Chromoplast Isolation and Its Proteomic Analysis from Cassava Storage Roots

DENG Chang-Zhe1,2 YAO Hui1 AN Fei-Fei1 LI Kai-Mian1 CHEN Song-Bi1

(1.Tropical Crops Genetic resources Institute, Chinese Academy of Tropical Agricultural Sciences/Key Laboratory of Ministry of Agriculture for Germplasm Resources Conservation and Utilization of Cassava, Danzhou, China 571737)
(2.Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, China 570228)

【Abstract】Chromoplasts are the sites to store carotenoids and regulate a variety of physiological and biochemical processes in the storage roots of cassava (Manihot esculenta Crantz). In the present study, it was found that Percoll density gradient centrifugation was suitable for isolating the chromoplasts from cassava storage roots. Rich and intact chromoplasts were located in 40% to 50% layer of Percoll, the expression level of Vdac1, a mitochondrial marker, was the lowest and the expression level of RbcL, a plastid marker, was the highest compared with those in other layers using Western blot. Thirty-four differentially expressed proteins were detected in SC9, in which 17 were up-regulated, and the others were down-regulated. The differential proteins related to carbohydrate and energy metabolism accounted for the highest proportion. The STRING protein-protein interaction network showed that Enolase 2 and Elongation Factor were the hub proteins, which played the key roles in the whole regulatory network. Quantitative analysis by qRT-PCR confirmed the Enolase 2 expression was more significantly up-regulated in the high carotenoid cassava cultivar than in the low carotenoid cassava cultivar SC6068. These two proteins may be the key points for affecting the carotenoid content between SC9 and SC6068.

【Keywords】 Cassava storage root; Chromoplast protein; Isolation method; Proteomics;

【DOI】

【Funds】 National Natural Science Foundation of China (31271776) supported by the National Natural Science Foundation of China (31271776) Initial Fund of High-level Creative Talents in Hainan Province (2012–2016) the Initial Fund of High-level Creative Talents in Hainan Province (2012–2016)

Download this article

    References

    [1] Zhang Z W, Li K M, Ye J Q, Xu R L. The study on photosynthetic characteristic of cassava. J Yunnan Univ (Nat Sci), 2007, 29: 628–632 (in Chinese with English abstract)

    [2] Chen G X, Li K M, Ye J Q, Xu R L. Growth and yield of 6 cassava varieties. J Trop Agric, 2009, 29: 26–29 (in Chinese with English abstract)

    [3] Li K, Zhu W, Zeng K, Zhang Z, Zhang Z W, Ye J Q, Ou W J, Rehman S, Heuer B, Chen S B. Proteome characterization of cassava (Manihot esculenta Crantz) somatic embryos, plantlets and tuberous roots. Proteome Sci, 2010, 8: 10

    [4] Nassar N M A, Junior O P, Sousa M V, Ortiz R. Improving carotenoids and amino-acids in cassava. Nutr Agric, 2009, 1: 32–38

    [5] Cazzonelli C, Pogson B. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci, 2010, 15: 266–274

    [6] Kris G, Joel V. Protein identification methods in proteomics. Electrophoresis, 2000, 21: 1145–1154

    [7] Thierry R. Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics, 2002, 2: 3–10

    [8] Fan P X, Wang X C, Kuang T Y, Li Y X. An efficient method for the extraction of chloroplast proteins compatible for 2-DE and MS analysis. Electrophoresis, 2009, 30: 3024–3033

    [9] Wang J H. Proteomic Analysis of Rice and Maize Chloroplast and Construction of Maize Chloroplast Transformation System. MS Thesis of Chinese Academy of Agricultural Sciences, 2011 (in Chinese with English abstract)

    [10] THIELLEMENT H. Plant proteomics methods and protocols. Totowa: Humana Press Inc, 2007. pp 43–48

    [11] Tanka N, Fujita M, Handa H, Murayama S, Uemura M, Kawamura Y, Mitsui T, Mikami S. Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments. Mol Genet Genom, 2004, 271: 566–576

    [12] Huang S B, Nicolas L T, Reena N, Holger E, James W, Harvey M. Experimental analysis of the rice mitochondrial proteome, its biogenesis, and heterogeneity. Plant Physiol, 2009, 149: 719–734

    [13] Taise S, Miwa O, Takashi S, Naoto M. Ikuko H, Kenichior S, Masayoshi M, Akiho Y, Kenichi T, Tetsuro M. Isolation of intact vacuoles and proteomic analysis of tonoplast from suspension-cultured cells of Arabidopsis thaliana. Plant Cell Physiol, 2004, 45: 672–683

