H63D variant of the homeostatic iron regulator (HFE) gene alters α-synuclein expression, aggregation, and toxicity.

Pathological features of Parkinson's disease include the formation of Lewy bodies containing α-synuclein and the accumulation of iron in the substantia nigra. Previous studies have suggested that iron accumulation contributes to the Parkinson's disease pathology through reactive oxygen species production and accelerated α-synuclein aggregation. This study examines the effects of commonly occurring H63D variant of the homeostatic iron regulatory (HFE) gene on α-synuclein pathology in cell culture and animal models. H63D HFE expression in SH-SY5Y cells lowered endogenous α-synuclein levels and significantly decreased pre-formed fibril-induced α-synuclein aggregation. H63D HFE cells were also protected from pre-formed fibril-induced apoptosis. Autophagic flux, a major pathway for α-synuclein clearance, was increased in H63D HFE cells. Expression of REDD1 was elevated and rapamycin treatment was unable to further induce autophagy, indicating mTORC1 inhibition as the main mechanism of autophagy induction. Moreover, siRNA knockdown of REDD1 in H63D HFE cells decreased autophagic flux and increased the sensitivity to PFF-mediated toxicity. While iron chelator (deferiprone) treatment rescued WT HFE cells from pre-formed fibril toxicity, it exacerbated or was unable to rescue H63D HFE cells. In the in vivo pre-formed fibril intracranial injection model, H67D Hfe (mouse homolog of the human H63D HFE variant) C57BL/6J × 129 mice showed less α-synuclein aggregation and less decline in motor function compared to WT Hfe. Collectively, this study suggests that H63D HFE variant modifies α-synuclein pathology through the induction of autophagy and has the potential to impact the pathogenesis and treatment response in Parkinson's disease.

Cell Death Triggered by the YUCCA-like Bs3 Protein Coincides with Accumulation of Salicylic Acid and Pipecolic Acid But Not of Indole-3-Acetic Acid.

The pepper (Capsicum annuum) resistance gene bacterial spot3 (Bs3) is transcriptionally activated by the matching Xanthomonas euvesicatoria transcription-activator-like effector (TALE) AvrBs3. AvrBs3-induced Bs3 expression triggers a rapid and local cell death reaction, the hypersensitive response (HR). Bs3 is most closely related to plant flavin monooxygenases of the YUCCA (YUC) family, which catalyze the final step in auxin biosynthesis. Targeted mutagenesis of predicted NADPH- and FAD-cofactor sites resulted in Bs3 derivatives that no longer trigger HR, thereby suggesting that the enzymatic activity of Bs3 is crucial to Bs3-triggered HR. Domain swap experiments between pepper Bs3 and Arabidopsis (Arabidopsis thaliana) YUC8 uncovered functionally exchangeable and functionally distinct regions in both proteins, which is in agreement with a model whereby Bs3 evolved from an ancestral YUC gene. Mass spectrometric measurements revealed that expression of YUCs, but not expression of Bs3, coincides with an increase in auxin levels, suggesting that Bs3 and YUCs, despite their sequence similarity, catalyze distinct enzymatic reactions. Finally, we found that expression of Bs3 coincides with increased levels of the salicylic acid and pipecolic acid, two compounds that are involved in systemic acquired resistance.

The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1.

BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.

Comparing Arabidopsis receptor kinase and receptor protein-mediated immune signaling reveals BIK1-dependent differences.

Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine-rich repeat receptor kinases (LRR-RK) FLS2 and EFR, and the LRR receptor protein (LRR-RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR-RK and LRR-RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP-mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23-regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2-signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP-type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.

Targeted manipulation of bZIP53 DNA-binding properties influences Arabidopsis metabolism and growth.

bZIP transcription factors regulate diverse processes in eukaryotic cells. Arabidopsis bZIP members of the C and S1 groups form heterodimers and synergistically control metabolic reprogramming during stress responses. However, their functional characterization is complicated due to an overlapping heterodimerization network and high redundancy. In this study, we develop a simple but powerful approach for generating dominant negative mutants of bZIP factors with high specificity. By applying in vitro DNA-binding, reporter gene and protoplast two-hybrid assays, and plant mutant analysis, we show that phosphorylation-mimicking substitution of conserved serines in the DNA-binding domain of bZIP monomeric subunits suffices for the disruption of the interaction of both bZIP homo- and heterodimers with cognate DNA. This results in the transcriptional inactivation of target genes. The dominant-negative effect is achieved by the unaltered function of the intrinsic nuclear localization signal and dimerization properties of the mutated bZIP protein. Our findings not only reveal an additional regulatory mechanism of bZIP10 intracellular localization, but also provide evidence of the involvement of bZIP53 in the diurnal adjustments of amino acid metabolism. Our data demonstrate the advantages and the suitability of this new approach for the artificial inactivation of bZIP transcription factors in plants, and it may also be of use for other organisms.

