ArticlePDF Available

Abstract and Figures

Background and aims Heterotrophic growth relies on remobilisation of seed reserves and mineral absorption. We used a compartmental model to investigate the fluxes of N absorption and remobilisation of N reserves in a legume seed with high protein content. Methods Seedling growth was studied during the heterotrophic stage in two genotypes of Medicago truncatula as a function of N supply. N absorption and seed remobilisation fluxes were distinguished in a 15 N labelling experiment. Results Remobilisation of seed N reserves was high during germination, but N uptake started as soon as the radicle protruded. Both sources contributed to high elongation rates of the radicle and hypocotyl. When organ lengths stabilised, there was an efflux of N from the cotyledons and roots indicating that seedling growth was limited by carbohydrate production. No significant differences between genotypes were observed except for early N uptake, which was lower in the genotype with the highest initial seed N content. Conclusions N fluxes were similar to those of other non-legume dicotyledonous species but differed from monocotyledonous species. These results improve our understanding of the effects of mineral fertilisation on crop establishment. The compartmental model is a useful tool to analyse N fluxes patterns within and between diverse species, in relation to seed characteristics and soil N availability.
Content may be subject to copyright.
REGULAR ARTICLE
Modelling the relative contribution of seed nitrogen reserves
and external nitrogen uptake during heterotrophic growth
in Medicago truncatula
Sophie Brunel-Muguet &Bruno Mary &Carolyne Dürr
Received: 19 March 2014 /Accepted: 23 June 2014
#Springer International Publishing Switzerland 2014
Abstract
Background and aims Heterotrophic growth relies on
remobilisation of seed reserves and mineral absorption.
We used a compartmental model to investigate the
fluxes of N absorption and remobilisation of N reserves
in a legume seed with high protein content.
Methods Seedling growth was studied during the het-
erotrophic stage in two genotypes of Medicago
truncatula as a function of N supply. N absorption and
seed remobilisation fluxes were distinguished in a
15
N
labelling experiment.
Results Remobilisation of seed N reserves was high
during germination, but N uptake started as soon as
the radicle protruded. Both sources contributed to high
elongation rates of the radicle and hypocotyl. When
organ lengths stabilised, there was an efflux of N from
the cotyledons and roots indicating that seedling growth
was limited by carbohydrate production. No significant
differences between genotypes were observed except
for early N uptake, which was lower in the genotype
with the highest initial seed N content.
Conclusions N fluxes were similar to those of other
non-legume dicotyledonous species but differed from
monocotyledonous species. These results improve our
understanding of the effects of mineral fertilisation on
crop establishment. The compartmental model is a use-
ful tool to analyse N fluxes patterns within and between
diverse species, in relation to seed characteristics and
soil N availability.
Keywords Nitrogen .Seedling .Absorption .
Remobilization .Compartment model .Medicago
truncatula
Introduction
During the heterotrophic stage, seedling growth is en-
abled by depletion of seed reserves until the seedling
emerges and photosynthesis starts. The supply of carbon
(C) depends exclusively on seed reserves during hetero-
trophic growth, but the contribution of other major
minerals such as nitrogen (N) from seed reserves and
from external sources merits further investigation, espe-
cially to assess the importance of early mineral
fertilisation for crop stand establishment. Early localised
mineral fertilisation has been shown to improve stand
establishment and favour early growth (Rosati and
Magnifico 2001; Andrews et al. 1991). But few studies
Plant Soil
DOI 10.1007/s11104-014-2182-x
Responsible Editor: Ad C. Borstlap.
S. Brunel-Muguet
INRA UMR 950 Ecophysiologie Végétale, Agronomie et
nutritions N, C, S,
Caen F-14032, France
e-mail: sbrmuguet@rennes.inra.fr
B. Mary
INRA UR Agro-Impact, Pôle du Griffon,
Barenton-Bugny F-02000, France
e-mail: bruno.mary@laon.inra.fr
C. Dürr (*)
INRA UMR 1345 Institute of Research on Horticulture and
seeds,
Beaucouzé F-49045, France
e-mail: durr@angers.inra.fr
have focused on the timing of early N uptake with
respect to the period of seed N remobilisation, or con-
sidered possible differences among species. In sugar
beet (Beta vulgaris L.), a starchy dicotyledonous seed,
absorption begins as soon as the radicle protrudes from
the teguments (Dürr and Mary 1998), which stimulates
hypocotyl elongation (Durrant and Mash 1989). By
contrast, in wheat (Triticum aestivum L.), which is also
a starchy but monocotyledonous seed, N absorption
begins later, when the leaves break through the coleop-
tile (Dürr and Mary 1998). The different categories of
species and seed morphology may thus influence early
patterns of N fluxes i.e. absorption and remobilisation.
As legume seeds are rich in reserve proteins (possibly
more than 35 % of seed dry weight, Monti and Grillo
1983), they are often expected to remain self-sufficient
longer to fulfil early seedling N requirements during the
heterotrophic stage and even after the beginning of
photosynthesis until efficient symbiotic N
2
fixation with
Rhizobium is established. Previous studies have shown
that nodule activity is efficient after about 200°-days
from emergence under low soil nitrate availability
(Pisum sativum L., Voisin et al. 2002;Medicago
truncatula G., Moreau et al. 2008). Studies on mineral
absorption by several forage legume species (Cooper
1977) suggested that the absorption of N and other
minerals begins early in seedling life, but the exact
timing of uptake has never been established. To this
end, we studied N absorption and remobilisation fluxes
in Medicago truncatula, one of the model legumes
which provides molecular tools to elucidate the genetic
determinism of legume characteristics (Cook 1999;
Thoquet et al. 2002; Tivoli et al. 2006; Young et al.
2011). In this study, we characterised two genotypes,
Jemalong A17, the reference for genomic studies, and
Paraggio, a cultivar mainly grown in Australia, which
have different seed N contents (Brunel et al. 2009). A
compartmental model was built to estimate the respec-
tive contribution of N sources, i.e. absorption and seed
reserves, to the different parts of the seedling. The
objectives of the present study were to (i) deter-
mine the timing of N absorption by seedlings in
two genotypes of Medicago truncatula under vary-
ing N supplies, (ii) estimate the respective contri-
bution of external N and N seed reserves to the
different seedling parts during heterotrophic
growth, and (iii) analyse legume N uptake and
allocation patterns with regard to seed characteris-
tics by comparison with non-legumes species.
