Expression of potential reference genes in response to macronutrient stress in rice and soybean.

Given the complexity of nutrient stress responses and the availability of a few validated reference genes, we aimed to identify robust and stable reference genes for macronutrient stress in rice and soybean. Ten potential reference genes were evaluated using geNorm, NormFinder, BestKeeper, Comparative ΔCt method, and RefFinder algorithms under low and completely starved conditions of nitrogen (N), phosphorus (P), potassium (K), and sulphur (S). Results revealed distinct sets of reference gene pairs, showing stable expression under different experimental conditions. The gene pairs TIP41/UBC(9/10/18) and F-box/UBC10 were most stable in rice and soybean, respectively under N stress. Under P stress, UBC9/UBC10 in rice and F-Box/UBC10 in soybean were most stable. Similarly, TIP41/UBC10 in rice and RING FINGER/UBC9 in soybean were the best gene pairs under K stress while F-Box/TIP41 in rice and UBC9/UBC10 in soybean were the most stable gene pairs under S stress. These reference gene pairs were validated by quantifying the expression levels of high-affinity transporters like NRT2.1/NRT2.5, PT1, AKT1, and SULTR1 for N, P, K, and S stress, respectively. This study reiterates the importance of choosing reference genes based on crop species and the experimental conditions, in order to obtain concrete answers to missing links of gene regulation in response to macronutrient deficiencies.

The Transcription Factor EIL1 Participates in the Regulation of Sulfur-Deficiency Response.

Sulfur, an indispensable constituent of many cellular components, is a growth-limiting macronutrient for plants. Thus, to successfully adapt to changing sulfur availability and environmental stress, a sulfur-deficiency response helps plants to cope with the limited supply. On the transcriptional level, this response is controlled by SULFUR LIMITATION1 (SLIM1), a member of the ETHYLENE-INSENSITIVE3-LIKE (EIL) transcription factor family. In this study, we identified EIL1 as a second transcriptional activator regulating the sulfur-deficiency response, subordinate to SLIM1/EIL3. Our comprehensive RNA sequencing analysis in Arabidopsis (Arabidopsis thaliana) allowed us to obtain a complete picture of the sulfur-deficiency response and quantify the contributions of these two transcription factors. We confirmed the key role of SLIM1/EIL3 in controlling the response, particularly in the roots, but showed that in leaves more than 50% of the response is independent of SLIM1/EIL3 and EIL1. RNA sequencing showed an additive contribution of EIL1 to the regulation of the sulfur-deficiency response but also identified genes specifically regulated through EIL1. SLIM1/EIL3 seems to have further functions (e.g. in the regulation of genes responsive to hypoxia or mediating defense at both low and normal sulfur supply). These results contribute to the dissection of mechanisms of the sulfur-deficiency response and provide additional possibilities to improve adaptation to sulfur-deficiency conditions.

Sulfur depletion induces autophagy through Ecl1 family genes in fission yeast.

Autophagy is an intracellular degradation system widely conserved among various species. Autophagy is induced by the depletion of various nutrients, and this degradation mechanism is essential for adaptation to such conditions. In this study, we demonstrated that sulfur depletion induces autophagy in the fission yeast Schizosaccharomyces pombe. Based on the finding that autophagy induced by sulfur depletion was completely abolished in a mutant in which the ecl1, ecl2 and ecl3 genes were deleted (Δecls), we report that these three genes are essential for the induction of autophagy by sulfur depletion. Furthermore, autophagy-defective mutant cells exhibited poor growth and short lifespan (compared with wild-type cells) under the sulfur-depleted condition. These results indicated that the mechanism of autophagy is necessary for the appropriate adaptation to sulfur depletion.

Transcriptomic analysis at organ and time scale reveals gene regulatory networks controlling the sulfate starvation response of Solanum lycopersicum.