Intergenerational environmental effects: functional signals in offspring transcriptomes and metabolomes after parental jasmonic acid treatment in apomictic dandelion.

Parental environments can influence offspring traits. However, the magnitude of the impact of parental environments on offspring molecular phenotypes is poorly understood. Here, we test the direct effects and intergenerational effects of jasmonic acid (JA) treatment, which is involved in herbivory-induced defense signaling, on transcriptomes and metabolomes in apomictic common dandelion (Taraxacum officinale). In a full factorial crossed design with parental and offspring JA and control treatments, we performed leaf RNA-seq gene expression analysis, LC-MS metabolomics and total phenolics assays in offspring plants. Expression analysis, leveraged by a de novo assembled transcriptome, revealed an induced response to JA exposure that is consistent with known JA effects. The intergenerational effect of treatment was considerable: 307 of 858 detected JA-responsive transcripts were affected by parental JA treatment. In terms of the numbers of metabolites affected, the magnitude of the chemical response to parental JA exposure was c. 10% of the direct JA treatment response. Transcriptome and metabolome analyses both identified the phosphatidylinositol signaling pathway as a target of intergenerational JA effects. Our results highlight that parental environments can have substantial effects in offspring generations. Transcriptome and metabolome assays provide a basis for zooming in on the potential mechanisms of inherited JA effects.

d-Amino Acids Are Exuded by Arabidopsis thaliana Roots to the Rhizosphere.

Proteinogenic l-amino acids (l-AAs) are essential in all kingdoms as building blocks of proteins. Their d-enantiomers are also known to fulfill important functions in microbes, fungi, and animals, but information about these molecules in plants is still sparse. Previously, it was shown that d-amino acids (d-AAs) are taken up and utilized by plants, but their ways to reduce excessive amounts of them still remained unclear. Analyses of plant d-AA content after d-Ala and d-Glu feeding opened the question if exudation of d-AAs into the rhizosphere takes place and plays a role in the reduction of d-AA content in plants. The exudation of d-Ala and d-Glu could be confirmed by amino acid analyses of growth media from plants treated with these d-AAs. Further tests revealed that other d-AAs were also secreted. Nevertheless, treatments with d-Ala and d-Glu showed that plants are still able to reduce their contents within the plant without exudation. Further exudation experiments with transport inhibitors revealed that d-AA root exudation is rather passive and comparable to the secretion of l-AAs. Altogether, these observations argued against a dominant role of exudation in the regulation of plant d-AA content, but may influence the composition of the rhizosphere.

A Combination of Structural and Empirical Analyses Delineates the Key Contacts Mediating Stability and Affinity Increases in an Optimized Biotherapeutic Single-chain Fv (scFv).

Fully-human single-chain Fv (scFv) proteins are key potential building blocks of bispecific therapeutic antibodies, but they often suffer from manufacturability and clinical development limitations such as instability and aggregation. The causes of these scFv instability problems, in proteins that should be theoretically stable, remains poorly understood. To inform the future development of such molecules, we carried out a comprehensive structural analysis of the highly stabilized anti-CXCL13 scFv E10. E10 was derived from the parental 3B4 using complementarity-determining region (CDR)-restricted mutagenesis and tailored selection and screening strategies, and carries four mutations in VL-CDR3. High-resolution crystal structures of parental 3B4 and optimized E10 scFvs were solved in the presence and absence of human CXCL13. In parallel, a series of scFv mutants was generated to interrogate the individual contribution of each of the four mutations to stability and affinity improvements. In combination, these analyses demonstrated that the optimization of E10 was primarily mediated by removing clashes between both the VL and the VH, and between the VL and CXCL13. Importantly, a single, germline-encoded VL-CDR3 residue mediated the key difference between the stable and unstable forms of the scFv. This work demonstrates that, aside from being the critical mediators of specificity and affinity, CDRs may also be the primary drivers of biotherapeutic developability.