Material and methods
Seedling growth experiments
Two genotypes were studied: Jemalong A17, the refer-
ence line used for the genome sequencing of Medicago
truncatula (Young et al. 2011) and Paraggio, a cultivar
mainly grown in southern Australia. Mother plants of
A17 seeds were grown in a greenhouse in INRA
Montpellier (43.61°N, 3.87°E, South of France) in
2005 and Paraggio seeds were produced under field
conditions in Australia in 2004 (Seedco Australia Co-
Operative Ltd). Pots were filled with 500 g of sand
(SIFRACO quality NE34: SiO2>99.7 %, solid density
2.65, mean particle size 200 μm). Before sowing, the
sand water gravimetric content was adjusted to
0.200 kg kg
1
with either deionised water or nutrient
solution (Saglio and Pradet 1980) and was maintained
constant throughout the experiment by watering regu-
larly with deionised water. Before dilution, the initial
nutrient solution contained 700 mmol L
-1
KNO
3
,
750 mmol L
-1
NH
4
NO
3
, 650 mmol L
-1
Ca (NO
3
)
2
,
275 mmol L
-1
KH
2
PO
4
, 50 mmol L
-1
NaCl,
157 mmol L
-1
MgSO
4
, plus micronutrient supplements.
We used a
15
N labelled solution to differentiate N
absorption from seed N remobilisation. Differences in
natural
15
N abundance are not usually sufficient to
ensure
15
N tracking which requires the external source
to be enriched with
15
N(Ducetal.1988). This is the
case in legumes, whose natural abundance in the seed is
close to that of atmospheric N
2
because of the ability of
mother plants to fix atmospheric N
2
. For that reason, we
used a
15
N labelled solution (2 atom % excess, APE) to
detect the exact time absorption began and to distinguish
the allocation of N originating from absorption to the
different seedling parts: radicle, hypocotyl and cotyle-
dons. The solution was diluted with deionised water at a
rate of 2, 3, 4 mL L
-1
to obtain N concentrations of 7
(S1), 10.5 (S2) and 14 (S3) mmol L
-1
, the last concen-
tration being usually used to grow plants in pots under
non-limiting mineral nutrition conditions. The experi-
ment with only deionised water and no added nutrient
solution is hereafter referred to as S0. Five seeds were
sown per pot (6.5 cm in diameter, 10 cm high) at a depth
of 1.5 cm in a pre-defined position in the pot so we were
able to refer the seedling weight to its initial dry seed
weight. Seeds were first rubbed to overcome seed phys-
ical dormancy (Uzun and Aydin 2004;Zengetal.2005)
and then individually weighed. Seed water content was
Plant Soil
measured in a sub-sample of 100 seeds taken from the
same seed lot to estimate seed dry weight from the initial
seed fresh weight. Pots were incubated in a growth
chamber at 20 °C in the dark to mimic heterotrophic
growth in the soil.
Phenological measurements and
15
Nanalyses
For each concentration of the nutrient solution and each
genotype, three pots were sampled 24, 48, 96, 144, 192
and 264 h (h) after sowing until maximum elongation was
reached. On each sampling occasion, the length of the
hypocotyl and radicle was measured. The seedling parts
were dried at 80 °C for 48 h and then weighed. Changes in
the dry mass (DM) of the different parts of the seedling
over time are expressed as the percentage of the initial
seed dry mass (SDMi) so as to account for the effects of
the initial seed mass on variations in the mass of the
different parts of the seedling. Total N content and
15
N
isotopic composition were measured using a mass spec-
trometer (VG SIRA 9) connected to an automatic com-
bustion analyser (Carlo Erba NA 1500). On each sam-
pling occasion, three replicates were obtained by pooling
the five plants grown in each sample pot. The seedling
parts (radicle, hypocotyl, cotyledons) were analysed sep-
arately after rinsing with deionised water to ensure there
was no
15
N left on the organs surface. Results are
expressed as
15
N content which is the product of N
content and
15
N atom excess, i.e.
15
N abundance of the
sample minus
15
N abundance in the air (0.3663 %
15
N).
Data fitting and statistical analyses
Two-way ANOVA was performed to test the effects of
genotype (G), N supply (S) and G x S interactions of the
measured variables (STATGRAPHICS Plus 3.1. soft-
ware). The N supply in each genotype was compared
using Bonferronis multiple comparison procedure. For
a given level of N supply, the genotype effect was tested
using a one-way ANOVA. A model was built to calcu-
late N fluxes to estimate the relative proportions of
nitrogen originating from mobilisation of seed reserves
and N absorption using
15
N labelling (Fig. 1). To esti-
mate N fluxes, the non-parametric Kruskal-Wallis pro-
cedure was performed to test the genotype and the N
supply effects because sample sizes were reduced (two
replicates per modality i.e. n=8 and n= 4 for testing the
genotype and the N supply effects respectively) and the
variance equality was verified (Levenestest).
Results
Seedling growth and the length of the radicle
and hypocotyl depend on nutrient supply
The hypocotyl and the radicle reached plateau values
150 h after germination independent of the amount of
nutrients in the solution. The final length of the hypo-
cotylrangedfrom52to76mm(Fig.2). No genotype
effect was observed, but in both genotypes, final lengths
were shorter in seeds supplied with the S0 solution
compared to those supplied with the S1, S2 and S3
solution. By contrast, the final length of the radicle
ranged from 45 to 57 mm and significant effects of the
nutrient supply and genotype were observed. Paraggio
radicles were longer than A17 radicles independent of
the nutrient solution (Fig. 2). The effect of the nutrient
supply was visible in the longer radicle in Paraggio
seeds grown in the S0 solution, leading to a significant
G x S interaction effect, since there was no significant S
effect on the final length of the radicle in A17
(Bonferronis comparison, data not shown). Regarding
changes in dry mass (DM) with time (Fig. 3), a slow
decrease in seedling DM was observed independent of
the genotype and solution. The decrease is expressed as
a percentage of the initial seed dry mass (SDMi) and
ranged between 10 % and 15 % except in seeds grown in
the S0 solution, in which case it reached about 20 %
(Fig. 3). Irrespective of the genotype and nutrient sup-
ply, there was a sharp decrease in cotyledon DM of from
25 % (after 24 h) to 75 % (after 264 h) of the initial
SDMi (Fig. 3). During the same period, the DM of the
hypocotyl and radicle increased (Fig. 3). Hypocotyl DM
reached between 30 % and 45 % of the SDMi 144 h
after sowing. Radicle DM reached its peak value, 10 %
of the SDMi, sooner i.e. 96 h after sowing. When
estimating the effect of different nutrient solutions and
genotypes, we found a significant effect of the nutrient
solution on seedling DM from 96 h after sowing
(P<0.001; Fig. 3). This effect was due to a decrease in
cotyledon DM (at 96 and 264 h after sowing) concom-
itant with an increase in hypocotyl DM (from 144 h after
sowing) and an increase in root DM (at 144 and 264 h
after sowing) when solution was added, independent of
the concentration (S1, S2 and S3). The decrease in
cotyledon DM at 96 h after sowing was not linked with
an increase in the DM of the hypocotyl or radicle,
meaning C losses from the cotyledons were only used
for respiration at this time. Genotype effects were
Plant Soil
mainly observed in root and cotyledon DM from 144 h
after sowing, when higher values were measured in
Paraggio irrespective of the solution.
