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Serine is an important precursor of various biomolecules. Here, we investigated the role of the phosphorylated pathway of serine biosynthesis in a non-vascular plant Marchantia polymorpha by analyzing knockout mutants of MpPGDH, a single gene encoding the first committed enzyme 3-phosphoglycerate dehydrogenase (PGDH), to assess functions of this pathway in relation to those of the other two pathways. Growth phenotypes of the mutants indicated that serine supply from the phosphorylated pathway in the dark was crucial for vegetative growth. Sperm formation required serine from this pathway, while egg formation did not depend on it. Knockout of MpPGDH in the maternal genome disrupted sporophyte development. When the mutants were grown in high CO2 where the photorespiratory glycolate pathway for serine biosynthesis is inhibited, thallus growth was suppressed and not fully recovered to wild-type level by exogenous serine supplement, suggesting that serine homeostasis involving both the phosphorylated and glycolate pathways was essential. Metabolome and lipidome analyses indicated that the phosphorylated pathway mainly influenced the tricarboxylic acid cycle, the amino acid and nucleotide metabolism, and lack of serine significantly perturbed lipid metabolism. Our results indicate the importance of serine from the phosphorylated pathway for sperm formation, sporophyte development, and metabolism in M. polymorpha.

Tropane alkaloids, including clinically important hyoscyamine and scopolamine, are produced in the roots of medicinal plant species, such as Atropa belladonna, from the Solanaceae family. Recent molecular and genomic approaches have advanced our understanding of the metabolic enzymes involved in tropane alkaloid biosynthesis. A noncanonical type III polyketide synthase, pyrrolidine ketide synthase (PYKS) catalyzes a two-step decarboxylative reaction, which involves imine–ketide condensation indispensable to tropane skeleton construction. In this study, we generated pyks mutant A. belladonna hairy roots via CRISPR/Cas9-mediated genome editing and analyzed the metabolic consequences of the loss of PYKS activity on tropane alkaloids, providing insights into a crucial role of the scaffold-forming reaction in the biosynthetic pathway.

A number of mutational changes in transcriptional regulators of defense metabolism have occurred during plant domestication and improvement. Plant domestication and improvement entail genetic changes that underlie divergence in development and metabolism, providing a tremendous model of biological evolution. Plant metabolism produces numerous specialized alkaloids, terpenoids, phenolics, and cyanogenic glucosides with indispensable roles in defense against herbivory and microbial infection. Many compounds toxic or deterrent to predators have been eliminated through domestication and breeding. Series of genes involved in defense metabolism are coordinately regulated by transcription factors that specifically recognize cis-regulatory elements in promoter regions of downstream target genes. Recent developments in DNA sequencing technologies and genomic approaches have facilitated studies of the metabolic and genetic changes in chemical defense that have occurred via human-mediated selection, many of which result from mutations in transcriptional regulators of defense metabolism. In this article, we review such examples in almond (Prunus dulcis), cucumber (Cucumis sativus), pepper (Capsicum spp.), potato (Solanum tuberosum), quinoa (Chenopodium quinoa), sorghum (Sorghum bicolor), and related species and discuss insights into the evolution and regulation of metabolic pathways for specialized defense compounds.

In tobacco, the homologous ETHYLENE RESPONSE FACTOR (ERF) transcription factors ERF199 and ERF189 coordinate the transcription of multiple metabolic genes involved in nicotine biosynthesis. Natural alleles at the NIC1 and NIC2 loci greatly affect alkaloid accumulation and overlap with ERF199 and ERF189 in the tobacco genome, respectively. In this study, we identified several low-nicotine tobacco varieties lacking ERF199 or ERF189 from a tobacco germplasm collection. We characterized the sequence of these new nic1 and nic2 alleles, as well as the previously defined alleles nic1-1 and nic2-1. Moreover, we examined the influence of different nic alleles on alkaloid contents and expression levels of genes related to nicotine biosynthesis. We also demonstrated that the deletion of a distal genomic region attenuates ERF199 expression, resulting in a moderately negative effect on the alkaloid phenotype. Our study provides new insights into the regulation of nicotine biosynthesis and novel genetic resources to breed low-nicotine tobacco.

