Catalase: A critical node in the regulation of cell fate.

Catalase (CAT) is an extensively studied if somewhat enigmatic enzyme that is at the heart of eukaryotic antioxidant systems with a canonical role in peroxisomal function. The CAT family of proteins exert control over a wide range of plant growth and defence processes. CAT proteins are subject to many types of post-translational modification (PTM), which modify activity, ligand binding, stability, compartmentation and function. The CAT interactome involves many cytosolic and nuclear proteins that appear to be essential for protein functions. Hence, the CAT network of roles extends far beyond those associated with peroxisomal metabolism. Some pathogen effector proteins are able to redirect CAT to the nucleus and recent evidence indicates CAT can traffic to the nucleus in the absence of exogenous proteins. While the mechanisms that target CAT to the nucleus are not understood, CAT activity in the cytosol and nucleus is promoted by interactions with nucleoredoxin. Here we discuss recent findings that have been pivotal in generating a step change in our understanding of CAT functions in plant cells.

Broad Specific Xyloglucan:Xyloglucosyl Transferases Are Formidable Players in the Re-Modelling of Plant Cell Wall Structures.

Plant xyloglucan:xyloglucosyl transferases, known as xyloglucan endo-transglycosylases (XETs) are the key players that underlie plant cell wall dynamics and mechanics. These fundamental roles are central for the assembly and modifications of cell walls during embryogenesis, vegetative and reproductive growth, and adaptations to living environments under biotic and abiotic (environmental) stresses. XET enzymes (EC 2.4.1.207) have the ß-sandwich architecture and the ß-jelly-roll topology, and are classified in the glycoside hydrolase family 16 based on their evolutionary history. XET enzymes catalyse transglycosylation reactions with xyloglucan (XG)-derived and other than XG-derived donors and acceptors, and this poly-specificity originates from the structural plasticity and evolutionary diversification that has evolved through expansion and duplication. In phyletic groups, XETs form the gene families that are differentially expressed in organs and tissues in time- and space-dependent manners, and in response to environmental conditions. Here, we examine higher plant XET enzymes and dissect how their exclusively carbohydrate-linked transglycosylation catalytic function inter-connects complex plant cell wall components. Further, we discuss progress in technologies that advance the knowledge of plant cell walls and how this knowledge defines the roles of XETs. We construe that the broad specificity of the plant XETs underscores their roles in continuous cell wall restructuring and re-modelling.

An ABHD17-like hydrolase screening system to identify de-S-acylation enzymes of protein substrates in plant cells.

Protein S-acylation is an important post-translational modification in eukaryotes, regulating the subcellular localization, trafficking, stability, and activity of substrate proteins. The dynamic regulation of this reversible modification is mediated inversely by protein S-acyltransferases and de-S-acylation enzymes, but the de-S-acylation mechanism remains unclear in plant cells. Here, we characterized a group of putative protein de-S-acylation enzymes in Arabidopsis thaliana, including 11 members of Alpha/Beta Hydrolase Domain-containing Protein 17-like acyl protein thioesterases (ABAPTs). A robust system was then established for the screening of de-S-acylation enzymes of protein substrates in plant cells, based on the effects of substrate localization and confirmed via the protein S-acylation levels. Using this system, the ABAPTs, which specifically reduced the S-acylation levels and disrupted the plasma membrane localization of five immunity-related proteins, were identified respectively in Arabidopsis. Further results indicated that the de-S-acylation of RPM1-Interacting Protein 4, which was mediated by ABAPT8, resulted in an increase of cell death in Arabidopsis and Nicotiana benthamiana, supporting the physiological role of the ABAPTs in plants. Collectively, our current work provides a powerful and reliable system to identify the pairs of plant protein substrates and de-S-acylation enzymes for further studies on the dynamic regulation of plant protein S-acylation.

The Jo-In protein welding system is a relevant tool to create CBM-containing plant cell wall degrading enzymes.

