Impaginato 233 Adv. Hort. Sci., 2020 34(2): 233­240 DOI: 10.13128/ahsc­7848 Extraction of total protein from shoots of Cereus morphological variants (Cactaceae) for proteomic analysis V.N.A. Fernandes 1, C.A. Mangolin 2, A.F. Neves 1, F.C. Sousa Nogueira 3, H. Zeni Neto 4, M.F.P.S. Machado 2 (*) 1 Postgraduate Program in Genetics and Breeding, Universidade Estadual de Maringá, Maringá PR, Brazil. 2 Department of Biotechnology, Genetics and Cell Biology, Universidade Estadual de Maringá, Maringá PR, Brazil. 3 Department of Chemistry of Proteins, Unidad Proteomics, Chemistry Institute, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil. 4 Department of Agronomy, Universidade Estadual de Maringá, Maringá PR, Brazil. Key words: cactus, phenotypic variants, protein extraction, SDS­PAGE, succulent tissues. Abstract: Since there is a hypothesis that qualitative and/or quantitative differ­ ences of specific proteins may be associated with morphological variants in cacti of the Cereus genus (phenotypes erect, tortuosus and monstruosus), in current study we tested three different methods for protein extraction from shoots of the phenotypic variants to obtain protein fractions for further pro­ teomic analysis. The TCA/acetone method for protein extraction revealed a larger number of well­defined bands in SDS­PAGE system than the methods with phenol. The quantification of protein extracted by TCA/acetone ranged between 0.488 (tortuosus) and 2.92 µg·mL­1 (monstruosus). Although the use of phenol is the most appropriate procedure for protein extraction from recalci­ trant tissues, results have shown that extraction buffer containing two antioxi­ dant agents (EDTA and β­mercaptoethanol) and PMSF to prevent protein degradation was efficient to avoid proteolysis and lower protein yield, than using TCA/acetone precipitation for protein extraction from shoots of Cereus sp. The use of extraction buffer with appropriate combinations of antioxidant agents, phenol­complexing agents, and protease inhibitors may be an efficient alternative for proteins extraction from succulent and recalcitrant tissues (such as cactus plants) using a simple protein extraction method. 1. Introduction Since the 1960s the study on proteins from plant tissues is frequently restricted by extraction procedures from different tissues (Loomis, 1969; 1974; Kelley and Adams, 1977). Different procedures for protein extrac­ tion have been reported for different tissues and plant species to obtain (*) Corresponding author: mfpsmachado@uem.br Citation: FERNANDES V.N.A., MANGOLIN C.A., NEVES A.F., SOUSA NOGUEIRA F.C., ZENI NETO H., MACHADO M.F. P.S., 2020 ­ Extraction of total protein from shoots of Cereus morphological variants (Cactaceae) for proteomic analysis. ‐ Adv. Hort. Sci., 34(2): 233­240. Copyright: © 2020 Fernandes V.N.A., Mangolin C.A., Neves A.F., Sousa Nogueira F.C., Zeni Neto H., Machado M.F.P.S. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 4 September 2019 Accepted for publication 16 January 2020 AHS Advances in Horticultural Science Short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2020 34(2): 233­240 234 protein fractions which are adequate for elec­ trophoresis and polypeptides characterization on gel. Taxa and tissues have their own set of endoge­ nous tannins, phenols and phenoloxidases which interfere differently with protein stability. Protein extraction from cactus tissues is particularly an arduous process due to the abundance of complex heteropolysaccharides, coupled to endogenous tan­ nins, phenols and phenoloxidases in the shoot tis­ sues of adult plants. Heteropolysaccharides confer high viscosity to the extraction solution after shoot­ tissue homogenization and hinder the solubilization of proteins. Protein extraction has been a primordial stage for the studies on proteomics. The detection of pro­ teins and protein variants found in cells of a given tissue under specific conditions (functional genom­ ic) (Wilkins et al., 1996; Westermeier and Naven, 2002) requires adequate solubilization and stability of protein fractions. The first step should establish a procedure for protein extraction from shoots, or rather, for the proteome analysis in shoots of phe­ notypic variants of cactus from genus Cereus. The Cereus plants have been mainly used as forage for ruminants (Silva et al., 2011) and fruits and pulp are used in the preparation of cookies, pies, and pastries (Almeida et al., 2007). Several studies have revealed the importance of the C. peruvianus species as fruit crop (Mizrahi, 2014). While the