Intronic MicroRNA-directed Regulation Of Mitochondrial Reactive Oxygen Species Enhances Plant Cadmium Stress Tolerance In Arabidopsis

Heavy metal pollution in soil is a global problem with serious impacts on human health and ecological security.Cadmium is one of the most biotoxic pollutants,which easily accumulates in plants and affects animal and human health through the food chain.Alternative splicing,which generates multiple transcripts from the same gene,is an important modulator of gene expression that can increase proteome diversity and regulate m RNA levels.Plant micro RNA（miRNA）is a kind of non-coding small RNA with a length of about 20-24 nt,which play important roles in regulating plant development and stress responses.MiRNAs can be classified as either intergenic miRNAs or intronic miRNAs;the former are located in noncoding regions between genes and transcribed by their own promoters,while the latter are located within intronic regions and are derived from the introns of their host genes.However,the functions and mechanism of intronic miRNAs in plants are poorly understood.Our previous study revealed that a specific AS event triggered under heat stress in the 306-bp miR400-containing intron.To investigate the splicing mechanism of the miR400-containing intron in detail,we constructed a reporter system consisting of the firefly luciferase（LUC）reporter gene to track intron splicing events in Arabidopsis.The main results are as follows:（1）Cd²⁺triggers miR400-containing intron retention,which inhibits the production of mature miR400.We measured the LUC activity of MIR400_LUC transgenic plants under different stress and high Cd²⁺concentration was found to significantly suppress MIR400_LUCderived luciferase activity.To investigate the relationship between miR400（mature miR400）levels and Cd²⁺stress in Arabidopsis,we performed the reverse-transcription quantitative PCR（RT-qPCR）using 7-d-old WT seedlings exposed to Cd²⁺stress.We found that the primary MIR400（primary miR400）transcript level increased significantly while mature miR400 levels decreased.（2）miR400 negatively regulates Cd²⁺stress response in Arabidopsis.Phenotypic analysis showed that OXmiR400 plants were more sensitive to Cd²⁺stress,root growth and fresh weight were reduced relative to the wild type（WT）plants,while STTM400（Short Tandem Target Mimic）were more tolerant to Cd²⁺stress,root growth and fresh weight were higher than that of the WT plants.These results illustrate that miR400 acts as a negative regulator in response to Cd²⁺stress.（3）PPR1（Pentatricopeptide Repeat Protein 1）and PPR2 are directly regulated by miR400.We performed 5’RACE PCR（5’Rapid amplification of c DNA ends Polymerase Chain Reaction）demonstrated that miR400 directs the cleavage of the PPR1 and PPR2transcripts.A transient co-expression assay in N.benthamiana leaves verified that miR400can direct the cleavage and subsequent degradation of PPR1 and PPR2 m RNAs in vivo.In addition,we measured the transcript levels of PPR1 and PPR2 in the OXmiR400 and STTM400 plants by RT-qPCR.PPR1 and PPR2 transcript levels were lower in the OXmiR400 plants than in the WT plants but much higher in the STTM400 plants.These results indicate that miR400 prevents the accumulation of PPR1 and PPR2 m RNAs.（4）Overexpression of PPR1 improves Cd²⁺resistance.Phenotypic analysis showed that overexpression of r PPR1 were more tolerant to Cd²⁺stress,root growth and fresh weight were higher than that of the WT plants,while ppr1 mutant were more sensitive to Cd²⁺stress,root growth and fresh weight were reduced relative to the WT plants.In addition,the phenotypes of overexpression r PPR2 and WT plants were not found to differ in response to Cd²⁺stress.These results indicate that only PPR1 plays a positive role in Cd²⁺tolerance.（5）miR400 and PPR1 mediate oxidative-responsive gene expression.We conducted transcriptome deep sequencing（RNA-Seq）by using 2-week-old seedlings of miR400 and r PPR1 overexpression plants and WT seedlings,all of which were subjected to Cd²⁺treatment.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses the DEGs（differentially expressed genes）between the miR400 and r PPR1 overexpression plants are closely related to hydrogen peroxide catabolic process.Taken together,miR400 and PPR1play pivotal roles in ROS signaling.（6）PPR1 enhances stress tolerance by maintaining of low ROS levels.To determine whether Cd²⁺affected the ROS levels,we visualized ROS accumulation in the roots of all genotypes grown in the absence or presence of Cd²⁺by using NBT,DAB,and the fluorescent dye APF.ROS accumulation in the STTM400 and r PPR1 overexpression seedlings was lower than that in the WT plants,whereas decreased of PPR1 function in miR400 overexpression seedlings and loss of ppr1 function in ppr1 mutant resulted in higher ROS accumulation.These results illustrate that overexpression of PPR1 reduced ROS content.（7）PPR1 affects the Complex III activity in the ETC and RNA editing of the mitochondrial transcript ccm B.Reactive oxygen species are generated in the mitochondria due to the oxidation of metabolic intermediates of the ETC.Complex III activity decreased with Cd²⁺treatment in all genotypes.Interestingly,Complex III activity was significantly reduced in the miR400 overexpression plants and ppr1 mutants but enhanced in STTM400and r PPR1 overexpression plants compared with the WT plants.We analyzed the C-to-U events of ccm B transcripts in the WT and ppr1 seedlings and found that 14 editing sites showed an altered pattern in the ppr1 mutants.These RNA editing defects in ppr1 could be rescued in a complemented transgenic plant.These results indicated that PPR1 is required for maintenance of Complex III activity and the normal C-to-U editing of ccm B RNA transcripts.（8）PPR1 acts genetically downstream of miR400 to modulate Cd²⁺stress response.Phenotypic analysis showed that the miR400/r PPR1 double overexpression plants restored the sensitive phenotype of miR400 overexpression lines under Cd²⁺stress conditions,the root length and fresh weight of the miR400/r PPR1 double overexpression plants was comparable to that of the r PPR1 overexpression plants.In addition,NBT and DAB staining,as well as APF fluorescence analysis,showed that ROS levels in the miR400/r PPR1 double overexpression plants and r PPR1 overexpression plants were similar.Taken together,these results indicate that miR400 acts genetically upstream of PPR1 to regulate Cd²⁺tolerance.In summary,we discovered intron splicing as a key step in intronic miR400 regulation and identified the role of intronic miRNAs as a’signal transducer’.MiR400 plays a crucial role in RNA-based signal transmission from the nucleus to mitochondria to enhance plant stress tolerance by downregulating the expression of its target pentatricopeptide repeat gene PPR1.Our results provide novel insights into the stress-responsive intron splicing that fine-tunes the intronic miRNA processing.