Salicylic acid and RNA interference mediate antiviral immunity of plant stem cells.

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

The Ninhydrin Reaction Revisited: Optimisation and Application for Quantification of Free Amino Acids.

The ninhydrin reaction is commonly used for the detection of amino acids. However, in the literature, different conditions with respect to the buffer system, its pH and concentration, type of organic solvent, incubation time, and temperature, as well as the concentrations of the reagents, are described. To identify the most suitable conditions, colour development with reagents of varying compositions and different reaction temperatures and times were investigated using asparagine as a model amino acid. Asparagine was selected since it is one of the most abundant free amino acids in many types of samples. The optimal reaction mixture consisted of 0.8 mol L-1 potassium acetate, 1.6 mol L-1 acetic acid, 20 mg mL-1 ninhydrin and 0.8 mg mL-1 hydrindantin in DMSO/acetate buffer 40/60 (v/v) (final concentrations). The best reaction condition was heating the samples in 1.5 mL reaction tubes to 90 °C for 45 min. Afterwards, the samples were diluted with 2-propanol/water 50/50 (v/v) and the absorbance was measured at 570 nm. The proteinogenic amino acids showed a similar response except for cysteine and proline. The method was highly sensitive and showed excellent linearity as well as intra-day and inter-day reproducibility.

Brassinosteroid-regulated bHLH transcription factor CESTA induces the gibberellin 2-oxidase GA2ox7.

Brassinosteroids (BRs) are plant steroids that have growth-promoting capacities, which are partly enabled by an ability to induce biosynthesis of gibberellins (GAs), a second class of plant hormones. In addition, BRs can also activate GA catabolism; here we show that in Arabidopsis (Arabidopsis thaliana) the basic helix-loop-helix transcription factor CESTA (CES) and its homologues BRASSINOSTEROID-ENHANCED EXPRESSION (BEE) 1 and 3 contribute to this activity. CES and the BEEs are BR-regulated at the transcriptional and posttranslational level and participate in different physiological processes, including vegetative and reproduction development, shade avoidance, and cold stress responses. We show that CES/BEEs can induce the expression of the class III GA 2-oxidase GA2ox7 and that this activity is increased by BRs. In BR signaling - and CES/BEE-deficient mutants, GA2ox7 expression decreased, yielding reduced levels of GA110, a product of GA2ox7 activity. In plants that over-express CES, GA2ox7 expression is hyper-responsive to BR, GA110 levels are elevated and amounts of bioactive GA are reduced. We provide evidence that CES directly binds to the GA2ox7 promoter and is activated by BRs, but can also act by BR-independent means. Based on these results, we propose a model for CES activity in GA catabolism where CES can be recruited for GA2ox7 induction not only by BR, but also by other factors.

Tissue-Specific Hormone Signalling and Defence Gene Induction in an In Vitro Assembly of the Rapeseed Verticillium Pathosystem.

Priming plants with beneficial microbes can establish rapid and robust resistance against numerous pathogens. Here, compelling evidence is provided that the treatment of rapeseed plants with Trichoderma harzianum OMG16 and Bacillus velezensis FZB42 induces defence activation against Verticillium longisporum infection. The relative expressions of the JA biosynthesis genes LOX2 and OPR3, the ET biosynthesis genes ACS2 and ACO4 and the SA biosynthesis and signalling genes ICS1 and PR1 were analysed separately in leaf, stem and root tissues using qRT-PCR. To successfully colonize rapeseed roots, the V. longisporum strain 43 pathogen suppressed the biosynthesis of JA, ET and SA hormones in non-primed plants. Priming led to fast and strong systemic responses of JA, ET and SA biosynthesis and signalling gene expression in each leaf, stem and root tissue. Moreover, the quantification of plant hormones via UHPLC-MS analysis revealed a 1.7- and 2.6-fold increase in endogenous JA and SA in shoots of primed plants, respectively. In roots, endogenous JA and SA levels increased up to 3.9- and 2.3-fold in Vl43-infected primed plants compared to non-primed plants, respectively. Taken together, these data indicate that microbial priming stimulates rapeseed defence responses against Verticillium infection and presumably transduces defence signals from the root to the upper parts of the plant via phytohormone signalling.

The acyltransferase PMAT1 malonylates brassinolide glucoside.

Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown. Here, we show that in Arabidopsis thaliana malonylation of BL-23-O-Glc is catalyzed by the acyltransferase phenolic glucoside malonyl-transferase 1 (PMAT1), which is also known to malonylate phenolic glucosides and lipid amides. Loss of PMAT1 abolished BL-23-O-malonylglucoside formation and enriched BL-23-O-Glc, showing that the enzyme acts on the glucoside. An overexpression of PMAT1 in plants where UGT73C6 was also overexpressed, and thus, BL-23-O-Glc formation was promoted, enhanced the symptoms of BR-deficiency of UGT73C6oe plants, providing evidence that PMAT1 contributes to BL inactivation. Based on these results, a model is proposed in which PMAT1 acts in the conversion of both endogenous and xenobiotic glucosides to adjust metabolic homeostasis in spatial and temporal modes.

Synergistic Effects of a Root-Endophytic Trichoderma Fungus and Bacillus on Early Root Colonization and Defense Activation Against Verticillium longisporum in Rapeseed.

Rhizosphere-competent microbes often interact with plant roots and exhibit beneficial effects on plant performance. Numerous bacterial and fungal isolates are able to prime host plants for fast adaptive responses against pathogen attacks. Combined action of fungi and bacteria may lead to synergisms exceeding effects of single strains. Individual beneficial fungi and bacteria have been extensively studied in Arabidopsis thaliana, but little is known about their concerted actions in the Brassicaceae. Here, an in-vitro system with oilseed rape (Brassica napus) was established. Roots of two different cultivars were inoculated with well-characterized fungal (Trichoderma harzianum OMG16) and bacterial (Bacillus velezensis FZB42) isolates alone or in combination. Microscopic analysis confirmed that OMG16 hyphae entered root hairs through root hair tips and formed distinct intracellular structures. Quantitative PCR revealed that root colonization of OMG16 increased up to 10-fold in the presence of FZB42. Relative transcript levels of the ethylene- and jasmonic acid-responsive genes PDF1.2, ERF2, and AOC3 were recorded in leaves by quantitative reverse transcription PCR to measure induced systemic resistance in tissues distant from the roots. Combined action of OMG16 and FZB42 induced transcript abundances more efficiently than single inoculation. Importantly, microbial priming reduced Verticillium longisporum root infection in rapeseed by approximately 100-fold compared with nonprimed plants. Priming also led to faster and stronger systemic responses of the defense genes PDF1.2, ERF2, AOC3, and VSP2.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Dual control of MAPK activities by AP2C1 and MKP1 MAPK phosphatases regulates defence responses in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades transmit environmental signals and induce stress and defence responses in plants. These signalling cascades are negatively controlled by specific Ser/Thr protein phosphatases of the type 2C (PP2C) and dual-specificity phosphatase (DSP) families that inactivate stress-induced MAPKs; however, the interplay between phosphatases of these different types has remained unknown. This work reveals that different Arabidopsis MAPK phosphatases, the PP2C-type AP2C1 and the DSP-type MKP1, exhibit both specific and overlapping functions in plant stress responses. Each single mutant, ap2c1 and mkp1, and the ap2c1 mkp1 double mutant displayed enhanced stress-induced activation of the MAPKs MPK3, MPK4, and MPK6, as well as induction of a set of transcription factors. Moreover, ap2c1 mkp1 double mutants showed an autoimmune-like response, associated with increased levels of the stress hormones salicylic acid and ethylene, and of the phytoalexin camalexin. This phenotype was reduced in the ap2c1 mkp1 mpk3 and ap2c1 mkp1 mpk6 triple mutants, suggesting that the autoimmune-like response is due to MAPK misregulation. We conclude that the evolutionarily distant MAPK phosphatases AP2C1 and MKP1 contribute crucially to the tight control of MAPK activities, ensuring appropriately balanced stress signalling and suppression of autoimmune-like responses during plant growth and development.

Plant Nutrition: Physiological and Metabolic Responses, Molecular Mechanisms and Chromatin Modifications.

Plant growth and crop yield highly depend on the availability of all required nutrients, ideally in well-balanced ratios [...].

Quantification of Arbutin in Cosmetics, Drugs and Food Supplements by Hydrophilic-Interaction Chromatography.

