吉本 尚子

Marasmin [S-(methylthiomethyl)-L-cysteine-4-oxide] is a pharmaceutically valuable sulfur-containing compound produced by the traditional medicinal plant, Tulbaghia violacea. Here, we report the identification of an S- oxygenase, TvMAS1, that produces marasmin from its corresponding sulfide, S-(methylthiomethyl)-L-cysteine. The amino acid sequence of TvMAS1 showed high sequence similarity to known flavin-containing S-oxygenating monooxygenases in plants. Recombinant TvMAS1 catalyzed regiospecific S-oxygenation at S4 of S-(methylthiomethyl)-L-cysteine to yield marasmin, with an apparent Km value of 0.55 mM. TvMAS1 mRNA accumulated with S-(methylthiomethyl)-L-cysteine and marasmin in various organs of T. violacea. Our findings suggest that TvMAS1 catalyzes the S-oxygenation reaction during the last step of marasmin biosynthesis in T. violacea.

S-Alk(en)ylcysteine sulfoxides (CSOs), such as methiin, alliin, and isoalliin, are health-beneficial natural products biosynthesized in the genus Allium. Here, we report the induction of multiple callus tissue lines from three Allium vegetables, onion (A. cepa), Welsh onion (A. fistulosum), and Chinese chive (A. tuberosum), and their ability to accumulate CSOs. Callus tissues were initiated and maintained in the presence of picloram and 2-isopentenyladenine as auxin and cytokinin, respectively. For all plant species tested, the callus tissues almost exclusively accumulated methiin as CSO, while the intact plants contained a substantial amount of isoalliin together with methiin. These results suggest that the cellular developmental conditions and the regulatory mechanisms required for the biosynthesis of methiin are different from those of alliin and isoalliin. The methiin content in the callus tissues of onion and Welsh onion was much higher compared to that in the intact plants, and its cellular concentration could be estimated as 1.9–21.7 mM. The activity of alliinase that degrades CSOs in the callus tissues was much lower than that of the intact plants for onion and Welsh onion, but at similar levels as in the intact plants for Chinese chive. Our findings that the callus tissues of onion and Welsh onion showed high methiin content and low alliinase activity highlighted their potential as a plant-based system for methiin production.

一般的に含硫化合物には化学反応性が高いものが多く，天然の含硫化合物の中にも特異な生物活性を示すものが多く存在する．アブラナ科植物が含有するグルコシノレートや，ヒガンバナ科ネギ属植物が含有するアリイン類は，これらの植物の特徴的な香味の原因となる含硫化合物である．近年の研究により，グルコシノレートが癌の予防や改善に，アリイン類が癌や循環器系疾患に加えて加齢による認知機能低下の予防や改善に役立つことが示された．本稿では，グルコシノレートとアリイン類の健康機能発揮のメカニズムと，これら含硫化合物を含む植物の生薬やサプリメントとしての利用状況について概説する．

ケシが生合成するモルヒナン型アルカロイドのうち，モルヒネはがんや外傷による疼痛の緩和に用いられる天然オピオイドとして，テバインは半合成オピオイドであるオキシコドンやオピオイド受容体拮抗薬であるナロキソン等の製造原料として，極めて重要な化合物である．気象災害や病害によるケシ収穫量減少に伴う医薬品の供給不足を避けるため，遺伝子組換え微生物を用いて，モルヒネやテバインを安定で効率的に生産する技術の開発が期待されている．今回，モルヒネ生合成において非酵素的に進むと考えられていたテバイン合成反応を触媒する酵素が同定され，効率的なテバインの異種生物生産が可能になったので紹介する．

The secret of chemical diversity and function of specialized metabolites in medicinal plants will be unveiled by study of functional genomics at an unprecedentedly rapid rate in the coming years. This is mostly ascribed to the remarkable advancement in the high-throughput DNA sequencing together with other omics technologies such as metabolomics, in particular, due to drastic reduction in the cost of acquiring, storing and analyzing massive omics datasets. Once the genes involved in a biosynthetic pathway of specialized compounds in plants are elucidated, synthetic biology or genome editing can be applied to produce the target compounds in an engineered organism or to manipulate the pathway in planta. Coupled with these advancements in pathway elucidation approaches, modern plant biotechnology strategies are bound to significantly contribute to the sustainable development goals set by United Nations.

連載第一回では、植物の硫黄の吸収と同化を担うトランスポーターや酵素の機能について紹介した。硫黄は全ての生物にとって不可欠な必須多量元素である。動物は硫黄の還元・同化の仕組みを体内に持たないため、植物による硫黄の還元・同化は全ての生物の生存にとって重要である。植物にとっても、生存に必要な硫黄源を獲得し、タンパク質生産に必要なシステインやメチオニンを合成することは必須である。しかしながら、植物が育つ環境は一定ではなく、硫黄源の量も土壌によって異なり、また環境に応じて変動する。このため、植物は様々な環境変化に応じて硫黄の輸送や同化の効率を最適化する仕組みを発達させている。連載第二回では、植物による硫黄の吸収や同化のうち、特に硫酸イオン吸収の制御機構について紹介する。

硫黄は地球上の全ての生物にとって不可欠な必須多量元素である。これは、生体内において硫黄が、タンパク質を構成するアミノ酸のうちシステインやメチオニン、ビタミンのうちビタミンB1（チアミン）やビオチン等、生命活動に必要な有機硫黄化合物に含まれるからである。環境中では、硫黄の多くは無機硫黄である硫酸イオンとして存在する。植物は、環境中の硫酸イオンを生体内に取り込み、システイン等の有機硫黄化合物を合成する。一方、人類を含む動物は、硫酸イオンから有機硫黄化合物を合成する経路を持たず、植物を摂取することで必要な有機硫黄源を得ている。すなわち、植物による硫酸イオンの獲得と有機硫黄化合物の合成は、植物自身のみならず我々人類を含む動物の生命活動にとっても不可欠な、極めて重要な代謝プロセスである。本連載では、植物における硫酸イオンの吸収とシステインへの同化の分子機構とその制御について、２回に分けて取り上げる。連載第一回では、植物の硫黄の吸収と同化を担うトランスポーターや酵素の機能について、分子生物学的研究のモデル植物であるシロイヌナズナに関する知見を例として紹介する。

S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RCSS)-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to -glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored -glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

Pueraria lobata (Willd.) Ohwi has a long and broad application in the treatment of disease. However, in the US and EU, it is treated as a notorious weed. The information to be gained from decoding the deep transcriptome profile would facilitate further research on P lobata. In this study, more than 93 million fastq format reads were generated by Illumina's next-generation sequencing approach using five types of P lobata tissue, followed by CLC de novo assembly methods, ultimately yielding about 83,041 contigs in total. Then BLASTx similarity searches against the NCBI NR database and UniProtKB database were conducted. Once the duplicates among BLASTx hits were eliminated, ID mapping against the UniProt database was conducted online to retrieve Gene Ontology information. In search of the putative genes relevant to essential biosynthesis pathways, all 1,348 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. Enzymes related to the isoflavonoid and flavonoid biosynthesis pathways were focused for detailed investigation and subsequently, quantitative real-time reverse transcription polymerase chain reaction was conducted for biological validation. Metabolites of interest, puerarin and daidzin were studied by HPLC. The findings in this report may serve as a footstone for further research into this promising medicinal plant.

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transeriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

S-Alk(en)yl-L-cysteine sulfoxides are sulfur-containing secondary metabolites characteristically found in the genus Allium. Upon tissue damage, they are converted to a variety of sulfur-containing compounds that have a range of pharmacological activities. Despite the pharmaceutical importance of S-alk(en)yl-L-cysteine sulfoxides, to date very little is known about the molecular details of their biosynthesis. Previous tracer experiments have indicated that S-oxygenation reactions to convert biosynthetic intermediate sulfide compounds to their respective sulfoxides are involved in the later stage of the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides. In order to obtain molecular insights into the biosynthesis of S-alk(en)-yl-L-cysteine sulfoxides, we searched for nucleotide sequences homologous to known genes encoding flavin-containing monooxygenases (FMOs) that have S-oxygenation activities, and identified two EST clones from onion (Allium cepa). The deduced amino acid sequences of these ESTs showed the high sequence similarity to Arabidopsis FMO GS-OX proteins catalyzing S-oxygenation of S-methylthioalkyl glucosinolates, and contained some sequence motifs typically found in plant FMOs. These observations suggest that the onion ESTs identified in this study were derived from genes encoding functional FMO proteins catalyzing the S-oxygenation reactions, which may be required for the biosynthesis of S-alk(en)-yl-L-cysteine sulfoxides. Future studies aimed at isolating full-length cDNAs corresponding to these ESTs and elucidating the functions of their encoded proteins may provide new insight into the molecular mechanisms underlying the biosynthesis of S-alk(en)yl-L-cysteine sulfoxides.