Irrespective of their biological origin, most proteins are composed of several elementary domains connected by linkers. These domains are either functionally independent units, or part of larger multidomain structures whose functions are defined by their spatial proximity. Carbohydrate-degrading enzymes provide examples of a range of multidomain structures, in which catalytic protein domains are frequently appended to one or more non-catalytic carbohydrate-binding modules which specifically bind to carbohydrate motifs. While the carbohydrate-binding specificity of these modules is clear, their function is not fully elucidated. Herein, an original approach to tackle the study of carbohydrate-binding modules using the Jo-In biomolecular welding protein pair is presented. To provide a proof of concept, recombinant xylanases appended to two different carbohydrate-binding modules have been created and produced. The data reveal the biochemical properties of four xylanase variants and provide the basis for correlating enzyme activity to structural properties and to the nature of the substrate and the ligand specificity of the appended carbohydrate-binding module. It reveals that specific spatial arrangements favour activity on soluble polymeric substrates and that activity on such substrates does not predict the behaviour of multimodular enzymes on insoluble plant cell wall samples. The results highlight that the Jo-In protein welding system is extremely useful to design multimodular enzyme systems, especially to create rigid conformations that decrease the risk of intermodular interference. Further work on Jo-In will target the introduction of varying degrees of flexibility, providing the means to study this property and the way it may influence multimodular enzyme functions.

Overexpression of AtBBD1, Arabidopsis Bifunctional Nuclease, Confers Drought Tolerance by Enhancing the Expression of Regulatory Genes in ABA-Mediated Drought Stress Signaling.

Drought is the most serious abiotic stress, which significantly reduces crop productivity. The phytohormone ABA plays a pivotal role in regulating stomatal closing upon drought stress. Here, we characterized the physiological function of AtBBD1, which has bifunctional nuclease activity, on drought stress. We found that AtBBD1 localized to the nucleus and cytoplasm, and was expressed strongly in trichomes and stomatal guard cells of leaves, based on promoter:GUS constructs. Expression analyses revealed that AtBBD1 and AtBBD2 are induced early and strongly by ABA and drought, and that AtBBD1 is also strongly responsive to JA. We then compared phenotypes of two AtBBD1-overexpression lines (AtBBD1-OX), single knockout atbbd1, and double knockout atbbd1/atbbd2 plants under drought conditions. We did not observe any phenotypic difference among them under normal growth conditions, while OX lines had greatly enhanced drought tolerance, lower transpirational water loss, and higher proline content than the WT and KOs. Moreover, by measuring seed germination rate and the stomatal aperture after ABA treatment, we found that AtBBD1-OX and atbbd1 plants showed significantly higher and lower ABA-sensitivity, respectively, than the WT. RNA sequencing analysis of AtBBD1-OX and atbbd1 plants under PEG-induced drought stress showed that overexpression of AtBBD1 enhances the expression of key regulatory genes in the ABA-mediated drought signaling cascade, particularly by inducing genes related to ABA biosynthesis, downstream transcription factors, and other regulatory proteins, conferring AtBBD1-OXs with drought tolerance. Taken together, we suggest that AtBBD1 functions as a novel positive regulator of drought responses by enhancing the expression of ABA- and drought stress-responsive genes as well as by increasing proline content.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

A mitochondria-targeted coenzyme Q peptoid induces superoxide dismutase and alleviates salinity stress in plant cells.

Salinity is a serious challenge to global agriculture and threatens human food security. Plant cells can respond to salt stress either by activation of adaptive responses, or by programmed cell death. The mechanisms deciding the respective response are far from understood, but seem to depend on the degree, to which mitochondria can maintain oxidative homeostasis. Using plant PeptoQ, a Trojan Peptoid, as vehicle, it is possible to transport a coenzyme Q10 (CoQ10) derivative into plant mitochondria. We show that salinity stress in tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow-2) can be mitigated by pretreatment with plant PeptoQ with respect to numerous aspects including proliferation, expansion, redox homeostasis, and programmed cell death. We tested the salinity response for transcripts from nine salt-stress related-genes representing different adaptive responses. While most did not show any significant response, the salt response of the transcription factor NtNAC, probably involved in mitochondrial retrograde signaling, was significantly modulated by the plant PeptoQ. Most strikingly, transcripts for the mitochondrial, Mn-dependent Superoxide Dismutase were rapidly and drastically upregulated in presence of the peptoid, and this response was disappearing in presence of salt. The same pattern, albeit at lower amplitude, was seen for the sodium exporter SOS1. The findings are discussed by a model, where plant PeptoQ modulates retrograde signalling to the nucleus leading to a strong expression of mitochondrial SOD, what renders mitochondria more resilient to perturbations of oxidative balance, such that cells escape salt induced cell death and remain viable.

