Comprehensive comparative assessment of the Arabidopsis thaliana MLO2-CALMODULIN2 interaction by various in vitro and in vivo protein-protein interaction assays.

Mildew resistance locus o (MLO) proteins are heptahelical integral membrane proteins of which some isoforms act as susceptibility factors for the powdery mildew pathogen. In many angiosperm plant species, loss-of-function mlo mutants confer durable broad-spectrum resistance against the fungal disease. Barley Mlo is known to interact via a cytosolic carboxyl-terminal domain with the intracellular calcium sensor calmodulin (CAM) in a calcium-dependent manner. Site-directed mutagenesis has revealed key amino acid residues in the barley Mlo calmodulin-binding domain (CAMBD) that, when mutated, affect the MLO-CAM association. We here tested the respective interaction between Arabidopsis thaliana MLO2 and CAM2 using seven different types of in vitro and in vivo protein-protein interaction assays. In each assay, we deployed a wild-type version of either the MLO2 carboxyl terminus (MLO2CT), harboring the CAMBD, or the MLO2 full-length protein and corresponding mutant variants in which two key residues within the CAMBD were substituted by non-functional amino acids. We focused in particular on the substitution of two hydrophobic amino acids (LW/RR mutant) and found in most protein-protein interaction experiments reduced binding of CAM2 to the corresponding MLO2/MLO2CT-LW/RR mutant variants in comparison with the respective wild-type versions. However, the Ura3-based yeast split-ubiquitin system and in planta bimolecular fluorescence complementation (BiFC) assays failed to indicate reduced CAM2 binding to the mutated CAMBD. Our data shed further light on the interaction of MLO and CAM proteins and provide a comprehensive comparative assessment of different types of protein-protein interaction assays with wild-type and mutant versions of an integral membrane protein.

Assessment of Biological Activity of 28-Homobrassinolide via a Multi-Level Comparative Analysis.

Brassinosteroids (BRs) play vital roles in the plant life cycle and synthetic BRs are widely used to increase crop yield and plant stress tolerance. Among them are 24R-methyl-epibrassinolide (24-EBL) and 24S-ethyl-28-homobrassinolide (28-HBL), which differ from brassinolide (BL, the most active BR) at the C-24 position. Although it is well known that 24-EBL is 10% active as BL, there is no consensus on the bioactivity of 28-HBL. A recent outpouring of research interest in 28-HBL on major crops accompanied with a surge of industrial-scale synthesis that produces mixtures of active (22R,23R)-28-HBL and inactive (22S,23S)-28HBL, demands a standardized assay system capable of analyzing different synthetic "28-HBL" products. In this study, the relative bioactivity of 28-HBL to BL and 24-EBL, including its capacity to induce the well-established BR responses at molecular, biochemical, and physiological levels, was systematically analyzed using the whole seedlings of the wild-type and BR-deficient mutant of Arabidopsis thaliana. These multi-level bioassays consistently showed that 28-HBL exhibits a much stronger bioactivity than 24-EBL and is almost as active as BL in rescuing the short hypocotyl phenotype of the dark-grown det2 mutant. These results are consistent with the previously established structure-activity relationship of BRs, proving that this multi-level whole seedling bioassay system could be used to analyze different batches of industrially produced 28-HBL or other BL analogs to ensure the full potential of BRs in modern agriculture.

Functional assessment of AtPAP17; encoding a purple acid phosphatase involved in phosphate metabolism in Arabidopsis thaliana.

PURPOSE: Purple acid phosphatases (PAPs) includ the largest classes of non-specific plant acid phosphatases. Most characterized PAPs were found to play physiological functions in phosphorus metabolism. In this study, we investigated the function of AtPAP17 gene encoding an important purple acid phosphatase in Arabidopsis thaliana. METHODS: The full-length cDNA sequence of AtPAP17 gene under the control of CaMV-35S promoter was transferred to the A. thaliana WT plant. The generated homozygote AtPAP17-overexpressed plants were compared by the types of analyses with corresponding homozygote atpap17-mutant plant and WT in both + P (1.2 mM) and - P (0 mM) conditions. RESULTS: In the + P condition, the highest and the lowest amount of Pi was observed in AtPAP17-overexpressed plants and atpap17-mutant plants by 111% increase and 38% decrease compared with the WT plants, respectively. Furthermore, under the same condition, APase activity of AtPAP17-overexpressed plants increased by 24% compared to the WT. Inversely, atpap17-mutant plant represented a 71% fall compared to WT plants. The comparison of fresh weight and dry weight in the studied plants showed that the highest and the lowest amount of absorbed water belonged to OE plants (with 38 and 12 mg plant-1) and Mu plants (with 22 and 7 mg plant-1) in + P and - P conditions, respectively. CONCLUSION: The lack of AtPAP17 gene in the A. thaliana genome led to a remarkable reduction in the development of root biomass. Thus, AtPAP17 could have an important role in the root but not shoot developmental and structural programming. Consequently, this function enables them to absorb more water and eventually associated with more phosphate absorption.

