Plants fighting back: to transport or not to transport, this is a structural question

Abstract not availableMaria Hrmova and Matthew Gilliha

Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism

Version of Record published: 2 June 2023Processive 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.Maria Hrmova and Julian G. Schwerd

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

Version of Record published: 11 August 2020Membrane 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.Maria Hrmova, Matthew Gilliham and Stephen D. Tyerma

Wheat drought-responsive WXPL transcription factors regulate cuticle biosynthesis genes

Published online: 4 February 2017The 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.Huihui Bi, Sukanya Luang, Yuan Li, Natalia Bazanova, Nikolai Borisjuk, Maria Hrmova, Sergiy Lopat

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+. Threedimensional 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 Km 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.Bo Xu, Maria Hrmova, Matthew Gilliha

Molecular interaction of the gamma-clade homeodomain-leucine zipper class I transcription factors during the wheat response to water deficit

The ᵧ-clade of class I homeodomain-leucine zipper (HD-Zip I) transcription factors (TFs) constitute members which play a role in adapting plant growth to conditions of water deficit. Given the importance of wheat (Triticum aestivum L.) as a global food crop and the impact of water deficit upon grain yield, we focused on functional aspects of wheat drought responsive HD-Zip I TFs. While the wheat ᵧ-clade HD-Zip I TFs share significant sequence similarities with homologous genes from other plants, the clade-specific features in transcriptional response to abiotic stress were detected. We demonstrate that wheat TaHDZipI- 3, TaHDZipI-4, and TaHDZipI-5 genes respond differentially to a variety of abiotic stresses, and that proteins encoded by these genes exhibit pronounced differences in oligomerisation, strength of DNA binding, and trans-activation of an artificial promoter. Three-dimensional molecular modelling of the protein-DNA interface was conducted to address the ambiguity at the central nucleotide in the pseudo-palindromic cis-element CAATNATTG that is recognised by all three HD-Zip I proteins. The coexpression of these genes in the same plant tissues together with the ability of HD-Zip I TFs of the ᵧ -clade to heterodimerise suggests a role in the regulatory mechanisms of HD-Zip I dependent transcription. Our findings highlight the complexity of TF networks involved in plant responses to water deficit. A better understanding of the molecular complexity at the protein level during crop responses to drought will enable adoption of efficient strategies for production of cereal plants with enhanced drought tolerance.John C. Harris, Pradeep Sornaraj, Mathew Taylor, Natalia Bazanova, Ute Baumann, Ben Lovell, Peter Langridge, Sergiy Lopato, Maria Hrmov

Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis.

Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development.Hongyu Zhang, Ming Luo, Robert C. Day, Mark J. Talbot, Aneta Ivanova, Anthony R. Ashton, Abed M. Chaudhury, Richard C. Macknight, Maria Hrmova, and Anna M. Koltuno

Molecular modeling of S-RNases involved in almond self-incompatibility

Gametophytic self-incompatibility (GSI) is a mechanism in flowering plants, to prevent inbreeding and promote outcrossing. GSI is under the control of a specific locus, known as the S-locus, which contains at least two genes, the RNase and the SFB. Active S-RNases in the style are essential for rejection of haploid pollen, when the pollen S-allele matches one of two S-alleles of the diploid pistil. However, the nature of their mutual interactions at genetic and biochemical levels remain unclear. Thus, detailed understanding of the protein structure involved in GSI may help in discovering how the proteins involved in GSI may function and how they fulfill their biological roles. To this end, 3D models of the SC (Sf) and two SI (S8 and S23) S-RNases of almond were constructed, using comparative modeling tools. The modeled structures consisted of mixed α and β folds, with six helices and six β-strands. However, the self-compatible (Sf) RNase contained an additional extended loop between the conserved domains RC4 and C5, which may be involved in the manifestation of self-compatibility in almond

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

OnlinePublAquaporins 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 2,993 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.Akshayaa Venkataraghavan, Julian G. Schwerdt, Stephen D. Tyerman, Maria Hrmov

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

Editorial. PUBLISHED 09 September 2022Raul A. Sperotto, Maria Hrmova, Steffen P. Graether and Luis Fernando S. M. Timmer