of gene expression. Table10.1shows mutants of
L. japonicus that carry mutations in specific
genes encoding enzymes of starch and sucrose
metabolism. Both forward genetic screens and
TILLING (see Chap. 22 ) have been used to
isolate mutants with altered starch content
(revealed by iodine staining). For the pathway of
starch biosynthesis, the mutant phenotypes indi-
cate that the same enzymes are required in this
model legume as in pea and thatL. japonicus
conforms to the standard dicotyledonous path-
way established inArabidopsis. However, the
effect of elimination of starch synthesis on plant
growth and development differs significantly
between the legumes andArabidopsis. Whereas
the almost starchlesspgmmutant ofArabidopsis
grows more slowly than wild-type plants in most
day lengths (Caspar et al. 1985 ), the equivalent
mutants of L. japonicus and pea (the rug3
mutant), which also lack starch, are not impaired
in growth (Harrison et al. 1998 ; Vriet et al. 2010 )
suggesting that the transitory storage of starch isdispensable in these legume species. They also
retain functional root nodules and—at least forL.
japonicus—mycorrhizal associations, showing
that starch storage and turnover is not essential
for the establishment of successfulRhizobium
and mycorrhizal symbioses (Vriet et al. 2010 ;
Gutjahr et al. 2011 ). It seems likely that, as in
Arabidopsis, the starchless mutants of legumes
have altered patterns of sugar export and util-
isation over the day (Stitt and Zeeman 2012 ).
Why these patterns are able to compensate
completely for the loss of starch turnover in
legumes, but not inArabidopsisremains to be
investigated.
The phenotypes of mutants lacking specific
enzymes of starch degradation show that at least
thefirst step in theArabidopsisstarch degrada-
tion pathway—the phosphorylation of the starch
granule surface—is also important for the pro-
cess inL. japonicus. Bothgwdandpwdmutants
ofL. japonicushave increased levels of starch in
their leaves (Vriet et al. 2010 ). However,
impaired starch degradation caused by loss of
GWD activity has a far greater effect on the plant
inL. japonicusthan it does inArabidopsis. The
L. japonicus gwdmutants grow more slowly in
comparison with wild-type plants thanArabid-
opsis gwdmutants, and unlike theArabidopsis
mutants, they are largely infertile.
As well as identifying genes already known to
encode enzymes of starch metabolism, forward
screens for abnormal starch levels inL. japonicus
also identified several loci not previously known
to encode proteins important for starch metabo-
lism (Vriet et al. 2010 ). Recent data from map-
based cloning indicate one of these encodes a
pentatricopeptide repeat-containing protein (Vri-
et, Welham, Brachmann, Edwards, Parniske,Fig. 10.3 Starch accumulation in embryos during devel-
opment. Embryos ofL. japonicusGifu were removed
from their seed coat and depigmented by heating in an
ethanol/chloroform/water mix. The starch was then
stained with Lugol’s iodine solution (indicated byblue/
blackcolouration). Stages of development fromleftto
right early heart shaped; mid-cotyledonary; maximum
fresh weight; drying, near-mature embryo (testa peelable).
Note the root cap staining as well as the cotyledons and
hypocotyl. Bar = ca. 1 mmFig. 10.2 Transcript levels (absolute) ofLotusstarch
genes in various organs. The expression of genes
identified in Table10.1were analysed using theLotus
japonicusgene expression atlas versus 3 from the Noble
Foundation (http://ljgea.noble.org/v3/). Where genes were
represented by more than one probeset, median values
were used. Organs and tissue used by the atlas:leaftri-
folia without their petioles from 28-d-old plants;stem
stems from 28-d-old plants;flowerfully developedflow-
ers;rootroots from 28-d-old plants;nod0dN-starvedroots at 0 dpi (control for nodules);nod21dmature N-
fixing nodules at 21 dpi;seed10ddeveloping seeds, 10
dpa (late embryogenesis);seed14ddeveloping seeds, 14
dpa (accumulation of storage compounds); seed20d
developing seeds, 20 dpa (physiologically mature seeds,
onset of dessication).aA comparison of the transcript
levels in different organs;ba comparison of transcript
levels following inoculation withRhizobium;ca com-
parison at three different stages of seed development.Dpa
days post-anthesis;dpidays post-inoculationb
10 Sucrose and Starch Metabolism 111