C. jamacaru plants constitute a wild natural resource in the semiarid region of Northeastern Brazil, an industrial and eco­ nomic importance has been attributed to the C. peru‐ vianus species cultivated in the Southern region of Brazil (Alvarez et al., 1995; Barros and Nozaki, 2002). Medicinal importance is also attributed to C. peru‐ vianus since arabinogalactan extracted from the gum has been indicated for the treatment of gastric ulcers (Tanaka et al., 2010). Phenotypic variants of cacti from the genus Cereus, tagged tortuosus and mon‐ struosus varieties, are frequently cultivated together with plants featuring typical columnar­erect shoots in home gardens and public parks and squares in Brazil´s southern region. Although the origin of the tortuosus and monstruosus varieties has not been reported in the literature, the qualitative and/or quantitative differences of specific proteins may be associated with the morphological variants. Since the success of a proteomic experiment depend on the correct identification of proteins, three different methods described by He and Wang (2008) for protein extraction from shoots of the three phenotypic variants of Cereus were employed in the current study. The methods described by He and Wang (2008) were used in protein extraction from Aloe vera tissues, a succulent and recalcitrant plant similar to cacti. It is expected that protocols described by these authors may be suitable for pro­ tein extraction and quantification from shoots of Cereus plants. 2. Materials and Methods Samples of Cereus plants with typically erect shoots and plants of the varieties tortuosus and mon‐ struosus (Fig. 1), cultivated in south Brazil (in Maringá PR Brazil, at 23°25’38” S; 51°56’15” W), were collect­ ed from home gardens and maintained in the Experimental Botanic Garden of the State University of Maringá (altitude 554.9 m; 23°25’S; 51°25ʹW). Pieces of shoots (2 g) from erect, tortuosus and mon‐ struosus plants (four plants of each morphology) were collected and used as samples for protein extraction. The cuticle and the fractions of cells with chlorophyll were removed from the pieces of shoot to minimize the contamination of the samples by polysaccharides. Fresh shoot sections (2 g) of each Cereus plant with erect shoots (E1­E4) and plants of the varieties tortuosus (T1­T4) and monstruosus (M1­M4) were ground to a fine powder in liquid nitrogen and homogenized in 2 mL buffer 0.02 M Tris­HCl pH 7.5, 0.25 M sucrose, 0.01M ethylene glycol tetraacetic acid (EDTA), 0.001 M phenylmethylsulfonyl fluoride (PMSF), 1% Triton X­100 and 2% ß­mercaptoethanol, following method by He and Wang (2008). Proteins were extracted from two samples of each E1­E4, T1­ Fig. 1 ­ Different morphologies of Cereus peruvianus plants showing stems: erect (A), tortuosus (B), monstruosus (C). Fernandes et al. ‐ Extraction of protein for proteomic analysis 235 T4, and M1­M4 plants (Table 1). After homogenization, 1.0­mL aliquots from each Cereus plants were transferred to 2 mL microtubes and prepared by three procedures: i) TCA­acetone precipitation; ii) phenol extraction, and iii) improved phenol extraction method according to described by He and Wang (2008). Protein was quantified by the fluorometric method in a Fluorometer Qubit® 1.0 using Qubit® Protein Assay kit from Life Technologies. Polyacrylamide gel (12%) was prepared with 16.2 mL of 30% acrylamide and 0.8% bis­acrylamide dissolved in 4 mL of 1.5 M Tris­HCl, pH 8.8, 107 µL 10% SDS, 5.7 mL twice­distilled water, 320 µL 2% ammonium per­ sulfate and 16 µL TEMED. Stack gel was prepared at a final concentration of 5%, pH 6.8: 3 mL of 10% acry­ lamide and 5% bis­acrylamide dissolved in 3 mL of 0.24 M Tris­HCl pH 6.8, 30 µL twice­distilled water, 250 µL 2% ammonium persulfate and 3 µL TEMED. Further, 25 mM Tris/200 mM glycine, pH 8.3, and 0.1% SDS were employed in the electrode chambers. Samples were taken from the freezer and 2 μL loading dye [20% glycerol 10 mM, Tris­HCl 1.5 M pH 8.8, 10% (w·v­1) bromophenol blue, 10% SDS (w·v­1), β­mercaptoethanol 2% (v·v­1), and twice­distilled water q.s.p.] were added to 8 μL of sample and applied to the gel. Electrophoresis was performed in Tris­glycine buffer at 200 volts, for thirty minutes, for the stacking gel, followed by two hours and thirty minutes for the separation of the proteins. After elec­ trophoresis, the gel was fixed in a fixation solution (40% methanol; 70% acetic acid) and stained in silver 20% following protocol by Laemmli (1970). After migration, gels were stained, photographed and the images were