群感信号AHLs调控杨树人工林短命细根衰老的微生态机制

Tree roots turnover is a critical process of terrestrial ecosystem carbon cycling. The senescence and death of ephemeral fine roots play an important role in determining the photosynthate allocation and forest productivity. phenolic acids are a group of important metabolic compounds via shikimic acid pathway in plant, and they could be released into soil via secretion from living roots or decomposition of plant dead residuals. Many studies indicated that phenolic acids played an important role in rhizosphere ecological process, and was especially considered as an important signal between plant-microbe interaction. Although it has been proved that phenolic acids rhizodepostion lead to heterogeneity of microbial community between rhizosphere and bulk soil, the relationship between phenolic acids rhizodepostion and fine roots senescence was still obscure. We hypotheses that the succession of microbial community on root-soil interface would affect the expression of quorum sensing signals, and then one dominant quorum sensing (QS) compounds, N-acyl-homoserine lactones (AHLs) which were metabolized by most gram-negative bacteria, would modulate programmed cell death (PCD) of ephemeral fine roots, and finally lead to the faster senescence and apoptosis death of fine roots. Based on the hypothesis, this study focuses on the micro-ecological mechanism of AHLs modulating senescence of fine roots in poplar plantation. Firstly, ephemeral fine roots (mainly 1,2 order root) in situ were sampled via ingrowth cores in spring (April), summer (July), autumn (September) separately. High-throughput sequencing technology would be employed to analyze the difference of microbial metagenome on root-soil interface between 1 and 2 orders, as well among three seasons. AHLs modules would also be detected via LC-ESI/MS analyzer to build the linkage between bacterial community succession and quorum sensing compounds metabolites. Secondly, we would design a series of controlled experiments to examine the effects of gradient concentration AHLs on poplar fine roots growth and development. In this part, we firstly employ transmission scanning electron microscopy (TSEM) technology to observe the structures and substructures of tissue and organells of fine roots, and then use fluorescent staining to examine the PCD charateristics under AHLs influence. Especially, we would employ multiple methods, such as real-time fluorescence quota PCR (RT-qPCR), proteins immunocytochemistry technology, to explore the regulation mechanism of AHLs on morphological development of fine root, including chalcone synthase (CHS) gene expression, CHS tissue positioning and chalcone content in roots. And finally, a scientific conceptual model about the relationship among AHLs -chalcone synthesis- PCD- root senescence- root apoptosis death, would be set up to give deeper insight into the regulation mechanism of fine roots senescence and death, and to clarify the relationship between trees rhizosphere proces

细根周转导致森林净初级生产力大量消耗，而衰老是细根周转最重要的阶段，短命细根尤其与林分生产力形成关系密切。本项目基于微生物-根系互作关系，在以往研究基础上开展杨树短命细根衰老调控机制研究，提出根土界面微生物群落趋异化导致细菌群感信号差异表达，进而调控短命细根细胞程序性死亡和细根衰老的科学假设。通过试验地原位获取杨树细根新生和衰老短命模块，基于宏基因组学技术对比分析根土界面细菌群落组成结构及群感信号(AHLs)通路相关基因表达差异，并检测AHLs在根土界面的原位表达量。然后通过室内布设AHLs浓度梯度试验，分析AHLs对杨树细根形态建成及细胞程序性死亡的影响，对查尔酮在细根组织的含量进行测定，同时对查尔酮合成酶进行组织定位并从转录水平定量检测查尔酮合成酶基因表达。建立微生物群落演变-群感信号表达-查尔酮合成-细胞程序性死亡-细根衰老关系模型，为探索人工林根际过程调控与生产力维持技术奠定基础。

树木根系周转是陆地生态系统C循环的核心，细根衰老和死亡决定树木光合产物分配和林分生产力。项目以土壤微生物和根系互作关系为出发点，通过固定试验地取样和室内布设控制试验两条途径，认真细致地开展了杨树细根生长的季节动态及代际差异、杨树细根寿命的代际差异及其影响因素、基于根序的杨树根际微生物组成和结构差异、杨树短命细根不同生长发育时期的形态特征及其根际微生物组特征、杨树根际高丝氨酸内脂的分离鉴定及含量检测、C8-HSL对杨树细根形态建成及细胞程序性死亡特征的影响、C8-HSL影响下根内查尔酮合成酶基因表达等8个方面的研究工作。研究发现，杨树细根数量特征具有明显的根序差异性，且随季节呈现明显变化动态。基于此，细根生长发育与人工林生产力关系极为密切。杨树细根累积生存率存在显著的根序差异性，并且根际微生物群落组成和结构亦差异显著，这与根际碳氮磷生态化学计量特征密切相关。为此，探究杨树短命细根根际微生物组的变化将可为揭示细根寿命的调控机制提供理论依据。杨树细根不同生长阶段根际碳组分的差异性可为根际微生物组成和结构变化提供支持，同时可影响根际养分有效性。基于液质联用色谱分析，树根际高丝氨酸内脂的种类主要是C8-HSL，其在土壤中的含量呈现随细根衰老的显著增加，该物质可能是杨树根际的主要群感信号分子并与短命细根的衰老密切相关。首先，C8-HSL可显著影响杨树细根生长的形态建成过程。其次，C8-HSL导致杨树细根内DNA断裂并加速细胞PCD过程，进而调控细根活力及生长。C8-HSL导致了杨树细根转录组分异，查尔酮合成酶基因表达量发生变化，导致与组织衰老重要的启动物质——查尔酮含量的变化，这可能是诱发杨树细根早衰的重要驱动力。基于上述研究结论构建了杨树短命细根衰老-群感信号表达的关系模型，深入揭示了树木细根衰老和凋亡的微生态调控机制，为今后深入探索人工林根际过程调控和生产力维持技术奠定了基础。