Patterns of seedling
15
N enrichment of seedling
highlights early absorption just after radicle protrusion
The initial N content in seeds was significantly higher in
Paraggio (7.6±0.1 % w/w) than in A17 (7.0±0.1 % w/w).
Initial %
15
N excess in seeds was also significantly differ-
ent but very low in the two genotypes (0.00012±
0.00003 % in Paraggio and 0.00025±0.00002 % in
A17 respectively). In both genotypes, as soon as the seeds
had germinated about 24 h after sowing,
15
N content
increased in all the seedling parts (radicle, hypocotyl and
cotyledons, Figs. 4a, b, c and d)intreatmentswithS1,S2
and S3 solutions, whereas it remained close to zero in S0.
The increase in
15
N content was earlier and higher in the
radicle than in the hypocotyl and was slowest and very
small in the cotyledons until 192 h after sowing. The effect
of the N supply was significant from 24 h after sowing in
the radicle and the cotyledons, and from 48 h after sowing
in the hypocotyl, with lower
15
N contents in S1 and S2
than in S3 (Figs. 4b, c and d). On each sampling occasion,
the differences in
15
N contents between treatments (S1, S2
and S3) were larger in A17 than in Paraggio. For a given
Nutrient Soluon
N, E
Cotyledons
C, Ec
Radicle
R, Er
Hypocotyl
H, Eh
Seedling S, Es
a
g
b
c
d
Measured variables
N : N amount (μg
15
N plant
-1
) in the nutrient soluon
C : N amount (μg
15
N plant
-1
)inthecotyledons
R : N amount (μg
15
N plant
-1
)intheradicle
H:Namount(μg
15
N plant
-1
) in the hypocotyl
E: isotopic excess in the nutrient soluon
Ec: isotopic excess in the cotyledons
Er: isotopic excess in the radicle
Eh: isotopic excess in the hypocotyl
Calculated variables
N* = N.E
15
Namountg
15
N plant
-1
) in the nutrient soluon
C* = C.Ec
15
Namount(μg
15
N plant
-1
)inthecotyledons
R* = R.Er
15
Namountg
15
N plant
-1
)intheradicle
H* = H.Eh
15
Namount(μg
15
N plant
-1
) in the hypocotyl
Esmated fluxes
a : N uptake flux (μg day
-1
plant
-1
)
g : N remobilizaon flux from the cotyledons (μg day
-1
plant
-1
)
day
-1
plant
-1
)
day
-1
plant
-1
)
day
-1
plant
-1
)
b : N remobilizaon flux from cotyledons to radicle (μg
c : N remobilizaon flux from cotyledons to hypocotyl (μg
d : N transfer flux fro m radicle to hypocotyl (μg
Equaons
g=b+c
ΔR/Δt = a+b-d ΔR*/Δt = a.E+b.Ec-d.Er
ΔH/Δt = c+d ΔH*/Δt = c.Ec
ΔS/Δt = a+g ΔS*/Δt = a.E+g.Ec
a = (ΔS*/Δt - ΔS/Δt.Ec)/ (E-Ec)
g= ΔS/Δt-a
Fig. 1 Compartmental model for estimating the fluxes and relative proportions of N originating from seed reserves and absorption
0
25
50
75
100
0 48 96 144 192 240
*† *† *†
0
25
50
75
100
0 48 96 144 192 240
*†
*RadicleHypocotyl
A17 S0
A17 S1
A17 S2
A17 S3
Paraggio S0
Paraggio S1
Paraggio S2
Paraggio S3
**
Length (mm)
Time (hours)
0 48 96 144 192 240 288
0 48 96 144 192 240 288
*
aer germinaon
Fig. 2 Time course of elongation of the radicle and the hypocotyl
after sowing in A17 (black symbols, solid line) and Parragio
(empty symbols, dashed line) genotypes according to the concen-
tration of the nutrient solution (S0, S1, S2, S3). Lines are fitted to a
Weibull function. The effects of N supply and of genotype are
denoted * and respectively when significant (P<0.05). Vertical
bars denote s.e. (n=15)
Plant Soil
genotype, the effect of N supply was only significant in
Paraggio radicle, hypocotyl, and cotyledons 24 h after
sowing, whereas in A17 it was significant at 24 h in all
three organs, at 144 h in the radicle and the hypocotyl, and
at 192 h in the hypocotyl and the cotyledons, which were
the last organs supplied with
15
N (Bonferronis compari-
son, data not shown).
15
N content stopped increasing
concomitantly with the end of elongation of the seedling
parts (Figs. 2and 4b and c).
Seed reserves are the main source of N for seedling
growth and are supported by exogenous N uptake
after germination
Figure 5shows daily fluxes of N from external uptake
and from remobilisation of seed reserves based on the
calculations performed with the compartmental model
in both genotypes (aand gfluxes respectively, Fig. 1).
The two genotypes followed the same general pattern
(Fig. 5). During the first 24 h after sowing (before the
mean germination time), a large daily flux of N came
from the seed reserve compartment (g), reaching about
30 μgday
1
plant
1
24 h after sowing, and remaining at
this level until 48 h after sowing in both genotypes
(Fig. 5). Absorption started about 24 h after sowing
and daily N amounts from absorption (a) increased until
96 h after sowing whereas the flux (g) from the seed
reserves started decreasing. From 24 to 96 h after sow-
ing, daily N absorption and N remobilisation allowed
the flow of N towards the growing organs i.e. radicle
and hypocotyl, to be maintained at about 30 μgday
1
plant
1
. After 96 h, both N remobilisation and absorp-
tion rates decreased, which was concomitant with the
end of hypocotyl elongation. Finally, the N
remobilisation flux (g) reached negative values in
Paraggio in the S3 treatment 264 h after sowing, and
in A17 in the S1, S2 and S3 treatments at 144, 192 and
264 h after sowing, meaning the plants released N when
the hypocotyl and root were no longer growing (Fig. 2).
No significant differences in the fluxes from seed
0%
25%
50%
75%
0 48 96 144 192 240 288
Hypocotyl
0%
25%
50%
75%
100%
0 48 96 144 192 240 288
Cotyledons
0%
5%
10%
15%
20%
0 48 96 144 192 240 288
Radicle
50%
60%
70%
80%
90%
100%
0 48 96 144 192 240 288
Whole seedling
Dry mass (in % of inial seed mass)
Time aer sowing (hours)
*†
*†
*†
*†
*† *
*
**
*
*
A17 S0
A17 S1
A17 S2
A17 S3
Paraggio S0
Paraggio S1
Paraggio S2
Paraggio S3
Fig. 3 Changes in dry mass with respect to the initial seed dry
mass with time after sowing in A17 (black symbols, solid line) and
Paraggio (empty symbols, dashed line) genotypes according to the
concentration of the nutrient solution (S0, S1, S2, S3) in the whole
seedling, cotyledons, hypocotyl and radicle. The solution and
genotype effects are denoted * and respectively when significant
(P<0.05). Vertical bars denote s.e. (n=15)
Plant Soil
reserves and N absorption were observed between the
two genotypes over the sampling period (data not
shown).
