annals of botany lecture - university of california, davis
TRANSCRIPT
Annals of Botany lecture:
Nitrogen recycling and translocation for plant development and grain filling
in Arabidopsisin Arabidopsis
Celine Masclaux‐DaubressesCeline Masclaux DaubressesCNRS‐INRA Versailles, France
Nitrogen recycling and translocation
for plant development and grain fillingfor plant development and grain filling
in Arabidopsis
Céline [email protected]
Laboratory of Plant Nitrogen Nutritiony gINRA Versailles France
Plants are static and most of the time nutrient starved
Senescence allows nutrient recycling
Remobilisation
•50%to 90% of N in rice spike from leaf N remobilisation (Van Sanford & Mackown. 1987)p
• 60% to 80% of N in maize kernel from leaf N remobilisation (Ta & Weiland. 1992)
Nutrient remobilisation in Arabidopsis(Himelblau &Amasino , 2000)
M i f ti f l fMain function of leaf senescenceis the recycling of nutrients( N, S, P and K)
> 80 % N contained in Arabidopsisleaves is exported during senescenceleaves is exported during senescence
Natural variation of N-remobilisation efficiencyN-remobilisation efficiency
in Arabidopsis
Genetic variation of leaf senescence and nutrient recycling at vegetative stage
Bay-0 x Shahdara Recombinant inbred line (RIL) Population (Loudet et al. 2002)
Early Late
35 DAS
yYellowing Yellowing
RIL310 RIL083 RIL232 RIL045 RIL272
When plants are grown at low nitrate (2 mM)When plants are grown at low nitrate (2 mM)
large variation of leaf Yellowing (i.e.senescence) among RILs
(Diaz et al. 2005 Plant Physiol., Diaz et al. 2006 Plant Cell Physiol., Diaz et al. 2008 Plant Physiol.)
15N abundance and total N YL
OLOL
DAS3 weeks 1 week
Ch i d
R
OL
R
15N Pulse/chase
Pulse period15N 2.5 %0.1 mM nitrate
T0 T1
Chase period
14N
16% 10% 12% 5% 7%
RIL310 RIL083 RIL232 RIL045 RIL272
Young leaves
Old leaves
15N fluxesbetween T0 et T1
(% T0)
2% 7% 2% 5% 5%
Old leaves
Roots
( )
Leaf-to-leaf N-remobilisation and leaf yellowing are linked
Diaz et al. Plant Physiol. 2008
At reproductive stageSame shape
Same biomassSame morphology
Diaz et al. Plant Physiology (2008)
Same morphologySame flowering time
Siliques
Flowering stem
RIL083 RIL272 Flowering stem
Siliques
stem
Rosette12% 12% 12% 24% 29%
Rosette
stem
RosetteNO DIFFERENCE
Roots 2%Roots Roots
NO DIFFERENCE
T1T0 T2T1T0 T2Leaf-to-stem/siliques N-remobilisation is not related to leaf senescence
During grain filling:
Total N in seeds, seeds DW,yield, 15N enrichement
seeds
yield, N enrichement
40DAS
15N labelling
Flowering
seeds
Long days
Short days 40DAS42 DAS
Flowering
56 DASg y
dry remains :DW, total N, 15N enrichement
As a prerequisite we assume 15NcontentWholePlant = total 15N absorbed (no
efflux))
Harvest Index = dry weightS d / dry weightWh l Pl tHarvest Index dry weightSeeds / dry weightWholePlant
15N P titi i i d 15NHI 15N t t /15N Partitioning in seeds =15NHI= 15NcontentSeeds / 15NcontentWholePlant
N remobilisation to the seeds
Harvest Index = DWSeeds / DWWholePlant
15NHI= 15NcontentSeeds / 15NcontentWholePlant
15NHI HI
RIL310
RIL083
RIL310 R
IL083 RIL232
5.4±7 .5
67 1
3.2±4.5
37 5RIL083
RIL232
RIL045 R
IL272
67.1±6.0
63.4±4.5
37.5±2.4
29.0ア 2.5
RIL045
RIL272
63.64ア 14.6
69.12 2
25.0±8.7
33.21 0±2.2 ±1.0
Rosette to seeds N-remobilisation is not related to leaf senescencebut might be related to harvest index
Natural variation of N remobilisation to the seeds
19 accessions of Arabidopsis at Low (LN) and Highnitrate (HN) supply in short days
LN (2mM nitrate) HN (10mM nitrate)
Col-0Col 0
N13
Ge-0
S k tSakata
Mr-0
Bur-0
4 plants per accessions4 plants per accessions2 biological repeats at two different times
Harvest index is correlated to flowering time
N+N-
R2 = 0.5857
R2 = 0.3065
0.30
0.35
0.40
0.10
0.15
0.20
0.25
0.00
0.05
50 60 70 80 90
Flowering time
Plant fertility is higher in early-flowering plants
early-flowering plants did not obligatory present early yellowing
0.40 a
Strong genetic variation effect onHarvest Index (HI) andPartitioning of 15N in seeds (15NHI). y = 0.6029x + 0.0738
0.20
0.30
I at N
+
N13
Stw-0St-0
Bl-1Mr-0
There is a correlation between N+ and N-measured trait
R2 = 0.62950.00
0.10
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40
HI at N-
HI
Bur-0
measured trait.
Correlations show that 15NHI is higher at N- than at N+.0.40
0.50
at N
+
Stw-0 St-0
Shahdarac
N than at N .
y = 0.4347x + 0.0696
R2= 0.56340.10
0.20
0.30
15N
HI a
Bur-0
N13
Stw 0 St 0
Bl-1Mr-0Globally N limitation did not change HI but favored N remobilisation to the seeds0.00
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80
15NHI at N-
Bur 0 but favored N-remobilisation to the seeds.