Arbutin, the glucoside of hydroquinone, exists in two isomers, α-arbutin and ß-arbutin. The synthetic α isomer is mainly used as a skin brightening agent, while ß-arbutin occurs naturally, for instance in bearberry, and is used in drugs for treatment of lower urinary tract infections and as a food supplement. Since both isomers can be harmful at high concentrations, methods for their quantification are required. Classically they have been determined by reversed-phase chromatography, but separation of both isomers is often unsatisfactory. Here we present a simple and reliable method for quantification of α- and ß-arbutin based on hydrophilic-interaction chromatography. Prior to analysis, interfering compounds that would frequently be present in cosmetics and drugs, particularly biopolymers, were efficiently removed by precipitation with acetonitrile. In this paper, for separation, a Cyclobond I 2000 5 µm 250 × 4.6 mm column was employed as stationary phase and acetonitrile/water 92/8 (v/v) was used as an eluent at a flow rate of 0.8 mL min−1. For quantification, a UV detector operating at 284 nm was applied. Although analysis took less than 10 min, baseline separation of α- and ß-arbutin was achieved. The response was highly linear (r > 0.999) and the method had, for both α- and ß-arbutin, a LOD of 0.003% (w/w) and a LOQ of 0.009% (w/w). Moreover, the method showed excellent intra-day and inter-day repeatability with relative standard deviations in the range of 0.5% to 2.3% and 1.0% to 2.2%, respectively, with cosmetics, drugs and food supplements as samples.

Comparative analysis of epigenetic inhibitors reveals different degrees of interference with transcriptional gene silencing and induction of DNA damage.

Repetitive DNA sequences and some genes are epigenetically repressed by transcriptional gene silencing (TGS). When genetic mutants are not available or problematic to use, TGS can be suppressed by chemical inhibitors. However, informed use of epigenetic inhibitors is partially hampered by the absence of any systematic comparison. In addition, there is emerging evidence that epigenetic inhibitors cause genomic instability, but the nature of this damage and its repair remain unclear. To bridge these gaps, we compared the effects of 5-azacytidine (AC), 2'-deoxy-5-azacytidine (DAC), zebularine and 3-deazaneplanocin A (DZNep) on TGS and DNA damage repair. The most effective inhibitor of TGS was DAC, followed by DZNep, zebularine and AC. We confirmed that all inhibitors induce DNA damage and suggest that this damage is repaired by multiple pathways with a critical role of homologous recombination and of the SMC5/6 complex. A strong positive link between the degree of cytidine analog-induced DNA demethylation and the amount of DNA damage suggests that DNA damage is an integral part of cytidine analog-induced DNA demethylation. This helps us to understand the function of DNA methylation in plants and opens the possibility of using epigenetic inhibitors in biotechnology.

Higher expression of the strawberry xyloglucan endotransglucosylase/hydrolase genes FvXTH9 and FvXTH6 accelerates fruit ripening.

Fruit softening in Fragaria (strawberry) is proposed to be associated with the modification of cell wall components such as xyloglucan by the action of cell wall-modifying enzymes. This study focuses on the in vitro and in vivo characterization of two recombinant xyloglucan endotransglucosylase/hydrolases (XTHs) from Fragaria vesca, FvXTH9 and FvXTH6. Mining of the publicly available F. vesca genome sequence yielded 28 putative XTH genes. FvXTH9 showed the highest expression level of all FvXTHs in a fruit transcriptome data set and was selected with the closely related FvXTH6 for further analysis. To investigate their role in fruit ripening in more detail, the coding sequences of FvXTH9 and FvXTH6 were cloned into the vector pYES2 and expressed in Saccharomyces cerevisiae. FvXTH9 and FvXTH6 displayed xyloglucan endotransglucosylase (XET) activity towards various acceptor substrates using xyloglucan as the donor substrate. Interestingly, FvXTH9 showed activity of mixed-linkage glucan:xyloglucan endotransglucosylase (MXE) and cellulose:xyloglucan endotransglucosylase (CXE). The optimum pH of both FvXTH9 and FvXTH6 was 6.5. The prediction of subcellular localization suggested localization to the secretory pathway, which was confirmed by localization studies in Nicotiana tabacum. Overexpression showed that Fragaria × ananassa fruits infiltrated with FvXTH9 and FvXTH6 ripened faster and showed decreased firmness compared with the empty vector control pBI121. Thus FvXTH9 and also FvXTH6 might promote strawberry fruit ripening by the modification of cell wall components.

Carbon isotope composition, water use efficiency, and drought sensitivity are controlled by a common genomic segment in maize.

