Malcolm J. Hawkesford

Systems rebalancing of metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants.

Sulfur is an essential macroelement in plant and animal nutrition. Plants assimilate inorganic sulfate into two sulfur-containing amino acids, cysteine and methionine. Low supply of sulfate leads to decreased sulfur pools within plant tissues. As sulfur-related metabolites represent an integral part of plant metabolism with multiple interactions, sulfur deficiency stress induces a number of adaptive responses, which must be coordinated. To reveal the coordinating network of adaptations to sulfur deficiency, metabolite profiling of Arabidopsis has been undertaken. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry techniques revealed the response patterns of 6,023 peaks of nonredundant ion traces and relative concentration levels of 134 nonredundant compounds of known chemical structure. Here, we provide a catalogue of the detected metabolic changes and reconstruct the coordinating network of their mutual influences. The observed decrease in biomass, as well as in levels of proteins, chlorophylls, and total RNA, gives evidence for a general reduction of metabolic activity under conditions of depleted sulfur supply. This is achieved by a systemic adjustment of metabolism involving the major metabolic pathways. Sulfur/carbon/nitrogen are partitioned by accumulation of metabolites along the pathway O-acetylserine to serine to glycine, and are further channeled together with the nitrogen-rich compound glutamine into allantoin. Mutual influences between sulfur assimilation, nitrogen imbalance, lipid breakdown, purine metabolism, and enhanced photorespiration associated with sulfur-deficiency stress are revealed in this study. These responses may be assembled into a global scheme of metabolic regulation induced by sulfur nutritional stress, which optimizes resources for seed production.

Biofortification of UK food crops with selenium

Se is an essential element for animals. In man low dietary Se intakes are associated with health disorders including oxidative stress-related conditions, reduced fertility and immune functions and an increased risk of cancers. Although the reference nutrient intakes for adult females and males in the UK are 60 and 75 microg Se/d respectively, dietary Se intakes in the UK have declined from >60 microg Se/d in the 1970s to 35 microg Se/d in the 1990s, with a concomitant decline in human Se status. This decline in Se intake and status has been attributed primarily to the replacement of milling wheat having high levels of grain Se and grown on high-Se soils in North America with UK-sourced wheat having low levels of grain Se and grown on low-Se soils. An immediate solution to low dietary Se intake and status is to enrich UK-grown food crops using Se fertilisers (agronomic biofortification). Such a strategy has been adopted with success in Finland. It may also be possible to enrich food crops in the longer term by selecting or breeding crop varieties with enhanced Se-accumulation characteristics (genetic biofortification). The present paper will review the potential for biofortification of UK food crops with Se.

Influence of Iron Status on Cadmium and Zinc Uptake by Different Ecotypes of the Hyperaccumulator Thlaspi caerulescens

We have previously identified an ecotype of the hyperaccumulator Thlaspi caerulescens (Ganges), which is far superior to other ecotypes (including Prayon) in Cd uptake. In this study, we investigated the effect of Fe status on the uptake of Cd and Zn in the Ganges and Prayon ecotypes, and the kinetics of Cd and Zn influx using radioisotopes. Furthermore, the T. caerulescens ZIP (Zn-regulated transporter/Fe-regulated transporter-like protein) genes TcZNT1-G andTcIRT1-G were cloned from the Ganges ecotype and their expression under Fe-sufficient and -deficient conditions was analyzed. Both short- and long-term studies revealed that Cd uptake was significantly enhanced by Fe deficiency only in the Ganges ecotype. The concentration-dependent kinetics of Cd influx showed that theV max of Cd was 3 times greater in Fe-deficient Ganges plants compared with Fe-sufficient plants. In Prayon, Fe deficiency did not induce a significant increase inV max for Cd. Zn uptake was not influenced by the Fe status of the plants in either of the ecotypes. These results are in agreement with the gene expression study. The abundance ofZNT1-G mRNA was similar between the Fe treatments and between the two ecotypes. In contrast, abundance of theTcIRT1-G mRNA was greatly increased only in Ganges root tissue under Fe-deficient conditions. The present results indicate that the stimulatory effect of Fe deficiency on Cd uptake in Ganges may be related to an up-regulation in the expression of genes encoding for Fe2+ uptake, possibly TcIRT1-G.

Vacuolar Sulfate Transporters Are Essential Determinants Controlling Internal Distribution of Sulfate in Arabidopsis

Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [35S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [35S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of low-sulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.

