【作者】 黄伟；

【导师】 王丽红； 张军毅；

【作者基本信息】 江南大学 ， 环境工程（专业学位）， 2025， 硕士

【摘要】 稀土元素(REEs)因其独特理化性质被广泛应用于农业以促进作物生长,但常规施用方式,如叶面喷施和土壤施用,导致其在环境中累积,并通过食物链传递,最终威胁人体健康。相比之下,REEs浸种可作为一种替代方式,减少其环境累积。光形态建成是植物依赖光来调控植物发育的过程,经历暗形态建成后转变的阶段,对幼苗后续发育至关重要。研究表明,乙烯(ETH)在植物形态建成(暗或光形态建成)中发挥重要调控作用,且土壤施用REEs可通过激活网格蛋白介导的胞吞作用(CME),诱导植物内源ETH合成和提高ETH信号正调控因子EIN3蛋白水平,进而调控形态建成。那么La(Ⅲ)浸种是否影响植物形态建成?该影响是否受ETH信号调控?La(Ⅲ)浸种对形态建成的调控是否也与La(III)激活CME有关?鉴于此,本文以模式植物拟南芥为研究对象,以La(Ⅲ)为REEs代表,研究REEs浸种对植物形态建成的影响,并从ETH信号角度揭示影响机制;对La(Ⅲ)浸种的效果及机制与Cd(Ⅱ)(有害元素代表)和Ca(Ⅱ)(有益元素代表)浸种作比较,以及REE常规施用方式-叶喷;从La(Ⅲ)激活CME和转录组学角度,揭示La(Ⅲ)浸种对植物ETH信号影响的机制,主要结果如下:(1)发现La(Ⅲ)浸种可促进拟南芥暗和光形态建成。发现La(III)浸种促进暗形态建成的表现:下胚轴长度缩短、顶端弯钩弯曲角度增大和Pchlide含量下降,且影响最大的浓度-时间条件为:60μM浸种12 h。La(Ⅲ)浸种促进了光形态建成的表现:叶绿素含量、净光合速率、蒸腾速率和胞间二氧化碳浓度上升,并在22 d时增幅达到最大。La(Ⅲ)浸种进而引起拟南芥抗氧化酶(过氧化物酶、超氧化物歧化酶、过氧化氢酶)活性上升和元素含量(Ca、K、P、Mg、Mn、Fe、Cu、Zn)增加。La(Ⅲ)浸种对暗形态建成的促进效果>Ca(Ⅱ)浸种>Cd(Ⅱ)浸种;对暗形态建成的促进效果>Ca(Ⅱ)浸种>Cd(Ⅱ)浸种,特别地,Cd(Ⅱ)浸种降低了光形态建成、抗氧化酶活性及元素含量。La(Ⅲ)浸种对拟南芥光形态建成、暗形态建成、抗氧化酶活性和元素含量的促进效果大于叶面喷施。(2)从ETH信号的角度揭示了La(Ⅲ)浸种对拟南芥形态建成的影响机制。发现La(III)浸种增加了ETH含量及ETH信号正向调控因子EIN3的基因表达量和合成量。外源添加ETH合成前体物质1-氨基环丙烷-1-羧酸后,La(Ⅲ)浸种引起的拟南芥暗形态建成、光形态建成、抗氧化酶酶活性以及元素含量增加程度加大。与野生型拟南芥相比,La(III)浸种处理对EIN3蛋白功能受抑制突变体暗形态建成、光形态建成、抗氧化酶酶活性以及元素含量的促进程度明显降低。这些结果表明,La(III)浸种通过增加形态建成过程中ETH合成量及后续的ETH信号来促进植物形态建成、抗氧化能力和元素含量。与La(III)浸种相比,Cd(Ⅱ)和Ca(Ⅱ)浸种未影响ETH合成量和信号转导,表明La(III)浸种对植物形态建成的影响机制具有ETH特异性。此外,La(Ⅲ)浸种对拟南芥ETH合成量和信号转导的促进作用大于叶面喷施。(3)利用激光共聚焦显微镜和转录组测序技术(RNA-seq),从胞吞作用和转录组角度揭示La(Ⅲ)浸种对拟南芥ETH信号的影响。发现La(Ⅲ)浸种激活叶细胞胞吞作用,同时增强根细胞胞吞作用。通过使用CEM功能缺陷突变体,进一步确定La(Ⅲ)浸种激活的胞吞作用为CME。Ca(Ⅱ)和Cd(Ⅱ)浸种未引起拟南芥叶细胞和根细胞胞吞作用变化,且La(Ⅲ)浸种对叶和根细胞胞吞作用的激活程度高于叶面喷施。La(Ⅲ)浸种激活的CME对ETH合成和信号转导起正向调控作用,具体表现为随着叶和根细胞中CME增强,ETH含量、EIN3蛋白相对表达水平和EIN3基因表达量上升。当CME功能被抑制后(CME功能缺陷突变体),上述效应消失,表明La(Ⅲ)浸种通过激活CME来调控EHT信号。此外,La(Ⅲ)浸种增加拟南芥基因DEGs数量,且明显多于Ca(Ⅱ)和Cd(Ⅱ)浸种。GO富集分析结果表明,经La(Ⅲ)浸种处理的拟南芥中DEGs主要富集在胞吞作用、细胞缺氧响应、细胞对ETH刺激的响应、CCR4-NOT核心复合体、离子通道复合体以及金属离子跨膜转运蛋白活性等条目。KEGG富集分析结果表明,经La(Ⅲ)浸种处理的拟南芥中DEGs主要富集在植物-病原体相互作用、甘氨酸、丝氨酸和苏氨酸代谢、苯丙烷类生物合成以及植物激素信号转导等条目。经相关性分析发现,这些条目所代表的信号通路对La(Ⅲ)浸种引起的ETH信号变化起到调控作用。更多还原

【Abstract】 Rare earth elements(REEs)are widely used in agriculture to promote crop growth due to their unique physical and chemical properties,but routine application methods,such as leaf spraying and soil application,cause them to accumulate in the environment and pass through the food chain,ultimately threatening human health.In contrast,REEs immersion can be used as an alternative way to reduce its environmental accumulation.Photomorphogenesis is a process in which plants rely on light to regulate plant development.It is a stage of transformation after dark morphogenesis,which is crucial for the subsequent development of seedlings.Studies have shown that ethylene(ETH)plays an important regulatory role in plant