Adriano Almeida

Carla Garcia

Danieli Cristina Gonçalves

DM Brown

Eduardo Camargo

Gonçalo Pereira

Jorge Lepikson-Neto

Leandro Costa do Nascimento

M Ranik

Marcela Salazar

MS Doblin

Wesley L Marques

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BMC Proceedings

Transgenic Arabidopsis thaliana and Nicotiana tabacum overexpressing the Eucalyptus grandis Cellulose Synthase 3 and its expression pattern in different Eucalyptusspecies and tissues

Marques Wesley L

Salazar Marcela

Camargo Eduardo

Lepikson-Neto Jorge

Gonçalves Danieli

Costa do Nascimento Leandro

Garcia Carla

Almeida Adriano

Pereira Gonçalo

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Transgenic <it>Arabidopsis thaliana</it> and <it>Nicotiana tabacum</it> overexpressing the <it>Eucalyptus grandis</it> Cellulose Synthase 3 and its expression pattern in different <it>Eucalyptus </it>species and tissues

Danieli Gonçalves

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POSTER PRESENTATION Open Access
Transgenic Arabidopsis thaliana and Nicotiana
tabacum overexpressing the Eucalyptus grandis
Cellulose Synthase 3 and its expression pattern
in different Eucalyptus species and tissues
Wesley L Marques1*, Marcela Salazar1*, Eduardo Camargo1*, Jorge Lepikson-Neto1*, Danieli Cristina Gonçalves1*,
Leandro Costa do Nascimento1*, Carla Garcia2*, Adriano Almeida2*, Gonçalo Pereira1*
From IUFRO Tree Biotechnology Conference 2011: From Genomes to Integration and Delivery
Arraial d’Ajuda, Bahia, Brazil. 26 June - 2 July 2011
Background
In Brazil, the forest industry accounts for 4,5% from the
Gross Domestic Product and the country is the biggest
Eucalyptus cellulose exporter. That’s really good news
because Eucalyptus forests are a competitive and effi-
cient alternative to convert carbon from the atmosphere
in cellulose, an important source for paper and bioe-
nergy production.
The cellulose biosynthesis happens through the Cellu-
lose Synthase Complex activity. This complex is com-
posed by different Cellulose Synthase genes (CesA) that
work together in a non redundant way [1]. It is also
known that some of these isoforms act in the primary
cell wall synthesis while others, in the secondary cell
wall. In this last group there is the gene Eucalyptus Cel-
lulose Synthase 3 (EgCesA3), the most expressed CesA
gene during xylogenesis [2]. Besides, knockout experi-
ments proved that the AtCesA7 (EgCesA3 ortholog in
Arabidopsis thaliana) activity is essential for the xylem
formation and for plant vertical growth [3].
In front of these evidences, the EgCesA3 gene had it
expression pattern evaluated in leaf and xylem tissues
among the three most economic important Eucalyptus
species in Brazil: E. grandis, E. globulus andE. urophyla.
As demonstrated in this work, the expression experi-
ment provided enough information about the EgCesA3
function, that’s why this gene was chosen to be overex-
pressed in model plants (Arabidopsis thaliana and
Nicotiana tabacum). It’s expected increased cellulose
content in the transgenic plants xylem.
Material and methods
The EgCesA3 expression pattern was examined trough
qRT-PCR and Northern-Blot in xylem and leaf from the
tree most commercially important Eucalyptus species: E.
grandis,E. urophyla andE. globulus. The qRT-PCR was
performed using SYBR Green and amplicons with
approximated size of 100pb. On the other hand, the
Northern-Blot was made with probes containing phos-
phorous-32 radioactive.
To create transgenic plants with improved cellulose
content this work overexpressed the gene EgCesA3
under the control of the CaMV 35S promoter in A.
thaliana and Nicotiana tobacum trough Agrobacterium
transformation by floral-spray.
Results and discussion
The results show that the EgCesA3 is strongly more
expressed in xylem than in leaves among the three most
important economical Eucalyptus species for the Brazi-
lian forest industry (Fig. 1). This data corroborate to the
theory that this gene is related to the secondary cell wall
formation during the xylem development.
However, it’s necessary to mention that the gene
expression data obtained through the northern-blot
related to the E. globulus leaf can’t be analyzed. This
because the amount of RNA used in this sample was
different from the others. It can be seen through the
rRNA amounts in the RNA electrophoresis (Fig. 1).
* Correspondence: goncalo@unicamp.br
