PhD Thesis proposal form
Discipline
Biology
Doctoral School
ED 145: Plant Sciences / Sciences du Végétal
http://www.ed-sciences-du-vegetal.u-psud.fr/en/ecoledoctorale.htm
Thesis subject title: Implication of Nitrogen Status for Plant Physiology and Metabolism:
Impact on Energy/Amino Acid Metabolisms and Biomass Production
 Laboratory name and web site:
o Metabolic Signalling and Regulation,
o Institute of Plant Biology (IBP),
o http://www.ibp.u-psud.fr
 PhD supervisor (contact person):
 Name: Bertrand Gakière
 Position: Associate-Professor
 email:bertrand.gakiere@u-psud.fr
 Phone number:+33 1 69 15 33 75
 Thesis proposal (max 1500 words):
Abstract
Plant growth and biomass production are strongly dependent on nitrogen availability in soils.
This explains why so many studies focused on nitrogen metabolism. However, interactions between
nitrogen status and primary metabolic pathways, such as amino acid metabolism together with
physiological processes such as respiration, are not as complete. In order to elucidate these
interactions and their dynamics, the present PhD project includes three parts, (i) characterization of
mutants exhibiting modified nitrogen status and disturbed respiratory metabolism, (ii) transcriptomic,
metabolomic and isotope-based fluxomic studies, (iii) physiological parameter measurements under
various environmental conditions.
Scientific context and goals
As sessile organisms, plants have adapted to environmental conditions that affect their
optimal growth and development. This adaptation relies on sensing and signalling mechanisms,
allowing plant organs to modify their physiology and morphology in response to various stimuli.
Nitrogen availability and plant nitrogen status are the most growth-limiting factors in crop
ecosystems (1). Once they are absorbed into plants, mineral nitrogen forms can be incorporated into
organic acids to produce amino acids. This step requires a tight coordination between nitrogen (N)
and carbon (C) metabolisms, which are under light and nitrogen status control (2). Aspartate,
asparagine, glutamate and glutamine are major organic nitrogen compounds. Not only are they
storage and transport forms for nitrogen in plants, but they are also precursors of many metabolic
pathways (3). Aspartate, whose synthesis is derived from respiration, is the precursor of three main
metabolic paths (4). The first leads to asparagine, a key compound used by plants to transport and
store organic nitrogen. The second produces pyridine nucleotides, among them is NAD (nicotinamide
adenine-dinucleotide) that plays a major role in redox recycling, a process that is central for many
physiological processes such as respiration (5). Aspartate is also a precursor for high nutritional value
amino acids such as lysine, threonine, isoleucine and methionine, which are also called “aspartatederived” amino acids. Among them, methionine is a metabolic hub at the crossroad of sulphur,
nitrogen, respiratory and photorespiratory metabolisms (6, 7).
The nutritional and metabolic plant status can be determined using metabolomic analyses.
When plants face non-optimal growth conditions, metabolic imbalances occur, and a reverse
correlation between sugars and amino acids levels can be measured. This is also observed for many
mutants whose metabolic functions are disturbed. In the case of a tobacco mitochondrial mutant
(called CMSII) produced in our laboratory, strong metabolic and physiological perturbations are
accompanied by a strong increase in free amino acid content, in particular that of asparagine and
arginine, and a strong decrease in free sugar levels (8, 9). These metabolomic changes are closely
correlated with high pyridine nucleotide levels, suggesting that the NAD pool exerts tight control on
carbon (C)/nitrogen (N) balance (8). We have recently initiated the study of Arabidopsis mutant
plants whose NAD synthesis, a major consumer of aspartate pool, was saltered.
Figure 1. Nitrate Reductase (NR) enrichment and decreased nitrate contents in an Arabidopsis mutant
line (Mutant N57) whose NAD synthesis, a major consumer of aspartate pool, has been altered. A, Leaf
NR activity. Col 0, wild type. N57, mutant. MgCl2, endogenous activity obtained with MgCl2. EDTA, maximal activity
obtained with EDTA. B, Leaf nitrate contents.
