Epitope detection chromatography: a method to dissect the structural heterogeneity and inter‐connections of plant cell‐wall matrix glycans

Plant cell walls are complex, multi‐macromolecular assemblies of glycans and other molecules and their compositions and molecular architectures vary extensively. Even though the chemistry of cell‐wall glycans is now well understood, it remains a challenge to understand the diversity of glycan configurations and interactions in muro, and how these relate to changes in the biological and mechanical properties of cell walls. Here we describe in detail a method called epitope detection chromatography analysis of cell‐wall matrix glycan sub‐populations and inter‐connections. The method combines chromatographic separations with use of glycan‐directed monoclonal antibodies as detection tools. The high discrimination capacity and high sensitivity for the detection of glycan structural features (epitopes) provided by use of established monoclonal antibodies allows the study of oligosaccharide motifs on sets of cell‐wall glycans in small amounts of plant materials such as a single organ of Arabidopsis thaliana without the need for extensive purification procedures. We describe the use of epitope detection chromatography to assess the heterogeneity of xyloglucan and pectic rhamnogalacturonan I sub‐populations and their modulation in A. thaliana organs.     

Arabidopsis plants expressing only the redox‐regulated Rca‐α isoform have constrained photosynthesis and plant growth

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca‐α and Rca‐β. Redox modulation of Rca‐α regulates the function of Rca‐α and Rca‐β acting together to control Rubisco activation. Although Arabidopsis Rca‐α alone less effectively activates Rubisco in vitro, it is not known how CO₂ assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca‐α in the absence of Rca‐β (‘Rca‐α only’ lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca‐α only lines, and they maintained a lower rate of CO₂ assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca‐α only are less effective in initiating and sustaining the activation of Rubisco than when Rca‐β is also present. Currently there are no examples of any plant species that naturally express Rca‐α only but numerous examples of species expressing Rca‐β only. That Rca‐α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.     

The Arabidopsis Tellurite resistance C protein together with ALB3 is involved in photosystem II protein synthesis

Assembly of photosystem II (PSII) occurs sequentially and requires several auxiliary proteins, such as ALB3 (ALBINO3). Here, we describe the role of the Arabidopsis thaliana thylakoid membrane protein Tellurite resistance C (AtTerC) in this process. Knockout of AtTerC was previously shown to be seedling‐lethal. This phenotype was rescued by expressing TerC fused C–terminally to GFP in the terc–1 background, and the resulting terc–1_TerC–GFP line and an artificial miRNA‐based knockdown allele (amiR‐TerC) were used to analyze the TerC function. The alterations in chlorophyll fluorescence and thylakoid ultrastructure observed in amiR‐TerC plants and terc–1_TerC–GFP were attributed to defects in PSII. We show that this phenotype resulted from a reduction in the rate of de novo synthesis of PSII core proteins, but later steps in PSII biogenesis appeared to be less affected. Yeast two‐hybrid assays showed that TerC interacts with PSII proteins. In particular, its interaction with the PSII assembly factor ALB3 has been demonstrated by co‐immunoprecipitation. ALB3 is thought to assist in incorporation of CP43 into PSII via interaction with Low PSII Accumulation2 (LPA2) Low PSII Accumulation3 (LPA3). Homozygous lpa2 mutants expressing amiR‐TerC displayed markedly exacerbated phenotypes, leading to seedling lethality, indicating an additive effect. We propose a model in which TerC, together with ALB3, facilitates de novo synthesis of thylakoid membrane proteins, for instance CP43, at the membrane insertion step.     