1State University of Campinas – UNICAMP, Brazil
2International Paper
Full list of author information is available at the end of the article
Marques et al. BMC Proceedings 2011, 5(Suppl 7):P167
http://www.biomedcentral.com/1753-6561/5/S7/P167
© 2011 Marques et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Besides, comparing theEgCesA3 expression pattern
between the xylem from the three species studied, it’s
possible to conclude that should exist a difference: the
EgCesA3 gene is apparently more expressed in E.
grandis and E. urophyla xylem than in E. globulus xylem
(Fig. 1). However, this conclusion isn’t distant from
doubts: the problem is the large error bars that hap-
pened in the experiment. In order to solve it, the qRT-
PCR repetition is being carried on.
In front of the EgCesA3 rolein the secondary cell wall
synthesis, this gene was overexpressed in model plants
(Fig. 2). The genetic modified plants were successfully
obtained. Thus, after the generation advance, the
homozygous transgenic plants will be evaluated to
measure the cell wall chemical composition and
morphology.
Figure 1 Eucalyptus Cellulose Synthase 3 expression pattern. On the top, expression pattern analyzed trough qRT-PCR and, below, the expression
analyzed trough Northern-Blot. The tissues studied were leaves and xylem from three different Eucalyptus species: E. globulus, E. grandis and E.
urophyla.
Figure 2 Transgenic model plants overexpressing the Eucalyptus
grandis Cellulose Synthase 3 gene. In “A” it’s possible to see one
transgenic A. thaliana near a nother non-transgenic seeds being
selected in the media containing hygromycin. In “B” it’s shown
transgenic Nicotiana tabacum growing in selection media with
hygromycin.
Marques et al. BMC Proceedings 2011, 5(Suppl 7):P167
http://www.biomedcentral.com/1753-6561/5/S7/P167
Page 2 of 3
Conclusion
The Eucalyptus Cellulose Synthase 3 function in the sec-
ondary cell wall synthesis was confirmed by this work;
The cellulose content variation trough the species stu-
died should be a consequence of the Eucalyptus CesA3
expression level;
Transgenic A. thaliana andN. tabacum overexpressing
the EgCesA3 gene were successfully obtained. The trans-
genic plants analyses are being performed; however it’s
already possible to notice, by empirical view, growth
improvement in the transgenic plants in comparison to
the wild type.
Author details
1State University of Campinas – UNICAMP, Brazil. 2International Paper.
Published: 13 September 2011
References
1. Doblin MS, Kurek I, Jacob-Wilk D, Delmer DP: Cellulose biosynthesis in
plants: from genes to rosettes. Plant & cell physiology 2002, 43:1407-20.
2. Ranik M, Myburg A: a: Six new cellulose synthase genes from Eucalyptus
are associated with primary and secondary cell wall biosynthesis. Tree
physiology 2006, 26:545-56.
3. Brown DM, Zeef LAH, Ellis J, Goodacre R, Turner SR: Identification of Novel
Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using
Expression Profiling and Reverse Genetics. Cellulose 2005, 17:2281-2295.
doi:10.1186/1753-6561-5-S7-P167
Cite this article as: Marques et al.: Transgenic Arabidopsis thaliana and
Nicotiana tabacum overexpressing the Eucalyptus grandis Cellulose
Synthase 3 and its expression pattern in different Eucalyptus species
and tissues. BMC Proceedings 2011 5(Suppl 7):P167.
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Marques et al. BMC Proceedings 2011, 5(Suppl 7):P167
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Page 3 of 3


Transgenic Arabidopsis thaliana and Nicotiana tabacum overexpressing the Eucalyptus grandis Cellulose Synthase 3 and its expression pattern in different Eucalyptus species and tissues

DP: Cellulose biosynthesis in plants: from genes to rosettes. Plant & cell physiology

Myburg A: a: Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cell wall biosynthesis. Tree physiology

SR: Identification of Novel Genes in Arabidopsis Involved in Secondary Cell Wall Formation Using Expression Profiling and Reverse Genetics. Cellulose

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance

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Transgenic Arabidopsis thaliana and Nicotiana tabacum overexpressing the Eucalyptus grandis Cellulose Synthase 3 and its expression pattern in different Eucalyptus species and tissues

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