18.3 Proteomics of Lotus Seeds and
Pods
Legumes have a high level of nutrients in mature
seeds, however, the molecular mechanisms
behind the accumulation of nutrients have not
been revealed. Model legumes with sequenced
genomes and the availability of mutant lines are
useful to obtain a more detailed knowledge of the
molecular mechanisms. InLotus, seeds fromfive
developmental stages and pods from three
developmental stages were analyzed using a
transcriptomics approach focusing on transcrip-
tion factors controlling/regulating the seed
development (Verdier et al. 2013 ). Furthermore,
allLotus Affymetrix transcriptomics data sets
from different mutants and tissues are combined
and searchable at the http://ljgea.noble.org/
homepage (Verdier et al. 2013 ). Thus, together
with the sequenced Lotus genome, several
transcriptomics, and microRNA studies (Verdier
et al. 2013 ; Sato et al. 2008 ;Høgslund et al.
2009 ; De Luis et al. 2012 ), proteomics may
provide new knowledge about protein networks
essential for the high nutrient value in Lotus
seeds.
Prior to proteomics of mature Lotus seed,
protein, starch, lipids, phytic acid, and ash levels
were determined (Dam et al. 2009 ). The mature
Lotusseeds contain approximately 43 % protein
and less than 1 % starch even though starch
granulates are visible during the seed filling
phase (Dam et al. 2009 ). This protein and starch
pattern is also seen for soybean andMedicago,
whereas mature pea seeds have a lower protein
level at ~25 % but ~50 % starch (Wilson et al.
1978 ; Prakash and Misra 1988 ; Djemel et al.
2005 ). The storage globulin fraction, which is
insoluble in H 2 O but soluble in 1 M of salt, can
be up to 80 % of the protein fraction in mature
legume seeds. The globulins were enriched and
separated using 2D gels. Spots corresponding to
legumin and convicilin, which are two of the
major globulins, were identified with an
approximate volume ratio 9:1 in mature wild-
type seeds (Dam et al. 2009 ). InLotus,80%of
the nitrogen transport from root to shoot via
xylem is linked to asparagine (Waterhouse et al.
1996 ). In line with this observation, transcripts of
the K+-dependent NSE1 asparaginase, that cata-
lyzes the hydrolysis of asparagine to aspartic
acid, was abundant inLotussink tissues (Credali
et al. 2011 ). Further, functional analysis supports
a role for the NSE1 asparaginase during seed
development and/or seedfilling. Seeds from the
NSE1 mutant have lower seed weight than wild
type together with a higher number of abnormal
seeds in the pods (Credali et al. 2013 ). For the
most severe mutant,nse1- 3 , the globulin fraction
is 20 times less abundant compared with wild-
type seeds, whereas the legumin/convicilin ratio
was not changed (Credali et al. 2013 ). In con-
clusion, proteomics of theLotusseed globulin
fraction is useful for composition and abundance
analysis of storage globulins from mutants
affected in the mature seed protein composition.
For a more elaborate proteomics study of
different developmental stages of Lotus seed
development, the switches between embryogen-
esis, seedfilling, and desiccation phases were
determined by calculating the water content
together with histological sections of seeds from
defined developmental stages (between 7 and
43 days afterflowering with a three days inter-
val) (Dam et al. 2009 ). Proteomics was initially
performed from two developmental stages cor-
responding to the seedfilling [between 19 and
25 days afterflowering (green seeds)] and des-
iccation [more than 43 days after flowering
(mature seeds)] phases using GeLC-MS/MS with
920 and 264 proteins identified from the two
developmental stages, respectively. The lower
number of proteins identified from mature seeds
represent probably the high abundant legumins
and convicilins more than that a lower number of
proteins present in mature seeds (Dam et al.
2009 ). Additionally, a quantitative and a more
systematic proteomics study of Lotus seed
development, using 2D gels within two pH ran-
ges; pH 4-7 and pH 6-11, was performed. Five
developmental stages, i.e., two stages of
embryogenesis and seedfilling phases together
with one desiccation phase, were analyzed to
determine the differences in the level of specific18 Proteomics 203