Saponins are the group of plant specialized metabolites which are widely distributed in angiosperm plants and have various biological activities. The present study focused on α-tomatine, a major saponin present in tissues of tomato (Solanum lycopersicum) plants. α-Tomatine is responsible for defense against plant pathogens and herbivores, but its biological function in the rhizosphere remains unknown. Secretion of tomatine was higher at the early growth than the green-fruit stage in hydroponically grown plants, and the concentration of tomatine in the rhizosphere of field-grown plants was higher than that of the bulk soil at all growth stages. The effects of tomatine and its aglycone tomatidine on the bacterial communities in the soil were evaluated in vitro, revealing that both compounds influenced the microbiome in a concentration-dependent manner. Numerous bacterial families were influenced in tomatine/tomatidine-treated soil as well as in the tomato rhizosphere. Sphingomonadaceae species, which are commonly observed and enriched in tomato rhizospheres in the fields, were also enriched in tomatine- and tomatidine-treated soils. Moreover, a jasmonate-responsive ETHYLENE RESPONSE FACTOR 4 mutant associated with low tomatine production caused the root-associated bacterial communities to change with a reduced abundance of Sphingomonadaceae. Taken together, our results highlight the role of tomatine in shaping the bacterial communities of the rhizosphere and suggest additional functions of tomatine in belowground biological communication.

Plants produce structurally diverse specialized metabolites, including bioactive alkaloids and terpenoids, in response to biotic and abiotic environmental stresses. The APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family of transcription factors (TFs) play key roles in regulating biosynthesis of specialized metabolites. Increasing genomic and functional evidence shows that a subset of the ERF genes occurs in clusters on the chromosomes. These jasmonate-responsive ERF TF gene clusters control the biosynthesis of many important metabolites, from natural products, such as nicotine and steroidal glycoalkaloids (SGAs), to pharmaceuticals, such as artemisinin, vinblastine, and vincristine. Here, we review the function, regulation, and evolution of ERF clusters and highlight recent advances in understanding the distinct roles of clustered ERF genes and their possible application in metabolic engineering.

The toxic alkaloid nicotine is produced in the roots of Nicotiana species and primarily accumulates in leaves as a specialized metabolite. A series of metabolic and transport genes involved in the nicotine pathway are coordinately upregulated by a pair of jasmonate-responsive AP2/ERF-family transcription factors, NtERF189 and NtERF199, in the roots of Nicotiana tabacum (tobacco). In this study, we explored the potential of manipulating the expression of these transcriptional regulators to alter nicotine biosynthesis in tobacco. The transient overexpression of NtERF189 led to alkaloid production in the leaves of Nicotiana benthamiana and Nicotiana alata. This ectopic production was further enhanced by co-overexpressing a gene encoding a basic helix-loop-helix-family MYC2 transcription factor. Constitutive and leaf-specific overexpression of NtERF189 increased the accumulation of foliar alkaloids in transgenic tobacco plants but negatively affected plant growth. By contrast, in a knockout mutant of NtERF189 and NtERF199 obtained through CRISPR/Cas9-based genome editing, alkaloid levels were drastically reduced without causing major growth defects. Metabolite profiling revealed the impact of manipulating the nicotine pathway on a wide range of nitrogen- and carbon-containing metabolites. Our findings provide insights into the biotechnological applications of engineering metabolic pathways by targeting transcription factors.

Steroidal glycoalkaloids (SGAs) are specialized anti-nutritional metabolites that accumulate in Solanum lycopersicum (tomato) and Solanum tuberosum (potato). A series of SGA biosynthetic genes is known to be upregulated in Solanaceae species by jasmonate-responsive Ethylene Response Factor transcription factors, including JRE4 (otherwise known as GAME9), but the exact regulatory significance in planta of each factor has remained unaddressed. Here, via TILLING-based screening of an EMS-mutagenized tomato population, we isolated a JRE4 loss-of-function line that carries an amino acid residue missense change in a region of the protein important for DNA binding. In this jre4 mutant, we observed downregulated expression of SGA biosynthetic genes and decreased SGA accumulation. Moreover, JRE4 overexpression stimulated SGA production. Further characterization of jre4 plants revealed their increased susceptibility to the generalist herbivore Spodoptera litura larvae. This susceptibility illustrates that herbivory resistance is dependent on JRE4-mediated defense responses, which include SGA accumulation. Ethylene treatment attenuated the jasmonate-mediated JRE4 expression induction and downstream SGA biosynthesis in tomato leaves and hairy roots. Overall, this study indicated that JRE4 functions as a primary master regulator of SGA biosynthesis, and thereby contributes toward plant defense against chewing insects.