Meta-Analysis of Arabidopsis KANADI1 Direct Target Genes Identifies a Basic Growth-Promoting Module Acting Upstream of Hormonal Signaling Pathways.

An intricate network of antagonistically acting transcription factors mediates the formation of a flat leaf lamina of Arabidopsis (Arabidopsis thaliana) plants. In this context, members of the class III homeodomain leucine zipper (HD-ZIPIII) transcription factor family specify the adaxial domain (future upper side) of the leaf, while antagonistically acting KANADI transcription factors determine the abaxial domain (future lower side). Here, we used a messenger RNA sequencing approach to identify genes regulated by KANADI1 (KAN1) and subsequently performed a meta-analysis combining our data sets with published genome-wide data sets. Our analysis revealed that KAN1 acts upstream of several genes encoding auxin biosynthetic enzymes. When exposed to shade, we found three YUCCA genes, YUC2, YUC5, and YUC8, to be transcriptionally up-regulated, which correlates with an increase in the levels of free auxin. When ectopically expressed, KAN1 is able to transcriptionally repress these three YUC genes and thereby block shade-induced auxin biosynthesis. Consequently, KAN1 is able to strongly suppress shade-avoidance responses. Taken together, we hypothesize that HD-ZIPIII/KAN form the basis of a basic growth-promoting module. Hypocotyl extension in the shade and outgrowth of new leaves both involve auxin synthesis and signaling, which are under the direct control of HD-ZIPIII/KAN.

Stinging insect identification: Are the allergy specialists any better than their patients?

BACKGROUND: It has been reported that the general population is not skillful at identifying stinging insects with the exception of the honeybee. No information is available to evaluate allergy physicians' accuracy with stinging insect identification. OBJECTIVE: To measure the accuracy of allergists' ability to identify stinging insects and assess their common practices for evaluating individuals with suspected insect hypersensitivity. METHODS: A picture-based survey and a dried specimen insect box were constructed to determine allergists' and nonallergists' accuracy in identifying insects. Allergists attending the 2013 American College of Allergy, Asthma, and Immunology meeting were invited to participate in the study. Common practice approaches for evaluating individuals with stinging insect hypersensitivity were also investigated using a brief questionnaire. RESULTS: Allergy physicians are collectively better at insect identification than nonallergists. Overall, the mean (SD) number of correct responses for nonallergists was 5.4 (2.0) of a total of 10. This score was significantly lower than the score for allergists (6.1 [2.0]; P = .01) who participated in the study. Most allergists (78.5%) test for all stinging insects and use skin testing (69.5%) as the initial test of choice for evaluating individuals with insect hypersensitivity. CONCLUSION: Overall, allergists are more skilled at Hymenoptera identification. Most allergy specialists reported testing for all stinging insects when evaluating insect hypersensitivity, and skin testing was the preferred testing method in nearly 70% of allergists. These data support the practice parameter's recommendation to consider testing for all flying Hymenoptera insects during venom evaluation, which most of the participating allergists surveyed incorporate into their clinical practice.

Aldehyde dehydrogenase inhibition as a pathogenic mechanism in Parkinson disease.

Parkinson disease (PD) is a neurodegenerative disorder particularly characterized by the loss of dopaminergic neurons in the substantia nigra. Pesticide exposure has been associated with PD occurrence, and we previously reported that the fungicide benomyl interferes with several cellular processes potentially relevant to PD pathogenesis. Here we propose that benomyl, via its bioactivated thiocarbamate sulfoxide metabolite, inhibits aldehyde dehydrogenase (ALDH), leading to accumulation of the reactive dopamine metabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL), preferential degeneration of dopaminergic neurons, and development of PD. This hypothesis is supported by multiple lines of evidence. (i) We previously showed in mice the metabolism of benomyl to S-methyl N-butylthiocarbamate sulfoxide, which inhibits ALDH at nanomolar levels. We report here that benomyl exposure in primary mesencephalic neurons (ii) inhibits ALDH and (iii) alters dopamine homeostasis. It induces selective dopaminergic neuronal damage (iv) in vitro in primary mesencephalic cultures and (v) in vivo in a zebrafish system. (vi) In vitro cell loss was attenuated by reducing DOPAL formation. (vii) In our epidemiology study, higher exposure to benomyl was associated with increased PD risk. This ALDH model for PD etiology may help explain the selective vulnerability of dopaminergic neurons in PD and provide a potential mechanism through which environmental toxicants contribute to PD pathogenesis.

