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Keywords:

  • ABI4;
  • abscisic acid (ABA);
  • Arabidopsis;
  • cell walls;
  • pectic arabinan;
  • quantitative fluorescence imaging;
  • rhamnogalacturonan-I;
  • root meristem

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • The hormonal and physiological regulations underpinning the cell contexts of structural features of the heterogeneous cell wall pectic polysaccharide rhamnogalacturonan-I are far from being understood.
  • The effect of the modulation of abscisic acid (ABA) concentrations and sensitivity on the detection of the LM6 1,5-arabinan epitope at the surface of Arabidopsis thaliana seedling root apices was assessed by means of fluorescence imaging.
  • Treatment with 50 nM ABA resulted in an increase in the detection of the LM6 epitope at the root surface in the region of the meristem. An inhibitor of ABA biosynthesis and introduction of the ABA synthesis mutation aba3-2 resulted in reduced epitope detection. The same ABA application resulted in an increase in the number of epidermal root meristem cells and both this and LM6 epitope detection were specifically disrupted in the abi4 ABA-insensitive mutant. These two effects were uncoupled with the application of higher ABA concentrations, which resulted in a reduction in the number of epidermal root meristem cells but increased LM6 epitope detection.
  • This work demonstrates a role for ABI4-mediated ABA signalling in the modulation of pectic arabinan occurrence at the A. thaliana root meristem.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Primary plant cell walls are dynamic cellular compartments that provide the mechanical strength and the cell adhesion that underpins both the defined morphologies and growth capacities of cells, meristems and organs. Primary cell walls are largely comprised of diverse sets of complex polysaccharides. These polysaccharides are currently classed as microfibrillar cellulose and the structurally complex hemicellulose and pectic groups of polymers (O’Neill & York, 2003). The pectic group of polymers are envisaged to form a matrix surrounding and interacting with a cellulose–hemicellulose network and influencing a wide range of functions, including cell wall assembly, cell wall porosity, cell extension and mechanical properties in addition to cell adhesion. Pectins contain a number of structurally diverse polysaccharide domains that include homogalacturonan (HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II and xylogalacturonan (Ridley et al., 2001; Willats et al., 2001; Caffell & Mohnen, 2009). How these pectic domains are integrated in both structural and functional terms is not well understood.

Rhamnogalacturonan-I polymers, which include a rhamnogalacturonan backbone and 1,4-galactan and 1,5-arabinan as major structural motifs, are highly heterogeneous sets of polysaccharides displaying considerable structural variation in both cell and taxonomic contexts (Willats et al., 2001; Caffell & Mohnen, 2009; Verhertbruggen et al., 2009). Two monoclonal antibodies to 1,4-galactan and 1,5-arabinan motifs – LM5 and LM6, respectively – have been instrumental tools for exploring RG-I structure and heterogeneity in cell wall contexts and have been indicative of complex and perhaps separate functional roles for galactan- and arabinan-rich RG-I polymers in plant systems, although what these roles are in mechanistic terms is as yet uncertain (Willats et al., 1999; McCartney et al., 2000, 2003; Ulvskov et al., 2005). Recent work with a widened set of arabinan-directed monoclonal antibodies has emphasized that 1,5-arabinan and metabolically related structures are highly dynamic components of primary cell walls (Verhertbruggen et al., 2009).

