| Almost all plant species form mutualistic symbioses with microbes,and the relationship between plant and microbes can affect many biological processes and induce some beneficial effects,such as promoting plant growth and nutrients uptake,enhancing the resistance to various biotic and abiotic stresses,transferring more nutrient to plants or improving the ability to capture nutrient from soil.Thus,these beneficial microorganisms show a great promise in the commercial development of microbial agents for agricultural sustainability.Endophytic fungi,an important component of plant-associated symbionts,have attracted increasing attention because of their remarkable species and functional diversity.Previous research showed that Phomopsis liquidambari B3,can develop a symbiotic relationship with rice,improve the N use in rice,promote the growth and yield of rice.The above results indicate the useful impacts of P.liquidambari on nitrogen uptake and metabolism in rice,however the underlying mechanism is very little known.To explain the potential mechanism of these effects caused by B3,we designed pot experiment and hydroponic experiment.Firstly,we used a fluorescence microscopy to observe the tissues of GFP-B3 infected rice,and images showed that fungal mycelia were visible in the epidermal and cortical cell layers,indicating that P.liquidambari had successfully established a mutualistic symbiotic relationship with rice.Secondly,a pot experiment was conducted to examine the N and phytohormone levels in response to endophyte infection at four whole growth durations during exposure to different N levels to examine the mechanism underlying the effect of B3 on nitrogen accumulation and metabolism in rice plants.Our results showed that the contents of auxin(IAA),cytokinin(CTK)and ethylene(ETH)in rice were significantly enhanced by B3 under low N levels at different growth durations;B3 symbiosis increased N accumulation and rice yield and induced the expression of some genes related to N uptake and metabolism.Thirdly,to further verify whether B3 symbiosis could enhance N use in rice by regulating phytohormones,we performed a hydroponic experiment in which exogenous phytohormones and their specific inhibitors were applied.The results showed that the application of exogenous auxin,cytokinin and ethylene increased the rice nitrogen content,and their inhibitors decreased the amount of nitrogen absorbed in rice.As expected,B3 infection alleviated the negative effect caused by inhibitors slightly.And the result of expression levels of genes related to auxin(IAA),cytokinin(CTK)and ethylene(ETH)synthesis indicated that B3 symbiosis can induce a higher expression level of genes related in auxin(IAA),cytokinin(CTK)and ethylene(ETH)synthesis.Above results reavel that B3 can regulate the production of phytohormone in rice to participate in N use of rice.Finally,relationship among IAA,CTK and ETH signaling pathway in the B3-induced N accumulation in rice were elucidated.Our results showed that P.liquidambari,promoted N accumulation of rice,also regulated IAA,CTK and ETH signaling pathway.Pretreatment of rice with the IAA inhibitor blocked not only the B3-induced N accumulation but also induced IAA and ETH generations;nevertheless,ETH inhibitor showed no significant suppression on the IAA production,implying that IAA acted on the upstream of ETH production.And the relationship between CTK and ETH is similar,that CTK acted on the upstream of ETH generation.And the relationship between IAA and CTK is antagonism.In conclusion,IAA,CTK and ETH signaling systems build a network and mutually regulate N transport and metabolic systems. |
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