Managing legume and
grower nitrogen needs
Presented by
Associate Professor Brent N. Kaiser
Centre, for Carbon Water and Food
School of Environmental and Life Sciences
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
©FAO / Illustration: Cecilia Sanchez
Acknowledgements
P. Loughlin
Sydney
E. Federova
Wageningen
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
D. Chiasson
Germany
T. Bisseling
Wageningen
D. Mazurkiewicz
Adelaide
E. Ovchinnikova
Sydney
M. Mohamidejeh
Adelaide
D. Day
Flinders
S. Tyerman
Adelaide
Introduction – Managing Nitrogen use in Agriculture
– Agriculture requires N inputs
– Yield and quality
– Global N use ~ 110 M tonne per
annum
• 60% of that is used for cereal
production
– N Fertiliser use can be inefficient
– Most cereals on average only
capture 30-50% of the applied N
fertilizers in the harvested seed
– Improving nitrogen use efficiencies
(NUE) is a global initiative
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
80 Kg N HA-1
0 Kg N HA-1
Introduction – Managing Nitrogen
NH3
– Where does the remaining N go?
– Loss through nitrate leaching
– Microbial competition for soil N
– Soil and plant N volatilization
• N2, N2O, NH3
NH3
N2 O
NOx
N2
Microbial N
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
NO3-
Hoogstraat, Wageningen NL - 2010
Legume N2-Fixation is important?
– Legume N2-fixation - an alternative N resource
– Legumes can develop a symbiosis with
Rhizobia spp.
• When inside a legume root the bacteria
can reduce N2 to NH3
• Plant provides carbon (energy) through
photosynthesis
2NH3
Nitrogenase
Fe/Mo Enzyme
catalyst
N2 + 8 e- + 8H+ + 16ATP → 2 NH3 + H2 + 16ADP + 16Pi
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Legume N2-Fixation Improves Plant Growth on Low N Soils
+ Rhizobia (N2 fixing)
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
- Rhizobia
Legume N in
Agriculture
– Legume derived N can be used
in multiple ways
– ~400 million tonnes / pa
– Direct protein transfer to
animals
• Legume feed, seeds
– Soil mineralisation upon
plant and animal decay
• Crop rotations, green
manures
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Managing the Legume N2-Fixation
Research Goals
– Increase N2-fixation
and assimilation?
• Legume yield
– Enhance fixed N
release to the soil?
• Rotational
benefits
Indirect
(N2-fix)
– Make legumes
tolerant of soil N
• Maximise direct
and indirect N
pathways
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
N fertiliser
Crop residue
N
Decompostion / Mineralisation
Root & Nodule N exudation?
? NH4+, AA
Direct
(NO 3-, NH4+)
Are we achieving maximum N2-fixation and N2 utilisation?
NO3- or NH4+
N2
NH4+
AA
NH4+
AA
N2
Nodule N2-Fixation – NH4+ Assimilation – N Export to Plant and Soil?
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
?
NH4+
AA
N release from legume nodules – does it matter?
NH4+- N
% of
Leachate N
AA
% of
Leachate N
Symbiotic N
% of total N
accumulation
Soybean
1-3%
0.1-0.4%
10.4%
Alfalfa
29-62%
0.7-17%
4.5%
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Phi Hung, N.V et al 2013, Soil science and Plant Nutrition 59:888-895
Phi Hung, N.V 2014, PhD Thesis
Nodule N management: following the ammonium pathway
– Bacteroid NH4+ (10 – 60 mM)
IR
PC
?NH4+
VC
?NH4+
?NH4+
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Biswas and Gresshoff, Int. J. Mol. Sci. 2014, 15, 7380-97
NH4+ transport across the Symbiosome Membrane
+
N2
Bact
Infected cells
SM
bact
Nitr
NH3
NH3 / NH4+
Soybean
nodules
H+
AMT, NOD26,
NSC Channel,
AMF, other?
ADP + Pi
+
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
NH4
GmbHLHm1
(SAT1)
ATP
GES (aa = 19)
3
bHLHm1 influences nodule development/activity
2 Hydrophillic
1
0
-2
– RNAi –
35S::GmbHLHm1
3’UTR
– Hairy-root, A.
