proteins during seed development. For a com-
parative analysis, pods without seeds for thefive
corresponding stages were included (Nautrup-
Pedersen et al. 2010 ). In total, 604 and 965
protein spots were identified for pods and seeds
where the pod proteins correspond to 567 dif-
ferent gene accessions and 263 of those were not
identified in seeds, indicating differences in the
level and composition of proteins between the
two tissues. The identification of different puta-
tive enzymes in the urea cycle pathway between
pods and seeds suggests that the ammonium of
degraded urea can be assimilated into amino
acids in the seed and, thus, a possible factor for
the high protein level in mature legume seeds
(Nautrup-Pedersen et al. 2010 ). All obtained
proteomics data from Lotusseed and pod are
stored and available at http://www.cbs.dtu.dk/
cgi-bin/lotus/db.cgi(Dam et al. 2009 ; Nautrup-
Pedersen et al. 2010 ). For the 2D gel analysis, a
master gel for pods and seeds is uploaded and by
clicking on the master gel, protein accessions and
spot numbers are visible. Furthermore, GO
annotations, identified peptides and quantitative
data are available for each of thefive develop-
mental stages analyzed.
The overall seed development for the two
model legumes Lotusand Medicagotogether
with soybean is similar with a transiently accu-
mulation of starch during seed development,
whereas in the mature seed the starch level is
lower than 1 % together with high protein level.
Thus, all proteomics data from these three spe-
cies were merged and available at http://
bioinfoserver.rsbs.anu.edu.au/utils/PathExpress/
pathexpress4legumes.php. This is useful for a
more broad analysis of seed development
between species to identify similarities and dif-
ferences in pathways important, for example, for
the accumulation of the high level of proteins in
the mature legume seed (Dam et al. 2009 ;
Nautrup-Pedersen et al. 2010 ; Gallardo et al.
2003 , 2007 ; Hajduch et al. 2005 ; Agrawal et al.
2008 ).
18.4 Proteomics of Lotus Nodules
and RootsThe majority of legumes, includingLotus, have
the ability to form symbiosis with rhizobia in
specialized root nodule organs.Lotuswith the
sequenced diploid genome andLotusretrotrans-
poson 1 (LORE1) mutant population, currently
with more than 80,000 lines, is an excellent
model plant to study symbiosis (Sato et al. 2008 ;
Urbanski et al. 2012 ). Using forward and reverse
genetics, several genes essential for symbiosis
have been identified inLotus,Medicago, soy-
bean, and pea (Madsen et al. 2003 ; Radutoiu
et al. 2003 ; Limpens et al. 2003 ; Kouchi et al.
2010 ). Currently, proteomics is less used to study
symbiosis; however, comparative proteomics of
wild-type nodules and nodulation mutants can be
essential to identify proteins affected/delayed in
the functional nodule formation. InLotus,2D
proteome reference maps of cytosolic nodule and
root proteins were obtained. In total, 780 and 790
spots were identified from nodule and root cor-
responding to more than 800 differentLotusgene
accessions with approximately 45 % intersection.
Nodule and root master gels together with
obtained data for each spot are available and can
be further examined athttps://www.cbs.dtu.dk/
cgi-bin/lotus2_5/db.cgi(Dam et al. 2014 ). Fur-
thermore, proteomics of the soybean cytosolic
nodule fraction together with the plant and bac-
terial fractions ofMedicagonodules were per-
formed (Oehrle et al. 2008 ; Larrainzar et al.
2007 ) and the intersection of homologous pro-
teins identified between nodule proteomics
studies was determined (Dam et al. 2014 ).
To identify proteins important for transferring
nutrients between the plant and symbiont, pro-
teomics of enriched symbiosome membranes
from Lotus, soybean, and pea was performed
(Wienkoop and Saalbach 2003 ; Panter et al. 2000 ;
Saalbach et al. 2002 ). One of the proteins identi-
fied from theLotussymbiosome membrane was a
predicted sulfate transporter and, subsequently,204 S. Dam and J. Stougaard