The hypocotyl is the main sink for N from seed reserves
and from external absorption
The compartmental model enabled us to calculate net
fluxes between the different seedling parts (b,c,din
Fig. 1). These calculations enabled finer estimation of
the two components of the daily N remobilisation flux
(g), i.e. b(from cotyledons to the radicle) and c(from the
cotyledons to the hypocotyl) and of the daily N transfer
between the radicle and the hypocotyl (d). Seed N re-
serves from the cotyledons were allocated to the growing
parts of the embryo i.e. radicle and hypocotyl (Fig. 6).
Total daily N fluxes from the cotyledons were mainly
allocated to the radicle during the first 48 h after sowing
(b,2040 μgday
1
plant
1
depending on N supplies and
genotypes, but only one genotype effect was observed at
24 h after sowing: mainly a higher value in A17 under no
N supply). N allocation to the hypocotyl was lower (c,
ca.10 μgday
1
plant
1
) and almost zero under no N
supply (Fig. 6). The radicle also absorbed N directly from
the solution as shown by the
15
N content (Fig. 3). Fig. 6
shows a huge flux from the radicle to the hypocotyl (d)
from germination (about 24 h after sowing) to 96 h after
sowing, when the radicle reached its final length (Fig. 2).
The flux from the radicle was supported by the N flux
from the cotyledons (c), both fluxes helped maintain the
total flux to the hypocotyl at about 30 μgday
1
plant
1
.
From 96 h after sowing onwards, N fluxes from the
cotyledons to the radicle (b) and to the hypocotyl (c)
decreased significantly. At 192 h after sowing, fluxes
from the cotyledons to the hypocotyl (c) and from the
radicle to the hypocotyl (d) were negative in A17 with the
S3 and S2 treatments, meaning that the radicle and the
cotyledons released N under high N supply conditions,
which was concomitant with the end of hypocotyl elon-
gation. Considering the sampling period as a whole, a
0
50
100
150
200
250
300
0 48 96 144 192 240 288
0
200
400
600
800
1 000
0 48 96 144 192 240 288
0
200
400
600
800
1 000
0 48 96 144 192 240 288
0
100
200
300
400
500
600
0 48 96 144 192 240 288
*† ***
*†
*
A17 S0
A17 S1
A17 S2
A17 S3
Paraggio S0
Paraggio S1
Paraggio S2
Paraggio S3
a
Whole seedling
15
Ncontent
(μg
15
N gDW
-1
)
(μg
15
N gDW
-1
)
(μg
15
N gDW
-1
)
(μg
15
N gDW
-1
)
*†
*
*†
*
*
*† **
b
Radicle
15
Ncontent
*
*
†*
*† *
Hypocotyl
15
Ncontent
cd
Time aer sowing (hours)
Cotyledons
15
Ncontent
Fig. 4 Changes in
15
N content (μg
15
NgDW
1
) in the whole
seedling (a)intheradicle(b), hypocotyl (c) and the cotyledons (d)
in A17 (black symbols, solid line) and Paraggio (empty symbols,
dashed line) genotypes according to the concentration of the
nutrient solution (S0, S1, S2, S3). The effects of N supply and of
genotype are denoted * and respectively when significant
(P<0.05). Vertical bars denote s.e. (n=15)
Plant Soil
significant genotype effect was only observed 24 h after
sowing in the daily N fluxes b,cand dwith lower values
in Paraggio (Fig. 6). A significant effect of N supply was
observed in daily N fluxes from the cotyledons to the
radicle 96 h after sowing, with lower values with solution
S0. Indeed, 96 h after sowing, the N from the solution
-20
-10
0
10
20
30
40
50
0 48 96 144 192 240 288
-20
-10
0
10
20
30
40
50
0 48 96 144 192 240 288
Time aer sowing (hours)
Daily N fluxes μg N day
-1
plant
-1
S0
S1
S2
S3
S0
S1
S2
S3
Paraggio A17
Fig. 5 Daily N uptake (a,μg N day
1
plant
1
, dotted line) and
remobilisation (g,μgNday
1
plant
1
, solid line) fluxes in
Paraggio (A)andA17(B) genotypes under S0, S1, S2 and S3
treatments. No significant effect of N supply on uptake and
remobilisation were observed (Kruskal-Wallis procedure, n=8).
Vertical bars denote s.e
60
80
100
120
140
0 48 96 144 192 240 288
-20
-10
0
10
20
30
40
50
0 48 96 144 192 240 288
-20
-10
0
10
20
30
40
50
0 48 96 144 192 240 288
Daily N fluxes μg N plant-1 day-1
Cotyledons towards hypocotyl (c)
Cotyledons towards radicle (b)
Time aer sowing (hours)
Amount of N in % of iniƟal seed N
*
*
-20
-10
0
10
20
30
40
50
0 48 96 144 192 240 288
*
*
A17 S0
A17 S1
A17 S2
A17 S3
Paraggio S0
Paraggio S1
Paraggio S2
Paraggio S3
Radicle towards hypocotyl (d)
Daily N fluxes μg N plant-1 day-1
Daily N fluxes μg N plant-1 day-1
Fig. 6 Amount of N in the whole seedling as a proportion of
initial seed N and daily N fluxes from the cotyledons to the radicle
(bflux,μg N day
1
plant
1
), from the cotyledons to the hypocotyl
(cflux, μg N day
1
plant
1
), and from the radicle to the hypocotyl
(dflux, μg N day
1
plant
1
) in Paraggio and A17 genotypes under
S0, S1, S2 and S3 treatments. The N supply and genotypes effects
are denoted with * and respectively when significant (Kruskal-
Wallis procedure, n=8 and n=4 for testing genotype and solution
effects, respectively). Vertical bars denote s.e
Plant Soil
(quantified by
15
N content) in the cotyledons started
being a source of N for this plant part with the S1, S2
andS3treatments(Fig.4d). Therefore, with the S0 solu-
tion, the amount of N in the cotyledons only came from
initial seed reserves with no additional N from the solu-
tion, leading to a net depletion of N from the cotyledons.
Figure 6shows that the global net N balance (ratio of
N seedling content to initial seed N content) decreased
progressively from 192 h after sowing with the S0
solution in both genotypes, indicating that, from then
on, there was a N net efflux from the seedling (i.e.
radicle and hypocotyl, Fig. 1), which reached about
20 % of the seed initial content. This efflux occurred
in both the cotyledons and the radicle as shown in Fig. 6.