Plant Cell Wall Proteins and Development.

Plant cell walls surround cells and provide both external protection and a means of cell-to-cell communication [...].

Biochemical and physiological aspects of chlorophyll breakdown / Biochemiczne i fizjologiczne aspekty rozkladu barwników chlorofilowych.

During leaf senescence and fruit ripening chlorophyll is broken down into nonfluorescent catabolites (NCCs). The chlorophyll degradation pathway includes a series of biochemical transformations ocurring sequentially in chloroplasts, cytosol and vacuoles. The path begins with enzymatic reduction of chlorophyll b to chlorophyll a. Next, the specific dechelatase and esterase remove the magnesium atom and the phytol chain resulting in the formation of pheophorbide a. In the next step, the porphyrin macroring is opened by pheophorbide a oxygenase and red catabolite reductase. The product of this transformation is an early fluorescent catabolite (pFCC), which after hydroxylation and species-specific modifications is imported into the vacuole. In acidic medium of the vacuole pFCC undergo isomerization to their respective colorless NCCs, which are final chlorophyll degradation products in higher plants. There are still no answers to a number of questions about the fate and significance of millions tons of chlorophyll catabolites released annually in the aquatic environment as a result of cellular senescence and death of phytoplankton. A few reports indicate that algae and cyanobacteria may metabolize their photosynthetic pigments in a similar way as higher plants do, however, the course of chlorophyll breakdown in these organisms has not been yet elucidated.

Plant Cell Polarity: Creating Diversity from Inside the Box.

Cell polarity in plants operates across a broad range of spatial and temporal scales to control processes from acute cell growth to systemic hormone distribution. Similar to other eukaryotes, plants generate polarity at both the subcellular and tissue levels, often through polarization of membrane-associated protein complexes. However, likely due to the constraints imposed by the cell wall and their extremely plastic development, plants possess novel polarity molecules and mechanisms highly tuned to environmental inputs. Considerable progress has been made in identifying key plant polarity regulators, but detailed molecular understanding of polarity mechanisms remains incomplete in plants. Here, we emphasize the striking similarities in the conceptual frameworks that generate polarity in both animals and plants. To this end, we highlight how novel, plant-specific proteins engage in common themes of positive feedback, dynamic intracellular trafficking, and posttranslational regulation to establish polarity axes in development. We end with a discussion of how environmental signals control intrinsic polarity to impact postembryonic organogenesis and growth.

Nitric oxide synthase inhibitor L-NAME affects Arabidopsis root growth, morphology, and microtubule organization.

The presence of evolutionarily conserved NOS or NOS-like enzymes in land plants different than those in animals is still unclear, despite their activity has been revealed in cytosol and some organelles. At the same time, the emerging evidence for the importance of L-arginine-dependent pathways of NO synthesis in plant cells is still accumulating. The aim of our study was to reveal physiological effects on growth and differentiation processes, and microtubular cytoskeleton organization of the competitive mammalian NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME). Thus, the treatment of Arabidopsis with L-NAME (50-1 mM) caused dose- and time-dependent inhibition of primary roots growth. Moreover, the morphology of primary roots under the influence of L-NAME also underwent changes. L-NAME (>100 µM) induced the formation of novel over-elongated root hairs in shortened elongation zone, while in higher concentrations (500 µM) it caused a slight swelling of epidermal cells in differentiation zone. L-NAME also provoked microtubule reorganization in epidermal cells of different root growth zones. Thus, L-NAME at concentrations of 50-1 mM induced cortical microtubules randomization and/or depolymerization in epidermal cells of the root apex, meristem, transition, elongation, and differentiation zones after 2 h of treatment. Disordered microtubules in trichoblasts could initiate the formation of actively elongating root hairs that reveals longitudinal microtubules ensuring their active growth at 24 h of treatment. Therefore, L-NAME inhibits primary root growth, induces the differentiation processes in roots, reorganizes cortical microtubules in epidermal root cells suggesting the importance of L-arginine-dependent pathways of NO synthesis in plants.

