Jingping Rao

Wax composition of ‘Red Fuji’ apple fruit during development and during storage after 1-methylcyclopropene treatment

Changes in surface wax composition of ‘Red Fuji’ apple (Malus domestica Borkh. ‘Red Fuji’) during development and during storage at 0 ± 1°C after 1-methylcyclopropene (1-MCP) treatments were studied by the means of gas chromatography -mass spectrometry. Total wax increased during fruit development. During the storage at 0 ± 1°C, total waxes declined, and the decline was significantly delayed in 1-MCP-treated fruit. Total waxes were chromatographically separated into nonpolar and polar components. Nonacosane was the most abundant nonpolar wax, comprising 95% of total hydrocarbons. Polar wax components were comprised of nonacosan-10-ol (29%) and nonacosan-10-one (16%), along with a series of fatty acids and derivatives. Distinct patterns of wax changes were observed. Nonacosane, heptacosane and nonacosene increased during the development and decreased over seven months of fruit storage at 0 ± 1°C. Declines were delayed or slightly suppressed in 1-MCP-treated fruit. By contrast, nonacosan-10-ol, nonacosan-10-one, and 9, 12-octadecadienoic acid showed variable accumulation trends during the development but significant increases during storage that were strongly suppressed in 1-MCP-treated fruit. The association of wax changes with ethylene production and the responses to 1-MCP indicate that ethylene strongly influences wax composition in ‘Red Fuji’ apple fruit.

Low-temperature conditioning induces chilling tolerance in 'Hayward' kiwifruit by enhancing antioxidant enzyme activity and regulating en-dogenous hormones levels.

BACKGROUND To understand the mechanisms leading to the enhanced chilling tolerance of kiwifruit by low-temperature conditioning (LTC, 12 °C for 3 days), this study investigated the effect of LTC on chilling tolerance and changes in antioxidant enzyme activities and endogenous hormones. RESULTS LTC significantly alleviated chilling injury in kiwifruit. Fruits treated with LTC maintained lower respiration and ethylene production and higher firmness. Furthermore, this treatment inhibited the accumulation of malondialdehyde, superoxide radicals and hydrogen peroxide and the increase in membrane permeability and increased the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase under chilling stress. The treatment also maintained higher levels of endogenous abscisic acid (ABA), indole-3-acetic acid (IAA) and zeatin riboside (ZR), lower gibberellic acid (GA3) levels and higher ABA/GA3 and ABA/IAA ratios. CONCLUSION The results suggested that LTC alleviated chilling injury in kiwifruit by improving antioxidant enzyme activities and maintaining higher levels of endogenous ABA, IAA and ZR, lower GA3 levels and higher ABA/GA3 and ABA/IAA ratios.

Analysis of Xyloglucan Endotransglycosylase/Hydrolase (XTH) Genes and Diverse Roles of Isoenzymes during Persimmon Fruit Development and Postharvest Softening

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes have played a role in the remodeling of cell wall hemicelluloses. To investigate the function of XTHs in persimmon (Diospyros kaki L.) fruit development and postharvest softening, five cDNAs (DkXTH1 to DkXTH5), whose putative proteins contained the conserved DEIDFEFLG motif of XTH, were cloned. Real time quantitative PCR analysis revealed that DkXTH1, DkXTH4, and DkXTH5 peaked in immature expanding fruit, and their higher expression was observed along with higher fruit firmness in cold-treated fruit or firmer cultivar fruit during storage. The opposite gene expression patterns were observed in DkXTH2 and DkXTH3, which reached maxima concomitance with pronounced fruit softening. Meanwhile, the xyloglucan endotransglycosylase (XET) enzymes play important roles in both the rapid growth and ripening of persimmon fruit. Furthermore, the recombined DkXTH1 and DkXTH2 proteins showed significant XET activity without any detected XEH activity. However, the XET activity of recombined DkXTH2 protein had a higher affinity for small acceptor molecules than that of recombined DkXTH1 protein. The former might prefer to participate in cell wall restructuring, and the latter is more inclined to participate in cell wall assembly. Besides, DKXTH proteins could function by targeting to the cell wall under regulation of a signal peptide. The data suggested that individual DKXTHs could exhibit different patterns of expression, and the encoded products possessed specific enzymatic properties conferring on their respective functions in growth and postharvest softening of persimmon fruit.