By contrast, where N absorption occurred in treatments
with the other solutions (S1, S2 and S3), the net balance
remained close to 100 %, meaning N uptake from
external solution compensated for N losses.
Discussion
The present study enabled us to calculate the relative
contribution of N from seed reserves and from absorp-
tion during the first stages of crop establishment in
Medicago truncatula, a legume species with high seed
N content. Previous studies reported early absorption in
legume species but without mentioning the exact time N
absorption began (Cooper 1977). In our experiment,
when the level of nutrient solution supply varied, the
concentrations of all the nutrients in the solution were
changed in the same time, which could have influenced
seedling growth. However, knowing this limit of the
experiment, by
15
N labelling the nutrient solution, we
were able to check the origin of the different sources of
N in the different seedling parts more precisely. Our
results demonstrate that with an external supply of N,
N absorption began as soon as the radicle protruded
from the teguments (i.e. at germination) and that
chronogically, N was first allocated to the radicle and
then to the hypocotyl for elongation. The cotyledons
were also supplied with external N but to a lesser extent
during the early days of elongation. When the radicle
and hypocotyl reached their final lengths, the amount of
external N absorbed decreased as a consequence of a
decrease in demand for N by both the radicle and the
hypocotyl. The release of N from the cotyledons and the
radicle observed when elongationstopped highlights the
fate of absorbed N, which was neither stored nor
assimilated for tissue growth. This could be linked to
the allocation of carbohydrate resources which might be
oriented towards more C demanding metabolic func-
tions such as respiration and/or cellulose accumulation
in the cell wall, and to the lack of newly produced
carbohydrate resources under heterotrophic conditions.
These observations in the legume species Medicago
truncatula are in agreement with results observed in
sugar beet (Beta vulgaris L.), another dicotyledonous
but non-legume species: N absorption in sugar beet
started just after germination with absorbed N cumulat-
ing first in the root and then in the hypocotyl (Dürr and
Mary 1998). These authors showed that absorption be-
gan later in wheat (Triticum aestivum L), a monocoty-
ledonous species, after the leaves broke out of the cole-
optile. Other studies on the allocation of remobilised
seed phosphorus (P) reserves and external P uptake
during early growth in maize, another monocotyledon-
ous species, showed that the timing of P uptake was
similar to that in wheat, since P uptake began as soon as
the seedling leaves developed (Nadeem et al. 2012,
Nadeem et al. 2013). These observed differences in
early external mineral uptake among groups of species
are also consistent with earlier studies in dicotyledonous
pasture species and grasses (MacWilliam et al. 1970),
showing that dicotyledonous species are more respon-
sive to an early external N supply (when available), than
monocotyledonous species, irrespective of the nature of
the seed reserves. Interestingly, Fayaud et al. (2014)
reported similar effects on early N supply to those
observed in monocotyledonous species, in pea (Pisum
sativum L.), which is a dicotyledonous legume species
but whose seedling growth and emergence resemble
those of monocotyledonous species (pea cotyledons
remain in the soil and the seedling has an epicotyl like
many monocotyledonous species rather than a hypocot-
yl like most dicotyledons). Finally, the timing of the
beginning of mineral absorption and its effects on seed-
ling growth could be linked to seed morphology and
embryo growth, and seed classification (as proposed for
instance in Finch-Savage and Leubner-Metzger 2006),
could help predict these effects.
Our study revealed no differences between the two
genotypes except for very early N uptake which was
lower in Paraggio, the genotype with the highest N
initial seed content. This genotype had a lower %
15
N
excess in the root just after germination, meaning the
uptake flux to the root was lower than in A17, perhaps
due to the larger amount of readily available N from the
Plant Soil
seed reserves. The larger amount of N in the seeds could
be due to the seed production conditions, which were
not the same for the two genotypes. However, this
difference in N content in the two genotypes was already
observed in other seed lots produced under the same
conditions and could also be a genotypic difference as
reported by Brunel et al. (2009). In agreement with these
observations, a study on maize seedlings also showed
that initial P seed contents affected the intensity of P
uptake and the allocation to roots just after the radicle
protruded (Nadeem et al. 2012).
Conclusion
This study describes patterns of early N uptake and
remobilisation during heterotrophic growth in the le-
gume species Medicago truncatula. Because of its high
seed N content, contrasting patterns with other non-
legume seeds might be expected, but the observed dif-
ferences were more pronounced between monocotyle-
donous and dicotyledonous species than between le-
gumes and dicotyledonous non-legumes. The modelling
approach used in this study allowed N uptake and N
remobilisation from seed reserve to be analysed sepa-
rately, and the seedling N budget to be interpreted using
both sources during early growth. The flux compartment
model we developed is thus a powerful tool to analyse
(i) differences among species in the allocation of internal
and external sources of N and of other minerals before
autotrophic growth and (ii) the putative effects of genet-
ic diversity in early mineral absorption related to initial
seed characteristics or to differences in seedling growth.
Acknowledgements This research was funded by the Region
Pays de Loire and INRA (French National Institute for Agricul-
tural Research). The authors thank MH. Wagner and L. Ledroit for
technical assistance with measurements on the plants, and O.
Delfosse for mass spectrometry analysis. The authors are also
grateful to E. Personeni for her helpful reading of the manuscript
and to Jean-Paul Maalouf for his help with statistical analyses.
References
Andrews M, Scott WR, McKenzie BA (1991) Nitrate effects on
pre-emergence growth and emergence percentage of wheat
(Triticum aestivum L.) from different sowing depths. J Exp
Bot 42:14491454
Brunel S, Teulat-Merah B, Wagner MH, Huguet T, Prosperi JM,
Durr C (2009)Using a model-based framework foranalysing
genetic diversity during germination and heterotrophic
growth of Medicago truncatula. Ann Bot 103:11031117
Cook DR (1999) Medicago truncatula - a model in the making!
Curr Opin Plant Biol 2:301304
Cooper CS (1977) Growth of the legume seedling. Adv Agron 29:
119139
Duc G, Mariotti A, Amarger N (1988) Measurements of genetic
variability for symbiotic dinitrogen fixation in field-grown
faba bean (Vicia faba L.) using a low level 15 N-tracer
technique. Plant Soil 106:269276
Dürr C, Mary B (1998) Effects of nutrient supply on pre-
emergence growth and nutrient absorption of wheat
(Triticum aestivum L.) and sugarbeet (Beta vulgaris L.).
