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Functional investigation of Arabidopsis
Fasciclin-Like Arabinogalactan-protein 16 (FLA16)
Previous studies revealed FLA11 and FLA12 are highly expressed in the stem (Schmid, 2005; Toufighi, 2005; Winter et al., 2007) (see Appendices Fig. A4.1, 4.2). Knockout mutants for Arabidopsis FLA11 and FLA12 were investigated for differences in the biomechanical properties, cell wall architecture and carbohydrate composition of the stems. Investigation of fla11 and fla12 single mutants did not reveal any major differences whereas fla11fla12 double mutants showed a reduction in tensile strength and stiffness, increased cellulose microfibril angle and reduced cellulose, galactose and arabinose content (MacMillan et al., 2010). This suggested FLA11 and FLA12 contribute to the maintenance of stem biomechanical properties via regulation of wall architecture and/or biosynthesis (MacMillan et al., 2010). Overexpression of Eucalyptus FLA2, orthologue of Arabidopsis FLA11/FLA12, resulted in a reduction of microfibril angle (MFA) by three degrees in the Eucalyptus xylem fibres, and the heterologous overexpression of Eucalyptus FLA3, orthologue of Arabidopsis FLA11/FLA12, in tobacco caused reduced flexural strength in the stem (MacMillan et al., 2015). Furthermore, suppression of Populus FLA6 (orthologue of Arabidopsis FLA11/FLA12) transcripts using an antisense RNAin Populus led to a reduction of flexural strength and stiffness in the stem, suggesting Populus FLA6 influences the biomechanics of the Populus stem (Wang et al., 2015).
Despite differences in the biomechanical properties in stems, no overall change in plant morphology was observed in fla11fla12 mutants suggesting that further redundancy with other FLA members might occur. Amongst all the FLAs, a member in group B, FLA16 (Fig. 1.6), is proposed to be a good candidate given it is the most highly expressed FLA in the stem after FLA11 and FLA12 (MacMillan et al., 2010). Based on the Arabidopsis eFP browser FLA16 is almost exclusively expressed in the stem (Schmid, 2005; Toufighi, 2005; Winter et al., 2007) (see Appendices Fig. 4.3). Unlike FLA11 and FLA12, FLA16 is predicted to contain two FAS domains and is not predicted to be GPI-anchored (Johnson et al., 2003). The biological roles of FLA16 in Arabidopsis are unknown and this research aims to determine if FLA16 plays a role in stem development. In this chapter the impact of loss of FLA16 is explored using a fla16 mutant.
4.2.1 Investigation of FLA16 expression in Arabidopsis
To confirm the levels of FLA16 transcript abundance, Q-PCR was undertaken using a range of Arabidopsis tissues. These included the flower, silique, cauline leaf, rosette leaf, branch and the top, middle and basal segments of the stem (Fig. 4.1A).
Transcripts of FLA16 were found in all tissues investigated. The highest expression was observed in RNA extracted from all branches, followed by siliques, flower, stem and root, but was barely detectable in the cauline and rosette leaves (Fig. 4.1A).
4.2.2 Identification and characterisation of a fla16 mutant
A T-DNA insertion in the intron of the FLA16 gene was identified in the Arabidopsis SALK collection (SALK_131248) (Fig. 4.1B). The insertion in FLA16 was confirmed using PCR genotyping and Q-PCR of FLA16 transcripts in the stem tissue. The expression levels of FLA16 in the fla16mutant was about one-sixth of the wild-type (wt) counterpart suggesting this was a severe knock-down mutant line (Fig 4.1C). Therefore, this fla16 mutant line was investigated further to determine if it showed phenotypic differences in growth and development compared to wt plants.