KEY MESSAGE: A genomic segment on maize chromosome 7 influences carbon isotope composition, water use efficiency, and leaf growth sensitivity to drought, possibly by affecting stomatal properties. Climate change is expected to decrease water availability in many agricultural production areas around the globe. Therefore, plants with improved ability to grow under water deficit are urgently needed. We combined genetic, phenomic, and physiological approaches to understand the relationship between growth, stomatal conductance, water use efficiency, and carbon isotope composition in maize (Zea mays L.). Using near-isogenic lines derived from a maize introgression library, we analysed the effects of a genomic region previously identified as affecting carbon isotope composition. We show stability of trait expression over several years of field trials and demonstrate in the phenotyping platform Phenodyn that the same genomic region also influences the sensitivity of leaf growth to evaporative demand and soil water potential. Our results suggest that the studied genomic region affecting carbon isotope discrimination also harbours quantitative trait loci playing a role in maize drought sensitivity possibly via stomatal behaviour and development. We propose that the observed phenotypes collectively originate from altered stomatal conductance, presumably via abscisic acid.

Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants.

Brassinosteroids (BRs) are growth-promoting plant hormones that play a role in abiotic stress responses, but molecular modes that enable this activity remain largely unknown. Here we show that BRs participate in the regulation of freezing tolerance. BR signaling-defective mutants of Arabidopsis thaliana were hypersensitive to freezing before and after cold acclimation. The constitutive activation of BR signaling, in contrast, enhanced freezing resistance. Evidence is provided that the BR-controlled basic helix-loop-helix transcription factor CESTA (CES) can contribute to the constitutive expression of the C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF) transcriptional regulators that control cold responsive (COR) gene expression. In addition, CBF-independent classes of BR-regulated COR genes are identified that are regulated in a BR- and CES-dependent manner during cold acclimation. A model is presented in which BRs govern different cold-responsive transcriptional cascades through the posttranslational modification of CES and redundantly acting factors. This contributes to the basal resistance against freezing stress, but also to the further improvement of this resistance through cold acclimation.

Pyrrolizidine Alkaloids: Biosynthesis, Biological Activities and Occurrence in Crop Plants.

Pyrrolizidine alkaloids (PAs) are heterocyclic secondary metabolites with a typical pyrrolizidine motif predominantly produced by plants as defense chemicals against herbivores. They display a wide structural diversity and occur in a vast number of species with novel structures and occurrences continuously being discovered. These alkaloids exhibit strong hepatotoxic, genotoxic, cytotoxic, tumorigenic, and neurotoxic activities, and thereby pose a serious threat to the health of humans since they are known contaminants of foods including grain, milk, honey, and eggs, as well as plant derived pharmaceuticals and food supplements. Livestock and fodder can be affected due to PA-containing plants on pastures and fields. Despite their importance as toxic contaminants of agricultural products, there is limited knowledge about their biosynthesis. While the intermediates were well defined by feeding experiments, only one enzyme involved in PA biosynthesis has been characterized so far, the homospermidine synthase catalyzing the first committed step in PA biosynthesis. This review gives an overview about structural diversity of PAs, biosynthetic pathways of necine base, and necic acid formation and how PA accumulation is regulated. Furthermore, we discuss their role in plant ecology and their modes of toxicity towards humans and animals. Finally, several examples of PA-producing crop plants are discussed.

Inhibitors of Brassinosteroid Biosynthesis and Signal Transduction.

Chemical inhibitors are invaluable tools for investigating protein function in reverse genetic approaches. Their application bears many advantages over mutant generation and characterization. Inhibitors can overcome functional redundancy, their application is not limited to species for which tools of molecular genetics are available and they can be applied to specific tissues or developmental stages, making them highly convenient for addressing biological questions. The use of inhibitors has helped to elucidate hormone biosynthesis and signaling pathways and here we review compounds that were developed for the plant hormones brassinosteroids (BRs). BRs are steroids that have strong growth-promoting capacities, are crucial for all stages of plant development and participate in adaptive growth processes and stress response reactions. In the last two decades, impressive progress has been made in BR inhibitor development and application, which has been instrumental for studying BR modes of activity and identifying and characterizing key players. Both, inhibitors that target biosynthesis, such as brassinazole, and inhibitors that target signaling, such as bikinin, exist and in a comprehensive overview we summarize knowledge and methodology that enabled their design and key findings of their use. In addition, the potential of BR inhibitors for commercial application in plant production is discussed.

Determination of the [15N]-Nitrate/[14N]-Nitrate Ratio in Plant Feeding Studies by GC⁻MS.

