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Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin profiles were examined in roots and nodules of the actinorhizal species Datisca glomerata and the model legume Medicago truncatula. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter DR5. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots vs. nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of cis -zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of D. glomerata , but not of M. truncatula. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the DR5 promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to DR5 activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in M. truncatula , but a negative effect in D. glomerata. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in D. glomerata does not follow that pattern. [ABSTRACT FROM AUTHOR]

Selection for deep rooting is critical for the development of genotypes that are able to explore deep soil water and nutrients, particularly as agricultural resources become more limited. However, current root phenotyping techniques demand significant investments of time, money, and effort, and measurements on very young plants or plants grown under soilless culture. This study introduced four novel and simple techniques for fast evaluation of root depth in tube rhizotrons, which enable root observation around the transparent tube walls, and allow roots growing to natural size in semi-field conditions. The first and second innovations involve the introduction of 15N tracer and herbicide to the roots, which estimated root activity by measuring the responses of the shoots aboveground. The third involves placement of a cone deep in the rhizotron, to increase chances to observe more deep roots along the tube walls. The fourth involves measurement of roots that emerge from the rhizotron bottom. The reliability of these techniques were assessed in a series of five experiments during 2014 and 2015. These tests compared two pairs of genotypes that previous studies had shown to have mutually distinctive root traits: the spring wheat pair of ‘April bearded’ vs. ‘Dacke’; and the winter wheat pair of ‘Tabasco’ vs. ‘Genius’, with the first of each pair being the genotype known for deep rooting. Results showed that the new techniques were as good as or better than existing alternatives at accurately measuring root traits. In eight of the nine comparisons, the measurements were consistent with the expectations of root characteristics for these known genotypes. Importantly, the indirect root activity measures (herbicide and 15N) showed the same trend as the direct root observation techniques in all experiments, but higher ability to distinguish the genotypes and more promise for future upscaling for plant breeding. [ABSTRACT FROM AUTHOR]

Gene amplification followed by functional diversification is a major force in evolution. A typical example of this is seen in the WUSCHEL-RELATED HOMEOBOX ( WOX ) gene family, named after the Arabidopsis stem cell regulator WUSCHEL . Here we analyze functional divergence in the WOX gene family. Members of the WUS clade, except the cambium stem cell regulator WOX4 , can substitute for WUS function in shoot and floral stem cell maintenance to different degrees. Stem cell function of WUS requires a canonical WUS-box, essential for interaction with TPL/TPR co-repressors, whereas the repressive EAR domain is dispensable and the acidic domain seems only to be required for female fertility. In contrast to the WUS clade, members of the ancient WOX13 and the WOX9 clades cannot support stem cell maintenance. Although the homeodomains are interchangeable between WUS and WOX9 clade members, a WUS-compatible homeodomain together with canonical WUS-box is not sufficient for stem cell maintenance. Our results suggest that WOX function in shoot and floral meristems of Arabidopsis is restricted to the modern WUS clade, suggesting that stem cell control is a derived function. Yet undiscovered functional domains in addition to the homeodomain and the WUS-box are necessary for this function. [ABSTRACT FROM AUTHOR]

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family. [ABSTRACT FROM AUTHOR]

Fructans are polymers of fructose and one of the main constituents of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates. Fructans are involved in cold and drought resistance, regrowth following defoliation and early spring growth, seed filling, have beneficial effects on human health and are used for industrial processes. Perennial ryegrass (Lolium perenne L.) serves as model species to study fructan metabolism. Fructan metabolism is under the control of both synthesis by fructosyltransferases (FTs) and breakdown through fructan exohydrolases (FEHs). The accumulation of fructans can be triggered by high sucrose levels and abiotic stress conditions such as drought and cold stress. However, detailed studies on the mechanisms involved in the regulation of fructan metabolism are scarce. Since different phytohormones, especially abscisic acid (ABA), are known to play an important role in abiotic stress responses, the possible short term regulation of the enzymes involved in fructan metabolism by the five classical phytohormones was investigated. Therefore, the activities of enzymes involved in fructan synthesis and breakdown, the expression levels for the corresponding genes and levels for water-soluble carbohydrates were determined following pulse treatments with ABA, auxin (AUX), ethylene (ET), gibberellic acid (GA), or kinetin (KIN). The most pronounced fast effects were a transient increase of FT activities by AUX, KIN, ABA, and ET, while minor effects were evident for 1-FEH activity with an increased activity in response to KIN and a decrease by GA. Fructan and sucrose levels were not affected. This observed discrepancy demonstrates the importance of determining enzyme activities to obtain insight into the physiological traits and ultimately the plant phenotype. The comparative analyses of activities for seven key enzymes of primary carbohydrate metabolism revealed no co-regulation between enzymes of the fructan and sucrose pool. [ABSTRACT FROM AUTHOR]

