Expression,regulation And Functional Analysis Of Superoxide Dismutase Family Genes In Kandelia Obovata Under Cadmium Stress

Mangrove wetland play vital ecological roles in tropical and subtropical coastal areas.Mangroves are found in areas that are muddy and have high organic matter,or in slow-flow streams that have high H2S contents.Mangrove wetlands are often enriched in heavy metals.As urbanization rapidly increases,heavy metal pollution in mangrove wetlands has received greater attention.Among them,Cadmium(Cd)is one of the heavy metal pollutants that seriously threaten mangrove wetlands.Kandelia obovata is a dominant mangrove species in south China and is widely distributed across Asia.This species has high resistance to Cd toxicity,but the mechanisms that confer this resistance are unclear.Previous studies revealed that the resistance of K.obovata to heavy metal stress can generally be ascribed to high superoxide dismutase(SOD)activity that reduces damage induced by heavy metal stress.To date,however,research on K.obovata has mainly focused on enzymatic changes and not SOD family genes.Knowledge of these internal protective physiological mechanisms could facilitate development of genetic engineering strategies that would improve tolerance of other plants to heavy metals.In this study,the role and regulatory mechanism of KoSODs was explored under heavy metal tolerance.SOD family gene transcripts,gene structure,protein motifs phylogenetic analysis,expression profiles and subcellular localization in K.obovata were characterized following exposure of plants to Cd.In addition,KoSODs were overexpressed in Nicotiana benthamiana to verify the functions of KoSOD in defense Cd stress in K.obovata.The conclusions as following:1.Seven KoSOD genes belonging to three SOD subtypes were obtained from K.obovata.The diversity of KoSOD gene transcripts was attributed to exon deletions,intron retention,and alternative polyadenylation events transcripts.Except for KoCSD3 and KoFSD2,KoSOD members and Arabidopsis.thaliana SOD family genes(AtSODs)had similar gene structures,such as exon numbers and intron phases,indicating that KoCSD3 and KoFSD2 may be unique members.2.Eleven KoSOD transcripts encoded 10 proteins.The protein size ranges from 13.44 to 33.46 kDa.Most KoSOD proteins were classified as stable and hydrophilic proteins.Sequence alignment of Bruguiera.gymnorrhiza,Kandelia.candel,A.thaliana and K.obovata SOD proteins suggested that KoSOD are highly conserved at active enzyme sites,metal binding sites,and N-/C-terminal domains.The phylogenetic analysis and the dN/dS values among three SOD subtypes indicated that MnSODs and Cu/ZnSODs have higher purifying selection during evolution relative to that for FeSODs,suggesting that FeSOD types may be species specific.Subcellular localization showed that KoCSD1 and KoCSD3 are mainly localize to cytoplasm,while KoCSD2,KoFSDs and KoMSD are more likely to localize to the chloroplasts and mitochondria.3.KoSODs were mainly expressed in the root,hypocotyl and stem after Cd treatment.Interestingly,expression profiles of KoSODs exihibted tissue-specific.For example,KoCSD2 had moderate expression in the stem and hypocotyl,but KoCSD3 was mainly expressed in roots.KoFSDs had mainly expression in roots.KoMSD expression was absent only in leaves.In addition,different KoSOD genes of the same subfamily expressed reciprocally in various treatment stages in response to Cd treatment.Among them,the expression levels of KoCSD3 and KoFSD2 increased most significantly during the Cd stress.4.We found that the thick,lignified and suberized epidermis and exodermis of K.obovata roots could act as a barrier to inhibit Cd entry.The highest Cd content was detected in the epidermis and exodermis,followed by the cortex and stele.Similarly,the epidermis and exodermis had the highest oxidative stress and SOD activity,followed by the root cortex and stele in Cd-treated palnts.qPCR data revealed that KoFSD2 gene was strongly expressed throughout the root,whereas KoCSD3 was mainly expressed in the epidermis and exodermis.5.The sequence analysis indicated that the KoFSD2 and KoCSD3 promoter sequences carried six types of regulatory cis-elements and two types of transcription factors that are related to ABA,SA and auxin responses,respectively.Furthermore,Assays on the effects of hormones showed that increased endogenous indole-3-acetic acid(IAA)was observed in the cortex and stele,whereas the abscisic acid(ABA)content was enhanced in the epidermis and exodermis in roots during Cd treatments.KoFSD2 and KoCSD3 expression was induced by treatment with exogenous hormones ABA,but KoCSD3 expression did not significantly change in response to IAA stimulation.6.Tobacco plants with transgenic expression of KoFSD2 and KoCSD3,T-KoFSD2 and T-KoCSD3,respectively,showed less severe inhibition of root elongation compared to WT plants after Cd stress,indicating that the transgenic plants had higher Cd tolerance than the WT plants T-KoFSD2 and T-KoCSD3 plants had different SOD-mediated H2O2 scavenging activities.Expression of the superoxide dismutase gene KoFSD2 could promote activity of antioxidant system enzymes,including catalase and glutathione reductase,to reduce oxidative damage.The related gene KoCSD3 was mainly expressed in root epidermis and exodermis,and cooperated with ascorbate peroxidase to maintain moderate hydrogen peroxide concentrations in roots,which may be related to the accumulation of lignin in the epidermis and exodermis.