Endogenous plant nitroxyl, a new component of nitric oxide biology.

Nitroxyl (HNO), a one-electron reduced and protonated congener of nitric oxide (•NO), was recently discovered in Arabidopsis thaliana. Due to its distinct chemical properties, we believe HNO must be further studied to determine how many physiological processes it impacts.

PEP444c encoded within the MIR444c gene regulates microRNA444c accumulation in barley.

MicroRNAs are small, noncoding RNA molecules that regulate the expression of their target genes. The MIR444 gene family is present exclusively in monocotyledons, and microRNAs444 from this family have been shown to target certain MADS-box transcription factors in rice and barley. We identified three barley MIR444 (MIR444a/b/c) genes and comprehensively characterised their structure and the processing pattern of the primary transcripts (pri-miRNAs444). Pri-microRNAs444 undergo extensive alternative splicing, generating functional and nonfunctional pri-miRNA444 isoforms. We show that barley pri-miRNAs444 contain numerous open reading frames (ORFs) whose transcripts associate with ribosomes. Using specific antibodies, we provide evidence that selected ORFs encoding PEP444a within MIR444a and PEP444c within MIR444c are expressed in barley plants. Moreover, we demonstrate that CRISPR-associated endonuclease 9 (Cas9)-mediated mutagenesis of the PEP444c-encoding sequence results in a decreased level of PEP444 transcript in barley shoots and roots and a 5-fold reduced level of mature microRNA444c in roots. Our observations suggest that PEP444c encoded by the MIR444c gene is involved in microRNA444c biogenesis in barley.

Dynamics of nitration during dark-induced leaf senescence in Arabidopsis reveals proteins modified by tryptophan nitration.

Nitric oxide (NO) is a critical molecule that links plant development with stress responses. Herein, new insights into the role of NO metabolism during leaf senescence in Arabidopsis are presented. A gradual decrease in NO emission accompanied dark-induced leaf senescence (DILS), and a transient wave of peroxynitrite (ONOO-) formation was detected by day 3 of DILS. The boosted ONOO- did not promote tryptophan (Trp) nitration, while the pool of 6-nitroTrp-containing proteins was depleted as senescence progressed. Immunoprecipitation combined with mass spectrometry was used to identify 63 and 4 characteristic 6-nitroTrp-containing proteins in control and individually darkened leaves, respectively. The potential in vivo targets of Trp nitration were mainly related to protein biosynthesis and carbohydrate metabolism. In contrast, nitration of tyrosine-containing proteins was intensified 2-fold on day 3 of DILS. Also, nitrative modification of RNA and DNA increased significantly on days 3 and 7 of DILS, respectively. Taken together, ONOO- can be considered a novel pro-senescence regulator that fine-tunes the redox environment for selective bio-target nitration. Thus, DILS-triggered nitrative changes at RNA and protein levels promote developmental shifts during the plant's lifespan and temporal adjustment in plant metabolism under suboptimal environmental conditions.

Epigenetic Landmarks of Leaf Senescence and Crop Improvement.

This review synthesizes knowledge on epigenetic regulation of leaf senescence and discusses the possibility of using this knowledge to improve crop quality. This control level is implemented by different but interacting epigenetic mechanisms, including DNA methylation, covalent histone modifications, and non-covalent chromatin remodeling. The genetic and epigenetic changes may act alone or together and regulate the gene expression, which may result in heritable (stress memory) changes and may lead to crop survival. In the review, the question also arises whether the mitotically stable epigenetic information can be used for crop improvement. The barley crop model for early and late events of dark-induced leaf senescence (DILS), where the point of no return was defined, revealed differences in DNA and RNA modifications active in DILS compared to developmental leaf senescence. This suggests the possibility of a yet-to-be-discovered epigenetic-based switch between cell survival and cell death. Conclusions from the analyzed research contributed to the hypothesis that chromatin-remodeling mechanisms play a role in the control of induced leaf senescence. Understanding this mechanism in crops might provide a tool for further exploitation toward sustainable agriculture: so-called epibreeding.

Physio-Genetic Dissection of Dark-Induced Leaf Senescence and Timing Its Reversal in Barley.

