A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis
Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.
KeywordsAgave CAM plants Protein extraction Protease Electrophoresis Mass spectrometry
Crassulacean acid metabolism
We are thankful to Dr. Salvador Arias from Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México for providing O. ficus-indica, L.marginatus, and M. magnimamma samples. We also thank Unidad de Proteómica and Unidad Universitaria de Apoyo Bioinformático, Instituto de Biotecnología, Universidad Nacional Autónoma de México for all mass spectrometry analysis and the production of our local Agave fasta database, respectively. This work was supported by research grants from PAPIIT/DGAPA/UNAM IN212116 (F Lledías) and IG200515 (J Nieto-Sotelo and G Cassab), UNAM-Allied/Domecq P-150 (J Nieto-Sotelo and G Cassab), and CONACyT PN-247732 (J Nieto-Sotelo and G Cassab).
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Human participants and animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- 4.García-Mendoza A, Galván VR (1995) Riqueza de las familias Agavaceae y Nolinaceae en México. Bol Soc Bot México 56:7–24Google Scholar
- 9.Ahumada-Santosa YP, Montes-Avila J, Uribe-Beltrána M, Díaz-Camachoa SP, López-Angulo G, Rito Vega-Aviña R, López-Valenzuela JA, Heredia JB, Delgado-Vargas F (2013) Chemical characterization, antioxidant and antibacterial activities of six Agave species from Sinaloa, México. Ind Crops Prod 49:143–149CrossRefGoogle Scholar
- 11.Nobel PS, Smith SD (1983) High and low temperature tolerances and their relationships to distribution of agaves. Plant Cell Environ 6:711–719Google Scholar
- 12.González-Cruz L, Jaramillo-Flores ME, Bernardino-Nicanor Mora-Escobedo R (2011) Influence of plant age on fructan content and fructosyltranserase activity in Agave atrovirens Karw leaves. Afr J Biotechnol 10:15911–15920Google Scholar
- 15.Gentry HS (1982) Agaves of continental North America. University of Arizona Press, TucsonGoogle Scholar
- 22.Tipton KF (1964) Agavain: a new plant proteinase. Biochim Biophys Acta 92:341–350Google Scholar
- 24.Du Toit PJ (1976) Isolation and partial characterization of a protease from Agave americana variegata. BBA-Enzymol 429:895–911Google Scholar
- 31.Charney J, Tomarelli RM (1947) A colorimetric method for the determination of the proteolytic activity of duodenal juice. J Biol Chem 171:501–505Google Scholar
- 32.Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- 36.Balen B, Krsnik-Rasol M, Zadro I, Simeon-Rudolph V (2004) Esterase activity and isoenzymes in relation to morphogenesis in Mammillaria gracillis Pfeiff tissue culture. Acta Bot Croat 63:83–91Google Scholar
- 37.O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021Google Scholar