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Background Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth. Results In this study, we report on transgenic hybrid aspen (Populus tremula × tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula × tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20–44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26–50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control. Conclusions The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.

Transport of monolignols into the apoplastic space is a major event in woody stems. This work identifies candidate transporters in Norway spruce through large-scale LC-MS/MS membrane proteomics and gene co-expression analyses., Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

Cellulose is the main structural component in the plant cell walls. We show that two glycosyltransferase family 31 (GT31) enzymes of Arabidopsis thaliana, here named cellulose synthesis associated glycosyltransferases 1 and 2 (CAGE1 and 2), influence both primary and secondary cell wall cellulose biosynthesis. cage1cage2 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. Single and double cage mutants also show increased sensitivity to the cellulose biosynthesis inhibitor isoxaben. The cage1cage2 phenotypes were associated with an approximately 30% reduction in cellulose content, an approximately 50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels in stems and reduced cellulose biosynthesis rate in seedlings. CESA transcript levels were not significantly altered in cage1cage2 mutants, suggesting that the reduction in CESA levels was caused by a post-transcriptional mechanism. Both CAGE1 and 2 localize to the Golgi apparatus and are predicted to synthesize β-1,3-galactans on arabinogalactan proteins. In line with this, the cage1cage2 mutants exhibit reduced levels of β-Yariv binding to arabinogalactan protein linked β-1,3-galactan. This leads us to hypothesize that defects in arabinogalactan biosynthesis underlie the cellulose deficiency of the mutants.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) share some common molecular deficits including disruption of protein homeostasis leading to disease-specific protein aggregation. While insoluble protein aggregates are the defining pathological confirmation of diagnosis, patient stratification based on early molecular etiologies may identify distinct subgroups within a clinical diagnosis that would respond differently in therapeutic development programs. We are developing targeted multiple reaction monitoring (MRM) mass spectrometry methods to rigorously quantify CSF proteins from known disease genes involved in lysosomal, ubiquitin-proteasomal, and autophagy pathways. Analysis of CSF from 21 PD, 21 ALS, and 25 control patients, rigorously matched for gender, age, and age of sample, revealed significant changes in peptide levels between PD, ALS, and control. In patients with PD, levels of two peptides for chromogranin B (CHGB, secretogranin 1) were significantly reduced. In CSF of patients with ALS, levels of two peptides from ubiquitin carboxy-terminal hydrolase like protein 1 (UCHL1) and one peptide each for glycoprotein non-metastatic melanoma protein B (GPNMB) and cathepsin D (CTSD) were all increased. Analysis of patients with ALS separated into two groups based on length of survival after CSF sampling revealed that the increases in GPNMB and UCHL1 were specific for short-lived ALS patients. While analysis of additional cohorts is required to validate these candidate biomarkers, this study suggests methods for stratification of ALS patients for clinical trials and identifies targets for drug efficacy measurements during therapeutic development. Electronic supplementary material The online version of this article (10.1007/s12031-019-01411-y) contains supplementary material, which is available to authorized users.

Arabinogalactan protein (AGP) glycan biosynthesis in the Golgi apparatus contributes to plant cell wall assembly, but the mechanisms underlying this process are largely unknown. Here, we show that two putative galactosyltransferases -named GALT7 and GALT8 -from the glycosyltransferase family 31 (GT31) of Arabidopsis thaliana participate in cellulose biosynthesis. galt7galt8 mutants show primary cell wall defects manifesting as impaired growth and cell expansion in seedlings and etiolated hypocotyls, along with secondary cell wall defects, apparent as collapsed xylem vessels and reduced xylem wall thickness in the inflorescence stem. These phenotypes were associated with a ∼30% reduction in cellulose content, a ∼50% reduction in secondary cell wall CELLULOSE SYNTHASE (CESA) protein levels and reduced cellulose biosynthesis rate. CESA transcript levels were not significantly altered in galt7galt8 mutants, suggesting that the reduction in CESA levels was caused by a post-transcriptional mechanism. We provide evidence that both GALT7 and GALT8 localise to the Golgi apparatus, while quantitative proteomics experiments revealed reduced levels of the entire FLA subgroup B in the galt7galt8 mutants. This leads us to hypothesize that a defect in FLA subgroup B glycan biosynthesis reduces cellulose biosynthesis rate in galt7galt8 mutants.

The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale., Miguel et al. identify a new enzyme belonging to the GDSL lipase-like family that is involved in the final stage of transformation of 5-CQA into 3,5-diCQA. This enzyme is able to realize an efficient transformation by over 60%, making the transformation process a valuable technological tool that can be easily transferred on an industrial scale.

