Propiconazole Is a Specific and Accessible Brassinosteroid (BR) Biosynthesis Inhibitor for Arabidopsis and Maize

Brassinosteroids (BRs) are steroidal hormones that play pivotal roles during plant development. In addition to the characterization of BR deficient mutants, specific BR biosynthesis inhibitors played an essential role in the elucidation of BR function in plants. However, high costs and limited availability of common BR biosynthetic inhibitors constrain their key advantage as a species-independent tool to investigate BR function. We studied propiconazole (Pcz) as an alternative to the BR inhibitor brassinazole (Brz). Arabidopsis seedlings treated with Pcz phenocopied BR biosynthetic mutants. The steady state mRNA levels of BR, but not gibberellic acid (GA), regulated genes increased proportional to the concentrations of Pcz. Moreover, root inhibition and Pcz-induced expression of BR biosynthetic genes were rescued by 24epi-brassinolide, but not by GA3 co-applications. Maize seedlings treated with Pcz showed impaired mesocotyl, coleoptile, and true leaf elongation. Interestingly, the genetic background strongly impacted the tissue specific sensitivity towards Pcz. Based on these findings we conclude that Pcz is a potent and specific inhibitor of BR biosynthesis and an alternative to Brz. The reduced cost and increased availability of Pcz, compared to Brz, opens new possibilities to study BR function in larger crop species

The Arabidopsis \u3cem\u3edwf/ste1\u3c/em\u3e Mutant is Defective in the Δ\u3csup\u3e7\u3c/sup\u3e Sterol C-5 Desaturation Step Leading to Brassinosteroid Biosynthesis

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Δ7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7

The Arabidopsis \u3cem\u3edwarf1\u3c/em\u3e Mutant is Defective in the Conversion of 24-Methylenecholesterol to Campesterol in Brassinosteroid Biosynthesis

Since the isolation and characterization of dwarf1-1 (dwf1-1) from a T-DNA insertion mutant population, phenotypically similar mutants, including deetiolated2 (det2),constitutive photomorphogenesis and dwarfism(cpd), brassinosteroid insensitive1 (bri1), and dwf4, have been reported to be defective in either the biosynthesis or the perception of brassinosteroids. We present further characterization of dwf1-1 and additional dwf1 alleles. Feeding tests with brassinosteroid-biosynthetic intermediates revealed that dwf1 can be rescued by 22α-hydroxycampesterol and downstream intermediates in the brassinosteroid pathway. Analysis of the endogenous levels of brassinosteroid intermediates showed that 24-methylenecholesterol in dwf1 accumulates to 12 times the level of the wild type, whereas the level of campesterol is greatly diminished, indicating that the defective step is in C-24 reduction. Furthermore, the deduced amino acid sequence of DWF1 shows significant similarity to a flavin adenine dinucleotide-binding domain conserved in various oxidoreductases, suggesting an enzymatic role for DWF1. In support of this, 7 of 10 dwf1 mutations directly affected the flavin adenine dinucleotide-binding domain. Our molecular characterization of dwf1 alleles, together with our biochemical data, suggest that the biosynthetic defect in dwf1 results in reduced synthesis of bioactive brassinosteroids, causing dwarfism

Genetics and biology of Arabidopsis brassinosteroid dwarf mutants

Brassinosteroids (BRs) have long been known to be effective in plant growth promotion. However, definitive evidence of BR's role in growth stimulation has remained unclear. Recently, genetic approaches using BR-deficient dwarf(dwf) mutants have begun to unravel the role of BRs in plant growth and development. BR dwarf mutants are characterized by multiple growth alterations: robust stem, reduced fertility, prolonged life cycle, dark-green appearance, round and curled leaves, and when grown in the dark, short hypocotyls and expanded cotyledons. Genetic analysis of the dwf mutants defined eight independent genetic loci defective in BR biosynthesis or perception. Allelism tests with previously reported genes revealed that d̲i̲m̲inuto 1 (dim1) was an allele of dwf1, and dwf2, dwf3, and dwf6 are allelic to b̲r̲assinosteroid i̲nsensitive (bri), c̲onstitutive p̲hotomorphogenesis and d̲warfism (cpd), and d̲e̲-e̲t̲iolated2 (det2), respectively. dwf4, dwf5, dwf7, and dwf8 were found to be novel and are the focus of this research. Anatomical analysis demonstrates that a reduction in cell length causes dwarf phenotype. Dwarfism was rescued by exogenous application of BRs. Feeding studies utilizing BR biosynthetic intermediates were employed to identify defective steps of BR biosynthesis in each of these dwarf mutants. dwf4 mutants were rescued only by 22α hydroxylated BRs, suggesting that the 22α hydroxylation reactions, putative rate-determining steps, are blocked. In fact, DWF4 has been cloned and shown to encode a cytochrome P450 steroid hydroxylase. Feeding studies also showed that dwf8 plants are rescued only by intermediates after 3 dehydrogenation reactions, indicating that the 3-dehydrogenase is defective in dwf8 plants. Gas Chromatography-Selective Ion Monitoring (GC-SIM) analysis of endogenous BRs in dwf5 plants showed that the level of 24-methylene cholesterol is greatly diminished as compared to wild type, suggesting that the biochemical defect occurs before 24-methylene cholesterol. Similar to dwf5, the biosynthetic defect in dwf7 is also shown to be in a step before 24-methylene cholesterol. The pleiotropic phenotypes in these dwf mutants due to biochemical defects in BR biosynthesis suggests that BRs are essential for proper growth and development of plants