The Persimmon 9-lipoxygenase Gene DkLOX3 Plays Positive Roles in Both Promoting Senescence and Enhancing Tolerance to Abiotic Stress.

The lipoxygenase (LOX) pathway is a key regulator for lipid peroxidation, which is crucial for plant senescence and defense pathways. In this study, the transcriptional expression patterns of three persimmon (Diospyros kaki L. ‘Fupingjianshi’) 9-lipoxygenase genes (DkLOX1, DkLOX3, and DkLOX4) were investigated. DkLOX1 was specifically expressed in fruit, particularly in young fruit, and showed little response to the postharvest environments. DkLOX4 was expressed in all tissues and slightly stimulated by mechanical damage and low temperature. DkLOX3 was expressed mainly in mature fruit, and the expression was extremely high throughout the storage period, apparently up-regulated by mechanical damage and high carbon dioxide treatments. Further functional analysis showed that overexpression of DkLOX3 in tomato (Solanum lycopersicum cv. Micro-Tom) accelerated fruit ripening and softening. This was accompanied by higher malondialdehyde (MDA) content and lycopene accumulation, advanced ethylene release peak and elevated expression of ethylene synthesis genes, including ACS2, ACO1, and ACO3. In addition, DkLOX3 overexpression promoted dark induced transgenic Arabidopsis leaf senescence with more chlorophyll loss, increased electrolyte leakage and MDA content. Furthermore, the functions of DkLOX3 in response to abiotic stresses, including osmotic stress, high salinity and drought were investigated. Arabidopsis DkLOX3 overexpression (DkLOX3-OX) transgenic lines were found to be more tolerant to osmotic stress with higher germination rate and root growth than wild-type. Moreover, DkLOX3-OX Arabidopsis plants also exhibited enhanced resistance to high salinity and drought, with similar decreased O2- and H2O2 accumulation and upregulation of stress-responsive genes expression, including RD22, RD29A, RD29B, and NCED3, except for FRY1, which plays a negative role in stress response. Overall, these results suggested that DkLOX3 plays positive roles both in promoting ripening and senescence through lipid peroxidation and accelerated ethylene production and in stress response via regulating reactive oxygen species accumulation and stress responsive genes expression.

ANTIOXIDANT ENZYME ACTIVITY AND CHILLING INJURY DURING LOW-TEMPERATURE STORAGE OF KIWIFRUIT CV. HONGYANG EXPOSED TO GRADUAL POSTHARVEST COOLING

Kiwifruits (Actinidia chinensis cv. Hongyang) were treated by direct cooling and gradual cooling to investigate the effect of cooling treatment on chilling injury. The direct cooling fruits were immediately cooled at 0°C after harvest. The gradual cooling fruits were held for 3 days at 5 °C (from 5 °C to 0°C), or for 7 days at 2°C (from 2°C to 0°C), or decreased in temperature from 15°C to 5°C by 5°C at 1 days intervals and then maintained at 5°C for 3 days plus a subsequent period of of 7 days at 2 (from 15°C to 0°C). After the above treatments, then those fruit were stored at 0 ± 0.5°C, 90% to 95% RH for 80 days. Gradual cooling (from 15°C to 0°C) significantly maintained higher percentage of accepted fruit and lower chilling injury index and chilling injury incidence of fruit compared with the direct cooling. Some attributes were then assayed in the fruits treated with gradual cooling (from 15°C to 0°C). Gradual cooling (from 15°C to 0°C) inhibited increases in membrane permeability, malondialdehyde content, superoxide anion production rate, and H2O2 content. At the same time, fruit cooled gradually (from 15°C to 0°C) exhibited higher superoxide dismutase, catalase, ascorbate peroxidase, and peroxidase activities than those treated by direct cooling during storage. The present study indicated that enhancement in antioxidant enzyme activity may be attributed to the reduction in CI symptoms by the gradual cooling treatment (from 15°C to 0°C).

