Editing of a Novel Cd Uptake-Related Gene CUP1 Contributes to Reducing Cd Accumulations in Arabidopsis thaliana and Brassica napus.

Brassica napus is a Cd hyperaccumulator, which is a serious threat to food and fodder safety. However, no related studies on developing Cd-safe B. napus have been reported yet. Here, we screened out a novel Cd uptake-related gene, AtCUP1, from the major facilitator superfamily in Arabidopsis thaliana. The mutation of AtCUP1 decreased Cd accumulation, both in roots and shoots of A. thaliana. Furthermore, the disruption of the AtCUP1 gene by the CRISPR/Cas9 system significantly reduced Cd accumulation in A. thaliana. Interestingly, the disruption of the BnCUP1 gene, an orthologous gene of AtCUP1, by the CRISPR/Cas9 system also diminished Cd accumulation in both roots and shoots of B. napus based on the hydroponics assay. Furthermore, for the field experiment, the Cd accumulations of BnCUP1-edited lines were reduced by 52% in roots and 77% in shoots compared to that of wild-type (WT) lines, and the biomass and yield of BnCUP1-edited lines increased by 42% and 47% of that of WT, respectively. Noteworthily, agronomic characteristics of B. napus were not apparently affected by BnCUP1-editing. Thus, BnCUP1-edited lines are excellent non-transgenic germplasm resources for reducing Cd accumulation without a distinct compromise in yield, which could be applied to agricultural production in Cd-contaminated soils.

The kinase CIPK11 functions as a positive regulator in cadmium stress response in Arabidopsis.

Cadmium (Cd) pollution in agricultural soil has always been a knotty problem, which made it necessary to find the mechanism related to Cd transport in plant. In this study, we found a novel character of the CIPK11 modulating the transport of Cd in Arabidopsis thaliana. Over-expression of CIPK11 (CIPK11OE#1-7, CIPK11OE#8-5) resulted in the increased tolerance to Cd stress, which embodied in higher fresh weight, lower Cd enrichment and reactive oxygen species (ROS) than the wild-type (WT) plants. qRT-PCR results showed a collective down-regulation of the expression of IRT1 and transcription factor genes FIT, bHLH039 in the CIPK11-overexpression plants after Cd stress. Overexpression of CIPK11 significantly increased the expression of ABA marker genes in Arabidopsis after Cd stress. With different concentrations of ABA treatment, the root length differences caused by Cd stress could be recovered. However the transcription levels of FIT and bHLH039 decreased in WT and cipk11 mutant when treated with ABA which indicated that ABA can inhibit the transcription of IRT1 by repressing FIT and bHLH039 expression. Taken together, our results demonstrated that the kinase CIPK11 responses to Cd stress by ABA signaling pathway.

A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering.

Reporter systems are routinely used in plant genetic engineering and functional genomics research. Most such plant reporter systems cause accumulation of foreign proteins. Here, we demonstrate a protein-independent reporter system, 3WJ-4 × Bro, based on a fluorescent RNA aptamer. Via transient expression assays in both Escherichia coli and Nicotiana benthamiana, we show that 3WJ-4 × Bro is suitable for transgene identification and as an mRNA reporter for expression pattern analysis. Following stable transformation in Arabidopsis thaliana, 3WJ-4 × Bro co-segregates and co-expresses with target transcripts and is stably inherited through multiple generations. Further, 3WJ-4 × Bro can be used to visualize virus-mediated RNA delivery in plants. This study demonstrates a protein-independent reporter system that can be used for transgene identification and in vivo dynamic analysis of mRNA.

Functional Analysis of Organic Acids on Different Oilseed Rape Species in Phytoremediation of Cadmium Pollution.

Cadmium (Cd) pollution in soil is becoming increasingly serious due to anthropogenic activities, which not only poses a threat to the ecological environment, but also causes serious damage to human health via the biological chain. Consequently, special concerns should be paid to develop and combine multiple remediation strategies. In this study, different subspecies of oilseed rape, Brassica campestris, Brassica napus and Brassica juncea were applied, combined with three organic acids, acetic acid, oxalic acid and citric acid, in a simulated Cd-contaminated soil. Various physiological and biochemical indexes were monitored in both plant seedling, growth period and mature stage. The results showed that organic acids significantly promoted the growth of Brassica campestris and Brassica juncea under Cd stress. The photosynthesis and antioxidant enzyme activities in Brassica campestris and Brassica juncea were induced at seedling stage, while that in Brassica napus were suppressed and disturbed. The enrichment of Cd in oilseed rape was also obviously increased. Brassica juncea contained relatively high resistance and Cd content in plant but little Cd in seed. Among the three acids, oxalic acids exhibited the most efficient promoting effect on the accumulation of Cd by oilseed rape. Here, a comprehensive study on the combined effects of oilseed rape and organic acids on Cd contaminated soil showed that Brassica juncea and oxalic acid possessed the best effect on phytoremediation of Cd contaminated soil. Our study provides an optimal way of co-utilizing oilseed rape and organic acid in phytoremediation of Cd contaminated soil.

