Barley Nodulin 26-like intrinsic protein permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification.

Aquaporins can facilitate the passive movement of water, small polar molecules, and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols, were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.

Engineering of substrate specificity in a plant cell-wall modifying enzyme through alterations of carboxyl-terminal amino acid residues.

Structural determinants of substrate recognition remain inadequately defined in broad specific cell-wall modifying enzymes, termed xyloglucan xyloglucosyl transferases (XETs). Here, we investigate the Tropaeolum majus seed TmXET6.3 isoform, a member of the GH16_20 subfamily of the GH16 network. This enzyme recognises xyloglucan (XG)-derived donors and acceptors, and a wide spectrum of other chiefly saccharide substrates, although it lacks the activity with homogalacturonan (pectin) fragments. We focus on defining the functionality of carboxyl-terminal residues in TmXET6.3, which extend acceptor binding regions in the GH16_20 subfamily but are absent in the related GH16_21 subfamily. Site-directed mutagenesis using double to quintuple mutants in the carboxyl-terminal region - substitutions emulated on barley XETs recognising the XG/penta-galacturonide acceptor substrate pair - demonstrated that this activity could be gained in TmXET6.3. We demonstrate the roles of semi-conserved Arg238 and Lys237 residues, introducing a net positive charge in the carboxyl-terminal region (which complements a negative charge of the acidic penta-galacturonide) for the transfer of xyloglucan fragments. Experimental data, supported by molecular modelling of TmXET6.3 with the XG oligosaccharide donor and penta-galacturonide acceptor substrates, indicated that they could be accommodated in the active site. Our findings support the conclusion on the significance of positively charged residues at the carboxyl terminus of TmXET6.3 and suggest that a broad specificity could be engineered via modifications of an acceptor binding site. The definition of substrate specificity in XETs should prove invaluable for defining the structure, dynamics, and function of plant cell walls, and their metabolism; these data could be applicable in various biotechnologies.

Enzymes in 3D: Synthesis, remodelling, and hydrolysis of cell wall (1,3;1,4)-ß-glucans.

Recent breakthroughs in structural biology have provided valuable new insights into enzymes involved in plant cell wall metabolism. More specifically, the molecular mechanism of synthesis of (1,3;1,4)-ß-glucans, which are widespread in cell walls of commercially important cereals and grasses, has been the topic of debate and intense research activity for decades. However, an inability to purify these integral membrane enzymes or apply transgenic approaches without interpretative problems associated with pleiotropic effects has presented barriers to attempts to define their synthetic mechanisms. Following the demonstration that some members of the CslF sub-family of GT2 family enzymes mediate (1,3;1,4)-ß-glucan synthesis, the expression of the corresponding genes in a heterologous system that is free of background complications has now been achieved. Biochemical analyses of the (1,3;1,4)-ß-glucan synthesized in vitro, combined with 3-dimensional (3D) cryogenic-electron microscopy and AlphaFold protein structure predictions, have demonstrated how a single CslF6 enzyme, without exogenous primers, can incorporate both (1,3)- and (1,4)-ß-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3;1,4)-ß-glucan endo- and exohydrolases have allowed the mechanisms of (1,3;1,4)-ß-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 ß-glucan exohydrolase recently revealed a previously unknown and remarkable molecular mechanism with reactant trajectories through which a polysaccharide exohydrolase can act with a processive action pattern. The availability of high-quality protein 3D structural predictions should prove invaluable for defining structures, dynamics, and functions of other enzymes involved in plant cell wall metabolism in the immediate future.

Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism.

Processive and distributive catalysis defines the conversion continuum, thus underpinning the transformation of oligo- and polymeric substrates by enzymes. Distributive catalysis follows an association-transformation-dissociation pattern during the formation of enzyme-reactant complexes, whereas during processive catalysis, enzymes partner with substrates and complete multiple catalytic events before dissociation from an enzyme-substrate complex. Here, we focus on processive catalysis in glycoside hydrolases (GHs), which ensures efficient conversions of substrates with high precision, and has the advantage over distributive catalysis in efficiency. The work presented here examines a recent discovery of substrate-product-assisted processive catalysis in the GH3 family enzymes with enclosed pocket-shaped active sites. We detail how GH3 ß-d-glucan glucohydrolases exploit a transiently formed lateral pocket for product displacement and reactants sliding (or translocation motion) through the catalytic site without dissociation, including movements during nanoscale binding/unbinding and sliding. The phylogenetic tree of putative 550 Archaean, bacterial, fungal, Viridiplantae, and Metazoan GH3 entries resolved seven lineages that corresponded to major substrate specificity groups. This analysis indicates that two tryptophan residues in plant ß-d-glucan glucohydrolases that delineate the catalytic pocket, and infer broad specificity, high catalytic efficiency, and substrate-product-assisted processivity, have evolved through a complex evolutionary process, including horizontal transfer and neo-functionalisation. We conclude that the definition of thermodynamic and mechano-structural properties of processive enzymes is fundamentally important for theoretical and practical applications in bioengineering applicable in various biotechnologies.

Definition of the Acceptor Substrate Binding Specificity in Plant Xyloglucan Endotransglycosylases Using Computational Chemistry.

Xyloglucan endotransglycosylases (XETs) play key roles in the remodelling and reconstruction of plant cell walls. These enzymes catalyse homo-transglycosylation reactions with xyloglucan-derived donor and acceptor substrates and hetero-transglycosylation reactions with a variety of structurally diverse polysaccharides. In this work, we describe the basis of acceptor substrate binding specificity in non-specific Tropaeolum majus (TmXET6.3) and specific Populus tremula x tremuloides (PttXET16A) XETs, using molecular docking and molecular dynamics (MD) simulations combined with binding free energy calculations. The data indicate that the enzyme-donor (xyloglucan heptaoligosaccharide or XG-OS7)/acceptor complexes with the linear acceptors, where a backbone consisted of glucose (Glc) moieties linked via (1,4)- or (1,3)-ß-glycosidic linkages, were bound stably in the active sites of TmXET6.3 and PttXET16A. Conversely, the acceptors with the (1,6)-ß-linked Glc moieties were bound stably in TmXET6.3 but not in PttXET16A. When in the (1,4)-ß-linked Glc containing acceptors, the saccharide moieties were replaced with mannose or xylose, they bound stably in TmXET6.3 but lacked stability in PttXET16A. MD simulations of the XET-donor/acceptor complexes with acceptors derived from (1,4;1,3)-ß-glucans highlighted the importance of (1,3)-ß-glycosidic linkages and side chain positions in the acceptor substrates. Our findings explain the differences in acceptor binding specificity between non-specific and specific XETs and associate theoretical to experimental data.

The evolutionary advantage of an aromatic clamp in plant family 3 glycoside exo-hydrolases.

In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.

Broad Specific Xyloglucan:Xyloglucosyl Transferases Are Formidable Players in the Re-Modelling of Plant Cell Wall Structures.

Plant xyloglucan:xyloglucosyl transferases, known as xyloglucan endo-transglycosylases (XETs) are the key players that underlie plant cell wall dynamics and mechanics. These fundamental roles are central for the assembly and modifications of cell walls during embryogenesis, vegetative and reproductive growth, and adaptations to living environments under biotic and abiotic (environmental) stresses. XET enzymes (EC 2.4.1.207) have the ß-sandwich architecture and the ß-jelly-roll topology, and are classified in the glycoside hydrolase family 16 based on their evolutionary history. XET enzymes catalyse transglycosylation reactions with xyloglucan (XG)-derived and other than XG-derived donors and acceptors, and this poly-specificity originates from the structural plasticity and evolutionary diversification that has evolved through expansion and duplication. In phyletic groups, XETs form the gene families that are differentially expressed in organs and tissues in time- and space-dependent manners, and in response to environmental conditions. Here, we examine higher plant XET enzymes and dissect how their exclusively carbohydrate-linked transglycosylation catalytic function inter-connects complex plant cell wall components. Further, we discuss progress in technologies that advance the knowledge of plant cell walls and how this knowledge defines the roles of XETs. We construe that the broad specificity of the plant XETs underscores their roles in continuous cell wall restructuring and re-modelling.

Potassium Alginate Oligosaccharides Alter Gut Microbiota, and Have Potential to Prevent the Development of Hypertension and Heart Failure in Spontaneously Hypertensive Rats.

Food-derived oligosaccharides show promising therapeutic potential in lowering blood pressure (BP), but the mechanism is poorly understood. Recently, the potential role of gut microbiota (GM) in hypertension has been investigated, but the specific GM signature that may participate in hypertension remains unclear. To test the potassium alginate oligosaccharides (PAO) mechanism in lowering BP and specific microbial signature changes in altering GM, we administered various dosages of PAO in 40 spontaneously hypertensive rats for a duration of six weeks. We analyzed BP, sequenced the 16S ribosomal DNA gene in the cecum content, and gathered RNA-seq data in cardiac tissues. We showed that the oral administration of PAO could significantly decrease systolic BP and mean arterial pressure. Transcriptome analyses demonstrated that the protective effects of developing heart failure were accompanied by down-regulating of the Natriuretic Peptide A gene expression and by decreasing the concentrations of angiotensin II and atrial natriuretic peptide in plasma. In comparison to the Vehicle control, PAO could increase the microbial diversity by altering the composition of GM. PAO could also decrease the ratio of Firmicutes to Bacteroidetes by decreasing the abundance of Prevotella and Phascolarctobacterium bacteria. The favorable effect of PAO may be added to the positive influence of the abundance of major metabolites produced by Gram-negative bacteria in GM. We suggest that PAO caused changes in GM, and thus, they played an important role in preventing the development of cardiovascular disease.

Special Issue: "Peter Biely, A Pioneering Researcher in the Enzymology of Plant Biomass Degradation".

As it has been outlined on the website of the Special Issue entitled "Peter Biely, a pioneering researcher in the enzymology of plant biomass degradation" in the journal Molecules (section Macromolecular Chemistry, ISSN 1420-3049), plant biomass is a key renewable resource [...].

Plant Transcription Factors Involved in Drought and Associated Stresses.

Transcription factors (TFs) play a significant role in signal transduction networks spanning the perception of a stress signal and the expression of corresponding stress-responsive genes. TFs are multi-functional proteins that may simultaneously control numerous pathways during stresses in plants-this makes them powerful tools for the manipulation of regulatory and stress-responsive pathways. In recent years, the structure-function relationships of numerous plant TFs involved in drought and associated stresses have been defined, which prompted devising practical strategies for engineering plants with enhanced stress tolerance. Vast data have emerged on purposely basic leucine zipper (bZIP), WRKY, homeodomain-leucine zipper (HD-Zip), myeloblastoma (MYB), drought-response elements binding proteins/C-repeat binding factor (DREB/CBF), shine (SHN), and wax production-like (WXPL) TFs that reflect the understanding of their 3D structure and how the structure relates to function. Consequently, this information is useful in the tailored design of variant TFs that enhances our understanding of their functional states, such as oligomerization, post-translational modification patterns, protein-protein interactions, and their abilities to recognize downstream target DNA sequences. Here, we report on the progress of TFs based on their interaction pathway participation in stress-responsive networks, and pinpoint strategies and applications for crops and the impact of these strategies for improving plant stress tolerance.

A single residue deletion in the barley HKT1;5 P189 variant restores plasma membrane localisation but not Na+ conductance.

Leaf Na+ exclusion, mediated by plasma membrane-localised Class 1 High-affinity potassium (K+) Transporters (HKTs), is a key mechanism contributing to salinity tolerance of several major crop plants. We determined previously that the leucine to proline residue substitution at position 189 (L189P) in barley HvHKT1;5 disrupts its characteristic plasma membrane localisation and Na+ conductance. Here, we focus on a surprising observation that a single residue deletion of methionine at position 372 (M372del) within the conserved VMMYL motif in plant HKTs, restores plasma membrane localisation but not Na+ conductance in HvHKT1;5 P189. To clarify why the singular M372 deletion regains plasma membrane localisation, we built 3D models and defined α-helical assembly pathways of the P189 M372del mutant, and compared these findings to the wild-type protein, and the HvHKT1;5 L189 variant and its M372del mutant. We find that α-helical association and assembly pathways in HvHKT1;5 proteins fall in two contrasting categories. Inspections of structural flexibility through molecular dynamics simulations revealed that the conformational states of HvHKT1;5 P189 diverge from those of the L189 variant and M372del mutants. We propose that M372del in HvHKT1;5 P189 instigates structural rearrangements allowing routing to the plasma membrane, while the restoration of conductance would require further interventions. We integrate the microscopy, electrophysiology, and biocomputational data and discuss how a profound structural change in HvHKT1;5 P189 M372del impacts its α-helical protein association pathway and flexibility, and how these features underlie a delicate balance leading to restoring plasma membrane localisation but not Na+ conductance.

Plant Xyloglucan Xyloglucosyl Transferases and the Cell Wall Structure: Subtle but Significant.

Plant xyloglucan xyloglucosyl transferases or xyloglucan endo-transglycosylases (XET; EC 2.4.1.207) catalogued in the glycoside hydrolase family 16 constitute cell wall-modifying enzymes that play a fundamental role in the cell wall expansion and re-modelling. Over the past thirty years, it has been established that XET enzymes catalyse homo-transglycosylation reactions with xyloglucan (XG)-derived substrates and hetero-transglycosylation reactions with neutral and charged donor and acceptor substrates other than XG-derived. This broad specificity in XET isoforms is credited to a high degree of structural and catalytic plasticity that has evolved ubiquitously in algal, moss, fern, basic Angiosperm, monocot, and eudicot enzymes. These XET isoforms constitute gene families that are differentially expressed in tissues in time- and space-dependent manners during plant growth and development, and in response to biotic and abiotic stresses. Here, we discuss the current state of knowledge of broad specific plant XET enzymes and how their inherently carbohydrate-based transglycosylation reactions tightly link with structural diversity that underlies the complexity of plant cell walls and their mechanics. Based on this knowledge, we conclude that multi- or poly-specific XET enzymes are widespread in plants to allow for modifications of the cell wall structure in muro, a feature that implements the multifaceted roles in plant cells.

High affinity Na+ transport by wheat HKT1;5 is blocked by K.

The wheat sodium transporters TmHKT1;5-A and TaHKT1;5-D are encoded by genes underlying the major shoot Na+ exclusion loci Nax2 and Kna1 from Triticum monococcum (Tm) and Triticum aestivum (Ta), respectively. In contrast to HKT2 transporters that have been shown to exhibit high affinity K+-dependent Na+ transport, HKT1 proteins have, with one exception, only been shown to catalyze low affinity Na+ transport and no K+ transport. Here, using heterologous expression in Xenopus laevis oocytes we uncover a novel property of HKT1 proteins, that both TmHKT1;5-A and TaHKT1;5-D encode dual (high and low) affinity Na+-transporters with the high-affinity component being abolished when external K+ is in excess of external Na+. Three-dimensional structural modeling suggested that, compared to Na+, K+ is bound more tightly in the selectivity filter region by means of additional van der Waals forces, which is likely to explain the K+ block at the molecular level. The low-affinity component for Na+ transport of TmHKT1;5-A had a lower K m than that of TaHKT1;5-D and was less sensitive to external K+. We propose that these properties contribute towards the improvements in shoot Na+-exclusion and crop plant salt tolerance following the introgression of TmHKT1;5-A into diverse wheat backgrounds.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Plant transporters involved in combating boron toxicity: beyond 3D structures.