4.2.3 Growth is delayed in the fla16 mutant
The timing of specific growth stages was analysed in fla16 compared to wt plants according to the method of Boyes et al. (2001). The emergence of even-numbered rosette leaves, root growth, the timing of bolting and appearance of the first flower were assessed (Table 4.1). Between seedling stages 0.5-1.0 (Boyes et al., 2001), a delay in root growth was observed in fla16 compared to wt. During early root growth, between day 2 to 5, a significantly shorter root length was observed in fla16 mutants (Table 4.1). At later stages (days 3-5) of root growth a reduced root length in fla16 was shown to result from the slower growth rate between days 1-3 with no significant difference in growth rate between days 3 to 5 (Table 4.1).
Counting analysis of rosette leaf number was carried out from the first true leaf until the first flower emerged. No significant differences between fla16 and wt occurred in rosette leaf development from the 2nd to the 9th leaf. Compared to wt, at the completion of rosette development fla16 had fewer rosette leaves, earlier bolting time of 3 days but showed no difference in the time of first flower emergence (Table 4.1).
4.2.4 Reduced stem height and transverse area was observed in fla16 mutants
Due to the expression of pFLA16:FLA16-VH in fibre cells in stems/branches (see Chapter 3, Fig. 3.8, 3.9), the stem height and area of fla16 mutants was of interest. Height of the total stem and first internode were measured in plants reached post-maturity (growth stage 6.9) grown in long day conditions (Fig 4.2). For area measurements, the base and first node positions were chosen as two developmentally comparative regions. The height of first internode versus total stem height and total transverse area at the base versus first node were used to assess if differences in the proportions of stem growth occurred.
The fla16 mutant was shown to develop a shorter stem with a significant reduction in the height of both the total stem and first internode. The average total stem height of fla16 mutants was reduced by 25%, and the reduction of first internode was by 54%, compared to wt. The ratio of the first internode to total stem height showed that the first internode of fla16 was proportionally shorter than wt (Table 4.2).
In addition to changes in the stem height, fla16 mutants have thinner stems. The average stem area of fla16 mutants was reduced by 14% and 31% at the base and first node, respectively, compared to wt. The ratio of the first node to basal area of fla16 () was also found to significantly differ from wt (Table 4.2).
4.2.5 The fla16 stem has morphological differences to wt
Histological analyses of transverse and longitudinal sections of the stem (growth stage 6.5) were undertaken to determine if cellular differences occur in fla16, which may contribute to the reduced stem height and area.
Transverse sections of fresh tissue (~0.5mm) and chemically fixed and resin embedded (~500 µm) stems of fla16 were obtained at the base and first node. Toluidine blue staining for morphological examination did not reveal obvious differences in cellular organisation and shape compared to wt. Mäule staining for distribution of lignin also did not show obvious differences between fla16 and wt (Fig. 4.3, 4.4).
To investigate the changes in stem area further, the stem was divided into three regions namely, cortex, pith and intra-vascular region (see Appendices Fig. A4.4) consisted of interfascicular fibre, phloem, cambium and xylem cells. Cell size analysis was undertaken for epidermis, cortex, interfascicular fibre, xylem vessel and pith using transverse sections. To assess changes in cell populations, total cell count was performed for the pith, interfascicular fibre and xylem vessel cells (Table 4.3).
Analyses of the tissue area revealed that pith area was significantly reduced in the base (30%) and first node (38%) of fla16 compared to wt. The total number of pith cells taken from the base and first node revealed fla16 had significantly fewer pith cells than wt. In addition, the fla16 stem showed a significantly reduced intra-vascular region area (32%) at the first node but not base, which likely resulted from a reduced interfascicular fibre cell population. Nevertheless, there were no significant changes observed in the cortex region at both stem positions (Table 4.3).
Analysis of cell size in the stem transverse sections showed fla16 had significantly smaller pith cells in the first node but not the base. Other cell types examined, including epidermal, xylem vessel and interfascicular fibre cells, did not show statistically significant differences in size at both stem positions. This suggested that the reduction in pith cell size and number in fla16 compared to wt is the major cause of the reduced stem area (Table 4.3).
Longitudinal sections at the base and first node of the stem were examined to determine if morphological differences could be observed in fla16 compared to wt. No significant differences in the length of epidermal, and pith cells were observed (Fig. 4.5A). Investigation of the angle of the first branch in relation to the stem also showed no differences between fla16 and wt (Fig. 4.5B).
