Journal of Life Sciences Volume 6 Number (8)
J LS
Journal of Life Sciences
Volume 6, Number 9, September 2012 (Serial Number 53)
Contents
Microbiology and Biochemistry
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations
Katerina Boberova, Eva Drozdova and Kristyna Pizova 970 A Comparative Study of Soluble Protein Extractions of Populus deltoides × (Trichocarpa ×
Deltoides) for 2-DE
Joke Dupae, Ann Cuypers, Jean-Paul Noben, Jana Boulet, Nele Weyens, Karen Verstraelen and Jaco Vangronsveld
Xanthophyll Cycle and Its Relative Enzymes
Xirui Xiong, Xuefei Wang and Ming’an Liao 985 Isolation and Identification of Cardenolide Compounds of Gomphocarpus sinaicus and Their
Fungicidal Activity Against Soil Borne and Post Harvest Fungi
Moustafa A. Abbassy, Ezzat A. Kadous, EL-Sayed A.M. Abd-Allah and Gehan I.Kh. Marei 995 Microbial Aspects in Comparing Modified Atmosphere Packaging and Vacuum Packaging on
Shelf Life of Fresh Bull Meat
Abbas Najafpourkhadem, Lamiya Rokouie, Firouz Oghabi, Naser Valaie and Ali Shafighi
Botany and Zoology
1003 Effect of Different Organic Nutrient Sources and Two NPK Rates on the Performance and
Nutrient Contents of A Newly Released Cassava Variety
Samson Adeola Odedina, Joy Nwakaego Odedina, Stephen Olusola Ojeniyi and Funmi Akinlana 1008 Nutritional Profile of Untraditional Colored Wheat Grains and Bread Making Utilisation Lenka Ducho ňová, Mária Vargovičová and Ernest Šturdík
1016 Different Growth Characteristics of Grewia mollis, Grewia tenax and Grewia villosa Under
Nursery and Field Conditions
Niemat Abdalla Saleem, Khalil Ayoub Adam Mohamed and Mohamed El Nour
1025 Feeding Enrichment and Leopard Pacing in ZOO Dv ůr Králové, a.s.
Ivana Gardiánová, Ivona Svobodová and Petra Stašáková
Interdisciplinary Researches
Health Impact Assessment of Air Pollution in Some Regions in Albania
Mirela Lika (Çekani), Anjeza Çoku and Erida Nelaj 1034 The Mulching Effect of the Olives Mill Wastewaters on the Soil Cohesion Improvement and Wind
Erosion Reduction in the Southern Tunisian Arid Zones
Mounir Abichou, Mohamed Labiadh, Nagwa Elnwishy and Hanane Abichou 1041 Predicting Potential Distribution of Gaur (Bos gaurus) in Tadoba-Andhari Tiger Reserve, Central
India
Ambica Paliwal and Vinod Bihari Mathur 1050 Management Guidelines for Calabrian Pine Reforestations Carried Out in Southern Italy in the
1950s-70s
Silvano Avolio, Vincenzo Bernardini, Erica Clerici and Matteo Tomaiuolo 1057 Adaptability of Permanent Grassland to Drought Ľuboš Vozár, Ján Jančovič, Peter Kovár and Slávka Bačová 1061 The Effect of Utilisation on the Floristic Composition of Meadow Communities Anna Kryszak, Jan Kryszak, Agnieszka Strychalska and Agnieszka Klarzynska 1068 Biotechnological Strategies for Phytoremediating Triazinic Herbicides in the Humid Pampa
(Argentine)
Luciano Jose Merini, Virginia Cuadrado and Ana María Giulietti
Journal of Life Sciences 6 (2012) 961-969
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations
Katerina Boberova, Eva Drozdova and Kristyna Pizova Laboratory of Biological and Molecular Anthropology, Institute of Experimental Biology, Faculty of Science, Masaryk University,
Brno 611 37, Czech Republic
Received: March 27, 2012 / Accepted: April 27, 2012 / Published: September 30, 2012.
Abstract: The purpose of this study was to demonstrate the application of molecular genetic methods in anthropology and paleodemography in case where the examined bone material is damaged and fragmented, and where the skeletal remains of children are investigated. The application of traditional anthropological methods is limited, especially if sex determination of children and fragmentary skeletal remains is detected. Sex typing genetic markers (SRY, amelogenin) were used for sex determination of children and undetermined fragmentary skeletal remains from the burial site Pohansko, south outer precincts (Czech Republic). This is an approximately 1,200 year-old burial site (the Great Moravian period), which was excavated during rescue excavations. After the genetic analysis, sex was determined in more than half of the investigated samples. The results of the genetic analysis were used for completion of demographic data of this archaeological site. The results of sex determination of several samples were independently verified by the Institute of Criminology in Prague (Czech Republic), using the PowerPlex ® ESX 17 System (Promega). This study showed the suitability of modern molecular genetic methods to skeletal anthropology and paleodemographic analyses.
Key words: Sex determination, aDNA, amelogenin, SRY, PowerPlex ® ESX 17 System, Pohansko.