morphogenesis(dark or light morphogenesis),and soil application of REEs can induce endogenous ETH synthesis by activating clathrin-mediated endocytosis(CME)and increase the level of EIN3 protein,a positive regulator of ETH signaling,thereby regulating morphogenesis.Does La(Ⅲ)soaking affect plant morphogenesis?Is the effect regulated by ETH signals?Is the regulation of morphogenesis by La(III)soaking also related to the activation of CME by La(III)?In view of this,in this paper,the model plant Arabidopsis Thaliana was selected as the research object,with La(Ⅲ)as the representative of REEs,to study the effect of REEs immersion on plant morphogenesis,and to reveal the mechanism from the perspective of ETH signal.The effect and mechanism of La(Ⅲ)seed soaking were compared with that of Cd(Ⅱ)(harmful element representative)and Ca(Ⅱ)(beneficial element representative),and the conventional application of REE(leaf spraying).From the perspective of La(Ⅲ)activation of CME and transcriptomics,the mechanism of the effect of La(Ⅲ)soaking on plant ETH signal was revealed.The main results were as follows:(1)It was found that La(Ⅲ)soaking could promote dark and light morphogenesis of Arabidopsis.The results showed that the length of hypocotyl was shortened,the bending Angle of tip hook was increased and the content of Pchlide was decreased,and the maximum effect was 60μM soaked seed for 12 h.The soaking of La(Ⅲ)promoted photomorphogenesis:chlorophyll content,net photosynthetic rate,transpiration rate and intercellular carbon dioxide concentration increased,and the increase reached the maximum at 22 days.The activity of antioxidant enzymes(peroxidase,superoxide dismutase,catalase)and the content of elements(Ca,K,P,Mg,Mn,Fe,Cu,Zn)were increased by La(Ⅲ)immersion.The effect of La(Ⅲ)soaking on dark morphogenesis was higher than that of Ca(Ⅱ)soaking and higher than that of Cd(Ⅱ)soaking.The promotion effect on dark morphogenesis was>Ca(Ⅱ)>Cd(Ⅱ).In particular,Cd(Ⅱ)decreased photomorphogenesis,antioxidant enzyme activity and element content.The effects of La(Ⅲ)leaching on light morphogenesis,dark morphogenesis,antioxidant enzyme activity and element content of Arabidopsis Thaliana were greater than those of foliar spraying.(2)From the perspective of ETH signal,the influence mechanism of La(Ⅲ)immersion on Arabidopsis morphogenesis was revealed.It was found that La(III)soaking increased ETH content and gene expression and synthesis of EIN3,a positive regulator of ETH signal.The dark morphogenesis,photomorphogenesis,antioxidant enzyme activity and element content of Arabidopsis thaliana induced by La(Ⅲ)soaking were significantly increased after the addition of 1-aminocyclopropane-1-carboxylic