Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia

ABSTRACTThe rhizobia-legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain <i>Ensifer medicae</i> MA11 in Cu contaminated substrates. The <i>copAB</i> genes from a Cu resistant <i>Pseudomonas fluorescens</i> strain were expressed in <i>E. medicae</i> MA11 under the control of the <i>nifH</i> promoter. The resulting strain, <i>E. medicae</i> MA11-<i>copAB</i> was able to alleviate the toxic effect of Cu in <i>Medicago truncatula</i>. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate 2-fold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered <i>E. medicae</i> strain increased Cu accumulation in roots and decreased the content in shoots. Thus, <i>E. medicae</i> MA11-<i>copAB</i> increased the capacity of <i>M. truncatula</i> to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the <i>nifH</i> promoter-<i>copAB</i> construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia mycrosymbionts of authoctonous legumes growing in Cu contaminated soils.Supplementary material

摘要：根瘤菌-豆科植物共生互作被认为是修复重金属污染土壤的高效且适宜的根际稳定化工具。应用该共生体系的核心前提之一，是获取兼具重金属耐受性与高效共生功能的根瘤菌菌株。本研究旨在提升耐砷野生型菌株苜蓿中华根瘤菌（Ensifer medicae）MA11在铜污染基质中的共生性能。将来自抗铜菌株荧光假单胞菌（Pseudomonas fluorescens）的copAB基因，在nifH启动子的调控下导入苜蓿中华根瘤菌MA11中，所得重组菌株苜蓿中华根瘤菌MA11-copAB可有效缓解铜对蒺藜苜蓿（Medicago truncatula）的毒害作用。
当铜浓度达到300 μM时，接种野生型菌株的蒺藜苜蓿植株，其根、茎干物质产量、氮含量、根瘤数量及光合速率均出现显著下降；而接种重组菌株的植株上述各项参数均未发生明显改变。此外，该重组菌株诱导形成的根瘤中铜积累量可达野生型菌株根瘤的2倍。进一步研究发现，工程化改造后的苜蓿中华根瘤菌菌株可促进蒺藜苜蓿根系对铜的积累，并降低铜向地上部的转运量。综上，苜蓿中华根瘤菌MA11-copAB可提升蒺藜苜蓿对铜的根际稳定化能力，降低铜的转运系数，避免重金属进入食物链。携带nifH启动子-copAB表达盒的质粒可作为实用的生物工具，用于依托豆科植物的铜污染土壤根际稳定化修复，该质粒可被转移至铜污染土壤中土著豆科植物的不同根瘤菌共生伙伴中。
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