Contrasting cytosolic glutathione redox dynamics under abiotic and biotic stress in barley as revealed by the biosensor Grx1-roGFP2.

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding ß-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Tomato MYB21 Acts in Ovules to Mediate Jasmonate-Regulated Fertility.

The function of the plant hormone jasmonic acid (JA) in the development of tomato (Solanum lycopersicum) flowers was analyzed with a mutant defective in JA perception (jasmonate-insensitive1-1, jai1-1). In contrast with Arabidopsis (Arabidopsis thaliana) JA-insensitive plants, which are male sterile, the tomato jai1-1 mutant is female sterile, with major defects in female development. To identify putative JA-dependent regulatory components, we performed transcriptomics on ovules from flowers at three developmental stages from wild type and jai1-1 mutants. One of the strongly downregulated genes in jai1-1 encodes the MYB transcription factor SlMYB21. Its Arabidopsis ortholog plays a crucial role in JA-regulated stamen development. SlMYB21 was shown here to exhibit transcription factor activity in yeast, to interact with SlJAZ9 in yeast and in planta, and to complement Arabidopsis myb21-5 To analyze SlMYB21 function, we generated clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9 (Cas9) mutants and identified a mutant by Targeting Induced Local Lesions in Genomes (TILLING). These mutants showed female sterility, corroborating a function of MYB21 in tomato ovule development. Transcriptomics analysis of wild type, jai1-1, and myb21-2 carpels revealed processes that might be controlled by SlMYB21. The data suggest positive regulation of JA biosynthesis by SlMYB21, but negative regulation of auxin and gibberellins. The results demonstrate that SlMYB21 mediates at least partially the action of JA and might control the flower-to-fruit transition..

Jasmonate Signaling during Arabidopsis Stamen Maturation.

The last stages of stamen development, collectively called stamen maturation, encompass pollen viability, filament elongation and anther dehiscence or opening. These processes are essential for male fertility in Arabidopsis and require the function of jasmonate signaling. There is a good understanding of jasmonate synthesis, perception and transcriptional outputs in Arabidopsis stamens. In addition, the spatiotemporal localization of jasmonate signaling components at the tissue and cellular levels has started to emerge in recent years. However, the ultimate cellular functions activated by jasmonate to promote stamen maturation remain unknown. The hormones auxin and gibberellin have been proposed to control the activation of jasmonate synthesis to promote stamen maturation, although we hypothesize that this action is rather indirect. In this review, we examine these different areas, attempt to clarify some confusing aspects found in the literature and raise testable hypothesis that may help to further understand how jasmonate controls male fertility in Arabidopsis.

Genes for seed longevity in barley identified by genomic analysis on near isogenic lines.

Genes controlling differences in seed longevity between 2 barley (Hordeum vulgare) accessions were identified by combining quantitative genetics "omics" technologies in near isogenic lines (NILs). The NILs were derived from crosses between the spring barley landraces L94 from Ethiopia and Cebada Capa from Argentina. A combined transcriptome and proteome analysis on mature, nonaged seeds of the 2 parental lines and the L94 NILs by RNA-sequencing and total seed proteomic profiling identified the UDP-glycosyltransferase MLOC_11661.1 as candidate gene for the quantitative trait loci on 2H, and the NADP-dependent malic enzyme (NADP-ME) MLOC_35785.1 as possible downstream target gene. To validate these candidates, they were expressed in Arabidopsis under the control of constitutive promoters to attempt complementing the T-DNA knockout line nadp-me1. Both the NADP-ME MLOC_35785.1 and the UDP-glycosyltransferase MLOC_11661.1 were able to rescue the nadp-me1 seed longevity phenotype. In the case of the UDP-glycosyltransferase, with high accumulation in NILs, only the coding sequence of Cebada Capa had a rescue effect.

Multilayered Organization of Jasmonate Signalling in the Regulation of Root Growth.