BACKGROUND: Sulfur is a major component of biological molecules and thus an essential element for plants. Deficiency of sulfate, the main source of sulfur in soils, negatively influences plant growth and crop yield. The effect of sulfate deficiency on plants has been well characterized at the physiological, transcriptomic and metabolomic levels in Arabidopsis thaliana and a limited number of crop plants. However, we still lack a thorough understanding of the molecular mechanisms and regulatory networks underlying sulfate deficiency in most plants. In this work we analyzed the impact of sulfate starvation on the transcriptome of tomato plants to identify regulatory networks and key transcriptional regulators at a temporal and organ scale. RESULTS: Sulfate starvation reduces the growth of roots and leaves which is accompanied by major changes in the organ transcriptome, with the response being temporally earlier in roots than leaves. Comparative analysis showed that a major part of the Arabidopsis and tomato transcriptomic response to sulfate starvation is conserved between these plants and allowed for the identification of processes specifically regulated in tomato at the transcript level, including the control of internal phosphate levels. Integrative gene network analysis uncovered key transcription factors controlling the temporal expression of genes involved in sulfate assimilation, as well as cell cycle, cell division and photosynthesis during sulfate starvation in tomato roots and leaves. Interestingly, one of these transcription factors presents a high identity with SULFUR LIMITATION1, a central component of the sulfate starvation response in Arabidopsis. CONCLUSIONS: Together, our results provide the first comprehensive catalog of sulfate-responsive genes in tomato, as well as novel regulatory targets for future functional analyses in tomato and other crops.

Proteasomal Degradation of Proteins Is Important for the Proper Transcriptional Response to Sulfur Deficiency Conditions in Plants.

Plants are continuously exposed to different abiotic and biotic stresses; therefore, to protect themselves, they depend on the fast reprogramming of large gene repertoires to prioritize the expression of a given stress-induced gene set over normal cellular household genes. The activity of the proteasome, a large proteolytic complex that degrades proteins, is vital to coordinate the expression of such genes. Proteins are labeled for degradation by the action of E3 ligases that site-specifically alter their substrates by adding chains of ubiquitin. Recent publications have revealed an extensive role of ubiquitination in the utilization of nutrients. This study presents the transcriptomic profiles of sulfur-deficient rosettes and roots of Arabidopsis thaliana rpt2a mutant with proteasomal malfunction. We found that genes connected with sulfur metabolism are regulated to the lesser extent in rpt2a mutant while genes encoding transfer RNAs and small nucleolar RNAs are highly upregulated. Several genes encoding E3 ligases are specifically regulated by sulfur deficiency. Furthermore, we show that a key transcription factor of sulfur deficiency response, Sulfur LIMitation1, undergoes proteasomal degradation and is able to interact with F-box protein, EBF1.

Silicon Regulates Source to Sink Metabolic Homeostasis and Promotes Growth of Rice Plants Under Sulfur Deficiency.

Being an essential macroelement, sulfur (S) is pivotal for plant growth and development, and acute deficiency in this element leads to yield penalty. Since the last decade, strong evidence has reported the regulatory function of silicon (Si) in mitigating plant nutrient deficiency due to its significant diverse benefits on plant growth. However, the role of Si application in alleviating the negative impact of S deficiency is still obscure. In the present study, an attempt was undertaken to decipher the role of Si application on the metabolism of rice plants under S deficiency. The results showed a distinct transcriptomic and metabolic regulation in rice plants treated with Si under both short and long-term S deficiencies. The expression of Si transporters OsLsi1 and OsLsi2 was reduced under long-term deficiency, and the decrease was more pronounced when Si was provided. The expression of OsLsi6, which is involved in xylem loading of Si to shoots, was decreased under short-term S stress and remained unchanged in response to long-term stress. Moreover, the expression of S transporters OsSULTR tended to decrease by Si supply under short-term S deficiency but not under prolonged S stress. Si supply also reduced the level of almost all the metabolites in shoots of S-deficient plants, while it increased their level in the roots. The levels of stress-responsive hormones ABA, SA, and JA-lle were also decreased in shoots by Si application. Overall, our finding reveals the regulatory role of Si in modulating the metabolic homeostasis under S-deficient condition.

