High‐throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray‐based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.     

Putrescine alleviates aluminum toxicity in rice (Oryza sativa) by reducing cell wall Al contents in an ethylene‐dependent manner

Aluminum (Al³⁺) toxicity in acidic soils limits crop productivity worldwide. In this study, we found that putrescine (PUT) significantly alleviates Al toxicity in rice roots. The addition of 0.1 mM PUT promoted root elongation and reduced the Al content in the root apices of Nipponbare (Nip) and Kasalath (Kas) rice under Al toxicity conditions. Exogenous treatment with PUT reduced the cell wall Al content by reducing polysaccharide (pectin and hemicellulose) levels and pectin methylesterase (PME) activity in roots and decreased the translocation of Al from the external environment to the cytoplasm by downregulating the expression of OsNRAT1, which responsible to encode an Al transporter protein Nrat1 (Nramp aluminum transporter 1). The addition of PUT under Al toxicity conditions significantly inhibited ethylene emissions and suppressed the expression of genes involved in ethylene biosynthesis. Treatment with the ethylene precursor 1‐aminocylopropane‐1‐carboxylic acid (ACC) significantly improved ethylene emission, inhibited root elongation, increased the Al accumulation in root tips and the root cell wall, and increased cell wall pectin and hemicellulose contents in both rice cultivars under Al toxicity conditions. The ethylene biosynthesis antagonist aminoethoxyvinylglycine (AVG, inhibitor of the ACC synthase) had the opposite effect and reduced PME activity. Together, our results show that PUT decreases the cell wall Al contents by suppressing ethylene emissions and decreases the symplastic Al levels by downregulating OsNRAT1 in rice.     

PARVUS affects aluminium sensitivity by modulating the structure of glucuronoxylan in Arabidopsis thaliana

Glucuronoxylan (GX), an important component of hemicellulose in the cell wall, appears to affect aluminium (Al) sensitivity in plants. To investigate the role of GX in cell‐wall‐localized xylan, we examined the Arabidopsis thaliana parvus mutant in detail. This mutant lacks α‐D‐glucuronic acid (GlcA) side chains in GX and has greater resistance to Al stress than wild‐type (WT) plants. The parvus mutant accumulated lower levels of Al in its roots and cell walls than WT despite having cell wall pectin content and pectin methylesterase (PME) activity similar to those of WT. Our results suggest that the altered properties of hemicellulose in the mutant contribute to its decreased Al accumulation. Although we observed almost no differences in hemicellulose content between parvus and WT under control conditions, less Al was retained in parvus hemicellulose than in WT. This observation is consistent with the finding that GlcA substitutions in WT GX, but not mutant GX, were increased under Al stress. Taken together, these results suggest that the modulation of GlcA levels in GX affects Al resistance by influencing the Al binding capacity of the root cell wall in Arabidopsis.     

Pectin methylesterase modulates aluminium sensitivity in Zea mays and Solanum tuberosum

Cell suspension cultures of Zeamays L. were adapted to grow under conditions of NaCl stress, which increased the cell‐wall pectin content of these cells by 31% compared with unadapted cells (controls). Both cultures were treated for 5 or 10 min with pectin methylesterase (PME) and afterwards incubated in the presence of Al for 2 h. The different capabilities of the cells to synthesise callose due to pre‐treatment were taken into account by calculating relative Al‐induced callose induction (digitonin=100%). Only in salt‐adapted cells with a degree of methylation of cell‐wall pectin (DM) decreasing from 34% (control) to 13%, did PME treatment enhance total and BaCl₂‐non‐exchangeable Al contents and Al sensitivity as indicated by increased callose formation. In a further step, a wider variation in DM was achieved by subculturing the NaCl‐adapted cells for up to 3 weeks without NaCl supply and adapting them to the cellulose‐synthesis inhibitor 2,6‐dichlorbenzonitrile (DCB). This reduced DM to 26%, while short‐term treatment with pectolyase resulted in the lowest DM (12%). After the 2 h Al treatment, there was a close negative relationship between DM and relative callose formation of Al contents, with the exception of pectolyase‐treated cells. In addition, intact plants of Solanumtuberosum L. genotypes were characterised for their Al sensitivity in hydroponics using root elongation, Al‐induced callose formation and Al contents of root tips as parameters. Based on all three parameters, the transgenic potato mutant overexpressing PME proved to be more Al‐sensitive than the wild type, the Al‐resistant and even the Al‐sensitive potato cultivar. Especially in the root tips (1 cm), Al treatment (2 h, 50 μM) increased the activity of PME more in the Al‐sensitive than in the Al‐resistant genotypes. The presented data emphasise the importance of the DM of the pectin matrix and the activity of PME for the expression of Al toxicity and Al resistance.     