In plants, different stimuli, both internal and external, activate production of reactive oxygen species (ROS). Photosynthesis is considered as high rate redox-metabolic process with rapid transients including light/photon capture, electron fluxes, and redox potentials that can generate ROS; thus, regulatory systems are required to minimize ROS production. Despite their potential for causing harmful oxidations, it is now accepted that redox homeostasis mechanisms that maintain the intracellular reducing environment make it possible to use ROS as powerful signaling molecules within and between cells. Redox and ROS information from the chloroplasts is a fine-tuning mechanism both inside the chloroplast and as retrograde signal to the cytosol and nucleus to control processes such as gene expression/transcription and translation. Wide repertoires of downstream target genes expression (activation/repression) is regulated by transcription factors. In many cases, transcription factors function through various mechanisms that affect their subcellular localization and or activity. Some post-translational modifications (PTMs) known to regulate the functional state of transcription factors are phosphorylation, acetylation, and SUMOylation, ubiquitylation and disulfide formation. Recently, oxPTMs, targeted in redox proteomics, can provide the bases to study redox regulation of low abundant nuclear proteins. This review summarizes the recent advances on how cellular redox status can regulate transcription factor activity, the implications of this regulation for plant growth and development, and by which plants respond to environmental/abiotic stresses.

Chlamydomonas reinhardtii is a unicellular green alga that can survive at a wide range of inorganic carbon (Ci) concentrations by regulating the activity of a CO2-concentrating mechanism (CCM) as well as other cellular functions. Under CO2 limited conditions, C. reinhardtii cells display a wide range of adaptive responses including changes in photosynthetic electron transport, mitochondria localization in the cells, the structure of the pyrenoid starch sheath, and primary metabolism. In addition to these functional and structural changes, gene and protein expression are also affected. Several physiological aspects of the CO2 response mechanism have been studied in detail. However, the regulatory components (transcription factors and transcriptional regulators) involved in this process are not fully characterized. Here we report a comprehensive analysis of the C. reinhardtii nuclear proteome using liquid chromatography electrospray ionization spectrometry (LC-ESI-MS). The study aims to identify the proteins that govern adaptation to varying CO2 concentrations in Chlamydomonas. The nuclear proteome of C. reinhardtii cells grown in the air at high (5%) and low (0.04%) CO2 concentrations were analyzed. Using this approach, we identified 1378 proteins in total, including 90 putative transcription factors and 27 transcriptional regulators. Characterization of these new regulatory components could shed light on the molecular mechanisms underlying acclimation to CO2 stress.

Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.

Background Secretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32–38 kDa proteins that are involved in membrane trafficking. A systems approach was taken to elucidate function of SCAMPs in wood formation of Populus trees. Phenotypic and multi-omics analyses were performed in woody tissues of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3; Potri.019G104000). Results The woody tissues of the transgenic trees displayed increased amounts of both polysaccharides and lignin oligomers, indicating increased deposition of both the carbohydrate and lignin components of the secondary cell walls. This coincided with a tendency towards increased wood density as well as significantly increased thickness of the suberized cork in the transgenic lines. Multivariate OnPLS (orthogonal projections to latent structures) modeling of five different omics datasets (the transcriptome, proteome, GC-MS metabolome, LC-MS metabolome and pyrolysis-GC/MS metabolome) collected from the secondary xylem tissues of the stem revealed systemic variation in the different variables in the transgenic lines, including changes that correlated with the changes in the secondary cell wall composition. The OnPLS model also identified a rather large number of proteins that were more abundant in the transgenic lines than in the wild type. Several of these were related to secretion and/or endocytosis as well as both primary and secondary cell wall biosynthesis. Conclusions Populus SCAMP proteins were shown to influence accumulation of secondary cell wall components, including polysaccharides and phenolic compounds, in the woody tissues of Populus tree stems. Our multi-omics analyses combined with the OnPLS modelling suggest that this function is mediated by changes in membrane trafficking to fine-tune the abundance of cell wall precursors and/or proteins involved in cell wall biosynthesis and transport. The data provides a multi-level source of information for future studies on the function of the SCAMP proteins in plant stem tissues. Electronic supplementary material The online version of this article (10.1186/s12864-017-4411-1) contains supplementary material, which is available to authorized users.

Metacaspases (MCs) are cysteine proteases that are implicated in programmed cell death of plants. AtMC9 (Arabidopsis thaliana Metacaspase9) is a member of the Arabidopsis MC family that controls the rapid autolysis of the xylem vessel elements, but its downstream targets in xylem remain uncharacterized. PttMC13 and PttMC14 were identified as AtMC9 homologs in hybrid aspen (Populustremulaxtremuloides). A proteomic analysis was conducted in xylem tissues of transgenic hybrid aspen trees which carried either an overexpression or an RNA interference construct for PttMC13 and PttMC14. The proteomic analysis revealed modulation of levels of both previously known targets of metacaspases, such as Tudor staphylococcal nuclease, heat shock proteins and 14-3-3 proteins, as well as novel proteins, such as homologs of the PUTATIVE ASPARTIC PROTEASE3 (PASPA3) and the cysteine protease RD21 by PttMC13 and PttMC14. We identified here the pathways and processes that are modulated by PttMC13 and PttMC14 in xylem tissues. In particular, the results indicate involvement of PttMC13 and/or PttMC14 in downstream proteolytic processes and cell death of xylem elements. This work provides a valuable reference dataset on xylem-specific metacaspase functions for future functional and biochemical analyses. Bio4Energy

In eukaryotic organisms, Elongator is a six‐subunit protein complex required for the formation of 5‐carbamoylmethyl (ncm5) and 5‐methylcarboxymethyl (mcm5) side chains on uridines present at the wobble position (U34) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in‐frame 5′ AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full‐length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC‐MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full‐length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N‐terminal truncation of Elp1p is not likely to regulate Elongator complex activity.