Proteomics of Campylobacter jejuni Growth in Deoxycholate Reveals Cj0025c as a Cystine Transport Protein Required for Wild-type Human Infection Phenotypes.

Campylobacter jejuni is a major cause of food-borne gastroenteritis. Proteomics by label-based two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified proteins associated with growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts), and system-wide validation was performed by data-independent acquisition (DIA-SWATH-MS). LC-MS/MS quantified 1326 proteins (∼82% of the predicted C. jejuni proteome), of which 1104 were validated in additional biological replicates by DIA-SWATH-MS. DOC resulted in a profound proteome shift with 512 proteins showing significantly altered abundance. Induced proteins were associated with flagellar motility and antibiotic resistance; and these correlated with increased DOC motility and resistance to polymyxin B and ciprofloxacin. DOC also increased human Caco-2 cell adherence and invasion. Abundances of proteins involved in nutrient transport were altered by DOC and aligned with intracellular changes to their respective carbon sources. DOC increased intracellular levels of sulfur-containing amino acids (cysteine and methionine) and the dipeptide cystine (Cys-Cys), which also correlated with reduced resistance to oxidative stress. A DOC induced transport protein was Cj0025c, which has sequence similarity to bacterial Cys-Cys transporters. Deletion of cj0025c (Δcj0025c) resulted in proteome changes consistent with sulfur starvation, as well as attenuated invasion, reduced motility, atypical morphology, increased antimicrobial susceptibility and poor biofilm formation. Targeted metabolomics showed Δcj0025c could use known C. jejuni amino and organic acid substrates commensurate with wild-type. Medium Cys-Cys levels however, were maintained in Δcj0025c relative to wild-type. A toxic Cys-Cys mimic (selenocystine) inhibited wild-type growth, but not Δcj0025c Provision of an alternate sulfur source (2 mm thiosulfate) restored Δcj0025c motility. Our data confirm that Cj0025c is a Cys-Cys transporter that we have named TcyP consistent with the nomenclature of homologous proteins in other species.

Omics Data Reveal Putative Regulators of Einkorn Grain Protein Composition under Sulfur Deficiency.

Understanding the molecular mechanisms controlling the accumulation of grain storage proteins in response to nitrogen (N) and sulfur (S) nutrition is essential to improve cereal grain nutritional and functional properties. Here, we studied the grain transcriptome and metabolome responses to postanthesis N and S supply for the diploid wheat einkorn (Triticum monococcum). During grain filling, 848 transcripts and 24 metabolites were differentially accumulated in response to N and S availability. The accumulation of total free amino acids per grain and the expression levels of 241 genes showed significant modifications during most of the grain filling period and were upregulated in response to S deficiency. Among them, 24 transcripts strongly responded to S deficiency and were identified in coexpression network analyses as potential coordinators of the grain response to N and S supply. Sulfate transporters and genes involved in sulfate and Met metabolism were upregulated, suggesting regulation of the pool of free amino acids and of the grain N-to-S ratio. Several genes highlighted in this study might limit the impact of S deficiency on the accumulation of grain storage proteins.

Sulfur Deficiency Increases Phosphate Accumulation, Uptake, and Transport in Arabidopsis thaliana.

Recent studies have shown various metabolic and transcriptomic interactions between sulfur (S) and phosphorus (P) in plants. However, most studies have focused on the effects of phosphate (Pi) availability and P signaling pathways on S homeostasis, whereas the effects of S availability on P homeostasis remain largely unknown. In this study, we investigated the interactions between S and P from the perspective of S availability. We investigated the effects of S availability on Pi uptake, transport, and accumulation in Arabidopsis thaliana grown under sulfur sufficiency (+S) and deficiency (-S). Total P in shoots was significantly increased under -S owing to higher Pi accumulation. This accumulation was facilitated by increased Pi uptake under -S. In addition, -S increased root-to-shoot Pi transport, which was indicated by the increased Pi levels in xylem sap under -S. The -S-increased Pi level in the xylem sap was diminished in the disruption lines of PHT1;9 and PHO1, which are involved in root-to-shoot Pi transport. Our findings indicate a new aspect of the interaction between S and P by listing the increased Pi accumulation as part of -S responses and by highlighting the effects of -S on Pi uptake, transport, and homeostasis.

