Cornelia Herschbach

S-Methylmethionine Plays a Major Role in Phloem Sulfur Transport and Is Synthesized by a Novel Type of Methyltransferase

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem (∼2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l–35S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain (∼300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5′-phosphate–dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

Consequences of air pollution on shoot-root interactions

Summary The impact of SO 2 , NO 2 and O 3 on physiological processes in plants and their consequences at the whole-plant level are discussed in the present paper. Ozon interacts with carbon allocation most likely by inhibiting sucrose export. This causes an accumulation of carbohydrates and starch in leaves and results in a reduction of photosynthesis. Thus, O 3 -exposure can diminish the availability of photosynthetate for growth and development and result in an increased shoot to root ratio and an overall reduction in biomass. By contrast, SO 2 and NO 2 can act as nutrients. SO 2 affects the sulfate and the organic sulfur pools of the leaves and will cause an enhanced export of sulfur. As a consequence, plants fumigated with SO 2 contain enhanced amounts of reduced sulfur, mainly glutathione, in the roots. Glutathione acts as a signal to control sulfate uptake from the soil and inhibits the process of xylem loading. Apparently, sulfur from atmospheric pollution can interact with the sulfur nutrition of plants. NO 2 may interact with the nitrogen nutrition of plants in a similar way. The absorbed NO 2 is used to synthesize amino acids which are translocated in the phloem to the roots. Since amino acids transported in the phloem can decrease nitrate uptake by roots, it is feasible that nitrogen taken up via the leaves can interact with whole nitrogen nutrition of plants as described for sulfur. The significance of SO 2 , NO 2 and O 3 in affecting root-shoot interactions, will depend on the availability of defence systems, the size of internal storage pools and the actual growth rate of the plant.

Functional Knockout of the Adenosine 5′-Phosphosulfate Reductase Gene in Physcomitrella patens Revives an Old Route of Sulfate Assimilation

The reduction of adenosine 5′-phosphosulfate (APS) to sulfite catalyzed by adenosine 5′-phosphosulfate reductase is considered to be the key step of sulfate assimilation in higher plants. However, analogous to enteric bacteria, an alternative pathway of sulfate reduction via phosphoadenosine 5′-phosphosulfate (PAPS) was proposed. To date, the presence of the corresponding enzyme, PAPS reductase, could be neither confirmed nor excluded in plants. To find possible alternative routes of sulfate assimilation we disrupted the adenosine 5′-phosphosulfate reductase single copy gene inPhyscomitrella patens by homologous recombination. This resulted in complete loss of the correct transcript and enzymatic activity. Surprisingly, the knockout plants grew on sulfate as the sole sulfur source, and the concentration of thiols in the knockouts did not differ from the wild type plants. However, when exposed to a sublethal concentration of cadmium, the knockouts were more sensitive than wild type plants. When fed [35S]sulfate, the knockouts incorporated 35S in thiols; the flux through sulfate reduction was ∼50% lower than in the wild type plants. PAPS reductase activity could not be measured with thioredoxin as reductant, but a cDNA and a gene coding for this enzyme were detected inP. patens. The moss Physcomitrella patens is thus the first plant species wherein PAPS reductase was confirmed on the molecular level and also the first organism wherein both APS- and PAPS-dependent sulfate assimilation co-exist.

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.

Transgenic trees as tools in tree and plant physiology

Abstract. Transgenic trees are major products of tree biotechnology. This relatively young field of both plant biotechnology and tree biology concentrates on (1) improvement of pathogen, pesticide, and stress resistance, (2) manipulation of lignin content and composition, and (3) improvement of growth. Transgenic trees also have a great potential in other areas of applied and environmental research, such as in the production of phytochemicals and in phytoremediation of polluted soils. However, genetically modified trees are also excellent tools for physiological research. Transgenic trees are indispensable in investigations of the regulation of wood formation, long-distance transport, and tree growth cycles. In addition, transgenic poplars contribute significantly to our understanding of the regulation of sulfur nutrition. In this review we concentrate on the use of transgenic tree species to improve knowledge in tree and, more generally, plant physiology rather than to cover extensively the field of commercial tree biotechnology or the biological safety of transgenic plant release.

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.

Significance of Phloem-Translocated Organic Sulfur Compounds for the Regulation of Sulfur Nutrition

Sulfur is an essential nutrient of all living organisms. In plants, it is fifth or sixth in order of elemental abundance, after hydrogen, oxygen, carbon, nitrogen and phosphorus (Pitman and Cram 1977; Raven 1980; Cram 1990). Reduced-sulfur, i.e. sulfur in the oxidation state -2, is the most important form of sulfur in living cells. It supports the specific conformations and functions of enzymes and structural proteins via reactive sulfide moieties and disulfide bonds. Sulfur is available for plants mainly as sulfate at the roots (Rennenberg 1984). Therefore, sulfate has to be activated, reduced to sulfide and incorporated into carbohydrate skeletons by assimilatory sulfate reduction before it can be used in protein synthesis (Brunold 1990, 1993). The final product of assimilatory sulfate reduction in plants is cysteine. From this amino acid, all other reduced-sulfur compounds, including methionine (Giovanelli 1990), glutathione (Bergmann and Rennenberg 1993), the S-alkylcysteine sulfoxides of Liliacea., the isothiocyanates of Brassicacea. (Schnug 1990) and phytochelatins (Rauser 1995), are synthesized in a whole set of metabolic pathways. In most plant species, a specific sulfur/nitrogen (S/N) ratio of approximately 1/20 reflects the relationship of these macro-nutrients in protein (Dijkshoorn and Van Wijk 1967).

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.

Phosphorus nutrition of woody plants: many questions – few answers

Phosphorus (P) acquisition, cycling and use efficiency has been investigated intensively with herbaceous plants. It is known that local as well as systemic signalling contributes to the control of P acquisition. Woody plants are long-lived organisms that adapt their life cycle to the changing environment during their annual growth cycle. Little is known about P acquisition and P cycling in perennial plants, especially regarding storage and mobilisation, its control by systemic and environmental factors, and its interaction with the largely closed ecosystem-level P cycle. The present report presents a view on open questions on plant internal P cycling in woody plants.

Long-Distance Transport and Plant Internal Cycling of N- and S-Compounds

A coordinated supply of the whole plant with sulfur (S) and nitrogen (N) requires mechanisms to regulate not only uptake and assimilation but also long-distance transport of both nutrients in the phloem and xylem as well as the plant internal cycling of S and N compounds. In trees, plant internal nutrient cycling which includes bidirectional exchange between phloem and xylem allows to partially uncouple nutrient demand from soil supply and needs to be highly coordinated with seasonal storage and remobilisation of S- and N-compounds. In both annual and perennial plants the pools of N and S compounds cycling within the plant provide an integrated signal to adapt the nutrient supply of the plant to the actual demand.