Heat Pre-Treatment Modified Host and Non-Host Interactions of Powdery Mildew with Barley Brassinosteroid Mutants and Wild Types.

High temperatures associated with climate change may increase the severity of plant diseases. This study investigated the effect of heat shock treatment on host and non-host barley powdery mildew interactions using brassinosteroid (BR) mutants of barley. Brassinosteroids are plant steroid hormones, but so far little is known about their role in plant-fungal interactions. Wild type barley cultivar Bowman and its near-isogenic lines with disturbances in BR biosynthesis or signalling showed high compatibility to barley powdery mildew race A6, while cultivar Delisa and its BR-deficient mutants 522DK and 527DK were fully incompatible with this pathogen (host plant-pathogen interactions). On the other hand, Bowman and its mutants were highly resistant to wheat powdery mildew, representing non-host plant-pathogen interactions. Heat pre-treatment induced shifts in these plant-pathogen interactions towards higher susceptibility. In agreement with the more severe disease symptoms, light microscopy showed a decrease in papillae formation and hypersensitive response, characteristic of incompatible interactions, when heat pre-treatment was applied. Mutant 527DK, but not 522DK, maintained high resistance to barley powdery mildew race A6 despite heat pre-treatment. By 10 days after heat treatment and infection, a noticeable shift became apparent in the chlorophyll a fluorescence and in various leaf reflectance parameters at all genotypes.

CRISPR enables sustainable cereal production for a greener future.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has become the most important tool for targeted genome editing in many plant and animal species over the past decade. The CRISPR/Cas9 technology has also sparked a flood of applications and technical advancements in genome editing in the key cereal crops, including rice, wheat, maize, and barley. Here, we review advanced uses of CRISPR/Cas9 and derived systems in genome editing of cereal crops to enhance a variety of agronomically important features. We also highlight new technological advances for delivering preassembled Cas9-gRNA ribonucleoprotein (RNP)-editing systems, multiplex editing, gain-of-function strategies, the use of artificial intelligence (AI)-based tools, and combining CRISPR with novel speed breeding (SB) and vernalization strategies.

Mutual dependence of brassinosteroid homeostasis and plasmodesmata permeability.

Brassinosteroids (BRs) are exceptional phytohormones: they do not undergo a long-distance transport between plant organs. However, the mechanism of short-distance (intercellular) transport of BRs remains poorly understood. Recently, Wang et al. provided a novel insight into the mutual dependence of BR homeostasis, their intercellular transport, and plasmodesmata permeability.

CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals - current status, improvements, and perspectives.

Cereal crops, including triticeae species (barley, wheat, rye), as well as edible cereals (wheat, corn, rice, oat, rye, sorghum), are significant suppliers for human consumption, livestock feed, and breweries. Over the past half-century, modern varieties of cereal crops with increased yields have contributed to global food security. However, presently cultivated elite crop varieties were developed mainly for optimal environmental conditions. Thus, it has become evident that taking into account the ongoing climate changes, currently a priority should be given to developing new stress-tolerant cereal cultivars. It is necessary to enhance the accuracy of methods and time required to generate new cereal cultivars with the desired features to adapt to climate change and keep up with the world population expansion. The CRISPR/Cas9 system has been developed as a powerful and versatile genome editing tool to achieve desirable traits, such as developing high-yielding, stress-tolerant, and disease-resistant transgene-free lines in major cereals. Despite recent advances, the CRISPR/Cas9 application in cereals faces several challenges, including a significant amount of time required to develop transgene-free lines, laboriousness, and a limited number of genotypes that may be used for the transformation and in vitro regeneration. Additionally, developing elite lines through genome editing has been restricted in many countries, especially Europe and New Zealand, due to a lack of flexibility in GMO regulations. This review provides a comprehensive update to researchers interested in improving cereals using gene-editing technologies, such as CRISPR/Cas9. We will review some critical and recent studies on crop improvements and their contributing factors to superior cereals through gene-editing technologies.

CRISPR/Cas9 boosts wheat yield by reducing brassinosteroid signaling.