analyzed with GelAnalyzer 19.1 softwa­ re (http://www.gelanalyzer.com/) to transform elec­ trophoretic bands into peaks, to calculate Rf and esti­ mate the molecular weight of proteins. The protein quantification (µg·µL­1) by the fluoro­ metric method in duplicate of morphological variants of Cereus with shoots erect (E1­E4), tortuosus (T1­ T4), and monstruosus (M1­M4) obtained by extrac­ tion with TCA/acetone method described by He and Wang (2008) were analyzed using the software R (R Core Team, 2019) with the packages: i) “nortest” (Gross and Ligges, 2015) to verify the normality of the errors by the Lilliefors test (Lilliefors, 1969), ii) “car” (Fox and Weisberg, 2019) to verify the indepen­ dence of errors by the Durbin­Watson test (Durbin and Watson, 1951), and iii) the “ExpDes” package (Ferreira et al., 2018) to verify the homogeneity of variances by the O´Neill and Matthews test (O´Neill and Matthews, 2000). All these procedures were admitting the error at 1% of significance. 3. Results and Discussion The TCA/acetone method for protein extraction from shoots of Cereus plants revealed a larger num­ ber of well­defined bands in SDS­PAGE system than the methods with phenol and improved­phenol extraction. In the TCA/acetone method, each homog­ enized tissue (plant tissues with shoots erect, tortuo‐ sus and monstruosus) in the extraction buffer was centrifuged at 15,000 rpm for 30 min at 4°C. The supernatant was then recovered and placed in a new tube where ¼ of the volume of acetone containing 50% trichloroacetic acid (TCA) was added. The solu­ tion was incubated for 2 h, at ­20°C and centrifuged again at 15,000 rpm for 40 min, at 4°C. The super­ natant was then discarded and the pellet was Table 1 ­ Protein quantification (µg µL­1) by the fluorometric method in duplicate of morphological variants of Cereus with shoots erect (E1­E4), monstruosus (M1­M4), and tortuosus (T1­T4) obtained by the extraction with TCA/acetone method described by He and Wang (2008) Erect Monstruosus Tortuosus sample µg·µL­1 sam µg·µL­1 sample µg·µL­1 E1 0.824 M1 2.64 T1 1.090 0.658 2.68 0.990 E2 0.732 M2 1.71 T2 0.768 0.766 1.91 1.150 E3 0.964 M3 2.26 T3 1.740 0.874 2.40 1.520 E4 1.100 M4 2.54 T4 0.488 1.480 2.92 0.592 Adv. Hort. Sci., 2020 34(2): 233­240 236 washed three times with acetone containing 0.2% 1,4­Dithiothreitol (DTT). Pellets were then dissolved in 50 µL lysis buffer (8 M urea, 4% NP­40 and 1% DTT) and stocked at ­20°C. The quantification of protein extracted by TCA/acetone method was made in duplicate (two samples of each shoot morphology: erect, tortuosus and monstruosus) and ranged between 0.488 (tortu‐ osus) and 2.92 µg·mL­1 (monstruosus) (Table 1). The normality of the errors by the Lilliefors test (Lilliefors, 1969), the independence of errors by the Durbin­Watson test (Durbin and Watson, 1951) and the homogeneity of variances by the O´Neill and Matthews test (O´Neill and Matthews, 2000) showed that there were no restrictions by p­values regarding analysis of variance (ANOVA) procedure (Table 2). Significant differences (at 1% level) in protein con­ centrations within and between Cereus plants with the erect, tortuosus and monstruosus morphologies were detected by ANOVA procedure (Table 3). The non­significant experimental error indicates that the sampling error (different morphologies) may justify the differences in protein concentrations between Cereus plant morphologies. Non­significant experimental error assures the researcher more reli­ able results because the effect of uncertainty is smaller (Patterson, 1946; Cochran and Cox, 1957; Snedecor and Cochran, 1980). A higher protein concentration in monstruosus plants than in erect and tortuosus plants has been detected by Tukey´s test (Tukey, 1953) employed to compare the averages of protein concentrations in erect, tortuosus and monstruosus plants (Table 4). Figure 2 also illustrates the highest protein concen­ tration in monstruosus plants and shows the ampli­ tude of concentration rates within each plant. Differences in protein concentrations in Cereus plants with the erect, tortuosus and monstruosus morphologies indicate that it is necessary to evaluate the protein concentration in more than one plant with the same morphology to specify the average protein concentration of each morphology. Differences in protein concentrations among Cereus plants with erect, tortuosus and monstruosus mor­ phologies detected in current study support the hypothesis that qualitative and/or quantitative differ­ ences of specific proteins may be associated with