Agrobacterium-tumefaciens mediated transformation of M. truncatula and expression studies of DOF1147

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Phylogenetic analysis of Dof proteins

The predicted 22 MtDof proteins were subjected to multiple sequence alignment using tree was reconstructed using CLC Sequence Viewer software (Version 6.7.1) with neighbour joining method and 1,000 replicates (Fig. 19). Based on the classification of Yanagisawa (2002), a total of seven major groups of Dof proteins were observed in the phylogenetic tree with the participation of MtDofs in all the groups.
The CrDof1 from Chlamydomonas being so far the only DOF found in an organism different from land plants displayed a distinct separate position in the phylogenetic tree. CrDof1 with some Dof proteins from moss are believed to be close to the origin of the evolutionary divergence of Dofs in higher plants (Shigyo et al., 2007). Up until now, there is limited information about the relationship between functional diversification and gene multiplication in various crop plants. However, the possibility that members in the same phylogenetic subgroup show redundant, overlapping or related functions cannot be ruled out.
A total of 7 paralogous proteins i.e. 3 from AtDof and 4 from MtDofs have been identified in Group II. Overall, 10 out of 22 MtDof proteins are classified in group II but none of them have yet been characterized for their physiological functions. Among them, MtDof5.2 and MtDof5.3, MtDof8.1 and MtDof0.2 as well as MtDof5.4 and MtDof5.5 are located in pairs close to one another in the phylogenetic tree and in view of their close similarity, probably reflect that they are paralogous. The evolution of these genes seems to be a result of recent duplication events. The sequence similarity among MtDofs located on chromosome 5 in close vicinity suggests that they are tendem repeats. On the other hand, the Arabidopsis group II Dof proteins are encoded by seven Dof genes i.e. AtDof2.3, AtDof1.5 (COG1), AtDof1.10, AtDof1.3, AtDof5.2 (CDF2), AtDof5.5 (CDF1) and AtDof3.3 (CDF3). The physiological functions of most of these genes have already been described. For example AtDof1.5 encoding COG1 is involved in the negative regulation of phytochrome signalling pathways (Park et al., 2003). AtDof5.5, AtDof5.2 and AtDof3.3 encoding CDF1, CDF2 and CDF3 proteins have been proposed to be involved in the regulation of photoperiodic dependent flowering (Imaizumi et al., 2005). Hence, Mt group II Dof domain proteins might be implicated in light regulated mechanisms, although the functional characterization of MtDof domain proteins will be necessary to fully support this possibility.
Group VII comprised of AtDof5.6 and its ortholog MtDof3.1, as well as all orthologs PBF proteins from barley (BPBF), wheat (WPBF) and maize (ZmPBF). In other studies, these PBF proteins have been shown to share a high degree of homology in their protein sequences and have also been reported to form a distinct phylogenetic group (Yanagisawa, 2002). The PBF proteins are specifically expressed during the grain filling stage and have been demonstrated to regulate the expression of seed storage protein genes in the endosperm of monocots by interacting with signature motifs present in their promoter regions (Vicente-Carbajosa et al., 1997, Mena et al., 1998). AtDof5.6, which encodes the HCA2 protein, has been shown to be expressed in the vascular tissues as well as in seeds and has been implicated in cambium formation and vascular tissue development (Guo et al., 2009) while its ortholog in M. truncatula i.e. MtDof3.1 has not yet been characterized.
Group III contains 16 Dof proteins, i.e. 10 from Arabidopsis, 4 from pea and 2 from M. truncatula. In this group, three paralogous pairs of AtDof proteins i.e. AtDof5.3 & AtDof3.2, AtDof3.7 & AtDof2.7 and AtDof4.6 & AtDof1.8 have been observed. The similarity in these proteins suggests that they might evolved as a result of gene duplications in A. thaliana. The AtDof3.7 and AtDof2.5 genes encode DAG1 and DAG2 proteins which have been involved in light-dependent seed germination (Gualberti et al., 2002). Recently, DAG1 has been shown to negatively regulate GA biosynthetic pathway during seed germination (Gabriele et al., 2010). The pea Dof genes PsDof1 and PsERDF encode elicitor-responsive Dof factors which are involved in the defence mechanism of plants (Seki et al., 2002). The Phylogenetic tree demonstrates that PsERDF pairs with MtDof2.5 which suggests that these are orthologs and have separated as a result of speciation.
Group V is quite heterogeneous, with 6 Dof proteins from Arabidopsis, 5 from M. truncatula and 3 from pea. The MtDof2.3 and MtDof2.4 in this group share homology with pea Dof proteins PsDof3 and PsDof6 respectively suggesting that these genes are orthologs and might have evolved in a legume specific manner. Group I contains only one legume Dof protein i.e. MtDof2.2. The MtDof2.2 encodes Dof protein also named as Dof1147 which has been shown to express in developing seed endosperm during the seed filling phase in M. truncatula. Group IV mainly contains the members of Arabidopsis Dof family with I from pea (PsDof5) and one from M. truncatula (MtDof0.1). Most Dof proteins in this group have not yet been characterized. Recently, AtDof1.7 from group IV has been shown to regulate the fatty acid metabolic pathway in A. thaliana seed (Yin et al., 2011).
Chapter 3 DOF identification & in silico promoter analysis Dof proteins. 22 Dof proteins from M. truncatula, 35 Dof proteins from A. thaliana, 8 Dof proteins from pea, 3 Dof proteins from wheat, barley and maize PBF, and one Dof protein from Chlamydomonas used as an outgroup. An unrooted tree is constructed by Neighbour-Joining method using CLC Squence Viewer (version 6.7.1). The resulting groups are indicated as I-VII. All the Dof protein sequences are used for conserved domain and duplication analysis using online MEME server. 26 conserved domains of different lengths are depicted on the protein map next to the phylogenetic tree. While the motif codes are indicated below the tree and the respective sequences of the domains are shown in Table 5.