The Mediator is a multi-protein complex that plays a key role in modulating gene expression. Our previous studies suggested that the MED10a, MED28, MED32 complex subunits could be subject to redox regulation. In this study we tested the capacity of different thioredoxins (TRXs) from poplar (TRX-H3 and TRX-H5) and Arabidopsis thaliana (TPR repeat-containing thioredoxin, TDX) as well as glutaredoxins (GRXs) from poplar (GRX-C3 and GRX-C4) to reduce MED28 oligomers in vitro and found that these proteins were less efficient than the the previously tested poplar TRX-H1 and Arabidopsis GRX-C1. Concerning the susceptibility of MED28 to oxidation, both hydrogen peroxide (H2O2) and glutathione disulfide (GSSG) are efficiently mediating the formation of intermolecular disulfides. In fact, MED28 forms homo- oligomers in vivo as assessed by yeast two-hybrid experiments but also in vitro in solution as shown by size-exclusion chromatography, the latter also demonstrated the formation of noncovalent homo-oligomers. These findings suggest that both the redox-dependent and - independent MED28 oligomerization could regulate its biological activities, could it be linked or not to the Mediator. In particular, it would be important to assess MED28 oligomerization state during senescence considering the previously observed phenotype of med28 plants.

Proteome analysis of tissues at various stages of tension wood and normal wood formation revealed signaling and lignocellulosic proteins important for tension wood developmental processes in Populus., Tension wood (TW) is a specialized tissue with contractile properties that is formed by the vascular cambium in response to gravitational stimuli. We quantitatively analysed the proteomes of Populus tremula cambium and its xylem cell derivatives in stems forming normal wood (NW) and TW to reveal the mechanisms underlying TW formation. Phloem-, cambium-, and wood-forming tissues were sampled by tangential cryosectioning and pooled into nine independent samples. The proteomes of TW and NW samples were similar in the phloem and cambium samples, but diverged early during xylogenesis, demonstrating that reprogramming is an integral part of TW formation. For example, 14-3-3, reactive oxygen species, ribosomal and ATPase complex proteins were found to be up-regulated at early stages of xylem differentiation during TW formation. At later stages of xylem differentiation, proteins involved in the biosynthesis of cellulose and enzymes involved in the biosynthesis of rhamnogalacturonan-I, rhamnogalacturonan-II, arabinogalactan-II and fasciclin-like arabinogalactan proteins were up-regulated in TW. Surprisingly, two isoforms of exostosin family proteins with putative xylan xylosyl transferase function and several lignin biosynthesis proteins were also up-regulated, even though xylan and lignin are known to be less abundant in TW than in NW. These data provided new insight into the processes behind TW formation.

Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance.

Plant genes that contain the G-box in their promoters are responsive to a variety of environmental stimuli. Bioinformatics analysis of transcriptome data revealed that the G-box element is significantly enriched in promoters of high light-responsive genes. From nuclear extracts of high light-treated Arabidopsis plants, we identified the AtbZIP16 transcription factor as a component binding to the G-box-containing promoter fragment of light-harvesting chlorophyll a/b-binding protein2.4 (LHCB2.4). AtbZIP16 belongs to the G-group of Arabidopsis basic region leucine zipper (bZIP) type transcription factors. Although AtbZIP16 and its close homologues AtbZIP68 and AtGBF1 bind the G-box, they do not bind the mutated half-sites of the G-box palindrome. In addition, AtbZIP16 interacts with AtbZIP68 and AtGBF1 in the yeast two-hybrid system. A conserved Cys residue was shown to be necessary for redox regulation and enhancement of DNA binding activity in all three proteins. Furthermore, transgenic Arabidopsis lines overexpressing the wild type version of bZIP16 and T-DNA insertion mutants for bZIP68 and GBF1 demonstrated impaired regulation of LHCB2.4 expression. Finally, overexpression lines for the mutated Cys variant of bZIP16 provided support for the biological significance of Cys(330) in redox regulation of gene expression. Thus, our results suggest that environmentally induced changes in the redox state regulate the activity of members of the G-group of bZIP transcription factors.

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-amino-acids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

Mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS) through a cytotoxic mechanism of unknown nature. A hallmark in ALS patients and transgenic mouse models carrying human SOD1 (hSOD1) mutations are hSOD1-immunoreactive inclusions in spinal cord ventral horns. The hSOD1 inclusions may block essential cellular functions or cause toxicity through sequestering of other proteins. Inclusions from four different transgenic mouse models were examined after density gradient ultracentrifugation. The inclusions are complex structures with heterogeneous densities and are disrupted by detergents. The aggregated hSOD1 was mainly composed of subunits that lacked the native stabilizing intra-subunit disulfide bond. A proportion of subunits formed hSOD1 oligomers or was bound to other proteins through disulfide bonds. Dense inclusions could be isolated and the protein composition was analyzed using proteomic techniques. Mutant hSOD1 accounted for half of the protein. Ten other proteins were identified. Two were cytoplasmic chaperones, four were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum (ER). The presence of ER proteins in inclusions containing the primarily cytosolic hSOD1 further supports the notion that ER stress is involved in ALS.

