The plant TOR kinase tunes autophagy and meristem activity for nutrient stress-induced developmental plasticity.

Plants, unlike animals, respond to environmental challenges with comprehensive developmental transitions that allow them to cope with these stresses. Here we discovered that antagonistic activation of the Target of Rapamycin (TOR) kinase in Arabidopsis thaliana roots and shoots is essential for the nutrient deprivation-induced increase in the root-to-shoot ratio to improve foraging for mineral ions. We demonstrate that sulfate limitation-induced downregulation of TOR in shoots activates autophagy, resulting in enhanced carbon allocation to the root. The allocation of carbon to the roots is facilitated by the specific upregulation of the sucrose-transporter genes SWEET11/12 in shoots. SWEET11/12 activation is indispensable for enabling sucrose to act as a carbon source for growth and as a signal for tuning root apical meristem activity via glucose-TOR signaling. The sugar-stimulated TOR activity in the root suppresses autophagy and maintains root apical meristem activity to support root growth to enhance mining for new sulfate resources in the soil. We provide direct evidence that the organ-specific regulation of autophagy is essential for the increased root-to-shoot ratio in response to sulfur limitation. These findings uncover how sulfur limitation controls the central sensor kinase TOR to enable nutrient recycling for stress-induced morphological adaptation of the plant body.

Differential modulation of Target of Rapamycin activity under single and combined iron and sulfur deficiency in tomato plants.

Over the past few decades, a close relationship between sulfur (S) and iron (Fe) in terms of functionality and nutrition was demonstrated in the tomato. However, very little is known about the regulatory mechanisms underlying S/Fe interactions. Recently, the potential role of citrate in plant adaptation to Fe deficiency and combined S and Fe deficiency has been described. It is known that an impaired organic acid metabolism may stimulate a retrograde signal, which has been proven to be linked to the Target of Rapamycin (TOR) signaling in yeast and animal cells. Recent reports provided evidence of TOR involvement in S nutrient sensing in plants. This suggestion prompted us to investigate whether TOR may play a role in the cross-talk of signaling pathway occurring during plant adaptation to combined nutrient deficiency of Fe and S. Our results revealed that Fe deficiency elicited an increase of TOR activity associated with enhanced accumulation of citrate. In contrast, S deficiency resulted in decreased TOR activity and citrate accumulation. Interestingly, citrate accumulated in shoots of plants exposed to combined S/Fe deficiency to values between those found in Fe- and S-deficient plants, again correlated with TOR activity level. Our results suggest that citrate might be involved in establishing a link between plant response to combined S/Fe deficiency and the TOR network.

Dynamic association of the plastid localized cysteine synthase complex is vital for efficient cysteine production, photosynthesis, and granal thylakoid formation in transgenic tobacco.

Cysteine biosynthesis is essential for translation and represents the entry point of reduced sulfur into plant metabolism. The two consecutively acting enzymes serine acetyltransferase (SAT) and O-acetylserine-thiol-lyase catalyse cysteine production and form the cysteine synthase complex, in which SAT is activated. Here we show that tobacco (Nicotiana tabacum) expressing active SAT in plastids (referred to as PSA lines) shows substantial cysteine accumulation in plastids. Remarkably, enhanced cysteine production in plastids entirely abolished granal stack formation, impaired photosynthesis capacity, and decreased the number of chloroplasts in mesophyll cells of the PSA lines. A transgenic tobacco line expressing active SAT in the cytosol accumulated comparable amounts of thiols but displayed no phenotype. To dissect the consequences of cysteine synthase complex formation from enhanced SAT activity in tobacco plastids, we expressed an enzymatically inactive SAT that can still form the cysteine synthase complex in tobacco plastids (PSI lines). The PSI lines were indistinguishable from the PSA lines, although the PSI lines displayed no increase in plastid-localized SAT activity. Neither PSA lines nor PSI lines suffered from an oxidized redox environment in plastids that could have been causative for the disturbed photosynthesis. From these findings, we infer that the association of the plastid cysteine synthase complex itself triggers a signaling cascade controlling sulfur assimilation and photosynthetic capacity in leaves.