In Solanum lycoperisicum (tomato), a transcription factor of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family, JASMONATE-RESPONSIVE ERF 3 (JRE3), is a closest homolog of JRE4, a master transcriptional regulator of steroidal glycoalkaloid (SGA) biosynthesis. In tomato genome, JRE3 resides in a gene cluster with JRE4 and related JRE1, JRE2, and JRE5, while JRE6 exists as a singleton on a different chromosome. All of the JREs are induced by jasmonates (JAs), whereas sodium chloride (NaCl) treatment drastically increases the expression of the JREs except for JRE4 and JRE6. In this study, to get insights into the regulatory function of the JA- and NaCl-inducible JRE3, a series of genes upregulated by β-estradiol-induced overexpression of JRE3 are identified with microarray analysis in transgenic tomato hairy roots. No gene involved in the SGA pathway has been identified through the screening, confirming the functional distinction between JRE3 and JRE4. Among the JRE3-regulated genes, we characterize the stress-induced expression of genes encoding malate synthase and tonoplast dicarboxylate transporter both involved in malate accumulation. In transient transactivation assay, we reveal that both terminal regions of JRE4, but not a central DNA-binding domain, are indispensable for the induction of a gene involved in the JRE4 regulon. Functional differentiation of the JREs is discussed.

The jasmonate-responsive transcription factor ERF189 in tobacco (Nicotiana tabacum) and its ortholog JRE4 in tomato (Solanum lycopersicum) regulate a series of biosynthetic genes involved in the nicotine and steroidal glycoalkaloid pathways. In tobacco, QUINOLINATE PHOSPHORIBOSYL TRANSFERASE 2 (NtQPT2) is regulated by ERF189; however, we found that the tomato QPT gene is not regulated by JRE4. Here, we explored whether and how NtQPT2 is regulated in a heterogenous tomato host. We used a NtQPT2 promoter-driven reporter gene to examine the cell type-specific and jasmonate-induced expression of this gene in transgenic tomato hairy roots. The downregulation of the reporter in the jre4 loss-of-function tomato mutant and its transactivation by JRE4 in transient expression experiments suggested that JRE4, like its ortholog ERF189 in tobacco, activates the NtQPT2 promoter in tomato. We discuss the evolution of QPT2 in the Nicotiana lineage, which mainly occurred through mutational changes in the promoter that altered the control of the functionally conserved transcription factors.

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.

The intracellular localization of enzymes provides key information for understanding complex metabolic pathways. Based on enzyme localization data, the involvement of multiple organelles and the movement of metabolites between cellular compartments have been suggested for a number of pathways. Transient expression of fluorescently tagged proteins in the leaves of Nicotiana benthamiana through Agrobacterium infiltration is a simple and versatile way to examine the intracellular localization of proteins of interest. Here, this method was applied to demonstrate the peroxisomal localization of a pair of homologous copper-containing amine oxidases (CuAOs) from tobacco with distinct substrate preferences: diamine oxidase (DAO), which mediates polyamine catabolism, and N-methylputrescine oxidase (MPO), which is involved in nicotine biosynthesis. Our results demonstrate that the Agrobacterium infiltration protocol can be effectively used to study the intracellular localization of oxidases that localize to the peroxisome.

In tobacco, the defense alkaloid nicotine is produced in roots and accumulates mainly in leaves. Signaling mediated by jasmonates (JAs) induces the formation of nicotine via a series of structural genes that constitute a regulon and are coordinated by JA-responsive transcription factors of the ethylene response factor (ERF) family. Early steps in the pyrrolidine and pyridine biosynthesis pathways likely arose through duplication of the polyamine and nicotinamide adenine dinucleotide (NAD) biosynthetic pathways, respectively, followed by recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon. Transcriptional regulation of nicotine biosynthesis by ERF and cooperatively-acting MYC2 transcription factors is implied by the frequency of cognate cis-regulatory elements for these factors in the promoter regions of the downstream structural genes. Indeed, a mutant tobacco with low nicotine content was found to have a large chromosomal deletion in a cluster of closely related ERF genes at the nicotine-controlling NICOTINE2 (NIC2) locus.

