Insights into the regulation of DMSP synthesis in the diatom Thalassiosira pseudonana through APR activity, proteomics and gene expression analyses on cells acclimating to changes in salinity, light and nitrogen

Despite the importance of dimethylsulphoniopropionate (DMSP) in the global sulphur cycle and climate regulation, the biological pathways underpinning its synthesis in marine phytoplankton remain poorly understood. The intracellular concentration of DMSP increases with increased salinity, increased light intensity and nitrogen starvation in the diatom Thalassiosira pseudonana. We used these conditions to investigate DMSP synthesis at the cellular level via analysis of enzyme activity, gene expression and proteome comparison. The activity of the key sulphur assimilatory enzyme, adenosine 5′- phosphosulphate reductase was not coordinated with increasing intracellular DMSP concentration. Under all three treatments coordination in the expression of sulphur assimilation genes was limited to increases in sulphite reductase transcripts. Similarly, proteomic 2D gel analysis only revealed an increase in phosphoenolpyruvate carboxylase following increases in DMSP concentration. Our findings suggest that increased sulphur assimilation might not be required for increased DMSP synthesis, instead the availability of carbon and nitrogen substrates may be important in the regulation of this pathway. This contrasts with the regulation of sulphur metabolism in higher plants, which generally involves upregulation of several sulphur assimilatory enzymes. In T. pseudonana changes relating to sulphur metabolism were specific to the individual treatments and, given that little coordination was seen in transcript and protein responses across the three growth conditions, different patterns of regulation might be responsible for the increase in DMSP concentration seen under each treatment

Genome-Wide Identification, Classification, and Expression Analysis of Autophagy-Associated Gene Homologues in Rice (Oryza sativa L.)

Autophagy is an intracellular degradation process for recycling macromolecules and organelles. It plays important roles in plant development and in response to nutritional demand, stress, and senescence. Organisms from yeast to plants contain many autophagy-associated genes (ATG). In this study, we found that a total of 33 ATG homologues exist in the rice [Oryza sativa L. (Os)] genome, which were classified into 13 ATG subfamilies. Six of them are alternatively spliced genes. Evolutional analysis showed that expansion of 10 OsATG homologues occurred via segmental duplication events and that the occurrence of these OsATG homologues within each subfamily was asynchronous. The Ka/Ks ratios suggested purifying selection for four duplicated OsATG homologues and positive selection for two. Calculating the dates of the duplication events indicated that all duplication events might have occurred after the origin of the grasses, from 21.43 to 66.77 million years ago. Semi-quantitative RT–PCR analysis and mining the digital expression database of rice showed that all 33 OsATG homologues could be detected in at least one cell type of the various tissues under normal or stress growth conditions, but their expression was tightly regulated. The 10 duplicated genes showed expression divergence. The expression of most OsATG homologues was regulated by at least one treatment, including hormones, abiotic and biotic stresses, and nutrient limitation. The identification of OsATG homologues showing constitutive expression or responses to environmental stimuli provides new insights for in-depth characterization of selected genes of importance in rice

Redox states of glutathione and ascorbate in root tips of poplar (Populus tremula×P. alba) depend on phloem transport from the shoot to the roots

Glutathione (GSH) and ascorbate (ASC) are important antioxidants that are involved in stress defence and cell proliferation of meristematic root cells. In principle, synthesis of ASC and GSH in the roots as well as ASC and GSH transport from the shoot to the roots by phloem mass flow is possible. However, it is not yet known whether the ASC and/or the GSH level in roots depends on the supply from the shoot. This was analysed by feeding mature leaves with [14C]ASC or [35S]GSH and subsequent detection of the radiolabel in different root fractions. Quantitative dependency of root ASC and GSH on shoot-derived ASC and GSH was investigated with poplar (Populus tremula×P. alba) trees interrupted in phloem transport. [35S]GSH is transported from mature leaves to the root tips, but is withdrawn from the phloem along the entire transport path. When phloem transport was interrupted, the GSH content in root tips halved within 3 d. [14C]ASC is also transported from mature leaves to the root tips but, in contrast to GSH, ASC is not removed from the phloem along the transport path. Accordingly, ASC accumulates in root tips. Interruption of phloem transport disturbed the level and the ASC redox state within the entire root system. Diminished total ASC levels were attributed mainly to a decline of dehydroascorbate (DHA). As the redox state of ASC is of particular significance for root growth and development, it is concluded that phloem transport of ASC may constitute a shoot to root signal to coordinate growth and development at the whole plant level

