When Plant Life Gets Tough Sulfur Gets Going

The Romans discovered the beneficial effects of sulfur as a potent antidote against plant pathogens and they used elemental sulfur to protect their vineyards. More than 2000 years later, scientists rediscovered the importance of adequate sulfur nutrition for plant defence against pathogens. Due to the clean air act, SO₂ emissions drastically declined during the late 1980s, resulting in mild sulfur deficiency in crops, a condition which apparently affected the plants's potential to effectively mount a defence against invading pathogens ([Schnug et al., 1995]). By that time, plant scientists had firmly established that several sulfur-containing metabolites and proteins were intimately involved in the plant responses to biotic and also abiotic stress. In basic science, the molecular mechanisms of sulfur assimilation and metabolism in stress defence-related compounds has been the focus of excellent research in the 1990s ([Leustek et al., 2000]) and into the new millennium ([Saito, 2004]). However, field observations in agricultural systems now indicate that the syndrome of reduced plant fitness due to low sulfur availability is likely to reduce crop yield. This perspective produced a strong motivation for much of the sulfur-related research in the past decade.

At the European level, a COST Action (COST 829: “Fundamental, agronomical and environmental aspects of sulfur nutrition and assimilation in plants”) was initiated in 1998, addressing diverse issues of plant sulfur metabolism. Motivated by the immense progress in sulfur-related plant research in the late 1990s, and under the impact of a growing awareness that sulfur-related defence operations of plants against pathogens might have an as yet overlooked relevance in agriculture, eight German research groups merged their sulfur-related research efforts and embarked on a joint research initiative in 2000, which was funded by the German Science Foundation (German Plant Sulfur Group, GPSG). The major goal of this initiative was to unravel how basic sulfur metabolism and its manifold links to stress defence, built on sulfur-containing compounds, were connected at the molecular level. Over a period of 6 years (2000 - 2006), GPSG has made important contributions to an internationally competitive research field by providing novel insight into molecular components and mechanisms involved in plant stress responses, by highlighting important aspects of sulfur nutrition, but also by taking up the challenge of establishing the link between controlled growth chamber experiments with the model plant Arabidopsis thaliana and field studies with its big relative, i.e., oilseed rape (Brassica napus). The progress of this research has found its way into a number of important original research papers. In October 2006, an international symposium was held in Heidelberg to sum up the ground-breaking highlights of this collaborative research effort and to present it to the sulfur research community, including scientists from all over Europe and the USA (“Sulfur-containing defence compounds: pivotal players in plant stress tolerance”, Heidelberg, organised by Thomas Rausch, Rüdiger Hell, and Malcolm Hawkesford).

This special issue of PLANT BIOLOGY reflects the progress made in a collection of eight reviews presented by GPSG members, but also includes some original research papers contributed by members of the European plant sulfur community. The purpose of this editorial is to point out the many links between the individual research areas and to underline the most important results for those not intimately familiar with the current stage of plant sulfur research. However, it will also identify some of the open questions which need to be addressed in the years to come, because, as in any such endeavour, new insights have revealed important unknowns that we were not even aware of.

This special issue is opened by an evolutionary perspective. Kopriva and coworkers ([Kopriva et al., 2007]) confront us with surprising results from a moss: Physcomitrella patens has recently developed into a valuable model system to study plant gene functions in general by offering the tool of gene replacement via homologous recombination. However, for plant sulfur research, Physcomitrella has also offered unexpected evolutionary insights. Thus, in contrast to higher plants, the moss Physcomitrella exhibits redundancy in the pathway of early sulfur assimilation by not only expressing an adenosine-5‘-phosphosulfate reductase (APS reductase) that is also found in higher plants, but also a 3′-phosphoadenosine-5’-phosphosulfate reductase (PAPS reductase), hitherto thought to be limited to fungi and certain bacteria. Starting from this surprising observation, the authors present a comprehensive picture of sulfur assimilation in higher and lower plants, drawing information from genome sequencing. As a result, we are forced to challenge our simplistic view of a general scheme for sulfur assimilation in the plant kingdom, and are confronted with an unexpected range of diversity.