Physical damage can strongly affect plant growth, reducing the biomass of developing organs situated at a distance from wounds. These effects, previously studied in leaves, require the activation of jasmonate (JA) signalling. Using a novel assay involving repetitive cotyledon wounding in Arabidopsis seedlings, we uncovered a function of JA in suppressing cell division and elongation in roots. Regulatory JA signalling components were then manipulated to delineate their relative impacts on root growth. The new transcription factor mutant myc2-322B was isolated. In vitro transcription assays and whole-plant approaches revealed that myc2-322B is a dosage-dependent gain-of-function mutant that can amplify JA growth responses. Moreover, myc2-322B displayed extreme hypersensitivity to JA that totally suppressed root elongation. The mutation weakly reduced root growth in undamaged plants but, when the upstream negative regulator NINJA was genetically removed, myc2-322B powerfully repressed root growth through its effects on cell division and cell elongation. Furthermore, in a JA-deficient mutant background, ninja1 myc2-322B still repressed root elongation, indicating that it is possible to generate JA-responses in the absence of JA. We show that NINJA forms a broadly expressed regulatory layer that is required to inhibit JA signalling in the apex of roots grown under basal conditions. By contrast, MYC2, MYC3 and MYC4 displayed cell layer-specific localisations and MYC3 and MYC4 were expressed in mutually exclusive regions. In nature, growing roots are likely subjected to constant mechanical stress during soil penetration that could lead to JA production and subsequent detrimental effects on growth. Our data reveal how distinct negative regulatory layers, including both NINJA-dependent and -independent mechanisms, restrain JA responses to allow normal root growth. Mechanistic insights from this work underline the importance of mapping JA signalling components to specific cell types in order to understand and potentially engineer the growth reduction that follows physical damage.

Axial and Radial Oxylipin Transport.

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.

Role of NINJA in root jasmonate signaling.

Wound responses in plants have to be coordinated between organs so that locally reduced growth in a wounded tissue is balanced by appropriate growth elsewhere in the body. We used a JASMONATE ZIM DOMAIN 10 (JAZ10) reporter to screen for mutants affected in the organ-specific activation of jasmonate (JA) signaling in Arabidopsis thaliana seedlings. Wounding one cotyledon activated the reporter in both aerial and root tissues, and this was either disrupted or restricted to certain organs in mutant alleles of core components of the JA pathway including COI1, OPR3, and JAR1. In contrast, three other mutants showed constitutive activation of the reporter in the roots and hypocotyls of unwounded seedlings. All three lines harbored mutations in Novel Interactor of JAZ (NINJA), which encodes part of a repressor complex that negatively regulates JA signaling. These ninja mutants displayed shorter roots mimicking JA-mediated growth inhibition, and this was due to reduced cell elongation. Remarkably, this phenotype and the constitutive JAZ10 expression were still observed in backgrounds lacking the ability to synthesize JA or the key transcriptional activator MYC2. Therefore, JA-like responses can be recapitulated in specific tissues without changing a plant's ability to make or perceive JA, and MYC2 either has no role or is not the only derepressed transcription factor in ninja mutants. Our results show that the role of NINJA in the root is to repress JA signaling and allow normal cell elongation. Furthermore, the regulation of the JA pathway differs between roots and aerial tissues at all levels, from JA biosynthesis to transcriptional activation.

The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding.

Jasmonates are lipid mediators that control defence gene expression in response to wounding and other environmental stresses. These small molecules can accumulate at distances up to several cm from sites of damage and this is likely to involve cell-to-cell jasmonate transport.Also, and independently of jasmonate synthesis, transport and perception, different long distance wound signals that stimulate distal jasmonate synthesis are propagated at apparent speeds of several cm min­1 to tissues distal to wounds in a mechanism that involves clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes. A search for jasmonate synthesis enzymes that might decode these signals revealed LOX6, a lipoxygenase that is necessary for much of the rapid accumulation of jasmonic acid at sites distal to wounds. Intriguingly, the LOX6 promoter is expressed in a distinct niche of cells that are adjacent to mature xylem vessels,a location that would make these contact cells sensitive to the release of xylem water column tension upon wounding. We propose a model in which rapid axial changes in xylem hydrostatic pressure caused by wounding travel through the vasculature and lead to slower,radially dispersed pressure changes that act in a clade 3 GLR-dependent mechanism to promote distal jasmonate synthesis.

The transcription factor HSFA7b controls thermomemory at the shoot apical meristem by regulating ethylene biosynthesis and signaling in Arabidopsis.

