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On-line Access: 2017-01-03

Received: 2016-03-27

Revision Accepted: 2016-09-12

Crosschecked: 2016-12-16

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Ling-li Lu


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Journal of Zhejiang University SCIENCE B 2017 Vol.18 No.1 P.85-88


Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts

Author(s):  Xiao-yu Gao, Xing-cheng Liao, Ruo-lai Wu, Ting Liu, Hai-xing Wang, Ling-li Lu

Affiliation(s):  MOE Key Laboratory of Environment Remediation and Ecological Health, College of Environmental & Resource Sciences, Zhejiang University, Hangzhou 310058, China

Corresponding email(s):   lulingli@zju.edu.cn

Key Words:  Hyperaccumulator plant, Sedum alfredii, Purified isolation, Protoplast, Vacuole

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Xiao-yu Gao, Xing-cheng Liao, Ruo-lai Wu, Ting Liu, Hai-xing Wang, Ling-li Lu. Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts[J]. Journal of Zhejiang University Science B, 2017, 18(1): 85-88.

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author="Xiao-yu Gao, Xing-cheng Liao, Ruo-lai Wu, Ting Liu, Hai-xing Wang, Ling-li Lu",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts
%A Xiao-yu Gao
%A Xing-cheng Liao
%A Ruo-lai Wu
%A Ting Liu
%A Hai-xing Wang
%A Ling-li Lu
%J Journal of Zhejiang University SCIENCE B
%V 18
%N 1
%P 85-88
%@ 1673-1581
%D 2017
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1600138

T1 - Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts
A1 - Xiao-yu Gao
A1 - Xing-cheng Liao
A1 - Ruo-lai Wu
A1 - Ting Liu
A1 - Hai-xing Wang
A1 - Ling-li Lu
J0 - Journal of Zhejiang University Science B
VL - 18
IS - 1
SP - 85
EP - 88
%@ 1673-1581
Y1 - 2017
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1600138

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.


方法:主要包括原生质体提取、液泡粗提和液泡纯化。原生质体提取:取东南景天叶片,切成1~2 mm的细条状后浸入经预热过的细胞裂解液中,震荡2 h后过滤,离心清洗后获得原生质体。液泡粗提:采用1-丙磺酸浓度为0.675 mmol/L的原生质体裂解液裂解原生质体,离心后获得粗提的液泡,并加入含0.8 mol/L甘露醇的液泡保护液。液泡纯化:往初提液泡的悬浮液下层加入质量体积比浓度为0.10 g/ml的Ficoll溶液,进行密度梯度离心,获取纯化的液泡。
结论:细胞裂解液的预热处理可加速细胞壁降解,裂解时间设置为2 h有利于原生质体的高效提取;通过对原生质体裂解液浓度、细胞保护液浓度和梯度离心等参数的改良,可有效提取叶片细胞原生质体中的液泡。


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[1]Huang, J., Zhang, Y., Peng, J.S., et al., 2012. Fission yeast HMT1 lowers seed cadmium through phytochelatin-dependent vacuolar sequestration in Arabidopsis. Plant Physiol., 158(4):1779-1788.

[2]Leitenmaier, B., Küpper, H., 2013. Compartmentation and complexation of metals in hyperaccumulator plants. Front. Plant Sci., 4:374.

[3]Ma, J.F., Ueno, D., Zhao, F.J., et al., 2005. Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of Thlaspi caerulescens. Planta, 220(1):731-736.

[4]Robert, S., Zouhar, J., Carter, C., et al., 2007. Isolation of intact vacuoles from Arabidopsis rosette leaf-derived protoplasts. Nat. Protoc., 2(2):259-262.

[5]Schuck, S., Honsho, M., Ekroos, K., et al., 2003. Resistance of cell membranes to different detergents. PNAS, 100(10):5795-5800.

[6]Shimaoka, T., Ohnishi, M., Sazuka, T., et al., 2004. Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. Plant Cell Physiol., 45(6):672-683.

[7]Song, W.Y., Mendoza-Cózatl, D.G., Lee, Y., et al., 2014. Phytochelatin-metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis. Plant Cell Environ., 37(6):1192-1201.

[8]Tian, S., Lu, L., Labavitch, J., et al., 2011. Cellular sequestration of cadmium in the hyperaccumulator plant species Sedum alfredii. Plant Physiol., 157(4):1914-1925.

[9]Yang, X., Long, X.X., Ye, H.B., et al., 2004. Cadmium tolerance and hyperaccumulation in a new Zn-hyperaccumulating plant species (Sedum alfredii Hance). Plant Soil., 259(1-2):181-189.

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