Unraveling Transcriptome Profile, Epigenetic Dynamics, and Morphological Changes in Psoriasis-like Keratinocytes: "Insights into Similarity with Psoriatic Lesional Epidermis".

Keratinocytes are one of the primary cells affected by psoriasis inflammation. Our study aimed to delve deeper into their morphology, transcriptome, and epigenome changes in response to psoriasis-like inflammation. We created a novel cytokine mixture to mimic mild and severe psoriasis-like inflammatory conditions in cultured keratinocytes. Upon induction of inflammation, we observed that the keratinocytes exhibited a mesenchymal-like phenotype, further confirmed by increased VIM mRNA expression and results obtained from confocal microscopy. We performed RNA sequencing to achieve a more global view, revealing 858 and 6987 DEGs in mildly and severely inflamed keratinocytes, respectively. Surprisingly, we found that the transcriptome of mildly inflamed keratinocytes more closely mimicked that of the psoriatic epidermis transcriptome than the severely inflamed keratinocytes. Genes involved in the IL-17 pathway were a major contributor to the similarities of the transcriptomes between mildly inflamed KCs and psoriatic epidermis. Mild and severe inflammation led to the gene regulation of epigenetic modifiers such as HATs, HDACs, DNMTs, and TETs. Immunofluorescence staining revealed distinct 5-hmC patterns in inflamed versus control keratinocytes, and consistently low 5-mC intensity in both groups. However, the global DNA methylation assay detected a tendency of decreased 5-mC levels in inflamed keratinocytes versus controls. This study emphasizes how inflammation severity affects the transcriptomic similarity of keratinocytes to psoriatic epidermis and proves dynamic epigenetic regulation and adaptive morphological changes in inflamed keratinocytes.

Phenol-Soluble Modulin α3 Stimulates Autophagy in HaCaT Keratinocytes.

BACKGROUND: Phenol-soluble modulins (PSMs) are pore-forming toxins (PFTs) produced by staphylococci. PSMs exert diverse cellular effects, including lytic, pro-apoptotic, pro-inflammatory and antimicrobial actions. Since the effects of PSMs on autophagy have not yet been reported, we evaluated the autophagic activity in HaCaT keratinocytes treated with recombinant PSMα3. METHODS: The autophagic flux and levels of autophagic marker proteins were determined using Western blot analysis. Subcellular localization of LC3B and Beclin-1 was investigated using an indirect immunofluorescence assay. The ultrastructural features of control and PSMα3-treated cells were evaluated via transmission electron microscopy. Cytoplasmic acidification was measured via acridine orange staining. Phosphorylation levels of protein kinases, implicated in autophagy regulation, were studied using a phospho-kinase array and Western blot analysis. RESULTS: PSMα3 facilitated the intracellular redistribution of LC3B, increased the average number of autophagosomes per cell, promoted the development of acidic vesicular organelles, elevated the levels of LC3B-II, stimulated autophagic flux and triggered a significant decrease in the net autophagic turnover rate. PSMα3 induced the accumulation of autophagosomes/autolysosomes, amphisomes and multilamellar bodies at the 0.5, 6 and 24 h time points, respectively. The phospho-Akt1/2/3 (T308 and S473), and phospho-mTOR (S2448) levels were decreased, whereas the phospho-Erk1/2 (T202/Y204 and T185/Y187) level was increased in PSMα3-treated cells. CONCLUSIONS: In HaCaT keratinocytes, PSMα3 stimulates autophagy. The increased autophagic activity elicited by sub-lytic PSM concentrations might be an integral part of the cellular defense mechanisms protecting skin homeostasis.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Manifestation of Triploid Heterosis in the Root System after Crossing Diploid and Autotetraploid Energy Willow Plants.