Sulfur Deficiency-Induced Glucosinolate Catabolism Attributed to Two ß-Glucosidases, BGLU28 and BGLU30, is Required for Plant Growth Maintenance under Sulfur Deficiency.

Sulfur (S) is an essential element for plants, and S deficiency causes severe growth retardation. Although the catabolic process of glucosinolates (GSLs), the major S-containing metabolites specific to Brassicales including Arabidopsis, has been recognized as one of the S deficiency (-S) responses in plants, the physiological function of this metabolic process is not clear. Two ß-glucosidases (BGLUs), BGLU28 and BGLU30, are assumed to be responsible for this catabolic process as their transcript levels were highly upregulated by -S. To clarify the physiological function of BGLU28 and BGLU30 and their roles in GSL catabolism, we analyzed the accumulation of GSLs and other S-containing compounds in the single and double mutant lines of BGLU28 and BGLU30 and in wild-type plants under different S conditions. GSL levels were highly increased, while the levels of sulfate, cysteine, glutathione and protein were decreased in the double mutant line of BGLU28 and BGLU30 (bglu28/30) under -S. Furthermore, transcript level of Sulfate Transporter1;2, the main contributor of sulfate uptake from the environment, was increased in bglu28/30 mutants under -S. With these metabolic and transcriptional changes, bglu28/30 mutants displayed obvious growth retardation under -S. Overall, our results indicate that BGLU28 and BGLU30 are required for -S-induced GSL catabolism and contribute to sustained plant growth under -S by recycling sulfate to primary S metabolism.

Photoautotrophic cultures of Chlamydomonas reinhardtii: sulfur deficiency, anoxia, and hydrogen production.

The aim of this work was a comparative study of S-repleted and S-depleted photoautotrophic cultures of Chlamydomonas reinhardtii under aerobic and anoxic conditions with the main focus on PSII activity. For that we used photobioreactor with short light path connected on-line to PAM fluorometer and cultivated microalgae in twice concentrated HS medium to avoid any uncontrolled limitation by mineral elements. Photoautotrophic cultures grown under Ar + CO2 gas mixture did not reach the same Chl (a + b) concentration as control culture (grown under air + CO2). At pO2 40% of air saturation (96 µM O2), the actual quantum yield of PSII started to decrease. Under microaerobic conditions when cultures stopped growing, the most significant changes in PSII function were observed. Maximum quantum yield Fv/Fm decreased significantly along with performance index, PIabs. It was accompanied by increase of fluorescence at J point, Vj. Results indicate that microaerobic conditions are stressful for photoautotrophic cultures. Photoautotrophic cultures of microalgae under S-deprivation in aerobic or anaerobic conditions showed similar behavior as photoheterotrophic ones described earlier. However, photoautotrophic cultures during anaerobiosis establishment did not show sharp "switch off" effect of actual quantum yield. We show also that S-deprivation under air or argon as well as the growth under Ar + CO2 cause significant increase of initial rise of fluorescence, which indicates that PSII and oxygen-evolving complex might be disintegrated.

Exogenous nitric oxide alleviates sulfur deficiency-induced oxidative damage in tomato seedlings.