The shoot apical meristem (SAM) is responsible for overall shoot growth by generating all aboveground structures. Recent research has revealed that the SAM displays an autonomous heat stress (HS) memory of a previous non-lethal HS event. Considering the importance of the SAM for plant growth, it is essential to determine how its thermomemory is mechanistically controlled. Here, we report that HEAT SHOCK TRANSCRIPTION FACTOR A7b (HSFA7b) plays a crucial role in this process in Arabidopsis, as the absence of functional HSFA7b results in the temporal suppression of SAM activity after thermopriming. We found that HSFA7b directly regulates ethylene response at the SAM by binding to the promoter of the key ethylene signaling gene ETHYLENE-INSENSITIVE 3 to establish thermotolerance. Moreover, we demonstrated that HSFA7b regulates the expression of ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-LIKE 1, both of which encode ethylene biosynthesis repressors, thereby ensuring ethylene homeostasis at the SAM. Taken together, these results reveal a crucial and tissue-specific role for HSFA7b in thermomemory at the Arabidopsis SAM.

Hordedane diterpenoid phytoalexins restrict Fusarium graminearum infection but enhance the colonization by Bipolaris sorkiniana of barley roots.

Plant immunity is a multi-layered process that includes recognition of patterns or effectors from pathogens to elicit defense responses. These include the induction of a cocktail of defense metabolites that typically restrict pathogen virulence. Here, we investigate the interaction between barley roots and the fungal pathogens Bipolaris sorokiniana (Bs) and Fusarium graminearum (Fg) at the metabolite level. We identify hordedanes, a previously undescribed set of labdane- related diterpenoids with antimicrobial properties, as critical players in these interactions. Infection of barley roots by Bs and Fg elicits hordedane synthesis from a 600-kb gene cluster. Heterologous reconstruction of the biosynthesis pathway in yeast and Nicotiana benthamiana produced several hordedanes, including one of the most functionally decorated products 19-ß-hydroxy- hordetrienoic acid (19-OH-HTA). Barley mutants in the diterpene synthase genes of the cluster are unable to produce hordedanes but, unexpectedly, show reduced Bs colonization. By contrast, colonization by Fusarium graminearum, another fungal pathogen of barley and wheat, is four-fold higher in mutants completely lacking hordedanes. Accordingly, 19-OH-HTA enhances both germination and growth of Bs, while it inhibits other pathogenic fungi, including Fg. Microscopy and transcriptomics suggest hordedanes delay the necrotrophic phase of Bs. Our data show that adapted pathogens such as Bs can subvert plant metabolic defenses to facilitate root colonization.

A GH81-type ß-glucan-binding protein enhances colonization by mutualistic fungi in barley.

Cell walls are important interfaces of plant-fungal interactions, acting as robust physical and chemical barriers against invaders. Upon fungal colonization, plants deposit phenolics and callose at the sites of fungal penetration to prevent further fungal progression. Alterations in the composition of plant cell walls significantly impact host susceptibility. Furthermore, plants and fungi secrete glycan hydrolases acting on each other's cell walls. These enzymes release various sugar oligomers into the apoplast, some of which activate host immunity via surface receptors. Recent characterization of cell walls from plant-colonizing fungi has emphasized the abundance of ß-glucans in different cell wall layers, which makes them suitable targets for recognition. To characterize host components involved in immunity against fungi, we performed a protein pull-down with the biotinylated ß-glucan laminarin. Thereby, we identified a plant glycoside hydrolase family 81-type glucan-binding protein (GBP) as a ß-glucan interactor. Mutation of GBP1 and its only paralog, GBP2, in barley led to decreased colonization by the beneficial root endophytes Serendipita indica and S. vermifera, as well as the arbuscular mycorrhizal fungus Rhizophagus irregularis. The reduction of colonization was accompanied by enhanced responses at the host cell wall, including an extension of callose-containing cell wall appositions. Moreover, GBP mutation in barley also reduced fungal biomass in roots by the hemibiotrophic pathogen Bipolaris sorokiniana and inhibited the penetration success of the obligate biotrophic leaf pathogen Blumeria hordei. These results indicate that GBP1 is involved in the establishment of symbiotic associations with beneficial fungi-a role that has potentially been appropriated by barley-adapted pathogens.

Auxin boosts energy generation pathways to fuel pollen maturation in barley.

