Nakako Shibagaki

An Arabidopsis thaliana high-affinity molybdate transporter required for efficient uptake of molybdate from soil

Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the Km value of MOT1 for molybdate is ≈20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

The Role of the STAS Domain in the Function and Biogenesis of a Sulfate Transporter as Probed by Random Mutagenesis

Sulfate transporters in plants represent a family of proteins containing transmembrane domains that constitute the catalytic part of the protein and a short linking region that joins this catalytic moiety with a C-terminal STAS domain. The STAS domain resembles an anti-sigma factor antagonist of Bacillus subtilis, which is one distinguishing feature of the SLC26 transporter family; this family includes transporters for sulfate and other anions such as iodide and carbonate. Recent work has demonstrated that this domain is critical for the activity of Arabidopsis thaliana sulfate transporters, and specific lesions in this domain, or the exchange of STAS domains between different sulfate transporters, can severely impair transport activity. In this work we generated a Saccharomyces cerevisiae expression library of the A. thaliana Sultr1;2 gene with random mutations in the linking region-STAS domain and identified STAS domain lesions that altered Sultr1;2 biogenesis and/or function. A number of mutations in the β-sheet that forms the core of the STAS domain prevented intracellular accumulation of Sultr1;2. In contrast, the linking region and one surface of the STAS domain containing N termini of the first and second α-helices have a number of amino acids critical for the function of the protein; mutations in these regions still allow protein accumulation in the plasmamembrane, but the protein is no longer capable of efficiently transporting sulfate into cells. These results suggest that the STAS domain is critical for both the activity and biosynthesis/stability of the transporter, and that STAS sub-domains correlate with these specific functions.

Insights into the acclimation of Chlamydomonas reinhardtii to sulfur deprivation

During sulfur deprivation, the photosynthetic green alga Chlamydomonas reinhardtii develops a high-affinity sulfate uptake system and increases the expression of genes encoding proteins involved in sulfur assimilation. Although two regulatory elements, SAC1 and SAC3, have been shown to be required for normal acclimation of C. reinhardtii to sulfur deprivation, a number of other regulatory elements appear to also be involved. The molecular mechanisms by which these regulatory elements function are largely unknown. This manuscript presents our current knowledge of sulfur deprivation responses and the regulation of these responses in C. reinhardtii. In addition, we present preliminary results of a sub-saturation screen for novel sulfur acclimation mutants of C. reinhardtii. A speculative model, incorporating the activities of established regulatory elements with putative novel components of the signal transduction pathway(s) is discussed.

Binding of Cysteine Synthase to the STAS Domain of Sulfate Transporter and Its Regulatory Consequences

The sulfate ion (SO42−) is transported into plant root cells by SO42− transporters and then mostly reduced to sulfide (S2−). The S2− is then bonded to O-acetylserine through the activity of cysteine synthase (O-acetylserine (thiol)lyase or OASTL) to form cysteine, the first organic molecule of the SO42− assimilation pathway. Here, we show that a root plasma membrane SO42− transporter of Arabidopsis, SULTR1;2, physically interacts with OASTL. The interaction was initially demonstrated using a yeast two-hybrid system and corroborated by both in vivo and in vitro binding assays. The domain of SULTR1;2 shown to be important for association with OASTL is called the STAS domain. This domain is at the C terminus of the transporter and extends from the plasma membrane into the cytoplasm. The functional relevance of the OASTL-STAS interaction was investigated using yeast mutant cells devoid of endogenous SO42− uptake activity but co-expressing SULTR1;2 and OASTL. The analysis of SO42− transport in these cells suggests that the binding of OASTL to the STAS domain in this heterologous system negatively impacts transporter activity. In contrast, the activity of purified OASTL measured in vitro was enhanced by co-incubation with the STAS domain of SULTR1;2 but not with the analogous domain of the SO42− transporter isoform SULTR1;1, even though the SULTR1;1 STAS peptide also interacts with OASTL based on the yeast two-hybrid system and in vitro binding assays. These observations suggest a regulatory model in which interactions between SULTR1;2 and OASTL coordinate internalization of SO42− with the energetic/metabolic state of plant root cells.

Characterization of the major bacterial–fungal populations colonizing dandruff scalps in Shanghai, China, shows microbial disequilibrium

Dandruff is a scalp disorder characterized by the formation of flaky white‐yellowish scales due to an altered proliferation and differentiation status; a disrupted barrier function; a decrease in the level of hydration and of natural moisturizing factors (NMF) in the scalp, with a persistent and relapsing inflammatory condition. It was recently reported that an imbalance between bacterial and fungal species colonizing the scalp of French volunteers was associated with dandruff condition. The purpose of the present study was to analyze the major bacterial and fungal species present on the scalp surface of Chinese volunteers and to investigate possible region‐related variation in the microbiota linked to dandruff condition. The data obtained from the Chinese populations were highly similar to those obtained in France, confirming that dandruff scalps are associated with a higher incidence of Malassezia restricta and Staphylococcal sp. The ratios of Malassezia to Propionibacterium and Propionibacterium to Staphylococcus were also significantly higher in the dandruff volunteers as compared to normal volunteers, suggesting that equilibrium between the major bacterial and fungal taxa found on the normal scalps is perturbed in the dandruff scalps. The main difference between the French and Shanghai subjects was in their Staphylococcal biota. The results obtained in China and in France suggest that targeting one particular Malassezia sp. by antifungals instead of using large spectrum antifungals and rebalancing the dandruff scalp microbiota could be common approach to improve dandruff condition in the two countries.

