| Phytoextraction is a potentially cost-effective and environmentally friendly technique to eliminate heavy metal contamination from environment mainly by plant uptake. Plant growth promoting bacteria (PGPB) are beneficial microbes isolated from rhizosphere soil or plant and have a variety of PGP features. Some PGPB with heavy metal tolerance, which can improve phytoextraction efficiency, have caused extensive attention. However, the effects and mechanisms of PGPB on heavy metal phytoextraction in different plant systems are not clear yet. In this research, hyperaccumulator Sedum alfredii (S. alfredii) and cadmium (Cd) accumulating Brassica napus were selected as plant materials, and we conducted a serious of pot and hydroponic experiments, combining with phytophysiology, genomics and proteomics, using high-throughput sequencing, Real-Time PCR (RT-PCR), Fluorescence in situ hybridization (FISH), Green fluorescent protein (GFP) labeling and isobaric tags for relative and absolute quantitation (iTRAQ), to elucidate the effects and mechanisms PGPB on plant growth as well as Cd uptake and accumulation. The main results were as follows:1) S. alfredii growth and the endophytic bacteria community structure in 4 different soils with artificial Cd contamination were investigated by pot experiments through high-throughput sequencing techniques. The results showed that S. alfredii growth and the endophytic bacteria community structure were significantly influenced by soil properties and Cd contaminated levels. In red soil, S. alfredii shared low biomass and high Cd concentration, and the bacteria community structure was simple. However, in alkaline black soil, S. alfredii grew well and had a large biomass as well as low Cd concentration, while the bacteria community structure was more complicated. The bacteria community structure was different in the same soil treated with different Cd treatment. At high Cd treatment, the bacteria community structure was more complicated in black soil, paddy soil and red soil. At all treatments, Acinetobacter, Escherichia, and Pseudomonas were the most common endophytic bacteria, indicating some special bacteria could be choose to colonize in hyperaccumulators.2) PGPB were isolated and identified from rhizosphere soil and inner tissue of 5. alfredii. By pot experiments, the effect of two rhizosphere bacteria and two endophytic bacteria on plant growth and Cd accumulation were compared. FISH and GFP were conducted to demonstrate endophytic bacteria could colonize successfully in the B. napus roots. The results showed that both rhizosphere and endophytic bacteria could promote plant growth and Cd uptake. The endophytic bacteria performed better in this respect than the rhizosphere bacteria, and there were apparent differences among different endophytic bacteria. Due to its potential to enhance Cd uptake by the plant and to restrict Cd accumulation in the seeds, SaMR12 was selected as the most promising microbial partner of B. napus when setting up a plant-microbe fortified remediation system.3) The effects 5. alfredii and B. napus rotation and SaMR12 on Cd phytoextraction in three typical soils by pot experiment. The results showed that the accumulation of S. alfredii and B. napus was 63.4-477.6 μg, and the effect was the best in paddy soil. SaMR12 could further improve Cd phytoextraction efficiency (6-55%), and the effect was the best in black soil. SaMR12 could improve the diversity of rhizosphere bacteria community structure of S. alfredii, but had little effects on B. napus. Therefore, inoculation with PGPB is an effective method to improve phytoextraction efficiency of S. alfredii and B. napus rotation system.4) A hydroponic experiment was conducted to verify that SaMR12 could enhance Cd accumulation in S. alfredii under Cd stress. The results showed that inoculation of SaMR12 improved Cd accumulation and upregulated glutathione synthase (GS) expression, but slightly reduced malondialdehyde (MDA) concentration and alleviated Cd-induced damage in roots. The expression of related genes, such as PERI, ATPS GS and GSH1 were influenced by SaMR12. However, it didn’t alter the activities of antioxidant enzymes. These results indicate that although SaMR12 has no significant effects on antioxidases activities, it reduces H2O2 concentration by enhancing GSH concentration and relevant genes expression, and subsequently improves Cd tolerance and accumulation.5) A hydroponic experiment was conducted to investigate the effects of an endophytic bacterium SaMR12 on metal ions uptake and the expression of three metal transporter families of S. alfredii at different Cd treatment levels. The results showed that at low Cd condition, SaMR12 increased Fe, Zn and Cu content in shoot, elevated expression of SaIRTl, SaHMA2 and SaNRAMP3 in shoot and SaHMA2, SaNARMPl in root; and at high Cd condition, SaMR12 increased leave chlorophyll concentration and Fe/Mg content in shoot, elevated expression of SalRTl, SaZIP3, SaHMA2, SaHMA3 and SaNRAMP6 in shoot and SaIRT1, SaZIP3, SaHMA3, SaNARMP6 in root. Those results indicated that SaMR12 can elevate essential metal ions uptake and regulate the expression of transport genes to promote plant growth, enhance Cd tolerance and improve Cd accumulation.6) By pot experiments, iTRAQ was conducted to study the effects of PGPB on proteins expression of S. alfredii. When inoculated with PGPB,67 differential proteins were identified, including 40 upregulated proteins and 27 downregulated proteins. Compared to the plant grown mined soil for long time,230 differential proteins were identified, including 113 upregulated proteins and 117 downregulated proteins. Among those proteins, there were 15 co-upregulated proteins and 13 co-downregulated proteins. The results indicated that PGPB may promote plant growth by acceleration of nucleotide metabolism. Those upregulated proteins mainly participated in ribosomal peptide synthesis, photosynthesis, oxidative phosphorylation and glutathione metabolism, which indicated that PGPB can accelerate plant photosynthesis and enhance antioxidative capacity of plant to strengthen the adaptive ability of S. alfredii. |
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