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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.html
Thesis subject title:
Dissection of the long distance signaling pathway associated to the PP2 phloem lectins
 Laboratory name and web site:
- Institut Jean Pierre Bourgin, Versailles [http://www-ijpb.versailles.inra.fr/en/index.htm],
- Group “Phloem, transport and Signaling” [http://wwwijpb.versailles.inra.fr/en/nap/equipes/Genomique_Phloeme/index.html]
 PhD supervisor (contact person):
 Name: Sylvie DINANT
 Position: Senior Scientist
 email: [email protected]
 Phone number: (33) 1 30 83 30 47
 Thesis proposal (max 1500 words):
Abstract: Phloem proteins 2 (PP2) are among the most abundant proteins in the phloem sieve
elements. PP2s present lectin activity, ability to bind to proteins and RNAs, and they move from cell
to cell. Moreover, several evidences suggest that PP2s behave like chaperones, acting on
macromolecules trafficking and in long distance signaling. In Arabidopsis two genes, PP2-A1 and
PP2-A2, specifically expressed in the phloem companion cell - sieve element complexes, encode
these proteins. The objectives of the PhD project are the precise the long distance signaling pathways
associated to the PP2s as well as the identification of the potential targets, protein and mRNA, with
which these proteins interact.
Introduction: In higher plants, the phloem tissue fulfills a multitude of functions. It transports
photoassimilates, especially sugars, from photosynthetic leaves to other organs. It also controls the
remobilization of nitrogen compounds and other nutrients accumulating in the leaves. Thus the
phloem integrates sink demands with respect to resource availability [1]. The phloem acts on interorgan coordination of many other physiologic and metabolic adjustments, in response to change in
the plant environment or to biotic and abiotic stresses [2]. For example it controls long-distance
signaling during developmental transitions such as flowering, systemic resistances against pests and
pathogens, or propagation of silencing throughout the plant. Phloem signals include a range of
metabolites, mineral nutrients, proteins, RNAs and hormones.
Long-distance transport occurs within living cells creating multicellular syncytia, the sieve
tubes, formed by a series of connected phloem sieve elements (SE) [3]. The differentiation of SE is
characterized by the degradation and removal of large cellular organelles, which could impede
assimilate flow. Mature SEs lack nuclei, vacuoles, Golgi bodies and ribosomes. The result of this
differentiation process is the formation of a continuous, membrane-lined compartment that provides a
pathway for nutrient flow and for signaling. Proteins and RNAs found in the SE are mostly
synthesized in the companion cells (CC), and then transported into SE via the plasmodesmata (PD),
specific cell wall openings that enable symplasmic continuity between plant cells [4].
Several models have been proposed for the translocation of these macromolecules from CC to
SE, via the PD. They distinguish mechanisms acting on targeted and non-targeted proteins through PD
(Fig. 1). They include 1) for non-targeted proteins a symplasmic transport depending exclusively on
the size of the protein and 2) for targeted proteins and RNAs, a transport through PD requiring the
activity of chaperones or lectins for unfolding during the passage through PD and shuttling and a
anchoring either to the plasma membrane or to the endoplasmic reticulum.
Figure 1. Models for the transport of macromolecules from CC to SE in the sieve elements
(CC: companion cells, SE: sieve element; PD: plasmodesmata; PM: plasma membrane, ER: endoplasmic
reticulum; TGN: trans golgi network)
We are interested in understanding the molecular mechanisms involved in the targetedtransport of macromolecules from CC to the SE. The dimeric phloem lectins, also known as PP2s, for
Phloem Proteins 2, have been proposed to be involved in the shuttling of glycoproteins from CC to
the SE [5, 6]. These proteins are synthesized in the CC then accumulate in the SE, therefore
presenting the characteristics of non-cell autonomous proteins (NCAP). The PP2 proteins have also
been shown to be RNA-binding proteins, suggesting that in addition to the shuttling of proteins from
companion cells to the sieve elements, they can also be part of large ribonucleoprotein phloem
complexes. PP2s accumulate in the SE as protein bodies, partially associated to the filamentous
phloem P-proteins [7]. We showed that PP2s bind to phloem sap proteins and are GlcNAc lectins [6].
In addition, binding properties of PP2s differ depending on their level of oligomerization, indicating
that the oligomerization status might be crucial for regulatory events. In plant cells, the role of
GlcNAc lectins and their oligomerization are still poorly understood. In animal cells, GlcNAc lectins
are involved in the nucleocytoplasmic shuttling through nuclear pores. It has been proposed that PP2s
could be involved in a similar mechanism acting in the phloem for cell-to-cell cytoplasmic shuttling
of macromolecules, proteins and RNAs, through plasmodesmata.
Objectives of the thesis: PP2 proteins, like other O-GlcNAc proteins binding-lectin, are proposed to
have a role in the shuttling of proteins and RNAs between phloem cells. In the plant model species
Arabidopsis thaliana, two genes, PP2-A1 and PP2-A2, specifically expressed in the phloem, encore
the phloem PP2 proteins. The developmental defects observed in PP2-A1 and PP2-A2 mutants
suggest that they target proteins and or RNAs involved in long distance signaling. The objectives are
to identify of the proteins and the RNAs present in the phloem sap requiring the PP2s for loading into
the translocation stream, and to determine the signaling pathways to which these macromolecules
participate.
Program:
1. Identification of the macromolecules present in Arabidopsis phloem sap
We previously showed that PP2-A1 interacts with phloem sap proteins and we carried out a first
identification of the phloem proteins present in the sap. We will use the same method to exudate
phloem sap and to analyze the population of RNAs present in the phloem sap and potentially moving
long distance for signaling. These RNAs will be analyzed using the CATMA microarrays.
