Cell-free protein synthesis of membrane (1,3)-beta-D-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.Agalya Periasamy, Nadim Shadiac, Amritha Amalraj, Soňa Garajová, Yagnesh Nagarajan, Shane Waters, Haydyn D.T. Mertens, Maria Hrmov

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

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+.R.W., M.S., and M.M. acknowledge support from ERC project 669182 ‘SHUFFLE’ to R.W. K.H., P.S., M.B., J.R., and R.W. acknowledge the Rural & Environment Science & Analytical Services Division of the Scottish Government. C.B., J.Q., and Y.Q. acknowledge support from Rutherford Fund Strategic Partner Grants 2018 - Award Reference: RF-2018-30 to C.H. and R.W. S.R. is grateful for financial assistance from The Australian Research Council Industrial Transformation Research Hub for Wheat in a Hot and Dry Climate (IH130200027), the Grains Research and Development Corporation (ACP00009) and The Waite Research Institute, University of Adelaide. C.B. acknowledges support from the Grains Research and Development Corporation (GRDC) and the Australian Research Council (FT180100476). We are thankful to the Australian Research Council for funding through CE140100008 to M.G. (J.Q. and Y.Q.), FT180100476 to C.S.B. and DE160100804 to S.W. (A.S.). D.E.S. acknowledges support from BBSRC Grant BB/L000113/1. MH acknowledges financial support from the Huaiyin Normal University, Chin

Plant High-Affinity Potassium (HKT) transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity

High-affinity Potassium Transporters (HKTs) belong to an important class of integral membrane proteins (IMPs) that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.Shane Waters, Matthew Gilliham and Maria Hrmov

Role of homeodomain leucine zipper (HD-Zip) iv transcription factors in plant development and plant protection from deleterious environmental factors

Homeobox genes comprise an important group of genes that are responsible for regulation of developmental processes. These genes determine cell differentiation and cell fate in all eukaryotic organisms, starting from the early stages of embryo development. Homeodomain leucine zipper (HD-Zip) transcription factors are unique to the plant kingdom. Members of the HD-Zip IV subfamily have a complex domain topology and can bind several cis-elements with overlapping sequences. Many of the reported HD-Zip IV genes were shown to be specifically or preferentially expressed in plant epidermal or sub-epidermal cells. HD-Zip IV TFs were found to be associated with differentiation and maintenance of outer cell layers, and regulation of lipid biosynthesis and transport. Insights about the role of these proteins in plant cuticle formation, and hence their possible involvement in plant protection from pathogens and abiotic stresses has just started to emerge. These roles make HD-Zip IV proteins an attractive tool for genetic engineering of crop plants. To this end, there is a need for in-depth studies to further clarify the function of each HD-Zip IV subfamily member in commercially important plant species.William Chew, Maria Hrmova and Sergiy Lopat

A two-staged model of Na+ exclusion in rice explained by 3D modeling of HKT transporters and alternative splicing

The HKT family of Na+ and Na+/K+ transporters is implicated in plant salinity tolerance. Amongst these transporters, the cereal HKT1;4 and HKT1;5 are responsible for Na+ exclusion from photosynthetic tissues, a key mechanism for plant salinity tolerance. It has been suggested that Na+ is retrieved from the xylem transpiration stream either in the root or the leaf sheath, protecting the leaf blades from excessive Na+ accumulation. However, direct evidence for this scenario is scarce. Comparative modeling and evaluation of rice (Oryza sativa) HKT-transporters based on the recent crystal structure of the bacterial TrkH K+ transporter allowed to reconcile transcriptomic and physiological data. For OsHKT1;5, both transcript abundance and protein structural features within the selectivity filter could control shoot Na+ accumulation in a range of rice varieties. For OsHKT1;4, alternative splicing of transcript and the anatomical complexity of the sheath needed to be taken into account. Thus, Na+ accumulation in a specific leaf blade seems to be regulated by abundance of a correctly spliced OsHKT1;4 transcript in a corresponding sheath. Overall, allelic variation of leaf blade Na+ accumulation can be explained by a complex interplay of gene transcription, alternative splicing and protein structure.Olivier Cotsaftis, Darren Plett, Neil Shirley, Mark Tester and Maria Hrmov

Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial

Background: Among people with diabetes, those with kidney disease have exceptionally high rates of cardiovascular (CV) morbidity and mortality and progression of their underlying kidney disease. Finerenone is a novel, nonsteroidal, selective mineralocorticoid receptor antagonist that has shown to reduce albuminuria in type 2 diabetes (T2D) patients with chronic kidney disease (CKD) while revealing only a low risk of hyperkalemia. However, the effect of finerenone on CV and renal outcomes has not yet been investigated in long-term trials. Patients and Methods: The Finerenone in Reducing CV Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial aims to assess the efficacy and safety of finerenone compared to placebo at reducing clinically important CV and renal outcomes in T2D patients with CKD. FIGARO-DKD is a randomized, double-blind, placebo-controlled, parallel-group, event-driven trial running in 47 countries with an expected duration of approximately 6 years. FIGARO-DKD randomized 7,437 patients with an estimated glomerular filtration rate >= 25 mL/min/1.73 m(2) and albuminuria (urinary albumin-to-creatinine ratio >= 30 to <= 5,000 mg/g). The study has at least 90% power to detect a 20% reduction in the risk of the primary outcome (overall two-sided significance level alpha = 0.05), the composite of time to first occurrence of CV death, nonfatal myocardial infarction, nonfatal stroke, or hospitalization for heart failure. Conclusions: FIGARO-DKD will determine whether an optimally treated cohort of T2D patients with CKD at high risk of CV and renal events will experience cardiorenal benefits with the addition of finerenone to their treatment regimen. Trial Registration: EudraCT number: 2015-000950-39; ClinicalTrials.gov identifier: NCT02545049