In tomato, perception of jasmonates by a receptor complex, which includes the F-box protein CORONATINE INSENSITIVE 1 (COI1), elicits biosynthesis of defensive steroidal glycoalkaloids (SGAs) via a jasmonate-responsive ERF transcription factor, JRE4/GAME9. Although JRE4 is upregulated by jasmonate and induces the expression of many metabolic genes involved in SGA biosynthesis, it is not known whether JRE4 alone is sufficient for increased SGA biosynthesis upon activation of jasmonate signaling. Here, we show that application of methyl jasmonate induces the expression of JRE4 and SGA biosynthesis genes in leaves and hairy roots of wild-type tomato, but not in jasmonic acid insensitive 1 (jai1), a loss-of-function mutant allele of the tomato COI1 gene. Induced overexpression of JRE4 increased the expression of SGA biosynthesis genes in transgenic hairy roots of both wild-type tomato and the jai1 mutant, suggesting that JRE4 is the primary transcription factor that functions downstream of the jasmonate signaling pathway.

In tobacco (Nicotiana tabacum), nicotine is the predominant alkaloid. It is produced in the roots and accumulated mainly in the leaves. Jasmonates play a central signaling role in damage-induced nicotine formation. The genome sequence of tobacco provides us an almost complete inventory of structural and regulatory genes involved in nicotine pathway. Phylogenetic and expression analyses revealed a series of structural genes of the nicotine pathway, forming a regulon, under the control of jasmonate-responsive ETHYLENE RESPONSE FACTOR (ERF) transcription factors. The duplication of NAD and polyamine metabolic pathways and the subsequent recruitment of duplicated primary metabolic genes into the nicotine biosynthesis regulon were suggested to be the drivers for pyridine and pyrrolidine ring formation steps early in the pathway. Transcriptional regulation by ERF and cooperatively acting MYC2 transcription factors are corroborated by the frequent occurrence of cognate cis-regulatory elements of the factors in the promoter regions of the downstream structural genes. The allotetraploid tobacco has homologous clusters of ERF genes on different chromosomes, which are possibly derived from two ancestral diploids and include either nicotine-controlling ERF189 or ERF199 A large chromosomal deletion was found within one allele of the nicotine-controlling NICOTINE2 locus, which is part of one of the ERF gene clusters, and which has been used to breed tobacco cultivars with a low-nicotine content.

The down-regulation of a tobacco (Nicotiana tabacum) plasma membrane-localized nicotine uptake permease, NUP1, was previously reported to reduce total alkaloid levels in tobacco plants. However, it was unclear how this nicotine transporter affected the biosynthesis of the alkaloid nicotine. When NUP1 expression was suppressed in cultured tobacco cells treated with jasmonate, which induces nicotine biosynthesis, the NICOTINE2-locus transcription factor gene ETHYLENE RESPONSE FACTOR189 (ERF189) and its target structural genes, which function in nicotine biosynthesis and transport, were strongly suppressed, resulting in decreased total alkaloid levels. Conversely, NUP1 overexpression had the opposite effect. In these experiments, the expression levels of the MYC2 transcription factor gene and its jasmonate-inducible target gene were not altered. Inhibiting tobacco alkaloid biosynthesis by suppressing the expression of genes encoding enzymes in the nicotine pathway did not affect the expression of ERF189 and other nicotine pathway genes, indicating that ERF189 is not regulated by cellular alkaloid levels. Suppressing the expression of jasmonate signaling components in cultured tobacco cells showed that NUP1 acts downstream of the CORONATINE INSENSITIVE1 receptor and MYC2, but upstream of ERF189. These results suggest that although jasmonate-activated expression of MYC2 induces the expression of both NUP1 and ERF189, expression of ERF189 may actually be mediated by NUP1. Furthermore, NUP1 overexpression in tobacco plants inhibited the long-range transport of nicotine from the roots to the aerial parts. Thus, NUP1 not only mediates the uptake of tobacco alkaloids into root cells, but also positively controls the expression of ERF189, a key gene in the biosynthesis of these alkaloids.