Ann Bot 81: 665672
Durrant MJ, Mash SJ (1989) Stimulation of sugarbeet hypocotyl
extension with potassium nitrate. Ann Appl Biol 115:367
374
Fayaud B, Coste F, Corre-Hellou G, Gardarin A, Dürr C (2014)
Modelling early growth under different sowing conditions: A
tool to predict variations in intercrop early stages. Eur J
Agron 52:180190
Finch-Savage WE, Leubner-Metzger G (2006) Seed dormancy
and the control of germination. New Phytol 171:501523
MacWilliam JR, Clements RJ, Dowling PM (1970) Some factors
influencing the germination and early seedling development
of pasture plants. Aust J Agric Res 21:1932
Monti LM, Grillo S (1983) Legume seed improvement for protein
content and quality. Qual Plant Foods Hum Nutr 32:253266
Moreau D, Voisin AS, Salon C, Munier-Jolain N (2008) The
model symbiotic association between Medicago truncatula
cv. Jemalong and Rhizobium meliloti strain 2011 leads to N-
stressed plants when symbiotic N2 fixation is the main N
source for plant growth. J Exp Bot 59:35093522
Nadeem M, Mollier A, Morel C, Shahid M, Aslam M, Zia-ur-
Rehman M, Ashfaq Wahid M, Pellerin S (2013) Maize
seedling phosphorus nutrition: Allocation of remobilized
seed phosphorus reserves and external phosphorus uptake
to seedling roots and shoots during early growth stages.
Plant Soil 371:327338
Nadeem M, Mollier A, Morel C, Vives A, Prudhomme L, Pellerin
S (2012) Seed phosphorus remobilization is not a major
limiting step for phosphorus nutrition during early growth
of maize. J Plant Nutr Soil Sci 175:805809
Rosati A, Magnifico V (2001) Effect of early and localised nitro-
gen fertilisation on growth, yield, earliness and fertiliser use
efficiency of field eggplant. Acta Horticult 563:187194
Saglio P, Pradet M (1980) Soluble sugars, respiration and energy
charge during aging of excised maize root tips. Plant Physiol
66:516519
Thoquet P, Ghérardi M, Journet ET, Kereszt A, Ané JM, Prospéri
JM, Huguet T (2002) The molecular genetic linkage map of
the model legume Medicago truncatula: an essential tool for
comparative legume genomics and the isolation of agronom-
ically important genes. BMC Plant Biol 2:113
Tivoli B, Barange A, Sivasithamparam K, Barbetti J (2006)
Annual Medicago: From a Model Crop Challenged by a
Spectrum of Necrotrophic Pathogens to a Model Plant to
Explore the Nature of Disease Resistance. Ann Bot 98:
11171128
Plant Soil
Uzun F, Aydin I (2004) Improving germination rate of
Medicago and Trifolium species. Asian J Plant Sci 3:
714717
Voisin AS, Salon C, Munier-Jolain N, Ney B (2002) Quantitative
effects of soil nitrate, growth potential and phenology on
symbiotic nitrogen fixation of pea (Pisum sativum L.). Plant
Soil 243:3142
Young ND, Debellé F, Oldroyd GE, Geurts R, Cannon SB,
Udvardi MK, Benedito VA, Mayer KFX, Gouzy J, Schoof
H et al (2011) The Medicago genome provides insight into
the evolution of rhizobial symbioses. Nature 480:520524
Zeng LW, Cocks PS, Kailis SG, Kuo J (2005) Structure of the seed
coat and its relationship to seed softening in Mediterranean
annual legumes. Seed Sci Technol 33:351362
Plant Soil
... In Brassicaceae species, radicle protrusion occurs approximately 2 days after sowing (Dell'Aquila et al. 2000;Russo et al. 2010). Radicle growth depends on both mineral seed reserves and nutrient uptake from the substrate (Brunel-Muguet et al. 2015). This is especially true in small seeds (Peñaloza and Durán 2015), such as mustard seeds. ...
Article
Root elongation method may be implemented using two internationally accepted protocols: exposing plants to either soil-water extract or whole soil. But which of the two protocols is more suitable for root elongation analysis undertaken for the quality assessment of metal-polluted soils? Soils were sampled at various distances from the site of the Middle Urals Copper Smelter located in Russia. White mustard was used as a bioindicator. We observed considerable differences in root elongation under the two protocols. In plants grown in whole soil, root length inversely correlated with pollution index, but in soil-water extract, metal concentrations had no effect on root length. Nutrient and metal concentrations in the soil-water extract were not buffered, due to the absence of the solid soil phase. It is for this reason that in highly polluted soils, root growth was greater in soil-water extracts rather than in whole soils, whereas in background soils (in the absence of toxicity), root growth was greater in whole soils compared with soil-water extracts. The quantity, intensity, and capacity factors are a plausible explanation for the differences in root length between the two protocols. The soil-water extract does not represent actual soil with respect to the desorption-dissolution reactions that take place between the soil solid phase and the soil solution. For this reason, whole soil protocol should be used for measuring root elongation given that only under this protocol, direct contact between metal-polluted soil and test organisms correctly replicates the risks inherent in the actual soil habitat.
... Gaining knowledge on genetic variation and architecture of traits associated with seedling performance such as traits optimizing N acquisition (Kiba and Krapp 2016), seed biomass allocation to the seedling organs, or nitrogen use efficiency during this phase is a first step necessary for the identification and incorporation of new traits and alleles in breeding programs. Only few works, on small number of genotypes, studied allocation of seed biomass between seedling organs during heterotrophic growth (Dürr and Mary 1998, Brunel et al. 2009, Brunel-Muguet et al. 2015. Phenotypic plasticity can also provide relevant adaptive traits in response to climatic variability or management actions (Wu et al. 2011, Yu et al. 2014, Casadebaig et al. 2015, Mašková and Herben 2018, Scheepens et al. 2018. ...
Article
Full-text available
Seedling pre‐emergence is a critical phase of development for successful crop establishment because of its susceptibility to environmental conditions. In a context of reduced use of inorganic fertilizers, the genetic bases of the response of seedlings to nitrate supply received little attention. This issue is important even in legumes where nitrate absorption starts early after germination, before nodule development. Natural variation of traits characterizing seedling growth in the absence or presence of nitrate was investigated in a core collection of 192 accessions of Medicago truncatula . Plasticity indexes to the absence of nitrate were calculated. The genetic determinism of the traits was dissected by genome‐wide association study (GWAS). The absence of nitrate affected seed biomass mobilization and root/shoot length ratio. However, the large range of genetic variability revealed different seedling performances within natural diversity. A principal component analysis (PCA) carried out with plasticity indexes highlighted four physiotypes of accessions differing in relationships between seedling elongation and seed biomass partitioning traits in response to the absence of nitrate. Finally, GWAS revealed 45 associations with single or combined traits corresponding to coordinates of accessions on PCA, as well as two clusters of genes encoding sugar transporters and glutathione transferases surrounding loci associated with seedling elongation traits. This article is protected by copyright. All rights reserved.
