, “Sabina” and four breeding selections. Cultivars/selections that were homozygous ACS1-2 but not ACO1-1 were: “Ambrosia”, “Aurora Golden Gala”, “CrimsonCrisp”, “Gala”, “GoldRush”, “Huaguan”, “Pacific Rose, “Pacific Queen”, “Pinova”, “Sansa”, “Sonja”, “Sundance”, “Zestar”, and 17 breeding selections. Cultivars with the heterozygous ACS1-1/2 genotype were “Arlet”, “Braeburn”, “Cameo”, “Delicious”, “Delorgue”, “Empire”, “Enterprise”, “Ginger Gold”, “Golden Delicious”, “Granny Smith”, “Honeycrisp”, “Orin”, “Pink Lady”, “Silken”, “Suncrisp”, “Sundowner”, “Sunrise” and 11 breeding selections. No cultivars were detected homozygous for both ACS1-1 and ACO1-1, or for both ACS1-2 and ACO1-2. This study is the first large-scale allelic genotyping of both ethylene synthesis genes for a comprehensive set of apple breeding parents used in an ongoing breeding project. The data reported here are important for informative selection of parent combinations and marker-assisted selection of progeny for breeding low ethylene-producing apple cultivars for better storability and improved consumer acceptance.     

Apple 1-Aminocyclopropane-1-Carboxylic Acid Synthase Genes, MdACS1 and MdACS3a, are Expressed in Different Systems of Ethylene Biosynthesis

1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is one of the key regulatory enzymes involved in the synthesis of ethylene. Climacteric fruit ripening is accompanied by increased ethylene production, in which ethylene biosynthesis is changed from system 1 to system 2. In apple, at least four members of the ACS gene family have been identified, two of which, MdACS1 and MdACS3a, have been studied extensively due to their specific expression in fruit tissue. However, the regulatory role of MdACS1 and MdACS3a in the ethylene biosynthesis system is unknown. Here we addressed this issue by investigating ACS expression in ripening apple fruits. Expression analysis in ‘Golden Delicious’ and ‘Red Fuji’ fruits, in combination with treatments of 1-MCP (1-methylcyclopropene, an ethylene inhibitor) and Ethephon (an ethylene releaser) has demonstrated that MdACS3a and MdACS1operate in system 1 and system 2 ethylene biosynthesis, respectively.     

Genetic Diversity of Apple Cultivars Growing in Kazakhstan

Identification of the varieties is the primary requirement for characterization of the gene pool in agricultural production and implementation of breeding programs. In the present work, a set of six SSR markers was used for identification of cultural material collected on various horticultural farms in Kazakhstan: 30 varieties of Kazakhstani selection, 40 foreign varieties, and 16 Dzhangaliev’s apple clones selected in wild apple populations. Values of expected (H_e) and observed (H_o) heterozygosity in groups of all analyzed varieties were high: from 0.735 to 0.812 and from 0.661 to 0.721 respectively. Cluster analysis and analysis of genetic distances (STRUCTURE, UPGMA) showed a distribution of all samples into four major groups with a set of small subgroups caused by origin diversity. The first group included Kazakhstani varieties originating from Reneitte Burchardt, the second group included varieties with Aporta in parentage, the third group was represented by subclusters with a majority of foreign varieties, and the last group was composed of Dzhangaliev’s apple clones and Kazakhstani varieties with wild apple as their ancestor. Genotyping revealed inconsistencies in individual samples (and/or parentage) with claimed names. Analysis by markers of the Md-ACS1 and Md-ACO1 genes responsible for ethylene levels in fruits, which according to the literature correlates with fruit hardness and storability, did not reveal among Kazakhstani varieties any ACS1-2/2 homozygotes, the genotype with the highest expression of these traits. A quarter of Kazakhstani varieties and about a half of foreign varieties were heterozygous in ACS1, an indication of medium hardness and relatively long period of fruit storage. In two Kazakhstani and two foreign varieties heterozygous in ACS1, an improvement of the traits is possible owing to homozygosity in ACO1-1/1.     

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is β-cyanoalanine synthase (β-CAS). As little is known about the molecular function of β-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple β-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as β-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, β-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Influence of Plant Hormones on Ethylene Production in Apple, Tomato, and Avocado Slices during Maturation and Senescence

Ethylene production by tissue slices from preclimacteric, climacteric, and postclimacteric apples was significantly reduced by isopentenyl adenosine (IPA), and by mixtures of IPA and indoleacetic acid, and of IPA, indoleacetic acid, and gibberellic acid after 4 hours of incubation. Ethylene production by apple (Pyrus malus L.) slices in abscisic acid was increased in preclimacteric tissues, decreased in climacteric peak tissues, and little affected in postclimacteric tissues. Indoleacetic acid suppressed ethylene production in tissues from preclimacteric apples but stimulated ethylene production in late climacteric rise, climacteric, and postclimacteric tissue slices. Gibberellic acid had less influence in suppressing ethylene production in preclimacteric peak tissue, and little influenced the production in late climacteric rise, climacteric peak, and postclimacteric tissues. IPA also suppressed ethylene production in pre- and postclimacteric tissue of tomatoes (Lycopersicon esculentum) and avocados (Persea gratissima). If ethylene production in tissue slices of ripening fruits is an index of aging, then IPA would appear to retard aging in ripening fruit, just as other cytokinins appear to retard aging in senescent leaf tissue.     