Despite the fact that fructans are the main constituent of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates, little knowledge is available on the regulation of the enzymes involved in fructan metabolism. The analysis of enzyme activities involved in this process has been hampered by the low affinity of the fructan enzymes for sucrose and fructans used as fructosyl donor. Further, the analysis of fructan composition and enzyme activities is restricted to specialized labs with access to suited HPLC equipment and appropriate fructan standards. The degradation of fructan polymers with high degree of polymerization (DP) by fructan exohydrolases (FEHs) to fructosyloligomers is important to liberate energy in the form of fructan, but also under conditions where the generation of low DP polymers is required. Based on published protocols employing enzyme coupled endpoint reactions in single cuvettes, we developed a simple and fast kinetic 1-FEH assay. This assay can be performed in multi-well plate format using plate readers to determine the activity of 1-FEH against 1-kestotriose, resulting in a significant time reduction. Kinetic assays allow an optimal and more precise determination of enzyme activities compared to endpoint assays, and enable to check the quality of any reaction with respect to linearity of the assay. The enzyme coupled kinetic 1-FEH assay was validated in a case study showing the expected increase in 1-FEH activity during cold treatment. This assay is cost effective and could be performed by any lab with access to a plate reader suited for kinetic measurements and readings at 340 nm, and is highly suited to assess temporal changes and relative differences in 1-FEH activities. Thus, this enzyme coupled kinetic 1-FEH assay is of high importance both to the field of basic fructan research and plant breeding. [ABSTRACT FROM AUTHOR]

Phytohormones are known as essential regulators of plant defenses, with ethylene, jasmonic acid, and salicylic acid as the central immunity backbone, while other phytohonnones have been demonstrated to interact with this. Only recently, a function of the classic phytohormone cytokinin in plant immunity has been described in Arabidopsis, rice, and tobacco. Although interactions of cytokinins with salicylic acid and auxin have been indicated, the complete network of cytokinin interactions with other immunity-relevant phytohormones is not yet understood. Therefore, we studied the interaction of kinetin and abscisic acid as a negative regulator of plant immunity to modulate resistance in tobacco against Pseudomonas syringae. By analyzing infection symptoms, pathogen proliferation, and accumulation of the phytoalexin scopoletin as a key mediator of kinetin-induced resistance in tobacco, antagonistic interaction of these phytohonnones in plant immunity was identified. Kinetin reduced abscisic acid levels in tobacco, while increased abscisic acid levels by exogenous application or inhibition of abscisic acid catabolism by diniconazole neutralized kinetin-induced resistance. Based on these results, we conclude that reduction of abscisic acid levels by enhanced abscisic acid catabolism strongly contributes to cytokinin-mediated resistance effects. Thus, the identified cytokinin-abscisic acid antagonism is a novel regulatory mechanism in plant immunity. [ABSTRACT FROM AUTHOR]

Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (olycystin, ipoxygenase, lpha-toxin and riacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty. [ABSTRACT FROM AUTHOR]

A letter is presented that proposes a rapid screening method for the parallel determination of various phytohormones such as abscisic acid (ABA) and indole-3-acetic acid (IAA), and phytoalexins like camalexin and scopoleptin in various plant species, notably "Arabidopsis thaliana" and "Nicotiana tabacum." It claims that this method can increase the process of physiological phenotyping in plants, thus helping researchers to develop effective strategies for the protection of agricultural crops.

