| Leaves are not only important plant photosynthesis organs but also vegetable product organs. Most of yellow leaves mutants appear to be reduced growth. However, the leaves mutant of Chinese cabbage grows well and improves the nutrition quality of Chinese cabbage. Therefore, it has a theoretical and practical guide for uncovering the molecular basis of the formation of different yellow/orange leaves. In this study, two leaves color mutants from Chinese cabbage and Arabidopsis were used for analysis. The key candidate genes for Chinese cabbage and Arabidopsis leaves color mutant phenotypes were obtained by fine mapping and map-based cloning. Gene structure, gene expression, functional complementation and metabolic pathways in Chinese cabbage and Arabidopsis leaves color mutants were conducted and these data can provide some explanations for the mutant phenotypes. All these information will enrich plant carotenoid accumulation theory, provide insights into genetic improvement of Chinese cabbage and lay a solid foundation for the function of PRPS5 on plant growth and development.The main results are as follows:1. The F2S4 mapping population consisting of 1724 individuals was developed from the cross between 11J16 and 11S39-2 parent lines by continuous selfing of a single heterozygous individual.Fine mapping and map-based cloning showed BrCRTISO was co-segregated with the Br-or locus.2. The whole length of Brcrtiso was obtained by sequence segment amplification and assembly. In comparison with the genomic sequence of WT, Br-or contained a large insertion of 501 bp at the 3’end, and two two amino acid substitutions in coding region and a deletion of 88 bp in the promoter region.3. Based on the polymorphic sequences with 88-bp deletion in the promoter region of BrCRITSO, an InDel co-dominant marker, Br-Pro-Indel, was developed. This genespecific marker will improve the efficiency of Chinese cabbage breeding.4. Br-or mainly accumulated lycopene and 7,9,9’,7’-tetra-cis-lycopene(prolycopene) However,lutein and β-carotene were the major carotenoids in WT.5. No great difference in the transcriptional levels of BrCRITSO was observed between WT and Br-or. All of these test genes involed in carotenoid accumulation showed similar transcript levels between WT and Br-or. Immunoblot analysis of PSY and ZEP reflected no dramatic difference in the amounts of these two proteins was observed, confirming that the mutation in BrCRITSO in general did not sharply affect carotenogenic enzyme expression.6. Phenotypic complementation tests in a newly isolated Atcrtiso mutant. The whole length cDNA of WT(BrCRITSO) and Br-or that contained the two SNPs but without the large insertion of 501 bp(Brcrtiso?) was introduced into the Atcrtiso mutant. As expected, BrCRITSOand Brcrtiso??complemented the Atcrtiso mutant phenotype and the transgenic lines exhibited wild-type green leaves. A complementation test was performed using whole length cDNA of Br-or. All of the positive transgenic lines overexpressing Brcrtiso did not recover the Atcrtiso mutant phenotype. These results indicated that a large insertion in the C-terminal end of Br CRITSO might produce a malfunction protein, which resulted in orange head leaves in Br-or.7. RNA-seq analysis was employed to profile gene expression difference in the nearisogenic lines(NILs). Statistical analysis identified 372 differentially expressed genes with at least 2-fold changes between WT control and Br-or mutant from the three biological replicates(adjusted P-values < 0.05). The significantly differentially expressed genes between WT and Br-or were catagorized into functional groups using MapMan. Notably, the mutation of BrCRITSO down-regulated the expression of a large number of transcription factors such as GOLDEN2-like, MYB, TCP, AP2/EREBP, WRKY and bHLH. The transcript abundances of many of them were dramatically reduced. These transcription factors are known to regulate multiple processes of plant growth and development, ranging from fruit chloroplast development to stress responses.8. In comparison with wild-type plants, first pairs of true leaves are small, narrow and some serrated edges and a delay of two days in first true leaf emergence in mutant plants was observed. In addition, the mutant had pale green mature leaves, stems, inflorescences, flower buds and siliques and mutant plants grew with reduced growth rate and were much smaller than wild-type plants. In an F2 mapping population, that the segregation ratio of wild-type and mutant was 3:1, which showed the yellow mutant phenotype was controlled by a single recessive gene.9. The total levels of chlorophyll pigments were dramatically reduced in prps5-1. The mutations also significantly lowered the chlorophyll a/b ratios. HPLC analysis revealed that the mutation did not alter carotenoid composition but dramatically reduced carotenoid content, especially β-carotene.10. The mutant plants showed shorter primary root lengths than wild-type plants when they were grown on MS medium. The application of 100 nM 1-naphthaleneacetic acid(NAA) could not restore root length of the mutant plants to the level found in the wild-type and the root length inhibition in mutant seedlings grown in NAA-containing MS medium was less serious than in the wild-type seedlings, indicating that the mutant seedlings were less sensitive to exogenous NAA treatment than wild-type.11. Map-based cloning of At2g33800 and a missense mutation was found in At2g33800 due to a G to A base substitution in the first exon in prps5, which resulted in an amino acid Gly to Glu change at position 180 of PRPS5. This point mutation was cosegregated with mutant phenotype.12. We investigated the subcellular location of PRPS5 by transiently expressing the 35S:PRPS5-GFP fusion construct in tobacco leaves. By merging the chlorophyll auto-?uorescence and green ?uorescence, the signal was clearly present in the chloroplasts. The 35S:prps5-GFP fusion construct also was infiltrated into tobacco leaves and expression of the fusion protein was examined. The signal pattern of prps5 fusion protein was identical to that of PRPS5-GFP. These results showed that the mutation of PRPS5 did not alter the subcellular location of the PRPS5 protein.13. The mature 16 S rRNA was dramatically reduced in the prps5-1 mutants compared with wild-type plants, whereas the 16 S r RNA precursors were increased in the mutant plants.14. No dramatic difference in expression of photosystem I and photosystem II was observed in mutant plants compared with wild-type plants. The protein level of chloroplast-related proteins were changed in mutant plants.15. Comparative proteomics analysis of total proteins from 4-week-old wild type and prps5-1 leaves was carried out using isobaric tags for relative and absolute quanti?cation(iTRAQ)-based method. Among the differentially expressed proteins, 58 proteins were up-regulated with at least 1.33-fold change and 86 proteins were downregulated with at least 0.75-fold change in all three biological replicates of prps5-1. Compared with wild-type plants, the PRPS5 protein level was also detected to have ~43% reduction in prps5-1. Two-dimensional(2D) gel-based proteomics analysis confirmed that PRPS5 protein amount was greatly reduced in prps5-1 plants. These differentially expressed proteins were categorized into functional groups using MapMan. Proteins were involved in photosynthesis, protein, stress, redox regulation and amino acid metabolism and so on. Proteins associated with photosynthesis and protein synthesis represented the most abundant groups. The down-regulated proteins included photosystem I polypeptidesubunits, photosystem II polypeptide subunits, development and stress-related proteins. Global suppression of specific protein synthesis was responsible for the mutant phenotype. |
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