Barley crop model was analyzed for early and late events during the dark-induced leaf senescence (DILS) as well as for deciphering critical time limit for reversal of the senescence process. Chlorophyll fluorescence vitality index Rfd was determined as the earliest parameter that correlated well with the cessation of photosynthesis prior to microautophagy symptoms, initiation of DNA degradation, and severalfold increase in the endonuclease BNUC1. DILS was found characterized by up-regulation of processes that enable recycling of degraded macromolecules and metabolites, including increased NH4 + remobilization, gluconeogenesis, glycolysis, and partial up-regulation of glyoxylate and tricarboxylate acid cycles. The most evident differences in gene medleys between DILS and developmental senescence included hormone-activated signaling pathways, lipid catabolic processes, carbohydrate metabolic processes, low-affinity ammonia remobilization, and RNA methylation. The mega-autophagy symptoms were apparent much later, specifically on day 10 of DILS, when disruption of organelles-nucleus and mitochondria -became evident. Also, during this latter-stage programmed cell death processes, namely, shrinking of the protoplast, tonoplast interruption, and vacuole breakdown, chromatin condensation, more DNA fragmentation, and disintegration of the cell membrane were prominent. Reversal of DILS by re-exposure of the plants from dark to light was possible until but not later than day 7 of dark exposure and was accompanied by regained photosynthesis, increase in chlorophyll, and reversal of Rfd, despite activation of macro-autophagy-related genes.

Insight into metabolic sensors of nitrosative stress protection in Phytophthora infestans.

Phytophthora infestans, a representative of phytopathogenic oomycetes, have been proven to cope with redundant sources of internal and host-derived reactive nitrogen species (RNS). To gain insight into its nitrosative stress resistance mechanisms, metabolic sensors activated in response to nitrosative challenge during both in vitro growth and colonization of the host plant were investigated. The conducted analyses of gene expression, protein accumulation, and enzyme activity reveal for the first time that P. infestans (avirulent MP946 and virulent MP977 toward potato cv. Sarpo Mira) withstands nitrosative challenge and has an efficient system of RNS elimination. The obtained data indicate that the system protecting P. infestans against nitric oxide (NO) involved the expression of the nitric oxide dioxygenase (Pi-NOD1) gene belonging to the globin family. The maintenance of RNS homeostasis was also supported by an elevated S-nitrosoglutathione reductase activity and upregulation of peroxiredoxin 2 at the transcript and protein levels; however, the virulence pattern determined the expression abundance. Based on the experiments, it can be concluded that P. infestans possesses a multifarious system of metabolic sensors controlling RNS balance via detoxification, allowing the oomycete to exist in different micro-environments flexibly.

Metal tolerance gene family in barley: an in silico comprehensive analysis.

Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. However, a comprehensive study of MTPs is still lacking in crop plants. The current study aimed to comprehensively identify and characterize the MTP gene family in barley (Hordeum vulgare, Hv), an important crop. In total, 12 HvMTPs were identified in the barley genome in this study. They were divided into three phylogenetic groups (Zn-cation diffusion facilitator proteins [CDFs], Fe/Zn-CDFs, and Mn-CDFs) and further subdivided into seven groups (G1, G5, G6, G7, G8, G9, and G12). The majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene duplication analysis of HvMTPs revealed one pair of segmental-like duplications in the barley genome. Evolutionary analysis suggested that barley MTPs underwent purifying natural selection. Additionally, the HvMTPs were analyzed in the pan-genome sequences of barley (20 accessions), which suggests that HvMTPs are highly conserved in barley evolution. Cis-acting regulatory elements, microRNA target sites, and protein-protein interaction analysis indicated the role of HvMTPs in a variety of biological processes. Expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle, and their expression levels were not significantly altered by abiotic stresses like cold, drought, or heat. The expression of barley HvMTP genes in the presence of heavy metals such as Zn2+, Cu2+, As3+, and Cd2+ revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance or transportation. The identification and comprehensive investigation of MTP gene family members will provide important gene resources for the genetic improvement of crops for metal tolerance, bioremediation, or biofortification of staple crops.

Genetic portrait of polyamine transporters in barley: insights in the regulation of leaf senescence.