NatB-Mediated N-Terminal Acetylation Affects Growth and Biotic Stress Responses.

N∝-terminal acetylation (NTA) is one of the most abundant protein modifications in eukaryotes. In humans, NTA is catalyzed by seven Nα-acetyltransferases (NatA-F and NatH). Remarkably, the plant Nat machinery and its biological relevance remain poorly understood, although NTA has gained recognition as a key regulator of crucial processes such as protein turnover, protein-protein interaction, and protein targeting. In this study, we combined in vitro assays, reverse genetics, quantitative N-terminomics, transcriptomics, and physiological assays to characterize the Arabidopsis (Arabidopsis thaliana) NatB complex. We show that the plant NatB catalytic (NAA20) and auxiliary subunit (NAA25) form a stable heterodimeric complex that accepts canonical NatB-type substrates in vitro. In planta, NatB complex formation was essential for enzymatic activity. Depletion of NatB subunits to 30% of the wild-type level in three Arabidopsis T-DNA insertion mutants (naa20-1, naa20-2, and naa25-1) caused a 50% decrease in plant growth. A complementation approach revealed functional conservation between plant and human catalytic NatB subunits, whereas yeast NAA20 failed to complement naa20-1 Quantitative N-terminomics of approximately 1000 peptides identified 32 bona fide substrates of the plant NatB complex. In vivo, NatB was seen to preferentially acetylate N termini starting with the initiator Met followed by acidic amino acids and contributed 20% of the acetylation marks in the detected plant proteome. Global transcriptome and proteome analyses of NatB-depleted mutants suggested a function of NatB in multiple stress responses. Indeed, loss of NatB function, but not NatA, increased plant sensitivity toward osmotic and high-salt stress, indicating that NatB is required for tolerance of these abiotic stressors.

NAA50 Is an Enzymatically Active N α-Acetyltransferase That Is Crucial for Development and Regulation of Stress Responses.

Nα-terminal acetylation (NTA) is a prevalent protein modification in eukaryotes. In plants, the biological function of NTA remains enigmatic. The dominant N-acetyltransferase (Nat) in Arabidopsis (Arabidopsis thaliana) is NatA, which cotranslationally catalyzes acetylation of ∼40% of the proteome. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. In human (Homo sapiens), fruit fly (Drosophila melanogaster), and yeast (Saccharomyces cerevisiae), this core NatA complex interacts with NAA50 to form the NatE complex. While in metazoa, NAA50 has N-acetyltransferase activity, yeast NAA50 is catalytically inactive and positions NatA at the ribosome tunnel exit. Here, we report the identification and characterization of Arabidopsis NAA50 (AT5G11340). Consistent with its putative function as a cotranslationally acting Nat, AtNAA50-EYFP localized to the cytosol and the endoplasmic reticulum but also to the nuclei. We demonstrate that purified AtNAA50 displays Nα-terminal acetyltransferase and lysine-ε-autoacetyltransferase activity in vitro. Global N-acetylome profiling of Escherichia coli cells expressing AtNAA50 revealed conservation of NatE substrate specificity between plants and humans. Unlike the embryo-lethal phenotype caused by the absence of AtNAA10 and AtNAA15, loss of NAA50 expression resulted in severe growth retardation and infertility in two Arabidopsis transfer DNA insertion lines (naa50-1 and naa50-2). The phenotype of naa50-2 was rescued by the expression of HsNAA50 or AtNAA50. In contrast, the inactive ScNAA50 failed to complement naa50-2 Remarkably, loss of NAA50 expression did not affect NTA of known NatA substrates and caused the accumulation of proteins involved in stress responses. Overall, our results emphasize a relevant role of AtNAA50 in plant defense and development, which is independent of the essential NatA activity.

Sulfate is Incorporated into Cysteine to Trigger ABA Production and Stomatal Closure.