Analysis of conserved domains and gene duplications

The complete sequence of Dof proteins used for construction of phylogenetic tree was subjected to MEME server for analysis of conserved domain and duplication events during the evolutionary process. After following the parameters described in M&M (cf. § 2.2.1.7), a total of 26 different conserved domains have been identified in the 69 Dof sequences (Table 6). The identified domains ranged from 8 to 52 residues in length (Table 6).
Some of the groups shown in phylogenetic tree contain single or double conserved domains, while others may contain multiple conserved domains. Group IV and VII contain 2 conserved domains each while I and III contain 4-5 conserved domains of different residues. Maximum number of conserved domains was observed in group II i.e. 10-12 conserved domains.
All Dof proteins in group II, either MtDofs or AtDofs, contain 22 amino acid domain at the extreme N terminal region near the Methionine (Motif 4 in Table 6) apart from Dof binding domain. The MtDofs in this group (i.e. MtDof4.2, MtDof5.2, MtDof5.3, MtDof5.4, MtDof5.5, MtDof6.1 and MtDof7.1) share the 11 amino acids long conserved domain (Motif11: PNSPTLGKHSR) near the C-terminal of the protein. Interestingly, MtDof5.2, MtDof5.3, MtDof5.4 and MtDof5.5 genes are localized on chromosome 5 of M. truncatula and, present in close vicinity, are likely to share the same loci, as is evident from Table 6. When further investigated, these proteins contain a highly conserved 22 amino acid sequence EHDAKRYKETSRKELTSVLDY (Motif 18 in Table 6) upstream of the Dof domain, as well as motif 6 and 8. The presence of multiple conserved domains in MtDof5s proteins suggests that the genes encoding these proteins originate from the same duplication events on the same chromosome.
In group I, the AtDof proteins share multiple conserved domains (Motif 14, 22, 26 in Table 6) with each other. These genes encoding these proteins are also localized on the same chromosomes. In M. truncatula, 9 Dof proteins i.e. MtDof0.2, MtDof4.2, MtDof5.2, MtDof5.3, MtDof5.5, MtDof6.1, MtDof7.1, MtDof7.3 and MtDof8.1 share the 43 residues conserved domain (motif 3) near the C terminal region of Dof proteins as well as motif 4 at the terminal region of the respective proteins. The presence of conserved domains among different Dof proteins especially in the same group is likely to reflect gene duplication events during evolution and their related functions. Recently, gene expression studies in A. thaliana have revealed that most of the closely related genes in the Dof phylogenetic tree had redundant expression patterns in the different tissues (Moreno-Risueno et al., 2007).