Successful use of woody species in reducing climatic and environmental risks of energy shortage and spreading pollution requires deeper understanding of the physiological functions controlling biomass productivity and phytoremediation efficiency. Targets in the breeding of energy willow include the size and the functionality of the root system. For the combination of polyploidy and heterosis, we have generated triploid hybrids (THs) of energy willow by crossing autotetraploid willow plants with leading cultivars (Tordis and Inger). These novel Salix genotypes (TH3/12, TH17/17, TH21/2) have provided a unique experimental material for characterization of Mid-Parent Heterosis (MPH) in various root traits. Using a root phenotyping platform, we detected heterosis (TH3/12: MPH 43.99%; TH21/2: MPH 26.93%) in the size of the root system in soil. Triploid heterosis was also recorded in the fresh root weights, but it was less pronounced (MPH%: 9.63-19.31). In agreement with root growth characteristics in soil, the TH3/12 hybrids showed considerable heterosis (MPH: 70.08%) under in vitro conditions. Confocal microscopy-based imaging and quantitative analysis of root parenchyma cells at the division-elongation transition zone showed increased average cell diameter as a sign of cellular heterosis in plants from TH17/17 and TH21/2 triploid lines. Analysis of the hormonal background revealed that the auxin level was seven times higher than the total cytokinin contents in root tips of parental Tordis plants. In triploid hybrids, the auxin-cytokinin ratios were considerably reduced in TH3/12 and TH17/17 roots. In particular, the contents of cytokinin precursor, such as isopentenyl adenosine monophosphate, were elevated in all three triploid hybrids. Heterosis was also recorded in the amounts of active gibberellin precursor, GA19, in roots of TH3/12 plants. The presented experimental findings highlight the physiological basics of triploid heterosis in energy willow roots.

Human pancreatic ductal organoids with controlled polarity provide a novel ex vivo tool to study epithelial cell physiology.

Epithelial ion and fluid secretion determine the physiological functions of a broad range of organs, such as the lung, liver, or pancreas. The molecular mechanism of pancreatic ion secretion is challenging to investigate due to the limited access to functional human ductal epithelia. Patient-derived organoids may overcome these limitations, however direct accessibility of the apical membrane is not solved. In addition, due to the vectorial transport of ions and fluid the intraluminal pressure in the organoids is elevated, which may hinder the study of physiological processes. To overcome these, we developed an advanced culturing method for human pancreatic organoids based on the removal of the extracellular matrix that induced an apical-to-basal polarity switch also leading to reversed localization of proteins with polarized expression. The cells in the apical-out organoids had a cuboidal shape, whereas their resting intracellular Ca2+ concentration was more consistent compared to the cells in the apical-in organoids. Using this advanced model, we demonstrated the expression and function of two novel ion channels, the Ca2+ activated Cl- channel Anoctamin 1 (ANO1) and the epithelial Na+ channel (ENaC), which were not considered in ductal cells yet. Finally, we showed that the available functional assays, such as forskolin-induced swelling, or intracellular Cl- measurement have improved dynamic range when performed with apical-out organoids. Taken together our data suggest that polarity-switched human pancreatic ductal organoids are suitable models to expand our toolset in basic and translational research.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that the expression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Orai1 calcium channel inhibition prevents progression of chronic pancreatitis.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Psoriatic Resolved Skin Epidermal Keratinocytes Retain Disease-Residual Transcriptomic and Epigenomic Profiles.

The disease-residual transcriptomic profile (DRTP) within psoriatic healed/resolved skin and epidermal tissue-resident memory T (TRM) cells have been proposed to be crucial for the recurrence of old lesions. However, it is unclear whether epidermal keratinocytes are involved in disease recurrence. There is increasing evidence regarding the importance of epigenetic mechanisms in the pathogenesis of psoriasis. Nonetheless, the epigenetic changes that contribute to the recurrence of psoriasis remain unknown. The aim of this study was to elucidate the role of keratinocytes in psoriasis relapse. The epigenetic marks 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) were visualized using immunofluorescence staining, and RNA sequencing was performed on paired never-lesional and resolved epidermal and dermal compartments of skin from psoriasis patients. We observed diminished 5-mC and 5-hmC amounts and decreased mRNA expression of the ten-eleven translocation (TET) 3 enzyme in the resolved epidermis. SAMHD1, C10orf99, and AKR1B10: the highly dysregulated genes in resolved epidermis are known to be associated with pathogenesis of psoriasis, and the DRTP was enriched in WNT, TNF, and mTOR signaling pathways. Our results suggest that epigenetic changes detected in epidermal keratinocytes of resolved skin may be responsible for the DRTP in the same regions. Thus, the DRTP of keratinocytes may contribute to site-specific local relapse.