To date, analyses of these mutants revealed high aspartate and nitrogen-rich amino acid contents, a
sharp decrease in nitrate and a significant increased nitrate reductase activity (Figure 1). These
observations show that it is possible to modify the nitrogen status of plants in a targeted manner by
altering aspartate catabolism.
The present project aims to better understand the involvement of nitrogen status in
plant growth and physiology and to identify regulatory mechanisms coordinating carbon,
nitrogen and amino acid metabolisms.
Research project
A- Obtaining stable Arabidopsis lines that exhibit altered nitrogen status and/or disturbed
energetic metabolism. Obtaining Arabidopsis mutant lines with altered metabolism in a targeted
manner allows the measurement of the real impact of nitrogen and energetic status on plant
metabolism, and as a consequence on plant physiology and growth. The N57 line with altered
nitrogen status will be used to analyse consequences of internal N stimuli, while many studies so far
had considered the impact of a change resulting from an external nitrogen (nitrate) deficiency (10).
The use of a mitochondrial complex I double mutant of Arabidopsis will allow us to use genomic
tools and crossings with other available mutants to dissect the consequences of an altered energy
balance under various nitrogen status conditions; this is not feasible with the tobacco CMSII mutant
that has the same type of mitochondrial alterations. Other Arabidopsis lines recently available in our
laboratory, and showing constitutively huge NAD pools, will be used to analyse consequences of a
targeted increase of pyridine nucleotides on energetic and nutritional plant main functions.
B- Profiling of transcripts encoding enzymes of metabolic pathways involved in C, N and
amino acid metabolisms. Expression levels of genes encoding enzymes and transporters of targeted
metabolic paths will be measured using quantitative PCR. This will allow us to identify the genes that
are most highly regulated by metabolic alterations. These measurements will be performed on
different organs (leaves/roots) and under various environmental conditions (especially under stress
conditions). Transcript levels of marker genes will provide information on physiological state of the
analysed samples.
C- Functional consequences of targeted manipulations. Plants will be grown under various
environmental conditions, under nitrogen or sulphur deficiency for example. We will also examine
the impact of increased CO2 levels in a disturbed respiratory mutant. The physiological consequences
of the mutations will be evaluated by analyses of photosynthesis and respiration. Metabolomic
profiling will be performed by GC-MS, HPLC and LC-MS techniques available in our institute. The
experiments will be designed to directly compare metabolomic, transcriptomic and physiological
analyses. Gas exchange analyses and metabolomics will inform us about the overall carbon flow and
the levels of metabolites. Based on these results, we will analyse the flow into specific metabolic
pathways. For example, we can analyse the incorporation of inorganic N into amino acids in the
transgenic lines. This can be done using stable isotope enrichment and IR-MS analysis. An isotope
tracing of metabolic pathways of sulphur from labelled sulphate will also be considered. The choice
of three different markings (carbon, nitrogen, sulphur) will allow us to compare and refine the
obtained fluxomic results. In addition to transcriptomic and metabolomic tools that will provide a
"snapshot" of metabolic disturbances, biochemical studies and especially results from the isotopic
fluxome experiments will achieve a dynamic image, i.e. a "movie" of the general metabolism
changed in these plants in relation to their nitrogen status with changing interdependencies governing
their metabolism and physiology.
The project is planned for a timescale of three to four years. Training will be provided in all
techniques, all of which are being used routinely in our institute. Overall, the study will provide new
information to emerging novel concepts on metabolic adaptations to nutritional and energetic stimuli
in higher plants; this study will allow the student to gain competence in a range of key techniques
(qPCR, DNA chip, HPLC, GC-MS, LC-MS, isotopic IR-MS, Gas exchange, Bioinformatics).
References
(1) Masclaux-Daubresse C, Daniel-Vedele F, Dechorgnat J, Chardon F, Gaufichon L, Suzuki A
(2010).Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive
agriculture. Ann Bot 105, 1141-57.