Nitrogen (N) is one of the most expensive nutrients to supply, therefore, improving the efficiency of N use is essential to reduce the cost of commercial fertilization in plant production. Since cells cannot store reduced N as NH3 or NH4 +, polyamines (PAs), the low molecular weight aliphatic nitrogenous bases, are important N storage compounds in plants. Manipulating polyamines may provide a method to increase nitrogen remobilization efficiency. Homeostasis of PAs is maintained by intricate multiple feedback mechanisms at the level of biosynthesis, catabolism, efflux, and uptake. The molecular characterization of the PA uptake transporter (PUT) in most crop plants remains largely unknown, and knowledge of polyamine exporters in plants is lacking. Bi-directional amino acid transporters (BATs) have been recently suggested as possible PAs exporters for Arabidopsis and rice, however, detailed characterization of these genes in crops is missing. This report describes the first systematic study to comprehensively analyze PA transporters in barley (Hordeum vulgare, Hv), specifically the PUT and BAT gene families. Here, seven PUTs (HvPUT1-7) and six BATs (HvBAT1-6) genes were identified as PA transporters in the barley genome and the detailed characterization of these HvPUT and HvBAT genes and proteins is provided. Homology modeling of all studied PA transporters provided 3D structures prediction of the proteins of interest with high accuracy. Moreover, molecular docking studies provided insights into the PA-binding pockets of HvPUTs and HvBATs facilitating improved understanding of the mechanisms and interactions involved in HvPUT/HvBAT-mediated transport of PAs. We also examined the physiochemical characteristics of PA transporters and discuss the function of PA transporters in barley development, and how they help barley respond to stress, with a particular emphasis on leaf senescence. Insights gained here could lead to improved barley production via modulation of polyamine homeostasis.

Plant Transglutaminases: New Insights in Biochemistry, Genetics, and Physiology.

Transglutaminases (TGases) are calcium-dependent enzymes that catalyse an acyl-transfer reaction between primary amino groups and protein-bound Gln residues. They are widely distributed in nature, being found in vertebrates, invertebrates, microorganisms, and plants. TGases and their functionality have been less studied in plants than humans and animals. TGases are distributed in all plant organs, such as leaves, tubers, roots, flowers, buds, pollen, and various cell compartments, including chloroplasts, the cytoplasm, and the cell wall. Recent molecular, physiological, and biochemical evidence pointing to the role of TGases in plant biology and the mechanisms in which they are involved allows us to consider their role in processes such as photosynthesis, plant fertilisation, responses to biotic and abiotic stresses, and leaf senescence. In the present paper, an in-depth description of the biochemical characteristics and a bioinformatics comparison of plant TGases is provided. We also present the phylogenetic relationship, gene structure, and sequence alignment of TGase proteins in various plant species, not described elsewhere. Currently, our knowledge of these proteins in plants is still insufficient. Further research with the aim of identifying and describing the regulatory components of these enzymes and the processes regulated by them is needed.

Fungal and oomycete pathogens and heavy metals: an inglorious couple in the environment.

Heavy metal (HM) contamination of the environment is a major problem worldwide. The rate of global deposition of HMs in soil has dramatically increased over the past two centuries and there of facilitated their rapid accumulation also in living systems. Although the effects of HMs on plants, animals and humans have been extensively studied, yet little is known about their effects on the (patho)biology of the microorganisms belonging to a unique group of filamentous eukaryotic pathogens, i.e., fungi and oomycetes. Much of the literature concerning mainly model species has revealed that HM stress affects their hyphal growth, morphology, and sporulation. Toxicity at cellular level leads to disturbance of redox homeostasis manifested by the formation of nitro-oxidative intermediates and to the induction of antioxidant machinery. Despite such adverse effects, published data is indicative of the fact that fungal and oomycete pathogens have a relatively high tolerance to HMs in comparison to other groups of microbes such as bacteria. Likely, these pathogens may harbor a network of detoxification mechanisms that ensure their survival in a highly HM-polluted (micro)habitat. Such a network may include extracellular HMs immobilization, biosorption to cell wall, and/or their intracellular sequestration to proteins or other ligands. HMs may also induce a hormesis-like phenomenon allowing the pathogens to maintain or even increase fitness against chemical challenges. Different scenarios linking HMs stress and modification of the microorganisms pathogenicity are disscused in this review.