A modern green revolution is needed to ensure global food security. Recently, Song et al. reported a new strategy to create high-yielding, semi-dwarf wheat varieties with improved nitrogen-use efficiency by inhibiting brassinosteroid (BR) signaling through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9)-mediated knockout of the ZnF-B gene encoding a zinc-finger RING-type E3 ligase.

Analyses of genes encoding the Glycogen Synthase Kinases in rice and Arabidopsis reveal mechanisms which regulate their expression during development and responses to abiotic stresses.

Plant Glycogen Synthase Kinases (GSKs) enable a crosstalk among the brassinosteroid signaling and phytohormonal- and stress-response pathways to regulate various physiological processes. Initial information about regulation of the GSK proteins' activity was obtained, however, mechanisms that modulate expression of the GSK genes during plant development and stress responses remain largely unknown. Taking into account the importance of the GSK proteins, combined with the lack of in-depth knowledge about modulation of their expression, research in this area may provide a significant insight into mechanisms regulating these aspects of plant biology. In the current study, a detailed analysis of the GSK promoters in rice and Arabidopsis was performed, including identification of the CpG/CpNpG islands, tandem repeats, cis-acting regulatory elements, conserved motifs, and transcription factor-binding sites. Moreover, characterization of expression profiles of the GSK genes in different tissues, organs and under various abiotic stress conditions was performed. Additionally, protein-protein interactions between products of the GSK genes were predicted. Results of this study provided intriguing information about these aspects and insight into various regulatory mechanisms that influence non-redundant and diverse functions of the GSK genes during development and stress responses. Therefore, they may constitute a reference for future research in other plant species.

Glycogen synthase kinases in model and crop plants - From negative regulators of brassinosteroid signaling to multifaceted hubs of various signaling pathways and modulators of plant reproduction and yield.

Glycogen synthase kinases, also known as SHAGGY-like Kinases (GSKs/SKs), are highly conserved serine/threonine protein kinases present both in animals and plants. Plant genomes contain multiple homologs of the GSK3 genes which participate in various biological processes. Plant GSKs/SKs, and their best known representative in Arabidopsis thaliana - Brassinosteroid Insentisive2 (BIN2/SK21) in particular, were first identified as components of the brassinosteroid (BR) signaling pathway. As phytohormones, BRs regulate a wide range of physiological processes in plants - from germination, cell division, elongation and differentiation to leaf senescence, and response to environmental stresses. The GSKs/SKs proteins belong to a group of several highly conserved components of the BR signaling which evolved early during evolution of this molecular relay. However, recent reports indicated that the GSKs/SKs proteins are also implicated in signaling pathways of other phytohormones and stress-response processes. As a consequence, the GSKs/SKs proteins became hubs of various signaling pathways and modulators of plant development and reproduction. Thus, it is very important to understand molecular mechanisms regulating activity of the GSKs/SKs proteins, but also to get insights into role of the GSKs/SKs proteins in modulation of stability and activity of various substrate proteins which participate in the numerous signaling pathways. Although elucidation of these aspects is still in progress, this review presents a comprehensive and detailed description of these processes and their implications for regulation of development, stress response, and reproduction of model and crop species. The GSKs/SKs proteins and their activity are modulated through phosphorylation and de-phosphorylation reactions which are regulated by various proteins. Importantly, both phosphorylations and de-phosphorylations may have positive and negative effects on the activity of the GSKs/SKs proteins. Additionally, the activity of the GSKs/SKs proteins is positively regulated by reactive oxygen species, whereas it is negatively regulated through ubiquitylation, deacetylation, and nitric oxide-mediated nitrosylation. On the other hand, the GSKs/SKs proteins interact with proteins representing various signaling pathways, and on the basis of the complicated network of interactions the GSKs/SKs proteins differentially regulate various physiological, developmental, stress response, and yield-related processes.

In-Silico Study of Brassinosteroid Signaling Genes in Rice Provides Insight Into Mechanisms Which Regulate Their Expression.