Chapter
Medicago truncatula seedling establishment is a multigenic trait responsive to exogenous nitrate. A quantitative genetic dissection of this trait allowed us to hypothesize that nitrate‐signaling pathway for the control of primary root elongation involves the double affinity nitrate transporter MtNPF6.8. This hypothesis was challenged by phenotyping wild type (M. truncatula, R108) and an RNAi mutant npf6.8 in the presence (5 mM NO3 −) and absence of exogenous nitrate. The phenotyping consisted in both of measuring the length of the primary roots of seedlings grown on vertical plates and measuring the length of the cortical cells. The results of this experiment supported the above‐mentioned hypothesis by showing that the inhibitory effect of nitrate on primary root and on cortical cell elongation was abolished in the mutant lines. This result supports the assumption that in M. truncatula, nitrate regulates primary root growth by controlling the elongation of cortical cells. As several works proposed that abscisic acid (ABA) may act downstream of nitrate for its perception as a signal in its role in the modulation of root architecture, we were interested to check for a potential involvement of ABA in MtNPF6.8‐mediated nitrate signaling. Treating the npf6.8 knockdown lines by exogenous ABA (10 μM) restored the inhibitory effect of nitrate on primary root growth. Accordingly, we proposed that ABA might intervene downstream of the nitrate transporter MtNPF6.8 in nitrate signaling pathway for the control of cortical cells elongation in M. truncatula. ABA action on root elongation was associated to another nitrate transporter of the NPF family, MtNPF1.7 (MtLATD/NIP) in M. truncatula. Thorough studies of the mutation affected in MtNPF1.7, showed that unlike the interplay between ABA and MtNPF6.8, ABA in concert with MtNPF1.7, exerted a positive effect on root elongation. Exogenous ABA, in the absence of an environmental nitrate signal rescued the primary and lateral root meristem arrest and root growth defect of npf1.7 mutants. Taking together these two facets of the interplay between two transporters of the NPF family and ABA for the control of primary root elongation we suggest that exogenous nitrate intervenes as a decisive partner in the determination of the ABA effect.
Article
Full-text available
Background and aims The growth of green plants depends not only on photosynthesis, but also on the successful remobilization and translocation of seed phosphorus (P) reserves to the vegetative parts of the developing seedling during early growth. Remobilization and photosynthesis are therefore two parallel and co-coinciding processes involved in better seedling establishment and early growth. Methods A study was conducted to evaluate the priority of developing maize seedlings to translocate the remobilized seed P reserves and external P uptake to seedling root and shoot sinks during 4 weeks of early growth. Two fluxes of P in growing seedlings, one from seed remobilized P reserves and one from external P uptake, were distinguished by labelling external nutrient solution P with 32P. Results The seedling phytomass was equally distributed between seedling roots and shoots for 530 cumulated degree days after sowing. Seedlings partitioned up to 71 % of P from seed reserves and up to 68 % of P acquired from the nutrient solution, to the shoots, depending on the seed P content and P concentration in the nutrient solution. It appears that accumulation of P slows down in seedling roots corresponds to the translocative functions of root P towards shoots for start of photosynthesis. Conclusions Our results suggest that the major part of seed P reserves and external P uptake were used in early development of the seedling and the preferred sink was seedling shoots.
Article
The effects of short-term deficiency of soil N at transplanting and the effectiveness of early and localised N fertilisation were studied on field eggplant (Solanum melongena L.). Pre -transplanting some of the plantlets (T1) were irrigated with water while the others (T2 and T3) were irrigated twice with a water based solution of urea, providing 0.25 kg N ha-1. In addition some of the plants (T3) receiving the urea solution were then fertigated at transplanting with 23 kg N ha -1 while all other were not. Eight days after transplanting (DAT) all plants were fertigated with 23 kg N ha-1. No other N fertilisation was applied until 30 DAT, after which plants (T1 and T2) were fertigated bi-weekly with N fertiliser until a total of 200 kg N ha-1 had been applied; T3 plants were dropped from this experiment at this time. Net CO 2 assimilation rate, measured 8 DAT (pre fertigation), was 9, 24 and 27 μmol m-2 s-1 respectively for the non-fertilised (T1), fertilised before transplanting (T2), and fertilised both be fore and at transplanting (T3) treatments. Plant dry weights and leaf area were measured and N uptake assessed at transplanting and at 8, 16 and 29 DAT. At eight DAT, non-fertilised plants were half the weight and had a third the leaf area of the plants fertilised both before and at transplanting. Plants fertilised only before transplanting had values of about 80% of those of plants fertilised both before and at transplanting. Although all plants were equally fertigated at 8 DAT with 23 kg N ha-1, the differences in dry weight and leaf area between treatments remained nearly constant at 16 and 29 DAT, and the initial delay in plant development was never recovered. This delay resulted in reduced yield and a later harvest. Crop N uptake was very low during the first 29 days reaching 14.5 kg N ha -1 in the most fertilised treatment. The importance of early and localised N fertilisation is discussed.
Chapter
This chapter focuses on the factors affecting the development and growth of the forage legume seedling. The chapter reveals that the growth of a legume seedling depends on its inherent vigor and the environmental conditions present during seed germination, maturation, and growth. Environmental conditions during seed formation could influence seed size and subsequent progeny performance. Growth responses—which could be traced to environmental conditions at some stage of previous development—are designated as physiological predetermination to distinguish them from those, which are because of hereditary causes. The chapter enlists three conditions, which could affect the potentiality of the seed or the capacity of the resulting plant for growth and yield: (1) parental conditions; (2) conditions immediately preceding germination, during germination, or in the early stages of the seedling's growth; and (3) harvesting conditions. Three stages of development of a seedling—that is, heterotrophic, transitional, and autotrophic are described in the chapter.
Article
Early growth is a critical phase of the crop cycle, which lasts from emergence to the beginning of competition between plants and is sensitive to sowing conditions and species characteristics. Providing tools to improve the management of this critical phase in intercrops is a challenge for agroecology as these cropping systems are the subject of renewed interest for their ability to maintain yields while requiring fewer inputs. The aim of the present study was to investigate variations in early growth under different sowing conditions in different species with contrasted seed and seedling characteristics (seed mass, hypogeal or epigeal emergence, and legume and non-legume species), especially species used as intercrops. Experiments were carried out in glasshouses using different sowing depths and levels of mineral nutrition, first for each species separately, then in mixed sowings. In the first set of experiments, biomass at emergence and relative growth rate after emergence were measured and then modelled as a function of seed mass, mineral nutrition, and time to emergence. Predictive equations were tested by comparing simulations with biomass measured in the second set of experiments, for two intercrops grown under varied sowing conditions. Finally, simulations were run to analyse variations in the early growth of two intercrops (durum wheat/pea or alfalfa) under a wider range of sowing conditions (seed mass, sowing depth, and with or without mineral nutrition). Biomass at emergence was positively correlated with seed mass, and in epigeal species, was also negatively impacted by time to emergence. Relative growth rate was highly stimulated by mineral nutrition whereas its response to time to emergence varied among species. The amount of seminal reserves at emergence (in hypogeal species) and the cotyledon specific mass (in epigeal species) were correlated with the establishment of the relative growth rate. When evaluated, the model was shown to satisfactorily predict the early growth of two intercrops. Used as a simulation tool, the model indicated that all the sowing techniques tested can have a major influence on total biomass and on the proportion of each component species when competition begins. This model can thus contribute to the management of sowing techniques for sole as well as combined crops whose effects are difficult to predict and are also difficult to analyse from field experiments alone because of the number of possible combinations and interactions.