1. Introduction where the skull or pelvis is damaged or missing, sex determination is difficult and often impossible. Sex
Sex determination is one of the most basic assessment of subadult skeletal remains is unreliable
anthropological characteristics important for any because the sexually dimorphic characters are not yet
reconstruction of the demographic structure of past developed. Anthropological methods of sex
populations. A large representation of subadult determination are limited in such cases [1, 2], and
skeletons or fragmentary skeletal remains of unknown genetic methods can be a suitable alternative.
sex is a frequent problem in demographic analysis of Sex can be determined from skeletal remains based
historic populations because it leads to inaccuracy of on aDNA (ancient DNA) analysis with the use of
results. The presence of fragmentary skeletal material appropriate sex markers. One of the most common
is often associated with rescue archaeological research methods utilized in PCR-based sex determination is
where there is a lack of time for careful excavation and amelogenin gene amplification. The amelogenin gene
it is necessary to use heavy equipment to uncover the lies on the X and Y chromosomes in humans (Yp11.2,
findings, which can damage skeletal material. In cases Xp22.31-p22.1) and produces a protein important in
Corresponding author: Katerina Boberova, M.Sc., Mgr., the development of the tooth enamel matrix. The X and research fields: molecular anthropology, physical anthropology.
E-mail: [email protected]. Y chromosomes contain a high degree of sequence
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations
homology at the amelogenin locus [3]. Using specific period of 1960-1962 [24]. During rescue excavation in amelogenin PCR primers, different base pair (bp)
the years 1960, 1962, 1975-1979, and 1991-1994, fragments are amplifiable from the X and Y
groups of graves and isolated graves were excavated chromosomes. Primer pairs used for sex identification
amongst the numerous settlement objects [25-27]. A are mainly those described by Sullivan et al. [4, 5],
total of 205 graves were explored [28]. The majority of exhibiting a length dimorphism for the X and Y
them contained the skeletons of children and chromosomes (106 and 112 bp). The presence of a
fragmentary skeletal remains of unknown sex. single band indicates that the sample came from a
Molecular sex identification of children and female, while two bands identify the sample’s source
undetermined skeletal remains was performed by PCR as male. An advantage of this approach is that the X
amplification of amelogenin and SRY markers. For chromosome product itself plays a role as a positive
verification, the results of genetic sex determination of control [3].
three samples were subsequently proved using the The amelogenin marker has been successfully
PowerPlex ® ESX 17 System (Promega). An employed in sex determination of skeletal remains
anthropological and demographic study of the from different archaeological sites [6-11]. However, in
population from Pohansko has been published [24], but some cases authors have reported observation of allelic
it was partially distorted due to the large number of drop-out, a frequent problem with amplification of
individuals without known sex. Application of degraded aDNA, potentially leading to incorrect sex
molecular genetic methods in anthropology offers a determination [7, 8, 12, 13]. Due to the relatively
solution to this limitation and helps to improve our frequent occurrence of this phenomenon, it is suitable
knowledge about historic populations. to use an additional genetic sex marker; for example,
2. Materials and Methods
SRY [14]. The SRY gene codes for the testes determining factor and is located on the short arm of
Skeletal remains from the archaeological site the Y chromosome (Yp11.3). The occurrence of a
Pohansko, the south outer precincts, were chosen for single 93 bp amplicon of the SRY gene would
DNA-based sex determination. The bones were in very distinguish authentic male DNA from a female DNA
poor condition, fragmented, and in most cases sample [14]. The SRY marker has been successfully
unidentifiable. Using morphometric and morphologic used for sex typing of skeletal materials [15-17], often
methods of sex estimation for the recognizable bones, combined with the amelogenin marker [18-23].
40 adult females and 27 adult males were identified. The objective of our study was to demonstrate the
The number of subadult (child) skeletons was 87, and application of genetic methods in anthropological and
36 skeletal remains were undetermined [24]. The sex of paleodemographic research when fragmentary adult
the children and unidentified skeletal remains could not and child skeletal remains are analyzed. The Pohansko
be determined by traditional anthropological methods, archaeological site, situated in the south-east part of the
so samples for aDNA analysis were taken. The samples Czech Republic near the town of Breclav, was the
were taken preferentially from skulls (74 samples), source of the samples in this study. Archeological
long bones (24 samples) and teeth (10 samples), which excavations at this site uncovered the foundations of a
are the most appropriate bone sources for aDNA fortified settlement, church, burial grounds and other
analysis [2, 11, 13, 29]. The remaining samples were buildings. One of the settlement units at Pohansko is
taken from vertebrae (7 samples), mandibles (3 the south outer precincts. It is dated approximately to
samples) and ribs (2 samples). All together, 120 the 9th-10th century, and was discovered during the
samples were analysed for sex determination.