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In silico motif analysis of putative target gene promoters

Using Affymetrix, 195 genes have been isolated whose expression has increased during seed development in the EMS109 dof-mutants in our research unit (M. Noguero and J. Verdier, pers. comm.). Among them, 34 overexpressed genes were identified having expression more than four times. Based on Affymetrix codes, the sequences of 29 of them were retrieved from M. truncatula genome. Using the CLC Sequence Viewer, the 2kb upstream promoter region from transcription start site of these genes were isolated. All the motifs for attachment of TFs to the promoters are generally present in this 2kb upstream region. The dataset was then subjected to PLACE (Higo et al., 1998, 1999) and PlantCARE database (Lescot et al., 2002) for identification of already experimentally described motifs. The analyses have shown that the promoter regions of all the over as well as under-expressed genes contain multiple copies of the basic elements required for promoter identity i.e. TATA-box and CAAT-box (Casimiro et al., 2008) and recruited by basal transcription apparatus before the start of transcription. In order to evaluate the significance of predicted motifs in the promoter regions, a controlled dataset was prepared by randomly choosing the thirty sequences of 2kb each. These control sequences were also subjected for analysis using PlantCARE and PLACE.

Table of contents :

Chapter 1: General Introduction
1.1 Legume Family
1.1.1 Characteristics of legumes
1.1.1.1 Impact on soil and environment
1.1.1.2 Impact as food and feed
1.1.2 Protein sector in Europe
1.1.3 Legume status in France
1.1.4 Constraints for lower production
1.1.5 Need for legume improvement
1.1.6 Need for a model plant
1.2 Medicago truncatula as a model legume
1.2.1 Biological and agronomic interests
1.2.2 Synteny with legumes
1.2.3 Availability of genomic and bioinformatics tools
1.3 Seed development
1.3.1 What is seed development
1.3.1.1 Cell division phase
1.3.1.2 Seed filling and maturation phase
1.3.1.3 Dehydration phase
1.3.1.4 Proteomics of seed development
1.4 Transcriptional control of Seed development
1.4.1 Plant transcription factors
1.4.2 Transcriptional regulation of seed development
1.4.3 Role of master regulators during SSP accumulation
1.4.4 Regulatory network of seed maturation
1.4.5 Repressors of maturation-related genes
1.4.6 DOF transcription factors
1.4.6.1 Structure of DOF proteins
1.4.6.2 Role of DOF in seed filling
1.5 Hormonal control of seed development
1.5.1 Role of Auxins
1.5.2 Role of Abscisic acid
1.5.3 Role of Gibberellins
1.5.4 Role of Cytokinins
1.6 Stable genetic transformation and functional genomics
1.6.1 Gene transfer methods
1.6.1.1 Agrobacterium-mediated gene transfer
1.6.1.1.1 Agrobacterium rhizogenes
1.6.1.1.2 Agrobacterium tumefaciens
1.6.1.2 Direct gene transfer
1.6.1.2.1 Electroporation
1.6.1.2.2 Biolistics
1.6.2 Gene transfer in Medicago truncatula
1.7 Flow cytometry in plant breeding
1.8 Rationale of the thesis
1.9 Scheme of work
Chapter 2: Materials and Methods
2.1 Materials
2.1.1 Plant material and growth conditions
2.1.2 Cloning vectors, bacterial strains and primers
2.1.2.1 Plant expression vectors
2.1.2.1.1 PMDC 83 vector
2.1.2.1.2 PMDC 107 vector
2.1.2.1.3 pDONR221 vector
2.1.2.2 E coli strains
2.1.2.3 Agrobacterium tumefaciens strains
2.1.2.3.1 LBA4404:
2.1.2.3.2 EHA105:
2.1.3 Laboratory chemicals and consumables
2.1.4 Solutions, media, buffers and antibiotics
2.1.4.1 Stock Solutions
2.1.4.2 Media
2.1.4.3 Buffers
2.1.4.4 Antibiotics and selection agents
2.2 Methods
2.2.1 In silico and bioinformatic analysis
2.2.1.1 Sequence data collection
2.2.1.2 Identification of Dof family members from M. truncatula genome
2.2.1.3 Sorting of identified DOF proteins
2.2.1.4 Mapping of MtDof genes on chromosomes
2.2.1.5 Comparison of conserved residues in the Dof DNA-binding domain
2.2.1.6 Construction of Phylogenetic tree
2.2.1.7 Identification of conserved domains and duplications in Dof proteins
2.2.1.8 Analysis of cis-regulatory elements in putative target gene promoters
2.2.1.9 In silico prediction of DOF1147 subcellular localization
2.2.2 Preparation of Constructs