Comparative genomic analysis of the aldehyde dehydrogenase gene superfamily in Arabidopsis thaliana - searching for the functional key to hypoxia tolerance.

Flooding entails different stressful conditions leading to low oxygen availability for respiration and as a result plants experience hypoxia. Stress imposed by hypoxia affects cellular metabolism, including the formation of toxic metabolites that dramatically reduce crop productivity. Aldehyde dehydrogenases (ALDHs) are a group of enzymes participating in various aspects of plant growth, development and stress responses. Although we have knowledge concerning the multiple functionalities of ALDHs in tolerance to various stresses, the engagement of ALDH in plant metabolism adjustment to hypoxia is poorly recognized. Therefore, we explored the ALDH gene superfamily in the model plant Arabidopsis thaliana. Genome-wide analyses revealed that 16 AtALDH genes are organized into ten families and distributed irregularly across Arabidopsis 5 chromosomes. According to evolutionary relationship studies from different plant species, the ALDH gene superfamily is highly conserved. AtALDH2 and ALDH3 are the most numerous families in plants, while ALDH18 was found to be the most distantly related. The analysis of cis-acting elements in promoters of AtALDHs indicated that AtALDHs participate in responses to light, phytohormones and abiotic stresses. Expression profile analysis derived from qRT-PCR showed the AtALDH2B7, AtALDH3H1 and AtALDH5F1 genes as the most responsive to hypoxia stress. In addition, the expression of AtALDH18B1, AtALDH18B2, AtALDH2B4, and AtALDH10A8 was highly altered during the post-hypoxia-reoxygenation phase. Taken together, we provide comprehensive functional information on the ALDH gene superfamily in Arabidopsis during hypoxia stress and highlight ALDHs as a functional element of hypoxic systemic responses. These findings might help develop a framework for application in the genetic improvement of crop plants.

N6-methyladenosine (m6A) RNA modification as a metabolic switch between plant cell survival and death in leaf senescence.

Crop losses caused by climate change and various (a)biotic stressors negatively affect agriculture and crop production. Therefore, it is vital to develop a proper understanding of the complex response(s) to (a)biotic stresses and delineate them for each crop plant as a means to enable translational research. In plants, the improvement of crop quality by m6A editing is believed to be a promising strategy. As a reaction to environmental changes, m6A modification showed a high degree of sensitivity and complexity. We investigated differences in gene medleys between dark-induced leaf senescence (DILS) and developmental leaf senescence in barley, including inter alia RNA modifications active in DILS. The identified upregulated genes in DILS include RNA methyltransferases of different RNA types, embracing enzymes modifying mRNA, tRNA, and rRNA. We have defined a decisive moment in the DILS model which determines the point of no return, but the mechanism of its control is yet to be uncovered. This indicates the possibility of an unknown additional switch between cell survival and cell death. Discoveries of m6A RNA modification changes in certain RNA species in different stages of leaf senescence may uncover the role of such modifications in metabolic reprogramming. Nonetheless, there is no such data about the process of leaf senescence in plants. In this scope, the prospect of finding connections between the process of senescence and m6A modification of RNA in plants seems to be compelling.

Plant homocysteine, a methionine precursor and plant's hallmark of metabolic disorders.

Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid, which arises from redox-sensitive methionine metabolism. In plants, Hcy synthesis involves both cystathionine ß-lyase and S-adenosylhomocysteine hydrolase activities. Thus, Hcy itself is crucial for de novo methionine synthesis and S-adenosylmethionine recycling, influencing the formation of ethylene, polyamines, and nicotianamine. Research on mammalian cells has shown biotoxicity of this amino acid, as Hcy accumulation triggers oxidative stress and the associated lipid peroxidation process. In addition, the presence of highly reactive groups induces Hcy and Hcy derivatives to modify proteins by changing their structure and function. Currently, Hcy is recognized as a critical, independent hallmark of many degenerative metabolic diseases. Research results indicate that an enhanced Hcy level is also toxic to yeast and bacteria cells. In contrast, in the case of plants the metabolic status of Hcy remains poorly examined and understood. However, the presence of the toxic Hcy metabolites and Hcy over-accumulation during the development of an infectious disease seem to suggest harmful effects of this amino acid also in plant cells. The review highlights potential implications of Hcy metabolism in plant physiological disorders caused by environmental stresses. Moreover, recent research advances emphasize that recognizing the Hcy mode of action in various plant systems facilitates verification of the potential status of Hcy metabolites as bioindicators of metabolism disorders and thus may constitute an element of broadly understood biomonitoring.

