| From 2006 to 2007, systematic researches were conducted on sugar and starch metabolism, cell wall degradation, changes of lipoxygenase (LOX) activity, and the gene expression properties of their key enzymes during fruit development, maturation and softening. These researches were carried out in the apple-producing areas of Yanshan, Hebei province, using three apple cultivars,'Fuji','Golden Delicious'and'Gala', which had different texture and storage characteristics. The post-harvest regulation by ethylene and low temperature (0℃) on fruit softening was also investigated. The results showed that fruit texture development and softening of apple was a complex physiological and biochemical process, regulated by a variety of factors, but there were significant differences among apple cultivars. The main results obtained were shown as follows:1. During fruit maturation and softening, the texture firmness decreased slowly in'Fuji'apple, and it could greatly remained its original texture during post-harvest storage, and was affected little by ethylene and low temperature. While the fruit texture softened quickly in'Golden Delicious'and'Gala'apple, and there was short storage time under room temperature, obviously affected by ethylene and low temperature. Respiration rate was very low in post-harvest fruit of'Fuji'apple, and was insensitive to ethylene and low temperature treatments. Both'Golden Delicious'and'Gala'fruit had higher respiration rate. Their respiratory rate increased rapidly under room temperature, and reached the respiratory peak soon. Both 1-MCP and low-temperature treatments strongly inhibited their fruits respiratory and delayed the climatic period.2. The changes of fruit starch content and amylase (AM) activity had significant effects on fruit texture of apple, had obvious relationship with fruit softening. They were the most significant factors of cell internal substance relating to fruit softening, whilch there was closely correlation to apple storage characteristics. Accordingly, the AM gene expression pattern had significant difference in different cultivars. AM gene of the'Golden Delicious'apple fruit expressed strongly during the early development stage and ripening period, but that of'Fuji'apple had much lower expression intensity. The post-harvest ethephon treatment had stronger enhancement in AM gene expression of'Golden Delicious'than that of'Fuji'. 1-MCP treatment dramatically inhibited the AM gene expression during the whole storage stage in'Fuji'apple, while only inhibited its expression during early storage period in'Golden Delicious'. There had same effects on AM gene expression both in'Fuji'and'Golden Delicious'under 0℃storage.3. The increase in sucrose, fructose and glucose content had obvious correlation with texture softening during fruit maturation. During fruit ripening and softening, sucrose accumulation had more relevance with fruit softening than other sugars, and affected more strongly by ethylene factors. The activity of sucrose phosphate synthase (SPS) was significantly correlated with sugar accumulation during fruit maturation and softening, but there was remarkable difference in different cultivars. The SPS activity was more strongly regulated by ethylene factors in'Golden Delicious'and'Gala'than that in'Fuji'apple. All these results indicated that SPS played an important role in sugar accumulation and metabolism during fruit ripening. But fruit sugar content and enzyme activity show smaller fluctuations under low temperature. The expression of SPS gene during fruit softening was stronger than that during fruit development, and had different expression pattern between'Fuji'and'Golden Delicious'. The SPS gene expression in'Fuji'was higher in the mid-and late fruit development, and lower at the harvest time, increased again during fruit storage. The SPS gene expression of post-harvest apple fruit was significantly regulated by ethylene factors, and could be remarkably inhibited by 1-MCP treatment. However, ethephon treatment had little influence on the gene expression of SPS. Low-temperature inhibited the SPS gene expression at the initial stage to a certain extent.4. The covalent binding pectin (CSP) was the main pectin component. There was the highest CSP content and the lowest water soluble pectin (WSP) content in'Fuji'apple fruit. The content of three kind of pectin in'Gala'was in the middle. There was the lowest in pectin content and had smaller fluctuation in'Golden Delicious'apple fruit. During fruit maturation and softening, both CSP and WSP played an important role in fruit softening. WSP content increased significantly in'Golden Delicious'and'Gala'apple fruit, with the decreased content in CSP. But WSP content in'Fuji'apple fruit increased slowly, with constant content of CSP. The change of ionic soluble pectin (ISP) content had little correlation with fruit softening and storage characteristics. There had similar trends in change of cellulose content, but different in hemi-cellulose content change among three apple cultivars. It seemed like that hemi-cellulose had a closer relationship with fruit storage characteristics. WSP and CSP content were significantly affected by ethylene factor and low temperature, and closely relating to fruit storage property. The main period, which hemi-cellulose content decreased and was regulated, was at the initial stage of storage, though cellulose was significantly regulated by ethylene factors and low temperature, there was the similar intensity in three apple cultivars.5. Cell wall-degrading enzymes were at the core in fruit texture softening of apple, and had close relationship with fruit storage characteristics. The results indicated thatβ-galactosidase (β-gal) andα-L-arabinofuranosidase (α-L-Af) activities had the closest correlation to fruit texture softening and the change of cell wall component. The activities ofβ-Gal andα-L-Af increased rapidly at the initial storage of'Golden Delicious'and'Gala'apple fruit. It seemed like that the two enzymes had stronger effects on fruit softening than the other cell wall enzymes. Furthermore, bothβ-gal andα-L-Af activities during post-harvest period were strongly inhibited by 1-MCP and low-temperature, while enhanced by ethephon, which had stronger effects on'Golden Delicious'and'Gala'than that on'Fuji'apple. Then it could make a conclusion that bothβ-gal andα-L-Af were closely related to fruit softening and storability of apple cultivars. Polygalacturonase (PG) and pectinesterase (PME) also showed activity during fruit maturation and softening. During the developmental stage, the activity of PG was very low, and was similar in different cultivars. PME activity was higher during fruit developmental stage in both'Golden Delicious'and'Gala'than that in'Fuji'apple. With fruit ripening and softening, PG and PME activities increased, and the increase rate of PG activity was much faster than that of PME, indicating that PG mainly worked at late stage of fruit ripening, and PME degraded pectin during the whole period of fruit development and ripening. The results showed that the regulated effects by ethylene and low temperature on PG and PME were different due to distinct storage characteristics between cultivars, and mainly affected fruit softening during the mid-and late stage of storage. Analysis of Cellulase (Cx) activity suggested that Cx was involved in cellulose degradation of apple fruit. Cx activity between cultivars and post-harvest regulation pattern were similar, illuminating that Cx affected fruit softening, but was not the key indicator of apple storage property.6. The gene expressions ofβ-Gal,α-L-Af, PG and PME could be detected during fruit development, maturation and ripening. But their expression patterns were different to different cultivars and developmental stages. During fruit development, PME gene expressed higher, andβ-Gal,α-L-Af and PG gene expressed lower, but all expressed strongly with fruit maturation and softening. Of the glycosidase,α-L-Af gene expression between'Fuji'and'Golden Delicious'was more difference than that ofβ-Gal. PME gene expression in'Fuji'was much higher than that in'Golden Delicious', and the difference was far more than PG. The results of post-harvest regulation showed that PME gene expression was more intensively influenced by ethylene factors and low temperature in'Golden Delicious'than those in'Fuji'. In'Golden Delicious'fruit, PG gene expression was also more strongly enhanced by ethephon and inhibited by 1-MCP than those in'Fuji'. But 0℃low temperature made little inhibited effect between cultivars.α-L-Af gene was significantly inhibited by 0℃treatment, and had more markably effect in'Golden Delicious'than that in'Fuji'. Ethephon promotedα-L-Af gene expression of the two apple cultivars at early stage of storage, and 1-MCP treatment significantly inhibited theα-L-Af gene expression in'Golden Delicious', but not in'Fuji'. 0℃treatment showed significant inhibitory effect onβ-Gal gene expression both in the two cultivars. 1-MCP treatment inhibited theβ-Gal gene expression of'Golden Delicious'fruit at early stage of storage, and inhibited its expression of'Fuji'fruits during the entire storage. The treatment by ethephon significantly increased in expression level ofβ-Gal gene of'Golden Delicious'apple, and with little effect on'Fuji'apple fruit. These results suggested that,β-Gal had different patterns of temporal and spatial expression in the two cultivars, and further confirmed that cell wall degradation played a central role in fruit softening; glycosidase was very important for fruit texture loosing and softening of apple, and the role of PME was stronger than that of PG.7. The lipoxygenase (LOX) showed similar change among the three apple cultivars, expressed higher during the early development and ripening stage. The results indicated that LOX had different physiological functions at different development stages and its effect on texture softening mainly appeared during ripening stage, and showed correlation with apple storage characteristics to some extent. LOX activity of apple fruit was significantly regulated by ethylene factors and low temperature. The influence of ethylene on LOX activity was mainly at the early storage, and had same effect on both'Fuji'and'Golden Delicious'apple. The low temperature more strongly inhibited LOX gene expression in'Fuji'than that in'Golden Delicious'apple, which had nearly no expression of LOX gene. LOX gene expressed highly at the early development and ripening stage, but the different expression between'Fuji'and'Golden Delicious'was at the early development stage. All above results also showed that LOX had different response patterns to ethylene and low temperature, and maybe play a role in fruit early softening.In the whole, cell wall degradation was at the central role during fruit texture development and softening, which was not only the main factor of fruit softening, but also had an important effect on cell wall loosing, cell growth and fruit morphogenesis. LOX involved in fruit growing and softening through modifying membrane permeability and starting lipid per-oxidation. Otherwise, the products from synthesis and metabolism of sugar and starch could provided energy and substrate for fruit growth and respiration, could maintain cell turgor pressure which affected the cell wall tensile strength ability, and maybe the other main reason for causing fruit softening.
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