Localization of molecular players in tension wood fibers of simarouba in comparison to tension wood fibers of poplar

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Molecular players potentially important for the G-layer properties

As presented in the previous chapters, the G-layer is characterized with particular properties, which are thought to be related to a specialized function in the generation of tensile stress (Mellerowicz and Gorshkova, 2012; Chang et al., 2015; Guedes, 2013). AGPF laboratory at INRA Orleans aims in deciphering a relation between molecular properties and generation of tensile stress in the G-layer. To date, the research involved studies of transcriptional changes in TW (Déjardin et al., 2004), lignin (Pilate et al., 2004), fasciclin-like arabinogalactan proteins (Lafarguette et al., 2004), monosaccharide composition in the G-layer and polysaccharide distribution along differentiating TW fibers (Guedes, 2013). The latest research, partially conducted within the project Stress In Trees, particularly approached in the identification of molecules potentially responsible for the G-layer properties. This thesis was conducted within the same project with the aim to continue in identification of molecular players in the G-layer. Research performed in frame of the thesis has put in focus only several molecular players – beta-galactosidase, fasciclin-like arabinogalactan proteins and chitinase-like protein. Further chapters have the aim to justify their choice according to current findings about their putative functions in plants, particularly during xylem development.

Beta-galactosidase

Beta-galactosidase (BGAL) is an enzyme potentially responsible for the modification of RG-I pectin in the G-layer, which was detected as progressive decrease of immunosignal of beta-(1,4)-galactan side chains and increase of RG-I backbone signal (Guedes, 2013). Debranching of RG-I may be related with a gel formation in the G-layer, since the action of beta-galactosidase (AtBGAL6) on side chains of RG-I pectin was shown to increase its hydrophilic potential necessary for the seed hydration (Macquet et al., 2007). Moreover, an epitope of beta-galactosidase was localized in the G-layer of poplar and G-type walls of hemp phloemic fibres (Mokshina et al., 2012). In addition, in situ incubation of poplar xylem sections with Gal-resorufin revealed higher beta-galactosidase activity in the cell walls of TW than in NW (Gorshkova et al., 2015).
Flax develops phloemic fibers, which have remarkable similarities with the G-fibers of tension wood. Remodelling of phloemic G-fibres during which galactan-rich layer (Gn-layer) turns to cellulose-rich layer (G-layer) involves hydrolysis of high-molecular mass galactans. The modification is related to beta-galactosidase enriched within developing flax fibres, localized in secreted Golgi vesicles and gelatinous SCWs, and encoded by LuBGAL1 and/or LuBGAL2 (Roach et al., 2011; Mokshina et al., 2012). Down-regulation of LuBGAL1 and LuBGAL2 increased the content of cell wall-associated galactans, decreased the ability of Gn- conversion to G-layer and reduced cellulose crystallinity and stem strength (Roach et al., 2011).
Beta-galactosidases (BGAL) are involved in the hydrolysis of terminal non-reducing beta-D-galactose residues (CAZy.org). In plants, they are found as a part of isofunctional families of glycoside hydrolases GH35 and GH42. Beta-galactosidase families were predicted in various plant species as Arabidopsis, rice, tomato, flax and chickpea (Hobson and Deyholos, 2013).
Beta-galactosidases have putative function in the cell wall biogenesis and remodelling, degradation of cell wall components during cell expansion, cell senescence, fruit ripening and storage mobilization (Martin et al., 2013). The activity of beta-galactosidase is highly correlated to the formation of secondary cell walls. -(1,4)-galactosidase activity was partially detected in the innermost secondary cell walls of sclerenchyma cells in Arabidopsis (Banasiak et al., 2014). Several BGALs were identified as highly-expressed in wood-forming xylem of poplar (Aspeborg et al., 2005). Transcripts of one BGAL were enriched in the transition between elongation and secondary cell wall deposition in hypocotyl of flax (Roach and Deyholos, 2008).
Cell wall targets of beta-galactosidase activity may be carbohydrates RG-I pectins, galactomannans and xyloglucans; arabinogalactan proteins and galactolipids (Ahn et al., 2007). Based on recombinant protein activity, members of the largest a1 subgroup from Arabidopsis were functionally characterized as cell wall exo-galactanases, with activities toward β-(1,4)- and β-(1,3)-galacto-oligosaccharides, except for AtBGAL12, which also cleaved β-(1,6)-galactosyl substrates (Ahn et al., 2007; Gantulga et al., 2008; Gantulga et al., 2009). Interestingly, LusBGAL1 and LusBGAL2 are homologous to this group. BGAL from radish (Raphanus sativus), also homologous to the a1 subgroup, was shown to specifically cut beta-(1,3)-galactosyl and beta-(1,6)-galactosyl residues of arabinogalactan protein, suggesting that structurally similar beta-galactosidases may have a specific function in different plant species (Kotake et al., 2005).

