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Grafting of commercial varieties onto transgenic stress-tolerant rootstocks is attractive approach, because fruit from the non-transgenic plant body does not contain foreign genes. RNA silencing can modulate gene expression and protect host plants from viruses and insects, and small RNAs (sRNAs), key molecules of RNA silencing, can move systemically. Here, to evaluate the safety of foods obtained from sRNA-recipient plant bodies, we investigated the effects of rootstock-derived sRNAs involved in mediating RNA-directed DNA methylation (RdDM) on non-transgenic scions. We used tobacco rootstocks showing RdDM against the cauliflower mosaic virus (CaMV) 35S promoter. When scions harboring CaMV 35S promoter sequence were grafted onto RdDM-inducing rootstocks, we found that RdDM-inducing sRNAs were only weakly transported from the rootstocks to the scion, and we observed a low level of DNA methylation of the CaMV 35S promoter in the scion. Next, wild-type (WT) tobacco scions were grafted onto RdDM-inducing rootstocks (designated NT) or WT rootstocks (designated NN), and scion leaves were subjected to multi-omics analyses. Our transcriptomic analysis detected 55 differentially expressed genes between the NT and NN samples. A principal component analysis of proteome profiles showed no significant differences. In the positive and negative modes of LC-ESI-MS and GC-EI-MS analyses, we found a large overlap between the metabolomic clusters of the NT and NN samples. In contrast, the negative mode of a LC-ESI-MS analysis showed separation of clusters of NT and NN metabolites, and we detected 6 peak groups that significantly differed. In conclusion, we found that grafting onto RdDM-inducing rootstocks caused a low-level transmission of sRNAs, resulting in limited DNA methylation in the scion. However, the causal relationships between sRNA transmission and the very slight changes in the transcriptomic and metabolomic profiles of the scions remains unclear. The safety assessment points for grafting with RdDM rootstocks are discussed.

Grafting of non-transgenic scion onto genetically modified (GM) rootstocks provides superior agronomic traits in the GM rootstock, and excellent fruits can be produced for consumption. In such grafted plants, the scion does not contain any foreign genes, but the fruit itself is likely to be influenced directly or indirectly by the foreign genes in the rootstock. Before market release of such fruit products, the effects of grafting onto GM rootstocks should be determined from the perspective of safety use. Here, we evaluated the effects of a transgene encoding β-glucuronidase (GUS) on the grafted tomato fruits as a model case. An edible tomato cultivar, Stella Mini Tomato, was grafted onto GM Micro-Tom tomato plants that had been transformed with the GUS gene. The grafted plants showed no difference in their fruit development rate and fresh weight regardless of the presence or absence of the GUS gene in the rootstock. The fruit samples were subjected to transcriptome (NGS-illumina), proteome (shotgun LC-MS/MS), metabolome (LC-ESI-MS and GC-EI-MS), and general food ingredient analyses. In addition, differentially detected items were identified between the grafted plants onto rootstocks with or without transgenes (more than two-fold). The transcriptome analysis detected approximately 18,500 expressed genes on average, and only 6 genes were identified as differentially expressed. Principal component analysis of 2,442 peaks for peptides in proteome profiles showed no significant differences. In the LC-ESI-MS and GC-EI-MS analyses, a total of 93 peak groups and 114 peak groups were identified, respectively, and only 2 peak groups showed more than two-fold differences. The general food ingredient analysis showed no significant differences in the fruits of Stella scions between GM and non-GM Micro-Tom rootstocks. These multiple omics data showed that grafting on the rootstock harboring the GUS transgene did not induce any genetic or metabolic variation in the scion.

Significance Hormone-like peptides derived from small coding genes (Arabidopsis , we showed that four genes conferred increased salinity stress tolerance when overexpressed in transgenic plants. One of the four genes ( AtPROPEP3 ) was found to induce salinity stress tolerance by treatment with a 13-peptide (KPTPSSGKGGKHN) fragment, providing unique functional evidence for enhanced salinity stress tolerance in plants in response to a peptide treatment. Although the 13-peptide fragment shares homology with known peptides associated with immune response, the other peptides may encode unique hormone-like peptides associated with salinity stress tolerance.

AtPEPTIDE RECEPTOR2 (AtPEPR2) is a member of leucine-rich repeat receptor-like kinase family and binds to a group of AtPROPEP gene-encoded endogenous peptides, AtPeps. Previously, we found that AtPEPR2 plays a moderate role in the AtPep1-mediated innate immunity responses in Arabidopsis leaf. In this study, we found that AtPEPR2 promoter has strong activity in the vascular tissues of the roots and the atpepr2 mutants showed a moderate but significantly shorter root phenotype. AtPEPR2 partially mediated AtPep1-induced root elongation inhibition. AtPep1-triggered cytosolic Ca(2+) transient rise in roots showed partial dependence on AtPEPR2 and fully on extracellular Ca(2+) ([Ca(2+) ]ext ). Transcriptional profiling analysis found that expression of 75% of AtPep1-modulated genes in roots was fully dependent on AtPEPR2, of which two dramatically induced genes showed partial dependence on the [Ca(2+) ]ext . Arabidopsis genome contains seven Glutamine Dumpers genes (AtGDUs), encoding amino acid exporters. Three of them (AtGDU2, 3, 5) were among the top 10 genes that were downregulated by AtPep1 through AtPEPR2 fully dependent pathway. Treatment with AtPep1 strongly suppressed promoter activity of AtGDU3 in roots, which was relieved by chelating [Ca(2+) ]ext . Arabidopsis overexpressing AtGDU3 showed a shorter root phenotype and decreased sensitivity to the AtPep1-mediated inhibition of root elongation. Taken together, this study demonstrated a significant role of AtPEPR2 in the AtPep1-mediated signaling in the roots.

Spinach (Spinacia oleracea L.) develops leaf rosettes under short-day conditions, and starts reproductive growth including bolting and flowering under long-day conditions. Japanese people prefer Oriental spinach that bolts easily with a shorter photoperiod than European spinach. This is one of the main reasons that Oriental spinach is difficult to grow year-round. In order to understand spinach flowering mechanisms and obtain knowledge for spinach breeding, we isolated one CONSTANS-like (COL) and two FLOWERING LOCUS T (FT) homologs, which are key components of photoperiodic regulation of flowering time, from a Japanese cultivar. The expression of SoCOL1 showed diurnal rhythm with the highest expression at the end of the dark cycle. This diurnal rhythm is similar to the expression of BvCOL1 from sugar beet (Beta vulgaris), whose flower-promoting effect was observed when overexpressed in Arabidopsis. Phylogenetic analysis showed that SoCOL1 is the closest homolog of BvCOL1, suggesting that SoCOL1 is an ortholog of BvCOL1. SoFT1 and SoFT2 are closely related to BvFT1 and BvFT2, respectively. The expression of SoFT1 and SoFT2 were induced in advance of flower bud formation after changing the photoperiod, but the expression level of SoFT1 was much lower than SoFT2. Currently, we are speculating that SoFT2 is a flower-promoting factor of spinach, and that SoFT1 has a role in light signaling because the expression of SoFT1 showed a diurnal rhythm.

Plant defense responses against invading organisms are initiated through the perception of molecules associated with attacking microbes and herbivores by pattern recognition receptors. In addition to elicitor molecules derived from attacking organisms, plants recognize host-derived molecules. These endogenous elicitors induce and amplify the defense responses against invading organisms both locally and systemically. Several classes of plant-derived molecules elicit defense, including cell wall fragments and peptides. Endogenous peptide elicitors have been discovered in species across the plant kingdom, and their role regulating immunity to both herbivores and pathogens is becoming increasingly appreciated. In this review, we will focus on the five known endogenous peptide elicitor families, summarize their properties, and discuss research goals to further understanding of plant innate immunity.

Only a handful of endogenous peptide defense signals have been isolated from plants. Herein, we report a novel peptide from soybean (Glycine max) leaves that is capable of alkalinizing the media of soybean suspension cells, a response that is generally associated with defense peptides. The peptide, DHPRGGNY, was synthesized and found to be active at 0.25 nm and requiring only 5 to 10 min to obtain a maximal pH change. The peptide is located on the carboxy-terminal end of a 52-amino acid precursor protein (Glyma12g00990) deduced from the soybean genome project. A search of the soybean databank revealed a homolog (Glyma09g36370) that contained a similar peptide, DLPRGGNY, which was synthesized and shown to have identical activity. The peptides, designated GmPep914 (DHPRGGNY) and GmPep890 (DLPRGGNY), were capable of inducing the expression of both Glyma12g00990 (GmPROPEP914) and Glyma09g36370 (GmPROPEP890) in cultured soybean suspension cells within 1 h. Both peptides induced the expression of defense genes, including CYP93A1, a cytochrome P450 gene involved in phytoalexin synthesis, chitinaseb1-1, a chitinase involved in pathogen defense, and Glycine max chalcone synthase1 (Gmachs1), chalcone synthase, involved in phytoalexin production. Both GmPROPEP914 and GmPROPEP890 were highly expressed in the roots, relative to the aerial portions of the plant. However, treatment of the aerial portion of soybean plants with hormones involved in elicitation of defense responses revealed a significant increase in expression levels of GmPROPEP914 and GmPROPEP890. A search of gene databases revealed homologous sequences in other members of the Fabales and also in the closely related Cucurbitales but not in any other order of plants.

GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1-11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4-12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.

A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogen-defense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor-like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2-dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes ( PDF1.2 , MPK3 , and WRKY33 ) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen-defense genes in a Ca 2+ -dependent manner.

Rapid Alkalinization Factor (RALF) is a 49-amino acid peptide initially isolated from tobacco leaves that is capable of arresting both root and pollen tube growth. With suspension cells, addition of RALF causes an elevation of the pH of the extracellular media, caused by the blockage of a proton pump. RALF associates with a putative receptor(s) on the surface of the plant cell, initiating a signal transduction pathway. Although the exact function(s) of RALFs are unknown, its presence throughout the plant kingdom attests to its importance in some type of basic regulatory role. In the present study, deletion and substitution analyses of RALF reveal a specific - YISY - motif located at positions 5 through 8 from the N-terminus, highly conserved within the plant kingdom, which is a requirement for productive binding of RALF to its putative receptor. Replacement of isoleucine with alanine in the - YISY - motif caused a severe reduction in alkalinization of suspension cell media and a loss of root growth inhibition with tomato seedlings.

Among the arsenal of plant-derived compounds activated upon attack by herbivores and pathogens are small peptides that initiate and amplify defense responses. However, only a handful of plant signaling peptides have been reported. Here, we have isolated a 12-aa peptide from soybean ( Glycine max ) leaves that causes a pH increase of soybean suspension-cultured cell media within 10 min at low nanomolar concentrations, a response that is typical of other endogenous peptide elicitors and pathogen-derived elicitors. The amino acid sequence was determined and was found to be derived from a member of the subtilisin-like protease (subtilase) family. The sequence of the peptide was located within a region of the protein that is unique to subtilases in legume plants and not found within any other plant subtilases thus far identified. We have named this peptide signal G lycine m ax Sub tilase Pep tide ( Gm SubPep). The gene (Glyma18g48580) was expressed in all actively growing tissues of the soybean plant. Although transcription of Glyma18g48580 was not induced by wounding, methyl jasmonate, methyl salicylate, or ethephon, synthetic Gm SubPep peptide, when supplied to soybean cultures, induced the expression of known defense-related genes, such as Cyp93A1 , Chib-1b , PDR12 , and achs . Gm SubPep is a unique plant defense peptide signal, cryptically embedded within a plant protein with an independent metabolic role, providing insights into plant defense mechanisms.

