Brassinosteroids fine-tune secondary and primary sulfur metabolism through BZR1-mediated transcriptional regulation

For adaptation to ever-changing environments,plants have evolved elaborate metabolic systems coupled to a regulatory network for optimal growth and defense. Regulation of plant secondary metabolic pathways such as glucosinolates(GSLs) by defense phytohormones in response to different stresses and nutrient deficiency has been intensively investigated, while how growth-promoting hormone balances plant secondary and primary metabolism has been largely unexplored. Here, we found that growth-promotin...     

Plant sulfur nutrition: From Sachs to Big Data

Together with water and carbon dioxide plants require 14 essential mineral nutrients to finish their life cycle. The research in plant nutrition can be traced back to Julius Sachs, who was the first to experimentally prove the essentiality of mineral nutrients for plants. Among those elements Sachs showed to be essential is sulfur. Plant sulfur nutrition has been not as extensively studied as the nutrition of nitrogen and phosphate, probably because sulfur was not limiting for agriculture. However, with the reduction of atmospheric sulfur dioxide emissions sulfur deficiency has become common. The research in sulfur nutrition has changed over the years from using yeast and algae as experimental material to adopting Arabidopsis as the plant model as well as from simple biochemical measurements of individual parameters to system biology. Here the evolution of sulfur research from the times of Sachs to the current Big Data is outlined.     

Herbicide safeners and glutathione metabolism

Herbicide safeners are chemicals which protect crop plants from injury by certain herbicides, without affecting weed control efficacy of the herbicides. The protective mechanism of herbicide safeners has not yet been fully elucidated, but there is increasing evidence that safeners act by selectively enhancing herbicide detoxification in crop plants. To date, two main detoxification pathways have been related to the mode of action of herbicide safeners. The first includes oxidation and subsequent glucose conjugation, mediated by cytochrome P450 -dependent monooxygenases and UDP-glucosyltransferases, respectively. This pathway appears to be important predominantly in safener protection to aryloxyphenoxypropionate and sulfonylurea herbicides. The second pathway represents the conjugation of thiocarbamate sulfoxides and chloroacetanilide herbicides with glutathione. This mechanism is accomplished by either elevating the levels of reduced glutathione or the activity of glutathione S-transferase, or both. Since glutathione has been reported to be involved in several stress situations of plants its function associated with safener-induced herbicide tolerance will be discussed in more detail in this review.     

Relation between primary and secondary metabolism in plant cell suspensions

Cell suspensions ofMorinda citrifolia are able to produce large amounts of anthraquinones (AQ) when they are cultivated on a B5-medium containing 1 mg 1^-1 naphtyl acetic acid (NAA); this production is inhibited by addition of 2,4-dichloro-phenoxyacetic acid (2,4-d). Also during cultivation on 1 mg 1^-1 2,4-d AQ-production is absent.It appeared that in the presence of NAA a kind of AQ-production program is switched on: cell division rate is low as well as metabolic activity, while endogenous sugar levels are high. The same properties develop in the presence of auxins like indolyl-acetic acid and p-chloro-phenylacetic acid. With 2,4-d and related auxins (like p-chloro-phenoxyacetic acid) AQ production is absent and emphasis is laid on a developmental program characterized by high cell division rates, high metabolic activity and low endogenous sugar contents. Independent of the type of auxin applied, the cells grow as a suspension consisting of finely dispersed cells. The AQ-producing differentiation program cannot be maintained during a consecutive series of subculturings: with increasing AQ-contents the viability of the cells and the cell division rate decrease.The possible mechanisms of regulation of AQ-production by auxins are discussed as well as the advantages of the use of theMorinda model system in the study of the relation between growth, primary and secondary metabolism.Abbreviations AQ anthraquinones - 2,4-d 2,4-dichloro-phenoxylacetic acid - DW dry weight - EFW extractive free weight - FW fresh weight - IAA indolyl acetic acid - NAA naphtyl acetic acid - pCP p-chloro-phenylacetic acid - pCPO p-chloro-phenoxy-acetic acid     

Flux control of sulphate assimilation in Arabidopsis thaliana: adenosine 5'-phosphosulphate reductase is more susceptible than ATP sulphurylase to negative control by thiols