At the carrot root meristem, the LM6 arabinan epitope is associated with walls of the central meristematic cells and there is a switch in relative abundance of the LM5 galactan and LM6 arabinan epitopes associated with a switch from cell proliferation to cell elongation and differentiation (Willats et al., 1999). At the surface of the Arabidopsis root apex, the LM5 galactan epitope has been shown to mark the transition zone, where the cells leave the meristem and begin to elongate (McCartney et al., 2003). In such studies the LM6 arabinan epitope is associated with meristematic regions of root apices (Willats et al., 2001; Verhertbruggen & Knox, 2007). Although in some studies reduced levels of pectic arabinan are not associated with growth phenotypes (Skjot et al., 2002; Harholt et al., 2006), a functional association of pectic arabinan with meristematic regions has been demonstrated in series of experiments involving overexpression of microbial arabinanases that resulted in serious defects to cell and meristem development (Oomen et al., 2002; Skjot et al., 2002; Borkhardt et al., 2005). It has also been suggested that pectic arabinan has a role in maintaining cell–cell linkages (Iwai et al., 2001; Orfila et al., 2001; Peňa & Carpita, 2004) and cell adhesion has long been established to be important in the organization, differentiation state and tissue patterning within meristems (van den Berg et al., 1997). In this context, cell wall structures and properties are clearly of high importance in meristems, where the specification and generation of cellular properties is crucial for successful organ initiation, growth and development. Although there is some knowledge of the hormonal regulation of factors that influence cell walls in A. thaliana (Sánchez-Rodríguez et al., 2010) there is little known about the pathways that can influence the composition of the hemicellulose and pectin classes of polysaccharides during meristem and cell development.

The root meristem is the source of all cells and patterning in the root, and the processes involved in its specification and function have been studied extensively. The phytohormones auxin, ethylene and cytokinin are known to play important roles in this. Auxin has a response maximum in the meristem, and is necessary for maintaining the potency of the stem cell niche and its fast dividing progenitor cells (Grieneisen et al., 2007). Cytokinin is involved in regulating the position of the transition zone, with exogenous application resulting in markedly less cell elongation (Ioio et al., 2007). Ethylene is known to be involved in the regulation of mitotic activity within the quiescent centre (Ortega-Martínez et al., 2007) and the spatial distribution of auxin within the root meristem (Růžička et al., 2007).

Detection of the LM5 galactan epitope at the Arabidopsis seedling root surface as cells begin to elongate has been shown to be responsive to phytohormone signals correlating with impacts upon cell elongation (McCartney et al., 2003). However, the specification of pectic arabinan, as imaged with LM6 arabinan monoclonal antibody, has not yet been placed in any physiological or signalling networks. Here we demonstrate that abscisic acid (ABA) signalling, which has recently been shown to have an impact on meristem functions (Zhang et al., 2010), leads to an increase in the detection of the LM6 arabinan epitope at the surface of Arabidopsis root apices and that this is dependent upon the ABI4 transcription factor.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant materials

Arabidopsis thaliana (L.) Heynh. lines used in the study were from the following sources: abi1-1, abi2-1, abi3-1 and abi5-1 from Ruth Finkelstein (University of California at Santa Barbara, CA, USA); aba3-2 from Maarten Koornneef (MPIZ Cologne); Col-0, abi4-1 and wox5-1 from the Nottingham Arabidopsis Stock Centre (NASC); and scr4 from Philip Benfey (Duke University). Seeds were germinated and grown vertically for 7 d on the surface of agar-solidified media in a growth room at 21°C with a 16 : 8 h light : dark regime. The agar solidified medium consisted of 0.4 mM KNO3; 0.2 mM MgSO4; 1.5 mM CaCl2; 1.0 mM NaH2PO4; 0.2 mM MnSO4; 0.09 mM KI; 0.97 mM H3BO4; 0.14 mM ZnSO4; 2 nM CuSO4; 20.6 nM Na2MoO4; 2.1 nM CoCl2; 3.6 mM Fe-EDTA; 0.5 g l−1 2-(N-morpholino)ethanesulfonic acid (MES); 5 g l−1 sucrose; 10 g l−1 agar-agar (Fisher Scientific, Loughborough, UK), which was adjusted to pH 5.7 with 1.0 M KOH. The medium was autoclaved for 20 min at 121°C and all hormone treatments were added after autoclaving. The hormones used were the naturally occurring auxin indole acetic acid (IAA); abscisic acid ((±)-ABA), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the cytokinin benzyl aminopurine (BAP) (all Sigma Aldrich). All seedlings were first germinated on untreated medium and grown for 4 d, before being transferred on to media containing hormone or control treatments for 3 d.