Despite numerous reports on the role of nitric oxide (NO) in regulating plants growth and mitigating different environmental stresses, its participation in sulfur (S) -metabolism remains largely unknown. Therefore, we studied the role of NO in S acquisition and S-assimilation in tomato seedlings under low S-stress conditions by supplying NO to the leaves of S-sufficient and S-deficient seedlings. S-starved plants exhibited a substantial decreased in plant growth attributes, photosynthetic pigment chlorophyll (Chl) and other photosynthetic parameters, and activity of enzymes involved in Chl biosynthesis (δ-aminolevulinic acid dehydratase), and photosynthetic processes (carbonic anhydrase and RuBisco). Also, S-deficiency enhanced reactive oxygen species (ROS) (superoxide and hydrogen peroxide) and lipid peroxidation (malondialdehyde) levels in tomato seedlings. Contrarily, foliar supplementation of NO to S-deficient seedlings resulted in considerably reduced ROS formation in leaves and roots, which alleviated low S-stress-induced lipid peroxidation. However, exogenous NO enhanced proline accumulation by increasing proline metabolizing enzyme (Δ1-pyrroline-5-carboxylate synthetase) activity and also increased NO, hydrogen sulfide (a gasotransmitter small signaling molecule) and S uptake, and content of S-containing compounds (cysteine and reduced glutathione). Under S-limited conditions, NO improved S utilization efficiency of plants by upregulating the activity of S-assimilating enzymes (ATP sulfurylase, adenosine 5-phosphosulfate reductase, sulfide reductase and O-acetylserine (thiol) lyase). Under S-deprived conditions, improved S-assimilation of seedlings receiving NO resulted in improved redox homeostasis and ascorbate content through increased NO and S uptake. Application of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy l-3-oxide (an NO scavenger) invalidated the effect of NO and again caused low S-stress-induced oxidative damage, confirming the beneficial role of NO in seedlings under S-deprived conditions. Thus, exogenous NO enhanced the tolerance of tomato seedlings to limit S-triggered oxidative stress and improved photosynthetic performance and S assimilation.

Do nitrogen- and sulphur-remobilization-related parameters measured at the onset of the reproductive stage provide early indicators to adjust N and S fertilization in oilseed rape (Brassica napus L.) grown under N- and/or S-limiting supplies?

MAIN CONCLUSION: Specific combinations of physiological and molecular parameters associated with N and S remobilization measured at the onset of flowering were predictive of final crop performances in oilseed rape. Oilseed rape (Brassica napus L.) is a high nitrogen (N) and sulphur (S) demanding crop. Nitrogen- and S-remobilization processes allow N and S requirements to reproductive organs to be satisfied when natural uptake is reduced, thus ensuring high yield and seed quality. The quantification of physiological and molecular indicators of early N and S remobilization could be used as management tools to correct N and S fertilization. However, the major limit of this corrective strategy is to ensure the correlation between final performances-related variables and early measured parameters. In our study, four genotypes of winter oilseed rape (OSR) were grown until seed maturity under four nutritional modalities combining high and/or low N and S supplies. Plant final performances, i.e., seed production, N- and S-harvest indexes, seed N and S use efficiencies, and early parameters related to N- or S-remobilization processes, i.e., photosynthetic leaf area, N and S leaf concentrations, leaf soluble protein and leaf sulphate concentrations, and leaf RuBisCO abundance at flowering, were measured. We demonstrated that contrasting final performances existed according to the N and S supplies. An optimal N:S ratio supply could explain the treatment-specific crop performances, thus justifying N and S concurrent managements. Specific combinations of early measured plant parameters could be used to predict final performances irrespective of the nutritional supply and the genotype. This work demonstrates the potential of physiological and molecular indicators measured at flowering to reflect the functioning of N- and S-compound remobilization and to predict yield and quality penalties. However, because the predictive models are N and S independent, instant N and S leaf analyses are required to further adjust the adequate fertilization. This study is a proof of a concept which opens prospects regarding instant diagnostic tools in the context of N and S mineral fertilization management.

Isolation and Characterization of Chlamydomonas Autophagy-Related Mutants in Nutrient-Deficient Conditions.

Autophagy is a recycling system for amino acids and carbon- and nitrogen (N)-containing compounds. To date, the functional importance of autophagy in microalgae in nutrient-deficient conditions has not been evaluated by using autophagy-defective mutants. Here, we provide evidence which supports the following notions by characterizing an insertional mutant of the autophagy-related gene ATG8, encoding a ubiquitin-like protein necessary for the formation of the autophagosome in the green alga, Chlamydomonas reinhardtii. First, ATG8 is required for maintenance of cell survival and Chl content in N-, sulfur- and phosphate-deficient conditions. Secondly, ATG8 supports the degradation of triacylglycerol and lipid droplets after the resupply of N to cells cultured in N-limiting conditions. Thirdly, ATG8 is also necessary for accumulation of starch in phosphate-deficient conditions. Additionally, autophagy is not essential for maternal inheritance of the organelle genomes in gametogenesis.