Plants close stomata when root water availability becomes limiting. Recent studies have demonstrated that soil-drying induces root-to-shoot sulfate transport via the xylem and that sulfate closes stomata. Here we provide evidence for a physiologically relevant signaling pathway that underlies sulfate-induced stomatal closure in Arabidopsis (Arabidopsis thaliana). We uncovered that, in the guard cells, sulfate activates NADPH oxidases to produce reactive oxygen species (ROS) and that this ROS induction is essential for sulfate-induced stomata closure. In line with the function of ROS as the second-messenger of abscisic acid (ABA) signaling, sulfate does not induce ROS in the ABA-synthesis mutant, aba3-1, and sulfate-induced ROS were ineffective at closing stomata in the ABA-insensitive mutant abi2-1 and a SLOW ANION CHANNEL1 loss-of-function mutant. We provided direct evidence for sulfate-induced accumulation of ABA in the cytosol of guard cells by application of the ABAleon2.1 ABA sensor, the ABA signaling reporter ProRAB18:GFP, and quantification of endogenous ABA marker genes. In concordance with previous studies, showing that ABA DEFICIENT3 uses Cys as the substrate for activation of the ABSCISIC ALDEHYDE OXIDASE3 (AAO3) enzyme catalyzing the last step of ABA production, we demonstrated that assimilation of sulfate into Cys is necessary for sulfate-induced stomatal closure and that sulfate-feeding or Cys-feeding induces transcription of NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3, limiting the synthesis of the AAO3 substrate. Consequently, Cys synthesis-depleted mutants are sensitive to soil-drying due to enhanced water loss. Our data demonstrate that sulfate is incorporated into Cys and tunes ABA biosynthesis in leaves, promoting stomatal closure, and that this mechanism contributes to the physiological water limitation response.

The Arabidopsis Nα -acetyltransferase NAA60 locates to the plasma membrane and is vital for the high salt stress response.

In humans and plants, N-terminal acetylation plays a central role in protein homeostasis, affects 80% of proteins in the cytoplasm and is catalyzed by five ribosome-associated N-acetyltransferases (NatA-E). Humans also possess a Golgi-associated NatF (HsNAA60) that is essential for Golgi integrity. Remarkably, NAA60 is absent in fungi and has not been identified in plants. Here we identify and characterize the first plasma membrane-anchored post-translationally acting N-acetyltransferase AtNAA60 in the reference plant Arabidopsis thaliana by the combined application of reverse genetics, global proteomics, live-cell imaging, microscale thermophoresis, circular dichroism spectroscopy, nano-differential scanning fluorometry, intrinsic tryptophan fluorescence and X-ray crystallography. We demonstrate that AtNAA60, like HsNAA60, is membrane-localized in vivo by an α-helical membrane anchor at its C-terminus, but in contrast to HsNAA60, AtNAA60 localizes to the plasma membrane. The AtNAA60 crystal structure provides insights into substrate-binding, the broad substrate specificity and the catalytic mechanism probed by structure-based mutagenesis. Characterization of the NAA60 loss-of-function mutants (naa60-1 and naa60-2) uncovers a plasma membrane-localized substrate of AtNAA60 and the importance of NAA60 during high salt stress. Our findings provide evidence for the plant-specific evolution of a plasma membrane-anchored N-acetyltransferase that is vital for adaptation to stress.

SULTR3s Function in Chloroplast Sulfate Uptake and Affect ABA Biosynthesis and the Stress Response.

