terrestrial plants annually assimilate 1.3    10 metric tons of co2 through the process of photosynthesis ( beer et al . , 2010 ) , and the majority of the resulting photosynthate is used to produce the polysaccharide - rich walls that surround plant cells ( pauly and keegstra , 2008 ) . these complex extracellular matrices are composed of cellulose microfibrils ( cmfs ) , neutral hemicelluloses , acidic pectins , and proteins and must simultaneously be strong enough to resist the substantial cellular osmotic pressure necessary to support turgor - mediated cell growth and dynamic enough to allow cell expansion to occur ( somerville et al . the ordered deposition of plant cell walls determines the patterning and extent of plant growth and development at the cellular level ( desnos et al . , 1996 ; arioli et al . , 1998 ; nicol et al . , 1998 despite the physiological importance of cell wall deposition and dynamics , relatively little is known about cell wall structure in living cells , primarily due to a paucity of effective labeling strategies for polysaccharides in their native environment . genetically encoded fluorescent tags for polysaccharides do not exist , necessitating novel approaches for the specific labeling of cell wall glycans . although chemical ( reiter et al . , 1997 ; brown et al . , 2007 ) and biochemical ( lerouxel et al . , 2002 ; barton et al . , 2006 ; bauer et al . , 2006 ) techniques can accurately determine the primary structures of polysaccharides , these methods provide few spatial details due to the necessity for sample homogenization . , 1991 ; lynch and staehelin , 1992 ) , raman microspectroscopy ( schmidt et al . , 2010 ) , and fluorescence microscopy with an array of polysaccharide - specific antibodies , lectins , and carbohydrate binding domains ( pattathil et al . , 2010 ) have provided qualitative information regarding the localization and relative abundance of numerous cell wall polysaccharides . however , these techniques are limited by long sample preparation times , potential fixation artifacts , loss of temporal information , lack of epitope specificity ( pattathil et al . , 2010 ) , and complications in interpretation due to epitope masking ( marcus et al . , 2008 ) . in addition , protein - based probes , particularly intact 160  kda igg molecules , are very large compared to the approximately 3  nm pore size of cell walls ( chesson et al . , 1997 ) and may not efficiently label some cell wall components ( marcus et al . , 2010 ) . as a result , many developmental changes in cell wall architecture can not be fully characterized using these approaches . polysaccharide - specific fluorescent probes and metabolic labels augment the currently available cell wall imaging toolbox , with the added benefit that these probes are compatible with live - cell imaging and/or pulse - chase experiments . in this review , we summarize the recent development of specific small molecule chemical probes for cell wall imaging , and the insights that these new probes have provided concerning plant cell wall structure . the structural complexity of the plant cell wall arises in part due to the variety of polysaccharides used to assemble this organelle , the spatial and temporal separation of their synthesis , and the various chemical modifications that occur within these polysaccharides . cell wall polysaccharides are often grouped into three functional categories : cellulose , hemicelluloses , and pectins . cellulose is composed of 3040 -(1    4)-linked glucan chains that associate to produce semi - crystalline cmfs ( somerville , 2006 ) . these structural polysaccharides are synthesized at the plasma membrane by 30  nm  rosette  complexes ( arioli et al . , 1998 ; kimura et al . , 1999 ) containing multiple cellulose synthase a ( cesa ) catalytic subunits ( taylor et al . , 2003 ; desprez et al . , 2007 live - cell imaging has indicated that cesa - containing complexes co - align with cortical microtubules underlying the plasma membrane as they produce nascent cmfs ( paredez et al . , 2006 ; li et al . , 2012 ) , which are deposited in the apoplast . in contrast to cmfs , hemicelluloses and pectins are synthesized in the golgi ( mohnen , 2008 ; caffall and mohnen , 2009 ) and delivered to the apoplast by vesicle - mediated exocytosis via an incompletely described pathway ( moore et al . once deposited in the apoplast , hemicelluloses , such as xyloglucan , are thought to associate with and crosslink cmfs in the cell wall ( vincken et al . , 1995 ; lima et al . , 2004 ) , the interaction between these polysaccharides is proposed to prevent individual cmfs from interacting with each other and to help organize cmfs into a cohesive , dynamic network , although this model has recently been challenged ( park and