Aluminium effects on mechanical properties of cell wall analogues

Aluminium (Al) toxicity adversely impacts plant productivity in acid soils by restricting root growth and although several mechanisms are involved the physiological basis of decreased root elongation remains unclear. Understanding the primary mechanisms of Al rhizotoxicity is hindered due to the rapid effects of soluble Al on root growth and the close proximity of many cellular components within the cell wall, plasma membrane, cytosol and nucleus with which Al may react. To overcome some of these difficulties, we report on a novel method for investigating Al interactions with Komagataeibacter xylinus bacterial cellulose (BC)‐pectin composites as cell wall analogues. The growth of K. xylinus in the presence of various plant cell wall polysaccharides, such as pectin, has provided a unique in vitro model system with which to investigate the interactions of Al with plant cell wall polysaccharides. The BC‐pectin composites reacted in a similar way with Al as do plant cell walls, providing insights into the effects of Al on the mechanical properties of the BC‐pectin composites as cell wall analogues. Our findings indicated that there were no significant effects of Al (4–160 μM) on the tensile stress, tensile strain or Young's modulus of the composites. This finding was consistent with cellulose, not pectin, being the major load bearing component in BC‐pectin composites, as is also the case in plant cell walls.     

Cell wall pectin content modulates aluminium sensitivity of Zea mays (L.) cells grown in suspension culture

The pectin content of the cell walls of maize suspension cells was modified to investigate its role in the expression of aluminium (Al) toxicity. Long‐term adaptation to NaCl or to the cellulose synthesis inhibitor 2,6‐dichlorbenzonitirle (DCB) increased the pectin content by 31 and 86%, respectively. Subculturing salt‐adapted cells for up to 3 weeks without NaCl supply or treatment of cells with pectolyase for up to 15 min reduced pectin contents by up to 46%. Such pre‐cultured cells were incubated for 2 h in presence of Al. There was a close positive correlation between pectin and both total and BaCl₂ non‐exchangeable Al contents. Aluminium‐induced callose formation as an indicator of Al injury was closely positively correlated to the loss of cell viability. In NaCl‐adapted and pectolyase‐treated cells, Al‐induced callose formation was reduced when compared with normal cells. However, there was a close positive relationship between pectin contents and relative callose induction (digitonin‐induced callose formation, reflecting the different capabilities of cells to synthesize callose set to 100%) indicating that cells with higher pectin contents are more Al‐sensitive. The results presented support our view that the binding of Al to the cell wall pectin‐matrix represents an important step in the expression of Al toxicity.     

Arabinoxylan, β‐glucan and pectin in barley and malt endosperm cell walls: a microstructure study using CLSM and cryo‐SEM

The architecture of endosperm cell walls in Hordeum vulgare (barley) differs remarkably from that of other grass species and is affected by germination or malting. Here, the cell wall microstructure is investigated using (bio)chemical analyses, cryogenic scanning electron microscopy (cryo‐SEM) and confocal laser scanning microscopy (CLSM) as the main techniques. The relative proportions of β‐glucan, arabinoxylan and pectin in cell walls were 61, 34 and 5%, respectively. The average thickness of a single endosperm cell wall was 0.30 µm, as estimated by the cryo‐SEM analysis of barley seeds, which was reduced to 0.16 µm after malting. After fluorescent staining, 3D confocal multiphoton microscopy (multiphoton CLSM) imaging revealed the complex cell wall architecture. The endosperm cell wall is composed of a structure in which arabinoxylan and pectin are colocalized on the outside, with β‐glucan depositions on the inside. During germination, arabinoxylan and β‐glucan are hydrolysed, but unlike β‐glucan, arabinoxylan remains present in defined cell walls in malt. Integrating the results, an enhanced model for the endosperm cell walls in barley is proposed.     