Different individual behaviors at N+ and/or N- can however be observed.
eds 0.8
Correlations on mean data
onin
g in
see
N- r2 = 0.9489
0.5
0.6
0.7
N-15
N p
artit
io
N+ r2 = 0.92710.2
0.3
0.4 N+
0.0
0.1
0.0 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45
Harvest Index
15NHI and HI highly correlated: 15N remobilisation seems mainly driven by the sink sizey y
[15NHI/HI] slope higher at N- than N+:N-remobilisation is increased at N-
Lemaître and Gaufichon et al. Plant Cell Physiol. 2008Masclaux-Daubresse et al. submitted
onin
g in
see
dsN- r2 = 0.9489
0.40.50.60.70.8
N-
Despite the strong [HI - 15NHI] correlation, outliers can be identified
15N
par
titio
N+ r2 = 0.9271
0.00.10.20.3
0.0 0.1 0.15 0.2 0.25 0.3 0.35 0.4
Harvest Index
N+
(15NHI / HI) slope was computed and normalised according to the mean as the Rem indicator
Rem can be understood as an “extra-remobilisation” indicator when >100% And as a “N-remobilisation disabled” indicator when <100%
Rem N+
100%
120%
140%
120%
140%
160% Rem N-
40%
60%
80%
40%
60%
80%
100%
0%
20%
0%
20%
Rem variation is independent of HI variation and genetically determinedRem is a valuable quantitative trait usable to map QTLs.
Relative Specific Abundance (RSA) is an indicator of 15N enrichment of plant tissues
RSA ratio N+1.8 RSA ratio N-1.2
The ratio RSASeeds/RSAWholePlant was considered according to Gallais et al. 2004.
RSA ratio N+
0.40.60.81.01.21.41.6
RSA ratio N
0.4
0.6
0.8
1.0
0.00.2
0.0
0.2
At N+ most of the accessions have RSA ratio >1 At N- most of the accessions have RSA ratio <1
N% in seeds 3.5%N% in seeds 4.2%
15N 15N
14N 14N
N% in dry remains 4% N% in dry remains 1%
15N labeling formutant screeningmutant screening
Our mutants of interest:
RespirationPhotorespiration
PhotosynthesisGLNASNGLU
Proteolysis
Dismanteling GLU5 GS1 genes
ChloroplastMitochondria
Photorespiration
N primary assimilation
2-OGNH +
GLU GOGAT
GLU g
NH4+
GLUGaba
GDH
g
Amino acidsNH4
+
NH4+
GLU GLN
GS2
GS1
GS1Amino acids 3 AS genesGLN
NH4+GLU
Vacuole
N remobilisation
NO3-
NH4+
Uptake
AS ASN
Cytosol
N remobilisation
ATG autophagy genes
GS1 cytosolic glutamine synthetase AS asparagine synthetase
Masclaux-Daubresse Plant Biology 2008
Total N in seeds, seeds DW,yield, 15N enrichement
Autophagy mutants provided by Pr Y Ohsumi and Pr D Bassham
30%
40%
Harvest Index: DWSeeds/ DWWholePlant
Short days 40DAS42 DAS
15N labelling
Flowering
seeds
0%
10%
20%Long days
42 DAS
dry remains :
56 DAS
DW, total N, 15N enrichement
0%RNAi atg18
Col8 atg-5 atg-9.2
4 5
N concentration in seeds (N%)15NHI: 15Nseeds / 15N whole plant
2,5
3,5
4,5 15NHI: 15Nseeds / 15N whole plant
60%
80%
0,5
1,5
RNAi atg18
Col8 atg-5 atg-9.20%
20%
40%
RNAiCol8 atg 5 atg 9 2g RNAi atg18
Col8 atg-5 atg-9.2
Unpublished
Main take-home messages:
At vegetative stage, leaf to leaf N-remobilisation is correlated with leaf senescence.
At reproductive stage, leaf to stem/silique N-remobilisation is no more linked to leaf senescence
At seed filling stage15N remobilisation from rosette to seeds is mainly driven by the sink and source sizes ratio:
P%15N = a.HI + ==> Main QTL for N-remobilisation will certainly be QTL for HI
During grain fillingN-uptake benefits to vegetative organs at N+N uptake benefits to vegetative organs at N+ N-uptake benefits to seeds at N-==> N% in dry remains is much lower at N- than N+ while N% in seeds weakly decreased.
O 15N t l t N bili ti t d i f l f t t iOur 15N protocole to measure N-remobilisation to seeds is useful for mutant screening==> as a first result we found that autophagy mutants are inefficient at N-remobilisation
Masclaux-Daubresse et al. submited New Phytol.
Conclusion/Perspectives
Natural variation
15N labelling
N-remobilisation
variation
N remobilisationLeaf senescence
ArabidopsisFunctional
Analysis of MutantsNUE
improvement
QTL
At the END of plant cycle
stover seeds whole plant Partition in seeds%N DM (g) %N DM (g) %N DM (g) P% DM P% N P% 15N
10 mM 4,2 1,9 4,6 0,5 4,3 2,3 0,22 0,24 0,282 mM 1 0 6 3 5 0 2 1 5 0 8 0 23 0 5 0 482 mM 1 0,6 3,5 0,2 1,5 0,8 0,23 0,5 0,48
x4 x3 x1,3 x2,5 x2,8 x2,8 similar x0,5 x0,610mM/2mM
Seed quality is weakly affected10 mM favoured biomass and yield
But partitioning of DM not different
2 mM : favoured N storage in seeds
But partitioning of DM not different
and N remobilisation
(Means of all plants, ecotypes and experiments repeats)