The sulfur pathway and diagnosis of sulfate depletion in grapevine

Sulfur is an essential nutrient to all plant species. Plants assimilate sulfur in a well-described pathway, which has been taken up by roots. Regulatory mech- anism has been the subject of many research papers. However, recent studies highlighted differences between crop plants and the model plant Arabidopsis thaliana. Our work focuses on the identification of genes involved in the sulfur metabolism in the Vitis vinifera genome, and their response to sulfur deficiency and other abiotic stress endured by grapevine in the field, namely water stress. Here, we describe the identification and brief characterization of the first assimilation enzymes involved in the sulfur pathway, the enzyme responsible for sulfur activa- tion, ATP sulfurylase (ATPS), and the two enzymes that reduce sulfate to sulfide, Adenosine 50-phosphosulate reductase (APR) and Sulfite reductase (SiR). A reduc- tion was observed in the number of ATPS and APR isoforms identified in V. vinifera genome when compared to A. thaliana or Glycine max genomes. Two ATPS isoforms were present in the Vitis genome, of which only ATPS1 transcript was detected in the tested tissues, and one APR isoform, suggesting an absence of redundancy in the role of both enzymes. ATPS1, APR and SiR transcript level was up-regulated in response to 2 days exposure to sulfur deficiency in V. vinifera cell cultures, which was completely reversed by the addition of GSH to the culture medium. Apparently, oxidative stress triggered GSH has a pivotal role in the regulation of ATPS1, APR and SiR transcription level, since their up-regulation was observed in mRNA from field grapevine berries under water stress, which is known to induce oxidative stress.info:eu-repo/semantics/publishedVersio

Sulphur flux through the sulphate assimilation pathway is differently controlled by adenosine 5′-phosphosulphate reductase under stress and in transgenic poplar plants overexpressing γ-ECS, SO, or APR

Sulphate assimilation provides reduced sulphur for the synthesis of cysteine, methionine, and numerous other essential metabolites and secondary compounds. The key step in the pathway is the reduction of activated sulphate, adenosine 5′-phosphosulphate (APS), to sulphite catalysed by APS reductase (APR). In the present study, [35S]sulphur flux from external sulphate into glutathione (GSH) and proteins was analysed to check whether APR controls the flux through the sulphate assimilation pathway in poplar roots under some stress conditions and in transgenic poplars. (i) O-Acetylserine (OAS) induced APR activity and the sulphur flux into GSH. (ii) The herbicide Acetochlor induced APR activity and results in a decline of GSH. Thereby the sulphur flux into GSH or protein remained unaffected. (iii) Cd treatment increased APR activity without any changes in sulphur flux but lowered sulphate uptake. Several transgenic poplar plants that were manipulated in sulphur metabolism were also analysed. (i) Transgenic poplar plants that overexpressed the γ-glutamylcysteine synthetase (γ-ECS) gene, the enzyme catalysing the key step in GSH formation, showed an increase in sulphur flux into GSH and sulphate uptake when γ-ECS was targeted to the cytosol, while no changes in sulphur flux were observed when γ-ECS was targeted to plastids. (ii) No effect on sulphur flux was observed when the sulphite oxidase (SO) gene from Arabidopsis thaliana, which catalyses the back reaction of APR, that is the reaction from sulphite to sulphate, was overexpressed. (iii) When Lemna minor APR was overexpressed in poplar, APR activity increased as expected, but no changes in sulphur flux were observed. For all of these experiments the flux control coefficient for APR was calculated. APR as a controlling step in sulphate assimilation seems obvious under OAS treatment, in γ-ECS and SO overexpressing poplars. A possible loss of control under certain conditions, that is Cd treatment, Acetochlor treatment, and in APR overexpressing poplar, is discussed

Sulphur limitation and early sulphur deficiency responses in poplar: significance of gene expression, metabolites, and plant hormones

The influence of sulphur (S) depletion on the expression of genes related to S metabolism, and on metabolite and plant hormone contents was analysed in young and mature leaves, fine roots, xylem sap, and phloem exudates of poplar (Populus tremula×Populus alba) with special focus on early consequences. S depletion was applied by a gradual decrease of sulphate availability. The observed changes were correlated with sulphate contents. Based on the decrease in sulphate contents, two phases of S depletion could be distinguished that were denominated as ‘S limitation’ and ‘early S deficiency’. S limitation was characterized by improved sulphate uptake (enhanced root-specific sulphate transporter PtaSULTR1;2 expression) and reduction capacities (enhanced adenosine 5′-phosphosulphate (APS) reductase expression) and by enhanced remobilization of sulphate from the vacuole (enhanced putative vacuolar sulphate transporter PtaSULTR4;2 expression). During early S deficiency, whole plant distribution of S was impacted, as indicated by increasing expression of the phloem-localized sulphate transporter PtaSULTR1;1 and by decreasing glutathione contents in fine roots, young leaves, mature leaves, and phloem exudates. Furthermore, at ‘early S deficiency’, expression of microRNA395 (miR395), which targets transcripts of PtaATPS3/4 (ATP sulphurylase) for cleavage, increased. Changes in plant hormone contents were observed at ‘early S deficiency’ only. Thus, S depletion affects S and plant hormone metabolism of poplar during ‘S limitation’ and ‘early S deficiency’ in a time series of events. Despite these consequences, the impact of S depletion on growth of poplar plants appears to be less severe than in Brassicaceae such as Arabidopsis thaliana or Brassica sp

Impact of SO2 on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing

Hamisch D, Randewig D, Schliesky S, et al. Impact of SO2 on Arabidopsis thaliana transcriptome in wildtype and sulfite oxidase knockout plants analyzed by RNA deep sequencing. New Phytologist. 2012;196(4):1074-1085.High concentrations of sulfur dioxide (SO2) as an air pollutant, and its derivative sulfite, cause abiotic stress that can lead to cell death. It is currently unknown to what extent plant fumigation triggers specific transcriptional responses. To address this question, and to test the hypothesis that sulfite oxidase (SO) is acting in SO2 detoxification, we compared Arabidopsis wildtype (WT) and SO knockout lines (SO-KO) facing the impact of 600 nl l (1) SO2, using RNAseq to quantify absolute transcript abundances. These transcriptome data were correlated to sulfur metabolism-related enzyme activities and metabolites obtained from identical samples in a previous study. SO-KO plants exhibited remarkable and broad regulative responses at the mRNA level, especially in transcripts related to sulfur metabolism enzymes, but also in those related to stress response and senescence. Focusing on SO regulation, no alterations were detectable in the WT, whereas in SO-KO plants we found up-regulation of two splice variants of the SO gene, although this gene is not functional in this line. Our data provide evidence for the highly specific coregulation between SO and sulfur-related enzymes like APS reductase, and suggest two novel candidates for involvement in SO2 detoxification: an apoplastic peroxidase, and defensins as putative cysteine mass storages

Structure, biogenèse et expression de la protéine T du complexe de la Glycine décarboxylase des plantes supérieures (Pisum sativum)

My present interest concerns the structure, the fonction and the biogenesis of the glycine decarboxylase complex. The glycine decarboxylase complex (GDC) is a multienzymatic system present in the mitochondria of both animal and plant cells, which catalyzes the oxidative decarboxylation of glycine into CO2, NH3, NADH and 5,10 methylenetetrahydroptaroylpoly glutamate (CH2-H4PteGlun) (Kikuchi and Hiraga, 1982; Walker and Oliver, 1986a; Bourguignon et al., 1988). * The aim of my work during my PhD thesis was first to determine the structure and to study biogenesis of the T protein (a tetrahydrofolate enzyme) implicated in the glycine decarboxylase complex. When purifying the T protein from pea (Pisum Sativum), I gained experience in cell biochemistry techniques such as plant cell fractionation, preparation of large amount of pure and intact mitochondria. In addition, I became familiar with the purification of protein using Pharmacia FPLC (Pharmacia) system and the general methods for handling proteins and antibodies. To study the primary structure of the T protein, I isolated a specific clone from a pea leaf cDNA library in *gt 11 using monospecific polyclonal antibodies raised against the T protein. Analysis with HPLC-MS (reverse-phase high-performance liquid chromatography/ mass spectrometry) and microsequenciing of some peptides obtained by chemical cleavage, confirmed the mass predicted from the isolated cDNA and 35 % of the total amino acid sequence. I could show that T protein and its transcript specifically accumulated in the leaf tissue with an additionnal induction by light. I am also studying the biogenesis of the GDC complex during the development of pea plants. For that purpose I measure several parameters such as glycine oxidation, GDC proteins and transcripts levels at different stages of development. * The second aim of my work was to isolate a gene encoding for the T protein. With the T cDNA as a probe, I screened a pea genomic library in EMBL 3. For the first time, the gene coding for the T protein with its promoter region was characterized and sequenced. The transcription start site has been determined by primer extension. Now, I am involved, in the study of the T gene regulation and in a project dealing with the expression of the T protein in Escherichia coli using a T7 RNA polymerase plasmid expression system. Indeed, cristallisation and site-directed mutagenesis will be necessary to understand how the T protein interacts with its cofactor and other proteins of the GDC complex such as the H protein.Nous avons utilisé des anticorps polyclonaux dirigés contre la protéine T du complexe de la glycine décarboxylase (GDC) pour isoler un ADNc de 1430 pb codant pour la protéine T de pois. L'analyse de la séquence nucléotidique a montré que la protéine T était synthétisée sous forme d'un précurseur comportant un peptide de transit mitochondriale de 30 aminoacides précédant la protéine T mature qui comporte 378 aminoacides. La masse déduite de la séquence primaire est de 40 961 Da et correspond exactement à la valeur mesurée au spectromètre de masse, ce qui indique que le cofacteur de la protéine T, le tétrahydrofolate n'est pas fixé de manière covalente sur la protéine. La structure primaire de la protéine T a pu également être confirmée par analyse en HPLC-ESI/MS et par microséquençage des fragments générés par protéolyse chimique de la protéine T purifiée. Des homologies de séquences avec les protéines T de foie de poulet, de boeuf et de pomme de terre, nous ont permis de localiser des régions hautement conservées comportant des résidus chargés positivement qui pourrait être impliquées dans les intéractions avec le tétrahydrofolate. L'étude de l'expression de la protéine T par Northern et Western-blot a montré que la protéine T et les messagers correspondants était principalement présents dans les tissus foliaires et subissaient une forte induction (8 à 10 fois) à la lumière. Disposant de tous les outils moléculaires nécessaires à l'étude de la biogénèse de la GDC, nous avons étudié les modalités de mise en place du complexe au cours du développement de la feuille de pois. Nous avons observé que la capacité d'oxydation des mitochondries de tissus foliaires durant les premiers stades était négligeable pour augmenter de façon dramatique lorsque la feuille était pleinement ouverte (9 jours). En effet, contrairement à la Rubisco qui est présente dans les chloroplastes dès les tout premiers stades du développement, les protéines de la GDC se mettent en place beaucoup plus tardivement au sein de la matrice mitochondriale. Ce chargement en GDC des mitochondries est tellement important qu'il se traduit par une augmentation de leur densité. L'analyse des transcrits des gènes codant pour les protéines de la GDC (P, H et T) et la petite sous unité de la Rubisco révèle qu'ils sont exprimés dès le 4ième jour de croissance, stade où les mitochondries sont incapables d'oxyder la glycine. Cela suggère l'existence d'un contrôle post-transcriptionnel de l'expression des gènes codant pour les protéines P, H et T de la GDC. Nous avons isolé le gène codant pour la protéine T qui est composé de 4 exons et dont la taille approximative est de 3 kpb. Nous avons détecté deux démarrages de transcription dont l'un présente une séquence riche en pyrimidine proche de la séquence Inr (Initiator element). L'analyse de la région promotrice du gène révèle essentiellement trois régions consensus, découvertes chez les gènes nucléaires codant pour la petite sous-unité de la Rubisco et pour la protéine liant les chlorophylles a/b : une région riche en nucléotide AT, un motif GATA en tandem et la boîte II (GGTTAA). Ces séquences semblent être impliquées dans la réponse à la lumière et dans la spécificité tissulaire. L'incubation de la région riche en nucléotides AT et de la boîte II avec un extrait nucléaire provenant de feuilles vertes a permis de caractériser certains facteurs de transcription similaires à ceux présents chez rbcS3,6 et rbcS3A. Dans la perspective de l'étude structurale de la protéine T, nous avons réussi à surexprimer chez E. coli la protéine T, seule ou en tandem avec la protéine H. La présence de nombreux codons rares dans la séquence nucléotidique, nous a amené à construire des vecteurs coexprimant l'ARNt codant pour l'arginine, de façon à permettre la traduction de la protéine dans la bactérie