Phytotyping4D: a light‐field imaging system for non‐invasive and accurate monitoring of spatio‐temporal plant growth

Integrative studies of plant growth require spatially and temporally resolved information from high‐throughput imaging systems. However, analysis and interpretation of conventional two‐dimensional images is complicated by the three‐dimensional nature of shoot architecture and by changes in leaf position over time, termed hyponasty. To solve this problem, Phytotyping^4D uses a light‐field camera that simultaneously provides a focus image and a depth image, which contains distance information about the object surface. Our automated pipeline segments the focus images, integrates depth information to reconstruct the three‐dimensional architecture, and analyses time series to provide information about the relative expansion rate, the timing of leaf appearance, hyponastic movement, and shape for individual leaves and the whole rosette. Phytotyping^4D was calibrated and validated using discs of known sizes, and plants tilted at various orientations. Information from this analysis was integrated into the pipeline to allow error assessment during routine operation. To illustrate the utility of Phytotyping^4D, we compare diurnal changes in Arabidopsis thaliana wild‐type Col‐0 and the starchless pgm mutant. Compared to Col‐0, pgm showed very low relative expansion rate in the second half of the night, a transiently increased relative expansion rate at the onset of light period, and smaller hyponastic movement including delayed movement after dusk, both at the level of the rosette and individual leaves. Our study introduces light‐field camera systems as a tool to accurately measure morphological and growth‐related features in plants.     

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β‐(1,4)‐linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one‐half of the xylosyl residues are O‐acetylated at C‐2 or C‐3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy.     

Cytosolic invertases contribute to cellulose biosynthesis and influence carbon partitioning in seedlings of Arabidopsis thaliana

In plants, UDP‐glucose is the direct precursor for cellulose biosynthesis, and can be converted into other NDP‐sugars required for the biosynthesis of wall matrix polysaccharides. UDP‐glucose is generated from sucrose by two distinct metabolic pathways. The first pathway is the direct conversion of sucrose to UDP‐glucose and fructose by sucrose synthase. The second pathway involves sucrose hydrolysis by cytosolic invertase (CINV), conversion of glucose to glucose‐6‐phosphate and glucose‐1‐phosphate, and UDP‐glucose generation by UDP‐glucose pyrophosphorylase (UGP). Previously, Barratt et al. (Proc. Natl Acad. Sci. USA, 106, 2009 and 13124) have found that an Arabidopsis double mutant lacking CINV1 and CINV2 displayed drastically reduced growth. Whether this reduced growth is due to deficient cell wall production caused by limited UDP‐glucose supply, pleiotropic effects, or both, remained unresolved. Here, we present results indicating that the CINV/UGP pathway contributes to anisotropic growth and cellulose biosynthesis in Arabidopsis. Biochemical and imaging data demonstrate that cinv1 cinv2 seedlings are deficient in UDP‐glucose production, exhibit abnormal cellulose biosynthesis and microtubule properties, and have altered cellulose organization without substantial changes to matrix polysaccharide composition, suggesting that the CINV/UGP pathway is a key metabolic route to UDP‐glucose synthesis in Arabidopsis. Furthermore, differential responses of cinv1 cinv2 seedlings to exogenous sugar supplementation support a function of CINVs in influencing carbon partitioning in Arabidopsis. From these data and those of previous studies, we conclude that CINVs serve central roles in cellulose biosynthesis and carbon allocation in Arabidopsis.     

ERIL1, the plant homologue of ERI‐1, is involved in the processing of chloroplastic rRNAs

Proteins belonging to the enhancer of RNA interference‐1 subfamily of 3′–5′ exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE‐1‐LIKE‐1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3′–5′ exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus‐encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.     

Inhibition of fucosylation of cell wall components by 2‐fluoro 2‐deoxy‐l‐fucose induces defects in root cell elongation

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2‐fluoro 2‐l ‐fucose (2F‐Fuc) reduces root growth at micromolar concentrations. The inability of 2F‐Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F‐Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N‐linked glycans is fully inhibited by 10 μm 2F‐Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F‐Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan‐II (RG‐II). At low concentrations, 2F‐Fuc induced a decrease in RG‐II dimerization. Both RG‐II dimerization and root growth were partially restored in 2F‐Fuc‐treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F‐Fuc was due to a deficiency of RG‐II dimerization. Closer investigation of the 2F‐Fuc‐induced growth phenotype demonstrated that cell division is not affected by 2F‐Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG‐II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG‐II cross‐linking, but that it might also be a signal molecule in the cell wall integrity‐sensing mechanism.     