Pep1 is a 23–amino acid peptide that enhances resistance to a root pathogen, Pythium irregulare. Pep1 and its homologs (Pep2 to Pep7) are endogenous amplifiers of innate immunity of Arabidopsis thaliana that induce the transcription of defense-related genes and bind to PEPR1, a plasma membrane leucine-rich repeat (LRR) receptor kinase. Here, we identify a plasma membrane LRR receptor kinase, designated PEPR2, that has 76% amino acid similarity to PEPR1, and we characterize its role in the perception of Pep peptides and defense responses. Both PEPR1 and PEPR2 were transcriptionally induced by wounding, treatment with methyl jasmonate, Pep peptides, and pathogen-associated molecular patterns. The effects of Pep1 application on defense-related gene induction and enhancement of resistance to Pseudomonas syringae pv tomato DC3000 were partially reduced in single mutants of PEPR1 and PEPR2 and abolished completely in double mutants. Photoaffinity labeling and binding assays using transgenic tobacco (Nicotiana tabacum) cells expressing PEPR1 and PEPR2 clearly demonstrated that PEPR1 is a receptor for Pep1-6 and that PEPR2 is a receptor for Pep1 and Pep2. Our analysis demonstrates differential binding affinities of two receptors with a family of peptide ligands and the corresponding physiological effects of the specific receptor–ligand interactions. Therefore, we demonstrate that, through perception of Peps, PEPR1 and PEPR2 contribute to defense responses in Arabidopsis.

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants.

AtPep1, a 23-amino acid peptide recently isolated from Arabidopsis leaves, induces the expression of the genes encoding defense proteins against pathogens. We investigated the structure–activity relationship of AtPep1 with its receptor, a 170 kDa leucine-rich repeat receptor kinase (AtPEPR1) by utilizing a suspension cell assay (the alkalinization assay). Binding of AtPep1 to AtPEPR1 on the cell surface is accompanied by an increase in the pH of Arabidopsis suspension cell media by 1 pH unit in 15 min with a half-maximal response of 0.25 nM. Sequential removal of N-terminal amino acids had little effect on activity until the peptide was reduced to 15 amino acids [AtPep1(9–23)], which decreased the activity by less than one order of magnitude. Activity was completely abolished when nine C-terminal amino acids remained. Removal of the C-terminal asparagine from AtPep1(9–23), resulted in a decrease in activity ( 1 2 max ∼ 100 nM). AtPep1(9–23) was used for alanine-substitution analysis and revealed two important residues for activity, a serine, [A15]AtPep1(9–23) ( 1 2 max ∼ 10 nM), and a glycine, [A17]AtPep1(9–23) ( 1 2 max ∼ 1000 nM). Neither [A17]AtPep1(9–23) nor the C-terminal truncated AtPep1, AtPep1(9–22), were able to compete with AtPep1(9–23) in the alkalinization assay. The importance of the glycine residue for binding to the AtPep receptor was also confirmed by competition assays using radiolabeled AtPep1. d -Alanine or 2-methylalanine substituted at the glycine position displayed only a slight decrease in activity whereas l - and d -proline substitution caused a loss of activity. Homologs of AtPep1 identified in Arabidopsis and other species revealed a strict conservation of the glycine residue.

Plants cope with pathogens with distinct mechanisms. One example is a gene-for-gene system, in which plants recognize the pathogen molecule by specified protein(s), this being called the R factor. However, mechanisms of interaction between proteins from the host and the pathogen are not completely understood. Here, we analyzed the mode of interaction between the N factor, a tobacco R factor, and the helicase domain (p50) of tobacco mosaic virus (TMV). To this end, domain dissected proteins were prepared and subjected to Agroinfiltration into intact leaves, followed by yeast two hybrid and pull-down assays. The results pointed to three novel features. First, the N factor was found to directly bind to the p50 of TMV, second, ATP was pre-requisite for this interaction, with formation of an ATP/N factor complex, and third, the N factor was shown to possess ATPase activity, which is enhanced by the p50. Moreover, we found that intra- and/or inter-molecular interactions take place in the N factor molecule. This interaction required ATP, and was disrupted by the p50. Based on these results, we propose a following model for the TMV recognition mechanism in tobacco plants. The N factor forms a complex with ATP, to which the helicase domain interacts, and enhances ATP hydrolysis. The resulting ADP/N factor complex then changes its conformation, thereby facilitating further interaction with the down-stream signaling factor(s). This model is consistent with the idea of 'protein machine'.

Hydrogen sulfide is a major environmental pollutant, highly toxic to living organisms at high concentrations. Even at low concentrations, it causes an unpleasant odor from wetlands, especially from wastewater. Plants can utilize hydrogen sulfide as a sulfur source to synthesize cysteine, which then serves as the principal substrate for synthesis of other sulfur containing compounds including glutathione and methionine. It was thus feasible to use aquatic plants, which possess high potential for sulfur assimilation, to remove hydrogen sulfide from the wetland. To this end, we have generated transgenic rice plants over-expressing cysteine synthase, a key enzyme in the sulfur assimilation pathway, and evaluated their capacity for sulfur uptake on hydrogen sulfide treatment. The obtained transgenic plants exhibited 3-fold elevated cysteine synthase activity, and incorporated more hydrogen sulfide into cysteine and glutathione than their wild type counterparts upon exposure to a high level of hydrogen sulfide. These observations suggest that over-expression of cysteine synthase in aquatic plants is a viable approach to remove hydrogen sulfide from polluted environments.