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies
of Fragmentary and Damaged Skeletal Material from Rescue Excavations
The most serious problem when working with ® supernatant using QIAamp DNA Mini Kit (Qiagen ) degraded aDNA from skeletal material is according to the manufacturer’s protocol. DNA
contamination [30]. It is therefore necessary to adopt extraction was performed in a laminar flow box. A many stringent precautions against contamination. To
minimum of 3 independent extractions per sample ensure the highest possible reliability of the present
were performed. During both the isolation and PCR work, the guidelines for aDNA analysis suggested by
steps, negative controls were performed to identify Cooper and Poinar [31] and Pääbo et al. [32] were
possible contamination by recent DNA. followed as closely as possible.
For molecular sex determination, the SRY marker Our laboratory is divided into three different
and the amelogenin marker were chosen because of laboratory rooms so that aDNA extraction, PCR
their relative small size. For amplification of the SRY amplification, and analysis of the PCR products are
marker, the protocol introduced by Cunha et al. [18] performed separately. The laboratory rooms, was adopted. The amelogenin marker was amplified workspaces and instruments were UV irradiated (254
using the slightly modified protocol introduced by nm) prior to use, and were cleaned with denatured
Hummel [13]. The PCR protocol for both markers ethanol and bleach solutions to destroy extraneous
included initial denaturation at 94 °C for 3 min, DNA molecules after each work step. Different sets of
followed by 20 cycles of 93 °C/45 s, 60 °C/1 min, and pipettes were used for aDNA extraction, PCR
72 °C/1 min; and 35 cycles of 93 °C/45 s, 66 °C/1 min, amplification, and analyses of PCR products. and 72 °C/1 min. Final extension at 72 °C was kept for Laboratory overalls with hood and face masks and
3 min. To avoid false negative and false positive results, disposable gloves were worn to decrease the risk of
each DNA sample was amplified at least three times, contamination with recent DNA. Separate personal
separately for the SRY and amelogenin markers. PCR protective equipment was used for each laboratory
products were separated using 3% agarose (SRY) or room. Only materials and chemicals of DNA-free
4.4% and 5% MetaPhor ® Agarose (amelogenin) gel quality or autoclaved were used, and all consumables
electrophoresis, and visualised by ethidium bromide were further UV irradiated for at least 20 minutes.
staining with UV illumination.
During the preparation of each sample, the bone Three bone samples were selected randomly and fragments were cleaned with household bleach (NaOCl)
sent to the Institute of Criminology in Prague (Czech and 96% ethanol. To remove contaminants from the
Republic) for independent sex determination. DNA bone sample surface, we ground away 1-2 mm of the
was extracted from bone powder using the MinElute
bone with micro tool (DREMEL ® Stylus PCR Purification Kit (Qiagen ) according to the Lithium-Ion). After grinding, the bone fragments were
TM
protocol described by Yang et al. [33] and Anderung et UV irradiated for 10 minutes on each side to eliminate ® al. [34]. The PowerPlex ESX 17 System (Promega)
contaminant DNA. Dried samples were powdered in a amplification kit was used to obtain the genetic profiles mortar or bone mill under sterile conditions. The bone
of the samples. Detection of PCR products using powder was decalcified using 0.5 M EDTA (pH 8.0) on
capillary electrophoresis was carried out using an ABI rotator at 4 °C overnight. The supernatant was ® PRISM 3130xl Genetic Analyzer (Applied
discarded and the decalcification step was repeated Biosystems). Data analysis was performed with (4 °C overnight). A new portion of 0.5 M EDTA was
GeneMapper ® ID-X software, version 1.2. added and the sample was decalcified at 4 °C for a
3. Results and Discussion
week. After decalcification, the supernatant was stored in a sterile tube. DNA was extracted from the
The main aim of our work was to demonstrate the
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations
application of aDNA analysis from unidentified bone
Table 1 The results of genetic sex determination (only valid data shown) fragments and child skeletal remains in anthropology a .
and paleodemography. Analysis of authentic DNA Grave No. Age at death b Genetic sex determination from historical bone material is problematic because
2A 2y
2B the preservation of DNA molecules depends on many nd F
nd F circumstances including environmental conditions, age
3/IV
11/IV
20-22 m
and preservation of bones, and handling of bones
16/IV
3y
during and after excavation [35, 36]. DNA from
17/IV
7y
ancient bones is fragmented and degraded by
29/IV
nd M
hydrolysis and oxidation over time, which complicates nd M
30/IV
34/IV B
3y
its analysis [37, 38]. Moreover, contamination with
37 7y
modern DNA is a serious problem, and many
39 40-50 y
precautions against contamination must be adopted
We analysed the skeletal remains of a population
50 B
35-40 y
from Pohansko, the south outer precincts. A total of 86 56 18 mF bone specimens of child skeletal remains and 34 bone
60 9y
specimens of unidentified skeletal remains were
62 8y
investigated for genetic sex determination (bone F
64 40-50 y
67 B
45-55 y
samples could not be removed from 3 skeletons, 1 child
69 7y
and 2 unidentified, because of their poor state of
75 B
2y
preservation). In general, the skeletal remains were 78 nd F badly preserved, which likely influenced the quality
80 3y
81 2y
and quantity of authentic DNA of the samples (data not 84 13-15 y F
shown).