Plants are major sulfur reducers in the global sulfur cycle. Sulfate, the major natural sulfur source in soil, is absorbed by plant roots and transported into plastids, where it is reduced and assimilated into Cys for further metabolic processes. Despite its importance, how sulfate is transported into plastids is poorly understood. We previously demonstrated using single Arabidopsis (Arabidopsis thaliana) genetic mutants that each member of the sulfate transporter (SULTR) subfamily 3 was able to transport sulfate across the chloroplast envelope membrane. To resolve the function of SULTR3s, we constructed a sultr3 quintuple mutant completely knocking out all five members of the subfamily. Here we report that all members of the SULTR3 subfamily show chloroplast membrane localization. Sulfate uptake by chloroplasts of the quintuple mutant is reduced by more than 50% compared with the wild type. Consequently, Cys and abscisic acid (ABA) content are reduced to ∼67 and ∼20% of the wild-type level, respectively, and strong positive correlations are found among sulfate, Cys, and ABA content. The sultr3 quintuple mutant shows obvious growth retardation with smaller rosettes and shorter roots. Seed germination of the sultr3 quintuple mutant is hypersensitive to exogenous ABA and salt stress, but is rescued by sulfide supplementation. Furthermore, sulfate-induced stomatal closure is abolished in the quintuple mutant, strongly suggesting that chloroplast sulfate is required for stomatal closure. Our genetic analyses unequivocally demonstrate that sulfate transporter subfamily 3 is responsible for more than half of the chloroplast sulfate uptake and influences downstream sulfate assimilation and ABA biosynthesis.

Sulfur Partitioning between Glutathione and Protein Synthesis Determines Plant Growth.

Photoautotrophic organisms must efficiently allocate their resources between stress-response pathways and growth-promoting pathways to be successful in a constantly changing environment. In this study, we addressed the coordination of sulfur flux between the biosynthesis of the reactive oxygen species scavenger glutathione (GSH) and protein translation as one example of a central resource allocation switch. We crossed the Arabidopsis (Arabidopsis thaliana) GSH synthesis-depleted cadmium-sensitive cad2-1 mutant, which lacks glutamate cysteine (Cys) ligase, into the sulfite reductase sir1-1 mutant, which suffers from a significantly decreased flux of sulfur into Cys and, consequently, is retarded in growth. Surprisingly, depletion of GSH synthesis promoted the growth of the sir1-1 cad2-1 double mutant (s1c2) when compared with sir1-1 Determination of GSH levels and in vivo live-cell imaging of the reduction-oxidation-sensitive green fluorescent protein sensor demonstrated significant oxidation of the plastidic GSH redox potential in cad2-1 and s1c2 This oxidized GSH redox potential aligned with significant activation of plastid-localized sulfate reduction and a significantly higher flux of sulfur into proteins. The specific activation of the serine/threonine sensor kinase Target of Rapamycin (TOR) in cad2-1 and s1c2 was the trigger for reallocation of Cys from GSH biosynthesis into protein translation. Activation of TOR in s1c2 enhanced ribosome abundance and partially rescued the decreased meristematic activity observed in sir1-1 mutants. Therefore, we found that the coordination of sulfur flux between GSH biosynthesis and protein translation determines growth via the regulation of TOR.

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress.

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch-activated ion channel with a slight preference for anions and provides protection against hypo-osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo-osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1-1 mutants under moderate heat- and heavy-metal-stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions.

System analysis of metabolism and the transcriptome in Arabidopsis thaliana roots reveals differential co-regulation upon iron, sulfur and potassium deficiency.

Deprivation of mineral nutrients causes significant retardation of plant growth. This retardation is associated with nutrient-specific and general stress-induced transcriptional responses. In this study, we adjusted the external supply of iron, potassium and sulfur to cause the same retardation of shoot growth. Nevertheless, limitation by individual nutrients resulted in specific morphological adaptations and distinct shifts within the root metabolite fingerprint. The metabolic shifts affected key metabolites of primary metabolism and the stress-related phytohormones, jasmonic, salicylic and abscisic acid. These phytohormone signatures contributed to specific nutrient deficiency-induced transcriptional regulation. Limitation by the micronutrient iron caused the strongest regulation and affected 18% of the root transcriptome. Only 130 genes were regulated by all nutrients. Specific co-regulation between the iron and sulfur metabolic routes upon iron or sulfur deficiency was observed. Interestingly, iron deficiency caused regulation of a different set of genes of the sulfur assimilation pathway compared with sulfur deficiency itself, which demonstrates the presence of specific signal-transduction systems for the cross-regulation of the pathways. Combined iron and sulfur starvation experiments demonstrated that a requirement for a specific nutrient can overrule this cross-regulation. The comparative metabolomics and transcriptomics approach used dissected general stress from nutrient-specific regulation in roots of Arabidopsis.

