| Cadmium(Cd),a carcinogen metal,is considered as one of the most toxic substances released into the environment.Increasing environmental issues due to Cd contamination of soils makes it imperative to encourage practices for public health safeguarding.Because of similarities in some characteristics with Cd hyperaccumulator species,rapeseed(Brassica napus L.)is regarded as one of the best choice for remediation of Cd contaminated soils.However,Cd detoxification mechanisms are yet to be fully elucidated at vegetative as well as reproductive stages,though this is prerequisite to making this crop species easily availed to phytoremediation technology.Investigations carried out during this thesis aimed at deciphering apoplastic and symplastic responses imparting tolerance abilities to Cd-accumulating B.napus genotypes,with emphasis on metabolites profiling.Analyses were extended on plant reproductive fitness that is needed to make phytoremediation more economically profitable.Therefore,both short term(hydroponic)and saison-long(soil-based)experiments were performed.To exploit natural variation of Cd tolerance,a screening of 102 B.napus genotypes with different genetic background was carried out.After two runs of screening,11 genotypes representing different ranges of Cd tolerance were selected and tested for Cd accumulation and root-to-shoot translocation.Notable variability of Cd tolerance and accumulation were observed,and two genotypes(CB671 and HL672)were identified with Cd-hyperaccumulator-like phenotype(tolerant high-Cd accumulating).Coincidentally,the two genotypes are white-colored-flower genotypes.The fact that only two out of 102 screened genotypes were identified with Cd-hyperaccumulator-like behavior showed that this phenotype might be of rare occurrence in B.napus species;hence these genotypes(high-Cd genotypes)were at focus in follow-up studies.The greater tolerance of high-Cd genotypes was examined in relation to the relative contribution of apoplastic and symplastic responses.To allow a comparative analysis,materials with distinct phenotype i.e.sensitive genotypes,tolerant low-Cd accumulating genotypes were integrated in the study.Results revealed comparable distribution of Cd between cell wall(CW)and symplast in high-Cd genotypes;comparatively,CWs of other categories of genotypes were poor in Cd immobilization,hence major proportion of Cd intruded the symplast.Further analyses revealed that irrespective of genotypes,Cd activity was modulated primarily through complexation with peptide ligands(NaCl-extracted fraction);however,the extent of interaction was significantly higher in CB671(high-Cd).Overall results demonstrated that the elevated Cd tolerance shown by high-Cd genotypes was a combined result of greater Cd sequestration at CW,and a more efficient complexation of Cd by intracellular organic ligands,whereas metal detoxification in other groups of genotypes primarily relied on symplastic events.Results further clarified that the elevated CW binding capacity shown by high-Cd genotypes was in connection with their ability to enhance production of CW polysaccharides,among which hemicellulose(HC)was revealed as the major storage site of CW-bound Cd.Interestingly,RNA-Seq data in CB671(representative of high-Cd genotypes)showed increased transcript abundance of HC biosynthetic genes i.e beta-mannan synthase,UDP-arabinose 4-epimerase upon Cd exposure,suggesting that the enhanced pool of HC was dictated at transcriptional level.Moreover,genes involved in xyloglucan fucosylation(alpha-L-fucosidase,beta-D-xylosidase 4)were regulated in the way to reduce HC fucosylation state,showing that in addition to quantitative changes,HC in CB671 was further remodeled to increase its biding capacity,which was opposite to observations in the Cd-sensitive genotype ZD22.Contrast to the trend of HC modification,a decline of pectin content was noted in leaves of CB671;this was interpreted in relation to its probable utilization as substract for ascorbic acid(AsA)production.Finally,RNA-Seq data indicated an active upregulation of genes encoding CW-localized chitinases in CB671,which was not noticed in ZD622.Together with the proposed role of pectin as substract for AsA production,observed induction of chitinases-related genes demonstrated that the high-Cd genotype(CB671)may be equipped with fine-tuned system to raise antioxidant power at CW level for effective mitigation of harms inherent to excessive Cd immobilisation in this cell compartment.For a holistic view of Cd tolerance in high-Cd genotypes,investigations were extended at whole metabolome level,using liquid chromatography/mass spectrometry.In response to Cd stress(100 μM),the two contrasting genotypes ZD622(Cd sensitive)and CB671(high-Cd genotype)differentially rearranged the flux of carbon.CB671 directed its carbon stock towards production of AsA,compatible solutes,sugar storage forms,as well as jasmonates,ethylene and vitamin B6,whereas related compounds were drastically depleted in ZD622.Interestingly,results indicated that CB671 diverted tryptophan from auxin(IAA)production and preferentially accumulated serotonin(3.48 fold rise),hiting at potential involvement of this compound for Cd toxification.Results suggested also ability for prompt triggering of detoxifying mechanisms in CB671,reflected by marked accumulation of unsaturated free fatty acids and oxylipins,paralleled by glycerophospholipids build-up and induction of inositol-derived signalling metabolites(up to 5.41-fold).Concomitantly,phytosteroids,monoterpenes and carotenoids were induced in this genotype,denoting primed system for membrane maintenance,which was not evident in ZD622 that accumulated more flavonoids instead.Most interstingly,metabolomic data showed increased abundance of mannans(HC),and lignans(CW-bound phenolics)in CB671 further supporting the view that CW of this high-Cd genotype was well conditioned to modulate Cd toxicity effects.Finally,data from long term experiments indicated that the greater Cd tolerance shown by high-Cd genotypes could be considered at whole plant level,since CB671 experienced lower reproductive cost than ZD622 despite accumulating higher amounts of Cd in reproductive organs.Overall,results pointed to post-fertilization events rather than failed pollination as possible factors of observed genotypic differences in reproductive cost.Particularly,results revealed that despite acting as sink of Cd,siliques wall(SW)of CB671 sustained photosynthesis activity,implying notable SW-based detoxifying mechanisms as compared to the Cd-sensitive genotype ZD622.To account for observed genotypic variation in SW performance,results highlighted a more efficient sulfur metabolism in SW of CB671 both in the reductive sulfate pathway and thiol metabolism,singling out the capacity of CB671 to sustain activity of SiR for sulfate reduction,GR for GSH recycling,and GST for direct modulation of Cd activity.Results further demonstrated the feasibility to rescue B.napus plants under Cd-contamination conditions through supplementation of serotonin,actions of which were shown to be in relation with better preservation of seed ultrastructural features,maintainance of SW photosynthesis,improved sulfur metalism through further induction of SiR and GST activities,and enhanced delivery of Mn and B to SW and seeds. |
PDF Full Text Request