Fasciclin-like arabinogalactan protein

Arabinogalactan proteins are glucuronic acid carriers, which potentially interact with negatively charged galacturonic acid of pectins (Lamport et al., 2014). Interactions with AGPs decrease pectin alignment and crosslinking in the cell wall and increase the porosity of pectin network (Lamport et al., 2005). In primary cell walls of Arabidopsis, Tan et al. (2013) evidenced the covalent link of AGPs to pectins and arabinoxylans in a complex structure. The interactions of AGPs with RG-I pectin may therefore influence the mesoporosity changes detected during the G-layer development (Chang et al., 2015). AGPs responsible for the gel structure would likely be retained between cellulose microfibrils, as detected for type II arabinogalactans in tension wood of poplar (Gorshkova et al., 2015).
Fasciclin-like arabinogalactan proteins (FLA) belong to a subgroup of AGPs with fasciclin domain (Fig. 10). It has been firstly reported in Drosophila melanogaster as a domain involved in cell adhesion (Gaspar et al., 2001). However, the exact function of FLA domain in plants is not known.
From the comparison of EST distribution and expression analysis in poplar, expression of FLA was detected in TW (Déjardin et al., 2004) and ten FLA genes were reported as differentially expressed in TW (Fig. 11; Lafarguette et al., 2004). Several FLAs were also found up-regulated in TW of eucalyptus (Mizratchi et al., 2015).
MacMillan et al., 2015 raised an idea of conserved sub-group A of FLAs, which is involved in secondary cell wall development and important for stem mechanical properties. Moreover, several reported mutant phenotypes (listed in the following text) indicate the group’s involvement in the orientation of cellulose microfibrils. FLAs are prominent candidates for a role in control of the axial orientation of cellulose microfibrils in the G-layer (MacMillan et al., 2015). In Arabidopsis, the double knockout of the group-A members FLA11 and FLA12 did not affect stem cell morphology, but it decreased polysaccharide and increased lignin content, increased microfibril angle and decreased stem tensile strength and stiffness (MacMillan et al., 2010). Several FLAs from eucalyptus, homologous to the group-A, were highly expressed in stems and overexpression of one (EgrFLA2) led to 3-fold reduction of CMF angle (MacMillan et al., 2015). GhFLA5 from cotton, which has common structural characteristics with the group-A, is highly expressed in fibers and was proposed to contribute to fiber strength by affecting cellulose synthesis and orientation of microfibrils (Liu et al., 2013a). In poplar, modification of TW-specific PtaFLA6 reduced stem flexural strength for 10-12% and flexural stiffness for 19-23%, reduced lignin and crystalline cellulose content, affected the expression of nine FLA homologous genes and genes involved in lignin and cellulose synthesis (Wang et al., 2015).
FLAs were also shown to have function other than in secondary cell wall development. AtFLA1 is involved in the early stages of lateral root and shoot development in tissue culture (Johnson et al., 2011). AtFLA3 was reported as involved in microspore development and cellulose deposition during pollen formation (Li et al., 2010). GhFLA1 from cotton is involved in fibre initiation and elongation through the modulation of the biosynthesis of primary cell wall components (Huang et al., 2013).

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Table of contents :

1. GROWING INTEREST FOR WOOD
1.1. Poplar as a model for wood studies
1.2. Wood properties
1.2.1. Formation and structure
1.2.2. Chemical composition
2. TENSION WOOD
2.1. Modulation of the tree orientation
2.2. The G-fiber
2.3. Peculiar properties of the G-layer
2.4. Molecular players potentially important for the G-layer properties
2.4.1. Beta-galactosidase
2.4.2. Fasciclin-like arabinogalactan protein
2.4.3. Chitinase-like protein
3. TOWARDS A MODEL OF THE TENSILE STRESS GENERATION
Objectives and strategy
MATERIALS & METHODS
I. Plant material
1.1. Poplar
Plant origin and manipulations
Sampling for diverse use
Acclimatization and sampling of transgenic lines for screening
Acclimatization and sampling of selected transgenic lines
1.2. Genetically modified poplars
Production of RNAi vectors
Plant transformation and regeneration
Selection of transgenic lines
1.3. Simarouba
II. Bioinformatic analyses
Alignments
Construction of primers
Phylogenetic analysis
III. Transcriptomic analyses
3.1. RNA isolation
3.2. RT-PCR
3.3. RT-qPCR
IV. Beta-galactosidase activity
4.1. Localization with X-Gal substrate
4.2. Quantification with MUG substrate
V. Wood characterization
5.1. Wood anatomy
Safranin/Alcian-blue staining
5.2. Immunohistolocalization
Fixation and embedding
Methylene blue/Azur II staining
Epitope detection
5.3. Wood chemical properties
Middle Infrared spectroscopy
5.4. Cellulose ultrastructure
X-ray diffractometry Identification and characterization of molecular players in tension wood of poplar
1. Article: CHITINASE-LIKE PROTEIN
2. FASCICLIN-LIKE ARABINOGALACTAN PROTEIN
2.1. FLA family
2.1.1. Phylogenetic analysis
2.1.2. Expression analysis in wood
2.2. Characterization of FLA1 and FLA14
2.2.1. Expression profile of FLA1 and FLA14
2.2.2. Production of FLA1 and FLA14 transgenic lines
2.2.3. Traits of FLA1 and FLA14 transgenic lines
2.3. Discussion
3. BETA-GALACTOSIDASE
3.1. Beta-galactosidase activity
3.1.1. Localization with X-Gal substrate
3.1.2. Quantification with MUG substrate
3.2. BGAL family
3.2.1. Phylogenetic analysis
3.2.2. Semi-quantitative expression analysis in OW and TW
3.2.3. Quantitative expression analysis in NW, OW and TW
3.3. Characterization of BGAL7
3.3.1. Expression profile of BGAL7
3.3.2. Production of BGAL7 transgenic lines
3.3.3. Traits of BGAL7 transgenic lines
3.4. Discussion
Localization of molecular players in tension wood fibers of simarouba in comparison to tension wood fibers of poplar
4. SIMAROUBA
4.1. Immunolocalization of RG-I and AGP structures in TW fibers of simarouba
4.2. Comparison of immunolocalization between S-G layer of simarouba and G-layer of poplar
4.3. Discussion
GENERAL DISCUSSION

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