Root growth as assessed by total root length was determined using a dissecting microscope on day 7 with 15 seedlings per treatment. For the assay of proximal root meristem size, seedlings were mounted intact on glass slides with 100% lactic acid and imaged using a Zeiss Axiovert 135TV microscope with the Openlab imaging system (Improvision, PerkinElmer, Massachusetts, USA). At least 15 root meristems were analysed per treatment. Cells, designated as belonging to the epidermal root meristem, were counted from the quiescent centre to the onset of rapid cell elongation at the transition zone (as indicated by a proximal cell being at least 50% greater in length than its distal neighbour).

Immunofluorescence analysis of root surfaces

In all cases, 7-d-old seedlings were fixed overnight in 4% paraformaldehyde in 50 mM piperazine-N,N′-bis(2-ethanesulphonic acid) (PIPES), 5 mM MgSO4, and 5 mM ethylene glycol tetraacetic acid (EGTA). Following fixation they were blocked with 5% (w/v) milk protein in phosphate-buffered saline (MP/PBS) for 1 h; then incubated in primary antibody (rat monoclonal antibody hybridoma cell culture supernatants LM5 (Jones et al., 1997), LM6 (Willats et al., 1998), LM2 (Smallwood et al., 1996) or LM16 or LM13 (Verhertbruggen et al., 2009)) diluted fivefold in MP/PBS for 1.5 h. They were then washed for three × 5 min in PBS, incubated for 1 h in darkness with anti-rat immunoglobulin G linked to fluorescein isothiocyanate (FITC; Sigma) diluted 100-fold in MP/PBS. After three 5 min washes in PBS the intact seedlings were mounted in the glycerol-based Citifluor AF1 antifade (Agar) and observed on an Olympus BX61 microscope equipped with a Hamamatsu ORCA285 camera and Volocity software (PerkinElmer, Massachusetts, USA). The fluorescence intensities at the surface of root apices were determined using a Volocity software protocol. Images were collected of all root apices, with the exposure times determined from the sample with the most intense fluorescence and all other images captured with the same exposure time. In all cases, total fluorescence intensity was assessed in the distal 1 mm of root apices and the protocol-generated ‘sum’ value of pixel intensity for the selected area of each image was used as the intensity value for that particular root. Fluorescence was quantified for at least nine root apices for each treatment and, in all cases, presented relative to the control treatment, which was assigned a value of 1.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Detection of the LM6 arabinan epitope at the surface of the Arabidopsis root meristem is increased by exogenous ABA

Comparative analysis of LM5 galactan and LM6 arabinan binding to the surface of intact root apices of A. thaliana indicates that the LM5 epitope is detected in a region back from the meristem that corresponds to the onset of cell elongation, as shown in Fig. 1 and as previously reported (McCartney et al., 2003). By contrast, the LM6 arabinan epitope is present in a distinct pattern and is most abundant in the more distal regions and over the root meristem (Fig. 1). Recent work has indicated that cell wall polysaccharide epitopes can be masked by pectic HG (Marcus et al., 2010). Removal of pectic HG from root surfaces by pectate lyase had no effect on the detection of the LM6 epitope (data not shown). The ability of exogenous phytohormones to modulate the detection of the LM6 arabinan epitope was explored. The incorporation of the auxin IAA (50 nM), the ethylene precursor ACC (500 nM) and the cytokinin BAP (500 nM) into solidified growth media had no effect on LM6 epitope detection (Fig. 2). By contrast, the addition of 50 nM of ABA (a concentration sufficient to promote stem cell maintenance; Zhang et al., 2010) resulted in an increase in LM6 arabinan epitope detection at the root surface in the region of the meristem (Fig. 2e).

image

Figure 1. Immunofluorescence analysis of the LM5 galactan and LM6 arabinan epitopes at the surface of intact seedling roots of Arabidopsis thaliana. (a) Bright field image of root apex. (b) The LM5 galactan epitope is most abundant in the region of the transition zone (black arrowhead and dotted line). (c) The LM6 arabinan epitope is most abundant in the region of the meristem. White arrowheads indicate root tips. Bar, 50 μm.