Unraveling the genetics of polyamine metabolism in barley for senescence-related crop improvement.

We explored the polyamine (PA) metabolic pathway genes in barley (Hv) to understand plant development and stress adaptation in Gramineae crops with emphasis on leaf senescence. Bioinformatics and functional genomics tools were utilized for genome-wide identification, comprehensive gene features, evolution, development and stress effects on the expression of the polyamine metabolic pathway gene families (PMGs). Three S-adenosylmethionine decarboxylases (HvSAMDCs), two ornithine decarboxylases (HvODCs), one arginine decarboxylase (HvADC), one spermidine synthase (HvSPDS), two spermine synthases (HvSPMSs), five copper amine oxidases (HvCuAOs) and seven polyamine oxidases (HvPAOs) members of PMGs were identified and characterized in barley. All the HvPMG genes were found to be distributed on all chromosomes of barley. The phylogenetic and comparative assessment revealed that PA metabolic pathway is highly conserved in plants and the prediction of nine H. vulgare miRNAs (hvu-miR) target sites, 18 protein-protein interactions and 961 putative CREs in the promoter region were discerned. Gene expression of HvSAMDC3, HvCuAO7, HvPAO4 and HvSPMS1 was apparent at every developmental stage. SPDS/SPMS gene family was found to be the most responsive to induced leaf senescence. This study provides a reference for the functional investigation of the molecular mechanism(s) that regulate polyamine metabolism in plants as a tool for future breeding decision management systems.

Dark-Induced Barley Leaf Senescence - A Crop System for Studying Senescence and Autophagy Mechanisms.

This review synthesizes knowledge on dark-induced barley, attached, leaf senescence (DILS) as a model and discusses the possibility of using this crop system for studying senescence and autophagy mechanisms. It addresses the recent progress made in our understanding of DILS. The following aspects are discussed: the importance of chloroplasts as early targets of DILS, the role of Rubisco as the largest repository of recoverable nitrogen in leaves senescing in darkness, morphological changes of these leaves other than those described for chloroplasts and metabolic modifications associated with them, DILS versus developmental leaf senescence transcriptomic differences, and finally the observation that in DILS autophagy participates in the circulation of cell components and acts as a quality control mechanism during senescence. Despite the progression of macroautophagy, the symptoms of degradation can be reversed. In the review, the question also arises how plant cells regulate stress-induced senescence via autophagy and how the function of autophagy switches between cell survival and cell death.

New Insight into Plant Signaling: Extracellular ATP and Uncommon Nucleotides.

New players in plant signaling are described in detail in this review: extracellular ATP (eATP) and uncommon nucleotides such as dinucleoside polyphosphates (NpnN's), adenosine 5'-phosphoramidate (NH2-pA), and extracellular NAD+ and NADP+ (eNAD(P)+). Recent molecular, physiological, and biochemical evidence implicating concurrently the signaling role of eATP, NpnN's, and NH2-pA in plant biology and the mechanistic events in which they are involved are discussed. Numerous studies have shown that they are often universal signaling messengers, which trigger a signaling cascade in similar reactions and processes among different kingdoms. We also present here, not described elsewhere, a working model of the NpnN' and NH2-pA signaling network in a plant cell where these nucleotides trigger induction of the phenylpropanoid and the isochorismic acid pathways yielding metabolites protecting the plant against various types of stresses. Through these signals, the plant responds to environmental stimuli by intensifying the production of various compounds, such as anthocyanins, lignin, stilbenes, and salicylic acid. Still, more research needs to be performed to identify signaling networks that involve uncommon nucleotides, followed by omic experiments to define network elements and processes that are controlled by these signals.

Polyamines - A New Metabolic Switch: Crosstalk With Networks Involving Senescence, Crop Improvement, and Mammalian Cancer Therapy.