Metabolic pathway engineering using the central signal processor PII.

BACKGROUND: PII signal processor proteins are wide spread in prokaryotes and plants where they control a multitude of anabolic reactions. Efficient overproduction of metabolites requires relaxing the tight cellular control circuits. Here we demonstrate that a single point mutation in the PII signaling protein from the cyanobacterium Synechocystis sp. PCC 6803 is sufficient to unlock the arginine pathway causing over accumulation of the biopolymer cyanophycin (multi-L-arginyl-poly-L-aspartate). This product is of biotechnological interest as a source of amino acids and polyaspartic acid. This work exemplifies a novel approach of pathway engineering by designing custom-tailored PII signaling proteins. Here, the engineered Synechocystis sp. PCC6803 strain with a PII-I86N mutation over-accumulated arginine through constitutive activation of the key enzyme N-acetylglutamate kinase (NAGK). RESULTS: In the engineered strain BW86, in vivo NAGK activity was strongly increased and led to a more than tenfold higher arginine content than in the wild-type. As a consequence, strain BW86 accumulated up to 57 % cyanophycin per cell dry mass under the tested conditions, which is the highest yield of cyanophycin reported to date. Strain BW86 produced cyanophycin in a molecular mass range of 25 to >100 kDa; the wild-type produced the polymer in a range of 30 to >100 kDa. CONCLUSIONS: The high yield and high molecular mass of cyanophycin produced by strain BW86 along with the low nutrient requirements of cyanobacteria make it a promising means for the biotechnological production of cyanophycin. This study furthermore demonstrates the feasibility of metabolic pathway engineering using the PII signaling protein, which occurs in numerous bacterial species.

Crystal structure of a human IκB kinase ß asymmetric dimer.

Phosphorylation of inhibitor of nuclear transcription factor κB (IκB) by IκB kinase (IKK) triggers the degradation of IκB and migration of cytoplasmic κB to the nucleus where it promotes the transcription of its target genes. Activation of IKK is achieved by phosphorylation of its main subunit, IKKß, at the activation loop sites. Here, we report the 2.8 Å resolution crystal structure of human IKKß (hIKKß), which is partially phosphorylated and bound to the staurosporine analog K252a. The hIKKß protomer adopts a trimodular structure that closely resembles that from Xenopus laevis (xIKKß): an N-terminal kinase domain (KD), a central ubiquitin-like domain (ULD), and a C-terminal scaffold/dimerization domain (SDD). Although hIKKß and xIKKß utilize a similar dimerization mode, their overall geometries are distinct. In contrast to the structure resembling closed shears reported previously for xIKKß, hIKKß exists as an open asymmetric dimer in which the two KDs are further apart, with one in an active and the other in an inactive conformation. Dimer interactions are limited to the C-terminal six-helix bundle that acts as a hinge between the two subunits. The observed domain movements in the structures of IKKß may represent trans-phosphorylation steps that accompany IKKß activation.

Pyroglutamate and O-linked glycan determine functional production of anti-IL17A and anti-IL22 peptide-antibody bispecific genetic fusions.

Protein biosynthesis and extracellular secretion are essential biological processes for therapeutic protein production in mammalian cells, which offer the capacity for correct folding and proper post-translational modifications. In this study, we have generated bispecific therapeutic fusion proteins in mammalian cells by combining a peptide and an antibody into a single open reading frame. A neutralizing peptide directed against interleukin-17A (IL17A) was genetically fused to the N termini of an anti-IL22 antibody, through either the light chain, the heavy chain, or both chains. Although the resulting fusion proteins bound and inhibited IL22 with the same affinity and potency as the unmodified anti-IL22 antibody, the peptide modality in the fusion scaffold was not active in the cell-based assay due to the N-terminal degradation. When a glutamine residue was introduced at the N terminus, which can be cyclized to form pyroglutamate in mammalian cells, the IL17A neutralization activity of the fusion protein was restored. Interestingly, the mass spectroscopic analysis of the purified fusion protein revealed an unexpected O-linked glycosylation modification at threonine 5 of the anti-IL17A peptide. The subsequent removal of this post-translational modification by site-directed mutagenesis drastically enhanced the IL17A binding affinity and neutralization potency for the resulting fusion protein. These results provide direct experimental evidence that post-translational modifications during protein biosynthesis along secretory pathways play critical roles in determining the structure and function of therapeutic proteins produced by mammalian cells. The newly engineered peptide-antibody genetic fusion is promising for therapeutically targeting multiple antigens in a single antibody-like molecule.