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image

Figure 2. LM6 arabinan immunofluorescence at the surface of intact seedling roots of Arabidopsis thaliana with exogenous phytohormones. (a) Untreated control seedling; (b) 500 nM 1-aminocyclopropane-1-carboxylic acid (ACC); (c) 50 nM indole acetic acid (IAA); (d) 500 nM benzyl aminopurine (BAP); (e) 50 nM abscisic acid (ABA); (f) 50 nM ABA with no LM6, negative control. Bar, 50 μm.

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In a comparative analysis of the impact of 50 nM ABA on the detection of related cell wall epitopes at the root surface, it was found that there was no impact on the detection of the LM5 galactan or the LM2 arabinogalactan-protein epitopes, as shown in Fig. 3. The LM16 epitope (a processed form of pectic arabinan; Verhertbruggen et al., 2009) was only present at the root surface in a proportion of primary root apices within a sample. The application of ABA did result in increased LM16 epitope detection in a region of the proximal root surface associated with the onset of cell elongation (Fig. 3). Over several series of experiments the response of the LM16 epitope to exogenous ABA was not found to be as consistent as that of the LM6 epitope, and the ABA induction of this epitope was not explored further. The LM13 arabinan epitope is specifically detected at the root cap at the root surface (Verhertbruggen et al., 2009) and showed no response to exogenous ABA (not shown).

image

Figure 3. Comparative immunofluorescence analysis of the impact of 50 nM abscisic acid (ABA) on the detection of the LM5 galactan, LM2 arabinogalactan-protein, LM16 processed arabinan and the LM6 arabinan epitopes at the surface of intact seedling roots of Arabidopsis thaliana. The top row shows untreated controls and the bottom row shows seedlings with exogenous ABA. Bar, 50 μm.

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The impact of ABA signalling on the detection of the LM6 arabinan epitope was explored further. Growth on medium with 10 μM fluridone, an inhibitor of ABA synthesis (Han et al., 2004), resulted in a decrease in LM6 epitope detection compared with untreated control roots (Fig. 4). Analysis of the LM6 epitope at the root surface of the ABA synthesis mutant aba3-2, which is defective in the final step of ABA synthesis (Koornneef et al., 1998; Bittner et al., 2001), also displayed reduced abundance of the LM6 arabinan epitope (Fig. 4). In terms of the lowest concentration of exogenous ABA required to induce an increase in LM6 epitope detection, it was found that application of 0.5 or 5 nM ABA did not produce any significant differences in LM6 epitope detection from untreated control plants (Fig. 4).

image

Figure 4. LM6 arabinan immunofluorescence analysis of the surface of intact seedling roots of Arabidopsis thaliana with modulated abscisic acid (ABA) concentrations. (a) 10 μM fluridone (Flu); (b) untreated control; (c) 50 nM ABA; (d) untreated aba3-2 mutant. Bar, 50 μm. (e) Chart showing quantified LM6 root surface immunofluorescence in response to 10 μM fluridone, the aba3-2 mutation and a range of concentrations of exogenous ABA shown relative to the untreated control. Error bars indicate SEM.