The morphological differences in the fla16 stem indicated potential alterations in the stem functionality such as biomechanical properties.
4.2.6 The fla16 stem has altered biomechanical properties
Cellulose is the major structural component of the cell wall and provides rigidity and strength (Doblin et al., 2010). How cellulose is deposited and integrated into the wall can therefore influence the walls biomechanical properties. To assess if the reduction of cellulose affects the strength of fla16 stems, biomechanical tests were carried out for the basal- and mid- stem segments, whereas the top of the stem was excluded due to the reduced stem height. Differences in the hydration status of stems can influence the biomechanical property measurements leading to more variable results, therefore dry stems were chosen for testing (Molina-Freaner et al., 1998). Flexure and tensile tests for the strength and stiffness, respectively, were undertaken. The strength indicates the maximum load required to break the stem whereas stiffness is a measure of the elasticity the stem (MacMillan et al., 2010). In additional, a ‘break’ profile was included to evaluate the response of stems under load. This allowed calculations of which stems underwent a sharp break and those without a sharp breakage. Since the stems were not of uniform shape, three flexure analytical models were compared covering the major geometrical shapes of the stem: circular, oval and rectangle (see Appendices Fig. 4.5). The circular model was chosen as the best model for tensile analyses according to Dr. MacMillan.
Flexure tests revealed that the basal stems of fla16 have significantly reduced strength (34.3%) compared to wt. None of the fla16 samples yielded a break profile compared to wt stems. No significant differences in flexure tests of the mid-stems were found between fla16 and wt (Table 4.2).
Tensile strength tests of basal- and middle-stems identified a significantly increased stiffness in the mid-stems, but not the basal-stems, of fla16 compared to wt (Table 4.2 andsee Appendices Fig. 4.6).
Cell walls are the exoskeleton of the stem cells, which consist of arrays of complex polysaccharides (Doblin et al., 2010), therefore any alteration of the wall biomechanical property is likely a result of the change in the composition, sequence and/or distribution of the component wall polysaccharides,.
4.2.7 Carbohydrate content is altered in fla16 stems
The polysaccharide composition was determined by linkage analyses of the alcohol insoluble residue (AIR) representing the cell wall fractions extracted from fla16 and wt stems (growth stage 6.9) (Pettolino et al., 2012). This was to determine whether cell wall compositional changes occur in the fla16 mutant stems. Linkage analyses of the cell wall polysaccharides revealed an approximately 8.96% reduction of cellulose in fla16 stems (Fig. 4.6A). The reduction in cellulose was also investigated using an acetic/nitric cellulose assay to determine the amount of crystalline cellulose (Updegraff, 1969) which recorded an approx. 16% reduction in the mutant line (Fig. 4. 6B, Table 4.2). Albeit the cellulose content in the fla16 stems showed varied reductions using these methods, results from polysaccharide linkage analyses were regarded more accurate due to higher sensitivity. However, both methods showed a consistent result of cellulose reduction in the fla16 stems concluding they have less cellulose compared to wt.
Thus far, the fla16 mutant line exhibited a number of phenotypes during the plant’s growth and development phases, particularly in the stem height and cellulose content. To verify that these phenotypes resulted specifically from the loss of FLA16 complementation of the fla16 mutant line was essential.
4.2.8 Complementation of the fla16 mutant
Two FLA16 endogenous promoter (pFLA16) driven complementation constructs were generated and transformed into the fla16 mutant. The first construct (pFLA16:FLA16) was made by inclusion of FLA16 coding region only whereas the second construct (pFLA16:FLA16-VH) was the reporter protein with Venus (V) and HIS (H) tag fusions at the C-terminus (see Chapter 3, Fig. 3.2). Homozygous plant lines for the transgenes in the fla16 mutant background were generated and examined for FLA16 expression by Q-PCR and complementation of the reduced cellulose content and stem height phenotypes.