The tyrosine-sulfated peptide receptors PSKR1 and PSY1R modify the immunity of Arabidopsis to biotrophic and necrotrophic pathogens in an antagonistic manner.

The tyrosine-sulfated peptides PSKα and PSY1 bind to specific leucine-rich repeat surface receptor kinases and control cell proliferation in plants. In a reverse genetic screen, we identified the phytosulfokine (PSK) receptor PSKR1 as an important component of plant defense. Multiple independent loss-of-function mutants in PSKR1 are more resistant to biotrophic bacteria, show enhanced pathogen-associated molecular pattern responses and less lesion formation after infection with the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. By contrast, pskr1 mutants are more susceptible to necrotrophic fungal infection with Alternaria brassicicola, show more lesion formation and fungal growth which is not observed on wild-type plants. The antagonistic effect on biotrophic and necrotrophic pathogen resistance is reflected by enhanced salicylate and reduced jasmonate responses in the mutants, suggesting that PSKR1 suppresses salicylate-dependent defense responses. Detailed analysis of single and multiple mutations in the three paralogous genes PSKR1, -2 and PSY1-receptor (PSY1R) determined that PSKR1 and PSY1R, but not PSKR2, have a partially redundant effect on plant immunity. In animals and plants, peptide sulfation is catalyzed by a tyrosylprotein sulfotransferase (TPST). Mutants lacking TPST show increased resistance to bacterial infection and increased susceptibility to fungal infection, mimicking the triple receptor mutant phenotypes. Feeding experiments with PSKα in tpst-1 mutants partially restore the defense-related phenotypes, indicating that perception of the PSKα peptide has a direct effect on plant defense. These results suggest that the PSKR subfamily integrates growth-promoting and defense signals mediated by sulfated peptides and modulates cellular plasticity to allow flexible adjustment to environmental changes.

Heterodimers of the Arabidopsis transcription factors bZIP1 and bZIP53 reprogram amino acid metabolism during low energy stress.

Control of energy homeostasis is crucial for plant survival, particularly under biotic or abiotic stress conditions. Energy deprivation induces dramatic reprogramming of transcription, facilitating metabolic adjustment. An in-depth knowledge of the corresponding regulatory networks would provide opportunities for the development of biotechnological strategies. Low energy stress activates the Arabidopsis thaliana group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 by transcriptional and posttranscriptional mechanisms. Gain-of-function approaches define these bZIPs as crucial transcriptional regulators in Pro, Asn, and branched-chain amino acid metabolism. Whereas chromatin immunoprecipitation analyses confirm the direct binding of bZIP1 and bZIP53 to promoters of key metabolic genes, such as ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE, the G-box, C-box, or ACT motifs (ACTCAT) have been defined as regulatory cis-elements in the starvation response. bZIP1 and bZIP53 were shown to specifically heterodimerize with group C bZIPs. Although single loss-of-function mutants did not affect starvation-induced transcription, quadruple mutants of group S1 and C bZIPs displayed a significant impairment. We therefore propose that bZIP1 and bZIP53 transduce low energy signals by heterodimerization with members of the partially redundant C/S1 bZIP factor network to reprogram primary metabolism in the starvation response.

The HIT study: Hymenoptera Identification Test--how accurate are people at identifying stinging insects?

BACKGROUND: Stinging insects in the order Hymenoptera include bees, wasps, yellow jackets, hornets, and ants. Hymenoptera sting injuries range from localized swelling to rarely death. Insect identification is helpful in the management of sting injuries. OBJECTIVE: To determine the accuracy of adults in identifying stinging insects and 2 insect nests. METHODS: This was a cross-sectional, multicenter study using a picture-based survey to evaluate an individual's success at identifying honeybees, wasps, bald-face hornets, and yellow jackets. Bald-face hornet and paper wasp nest identification also was assessed in this study. RESULTS: Six hundred forty participants completed the questionnaire. Overall, the mean number of correct responses was 3.2 (SD 1.3) of 6. Twenty participants (3.1%) correctly identified all 6 stinging insects and nests and only 10 (1.6%) were unable to identify any of the pictures correctly. The honeybee was the most accurately identified insect (91.3%) and the paper wasp was the least correctly identified insect (50.9%). For the 6 questions regarding whether the participant had been stung in the past by any of the insects (including an unidentified insect), 91% reported being stung by at least 1. Men were more successful at identify stinging insects correctly (P = .002), as were participants stung by at least 4 insects (P = .018). CONCLUSION: This study supports the general perception that adults are poor discriminators in distinguishing stinging insects and nests with the exception of the honeybee. Men and those participants who reported multiple stings to at least 4 insects were more accurate overall in insect identification.