Polyamines (PAs) are low molecular weight organic cations comprising biogenic amines that play multiple roles in plant growth and senescence. PA metabolism was found to play a central role in metabolic and genetic reprogramming during dark-induced barley leaf senescence (DILS). Robust PA catabolism can impact the rate of senescence progression in plants. We opine that deciphering senescence-dependent polyamine-mediated multidirectional metabolic crosstalks is important to understand regulation and involvement of PAs in plant death and re-mobilization of nutrients during senescence. This will involve optimizing the use of PA biosynthesis inhibitors, robust transgenic approaches to modulate PA biosynthetic and catabolic genes, and developing novel germplasm enriched in pro- and anti-senescence traits to ensure sustained crop productivity. PA-mediated delay of senescence can extend the photosynthesis capacity, thereby increasing grain starch content in malting grains such as barley. On the other hand, accelerating the onset of senescence can lead to increases in mineral and nitrogen content in grains for animal feed. Unraveling the "polyamine metabolic switch" and delineating the roles of PAs in senescence should further our knowledge about autophagy mechanisms involved in plant senescence as well as mammalian systems. It is noteworthy that inhibitors of PA biosynthesis block cell viability in animal model systems (cell tumor lines) to control some cancers, in this instance, proliferative cancer cells were led toward cell death. Likewise, PA conjugates work as signal carriers for slow release of regulatory molecule nitric oxide in the targeted cells. Taken together, these and other outcomes provide examples for developing novel therapeutics for human health wellness as well as developing plant resistance/tolerance to stress stimuli.

Teaching about Water Relations in Plant Cells: An Uneasy Struggle.

University students often struggle to understand the role of water in plant cells. In particular, osmosis and plasmolysis appear to be challenging topics. This study attempted to identify student difficulties (including misconceptions) concerning osmosis and plasmolysis and examined to what extent the difficulties could be revised during a plant physiology course. A questionnaire was developed to monitor university students' conceptual knowledge before and after a course, and both qualitative and quantitative data were obtained. The data were analyzed using the constant comparison technique and descriptive statistics. Students were found to come to university with many misconceptions that had accumulated during their education. These misconceptions are extremely difficult to change during the traditional course, which comprises lectures and practical exercises. Students' misconceptions originate from commonly used sources such as textbooks, which are perceived as being reliable. Effective teaching of water relations in plant cells could include such didactic methods as "questioning the author," which allow teachers to monitor students' knowledge and help students acquire a more scientific understanding of key concepts.

Lead-stress induced changes in the content of free, thylakoid- and chromatin-bound polyamines, photosynthetic parameters and ultrastructure in greening barley leaves.

The aim of this study was to determine the impact of lead (Pb) stress as 0.6mM Pb(NO3)2 on the content of free, thylakoid- and chromatin-bound polyamines (PAs) and diamine oxidase (DAO) activity in detached greening barley leaves. Additionally, photosynthetic-related parameters, generation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content and ultrastructural changes under Pb-stress were studied. The level of putrescine (Put) was reduced progressively to 56% at 24h of Pb stress, and it was correlated with 38% increase of DAO activity. Spermidine (Spd) content was not affected by Pb-stress, while the free spermine (Spm) level significantly increased by about 83% at 6h, and in that time the lowest level of H2O2 was observed. The exogenous applied Spm to Pb-treated leaves caused a decrease in the content of H2O2. In greening leaves exposed to Pb an accumulation of chlorophylls a and b was inhibited by about 39 and 47%, respectively, and photosynthetic parameters of efficiency of electron transport and photochemical reaction in chloroplasts as ΦPSII, ETR and RFd were lowered by about 23-32%. The level of thylakoid-bound Put decreased by about 22%. Moreover, thylakoids isolated from chloroplasts of Pb-treated leaves were characterized with lower Put/Spm ratio as compared to control leaves. In the presence of Pb the significant decrease in the number of thylakoids per granum and cap-shape invaginations of cytoplasmic material were noticed. In Pb-stressed leaves the level of chromatin-bound Spm increased by about 48% and sometimes condensed chromatin in nuclei was observed. We conclude that in greening barley leaves exposed to Pb-stress changes in free, thylakoid- and chromatin-bound PAs play some role in the functioning of leaves or plants in heavy metal stress conditions.