M Using the two common sex markers, SRY and
91 12 y
92 8y
amelogenin, sex was determined in 66 samples (55%): 95 nd M
96 31 male and 35 female. Only the concordant results of nd F 98 18 mF
nd M summarized in Table 1. In 38 cases, the results of the
both sex markers were considered as valid. Results are
20-24 y
SRY and amelogenin marker analyses were not
nd F
concordant; in 15 cases only either amelogenin or SRY nd F
104 A
18-22 y
was revealed; and in one sample the analysis yielded no
104 B
16-20 y
markers. All these data were excluded from the overall
2y
evaluation. The failure in SRY and amelogenin locus
60-70 y
detection may have been due to a high rate of aDNA
109 15 y
nd M fragmentation. The skeletal remains from Pohansko
M were treated with standard anthropological methods
116 11 y
nd F and kept in deposit for approximately 20-40 years,
which could have caused the degradation of aDNA.
121 B
6y
According to Pruvost et al. [35], freshly excavated F
6y
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies
of Fragmentary and Damaged Skeletal Material from Rescue Excavations
(Table 1 continued) male. As in the case of the skeletal remains from grave
Grave No. Age at death b Genetic sex determination
103, the genetic profile was not complete because of 136 6 y
F low concentration and degradation of DNA, but X and 139 14 y
138 12 y
F Y amelogenin alleles were detected (Fig. 2). The sex of 140 4 y
the infant remains from grave 155 was therefore 142 11 y
F confirmed as male.
143 nd
F The fragmentary skeletal remains from grave 160 147 18 m
146 nd
F probably belong to an adult individual (age not 151 20-24 y F
determined). By genetic analysis, the sex was 153 nd
determined as male (SRY positive, Y amelogenin 154 7 y
allele present). Unfortunately, using the PowerPlex 158 40-50 y F
155 18 m
ESX 17 System amplification kit, the genetic profile 160 nd
was of poor quality and amelogenin loci were not 162 3 y
F detected (Fig. 3). This may be attributed to ongoing 164 12 y
degradation of authentic DNA of the sample 165 18 m
174 nd
F (investigations in our laboratory and the laboratory of 190 8 y
the Institute of Criminalogy were time-separated by 206 3 y
more than 1 year). In this case, the results of sex Abbreviations: nd, not determined; M, male; F, female; y, years;
m, months. b Age determination was adopted from Drozdova determination with SRY and amelogenin markers
failed to verify independently.
The success rate of sex determination (55%) based bones are better for aDNA amplification than bones
on the combination of SRY and amelogenin markers in from collections because ancient DNA can degrade
the present study are comparable to other aDNA quickly after bones are removed from the preserving studies. In a similar study, Vanharova and Drozdova conditions of their original setting. [23] determined the gender in 4,000 year-old samples For verification of the concordant results, 3 bone up to 40% (21 of 53 samples). Zink et al. [20] identified samples (from graves 103, 155 and 160) were sent for the gender of 2,500-5,000 year-old samples by analysis evaluation to the Institute of Criminology in Prague. of SRY and amelogenin genes with a success rate of The skeletal remains from grave 103 belong to an
43.9% (18 out of 41 cases).
adult individual (age not determined). The bones Using the results of genetic analysis of the
were fragmented and in most cases unidentified. investigated samples in this study, the demographic
Genetic analysis was negative for SRY, and only the data of the Pohansko population could be completed.
X allele of amelogenin was present. Sex determination as female was confirmed by PCR
From the 86 subadult skeletal remains, we determined amplification using the PowerPlex ® ESX 17 System
the sex of 48 samples: 28 male and 20 female children. (Promega). The genetic profile was incomplete, but it
These findings correspond with demographic data of was clearly visible that only the X allele of
historical and recent populations. Infant mortality is amelogenin was detected (Fig. 1).
greater for males than for females in all age categories The skeleton from grave 155 belongs to an
[40]. The sex determination of the remaining 38 child 18-month-old child and was badly preserved. Genetic
individuals was not successful, likely due to the poor analysis was positive for SRY, and the Y allele of
state of preservation of the samples (degradation amelogenin was revealed, so the sex was determined as
of authentic DNA). From the 34 unidentified skeletal
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations
Fig. 1 Electropherogram of DNA typing results for sample 103 using PowerPlex ESX 17 System (Promega) amplification kit.
Fig. 2 Electropherogram of DNA typing results for sample 155 using PowerPlex ESX 17 System (Promega) amplification kit.
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies
of Fragmentary and Damaged Skeletal Material from Rescue Excavations
Fig. 3 Electropherogram of DNA typing results for sample 160 using PowerPlex ESX 17 System (Promega) amplification kit.