Drought stress in maize causes differential acclimation responses of glutathione and sulfur metabolism in leaves and roots.

BACKGROUND: Drought is the most important environmental stress that limits crop yield in a global warming world. Despite the compelling evidence of an important role of oxidized and reduced sulfur-containing compounds during the response of plants to drought stress (e.g. sulfate for stomata closure or glutathione for scavenging of reactive oxygen species), the assimilatory sulfate reduction pathway is almost not investigated at the molecular or at the whole plant level during drought. RESULTS: In the present study, we elucidated the role of assimilatory sulfate reduction in roots and leaves of the staple crop maize after application of drought stress. The time-resolved dynamics of the adaption processes to the stress was analyzed in a physiological relevant situation -when prolonged drought caused significant oxidation stress but root growth should be maintained. The allocation of sulfate was significantly shifted to the roots upon drought and allowed for significant increase of thiols derived from sulfate assimilation in roots. This enabled roots to produce biomass, while leaf growth was stopped. Accumulation of harmful reactive oxygen species caused oxidation of the glutathione pool and decreased glutathione levels in leaves. Surprisingly, flux analysis using [35S]-sulfate demonstrated a significant down-regulation of sulfate assimilation and cysteine synthesis in leaves due to the substantial decrease of serine acetyltransferase activity. The insufficient cysteine supply caused depletion of glutathione pool in spite of significant transcriptional induction of glutathione synthesis limiting GSH1. Furthermore, drought impinges on transcription of membrane-localized sulfate transport systems in leaves and roots, which provides a potential molecular mechanism for the reallocation of sulfur upon prolonged water withdrawal. CONCLUSIONS: The study demonstrated a significant and organ-specific impact of drought upon sulfate assimilation. The sulfur metabolism related alterations at the transcriptional, metabolic and enzyme activity level are consistent with a promotion of root growth to search for water at the expense of leaf growth. The results provide evidence for the importance of antagonistic regulation of sulfur metabolism in leaves and roots to enable successful drought stress response at the whole plant level.

SULTR3;1 is a chloroplast-localized sulfate transporter in Arabidopsis thaliana.

Plants play a prominent role as sulfur reducers in the global sulfur cycle. Sulfate, the major form of inorganic sulfur utilized by plants, is absorbed and transported by specific sulfate transporters into plastids, especially chloroplasts, where it is reduced and assimilated into cysteine before entering other metabolic processes. How sulfate is transported into the chloroplast, however, remains unresolved; no plastid-localized sulfate transporters have been previously identified in higher plants. Here we report that SULTR3;1 is localized in the chloroplast, which was demonstrated by SULTR3;1-GFP localization, Western blot analysis, protein import as well as comparative analysis of sulfate uptake by chloroplasts between knockout mutants, complemented transgenic plants, and the wild type. Loss of SULTR3;1 significantly decreases the sulfate uptake of the chloroplast. Complementation of the sultr3;1 mutant phenotypes by expression of a 35S-SULTR3;1 construct further confirms that SULTR3;1 is one of the transporters responsible for sulfate transport into chloroplasts.

Targeted systems biology profiling of tomato fruit reveals coordination of the Yang cycle and a distinct regulation of ethylene biosynthesis during postclimacteric ripening.

The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the postclimacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid oxidase. At the same time, 1-aminocyclopropane-1-carboxylic acid synthase activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during postclimacteric ripening. Postclimacteric red tomatoes on the plant showed only a moderate regulation of 1-aminocyclopropane-1-carboxylic acid synthase and Yang cycle genes compared with the regulation in detached fruit. Treatment of red fruit with 1-methylcyclopropane and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene biosynthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal.

A Low Level of NaCl Stimulates Plant Growth by Improving Carbon and Sulfur Assimilation in Arabidopsis thaliana.

High-salinity stress represses plant growth by inhibiting various metabolic processes. In contrast to the well-studied mechanisms mediating tolerance to high levels of salt, the effects of low levels of salts have not been well studied. In this study, we examined the growth of Arabidopsis thaliana plants under different NaCl concentrations. Interestingly, both shoot and root biomass increased in the presence of 5 mM NaCl, whereas more than 10 mM NaCl decreased plant biomass. To clarify the biological mechanism by which a low level of NaCl stimulated plant growth, we analyzed element accumulation in plants grown under different NaCl concentrations. In addition to the Na and Cl contents, C, S, Zn, and Cu contents were increased under 5 mM NaCl in shoots; this was not observed at higher NaCl concentrations. Adverse effects of high salinity, such as decreased levels of nitrate, phosphate, sulfate, and some cations, did not occur in the presence of 5 mM NaCl. An increase in C was possibly attributed to increased photosynthesis supported by Cl, Zn, and Cu, which also increased in shoots after NaCl application. Salt stress-responsive gene expression was enhanced under 20 mM NaCl but not at lower doses. Among the S metabolites analyzed, cysteine (Cys) was increased by 5 mM NaCl, suggesting that S assimilation was promoted by this dose of NaCl. These results indicate the usefulness of NaCl for plant growth stimulation.

Overexpression of serine acetlytransferase produced large increases in O-acetylserine and free cysteine in developing seeds of a grain legume.

There have been many attempts to increase concentrations of the nutritionally essential sulphur amino acids by modifying their biosynthetic pathway in leaves of transgenic plants. This report describes the first modification of cysteine biosynthesis in developing seeds; those of the grain legume, narrow leaf lupin (Lupinus angustifolius, L.). Expression in developing lupin embryos of a serine acetyltransferase (SAT) from Arabidopsis thaliana (AtSAT1 or AtSerat 2;1) was associated with increases of up to 5-fold in the concentrations of O-acetylserine (OAS), the immediate product of SAT, and up to 26-fold in free cysteine, resulting in some of the highest in vivo concentrations of these metabolites yet reported. Despite the dramatic changes in free cysteine in developing embryos of SAT overexpressers, concentrations of free methionine in developing embryos, and the total cysteine and methionine concentrations in mature seeds were not significantly altered. Pooled F(2) seeds segregating for the SAT transgene and for a transgene encoding a methionine- and cysteine-rich sunflower seed storage protein also had increased OAS and free cysteine, but not free methionine, during development, and no increase in mature seed total sulphur amino acids compared with controls lacking SAT overexpression. The data support the view that the cysteine biosynthetic pathway is active in developing seeds, and indicate that SAT activity limits cysteine biosynthesis, but that cysteine supply is not limiting for methionine biosynthesis or for storage protein synthesis in maturing lupin embryos in conditions of adequate sulphur nutrition. OAS and free methionine, but not free cysteine, were implicated as signalling metabolites controlling expression of a gene for a cysteine-rich seed storage protein.

MTHFD1 controls DNA methylation in Arabidopsis.

DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases.

Diversity and regulation of ATP sulfurylase in photosynthetic organisms.

