title: Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts.
abstract: This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h-long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.
authors.affiliation: MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xiao-Yu
authors.initials: XY
authors.lastname: Gao
authors.affiliation: MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Xing-Cheng
authors.initials: XC
authors.lastname: Liao
authors.affiliation: MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Ruo-Lai
authors.initials: RL
authors.lastname: Wu
authors.affiliation: MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Liu
authors.affiliation: MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Hai-Xing
authors.initials: HX
authors.lastname: Wang
authors.affiliation: MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China.
authors.forename: Ling-Li
authors.initials: LL
authors.lastname: Lu
mesh_terms: D002104:Cadmium / Q000378:metabolism; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D002681:China; D018525:Germination; D008293:Malates / Q000378:metabolism; D008353:Mannitol / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism*; D018517:Plant Roots / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism*; D031963:Sedum / Q000378:metabolism*; D013696:Temperature; D014617:Vacuoles
chemical_list: D008293:Malates; D002104:Cadmium; D008353:Mannitol; C030298:malic acid
keywords: Hyperaccumulator plant; Sedum alfredii; Purified isolation; Protoplast; Vacuole