【作者】 张勇；

【导师】 胡斌； Heinz Rennenberg；

【作者基本信息】 西南大学 ， 土壤学， 2023， 硕士

【摘要】 氮作为植物生长发育所必须的矿质营养元素之一,在植物生长过程中发挥重要作用。农业和园艺中大量添加氮肥以实现和维持粮食及生物量的高产。尿素占全球氮肥施用量50%以上,但有近一半尿素施用后不能被植物利用,而是释放到环境中不断积累并引发氮沉降等多种问题。研究发现我国已经成为仅次于欧洲和美国的全球第三大氮沉降区,随着我国工农业的不断发展,大气氮沉降量还会继续升高,大剂量的氮沉降势必会对我们生活生产的自然环境产生影响,而植物经常暴露在高浓度的氮环境下会产生一系列潜在的负面影响。豆科植物根系能够与土壤中的根瘤菌共生结瘤生成氮素供植物使用。刺槐（Robinia pseudoacacia L.）作为典型的固氮木本豆科植物,具有分布广泛、根系发达、抗逆性强等特点,是我国固沙保土、绿化荒山的先锋树种。因此我们选用来自东北种源的刺槐幼苗,通过接种根瘤菌和施加不同浓度尿素的处理并测定其光合和生理响应,揭示刺槐在共生固氮条件下对高氮胁迫的生理响应机制。选择温室中正常培养的3月龄刺槐幼苗（已接种中慢生根瘤菌Mesorhizobium huakuii QD9）为试验材料,进行氮胁迫处理。氮胁迫处理水平包括正常氮浓度(7 mmol·L^-1)、高氮浓度(57 mmol·L^-1)、极高氮浓度(107 mmol·L^-1)。处理方式为,将正常施氮处理组的幼苗每隔5天施加一次50 mmol·L^-1浓度的尿素,连续施加两次。将施用高浓度尿素前一天的采样作为对照组CK。每次施用尿素后的1、3、5天进行采样。采样结束后,对刺槐生物量、叶片光合作用相关参数、叶片和根系中氮含量及分配、叶片和根系中氮代谢和抗氧化代谢相关酶活性进行测定和比较研究。主要研究结果如下:（1）不同尿素施用量和暴露时间对刺槐叶片和根系生长状况和光合作用具有显著影响。与正常施氮刺槐相比,每次施用尿素后,刺槐叶片的光合效率均在第1、3天显著增强,并在第5天时恢复到CK水平。第一次尿素施用后根和根瘤生物量显著升高,而叶片和植株总生物量没有显著变化;第二次尿素施用后刺槐叶片、根、根瘤和总生物量均升高。（2）不同尿素施用量和暴露时间显著影响了刺槐叶片和根系中氮含量及分配。与正常施氮刺槐相比,不同浓度尿素施用后刺槐根系中总氮含量出现一个动态上升的过程。第一次施用尿素短暂增加了刺槐根系中总氮含量,这种影响随着时间推移逐渐消失,第二次施用尿素后再次出现。但尿素施用没有显著影响刺槐叶片中的总氮含量,且根系氮素含量显著低于叶片。第一次施用尿素可以显著增加刺槐叶片中可溶性蛋白氮的含量,这种效应是短暂的,并在第二次尿素施用后再次出现。第一次施用尿素刺槐叶片中总氨基酸氮含量没有显著变化,仅在第二次施用尿素后短暂升高。尿素施用还显著降低了刺槐叶片中硝态氮含量,但铵态氮和结构氮含量无显著变化。尿素施用首先提高了刺槐根系中可溶性蛋白氮含量,随后才提高根系中总氨基酸氮含量。第一次施用尿素可以显著增加刺槐根系总氮和结构氮的含量,这种效应是短暂的,并在第二次尿素施用时再次出现。（3）尿素施用和暴露时间对刺槐叶片和根系氮代谢及抗氧化代谢过程产生显著影响。与正常施氮刺槐相比,两次尿素施用后刺槐叶片和根系中硝酸还原酶（NR）活性均瞬时增强。与NR变化不同,不同尿素施用量和暴露时间下叶片谷氨酰胺合成酶（GS）和NADH型谷氨酸合成酶（NADH-GOGAT）活性没有显著变化,且随着尿素施用量增加和处理时间的延长,叶片中FD型谷氨酸合成酶（FD-GOGAT）活性不断降低。而根系中GS活性仅在第一次施用尿素后出现短暂升高,第二次施用尿素后无显著变化;根系中NADH-GOGAT活性在两次尿素施用后均出现短暂升高,但这种影响随着时间推移逐渐消失。第一次施用尿素后刺槐叶片和根系中谷胱甘肽还原酶（GR）活性均升高,并在第二次施用尿素后保持不变。然而尿素施用后刺槐叶片和根系中脱氢抗坏血酸还原酶（DHAR）活性并没有增强,反而随着暴露时间延长出现短暂降低趋势。综上所述,过量尿素施用可以显著促进刺槐的光合作用。尿素施用可以促进刺槐的生长,但对刺槐地上部生物量的影响显著滞后于地下部。过量尿素施用下刺槐叶片和根系中的氮会优先用于合成能有效抵御高氮胁迫的氮形态,以提高刺槐的抗逆性。不同尿素施用量对刺槐叶片和根系中氮素代谢都有促进作用,此外一定程度过量尿素施用下会增强刺槐的抗氧化能力。该结果可以为建立刺槐-根瘤菌这一林木微生物互作抗逆体系生理响应机制提供理论依据,同时为应对氮沉降可能带来的高氮胁迫提供科学指导。更多还原