rhizogenes (K599)
HLH
-1
--3
H
Hydrophobic
50
100
150
bhlhm1 vector
200
250
300
bhlhm1 vector
-N, + B. japonicum USDA110, 28 days
bhlhm1
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
vector
bhlhm1
vector
D. Chiasson et al 2014 PNAS 111: 4814-4819
bHLHm1 expression enhanced in nodules
12 days
12 days
IR
PC
36 days
GmbHLHm1;1P::GUS
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
VB
-N, + B. japonicum USDA110, 28 days after planting
D. Chiasson et al 2014 PNAS 111: 4814-4819
MtSAT1, MtSAT2 promoter:GUS
MtSAT1 Promoter:GUS
MtSAT2 Promoter:GUS
Medicago Nodule (14 Day)
Medicago Nodule (14 Day)
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
D. Chiasson, unpublished results
bHLHm1 & AMF1 are part of a gene cluster in dicot plants
AMF1
60
66
0
5g ;1)
a1 F1
lym M
G mA
(G
;1
1
FS
M
0
63
6
g0
5
a1
lym
G
6
22
2G
AT
80
90
40
20
00
10
66
0
66
67
67
67
67
g
0
0
0
0
0
)
15 1;1
5g
5g
5g
5g
5g
5g
a1 yma AT
a1
a1
a1
a1
a1
m
m
m
m
m
m
l
ly
ly
ly
ly
ly
ly
G mS
G
G
G
G
G
G
(G
70
6
06
0
80
10
30
20
00
04
05
05
05
05
1
1
1
1
1
g
g0 1)
g0
g0
g0
g0
tr2
tr2 T1;
tr2
tr2
tr2
tr2
d
d
d
d
d
d
e
e A
e
e
e
e
M
M tS
M
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(M
0
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5g ;1)
5g ;1)
a1 T1
a1 F1
m
A
ly
lym M
G mS
G mA
G
(
G
(
0
73 )
22 1;1
2G F
AT tAM
(A
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
0
75
2
AT
G
22
G
2
AT
0
0
Medicago Chr2
2.44-2.54 Mb
g
r2
t
ed
0
01
Soybean Chr15
4.66-4.76 Mb
M
Soybean Chr15
4.66 – 4.76 Mb
Arabidopsis Chr2
9.63-9.73 Mb
77
76
22
0
55
46
0
01
tM
90
bHLHm1
22
2G
AT
D. Chiasson et al 2014 PNAS 111: 4814-4819
GmAMF3 – Is an ammonium channel
Current (nA)
2000
0
**
**
**
**
**
**
water, 5NH4+
ScAMF1, 5NH4+
GmAMF3, 5NH4+
-3000
-150 -100 -50 0
Voltage (mV)
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Xenopus leavis
50
D. Chiasson et al 2014 PNAS 111: 4814-4819
GmAMF3 (qPCR, GmAMF3 prom::GUS, AMF3::YFP)
GmAMF3
Nodule
Root (-Rhizobia +N)
VB
0.10
0.05
IR
Soybean Nodule:
AMF3 Prom:GUS.
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Lateral Root
Main Root
Nodule
Flower
Stem
Inner
Cortex
0.00
Leaf
Relative to con6
0.15
Outer
Cortex
Ph
Xy
SC
Soybean Root:
AMF3 Prom:GUS.
Onion: 35S:AMF3-YFP
D. Chiasson et al 2014 PNAS 111: 4814-4819
Anti-GmAMF3 (green) localization in the 21
day-old soybean nodule contrasted with
Propidium Iodide
vector
RNAi – Gmamf3
Soybean
Medic
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
vector
Mtamf3
Evgenia Ovchinnikova, unpublished results
Other ammonium transport pathways (AMTs)
– High affinity ammonium
transporters (AMT) localisation
indicates a need by the plant to
manage nodule generated ammonium
– Plant uptake
– NH4+ compartmentalisation
– N homeostasis
AMT expression
LjAMT1;2
LjAMT1;1
– Loss of LjAMT1;1activity
– Uninfected cells
– Disrupts N2-fixation
– Increases nodule numbers
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
D’Appuzo et al., Plant Physiol. Vol. 134, 2004
Rogato et al., Plant Mol Biol (2008) 68:585–595 589
Ljamt1;1 - enhances nodulation
Questions: Ammonium assimilation
MtGS1a
MtGS1b
– Glutamine synthetase (GS) localisation may
also suggest alternative locations where
ammonium capture/assimilation is required
– Infected cell ammonium assimilation
– Assimilation of ammonium in cells where
no bacteria exist
– Nodule GS
KM (NH4+)
Medic
– low µM range
Pvgln- 𝜸
Pvglnβ
GS
Glutamate +
NH4+
+ ATP
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
Phaseolus
Glutamine + ADP + Pi + H+
Carvalho et al. 2000 PMB 42: 741-756
Forde et al 1989, Plant Cell 1(4):391-401
Concluding Remarks
– Require further investigation into legume N2-fixation
– Benefits to the host plant
– Benefits to local environment through N-fertilisation
?
– Evidence suggests nodule ammonium management may
involve multiple mechanisms
– Assimilation and redistribution to the shoots
– Recovery of ammonium from the apoplast
• AMF, AMT, GS activity
– Potential export of ammonium out of the nodule?
– Total N2-fixation activity needs to be quantified
– Effective distribution of fixed N to the shoots
– Distribution of fixed N elsewhere in the root
– Direct loss of fixed N from the nodule
– Genetic diversity amongst legumes for N2fixation
efficiencies
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney
N2
NH3 ?
AA
NH3
bact
C
14N
15N
NO3ROOT
NODULE
Legume Solutions
How do we fortress Australian pulses to be a reliable and profitable
seed commodity and a secure N resource
– Legumes for Sustainable Agriculture
– ARC Industrial Translational Research Hub
– July 2016 - June 2021
– Research Aims:
– Managing legume response to abiotic stress
• Drought, Heat, Salinity
– Improving legume functionality
• N utilisation and N2-fixation
On the Pulse
A/Prof Brent N. Kaiser, The University of Sydney