    [14] Barsan C, Sanchez-Bel P, Rombaldi C, Egea I, Rossignol M, Kuntz M, Zouine M, Latche A, Bouzayen M, Pech J C. Characteristics of the tomato chromoplast revealed by proteomic analysis. J Exp Bot, 2010, 61: 2413–2431

    [15] Siddique M A, Grossmann J, Gruissem W, Baginsky S. Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts. Plant Cell Physiol, 2006, 47: 1663–1673

    [16] Zeng Y, Pan Z, Ding Y, Zhu A, Cao H, Xu Q, Deng X X. A proteomic analysis of the chromoplasts isolated from sweet orange fruits (Citrus sinensis L. Osbeck). J Exp Bot, 2011, 62: 5297–5309

    [17] Wang Y Q, Yong Y, Fei Z J, Hui Y, Tara F, Theodre W, Michael M, Leao V, Wang X W, Li L. Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplasts function and development. J Exp Bot, 2013, 64: 949–961

    [18] An F F, Fan J, Li G H, Jian C P, Li K M. Comparison of leaves proteome and chlorophyll fluorescence of cassva cv. SC8 and its tetraploid mutants. Sci Agric Sin, 2013, 46: 3978–3987 (in Chineses with English abstract)

    [19] Song Y C, An F F, Xue J J, Qin Y L, Li K M, Chen S B. Protemic analysis on tuberous roots of cassava cultivar ZM-Seaside and Mosaic-leaf mutantion. Boll Biol, 2017, 33 (3): 78–85 (in Chineses with English abstract)

    [20] Sánchez T, Salcedo E, Ceballos H, Dufour D, Mafla G, Morante N, Calle F, Pérez J C, Debouck D, Jaramillo G, Moreno I X. Screening of starch quality traits in cassava (Manihot esculenta Crantz). Starch/Stärke, 2009, 61: 12–19

    [21] An F F, Jie F, Jun L, Li K M, Zhu W L, Wen F, Luzi J C B, Songbi C. Comparison of leaf proteomes of cassava (Manihot esculenta Crantz) cultivar NZ199 diploid and autotetraploid genotypes. PLoS One, 2014, 9 (4): e85991

    [22] Cristina M S, Petersen M, Mundy J. Mitogen-activated protein kinase signaling in plants. Annu Rev Plant Biol, 2010, 61: 621–649

    [23] Neuhaus H E, Emes M J. Nonphotosynthetic metabolism in plastid. Plant Physiol, 2000, 51: 111–140

    [24] Eage I, Brasan C, Bian W, Purgatto E, Purgatto E, Latche A, Chervin C, Bouzayen M, Pech J C. Chromoplast differentiation: current status and perspectives. Plant Cell Physiol, 2010, 51: 1601–1611

    [25] Reiser J, Linka N, Lemke L, Jeblick W, Neuhaus H E. Molecular physiological analysis of the two plastidic ATP/ADP transporters from Arabidopsis. Plant Physiol, 2004, 136: 3524–3536

    [26] Li L, Van Eck J. Metabolic engineering of carotenoid accumulation by creating a metabolic sink. Transgen Res, 2007, 16: 581–585

    [27] Pojidaeva E, Zinchenko V, Shestakov S, Sokolenko A. Involvement of the SppAl peptidase in acclimation to saturating light intensities in Synechocystis sp. strain PCC 6803. J Bacteriol, 2004, 186: 3991–3999

    [28] Jarvis P. Targeting of nucleus-encoded proteins to chloroplasts in plants. New Phytol, 2008, 179: 257–285

    [29] Sun W, Montagu M W, Verbruggen N. Small heat shock protein and stress tolerance in plants. Biochim Biophys Acta, 2002, 1577: 1–9

    [30] Wang W X, Vinocur B, Shoseyov O. Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Altman, Trends Plant Sci, 2004, 9: 244–252

    [31] Sakamoto W. Protein degradation machineries in plastids. Annu Rev Plant Biol, 2006, 57: 599–621

    [32] Peltier J B, Ripon D R, Friso G, Rudella A, Cai Y, Ytterberg J, Giacomelli L, Pillardy J, van Wijk K J. Clp protease complexes from photosynthetic and non-photosynthetic plastids and mitochondria of plants, their predicted three-dimensional structures, and functional implications. J Biol Chem, 2004, 279: 4768–4781

    [33] Veronica A, Ingenfeld A, Klaus A. Characterization of the snow cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality. Plant Mol Biol, 2006, 60: 507–518

This Article

ISSN:0496-3490

CN: 11-1809/S

Vol 43, No. 09, Pages 1290-1299

September 2017

Downloads:0

Share
Article Outline

Abstract

  • 1 Materials and methods
  • 2 Results and analyses
  • 3 Discussion
  • 4 Conclusions
  • References