International audience; The recent genome sequencing of Populus trichocarpa and Vitis vinifera, two models of woody plants, of Sorghum bicolor, a model of monocot using C4 metabolism, and of the moss Physcomitrella patens, together with the availability of photosynthetic organism genomes allows performance of a comparative genomic study with organisms having different ways of life, reproduction modes, biological traits, and physiologies. Thioredoxins (Trxs) are small ubiquitous proteins involved in the reduction of disulfide bridges in a variety of target enzymes present in all sub-cellular compartments and involved in many biochemical reactions. The genes coding for these enzymes have been identified in these newly sequenced genomes and annotated. The gene content, organization and distribution were compared to other photosynthetic organisms, leading to a refined classification. This analysis revealed that higher plants and bryophytes have a more complex family compared to algae and cyanobacteria and to non-photosynthetic organisms, since poplar exhibits 49 genes coding for typical and atypical thioredoxins and thioredoxin reductases, namely one-third more than monocots such as Oryza sativa and S. bicolor. The higher number of Trxs in poplar is partially explained by gene duplication in the Trx m, h, and nucleoredoxin classes. Particular attention was paid to poplar genes with emphasis on Trx-like classes called Clot, thioredoxin-like, thioredoxins of the lilium type and nucleoredoxins, which were not described in depth in previous genomic studies.

Recent evidence has shown that alternative splicing (AS) is widely involved in the regulation of gene expression, substantially extending the diversity of numerous proteins. In this study, a subset of expressed sequence tags representing members of the reactive oxygen species gene network was selected from the PopulusDB database to investigate AS mechanisms in Populus. Examples of all known types of AS were detected, but intron retention was the most common. Interestingly, the closest Arabidopsis (Arabidopsis thaliana) homologs of half of the AS genes identified in Populus are not reportedly alternatively spliced. Two genes encoding the protein of most interest in our study (high-isoelectric-point superoxide dismutase [hipI-SOD]) have been found in black cottonwood (Populus trichocarpa), designated PthipI-SODC1 and PthipI-SODC2. Analysis of the expressed sequence tag libraries has indicated the presence of two transcripts of PthipI-SODC1 (hipI-SODC1b and hipI-SODC1s). Alignment of these sequences with the PthipI-SODC1 gene showed that hipI-SODC1b was 69 bp longer than hipI-SODC1s due to an AS event involving the use of an alternative donor splice site in the sixth intron. Transcript analysis showed that the splice variant hipI-SODC1b was differentially expressed, being clearly expressed in cambial and xylem, but not phloem, regions. In addition, immunolocalization and mass spectrometric data confirmed the presence of hipI-SOD proteins in vascular tissue. The functionalities of the spliced gene products were assessed by expressing recombinant hipI-SOD proteins and in vitro SOD activity assays.

Mediator, a central coregulator of transcription, has been identified as a large protein complex in eukaryotes ranging from yeast to man. It is therefore remarkable that Mediator has not yet been identified within the plant kingdom. Here we identify Mediator in a plant, Arabidopsis thaliana. The plant Mediator subunits typically show very low homology to other species, but our biochemical purification identifies 21 conserved and six A. thaliana-specific Mediator subunits. Most notably, we identify the A. thaliana proteins STRUWWELPETER (SWP) and PHYTOCHROME AND FLOWERING TIME 1 (PFT1) as the Med14 and Med25 subunits, respectively. These findings show that specific plant Mediator subunits are linked to the regulation of specialized processes such as the control of cell proliferation and the regulation of flowering time in response to light quality. The identification of the plant Mediator will provide new tools and insights into the regulation of transcription in plants.

The effects of ultraviolet (UV) radiation on the photosynthetic and UV-screening pigments in needles of Scots pine (Pinus sylvestris L.) saplings were studied in a UV-exclusion field chamber experiment in northern Finland (67°N) during 2001–2002. The chambers held filters that excluded both UVB and UVA, only UVB, transmitted all UV, or lacked filters. Analyses of control needles (no filter and polyethene filter) showed that the first changes to occur in spring (end of April) was an abrupt increase in the epoxidation state (EPS) of the xanthophyll cycle pigments, likely in relation with the beginning of the photosynthetic activity. The concentration of chlorophyll, lutein, neoxanthin, α-carotene, β-carotene, and the size of the xanthophyll cycle pool (violaxanthin+antheraxanthin+zeaxanthin=VAZ) changed only later when needles reached their summer photosynthesis state. Exclusion of UV radiation significantly affected the xanthophyll cycle but not the other photosynthetic pigments analysed. Interestingly, the effects on xanthophylls were dependent on the sampling date. Under UVA/B-exclusion, the EPS was increased and VAZ pool size was unchanged in April, whereas EPS remained unchanged and the VAZ pool size was reduced in May and June. The existence of two sustained and active antenna modes during winter and summer could be an explanation for the specific UV-exclusion effect in the different season. A high-performance liquid chromatography analysis of soluble phenolics showed that the exclusion of UVA/B radiation caused a significant effect on five compounds out of 46 studied, without affecting the concentration of the total soluble phenolics. Under UVA/B-exclusion, the concentration of three of them (secoisolariciresinol-glucopyranoside, two unknown) was reduced while the concentration of dicoumaroyl-astragalin and pinosylvin monomethylether was increased compared with both controls separately. In general, the exclusion of UVA/B caused a stronger effect than the exclusion of UVB on both photosynthetic and UV screening pigments. The effects of UV radiation on xanthophyll cycle pigments were season-specific and detectable only under stressful spring conditions (freezing temperatures and high irradiance due to snow reflection). The effect on the xanthophyll cycle could be a direct consequence of UV treatments, or an indirect consequence of the changed flavonoid composition, or a combination of both.

Background Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. Results We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20–160 μm thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease in early xylem zones. We observed upregulation of two forms of xylem cysteine protease (Potri.002G005700.1 and Potri.005G256000.2; Pt-XCP2.1) in early xylem and their downregulation in late maturing xylem. Our data also show that Pt-KOR1.3 (Potri.003G151700.2) exhibits an expression pattern that supports the hypothesis put forward in previous studies that this is a key xyloglucanase involved in cellulose biosynthesis in primary cell walls and reduction of cellulose crystallinity in secondary walls. Conclusion Our novel multivariate approach highlights important processes and provides confirmatory insights into the molecular foundations of wood development. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2458-z) contains supplementary material, which is available to authorized users.

Mediator is a conserved multi-protein complex that acts as a bridge between promoter-bound transcriptional regulators and RNA polymerase II. While redox signaling is important in adjusting plant metabolism and development, the involvement of Mediator in redox homeostasis and regulation only recently started to emerge. Our previous results show that the MED10a, MED28, and MED32 Mediator subunits form various types of covalent oligomers linked by intermolecular disulfide bonds in vitro. To link that with biological significance we have characterized Arabidopsis med32 and med28 mutants and found that they are affected in root development and senescence, phenotypes possibly associated to redox changes.

The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combination with specific TFs as GeBPL.

Transgenic hybrid aspen (Populus tremula L. x P. tremuloides Michx.) plants expressing a high-isoelectric-point superoxide dismutase (hipI-SOD) gene in antisense orientation were generated to investigate its function. Immunolocalization studies showed the enzyme to be localized extracellularly, in the secondary cell wall of xylem vessels and phloem fibers. The antisense lines of hipI-SOD exhibited a distinct phenotype; growth rate was reduced, stems were thinner and leaves smaller than in wild-type (WT) plants. The abundance of hipI-SOD was reduced in the bark and xylem of plants from these antisense lines. The vascular tissue of transgenic lines became lignified earlier than in WT plants and also showed an increased accumulation of reactive oxygen species (ROS). Xylem fibers and vessels were shorter and thinner in the transgenic lines than in WT plants. The total phenolic content was enhanced in the antisense lines. Furthermore, microarray analysis indicated that several enzymes involved in cell signaling, lignin biosynthesis and stress responses were upregulated in apical vascular tissues of transgenic plants. The upregulation of selected genes involved in lignin biosynthesis was also verified by real-time PCR. The results suggest that, in the transgenic plants, a premature transition into maturation occurs and the process is discussed in terms of the effects of increased accumulation of ROS due to reduced expression of hipI-SOD during development and differentiation.

Populus euphratica is a light-demanding species ecologically characterized as a pioneer. It grows in shelter belts along riversides, being part of the natural desert forest ecosystems in China and Middle Eastern countries. It is able to survive extreme temperatures, drought and salt stress, marking itself out as an important plant species to study the mechanisms responsible for survival of woody plants under heat stress.Heat effects were evaluated through electrolyte leakage on leaf discs, and LT(50) was determined to occur above 50 degrees C. Protein accumulation profiles of leaves from young plants submitted to 42/37 degrees C for 3 d in a phytotron were determined through 2D-PAGE, and a total of 45 % of up- and downregulated proteins were detected. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)/TOF analysis, combined with searches in different databases, enabled the identification of 82 % of the selected spots.Short-term upregulated proteins are related to membrane destabilization and cytoskeleton restructuring, sulfur assimilation, thiamine and hydrophobic amino acid biosynthesis, and protein stability. Long-term upregulated proteins are involved in redox homeostasis and photosynthesis. Late downregulated proteins are involved mainly in carbon metabolism.Moderate heat response involves proteins related to lipid biogenesis, cytoskeleton structure, sulfate assimilation, thiamine and hydrophobic amino acid biosynthesis, and nuclear transport. Photostasis is achieved through carbon metabolism adjustment, a decrease of photosystem II (PSII) abundance and an increase of PSI contribution to photosynthetic linear electron flow. Thioredoxin h may have a special role in this process in P. euphratica upon moderate heat exposure.

Hypersensitivity responses (HR) play a major role in plant resistance to pathogens. It is often claimed that HR is also important in plant resistance to insects, although there is little unambiguous documentation. Large genotypic variation in resistance against the gall midge Dasineura marginemtorquens is found in Salix viminalis. Variation in larval performance and induced responses within a full-sib S. viminalis family is reported here; 36 sibling plants were completely resistant (larvae died within 48 h after egg hatch, no gall induction), 11 plants were totally susceptible, 25 plants were variable (living and dead larvae present on the same plant). Resistance was associated with HR, but to different degrees; 21 totally resistant genotypes showed typical HR symptoms (many distinct necrotic spots) whereas the remaining 15 genotypes showed no, or very few, such symptoms. Hydrogen peroxide, used as a marker for HR, was induced in genotypes expressing HR symptoms but not in resistant genotypes without symptoms, or in susceptible genotypes. These data suggest that production of hydrogen peroxide, and accompanying cell death, cannot explain larval mortality in the symptomless reaction. Another, as yet unknown, mechanism of resistance may be present. If so, then it is possible that this unknown mechanism also contributes to resistance in plants displaying HR. The apparent complexity observed in this interaction, with both visible and invisible plant responses associated with resistance against an adapted insect species, may have implications for the study of resistance factors in other plant-insect interactions.

A measurement campaign to assess the feasibility of remote sensing of sunlight-induced chlorophyll fluorescence (ChlF) from a coniferous canopy was conducted in a boreal forest study site (Finland). A Passive Multi-wavelength Fluorescence Detector (PMFD) sensor, developed in the LURE laboratory, was used to obtain simultaneous measurements of ChlF in the oxygen absorption bands, at 687 and 760 nm, and a reflectance index, the PRI (Physiological Reflectance Index), for a month during spring recovery. When these data were compared with active fluorescence measurements performed on needles they revealed the same trend. During sunny days fluorescence and reflectance signals were found to be strongly influenced by shadows associated with the canopy structure. Moreover, chlorophyll fluorescence variations induced by rapid light changes (due to transient cloud shadows) were found to respond more quickly and with larger amplitude under summer conditions compared to those obtained under cold acclimation conditions. In addition, ChlF at 760 nm was observed to increase with the chlorophyll content. During this campaign, the CO2 assimilation was measured at the forest canopy level and was found remarkably well correlated with the PRI index.

Several CuZn-superoxide dismutases (SODs; EC 1.15.1.1) were cloned from hybrid aspen (Populus tremula L. x tremuloides Michx.). Two of the cloned genes encode representatives of a novel type of CuZn-SOD and we named it HipI-SOD because of its high isoelectric point (or =9). The SODs were cloned by screening a cDNA library with a probe based on a Scots pine (Pinus sylvestris L.) CuZn-SOD that is predominantly located extracellularly. The expression pattern of HipI-SOD was examined using a Northern blot technique and compared with the expression patterns of cytosolic and chloroplastic SODs. Distinct expression patterns were observed for the three types of CuZn-SOD, with HipI-SODs showing strong expression in apical tissues. Southern blots as well as protein analysis suggest that these novel HipI-SODs belong to a small gene family, one member of which might be monomeric.

Land plants are sessile and have developed sophisticated mechanisms that allow for both immediate and acclimatory responses to changing environments. Partial exposure of low light–adapted Arabidopsis plants to excess light results in a systemic acclimation to excess excitation energy and consequent photooxidative stress in unexposed leaves. Thus, plants possess a mechanism to communicate excess excitation energy systemically, allowing them to mount a defense against further episodes of such stress. Systemic redox changes in the proximity of photosystem II, hydrogen peroxide, and the induction of antioxidant defenses are key determinants of this mechanism of systemic acquired acclimation.

Freezing injury of plants may be caused by the deleterious reactions of active oxygen species, and free-radical scavenging systems may be important in the alleviation of freezing stress. To test the feasibility of this hypothesis, enzymes and metabolites that cooperatively scavenge O-2(-) and H2O2 were analyzed in Scots pine (Pinus sylvestris L.) seedlings during a stepwise cold acclimation procedure. Elevated levels of enzymatic scavengers such as ascorbate peroxidase, glutathione reductase, monodehydroascorbate reductase, and dehy droascorbate reductase were found, along with increased freezing tolerance during cold acclimation, supporting the hypothesis. Induction of the scavenging systems during acclimation is discussed in relation to freezing tolerance. (C) 1998 Academic Press.

which is involved in lignification of cell walls (Dean and Eriksson, 1992), defence reactions against microbial infecExtracellular superoxide dismutase was found in extra- tions (Levine et al., 1994) and cross-linking of structural xylematic bark tissues and seedlings of Pinus syl- proteins of the cell wall (Bradley et al., 1992). Its generavestris L. The isozymes were partially purified by tion in cell walls of diVerentiating xylem and in cell walls infiltration and analysed by polyacrylamide gel electro- of phloem fibres has been demonstrated with histochemphoresis. One of the major isoforms was purified to ical assays (Cassab, 1993; Czaninski et al., 1993). homogeneity and the N-terminal amino acid sequence Superoxide dismutase (SOD; EC1.15.1.1), which catawas determined. The isoelectric point of these extra- lyses the dismutation of superoxide anion radicals (O’ 2 )

Here we report on the quantitative analysis of tert.-butyldimethylsilyl derivatives, of ascorbate and dehydroascorbate in plant extracts by high-resolution selected ion monitoring gas chromatography-mass spectrometry. The analysis involved an isotope dilution assay using [13C1]ascorbate and [13C1]dehydroascorbate. The limit of detection was approximately 0.5 pg for ascorbate and 5 pg for dehydroascorbate. The reproductibility of the whole quantification procedure, from extraction of Pinus sylvestris needles with 5% trichloroacetic acid to the final analysis, was very high: giving a relative standard deviation (R.S.D.) of 4% and 10% for ascorbate and dehydroascorbate, respectively. The reproducibility of the analysis of the redox ratio of ascorbate/(ascorbate + dehydroascorbate) was even higher (R.S.D. = 1%). The high-resolution selected ion monitoring gas chromatography-mass spectrometry methodology developed here with very high sensitivity and specificity for ascorbate and dehydroascorbate opens new perspectives for investigations on distribution patterns of these substances in plants.

Background Reactive oxygen species (ROS) are involved in the regulation of diverse physiological processes in plants, including various biotic and abiotic stress responses. Thus, oxidative stress tolerance mechanisms in plants are complex, and diverse responses at multiple levels need to be characterized in order to understand them. Here we present system responses to oxidative stress in Populus by integrating data from analyses of the cambial region of wild-type controls and plants expressing high-isoelectric-point superoxide dismutase (hipI-SOD) transcripts in antisense orientation showing a higher production of superoxide. The cambium, a thin cell layer, generates cells that differentiate to form either phloem or xylem and is hypothesized to be a major reason for phenotypic perturbations in the transgenic plants. Data from multiple platforms including transcriptomics (microarray analysis), proteomics (UPLC/QTOF-MS), and metabolomics (GC-TOF/MS, UPLC/MS, and UHPLC-LTQ/MS) were integrated using the most recent development of orthogonal projections to latent structures called OnPLS. OnPLS is a symmetrical multi-block method that does not depend on the order of analysis when more than two blocks are analysed. Significantly affected genes, proteins and metabolites were then visualized in painted pathway diagrams. Results The main categories that appear to be significantly influenced in the transgenic plants were pathways related to redox regulation, carbon metabolism and protein degradation, e.g. the glycolysis and pentose phosphate pathways (PPP). The results provide system-level information on ROS metabolism and responses to oxidative stress, and indicate that some initial responses to oxidative stress may share common pathways. Conclusion The proposed data evaluation strategy shows an efficient way of compiling complex, multi-platform datasets to obtain significant biological information.

Exposure of plants to light intensities that exceed the electron utilization capacity of the chloroplast has a dramatic impact on nuclear gene expression. The photoreceptor Cryptochrome 1 (cry1) is essential to the induction of genes encoding photoprotective components in Arabidopsis thaliana. Bioinformatic analysis of the cry1 regulon revealed the putative cis-element CryR1 (GnTCKAG), and here we demonstrate an interaction between CryR1 and the zinc finger GATA-type transcription factors ZINC FINGER PROTEIN EXPRESSED IN INFLORESCENCE MERISTEM LIKE1 (ZML1) and ZML2. The ZML proteins specifically bind to the CryR1 cis-element as demonstrated in vitro and in vivo, and TCTAG was shown to constitute the core sequence required for ZML2 binding. In addition, ZML2 activated transcription of the yellow fluorescent protein reporter gene driven by the CryR1 cis-element in Arabidopsis leaf protoplasts. T-DNA insertion lines for ZML2 and its homolog ZML1 demonstrated misregulation of several cry1-dependent genes in response to excess light. Furthermore, the zml1 and zml2 T-DNA insertion lines displayed a high irradiance-sensitive phenotype with significant photoinactivation of photosystem II (PSII), indicated by reduced maximum quantum efficiency of PSII, and severe photobleaching. Thus, we identified the ZML2 and ZML1 GATA transcription factors as two essential components of the cry1-mediated photoprotective response.

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M−1·s−1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44–Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.

This paper presents data on the purification and N -terminal sequence of S -adenosyl- l -methionine: theobromine 1- N -methyltransferase (STM

A differential analysis of CuZn-superoxide dismutase (SOD. EC 1.15.1.1) isozymes after native-polyacry lamide gel elecrrophoresis (PAGE) and isoelectric focusing (IEF) indicated that germinating seeds of Scots pine (Pinus sylvestris L.) 3 days after the start of imbibition (3 DAI) contain five CuZn-SOD isozymes. Two isozymes co-migrated on native–PAGE but were separated after IEF. CuZn-SODs of Scots pine were purified from germinating seeds (3 DAI) by anion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The final separation of CuZn-SOD isozymes was accomplished by native-PAGE. CuZn-SOD isozymes were electroblotted and their NH2-terminal amino acid sequence was determined. Comparisons of the amino acid sequences with sequences of CuZn-SOD isozymes from other plant sources indicated that one CuZn-SOD isozyme was of the chloroplastic type whereas the other four isozymes belonged to the cytosolic-type CuZn-SODs, The NH2-terminal amino acid sequence of the chloroplastic CuZn-SOD and of one cytosolic-type CuZn-SOD were identical to those of two previously isolated, sequenced and localized CuZn-SOD isozymes from Scots pine needles. Two cytosolic-type CuZn-SOD isozymes showed a homology at 20 out of 21 NH2-terminal amino acids. Mitochondria and glyoxysomes were isolated by differential and Percoll density-gradient centrifugation from germinating seeds (3 DAI). The cell fractionation experiments did not suggest that a major part of the CuZn-SOD activity in germinating seeds was derived from glyoxysomes or mitochondria.