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ABA induction of the LM6 arabinan epitope is not dependent on WOX5 or SCR gene pathways

The observations on the impact of ABA application on the detection of the LM6 epitope at the root surface indicated that ABA signalling is involved in the modulation of meristem-associated LM6 epitope and implicated pectic arabinan in some aspect of root meristem function. Two of the known pathways that influence cell proliferation in root meristems are the SCR/SHR and the WOX5 pathways. Analysis of the surface of the wox5-1 (Sarkar et al., 2007) and scr4 (Sabatini et al., 2003) mutants, with or without exogenous ABA, demonstrated that the ability of ABA to modulate LM6 epitope abundance did not differ from wild-type, indicating that the ABA-induced increase of the LM6 arabinan epitope does not require the SCR/SHR or the WOX5 gene pathways or their functions in the establishment and maintenance of the root meristem (Fig. 5). WOX5 acts to suppress differentiation of the quiescent centre (Sarkar et al., 2007) and the scr-4 mutation results in shrinkage of the stem cell population (Sabatini et al., 2003). The retention of LM6 arabinan epitope abundance and its ability to be elevated by exogenous ABA in the presence of these mutations indicate that pectic arabinan modulation is associated with factors or meristem functions that are independent of the SCR/SHR or WOX5 pathways.

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Figure 5. Seedling root surfaces of Arabidopsis thaliana wild-type (WT), wox5-1 and scr4 immunolabelled with the LM6 arabinan monoclonal antibody. The top row shows LM6 immunofluorescence analysis with no exogenous abscisic acid (ABA). The bottom row shows LM6 immunofluorescence analysis with 50 nM ABA. Bar, 50 μm.

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ABI4 is specifically involved in ABA signalling to modulate both the LM6 arabinan epitope and epidermal root meristem cell number

A series of abi mutations have been identified that result in insensitivity to ABA (Koornneef et al., 1984; Finkelstein, 1994) and these are useful to study components of the ABA signalling pathway that may act in the ABA-induced elevation of the LM6 arabinan epitope. ABA-insensitive mutants were grown without treatment or in the presence of fluridone or 50 nM ABA, and abundance of the LM6 arabinan epitope was determined at the surface of the root apices as shown in Fig. 6. At the surface of wild-type root apices, ABA treatment resulted in an increase in the abundance of the LM6 arabinan epitope and similar responses were found in the abi2-1, abi3-1 and abi5-1 mutants (Fig. 6). However, the abi4-1 mutant had significantly reduced detection of arabinan at the root meristem surface relative to the control and other abi mutants. The low abundance of LM6 epitope in abi4-1 roots was modulated by ABA and fluridone application, indicating that it retained some responsiveness to ABA signalling. The treatment of abi4-1 roots with fluridone resulted in the complete loss of LM6 binding (Fig. 6). The significant reduction in the LM6 arabinan epitope in all abi4 roots relative to wild-type and other abi roots indicated a clear and specific role for ABI4 in ABA signalling to modulate arabinan levels.

image

Figure 6. Immunofluorescence analysis of LM6 binding to Arabidopsis thaliana root surfaces. (a) Micrographs showing the impact of 10 μM fluridone (Flu) and 50 nM abscisic acid (ABA) on the surface of wild-type (WT) and abi4-1 seedlings. Bar, 50 μm. (b) Chart showing quantified relative LM6 arabinan immunofluorescence at the surface of abi2-1, abi3-1, abi4-1 and abi5-1 mutants in response to 10 μM fluridone (dotted bars) and 50 nM ABA (closed bars) (control, open bars). Error bars indicate SEM.

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A study of the impact of 50 nM ABA on A. thaliana roots indicated that it resulted in a small increase in root length after 3 d of ABA application and that this was associated with an increase in the number of cells in the proximal root meristem, as indicated by epidermal cell number before the transition zone and as previously reported (Zhang et al., 2010). Epidermal cell number increased from c. 30 to c. 35 cells in the epidermal root meristem in response to 50 nM ABA application (Fig. 7). In a related series of experiments, the impact of the abi2-1, abi3-1, abi4-1 and abi5-1 mutations on the capacity of 50 nM ABA to increase the number of epidermal root meristem cells was determined. The number of epidermal cells within the meristem was increased by exogenous ABA in a similar manner to the wild-type in abi2-1, abi3-1 and abi5-1, but not in abi4-1 where there was no response to ABA (Fig. 7). Of the known ABI factors, ABI4 appeared to be specifically involved in both ABA signalling to regulate LM6 arabinan epitope abundance and epidermal root meristem cell number.