Arabidopsis SNRK2.3 protein kinase is involved in the regulation of sulfur-responsive gene expression and O-acetyl-l-serine accumulation under limited sulfur supply

Abstract The role of the Arabidopsis thaliana genes SNRK2s, which are similar to the sulfur-regulatory gene SAC3 of Chlamydomonas reinhardtii, in the response of plants to sulfur was studied. The Arabidopsis genome contains 10 genes (SNRK2.1 to SNRK2.10) similar to SAC3. Among these genes, transcript accumulation of several genes including SNRK2.3 was induced in response to sulfur starvation. Independently isolated transgenic A. thaliana lines carrying T-DNA insertions in SNRK2.3 exhibited reduced sulfur-starvation induction of the transcript of the sulfate transporter SULTR2;2 gene, whereas the accumulation of O-acetyl-l-serine under sulfur deficiency conditions increased. These results suggest that SNRK2.3 plays a beneficial role in the regulation of gene expression and metabolism in response to sulfur starvation.

The State of Sulfur Metabolism in Algae: From Ecology to Genomics

232 I. Algae and the Global Sulfur Cycle 232 II. Sulfur Metabolism 233 A. SO4 2− Transport Systems 233 B. ATP Sulfurylase 237 C. APS Kinase 237 D. APS and PAPS Reductases 237 E. Sulfite Reductase 238 F. Serine Acetyltransferase 238 G. OASTL 239 H. Methionine Synthesis 239 I. Glutathione Synthesis 239 J. Phytochelatin Synthase 240 K. DMSP Synthesis 240 L. DMSP Degradation and Its Consequences 241 III. Non-Protein S Compounds 242 A. Sulpholipids 242 B. Sulfated Oligosaccharides 243 C. Thioredoxin and Glutaredoxin 243 D. Glutathione/Phytochelatin 245 E. DMSP and DMS 247 F. S-Containing Cofactors 247 IV. Selenocysteine Metabolism 248 V. Adaptation and Acclimation to S Deficiency 248 A. Cell Growth and Division 249 B. Changes Extracellular Proteins 249 C. Arylsulfatase and Hydrolytic Activity 249 D. SO 4 2− Transport 250 E. SO4 2− Assimilation 250 F. Metabolic Changes Elicited by S Deprivation 251 1. Down-Regulating Photosynthesis 251 2. Accumulation of Starch 252 3. H2 Production in S-Starved Cells 252 VI. Regulatory Mechanisms Controlling Responses to Sulfur Deficiency 253 A. SAC1 253 B. SAC2 255 C. SAC3 255 D. Other Mutants 255

Responses to Macronutrient Deprivation

Photosynthetic organisms have developed elaborate mechanisms to acquire macronutrients and to adjust to conditions in which those nutrients become limiting to growth. Some of the responses of photosynthetic organisms to macronutrient limitation may be specific for a particular nutrient and involve the development of various mechanisms to scavenge the limiting nutrient from the external milieu, which may require elevated synthesis of high affinity transport systems, the redistribution of internal nutrient stores and the synthesis of hydrolytic enzymes that release the nutrient from organic substrates in the soil. Other responses may be of a more general nature, occurring during a number of different nutrient limitation conditions, and involve modifying the biosynthetic machinery of the cell, including the photosynthetic apparatus. In this review we focus on the acquisition of the macronutrients nitrogen, sulfur and phosphorus from the environment, and the ways in which the unicellular green alga Chlamydomonas reinhardtii acclimates to changes in its nutrient environment.

Aging-related changes in the diversity of women’s skin microbiomes associated with oral bacteria

Skin aging is associated with changes in cutaneous physiology including interactions with a skin microbial community. A striking alteration and diversification in the skin microbiome with aging was observed between two different age groups of 37 healthy Japanese women, i.e. younger adults of 21–37 years old and older adults of 60–76 years old, using bacterial 16S rRNA gene sequencing. The analyses revealed that the alpha diversity/species richness was significantly higher in the older than the younger group for the cheek and forehead microbiomes, while the beta diversity in the overall structure significantly differed particularly for the forearm and scalp microbiomes between the two age groups. Taxonomic profiling showed a striking reduction in the relative abundance of the majority skin genus Propionibacterium in the cheek, forearm and forehead microbiomes of the older adults, and identified 38 species including many oral bacteria that significantly differentiated the two age groups with a skin site dependency. Furthermore, we found chronological age-related and unrelated skin clinical parameters that correlate with the observed changes in the skin microbiome diversity. Thus, our data suggested that the diversification of skin microbiomes in adult women was largely affected by chronological and physiological skin aging in association with oral bacteria.

Homomeric Interaction of the STAS Domain in Sultr1;2

We recently reported the interaction of the STAS domain of SULTR1;2 and O-acetylserine(thiol)lyase (OASTL). This inter-protein interaction was initially identified in the yeast two hybrid system and was confirmed using other methods. Interestingly, we also found that the STAS domain (comprised of both the L and STAS’ regions) of SULTR1;2 undergoes a homomeric interaction; an association between two STAS domains occurs as a consequence of a specific interaction between the L and STAS’ regions of the domain. The strength of this interaction depends on whether one or both of the interacting STAS partners contains an L region. A similar interaction was observed for the L and STAS’ regions of SULTR3;1 but not of SULTR1;1. Lesions that alter the SULTR1;2 L and STAS’ interactions do not appear to markedly impact sulfate transport activity, at least in yeast cells, although they may impact transport activity of SULTR1;2 in Arabidopsis. Hence, while regulation of sulfate transporter activity occurs through interactions of the transporter with OASTL, transporter activity may also be modulated by homomeric interactions in the STAS domain.