Challenging the water stress index concept: Thermographic assessment of Arabidopsis transpiration.

Water stress may greatly limit plant functionality and growth. Stomatal closure and consequently reduced transpiration are considered as early and sensitive plant responses to drought and salinity stress. An important consequence of stomatal closure under water stress is the rise of leaf temperature (Tleaf ), yet Tleaf is not only fluctuating with stomatal closure. It is regulated by several plant parameters and environmental factors. Thermal imaging and different stress indices, incorporating actual leaf/crop temperature and reference temperatures, were developed in previous studies toward normalizing for effects unassociated to water stress on Tleaf , aiming at a more efficient water stress assessment. The concept of stress indices has not been extensively studied on the model plant Arabidopsis thaliana. Therefore, the aim of this study was to examine the different indices employed in previous studies in assessing rosette transpiration rate (E) in Arabidopsis plants grown under two different light environments and subjected to salinity. After salinity imposition, E was gravimetrically quantified, and thermal imaging was employed to quantify rosette (Trosette ) and artificial reference temperature (Twet, Tdry ). Trosette and several water stress indices were tested for their relation to E. Among the microclimatic growth conditions tested, RWSI1 ([Trosette - Twet ]/[Tdry - Twet ]) and RWSI2 ([Tdry - Trosette ]/[Tdry - Twet ]) were well linearly-related to E, irrespective of the light environment, while the sole use of either Twet or Tdry in different combinations with Trosette returned less accurate results. This study provides evidence that selected combinations of Trosette , Tdry , and Twet can be utilized to assess E under water stress irrespective of the light environment.

Assessment of functional relevance of genes associated with local temperature variables in Arabidopsis thaliana.

How likely genetic variations associated with environment identified in silico from genome wide association study are functionally relevant to environmental adaptation has been largely unexplored experimentally. Here we analyzed top 29 genes containing polymorphisms associated with local temperature variation (minimum, mean, maximum) among 1129 natural accessions of Arabidopsis thaliana. Their loss-of-function mutants were assessed for growth and stress tolerance at five temperatures. Twenty genes were found to affect growth or tolerance at one or more of these temperatures. Significantly, genes associated with maximum temperature more likely have a detect a function at higher temperature, while genes associated with minimum temperature more likely have a function at lower temperature. In addition, gene variants are distributed more frequently at geographic locations where they apparently offer an enhanced growth or tolerance for five genes tested. Furthermore, variations in a large proportion of the in silico identified genes associated with minimum or mean-temperatures exhibited a significant association with growth phenotypes experimentally assessed at low temperature for a small set of natural accessions. This study shows a functional relevance of gene variants associated with environmental variables and supports the feasibility of the use of local temperature factors in investigating the genetic basis of temperature adaptation.

Critical assessment of genome-scale metabolic models of Arabidopsis thaliana.

Genome-scale metabolic models (GEMs) have enabled researchers to perform systems-level studies of living organisms. Flux balance analysis (FBA), as a constraint-based technique, enables computation of reaction fluxes and prediction of the metabolic phenotypes of a cell under a set of specified conditions. The quality of a GEM is important for obtaining accurate predictions. In this study, we evaluated the quality of five available GEMs for Arabidopsis thaliana from various points of views. To do this, we inspected some of their important features, including the number of reactions with well-defined gene-protein-reaction rules, number of blocked reactions, mass-unbalanced reactions, prediction accuracy in the simulation of key metabolic functions and existence of erroneous energy generating cycles (EGCs). All of the models were found to include some mass-unbalanced reactions. Moreover, four out of five models were found to include EGCs. However, Aracell includes the maximum number of blocked reactions, which suggests the presence of several incomplete pathways. These results clearly show that simulation by using these models may result in erroneous predictions and all of the publicly available GEMs for A. thaliana require extensive curations before being applied in practice.

Assessment of Mitochondrial DNA Copy Number, Stability, and Repair in Arabidopsis.