A) B) Sex determ ination based on concordance in both genetic B) sex determination based on concor dance in
sex determination based on anthropological A) Sex determ ination based on anthropological
analysis only m arkers
analysis only both genetic Markers
Unidentified children; 39
Males ; 30 Unidentified individuals; 36
Fig. 4 The sex structure of individuals from Pohansko, Fig. 5 The sex structure of individuals from Pohansko, south outer precincts, before genetic analysis.
south outer precincts, after genetic analysis.
remains, we identified 3 males and 15 females. The sex
4. Conclusion
identification of the remaining 16 individuals was not successful. Figs. 4 and 5 compare the number of adult
Damaged and fragmentary bones constitute a males, adult females, children without known sex and
significant portion of skeletal material from rescue unidentified individuals before and after genetic sex
excavations. The traditional anthropological approach assessment.
to demographic analysis is limited if poorly preserved
Application of Molecular Genetic Methods in Anthropological and Paleodemographic Studies of Fragmentary and Damaged Skeletal Material from Rescue Excavations
ancient bones are investigated, especially when sex test by amplification of the X-Y homologous gene determination is part of the analysis. In the present amelogenin, International Journal of Legal Medicine 106
(4) (1994) 190-193.
study, we demonstrate the utility of molecular genetic [6] C. Lassen, S. Hummel, B. Herrmann, Molekulare methods, specifically the application of genetic sex
Geschlechtsbestimmung an Skelettresten früh- und markers, in the analysis of fragmentary ancient skeletal neugeborener Individuen des Gräberfeldes Aegerten, Schweiz, Anthropologischer Anzeiger 55 (2) (1997)
remains. The SRY and amelogenin sex markers were
183-191.
used for genetic sex determination of fragmentary and [7] E. Meyer, M. Wiese, H. Bruchhaus, M. Claussen, A. Klein, child skeletal remains from rescue excavations at the
Extraction and amplification of authentic DNA from ancient human remains, Forensic Science International
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113 (2000) 87-90.
determined in more than half of the investigated cases. [8] D. Schmidt, S. Hummel, B. Herrmann, Brief Moreover, the results of sex determination of some
communication: multiplex X/Y-PCR improves sex skeletal remains were independently verified by the identification in aDNA analysis, American Journal of Physical Anthropology 121 (2003) 337-341.
Institute of Criminology in Prague. The results of this [9] M. Cipollaro, G. Di Bernardo, G. Galano, U. Galderisi, F.
study allowed the demographic profile of the Pohansko Guarino, F. Angelini, et al., Ancient DNA in human bone population to be completed with more accurate
remains from Pompeii archaeological site, Biochemical knowledge about the mortality of this population.
and Biophysical Research Communications 247 (3) (1998) 901-904.
Acknowledgments
[10] T. Waldron, G.M. Taylor, D. Rudling, Sexing of Romano-British baby burials from the Beddingham and This research was supported by the Ministry of
Bignor villas, Sussex Archaeological Collections 137 Education (Project No. MSM 0021622427), Czech (1999) 71-79. [11] M. Faerman, D. Filon, G. Kahila, C.L. Greenblatt, P.
Republic. We thank the Institute of Criminology in Smith, A. Oppenheim, Sex identification of archaeological
Prague for their analysis of the bone fragments from human remains based on amplification of the X and Y the Pohansko archaeological site. The authors thank
amelogenin alleles, Gene 167 (1995) 327-332. Kristina Kovarik, MSc. for language corrections. [12] M.A. Jobling, P. Gill, Encoded evidence: DNA in forensic analysis, Nature Reviews Genetics 5 (2004) 739-751.
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Journal of Life Sciences 6 (2012) 970-979
A Comparative Study of Soluble Protein Extractions of Populus deltoides × (Trichocarpa × Deltoides) for 2-DE
1 1 2 1 1 Joke Dupae 1 , Ann Cuypers , Jean-Paul Noben , Jana Boulet , Nele Weyens , Karen Verstraelen and Jaco Vangronsveld 1
1. Centre for Environmental Sciences, Hasselt University, Agoralaan Building D, B-3590 Diepenbeek, Belgium 2. Biomedical Institute, Hasselt University, Agoralaan, Building A, 3590 Diepenbeek, Belgium
Received: November 21, 2011 / Accepted: April 27, 2012 / Published: September 30, 2012.
Abstract: Background: The disclosure of the poplar genome strengthens its position as well-established model organism. Populus has been subject of several proteome studies, but up to date no comparative study was performed on the extraction method of soluble proteins for this species. The extraction is the most critical step in two-dimensional gel electrophoresis and each extraction method has its advantages, disadvantages and limitations. Therefore protein extraction methods should be optimized for each tissue before starting an experimental setup. In prospect of future DIGE (Differential Gel electrophoresis) experiments for the investigation of the effects of cadmium and inoculation with plant growth promoting bacteria at the proteome level, the aim of this study was to optimize an extraction method for soluble proteins of poplar leaves and roots. Results: The acetone-phenol extraction method was found to be the most suited, rendering a high spot number and low background interference. During further optimization, several critical steps in the extraction method were revealed. Conclusion: Aiming to optimize the extraction of soluble leaf and root proteins of Populus deltoides × (trichocarpa × deltoides) compatible with DIGE analysis, a protocol rendering high reproducibility, low background interference and a high spot number was established, however no novel insights were acquired.