Abstract: Phytoalexins are pathogen induced low molecular weight compounds with antimicrobial activities derived from secondary metabolism. Following their identification, phytoalexins were directly incorporated into the network of plant defense responses. Due to their heterogeneity, the metabolic pathways involved in phytoalexin formation and in particular the regulatory mechanisms remained elusive. Consequently, research focus shifted to the characterization of other components of plant immunity such as defense signaling and resistance mechanisms, including components of systemic acquired and induced systemic resistance, effector and pathogen-associated molecular pattern triggered immunity as well as R-gene resistance. Despite the obtained knowledge on these immunity mechanisms, genetic engineering employing these mechanisms and classical breeding reached too low improvements in crop protection, probably because classical breeding focused on yield performance and taste, rather than pathogen resistance. The increasing demand for disease resistant crop species and the aim to reduce pesticide application therefore requires alternative approaches. Recent advances in the understanding of phytoalexin function, biosynthesis and regulation, in combination with novel methods of molecular engineering and advances in instrumental analysis, returned attention to phytoalexins as a potent target for improving crop protection. Based on this, the advantages as well as potential bottlenecks for molecular approaches of modulating inducible phytoalexins to improve crop protection are discussed. [Copyright &y& Elsevier]

Cytokinins are phytohormones that are involved in various regulatory processes throughout plant development, but they are also produced by pathogens and known to modulate plant immunity. A novel transgenic approach enabling autoregulated cytokinin synthesis in response to pathogen infection showed that cytokinins mediate enhanced resistance against the virulent hemi-biotrophic pathogen Pseudomonas syringae pv tabaci. This was confirmed by two additional independent transgenic approaches to increase endogenous cytokinin production and by exogenous supply of adenine- and phenylurea-derived cytokinins. The cytokinin-mediated resistance strongly correlated with an increased level of bactericidal activities and up-regulated synthesis of the two major antimicrobial phytoalexins in tobacco (Nicotiana tabacum), scopoletin and capsidiol. The key role of these phytoalexins in the underlying mechanism was functionally proven by the finding that scopoletin and capsidiol substitute in planta for the cytokinin signal: phytoalexin pretreatment increased resistance against P. syringae. In contrast to a cytokinin defense mechanism in Arabidopsis (Arabidopsis thaliana) based on salicylic acid-dependent transcriptional control, the cytokinin-mediated resistance in tobacco is essentially independent from salicylic acid and differs in pathogen specificity. It is also independent of jasmonate levels, reactive oxygen species, and high sugar resistance. The novel function of cytokinins ill the primary defense response of solanaceous plant species is rather mediated through a high phytoalexin-pathogen ratio in the early phase of infection, which efficiently restricts pathogen growth. The implications of this mechanism for the coevolution of host plants and cytokinin-producing pathogens and the practical application in agriculture are discussed. [ABSTRACT FROM AUTHOR]

The complex dominant Arabidopsis thaliana mutant lettuce (let) shows the conversion of the leaf petiole into a leaf blade caused by an ectopic leaf blade formation. This is the result of the activation tagging of the LEAFY PETIOLE (LEP) gene encoding an AP2/EREBP-like transcription factor. Here, we report that in addition to this leafy petiole phenotype, the size of the vascular bundles is increased in all aerial organs in letas a result of an increase in the number of xylem, phloem (pro)cambial and pericycle cells. This vascular phenotype is caused by activation tagging of the two genes VASCULAR TISSUE SIZE (VAS) and LEP. These genes are closely linked and arranged in tandem. Activation tagging of LEP only caused a specific increase in the number of xylem cells. This increased xylem cell number, together with the ectopic leaf blade formation, indicates that LEP functions as a cell division-promoting factor. The activation tagging of VAS only resulted in a specific increase in phloem (pro)cambial and pericycle cells. We conclude that activation tagging of LEP and VAS results in additive phenotypes. Insertional mutants for LEP and VAS display wildtype vascular development, indicating the relevance of activation tagging for functional analysis of novel genes involved in plant development. [ABSTRACT FROM AUTHOR]