Brassinosteroids (BRs) regulate a diverse spectrum of processes during plant growth and development and modulate plant physiology in response to environmental fluctuations and stress factors. Thus, the BR signaling regulators have the potential to be targeted for gene editing to optimize the architecture of plants and make them more resilient to environmental stress. Our understanding of the BR signaling mechanism in monocot crop species is limited compared to our knowledge of this process accumulated in the model dicot species - Arabidopsis thaliana. A deeper understanding of the BR signaling and response during plant growth and adaptation to continually changing environmental conditions will provide insight into mechanisms that govern the coordinated expression of the BR signaling genes in rice (Oryza sativa) which is a model for cereal crops. Therefore, in this study a comprehensive and detailed in silico analysis of promoter sequences of rice BR signaling genes was performed. Moreover, expression profiles of these genes during various developmental stages and reactions to several stress conditions were analyzed. Additionally, a model of interactions between the encoded proteins was also established. The obtained results revealed that promoters of the 39 BR signaling genes are involved in various regulatory mechanisms and interdependent processes that influence growth, development, and stress response in rice. Different transcription factor-binding sites and cis-regulatory elements in the gene promoters were identified which are involved in regulation of the genes' expression during plant development and reactions to stress conditions. The in-silico analysis of BR signaling genes in O. sativa provides information about mechanisms which regulate the coordinated expression of these genes during rice development and in response to other phytohormones and environmental factors. Since rice is both an important crop and the model species for other cereals, this information may be important for understanding the regulatory mechanisms that modulate the BR signaling in monocot species. It can also provide new ways for the plant genetic engineering technology by providing novel potential targets, either cis-elements or transcriptional factors, to create elite genotypes with desirable traits.

Comprehensive Overview of the Brassinosteroid Biosynthesis Pathways: Substrates, Products, Inhibitors, and Connections.

Brassinosteroids (BRs) as a class of steroid plant hormones participate in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, flowering, and seed germination, as well as in responding to environmental stresses. During four decades of research, the BR biosynthetic pathways have been well studied with forward- and reverse genetics approaches. The free BRs contain 27, 28, and 29 carbons within their skeletal structure: (1): 5α-cholestane or 26-nor-24α-methyl-5α-cholestane for C27-BRs; (2) 24α-methyl-5α-cholestane, 24ß-methyl-5α-cholestane or 24-methylene-5α-cholestane for C28-BRs; (3) 24α-ethyl-5α-cholestane, 24(Z)-ethylidene-5α-cholestane, 25-methyl-5α-campestane or 24-methylene-25-methyl-5α-cholestane for C29-BRs, as well as different kinds and orientations of oxygenated functions in A- and B-ring. These alkyl substituents are also common structural features of sterols. BRs are derived from sterols carrying the same side chain. The C27-BRs without substituent at C-24 are biosynthesized from cholesterol. The C28-BRs carrying either an α-methyl, ß-methyl, or methylene group are derived from campesterol, 24-epicampesterol or 24-methylenecholesterol, respectively. The C29-BRs with an α-ethyl group are produced from sitosterol. Furthermore, the C29 BRs carrying methylene at C-24 and an additional methyl group at C-25 are derived from 24-methylene-25-methylcholesterol. Generally, BRs are biosynthesized via cycloartenol and cycloartanol dependent pathways. Till now, more than 17 compounds were characterized as inhibitors of the BR biosynthesis. For nine of the inhibitors (e.g., brassinazole and YCZ-18) a specific target reaction within the BR biosynthetic pathway has been identified. Therefore, the review highlights comprehensively recent advances in our understanding of the BR biosynthesis, sterol precursors, and dependencies between the C27-C28 and C28-C29 pathways.

Insights into Metabolic Reactions of Semi-Dwarf, Barley Brassinosteroid Mutants to Drought.

