Comparative transcriptomic analysis of plum fruit treated with 1-MCP

Microarray technology has allowed the large scale transcriptomic analysis of fruit ripening. The μPEACH1.0 microarray containing 4,806 probes corresponding to genes expressed in peach fruit tissues has been used in a heterologous fashion in two studies of plums ripening behavior. Gene expression of different cultivars of plums treated with the ethylene antagonist, 1-methylcyclopropene (1-MCP) and stored for short periods at room temperature or for longer periods of cold storage was examined. In the first study, mature fruit of a suppressed ethylene climacteric cultivar 'Shiro' and a cultivar characterized by a typical increase of ethylene production during ripening ('Santa Rosa') were harvested and incubated for 24h in air (control) or 1-MCP and allowed to ripen at room temperature. Different levels of transcripts of genes implicated in cell wall metabolism, hormone (ethylene and auxin) regulation, stress and defense, and in the transcription/translation machinery, as well as others involved with ripening were identified. In the second study, the effects of 1-MCP on gene expression in relation to the development of chilling injury (CI) in the climacteric cultivars 'Ruby Red' (RR) and 'October Sun' (OS) and 'Zee Lady' peaches (ZP) were analyzed. The fruit were treated for 24h at room temperature with 1-MCP prior to storage at 0°C. For RR, there was no significant effect of 1-MCP on the level of CI symptoms, while 1-MCP significantly reduced CI symptoms in OS fruit and an increase of CI in treated ZP fruit. Microarray analysis showed that immediately following treatment, 186, 134 and 56 genes were differentially expressed between the control and 1-MCP-treated fruit of these cultivars, respectively: after 4 weeks cold storage, 311, 52 and 224 genes for RR, OS and ZP, respectively, were differentially expressed between control and treated fruit. Thus, for OS, the number of differentially expressed genes reduced during storage while the number increased in RR and ZP. Comparisons of the data suggest that the transcript profile is altered by 1-MCP more in plums than peaches. These studies, carried out within an international collaborative network, will increase our understanding of the regulation of pathways involved in plum fruit ripening and in metabolic processes related to storage and shelf lif

Mechanism of action of VP1-001 in cryAB(R120G)-associated and age-related cataracts

PurposeWe previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.MethodsWe compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.ResultsDocking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.ConclusionsThe ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts

Detection of ripening-related proteins in stone fruit

The objective of this research was to identify ripening-related proteins in stone fruit that can be used as an index of ripening and whose expression is not affected by growing conditions. The initial research was conducted with Japanese type plums. Fruit were harvested at several stages during development, including optimum commercial harvest maturity, as determined by changes in skin colour, firmness, titratable acidity, soluble solids concentrations and storage life at 00C. Total proteins were extracted from the fruit and separated by 2-D polyacrylamide gel electrophoresis. Four proteins were detected that are synthesised a few days before ideal commercial maturity. Closely related proteins were also found in extracts of maturing peaches and nectarines. Monoclonal antibodies (mAbs) were raised against conjugated oligopeptides designed from amino acid sequence data from two of the proteins. However, the mAbs could not reliably detect the proteins either in protein extracts or juice samples from fresh fruit. Research is continuing on the properties of the ripening-related proteins that should enable the development of a highly sensitive immunological field test of maturity

Changes in total proteins related to chilling injury of nectarine

Low-temperature disorders in nectarines (Prunus persica) can be delayed by storage in atmospheres containing elevated CO₂; however, this benefit is limited to a few cultivars. The storage of produce at low temperatures itself leads to changes in protein synthesis; therefore, this study examined the applicability of 2-D electrophoresis to assess differences in protein production in cultivars of nectarine that either do ('Arctic Snow') or do not ('Red Gold') respond to elevated CO₂ levels during storage. In addition, the effect of harvest maturity and their response to added ethylene was assessed. Fruit of the two cultivars were either ripened immediately after harvest at 20°C before protein extraction or stored in air, air + CO₂, air + ethylene or air + CO₂ + ethylene then ripened at 20 °C before protein extraction. 2-D PAGE showed that the expression of approximately 30 proteins varied among cultivars and treatments. Two proteins showed clear differences in expression in both cultivars between fruit ripened immediately after harvest and those that had been stored. A second set of differences in protein expression was found in fruit of 'Red Gold' only and involved 12 proteins whose expression was either reduced or increased by storage: these proteins did not vary in expression in 'Arctic Snow'. A third set of differences in protein expression was observed in 'Arctic Snow' only in atmospheres containing ethylene. Several proteins were down-regulated by ethylene and this down-regulation was negated by the addition of CO₂ to the storage atmosphere. Thus, differences in protein expression can be found between cultivars of nectarines that do or do not respond to the addition of CO₂ to the storage atmosphere and in the cultivar that is known to respond to CO₂, this gas was able to antagonise protein changes mediated by ethylene