Identification of brassinosteroid genes in Brachypodium distachyon

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Background Brassinosteroids (BRs) are steroidal phytohormones that are involved in diverse physiological processes and affect many important traits, such as plant stature, stress tolerance, leaf angle, fertility, and grain filling. BR signaling and biosynthetic pathways have been studied in various plants, such as the model dicot Arabidopsis thaliana; however, relatively little is known about these pathways in monocots. Results To characterize BR-related processes in the model grass Brachypodium distachyon, we studied the response of these plants to the specific BR biosynthesis inhibitor, propiconazole (Pcz). We found that treatments with Pcz produced a dwarf phenotype in B. distachyon seedlings, similar to that observed in Pcz-treated Arabidopsis plants and in characterized BR-deficient mutants. Through bioinformatics analysis, we identified a list of putative homologs of genes known to be involved in BR biosynthesis and signaling in Arabidopsis, such as DWF4, BR6OX2, CPD, BRI1, and BIN2. Evaluating the response of these genes to Pcz treatments revealed that candidates for BdDWF4, BR6OX2 and, CPD were under feedback regulation. In addition, Arabidopsis plants heterologously expressing BdDWF4 displayed tall statures and elongated petioles, as would be expected in plants with elevated levels of BRs. Moreover, heterologous expression of BdBIN2 in Arabidopsis resulted in dwarfism, suggesting that BdBIN2 functions as a negative regulator of BR signaling. However, the dwarf phenotypes of Arabidopsis bri1-5, a weak BRI1 mutant allele, were not complemented by overexpression of BdBRI1, indicating that BdBRI1 and BRI1 are not functionally equivalent. Conclusion We identified components of the BR biosynthetic and signaling pathways in Brachypodium, and provided examples of both similarities and differences in the BR biology of these two plants. Our results suggest a framework for understanding BR biology in monocot crop plants such as Zea mays (maize) and Oryza sativa (rice)

Plant-Based COVID-19 Vaccines: Current Status, Design, and Development Strategies of Candidate Vaccines

The prevalence of the coronavirus disease 2019 (COVID-19) pandemic in its second year has led to massive global human and economic losses. The high transmission rate and the emergence of diverse SARS-CoV-2 variants demand rapid and effective approaches to preventing the spread, diagnosing on time, and treating affected people. Several COVID-19 vaccines are being developed using different production systems, including plants, which promises the production of cheap, safe, stable, and effective vaccines. The potential of a plant-based system for rapid production at a commercial scale and for a quick response to an infectious disease outbreak has been demonstrated by the marketing of carrot-cell-produced taliglucerase alfa (Elelyso) for Gaucher disease and tobacco-produced monoclonal antibodies (ZMapp) for the 2014 Ebola outbreak. Currently, two plant-based COVID-19 vaccine candidates, coronavirus virus-like particle (CoVLP) and Kentucky Bioprocessing (KBP)-201, are in clinical trials, and many more are in the preclinical stage. Interim phase 2 clinical trial results have revealed the high safety and efficacy of the CoVLP vaccine, with 10 times more neutralizing antibody responses compared to those present in a convalescent patient’s plasma. The clinical trial of the CoVLP vaccine could be concluded by the end of 2021, and the vaccine could be available for public immunization thereafter. This review encapsulates the efforts made in plant-based COVID-19 vaccine development, the strategies and technologies implemented, and the progress accomplished in clinical trials and preclinical studies so far

Additional file 1: Figure S1. of Identification of brassinosteroid genes in Brachypodium distachyon

Brachypodium seedlings exhibit dwarfism in response to propiconazole treatment in dark. Morphology of 7-days-old seedlings subjected to the mock treatment and 50 μM Pcz. (PPTX 1022 kb