The biosynthesis of the tripeptide, glutathione, the major thiol compound in most eukaryotes and many prokaryotes, has been the research focus of Rausch and coworkers ([Rausch et al., 2007]). Earlier studies had already indicated that the biosynthesis of this major anti-stress metabolite is not just regulated at the transcriptional level, but the molecular mechanism of such post-transcriptional control(s) had remained unknown. This review demonstrates how subcellular compart-mentation, i.e., of the two enzymes GSH1 and GSH2, could impact on the regulation of GSH biosynthesis. While GSH1 is exclusively localised in the plastids, GSH2 is to a larger part cytosolic, with less than 10 % of GSH2 transcripts coding for a protein with a functional transit peptide for import into plastids. The contribution of the two compartments to GSH biosynthesis could be an integral part of redox communication between the plastids and cytosol, including the nucleus. Furthermore, this review discusses recent progress achieved in our understanding of post-transcriptional GSH1 regulation. The protein structure of this enzyme, which catalyses the rate-limiting step in GSH biosynthesis, has been solved and a possible redox switch for enzyme activity has been discovered, which operates via reversible formation of an intramolecular disulfide bridge. Future research will have to determine the in vivo relevance of this apparent post-translational regulation. Apart from this, solving the protein structure of plant GSH1 has allowed us to explain how different mutations in GSH1 impact on plant GSH biosynthesis. Thus, an impaired response of certain Arabidopsis mutants to plant pathogens can now be attributed to defined changes in GSH1 structure.

The biosynthesis of an entire group of defence molecules, i.e., the glucosinolates of the Brassicales, and the impact of sulfur nutrition on their formation in planta has been the research focus of [Falk et al. (2007)]. In their studies on glucosinolate synthesis and its regulation, Gershenzon and colleagues have made important contributions to our understanding of the entire biosynthetic pathway and have revealed how different groups of glucosinolates are differentially affected by plant nutrition. As expected, the impact of sulfur nutrition was strongest on those glucosinolates derived from the sulfur-containing amino acid, methionine. Interestingly, under sulfur deprivation the genes involved in glucosinolate biosynthesis were coordinately downregulated, a process which may result from changed expression (or activity) of the recently discovered Myb-type transcription factors shown to regulate the entire glucosinolate biosynthetic pathway ([Hirai et al., 2007]). This review also addresses the important question to what extent glucosinolates are used by the plant as a source of sulfur under S-limiting conditions. Clearly, this aspect of glucosinolate function needs more research efforts in the future.

While the first three reviews focus on important aspects of sulfur assimilation and biosynthesis of sulfur-containing defence compounds, i.e., glutathione and the glucosinolates, the following reviews of [Papenbrock et al. (2007)] and [Hänsch et al. (2007)] address molecular aspects of so far neglected dissimilatory reactions, i.e., the action of H₂S-releasing L/D cysteine desulfhydrases and sulfite oxidase, respectively. Papenbrock and colleagues have characterised several L/D cysteine desulfhydrases (CDS) from Arabidopsis thaliana. These enzymes are involved in the formation of H₂S, a compound thought to contribute to the plant's defence against fungal pathogens. The authors have extended their studies to field-grown Brassica napus and have discovered genetically determined differences in CDS activities. While it appears likely that CDS activities are largely responsible for the release of H₂S from plant tissues, the possible contribution of side activities of O-acetylserine(thiol)lyase remains uncertain. However, even after many years of intense research efforts, the role of H₂S as a significant contributor to plant defence against pathogens still remains to be unequivocally established. An important technical advance would be to quantify the amounts of H₂S present in and released by different plant tissues or even cell types. To achieve this goal, [Papenbrock et al. (2007)] have explored the potential of specific H₂S microsensors, but the results obtained thus far have not been entirely promising. Clearly, mutant plants affected in their potential to release H₂S, either constitutively or in response to pathogen challenge, are needed to answer some of the open questions.

In their focused studies on the enzyme sulfite oxidase (SO), [Hänsch et al. (2007)] have shed light on the possible role of this still enigmatic enzyme. The structure of plant SO has been solved by Mendel and colleagues, its biochemical properties have been largely elucidated, and its subcellular compartmentation in peroxisomes has been established. As a surprising observation, it was found that molecular oxygen acts as an electron acceptor and H₂O₂ is formed as a reaction product. While the localisation of SO in peroxisomes apparently fitted well with the presence of catalase in the same organelle, it was demonstrated that H₂O₂ could also non-enzymatically oxidise sulfite. The emerging picture from studies with plants overexpressing SO assigns a role to this enzyme as a “safety valve” for removal of access sulfite; however, other possibilities may exist. Obviously, the co-regulation of SO with sulfate assimilation activities still poses a number of questions. More studies are needed with SO-deficient plants to understand SO functions in vivo in the absence of stress exposure, since SO appears to be constitutively expressed throughout plant development. Taken together, the reviews of Papenbrock et al. and Hänsch et al. have directed our attention to dissimilatory reactions in sulfur metabolism, which might even procede side by side with sulfur assimilation. While many molecular details of the reactions catalysed by CDS and SO have been addressed, their roles in a physiological context are only beginning to be explored.