Establishment and characterization of a novel human induced pluripotent stem cell line stably expressing the iRFP720 reporter.

Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy's success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

Viable protoplast formation of the coral endosymbiont alga Symbiodinium spp. in a microfluidics platform.

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.

Gene loss and compensatory evolution promotes the emergence of morphological novelties in budding yeast.

Deleterious mutations are generally considered to be irrelevant for morphological evolution. However, they could be compensated by conditionally beneficial mutations, thereby providing access to new adaptive paths. Here we use high-dimensional phenotyping of laboratory-evolved budding yeast lineages to demonstrate that new cellular morphologies emerge exceptionally rapidly as a by-product of gene loss and subsequent compensatory evolution. Unexpectedly, the capacities for invasive growth, multicellular aggregation and biofilm formation also spontaneously evolve in response to gene loss. These multicellular phenotypes can be achieved by diverse mutational routes and without reactivating the canonical regulatory pathways. These ecologically and clinically relevant traits originate as pleiotropic side effects of compensatory evolution and have no obvious utility in the laboratory environment. The extent of morphological diversity in the evolved lineages is comparable to that of natural yeast isolates with diverse genetic backgrounds and lifestyles. Finally, we show that both the initial gene loss and subsequent compensatory mutations contribute to new morphologies, with their synergistic effects underlying specific morphological changes. We conclude that compensatory evolution is a previously unrecognized source of morphological diversity and phenotypic novelties.

Small paraquat resistance proteins modulate paraquat and ABA responses and confer drought tolerance to overexpressing Arabidopsis plants.

Adaptation of higher plants to extreme environmental conditions is under complex regulation. Several small peptides have recently been described to modulate responses to stress conditions. The Small Paraquat resistance protein (SPQ) of Lepidium crassifolium has previously been identified due to its capacity to confer paraquat resistance to overexpressing transgenic Arabidopsis plants. Here, we show that overexpression of the closely related Arabidopsis SPQ can also enhance resistance to paraquat, while the Arabidopsis spq1 mutant is slightly hypersensitive to this herbicide. Besides being implicated in paraquat response, overexpression of SPQs enhanced sensitivity to abscisic acid (ABA), and the knockout spq1 mutant was less sensitive to ABA. Both Lepidium- and Arabidopsis-derived SPQs could improve drought tolerance by reducing water loss, stabilizing photosynthetic electron transport and enhancing plant viability and survival in a water-limited environment. Enhanced drought tolerance of SPQ-overexpressing plants could be confirmed by characterizing various parameters of growth, morphology and photosynthesis using an automatic plant phenotyping platform with RGB and chlorophyll fluorescence imaging. Our results suggest that SPQs can be regulatory small proteins connecting ROS and ABA regulation and through that influence responses to certain stresses.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin.

The cellular prion protein (PrPC) is renowned for its infectious conformational isoform PrPSc, capable of templating subsequent conversions of healthy PrPCs and thus triggering the group of incurable diseases known as transmissible spongiform encephalopathies. Besides this mechanism not being fully uncovered, the protein's physiological role is also elusive. PrPC and its newest, less understood paralog Shadoo are glycosylphosphatidylinositol-anchored proteins highly expressed in the central nervous system. While they share some attributes and neuroprotective actions, opposing roles have also been reported for the two; however, the amount of data about their exact functions is lacking. Protein-protein interactions and membrane microdomain localizations are key determinants of protein function. Accurate identification of these functions for a membrane protein, however, can become biased due to interactions occurring during sample processing. To avoid such artifacts, we apply a non-detergent-based membrane-fractionation approach to study the prion protein and Shadoo. We show that the two proteins occupy similarly raft and non-raft membrane fractions when expressed in N2a cells and that both proteins pull down the chaperone calnexin in both rafts and non-rafts. These indicate their possible binding to calnexin in both types of membrane domains, which might be a necessary requisite to aid the inherently unstable native conformation during their lifetime.

Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula.

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells.

IL-36α and Lipopolysaccharide Cooperatively Induce Autophagy by Triggering Pro-Autophagic Biased Signaling.