Key words: Proteomics, two-dimensional gel electrophoresis, soluble proteins, protein extraction, Populus.
1. Introduction proposed by Plomion et al. [4]. The genome of Populus trichocarpa has been sequenced by Tuskan et
Due to their low protein content and high protease al. [5], paving the way for poplar proteomics. activity, proteomic analysis of plant tissues appears Proteome studies on poplar have been published by to be particularly challenging. Moreover, other Bohler et al. and Kieffer et al. [6-8], but up to date no compounds such as phenols, terpenes, organic acids, comparative study on protein extraction of poplar has pigments, oxidative enzymes, lipids, etc. can been released. As protein extraction is critical in 2-DE interfere with two dimensional gel electrophoresis and since every extraction method has its limitations, (2-DE) and can cause vertical or horizontal streaking, advantages and disadvantages as stated by Carpentier smearing or reduction in the number of distinctly et al. [1], it is essential to find an optimal extraction resolved spots as indicated by Carpentier et al., protocol before starting an experimental set-up. Saravanan et al. and Wang et al. [1-3]. Protein Therefore the aim of this study was to optimize an extraction is therefore a crucial step in two- extraction method for soluble proteins of poplar leaves dimensional gel electrophoresis.
and roots.
Populus is a well-established model organism to
A literature review demonstrated that a elucidate the biological function unique to trees, as TCA/acetone extraction, a phenol extraction or a
Corresponding author: Joke Dupae, Ph.D. candidate, combination of both, are often used for the extraction research fields: environmental biology, proteomics. E-mail: [email protected].
of soluble plant proteins. In this study, 3 protocols
A Comparative Study of Soluble Protein Extractions of Populus
deltoides × (Trichocarpa × Deltoides) for 2-DE
were selected for comparison: (1) a combination of
2.2 Extraction Methods
acetone and phenol; a slightly changed protocol based At first we compared the differences between on Carpentier et al. [1], (2) a combination of extraction of soluble proteins within the same leaf TCA/acetone and phenol; this protocol was proposed during 1 h and overnight for each extraction method. by Wang et al. [9] to be universal, rapid and especially Subsequently we compared the three methods to each suited for recalcitrant plant tissues. And finally, (3) a other in a loop design and one phenol based method, TCA/DTT/acetone extraction; a protocol already described by Sarma et al. [12], was included applied on Populus tremula L. × P. alba L. (Populus ×
afterwards.
Canescens (Aiton) Smith)—clone INRA 717-1-B4
2.2.1 TCA/Acetone-Phenol Extraction and Populus tremula L. leaves described by Bohler et Prior to grinding in liquid nitrogen, the primary and
al. and Kieffer et al. [6-8]. By comparing these secondary veins of the leaves were removed. The
protocols and by further improving the spot pattern, an extraction was performed according to Wang et al. [9]
optimized extraction protocol for soluble leaf and root (tested on bamboo (Bambusa vulgaris), grape (Vitis
proteins of Populus deltoides × (trichocarpa × vinifera ), iris (Iris pseudacorus), olive (Olea europea),
deltoides ) was established, however no fundamentally lemon (Citrus limonum), pine (Pinus nigra), redwood
novel insights were acquired. In prospect of future (Sequoia sempervirens), sugarcane (Saccharum
experiments, buffer compatibility was checked for officinarum ), and tobacco (Nicotiana tabacum)) with
DIGE analysis. some modifications. Because of the much higher
2. Materials and Methods
amount of starting material, we used 10% TCA/acetone; 80% methanol-0.1M ammonium
2.1 Plant Material acetate and 80% acetone at 10 mL per gram ground
Cuttings (30 cm, with an average shoot height of 10 tissue, a phenol-Tris buffer (a pH of 8.0 is required for cm) of Populus deltoides × (trichocarpa × deltoides)
optimal results, as described by Thiellement et al. were grown on sand in 4l pots during 10 weeks. They
[11]), was used instead of a phenol-SDS buffer and were watered three times a week with 1/2 strength
our samples were allowed to precipitate during 1 h or Hoagland’s solution [10], until they had sufficient
overnight at – 20 °C. The dry pellet was finally expanded leaves. Of each plant three fully expanded
resuspended in resuspension buffer (7 M urea, 2 M leaves, developed after transfer to the pot system,
thio-urea, 4% chaps and 30 mM Tris) (incubation at were harvested. To minimize biological variation,
18 °C, 1,200 rpm, 2 h (Eppendorf mixer)), centrifuged methods were evaluated “within leaf”, meaning that
(70,000 g, 90 min, 18 °C) and stored at – 80 °C. every possible combination of two out of three
2.2.2 Acetone-Phenol Extraction extraction methods was performed on separate leaf
This protocol is based on the work of Carpentier et halves derived from same leaf. This design allowed
al. [1] (tested on banana (Musa spp.), apple (Malus for comparing the three methods within one plant. For
domestica ) and potato (Solanum tuberosum)) with each combination three biological replicates were
some modifications. The same handlings prior to performed using three plants in total. In future
grinding were performed as described above. Plant experiments poplar cuttings will be exposed to
tissue was ground in liquid nitrogen. Per gram fresh different treatments and proteins obtains from roots
weight (FW) 10 volumes of ice cold acetone were and leaves will be compared. Therefore, highest
added. After 1 h precipitation at – 20 °C, a reproducible protocol was tested for its applicability
centrifugation at 10,000 g during 10 min at 4 °C was on roots as well.