Plants assimilate inorganic sulfate into sulfur-containing vital metabolites. ATP sulfurylase (AIRS) is the enzyme catalyzing the key entry step of the sulfate assimilation pathway in both plastids and cytosol in plants. Arabidopsis thaliana has four ATPS genes (ATPS1, 2, 3, and 4) encoding AIRS pre-proteins containing N-terminal transit peptide sequences for plastid targeting, however, the genetic identity of the cytosolic AIRS has remained unverified. Here we show that Arabidopsis ATPS2 dually encodes plastidic and cytosolic AIRS isoforms, differentiating their subcellular localizations by initiating translation at AUGmeti to produce plastid-targeted ATPS2 pre-proteins or at AUGmet52 or AUGmet58 within the transit peptide to have ATPS2 stay in cytosol. Translational initiation of ATPS2 at AUGMet52 or AUGmet58 was verified by expressing a tandem-fused synthetic gene, ATPS2(5/ UTR Hisl 2) :Renilla luciferase:ATPS2(//e13 Va177): firefly luciferase, under a single constitutively active CaMV 35S promoter in Arabidopsis protoplasts and examining the activities of two different luciferases translated in-frame with split N-terminal portions of ATPS2. Introducing missense mutations at AUG Met52 and AUG Met58 significantly reduced the firefly luciferase activity, while AUGmet52 was a relatively preferred site for the alternative translational initiation. The activity of luciferase fusion protein starting at AUGMet52 or AUGmet58 was not modulated by changes in sulfate conditions. The dual localizations of ATPS2 in plastids and cytosol were further evidenced by expression of ATPS2-GFP fusion proteins in Arabidopsis protoplasts and transgenic lines, while they were also under control of tissue-specific ATPS2 promoter activity found predominantly in leaf epidermal cells, guard cells, vascular tissues and roots.

Contamination of soil by heavy metals such as Cd causes a serious negative impact on agricultural production and human health. Thus, improvement of tolerance to Cd is one of the major challenges in plant biotechnology. In the present study, we have generated transgenic Nicotiana tabacum (tobacco) plants overexpressing both serine acetyltransferase (SAT) and cysteine synthase (CS) [O-acetylserine (thiol)-lyase], which are committed in the last two steps of cysteine (Cys) biosynthesis, by crossing the respective single-gene transgenic plants. Two enzymatic activities were high in these two-gene overexpressing plants, and these plants exhibited more resistance to Cd stress than wild-type and single-gene transgenic plants. The two-gene transgenic plants also exhibited a higher production of phytochelatins (PCs) in an inducible manner by the Cd stress. The levels of free non-chelated Cd were lower in the two-gene transgenic plants than the wild-type and single-gene transformants. The levels of Cys and gamma-glutamylcysteine (gamma-EC) were also increased in the dual transgenic plants, presumably enhancing the metabolic flow of Cys biosynthesis leading to the ultimate synthesis of PCs which detoxify Cd by chelating. These results suggested that the overexpression of two genes, SAT and CS, could be a promising strategy for engineering Cd resistant plants.

The essential macronutrient sulfur enters plants as inorganic sulfate, which is assimilated by reduction to sulfide and incorporation into cysteine or by transfer to various metabolites as organic sulfo-group. The two pathways share the activation of sulfate by adenylation to adenosine 5'-phosphosulfate (APS). The further fate of APS is decided in interplay of two enzymes, APS reductase and APS kinase. Here we summarize recent progress in analysis of APS kinase and its role in plant sulfur metabolism as well as the contribution of the two enzymes to control of flux through sulfate assimilation and partitioning of sulfur between primary and secondary metabolism.

Sulfur is an essential macronutrient for plants with important roles in biological structure and function. Although it has long been known that sulfate uptake, assimilation and metabolism are highly controlled by sulfur availability, the detailed mechanism of regulation has only recently begun to be elucidated. In this review, we highlight recent advances in our understanding of the molecular and cellular basis of plant response to sulfur limitation.