Arabidopsis γ-glutamylcyclotransferase affects glutathione content and root system architecture during sulfur starvation.

γ-Glutamylcyclotransferase initiates glutathione degradation to component amino acids l-glutamate, l-cysteine and l-glycine. The enzyme is encoded by three genes in Arabidopsis thaliana, one of which (GGCT2;1) is transcriptionally upregulated by starvation for the essential macronutrient sulfur (S). Regulation by S-starvation suggests that GGCT2;1 mobilizes l-cysteine from glutathione when there is insufficient sulfate for de novo l-cysteine synthesis. The response of wild-type seedlings to S-starvation was compared to ggct2;1 null mutants. S-starvation causes glutathione depletion in S-starved wild-type seedlings, but higher glutathione is maintained in the primary root tip than in other seedling tissues. Although GGCT2;1 is induced throughout seedlings, its expression is concentrated in the primary root tip where it activates the γ-glutamyl cycle. S-starved wild-type plants also produce longer primary roots, and lateral root growth is suppressed. While glutathione is also rapidly depleted in ggct2;1 null seedlings, much higher glutathione is maintained in the primary root tip compared to the wild-type. S-starved ggct2;1 primary roots grow longer than the wild-type, and lateral root growth is not suppressed. These results point to a role for GGCT2;1 in S-starvation-response changes to root system architecture through activity of the γ-glutamyl cycle in the primary root tip. l-Cysteine mobilization from glutathione is not solely a function of GGCT2;1.

Melatonin alleviates low-sulfur stress by promoting sulfur homeostasis in tomato plants.

Despite involvement of melatonin (MT) in plant growth and stress tolerance, its role in sulfur (S) acquisition and assimilation remains unclear. Here we report that low-S conditions cause serious growth inhibition by reducing chlorophyll content, photosynthesis and biomass accumulation. S deficiency evoked oxidative stress leading to the cell structural alterations and DNA damage. In contrast, MT supplementation to the S-deprived plants resulted in a significant diminution in reactive oxygen species (ROS) accumulation, thereby mitigating S deficiency-induced damages to cellular macromolecules and ultrastructures. Moreover, MT promoted S uptake and assimilation by regulating the expression of genes encoding enzymes involved in S transport and metabolism. MT also protected cells from ROS-induced damage by regulating 2-cysteine peroxiredoxin and biosynthesis of S-compounds. These results provide strong evidence that MT can enhance plant tolerance to low-S-induced stress by improving S uptake, metabolism and redox homeostasis, and thus advocating beneficial effects of MT on increasing the sulfur utilization efficiency.

Sulfur nutrition stimulates lead accumulation and alleviates its toxicity in Populus deltoides.

Sulfur (S) can modulate plant responses to toxic heavy metals, but the underlying physiological and transcriptional regulation mechanisms remain largely unknown. To investigate the effects of S supply on lead (Pb)-induced toxicity in poplars, Populus deltoides monilifera (Aiton) Eckenw. saplings were exposed to 0 or 50 µM Pb together with one of the three S concentrations (0 (low S), 100 (moderate S) or 1500 (high S) µM Na2SO4). Populus deltoides roots absorbed Pb and it was partially translocated to the aerial organs, thereby decreasing the CO2 assimilation rate and leaf growth. Lead accumulation in poplars caused the overproduction of O2- and H2O2 to induce higher levels of total thiols (T-SH) and glutathione (GSH). Lead uptake by the roots and its accumulation in the aerial organs were repressed by low S application, but stimulated by high S supply. Lead-induced O2- and H2O2 production were exacerbated by S limitation, but alleviated by high S supply. Moreover, the concentrations of S-containing antioxidants including T-SH and GSH were reduced in S-deficient poplars, but increased in high S-treated plants, which corresponded well to the changes in the activities of enzymes involved in S assimilation and GSH biosynthesis. The transcript levels of both genes encoding sulfate transporters, i.e., SULTR1.1 and SULTR2.2, were elevated by low S application or high S supply in the roots, and the transcriptional upregulation of both genes was more pronounced under Pb exposure. Furthermore, the mRNA levels of several genes involved in S assimilation and the biosynthesis of GSH and phytochelatins, i.e., ATPS1, ATPS3, GSHS1, GSHS2 and PCS1, were upregulated in poplar roots with high S supply, particularly under Pb exposure. These results indicate that a high S supply can stimulate Pb accumulation and reduce its toxicity in poplars by improving S assimilation and stimulating the biosynthesis of S-containing compounds including T-SH and GSH.