Application of two-dimensional gel electrophoresis to detect proteins associated with harvest maturity in stonefruit

The correct assessment of harvest maturity stage of stonefruit is an important determinant of their quality when they reach the consumer. However, the current indices of ripening used (e.g. skin colour, firmness and soluble solids concentration) vary with cultivar, rootstock, growing conditions and seasonal climatic factors. To develop an index that is independent of environmental factors, total proteins were extracted from fruit and separated by 2D-PAGE. Four proteins (Z1, Z2, Y and X) that were synthesised in fruit a few days before optimum harvest maturity were identified in Japanese-type plums (Prunus salicina), peaches and nectarines (Prunus persica), and European plums (Prunus domestica). These proteins are first expressed a few days before optimum harvest date and their expression increases as the fruit mature. The N-termini of the three most prominent proteins from plums (Z1, Z2 and Y) were sequenced. There appears to be close homology between these proteins and those from a range of species including almond, grape and rubber. It appears that these proteins belong to a family of allergens common to plants whose function may be to confer some form of protection to the plant during periods of stress. These proteins may be used for the development of a test kit that will assist growers to determine optimum harvest maturity for cultivars of plums, peaches and nectarines

Changes in ACC and conjugated ACC following controlled atmosphere storage of nectarine

Low temperature disorders of nectarines are thought to be expressions of chilling injury. Chilling injury is a form of stress usually associated with increased synthesis of ethylene and its immediate precursor, aminocyclopropane-1-carboxylic acid (ACC). However, other mechanisms for the development of chilling injury have been proposed. To help determine the nature of the processes leading to chilling injury in nectarines (Prunus persica) and how the gaseous composition of the storage atmosphere effects the development of low temperature disorders, levels of ACC and conjugated ACC were measured in fruit of the cv. Arctic Snow. These compounds were measured in fruit ripened at 20°C immediately after harvest, in fruit on removal from cold storage and in fruit ripened at 20°C following cold storage. During storage, fruit were kept at 0°C in the 4 following atmospheres: air; air + 15% CO2; air + 15 µL/L ethylene; and air + 15% CO2 + 15 µL/L ethylene. Concentrations of ACC remained low in all treatments and no significant changes in ACC levels due to added ethylene or CO2 were observed. Concentrations of conjugated ACC were about 10-times that of ACC and again were not influenced by the composition of the storage atmosphere. No significant changes in either ACC or conjugated ACC were observed until after flesh bleeding, the major symptoms of low temperature disorder expressed in these fruit, had begun to appear. It was concluded that disorders in nectarines stored at low temperatures are not a stress response involving a disruption of ethylene metabolism but may be associated with differential changes in the metabolism of enzymes associated with normal ripening

Improving the dessert quality of stone fruits

The major postharvest diseases and methods of control are briefly reviewed. Since the application of fungicides is being restricted worldwide, close attention must be given to handling practices that reduce the need for fungicide treatments. These include an initial wash with a hypochlorite solution, the addition of ethanol to hot water and the use of a warm iodine solution. The use of biological control agents has received considerable attention but none has achieved acceptable levels of control. Rapid advances in genomics, proteomics and metabolomics have enabled genes controlling quality traits and to derive molecular markers for others to be identified. Marker production is facilitated by the small genome size of members of the Rosaceae and the lack of major chromosomal rearrangements among species of Prunus. Peach has been selected as the reference species for Prunus and will be the first member of the Rosaceae to have its genome sequenced and a physical map of the genome is nearing completion. A Prunus microarray (PEACH1.0) based on 4806 unigenes is now available. These molecular approaches will allow genetic markers for traits that affect tree and fruit growth, susceptibility to pests and diseases, and dessert and storage qualities to be developed. The major challenge for physiologists is now to determine how to overcome the large variation in fruit composition and hence eating quality that occurs within trees and among batches from the same orchards

