Maria Hrmova

DREB/CBF expression in wheat and barley using the stress‐inducible promoters of HD‐Zip I genes: impact on plant development, stress tolerance and yield

Publisher: Wiley

Date: 25-09-2020

DOI: 10.1111/PBI.13252

Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes

Publisher: Springer Science and Business Media LLC

Date: 04-02-2017

DOI: 10.1007/S11103-017-0585-9

Abstract: The cuticle forms a hydrophobic waxy layer that covers plant organs and provides protection from biotic and abiotic stresses. Transcription of genes responsible for cuticle formation is regulated by several types of transcription factors (TFs). Five orthologous to WAX PRODUCTION (WXP1 and WXP2) genes from Medicago truncatula were isolated from a cDNA library prepared from flag leaves and spikes of drought tolerant wheat (Triticum aestivum, breeding line RAC875) and designated TaWXP-like (TaWXPL) genes. Tissue-specific and drought-responsive expression of TaWXPL1D and TaWXPL2B was investigated by quantitative RT-PCR in two Australian wheat genotypes, RAC875 and Kukri, with contrasting glaucousness and drought tolerance. Rapid dehydration and/or slowly developing cyclic drought induced specific expression patterns of WXPL genes in flag leaves of the two cultivars RAC875 and Kukri. TaWXPL1D and TaWXPL2B proteins acted as transcriptional activators in yeast and in wheat cell cultures, and conserved sequences in their activation domains were localised at their C-termini. The involvement of wheat WXPL TFs in regulation of cuticle biosynthesis was confirmed by transient expression in wheat cells, using the promoters of wheat genes encoding two cuticle biosynthetic enzymes, the 3-ketoacyl-CoA-synthetase and the cytochrome P450 monooxygenase. Using the yeast 1-hybrid (Y1H) assay we also demonstrated the differential binding preferences of TaWXPL1D and TaWXPL2B towards three stress-related DNA cis-elements. Protein structural determinants underlying binding selectivity were revealed using comparative 3D molecular modelling of AP2 domains in complex with cis-elements. A scheme is proposed, which links the roles of WXPL and cuticle-related MYB TFs in regulation of genes responsible for the synthesis of cuticle components.

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

Publisher: MDPI AG

Date: 11-09-2021

DOI: 10.3390/IJMS22189823

Abstract: 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 ersity 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.

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

Publisher: MDPI AG

Date: 05-10-2022

DOI: 10.3390/IJMS231911838

Abstract: 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 erse 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)-β-glycosidic linkages, were bound stably in the active sites of TmXET6.3 and PttXET16A. Conversely, the acceptors with the (1,6)-β-linked Glc moieties were bound stably in TmXET6.3 but not in PttXET16A. When in the (1,4)-β-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 ,3)-β-glucans highlighted the importance of (1,3)-β-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.

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

Publisher: MDPI AG

Date: 11-08-2021

DOI: 10.3390/MOLECULES26164857

Abstract: 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 [...]

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

Publisher: Wiley

Date: 22-07-2020

DOI: 10.1111/PCE.13841

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

Publisher: Wiley

Date: 19-09-2020

DOI: 10.1111/TPJ.14964

Overexpression of the class I homeodomain transcription factor TaHDZipI‐5 increases drought and frost tolerance in transgenic wheat

Publisher: Wiley

Date: 27-12-2018

DOI: 10.1111/PBI.12865

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

Publisher: Wiley

Date: 10-2020

DOI: 10.1002/PLD3.275

Discovery of processive catalysis by an exo-hydrolase with a pocket-shaped active site

Publisher: Springer Science and Business Media LLC

Date: 20-05-2019

DOI: 10.1038/S41467-019-09691-Z

Abstract: Substrates associate and products dissociate from enzyme catalytic sites rapidly, which h ers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl β- d -glucoside and methyl 6-thio-β-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-β-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.

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

Publisher: Elsevier BV

Date: 10-2021

DOI: 10.1016/J.BBAMEM.2021.183669

Identification and characterization of wheat drought-responsive MYB transcription factors involved in the regulation of cuticle biosynthesis

Publisher: Oxford University Press (OUP)

Date: 03-08-2016

DOI: 10.1093/JXB/ERW298

Abstract: A plant cuticle forms a hydrophobic layer covering plant organs, and plays an important role in plant development and protection from environmental stresses. We examined epicuticular structure, composition, and a MYB-based regulatory network in two Australian wheat cultivars, RAC875 and Kukri, with contrasting cuticle appearance (glaucousness) and drought tolerance. Metabolomics and microscopic analyses of epicuticular waxes revealed that the content of β-diketones was the major compositional and structural difference between RAC875 and Kukri. The content of β-diketones remained the same while those of alkanes and primary alcohols were increased by drought in both cultivars, suggesting that the interplay of all components rather than a single one defines the difference in drought tolerance between cultivars. Six wheat genes encoding MYB transcription factors (TFs) were cloned four of them were regulated in flag leaves of both cultivars by rapid dehydration and/or slowly developing cyclic drought. The involvement of selected MYB TFs in the regulation of cuticle biosynthesis was confirmed by a transient expression assay in wheat cell culture, using the promoters of wheat genes encoding cuticle biosynthesis-related enzymes and the SHINE1 (SHN1) TF. Two functional MYB-responsive elements, specifically recognized by TaMYB74 but not by other MYB TFs, were localized in the TdSHN1 promoter. Protein structural determinants underlying the binding specificity of TaMYB74 for functional DNA cis -elements were defined, using 3D protein molecular modelling. A scheme, linking drought-induced expression of the investigated TFs with downstream genes that participate in the synthesis of cuticle components, is proposed.

Alluminating structure key to stress tolerance

Publisher: Springer Science and Business Media LLC

Date: 15-12-2021

DOI: 10.1038/S41422-021-00604-8

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

Publisher: Springer Science and Business Media LLC

Date: 22-05-2020

DOI: 10.1038/S42003-020-0990-5

Abstract: 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 )-encoding gene responsible for Na + content variation under these conditions. A leucine to proline substitution at position 189 (L189P) in HvHKT1 disturbs its characteristic plasma membrane localisation and disrupts Na + transport. Under low and moderate soil Na + , genotypes containing HvHKT1:5 P189 accumulate high concentrations of Na + but exhibit no evidence of toxicity. As the frequency of HvHKT1:5 P189 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 + .

Barley HvNIP2;1 aquaporin permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification

Publisher: Cold Spring Harbor Laboratory

Date: 18-04-2023

DOI: 10.1101/2023.04.17.537278

Abstract: Aquaporins can facilitate the passive movement of water and small polar molecules and some ions. The barley Nodulin 26-like Intrinsic Protein (HvNIP2 ) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus oocyte swelling assays was found to permeate water, boric and germanic acids, sucrose and L-arabinose 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 (lactose, cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols were permeated to a much lower extent. Apparent permeation of hydrated KCl and MgSO 4 ion pairs was observed, while CH 3 COONa and NaNO 3 permeated at significantly lower rates. Experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO 3 blocked the permeation of all solutes. Full-scale steered molecular dynamics simulations of HvNIP2 and spinach SoPIP2 revealed possible rectification for water, boric acid, and sucrose transport, and defined key residues interacting with permeants. In a biological context, the simulated sucrose rectification could mediate its apoplastic-to-intracellular transport but not the reverse, thus, constituting a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2,993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalised solute poly-selectivity in NIP3 sub-clade entries and suggested that they ersified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for sustained food production. Aquaporins are fundamental to water and solute movements in nearly all living organisms. Solute selectivity inspections of the HvNIP2 aquaporin revealed that it transported water, hydroxylated metalloids boric and germanic acids, sucrose, L-arabinose, KCl, and MgSO 4 ion pairs, but not D-glucose or D-fructose and to lesser extent urea, and acyclic polyols. This poly-selective transport by HvNIP2 classified in the NIP3 sub-clade aquaporins may afford nutritional and protective roles during plant development and in response to abiotic stresses. It is anticipated that the solute specificity definition of HvNIP2 inspires protein engineering and in silico mining to develop plants, which when exposed to suboptimal soil conditions of high soil metalloids, would overcome toxicity for sustained food production.

Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins

Publisher: Springer Science and Business Media LLC

Date: 10-06-2019

DOI: 10.1007/S11103-019-00889-9

Abstract: Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.

The wheat TabZIP2 transcription factor is activated by the nutrient starvation-responsive SnRK3/CIPK protein kinase

Publisher: Springer Science and Business Media LLC

Date: 21-03-2018

DOI: 10.1007/S11103-018-0713-1

Abstract: The understanding of roles of bZIP factors in biological processes during plant development and under abiotic stresses requires the detailed mechanistic knowledge of behaviour of TFs. Basic leucine zipper (bZIP) transcription factors (TFs) play key roles in the regulation of grain development and plant responses to abiotic stresses. We investigated the role and molecular mechanisms of function of the TabZIP2 gene isolated from drought-stressed wheat plants. Molecular characterisation of TabZIP2 and derived protein included analyses of gene expression and its target promoter, and the influence of interacting partners on the target promoter activation. Two interacting partners of TabZIP2, the 14-3-3 protein, TaWIN1 and the bZIP transcription factor TaABI5L, were identified in a Y2H screen. We established that under elevated ABA levels the activity of TabZIP2 was negatively regulated by the TaWIN1 protein and positively regulated by the SnRK3/CIPK protein kinase WPK4, reported previously to be responsive to nutrient starvation. The physical interaction between the TaWIN1 and the WPK4 was detected. We also compared the influence of homo- and hetero-dimerisation of TabZIP2 and TaABI5L on DNA binding. TabZIP2 gene functional analyses were performed using drought-inducible overexpression of TabZIP2 in transgenic wheat. Transgenic plants grown under moderate drought during flowering, were smaller than control plants, and had fewer spikes and seeds per plant. However, a single seed weight was increased compared to single seed weights of control plants in three of four evaluated transgenic lines. The observed phenotypes of transgenic plants and the regulation of TabZIP2 activity by nutrient starvation-responsive WPK4, suggest that the TabZIP2 could be the part of a signalling pathway, which controls the rearrangement of carbohydrate and nutrient flows in plant organs in response to drought.

Correction to: Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins

Publisher: Springer Science and Business Media LLC

Date: 07-2019

DOI: 10.1007/S11103-019-00893-Z

Abstract: Due to an unfortunate turn of events, the panels O to S are missing in Fig. 8 of the original publication. The correct Fig. 8 and its caption is published here and should be treated as definitive.

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

Publisher: Portland Press Ltd.

Date: 11-08-2020

DOI: 10.1042/BST20200164

Abstract: 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.

Streltsov-et-al_movie.mp4

Publisher: figshare

Date: 2022

DOI: 10.6084/M9.FIGSHARE.8075504

A Barley Efflux Transporter Operates in a Na⁺-Dependent Manner, as Revealed by a Multidisciplinary Platform

Publisher: Oxford University Press (OUP)

Date: 15-12-2015

DOI: 10.1105/TPC.15.00625

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

Publisher: MDPI AG

Date: 31-01-2022

DOI: 10.3390/IJMS23031656

Abstract: 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 β-sandwich architecture and the β-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 ersification 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.

Plant Transcription Factors Involved in Drought and Associated Stresses

Publisher: MDPI AG

Date: 26-05-2021

DOI: 10.3390/IJMS22115662

Abstract: 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.

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

Publisher: figshare

Date: 2022

DOI: 10.6084/M9.FIGSHARE.20469747

Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism

Publisher: Portland Press Ltd.

Date: 02-06-2023

DOI: 10.1042/BST20230136

Abstract: 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 β-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 β-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.

Overexpression of the TaSHN1 transcription factor in bread wheat leads to leaf surface modifications, improved drought tolerance, and no yield penalty under controlled growth conditions

Publisher: Wiley

Date: 02-08-2018

DOI: 10.1111/PCE.13339

Abstract: Transcription factors regulate multiple networks, mediating the responses of organisms to stresses, including drought. Here, we investigated the role of the wheat transcription factor TaSHN1 in crop growth and drought tolerance. TaSHN1, isolated from bread wheat, was characterized for molecular interactions and functionality. The overexpression of TaSHN1 in wheat was followed by the evaluation of T

Editorial: Structural bioinformatics and biophysical approaches for understanding the plant responses to biotic and abiotic stresses

Publisher: Frontiers Media SA

Date: 09-09-2022

DOI: 10.3389/FPLS.2022.1012584

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

Publisher: MDPI AG

Date: 29-11-2020

DOI: 10.3390/MOLECULES25235619

Abstract: 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 ersity 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.

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

Publisher: Wiley

Date: 02-09-2023

DOI: 10.1111/TPJ.16435

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

Publisher: Springer Science and Business Media LLC

Date: 23-09-2022

DOI: 10.1038/S41467-022-33180-5

Abstract: In the barley β- d -glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 cl ensures β- 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 β- 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)-β- d -glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan cl . 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 cl that confers broad specificity, high catalytic efficiency, and processivity.