Mitochondria dysfunctions under Fe and S deficiency: is citric acid involved in the regulation of adaptive responses?

Within the last years, extensive information has been accumulated on the reciprocal influence between S and Fe nutrition at both physiological and molecular level in several plant species, but the mechanisms regulating S and Fe sensing and signaling are not fully understood. Fe and S interact for the building of Fe-S clusters, and mitochondria is one of the cellular compartments where Fe-S cluster assembly takes place. Therefore, it would be expected that mitochondria might play a central role in the regulation of Fe and S interaction. The Fe deficiency-induced alteration in the synthesis of mitochondria-derived carboxylic acids, such as citric acid, and the evidence that such molecules have already been identified as important players of metabolite signaling in several organisms, further support this hypothesis. Tomato plants were grown under single or combined Fe and S deficiency with the aim of verifying whether mitochondria activities played a role in Fe/S interaction. Both Fe and S deficiencies determined similar alteration of respiratory chain activity: a general decrease of Fe-S containing complexes as well as an increase of alternative NAD(P)H activities was observed in both Fe and S deficient-plants. However, the content of Krebs cycle-related organic acids in roots was substantially different in response to treatments, being the accumulation of citric acid always increased, while the others (i.e. succinic, malic, fumaric acids) always decreased. Interestingly, citric acid levels significantly correlated with the expression of some Fe and S deficiency induced genes. Our results contribute to existing knowledge on the complexity of the S/Fe interaction, suggesting a model in which endogenous alteration of citric acid content in plant tissues might act as signal molecule for the regulation of some nuclear-encoded and nutrient-responsive genes and also provide a basis for further study of the mechanism underlying S and Fe sensing and signalling.

The mechanism of photosystem-II inactivation during sulphur deprivation-induced H2 production in Chlamydomonas reinhardtii.

Sulphur limitation may restrain cell growth and viability. In the green alga Chlamydomonas reinhardtii, sulphur limitation may induce H2 production lasting for several days, which can be exploited as a renewable energy source. Sulphur limitation causes a large number of physiological changes, including the inactivation of photosystem II (PSII), leading to the establishment of hypoxia, essential for the increase in hydrogenase expression and activity. The inactivation of PSII has long been assumed to be caused by the sulphur-limited turnover of its reaction center protein PsbA. Here we reinvestigated this issue in detail and show that: (i) upon transferring Chlamydomonas cells to sulphur-free media, the cellular sulphur content decreases only by about 25%; (ii) as demonstrated by lincomycin treatments, PsbA has a significant turnover, and other photosynthetic subunits, namely RbcL and CP43, are degraded more rapidly than PsbA. On the other hand, sulphur limitation imposes oxidative stress early on, most probably involving the formation of singlet oxygen in PSII, which leads to an increase in the expression of GDP-L-galactose phosphorylase, playing an essential role in ascorbate biosynthesis. When accumulated to the millimolar concentration range, ascorbate may inactivate the oxygen-evolving complex and provide electrons to PSII, albeit at a low rate. In the absence of a functional donor side and sufficient electron transport, PSII reaction centers are inactivated and degraded. We therefore demonstrate that the inactivation of PSII is a complex and multistep process, which may serve to mitigate the damaging effects of sulphur limitation.