The roles of endogenous brassinosteroids (BRs) in the modulation of reaction to drought and genetic regulation of this process are still obscure. In this study, a multidirectional analysis was performed on semi-dwarf barley (Hordeum vulgare) Near-Isogenic Lines (NILs) and the reference cultivar "Bowman" to get insights into various aspects of metabolic reaction to drought. The NILs are defective in BR biosynthesis or signaling and displayed an enhanced tolerance to drought. The BR metabolism perturbations affected the glucose and fructose accumulation under the control and stress conditions. The BR metabolism abnormalities negatively affected the sucrose accumulation as well. However, during drought, the BR-deficient NILs accumulated higher contents of sucrose than the "Bowman" cultivar. Under the control conditions, accumulation of transcripts encoding antioxidant enzymes ascorbate peroxidase (HvAPX) and superoxide dismutase (HvSOD) was BR-dependent. However, during drought, the accumulation of HvAPX transcript was BR-dependent, whereas accumulations of transcripts encoding catalase (HvCAT) and HvSOD were not affected by the BR metabolism perturbations. The obtained results reveal a significant role of BRs in regulation of the HvAPX and HvCAT enzymatic activities under control conditions and the HvAPX and HvSOD activities during physiological reactions to drought.

The Impact of Mutations in the HvCPD and HvBRI1 Genes on the Physicochemical Properties of the Membranes from Barley Acclimated to Low/High Temperatures.

(1) Background: The study characterized barley mutants with brassinosteroid (BR) biosynthesis and signaling disturbances in terms of the physicochemical/structural properties of membranes to enrich the knowledge about the role of brassinosteroids for lipid metabolism and membrane functioning. (2) Methods: The Langmuir method was used to investigate the properties of the physicochemical membranes. Langmuir monolayers were formed from the lipid fractions isolated from the plants growing at 20 °C and then acclimated at 5 °C or 27 °C. The fatty acid composition of the lipids was estimated using gas chromatography. (3) Results: The BR-biosynthesis and BR-signaling mutants of barley were characterized by a temperature-dependent altered molar percentage of fatty acids (from 14:0 to 20:1) in their galactolipid and phospholipid fractions in comparison to wild-type (WT). For example, the mutants had a lower molar percentage of 18:3 in the phospholipid (PL) fraction. The same regularity was observed at 5 °C. It resulted in altered physicochemical parameters of the membranes (Alim, πcoll, Cs-1). (4) Conclusions: BR may be involved in regulating fatty acid biosynthesis or their transport/incorporation into the cell membranes. Mutants had altered physicochemical parameters of their membranes, compared to the WT, which suggests that BR may have a multidirectional impact on the membrane-dependent physiological processes.

HSP Transcript and Protein Accumulation in Brassinosteroid Barley Mutants Acclimated to Low and High Temperatures.

In temperature stress, the main role of heat-shock proteins (HSP) is to act as molecular chaperones for other cellular proteins. However, knowledge about the hormonal regulation of the production of the HSP is quite limited. Specifically, little is known about the role of the plant steroid hormones-brassinosteroids (BR)-in regulating the HSP expression. The aim of our study was to answer the question of how a BR deficit or disturbances in its signaling affect the accumulation of the HSP90, HSP70, HSP18, and HSP17 transcripts and protein in barley growing at 20 °C (control) and during the acclimation of plants at 5 °C and 27 °C. In barley, the temperature of plant growth modified the expression of HSPs. Furthermore, the BR-deficient mutants (mutations in the HvDWARF or HvCPD genes) and BR-signaling mutants (mutation in the HvBRI1 gene) were characterized by altered levels of the transcripts and proteins of the HSP group compared to the wild type. The BR-signaling mutant was characterized by a decreased level of the HSP transcripts and heat-shock proteins. In the BR-deficient mutants, there were temperature-dependent cases when the decreased accumulation of the HSP70 and HSP90 transcripts was connected to an increased accumulation of these HSP. The significance of changes in the accumulation of HSPs during acclimation at 27 °C and 5 °C is discussed in the context of the altered tolerance to more extreme temperatures of the studied mutants (i.e., heat stress and frost, respectively).

Exploring the Brassinosteroid Signaling in Monocots Reveals Novel Components of the Pathway and Implications for Plant Breeding.