Functions of Macronutrients

Publisher Summary This chapter focuses on the role played by various macronutrients such as nitrogen (N), sulfur (S), phosphorus (P), magnesium (Mg), calcium (Ca), and potassium (K) in plant metabolism and growth and describes the symptoms of deficiency and toxicity of these macronutrients. N is the most essential element required after carbon, and it plays a central role in plant metabolism as a constituent of proteins, nucleic acids, chlorophyll, coenzymes, phytohormones, and secondary metabolites. When it is taken as ammonium or nitrate, it is assimilated into amino acids either in the roots or shoots and within the plant, it is translocated as nitrate or amino acids. Sulfur is taken up as sulphate and assimilated into S-containing amino acids such as cysteine that are used to synthesize S-containing enzymes and coenzymes as well as secondary compounds such as phytochelatins (detoxification of metals) or aliins and glucosinolates (feeding deterrents). Phosphorus is a structural element in nucleic acids, and as a component of adenosine phosphates, it plays an important role in energy transfer, and it is also essential for transfer of carbohydrates in leaf cells. Magnesium is a component of chlorophyll, and it is required for photosynthesis and protein synthesis. Calcium is important for cell wall and membrane stabilization, osmoregulation, and as second messenger, thereby allowing plants to regulate developmental processes in response to environmental stimuli. The main role of K is osmoregulation, which is important for cell extension and stomata movement, and it affects loading of sucrose and the rate of mass flow-driven solute movement within the plant.

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H2S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H2S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H2S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.

Variation in Molybdenum Content Across Broadly Distributed Populations of Arabidopsis thaliana Is Controlled by a Mitochondrial Molybdenum Transporter (MOT1)

Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 - MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence and expression of MOT1 tagged with GFP in protoplasts and transgenic plants, establishing the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants and shows that novel loci can be detected by association mapping.

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane.

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)+ RNA. This clone contains an insert which is 2775 by in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (Km = 7.5 ± 0.6 μM for SO42−) was observed. A genomic mutant of S. cerevisiae in which 1096 by were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.

Molecular mechanisms of phosphate and sulphate transport in plants

The application of molecular techniques in recent years has advanced our understanding of phosphate and sulphate transport processes in plants. Genes encoding phosphate and sulphate transporters have been isolated from a number of plant species. The transporters encoded by these genes are related to the major facilitator superfamily of proteins. They are predicted to contain 12 membrane-spanning domains and function as H(+)/H(2)PO(-4) or H(+)/SO(2/-4) cotransporters. Both high-affinity and low-affinity types have been identified. Most research has concentrated on genes that encode transporters expressed in roots. The expression of many of these genes is transcriptionally regulated by signals that respond to the nutrient status of the plant. Nutrient demand and the availability of precursors needed in the assimilatory pathways also regulate transcription of some of these genes. Information on the cell types in which phosphate and sulphate transporters are expressed is becoming available. These data, together with functional characterisation of the transporters, are enabling the roles of various transporters in the overall phosphate and sulphate nutrition of plants to be defined.

Sulphate/proton cotransport in plasma-membrane vesicles isolated from roots of Brassica napus L. : increased transport in membranes isolated from sulphur-starved plants

The characteristics of sulphate uptake into right-side-out plasma-membrane vesicles isolated from roots of Brassica napus L., Metzger, cv. Drakkar, and purified by aqueous polymer two-phase partitioning, were investigated. Sulphate uptake into the vesicles was driven by an artificially imposed pH gradient (acid outside), and could be observed for 5–10 min before a plateau was reached and no further net uptake occurred. The uptake was partially inhibited in the presence of depolarizing agents and little uptake was observed in the absence of an imposed pH gradient. Uptake was strongly pH-dependent, being greatest at more acidic pH. After imposition of a pH gradient, the capacity for uptake decreased slowly (t1/2>10 min). The uptake had a high-affinity component which was strongly dependent on the external proton concentration (Km=10μM at pH 5.0, 64 μM at pH 6.5). The Km for protons varied from 0.4–1.9 μM as the sulphate concentration was reduced from 33 to 1 μM. A low-affinity component was observed which could be resolved at low temperatures (0 °C). Microsomal membranes that partitioned into the lower phase of the two-phase system gave no indication of high-affinity sulphate transport. Sulphate uptake into plasma-membrane vesicles isolated from sulphur-starved plant material was approximately twofold greater than that observed in those isolated from sulphate-fed plant material. Isolated vesicles therefore mirror the well-known in-vivo response of roots, indicating an increase in the number of transporters to be, at least in part, the underlying cause of derepression.