Plants produce a multitude of secondary metabolites, including alkaloids with biological activities, and many alkaloids have been used for medicinal purposes. The biosynthetic enzymes and genes involved in alkaloid metabolic pathways exhibit divergent localizations, implying that alkaloid metabolites, including pathway products and intermediates, travel from organelle to organelle, cell to cell, and organ to organ. Biochemical studies have indicated that specific transporters move these metabolites. Indeed, molecular and cellular approaches have identified alkaloid transporters of the ATP-binding cassette (ABC) protein, multidrug and toxic compound extrusion (MATE), and purine permease (PUP) families. Interestingly, some of these transporters were found to be required for the efficient biosynthesis of alkaloids in plants. Here, we provide an updated inventory of alkaloid transporters and discuss the possibility of genetically manipulating the expression of these transporters to increase the accumulation of valuable alkaloid compounds.

Transcription factors (TFs) recognize target DNA sequences with distinct DNA-binding domains (DBDs). The DBD of Arabidopsis (Arabidopsis thaliana) ETHYLENE RESPONSE FACTOR1 (AtERF1) uses three consecutive β-strands to recognize a GCC-containing sequence, but tobacco (Nicotiana tabacum) ERF189 and periwinkle (Catharanthus roseus) Octadecanoid-derivative Responsive Catharanthus AP2-domain protein3 (ORCA3) of the same TF subgroup appear to target similar but divergent DNA sequences. Here, we examined how DNA-binding specificities of these TFs have diverged in each plant lineage to regulate distinct defense metabolisms. Extensive mutational analyses of these DBDs suggest that two modes of protein-DNA interactions independently contribute to binding specificity and affinity. Substitution of a conserved arginine to lysine in the first β-strand of ERF189 relaxes its interaction with the second GC pair of the GCC DNA sequence. By contrast, an increased number of basic amino acids in the first two β-strands of ORCA3 allows this TF to recognize more than one GCC-related target, presumably via increased electrostatic interactions with the negatively charged phosphate backbone of DNA. Divergent DNA-binding specificities of the ERFs may have arisen through mutational changes of these amino acid residues.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived specialized metabolites produced in species of the Solanaceae. Here, we report that a group of jasmonate-responsive transcription factors of the ETHYLENE RESPONSE FACTOR (ERF) family (JREs) are close homologs of alkaloid regulators in Cathranthus roseus and tobacco, and regulate production of SGAs in tomato. In transgenic tomato, overexpression and dominant suppression of JRE genes caused drastic changes in SGA accumulation and in the expression of genes for metabolic enzymes involved in the multistep pathway leading to SGA biosynthesis, including the upstream mevalonate pathway. Transactivation and DNA-protein binding assays demonstrate that JRE4 activates the transcription of SGA biosynthetic genes by binding to GCC box-like elements in their promoters. These JRE-binding elements occur at significantly higher frequencies in proximal promoter regions of the genes regulated by JRE genes, supporting the conclusion that JREs mediate transcriptional co-ordination of a series of metabolic genes involved in SGA biosynthesis.

In tobacco (Nicotiana tabacum), several transcription factors belonging to the IXa group of APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) cluster at the regulatory NIC2 locus for the leaf nicotine level, and directly activate structural genes required for nicotine biosynthesis and transport. Solanaceous Atropa and Hyoscyamus plants synthesize medicinal tropane alkaloids by utilizing enzymes that partially overlap with the nicotine pathway. We tested whether the tobacco NIC2-locus ERF189 binds to potential ERF binding sites in the promoters of various structural genes involved in biosynthesis of nicotine and tropane alkaloids by an electrophoresis mobility shift assay. ERF189 bound four new GCC-box-like sequences in three nicotine structural genes, but did not recognize other candidate binding sites. This binding preference was used to optimize the consensus binding sequence of ERF189 as 5′-(A/C)GC(A/C)NNCC(A/T)-3′. Five group IXa ERFs (tobacco ERF189, tobacco ERF163, Catharanthus ORCA3, Arabidopsis AtERF13, and Arabidopsis AtERF1) were examined whether they bind to the ERF189-recognition site in the promoter of the tobacco putrescine N-methyltransferase gene in vitro, and transactivate the promoter in a transient expression assay using cultured tobacco cells. The more the protein sequences were similar to ERF189, the more effective these ERFs were in these functional assays. The transient expression assay also identified transactivation domains in an N-terminal acidic region of ERF189, and in a C-terminal Ser-rich region of AtERF13.