Inhibition of photosynthesis plays an important role during winter-spring stress in evergreen plants such as Scots pine (Pinus sylvestris L.). In this experiment, naturally established three-year-old Scots pine seedlings were studied during recovery from winter stress, under natural conditions from May to the beginning of June. A comparison was made between top shoot needles exposed to sun and, prior to winter stress recovery snow-covered, and thus more shaded side shoot needles. Chlorophyll fluorescence, photochemical and non-photochemical quenching, pigment composition together with the Lhca2*l, Lhcbl*2, RbcS, rbcL, psbA, nad3 and cox2 gene expression were analysed. During the recovery, a general increase in the Fo/Fm ratio of chlorophyll fluorescence and in pigments was observed for both types of shoots. At the beginning of May, the Fo/Fm ratio of chlorophyll fluorescence showed that exposed top shoot needles were more photoinhibited than the snow-covered side shoot needles. The levels of chlorophyll a and b, neoxanthin, α- and β-carotene were lower, while the level of zeaxanthin was higher in top shoot needles than in side shoot needles during the whole period. On May 6, significantly higher (two-fold) mRNA levels for Lhca2*l and Lhcbl*2 genes were detected in top shoot needles than in side shoot needles. The reverse situation for these genes was observed on May 19 with a two-fold higher level of mRNA in side shoot needles. The transcript level was significantly higher for the Lhcbl*2 gene than for the Lhca2*l gene in both types of shoots only at the beginning of May. The mRNA level for the RbcS gene on May 4 and 6 was two- and four-fold higher, respectively, in top than in side shoot needles. The transcript levels for the plastid-encoded rbcL and psbA and for the mitochondrion-encoded nad3 and cox2 genes were constant and similar in top and side shoot needles. The results are discussed in relation to recovery from winter-spring stress and observed differential expression of nuclear- and organellar-encoded genes.

Cisplatin (cisPt), Pt(NH 3 ) 2 Cl 2 , is a cancer drug believed to kill cells via DNA binding and damage. Recent work has implied that the cellular copper (Cu) transport machinery may be involved in cisPt cell export and drug resistance. Normally, the Cu chaperone Atox1 binds Cu(I) via two cysteines and delivers the metal to metal-binding domains of ATP7B; the ATP7B domains then transfer the metal to the Golgi lumen for loading on cuproenzymes. Here, we use spectroscopic methods to test if cisPt interacts with purified Atox1 in solution in vitro. We find that cisPt binds to Atox1’s metal-binding site regardless of the presence of Cu or not: When Cu is bound to Atox1, the near-UV circular dichroism signals indicate Cu-Pt interactions. From NMR data, it is evident that cisPt binds to the folded protein. CisPt-bound Atox1 is however not stable over time and the protein begins to unfold and aggregate. The reaction rates are limited by slow cisPt dechlorination. CisPt-induced unfolding of Atox1 is specific because this effect was not observed for two unrelated proteins that also bind cisPt. Our study demonstrates that Atox1 is a candidate for cisPt drug resistance: By binding to Atox1 in the cytoplasm, cisPt transport to DNA may be blocked. In agreement with this model, cell line studies demonstrate a correlation between Atox1 expression levels, and cisplatin resistance.

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales.

The influence of photooxidative stress on genes expressing superoxide dismutase (Sod) and glutathione reductase (Gor) was analyzed in needles of top and side shoots of 3-year-old Pinus sylvestris (L.) seedlings. The study was carried out in the field during spring recovery. From mid-April the top shoots of seedlings protruded above the snow and thus were exposed to sunlight, whereas the side shoots were covered with snow until May 4. Needles were sampled from top and side shoots on five different occasions. At the beginning of May the mRNA levels for cytosolic CuZn-Sod were significantly higher in top-shoot needles than in side-shoot needles. Similar results were obtained for chloroplastic CuZn-Sod mRNA. After May 6 we could not detect any significant differences between top- and side-shoot needles for either CuZn-Sod mRNA level. Transcript accumulation for the chloroplastic CuZn-Sod was up to 4-fold higher than for cytosolic CuZn-Sod in both types of shoots. On June 1 minimum transcript levels were observed for both CuZn-SOD isoforms. Protein activity analysis for CuZn-SOD isozymes did not reveal any significant differences between top- and side-shoot needles during the whole period of measurements. The mRNA level for chloroplastic Gor was similar in both types of shoots. However, the total GR activity was significantly higher in top-shoot needles than in side-shoot needles at the beginning of May. The analysis of mRNA accumulation for chloroplastic CuZn-Sod and Gor indicates that transcript levels were at least 5- to 20-fold higher for CuZn-Sod than for chloroplastic Gor. The differential expressions of Sod and Gor genes are discussed in relation to regulation of the enzymic scavenging system during photooxidative stress conditions.

A 50-year-old Pinus sylvestris L. stand was exposed for 2 years to low concentrations of SO 2 and NO 2 in an open-air exposure experiment in northern Sweden. The mean SO 2 concentrations in the centre of the exposed plot during the 1988 exposure from 14 June to 25 September, and during the 1989 exposure from 6 June to 30 September were 15 nl l -1 and 12 nl l -1 , respectively. The corresponding values for NO 2 were 15 nl l -1 and 10 nl l -1 , respectively