Mitochondrial functions depend on the proper maintenance and expression of the mitochondrial genome (mtDNA). Therefore, understanding mtDNA replication and repair requires methods to assess its integrity. Mutations or chemical treatments that affect processes involved in the maintenance or stability of the mtDNA can affect its global copy number, but also the relative abundance of different genomic regions or the frequency of illegitimate recombination across repeated sequences. These can be conveniently tested by quantitative PCR (qPCR). Arabidopsis thaliana offers several advantages for studying these processes, because of the extensive collections of mutants, natural accessions and other genetic resources available from stock centers. Here we describe protocols we routinely use to explore changes in mtDNA copy number and relative stoichiometry in Arabidopsis mutants of genes involved in the replication, repair and recombination of the mtDNA.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

A central circadian oscillator confers defense heterosis in hybrids without growth vigor costs.

Plant immunity frequently incurs growth penalties, which known as the trade-off between immunity and growth. Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits but rarely for disease resistance. Here, we report that the central circadian oscillator, CCA1, confers heterosis for bacterial defense in hybrids without growth vigor costs, and it even significantly enhances the growth heterosis of hybrids under pathogen infection. The genetic perturbation of CCA1 abrogated heterosis for both defense and growth in hybrids. Upon pathogen attack, the expression of CCA1 in F1 hybrids is precisely modulated at different time points during the day by its rhythmic histone modifications. Before dawn of the first infection day, epigenetic activation of CCA1 promotes an elevation of salicylic acid accumulation in hybrids, enabling heterosis for defense. During the middle of every infection day, diurnal epigenetic repression of CCA1 leads to rhythmically increased chlorophyll synthesis and starch metabolism in hybrids, effectively eliminating the immunity-growth heterosis trade-offs in hybrids.

Knockout of the entire family of AITR genes in Arabidopsis leads to enhanced drought and salinity tolerance without fitness costs.

BACKGORUND: Environmental stresses including abiotic stresses and biotic stresses limit yield of plants. Stress-tolerant breeding is an efficient way to improve plant yield under stress conditions. Genome editing by CRISPR/Cas9 can be used in molecular breeding to improve agronomic traits in crops, but in most cases, with fitness costs. The plant hormone ABA regulates plant responses to abiotic stresses via signaling transduction. We previously identified AITRs as a family of novel transcription factors that play a role in regulating plant responses to ABA and abiotic stresses. We found that abiotic stress tolerance was increased in the single, double and triple aitr mutants. However, it is unclear if the increased abiotic stress tolerance in the mutants may have fitness costs. RESULTS: We report here the characterization of AITRs as suitable candidate genes for CRISPR/Cas9 editing to improve plant stress tolerance. By using CRISPR/Cas9 to target AITR3 and AITR4 simultaneously in the aitr256 triple and aitr1256 quadruple mutants respectively, we generated Cas9-free aitr23456 quintuple and aitr123456 sextuple mutants. We found that reduced sensitivities to ABA and enhanced tolerance to drought and salt were observed in these mutants. Most importantly, plant growth and development was not affected even in the aitr123456 sextuple mutants, in whom the entire AITR family genes have been knocked out, and the aitr123456 sextuple mutants also showed a wild type response to the pathogen infection. CONCLUSIONS: Our results suggest that knockout of the AITR family genes in Arabidopsis enhanced abiotic stress tolerance without fitness costs. Considering that knock-out a few AITRs will lead to enhanced abiotic stress tolerance, that AITRs are widely distributed in angiosperms with multiple encoding genes, AITRs may be targeted for molecular breeding to improve abiotic stress tolerance in plants including crops.

Molecular Assessment of the Toxic Mechanism of the Latest Neonicotinoid Dinotefuran with Glutathione Peroxidase 6 from Arabidopsis thaliana.

With widespread applications of the latest neonicotinoid in agriculture, dinotefuran has gradually become a hazardous contaminant for plants through the generation of excessive reactive oxygen species. However, the potential toxic mechanisms of oxidative damages to plants induced by dinotefuran are still unknown. As a core component of the glutathione antioxidant enzyme system, glutathione peroxidases have been used as biomarkers to reflect excessive oxidative stress. In this study, the hazardous effects of dinotefuran on AtGPX6 were investigated at the molecular level. The intrinsic fluorescence intensity of AtGPX6 was quenched using the static quenching mechanism upon binding with dinotefuran. Moreover, a single binding site was predicted for AtGPX6 toward dinotefuran, and the complex formation was presumed to be driven by hydrogen bonds or van der Waals forces, which conformed with the molecular docking results. In addition, AtGPX6 exhibited moderate binding affinity with dinotefuran based on the bio-layer interferometry assay. In addition, the loosening and unfolding of the protein skeleton of AtGPX6 with the addition of dinotefuran were explored along with the increase of hydrophobicity around tryptophan residues. Lastly, the toxic effects of dinotefuran on the root growth of Arabidopsis seedlings were also examined. The exploration of the binding mechanism of dinotefuran with AtGPX6 at the molecular level would provide the toxicity assessment of dinotefuran on plants.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

Determinants of enrollment in community based health insurance among Households in Tach-Armachiho Woreda, North Gondar, Ethiopia, 2019.