DkXTH8, a novel xyloglucan endotransglucosylase/hydrolase in persimmon, alters cell wall structure and promotes leaf senescence and fruit postharvest softening

Fruit softening is mainly associated with cell wall structural modifications, and members of the xyloglucan endotransglucosylase/hydrolase (XTH) family are key enzymes involved in cleaving and re-joining xyloglucan in the cell wall. In this work, we isolated a new XTH gene, DkXTH8, from persimmon fruit. Transcriptional profiling revealed that DkXTH8 peaked during dramatic fruit softening, and expression of DkXTH8 was stimulated by propylene and abscisic acid but suppressed by gibberellic acid and 1-MCP. Transient expression assays in onion epidermal cells indicated direct localization of DkXTH8 to the cell wall via its signal peptide. When expressed in vitro, the recombinant DkXTH8 protein exhibited strict xyloglucan endotransglycosylase activity, whereas no xyloglucan endohydrolase activity was observed. Furthermore, overexpression of DkXTH8 resulted in increased leaf senescence coupled with higher electrolyte leakage in Arabidopsis and faster fruit ripening and softening rates in tomato. Most importantly, transgenic plants overexpressing DkXTH8 displayed more irregular and twisted cells due to cell wall restructuring, resulting in wider interstitial spaces with less compact cells. We suggest that DkXTH8 expression causes cells to be easily destroyed, increases membrane permeability and cell peroxidation, and accelerates leaf senescence and fruit softening in transgenic plants.

Genome-wide identification of jasmonate biosynthetic genes and characterization of their expression profiles during apple (Malus × domestica) fruit maturation

AbstractThe plant hormones regulate most physiological processes including apple fruit ripening by integrating diverse developmental cues and environmental signals. In addition to the well-characterized role of ethylene, jasmonic acid (JA) and its derivatives have also been suggested to play an important role during apple fruit maturation and ripening. Till now, the role JA on apple fruit ripening was only observed through exogenous application of JA or its derivatives. The de novo JA biosynthesis and signal transduction in apple fruit tissues have not been studied. In this study, the members of major gene families implicated in JA biosynthesis were identified from apple genome sequences; and their expression profiles were characterized in ‘Golden Delicious’ using quantitative reverse transcription polymerase chain reactions. Our data indicated that the expression patterns of MdLOX23, MdAOS2 and MdJMT2 in JA biosynthesis pathway are coordinated with those of MdACS3, MdERF1 and MdERF2 genes during 12 consecutive weeks of apple fruit maturation toward commercial harvest. Both MdLOX23 and MdAOS2 showed comparable expression patterns in both core and cortex suggesting localized synthesis of JA in different fruit tissues. Most of JA biosynthesis genes were transiently up-regulated in response to exogenous application of methyl jasmonate to on-tree maturing apple fruit, though in a maturity dependent manner. Our results suggested regulating roles of JA biosynthesis and its signaling on apple fruit maturation and ripening.

Isolation and Characterization of Two Persimmon Xyloglucan Endotransglycosylase/Hydrolase (XTH) Genes That Have Divergent Functions in Cell Wall Modification and Fruit Postharvest Softening