cosgrove , 2012b ) . hemicelluloses and cmfs are embedded in a gel - like matrix composed of the pectic polysaccharides homogalacturonan , xylogalacturonan ( xga ) , rhamnogalacturonan - i ( rg - i ) , and rhamnogalacturonan - ii ( rg - ii ) which together control aspects of cell adhesion , wall extensibility , and wall porosity ( krupkova et al . , 2007 ; mohnen , 2008 ) . plant cell wall polysaccharides can also be modified during synthesis or after deposition in the apoplast by the activity of various enzymes . pectins can be reversibly acetylated at the o-2 or o-3 hydroxyl groups of their galacturonic acid backbone sugars and/or methylesterified at the o-6 carboxyl group ( ishii , 1997 ; manabe et al . , 2011 ) . 2011 ) or have their glycan side chains modified during plant development ( gnl et al . , 2011 ) . finally , transglycosylase enzymes , such as xyloglucan endotransglycosylase ( xet ) , cut and religate individual xyloglucan chains during wall extension ( xu et al . , 1995 ; maris et al . , 2009 ) . these modifications can significantly affect the physical properties of individual wall polymers ( huang et al . , 2002 ; ngouemazong et al . , 2012 ) and of the cell wall as a whole ( krupkova et al . , 2007 ; peaucelle et al . , cell wall polysaccharides are structurally diverse , exist in complex three - dimensional arrangements , and are synthesized , deposited , modified , and degraded in highly dynamic processes . therefore , probes that are highly specific for an individual polysaccharide type and compatible with live - cell imaging can effectively capture this complexity . various polysaccharide - binding dyes , including calcofluor white ( fagard et al . , 2000 ; , 2007 ; harpaz - saad et al . , 2011 ) , congo red ( kerstens and verbelen , 2002 ) , ruthenium red ( western et al . , 2001 ; marks et al . , 2008 ; harpaz - saad et al . , 2011 ) , and aniline blue ( adam and somerville , 1996 ; nishikawa et al . , 2005 ; nguyen et al . , 2010 ; xie et al . , 2011 ) , have been used as imaging tools for plant cell walls , but these dyes cross - react with multiple polysaccharides , and their direct glycan targets are often unknown . to address this issue , anderson et al . ( 2010 ) assayed the previously described fungal cell wall - binding dyes pontamine fast scarlet s4b ( s4b ) and solophenyl flavine 7gfe ( 7gfe ; hoch et al . , s4b ( figure 1a ) exhibits several useful imaging properties , including increased fluorescence intensity in the presence of cellulose , increased glycan specificity over calcofluor white , and spectral properties compatible with 532 and 561  nm lasers . s4b is also non - toxic at concentrations that are compatible with fluorescent imaging and can thus be used as a specific probe to dynamically image cellulose in living plant tissues . 7gfe ( figure 1b ) exhibits a characteristic shift in its fluorescence emission maximum in the presence of xyloglucan , but not cellulose . whereas 7gfe is slightly more toxic than s4b , this dye could potentially serve as an orthogonal fluorescent label for xyloglucan because its excitation and emission maxima do not overlap with those of s4b ( anderson et al . , the structures of the cellulose - binding dye pontamine fast scarlet 4b ( s4b ) ( a ) and the xyloglucan - binding dye solophenyl flavine 7gfe ( b ) . ( c ) a generalized version of the copper - catalyzed huisgen [ 2  +  3 ] cycloaddition reaction . this reaction is useful for coupling molecules containing terminal alkynyl or azido groups , such as per - acetylated fucose alkyne ( fucal ) ( d ) and alexa-488 azide ( e ) . sugar analogs such as fucal can be metabolically incorporated into glycans via the action of sugar salvage pathways and cognate glycosyltransferases , producing modified polysaccharides that can be labeled with azido - containing fluorophores by the click reaction . ( 2012 ) to metabolically label pectins by incorporating fucal into the root tissue of arabidopsis followed by copper - catalyzed labeling as shown in ( f ) . a representative image of root epidermal cells in the late elongation zone after labeling is shown in the right panel . s4b , 7gfe , and other fluorescent polysaccharide - binding probes are valuable tools that should be explored further , but the biophysical details of many dye  polysaccharide interactions are currently unknown , preventing the rational design of new dyes . however , alternative labeling strategies can be employed to design metabolic probes that can be targeted to individual cell wall polysaccharide networks . for example , fluorescently labeled xyloglucan fragments are metabolically incorporated into subdomains of xyloglucan by xets , and these oligosaccharides have been used to image the sites of xet activity during cell expansion ( vissenberg et al . , 2000 ) . additionally , molecules containing terminal alkynyl or azido functional groups can participate in a copper - catalyzed [ 2  +  3 ] cycloaddition referred to as the huisgen click  reaction ( figure 1c ) , which is bio - orthogonal , proceeds rapidly at room temperature with high specificity , and results in the formation of a stable triazole ring ( kolb et al . , 2001 ; rostovtsev et al . , 2002 ; tornoe et al . , 2002 ) . exploiting this reaction , small , biologically relevant molecules containing these functional groups have been synthesized and incorporated into proteins , fatty acids , nucleic acids , and sugars which are then covalently labeled with useful probes , such as fluorophores , epitope tags , or other affinity reagents that contain the opposite functional group ( chin et al . 2002 ; deiters et al . , 2003 ; kho et al . , 2004 ; link et al . , 2006 ; rabuka et al . , 2006 ; hang et al . , 2007 ; hsu et al . , 2007 ; jao and salic , 2008 ; salic and mitchison , 2008 ; chang et al . , 2009 ; kaschani et al . , 2009 these chemical methods have been particularly useful for imaging glycan conjugates in animal , bacterial , and fungal systems ( prescher et al . , 2004 ; dube et al . , 2006 ; laughlin et al . , 2008 ) , but had not previously been explored in the context of the plant cell wall . anderson et al . ( 2012 ) investigated the potential of click chemistry to label cell wall polysaccharides using per - acetylated fucose alkyne ( fucal ; figure 1d ) as a suitable click sugar monosaccharide analog . fucal was metabolically incorporated into arabidopsis root tissue and labeled with alexa-488 azide using the copper - catalyzed click reaction ( figures 1d  f ) . cytological and biochemical analyses indicated that the majority of the fucal label was incorporated into the pectic fraction of arabidopsis cell walls , most likely in arabinogalactan side chains of rhamnogalacturonan - i ( rg - i ) . fucose is present in multiple cell wall polysaccharides ( nakamura et al . , 2001 ; lerouxel et al . , 2002 ; , 2004 ; wu et al . , 2010 ) , but arabidopsis mutants lacking fucosyltransferases for xyloglucan ( vanzin et al . , 2002 ) , arabinogalactan protein ( wu et al . , 2010 ) , and n - linked glycoproteins ( strasser et al . , 2004 ) incorporated fucal at levels similar to wild - type plants , suggesting that fucal does not evenly label all fucosylated cell wall glycans ( anderson et al . , 2012 ) . we hypothesize that this selectivity might arise from conformational differences in the active sites of different fucosyltransferases , suggesting that click chemistry - compatible sugar analogs might be selectively incorporated into specific polysaccharide networks even if the parent monosaccharide is present in several polymers . structure  activity relationships of click sugar variants containing the alkyne or azide at different positions may elucidate the structural requirements for selective monosaccharide analog incorporation . s4b staining and spinning disk confocal microscopy were used to image cmfs in arabidopsis root tissues ( anderson et al . , 2010 ) , which represent a developmental continuum of dividing , expanding , and differentiated cells ( dolan et al . , s4b - stained cmfs were transversely oriented with respect to the major cellular expansion axis in recently divided cells near the root cap and cells in the early elongation zone . however , older cells in the elongation zone exhibited diagonally oriented cmfs , and fully differentiated cells that were no longer expanding contained longitudinally oriented cmfs ( figure 2a ) . this developmental gradient of cmf orientation agrees with previous electron microscopy studies describing developmental changes in cmf orientation along arabidopsis roots and hypocotyls ( sugimoto et al . , 2000 ; refregier et al . , 2004 ) , suggesting that s4b staining provides a rapid and specific method for cmf imaging . ( a ) a model for cellulose deposition and reorientation in anisotropically elongating root epidermal cells . shortly after division in the root meristematic region , cellulose synthase complexes in epidermal cells deposit cellulose ( red lines ) in a transverse orientation with respect to the elongation axis . this deposition pattern constrains radial expansion of the cell but permits axial expansion as indicated by the arrows above each cell . as epidermal cells elongate , new cellulose layers ( magenta lines , blue lines ) are deposited transversely , while older layers in the apoplast passively reorient from transverse to diagonal , then longitudinal orientations . the reorientation of older cellulose layers reduces the ability of differentiating cells to expand in the axial direction . ( b ) a model for pectin deposition and remodeling in the apoplast . in recently divided cells , putative sites of pectin delivery ( green dots ) can be imaged with fucal and are initially distributed evenly across the cell . as epidermal cells stop elongating , diagonal fibrils begin to appear that eventually reorient toward the longitudinal axis , potentially constraining further axial expansion . fucal incorporation and labeling has been used to image pectic networks along the same root developmental continuum . after 1  h of incorporation , fucal labeling in the root elongation zone appeared as small puncta in the cell wall ( anderson et al . , 2012 ) , potentially representing the locations of vesicle - mediated pectin delivery to the apoplast . after longer incorporation times ( e.g. , 12  h ) , the pattern of fucal labeling in arabidopsis roots exhibited a developmental gradient from the early elongation zone to the differentiation zone . in recently divided cells , fucal - associated fluorescence was homogeneous , likely as a result of cumulative evenly distributed vesicle - mediated polysaccharide delivery . however , cells in the late elongation zone exhibit more organized patterns of puncta that transition to diagonally oriented fibrillar structures in the differentiation zone ( anderson et al . , 2012 ) . these observations suggest that pectic wall material is delivered and organized in distinct patterns during cellular development and that this developmental gradient parallels the reorientation of cmfs during cell elongation ( figure 2b ) . polysaccharide - specific probes can also serve as useful tools for the detailed characterization of mutants affecting cell wall polymer networks . for example , s4b staining has revealed new aspects of wall morphology in previously described arabidopsis mutants defective in cellulose and xyloglucan biosynthesis . the cesa6 mutant procuste1 - 1 ( prc1 - 1 ; desnos et al . , 1996 ) ( fagard et al . , 2000 ) , and prc1 - 1 seedlings exhibited reduced s4b staining compared to wild - type seedlings ( anderson et al . , 2010 ) . additionally , microfibrils in the root elongation zone were more defined but less prevalent , suggesting that fewer intact microfibrils were synthesized , leading to compromised cell walls and increased isotropic expansion of root cells . s4b staining of the xxt1 ; xxt2 mutant , which lacks detectable amounts of xyloglucan ( cavalier et al . , 2008 ; park and cosgrove , 2012a ) , labeled cmf fibers that were more prominently defined and thicker than wild - type cmf fibers , supporting the hypothesis that hemicelluloses prevent cmfs from interacting with each other in wild - type primary cell walls . furthermore , s4b was recently used to characterize cellulose architecture in arabidopsis seed coat mucilage ( harpaz - saad et al . , 2011 ) , leading to the identification of cellulose synthase a5 ( cesa5 ) , fei2 , and salt overly sensitive 5 ( sos5 ) as genes responsible for mucilage cellulose deposition . this study indicates that fluorescent probes like s4b will be useful for cell wall imaging and mutant phenotype identification in multiple developmental contexts . due to the load - bearing function of cmfs in the cell wall , differences in the orientation of these polymers with respect to the growth axis of anisotropically expanding cells has been proposed to critically affect cell shape ( green , 1960 ) . when cmfs are deposited transversely , they resist radial expansion but allow cells to expand axially . however , as cmfs become longitudinally oriented and parallel to the elongation axis , cells are also expected to encounter expansion resistance in the axial direction ( figure 2a ) . previous observations suggest that varying cmf angles may result from reorientation of these polysaccharides after deposition in the apoplast ( refregier et al . , 2004 ) . optical sectioning of s4b - stained tissue indicates that cmfs in the outer cell wall layers of differentiated root epidermal cells are oriented longitudinally with respect to the cellular growth axis , whereas cmfs near the inner face of the wall are oriented transversely ( anderson et al . , 2010 ) . time - lapse imaging of s4b - stained cmfs revealed that the observed changes in orientation are attributable to the rotation of cmfs after they are deposited in the apoplast of root epidermal cells . fucal incorporation and labeling was also used to examine the dynamics of pectic networks in arabidopsis ( anderson et al . , 2012 ) . whereas the amounts of fucal required for incorporation experiments had no effect on normal arabidopsis seedling development , the cu ( i ) necessary to catalyze the cycloaddition reaction was toxic , in agreement with previous in vitro and in vivo observations in other organisms ( agard et al . , 2004 ; brewer , 2010 ; lallana et al . , 2011 ) . as an alternative to in vivo imaging , plants were pulsed with fucal for 1  h and labeled at various chase times ( anderson et al . , 2012 ) . these experiments revealed that fucal - associated fluorescence was initially punctate in the apoplast , but appeared as longitudinal fibers after 12 or 24  h chase periods , suggesting that , like cmfs , pectins become longitudinally oriented over time . as a whole , these observations suggest that polysaccharide networks are highly dynamic during cellular development and highlight the importance of specific polysaccharide - directed probes that are capable of capturing these events . the use of multiple labeling strategies for plant cell wall polysaccharides is altering the perception of the cell wall from that of a rigid case to that of a highly dynamic extracellular organelle that changes during development . however , the complexity of the cell wall necessitates the design of specific imaging probes for these dynamic events . polysaccharide - binding dyes , such as s4b and 7gfe , highlight the utility of fluorescent dyes that are compatible with live - cell imaging and exemplify one avenue for the implementation of new cell wall imaging tools for the future . indeed , we believe that novel cell wall imaging probes will most likely be isolated via high - throughput fluorescent interaction assays against isolated wall components , structure  activity studies of existing fluorophores , or array - based selectivity analyses that have been described for polysaccharide - binding antibodies ( moller et al . , 2008 ; these experiments may lead to the identification of novel fluorophores that label unique polysaccharide populations or networks , so that multiple cell wall polysaccharides can be imaged simultaneously . monosaccharide analogs compatible with click chemistry have some advantages over polysaccharide - specific fluorophores that suggest their utility in a variety of experiments . because click sugars are incorporated metabolically , they label only those polysaccharides synthesized during the incorporation period and are therefore distinct from probes that label all of the existing polysaccharides of a given type . these sugars have the potential to serve as valuable tools for examining the trafficking of polysaccharides from the golgi to the apoplast , elucidating extracellular events that occur after deposition of polysaccharides in the wall , and the isolation of mutants that affect these processes . these experimental goals will be furthered by the application of click sugars that label other polysaccharide networks , and it may be possible to tune the selective incorporation of a given sugar analog by changing the position or identity of the alkynyl or azido functional group . membrane permeable click - compatible coumarin and napthalamide fluorescent probes have been described ( sawa et al . , 2006 ; hsu et al . , 2007 ) , and could be used to image intracellular sites of polysaccharide synthesis to complement surface - specific labeling by membrane impermeable fluorophores , such as alexa fluor dyes . additionally , in vivo click labeling using either copper - free strain - promoted [ 2  +  3 ] cycloaddition reactions ( prescher et al . , 2004 ; dube et al . , 2006 ; laughlin et al . , 2008 ) or chelators that sequester toxic cu ( i ) ( chan et al . , 2004 ; lewis et al . , 2004 ) might be applicable to plant systems and could further facilitate the imaging of cell wall glycans in living cells . by adding a temporal dimension to studies of cell wall architecture , the approaches described here provide new ways of examining the synthesis , deposition , and dynamics of plant cell walls that will further our understanding of these remarkable bio - materials , both in the context of plant development and as renewable resources for human use . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
plant cell walls are composed of interlinked polymer networks consisting of cellulose , hemicelluloses , pectins , proteins , and lignin . 
 the ordered deposition of these components is a dynamic process that critically affects the development and differentiation of plant cells . 
 however , our understanding of cell wall synthesis and remodeling , as well as the diverse cell wall architectures that result from these processes , has been limited by a lack of suitable chemical probes that are compatible with live - cell imaging . in this review , 
 we summarize the currently available molecular toolbox of probes for cell wall polysaccharide imaging in plants , with particular emphasis on recent advances in small molecule - based fluorescent probes . 
 we also discuss the potential for further development of small molecule probes for the analysis of cell wall architecture and dynamics .