Accumulation Of Chromium And Cadmium In Coptis Chinensis Franch.

Coptidis rhizoma are the dry rhizome of the Ranunculaceae plant of Coptis chinensis Franch.,Coptis deltoidea C.Y.Cheng et Hsiao,and Coptis teeta Wall.,which are commonly referred to as“weilian”,“yalian”,and“yunlian”,respectively.Coptidis rhizoma is one of the common traditional medicinal materials in China and it is famous and precious.It has a medicinal usage history of more than 2000 years in China.In recent years,with the increasing recognition of traditional Chinese medicine(TCM)in the world,the safety of TCM has also received worldwide attention.The problem of excessive heavy metals in TCM,including coptidis rhizoma,is frequently reported,which not only affects its safety,but also seriously affects its export.At present,most of the studies on the heavy metals in coptidis rhizoma are only in the content detection,while,the thorough and systematic study on the the distribution,morphology and accumulation and detoxification mechanism of heavy metals in plants have not been reported.In this paper,we systematically studied the distribution of Cr and Cd,their morphology,and the expression of metallothionein genes related to heavy metal absorption in C.chinensis Franch.by using some modern advanced technology,including synchrotron radiation microscopic X-ray fluorescence(SR-?XRF),laser ablation inductively coupled plasma mass spectrometry(LA-ICP-MS),inductively coupled plasma mass spectrometry(ICP-MS),X-ray absorption near-edge spectroscopy(XANES),and molecular biology techniques.The main contents and results of this paper are as follows:1.We have studied the spatial distribution of Cr in the root,rhizome and petiole in C.chinensis Franch.by SR-?XRF and LA-ICP-MS imaging techniques.Our imaging results show that Cr distribution patterns vary among tissues in C.chinensis Franch..In roots,Cr principally accumulates in the central vascular cylinder,while in rhizomes,Cr occurs mostly in external layers that correspond to the periderm and some outer cortex.Our LA-ICP-MS results show that Cr in petioles is mostly accumulated in the cortex and some vascular bundles.Our?-XRF data suggest Cr enters the central vascular cylinder after absorption from the root;we therefore speculate that Cr may cross the endodermis via symplast within the root before being up-transported along the xylem and then principally accumulate in the rhizome periderm and some outer cortex,as well as in the cortex and some vascular bundles of petioles.The distribution pattern of Cr is very similar to that of Ca,suggesting that Cr absorption may be related to Ca²⁺channels.As Cr is structurally similar to certain essential elements,it may affect plant mineral nutrition in a complex manner.Our LA-ICP-MS results show that,compared with Cr0h group petioles,levels of both Cu and Mn markedly increased under Cr stress suggesting that Cr affected nutrient uptake by C.chinensis Franch..2.We have studied the spatial distribution of Cd in the root,rhizome and petiole in C.chinensis Franch.by LA-ICP-MS imaging techniques.From the rhizome LA-ICP-MS images,Cd was found mainly accumulated in the periderm,cortex,pith,and root trace vascular bundle,with higher Cd concentration in the periderm and cortex and the lower in the pith and root trace vascular bundle.Cd shares many physical similarities(charge and ionic radius)with Ca.In addition,Ca²⁺channel is the main pathway for Cd into plants.Thus,the similar distribution pattern between Cd and Ca might suggested that Ca²⁺channel might be a pathway for Cd into C.chinensis Franch..Our rhizome LA-ICP-MS imaging result showed that Cd affected nutrient elements uptake by C.chinensis Franch.,eg.Cu,Mn,and P.3.We have studied the subcellular distribution of Cr and Cd in different tissues of C.chinensis Franch.by using ICP-MS.The results showed that the cell wall was the principal Cr and Cd storage compartments in C.chinensis Franch.,and after crossing the plant cell walls,Cr and Cd were sequestered predominantly within vacuoles when the plant was subjected to Cr and Cd stress,functioning to reduce the toxic effects of this metal.That is,Cr and Cd accumulated preferentially in cell wall and then in vacuole in C.chinensis Franch..4.We have studied the Cr speciation in different tissues of C.chinensis Franch.by using XANES.The result showed that a reduction of Cr(VI)to Cr(III)occurred when C.chinensis Franch.treated with Cr(VI).This reduction is thought to represent an important detoxification pathway.Both histidine and phosphate may play important roles in Cr accumulation and detoxification in C.chinensis Franch..Our XANES liner combination fitting(LCF)data revealed an interesting result,that the medicinal plant C.chinensis Franch.contains the elemental chromium Cr(0)form.5.We have studied the chemical forms of Cd in C.chinensis Franch.by using ethanol,d-H₂O,NaCl,HAc,and HCl to extract different Cd forms.Results demonstrated that large part of Cd existed in less mobile and less toxic forms in all C.chinensis Franch.tissues.Therefore,we can deduce that higher proportions of Cd with lower migratory and less toxic to plant cells could be responsible for the adaptation of C.chinensis Franch..The proportion of Cd extracted by HAc and HCl increased in rhizomes and leaves after Cd exposure.This demonstrated that combining with phosphate and oxalate to give undissolved complex may be the detoxification response.The study on subcellular distribution of Cr and Cd,and chemical forms of Cd all showed that the level of Cr and Cd were both higher in roots than that in rhizomes and leaves.That is,most of Cd was fixed in roots,which suggested retention of Cd in its long-distance translocation from roots to rhizomes and leaves,so as to reduce the toxicity to plants.This is the tolerance mechanism of C.chinensis Franch.to Cr and Cd accumulation.6.Through the study on the speciation of Cr and the chemical forms of Cd in C.chinensis Franch.,we found that toxic Cr(VI)was not detected in the rhizome without Cr treatment,and that the concentration of Cd in ethanol and water,which were commonly used as extraction solvents of TCM,was far lower than the national standard of Cd?0.3 mg kg^-1.Therefore,we considered that the existing heavy metal limit standard of TCM is not scientific and reasonable enough.Different valence state and different chemical form of the same element represent the different toxicity,so with a single total content of an element to represent its toxicity is obviously unreasonable.It is necessary to establish scientific and reasonable limit standards of heavy metals in TCM by considering the valence state and chemical form of heavy metals and other aspects related to its toxicity.7.The metallothionein gene(CcMT2)was cloned from C.chinensis Franch.for the first time.In addition,the tissue specific expression of CcMT2 and the effect of Cr and Cd on its expression were studied.The results showed that CcMT2 was expressed in all tissues,and the order of expression level in different tissues was leaf>root>rhizome.Both Cr and Cd treatments induced up-regulation of CcMT2 expression.This suggested that the metallothionein encoded by CcMT2 may be involved in the accumulation and detoxification mechanism of Cr and Cd in C.chinensis Franch..Through this study,the accumulation and detoxification mechanism of Cr and Cd in C.chinensis Franch.was explored,which could provid a theoretical reference for reducing the content of Cr and Cd.At the same time,this study also provides the data basis for fomulating the scientific and reasonable heavy metal limit standards of TCM,and also provides the theoretical basis for other heavy metal research of TCM.