The following two reviews together reflect the progress in attempts to bridge the gap between controlled growth chamber experiments and field studies. [Bloem et al. (2007)] performed extended field studies on oilseed rape, with the goal to provide first links between sulfur nutrition, pathogen defence, and the formation of individual sulfur-containing defence compounds. [Kruse et al. (2007 a)] were successful in setting up a controlled infection system for the related model plant Arabidopsis and studied the effect of pathogen challenge on the expression of genes related to the formation of sulfur-based defence compounds under different sulfur nutrition regimes. Based on a number of field trials at different locations, [Bloem et al. (2007)] came to the conclusion that, while a benefical effect of appropriate sulfur nutrition for plant defense under field conditions is largely supported by circumstantial evidence, establishing a clear causal link suffers from the complexity of other environmental factors, equally impacting on plant defence. Conversely, results from the model system explored by Kruse et al., addressing the impact of pathogen attack under strictly controlled conditions, supports the role of sulfur nutrition in strengthening the plant's defence potential. However, this appears to be limited to fungal pathogens and could not be confirmed for pathogenic bacteria. A clear difference was found for the role of nitrogen in the investigated pathosystems. In marked contrast to sulfur, an increased nitrogen supply usually resulted in stronger infection rates. Using macroarray analysis, it could be shown that jasmonic acid signalling is involved in both sulfur metabolism and plant-pathogen interactions. Upon closer inspection of the data from field studies and from controlled growth chamber experiments, it becomes apparent that current research still has a long way to go until it provides meaningful links between the two approaches. The experimental hurdles have become very clear in recent years. First, results from Arabidopsis cannot be directly transferred to a crop plant like oilseed rape, despite the evolutionary relatedness between these species, due to different growth patterns, nutrient allocation strategies, and even differences in the regulation of orthologous genes. Second, under field conditions a complex set of environmental cues impinge to different degrees on sulfur metabolism and plant performance in general. Therefore, to evaluate to what extent sulfur nutrition is a major factor in plant pathogen defence in agricultural ecosytems, these other factors must not be neglected. Does that mean that we will not find a clear answer at all? No, but it means that when we wish to move beyond circumstantial evidence, extended molecular studies with different oilseed rape genotypes, including mutants in the genes of interest, will have to be included in the analysis. Also, when going for the molecular mechanisms behind the proposed sulfur-induced resistance (or sulfur-enhanced defence), it will not be sufficient to focus on gene expression profiles but comprehensive metabolite profiles will have to be included. Likewise, the temporal dynamics of the molecular changes during pathogen defence have to be taken into account. Clearly, a systems biology approach is required to understand the link between sulfur nutrition and defence potential.

This conclusion is also supported by the studies of Rennenberg et al., presented here as a review and a research paper. The authors have studied sulfur metabolism in trees, using gene expression profiles and metabolite analysis ([Rennenberg et al., 2007]). While, as expected, a comparison of sulfur metabolism in herbaceous plants and in trees revealed that the same overall routes and signalling pathways appear to operate in both plant forms (see also [Kopriva et al., 2007]), trees perform much more robust and flexible sulfur metabolism due to a higher buffering capacity, but also as a consequence of the organisms' longevity. In their experimental study on the interaction between nitrogen and sulfur metabolism in tobacco, [Kruse et al. (2007 b)] made use of transformant plants expressing nitrate reductase only in leaves, but not in roots, in order to address the importance of nitrate reductase for sulfur assimilation. Wild-type and transformant plants were grown on different nitrogen sources (nitrate/ammonium nitrate) and sulfur assimilation activities were monitored in roots and shoots. While the links observed between changed APS reductase activities and sulfur-containing metabolites in WT plants as compared to transformants clearly point to a strong mechanistic cross talk between nitrate reduction and sulfur assimilation, again a full assessment of this interaction will require a more comprehensive systems biology approach. A central aspect of both contributions are the many hints to the important role of sulfate transport and reallocation within the plant. Clearly, these processes are differently regulated in different plant forms. Major sulfate fluxes, but also the dynamics of long-distance transport of sulfur metabolites, e.g., glutathione, have to be understood before a comprehensive whole plant picture of sulfur metabolism can be drawn.

The research contributions of [Parmar et al. (2007)] and [Koralewska et al. (2007)] address different aspects of sulfate transport and allocation in Brassica napus and Brassica oleracea, respectively. The study of [Parmar et al. (2007)] directs our attention to the developmental component of the plant's response to sulfate deprivation. Thus, leaves of different physiological age respond very differently to sulfur depletion. This phenomenon is linked to the developmental gradient, reflecting active metabolism during expansion growth in young leaves and maintenance functions in fully expanded leaves. When comparing sulfate concentrations with nitrate and phosphate contents under conditions of sulfur depletion, phosphate contents remained unaffected, while nitrate contents declined in the oldest leaves. The authors also analysed the expression of the different members of the sulfate transporter family, including high-affinity transporters at the plasma membrane, but also putative vacuolar transporter isoforms, and observed wide variations in their individual response to sulfur depletion. An important conclusion from this study is that, in addition to the variation of external sulfate availability, the dynamic changes of intracellular (vacuolar) sulfate pools are important cues for overall regulation of sulfate allocation in the plant. Of course, a limitation of this type of study is the absence of dynamic flux data. Thus, in young developing leaves a greater proportion of sulfate is directly assimilated and incorporated into proteins, as compared to fully expanded leaves. A comprehensive study of these dynamic aspects remains an experimental challenge but such data would certainly improve our understanding of sulfur allocation.

