ABSTRACT
Sprouting regulation in potato tubers is important for improving commercial value and producing new plants. Camphor shows flexible inhibition of tuber sprouting and prolongs the storage period of potato, but its underlying mechanism remains unknown. The results of the present study suggest that camphor inhibition caused bud growth deformities and necrosis, but after moving to more ventilated conditions, new sprouts grew from the bud eye of the tuber. Subsequently, the sucrose and fructose contents as well as polyphenol oxidase (PPO) activity were assessed after camphor inhibition. Transcription and proteomics data from dormancy (D), sprouting (S), camphor inhibition (C), and recovery sprouting (R) samples showed changes in the expression levels of approximately 4000 transcripts, and 700 proteins showed different abundances. KEGG (Kyoto encyclopaedia of genes and genomes) pathway analysis of the transcription levels indicated that phytohormone synthesis and signal transduction play important roles in tuber sprouting. Camphor inhibited these processes, particularly for gibberellic acid, brassinosteroids, and ethylene, leading to dysregulation of physiological processes such as cutin, suberine and wax biosynthesis, fatty acid elongation, phenylpropanoid biosynthesis, and starch and sucrose metabolism, resulting in bud necrosis and prolonged storage periods. The KEGG pathway correlation between transcripts and proteins revealed that terpenoid backbone biosynthesis and plant-pathogen interaction pathways showed significant differences in D vs. S samples, but 13 pathways were remarkably different in the D vs. C groups, as camphor inhibition significantly increased both the transcription levels and protein abundance of pathogenesis-related protein PR-10a (or STH-2), the pathogenesis-related P2-like precursor protein, and the kirola-like protein as compared to sprouting. In recovery sprouting, these genes and proteins were decreased at both the transcriptional level and in protein abundance. It was important to find that the inhibitory effect of camphor on potato tuber sprout was reversible, revealing the action mechanism was similar to resistance to pathogen infection. The present study provides a theoretical basis for the application of camphor in prolonging seed potato storage.
Subject(s)
Camphor/pharmacology , Gene Expression Profiling , Plant Tubers/drug effects , Plant Tubers/physiology , Proteomics , Solanum tuberosum/drug effects , Solanum tuberosum/physiology , Computational Biology/methods , Fructose/metabolism , Gene Expression Regulation, Plant/drug effects , Gene Regulatory Networks , Phenotype , Proteome , Proteomics/methods , Signal Transduction , Solanum tuberosum/cytology , Sucrose/metabolism , TranscriptomeABSTRACT
Objective To observe the effect of zinc-dependent metalloprotease 1 (Zmp1) of Bacillus Calmette Guerin vaccine (BCG) on the proliferation and phagosome-lysosome fusion of RAW264.7 mouse macrophage. Methods Zmp1 was expressed in E.coli BL21 (DE3) and purified by metal chelate magnetic beads. RAW264.7 cells were incubated with the purified Zmp1. Cell proliferation was detected at 0, 24, 48, 72 and 96 hours by cell counting kit-8 (CCK-8) assay and cell cycle distribution was detected by flow cytometry (FCM). The formation of phagosome was induced after RAW264.7 cells were infected with attenuated Salmonella. Attenuated Salmonella in phagosomes and lysosome associated membrane protein 1 (LAMP1) were marked by Alexa FluorR350-anti-Salmonella antibodies (blue fluorescence) and Cy5-anti-LAMP1 antibodies (red fluorescence), respectively. Two kind of fluorescence in RAW264.7 cells were observed under a fluorescent microscope and the fusion of phagosome and lysosome was analyzed by overlaying two kind of fluorescence (purple fluorescence). Results RAW264.7 cell proliferation decreased obviously 48 hours after treated with Zmp1, and the cell count in G1 phase declined, but the number of S-phase cells increased. In RAW264.7 cells infected with attenuated Salmonella and labeled by double immunofluorescent staining, phagolysosomes exhibiting purple fluorescence were reduced after treated by Zmp1. Conclusion Zmp1 can inhibit mouse RAW264.7 cell proliferation and phagosome-lysosome fusion.
Subject(s)
BCG Vaccine/pharmacology , Bacterial Proteins/pharmacology , Cell Proliferation/drug effects , Lysosomes/physiology , Macrophages/cytology , Membrane Fusion/drug effects , Metalloproteases/pharmacology , Phagosomes/physiology , Animals , Lysosomes/drug effects , Macrophages/drug effects , Mice , Phagosomes/drug effects , RAW 264.7 CellsABSTRACT
OBJECTIVE: To construct the eukaryotic expression vector of zinc-dependent metalloprotease 1 (zmp1) gene from Bacillus Calmette-Guerin (BCG) and investigate its impact on the apoptosis of RAW264.7 macrophages. METHODS: Zmp1 gene was amplified from the genome of BCG by PCR. The zmp1 gene fragment was inserted into multiple cloning sites of pEGFP-N1 to construct the eukaryotic expression vector pEGFP-N1-zmp1. The constructed pEGFP-N1-zmp1 was transfected into RAW264.7 cells by Lipofectamine(TM) 2000. The expression of green fluorescent protein (GFP) was observed by fluorescence microscopy. The zmp1 mRNA was detected by quantitative real-time PCR (qR-PCR). The effect of Zmp1 protein on the apoptosis of RAW264.7 macrophages was detected by flow cytometry (FCM). RESULTS: With zmp1 gene amplified by PCR, we successfully constructed the recombinant vector pEGFP-N1-zmp1 as demonstrated by restriction enzyme analysis and sequencing. GFP was seen in RAW264.7 cells 24 hours after transfected with the recombinant plasmid. As qRT-PCR showed, the expression level of zmp1 mRNA was up-regulated. The early apoptotic rate increased 48 hours after transfection. CONCLUSION: The increased expression of Zmp1 in RAW264.7 cells promotes the apoptosis of RAW264.7 cells.