IDL6‐HAE/HSL2 impacts pectin degradation and resistance to Pseudomonas syringae pv tomato DC3000 in Arabidopsis leaves

Plant cell walls undergo dynamic structural and chemical changes during plant development and growth. Floral organ abscission and lateral root emergence are both accompanied by cell‐wall remodeling, which involves the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)‐derived peptide and its receptors, HAESA (HAE) and HAESA‐LIKE2 (HSL2). Plant cell walls also act as barriers against pathogenic invaders. Thus, the cell‐wall remodeling during plant development could have an influence on plant resistance to phytopathogens. Here, we identified IDA‐like 6 (IDL6), a gene that is prominently expressed in Arabidopsis leaves. IDL6 expression in Arabidopsis leaves is significantly upregulated when the plant is suffering from attacks of the bacterial Pseudomonas syringae pv. tomato (Pst) DC3000. IDL6 overexpression and knockdown lines respectively decrease and increase the Arabidopsis resistance to Pst DC3000, indicating that the gene promotes the Arabidopsis susceptibility to Pst DC3000. Moreover, IDL6 promotes the expression of a polygalacturonase (PG) gene, ADPG2, and increases PG activity in Arabidopsis leaves, which in turn reduces leaf pectin content and leaf robustness. ADPG2 overexpression restrains Arabidopsis resistance to Pst DC3000, whereas ADPG2 loss‐of‐function mutants increase the resistance to the bacterium. Pst DC3000 infection elevates the ADPG2 expression partially through HAE and HSL2. Taken together, our results suggest that IDL6‐HAE/HSL2 facilitates the ingress of Pst DC3000 by promoting pectin degradation in Arabidopsis leaves, and Pst DC3000 might enhance its infection by manipulating the IDL6‐HAE/HSL2‐ADPG2 signaling pathway.     

Genotypic differences in Al resistance and the role of cell-wall pectin in Al exclusion from the root apex in Fagopyrum tataricum

Background and Aims

Aluminium (Al) toxicity is one of the factors limiting crop production on acid soils. However, genotypic differences exist among plant species or cultivars in response to Al toxicity. This study aims to investigate genotypic differences among eight cultivars of tatary buckwheat (Fagopyrum tataricum) for Al resistance and explore the possible mechanisms of Al resistance.

Methods

Al resistance was evaluated based on relative root elongation (root elongation with Al/root elongation without Al). Root apex Al content, pectin content and exudation of root organic acids were determined and compared.

Key Results

Genotypic differences among the eight cultivars were correlated with exclusion of Al from the root apex. However, there was a lack of correlation between Al exclusion and Al-induced oxalate secretion. Interestingly, cell-wall pectin content of the root apex was generally lower in Al-resistant cultivars than in Al-sensitive cultivars. Although we were unable to establish a significant correlation between Al exclusion and pectin content among the eight cultivars, a strong correlation could be established among six cultivars, in which the pectin content in the most Al-resistant cultivar ‘Chuan’ was significantly lower than that in the most Al-sensitive cultivar ‘Liuku2’. Furthermore, root apex cell-wall pectin methylesterase activity (PME) was similar in ‘Chuan’ and ‘Liuku2’ in the absence of Al, but Al treatment resulted in increased PME activity in ‘Liuku2’ compared with ‘Chuan’. Immunolocalization of pectins also showed that the two cultivars had similar amounts of either low-methyl-ester pectins or high-methyl-ester pectins in the absence of Al, but Al treatment resulted in a more significant increase of low-methyl-ester pectins and decrease of high-methyl-ester pectins in ‘Liuku2’.

