Mechanism Of Nitrogen Application Enhancing Cadmium Accumulation Capacity In Lolium Multiflorum Lam.

Cadmium(Cd)is the most toxic heavy metal contaminant.Cd rice,which is produced by accumulating Cd contaminant into rice in paddy field,has become a serious food safety problem.How to remove Cd contaminant from paddy field to maintain food safety has been attracted more and more attention from researchers.Among various Cd remediation technologies,phytoextraction,one of phytoremediation,is likely to be more acceptable to the public because of its advantages of small environmental disturbance.At present,several Cd hyperaccumulators have been selected and used in phytoextraction.However,their efficiency of phytoremediation is limited by small biomass,poor environmental adaptability and other drawbacks.Therefore,researchers began to explore the phytoremediation potential with some crops featured in large biomass and certain Cd accumulation capacity.In addition,appropriate agronomic techniques,such as fertilization,are adopted to improve the Cd phytoremediation efficiency.Italian ryegrass(Lolium multifolorum Lam.)is widely planting gramineous forage after rice(Oryza sativa L.)in paddy field characterized by Cd accumulation.It has the potential of Cd phytoremediation in paddy field.At present,there are few studies on Cd tolerance and accumulation capabilities in Italian ryegrass,especially the effect and mechanism of N application on these capabilities,compared with other crop plants.In this study,Cd tolerance and accumulation capabilities of Italian ryegrass and its potential for application to phytoextraction were comprehensively evaluated in the two-year pot experiment by comparison with perennial ryegrass(Lolium perenne L.),which was featured with stronger Cd tolerance and accumulation capacities and learned from previous studies.Moreover,the effects of N application on Cd tolerance and accumulation capacities as well as the uptake mechanism were investigated by pot experiment and absorption experiment in Italian ryegrass.The results are as follows.1.Italian ryegrass is more tolerant to Cd,and it is a potential phytoremediation plant for Cd-contaminated paddy fieldThe results in 2016 showed that compared with perennial ryegrass,the inhibition ratio of Cd on the growth in Italian ryegrass was lower,and the toxicity as well as death threshold of Cd was higher.There was a significant effect on malondialdehyde(MDA)content,peroxidase(POD)and catalase(CAT)activities in perennial ryegrass under 120 mg·kg-1 Cd treatment,while there was no significant effect on those in Italian ryegrass.Similarly,120 mg·kg-1 Cd treatment significantly reduced the chlorophyll content,net photosynthetic rate(Pn),maximum light energy conversion efficiency(Fv/Fm)and actual photochemical quantum yield(ΦPSII)in perennial ryegrass,while the effect on those in Italian ryegrass was insignificant.The chlorophyll content was even improved by 40 and 80 mg·kg-1 Cd treatment in Italian ryegrass.The results in 2017 were similar to those in 2016,while the results of antioxidant enzyme activity in 2017 were slightly different.There was no significant difference in the POD and CAT activities of Italian ryegrass under Cd treatment,while the SOD activity was significantly decreased in 2017.Meanwhile,the enzyme activities of perennial ryegrass were significantly increased.These results of twoyear pot experiment suggested that Italian ryegrass was able to tolerate higher Cd concentrations.Higher Cd tolerance of Italian ryegrass was contributed by its stronger antioxidant enzyme system and more stable photosynthesis,and indicated its potential as a phytoremediation plant for Cd-contaminated.2.Italian ryegrass is more accumulative to Cd,and its root uptake pattern of Cd2+ is both active uptake and passive uptake through Ca2+ channelThe results of two-year pot experiment showed that the shoot Cd concentration and accumulation as well as TF and TAF values in Italian ryegrass were higher than those of perennial ryegrass.In contrast,the root Cd concentration in Italian ryegrass was lower than perennial ryegrass in two-year pot experiment.It indicated that Italian ryegrass had a higher root to shoot translocation and shoot accumulation capacity of Cd,and thus obtained higher Cd phytoextraction efficiency.The uptake experiment showed that DNP significantly reduced the root Cd concentration in Italian ryegrass.The results of twice uptake experiments confirmed that LaCl3 significantly inhibited the root Cd concentration.Compared with control,low Ca2+significantly increased the root Cd concentration,while high Ca2+ was the opposite.TEAC had no significant effect on the root Cd concentration.These results suggested that both active uptake and passive uptake through Ca2+channels participated in the uptake of Cd2+by root in Italian ryegrass,but no uptake through K+channels was found.3.N application is an effective meansure to enhance Cd accumulation capacity,which is contributed by the accelerated active uptake of Cd2+by the root in Italian ryegrassThe pot experiment results showed that N application effectively promoted the growth of Italian ryegrass under Cd stress.N application significantly increased SOD,POD and CAT activities,chlorophyll content,Pn,Fv/Fm and ΦPSII in Italian ryegrass under Cd stress.N application significantly increased the shoot Cd concentration and accumulation as well as the TF and TAF values in Italian ryegrass.N application significantly improved the inhibition ratio of DNP rather than LaCl3.N application significantly decreased the Km value and improved the a value,but no significant effect on the Vmax value in MichaelisMenten equation.These results suggested that N application not only increased the shoot biomass,but also promoted the root to shoot translocation of Cd and increased shoot Cd concentration in Italian ryegrass.According to the results of Michaelis-Menten equation,we proposed that the higher affinity to Cd2+of the membrane transporter after N application enhanced the active uptake of Cd2+,which contributed to the faster rate of Cd2+uptake by the root in Italian ryegrass.Therefore,the reason why N application enhanced the Cd accumulation of Italian ryegrass was it promoted Cd2+ uptake by the root,and enhanced the Cd tolerance thus allowing a higher Cd concentration in Italian ryegrass.In conclusion,Italian ryegrass was more tolerant,accumulative and transportable to Cd than perennial ryegrass and was a very promising plant for Cd phytoextraction.Moreover,N application not only enhanced the Cd tolerance and the biomass under Cd stress,but also promoted the uptake of Cd2+ by the root and increased the Cd concentration,thus enhanced the Cd accumulation capacity in Italian ryegrass.Therefore,Italian ryegrass had great potential for application in the phytoremediation of Cd-contaminated soils,and N application was an effective measure to enhance its Cd phytoremediation efficiency.