Feeding experiments with stable isotopes are helpful tools for investigation of metabolic fluxes and biochemical pathways. For assessing nitrogen metabolism, the heavier nitrogen isotope, [15N], has been frequently used. In plants, it is usually applied in form of [15N]-nitrate, which is assimilated mainly in leaves. Thus, methods for quantification of the [15N]-nitrate/[14N]-nitrate ratio in leaves are useful for the planning and evaluation of feeding and pulse-chase experiments. Here we describe a simple and sensitive method for determining the [15N]-nitrate to [14N]-nitrate ratio in leaves. Leaf discs (8 mm diameter, approximately 10 mg fresh weight) were sufficient for analysis, allowing a single leaf to be sampled multiple times. Nitrate was extracted with hot water and derivatized with mesitylene in the presence of sulfuric acid to nitromesitylene. The derivatization product was analyzed by gas chromatography-mass spectrometry with electron ionization. Separation of the derivatized samples required only 6 min. The method shows excellent repeatability with intraday and interday standard deviations of less than 0.9 mol%. Using the method, we show that [15N]-nitrate declines in leaves of hydroponically grown Crassocephalum crepidioides, an African orphan crop, with a biological half-life of 4.5 days after transfer to medium containing [14N]-nitrate as the sole nitrogen source.

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis.

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway.

Brassinosteroids Are Master Regulators of Gibberellin Biosynthesis in Arabidopsis.

Plant growth and development are highly regulated processes that are coordinated by hormones including the brassinosteroids (BRs), a group of steroids with structural similarity to steroid hormones of mammals. Although it is well understood how BRs are produced and how their signals are transduced, BR targets, which directly confer the hormone's growth-promoting effects, have remained largely elusive. Here, we show that BRs regulate the biosynthesis of gibberellins (GAs), another class of growth-promoting hormones, in Arabidopsis thaliana. We reveal that Arabidopsis mutants deficient in BR signaling are severely impaired in the production of bioactive GA, which is correlated with defective GA biosynthetic gene expression. Expression of the key GA biosynthesis gene GA20ox1 in the BR signaling mutant bri1-301 rescues many of its developmental defects. We provide evidence that supports a model in which the BR-regulated transcription factor BES1 binds to a regulatory element in promoters of GA biosynthesis genes in a BR-induced manner to control their expression. In summary, our study underscores a role of BRs as master regulators of GA biosynthesis and shows that this function is of major relevance for the growth and development of vascular plants.

Assay for abscisic acid 8'-hydroxylase activity of cloned plant cytochrome P450 oxidases in Saccharomyces cerevisiae.

The plant hormone abscisic acid (ABA) regulates many processes, including response to drought, seed dormancy and abscission of leaves and fruits. For maintenance of ABA homeostasis, catabolism of ABA by 8'-hydroxylation and subsequent cyclisation to phaseic acid (PA) is crucial. However, detection of ABA 8'-hydroxylation activity is tedious. We present a simple and rapid method for detection of ABA 8'-hydroxylase activity by cloning cDNAs of interest and expressing the respective protein in yeast. Upon addition of ABA, PA is formed and subsequently quantified in the yeast cell culture supernatant by heart cutting 2D-HPLC or GC-MS.

Quantification of the Pyrrolizidine Alkaloid Jacobine in Crassocephalum crepidioides by Cation Exchange High-Performance Liquid Chromatography.

INTRODUCTION: Pyrrolizidine alkaloids (PAs) are secondary plant metabolites with considerable hepatoxic, tumorigenic and genotoxic potential. For separation, reversed phase chromatography is commonly used because of its excellent compatibility with detection by mass spectrometry. However, reversed phase chromatography has a low selectivity for PAs. OBJECTIVE: The objective of this work was to investigate the suitability of cation exchange chromatography for separation of PAs and to develop a rapid method for quantification of jacobine in Crassocephalum crepidioides that is suitable for analysis of huge sample numbers as required for mutant screening procedures. RESULTS: We demonstrate that cation exchange chromatography offers excellent selectivity for PAs allowing their separation from most other plant metabolites. Due to the high selectivity, plant extracts can be directly analysed after simple sample preparation. Detection with UV at 200 nm instead of mass spectrometry can be applied, which makes the method very simple and cost-effective. The recovery rate of the method exceeded 95%, the intra-day and inter-day standard deviations were below 7% and the limit of detection and quantification were 1 mg/kg and 3 mg/kg, respectively. CONCLUSION: The developed method is sufficiently sensitive for reproducible detection of jacobine in C. crepidioides. Simple sample preparation and rapid separation allows for quantification of jacobine in plant material in a high-throughput manner. Thus, the method is suitable for genetic screenings and may be applicable for other plant species, for instance Jacobaea maritima. In addition, our results show that C. crepidioides cannot be considered safe for human consumption. Copyright © 2017 John Wiley & Sons, Ltd.