Conclusions

Cell-wall pectin content may contribute, at least in part, to differential Al resistance among tatary buckwheat cultivars.     

UDP‐Api/UDP‐Xyl synthases affect plant development by controlling the content of UDP‐Api to regulate the RG‐II‐borate complex

Rhamnogalacturonan‐II (RG‐II) is structurally the most complex glycan in higher plants, containing 13 different sugars and 21 distinct glycosidic linkages. Two monomeric RG‐II molecules can form an RG‐II‐borate diester dimer through the two apiosyl (Api) residues of side chain A to regulate cross‐linking of pectin in the cell wall. But the relationship of Api biosynthesis and RG‐II dimer is still unclear. In this study we investigated the two homologous UDP‐D‐apiose/UDP‐D‐xylose synthases (AXSs) in Arabidopsis thaliana that synthesize UDP‐D‐apiose (UDP‐Api). Both AXSs are ubiquitously expressed, while AXS2 has higher overall expression than AXS1 in the tissues analyzed. The homozygous axs double mutant is lethal, while heterozygous axs1/+ axs2 and axs1 axs2/+ mutants display intermediate phenotypes. The axs1/+ axs2 mutant plants are unable to set seed and die. By contrast, the axs1 axs2/+ mutant plants exhibit loss of shoot and root apical dominance. UDP‐Api content in axs1 axs2/+ mutants is decreased by 83%. The cell wall of axs1 axs2/+ mutant plants is thicker and contains less RG‐II‐borate complex than wild‐type Col‐0 plants. Taken together, these results provide direct evidence of the importance of AXSs for UDP‐Api and RG‐II‐borate complex formation in plant growth and development.     

Arabidopsis PME17 Activity can be Controlled by Pectin Methylesterase Inhibitor4

The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.     

Disruption of cellulose synthesis by 2,6‐dichlorobenzonitrile affects the structure of the cytoskeleton and cell wall construction in Arabidopsis

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6‐dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose‐ and time‐dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N‐ST‐YFP and TGN labelled with VHA‐a1‐GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.     

Altered cell wall properties are responsible for ammonium‐reduced aluminium accumulation in rice roots

The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH₄⁺) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al‐binding assay revealed that neither NH₄⁺ nor NO₃^? altered the Al‐binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH₄⁺‐supplied roots displayed lower Al‐binding capacity than those from NO₃^?‐supplied roots when grown in non‐buffered solutions. Fourier‐transform infrared microspectroscopy analysis revealed that, compared with NO₃^?‐supplied roots, NH₄⁺‐supplied roots possessed fewer Al‐binding groups (‐OH and COO‐) and lower contents of pectin and hemicellulose. However, when grown in pH‐buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al‐binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH₄⁺‐reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH₄⁺ uptake rather than direct competition for the cell wall binding sites between Al³⁺ and NH₄⁺.     

Plant cell wall imaging by metabolic click‐mediated labelling of rhamnogalacturonan II using azido 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid

In plants, 3‐deoxy‐d ‐manno‐oct‐2‐ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan‐II (RG‐II). Incubation of 4‐day‐old light‐grown Arabidopsis seedlings or tobacco BY‐2 cells with 8‐azido 8‐deoxy Kdo (Kdo‐N₃) followed by coupling to an alkyne‐containing fluorescent probe resulted in the specific in muro labelling of RG‐II through a copper‐catalysed azide–alkyne cycloaddition reaction. CMP‐Kdo synthetase inhibition and competition assays showing that Kdo and D‐Ara, a precursor of Kdo, but not L‐Ara, inhibit incorporation of Kdo‐N₃ demonstrated that incorporation of Kdo‐N₃ occurs in RG‐II through the endogenous biosynthetic machinery of the cell. Co‐localisation of Kdo‐N₃ labelling with the cellulose‐binding dye calcofluor white demonstrated that RG‐II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo‐N₃ and an alkynated derivative of L‐fucose that incorporates into rhamnogalacturonan I, co‐localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click‐mediated labelling with Kdo‐N₃ provides an efficient method to study the synthesis and redistribution of RG‐II during root growth.     

