网格蛋白介导的内吞作用参与银合欢适应铁缺乏胁迫机制研究

The mechanism of the adaptation in Leucaena leucocephala to iron deficient conditions [named as Fe(d)], where iron becomes insoluble and unavailable, needs to be investigated. Our previous study showed that the mimosine contents in roots and root exudates were significantly increased in Leucaena plants in response to iron deficiency. The expression of gene LlCSase encoding an enzyme catalyzing the biosynthesis of mimosine,was also markedly enhanced in Leucaena roots in response to iron deficiency..Mimosine is a strong iron chelator with a binding constant of ten to the power of 36. Mimosine, biosynthesized and secreted by Leucaena plants, can act as an efficient natural phytosiderophore and bind with trace amounts of iron to form a chelate Fe(III)-mimosine, which may then be taken up as iron-phytosiderophore intact molecule like in some Gramineae grasses..In the present study, with the treatments of iron deficiency [Fe(d)], or Fe(d) plus ferric reductase inhibitors, or Fe(d) plus ferric reductase inhibitors and specific inhibitors of clathrin-mediated endocytosis (CME), the mimosine content determination by HPLC, the expression detection of LlCHC and LlCLC genes,encoding Leucaena clathrin heavy chain and light chain respectively, by real-time RT-PCR, the counting of CCP (clathrin-coated pits) number in cell defined by TEM (Transmission electron microscope) observation and Image J analysis, the CME intensity analysis by laser scanning confocal microscope (LSCM), the iron content determination by ICP/AES (Inductively coupled plasma/Atomic emission spectrometry), and the leaf growth and development assay evaluated by the appearance of typical iron chlorosis symptoms or not, in hydroponically cultured Leucaena seedlings will be performed to demonstrate physiologically, biochemically and ultracytologically the uptake of the chelate Fe(III)-mimosine by clathrin-mediated endocytosis (CME) and the release of available iron from the chelate. Transgenic Leucaena plants constitutively expressing LlCHC gene hub (constitutive expression of CHC gene hub has a dominant negative effect on the assembly of the clathrin coat, thereby inhibiting its endocytic function) and transgenic Leucaena plants expressing LlCLC gene RNAi construct (interfering the assembly of clathrin) are to be generated, and the expression of LlCHC and LlCLC genes, the number of CCP in the cell, the CME intensity, the iron content, and the leaf growth and development assay in those Leucaena plants will be determined to demonstrate genetically and ultracytologically that clathrin-mediated endocytosis of the chelate Fe(III)-mimosine is involved in the adaptation to iron deficiency in Leucaena leucocephala. These investigations play an important role in getting further knowledge of the role of clathrin-mediated endocytosis in nutrient uptake in plant cells, and in gaining further insight into the iron uptake mechanism in plants.

银合欢能适应铁缺乏环境，其机制不清楚。我们前期研究发现，铁缺乏胁迫提高银合欢根及根系分泌物中含羞草素含量、增强根中含羞草素合成相关酶基因表达。已知含羞草素是强铁螯合剂，可作为高效植物铁载体，与铁缺乏环境中三价铁形成三价铁-含羞草素螯合物。本研究进而在铁缺乏胁迫下借助三价铁还原酶抑制剂和网格蛋白介导的内吞作用(CME)特异抑制剂处理、以及在银合欢中组成型表达LlCHC hub(对网格蛋白装配有显性负效应)、在银合欢中表达网格蛋白轻链基因(LlCLC) RNAi构建的遗传学手段，阻碍网格蛋白装配和其功能，抑制银合欢细胞中CME，分析各种处理下及各种基因型银合欢根细胞的含羞草素含量、LlCHC与LlCLC表达水平、网格蛋白包被小窝(CCP)数目、CME强度、铁含量、以及植株是否有缺铁失绿症状，从生理生化、遗传学与细胞超微结构上明确网格蛋白介导三价铁-含羞草素螯合物的内吞参与银合欢适应铁缺乏胁迫。

本项目参与人美国夏威夷大学Borthakur教授多年的研究发现，银合欢能适应铁缺乏（iron deficiency）环境，能在盐碱地和干旱地区生长良好。然而，银合欢适应铁缺乏环境的机制研究尚未见报道，我们认为这种适应性与银合欢在进化过程中形成的某种高效铁吸收机制有关，银合欢中这种高效的铁吸收机制不清楚。本项目在铁缺乏胁迫下，借助三价铁还原酶抑制剂和网格蛋白介导的内吞作用（CME）特异抑制剂单独或结合处理，发现各种不同组合处理下银合欢根细胞中含羞草素含量、LlCHC及LlCLC基因表达水平、CCP数目、以及CME强度在变化趋势上呈现高度一致性，且铁缺乏胁迫下，三价铁还原酶抑制剂和CME特异抑制剂共同处理时，银合欢植株出现新叶缺铁失绿症状，而三价铁还原酶抑制剂或CME特异抑制剂单独处理时，植株均未出现缺铁失绿症状。从生理生化与细胞超微结构上说明CME参与了三价铁-含羞草素螯合物进入银合欢细胞，为植株生长提供所需的铁，以使银合欢适应铁缺乏环境。.另外，通过在银合欢中组成型表达LlCHC hub的遗传学手段，阻碍网格蛋白蛋白装配从而抑制银合欢中网格蛋白介导的内吞作用，结果发现，铁缺乏胁迫能显著增强野生型银合欢根细胞对胞吞作用示踪剂FM4-64的内吞，但不能增强组成型表达LlCHC hub转基因银合欢（35S::Hub）对FM4-64的内吞作用，从遗传学上说明铁缺乏胁迫促进银合欢根细胞的内吞作用是由网格蛋白介导的；铁缺乏胁迫下，35S::Hub转基因银合欢植株中铁含量显著低于野生型的，35S::Hub植株新复叶出现明显的缺铁失绿症状，而野生型植株适应铁缺乏胁迫，从遗传学上进一步说明网格蛋白介导三价铁-含羞草素螯合物的内吞作用参与银合欢适应铁缺乏胁迫。.本研究结果对于阐明银合欢适应铁缺乏环境的机制有重要的科学意义，并可丰富根据铁吸收机制划分的策略II植物的类型（即银合欢虽不是禾本科植物，铁缺乏胁迫却能促进其合成一种天然高效植物铁载体-含羞草素，并分泌到根际周围，与铁缺乏环境中的三价铁形成Fe(III)-含羞草素螯合物，通过CME转运入细胞内，为细胞提供所需要的铁）。