The role of COBRA‐LIKE 2 function,as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization

The production of hydrophilic mucilage along the course of seed coat epidermal cell differentiation is a common adaptation in angiosperms. Previous studies have identified COBRA‐LIKE 2 (COBL2), a member of the COBRA‐LIKE gene family, as a novel component required for crystalline cellulose deposition in seed coat epidermal cells. In recent years, Arabidopsis seed coat epidermal cells (SCEs), also called mucilage secretory cells, have emerged as a powerful model system for the study of plant cell wall components biosynthesis, secretion, assembly and de muro modification. Despite accumulating data, the molecular mechanism of COBL function remains largely unknown. In the current research, we utilized genetic interactions to study the role of COBL2 as part of the protein network required for seed mucilage production. Using correlative phenotyping of structural and biochemical characteristics, unique features of the cobl2 extruded mucilage are revealed, including: ‘unraveled’ ray morphology, loss of primary cell wall ‘pyramidal’ organization, reduced Ruthenium red staining intensity of the adherent mucilage layer, and increased levels of the monosaccharides arabinose and galactose. Examination of the cobl2cesa5 double mutant provides insight into the interface between COBL function and cellulose deposition. Additionally, genetic interactions between cobl2 and fei1fei2 as well as between each of these mutants to mucilage‐modified 2 (mum2) suggest that COBL2 functions independently of the FEI‐SOS pathway. Altogether, the presented data place COBL2 within the complex protein network required for cell wall deposition in the context of seed mucilage and introduce new methodology expending the seed mucilage phenotyping toolbox.     

Serine and threonine residues of plant STN7 kinase are differentially phosphorylated upon changing light conditions and specifically influence the activity and stability of the kinase

STN7 kinase catalyzes the phosphorylation of the globally most common membrane proteins, the light‐harvesting complex II (LHCII) in plant chloroplasts. STN7 itself possesses one serine (Ser) and two threonine (Thr) phosphosites. We show that phosphorylation of the Thr residues protects STN7 against degradation in darkness, low light and red light, whereas increasing light intensity and far red illumination decrease phosphorylation and induce STN7 degradation. Ser phosphorylation, in turn, occurs under red and low intensity white light, coinciding with the client protein (LHCII) phosphorylation. Through analysis of the counteracting LHCII phosphatase mutant tap38/pph1, we show that Ser phosphorylation and activation of the STN7 kinase for subsequent LHCII phosphorylation are heavily affected by pre‐illumination conditions. Transitions between the three activity states of the STN7 kinase (deactivated in darkness and far red light, activated in low and red light, inhibited in high light) are shown to modulate the phosphorylation of the STN7 Ser and Thr residues independently of each other. Such dynamic regulation of STN7 kinase phosphorylation is crucial for plant growth and environmental acclimation.     

The Arabidopsis thylakoid transporter PHT4;1 influences phosphate availability for ATP synthesis and plant growth

The Arabidopsis phosphate transporter PHT4;1 was previously localized to the chloroplast thylakoid membrane. Here we investigated the physiological consequences of the absence of PHT4;1 for photosynthesis and plant growth. In standard growth conditions, two independent Arabidopsis knockout mutant lines displayed significantly reduced leaf size and biomass but normal phosphorus content. When mutants were grown in high‐phosphate conditions, the leaf phosphorus levels increased and the growth phenotype was suppressed. Photosynthetic measurements indicated that in the absence of PHT4;1 stromal phosphate was reduced to levels that limited ATP synthase activity. This resulted in reduced CO₂ fixation and accumulation of soluble sugars, limiting plant growth. The mutants also displayed faster induction of non‐photochemical quenching than the wild type, in line with the increased contribution of ΔpH to the proton‐motive force across thylakoids. Small‐angle neutron scattering showed a smaller lamellar repeat distance, whereas circular dichroism spectroscopy indicated a perturbed long‐range order of photosystem II (PSII) complexes in the mutant thylakoids. The absence of PHT4;1 did not alter the PSII repair cycle, as indicated by wild‐type levels of phosphorylation of PSII proteins, inactivation and D1 protein degradation. Interestingly, the expression of genes for several thylakoid proteins was downregulated in the mutants, but the relative levels of the corresponding proteins were either not affected or could not be discerned. Based on these data, we propose that PHT4;1 plays an important role in chloroplast phosphate compartmentation and ATP synthesis, which affect plant growth. It also maintains the ionic environment of thylakoids, which affects the macro‐organization of complexes and induction of photoprotective mechanisms.     