morphological variants in cacti of the Cereus genus, and may be used to justify a proposal for further pro­ teomic analysis. The phenol extraction method described by He and Wang (2008) revealed a number of protein frac­ Table 2 ­ p­values of the normality of errors by the Lilliefors test (Lilliefors, 1969), independence of errors by the Durbin­Watson test (Durbin and Watson, 1951) and the homogeneity of variances by the O´Neill and Matthews test (O´Neill and Matthews, 2000) Procedure Normality Independence Variance Lilliefors 0.8897 Durbin­Watson 0.0379 O´Neill and Matthews 0.4089 Table 3 ­ Mean Squares (MS) of the comparison of protein con­ centrations from the three Cereus morphologies (erect, tortuosus and monstruosus shoots) and their respective sampling and experimental mistakes ** p­value <0.01. MS Morphologies 5.2477 ** Sample error 0.2969 ** Experimental error 0.0251 Table 4 ­ Mean protein concentrations (µg µL­1) from the three Cereus morphologies (erect, tortuosus and monstruo‐ sus) revealed by the Tukey´s test Morphology Mean protein concentraction (µg·µL­1) Monstruosus 2.3825 a Tortuosus 1.0423 b Erect 0.9248 b Fig. 2 ­ Amplitude of the concentration values observed within monstruosus (M), tortuosus (T), and erect (E) plants of Cereus. Fernandes et al. ‐ Extraction of protein for proteomic analysis 237 tions smaller than the number of protein fractions detected by the TCA/acetone precipitation method in Cereus plants, while protein fractions were absent with the improved phenol extraction method described by He and Wang (2008)(Fig. 3). The GelAnalyzer software was useful to create calibration curves and to estimate the quantity of the protein from a band. Figure 4 shows the calibration curve that make a correspondence between the quantity of the protein loaded on each lane and the areas of the peaks of each lane (the software name the area of the peak as raw volume and the conventional units are in pixels). The correlation coefficients of such cali­ bration curves were higher than 0.99 for proteins with molecular masses ranging from 24 until 180 kDa (Table 5). However, data obtained by MS (Mass Spectrometry analysis) indicated proteins with mole­ cular weights varying from 220 to 15 kDa. In gel regions without clear band distinction in 1DE SDS­ PAGE were detected considerable amounts of pro­ tein identified by MS. Preliminary Mass Spectrometry analysis identified 753 proteins extracted by the TCA/acetone precipitation method in the Cereus plants (unpublished results). Improved phenol extraction method was described as the most appropriate procedure for pro­ Fig. 3 ­ Protein extraction from shoots of Cereus with erect (samples 1­2, 5­6, 9­10), tortuosus (samples 3, 7, 11) and monstruosus (samples 4, 8, 12) morphologies, with TCA/acetone method (samples 1­4), with phenol extrac­ tion method (samples 5­8) and with the improved­phe­ nol extraction method by He and Wang (2008) (samples 9­12), in the SDS­PAGE system 12%. Fig. 4 ­ The main panels of Gel Analyser software showing the image of the entire electrophoresis gel (panel A); the bands from the selected lane from panel A are transformed in peaks (panel B) and their area are computed in panel C, as raw volume (conventio­ nal units). Based on the molecular weights of the markers (in panel D), the correspondence between the molecular mass and the migration (as Rf) of the protein is presented. 238 Adv. Hort. Sci., 2020 34(2): 233­240 tein extraction from Aloe vera tissues, a succulent plant, similar to cacti. Phenol has also been consid­ ered as the most effective for the removal of unwanted interfering substances from the protein samples of tissues from other recalcitrant plants (He and Wang, 2008; Pavoković et al., 2012; Riffel et al., 2012). It has been postulated that, in the case of par­ ticularly recalcitrant tissues, acetone and TCA/ace­ tone precipitation do not sufficiently remove nucleic acids, carbohydrates and polyphenols, which cause co­precipitation and degradation of proteins. The phenol method, although more laborious and time­ consuming, resulted in higher protein yield and with the lowest contamination rate of samples than the TCA/acetone precipitation method alone for protein extraction from recalcitrant tissues (He and Wang, 2008; Pavoković et al., 2012; Wu et al., 2014). However, results from the current study are indicative that the use of an extraction solution con­ taining two antioxidant agents (EDTA and β­mercap­ toethanol) and PMSF to prevent protein degradation was efficient to avoid proteolysis and lower