Sulfate is an essential nutrient cycled in nature. Ion transporters that specifically facilitate the transport of sulfate across the membranes are found ubiquitously in living organisms. The phylogenetic analysis of known sulfate transporters and their homologous proteins from eukaryotic organisms indicate two evolutionarily distinct groups of sulfate transport systems. One major group named Tribe 1 represents yeast and fungal SUL, plant SULTR, and animal 5LC26 families.The evolutionary origin of SULTR family members in land plants and green algae is suggested to be common with yeast and fungal SUL and animal anion exchangers (5LC26). The lineage of plant SULTR family is expanded into four subfamilies (SULTR1 SULTR4) in land plant species. By contrast, the putative SULTR homologs from Chlorophyte green algae are in two separate lineages; one with the subfamily of plant tonoplast-localized sulfate transporters (SULTR4), and the other diverged before the appearance of lineages for SUL, SULTR, and 5LC26. There also was a group of yet undefined members of putative sulfate transporters in yeast and fungi divergent from these major lineages in Tribe 1. The other distinct group is Tribe 2, primarily composed of animal sodium-dependent sulfate/carboxylate transporters (SLC13) and plant tonoplastlocalized dicarboxylate transporters (TDT). The putative sulfur-sensing protein (SAC1) and SAC1-like transporters (SLT) of Chlorophyte green algae, bryophyte, and lycophyte show low degrees of sequence similarities with SLC13 and TDT. However, the phylogenetic relationship between SAC1/SLT and the other two families, SLC13 and TDT in Tribe 2, is not clearly supported. In addition, the SAC1/SLT family is absent in the angiosperm species analyzed. The present study suggests distinct evolutionary trajectories of sulfate transport systems for land plants and green algae.

MicroRNAs play a key role in the control of plant development and response to adverse environmental conditions. For example, microRNA395 (miR395), which targets three out of four isoforms of ATP sulfurylase, the first enzyme of sulfate assimilation, as well as a low-affinity sulfate transporter, SULTR2;1, is strongly induced by sulfate deficiency. However, other components of sulfate assimilation are induced by sulfate starvation, so that the role of miR395 is counterintuitive. Here, we describe the regulation of miR395 and its targets by sulfate starvation. We show that miR395 is important for the increased translocation of sulfate to the shoots during sulfate starvation. MiR395 together with the SULFUR LIMITATION 1 transcription factor maintain optimal levels of ATP sulfurylase transcripts to enable increased flux through the sulfate assimilation pathway in sulfate-deficient plants. Reduced expression of ATP sulfurylase (ATPS) alone affects both sulfate translocation and flux, but SULTR2;1 is important for the full rate of sulfate translocation to the shoots. Thus, miR395 is an integral part of the regulatory circuit controlling plant sulfate assimilation with a complex mechanism of action.

硫黄は自然界に多く存在する多量元素であり、生物においては生命維持に欠かせない必須元素の一つである。植物は土壌液中に含まれる硫酸イオンを吸収し、システインやメチオニン、ビタミンや補酵素、グルコシノレートやアリイン等に代表される含硫代謝物を生合成するための硫黄源として利用する。このため、植物は硫黄欠乏土壌では含硫代謝物を十分に合成できず生育が阻害される。また、硫黄欠乏植物はアミノ酸代謝バランスが変化しアスパラギンを過剰蓄積するが、これは加熱処理により発がん性物質であるアクリルアミドの蓄積を引き起こす。すなわち、植物の硫酸イオンの利用効率は、質的・量的に優れた作物を生産する上で重要な因子の1つである。近年、様々な植物種から硫酸イオンの輸送に関わるトランスポーターやその制御因子が同定され、植物における硫酸イオンの輸送と調節に関する分子レベルでの理解が進められている。本稿ではシロイヌナズナを中心に、植物の硫酸イオントランスポーター群の機能分担と発現制御について最近の知見を含めて紹介する。

Plants play an important role in the global sulphur cycle because they assimilate sulphur from the environment and build it into methionine and cysteine. Several genes of the sulphur assimilation pathway are regulated by microRNA-395 (miR395) that is itself induced by a low-sulphur (-S) environment. Here, we show that the six Arabidopsis miR395 loci are induced differently. We find that MIR395 loci are expressed in the vascular system of roots and leaves and root tips. Induction of miR395 by a -S environment in both roots and leaves suggests that translocation of miR395 from leaves to roots through the phloem is not necessary for plants growing on -S soil/medium. We also demonstrate that induction of miR395 is controlled by SLIM1, a key transcription factor in the sulphur assimilation pathway. Unexpectedly, the mRNA level of a miR395 target gene, SULTR2;1, strongly increases during miR395 induction in roots. We show that the spatial expression pattern of MIR395 transcripts in the vascular system does not appear to overlap with the expression pattern previously reported for SULTR2;1 mRNA. These results illustrate that negative temporal correlation between the expression level of a miRNA and its target gene in a complex tissue cannot be a requirement for target gene validation.

Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles.

硫黄は生体反応に必要な様々な物質に含まれており、動植物の生育に不可欠な元素である。植物は無機硫黄を有機硫黄に同化する硫黄同化系を有するのに対し、動物は硫黄同化系を持たず植物が合成した硫黄代謝物を摂取して有機硫黄源として利用するため、植物の硫黄同化系は植物のみならず動物にとっても重要な代謝経路であるといえる。植物の硫黄同化に関与する遺伝子については1990年代より大腸菌や酵母の変異株の機能相補スクリーニングによる同定が進み、その分子機能について解析が行われてきた。一方、2000年にシロイヌナズナのゲノム解読が完了した後は、ポストゲノム的手法を用いた研究によりイソ酵素群の機能分担の解明や、同化系の制御因子の同定が進んでいる。本稿では、植物の硫黄同化系とその制御についてモデル植物のシロイヌナズナを材料として進められた研究を中心に紹介する。

High-affinity sulfate transporters SULTR1;1 and SULTR1;2 are expressed at epidermis and cortex of Arabidopsis (Arabidopsis thaliana) roots during sulfur limitation. Here, we report that SULTR1;1 and SULTR1;2 are two essential components of the sulfate uptake system in roots and are regulated at posttranscriptional levels together with the previously reported transcriptional control. Double knockout of SULTR1; 1 and SULTR1;2 by T-DNA insertion gene disruption resulted in complete lack of sulfate uptake capacity and severely affected plant growth under low-sulfur conditions. Expression of epitope-tagged proteins SULTR1;1mycHis and SULTR1;2mycHis, under the control of the cauliflower mosaic virus 35S promoter, rescued the uptake of sulfate and the growth of the sultr1;1 sultr1;2 double knockout mutant. The recovery of the double knockout phenotypes was attributable to the posttranscriptional accumulation of sulfate transporter proteins that derive from the epitope-tagged transgenic constructs. Both SULTR1;1mycHis and SUTLR1;2mycHis mRNAs were predominantly found in roots and slightly induced by long-term sulfur limitation. SULTR1;1mycHis and SULTR1;2mycHis proteins were found exclusively in roots, and significantly accumulated by sulfur limitation, correlating with the induction of sulfate uptake activities. In the time course of short-term sulfate starvation treatment, SULTR1; 1mycHis and SULTR1;2mycHis proteins were significantly accumulated during the 8- to 72-h period, causing substantial induction of sulfate uptake activities, while their corresponding mRNAs were expressed constantly around the initial levels, except for the transient induction in the first 2 h. This study suggested the importance of root-specific and sulfur deficiency-inducible accumulation of SULTR1;1 and SULTR1; 2 sulfate transporter proteins for the acquisition of sulfate from low-sulfur environment.

For the effective recycling of nutrients, vascular plants transport pooled inorganic ions and metabolites through the sieve tube. A novel sulfate transporter gene, Sultr1;3, was identified as an essential member contributing to this process for redistribution of sulfur source in Arabidopsis. Sultr1;3 belonged to the family of high-affinity sulfate transporters, and was able to complement the yeast sulfate transporter mutant. The fusion protein of Sultr1;3 and green fluorescent protein was expressed by the Sultr1;3 promoter in transgenic plants, which revealed phloem-specific expression of Sultr1;3 in Arabidopsis. Sultr1;3-green fluorescent protein was found in the sieve element-companion cell complexes of the phloem in cotyledons and roots. Limitation of external sulfate caused accumulation of Sultr1;3 mRNA both in leaves and roots. Movement of (35)S-labeled sulfate from cotyledons to the sink organs was restricted in the T-DNA insertion mutant of Sultr1;3. These results provide evidence that Sultr1;3 transporter plays an important role in loading of sulfate to the sieve tube, initiating the source-to-sink translocation of sulfur nutrient in Arabidopsis.

Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1,2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9+/-1.0 muM). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1,2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 muM sulfate), whereas Sultr1,2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 muM sulfate), indicating that Sultr1,2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.