performed. Subsequently, the pellet was freeze-dried,
A Comparative Study of Soluble Protein Extractions of Populus
deltoides × (Trichocarpa × Deltoides) for 2-DE
and 10 volumes of extraction buffer (50 mM Tris pH gel, an observation first described by Espagne et al.
8.5, 25 mM Na 2 EDTA, 100 mM KCl, 30% w/v [13]. Comparing the buffers used, adding 0.5% sucrose, 2% -mercapto-ethanol and 0.4 mM PMSF)
ampholine pH 4-7 resulted in a lower RuBisCO were added per gram FW. Ten minutes later, the same
resolubilization. In order to control the RuBisCO in volume of phenol was added and the solution was
poplar leaf extracts, 1.25% (v/v) IPG 4-7 NL buffer mixed thoroughly. After centrifugation (8,000 rpm, 5
(GE Healthcare) was added to the resuspension buffer, min, 4 °C) the phenolic phase was collected, 5
samples were mixed (1,200 rpm) for 2 h at 18 °C and volumes of ammonium acetate in proportion to the
finally centrifuged (70,000 g, 90 min, 18 °C) to volume of collected phenol, were added and the
remove the unsolubilized proteins. sample was allowed to precipitate at – 20 °C for 1 h or
2.4 Protein Quantification
overnight. After centrifugation (10,000 g, 10 min,
4 °C) the pellet was washed three times with The protein concentration of each sample was ammonium acetate and once with acetone/DTT (0.2%
determined using the RC DC protein assay kit II w/v). Finally, the pellets were resuspended in (BIORAD, California) using BSA (1.54 mg/mL) as resuspension buffer as described in 2.2.1.
the standard. This protein assay is an improved
2.2.3 TCA/DTT/Acetone Extraction version of the Lowry assay [14], modified to be This protocol is based on the work of Bohler et al.
reducing agent compatible (RC) and detergent [6] (tested on poplar ([Populus tremula L. × P. alba L.
compatible (DC). Color development is achieved by a (Populus × Canescens (Aiton) Smith)]—clone INRA
two step reaction: (1) a reaction between proteins and 717-1-B4)) without modifications. As mentioned
copper in an alkaline medium followed by (2) a above, the same handlings prior to grinding were
reduction of Folin reagent by copper-treated proteins performed and the samples were allowed to precipitate
[14]. The Microfuge Tube Assay Protocol, provided for 1 h or overnight at – 20 °C. The final pellet was
by BIORAD, was used with a repetition of step 4 and treated as described in 2.2.1.
5. Leaf samples and root samples were 1/2 diluted
2.2.4 A Phenol Extraction Protocol Rendering High prior to quantification and all solutions were brought Resolution and Reproducibility of 2-DE
to 27 °C prior to use.
The extraction was carried out as described by
2.5 2-DE
Sarma et al. [12] (tested on soybean) with some modifications. They proposed a protocol based on
For the IEF, 24 cm strips with a non-linear pH phenol extraction of soluble proteins, rendering a high
gradient ranging from 3 to 10 (GE Healthcare) were resolution of 2-DE gels and a high reproducibility.
used in the IPGphor system (Amersham Biosciences, Protease inhibitors were excluded from the extraction
Uppsala, Sweden). The strips were rehydrated buffer since no problems with proteases were overnight in rehydration buffer (7 M urea, 2 M observed in the previous extraction methods. The final
thiourea, 4% w/v chaps, 65 mM DTT, 0.8% v/v IPG pellet was treated as described in 2.2.1.
buffers, 0.002% v/v bromophenol blue) containing proteins. The amount of proteins loaded onto the strip,
2.3 RuBisCO Interference depended on the used staining method (see section Addition of ampholine to the resuspension buffer
2.6). IEF was carried out on an Ettan IPGphor caused a position shift of the RuBisCO Large Subunit
Manifold (GE Healthcare) with the following settings: (LS) in the first dimension, resulting in lower
gradient step of 100 V for 3 h, constant step of 100 V interference of RuBisCO LS in the second dimension
for 3 h, gradient step of 500 V for 3 h, constant step of
A Comparative Study of Soluble Protein Extractions of Populus
deltoides × (Trichocarpa × Deltoides) for 2-DE
500 V for 3 h, gradient step of 1,000 V for 3 h, reproducibility of DIGE gels (Bohler S., personal constant step of 1,000 V for 3 h, gradient step of 8,000
comments).