Ethylene and fruit ripening

The latest advances in our understanding of the relationship between ethylene and fruit ripening are reviewed. Considerable progress has been made in the characterisation of genes encoding the key ethylene biosynthetic enzymes, ACC synthase (ACS) and ACC oxidase (ACO) and in the isolation of genes involved in the ethylene signal transduction pathway, particularly those encoding ethylene receptors ( ETR ). These have allowed the generation of transgenic fruit with reduced ethylene production and the identification of the Nr tomato ripening mutant as an ethylene receptor mutant. Through these tools, a clearer picture of the role of ethylene in fruit ripening is now emerging. In climacteric fruit, the transition to autocatalytic ethylene production appears to result from a series of events where developmentally regulated ACO and ACS gene expression initiates a rise in ethylene production, setting in motion the activation of autocatalytic ethylene production. Differential expression of ACS and ACO gene family members is probably involved in such a transition. Finally, we discuss evidence suggesting that the NR ethylene perception and transduction pathway is specific to a defined set of genes expressed in ripening climacteric fruit and that a distinct ETR pathway regulates other ethylene-regulated genes in both immature and ripening climacteric fruit as well as in non-climacteric fruit. The emerging picture is one where both ethylene-dependent and -independent pathways coexist in both climacteric and non-climacteric fruits. Further work is needed in order to dissect the molecular events involved in individual ripening processes and to understand the regulation of the expression of both ethylene-dependent and -independent genes.     

Growth and respiration patterns of snap bean fruits

The relationship of respiration and growth of seed, pericarp tissue and whole fruit of snap beans (Phaseolus vulgaris L.) was studied. The whole fruit exhibited an apparent climacteric type of respiration pattern. This pattern resulted from an increase in CO₂ production by the enlarging seed followed by a rapid decrease in CO₂ evolution by the pericarp tissue, and the pattern was not associated with any concomitant increase in ethylene production. Therefore, the apparent climacteric respiration pattern of a developing bean fruit is not comparable to the phenomenon that occurs in other ripening fruits.     

Ethylene production in relation to the initiation of respiratory climacteric in fruit

Respiration of cherimoya fruit shows two peaks of CO₂ production.The IEC is less than 0.05 ppm until the midpoint of the firstrespiratory rise. The fruit softens, develops pleasant aromaand flavor, and is considered ripe at the beginning of the secondrespiratory rise. Ripening and CO₂ production in cherimoya andavocado fruit are initiated with no increase of internal ethyleneand at ethylene levels which do not normally induce ripeningin fruit. The ratio of the internal concentration of ethylene to its productionrate varied with the stage of the climacteric and was less thanone except for the very early stage in cherimoyas. ¹Present address: Dept. of Plant Sciences, Kasetsart Univ.,Bangkok, Thailand. (Received February 6, 1975; )     

The Effect of Ethylene and Propylene Pulses on Respiration, Ripening Advancement, Ethylene-Forming Enzyme, and 1-Aminocyclopropane-1-carboxylic Acid Synthase Activity in Avocado Fruit

When early-season avocado fruit (Persea americana Mill. cv Hass) were treated with ethylene or propylene for 24 hours immediately on picking, the time to the onset of the respiratory climacteric, i.e. the lag period, remained unchanged compared with that in untreated fruit. When fruit were pulsed 24 hours after picking, on the other hand, the lag period was shortened. In both cases, however, a 24 hour ethylene or propylene pulse induced a transient increase in respiration, called the pulse-peak, unaccompanied by ethylene production (IL Eaks [1980] Am Soc Hortic Sci 105: 744-747). The pulse also caused a sharp rise in ethylene-forming enzyme activity in both cases, without any increase in the low level of 1-aminocyclopropane-1-carboxylic acid synthase activity. Thus, the shortening of the lag period by an ethylene pulse is not due to an effect of ethylene on either of the two key enzymes in ethylene biosynthesis. A comparison of two-dimensional polyacrylamide gel electrophoresis polypeptide profiles of in vitro translation products of poly(A⁺) mRNA from control and ethylene-pulsed fruit showed both up- and down-regulation in response to ethylene pulsing of a number of genes expressed during the ripening syndrome. It is proposed that the pulse-peak or its underlying events reflect an intrinsic element in the ripening process that in late-season or continuously ethylene-treated fruit may be subsumed in the overall climacteric response. A computerized system that allows continuous readout of multiple samples has established that the continued presentation of exogeneous ethylene or propylene to preclimacteric fruit elicits a dual respiration response comprising the merged pulse-peak and climacteric peak in series. The sequential removal of cores from a single fruit has proven an unsatisfactory sampling procedure inasmuch as coring induces wound ethylene, evokes a positive respiration response, and advances ripening.