BACKGROUND: Recently in Ethiopia, there is an increasing movement to implement community based health insurance scheme as integral part of health care financing and remarkable movements has resulted in the spread of the scheme in different parts of the country. Despite such increasing effort, recent empirical evidence shows enrolment has remained low. To identify determinants of enrollment in community based health insurance among households in Tach-Armachiho Woreda, North Gondar, Ethiopia, 2019. METHODS: A community based unmatched case control study was conducted Tach-Armachiho Woreda from March to May 2019 among 262 participants (88 cases and 174 controls with case control ratio of 1:2). Study subjects were selected using multi-stage sampling technique. Data were collected using a pretested, structured interviewer administered questioner. Data were entered to Epi-info 7 and exported to SPSS version 20 for analysis. Bivariable and multivariable logistic regression model were used to see the determinants of enrollment in community based health insurance. Adjusted odds ratio with 95% CI at p-value <0.05 in multivariable logistics regression analysis factors were identified as statistically significantly associated. RESULT: Female headed households (AOR = 2.79, 95% CI = 1.16, 6.69), Increase in Age (AOR = 1.09, 95% CI = 1.05, 1.13) and negative perception towards community based health insurance (AOR = 0.062, 95% CI = .030, .128) were found to be significant predictors. CONCLUSION: This study provides evidence that the decision to enroll in the scheme is shaped by age and a combination of household head sex and perception towards community based health insurance. Implementers aimed at enhancing enrolment ought to act on the bases of this findings.

Low-cost and efficient confocal imaging method for arabidopsis flower.

For an extensive period of time apical meristem (SAM) has been considered as a mysterious organ, due to its small, hidden and dynamic structure. Confocal imaging, combined with fluorescent reporters, enables researchers to unveil the mechanisms underlying cellular activities, such as gene expression, cell division, growth patterns and cell-cell communications. Recently, a series of protocols were developed for confocal imaging of inflorescence meristem (IM) and floral meristem (FM). However, the requirement of high configuration, such as the need of a water-dipping lens without coverslip and the specialized turrets associated with fixed-stage microscopes, impedes the wide adoption of these methods. We exploited an improved object slide and matching method aiming to decrease the configuration requirement. Following this protocol, various dry microscope lenses can be selected with flexibility for building 3D images of IM and FM.

Hoechst-tagged Fluorescein Diacetate for the Fluorescence Imaging-based Assessment of Stomatal Dynamics in Arabidopsis thaliana.

In plants, stomata regulate water loss through transpiration for plant growth and survival in response to various environmental stressors; and simple methods to assess stomatal dynamics are needed for physiological studies. Herein, we report a fluorescence-imaging-based method using fluorescein diacetate tagged with Hoechst 33342, a nuclear staining chemical probe (HoeAc2Fl) for the qualitative assessment of stomatal dynamics. In our method, the stomatal movement is inferred by simple monitoring of the fluorescence intensity in the nucleus of the stomata.

COST1 regulates autophagy to control plant drought tolerance.

Plants balance their competing requirements for growth and stress tolerance via a sophisticated regulatory circuitry that controls responses to the external environments. We have identified a plant-specific gene, COST1 (constitutively stressed 1), that is required for normal plant growth but negatively regulates drought resistance by influencing the autophagy pathway. An Arabidopsis thaliana cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes, while overexpression of COST1 confers drought hypersensitivity and reduced autophagy. The COST1 protein is degraded upon plant dehydration, and this degradation is reduced upon treatment with inhibitors of the 26S proteasome or autophagy pathways. The drought resistance of a cost1 mutant is dependent on an active autophagy pathway, but independent of other known drought signaling pathways, indicating that COST1 acts through regulation of autophagy. In addition, COST1 colocalizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and affects the level of ATG8e protein through physical interaction with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. Under drought, COST1 is degraded, enabling activation of autophagy and suppression of growth to enhance drought tolerance. Our research places COST1 as an important regulator controlling the balance between growth and stress responses via the direct regulation of autophagy.