The effect of externally applied L-cysteine and glutathione (GSH) on ATP sulphurylase and adenosine 5'-phosphosulphate reductase (APR), two key enzymes of assimilatory sulphate reduction, was examined in Arabidopsis thaliana root cultures. Addition of increasing L-cysteine to the nutrient solution increased internal cysteine, gamma-glutamylcysteine and GSH concentrations, and decreased APR mRNA, protein and extractable activity. An effect on APR could already be detected at 0.2 mm L-cysteine, whereas ATP sulphurylase was significantly affected only at 2 mm L-cysteine. APR mRNA, protein and activity were also decreased by GSH at 0.2 mm and higher concentrations. In the presence of L-buthionine-S, R-sulphoximine (BSO), an inhibitor of GSH synthesis, 0.2 mm L-cysteine had no effect on APR activity, indicating that GSH formed from cysteine was the regulating substance. Simultaneous addition of BSO and 0.5 mm GSH to the culture medium decreased APR mRNA, enzyme protein and activity. ATP sulphurylase activity was not affected by this treatment. Tracer experiments using (35)SO(4)(2-) in the presence of 0.5 mm L-cysteine or GSH showed that both thiols decreased sulphate uptake, APR activity and the flux of label into cysteine, GSH and protein, but had no effect on the activity of all other enzymes of assimilatory sulphate reduction and serine acetyltransferase. These results are consistent with the hypothesis that thiols regulate the flux through sulphate assimilation at the uptake and the APR step. Analysis of radioactive labelling indicates that the flux control coefficient of APR is more than 0.5 for the intracellular pathway of sulphate assimilation. This analysis also shows that the uptake of external sulphate is inhibited by GSH to a greater extent than the flux through the pathway, and that the flux control coefficient of APR for the pathway, including the transport step, is proportionately less, with a significant share of the control exerted by the transport step.     

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5′ phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using ³⁴SO₄^2– also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non‐stress and oxidative stress conditions. However, when germinating on sulfur‐deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.     

Interaction of sulfur and nitrogen nutrition in tobacco (Nicotiana tabacum) plants: significance of nitrogen source and root nitrate reductase

Abstract: The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (Nicotiana tabacum) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.     

Whole Plant Regulation of Sulfur Nutrition of Deciduous Trees-Influences of the Environment

Abstract: The current view of sulfur nutrition is based on the source‐to‐sink relationship of carbohydrates. SO₄^2‐ reduction is thought to occur mainly in leaves. Surplus reduced sulfur must be transported out of the leaves, loaded into the phloem and transported to other tissues, in particular tissues assumed to be sink organs. However, it has not been proved that tissues which are sinks for carbohydrates are also sink organs for reduced sulfur. It is evident that sinks must communicate with sources, and vice versa, to signal demand and to transport the surplus of reduced sulfur that is produced. The demand‐driven control model of sulfur nutrition proposes that the tripeptide glutathione is the signal which regulates S nutrition of the whole plant at the level of SO₄^2‐ uptake. Acclimatization to environmental changes has been shown to result in several changes in S nutrition of deciduous trees: (i) Drought stress diminished SO₄^2‐ transport into the xylem, although the GSH content in lateral roots remained unaffected, possibly due to an overall reduction in water status. (ii) Flooding decreased APS reductase activity in the anoxic roots. This may be due to enhanced GSH transport to the roots, but it is more likely to be the result of a change in metabolism leading to diminished energy gain in the roots. (iii) Mycorrhization enhanced the GSH content in the phloem, while SO₄^2‐ uptake was not affected. This clearly goes against the demand‐driven control model. (iv) Under both short‐ and long‐term exposure to elevated pCO₂, the APS reductase activity in leaves and lateral roots did not correlate with the GSH contents therein. Therefore, it must be assumed that, under these conditions, regulation of S nutrition goes beyond the demand‐driven control model, and occurs within the network of other nutrient metabolism. (v) Atmospheric S in the form of H₂S enhanced the reduced sulfur content of the phloem and lateral roots. Under these conditions, the SO₄^2‐ loaded into the xylem decreased. It would appear that the demand‐driven control model of sulfur nutrition is not always valid in the case of deciduous trees.     

Sulfur-enhanced defence: effects of sulfur metabolism, nitrogen supply, and pathogen lifestyle

Evidence from field experiments indicates differential roles of sulfur and nitrogen supply for plant resistance against pathogens. Dissection of these observations in defined pathosystems and controlled nutritional conditions indicates an activation of plant sulfur metabolism in several incompatible and compatible interactions. Contents of cysteine and glutathione as markers of primary sulfate assimilation and stress response show increases in ARABIDOPSIS THALIANA upon infection, coinciding with the synthesis of sulfur-containing defence compounds. Similar increases of thiols were observed with necrotrophic, biotrophic, and hemibiotrophic pathogens. Sulfate supply was found to be neutral or beneficial for tolerance against fungal but neutral for bacterial pathogens under IN VITRO conditions. According to various reports and own observations the effects of nitrogen supply appeared to be neutral or harmful, depending on the pathogen. The activation of sulfur metabolism was a consequence of activation of gene expression as revealed by macroarray analysis of an A. THALIANA/ALTERNARIA BRASSICICOLA pathosystem. This activation appeared to be largely independent from sufficient or optimal sulfate supply and from the established sulfate deficiency response. The data suggest that plant-pathogen interactions and sulfur metabolism are linked by jasmonic acid as signal.     

