Ethylene Regulation On The Growth,Development,and Postharvest Senescence Of Agaricus Bisporus Frutiting Bodies And Investigation On Its Biosynthesis Pathway

Ethylene, which is the simplest plant hormone, plays very important roles in many aspects of plant growth, development, fruit ripening and senescence, and defense responses. The physiological role, biosynthesis pathway, and molecular action mechanism of ethylene in higher plants has been systematicly and intensively researched. Edible mushrooms, which have nutritional and medicinal values, are favored by most consumers. By contrast with higher plants, researches, however, on the ethylene production and action in edible fungi are very few. In the present study, Agaricus bisporus Lange Sing, the model of edible mushrooms was used to investigate the effects of ethylene on the growth, development, and postharvest senescence of fruiting bodies as well as ethylene biosynthesis pathway and its regulation, which will provide theory reference for resolving difficult problems in postharvest senescence of edible mushrooms. The major results of this research were as follows:Ethylene production of Agaricus bisporus fruiting bodies during development stages and postharvest storage was detected by gas chromatography. Results showed that ethylene was detectable in sporophores at each stage but was very low at development stages. However, ethylene production gradually increased with the continued mushroom maturation and senescence during postharvest storage and exibited2peaks in sporophores with protection veil starting to break and spores release at initial stage, respectively. The2peak values were as83and209times, respectively, high as that of sporophores before storage. The above mentioned indicated that ethylene probably participated in the postharvest senescence of fruiting bodies.Sporophores at commercial harvest time were treated with0.05%ethephon to research its effects on endogenous ethylene production and mushroom senescence. It was found that ethephon treatment speeded up the arrival of endo-ethylene production peak, increased the rate and degree of cap opening, raised the degree of cell membrane permeability and peroxidation, accelerated the degradation of soluble protein and sugar, and inhibited the increase of antioxidant enzyme activities and antioxidant compounds accumulation. These results suggested that ethylene could accelerate mushroom senescence.The supplement of L-MET (methionine), SAM (S-adenosylmethionine) and ACC (1-aminocyclopropane-1-carboxylic acid), precursors of ethylene biosynthesis in higher plants, accelerated the arrival of ethylene production peak and increased ethylene content in varying extents. In comparison, ACS (ACC synthase) inhibitor AOA (aminooxyacetic acid) and ACO (ACC oxidase) inhibitor CoCl2and sodium azide significantly lowered ethylene production. We successfully detected the activities of ACS and ACO, and ACC content in Agaricus bisporus fruiting bodies. Moreover, genes homologous to ethylene biosynthesis genes in higher plants were isolated and cloned. We hold that there is at least one pathway in Agaricus bisporus similar with the ethylene biosynthesis pathway in higher plants, that is Metâ†'SAMâ†'ACCâ†'ethylene. In addition, the increase amplitude of ACO activity was larger than ACS activity both in development stages and postharvest storage time, and the rise of ACO activity preceded ACS activity induced by ethephon treatment. It was demonstrated that ACO was likely to play important roles in the regulation of ethylene biosynthesis in Agaricus bisporus. The expression pattern of ethylene biosynthesis components in Agaricus bisporus was detected by real time PCR and Western blotting. Results showed that the low ethylene production in fruiting bodies at development stages was mainly regulated by AbACS2gene, and the other five genes had extremeiy low expression levels. However, ethylene production was coordinatedly regulated by AbACS1, AbACS2and AbACO genes during postharvest storage, and presented temporal expression differences. Futhermore, the expression of AbACO was induced earlier than that of AbACSl and AbACS2by ethephon, and the extent of ethephon-induce AbACO expression was considerably larger than that of AbACSl and AbACS2. The result suggested that different patterns of induction of ethylene biosynthesis genes existed during senescence in Agaricus bisporus. ACO protein gradually accumulated with the mushroom senescence during postharvest storage and had the highest expression level in the gill tissue, which manifested that ACO protein was regulated in a tissue-specific pattern. It can be seen from the above that ethylene biosynthesis in Agaricus bisporus was regulated similarly with higher plants at the levels of transcription and protein.Sequence analysis combined with homology modeling, site-directed mutagenesis and kinetic analysis were used to investigate the property of Agaricus bisporus AbACO. Results indicated that the difference of289th amino acid in active site between AbACO and plant ACOs was the primary reason for their different substrate binding characteristics.