yield of proteins using TCA/acetone precipitation for protein extraction from shoots of Cereus sp. Although the use of phenol is reported to be the most appropriate procedure for protein extraction from recalcitrant tis­ sues, a significant proportion on cellular proteins may be lost from the extracts during the extraction proce­ dure with phenol. The phenol extraction method involves more steps than the TCA/acetone method while a ‘minimum­steps´ method is preferred with protein extraction procedures (Isaacson et al., 2006). The TCA/acetone method described by He and Wang (2008) involved only one precipitation, two centrifu­ gations and three washes after homogenization of the tissues, whereas nine steps (including one overnight precipitation at ­20°C) were reported for phenol method. Thus, the use of the TCA/acetone method described by He and Wang (2008) represent­ ed a minimum­steps method and, thus, a shorter time for the extraction of proteins from shoot tissues of Cereus plants. A relatively simple extraction buffer containing one phenol­complexing agent (PVP) and two antiox­ idant agents (β­mercaptoethanol and EDTA) was also used for electrophoresis of the several isozymes in shoot tissues of the cactus Cereus peru‐ vianus (Mangolin and Machado, 1997), while phenol and ammonium acetate were employed for the pro­ tein extraction from callus tissues of C. peruvianus (Mangolin et al., 1999) for two­dimensional elec­ trophoresis of callus tissues grown in culture media containing different concentrations of auxin and cytokinin. Only one phenol­complexing agent (PVP) and a cocktail of protease inhibitors (Roche, USA) were used for protein extraction from tissues of the cactus Mammillaria gracilis grown in vitro (Rogic´ et al., 2015). The TCA/acetone/β­mercaptoethanol method plus protease inhibitors (Roche, USA) was also employed for protein extraction from several species of succulent plants and some cactus species (Lophocereus marginatus, Mammillaria magnimam‐ ma and Opuntia fícus‐indica) instead of phenol pre­ cipitation method (Lledías et al., 2017). The evidence from these studies actually demonstrates that, in the case of tissues with different levels of interfering sub­ stances, different phenol complexing agents and dif­ ferent antioxidant agents are required. The physio­ logical state of each tissue is the decisive factor of a greater or lesser complexity in protein extraction processes. As types and concentrations of polysac­ charides, polyphenols and other secondary metabo­ lites may be induced in response to environmental factors, different types and concentrations of phenol­ complexing agents and antioxidant agents may be needed for the same tissue in different environmen­ tal conditions. According to Wendel and Weeden (1989), extraction solutions for tissues with moderate Table 5 ­ Quantity of the protein loaded on each lane detected with TCA/acetone method (samples 1­4) and with phenol extraction method (samples 5­8) in the SDS­PAGE system 12% from the Cereus plants Lane MW of each band (KDa) Lane 1 ­ ­ ­ ­ ­ ­ 45 ­ 24 Lane 2 ­ ­ ­ 77 ­ ­ 45 ­ 24 Lane 3 ­ 145 90 77 ­ ­ 45 39 ­ Lane 4 180 145 90 77 ­ ­ 45 39 ­ Lane 5 ­ ­ ­ ­ ­ ­ 45 ­ ­ Lane 6 ­ 145 90 77 61 ­ 45 39 24 Lane7 ­ ­ ­ ­ ­ ­ 45 ­ ­ Lane 8 ­ ­ ­ ­ ­ 58 45 ­ ­ Fernandes et al. ‐ Extraction of protein for proteomic analysis 239 levels of interfering substances require at least two phenol­complexing and two antioxidant agents, while at least four phenol­complexing and three antioxi­ dant agents are needed for tissues with high levels of interfering substances. 4. Conclusions The simplest method (TCA/acetone precipitation), described by He and Wang (2008), and the use of two antioxidant agents and a protease inhibitor showed a number of protein fractions greater than the number of protein fractions detected with the phenol method in shoots of Cereus analyzed in cur­ rent study (plants with erect shoots and plants of the varieties tortuosus and monstruosus). The authors´ expectation is to use this simple method of extracting proteins for proteomic analysis of the phenotypic variants from the genus Cereus. However, the use of phenol or other phenol­complexing and antioxidant agents to extract proteins from shoots of other Cereus plants grown in different regions or in differ­ ent environmental conditions (different seasons of the year, e.g.) may be needed. Consequently, the proposal to establish a ‘universal protocol’ for succu­ lent plants, such as cacti, seems unattainable. 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