Brassinosteroids (BRs) are a class of steroidal phytohormones which are key regulators of diverse processes during whole life cycle of plants. Studies conducted in the dicot model species Arabidopsis thaliana have allowed identification and characterization of various components of the BR signaling. It is currently known that the BR signaling is interconnected at various stages with other phytohormonal and stress signaling pathways. It enables a rapid and efficient adaptation of plant metabolism to constantly changing environmental conditions. However, our knowledge about mechanism of the BR signaling in the monocot species is rather limited. Thus, identification of new components of the BR signaling in monocots, including cereals, is an ongoing process and has already led to identification of some monocot-specific components of the BR signaling. It is of great importance as disturbances in the BR signaling influence architecture of mutant plants, and as a consequence, the reaction to environmental conditions. Currently, the modulation of the BR signaling is considered as a target to enhance yield and stress tolerance in cereals, which is of particular importance in the face of global climate change.

Role of Strigolactones: Signalling and Crosstalk with Other Phytohormones.

Plant hormones play important roles in controlling how plants grow and develop. While metabolism provides the energy needed for plant survival, hormones regulate the pace of plant growth. Strigolactones (SLs) were recently defined as new phytohormones that regulate plant metabolism and, in turn, plant growth and development. This group of phytohormones is derived from carotenoids and has been implicated in a wide range of physiological functions including regulation of plant architecture (inhibition of bud outgrowth and shoot branching), photomorphogenesis, seed germination, nodulation, and physiological reactions to abiotic factors. SLs also induce hyphal branching in germinating spores of arbuscular mycorrhizal fungi (AMF), a process that is important for initiating the connection between host plant roots and AMF. This review outlines the physiological roles of SLs and discusses the significance of interactions between SLs and other phytohormones to plant metabolic responses.

Molecular Dynamics of Chloroplast Membranes Isolated from Wild-Type Barley and a Brassinosteroid-Deficient Mutant Acclimated to Low and High Temperatures.

Plants have developed various acclimation strategies in order to counteract the negative effects of abiotic stresses (including temperature stress), and biological membranes are important elements in these strategies. Brassinosteroids (BR) are plant steroid hormones that regulate plant growth and development and modulate their reaction against many environmental stresses including temperature stress, but their role in modifying the properties of the biological membrane is poorly known. In this paper, we characterise the molecular dynamics of chloroplast membranes that had been isolated from wild-type and a BR-deficient barley mutant that had been acclimated to low and high temperatures in order to enrich the knowledge about the role of BR as regulators of the dynamics of the photosynthetic membranes. The molecular dynamics of the membranes was investigated using electron paramagnetic resonance (EPR) spectroscopy in both a hydrophilic and hydrophobic area of the membranes. The content of BR was determined, and other important membrane components that affect their molecular dynamics such as chlorophylls, carotenoids and fatty acids in these membranes were also determined. The chloroplast membranes of the BR-mutant had a higher degree of rigidification than the membranes of the wild type. In the hydrophilic area, the most visible differences were observed in plants that had been grown at 20 °C, whereas in the hydrophobic core, they were visible at both 20 and 5 °C. There were no differences in the molecular dynamics of the studied membranes in the chloroplast membranes that had been isolated from plants that had been grown at 27 °C. The role of BR in regulating the molecular dynamics of the photosynthetic membranes will be discussed against the background of an analysis of the photosynthetic pigments and fatty acid composition in the chloroplasts.

Plasma membrane ATPase and the aquaporin HvPIP1 in barley brassinosteroid mutants acclimated to high and low temperature.