The expression of FLA16 transcripts in the total stem of fla16 was 21% of wt (Fig 4.1). In the fla16 pFLA16:FLA16 and fla16 pFLA16:FLA16-VH lines the transcript levels were 73% and 123% of wt levels, respectively. This indicated that FLA16 expression was likely to be partially recovered in fla16 pFLA16:FLA16 lines and increased in fla16 pFLA16:FLA16-VH lines. Based on acetic/nitric cellulose assays, the stem cellulose content in fla16 pFLA16:FLA16 and fla16 pFLA16:FLA16-VH stems was 98% and 93%, respectively, of wt levels. This is a significant increase in crystalline cellulose from the fla16 line whose content was reduced to 83% of wt. Both complementation lines partially recovered the stem height phenotype compared to wt whereas the height of first internode was not recovered and was comparable to fla16 (Fig. 4.7).
4.2.9 Expression of FLA16 in the stem overlaps with the expression of the primary and secondary cellulose synthases (CesAs).
The above data suggests that fla16 impacts upon the levels of crystalline cellulose in the stem. This could be due either to reduced expression or activity of cellulose synthases (CesAs), disruption of trafficking of cellulose synthase complexes (CSC), or altered cellulose polymerisation in the wall. To determine if the expression of CesAs involved in primary and secondary cell wall synthesis was altered in fla16 mutants, Q-PCR analysis was undertaken. Expression of the primary wall CesAs (1, 3 and 6) and secondary wall CesAs (4, 7 and 8) (Persson et al., 2007) was investigated at the base-, middle- and top-stem portions of fla16 and wt (growth stage 6.5).
In the top portion of fla16 stems CesAs (1, 3, 4, 7 and 8) were up-regulated and CesA6 was down-regulated compared to wt. In the fla16 mid stem, CesAs (1, 6 and 7) were up-regulated, CesAs (4 and 8) were down-regulated, and CesA3 was unchanged compared to wt. In the fla16 basal stem, CesA7 was up-regulated, CesAs (1, 3 and 6) were down-regulated and CesA8 was unchanged (Fig. 4.8).
The consistent up-regulation of CesA7 in all stem sections of the fla16 mutants compared to wt suggests FLA16 may be indirectly involved in the regulation of CesA7 (Fig. 4.8).
4.2.10 The fla16 mutant is more sensitive to isoxaben treatment than wt.
Transcripts of FLA16 were found to be present in the hypocotyl during early development (Schmid, 2005; Toufighi, 2005; Winter et al., 2007). Compared to wt, mutants with reduced cellulose levels frequently display higher sensitivity to the herbicide isoxaben, displaying reduced hypocotyl growth and swelling (Desprez et al., 2002). Isoxaben has been shown to inhibit cellulose synthesis in plants (Desprez et al., 2002). The fla16 mutants and wt were tested for sensitivity to isoxaben during dark-grown seedling development. Hypocotyls of fla16 mutants were found to be significantly shorter than wt with swelling onset suggesting that cellulose synthesis is compromised in fla16 (Fig. 4.9, Table 4.1).
Confirmation of the expression status and the biological influences on the plant growth and development is essential in the characterisation of FLA16. In current study, we confirmed the transcript expression status of FLA16 with a more quantitative method independently from that obtained from public database. In addition, we identify the loss of FLA16 leads to a number of growth deficiency in the plant, which allows us to further our understanding in the biological roles of FLA16.
4.3.1 FLA16 expression potentially affects plant growth and development via certain sensing mechanisms in the cell wall.
The cell wall is an extracellular matrix undergoing dynamic changes responding to stimuli during the growth/development of the plant and from environment (Xu et al., 2008). Despite the developmental phenotypes, a key finding in current study is the cellulose production and expression of CesA7 are related to the expression of FLA16. Since cellulose is the major component in the wall (Doblin et al., 2010), produced by CesAs (Persson et al., 2005), the correlation between cellulose, CesA7 and FLA16 suggests FLA16 is possibly acting as a regulator for CesA7, and hence affecting the wall composition and integrity. We noticed the reduction of cellulose is not correlated to the increased expression level of CesA7 that should be reduced. Transcript expression is not always correlating with the protein level and the gap could be significantly large (Edfors et al., 2016). Thereby, it is still unclear whether the reduction of cellulose in the fla16 mutant is due to reduced CesA protein copies merely based on our transcriptional analyses. Nevertheless, the cellulose reduction must be a consequence of either malfunction of the or reduced CSCs copies that could only be verified by quantitative proteomic analyses.
