Study On Elemental Defense Of Cd Accumulation In Brassica Juncea

The elemental defense hypothesis describes a new defensive strategy that hyperaccumulators defense the attacks from herbivores or pathogens by accumulating heavy metals.This makes the hyperaccumulators that grew from metal-contaminated sites a better fitness compared to non-hyperaccumulators.Brassica juncea has the ability in cadmium accumulation.However,the elemental defense effect and its regulation mechanism in B.juncea remain unclear.In this study,the elemental defense effect in Cd-accumulated Brassica juncea after Alternaria brassicicola infection was investigated.And the expression changes of the defense-related genes and microRNAs related to disease resistance during elemental defense were analyzed.The results are listed below:(1)The pathogenic effect of Alternaria brassicicola on B.juncea.The spore suspension of Aleternaria brassicicola was dropped on the upper surface of mustard leaves,and the development of disease spots and spore were recorded regularly.Results showed that Alternaria brassicicola had no obvious pathogenicity to mustard during the first 24 h;there was slight pathogenicity at 48h.When the time went to 72 h,the pathogenicity of the pathogen was quite obvious,and the degree of spore development at the lesion site was higher than that in other time points.This indicates that Alternaria brassicicola has pathogenic ability to mustard in 48h after inoculation,and time-dependent disease development of mustard was observed.(2)The elemental defense effect of mustard enriched with cadmium on Alternaria brassicicola.Taking Cd-accumulated-mustard-pathogenic fungus system as the research object,and a gradient of 0,180,360,720 mg/Kg DW Cd culture environment was set for mustard and named Control,T-1,T-2,and T-3 lines respectively.Spore coverage area and spores’development showed that differences in lesion area,diameter,and circumference were not significant among each group at 24 h.At 72 h,parameters of the lesions of all groups were larger than their respective 24 h lines.In T-1 and T-2 treated lines,lesions were significantly smaller than the control,and the degree of spore development at the lesion site was lower than that of the control.Together,the results indicate that the mustard in T-1 and T-2 lines show a certain kind of elemental defense in the process of resisting pathogens through the accumulated Cd.However,the elemental defense brought by heavy metals can only slow the process of the disease exacerbation,but could not change the direction of the disease development.(3)Changes in the expression of genes related to mustard defense in the process of elemental defense.T-1 and T-2 lines were inoculated with Alternaria brassicicola.72 hours later,total RNAs were extracted from samples of each line,and cDNAs were obtained by reverse transcription.After that,the expression of NPR1,ICS,DEF,and GL U were analyzed by qPCR.Results showed that 72 hours after inoculation with Alternaria brassicicola,an up-regulated expression levels of the systemic acquired resistance marker genes DEF and GLU were detected in samples of T-1 and T-2 lines.The expression of NPR1 and ICS were also up-regulated under the treatment of T-2.These may indicate that Cd-accumulated mustard may participate in the defense process against pathogens by coordinating SA-mediated SAR and other defense responses in a synergistic joint effect.(4)Changes in the expression of miRNAs related to mustard in the process of element defense.Total RNAs were extracted from leaves of the 72 h lines under T-1 and T-2 treatment.After obtaining cDNA,the relative expression of miR156,miR397,miR398a,miR398bc,miR408,miR393,miR395a,miR395b,miR396a,miR396b,as well as some of their target genes was determined by qPCR.Results showed that under the condition of Cd enrichment,Alternaria brassicicola infections could ultra the up-regulated relative expression of almost all miRNAs except miR393 in mustard leaves.Analysis of target genes showed that target genes of differentially express miRNAs were enriched in SQUAMOSA promoter-binding proteins,laccase,ribosomal proteins,ATP sulfurylase,growth-regulating factors.Moreover,the expression of miR396 and its target genes at the transcriptional level were not always exactly opposed.Together,the results indicate that Cd-accumulated mustard may balance its development and respond to biotic stress by coordinating multiple factors such as heavy metal enrichment and the expression of related miRNAs.