【Abstract】 Nitrogen as one of the essential mineral nutrients for plant growth and development,plays an important role in plant growth.Large amounts of nitrogen fertilizer are added to agriculture and horticulture to achieve and maintain high yields of food and biomass.Urea accounts for more than 50%of the global nitrogen fertilizer application.Nearly half of urea cannot be used by plants after application,but is released into the environment and accumulates continuously,causing a variety of problems such as nitrogen deposition.It was found that China has become the third largest nitrogen deposition area only after Europe and America.With the development of our country’s industry and agriculture,the atmospheric nitrogen deposition will be increased.Large dose of nitrogen deposition is certainly to affect the natural environment where plants are exposed to high concentration nitrogen,resulting in a series of potential negative effects.The roots of leguminous plants can symbiosis and nodulate with the rhizobia in soil to produce nitrogen for plant use.Robinia pseudoacacia L.is a typical nitrogen-fixing woody leguminous plant,has a wide distribution,developed root system and strong resistance to stess.It is a pioneer tree species of sand fixation to protect soil and afforestation barren mountain in our country.Therefore,we selected Robinia seedlings from Northeast China and tested their photosynthetic and physiological responses by inoculating rhizobia and applying different concentrations of urea to reveal the physiological response mechanism of Robinia to high nitrogen stress under symbiotic nitrogen fixation conditions.Three-month-old Robinia seedlings（inoculated with Mesorhizobium huakuii QD9）that were cultured normally in a greenhouse were selected as experimental materials for nitrogen stress treatment.Nitrogen stress treatment levels included normal nitrogen concentration(7 mmol·L^-1),high nitrogen concentration(57 mmol·L^-1)and extremely high nitrogen concentration(107 mmol·L^-1).The treatment method was to apply 50 mmol·L^-1 urea to the seedlings in the normal nitrogen treatment group once every 5 days for two consecutive times.Take the sampling from the day before the application of high concentration urea as the control group CK.Samples were collected 1,3,and 5 days after each urea application.After sampling,the biomass,leaf photosynthetic parameters,nitrogen content and distribution in leaves and roots,nitrogen metabolism and antioxidant metabolism related enzyme activities in leaves and roots of Robinia were determined and compared.The main research results are as follows:（1）Different urea application rates and exposure time had significant effects on the growth and photosynthesis of Robinia leaves and roots.Compared with normal nitrogen Robinia,the photosynthetic efficiency of Robinia leaves was significantly enhanced on the 1st and 3rd days after each urea application,and recovered to CK level on the 5th day.The root and nodule biomass increased significantly after the first urea application,but the total leaf and plant biomass did not change significantly.The leaves,roots,nodules and total biomass of Robinia increased after the second urea application.（2）Nitrogen content and distribution in leaves and roots of Robinia were significantly affected by different urea application rates and exposure time.Compared with normal nitrogen application,the total nitrogen content of root showed a dynamic increase after urea application of different concentrations.The first application of urea briefly increased the total nitrogen content in the roots of Robinia,but this effect gradually disappeared with the passage of time,and appeared again after the second application of urea.However,urea application had no significant effect on the total nitrogen content in leaves,and the root nitrogen content was significantly lower than that in leaves.The first application of urea significantly increased the content of soluble protein nitrogen in leaves.This effect was short-lived and reappeared after the second application of urea.The content of total amino acid nitrogen in leaves did not change significantly after the first application of urea,but only increased briefly after the second application of urea.Urea application also significantly reduced the nitrate nitrogen content in leaves,but the ammonium nitrogen and structural nitrogen contents had no significant changes.Urea application firstly increased soluble protein nitrogen content in root system,and then increased total amino acid nitrogen content in root system.The first application of urea significantly increased the content of total nitrogen and structural nitrogen in roots.This effect was short-lived and was repeated with the second application of urea.（3）Urea application and exposure time had significant effects on nitrogen metabolism and antioxidant metabolism of Robinia leaves and roots.Compared with normal nitrogen Robinia,NR activity in Robinia leaves and roots increased instantaneously after two urea applications.Different from NR,the activities of GS and NADH-GOGAT in leaves did not change significantly under different urea application rate and exposure time,and FD-GOGAT activities in leaves decreased with the increase of urea application rate and extension of treatment time.However,the activity of GS in roots increased only briefly after the first application of urea,but did not change significantly after the second application of urea.The activity of NADH-GOGAT in roots increased briefly after both urea application,but this effect disappeared over time.GR activity in leaves and roots was increased after the first urea application,and remained unchanged after the second urea application.However,the DHAR activity in leaves and roots did not increase after urea application,but showed a temporary decrease trend with the extension of exposure time.In conclusion,excessive urea application can significantly promote photosynthesis of Robinia.Urea application can promote the growth of Robinia,but its effect on overground biomass lags behind that of underground.Nitrogen from leaves and roots of Robinia under excessive urea application will be preferentially used to synthesize nitrogen forms that can effectively resist high nitrogen stress,so as to improve the stress resistance of Robinia.Different urea application rates can promote nitrogen metabolism in Robinia leaves and roots.In addition,excessive urea application to a certain extent can enhance antioxidant capacity of Robinia.The results can provide a theoretical basis for establishing the physiological response mechanism of robinia-rhizobia microbial interaction resistance system in forest trees,and provide scientific guidance for coping with the possible high nitrogen stress caused by nitrogen deposition.更多还原