Gene duplication is a powerful source of phenotypic diversity in plants, but the molecular mechanisms that generate new functions in duplicated genes are not fully documented. Here, we analyzed how duplicated genes encoding quinolinate phosphoribosyltransferase (QPT), an enzyme involved in the synthesis of nicotinamide adenine dinucleotide (NAD) and the pyridine moiety of nicotine, are regulated by the jasmonate-responsive transcriptional factor ERF189 that functions critically for nicotine biosynthesis in tobacco (Nicotiana tabacum). The tobacco genome contains duplicated QPT genes; QPT1 is expressed at a constitutive basal level, whereas QPT2 is regulated coordinately with other structural genes involved in nicotine biosynthesis, in terms of tissue specificity, jasmonate induction, and regulation by ERF189. The binding-site specificity of ERF189 was defined as 5'-(A/C)GC(A/C)(A/C)NCC-3' by using a characterized tobacco putrescine N-methyltransferase promoter, and was then used to search for potential binding sites in the QPT promoters. Assays involving in vitro DNA binding, transient transactivation, and transgenic hairy roots revealed that the QPT2 promoter contains three functional ERF189-binding sites, which individually confer incremental ERF189-mediated activation to the promoter. The QPT1 promoter is not bound and regulated by ERF189. These results indicate that one copy of the duplicated QPT genes was recruited to a tobacco alkaloid regulon by evolving multiple target cis-regulatory elements of ERF189 in its promoter, to cope with an increased metabolic demand for pyridine precursors during active alkaloid biosynthesis.

In Arabidopsis, the MYC2-family basic helix-loop-helix transcription factors mediate transcriptional regulation of jasmonate-responsive genes, and their transcriptional activities are suppressed by physical interactions with jasmonate-ZIM domain (JAZ) proteins. Jasmonate-inducible nicotine formation in Nicotiana plants has been shown to be suppressed by tobacco JAZ proteins, and be regulated by both MYC2-related and NIC2-locus ethylene response factor (ERF) transcription factors. We here show that tobacco MYC2 (NtMYC2) recognizes the G-box sequences, 5'-CAC(G/A)T(G/T)-3', found in the proximal promoter regions of several nicotine biosynthesis genes, including Putrescine N-Methyltransferase 2 (PMT2) and Quinolinate Phosphoribosyltransferase 2 (QPT2). Transient transactivation assays using cultured tobacco cells showed that NtMYC2 and NIC2-locus ERF189 additively activated the PMT2 and QPT2 promoters depending on their cognate binding sites. RNA interference (RNAi) silencing of NtMYC2 in tobacco hairy roots strongly decreased transcript levels of jasmonate-responsive structural genes, including those involved in nicotine biosynthesis, as well as the NIC2-locus ERF genes. Conversely, ERF189 was not required for the expression of NtMYC2. NtMYC2, but not ERF189, interacted with tobacoo JAZs in a yeast two-hybrid assay. These results indicate that NtMYC2 controls nicotine biosynthesis genes in two combinatorial ways, by directly binding the G-box in the target promoters and by up-regulating the NIC2-locus ERF genes.

Pollen tube growth is crucial for the delivery of sperm cells to the ovule during flowering plant reproduction. Previous in vitro imaging of Lilium longiflorum and Nicotiana tabacum has shown that growing pollen tubes exhibit a tip-focused Ca2+ concentration ([Ca2+]) gradient and regular oscillations of the cytosolic [Ca2+] ([Ca2+]cyt) in the tip region. Whether this [Ca2+] gradient and/or [Ca2+]cyt oscillations are present as the tube grows through the stigma (in vivo condition), however, is still not clear. We monitored [Ca2+]cyt dynamics in pollen tubes under various conditions using Arabidopsis (Arabidopsis thaliana) and N. tabacum expressing yellow cameleon 3.60, a fluorescent calcium indicator with a large dynamic range. The tip-focused [Ca2+]cyt gradient was always observed in growing pollen tubes. Regular oscillations of the [Ca2+]cyt, however, were rarely identified in Arabidopsis or N. tabacum pollen tubes grown under the in vivo condition or in those placed in germination medium just after they had grown through a style (semi-in vivo condition). On the other hand, regular oscillations were observed in vitro in both growing and nongrowing pollen tubes, although the oscillation amplitude was 5-fold greater in the nongrowing pollen tubes compared with growing pollen tubes. These results suggested that a submicromolar [Ca2+]cyt in the tip region is essential for pollen tube growth, whereas a regular [Ca2+] oscillation is not. Next, we monitored [Ca2+] dynamics in the endoplasmic reticulum ([Ca2+]ER) in relation to Arabidopsis pollen tube growth using yellow cameleon 4.60, which has a lower affinity for Ca2+ compared with yellow cameleon 3.60. The [Ca2+]ER in pollen tubes grown under the semi-in vivo condition was between 100 and 500 μ m. In addition, cyclopiazonic acid, an inhibitor of ER-type Ca2+-ATPases, inhibited growth and decreased the [Ca2+]ER. Our observations suggest that the ER serves as one of the Ca2+ stores in the pollen tube and cyclopiazonic acid-sensitive Ca2+-ATPases in the ER are required for pollen tube growth.

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein–CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.

Nicotine biosynthesis in Nicotiana species requires an oxidative deamination of N-methylputrescine, catalyzed by N-methylputrescine oxidase (MPO). In a screen for tobacco genes that were down-regulated in a tobacco mutant with altered regulation of nicotine biosynthesis, we identified two homologous MPO cDNAs which encode diamine oxidases of a particular subclass. Tobacco MPO genes were expressed specifically in the root, and up-regulated by jasmonate treatment. Recombinant MPO protein expressed in Escherichia coli formed a homodimer and deaminated N-methylputrescine more efficiently than symmetrical diamines. These results indicate that MPO evolved from general diamine oxidases to function effectively in nicotine biosynthesis.

Cortical microtubule arrays are critical in determining the growth axis of diffusely growing plant cells, and various environmental and physiological factors are known to affect the array organization. Microtubule organization is partly disrupted in the spiral1 mutant of Arabidopsis thaliana, which displays a right-handed helical growth phenotype in rapidly elongating epidermal cells. We show here that mutations in the plasma membrane Na(+)/H(+) antiporter SOS1 and its regulatory kinase SOS2 efficiently suppressed both microtubule disruption and helical growth phenotypes of spiral1, and that sos1 and sos2 roots in the absence of salt stress exhibited altered helical growth response to microtubule-interacting drugs at low doses. Salt stress also altered root growth response to the drugs in wild-type roots. Suppression of helical growth appeared to be specific to spiral1 since other helical growth mutants were not rescued. The effects of sos1 in suppressing spiral1 defects and in causing abnormal drug responses were nullified in the presence of the hkt1 Na(+) influx carrier mutation in roots but not in hypocotyls. These results suggest that cytoplasmic salt imbalance caused by insufficient SOS1 activity compromises cortical microtubule functions in which microtubule-localized SPIRAL1 is specifically involved.

Plants contain a large number of proteins homologous to isoflavone reductase, an NADPH-dependent reductase involved in the biosynthesis of isoflavonoid phytoalexins in legumes. Although some are bona fide isoflavone reductases, others may catalyze distinct reductase reactions. Two tobacco genes, TP7 and A622, encoding isoflavone reductase-like proteins, had been previously identified from their unique expression patterns, but their functions were not known. We show here that TP7 is a tobacco phenylcoumaran benzylic ether reductase involved in lignan biosynthesis, but that A622 is not. To gain insight into the possible function of A622, we analyzed in detail the expression patterns of the A622 gene by RNA and protein blots, immunohistochemistry, and its promoter expression in transgenic Nicotiana sylvestris roots. The A622 expression patterns were qualitatively similar to those of putrescine N-methyltransferase, the first enzyme in nicotine biosynthesis, suggesting that A622 may function in the metabolism of nicotine or related alkaloids.

Alkaloids are nitrogen-containing specialized metabolites generated from primary metabolites, including polyamines, in plants. Alkaloids include many biologically active chemicals and confer adaptive advantages to plants producing them. The tropane alkaloids include cocaine and calystegines, whereas pyrrolizidine alkaloids are a group of defense compounds that contain a necine base moiety derived from homospermidine. Nicotine and tropane alkaloids are commonly derived from the diamine putrescine, which is incorporated into the pyrrolidine ring of nicotine or the tropane ring in tropane alkaloids. Here we review the progress that has been made in understanding the biosynthesis of putrescine-derived alkaloids, which has been intensively studied in tobacco and medicinal plants of the Solanaceae. In solanaceous species, alkaloids, including clinically important hyoscyamine and scopolamine, are synthesized in specific cells in the roots and then translocated to shoots through the xylem. Homospermidine synthase, which catalyzes homospermidine formation from putrescine and spermidine, repeatedly evolved in multiple plant lineages from deoxyhypusine synthase involved in posttranslational activation of eIF5A protein. The evolution of these alkaloid biosynthesis enzymes and pathways is discussed in light of the newly available information in this field.

