Qiuyan Ban

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.

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.

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.

Overexpression of persimmon 9-lipoxygenase DkLOX3 confers resistance to Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea in Arabidopsis

Lipoxygenase (LOX) pathway initiates lipid peroxidation of cell membrane, which is crucial for plant senescence and defense pathways. Our previous study suggested that the persimmon 9-LOX gene, DkLOX3, promoted leaves senescence and enhanced tolerance to abiotic stresses. Here, the function of DkLOX3 in biotic stresses was investigated in DkLOX3-overexpression (DkLOX3-OX) Arabidopsis. The results showed that inoculation with Pseudomonas syringae pv. Tomato (Pst) DC3000 in transgenic leaves exhibited more ROS accumulation, lower activity of several antioxidant enzymes and cell death phenotype in early stage, in addition, less lesions and bacterial growth were observed as compared to wild type. Quantification of jasmonic acid (JA) and salicylic acid (SA) indicated SA contents and expression of its marker gene PR1 were increased in transgenic leaves with Pst DC3000 infection. Furthermore, inoculation with Botrytis cinerea in transgenic leaves caused more ROS accumulation, lower activity of antioxidant enzymes in early stage, however, there are no significant differences in both JA and SA contents and their marker genes expression profiles between transgenic lines and wild type. Overall, all these results indicated that DkLOX3 plays positive roles in reducing the sensitivity to Pst DC3000 via regulating ROS accumulation, cell death and SA sythesis pathways, and enhancing resistance to B. cinerea probably by regulating ROS accumulation, but not SA or JA metabolic pathway. Our preliminary results indicated DkLOX3 play an important role in different types of pathogens resistance.

Role of Brassinosteroids in Persimmon (Diospyros kaki L.) Fruit Ripening

Brassinosteroids (BRs) are phytohormones that regulate numerous processes including fruit ripening. In this study, persimmon ( Diospyros kaki L.) fruits were treated with 24-epibrassinolide (EBR) or brassinazole (Brz, a BR biosynthesis inhibitor) and then stored at ambient temperature. The results show that endogenous BR contents gradually increased during persimmon fruit ripening. EBR treatment significantly increased both the content of water-soluble pectin and the activities of polygalacturonase, pectate lyase, and endo-1,4-beta-glucanase but significantly reduced the content of acid-soluble pectin and cellulose, resulting in rapid fruit softening. The EBR treatment also promoted ethylene production and respiration rate. In contrast, Brz treatment delayed persimmon fruit ripening. qRT-PCR analysis showed that DkPG1, DkPL1, DkPE2, DkEGase1, DkACO2, DkACS1, and DkACS2 were up-regulated (especially a 38-fold increase in DkEGase1) in the fruit of the EBR-treated group. These results suggest that BRs are involved in persimmon fruit ripening by influencing cell-wall-degrading enzymes and ethylene biosynthesis.

Molecular Cloning, Structural Characterization, and Ripening-Related Expression of Lipoxygenase Genes from Three Persimmon Cultivars Differing in Postharvest Ripening Rate

A novel lipoxygenase (LOX) gene, DkLOX4 (KF035132), and the full-length DkLOX3 (KF035131) gene were identified from persimmon fruit, and transcriptional profiles of both genes in three persimmon cultivars with different ripening rates were investigated using quantitative real-time PCR (qRT-PCR). We also studied their expression in response to propylene and 1-methylcyclopropene (1-MCP), which can promote or impede fruit ripening. Phylogenetic analysis indicated that both DkLOX3 and DkLOX4 belong to the 9-LOX family. The identity of these DkLOX genes was confirmed by functional expression in bacteria. The results showed that both DkLOX3 and DkLOX4 possessed the expected LOX activity. qRT-PCR analysis showed that all DkLOX genes were expressed abundantly in persimmon fruit during ripening, but distinctly different expression patterns were evident. Expression patterns of all DkLOXs generally paralleled the climacteric rise and maxima in ethylene production. The trajectories of transcript accumulation and the expression peaks also paralleled the declines in fruit firmness and increase in LOX activity and malondialdehyde (MDA) content. The expression level of DkLOX3 was significantly higher than that of the other two DkLOXs, its expression maxima followed the order from high to low and early to late in ‘Fupingjianshi’, ‘Huoshi’ and ‘Ganmaokui’. And expression of DkLOX3 was particularly up-regulated by propylene treatment, and down-regulated by 1-MCP treatment. Overall, the results reported here indicate that LOX genes, especially DkLOX3, may play an important role in persimmon fruit ripening.

Functional characterization of persimmon β-galactosidase gene DkGAL1 in tomato reveals cell wall modification related to fruit ripening and radicle elongation

Cell wall metabolism during fruit ripening is a highly organized process that involves complex interplay among various cell wall hydrolases. Among these cell wall hydrolases, β-galactosidase has been identified to participate in cell wall metabolism via its ability to catalyze galactosyl metabolism from the large and complex side chains of cell walls. In this study, the galactose content in the pericarp increased during persimmon fruit ripening, but cell wall galactosyl residues decreased, indicating a relationship between galactose metabolism and persimmon fruit ripening. Expression of a previously isolated β-galactosidase gene, DkGAL1, increased 25.01-fold during fruit ripening. Heterologous expression of DkGAL1 under the CaMV 35S promoter in tomato accelerated on-plant and postharvest fruits ripening. The fruit firmness of one of transgenic line, OE-18, was 23.83% lower than that of WT at the breaker stage. The transgenic fruits produced more ethylene by promoting the expression of ethylene synthesis-related genes and cell wall degradation-related genes. Overexpression of DkGAL1 in tomato also reduced cell-to-cell adhesion and promoted both wider intercellular spaces and less cell compaction in transgenic fruit structures. Moreover, DkGAL1 was involved in seed germination and radicle elongation in transgenic tomato seeds. These results confirm the role of DkGAL1 in fruit ripening and suggest that this gene alters galactose metabolism in the fruit, which can promote ripening and reduce cellular adhesion. In addition, the role of DkGAL1 is not limited to fruit softening; DkGAL1 was also involved in seed germination and radicle elongation in transgenic tomato seeds.