Identification And Characterization Of Maize Dek33 And Dek37

Maize(Zea mays)is one of the most important crops in the world and also an important organism for genetic and molecular biology studies.The primary nutrient components of maize kernel are starch,proteins and oil,which decide the yield and quality.Thus,the research on maize kernel development provides possibilities for improving kernel yield and quality.The maize classic kernel mutant defective kernel 33(dek33)produced defective and occasionally viviparous kernel phenotype.The 100-kernel weight of dek33 was 63.7% that of the WT.Analysis of major storage components indicated that the content of total fatty acid and total protein was reduced in dek33.Paraffin section showed that dek33 embryos are much smaller compared with the WT.In addition,fewer oilbodies were observed in both dek33 embryo and aleurone layer cells compared with the WT using TEM.In this study,we cloned Dek33 by positional cloning and found that it encodes the pyrimidine reductase in riboflavin biosynthesis.In dek33,a single base mutation(G-to-A)in the C-terminal COG3236 domain caused a premature stop codon(TGA),producing a weak mutant allele.The dek33 mutation resulted in significant reduction of truncated DEK33 protein in dek33.The dek33 mutation did not affect the DEK33 reductase activity,but the riboflavin content decreased by 48% in dek33.dek33 kernels showed low level of germination rate(?15%),and dek33 seedlings supplemented with 0.3 m M riboflavin partially reverted the mutant phenotype.Seeling phenotype analysis indicated that the dek33 mutation severely affected chloroplast development and photosynthesis.During seed development,expression of Dek33 occurs before 3 DAP and continues later than 30 DAP,with a peak at 9 DAP.An immunoblot analysis with the DEK33 specific antibody showed that DEK33 is highly accumulated in kernel and leaf.Subcellular localization analysis indicated that DEK33 targets chloroplasts.RNA-seq data showed that the dek33 mutation caused 855 significantly expressed genes,including genes related to metabolic process,nucleosome assembly,monolayer-surrounded lipid storage body and nutrient reservoir activity.SDS-PAGE analysis of isolated oilbody-related proteins indicated significant reduction of oleosins in dek33 compared with that in the WT.Flow cytometric analysis indicated that the endoreduplication was suppressed in dek33 endosperm.The dek33 mutation also disrupted ABA biosynthesis,resulting in less ABA content,which might be responsible for the viviparous embryo.In addition,our results indicated that the COG3236 domain is important for the protein stability of DEK33.The yeast two-hybrid experiment identified several proteins that interact with DEK33,including RGLG2 and Sn RK1,suggesting possible post-translational regulation to DEK33 stability.This study provided a weak mutant allele to explore cellular responses due to impaired riboflavin biosynthesis during seed development.Our findings indicated that the COG3236 domain might be an essential regulatory structure for DEK33 stability in maize.The maize kernel mutant defective kernel 37(dek37)was isolated from maize active Mutator line.The mature dek37 kernels are small and mishappen,and the 100-kernel weight is only 18% that of the WT.Paraffin and resin sections of dek37 immature kernels showed developmental delay compared with the WT.Using Mutator tag isolation,we cloned Dek37 and found it encodes a classic P-type PPR protein that targets mitochondria.The dek37 mutation causes no detectable DEK37 protein in mutant kernels.During seed development,expression of Dek37 occurs before 3 DAP and continues later than 30 DAP.Subcellular localization analysis indicated that DEK37 targets mitochondria.Mitochondrial transcripts analysis indicated that dek37 mutation decreases splicing efficiency of mitochondrial nad2 intron 1.Severe defects of mitochondrial complex I assembly and activity was detected in dek37 by Blue Native Polyacrylamide Gel Electrophoresis(BN-PAGE)and in-gel NADH dehydrogenase activity test.In addition,q RT-PCR analysis of genes related to alternative respiratory pathway also showed significant up-regulation in dek37.TEM observation showed abnormal mitochondria phenotype with severe deformations and voided internal assembly.Transcriptome analysis of dek37 kernels showed extensive differentially expressed genes related to mitochondrial function.These results indicated that mitochondrial function was severely affected in dek37.Thus,our results indicated that Dek37 encodes a classic P-type PPR protein participating in the cis-splicing of mitochondrial nad2 intron 1,and is required for mitochondrial function and seed development.The study of DEK37 extended our knowledge of the essential roles for PPR proteins during maize seed development.