ATP sulfurylase (ATPS) catalyzes the first committed step in the sulfate assimilation pathway, the activation of sulfate prior to its reduction. ATPS has been studied in only a few model organisms and even in these cases to a much smaller extent than the sulfate reduction and cysteine synthesis enzymes. This is possibly because the latter were considered of greater regulatory importance for sulfate assimilation. Recent evidences (reported in this paper) challenge this view and suggest that ATPS may have a crucial regulatory role in sulfate assimilation, at least in algae. In the ensuing text, we summarize the current knowledge on ATPS, with special attention to the processes that control its activity and gene(s) expression in algae. Special attention is given to algae ATPS proteins. The focus on algae is the consequence of the fact that a comprehensive investigation of ATPS revealed that the algal enzymes, especially those that are most likely involved in the pathway of sulfate reduction to cysteine, possess features that are not present in other organisms. Remarkably, algal ATPS proteins show a great diversity of isoforms and a high content of cysteine residues, whose positions are often conserved. According to the occurrence of cysteine residues, the ATPS of eukaryotic algae is closer to that of marine cyanobacteria of the genera Synechococcus and Prochlorococcus and is more distant from that of freshwater cyanobacteria. These characteristics might have evolved in parallel with the radiation of algae in the oceans and the increase of sulfate concentration in seawater.

Transcriptome profiling of genes differentially modulated by sulfur and chromium identifies potential targets for phytoremediation and reveals a complex S-Cr interplay on sulfate transport regulation in B. juncea.

A differential display cDNA-AFLP derived technique was used to identify gene transcripts regulated by chromium (Cr) in relation to sulfur (S) nutrition in Brassica juncea. Twelve-day old plants were grown with 200 µM sulfate (+S), without sulfate (-S), with 200 µM sulfate plus 200 µM chromate (+S+Cr), or without sulfate plus 200 µM chromate (-S+Cr). Forty-four combinations of degenerate primers were assayed, which allowed the detection of 346 Transcript-Derived Fragments (TDFs) differentially regulated by Cr and S at times 0, 10 min, 1 h, 24 h. Eight sulfate transporters were identified, whose transcript abundance was dependent on the levels of plant S-compounds. For some of these transporters, a tight coordinated regulation of gene expression was observed in response to Cr. The MapMan analysis revealed a differential pattern of gene expression between +S+Cr and -S+Cr plants for several other transcripts and highlighted an overlap among responses to metals, defence against pathogens and senescence, hence suggesting the existence of common mechanisms of gene regulation. Among the identified gene transcripts, those involved in S metabolism and proteolitic processes may represent potential targets of genetic engineering in efforts to increase Cr accumulation and tolerance in plant species employed in phytoremediation techniques.

A mechanistic model of the cysteine synthase complex.

Plants and bacteria assimilate and incorporate inorganic sulfur into organic compounds such as the amino acid cysteine. Cysteine biosynthesis involves a bienzyme complex, the cysteine synthase (CS) complex. The CS complex is composed of the enzymes serine acetyl transferase (SAT) and O-acetyl-serine-(thiol)-lyase (OAS-TL). Although it is experimentally known that formation of the CS complex influences cysteine production, the exact biological function of the CS complex, the mechanism of reciprocal regulation of the constituent enzymes and the structure of the complex are still poorly understood. Here, we used docking techniques to construct a model of the CS complex from mitochondrial Arabidopsis thaliana. The three-dimensional structures of the enzymes were modeled by comparative techniques. The C-termini of SAT, missing in the template structures but crucial for CS formation, were modeled de novo. Diffusional encounter complexes of SAT and OAS-TL were generated by rigid-body Brownian dynamics simulation. By incorporating experimental constraints during Brownian dynamics simulation, we identified complexes consistent with experiments. Selected encounter complexes were refined by molecular dynamics simulation to generate structures of bound complexes. We found that although a stoichiometric ratio of six OAS-TL dimers to one SAT hexamer in the CS complex is geometrically possible, binding energy calculations suggest that, consistent with experiments, a ratio of only two OAS-TL dimers to one SAT hexamer is more likely. Computational mutagenesis of residues in OAS-TL that are experimentally significant for CS formation hindered the association of the enzymes due to a less-favorable electrostatic binding free energy. Since the enzymes from A. thaliana were expressed in Escherichia coli, the cross-species binding of SAT and OAS-TL from E. coli and A. thaliana was explored. The results showed that reduced cysteine production might be due to a cross-binding of A. thaliana OAS-TL with E. coli SAT. The proposed models of the enzymes and their complexes provide mechanistic insights into CS complexation.