Dataset Information

Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus.

ABSTRACT: Cyclophilins (CYPs) are a group of ubiquitous prolyl cis/trans isomerases (PPIases). It was shown that plants possess the most diverse CYP families and that these are abundant in the phloem long-distance translocation stream. Since phloem exudate showed PPIase activity, three single-domain CYPs that occur in phloem samples from Brassica napus were characterised on functional and structural levels. It could be shown that they exhibit isomerase activity and that this activity is controlled by a redox regulation mechanism, which has been postulated for divergent CYPs. The structure determination by small-angle X-ray scattering experiments revealed a conserved globular shape. In addition, the high-resolution crystal structure of BnCYP19-1 was resolved and refined to 2.0?Å resolution, and the active sites of related CYPs as well as substrate binding were modelled. The obtained data and results support the hypothesis that single domain phloem CYPs are active phloem PPIases that may function as chaperones.

SUBMITTER: Hanhart P

PROVIDER: S-EPMC6597583 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus.

Hanhart Patrizia P Falke Sven S Garbe Marcel M Rose Victoria V Thieß Melanie M Betzel Christian C Kehr Julia J

Scientific reports 20190627 1

Cyclophilins (CYPs) are a group of ubiquitous prolyl cis/trans isomerases (PPIases). It was shown that plants possess the most diverse CYP families and that these are abundant in the phloem long-distance translocation stream. Since phloem exudate showed PPIase activity, three single-domain CYPs that occur in phloem samples from Brassica napus were characterised on functional and structural levels. It could be shown that they exhibit isomerase activity and that this activity is controlled by a re ...[more]

PMID: 31249367

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Project description:In plants, phloem is not only a vital structure that is used for nutrient transportation, but it is also the location of a response that defends against various stresses, named phloem-based defense (PBD). Phloem proteins (PP2s) are among the predominant proteins in phloem, indicating their potential functional role in PBD. Sclerotinia disease (SD), which is caused by the necrotrophic fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum), is a devastating disease that affects oil crops, especially Brassica napus (B. napus), mainly by blocking nutrition and water transportation through xylem and phloem. Presently, the role of PP2s in SD resistance is still largely estimated. Therefore, in this study, we identified 62 members of the PP2 gene family in the B. napus genome with an uneven distribution across the 19 chromosomes. A phylogenetic analysis classified the BnPP2s into four clusters (I-IV), with cluster I containing the most members (28 genes) as a consequence of its frequent genome segmental duplication. A comparison of the gene structures and conserved motifs suggested that BnPP2 genes were well conserved in clusters II to IV, but were variable in cluster I. Interestingly, the motifs in different clusters displayed unique features, such as motif 6 specifically existing in cluster III and motif 1 being excluded from cluster IV. These results indicated the possible functional specification of BnPP2s. A transcriptome data analysis showed that the genes in clusters II to IV exhibited dynamic expression alternation in tissues and the stimulation of S. sclerotiorum, suggesting that they could participate in SD resistance. A GWAS analysis of a rapeseed population comprising 324 accessions identified four BnPP2 genes that were potentially responsible for SD resistance and a transgenic study that was conducted by transiently expressing BnPP2-6 in tobacco (Nicotiana tabacum) leaves validated their positive role in regulating SD resistance in terms of reduced lesion size after inoculation with S. sclerotiorum hyphal plugs. This study provides useful information on PP2 gene functions in B. napus and could aid elaborated functional studies on the PP2 gene family.

Project description:BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation. Identification of large numbers of SNPs is helpful for genetic diversity analysis, map-based cloning, genome-wide association analyses and marker-assisted breeding. Recently, identifying genome-wide SNPs in allopolyploid Brassica napus (rapeseed, canola) by resequencing many accessions has become feasible, due to the availability of reference genomes of Brassica rapa (2n = AA) and Brassica oleracea (2n = CC), which are the progenitor species of B. napus (2n = AACC). Although many SNPs in B. napus have been released, the objective in the present study was to produce a larger, more informative set of SNPs for large-scale and efficient genotypic screening. Hence, short-read genome sequencing was conducted on ten elite B. napus accessions for SNP discovery. A subset of these SNPs was randomly selected for sequence validation and for genotyping efficiency testing using the Illumina GoldenGate assay. RESULTS: A total of 892,536 bi-allelic SNPs were discovered throughout the B. napus genome. A total of 36,458 putative amino acid variants were located in 13,552 protein-coding genes, which were predicted to have enriched binding and catalytic activity as a result. Using the GoldenGate genotyping platform, 94 of 96 SNPs sampled could effectively distinguish genotypes of 130 lines from two mapping populations, with an average call rate of 92%. CONCLUSIONS: Despite the polyploid nature of B. napus, nearly 900,000 simple SNPs were identified by whole genome resequencing. These SNPs were predicted to be effective in high-throughput genotyping assays (51% polymorphic SNPs, 92% average call rate using the GoldenGate assay, leading to an estimated >450 000 useful SNPs). Hence, the development of a much larger genotyping array of informative SNPs is feasible. SNPs identified in this study to cause non-synonymous amino acid substitutions can also be utilized to directly identify causal genes in association studies.

Dataset Information

Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus.

Publications

Enzyme activity and structural features of three single-domain phloem cyclophilins from Brassica napus.

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