second most highly expressed gene encoding a
large subunit in every other organ. The transcript
level ofLjAPL4was low or at the threshold of
detection in all organs analysed (Fig.10.2).
Tissue-specific expression patterns were also
observed for genes encoding members of several
families of starch-synthesising and starch-
degrading enzymes. Thus, transcript levels of
GBSS1aandGBSS1bwere similar in shoots, but
levels forGBSS1awere much greater than those
ofGBSS1bin most other organs including roots
and seeds.LjBAM1was the main gene encoding
aβ-amylase expressed in leaves, whereas BAM3
was expressed only at low levels. Thisfinding
(together with the one mentioned in Sect.10.3
regarding family members) points to major dif-
ferences betweenArabidopsisandL. japonicus
in the types of BAM responsible for starch
degradation. InArabidopsis, BAM1 is a minor
isoform that may be expressed largely in guard
cells rather than mesophyll cells, whereas BAM3
is the major isoform responsible for starch deg-
radation in the chloroplast (Sparla et al. 2006 ;
Fulton et al. 2008 ).LjDPE1,LjDPE2,LjpGlcT
andLjMEX1genes were ubiquitously expressed,
suggesting that the encoded glucanotransferases
and glucose and maltose transporters are
involved in starch degradation in all organs.
However, in contrast to leaves in which their
expression was approximately equal, LjpGlcT
expression was ca. threefold higher than that of
LjMEX1in seeds, roots and nodules.
The expression of several genes was higher in
nodules at 21 days after inoculation of roots with
Rhizobiumbacteria (Fig.10.2b) than in roots at
the time of inoculation, suggesting that the
encoded proteins may be involved in starch
metabolism in nodules. This was the case for the
starch synthesis genesLjPGM1, LjAPS1, LjA-
PL2a and LjAPL3 as well as LjGBSS1a,
LjGBSS1b, LjSS5andLjSBE2. The expression of
LjSS5,in particular, was higher in nodules than
in any other organ (Fig.10.2). InArabidopsis,
this starch-synthase-like protein has not been
shown to have starch synthase activity or to be
involved in starch synthesis. It would be inter-
esting to investigate whether the protein makes a
contribution to nodule starch metabolism inL.
japonicus. Transcript levels of several genes
encoding enzymes involved in starch degradation
also significantly differed between nodules and
roots. ThusLjBAM3transcript levels were higher
in nodules than in uninoculated roots, whereas
the opposite was true forLjBAM1.
There was little change in transcript levels of
genes encoding enzymes of starch synthesis and
degradation during seed development
(Fig.10.2c). Starch turnover has not been studied
in detail inL. japonicusseeds, but starch is not a
major component of the seed once it is mature
(Dam et al. 2009 ). Of especial note, however, is
the high level ofBAM5 expression. As previ-
ously mentioned, it is not believed to have a role
in starch degradation in Arabidopsis leaves
because it is extra-plastidial (Streb and Zeeman
2012 ), but the high transcript level inL. japoni-
cus seeds indicates that the protein may be
abundant. BAM activity is also high during seed
development in soybean; this activity is largely
attributable to a gene with 85 % identity to
LjBAM5 (Kim and Krishnan 2010 ). Both L.
japonicus and soybean (Monma et al. 1991 )
produce starch during seed development, and
then towards the end of development, the starch
is degraded (Fig.10.3). The dry seed of Lotus
contains less than 1 % starch (Dam et al. 2009 ).
However, a soybean mutant that lacksβ-amylase
activity in the seed because of a deletion in the
BAM5-like gene is not defective in starch deg-
radation or in oil or total protein accumulation
(Hildebrand and Hymowitz 1981 ). The fact that
β-amylase is not required for seed starch degra-
dation suggests that it may function as a seed
storage protein. A storage function has also been
proposed for the large amounts ofβ-amylase that
accumulate in the tap roots of alfalfa (Gana et al.
1998 ).10.5 Lotus japonicusMutants
Defective in Starch or Sucrose
MetabolismAlthough transcript profiling can provide clues
about the function of genes, direct evidence
requires the isolation of plants with altered levels10 Sucrose and Starch Metabolism 109