Root zone temperature influences growth, partitioning, leaf morphology and physiology of the peach rootstock, Green Leaf Nemaguard

This paper examines the hypothesis that root zone temperature (RZT) affects the growth of stone fruit plants. This hypothesis was tested by growing plants of the peach rootstock, Green Leaf Nemaguard (Prunus persica L. Batsch), at a diurnally variable (26/15°C) or at constant (5, 15, 20°C) RZTs; the plants were either actively growing or emerging from dormancy when the treatments were applied. These trials demonstrated that RZT, independently of air temperature and light intensity, influences growth, dry matter partitioning, leaf morphology and physiological processes. The growth of plants emerging from dormancy was more sensitive to RZT than that of actively growing plants, therefore, phenology can influence sensitivity to RZT. The area, numbers and daily rates of expansion of leaves, correlated positively with RZT for both sets of plants. However, plants exposed to a diurnally variable 26/15°C RZT were smaller with respect to overall growth and aspects of leaf morphology than plants exposed to a constant 20°C RZT, despite the daily mean RZTs for both treatments being similar. This could be due to supraoptimal RZTs during the day and/or suboptimal RZTs at night. Root mass ratio, in both actively growing and plants exiting dormancy was highest at 20° RZT. In contrast, the stem mass ratio of actively growing plants was maximised at 5°C, and for plants exiting dormancy, the stem mass ratio was minimised at this RZT. RZTs influence the rate of leaf expansion, which in turn affects the total number of leaves and leaf area and, along with its effect on CO2 assimilation rates, results in reductions in DM production. This research illustrates the importance of RZTs, particularly in the spring, on growth and leaf development and suggests the need to incorporate RZT into development models for peaches

Temperature and seed weight affect the germination of peach rootstock seeds and the growth of rootstock seedlings

Seeds of five cultivars of peach rootstocks were assessed to determine the effect of seed quality and temperature on germination and seedling growth. The weight of each seed and its surrounding endocarp was determined. Based on weight, the seeds were divided into four quartiles, stratified, and grown at 23 °C with a 16 h photoperiod. The percentage of seeds germinating over an 8-week period was then recorded. Seed weight significantly affected germination characteristics of the rootstock seeds. The final percentage, time and duration of germination correlated with quartile. In addition, relative growth rate during the first 3 weeks of growth and final plant height also correlated with quartile. Therefore, the heaviest seeds produced the most vigorous seedlings. In a second trial, seeds were grown at constant temperatures (10, 15, 20 or 25 °C) or under ambient conditions. For seeds germinated at 15–25 °C and ambient temperatures, there was no difference in either their speed or duration of germination. However, those placed at 10 °C were both slower to germinate and had a greater duration of germination. At constant temperatures, there was a trend for percentage germination to increase with temperature. However, the highest germination was found in seeds germinated at ambient temperatures that varied diurnally. For each individual cultivar, there was a strong correlation between seed size and the size of the endocarp or stone. Therefore, the grading of either stones or seeds on the basis of size or weight and the removal of the smallest individuals will help improve germination, seedling quality and uniformity

Variety and temperature affect the production of multiple stems from peach seedling rootstocks

Seeds of different cultivars of peach rootstocks were assessed to determine the causes of several deleterious traits that affect the quality of the seedlings that result from them. The incidence of triple cotyledons was significantly influenced by variety. It was also found that that the production of multiple stems could be caused by either the production of multiple seeds or by multiple stems originating from cotyledonary axils. The incidence of both traits was heavily influenced by the genotype of the variety. The production of multiple shoots was also affected by temperature with low temperatures favouring their appearance. The incidence of double seeds also varied among cultivars with the average size of seeds from stones that contained double seeds being significantly smaller than for single seeds. The smaller size of double seeds was associated with poorer germination, reduced speed of germination and reduced growth rates. Although there was a strong relationship between seed size and stone (endocarp) size for stones that contained single seeds, no such relationship existed for those stones that contained double seeds. Therefore, selection on the basis of stone size cannot be used to select out stones containing double kernels. The elimination of multiple shoots and double seeds from peach seedlings would reduce the costs associated with rootstock production and could be achieved by removing plants that exhibit these traits from the production system and clonally propagating those trees that are free of such traits