Boron bridging of rhamnogalacturonan‐II,monitored by gel electrophoresis,occurs during polysaccharide synthesis and secretion but not post‐secretion

The cell‐wall pectic domain rhamnogalacturonan‐II (RG‐II) is cross‐linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross‐linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron‐bridged) RG‐II, we confirmed that Pb²⁺ promotes H₃BO₃‐dependent dimerisation in vitro. H₃BO₃ concentrations as high as 50 mm did not prevent cross‐linking. For in‐vivo experiments, we successfully cultured ‘Paul's Scarlet’ rose (Rosa sp.) cells in boron‐free medium: their wall‐bound pectin contained monomeric RG‐II domains but no detectable dimers. Thus pectins containing RG‐II domains can be held in the wall other than via boron bridges. Re‐addition of H₃BO₃ to 3.3 μm triggered a gradual appearance of RG‐II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG‐II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG‐II dimers. We conclude that RG‐II normally becomes boron‐bridged during synthesis or secretion but not post‐secretion. Supporting this conclusion, exogenous [³H]RG‐II was neither dimerised in the medium nor cross‐linked to existing wall‐associated RG‐II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG‐II domains have a brief window of opportunity for boron‐bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron‐bridging does not readily occur.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

Symbiont strain is the main determinant of variation in Wolbachia‐mediated protection against viruses across Drosophila species

Wolbachia is a common heritable bacterial symbiont in insects. Its evolutionary success lies in the diverse phenotypic effects it has on its hosts coupled to its propensity to move between host species over evolutionary timescales. In a survey of natural host–symbiont associations in a range of Drosophila species, we found that 10 of 16 Wolbachia strains protected their hosts against viral infection. By moving Wolbachia strains between host species, we found that the symbiont genome had a much greater influence on the level of antiviral protection than the host genome. The reason for this was that the level of protection depended on the density of the symbiont in host tissues, and Wolbachia rather than the host‐controlled density. The finding that virus resistance and symbiont density are largely under the control of symbiont genes in this system has important implications both for the evolution of these traits and for public health programmes using Wolbachia to prevent mosquitoes from transmitting disease.     

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.     

An Arabidopsis berberine bridge enzyme‐like protein specifically oxidizes cellulose oligomers and plays a role in immunity

The plant cell wall is the barrier that pathogens must overcome to cause a disease, and to this end they secrete enzymes that degrade the various cell wall components. Due to the complexity of these components, several types of oligosaccharide fragments may be released during pathogenesis and some of these can act as damage‐associated molecular patterns (DAMPs). Well‐known DAMPs are the oligogalacturonides (OGs) released upon degradation of homogalacturonan and the products of cellulose breakdown, i.e. the cellodextrins (CDs). We have previously reported that four Arabidopsis berberine bridge enzyme‐like (BBE‐like) proteins (OGOX1–4) oxidize OGs and impair their elicitor activity. We show here that another Arabidopsis BBE‐like protein, which is expressed coordinately with OGOX1 during immunity, specifically oxidizes CDs with a preference for cellotriose (CD3) and longer fragments (CD4–CD6). Oxidized CDs show a negligible elicitor activity and are less easily utilized as a carbon source by the fungus Botrytis cinerea. The enzyme, named CELLOX (cellodextrin oxidase), is encoded by the gene At4 g20860. Plants overexpressing CELLOX display an enhanced resistance to B. cinerea, probably because oxidized CDs are a less valuable carbon source. Thus, the capacity to oxidize and impair the biological activity of cell wall‐derived oligosaccharides seems to be a general trait of the family of BBE‐like proteins, which may serve to homeostatically control the level of DAMPs to prevent their hyperaccumulation.