Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007 1
THE STUDY OF HYDROPHOBIC PROTEINS OF ARABI-
DOPSIS THALIANA AND BANANA, A MODEL AND NON
VERTOMMEN A.1, PANIS B.1 , SWENNEN R.1 & CARPENTIER S.C.1
Laboratory of Tropical Crop Improvement. Division of Crop Biotechnics. Katholieke Universiteit
Musa spp. is a genus consisting of a wide range of banana and plantain va-
rieties. They are classified according to the relative proportion of Musa acu-
minata (A) and Musa balbisiana (B) in their genomes. As they are main staple
food crops, it is important to preserve this large diversity. Since banana is
mainly vegetatively propagated, the best method for safe storage is cryopre-
servation of meristems. Successful cryopreservation requires that cells are
dehydrated to a minimal cellular water content to avoid the formation of
intracellular ice crystals. This implies that the preserved tissue is drought
tolerant. Drought tolerance is not only important for cryopreservation, but is
also crucial for crop production. The yield loss due to drought is estimated at
40 % with annual yearly rainfall lower than 1200 mm (Van Asten, personal
communication). Despite the growing need for water in banana production,
the fundamental knowledge of drought tolerance in banana is very limited.
Some physiological studies indicate that the B genome is associated with
higher tolerance to drought (Thomas et al., 1998), but the molecular back-
ground is unknown. Therefore, we aim to characterize the molecular mecha-
nisms leading to drought tolerance. Since banana, like other non model
crops, is poorly sequenced, we study the proteome of banana varieties with
different genome compositions and different tolerances towards drought
(Carpentier et al., 2005; Carpentier et al., 2008a, Carpentier et al., 2008b). How-
ever, the proteome can not be studied at once since hydrophilic and hydro-
phobic proteins require different approaches. In the presented study we fo-
cus on the analysis of hydrophobic proteins in the non-model organism ba-
MATERIALS AND METHOD
The applied methods were first evaluated on leaves of the model organism
Arabidopsis thaliana var. Columbia., kindly provided by prof. P. Van Dyck
(VIB, K.U.Leuven, Belgium). The banana plants were received from the Bio-
versity International Musa collection at K.U. Leuven, Belgium. Banana
plants were grown under ideal growth conditions in the greenhouse.
After grinding in liquid nitrogen, 200-400 mg (fresh weight) was transferred
to 1 mL ice cold extraction buffer (Carpentier et al., 2005).
Sample was added to an ice-cold chloroform/methanol mixture in a ratio
1/9 as described by Seigneurin-Berny et al.(1999) and carefully mixed.
Different ratios chloroform to methanol were tested. Samples were incubated
on ice for 30 min and centrifuged at 16 000 g (4 °C°) for 1hr. Organic phases
were collected and proteins soluble in these phases were precipitated over-
night at -20°C by addition of 1 mL cold di-ethylether. Precipitated proteins
were recovered after centrifugation at 16 000g for 1 hr. (4°C°) and dissolved
in Laemmli buffer supplemented with 1 % DTT. Before loading, samples were
heated for 30 min at 37°C.
To test whether TCA enhances the yield of high molecular weight hydropho-
bic proteins, the same extraction was done with a C/M mixture supple-
mented with 0.1% TCA.
Proteins were separated by SDS PAGE on a 13-15 % gradient gel (24 cm,
hyperbolic gradient), produced by a 2DE optimizer (Nextgen) to test the reso-
lution of one-dimensional separation. For double SDS, a 10 % gel was used
in the first dimension and a 15 % gel in the second dimension. After the first
dimension, gel lanes were excised and equilibrated as described by Rais et
al. (2004). Separation was done at 2W per gel at 12°C.
After Coomassie blue staining, spots were manually picked. In-gel digestion
with trypsin and analysis of the tryptic peptides by MALDI TOF-TOF was
executed at the Centre de Recherche Public Gabriel Lipmann in Luxem-
bourg. Mascot was used for database searching. For Arabidopsis, proteins
that were identified with a protein score above the Mascot score (significance
threshold 0.05) and at least two peptides showing a significant ion score
were automatically validated. If only one ion score was significant, spectra
were manually checked. For banana, protein identifications were accepted if
protein score was significant and at least two peptides after MS-MS showed
an ion score.
Protein names were retrieved from Swissprot or NCBI. The TMHMM Server v
2.0 (http://protfun.net/services/TMHMM/) was used to calculate the
transmembrane domains. The ExPASy server (http://ca.expasy.org/) was
used to calculate the GRAVY (grand average of hydropathicity) score and
other parameters as pI and molecular mass.
RESULTS AND DISCUSSION
Chloroform/methanol extraction on Arabidopsis thaliana
First analyses were done on the model plant Arabidopsis thaliana var Co-
lumbia since protein identification is much easier with a fully sequenced
organism. To test the optimal ratio chloroform to methanol (C/M), several
ratios were tested. Since 5/4 (C/M) yielded most proteins, this ratio was
chosen for further analyses (data not shown).
Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007 3
Test addition of 0.1% TCA to enhance the yield of high molecular weight
The chloroform/methanol extraction protocol proved to be selective for pro-
teins with a low molecular weight (figure 1, lane 2). Addition of a halogene
rich organic acid, as proposed by Shröder and Hasilik, was therefore tested
(Schroder and Hasilik, 2006). As shown in figure 1 (lane 6) the presence of
trichloro-acetic acid (TCA) obviously enhanced the recuperation of proteins
with a high molecular weight. However, after analysis of the identified pro-
teins, it became clear that addition of TCA brought hydrophilic proteins back
in solution. Indeed, by addition of TCA, the amount of identified membrane
proteins was significantly decreased.
Figure 1. Proteins present in Arabidopsis
leaves separated on a 10% gel, Coomassie blue
stained. Lane 1 and 5: protein standards; lane
2: proteins soluble in 5/4 C/M, lane 3: total
protein fraction, lane 4, proteins insoluble in
5/4 C/M, lane 6: proteins soluble in 5/4 C/M
+ 0.1 % TCA, lane 7: proteins insoluble in 5/4
C/M + 0.1 % TCA
Double SDS on Arabidopsis thaliana
To check whether adding a second dimension improves resolution, the suc-
cess rate of proteins identified through MS/MS after one dimensional and
double SDS separation was compared.
Additionally, the amount of spots yielding more than one ID was calculated.
These analyses clearly showed that double SDS enhances resolution.
Test C/M extraction combined with dSDS on banana
Since chloroform/ methanol extraction combined with dSDS proved to be a
valuable technique to study the hydrophobic proteome of plants, the method
was applied to banana. This study clearly demonstrates the difficulties that
are encountered when working with non-model plants. The success rate of
Arabidopsis was 90% after one dimensional separation, while the spots
picked from banana gels gave only 58 % of reliable identifications.
When analyzing the proteome of non-model organisms, researchers have to
rely on gel-based technique like two dimensional electrophoresis (2DE) (Car-
pentier et al., 2005). Since hydrophobic proteins largely escape during such
classical 2DE studies, alternatives have to be investigated. However, these
alternative techniques demand an enrichment step prior to protein separa-
tion since resolution, as obtained by classical 2DE, can never be reached.
We decided to enrich hydrophobic proteins in our samples by extracting
them in a chloroform/methanol mixture. After careful analysis of different
ratios chloroform to methanol, we selected 5/4 as optimal ratio.
Chloroform/methanol extraction only yielded proteins with a low molecular
mass. Therefore, we added TCA to the C/M mixture. The addition of an acid
indeed raised the number of high molecular mass proteins but we observed
that it was not selective for hydrophobic proteins.
We tested whether the resolution of a 24 cm one dimensional separation was
sufficient for MS/MS based protein identification. As expected, analysis of
Arabidopsis samples gave a success rate of 90 %, while for banana only 58%
of the picked bands gave reliable protein identification. To raise the number
of identifications, we decided to add a second dimension to improve resolu-
tion. Double SDS indeed enhanced resolution and proved to be a valuable
alternative to study the hydrophobic proteome of non-model plants.
We will apply this method to search for hydrophobic proteins which are dif-
ferentially regulated in banana varieties that respond differently to drought
CARPENTIER, S.C., COEMANS, B., PODEVIN, N., LAUKENS, K., WITTERS, E., MATSUMURA, H.,
TERAUCHI, R., SWENNEN, R., AND PANIS, B. (2008a). Functional genomics in a
non-model crop: transcriptomics or proteomics? Physiologia Plantarum,
CARPENTIER, S.C., PANIS, B., VERTOMMEN, A., SWENNEN, R., SERGEANT, K., RENAUT, J.,
LAUKENS, K., WITTERS, E., SAMYN, B., AND DEVREESE, B. (2008b). Proteome
analysis of non-model plants: a challenging but powerful approach. Mass
Spectrometry Reviews, 27: 354-377.
CARPENTIER, S., WITTERS, E., LAUKENS, K., DECKERS, P., SWENNEN, R., AND PANIS, B.
(2005). Preparation of protein extracts from recalcitrant plant tissues: An
evaluation of different methods for two-dimensional gel electrophoresis anal-
ysis. Proteomics, 5: 2497-2507.
RAIS, I., KARAS, M., AND SCHAGGER, H. (2004). Two-dimensional electrophoresis for the
isolation of integral membrane proteins and mass spectrometric identifica-
tion. Proteomics, 4: 2567-2571.
SCHRODER, B AND HASILIK, A.(2006) A protocol for combined delipidation and subfrac-
tionation of membrane proteins using organic solvents. Analytical Biochemi-
stry, 357: 144-146.
SEIGNEURIN-BERNY, D., ROLLAND, N., GARIN, J., AND JOYARD, J. (1999). Differential ex-
traction of hydrophobic proteins from chloroplast envelope membranes: a
subcellular-specific proteomic approach to identify rare intrinsic membrane
proteins. The Plant Journal; 19: 217-228.
THOMAS, D.S., TURNER, D., AND EAMUS, D. (1998). Independent effects of the environ-
ment on the leaf gas exchange of three banana (Musa spp.) cultivars of differ-
ent genomic constitution. Scientia Horticulturae, 75: 41-57.
Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007 5