Fruit cell wall modification is the primary factor affecting fruit softening. Xyloglucan endotransglycosylase/hydrolase (XTH), a cell wall-modifying enzyme, is involved in fruit softening. In this study, two novel XTH genes (DkXTH6 and DkXTH7) were identified from persimmon fruit. Transcriptional profiles of both of the two genes were analyzed in different tissues of persimmon, and in response to multiple hormonal and environmental treatments [gibberellic acid (GA3), abscisic acid (ABA), propylene, and low temperature]. Expression of DkXTH6 was positively up-regulated during ethylene production and by propylene and ABA treatments, and suppressed by GA3 and cold treatment. In contrast, DkXTH7 exhibited its highest transcript levels in GA3-treated fruit and cold-treated fruit, which had higher fruit firmness. We found that DkXTH6 protein was localized in cell wall by its signal peptide, while cytoplasmic DkXTH7 protein contained no signal peptide. When expressed in vitro, the recombinant proteins of both DkXTH6 and DkXTH7 exhibited strict xyloglucan endotransglycosylase (XET) activity but no xyloglucan endohydrolase (XEH) activity. The recombinant protein of DkXTH6 showed a higher affinity with small acceptor molecules than the recombinant DkXTH7. Taken together with their opposing expression patterns and subcellular localizations, these results suggested that DkXTH6 might take part in cell wall restructuring and DkXTH7 was likely to be involved in cell wall assembly, indicating their special roles in persimmon fruit softening.

Characterization of β-Galactosidase Genes Involved in Persimmon Growth and Fruit Ripening and in Response to Propylene and 1-Methylcyclopropene

Fruit cell wall modification is the main factor affecting fruit softening. β-Galactosidase (EC 3.2.1.23) is one of the cell wall modifying enzymes that are involved in fruit ripening. Although β-galactosidase genes have been characterized in multiple fruits, the relationship between β-galactosidase genes and persimmon (Diospyros kaki) ripening remains unknown. In this study, four persimmon β-galactosidase genes, designated DkGAL1 to DkGAL4, were isolated from ‘Fupingjianshi’ persimmon fruit. Transcriptional profiles of DkGAL genes during fruit development and in response to ethylene were investigated. β-Galactosidase activity increased during postharvest ripening and was considerably induced by propylene. The accumulation of DkGAL1 was relatively low during fruit growth but significantly increased throughout fruit ripening. In contrast, DkGAL2 mRNA levels were the highest at the early phase of fruit growth and decreased towards fruit maturity. DkGAL3 and DkGAL4 exhibited very low levels of expression in both growing fruit and postharvest fruit but were predominantly expressed in the leaf and stem. The expression pattern of DkGAL1 was positively regulated by ethylene, up-regulated under propylene treatment and suppressed by 1-MCP treatment. Ethylene appears to have little effect on the transcription of DkGAL2, DkGAL3 and DkGAL4. These results demonstrate that multiple β-galactosidase genes have differential transcript and hormonal regulation characteristics in ripening persimmon fruit. The functions of DkGAL3 and DkGAL4 are limited in fruit. However, DkGAL2 may play a crucial role in fruit development. Furthermore, DkGAL1 is a predominant β-galactosidase gene that is involved in persimmon fruit softening and regulated by ethylene.

Overexpression of persimmon DkXTH1 enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants

Key messageDkXTH1promoted cell elongation and more strength to maintain structural integrity by involving in cell wall assembly, thus enhanced tolerance to abiotic stress with broader phenotype in transgenic plants.AbstractXyloglucan endotransglucosylase/hydrolase (XTH) is thought to play a key role in cell wall modifications by cleaving and re-joining xyloglucan, and participates in the diverse physiological processes. DkXTH1 was found to peak in immature expanding persimmon fruit, and its higher expression level exhibited along with firmer fruit during storage. In the present study, transgenic Arabidopsis and tomato plants were generated with DkXTH1 constitutively expressed. Overexpression of DkXTH1 enhanced tolerance to salt, ABA and drought stresses in transgenic Arabidopsis plants with respect to root and leaf growth, and survival. Transgenic tomatoes collected at the mature green stage, presented delayed fruit softening coupled with postponed color change, a later and lower ethylene peak, and higher firmness in comparison with the wild-type tomatoes during storage. Furthermore, broader leaves and tomato fruit with larger diameter were gained in transgenic Arabidopsis and tomato, respectively. Most importantly, transgenic plants exhibited more large and irregular cells with higher density of cell wall and intercellular spaces, resulting from the overactivity of XET enzymes involving in cell wall assembly. We suggest that DkXTH1 expression resulted in cells with more strength and thickness to maintain structural integrity, and thus enhanced tolerance to abiotic stress and delayed fruit softening in transgenic plants.