Planta (2008) 228:281–292
DOI 10.1007/s00425-008-0734-0
123
ORIGINAL ARTICLE
Cu/Zn superoxide dismutases in developing cotton Wbers:
evidence for an extracellular form
Hee Jin Kim · Naohiro Kato · Sunran Kim ·
Barbara Triplett
Received: 18 February 2008 / Accepted: 31 March 2008 / Published online: 19 April 2008
© The Author(s) 2008
Abstract Hydrogen peroxide and other reactive oxygen
species are important signaling molecules in diverse physi-
ological processes. Previously, we discovered superoxide
dismutase (SOD) activity in extracellular protein prepara-
tions from Wber-bearing cotton (Gossypium hirsutum L.)
seeds. We show here, based on immunoreactivity, that the
enzyme is a Cu/Zn-SOD (CSD). Immunogold localization
shows that CSD localizes to secondary cell walls of devel-
oping cotton Wbers. Five cotton CSD cDNAs were cloned
from cotton Wber and classiWed into three subfamilies
(Group 1: GhCSD1; Group 2: GhCSD2a and GhCSD2b;
Group 3: GhCSD3 and GhCSD3s). Members of Group 1
and 2 are expressed throughout Wber development, but pre-
dominant during the elongation stage. Group 3 CSDs are
also expressed throughout Wber development, but tran-
siently increase in abundance at the transition period
between cell elongation and secondary cell wall synthesis.
Each of the three GhCSDs also has distinct patterns of
expression in tissues other than Wber. Overexpression of
cotton CSDs fused to green Xuorescent protein in trans-
genic Arabidopsis demonstrated that GhCSD1 localizes to
the cytosol, GhCSD2a localizes to plastids, and GhCSD3 is
translocated to the cell wall. Subcellular fractionation of
proteins from transgenic Arabidopsis seedlings conWrmed
that only c-myc epitope-tagged GhCSD3 co-puriWes with
cell wall proteins. Extracellular CSDs have been suggested
to be involved in lignin formation in secondary cell walls of
other plants. Since cotton Wbers are not ligniWed, we sug-
gest that extracellular CSDs may be involved in other plant
cell wall growth and development processes.
Keywords Cell wall · Cotton Wbers · Cu/Zn superoxide
dismutase · Gossypium hirsutum · Hydrogen peroxide ·
Reactive oxygen species
Abbreviations
CSD Cu/Zn-type superoxide dismutase
DOA Day of anthesis
DPA Days post-anthesis
EC Extracellular
FSD Fe-type superoxide dismutase
GFP Green Xuorescent protein
MSD Mn-type superoxide dismutase
PCW Primary cell wall
Q-RT-PCR Quantitative, reverse-transcription polymer-
ase chain reaction
ROS Reactive oxygen species
SCW Secondary cell wall
SOD Superoxide dismutases
Accession numbers: The nucleotide sequence data reported here were
deposited in the GenBank database under the following accession
numbers: GhCSD1, AI727694; GhCSD2a, EU597268; GhCSD2b,
EU597269; GhCSD3, EU597270; GhCSD3s, EU597271.
S. Kim · B. Triplett (&)
USDA-ARS, Southern Regional Research Center,
1100 Robert E. Lee Blvd, New Orleans, LA 70124, USA
e-mail: Barbara.Triplett@ars.usda.gov
H. J. Kim
Department of Biological Sciences, University of New Orleans,
New Orleans, LA 70148, USA
N. Kato
Department of Biological Sciences, Louisiana State University,
Baton Rouge, LA 70803, USA
Present Address:
S. Kim
Department of Biochemistry, University of Missouri,
Columbia, MO 65211, USA