(2) Faure J-D, Meyer C, Caboche M (1997). Assimilation du nitrate : nitrate et nitrite réductases. In
Assimilation de l’azote chez les plantes. Aspect physiologique, biochimique et moléculaire. Morot-Gaudry JF,
(ed). INRA Editions, pp.199-219.
(3) Morot-Gaudry J-F (1997). Synthèse des acides amines. In Assimilation de l’azote chez les plantes. Aspect
physiologique, biochimique et moléculaire. Morot-Gaudry JF, (ed). INRA Editions, pp.199-219.
(4) Azevedo RA, Lancien M, Lea PJ (2006). The aspartic acid metabolic pathway, an exciting and essential
pathway in plants. Amino Acids 30, 143-62.
(5) Noctor G, Queval G, Gakière B (2006). NAD(P) synthesis and pyridine nucleotide cycling in plants and
their potential importance in stress conditions. J Exp Bot. 57, 1603-1620.
(6) Gakière B, Ravanel S, Droux M, Douce R, Job D. (2000). Mechanisms to account for maintenance of the
soluble methionine pool in transgenic Arabidopsis plants expressing antisense cystathionine gamma-synthase
cDNA. C R Acad Sci III. 323, 841-51.
(7) Ravanel S, Gakière B, Job D, Douce R (1998). The specific features of methionine biosynthesis and
metabolism in plants. Proc Natl Acad Sci U S A. 95, 7805-12.
(8) Dutilleul C, Lelarge C, Prioul JL, De Paepe R, Foyer CH, Noctor G (2005). Mitochondria-driven changes
in leaf NAD status exert a crucial influence on the control of nitrate assimilation and the integration of carbon
and nitrogen metabolism. Plant Physiol. 139, 64-78.
(9) Gutierres S, Sabar M, Lelandais C, Chetrit P, Diolez P, Degand H, Boutry M, Vedel F, de Kouchkovsky Y,
De Paepe R (1997). Lack of mitochondrial and nuclear-encoded subunits of complex I and alteration of the
respiratory chain in Nicotiana sylvestris mitochondrial deletion mutants. Proc Natl Acad Sci U S A. 94, 343641.
(10) Gojon A, Nacry P, Davidian JC (2009). Root uptake regulation: a central process for NPS homeostasis in
plants. Curr Opin Plant Biol. 12, 328-38.
Publications of the laboratory in the field (max 5):

Pétriacq P, de Bont L, Hager J, Didierlaurent L, Mauve C, Guérard F, Noctor G, Pelletier S, Renou JP,
Tcherkez, G, Gakière B (2012). Inducible NAD overproduction in Arabidopsis alters metabolic pools and gene
expression correlated with increased salicylate content and resistance to Pst-AvrRpm1. Plant J Accepted
manuscript online: 23 JAN 2012 06:39AM EST | DOI: 10.1111/j.1365-313X.2012.04920.x.
Djebbar R, Rzigui T, Pétriacq P, Fresneau C, De Paepe M, Benhassaine-Kesri G, Streb P, Gakière B, Cornic G,
De Paepe R (2011). Respiratory complex I deficiency induces drought tolerance by impacting leaf stomatal
and hydraulic conductances. Planta DOI 10.1007/s00425-011-1524-7.
Guérard F, Pétriacq P, Gakière B, Tcherkez G (2011). Liquid chromatography/time-of-flight mass
spectrometry for the analysis of plant samples: a method for simultaneous screening of common cofactors or
nucleotides and application to an engineered plant line. Plant Physiol Biochem. 49,1117-25.
Mainguet SE, Gakière B, Majira A, Pelletier S, Bringel F, Guérard F, Caboche M, Berthomé R, Renou
JP.(2009). Uracil salvage is necessary for early Arabidopsis development. Plant J. 60, 280-91.
Wang X, Lopez-Valenzuela JA, Gibbon BC, Gakière B, Galili G, Larkins BA (2007).Characterization of
monofunctional aspartate kinase genes in maize and their relationship with free amino acid content in the
endosperm. J. Exp. Bot. 58, 2653-60.
 Specific requirements to apply, if any:
Basic level of competence in English or French. Masters degree in biochemistry and/or molecular
biology.