Assessment of indium toxicity to the model plant Arabidopsis.

The use of indium in semiconductor products has increased markedly in recent years. The release of indium into the ecosystem is inevitable. Under such circumstances, effective and accurate assessment of indium risk is important. An indispensable aspect of indium risk assessment is to understand the interactions of indium with plants, which are fundamental components of all ecosystems. Physiological responses of Arabidopsis thaliana exposed to indium were investigated by monitoring toxic effects, accumulation and speciation of indium in the plant. Indium can be taken up by plants and is accumulated mainly in roots. Limited indium root-to-shoot translocation occurs because of immobilization of indium in the root intercellular space and blockage of indium by the Casparian band in the endodermis. Indium caused stunted growth, oxidative stress, anthocyanization and unbalanced phosphorus nutrition. Indium jeopardizes phosphate uptake and translocation by inhibiting the accumulation of phosphate transporters PHOSPHATE TRANSPORTER1 (PHT1;1/4), responsible for phosphate uptake, and PHOSPHATE1 (PHO1), responsible for phosphate xylem loading. Organic acid secretion is stimulated by indium exposure. Secreted citrate could function as a potential detoxifier to lower indium uptake. Our findings provide insights into the potential fate and effects of indium in plants and will aid the evaluation of risks with indium contamination.

Structural Aspects and Prediction of Calmodulin-Binding Proteins.

Calmodulin (CaM) is an important intracellular protein that binds Ca2+ and functions as a critical second messenger involved in numerous biological activities through extensive interactions with proteins and peptides. CaM's ability to adapt to binding targets with different structures is related to the flexible central helix separating the N- and C-terminal lobes, which allows for conformational changes between extended and collapsed forms of the protein. CaM-binding targets are most often identified using prediction algorithms that utilize sequence and structural data to predict regions of peptides and proteins that can interact with CaM. In this review, we provide an overview of different CaM-binding proteins, the motifs through which they interact with CaM, and shared properties that make them good binding partners for CaM. Additionally, we discuss the historical and current methods for predicting CaM binding, and the similarities and differences between these methods and their relative success at prediction. As new CaM-binding proteins are identified and classified, we will gain a broader understanding of the biological processes regulated through changes in Ca2+ concentration through interactions with CaM.

Glutathione peroxidase 6 from Arabidopsis thaliana as potential biomarker for plants exposure assessment to di-(2-ethylhexyl) phthalate.

As a most abundant plasticizer, Di-(2-ethylhexyl) phthalate (DEHP) has been widely used in agriculture with an associated potential toxicity to many species including plants via the production of the excessive reactive oxygen species (ROS). However, the potential toxic mechanisms of the plasticizer DEHP-induced oxidative damage to plants remain unknown. The antioxidant enzyme glutathione peroxidase has been suggested as biomarkers to reflect over excessive oxidative stress. In this study, the effect of DEHP on AtGPX6 was evaluated by multi-spectroscopic techniques and molecular docking method. The fluorescence intensity of AtGPX6 was reduced by the static quenching mechanism upon the addition of DEHP. The predominant forces in complex formation was mainly impelled by hydrogen bonding and Van der Waals forces based on the negative ΔH and ΔS, which was in accordance with the molecular docking results. In addition, the secondary structural changes resulted from the complex formation were investigated in presence of different amounts of DEHP by the combination of fluorescence, UV-vis absorption and Circular dichroism spectra, which revealed the loosening and unfolding of the framework of AtGPX6 accompanied with the enhancement of the hydrophilicity around the tryptophan residues. The exploration of the interaction mechanism of DEHP with AtGPX6 at molecular level would help to evaluate the toxicity of the plasticizers and forecast the related adverse effects on plants.

The alternative splicing of SKU5-Similar3 in Arabidopsis.

Alternative splicing largely enhanced the diversity of transcriptome and proteome in eukaryas. Along with technological development, more and more genes are reported to be alternatively spliced during mRNA maturation. Here, I report the alternative splicing of SKU5-Similar 3 (SKS3) and its special splicing site in Arabidopsis. SKS3 was predicted to be alternatively transcribed into two variants, SKS3.1 and SKS3.2, which encoded a GPI-anchored protein and a soluble secretory protein, respectively. But, according to experimental data, instead of SKS3.2, a novel variant, SKS3.3, which encodes a protein with a transmembrane region at its C-terminus, was demonstrated. Interestingly, it exhibites a different organ-specific expression pattern with SKS3.1, and an unusual intron splicing site not following 'GT-AG' rule or any reported rule.