The functional influences on the plant as the result of loss of FLA16 expression can be seen as a common delay of growth. Factors causing such biological impacts are possibly related to certain sensing mechanism in which the FLA16 protein might play a role as a wall sensor. However, whether abiotic stress responses and hormone control are also involved it ought to be investigated further as they have also been shown to partake in the regulation of the growth of plants (Gómez-Mena et al., 2001; Huang et al., 2008).
The change in the wall integrity of cells specific for FLA16 in the fla16 mutant might direct the rearrangement of these cells. Such rearrangement is likely the factor causing changes (i.e. reduced area and cell number) in the pith cells as they are encrusted by the primary wall only (Wang and Dixon, 2012) and more flexible (Cooper, 2000). Since FLA16 is not expressed in the pith such change is possibly due to indirect effect of the neighbouring tissue/cells, i.e. the intra-vascular region that restricts the growth of pith. However, the exact mechanism of how FLA16 signalling works is unknown but previous study on two LRR RLKs gives us some cues. These LRR RLKs are FEI1/2, are known to regulate cellulose synthesis through interaction with FLA4 (Xu et al., 2008) consisting of 2 FASs (Johnson et al., 2003) and localised to the PM (Xue et al., 2017). Although the domain constitution and arrangement of FLA4 and FLA16 are not highly comparable (Johnson et al., 2003), the common subcellular localisation and functional impact on the wall cellulose synthesis make suggestive indications for FLA16 as a wall signalling molecule. To verify the potential signalling role of FLA16, future works are needed.
- FLA16 is contributory to the biosynthesis and biomechanical properties of the cell wall.
The cell wall is the exoskeleton of plant cells, it bears most internal and external physical impacts, and responds to most of the mechanical changes (Doblin et al., 2010). The wall is a matrix in an array of complex components, mainly consisted of cellulose, and mechanical force is passed from such matrix to the cellulose microfibrils via interfacial shear stress. The composite stiffness is an overall averaged stiffness of each wall component. The microfibril angle (MFA) of cellulose is a crucial factor determining stiffness (Cave and Walker, 1994; Keckes et al., 2003) in addition to lignin and hemicellulose (Kohler and Spatz, 2002). When the plant material undergoes mechanical impacts such as the tensile action, the increased extension results in a decrease of MFA (Keckes et al., 2003). When the mechanical load passes the elastic extent of the sample, the weakest part of the sample starts to break causing plastic/viscoelastic deformation. Besides this, during the increase of shear stress at the matrix/fibril interface, breakage of the interface can occur leading to the ‘slip-stick Velcro’ mechanism (Keckes et al., 2003). As the load increased, the mechanical load passes the sample’s load-carrying limit and breaks, and the mechanical strength of the plant material is mainly influenced by the cellulose content (Turner, 1997; Li et al., 2003 ). The breakage occurrence could happen in either order depending on the property of the sample. When breakage takes place in the microfibril first, higher load is exposed to the matrix. Otherwise, when the matrix breaks first and the microfibril becomes the carrier of higher loads. In addition, when the MFA increases, breakage is easier to occur in the matrix causing the microfibril to peel apart and a reduction of the stiffness (MacMillan et al., 2010).
The changed biomechanics of the fla16 stems is likely related to the cellulose reduction that is potentially due to malfunction of CSCs (see Chapter 4.3.1) and/or miscommunication between the relevant protein components involved in cellulose biosynthesis.