acid,the precursor of synthesis of ETH.Compared with wild-type Arabidopsis thaliana,the effects of La(III)soaking on dark morphogenesis,light morphogenesis,activity of antioxidant enzyme and element content of EIN3 protein inhibited mutant were significantly reduced.These results suggest that La(III)soaking promotes plant morphogenesis,antioxidant capacity and element content by increasing the amount of ETH synthesized during morphogenesis and the subsequent ETH signal.Compared with La(III)soaking,Cd(Ⅱ)and Ca(Ⅱ)soaking did not affect ETH synthesis and signal transduction,indicating that the mechanism of La(III)soaking on plant morphogenesis was ETH specific.In addition,La(Ⅲ)soaking had a greater effect on ETH synthesis and signal transduction than foliar spraying.(3)Laser confocal microscopy and transcriptome sequencing(RNA-seq)were used to reveal the effects of La(Ⅲ)immersion on ETH signaling in Arabidopsis Thaliana from the perspective of entosis and transcriptome.It was found that La(Ⅲ)soaking activated endocytosis in leaf cells and enhanced endocytosis in root cells.The endocytosis induced by La(III)immersion was further identified as CME by the use of CEM functionally deficient mutants.Ca(Ⅱ)and Cd(Ⅱ)soaking did not cause any change in endocytosis of Arabidopsis leaf and root cells,and La(Ⅲ)soaking was more active than foliar spraying.CME activated by La(Ⅲ)seed soaking had positive regulatory effects on ETH synthesis and signal transduction.Specifically,with the increase of CME in leaf and root cells,the ETH content,the relative expression level of EIN3 protein and the expression level of EIN3 gene increased.When CME function was inhibited(CME dysfunction mutant),the above effect disappeared,indicating that La(III)impregnated seed regulates EHT signaling by activating CME.In addition,the number of Arabidopsis gene DEGs was increased by La(Ⅲ)soaking,which was significantly higher than that by Ca(Ⅱ)and Cd(Ⅱ)soaking.The results of GO enrichment analysis showed that the concentration of DEGs in Arabidopsis thaliana treated with La(Ⅲ)was mainly related to endocytosis,cell hypoxia response,cell response to ETH stimulation,CCR4-NOT core complex,ion channel complex,and metal ion transmembrane transporter activity.The results of KEGG enrichment analysis showed that DEGs was mainly concentrated in plant-pathogen interaction,glycine,serine and threonine metabolism,phenylpropanoid biosynthesis and plant hormone signal transduction in Arabidopsis thaliana treated with La(Ⅲ).Through correlation analysis,it was found that the signal pathways represented by these items played a regulatory role in the changes of ETH signal induced by La(Ⅲ)immersion.更多还原