| In higher plants, healthy leaves are important for plant photosynthesis. However, leafsenescence leads to a decline in photosynthetic activity. Senescence is the final stage of leafdevelopment, ultimately leading to the death of the entire leaf. It has been defined as a highlyorganized and genetically programmed process. The molecular catabolic events that featurethis process include chlorophyll degradation, proteolysis and nucleic acid hydrolysis, theaccumulation of dissociative amino acid residues and the up-regulation of proteinase andgrowth repressive factors. Chlorophyll breakdown is the most obvious event during leafsenescence. Pheophorbide a oxygenase (PaO) has been taken as the most important enzymeduring chlorophyll breakdown. PaO catalyzes the oxygenolytic opening of the porphyrin ringof pheophorbide a, forming the primary red chlorophyll catabolite (RCC). PaO was fristlydiscovered in maize. Since then, it has been found in several different plant species. Here, weselect hexaploid wheat (Triticum aestivum) as the experiment material, and a series ofresearch have been conducted on the isolation, sequence and expression pattern analysis,which can contribute to understand the function and mechanism of PaO. The main results areas follows:(1) A novel PaO gene, named as TaPaO (GenBank accession number: JN689384), wascloned from wheat using the bioinformatics analysis and RACE techniques. The full-lengthcDNA sequence of TaPaO consisted of1841nucleotides, containing a1572bp complete openreading frame (ORF), a48bp5’ untranslated region (UTR) and a221bp3’ UTR. TaPaOencoded523amino acids with a theoretical molecular mass of58.3kDa and a calculated pI of7.17. Further analysis indicated that TaPaO was located in chloroplast and the predictedchloroplast transit peptide of TaPaO had a length of38amino acids and a predicted cleavagesite location between R38and V39. The encoded protein contained the typical Rieske (2Fe-2S)and mononuclear iron-binding domains as well as the C-terminus CxxC motif that were conserved in and featured PaO homologues. Amino acid sequence alignment revealed thatTaPaO shared a high degree of identity with other higher plant PaO proteins. TaPaO sharedidentity of93%,83%,79%,69%and65%with HvPaO, SbPaO, ZmPaO, AtPaO and GmPaO,respectively. Phylogenetic analysis indicated that TaPaO was more closely related to HvPaOthan PaO proteins from other plants.(2) The expression profile of TaPaO in different tissues was studied by quantitativereal-time PCR analysis. The result showed that TaPaO was expressed in roots, stems andleaves and the expression level of TaPaO was obviously higher in leaves than in stems androots.(3) The expression profiles of TaPaO under various hormones was studied byquantitative real-time PCR analysis. The results indicated that exogenous hormones includingabscisic acid (ABA) and methyl jasmonate (MeJA) could enhance the expression levels ofTaPaO to different extents. However, gibberellin (GA) and salicylic acid (SA) didn’tsignificantly affect the expression of TaPaO. These results suggest that the expression ofTaPaO may be regulated by ABA-and MeJA-dependent defense signaling pathways.(4) The expression profiles of TaPaO under abiotic and biotic stresses was studied byquantitative real-time PCR analysis. The results showed that the expression levels of TaPaOwere all up-regulated under the abiotic stress conditions including wounding, low temperature(4C) and high salinity (200mM NaCl). when the wheat seedlings were infected with leaf rust,the expression level of TaPaO was also up-regulated. However, the expression of TaPaO wasnot affected obviously after infection with stripe rust. Overall, the results indicate that thatTaPaO may be involved in plant responses to various stresses. |
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