Comprehensive construction strategy of bidirectional green tissue-specific synthetic promoters.

Bidirectional green tissue-specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue-specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequence selection and the bidirectional green tissue-specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium-mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activities in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiencies specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters, and we successfully created four bidirectional green tissue-specific synthetic promoters. It is the first report on bidirectional green tissue-specific promoters that could be efficiently applied in genetic engineering.

Overexpression of Three Duplicated BnPCS Genes Enhanced Cd Accumulation and Translocation in Arabidopsis thaliana Mutant cad1-3.

Phytochelatins are widely known to chelate heavy metal in vacuole and decrease plant damage. Phytochelatin synthase gene (PCS), which is involved in phytochelatins synthesis, is commonly designated as a key gene for phytoremediation. In our study, we cloned three duplicated BnPCS genes from Brassica napus and transformed them into Arabidopsis thaliana AtPCS1 mutant cad1-3, respectively. Three transgene lines and cad1-3 were subjected to a cascade of concentrations of cadmium (Cd) treatment. Evaluation of morphological and physiological measurement results show that transgene lines possess higher Cd tolerance and resistance than A. thaliana mutant cad1-3. The analysis of PCs and Cd contents in root and shoot collectively indicated that transgenic plants promoted Cd accumulation and translocation. In conclusion, all the three BnPCS transgene lines enhanced Cd tolerance, accumulation and translocation, which could provide gene resources for phytoremediation.

Variations in cadmium accumulation and distribution among different oilseed rape cultivars in Chengdu Plain in China.

Cadmium (Cd) is a widespread toxic heavy metal trace pollutant worldwide. The ability of Cd absorption and accumulation highly varies among different species and varieties. In order to screen oilseed rape cultivars which are appropriate for cultivation and application in Cd-contaminated soils, we conducted the field trial of 32 oilseed rape varieties in Shifang County of Chengdu Plain. The various biomass, Cd accumulation, and distribution patterns were investigated via determining the Cd concentration in different plant tissues. Moreover, the food safety risks of rapeseeds were finally assessed. The results indicated diverse responses to Cd stress appeared in various tested varieties, including plant biomass, seed yield, Cd concentration, and proportion in different tissues. And most Cd were concentrated in non-edible parts. Through cluster analysis, we found that Nanchongjie, Pengzhoubai, and J-25 belong to high-biomass and high-Cd-accumulated groups in experimental cultivars, which indicated that they could possess more biomass and gather higher Cd content in overground part, so they could be great materials for phytoremediation in Cd-polluted area. Besides, combined with the risk assessment of food safety in rapeseeds, cultivars 72A and 47 with the traits of high yield, low-Cd concentration, and low food safety risk can be considered as suitable materials to widely plant as cash crop. These results provide valuable reference for practical planting and application of oilseed rape in Cd-polluted areas.

Metagenomic analysis of microbial community and function involved in cd-contaminated soil.

BACKGROUND: Soil contaminated with the heavy metal Cadmium (Cd) is a widespread problem in many parts of the world. Based on metagenomic analysis, we investigated the functional potential and structural diversity of the microbial community in Cd-contaminated and non-contaminated soil samples and we explored the associated metabolic pathway network in cluster of orthologous groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: The results showed that microorganisms in these soils were quite abundant, and many of them possessed numerous physiological functions. However, Cd-contamination has the potential to reduce the microbial diversity and further alter the community structure in the soil. Notably, function analysis of the crucial microorganisms (e. g. Proteobacteria, Sulfuricella and Thiobacillus) indicated that these bacteria and their corresponding physiological functions were important for the community to cope with Cd pollution. The COG annotation demonstrated that the predominant category was the microbial metabolism cluster in both soil samples, while the relative abundance of metabolic genes was increased in the Cd-contaminated soil. The KEGG annotation results exhibited that the non-contaminated soil had more genes, pathways, modules, orthologies and enzymes involved in metabolic pathways of microbial communities than the Cd-contaminated soil. The relative abundance of some dominant KEGG pathways increased in the Cd contaminated soil, and they were mostly enriched to the metabolism, biosynthesis and degradation of amino acids, fatty acids and nucleotides, which was related to Cd tolerance of the microorganisms. CONCLUSIONS: Cd-contamination can decrease the taxonomic species of microbes in soil and change the soil microbial composition. The functional pathways involved in the soil change with microbial structure variation, many of which are related to the heavy metal tolerance of soil microbes. The Cd-contaminated soil microbes is a potential resource for exploring cadmium resistant or tolerant bacteria.