Alternative oxidase (AOX) over-expression improves cell expansion and elongation in cotton seedling exposed to cool temperatures.

KEY MESSAGE: Evidence that supports a relation between AOX expression and improvement in plant height, internode length, and total leaf area under cool temperature is shown. Cell expansion and elongation appear to be enhanced when AOX expression was increased. Cotton growth is sensitive to cool temperature during germination and early seedling development. Delayed emergence, seedling damage, and increased risk to disease are common. Late seasonal cool weather is a major factor limiting the consistent production of high-quality cotton lint in West Texas. Alternative oxidase functions in the inner membrane of the mitochondria via an alternative respiration pathway and serves as a multifunctional system for amelioration of abiotic and biotic stresses. Cotton seedling emergence and growth exposed to cool temperatures was examined in plants with enhanced AOX expression. Thirteen T1 seed lines showed 3 to 1 segregation for the T-DNA containing the tobacco AOX1 gene. Two over-expressing, single-copy, homozygous AOX lines (94-20T and 66-6T) and Null line (94-3N) were selected for examination. The transcript levels were ≈ 2 to 6 fold higher in the AOX lines compared to those of the Null line and wild-type in stem, leaf, root and boll tissues. The research examined the hypothesis that transgenic cotton with enhanced AOX expression will have enhanced growth traits under suboptimal cool temperatures. Improved plant height, internode length, plant height and internode length from second node, and total leaf area under cool temperatures were observed in AOX over-expression lines. This may be attributed to improved cell expansion and elongation characteristics in the AOX line.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis.

Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root-meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels.

Rho GTPases play crucial roles in cell polarity and pattern formation. ROPs, Rho of plant GTPases, are widely involved in cell wall patterning in plants, yet the molecular mechanism underlying their action remains unknown. Arabidopsis ROP11 is locally activated to form plasma membrane domains, which direct formation of cell wall pits in metaxylem vessel cells through interaction with cortical microtubules. Here, we show that the pattern formation of cell wall pits is governed by ROP activation via a reaction-diffusion mechanism. Genetic analysis and reconstructive assays revealed that ROPGEF4/7 and ROPGAP3/4, which encode ROP activators and inactivators, respectively, regulated the formation of ROP-activated domains; these in turn determined the pattern of cell wall pits. Mathematical modelling showed that ROP-activation cycle generated ROP domains by reaction-diffusion mechanism. The model predicted that a positive feedback and slow diffusion of ROP11-ROPGEF4 complex were required to generate ROP-activated domains. ROPGEF4 formed a dimer that interacted with activated ROP11 in vivo, which could provide positive feedback for ROP activation. ROPGEF4 was highly stable on the plasma membrane and inhibited ROP11 diffusion. Our study indicated that ROP-based reaction-diffusion system self-organizes ROP-activated domains, thereby determines the pit pattern of metaxylem vessels.

Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.

Molecular cloning and biotic elicitation response of phenylalanine ammonia-lyase gene of Astragalus chrysochlorus.