Pollen grains become increasingly independent of the mother plant as they reach maturity through poorly understood developmental programs. We report that the hormone auxin is essential during barley pollen maturation to boost the expression of genes encoding almost every step of heterotrophic energy production pathways. Accordingly, auxin is necessary for the flux of sucrose and hexoses into glycolysis and to increase the levels of pyruvate and two tricarboxylic (TCA) cycle metabolites (citrate and succinate). Moreover, bioactive auxin is synthesized by the pollen-localized enzyme HvYUCCA4, supporting that pollen grains autonomously produce auxin to stimulate a specific cellular output, energy generation, that fuels maturation processes such as starch accumulation. Our results demonstrate that auxin can shift central carbon metabolism to drive plant cell development, which suggests a direct mechanism for auxin's ability to promote growth and differentiation.

Control of sexuality by the sk1-encoded UDP-glycosyltransferase of maize.

Sex determination in maize involves the production of staminate and pistillate florets from an initially bisexual floral meristem. Pistil elimination in staminate florets requires jasmonic acid signaling, and functional pistils are protected by the action of the silkless 1 (sk1) gene. The sk1 gene was identified and found to encode a previously uncharacterized family 1 uridine diphosphate glycosyltransferase that localized to the plant peroxisomes. Constitutive expression of an sk1 transgene protected all pistils in the plant, causing complete feminization, a gain-of-function phenotype that operates by blocking the accumulation of jasmonates. The segregation of an sk1 transgene was used to effectively control the production of pistillate and staminate inflorescences in maize plants.

Identifying resistance gene analogs associated with resistances to different pathogens in common bean.

ABSTRACT A polymerase chain reaction approach using degenerate primers that targeted the conserved domains of cloned plant disease resistance genes (R genes) was used to isolate a set of 15 resistance gene analogs (RGAs) from common bean (Phaseolus vulgaris). Eight different classes of RGAs were obtained from nucleotide binding site (NBS)-based primers and seven from not previously described Toll/Interleukin-1 receptor-like (TIR)-based primers. Putative amino acid sequences of RGAs were significantly similar to R genes and contained additional conserved motifs. The NBS-type RGAs were classified in two subgroups according to the expected final residue in the kinase-2 motif. Eleven RGAs were mapped at 19 loci on eight linkage groups of the common bean genetic map constructed at Centro Internacional de Agricultura Tropical. Genetic linkage was shown for eight RGAs with partial resistance to anthracnose, angular leaf spot (ALS) and Bean golden yellow mosaic virus (BGYMV). RGA1 and RGA2 were associated with resistance loci to anthracnose and BGYMV and were part of two clusters of R genes previously described. A new major cluster was detected by RGA7 and explained up to 63.9% of resistance to ALS and has a putative contribution to anthracnose resistance. These results show the usefulness of RGAs as candidate genes to detect and eventually isolate numerous R genes in common bean.

Jasmonates.

ARABIDOPSIS IS A SUPERB MODEL FOR THE STUDY OF AN IMPORTANT SUBGROUP OF OXYLIPINS: the jasmonates. Jasmonates control many responses to cell damage and invasion and are essential for reproduction. Jasmonic acid (JA) is a prohormone and is conjugated to hydrophobic amino acids to produce regulatory ligands. The major receptor for active jasmonate ligands is closely related to auxin receptors and, as in auxin signaling, jasmonate signaling requires the destruction of repressor proteins. This chapter uses a frequently asked question (FAQ) approach and concludes with a practical section.

tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize.

Sex determination in maize is controlled by a developmental cascade leading to the formation of unisexual florets derived from an initially bisexual floral meristem. Abortion of pistil primordia in staminate florets is controlled by a tasselseed-mediated cell death process. We positionally cloned and characterized the function of the sex determination gene tasselseed1 (ts1). The TS1 protein encodes a plastid-targeted lipoxygenase with predicted 13-lipoxygenase specificity, which suggests that TS1 may be involved in the biosynthesis of the plant hormone jasmonic acid. In the absence of a functional ts1 gene, lipoxygenase activity was missing and endogenous jasmonic acid concentrations were reduced in developing inflorescences. Application of jasmonic acid to developing inflorescences rescued stamen development in mutant ts1 and ts2 inflorescences, revealing a role for jasmonic acid in male flower development in maize.