Autophagy is an intracellular catabolic process that controls infections both directly and indirectly via its multifaceted effects on the innate and adaptive immune responses. It has been reported that LPS stimulates this cellular process, whereas the effect of IL-36α on autophagy remains largely unknown. We therefore investigated how IL-36α modulates the endogenous and LPS-induced autophagy in THP-1 cells. The levels of LC3B-II and autophagic flux were determined by Western blotting. The intracellular localization of LC3B was measured by immunofluorescence assay. The activation levels of signaling pathways implicated in autophagy regulation were evaluated by using a phosphokinase array. Our results showed that combined IL-36α and LPS treatment cooperatively increased the levels of LC3B-II and Beclin-1, stimulated the autophagic flux, facilitated intracellular redistribution of LC3B, and increased the average number of autophagosomes per cell. The IL36α/LPS combined treatment increased phosphorylation of STAT5a/b, had minimal effect on the Akt/PRAS40/mTOR pathway, and reduced the levels of phospho-Yes, phospho-FAK, and phospho-WNK1. Thus, this cytokine/PAMP combination triggers pro-autophagic biased signaling by several mechanisms and thus cooperatively stimulates the autophagic cascade. An increased autophagic activity of innate immune cells simultaneously exposed to IL-36α and LPS may play an important role in the pathogenesis of Gram-negative bacterial infections.

Superhydrophobic self-similar nonwoven-titanate nanostructured materials.

HYPOTHESIS: Self-similarity is a scale-invariant irregularity that can assist in designing a robust superhydrophobic material. A combinatorial design strategy involving self-similarity and dual-length scale can be employed to create a new library of a doubly re-entrant, disordered, and porous network of superhydrophobic materials. Asymmetric wettability can be engineered in nonwoven materials by rendering them with superhydrophobic characteristics on one side. EXPERIMENTS: A facile, scalable, and inexpensive spray-coating technique was used to decorate the weakly hydrophobicstearate-treatedtitanate nanowires (TiONWs)over the self-similar nonwoven material. Laser scanning confocal microscopy was employed to image the impalement dynamics in three dimensions. With the aid of X-ray microcomputed tomography analysis, the three-dimensional (3D) nonwoven structural parameters were obtained and analyzed. The underwater superhydrophobic behavior of the prepared samples was investigated. FINDINGS: A classic 'lotus effect' has been successfully endowed in self-similar nonwoven-titanate nanostructured materials (SS-Ti-NMs) from a nonwoven material that housed the air pockets in bulk and water repellent TiONWs on the surface. The finer fiber-based SS-Ti-NMs exhibited lower roll-off angles and a thinner layer of water on its surface. An asymmetric wettability and the unusual display of underwater superhydrophobic behavior of SS-Ti-NMs have been uncovered.

Proline is a quencher of singlet oxygen and superoxide both in in vitro systems and isolated thylakoids.

Proline is a versatile plant metabolite, which is produced in large amounts in plants exposed to osmotic and oxidative stress. Proline has been shown to provide protection against various reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals. On the other hand, its protective effect against singlet oxygen has been debated, and it is considered ineffective against superoxide. Here we used various methods for the detection of singlet oxygen (electron paramagnetic resonance, EPR, spin trapping by 2,2,6,6-tetramethyl-4-piperidone, fluorescence probing by singlet oxygen sensor green, SOSG, and oxygen uptake due to chemical trapping) and superoxide (oxygen uptake due to oxygen reduction) in vitro and in isolated thylakoids. We demonstrated that proline does quench both singlet oxygen and superoxide in vitro. By comparing the effects of chemical scavengers and physical quenchers, we concluded that proline eliminates singlet oxygen via a physical mechanism, with a bimolecular quenching rate of ca. 1.5-4 106 M-1 s-1 . Our data also show that proline can eliminate superoxide in vitro in a process that is likely to proceed via an electron transfer reaction. We could also show that proline does quench both singlet oxygen and superoxide produced in isolated thylakoids. The scavenging efficiency of proline is relatively small on a molar basis, but considering its presence in high amounts in plant cells under stress conditions it may provide a physiologically relevant contribution to ROS scavenging, supplementing other nonenzymatic ROS scavengers of plant cells.

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation.

The mitogen-activated protein kinase 4-phosphorylated heat shock factor A4A regulates responses to combined salt and heat stresses.

Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes.