Iron And Glutathione Mediated Physiological And Molecular Regulation Mechanisms Of Cadmium Accumulation In Poplars

Anthropogenic activities,such as mining,smelting,waste incinerating,combustion of fossil fuels and application of pesticides and fertilizers,have led to release of heavy metals（HMs）to the environment.Many countries,such as India,Japan and Nigeria,are polluted by heavy metals.And nearly 10,000,000 hectares of soil in our country are HMs-polluted.HMs contamination has posed a serious threat to the health of natural ecosystems and humans.Cadmium（Cd）is a toxic and non-essential element which can enter the food chain and be accumulated in organisms,causing serious damage to human beings.Therefore,it is necessary to repair heavy metal contaminated soil.Phytoremediation is referred to using plants to reduce concentrations of HMs in soils.Obviously,phytoremediation is an environmental friendly and cost effective method in comparison with physical and chemical remediation approaches.Populus species have been proposed for phytoremediation because of their high growth rates,large aboveground biomass compared with herbaceous plants.Iron（Fe）is essential for the synthesis of chlorophyll and the formation of thylakoids.GSH（glutathione）is functioned in the elimination of reactive oxygen species（ROS）and the chelation of toxic substances in cells.Fe and GSH play an important role in plant growth.The objectives of this study were to investigate the difference of Cd accumulation in different poplar genotypes under Fe-deficient condition,elucidate the mechanism of Cd uptake,migration and accumulation under different Fe supply levels and uncover the role of GSH in uptake and transport of Cd in poplar.For these purposes,we compared the ability to accumulate Cd of two poplar genotypes under Fe-deficient condition.And then,the physiological and transcriptional responses of poplar to Cd with Fe-deficiency or Fe-sufficiency were studied;Finally,we investigated the physiological and transcriptional responses of exogenous GSH-mediated Cd accumulation in poplar.In this study,we investigated the intraspecific variations of Cd accumulation in Populus nigra and P.cathayana with 0 or 50μM Fe Cl₃ in combination of Cd Cl₂.The photosynthetic rate,biomass,total root length,total root surface area,total root volume,Cd content in the aerial parts and total Cd amount in P.cathayana were higher than those in P.nigra.The photosynthesis rate,biomass,total root length,total root surface area,total root volume and Fe content in aerial parts were reduced under Cd treatment with normal Fe supply.The Fe content in roots was enhanced under Cd treatment.The inhibition of Cd on the photosynthetic rate of two poplar genotypes were aggravated under Fe-deficient condition.However,the Fe deficiency increased the Cd content in the tissues of P.cathayana.In Cd-stressed leaves of P.cathayana and P.nigra,the proportion of Cd in the cell wall was reduced by Fe deficiency.The proportion of Cd in organelles except the vacuole was increased in P.nigra.While the proportion of Cd in vacuole and soluble fractions was decreased in P.cathayana.This part of Cd was flowed to the organelles in P.nigra and soluble fraction in P.cathayana respectively.The increased proportion of Cd in organelle fractions aggravated the Cd toxicity in P.nigra.This difference may be related to the serious inhibition of photosynthetic rate of P.nigra by Cd in the absence of Fe.P.cathayana has greater biomass,stronger Cd accumulation ability,and superior regulatory ability under Cd toxicity.These results indicate that the difference of Cd proportion in subcellular fractions were related to the Cd tolerance.P.cathayana exhibited higher Cd accumulation ability.To clarify the physiological and transcriptional regulation mechanism of Fe-mediated Cd accumulation in poplar plants,P.cathayana were treated with different Fe levels（0,50 or 200μM Fe Cl₃）in combination of 0 or 120μM Cd Cl₂.Cd caused chlorosis of leaves.The photosynthetic rate,biomass,foliar Fe content were reduced,while the content of ROS and malondialdehyde（MDA）were increased in roots with Cd supply.The Cd-induced chlorosis and the production of ROS and MDA were exacerbated by Fe deficiency,but these were alleviated by high Fe supply.The biomass,foliar Fe content and total Cd,were reduced by Fe deficiency,but these were increased by high Fe supply.In total,213 different expressed lnc RNAs,87 different expressed mi RNAs and 1762 different expressed m RNAs were found.The m RNAs of IRT3,ZIP2,ZIP11 and NRAMP1 which encoding divalent ion transporters are involved in the transport of Cd and Fe.CAX1 and YSL3 are involved in the storage and transport of Cd and Fe.MYB72 regulated the expression of the nicotinamide synthase gene（NAS4）which funcationed in the transport of Fe to the aerial parts.Under high Fe supply condition,the upregulation of cme-mi R858＿R+1 and its cis-target gene MYB72 enhanced the transport of Fe to the leave.The upregulation of Cytochrome b561 with high Fe supply increases the efficiency of As A cycle,enhanced the ability to scavenge ROS,and thus reduced the oxidative stress caused by Cd.The upregulation of MSTRG.21421.1 under high Fe condition can reduce the molecules of ptc-mi R156g and roc-mi R156f for targeting SPL12,resulting in increasing transcript level of SPL12,which is function in the eliminating of ROS.These results indicate that the Cd-induced oxidative damage was aggravated by Fe deficiency and it was alleviated by high Fe supply.Poplar plants with high Fe supply have higher Cd enrichment ability.And the regulatory network of lnc RNAs-mi RNAs-m RNAs plays a role in the absorption,transport and accumulation of Cd in poplars.To investigate the physiological and molecular mechanism of exogenous GSH-mediated Cd accumulation,poplar plants were treated with Cd Cl₂ and exogenous GSH.The maximal net Cd²⁺influxes occurred at 600μm from the root tip irrespective of preceding Cd exposure and exogenous GSH treatments.Net Cd²⁺influx in the roots was stimulated by external GSH application and inhibited by BSO addition.The Cd-induced production of H₂O₂ was exacerbated by BSO addition,but it was alleviated by the exogenous application of GSH in the roots.Under Cd stress,the endogenous GSH concentration was reduced by BSO addition,but it was enhanced by the exogenous application of GSH.Under Cd exposure,the m RNA levels of ZIP6.2、NRAMP1.3、GS2、MTP1、ABCC3 in roots and ECS、OPT7、PCS1 in leaves were upregulated.These genes were involved in Cd uptake,detoxification and accumulation process.These results indicate that exogenous GSH induced a higher net Cd²⁺influxes,enhanced the translocation of Cd to the aerial part,and accumulated much more Cd.The addition of exogenous GSH increased the level of endogenous GSH,enhanced the ability to scavenge ROS,induced the upregulation of genes involved in Cd uptake,transport,detoxification and accumulation,thereby alleviated the Cd toxicity and enhanced the Cd accumulation.The above studies show that high concentration of Fe and exogenous GSH can enhance the ability of poplar to absorb and accumulate Cd.Therefore,it is possible to increase the Fe concentration or apply exogenous GSH to improve the efficiency of phytoremediation.