V for 3 h and finally a constant step of 8,000 V for 7 h
2.7 Image Analysis
at 20 °C with a maximum current setting of 50 µA/strip. On the paper wicks at the negative electrode,
Gel analysis was performed using ImageMaster 2D 150 µL DeStreak rehydration solution (GE Healthcare)
Platinum 5.0. For quantitative comparison, two was added to reduce streaking. After the IEF, the IPG
parameters were used: the total spot number and the strips were equilibrated at 18 °C for 15 min in
ratio of the number of automatically detected spots to equilibration buffer (75 mM Tris pH 8.8, 6 M urea,
the number of spots after manual verification. The last 30% v/v glycerol, 2% w/v SDS and a trace of
parameter is included to quantify background bromophenol blue) supplemented with 1% w/v DTT.
interference; the closer the ratio is to 1, the less
A second equilibration step of 15 min with the same background interference in the gel. Further on, this equilibration buffer, now containing 2.5% w/v ratio will be referred to as the “background ratio”. iodoacetamide, was carried out in the dark afterwards.
Values are the means ± standard deviation. The first equilibration step provides a completely
2.8 Statistical Analysis
reduced state of unalkylated, denaturated proteins. Whereas, in the second step, iodoacetamide alkylates
Statistical analysis of all data was performed using SAS 9.1. All tests included 3 biological replicates.
thiol groups in order to prevent reoxidation during Since all data had a normal distribution, a student’s
electrophoresis. On top, the Immobiline DryStrips are t -test was performed to determine statistically
saturated with the SDS buffer system, required for the significant differences between the groups. The
second dimension separation. The IPG strips were significance level was set at α = 0.05. then sealed on top of 200 mm × 260 mm × 1 mm,
12.5% polyacrylamide gels with 0.5% agarose in SDS
2.9 Spot Picking and Identification running buffer. The SDS-PAGE step was performed
Selected spots were picked manually using the at 15 °C in Ettan Dalt II tanks (GE Healthcare) at
OMX-S pro pickers. The standard protocol provided
0.5 W per gel for 20 h. by the manufacturers was used (OMX GmbH,
2.6 Staining We βling) for destaining, reduction, alkylation and
trypsine digestion. Tryptic digests were analysed by To visualize the protein spots, silver staining
LC-ESI-MS/MS on a LCQ Classic (Thermo Electron, (according to Shevchenko [15]) or Gel Code Blue
San Jose, CA, USA) ion trap mass spectrometer staining (according to the manufacturer guidelines;
equipped with a nano-LC column switching system Thermo Scientific) were performed. After staining, the
as described by Dumont et al. [16]. MS/MS data gels were scanned using a flatbed scanner (CanoScan
were searched against the Populus trichocarpa v1.1 4400F, Canon) at highest resolution. Comparing the
protein database (45555 entries) using Mascot extraction methods and testing the highest (version 2.1; Matrix Sciences, London, U.K.) and reproducible protocol on root samples, 100 µg soluble
Sequest (version 27, rev. 12 within Proteome protein was loaded onto silver stained gels. For the
Discoverer version 1.0; Thermo Fisher Scientific). comparison of RuBisCO focusing and the Sequest and Mascot parent ion mass tolerance was reproducibility test, Gel Code Blue staining was used
set to 3 Da and 1.4 Da, respectively; fragment ion (50 or 300 µg proteins, see results). Staining the gels
tolerance was 1 Da. Carbamidomethylation of with Gel Code Blue gives an idea about cysteine and oxidation of methionine, tryptophan and
A Comparative Study of Soluble Protein Extractions of Populus
deltoides × (Trichocarpa × Deltoides) for 2-DE
histidine were set as fixed and variable modifications,
3. Results and Discussion
respectively. Maximally one missed cleavage was allowed. Additional information (e.g. peptide
In order to find an optimal protocol for the sequence, charge state of each peptide) will be
extraction of soluble proteins from poplar leaves, a provided in supplementary data.
comparison was made between three extraction Resulting peptide identifications were accepted if
methods: (1) a combination of acetone and phenol, (2) they could be established at greater than 95.0%
a combination of TCA/acetone and phenol and (3) a probability as specified by the Peptide Prophet
TCA/DTT/acetone extraction. At first, the extraction algorithm described by Keller et al. [17] within
time was optimized. Samples precipitated overnight Scaffold version 2_05_02 (Proteome Software Inc.,
resulted in better spot patterns and less background Portland, OR). Protein identifications were accepted if
interference than samples precipitated during one hour they could be established at greater than 99.9%
(data not shown). Subsequently, the three extraction probability and contained at least 2 identified peptides.
methods were compared within the leaves in a loop Protein probabilities were assigned by the Protein
design. Since the total spot number was discernibly Prophet algorithm within Scaffold according to
lower after the TCA/acetone-phenol extraction, no Nesvizhskii et al. [18]. To assign a function, the
further analysis was performed at this stage and the accepted protein identifications were searched for