Summary Stem cells in plants and animals are maintained pluripotent by signals from adjacent niche cells. In plants, WUSCHEL HOMEOBOX (WOX) transcription factors are central regulators of stem cell maintenance in different meristem types, yet their molecular mode of action has remained elusive. Here we show that in the Arabidopsis root meristem, the WOX5 protein moves from the root niche organizer, the quiescent center, into the columella stem cells, where it directly represses the transcription factor gene CDF4 . This creates a gradient of CDF4 transcription, which promotes differentiation opposite to the WOX5 gradient, allowing stem cell daughter cells to exit the stem cell state. We further show that WOX5 represses CDF4 transcription by recruiting TPL/TPR co-repressors and the histone deacetylase HDA19, which consequently induces histone deacetylation at the CDF4 regulatory region. Our results show that chromatin-mediated repression of differentiation programs is a common strategy in plant and animal stem cell niches. [ABSTRACT FROM AUTHOR]

Plant genomes contain a large number of genes encoding for berberine bridge enzyme (BBE)-like enzymes. Despite the widespread occurrence and abundance of this protein family in the plant kingdom, the biochemical function remains largely unexplored. In this study, we have expressed two members of the BBE-like enzyme family from Arabidopsis thaliana in the host organism Komagataella pastoris. The two proteins, termed AtBBE-like 13 and AtBBE-like 15, were purified, and their catalytic properties were determined. In addition, AtBBE-like 15 was crystallized and structurally characterized by x-ray crystallography. Here, we show that the enzymes catalyze the oxidation of aromatic allylic alcohols, such as coumaryl, sinapyl, and coniferyl alcohol, to the corresponding aldehydes and that AtBBE-like 15 adopts the same fold as vanillyl alcohol oxidase as reported previously for berberine bridge enzyme and other FAD-dependent oxidoreductases. Further analysis of the substrate range identified coniferin, the glycosylated storage form of coniferyl alcohol, as a substrate of the enzymes, whereas other glycosylated monolignols were rather poor substrates. A detailed analysis of the motifs present in the active sites of the BBE-like enzymes in A. thaliana suggested that 14 out of 28 members of the family might catalyze similar reactions. Based on these findings, we propose a novel role of BBE-like enzymes in monolignol metabolism that was previously not recognized for this enzyme family. [ABSTRACT FROM AUTHOR]

Establishing the embryonic body plan of multicellular organisms relies on precisely orchestrated cell divisions coupled with pattern formation, which, in animals, are regulated by Polycomb group (PcG) proteins. The conserved Polycomb Repressive Complex 2 (PRC2) mediates H3K27 trimethylation and comes in different flavors in Arabidopsis. The PRC2 catalytic subunit MEDEA is required for seed development; however, a role for PRC2 in embryonic patterning has been dismissed. Here, we demonstrate that embryos derived from medea eggs abort because MEDEA is required for patterning and cell lineage determination in the early embryo. Similar to PcG proteins in mammals, MEDEA regulates embryonic patterning and growth by controlling cell-cycle progression through repression of CYCD1;1, which encodes a core cell-cycle component. Thus, Arabidopsis embryogenesis is epigenetically regulated by PcG proteins, revealing that the PRC2-dependent modulation of cell-cycle progression was independently recruited to control embryonic cell proliferation and patterning in animals and plants. [Display omitted] • MEDEA , a Polycomb Repressive Complex 2 (PRC2) subunit, is required in embryo and endosperm • MEDEA links cell proliferation and differentiation during embryonic pattern formation • The PRC2 protein MEDEA directly regulates core cell-cycle components, i.e., cyclin CYCD1;1 • Body plan and cell proliferation are epigenetically regulated in both animals and plants In animals, Polycomb group (PcG) proteins are essential for embryogenesis. Simonini et al. show that growth and patterning of plant embryos are also epigenetically regulated by PcG proteins, with the PRC2 histone methyltransferase MEDEA directly repressing the core cell-cycle component CYCD1;1 in the Arabidopsis embryo. [ABSTRACT FROM AUTHOR]