Cross-talk between sulfur assimilation and ethylene signaling in plants

Sulfur (S) deficiency is prevailing all over the world and becoming an important issue for crop improvement through maximising its utilization efficiency by plants for sustainable agriculture. Its interaction with other regulatory molecules in plants is necessary to improve our understanding on its role under changing environment. Our knowledge on the influence of S on ethylene signaling is meagre although it is a constituent of cysteine (Cys) required for the synthesis of reduced glutathione (GSH) and S-adenosyl methionine (SAM), a precursor of ethylene biosynthesis. Thus, there may be an interaction between S assimilation, ethylene signaling and plant responses under optimal and stressful environmental conditions. The present review emphasizes that responses of plants to S involve ethylene action. This evaluation will provide an insight into the details of interactive role of S and ethylene signaling in regulating plant processes and prove profitable for developing sustainability under changing environmental conditions.     

The partitioning of nitrate assimilation between root and shoot of higher plants

Abstract The partitioning of nitrate assimilation between root and shoot of higher plant species is indicated by the relative proportions of total plant nitrate reductase activity (NRA) in the two plant parts and the relative concentrations of nitrate and reduced N in the xylem sap. These have been collated here from the literature and temperate and tropical species compared. Both the distribution of NRA and xylem sap nitrate: reduced N indicate that the following four generalizations can be made.

• 1 Temperate, perennial species growing in low external nitrate concentrations (about 1 mol m^?3) carry out most of their nitrate assimilation in the root. As external nitrate concentration increases (in the range found in agricultural soils, 1–20 mol m^?3), shoot nitrate assimilation becomes increasingly important.
• 2 Temperate, annual legume species growing in low external nitrate concentrations carry out most of their nitrate assimilation in the root. Shoot nitrate assimilation increases in importance as external nitrate concentration is increased.
• 3 Temperate, annual non-legume species vary greatly in their partitioning of nitrate assimilation between root and shoot when growing in low external nitrate concentrations. Regardless of the proportion carried out in the root at low external nitrate concentrations, nitrate assimilation in the shoot becomes increasingly important as external nitrate concentration is increased.
• 4 Tropical and subtropical species, annual and perennial, carry out a substantial proportion of their nitrate assimilation in the shoot when growing in low external nitrate concentrations. The partitioning of nitrate assimilation between root and shoot remains constant as external nitrate concentration increases.

It is proposed that a greater proportion of nitrate assimilation occurs in the shoot when an increase in the rate of nitrate uptake does not induce an increase in NR level in the root. Thus, a greater proportion of the nitrate taken up remains unassimilated and is passed into the xylem. A constant partitioning of nitrate assimilation between root and shoot is achieved by balancing NR levels in the root with rates of nitrate uptake. The advantages and disadvantages of assimilating nitrate in either the root or shoot are discussed in relation to temperate and tropical habitats.     

Genes involved in hydrogen and sulfur metabolism in phototrophic sulfur bacteria

The dsr genes and the hydSL operon are present as separate entities in phototrophic sulfur oxidizers of the genera Allochromatium, Marichromatium, Thiocapsa and Thiocystis and are organized similarly as in Allochromatium vinosum and Thiocapsa roseopersicina, respectively. The dsrA gene, encoding the alpha subunit of 'reverse' siroheme sulfite reductase, is also present in two species of green sulfur bacteria pointing to an important and universal role of this enzyme and probably other proteins encoded in the dsr locus in the oxidation of stored sulfur by phototrophic bacteria. The hupSL genes are uniformly present in the members of the Chromatiaceae family tested. The two genes between hydS and hydL encode a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, resembling subunits of heterodisulfide reductase from methanogenic archaea. These genes are similar but not identical to dsrM and dsrK, indicating that the derived proteins have distinct functions, the former in hydrogen metabolism and the latter in oxidative sulfur metabolism.     

Isolation and characterisation of genes for sulphate activation and reduction in Aspergillus nidulans: implications for evolution of an allosteric control region by gene duplication

A region of the Aspergillus nidulans genome carrying the sA and sC genes, encoding PAPS reductase and ATP sulphurylase, respectively, was isolated by transformation of an sA mutant with a cosmid library. The genes were subcloned and their functions confirmed by retransformation and complementation of A. nidulans strains carrying sA and sC mutations. The physical distance of 2 kb between the genes corresponds to a genetic distance of 1 cM. While the deduced amino acid sequence of the sA gene product shows homology with the equivalent MET16 gene product of Saccharomyces cerevisiae, the sC gene product resembles the equivalent MET3 yeast gene product at the N-terminal end, but differs markedly from it at the C-terminal end, showing homology to the APS kinases of several microorganisms. It is proposed that this C-terminal region does not encode a functional APS kinase, but is responsible for allosteric regulation by PAPS of the sulphate assimilation pathway in A. nidulans, and that the ATP sulphurylase encoding-gene (sC) of filamentous ascomycetes may have evolved from a bifunctional gene similar to the nodQ gene of Rhizobium meliloti.     

Thiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world

Abstract Among sulfur compounds, thiosulfate and polythionates are present at least transiently in many environments. These compounds have a similar chemical structure and their metabolism appears closely related. They are commonly used as energy sources for photoautotrophic or chemolithotrophic microorganisms, but their assimilation has been seldom studied and their importance in bacterial physiology is not well understood. Almost all bacterial strains are able to cleave these compounds since they possess thiosulfate sulfur transferase, thiosulfate reductace or S -sulfocysteine synthase activities. However, the role of these enzymes in the assimilation of thiosulfate or polythionates has not always been clearly established.
Elemental sulfur is, on the contrary, very common in the environmental. It is an energy source for sulfur-reducing eubacteria and archaebacteria and many sulfur-oxidizing archaebacteria. A phenomenon still not well understood is the 'excessive assimilatory sulfur metabolism' as observed in methanogens which perform a sulfur reduction which exceeds their anabolic needs without any apparent benefit. In heterotrophs, assimilation of elemental sulfur is seldom described and it is uncertain whether this process actually has a physiological significance.
Thus, reduction of thiosulfate and elemental sulfur is a common by incompletely understood feature among bacteria. These activities could give bacteria a selective advantage, but futher investigations are needed to clarify this possibility. Presence of thiosulfate, polythionates and sulfur reductase activities does not imply obligatorily that these activities play a role in thiosulfate, polythionates or sulfur assimilation as these compounds could be merely intermediates in bacterial metabolism. The possibility also exists that the assimilation of these sulfur compounds is just a side effect of an enzymatic activity with a completely different function.     

Analysis of sulfur and selenium assimilation in Astragalus plants with varying capacities to accumulate selenium

Several Astragalus species have the ability to hyperaccumulate selenium (Se) when growing in their native habitat. Given that the biochemical properties of Se parallel those of sulfur (S), we examined the activity of key S assimilatory enzymes ATP sulfurylase (ATPS), APS reductase (APR), and serine acetyltransferase (SAT), as well as selenocysteine methyltransferase (SMT), in eight Astragalus species with varying abilities to accumulate Se. Se hyperaccumulation was found to positively correlate with shoot accumulation of S-methylcysteine (MeCys) and Se-methylselenocysteine (MeSeCys), in addition to the level of SMT enzymatic activity. However, no correlation was observed between Se hyperaccumulation and ATPS, APR, and SAT activities in shoot tissue. Transgenic Arabidopsis thaliana overexpressing both ATPS and APR had a significant enhancement of selenate reduction as a proportion of total Se, whereas SAT overexpression resulted in only a slight increase in selenate reduction to organic forms. In general, total Se accumulation in shoots was lower in the transgenic plants overexpressing ATPS, PaAPR, and SAT. Root growth was adversely affected by selenate treatment in both ATPS and SAT overexpressors and less so in the PaAPR transgenic plants. Such observations support our conclusions that ATPS and APR are major contributors of selenate reduction in planta. However, Se hyperaccumulation in Astragalus is not driven by an overall increase in the capacity of these enzymes, but rather by either an increased Se flux through the S assimilatory pathway, generated by the biosynthesis of the sink metabolites MeCys or MeSeCys, or through an as yet unidentified Se assimilation pathway.     

Gibberellic acid-induced changes in glutathione metabolism and anthocyanin content in plant tissue

Literature data point to a possible link between gibberellic acid (GA₃) and glutathione metabolism in plant tissue, as both are connected to dormancy breakage. In order to study the influence of GA₃ on glutathione metabolism, we treated an anthocyanin accumulating cell culture of periwinkle (Catharanthus roseus) and a shoot differentiated culture of pea (Pisum sativum) with GA₃. Glutathione reductase (GR; E.C. 1.6.4.2) activity increased to 135% and 190% of the control in C. roseus and P. sativum, respectively. The level of oxidized glutathione (GSSG) decreased to 60% of the control in the C. roseus culture while no change in GSSG was observed in the P. sativum culture. No changes in the tissue concentration of total glutathione was observed in the cultures after GA₃ treatment. Concomitant to the changes in GSSG and GR, an increase in anthocyanin accumulation was observed in the C. roseus culture in association with a strong increase in phenylalanine ammonia-lyase (PAL; E.C. 4.3.1.5) activity in response to GA₃. These data strongly suggest a link between GA₃ and glutathione metabolism.