Characterization And Mutational Analysis Of Cadmium Transport Genes OsLCT1 And OsNramp5 In Rice

Rice（Oryza sativa L.）is one of the major crops in the world,which is the staple food of 60%Chinese people.With the improvement of people’s living standard,the requirement of rice quality is increasing day by day.Cadmium（Cd）is a non-essential element and has toxic effects on the growth and development of plants and animals,including human beings.The pollution from industrial sewage,the discharge of mineral waste and the use of mineral fertilizers have resulted in excessive Cd content in paddy field,which has in turn caused excessive Cd accumulation in rice grain,posing a serious threat to people’s health.Government has adopted various soil remediation measures to reduce the impact of soil Cd pollution,but progress is not satisfactory.In order to produce rice safely in Cd-polluted soil,OsLCT1 and OsNramp5 mutant lines were generated by CRISPR/Cas9-mediated mutagenesis to prevent Cd absorption and translocation to rice grain.Two mutant lines of OsLCT1（lct1x1,lct1x3）and two mutant lines of OsNramp5（nramp5x7,nramp5x9）were evaluated for grain Cd accumulation and agronomic performance in pot and field test,and four osnramp5 mutant lines（nramp5x7+1,nramp5x7-2,nramp5x9+1,nramp5x9-33）were analyzed for physiological and transcriptomic changes in hydroponic growth.The main results are summarized as follows:1.Rice mutant lines of OsLCT1 and OsNramp5 with different types of mutations were generated by the CRSPR/Cas9 system.15 lct1x1,70 lct1x3,1 nramp5x7 and 4nramp5x9,targeting on 1^st and 3^rd exon of OsLCT1,7^th and 9^th exon of OsNramp5,were created by employing CRISPR-S gene editing vector developed in our lab.The mutants were initially screened by HRM and confirmed by Sanger sequencing.Two-thirds of oslct1x1 T₀ plants were heterozygous and the remaining mutants carried biallelic mutations,11.4%,62.9%and 25.7%of oslct1x3 T₀ plants had heterozygous,biallelic and homozygous mutants respectively.Different length（from 1 bp to 49 bp）of insertion or deletion mutation types are produced,and the most common mutation types are single base insertion or deletion.2.All the oslct1 and osnramp5 mutations result in significant reduction of Cd concentration in rice grain in different extent.In paddy field soil containing⁰.9 mg Cd/kg,lct1x1 grains contained only about⁴0%（0.18 mg/kg）of the Cd concentration present in the grains of the wild-type parental line,which is less than the China National Food Safety Standard（0.2 mg/kg）.Similarly,the Cd concentration in lct1x3 grains was about⁸0%of the wild type.In paddy field soil containing⁰.85 mg Cd/kg and⁰.9mg Cd/kg,the Cd concentration in lct1x3 grains was 0.28 mg/kg and 0.37 mg/kg respectively,which were more than safety level set by the China National Food Safety Standard.Both osnramp5 mutants showed low grain Cd accumulation（<0.06 mg/kg）in grains produced in both in the paddy field（⁰.9 mg Cd/kg）and in pots with soil spiked with 2 mg Cd/kg.However,only nramp5x7+1 plants showed normal growth and yield,whereas growth of nramp5x9-33plants was severely impaired.The study showed that rice grains could be safely produced for human consumption in lightly contaminated paddy soil using the knockout mutants of OsLCT1 in 1^st exon,while osnramp5 mutant could be used to produce safe rice in soils with cadmium pollution higher than the third grade of national soil environmental quality standard（1.0 mg/kg）.3.OsNramp5 plays an important role in Cd and Mn transport and mutations inhibit their transport.When plants grown hydroponically with 5μM Cd for 3 weeks,Cd concentration in 4 osnramp5 mutants（nramp5x7+1,nramp5x7-2,nramp5x9+1 and nramp5x9-33）was drastically decreased by 70.3%⁷4.4%,67.4%⁸0.5%,60.3%⁷7.7%and 56.8%⁷7.8%in shoot and 71.4%⁷7.6%,69.8%⁷6.7%,75.2%⁸2.4%and 63.2%⁶6.4%in root compared to wild type.Mn concentration of mutants were dropped by⁹4%in shoot and 80.2%⁹0.9%in root compared to wild type with excessive Mn supply and without Cd.4.Different phenotypes were revealed for the four osnramp5 mutants grown in culture medium with different Mn concentrations.In plants grown hydroponically with0.35μM Mn for 3 weeks,the growth of mutant line nramp5x7+1 was similar with wild type,and there was no difference in fresh weight of shoot and root compared with wild type Xidao#1,while the fresh weight of shoot and root in mutant lines nramp5x7-2,nramp5x9+1 and nramp5x9-33 were decreased by 36.5%and 33.1%,27.6%and 18%,16.1%and 5.8%,respectively;In plants grown hydroponically with 7μM Mn for 3weeks,compared to wild type Xidao#1,the fresh weight of shoot and root in mutant lines nramp5x7+1 were increased by 12.2%and 10.0%,while the fresh weight of shoot and root in mutant lines nramp5x7-2 were decreased by 21.6%and 15.5%,the fresh weight of shoot in nramp5x9+1 and nramp5x9-33 were decreased by 12.6%and18%,whereas increased by 6.6%and 0.8%in root,respectively;In plants grown hydroponically with 140μM Mn for 3 weeks,compared to wild type Xidao#1,the fresh weight of shoot and root in mutant lines nramp5x7+1,nramp5x9+1 and nramp5x9-33were increased by 27.5%and 15.4%,12.6%and 18%,6.6%and 0.8%,respectively,while the fresh weight of shoot in mutant lines nramp5x7-2 were decreased by 6.4%and 12.2%.5.The mutant line nramp5x7+1 might contain an enhanced redox homeostasis system.Transcriptome sequencing of the wild type Xidao#1 and its 3 osnramp5 mutant lines grown hydroponically with 7μM Mn for 3 weeks were conducted.The analysis of gene ontology（GO）for all differentially expressed genes（DEGs）revealed that the GO terms involved in biosynthetic and metabolic process of oligosaccharide,disaccharide,trehalose,cellular carbohydrate were up regulated in both leaf and root of all the 3 mutants（nramp5x7+1,nramp5x7-2 and nramp5x9+1）.Compared with nramp5x9+1,the up-regulated DEGs were significantly enriched in redox homeostasis in leaf and root of mutant line nramp5x7+1.Furthermore,there were several DEGs involved in response to abiotic stress in nramp5x7+1 compared to other 2 osnramp5mutants and wild type in root and leaf analyzed by Venn diagram,indicating that enhanced redox homeostasis contributed to better growth in nramp5x7+1 when grown with low Mn supply and Cd stress.In summary,we generated OsLCT1 and OsNramp5 mutant lines by CRISPR/Cas9-mediated mutagenesis,and assessed the Cd accumulation and agronomic traits of plants cultivated in pot and paddy field.The reduction in Cd accumulation in grains of osnramp5 mutants were significantly higher than oslct1mutants,and only nramp5x7+1 performed well in growth among all the osnramp5mutants,and hence,could be used for rice production for safe consumption.The physiology and transcriptome analysis of osnramp5 mutants revealed that the enhanced redox homeostasis contribute to significant growth of nramp5x7+1 while part of OsNramp5 protein was truncated,indicating that nramp5x7+1 was not only appropriate for rice safety produce in Cd contaminated soil,but also in Mn-excess polluted paddy field.Meanwhile,the genetic analysis of OsLCT1 and OsNramp5 in this study make great sense for metal transport and regulation in rice.