Adenosine phosphosulfate (APS) reductase is one of key enzymes in the sulfur assimilation pathway in higher plants, catalyzing the formation of adenosine 5′-phosphosulfate from sulfate and ATP. In order to improve sulfur uptake capacity of water spinach (Ipomea aquatica), a plant which commonly grows wild in Southern Asia and has good potential for sequestration of environmental pollutants like sulfuric compounds, an Arabidopsis gene (APR1), encoding a plastid-resident APS reductase, was introduced into cut cotyledons via Agrobacterium-mediated transformation. Among 267 regenerated shoots initially obtained from 2,119 cotyledon explants, two were found to efficiently express the introduced gene and could be grown to maturity. APS reductase activity in leaves was estimated to be over 2-fold the wild-type level. Upon cultivation in the presence of 2 mM sodium sulfate, a 2.5-fold higher sulfate uptake was observed in comparison with wild-type plants. When grown in the presence of toxic levels of sulfide or cadmium, they showed a higher tolerance with increased fresh weight as compared with controls. These results suggest that transcription from the introduced gene indeed strengthened the sulfur assimilation pathway, and that the generated plants may be practically useful for phytoremediation.

Immediate early responsive genes were screened by the differential display method during the hypersensitive response upon tobacco mosaic virus infection of tobacco ( Nicotiana tabacum L.) plants carrying the N gene. Three hours after temperature shift from 30 degrees C to 20 degrees C, an increase in transcripts of a particular clone was observed. The cDNA encoded a polypeptide of 330 amino acids, whose topology indicated it to be a seven-transmembrane protein, designated as Nt7TM1. This was confirmed by direct observation of cultured tobacco cells expressing an Nt7TM1-green fluorescent protein fusion protein, which migrated exclusively to the plasma membrane and the endoplasmic reticulum. RNA blot hybridization analysis indicated that Nt7TM1 transcripts were not induced by salicylic or jasmonic acids, ethylene or hydrogen peroxide. The results suggested the presence of a unique system for pathogen response involving a novel seven-transmembrane protein.

Caffeine is synthesized from xanthosine throughN-methylation and ribose removal steps. In the present study, three types of cDNAs encodingN-methyltransferases were isolated from immature fruits of coffee (Coffea arabica) plants, and designated asCaXMT1, CaMXMT2, andCaDXMT1, respectively. The bacterially expressed encoded proteins were characterized for their catalytic properties. CaXMT1 catalyzed formation of 7-methylxanthosine from xanthosine with aK m value of 78 μm, CaMXMT2 catalyzed formation of 3,7-dimethylxanthine (theobromine) from 7-methylxanthine with a K m of 251 μm, and CaDXMT1 catalyzed formation of 1,3,7-trimethylxanthine (caffeine) from 3,7-dimethylxanthine with aK m of 1,222 μm. The crude extract of Escherichia coli was found to catalyze removal of the ribose moiety from 7-methylxanthosine, leading to the production of 7-methylxanthine. As a consequence, when all three recombinant proteins and E. coli extract were combined, xanthosine was successfully converted into caffeine in vitro. Transcripts for CaDXMT1 were predominantly found to accumulate in immature fruits, whereas those for CaXMT1and CaMXMT2 were more broadly detected in sites encompassing the leaves, floral buds, and immature fruits. These results suggest that the presently identified threeN-methyltransferases participate in caffeine biosynthesis in coffee plants and substantiate the proposed caffeine biosynthetic pathway: xanthosine → 7-methylxanthosine → 7-methylxanthine → theobromine → caffeine.

Tobacco (Nicotiana tabacum) genes regulated during the early stage of responses to wounding were screened by a modified fluorescence differential display method. Among 28 genes initially identified, a particular clone designatedNtC7 was subjected to further analysis. Its transcripts were found to accumulate rapidly and transiently within 1 h upon treatments with not only wounding but also salt and osmotic stresses. However, jasmonic and abscisic acids and ethylene did not effectively induce NtC7 transcripts. Amino acid sequence analysis suggested NtC7 to be a new type of transmembrane protein that belongs to the receptor-like protein family, and a membrane location was confirmed in onion (Allium cepa) epidermis cells transiently expressing an NtC7-green fluorescent protein fusion protein. Seeds of transgenic tobacco overexpressing NtC7normally germinated and grew in the presence of 500 mmmannitol, but not in the presence of 220 mm sodium chloride or 60 mm lithium chloride. Cuttings of mature transgenic leaf exhibited a marked tolerance upon treatment with 500 mm mannitol for 12 h, at which concentration wild-type counterparts were seriously damaged. These results suggested that NtC7 predominantly functions in maintenance of osmotic adjustment independently of ion homeostasis.

Partially normalized complementary DNA (cDNA) libraries were generated from root and leaf tissues of a diploid tobacco species, Nicotiana sylvestris. Single-pass sequences were obtained from a total of 13, 019 cDNA clones and were assembled into 6, 513 non-redundant cDNA groups. We estimate that these cDNA sequences represent approximately 3, 500 unique tobacco genes. The EST database described here will become a valuable resource for future research using Nicotiana species.(Communicated by Yasuyuki YAMADA, M. J. A., June 10, 2003)

Water spinach (Ipomoea aquatica) has high nutritional value and is considered one of the most important vegetables in Southeast Asia. Because of its quick growth and efficient absorption of various substances, it has been suggested to be useful for sequestration of environmental pollutants as well as offering a source of medical materials. We have developed and established a system for stable genetic transformation by infecting cut cotyledons with Agrobacterium harboring the GUS gene as a model case after evaluating conditions of bacterial cell density, growth phase and concentrations of acetosyringone. The resulting transgenic plants grew normally to maturity, and exhibited stable GUS activity. Thus, genetic modification of I. aquatica can be readily achieved, thereby improving its quality for whatever traits desired.

When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990) Mol. Gen. Genet. 220, 441-447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.

Heavy metals are potentially highly toxic for organisms. Plants possess the ability to minimize damage but the underlying molecular mechanisms have yet to be detailed. Screening Cd-responsive genes in Arabidopsis, we previously identified a gene encoding a putative metal binding protein CdI19, which, upon introduction into yeast cells, conferred marked toleration of Cd exposure. Here we describe that bacterially expressed CdI19 directly interacts with Cd at its CXXC motif, as revealed by circular dichroism analysis, and that it is exclusively localized at plasma membranes, as revealed by heterologous expression of fusion product with a green fluorescent protein in BY2 cells. Northern blot analyses indicated that CdI19 transcripts were induced not only by Cd, but also by dicationic forms of Hg, Fe and Cu. Histochemical assays using transgenic Arabidopsis expressing the CdI19 promoter::GUS showed CdI19 to be expressed in petiole, hypocotyl, peduncle and vascular bundles in root tissues. Overexpression of the CdI19 cDNA conferred Cd tolerance in transgenic Arabidopsis. These results suggest that CdI19 plays an important role in the maintenance of heavy metal homeostasis and/or in detoxification by endowing plasma membranes with the capacity to serve as an initial barrier against inflow of free heavy metal ions into cells.

The tbzF gene of tobacco ( Nicotiana tabacum ) encodes a basic region leucine zipper protein (bZIP) that belongs to the LIP19 subfamily. It was previously shown that tbzF transcripts accumulate on cold, abscisic acid (ABA) or ethylene treatment. They were also abundant in senescing leaves and flower buds, suggesting tbzF to possess multiple functions. In order to analyze the transcript induction profile, a 1740 bp promoter region was isolated, fused to the β-glucuronidase (GUS) gene, and introduced into tobacco plants. Resulting transgenic plants exhibited GUS activity in the stomatal guard cells of senescing leaves and in flower buds, and also in ABA- and ethylene-treated young leaves. To identify promotion responsive regions, four deletion constructs were transformed into tobacco plants. As a result no response was observed with the −720 and −220 constructs in senescing leaves, or in ABA- and ethylene-treated young leaves. In contrast, response was only reduced with the −220 construct in flower buds. These observations indicate the 500 bp between −1220 and −720 to contain cis -element(s) for senescence-associated expression, and the 500 bp between −720 and −220 to be responsible for flower-specific expression. It is concluded that, although the promoter region within −220 is essential for tbzF expression in both senescing leaves and flower buds, additional but independent elements are necessary for full activation in each organ.

Following endoplasmic reticulum (ER) stress that prevents correct folding or assembly of ER proteins, at least three responses occur to maintain cell homeostasis: induction of chaperones, attenuation of protein synthesis, and enhancement of lipid synthesis. Transducers that transmit ER stress to the nucleus have already been identified in yeast and mammals. We report here isolation of a cDNA, OsIre1, from rice encoding a putative homolog of Ire1p, a yeast transducer of ER stress. OsIre1 encodes a polypeptide consisting of 893 amino acids, in which two hydrophobic stretches are present in the amino-terminal (N-terminal) and middle regions, possibly serving as a signal peptide and a transmembrane domain, respectively. The carboxyl-terminal (C-terminal) domain was found to possess serine/threonine protein kinase and ribonuclease-like domains showing high similarities with regions in Ire1 homologs from other organisms. A fusion protein of OsIre1 and green fluorescent protein (GFP) expressed in tobacco BY2 cells could be demonstrated to localize to the ER and the N-terminal domain of OsIre1 could substitute for yeast Ire1p in yeast cells. When produced in bacteria as a fusion protein, the C-terminal region of OsIre1 showed autophosphorylation activity. These results thus indicate that OsIre1 encodes a putative plant transducer of ER stress.

Chloroplast DNA of the green alga Chlamydomonas reinhardtii is maternally inherited. Methylation mapping directly revealed that, before mating, chloroplast DNA of maternal (mating type plus; mt + ) gametes is heavily methylated whereas that of paternal (mating type minus; mt − ) gametes is not. Indirect immunofluorescence analyses with anti-5-methylcytosine mAbs visually showed methylation to occur exclusively in chloroplast DNA of mt + gametes, and not in mt − gametes or nuclear DNA of either mt. To clarify the relationship between methylation and maternal inheritance of chloroplast DNA, we have isolated and characterized a cDNA encoding a DNA methyltransferase. The deduced protein, CrMET1, consists of 1,344 aa and contains a conserved catalytic domain at the C terminal and a nonconserved N-terminal region. The predicted N-terminal region has an arginine-rich domain, suggesting CrMET1 is transferred to chloroplasts. This finding could be directly shown by green fluorescent protein epifluorescence microscopy analyses. CrMET1 transcripts were found to be absent in both mt + and mt − vegetative cells. Upon gametogenesis, however, transcript levels clearly increased in mt + but not mt − cells. These experiments suggest that the CrMET1 protein is located in chloroplasts and that it specifically methylates cytosine residues of chloroplast DNA in mt + gametes. This conclusion was further strengthened by the observation that, during gametogenesis, CrMET1 is expressed in a mt − mutant, mat-1 , whose chloroplast DNA is heavily methylated in gametes and paternally inherited. The results provide evidence that cytosine methylation plays a critical role in maternal inheritance of chloroplast genes in C. reinhardtii .

The sulfur atom is an essential nutrient for living organisms because it plays a central role in protein folding, enzyme catalysis and maintenance of the redox status of cells. Microorganisms and plants can synthesize organic sulfur compounds, including cysteine, methionine and glutathione, from inorganic sulfur compounds such as sulfates. In contrast, animals utilize organic sulfur compounds that are mainly synthesized by plants. In the last decade, many genes whose products are involved in sulfate assimilation have been isolated from higher plants, and it has been revealed that there exist multiple isoforms for each step. The different properties of isoforms has been examined for sulfate transporters, which serve at the first step of sulfate assimilation, and for O-acetylserine(thiol)lyases, which catalyze the incorporation of sulfide into cysteine. Currently, however, it is not clear why plants developed multiple forms with similar catalytic properties. Mechanisms of regulation of the sulfate assimilation pathway have also attracted much attention, since metabolites such as cysteine and glutathione affect related enzymatic activity or corresponding gene expression. The precursor of cysteine, O-acetylserine, is a key compound in maintaining the balance between sulfate and nitrate assimilation in higher plants. In addition to nutrition, sulfate assimilation is important for conferring tolerance against environmental stresses including heavy metals. Genetic engineering of components of the sulfate assimilation pathway is a useful approach to generate useful plants for agriculture and phytoremediation.

AtPEPTIDE RECEPTOR2 (AtPEPR2) is a member of leucine-rich repeat receptor-like kinase family and binds to a group of AtPROPEP gene-encoded endogenous peptides, AtPeps. Previously, we found that AtPEPR2 plays a moderate role in the AtPep1-mediated innate immunity responses in Arabidopsis leaf. In this study, we found that AtPEPR2 promoter has strong activity in the vascular tissues of the roots and the atpepr2 mutants showed a moderate but significantly shorter root phenotype. AtPEPR2 partially mediated AtPep1-induced root elongation inhibition. AtPep1-triggered cytosolic Ca(2+) transient rise in roots showed partial dependence on AtPEPR2 and fully on extracellular Ca(2+) ([Ca(2+) ]ext ). Transcriptional profiling analysis found that expression of 75% of AtPep1-modulated genes in roots was fully dependent on AtPEPR2, of which two dramatically induced genes showed partial dependence on the [Ca(2+) ]ext . Arabidopsis genome contains seven Glutamine Dumpers genes (AtGDUs), encoding amino acid exporters. Three of them (AtGDU2, 3, 5) were among the top 10 genes that were downregulated by AtPep1 through AtPEPR2 fully dependent pathway. Treatment with AtPep1 strongly suppressed promoter activity of AtGDU3 in roots, which was relieved by chelating [Ca(2+) ]ext . Arabidopsis overexpressing AtGDU3 showed a shorter root phenotype and decreased sensitivity to the AtPep1-mediated inhibition of root elongation. Taken together, this study demonstrated a significant role of AtPEPR2 in the AtPep1-mediated signaling in the roots.

The term innate immunity has been described as '. . . the surveillance system that detects the presence and nature of the infection and provides the first line of host defense . . .' (Medzhitov, 2001; Nat Rev Immunol 1: 135-145). The strategy of innate immunity is based on the recognition of constitutive and conserved molecules from pathogens by specific receptors, triggering defence responses (Medzhitov and Janeway, 2002; Science 296: 298-300). It has been only within the past few years that studies of plant innate immunity, especially in Arabidopsis, have provided important insights into molecular details that define innate immunity in plants. Here we review the innate immune response in Arabidopsis, where leucine-rich repeat (LRR) cell surface receptors play central roles in monitoring the presence of pathogen (microbe) associated molecules to initiate the rapid expression of defence genes. The PAMPS also activate the expression of genes encoding a family of endogenous peptides (AtPep1 paralogues) and their receptor (PEPR1) that amplify defence signalling through a feedback loop initiated by PAMPS. The concept of innate immunity has provided a valuable framework for researchers to re-evaluate the roles of exogenous and endogenous signals that regulate the expression of plant defensive genes.

At Pep1 is a 23-aa endogenous peptide elicitor from Arabidopsis leaves that signals the activation of components of the innate immune response against pathogens. Here, we report the isolation of an At Pep1 receptor from the surface of Arabidopsis suspension-cultured cells. An 125 I-labeled At Pep1 analog interacted with suspension-cultured Arabidopsis with a K d of 0.25 nM, and an 125 I-labeled azido-Cys- At Pep1 photoaffinity analog specifically labeled a membrane-associated protein of ≈170 kDa. The labeled protein was purified to homogeneity, and its tryptic peptides were identified as gene At1g73080, which encodes a leucine-rich repeat receptor kinase, here called PEPR1. Verification of the binding protein as the receptor for At Pep1 was established by demonstrating the loss of function of microsomal membranes of two SALK insertional mutants and by a gain in function of the alkalinization response to At Pep1 by tobacco suspension-cultured cells expressing the At1g73080 transgene. Synthetic homologs of At Pep1, deduced from the C termini of six known paralogs of PROPEP1 , were biologically active and were competitors of the interaction of an At Pep1 radiolabeled analog with the receptor. The data are consistent with a role for PEPR1 as the receptor for At Pep1 to amplify innate immunity in response to pathogen attacks.

AtPepl is a 23-amino-acid defense-related peptide isolated from leaves of Arabidopsis thaliana that is derived from the carboxyl terminal of a 92-amino-acid precursor polypeptide called proAtPep1. The gene encoding proAtPep1 is expressed in leaves in response to several defense-related stimuli, including wounding, methyl jasmonate (MeJA), and ethylene. AtPep1 regulates the expression of plant defense genes against pathogens, including those encoding the antifungal proteins PDF1.2 (defensin) and several pathogen-related (PR) proteins that are known to be regulated through the jasmonate/ethylene defense signaling pathway. Constitutive overexpression of the proAtPep1 gene in Arabidopsis plants causes a constitutive expression of defense genes and enhances plant resistance to infection by an oomycete pathogen.

Wound-induced protein kinase (WIPK) is a tobacco (Nicotiana tabacum) mitogen-activated protein kinase known to play an essential role in defense against wounding and pathogens, although its downstream targets have yet to be clarified. This study identified a gene encoding a protein of 648 amino acids, which directly interacts with WIPK, designated as N. tabacum WIPK-interacting factor (NtWIF). The N-terminal region with approximately 250 amino acids showed a high similarity to the plant-specific DNA binding domain, B3, but no other similarity with known proteins. The C terminus of approximately 200 amino acids appeared to be essential for the interaction with WIPK, and a Luciferase-reporter gene assay using Bright Yellow 2 cells indicated the full-length protein to possess trans-activation activity, located to the middle region of approximately 200 amino acids. In vitro phosphorylation assays indicated that WIPK efficiently phosphorylates the full-length protein and the N terminus but not the C terminus. When full-length NtWIF was coexpressed with WIPK in Bright Yellow 2 cells, the Luciferase transcriptional activity increased up to 5-fold that of NtWIF alone, whereas no effect was observed with a kinase-deficient WIPK mutant. Transcripts of NtWIF began to simultaneously accumulate with those of WIPK 30 min after wounding and 1 h after the onset of hypersensitive response upon tobacco mosaic virus infection. These results suggest that NtWIF is a transcription factor that is directly phosphorylated by WIPK, thereby being activated for transcription of target gene(s) involved in wound and pathogen responses.