2. Identification of the PP2 partners, proteins and RNAs, sin the phloem sap
We previously identified in the phloem sap of Arabidopsis more than 300 proteins, some of which
such as TCTP, FT or other transcription factors or RNA-binding proteins are potential targets of
PP2s. For the identification of PP2 protein partners, we will use transgenic lines that express PP2
with a GFP tag for co-immunoprecipitation studies, using either GFP or PP2 antibodies. Proteins
partners will be identified by mass spectrometry. In parallel we will analyze by LC_MC/MS the
changes in phloem sap proteome in transgenic lines in which PP2s are downregulated. This approach
should enable us to identify the proteins whose accumulation is affected in the PP2 mutants. The
identification of the RNAs interacting with PP2s will be realized by the technique of RIP-Chip
(RNA-Binding Protein Immunoprecipitation for Microarray) using Immunoprecipitation of the PP2
proteins. The RNAs interacting with the PP2s will be analyzed using the Arabidopsis CATMA
expression microarray. These analysis will allow the identification of macromolecules transported in
the phloem and requiring PP2 for cell-to-cell transport.
3. Identification of the signaling pathways based on PP2-shuttling
The phenotype of the mutants downregulated in the expression of PP2-A1 or PP2-A2 or both shows a
dramatic alteration of flowering time and plant architecture. These observations suggest that the long
distance signaling pathway to the shoot apical meristem is altered. A number of factors involved in
this developmental transitions, such as the transcription factor FT, the zinc finger CONSTANS or
the circadian oscillator GIGANTEA, have been characterized. Interestingly, those three genes are
expressed in the phloem. We will analyzed in details the relationships between PP2 and these factors,
as well as the one identified by the previous approaches, to identify the PP2 signaling pathway.
Work plan
1. Year 1: Analysis of the phloem sap transcriptome and proteome; Set up of
immunoprecipitation and RIP-Chip, Crossing between PP2 mutants and ft, constans, gigantea
and other mutants.
2. Year 2 : Experiences if immunoprécipitation and RIP-CHIP ; Identification of protein and
RNA partners, genetic analysis of the double mutants produced during year 1. Crossing
between PP2 mutants and mutants corresponding to the partners identified.
3. Year 3 Analysis of the phenotype of all transgenic lines (including crosses) ; characterization
of the protein and RNA partners and preparation of the scientific articles and PhD thesis.
Ongoing work on PP2 and international collaborations
Our group studies several families of phloem proteins, including PP2s. The PhD project is following
previous important work on PP2s, including PP2 subcellular localization, PP2 biochemistry and
structural analysis, characterization of several mutants affected in the expression of these genes. The
PP2 project will benefit from two major collaborations, one with William Lucas (University of
California, Davis – USA), and one with Gary Thompson (Pennsylvania State University– USA). In
particular, part of the pull-down studies will be realized with the support of W. Lucas’ group.
References
1 van Bel, A.J. (2003) The phloem, a miracle of ingenuity. Plant Cell Environ 26, 125-149
2 Turgeon, R. and Wolf, S. (2009) Phloem transport: cellular pathways and molecular trafficking. Annu Rev
Plant Biol 60, 207-221
3 Sjölund, R.D. (1997) The phloem sieve slement: a river runs through it. Plant Cell 9, 1137-1146
4 Lough, T.J. and Lucas, W.J. (2006) Integrative plant biology: role of phloem long-distance macromolecular
trafficking. Annu Rev Plant Biol 57, 203-232
5 Dinant, S., et al. (2003) Diversity of the superfamily of phloem lectins (phloem protein 2) in angiosperms.
Plant Physiology 131, 114-128
6 Beneteau, J., et al. (2010) Binding properties of the N-acetylglucosamine and high-mannose N-glycan PP2A1 phloem lectin in Arabidopsis. Plant Physiology 153, 1345-1361
7 Batailler, B., et al. (in press) Soluble and filamentous proteins in Arabidopsis sieve elements. Plant Cell
Environ
 Publications of the laboratory in the field (max 5):
Batailler B, Lemaître T, Vilaine F, Sanchez C, Cayla T, Beneteau J, Renard D, Dinant S. (2012) Soluble and
filamentous proteins in Arabidopsis sieve elements. Plant Cell and Environment Sous presse
Dinant S., Lucas W.J. (2012) in: G.A. Thompson, A.J.E. van Bel (Eds.), Biochemistry of Phloem. Chapter 8:
Sieve Elements: Puzzling Activities Deciphered by Proteomics Studies, Wiley & Sons, Cichester, UK
Beneteau J, Renard D, Marche L, Douville E, Lavenant L, Rahbé Y, Vilaine F, Dinant S (2010) Binding
properties of the N-acetylglucosamine and high-mannose N-glycan PP2-A1 phloem lectin in Arabidopsis.
Plant Physiology. 153:1345-1361
Bencharki B, Boissinot S, Revollon S, Ziegler-Graff V, Erdinger M, Wiss L, Dinant S, Renard D, Beuve M,
Lemaitre-Guillier C, Brault V (2010) Phloem Protein Partners of Cucurbit aphid borne yellows virus:
Possible Involvement of Phloem Proteins in Virus Transmission by Aphids. Molecular Plant Microbe
Interaction. 23(6):799-810.
Dinant S., Clark A.M., Zhu Y., Vilaine F., Palauqui J.C., Kusiak C. & Thompson G.A. (2003) Diversity of
the superfamily of phloem lectins (phloem protein 2) in angiosperms. Plant Physiology, 131, 114-128.
 Specific requirements to apply, if any:
A strong background in molecular biology and biochemistry is required.