2.2.2.1 Primers designing
2.2.2.1.1 Calculation of annealing temperature by temperature gradient PCR
2.2.2.1.2 Designing of AttB-PCR primers
2.2.2.1.3 Amplifying attB-PCR Products
2.2.2.1.4 Agarose Gel Electrophoresis
2.2.2.1.5 Separation and purification of amplified attB-PCR Product
2.2.2.2 BP Recombination Reaction
2.2.2.2.1 Transformation of entry clone into E. Coli
2.2.2.2.2 Entry clone verification by PCR
2.2.2.2.3 Plasmid preparation from E.coli colonies
2.2.2.2.4 Sequencing of entry clones
2.2.2.3 LR Recombination Reaction
2.2.2.3.1 Expression clone verification and sequencing
2.2.3 Manipulations with Agrobacterium tumefaciens
2.2.3.1 Preparation of competent A. tumefaciens
2.2.3.2 Transformation of Agrobacteria by heat shock
2.2.3.3 Transformation of electro-competent Agrobacteria
2.2.4 Stable transformation of Medicago truncatula
2.2.4.1 Preparation of Agrobacterium culture
2.2.4.2 Preparation of explants
2.2.4.3 Inoculation of explants
2.2.4.4 Co-cultivation of explants
2.2.4.5 Callus induction and somatic embryogenesis
2.2.5 Analysis of putative transformants
2.2.5.1 Flow cytometric analysis of putative transformants
2.2.5.2 Molecular verification of putative transformants
2.2.5.2.1 Genomic DNA extraction
2.2.5.2.2 Verification of transformants by Polymerase Chain Reaction
2.2.5.3 Germination and transgene segregation test in the progeny
2.2.6 Transient expression assays
2.2.6.1 Transient expression in Nicotiana tabacum through agro-infiltration
2.2.7 Microscopic studies
2.2.7.1 Laser scanning confocal microscopy
2.2.7.2 Fluorescence microscopy
Chapter 3: Genome wide identification of DOF transcription factors in M. truncatula and in silico promoter analysis
3.1 Introduction
3.2 In-silico subcellular localization of DOF1147
3.3 Genome wide identification of MtDof family members
3.4 New annotations of M. truncatula Dof genes
3.5 Mapping MtDOF genes on M. truncatula chromosomes
3.6 Comparison of DNA-binding Dof domains of MtDofs
3.7 Phylogenetic analysis of Dof proteins
3.8 Analysis of conserved domains and gene duplications
3.9 In silico motif analysis of putative target gene promoters
Chapter 4: Agrobacterium-tumefaciens mediated transformation of M. truncatula and expression studies of DOF1147
4.1 Introduction
4.2 Results
4.2.1 Use of Gateway cloning technology for preparation of constructs
4.2.1.1 Preparation of promoter reporter construct (pdof::gfp)
4.2.1.2 Preparation of translational fusion construct (p35S::dof-gfp)
4.2.2 Transformation of expression clones into A. tumefaciens
4.3 Genetic transformation of Medicago truncatula
4.3.1 Generation of pdof::gfp cassette containing plants
4.3.1.1 Verification of pdof::gfp cassette at molecular level
4.3.2 Generation of 35S::dof-gfp cassette containing plants
4.3.2.1 Verification of 35S::dof-gfp cassette at molecular level
4.3.3 Comparison of transformation efficiency
4.3.4 Flow cytometric characterization of transformants
4.3.5 Phenotyping of transformants
4.3.6 Transgene segregation in the progeny
4.4 Laser scanning confocal microscopy
4.4.1 Subcellular localization of Dof1147
4.4.2 DOF1147 subcellular localization studies using Agroinfiltration
4.4.3 Expression of Dof1147 during seed development
Chapter 5: Role of auxin in in vitro seed development in M. truncatula
5.1 Introduction
5.2 In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula.
5.2.1 Abstract
5.2.2 Introduction
5.2.3 Materials and methods
5.2.3.1 In vitro culture
5.2.3.2 Determination of seed size, fresh weight and viability
5.2.3.3 Flow cytometry
5.2.4 Results
5.2.4.1 Development on auxin-free medium (MS130)
5.2.4.2 Effect of auxin on seed size
5.2.4.3 Effect of auxin on seed fresh weight
5.2.4.4 Flow cytometry of in vitro developing immature seeds
5.2.5 Discussion
Chapter 6: General Discussion
6.1 Construction of plant transformation vectors using Gateway technology
6.2 Stable genetic transformation of M. truncatula
6.3 Genome-wide identification of MtDOF TFs and their phylogenetic analysis
6.4 Expression of Dof1147 in the seed endosperm
6.5 Subcellular localization of Dof1147 protein
6.6 Role of auxin in seed development
Conclusions and Perspectives

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