Proteome
Proteome characterization of the primary cell wall from
sugarcane leaves: testing two different methods
Maria Juliana Calderan Rodrigues1, Thibaut Douché2, Amanda
Carmanhanis Begossi1, Laís Vaz Setem1, Elisabeth Jamet2, Rafael PontLezica2, Carlos Alberto Labate1
calabate@esalq.usp.br
Escola Superior de Agricultura “Luiz de Queiroz”-Universidade de São Paulo.
Laboratótio Max Feffer de Genética de Plantas. Brazil.
2
Université Paul Sabatier. Laboratoire de Recherche en Sciences Végétales
(LRSV). France.
1
This work was developed to produce information to support the use of plant cell
wall, component of sugarcane bagasse, in order to produce cellulosic ethanol,
eliminating residues and cooperating with the energy generation. Two different
methods of cell wall proteins extraction were tested, and their efficiencies were
evaluated. Using protocol one (based on [1]), leaves from 2-month-old plants were
collected and submerged into solution containing 5mM acetate buffer, 0,3M mannitol
and 0,2M CaCl2. Then, they were submitted twice to a 10 minutes vacuum infiltration
and the same was done replacing the calcium chloride for 2M LiCl. After that, tissues
were centrifuged and extracted proteins were separated in a SDS-PAGE (one
dimension), digested and sequenced through ESI-Q-TOF (LC-MS/MS), Waters. Protocol
two (based on [2]) relied on the trituration of sugarcane leaves in a blender with 5 mM
acetate buffer and centrifugation using a sucrose gradient. The extract was then
filtered on a nylon net and the walls were submitted to extraction using 0,2 M CaCl2
and 2M LiCl. Proteins were separated in a SDS-PAGE and sequenced in the same way
as protocol one. Therefore, proteins from both protocols were analyzed using
programs, such as X!Tandem [3] and TargetP [4] to identify them and predict the
subcellular location. Regarding protocol one, of the 59 identified proteins, the biggest
proportion, 33 (55,93%), had a signal peptide, whereas 26 (44,07%) were predicted
as contaminants. Protocol two showed that from the 66 proteins found, only 19
(28,79%) were predicted as cell wall proteins and most of them, 47 (71,21%), were
contaminant proteins. With these results we can conclude that the more suitable
method to extract cell wall proteins from sugarcane is protocol one, based on vacuum
infiltration. From the apoplastic proteins that we have found in protocol one, most of
them were cell wall modifying enzymes, such as hydrolases, which act on the
rearrangement of the polysaccharides during development, allowing certain flexibility.
Numerous glycoside hydrolases and proteinases were found, in agreement with the
expected role of the extracellular matrix in polysaccharide metabolism and signaling
phenomena. . We were able to generate valuable information about sugarcane cell wall
that can lead to future studies to enhance cellulosic ethanol production. Support:
Fapesp 2008/56100-5 and 2007/59327-8. CNPq/FAPESP INCT do Bioetanol.
[1] Boudart, G.; Jamet, E.; Rossignol, M.; Lafitte, C.; Borderies, G.; Jauneau, A.; Esquerré-Tugayé,
M.-T.; Pont-Lezica. Proteomics. 2005, 5, 212 - 221.
[2] Feiz, L.; Irshad, M.; Pont-Lezica, R.F.; Canut, H.; Jamet, E. Plant Methods. 2006, 2, 10 - 23.
[3] Bjornson et. al. Journal of Proteome Research. 2007.
[4] Emanuelsson, O.; Brunak, S.; von Heijne, G.; Nielsen, H. Nature Protocols. 2007, 2, 953 - 971.