Membrane transporters control the movement and distribution of solutes, including the disposal or compartmentation of toxic substances that accumulate in plants under adverse environmental conditions. In this minireview, in the light of the approaching 100th anniversary of unveiling the significance of boron to plants (K. Warington, 1923; Ann. Bot.37, 629) we discuss the current state of the knowledge on boron transport systems that plants utilise to combat boron toxicity. These transport proteins include: (i) nodulin-26-like intrinsic protein-types of aquaporins, and (ii) anionic efflux (borate) solute carriers. We describe the recent progress made on the structure-function relationships of these transport proteins and point out that this progress is integral to quantitative considerations of the transporter's roles in tissue boron homeostasis. Newly acquired knowledge at the molecular level has informed on the transport mechanics and conformational states of boron transport systems that can explain their impact on cell biology and whole plant physiology. We expect that this information will form the basis for engineering transporters with optimised features to alleviate boron toxicity tolerance in plants exposed to suboptimal soil conditions for sustained food production.

A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in bread wheat.

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+ ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.

Barley sodium content is regulated by natural variants of the Na+ transporter HvHKT1;5.

During plant growth, sodium (Na+) in the soil is transported via the xylem from the root to the shoot. While excess Na+ is toxic to most plants, non-toxic concentrations have been shown to improve crop yields under certain conditions, such as when soil K+ is low. We quantified grain Na+ across a barley genome-wide association study panel grown under non-saline conditions and identified variants of a Class 1 HIGH-AFFINITY-POTASSIUM-TRANSPORTER (HvHKT1;5)-encoding gene responsible for Na+ content variation under these conditions. A leucine to proline substitution at position 189 (L189P) in HvHKT1;5 disturbs its characteristic plasma membrane localisation and disrupts Na+ transport. Under low and moderate soil Na+, genotypes containing HvHKT1:5P189 accumulate high concentrations of Na+ but exhibit no evidence of toxicity. As the frequency of HvHKT1:5P189 increases significantly in cultivated European germplasm, we cautiously speculate that this non-functional variant may enhance yield potential in non-saline environments, possibly by offsetting limitations of low available K+.

Structural characterization of the Pet c 1.0201 PR-10 protein isolated from roots of Petroselinum crispum (Mill.) Fuss.

The native dimeric Petroselinum crispum (Mill.) Fuss protein Pet c 1.0201 and a monomeric xyloglucan endotransglycosylase enzyme (Garajova et al., 2008) isolated from the root cells co-purify and share similar molecular masses and acidic isoelectric points. In this work, we determined the complete primary structure of the parsley Pet c 1.0201 protein, based on tryptic and chymotryptic peptides followed by the manual micro-gradient chromatographic separation coupled with offline MALDI-TOF/TOF mass spectrometry. The bioinformatics approach enabled us to include the parsley protein into the PR-10 family, as it exhibited the highest protein sequence identity with the Apium graveolens Api g 1.0201 allergen and the major Daucus carota allergen Dau c 1.0201. Hence, we designated the Petroselinum crispum protein as Pet c 1.0201 and deposited it in the UniProt Knowledgebase under the accession C0HKF5. 3D protein homology modelling and molecular dynamics simulations of the Pet c 1.0201 dimer confirmed the typical structure of the Bet v 1 family allergens, and the potential of the Pet c 1.0201 protein to dimerize in water. However, the behavioural properties of Pet c 1.0201 and the celery allergen Api g 1.0101 differed in the presence of salts due to transiently and stably formed dimeric forms of Pet c 1.0201 and Api g 1.0101, respectively.