image

Figure 7. Effect of exogenous abscisic acid (ABA) addition on the number of cells in the Arabidopsis thaliana root meristem. (a) Representative micrographs showing region of epidermal root meristem (RM, dotted line) for untreated wild-type (WT) in comparison with fluridone (Flu)- and ABA-treated WT. (b) Representative micrographs showing the RM in abi4-1 and ABA-treated abi4-1. Proximal elongation zones (EZ) are indicated by solid lines. Bar, 25 μm. (c) Chart showing the impact of abi2-1, abi3-1, abi4-1 and abi5-1 mutations upon ABA-induced increase in the number of root meristem cells (ABA, closed bars; control, open bars). Error bars indicate SEM.

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ABA impacts on LM6 arabinan epitope detection and root meristem cell number are uncoupled at higher ABA concentrations

The possible linkage between the abundance of the LM6 arabinan epitope and epidermal root meristem cell number was explored further. The epidermal root meristem cell number and the consequent impact on root length by exogenous ABA had a concentration optimum of 50 nM with reductions in these parameters by the higher concentrations of 500 nM and 5 μM ABA (Fig. 8). By contrast, exogenous ABA did not display the same concentration optimum in its ability to increase the detection of the LM6 arabinan epitope, and higher concentrations of ABA resulted in increased detection of the LM6 arabinan epitope at the surface of root apices (Fig. 8).

image

Figure 8. Modulated responses in the Arabidopsis thaliana root to exogenous abscisic acid (ABA). Seedlings were transferred 4 d after germination (dag) on to different ABA treatments for 3 d. (a) Root length at 7 dag. (b) The number of cells in the epidermal root meristem (RM) at 7 dag. (c) Relative LM6 arabinan immunofluorescence at root surface at 7 dag. Error bars indicate SEM.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Pectic arabinan and meristems

Abscisic acid signalling is shown here to influence the detection of the LM6 arabinan epitope at the surface of Arabidopsis root apices. The mechanistic basis of the modulated detection of the LM6 arabinan epitope is not yet known but could be altered pectic arabinan synthesis (Harholt et al., 2006), altered arabinan metabolism (Chávez-Montes et al., 2008; Gomez et al., 2009) or even altered cell wall properties allowing increased access of LM6 to its epitope. That the LM6 epitope was the only cell wall epitope, of the several examined, to be modulated by exogenous ABA suggests the latter basis is unlikely, and moreover removal of pectic HG indicated that it was not masked by this polysaccharide. This is the first indication of phytohormone signalling that can modulate the level of pectic arabinan in a specific set of cell walls and is suggestive of another aspect of the mechanisms that regulate the structure of RG-I. Although the ABA signalling that impacts on LM6 epitope detection shares components with the impact of ABA on the root meristem, and specifically epidermal root meristem cell number, the level of LM6 epitope detection does not appear to be directly coupled to this feature in the meristem. This is in contrast to the detection of the LM5 galactan RG-I epitope, which is correlated with the onset of cell elongation and is responsive to hormonal and genetic factors in line with root growth (McCartney et al., 2003). The LM5 galactan epitope has been shown to be similarly modulated in relation to root growth in studies of the impact of the target of rapamycin (TOR) pathway on cell walls (Leiber et al., 2010).

