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 Masclaux-Daubressemasclaux@versailles.inra.fr

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)