The biosynthesis of cellulose requires cooperation/association of different CesAs and certain proteins (Schneider et al., 2016), and FLA16 might participate in such cooperation/association. The CSCs consist of a core with catalytic activity commonly in a configuration of CesA heterotrimer (Endler et al., 2016). For instance, CesAs1, 3 and 6 form the catalytic core of the CSC active in primary wall (Desprez et al., 2007), and CesAs4, 7 and 8 do the same for CSC active in secondary wall biosynthesis (Persson et al., 2007). After assembly in the Golgi apparatus, the CSCs are delivered to the PM coincidentally and spatially with the microtubules (Gutierrez et al., 2009). Once the CSCs are embedded in the PM, they start to synthesise cellulose microfibrils that are deposited in the wall (Endler et al., 2016). Following this, the cellulose microfibrils are believed to stabilised in the wall locally and the ongoing cellulose microfibril synthesis would cause the CSCs moving in the PM (McFarlane et al., 2014). The guidance of how CSCs move is proposed to be fulfilled by microtubules through cellulose synthase interacting 1 (CSI1) that is capable of binding both the microtubules and CesAs (Gu et al., 2010; Bringmann et al., 2012; Li et al., 2012). In addition to this, a number of protein components have been found to also take part in the synthesis of cellulose. These are the GPI-anchored COBRA (Roudier et al., 2005), endoglucanase KORIGAN (Vain et al., 2014), chitinase-like (CTL) proteins and CTLs1 and 2 (Sanchez-Rodriguez et al., 2012), companions of CesA (CCs) (Endler et al., 2016). Albeit not presenting a central catalytic activity in cellulose synthesis, these proteins might associate with the CSCs closely (Endler et al., 2016).
Considering the correlation between its impact on cellulose production (i.e. a great influence of the stem biomechanics) and the possession of the well-known cell adhesion FAS domains (Elkins et al., 1990a; Elkins et al., 1990b), FLA16 could be another candidate partaking in the process of cellulose synthesis. In addition to this, the AGP domain might also be contributory for its capability to facilitate erection of FLA16 on the PM thus hence interaction with other extracellular molecules (Jentoft, 1990) such as the ones mentioned in the above section.
We believe FLA16 also partakes in maintenance of the stem biomechanics through its influence of the MFA as we noticed a significant increase in the tensile stiffness of the fla16 stem compared to wt. This is consistent to the previous single fla11 and fla12 mutant study (MacMillan et al., 2010), and such increase suggests a decrease of MFA in the fla16 stems. By far, it is hard to elucidate the exact mechanism and it is likely through either direct contact with the microfibrils or indirect action to other relevant protein/wall components. As regards direct interaction with microfibrils, the FAS domains of FLA16 are potentially contributory but the existence of such FAS-microfibril interaction and its strength need to be investigated further (see Chapter 6). For indirect action, both the FAS and AGP domains could be responsible. There is no doubt about the protein-protein interaction potential of the FAS domain (Bastiani et al., 1987; Kolodkin et al., 1992; Litvin et al., 2004; Kannabiran and Klintworth, 2006; Liu et al., 2009). Because of this, FLA16 could associate with the proteins guiding the movement of CSCs that ought to synthesise and extend the microfibril chain continuously (Schneider et al., 2016). Such action might provide certain anchoring effect for the movement of CSCs and thus for the orientation of the microfibrils, which eventually lead to certain control of the MFA. In addition to the FAS, the AGP domains could also indirectly influence the MFA. Some AGPs show their ability to cross-link with the wall hemicellulose and pectin (Tan et al., 2013), and the wall cellulose is embedded in a mixed array of polysaccharide matrix including those mentioned (Doblin et al., 2010). The cross-linking might cause some shaping effect for the polysaccharide matrix and the microfibrils accommodated in such pre-shaped spaces, leading to a defined MFA.
Together with FLA11/FLA12 and FLA16, highly stem specific expression and similar roles in the stem are found. However, how these FLAs are interrelated as regards genetic interaction becomes our next research interest, and this is investigated in Chapter 5.
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