Phenylalanine ammonia lyase (PAL) is the first enzyme of the phenylpropanoid pathway, and it is necessary to upregulate flavonoid biosynthesis in most of the plant species. In this study, we have cloned PAL gene from endemic Astragalus chrysochlorus which is a producer of phenolic nicotiflorin (kaempferol-3-O-rutinoside). The cDNA encoding PAL was cloned from A. chrysochlorus using RT-PCR (reverse transcription-polymerase chain reaction) with conserved primer pairs. Amino acid sequence alignments showed that AcPAL (2160 bp, Accession number: KM189182) has more than 95% amino acid identity with their homologues in other Astragalus species. The coding sequence for the protein of AcPAL is 720 amino acids with a calculated molecular weight of 78.53 kDa. Full length AcPAL was cloned and expressed in Escherichia coli. qPCR (quantitative real-time PCR) analysis of the expression of PAL gene of A. chrysochlorus suggested that maximum transcript level was observed in 3 h yeast extract elicited suspension cells. Our findings suggest that AcPAL plays role in early response for yeast extract treatment. The isolation of AcPAL gene could be result in further studies for overproduction of secondary metabolite, nicotiflorin.

Cell and Developmental Biology of Plant Mitogen-Activated Protein Kinases.

Plant mitogen-activated protein kinases (MAPKs) constitute a network of signaling cascades responsible for transducing extracellular stimuli and decoding them to dedicated cellular and developmental responses that shape the plant body. Over the last decade, we have accumulated information about how MAPK modules control the development of reproductive tissues and gametes and the embryogenic and postembryonic development of vegetative organs such as roots, root nodules, shoots, and leaves. Of key importance to understanding how MAPKs participate in developmental and environmental signaling is the characterization of their subcellular localization, their interactions with upstream signal perception mechanisms, and the means by which they target their substrates. In this review, we summarize the roles of MAPK signaling in the regulation of key plant developmental processes, and we survey what is known about the mechanisms guiding the subcellular compartmentalization of MAPK modules.

Rosa hybrida orcinol O-methyl transferase-mediated production of pterostilbene in metabolically engineered grapevine cell cultures.

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties.

Combined enzymatic and metabolic analysis of grapevine cell responses to elicitors.

Elicitors trigger plant defense responses, including phytoalexin production and cell-wall reinforcement. Primary metabolism plays an important role in these responses as it fuels the associated energetic costs and provides precursors for the synthesis of the numerous secondary metabolites involved in defenses against pathogens. In this context, we aimed to determine whether oligosaccharidic elicitors differing in their capacity to activate defense-associated secondary metabolism in grapevine would differently impact primary metabolism. To answer this question, cell suspensions were treated with two elicitors: an oligogalacturonide, and the ß-glucan laminarin. Enzymatic activity assays together with targeted (HPLC) and global (GC-MS) analyses of metabolites were next performed to compare their impact on plant primary or secondary metabolism. The results showed that the oligogalacturonide, which induced the highest level of the phytoalexin resveratrol and the highest activity of stilbene synthase, also induced the highest activity of shikimate hydroxycinnamoyltransferase, a key enzyme involved in the synthesis of lignin. The oligogalacturonide-induced defenses had a significant impact on primary metabolism 24 h following elicitor treatment, with a reduced abundance of pyruvate and 2-oxoglutarate, together with an increase of a set of metabolites including carbohydrates and amino acids. Interestingly, an accumulation of galacturonate and gentiobiose was observed in the oligogalacturonide- and laminarin-treated cells, respectively, suggesting that both elicitors are rapidly hydrolyzed in grapevine cell suspension cultures.

Identification of the actual coding region for polyamine oxidase 6 from rice (OsPAO6) and its partial characterization.

Polyamines (PA) in plant play roles in growth and development and in responses to environmental stresses. The family of polyamine oxidases (PAO) contributes to a balanced homeostasis of PAs catalyzing two different reactions, terminal catabolic (TC) and back-conversion (BC) pathway, in PA catabolism. From the seven PAOs encoded by the rice genome (OsPAO1 - OsPAO7) OsPAO6 could so far not be characterized due to failure in obtaining the coding cDNA based on accessions in the genomic databases. We report cloning and characterization of the correct OsPAO6 cDNA with a length of 1,742 bp. The 1,491 bp long open reading frame codes for a 497-amino acid protein from nine exons. The protein which has 92% identity to OsPAO7 localizes to plasma membrane.