In the green alga, Chlamydomonas, chloroplast DNA is maternally transmitted to the offspring. We previously hypothesized that the underlying molecular mechanism involves specific methylation of maternal gamete DNA before mating, protecting against degradation. To obtain direct evidence for this, we focused on a DNA methyltransferase, DMT1, which was previously shown to be localized in chloroplasts. The full-length DMT1 protein with a molecular mass of 150 kD was expressed in insect cells, and its catalytic activity was determined. In vitro assays using synthetic DNA indicated methylation of all cytosine residues, with no clear selectivity in terms of the neighboring nucleotides. Subsequently, transgenic paternal cells constitutively expressing DMT1 were constructed and direct methylation mapping assays of their DNA showed a clear nonselective methylation of chloroplast DNA. When transgenic paternal cells were crossed with wild-type maternal cells, the frequency of biparental and paternal offspring of chloroplasts increased up to 23% while between wild-type strains it was ∼3%. The results indicate that DMT1 is a novel type of DNA methyltransferase with a nonselective cytosine methylation activity, and that chloroplast DNA methylation by DMT1 is one of factors influencing maternal inheritance of chloroplast genes.

The hypersensitive response (HR) is one of the most critical defense systems in higher plants. In order to understand its molecular basis, we have screened tobacco genes that are transcriptionally activated during the early stage of the HR by the differential display method. Among six genes initially identified, one was found encoding a 57 kDa polypeptide with 497 amino acids not showing significant similarity to any reported proteins except for the AAA domain (ATPase associated with various cellular activities) spanning over 230 amino acids. The bacterially expressed protein exhibited ATP hydrolysis activity, and a green fluorescent protein-fusion protein localized in the cytoplasm of onion epidermis cells. The protein was subsequently designated as NtAAA1 (Nicotiana tabacum AAA1). NtAAA1 transcripts were induced 6 h after HR onset not only by TMV but also by incompatible Psuedomonas syringae, indicating that NtAAA1 is under the control of the N-gene with a common role in pathogen responses. Expression of NtAAA1 was induced by jasmonic acid and ethylene, but not by salicylic acid (SA). It also occurred at a high level in SA-deficient tobacco plants upon TMV infection. When NtAAA1 was silenced by the RNAi method, accumulation of transcripts for PR-1a significantly increased during the HR. Treatments with SA induced higher expression of PR-1a and acidic PR-2 in RNAi transgenic plants than in wild-type counterparts. These results suggest that NtAAA1 mitigates the SA signaling pathway, and therefore that NtAAA1 modulates the pathogen response of the host plants by adjusting the HR to an appropriate level.

In plant DNA, cytosines in symmetric CpG and CpNpG (N is A, T, or C) are thought to be methylated by DNA methyltransferases, MET1 and CMT3, respectively. Cytosines in asymmetric CpNpN are also methylated, and genetic analysis has suggested the responsible enzyme to be domains rearranged methyltransferase (DRM). We cloned a tobacco cDNA, encoding a novel protein consisting of 608 amino acids, that resembled DRMs found in maize and Arabidopsis and designated this as NtDRM1. The protein could be shown to be localized exclusively in the nucleus and exhibit methylation activity toward unmethylated synthetic as well as native DNA samples upon expression in Sf9 insect cells. It also methylated hemimethylated DNA, but the activity was lower than that for unmethylated substrates. Methylation mapping of a 962-bp DNA, treated with NtDRM1 in vitro, directly demonstrated methylation of approximately 70% of the cytosines in methylatable CpNpN and CpNpG sequences but only 10% in CpG. Further analyses indicated that the enzyme apparently non-selectively methylates any cytosines except in CpG, regardless of the adjacent nucleotide at both 5' and 3' ends. Transcripts of NtDRM1 ubiquitously accumulated in all tissues and during the cell cycle in tobacco cultured BY2 cells. These results indicate that NtDRM1 is a de novo cytosine methyltransferase, which actively excludes CpG substrate.

Screening immediate-early responding genes during the hypersensitive response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana tabacum) plants, we identified a gene encoding ornithine decarboxylase. Subsequent analyses showed that other genes involved in polyamine biosynthesis were also up-regulated, resulting in the accumulation of polyamines in apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine biosynthesis, α-difluoromethyl-ornithine, however, suppressed accumulation of polyamines, and the rate of HR was reduced. In contrast, polyamine infiltration into a healthy leaf induced the generation of hydrogen peroxide and simultaneously caused HR-like cell death. Polyamine oxidase activity in the apoplast increased up to 3-fold that of the basal level during the HR, and its suppression with a specific inhibitor, guazatine, resulted in reduced HR. Because it is established that hydrogen peroxide is one of the degradation products of polyamines, these results indicate that one of the biochemical events in the HR is production of polyamines, whose degradation induces hydrogen peroxide, eventually resulting in hypersensitive cell death.

