| Cadmium(Cd)has been characterized as a hazardous element that causes distinct toxicities in both plants and animals.Unfortunately,a large amount of Cd has been released into the environment,especially in China,threatening crop safety and human health.Phytoremediation is considered as a promising practice to cope with the soil Cd pollution,whose efficiency is restricted by our limited understanding of Cd tolerance and accumulation mechanisms in plants.Cadmium hyperaccumulators are a special class of plants that can not only tolerate high Cd in the soil,but also accumulate considerable Cd in aboveground parts.Previously,great progresses have been made in Cd uptake,transport and accumulation mechanisms in hyperaccumulating plants,but few investigations focus on the short-term responses of plants to Cd stress,as well as the responses regarding Cd distribution and retranslocation under long-term Cd stress.With the combination of various technologies(such as ?-XRF,ICP-MS,LCM,RNA-seq),we here investigated multi-processes of Cd management in the Cd hyperaccumulating(HE)Sedum alfredii Hance and its non-hyperaccumulating ecotype(NHE),including the role of NO in root Cd tolerance under short-term Cd stress,aboveground Cd distribution and storage under long-term Cd treatment,phloem transport and Cd remobilization,site-specific transcriptional responses of stem parenchymal and vascular cells to Cd stress,as well as the interaction between Cd hyperaccumulation and biotic defence in plants.By comparing the two ecotypes,we tried to reveal some key characteristics that involves in metal hyperaccumulation to further our understanding of hyperaccumulators.Here are the main results.1.The role of nitric oxide(NO)in Cd tolerance under short-term Cd stress was investigated in roots of the two ecotypes of S.alfredii.Differing from that of NHE,Cd stress triggered a strong NO burst in HE roots,with a peak at 36 h.Consistently,Cd stress elevated the activities of nitrate reductase(NR)and nitrix oxide synthase(NOS),and NR and NOS inhibitors significantly reduced the NO level in roots.Elimination of endogenous NO did not affect Cd levels in roots and shoots,but greatly aggravated the metal toxicity,including increased ROS accumulation,oxidative damage and cell ultrastructure injury.Cd stress in HE roots triggered up-regulated SOD but down-regulated POD,CAT and APX activities,which were significantly inverted by NO scavenger.The NO burst also expanded the GSH pool in HE roots by activation of GR,GSNOR and y-ECS,but had no effects on the AsA cycling.Similar to that of NO,preferential localizations of ROS and GSH to meristem and cylinder were noted in root tips of HE.These findings suggest that NR/NOS-co-mediated NO burst activated a GSH-involved strategy,instead of altering Cd accumulation,to protect roots of HE S.alfredii against Cd toxicity at early stage.2.As to reveal the relationship between Cd management and plant development,we investigated Cd distribution in different stems and leaves of two ecotypes of S.alfredii.The results showed that Cd preferentially accumulated in younger organs compared to the older one in the HE seedlings,while in the NHE seedlings,Cd was trapped in old stems and the Cd concentrations of leaves were very low.In HE,in vivo imaging by ?-XRF showed that the primary distribution sites of Cd changed from parenchyma to vascular/epidermal cells with increased organ age.Particularly,strong Cd signals in phloem cells were observed in old stems of HE.By using TEM technology,we found that the disorder of phloem cell ultrastrucute was caused by Cd in NHE stem,but not in HE seedlings,which may afford Cd transport in HE phloem.All those findings suggest that Cd accumulation in aboveground part was both ecotype-and age-dependent,where phloem-mediated Cd transported could be involved.3.Our knowledge of Cd in hyperaccumulators mainly concerns root uptake,xylem translocation and foliar detoxification,while little attention has been paid to the role of phloem remobilization.Here we compared the characteristics of phloem-mediated Cd transport in two ecotypes of S.alfredii.Short-term leaf feeding experiments showed that Cd could be observed in the stem by ?-XRF just after foliar application for 6 h,indicating that phloem-mediated exportation of Cd in treated leaves was very rapid.After 7 d's leaf Cd treatment,HE relocated 44%of the total leaf-absorbed Cd to other organs,while over 90%Cd was retained in treated leaves of NHE.High Cd was detected in HE phloem exudates but not in those from NHE leaves.After long-term Cd treatment,just after K and Ca,Cd ranked the third most abundant element in the HE phloem exudate,the abundance of which was 10-fold higher than that of Fe.Moreover,pre-stored Cd was readily exported from older leaves and stems to growing shoots instead of roots,and relocation of Cd was accelerated by leaf senescence.In conclusion,phloem-mediated Cd transport is rapid,efficient and directional,playing a significant role in remobilizing Cd from old organs to growing ones,which may relate to the Cd tolerance and hyperaccumulation in S.alfredii and its great potential in phytoremediation.4.Cadmium hyperaccumulation in plants is a complex biological process controlled by gene regulatory networks.Efficient transport through vascular systems and storage by parenchymal cells are vital for Cd hyperaccumulation by S.alfredii,but the genes involved are poorly understood.We investigated the spatial gene expression profiles at S.alfredii stem transport and storage sites using laser capture microdissection coupled with RNA sequencing.Gene expression patterns in response to Cd were distinct in vascular and parenchymal cells,indicating functional divisions that corresponded to Cd transportation and storage,respectively.In vascular cells,plasma membrane-related GO terms enriched a large number of differentially expressed genes(DEGs)for foundational roles in Cd transportation.Parenchymal cells contained considerable DEGs specifically concentrated on vacuole-related terms associated with Cd sequestration and detoxification.In both cell types,DEGs were classified into different metabolic pathways in a similar way,indicating the role of Cd in activating a systemic stress signalling network involving ABC transporters and Ca2+ signal pathways.This study identified site-specific regulation of transcriptional responses to Cd stress in S.alfredii and selected a collection of genes that possibly function in Cd transport and storage,vacuole sequestration,phloem transport,and Cd detoxification,thus providing systemic information about molecular strategies associated with Cd hyperaccumulation.5.Although profound progresses have been made in understanding Cd transport and accumulation in the hyperaccumulators,the information regarding the possible ecological significance of Cd hyperaccumulation in plants is still limited.In this study,we investigated the effect of Cd on the biotic stress defence in HE S.alfredii.In long-term experiment,Cd treatment significantly increased the Cd content in HE seedlings and those HE seedlings grew healthly in the pathogenic environment full of black bean aphid(Aphis fabae Scopoli,Hemiptera,Aphididae).In short-term experiment,the accumulation of Cd in plant didn't affect the choice of aphids for cololization,but significantly increased Cd content in aphids and reduced the numbers of artificially released aphids in seedlings.By employing ?-XRF,the Cd distribution in aphids was clearly observed.In the pathogenic environment with unknown pathogen,control HE seedlings without Cd showed powdery mildew symptoms,which was further confirmed by internal transcribed spacer(ITS)sequence analysis.The pathogenic fungus was identified to belong to Erysiphacea Pseudoidium,a kind of biotrophic fungi which rooted in foliar epidermis cells for obtaining nutrients.The Cd treatement inhibited the colonization of this pathogenic fungi in the leaves of HE seedlings.In conclusion,Cd accumulation in its aboveground part armars the hyperaccumulating S.alfredii to against aphids and powdery mildew. |
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