While [Parmar et al. (2007)] exposed their plants to complete sulfate removal, the study of [Koralewska et al. (2007)] compares the effect of transferring plants from high to low sulfate availability, using Brassica oleracea, a species known for its high-sulfate content in shoots and roots. An important observation of this study was the absence of significant changes in plant growth and shoot to root biomass allocation in response to shifting the plants to low-external sulfate. This result was in marked contrast to the effects observed upon complete sulfate depletion treatment. Thus, the plant's behaviour with respect to sulfate uptake from the medium and allocation between root and shoot appears to dynamically respond to changed sulfate availability and reveals high adaptability. Again, the results confirm that internal sulfate contents are probably measured by the plant and are impacting on transport activities.

The last two research papers in this special issue address a topic of applied plant research, i.e., the possible use of plants for the phytoremediation of heavy metal-contaminated soils. While toxic heavy metal ions may be taken up by the plant via IRT- or Nramp-type transporters, oxidised forms like chromate have been shown to interfere with sulfate uptake. In their paper, [Schiavon et al. (2007)] studied the impact of chromate on sulfate transporter expression and sulfate content in maize seedlings. Maximal chromate was accumulated in sulfate-deprived seedlings. Interestingly, the expression of the root high-affinity sulfate transporter ZmST1;1 was strongly suppressed by exposure of seedlings to 200 µM chromate, and this effect was shown to be rather specific. Thus, it may be speculated that chromate, upon entering the cells (possibly via sulfate transporters), mimics availability of sulfate and therefore causes the repression of sulfate transporter expression.

While the work of Malagoli and coworkers addressed the interference of an anionic oxidised chromium species with sulfate uptake, [Pajuelo et al. (2007)] based their study on the well-established role of phytochelatin-mediated heavy metal sequestration, and directed their focus to O-acetylserine (thiol)lyase (OASTL), an enzyme forming the cysteine synthase complex, together with serine acetyl transferase. Previous studies had indicated that in several plant species the expression of OASTL is up-regulated in response to heavy-metal exposure, and this phenomenon was explained by the limiting role of cysteine for the synthesis of glutathione, the precursor of phytochelatins. In their contribution, Gotor and coworkers evaluated the potential role of increased OASTL expression and enzyme activity for heavy metal detoxification in several legume species. While presenting evidence for a possible involvement of increased OASTL activity and expression in heavy-metal detoxification (including As, Cd, Cu, and Pb), the results do not yet assign a decisive role to OASTL. As many other genes contribute to the increased sulfur assimilation and glutathione synthesis under heavy-metal exposure, demonstration of a fundamental contribution of OASTL to heavy-metal detoxification in legumes remains to be validated.

This collection of reviews and original research papers on different aspects of sulfur nutrition-dependent stress defence mechanisms and the underlying principles of sulfur assimilation and turnover in higher plants is nothing more than a photographic shot at a particular time. However, as it presents the progress during a particularly active period in plant sulfur research, it will be a valuable reference to the specialist, while also serving as a primer for those who enter the field. The presented work has covered the entire range of plant sulfur research, extending from the analysis of individual molecular components to the performance of crop plants under field conditions. Careful scrutiny by the reader will expose the many open questions remaining in this exciting research area, as has been attempted to some extent in this editorial. Thus, while the importance of several sulfur-containing compounds for plant defence against pathogens has been repeatedly claimed in the literature, exact causal relationships have not always been established ([Rausch and Wachter, 2005], and literature cited therein). It has become obvious that to increase our understanding of sulfur-based defence strategies in plants, much more work is needed to uncover the complexities of individual cellular components. Hence, launching a comprehensive systems biology approach will be a necessary complementary research strategy. In both approaches, mutant and transgenic plants affected in individual sulfur-related traits will play a major role, but clearly research will have to move beyond Arabidopsis when meaningful predictions for agriculture are required. Many more exciting contributions from both research areas are expected and, hopefully, will result in the establishment of clear causal relationships between sulfur nutrition and pathogen defence in particular (and, perhaps, stress tolerance in general).

References

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Editor: H. Rennenberg