In order to identify genes that are involved in the defense reaction against pathogen attack, we screened for examples that are regulated during the hypersensitive response (HR) to infection by tobacco mosaic virus (TMV) of tobacco ( Nicotiana tabacum cv. Xanthi nc) carrying the N gene, which confers resistance to TMV. Among seven genes initially identified by fluorescent differential display, one clone was further characterized because its transcripts accumulated rapidly and transiently after the onset of HR. Its full-length cDNA of 1346 bp encoded a polypeptide consisting of 258 amino acids. The deduced protein contained a single WRKY domain, a Cys(2)His(2) zinc-finger motif and a leucine-zipper motif, showing high similarity to WIZZ, a member of the family of WRKY transcription factors in tobacco. The gene was thus designated TIZZ. A GFP-TIZZ fusion protein was found to localize to the nucleus upon introduction into epidermal cells of onion. Bacterially expressed TIZZ was able to bind to the W-box (TTGAC) element that is recognized by other WRKY proteins, but transactivation assays showed it to be unable to activate reporter gene expression by itself. TIZZ transcripts were induced in TMV-infected nahG transgenic tobacco plants, in which salicylic acid fails to accumulate. Neither exogenously applied salicylic acid nor mechanical wounding induced TIZZ transcript accumulation. These results indicate the presence of salicylic acid-independent pathways for HR signal transduction, in which a novel type of WRKY protein(s) may play a critical role for the activation of defense.

Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and thiosulfate sulfurtransferase (TST, rhodanese, EC 2.8.1.1) are evolutionarily related enzymes that catalyze the transfer of sulfur ions from mercaptopyruvate and thiosulfate, respectively, to cyanide ions. We have isolated and characterized two cDNAs, AtMST1 and AtMST2, that are Arabidopsis homologs of TST and MST from other organisms. Deduced amino-acid sequences showed similarity to each other, although MST1 has a N-terminal extension of 57 amino acids containing a targeting sequence. MST1 and MST2 are located in mitochondria and cytoplasm, respectively, as shown by immunoblot analysis of subcellular fractions and by green fluorescent protein (GFP) analysis. However, some regions of MST1 fused to GFP were found to target not only mitochondria, but also chloroplasts, suggesting that the regions on the targeting sequence recognized by protein import systems of mitochondria and chloroplasts are not identical. Recombinant proteins, expressed in Escherichia coli, exhibited MST/TST activity ratios determined from kcat/Km values of 11 and 26 for MST1 and MST2, respectively. This indicates that the proteins encoded by both AtMST1 and AtMST2 are MST rather than TST type. One of the hypotheses proposed so far for the physiological function of MST and TST concerns iron–sulfur cluster assembly. In order to address this possibility, a T-DNA insertion Arabidopsis mutant, in which the AtMST1 was disrupted, was isolated by PCR screening of T-DNA mutant libraries. However, the mutation had no effect on levels of iron–sulfur enzyme activities, suggesting that MST1 is not directly involved in iron–sulfur cluster assembly.

Three cDNA clones encoding putative cysteine synthases (O-acetylserine (thiol) lyase, EC 4.2.99.8) were isolated from Arabidopsis thaliana and designated AtcysC1, AtcysD1 and AtcysD2, respectively. Southern blot analyses suggested that the corresponding genes were present as a single copy, or at most two copies, in the A. thaliana genome. Escherichia coli complementation analyses confirmed that the cDNAs encode cysteine synthase and the corresponding proteins produced in E. coli clearly showed cysteine synthase activity. In addition, AtcysC1 protein showed beta-cyanoalanine synthase (EC 4.4.1.9) activity, but the other two did not. Kinetic analysis suggests that AtcysC1 actually functions as beta-cyanoalanine synthase rather than cysteine synthase in vivo. The mRNA accumulation of AtcysC1, AtcysD1 and AtcysD2 differed in various organs, but did not change markedly when A. thaliana seedlings were subjected to various stresses, including nutrient deprivation. In vivo targeting experiments indicated that AtcysD1 and AtcysD2 are cytoplasmic isozymes, and AtcysC1 is a mitochondrial isozyme.

Expression of nine genes encoding enzymes involved in the sulfur assimilation pathway was examined by RNA blot hybridization. Significantly increased levels of transcripts encoding ATP sulfurylase and APS reductase were apparent under sulfur deprivation. However, in the absence of nitrogen, their responsiveness to sulfur deprivation was markedly reduced. Results suggest that the sulfur assimilation pathway is regulated at the transcriptional level by both nitrogen and sulfur sources.

Four cDNA clones, rcs1 , rcs2 , rcs3 and rcs4 , encoding cysteine synthase [ O -acetylserine(thiol)lyase] were isolated from rice. The predicted amino acid sequences contain the conserved PXXSVKDR region characteristic of cysteine synthase, which includes the lysine residue that binds the cofactor, pyridoxal 5′-phosphate. Molecular phylogenic analysis suggests that, whereas rcs1 and rcs3 belong to the cytosolic isoform family, rcs2 and rcs4 form a new family of cysteine synthase. Transcript accumulation of each gene was examined for organ specificity, and also for response to sulfur, nitrogen and light. The rcs1 transcript accumulated in all organs examined, and was induced in shoots and roots upon sulfur starvation under non-limiting nitrogen conditions. The rcs2 transcript accumulated in shoots grown in the light, but disappeared almost completely by dark treatment. The rcs3 transcript was found more abundantly in roots than in shoots, and was reduced in the dark, as well as under sulfur and nitrogen deprivation. The rcs4 transcript was scarce in all organs examined. These observations indicate that cysteine synthase genes encode functionally distinct cysteine synthase isoforms, and that they are coordinately regulated by the availability of sulfur, nitrogen, and light.

The demand for decaffeinated coffee is increasing because the stimulatory effects of caffeine can adversely affect sensitive individuals by triggering palpitations, increased blood pressure and insomnia1. Three N-methyltransferase enzymes are involved in caffeine biosynthesis in coffee plants — CaXMT1, CaMXMT1 (theobromine synthase) and CaDXMT1 (caffeine synthase), which successively add methyl groups to xanthosine in converting it into caffeine2,3,4. Here we describe the construction of transgenic coffee plants in which expression of the gene encoding theobromine synthase (CaMXMT1) is repressed by RNA interference (RNAi). The caffeine content of these plants is reduced by up to 70%, indicating that it should be feasible to produce coffee beans that are intrinsically deficient in caffeine.