Multiple cyclic nucleotide‐gated channels coordinate calcium oscillations and polar growth of root hairs

Calcium gradients underlie polarization in eukaryotic cells. In plants, a tip‐focused Ca²⁺‐gradient is fundamental for rapid and unidirectional cell expansion during epidermal root hair development. Here we report that three members of the cyclic nucleotide‐gated channel family are required to maintain cytosolic Ca²⁺ oscillations and the normal growth of root hairs. CNGC6, CNGC9 and CNGC14 were expressed in root hairs, with CNGC9 displaying the highest root hair specificity. In individual channel mutants, morphological defects including root hair swelling and branching, as well as bursting, were observed. The developmental phenotypes were amplified in the three cngc double mutant combinations. Finally, cngc6/9/14 triple mutants only developed bulging trichoblasts and could not form normal root hair protrusions because they burst after the transition to the rapid growth phase. Prior to developmental defects, single and double mutants showed increasingly disturbed patterns of Ca²⁺ oscillations. We conclude that CNGC6, CNGC9 and CNGC14 fulfill partially but not fully redundant functions in generating and maintaining tip‐focused Ca²⁺ oscillations, which are fundamental for proper root hair growth and polarity. Furthermore, the results suggest that these calmodulin‐binding and Ca²⁺‐permeable channels organize a robust tip‐focused oscillatory calcium gradient, which is not essential for root hair initiation but is required to control the integrity of the root hair after the transition to the rapid growth phase. Our findings also show that root hairs possess a large ability to compensate calcium‐signaling defects, and add new players to the regulatory network, which coordinates cell wall properties and cell expansion during polar root hair growth.     

Cell wall polysaccharides are specifically involved in the exclusion of aluminum from the rice root apex

Rice (Oryza sativa) is the most aluminum (Al)-resistant crop species among the small-grain cereals, but the mechanisms responsible for this trait are still unclear. Using two rice cultivars differing in Al resistance, rice sp. japonica 'Nipponbare' (an Al-resistant cultivar) and rice sp. indica 'Zhefu802' (an Al-sensitive cultivar), it was found that Al content in the root apex (0-10 mm) was significantly lower in Al-resistant 'Nipponbare' than in sensitive 'Zhefu802', with more of the Al localized to cell walls in 'Zhefu802', indicating that an Al exclusion mechanism is operating in 'Nipponbare'. However, neither organic acid efflux nor changes in rhizosphere pH appear to be responsible for the Al exclusion. Interestingly, cell wall polysaccharides (pectin, hemicellulose 1, and hemicellulose 2) in the root apex were found to be significantly higher in 'Zhefu802' than in 'Nipponbare' in the absence of Al, and Al exposure increased root apex hemicellulose content more significantly in 'Zhefu802'. Root tip cell wall pectin methylesterase (PME) activity was constitutively higher in 'Zhefu802' than in 'Nipponbare', although Al treatment resulted in increased PME activity in both cultivars. Immunolocalization of pectins showed a higher proportion of demethylated pectins in 'Zhefu802', indicating a higher proportion of free pectic acid residues in the cell walls of 'Zhefu802' root tips. Al adsorption and desorption kinetics of root tip cell walls also indicated that more Al was adsorbed and bound Al was retained more tightly in 'Zhefu802', which was consistent with Al content, PME activity, and pectin demethylesterification results. These responses were specific to Al compared with other metals (CdCl(2), LaCl(3), and CuCl(2)), and the ability of the cell wall to adsorb these metals was also not related to levels of cell wall pectins. All of these results suggest that cell wall polysaccharides may play an important role in excluding Al specifically from the rice root apex.