Article
The effects of a range of applied nitrate (NO3−) concentrations (0–20 mol m3) on germination and emergence percentage of Triticum aestivum L. cv. Otane were examined at 30, 60, 90 and 120 mm sowing depths. Germination percentage was not affected by either sowing depth or applied NO3− concentration whereas emergence percentage decreased with increased sowing depth regardless of applied NO3− concentration. Nitrate did not affect emergence percentage at 30 mm sowing depth, but at 60 to 120 mm depth, emergence percentage decreased sharply with an increased applied NO3− concentration of 0 to 1·0 mol m−3 then decreased only slightly with further increases in applied NO3− of about 5·0 mol m−3. Root and shoot growth, NO3− accumulation and nitrate reductase activity (NRA) of plants supplied with 0, 1·0 and 1·0 mol m−3 NO3− at a sowing depth of 60 mm were measured prior to emergence. The coleoptile of all seedlings opened within the substrate. Prior to emergence from the substrate, shoot extension growth was unaffected by additional NO3− but shoot fr. wt. and dry wt. were both greater at 1·0 and 1·0 mol m−3 NO3− than with zero NO3−. Root dry wt. was unaffected by NO3−. Nitrate concentration and NRA in root and shoot were always low without NO3−. At 1·0 and 10 mol m3 NO3−, NO3− accumulated in the root and shoot to concentrations substantially greater than that applied and caused the induction of NRA. Regardless of the applied NO3− concentration, seedlings which failed to emerge still had substantial seed reserves one month after planting. Coleoptile length was substantially less for seedlings which did not emerge than for seedlings which emerged, but was not affected by NO3−. It is proposed that (a) decreased emergence percentage with increased sowing depth was due to the emergence of leaf I from the coleoptile within the substrate and (b) decreased emergence percentage with additional NO3− was due to the increased expansion of leaf 1 within the substrate resulting in greater folding and damage of the leaf.
Some of the important environmental and plant factors influencing the germination and early seedling development of a number of temperate grasses and legumes have been investigated under controlled conditions. Significant differences were found between legumes and grasses in a number of characters which appear to be important for establishment under field conditions, including quantitative aspects of water absorption, rates of germination, and early root elongation. Ryegrass was superior to all other species in its ability to germinate under conditions of moisture stress. The lower limit for germination was from 4 to 6 bars below the limiting potential found for the other species, and thus may be one of the important factors contributing to the ease of establishment of this species in the field. The early onset of autotrophic growth in young seedlings, as judged by the utilization of external nutrients and the attainment of positive net photosynthesis, occurs within 5 days of imbibition under favourable conditions, well before the exhaustion of endogenous reserves. This highlights the need for adequate water, nutrients, and light during this early stage of seedling development. It also suggests that reserves available to the seedling are probably in excess of requirements under favourable conditions, and may be of potential value under adverse conditions. These results are discussed in relation to the definition of field problems, and to some likely breeding objectives to improve establishment.
Article
Before starting a breeding program aimed at improving the nitrogen nutrition ofVicia faba, the authors tried an alternative technique to the acetylene reduction assay, to measure some genetic variability in the plant material. The quantity of dinitrogen fixed by several cultivars ofVicia faba was estimated using a low enrichment15N tracer method and high precision15N mass spectrometry. The fababeans were cultivated for two years in two different soils. The percentage of fixed dinitrogen in the seed varied between genotypes from 40 to 83% of the total nitrogen and was positively correlated with the total seed nitrogen (r=0.64 to 0.86). A highly significant positive correlation was also found between the total seed nitrogen and the quantity of fixed dinitrogen in the seed (r=0.95 to 0.99). The technique used to measure dinitrogen fixation proved to be useful and reliable enough to discriminate between various genotypes, grown over a period of two years in two different soils. However, several non-fixing control plants showed significant differences in their15N enrichment and the problem of choosing a good reference plant was raised and discussed.
Article
Phosphorus (P) is the least mobile nutrient in the soil as compared to other macronutrients and therefore frequently limits crop growth. During germination and early growth, seed-phytate hydrolysis and seed-P remobilization is the major P source for developing seedlings. The objective of this paper was to investigate whether seed-P hydrolysis and remobilization of nonphytate P are sufficient for seedling P nutrition during early growth stages of maize. A large part of initial maize endogenous seed P reserves are mainly in the form of phytate. Till 70 cumulated degree days after sowing, nearly all the phytate (98%) was hydrolyzed and caused an increase in nonphytate P in seeds. Phytate hydrolysis and remobilization of nonphytate P was the main source of P supply for the newly growing seedlings and was not a limiting step for seedling P nutrition during the first four weeks of early growth.
Article
The softening of the hard seeds of six legumes (five clovers and one medic) were measured over one year and reverse logistic equations were used to measure three parameters (hardseededness after exposure to one year of field conditions, the reduction in hardseededness during that time and the time taken for half of the seeds that softened in one year to soften [half life]). All three parameters were related to seed coat and outer layer thickness (surface to the light line). Histochemistry was used to determine the locations of several chemicals found in the seeds of legumes, including polycarboxylic acids, polyphenols, proteins, lipids and polysaccharides.Seeds of the six species varied in radius with a range from 0.52 to 1.46 mm. The seeds of Trifolium subterraneum and Medicago clypeatum were large, while seeds of T. spumosum, T. lappaceum and T. glanduliferum were small. Seeds of M. polymorpha were kidney-shaped while those of the clovers were round. Seed coat morphology was similar for all six species, but the species differed in seed coat (51-150 μm) and outer layer thickness (2.2-10.7 μm) and, in the other layers, shape, size and arrangement of the cells. Lignin, polyphenols and lipids were found mainly in the seed coat. In particular the cuticle on the seed surface stained strongly with Sudan Black B, indicating that lipids were confined to the outer layers of the seed coats. Hardseededness after one year was predicted by outer layer thickness (r2=0.81). This relationship was improved (r2=0.92) by using the ratio of outer layer thickness to seed radius. However, neither outer layer thickness nor its ratio with seed radius predicted half life.The results are discussed in terms of conflicting reports in the literature about the role of the cuticle in the softening of hard seeds. The concentration of lipids in the cuticle lends support to the hypothesis that these compounds may be involved in the process of seed softening. There is a clear need for further research to clarify the roles of different chemical groups and different layers within the seed coat.