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

Publisher: Cold Spring Harbor Laboratory

Date: 12-03-2018

DOI: 10.1101/280453

Abstract: The wheat sodium transporters TmHKT1 -A and TaHKT1 -D are encoded by genes underlying 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 catalyse low affinity Na + transport and no K + transport. Here, using heterologous expression in Xenopus laevis oocytes we show that both TmHKT1 -A and TaHKT1 -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 + . Based on 3-D structural modelling we propose that tighter binding of K + , compared to that of Na + in the selectivity filter region by means of additional van der Waals forces, explains the K + block at the molecular level. The low-affinity component for Na + transport of TmHKT1 -A had a lower K m than that of TaHKT1 -D and was less sensitive to external K + . We propose that these properties underpin the improvements in shoot Na + -exclusion and crop plant salt tolerance following the introgression of TmHKT1 -A into erse wheat backgrounds.

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

Publisher: Oxford University Press (OUP)

Date: 18-08-2023

DOI: 10.1093/PLPHYS/KIAD415

Abstract: 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 ,4)-β-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 ,4)-β-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 ,4)-β-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)-β-linkages into the nascent polysaccharide chain. Similarly, 3D structures of xyloglucan endo-transglycosylases and (1,3 ,4)-β-glucan endo- and exohydrolases have allowed the mechanisms of (1,3 ,4)-β-glucan modification and degradation to be defined. X-ray crystallography and multi-scale modeling of a broad specificity GH3 β-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.

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

Publisher: Elsevier BV

Date: 10-2023

DOI: 10.1016/J.JBC.2023.105410

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

Publisher: Cold Spring Harbor Laboratory

Date: 24-01-2020

DOI: 10.1101/2020.01.21.909887

Abstract: 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 6 folds greater leaf and sheath sodium (Na + ) than two Australian cultivars, Gladius and Scout, under salt stress. 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 -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.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

Publisher: Springer Science and Business Media LLC

Date: 13-03-2019

DOI: 10.1007/S11103-019-00852-8

Abstract: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3 ,4)-, (1,6)- and (1,4)-β-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-β-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-β-D-glucooligosaccharides, while those with (1,3 ,4)- and (1,6)-β-D-glucooligosaccharides were suppressed the incorporation of xyloglucan to (1,4)-β-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.

Variation among S-locus haplotypes and among stylar RNases in almond

Publisher: Springer Science and Business Media LLC

Date: 17-01-2020

DOI: 10.1038/S41598-020-57498-6

Abstract: In many plant species, self-incompatibility systems limit self-pollination and mating among relatives. This helps maintain genetic ersity in natural populations but imposes constraints in agriculture and plant breeding. In almond [ Prunus dulcis (Mill.) D.A. Webb], the specificity of self-incompatibility is mainly determined by stylar ribonuclease (S-RNase) and S -haplotype-specific F-box (SFB) proteins, both encoded within a complex locus, S . Prior to this research, a nearly complete sequence was available for one S -locus haplotype. Here, we report complete sequences for four haplotypes and partial sequences for 11 haplotypes. Haplotypes vary in sequences of genes (particularly S-RNase and SFB ), distances between genes and numbers and positions of long terminal repeat transposons. Haplotype variation outside of the S-RNase and SFB genes may help maintain functionally important associations between S-RNase and SFB alleles. Fluorescence-based assays were developed to distinguish among some S-RNase alleles. With three-dimensional modelling of five S-RNase proteins, conserved active sites were identified and variation was observed in electrostatic potential and in the numbers, characteristics and positions of secondary structural elements, loop anchoring points and glycosylation sites. A hypervariable region on the protein surface and differences in the number, location and types of glycosylation sites may contribute to determining S-RNase specificity.

Definition of the acceptor aubstrate binding specificity in plant xyloglucan endotransglycosylases using computational chemistry

Publisher: figshare

Date: 2022

DOI: 10.6084/M9.FIGSHARE.21291684

University Of Adelaide

Location: Australia

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Huaiyin Normal University

Location: China

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Comenius University

Location: Slovakia

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Linkage Projects - Grant ID: LP120100201

Start Date: 03-2012

End Date: 12-2016

Amount: $431,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0452650

Start Date: 2004

End Date: 12-2004

Amount: $696,005.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE140100193

Start Date: 2014

End Date: 12-2014

Amount: $700,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP120100900

Start Date: 06-2012

End Date: 06-2016

Amount: $460,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989336

Start Date: 08-2009

End Date: 12-2010

Amount: $560,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0347746

Start Date: 2002

End Date: 12-2003

Amount: $199,000.00

Funder: Australian Research Council