Wild-growing lignicolous mushroom species as sources of novel agents with antioxidative and antibacterial potentials.

Crude extracts of two edible and two medicinal lignicolous mushroom species: Meripilus giganteus, Agrocybe aegerita, Fomes fomentarius and Xylaria polymorpha, growing wild in Serbia, were analyzed for their antioxidative and antibacterial potentials. Free radical scavenging capacity (RSC) on DPPH(•) and (•)OH was evaluated both by spectrophotometer and by Electron Spin Resonance (ESR) spectroscopy against DPPH(•). The highest antioxidant and antibacterial bioactivity was obtained with F. fomentarius extracts (IC50 ≈ 10.7 µg/ml in DPPH(•) assay; 136.6 mg ascorbic acid equivalents (AAE)/g dry weight (d.w.) for ferric reducing antioxidant power FRAP). It also showed the highest total phenol (TP) (82.54 mg gallic acid equivalents (GAE)/g dry weight (d.w.)) and total flavonoid (TF) content (76.8 µg rutin equivalents (RE)/g dry weight (d.w.)). A. aegerita showed the best antioxidant activity (IC50 = 0.87 mg/ml) against DPPH(•) in ESR analysis. Total redox potential of extracts was in direct positive correlation with TP content (r(2 )= 0.98) and TF content (r(2 )= 0.58). GC/MS analysis detected major constituents of extracts, confirming the presence of the following organic and phenolic acids: fumaric, succinic, mallic, 4-hydroxy benzoic, gentisic, protocatechuic, vanillic, gallic and p-coumaric acid.

Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses.

Unlike the situation in animals, the final morphology of the plant body is highly modulated by the environment. During Arabidopsis development, intrinsic factors provide the framework for basic patterning processes. CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) transcription factors are involved in embryo, shoot and root patterning. During vegetative growth HD-ZIPIII proteins control several polarity set-up processes such as in leaves and the vascular system. We have identified several direct target genes of the HD-ZIPIII transcription factor REVOLUTA (REV) using a chromatin immunoprecipitation/DNA sequencing (ChIP-Seq) approach. This analysis revealed that REV acts upstream of auxin biosynthesis and affects directly the expression of several class II HD-ZIP transcription factors that have been shown to act in the shade-avoidance response pathway. We show that, as well as involvement in basic patterning, HD-ZIPIII transcription factors have a critical role in the control of the elongation growth that is induced when plants experience shade. Leaf polarity is established by the opposed actions of HD-ZIPIII and KANADI transcription factors. Finally, our study reveals that the module that consists of HD-ZIPIII/KANADI transcription factors controls shade growth antagonistically and that this antagonism is manifested in the opposed regulation of shared target genes.

Siliques are Red1 from Arabidopsis acts as a bidirectional amino acid transporter that is crucial for the amino acid homeostasis of siliques.

Many membrane proteins are involved in the transport of nutrients in plants. While the import of amino acids into plant cells is, in principle, well understood, their export has been insufficiently described. Here, we present the identification and characterization of the membrane protein Siliques Are Red1 (SIAR1) from Arabidopsis (Arabidopsis thaliana) that is able to translocate amino acids bidirectionally into as well as out of the cell. Analyses in yeast and oocytes suggest a SIAR1-mediated export of amino acids. In Arabidopsis, SIAR1 localizes to the plasma membrane and is expressed in the vascular tissue, in the pericycle, in stamen, and in the chalazal seed coat of ovules and developing seeds. Mutant alleles of SIAR1 accumulate anthocyanins as a symptom of reduced amino acid content in the early stages of silique development. Our data demonstrate that the SIAR1-mediated export of amino acids plays an important role in organic nitrogen allocation and particularly in amino acid homeostasis in developing siliques.