The integral parts of the cell membranes are the functional proteins, which are crucial for cell life. Among them, proton-pumping ATPase and aquaporins appear to be of particular importance. There is some knowledge about the effect of the temperature during plant growth, including stress-inducing temperatures, on the accumulation of the membrane proteins: plasma membrane H+-ATPase and aquaporins, but not much is known about the effect of the phytohormones (i.e. brassinosteroids (BR)) on control of accumulation of these proteins. The aim of our study was to answer the question of how a BR deficit and disturbances in the BR perception/signalling affect the accumulation of plasma membrane H+-ATPase (PM H+-ATPase), the aquaporin HvPIP1 transcript and protein in barley growing at 20 °C and during its acclimation at 5 °C and 27 °C. For the studies, the BR-deficient mutant 522DK (derived from the wild-type Delisa), the BR-deficient mutant BW084 and the BR-signalling mutant BW312 and their wild-type Bowman were used. Generally, temperature of growth was significant factor influencing on the level of the accumulation of the H+-ATPase and HvPIP1 transcript and the PM H+-ATPase and HvPIP1 protein in barley leaves. The level of the accumulation of the HvPIP1 transcript decreased at 5 °C (compared to 20 °C), but was higher at 27 °C than at 20 °C in the analyzed cultivars. In both cultivars the protein HvPIP1 was accumulated in the highest amounts at 27 °C. On the other hand, the barley mutants with a BR deficiency or with BR signalling disturbances were characterised by an altered accumulation level of PM H+-ATPase, the aquaporin HvPIP1 transcript and protein (compared to the wild types), which may suggest the involvement of brassinosteroids in regulating PM H+-ATPase and aquaporin HvPIP1 at the transcriptional and translational levels.

The dmc1 Mutant Allows an Insight Into the DNA Double-Strand Break Repair During Meiosis in Barley (Hordeum vulgare L.).

Meiosis is a process of essential importance for sexual reproduction, as it leads to production of gametes. The recombination event (crossing-over) generates genetic variation by introducing new combination of alleles. The first step of crossing-over is introduction of a targeted double-strand break (DSB) in DNA. DMC1 (Disrupted Meiotic cDNA1) is a recombinase that is specific only for cells undergoing meiosis and takes part in repair of such DSBs by searching and invading homologous sequences that are subsequently used as a template for the repair process. Although role of the DMC1 gene has been validated in Arabidopsis thaliana, a functional analysis of its homolog in barley, a crop species of significant importance in agriculture, has never been performed. Here, we describe the identification of barley mutants carrying substitutions in the HvDMC1 gene. We performed mutational screening using TILLING (Targeting Induced Local Lesions IN Genomes) strategy and the barley TILLING population, HorTILLUS, developed after double-treatment of spring barley cultivar 'Sebastian' with sodium azide and N-methyl-N-nitrosourea. One of the identified alleles, dmc1.c, was found independently in two different M2 plants. The G2571A mutation identified in this allele leads to a substitution of the highly conserved amino acid (arginine-183 to lysine) in the DMC1 protein sequence. Two mutant lines carrying the same dmc1.c allele show similar disturbances during meiosis. The chromosomal aberrations included anaphase bridges and chromosome fragments in anaphase/telophase I and anaphase/telophase II, as well as micronuclei in tetrads. Moreover, atypical tetrads containing three or five cells were observed. A highly increased frequency of all chromosome aberrations during meiosis have been observed in the dmc1.c mutants compared to parental variety. The results indicated that DMC1 is required for the DSB repair, crossing-over and proper chromosome disjunction during meiosis in barley.

Crosstalk of the Brassinosteroid Signalosome with Phytohormonal and Stress Signaling Components Maintains a Balance between the Processes of Growth and Stress Tolerance.

Brassinosteroids (BRs) are a class of phytohormones, which regulate various processes during plant life cycle. Intensive studies conducted with genetic, physiological and molecular approaches allowed identification of various components participating in the BR signaling-from the ligand perception, through cytoplasmic signal transduction, up to the BR-dependent gene expression, which is regulated by transcription factors and chromatin modifying enzymes. The identification of new components of the BR signaling is an ongoing process, however an emerging view of the BR signalosome indicates that this process is interconnected at various stages with other metabolic pathways. The signaling crosstalk is mediated by the BR signaling proteins, which function as components of the transmembrane BR receptor, by a cytoplasmic kinase playing a role of the major negative regulator of the BR signaling, and by the transcription factors, which regulate the BR-dependent gene expression and form a complicated regulatory system. This molecular network of interdependencies allows a balance in homeostasis of various phytohormones to be maintained. Moreover, the components of the BR signalosome interact with factors regulating plant reactions to environmental cues and stress conditions. This intricate network of interactions enables a rapid adaptation of plant metabolism to constantly changing environmental conditions.