The function of cell wall arabinans in meristems remains unknown. Studies from other systems indicate that RG-I arabinans are required for maintenance of cell wall flexibility and RG-I galactans may be associated with stiffening of cell walls (McCartney et al., 2000; Jones et al., 2003; Ulvskov et al., 2005). The switch from predominantly arabinan-rich to predominantly galactan-rich RG-I concurrent with the switch in a meristem from cell proliferation to cell expansion could therefore reflect local remodelling of cell wall mechanical properties. Of course, such remodelling of RG-I structures in the context of developmental processes will need to be integrated with the complex phytohormone signalling pathways that maintain meristem structures and functions. The responsiveness of the LM6 arabinan epitope to ABA is likely to be a component of this. In the experiments described here, the impact of exogenous ABA may be of no direct consequence for root growth as the importance of cell wall flexibility at the distal region of a root apex may only become apparent during growth in varied soil environments. Nevertheless, the observations clearly demonstrate a link between ABA signalling and LM6 epitope abundance within the root. Our attempts to manipulate root cell wall arabinan with exogenous arabinan oligosaccharides or an endo-arabinanase had no impact on root growth or epidermal root meristem cell number (data not shown), although there must be caution in interpreting such experimental interventions, as these exogenous factors will be unlikely to access inner cell walls. Although pectic arabinans have been implicated in cell adhesion and in cell wall mechanical properties, both of which are of importance at root meristems, it is also possible that arabinan metabolism could contribute to some aspect of the signalling networks that underpin root meristem structures and functions. Such a role is suggested by the observed impact of microbial arabinanases on meristem function (Oomen et al., 2002; Skjot et al., 2002; Borkhardt et al., 2005).

ABA signalling

It is of interest that the modulation of the LM6 epitope by 50 nM exogenous ABA occurs concurrently with an increase in both root growth and the number of epidermal root meristem cells. The observation that mutation in ABI4 specifically blocks the response to ABA in terms of increased epidermal root meristem cell number and results in reduced LM6 epitope detection also suggests a possible linkage between these factors. However, the observation that detected LM6 epitope abundance of abi4-1 plants is still responsive to ABA rules out a causal connection of epidermal root meristem cell number and arabinan. The observations also suggest that two ABA signalling pathways may influence LM6 epitope abundance: one involving ABI4, which maintains LM6 epitope abundance, and the other pathway which results in the LM6 epitope being responsive to exogenous ABA as revealed in abi4-1. At higher concentrations of exogenous ABA, the apparent correlation between ABA signalling and epidermal root meristem cell number and LM6 epitope abundance was not observed, indicating that these two processes are not related. ABI4 is an ERF/AP2 type transcription factor which binds to the CE1 (coupling element) motif (Niu et al., 2002). Before this work, the described phenotype of abi4 was defective seed maturation and germination (Finkelstein et al., 1998), although ABI4 is known to be expressed at low levels throughout the root (Bossi et al., 2009). A functioning ethylene signalling cascade is known to be required for the inhibition of root growth by high concentrations of ABA (Beaudoin et al., 2000). However, in our experiments the application of 500 nM ACC did not cause any significant difference in LM6 epitope detection. This leads us to conclude that ethylene signalling is not involved in the modulation of the LM6 epitope. Moreover, this also provides additional evidence for the lack of a direct coupling of the two effects of ABA application that we describe here: LM6 detection and root growth.

There are several indications that cell wall molecules can influence ABA signalling and sensitivity to exogenous ABA, including an extensin epitope (Wang et al., 1995), an arabinogalactan-protein (van Hengel & Roberts, 2003) and a putative family 64 glycosyltransferase (Bown et al., 2007). None of these cell wall components has as yet been specifically linked with other ABA signalling components, or with arabinan detection or function. The demonstration here that ABA signalling through ABI4 can promote the detection of the LM6 arabinan epitope at the surface of Arabidopsis root apices offers a novel insight for future work aimed at an integrated molecular understanding of meristem development.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We acknowledge the award of a UK Biotechnology and Biological Sciences Research Council (BBSRC) postgraduate studentship to P.J.T.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References