The purine permeases (PUPs) constitute a large plasma membrane-localized transporter family in plants that mediates the proton-coupled uptake of nucleotide bases and their derivatives, such as adenine, cytokinins, and caffeine. A Nicotiana tabacum (tobacco) PUP-family transporter, nicotine uptake permease 1 (NtNUP1), was previously shown to transport tobacco alkaloids and to affect both nicotine biosynthesis and root growth in tobacco plants. Since Arabidopsis PUP1, which belongs to the same subclade as NtNUP1, was recently reported to transport pyridoxine and its derivatives (vitamin B6), it was of interest to examine whether NtNUP1 could also transport these substrates. Direct uptake measurements in the yeast Saccharomyces cerevisiae demonstrated that NtNUP1 efficiently promoted the uptake of pyridoxamine, pyridoxine, anatabine, and nicotine. The naturally occurring (S)-isomer of nicotine was preferentially transported over the (R)-isomer. Transport studies using tobacco BY-2 cell lines overexpressing NtNUP1 or PUP1 showed that NtNUP1, similar to PUP1, transported various compounds containing a pyridine ring, but that the two transporters had distinct substrate preferences. Therefore, the previously reported effects of NtNUP1 on tobacco physiology might involve bioactive metabolites other than tobacco alkaloids.

Plants have developed elaborate detoxification mechanisms to cope with a large number of potentially toxic compounds, which include exogenous xenobiotics and endogenous metabolites, especially secondary metabolites. After enzymatic modification or synthesis, such compounds are transported and accumulated in apoplastic cell walls or central vacuoles in plant cells. Membrane transporters actively catalyze translocation of a diverse range of these compounds across various membranes within cells. Biochemical, molecular, and genetic studies have begun to reveal functions of a handful of ATP-binding cassette and multidrug and toxic compound extrusion family transporters engaged in transport of organic xenobiotics, heavy metals, metalloids, aluminum, alkaloids, flavonoids, terpenoids, terpenoid-derived phytohormones, cuticle lipids, and monolignols in plants. This detoxification versatility and metabolic diversity may underlie the functional diversification in plants of these families of transporters, which are largely involved in multidrug resistance in microorganisms and animals.

Plants have evolved diverse defense metabolites as adaptations to biotic and abiotic stresses. The defense alkaloid nicotine is produced in Nicotiana tabacum (tobacco) and its biosynthesis is elicited by jasmonates in the roots. At least seven jasmonate-responsive genes that encode transcription factors of the Ethylene Response Factor (ERF) family are clustered at the nicotine-regulatory locus NICOTINE2 (NIC2) in the tobacco genome. A subset of the NIC2-locus ERFs and their homologs, including ERF189 and ERF199, have been shown to be most effective in controlling nicotine biosynthetic pathway genes. Herein reported is that the ERF genes of this group, other than ERF189 and ERF199, were strongly induced by NaCl in tobacco hairy roots, although salt stress had no effect on expression of nicotine biosynthesis genes. Abscisic acid and osmotic stress also increased expression of a subset of these NaCl-inducible ERF genes. Promoter expression analysis in transgenic tobacco hairy roots confirmed that while methyl jasmonate (MJ) activated the promoters of ERF29, ERF210 and ERF199, salt stress up-regulated the promoters of only ERF29 and ERF210, but not ERF199. The protein biosynthesis inhibitor cycloheximide induced expression of the ERFs, and simultaneous addition of MJ and cycloheximide showed synergistic effects. These results indicate that, after several gene duplication events, the NIC2-locus ERFs and possibly their homologs appear to have diverged in their responses to jasmonates and various environmental inputs, including salt stress, and may have evolved to regulate distinct metabolic processes and cellular responses.

Plants have developed elaborate detoxification mechanisms to cope with a large number of potentially toxic compounds, which include exogenous xenobiotics and endogenous metabolites, especially secondary metabolites. After enzymatic modification or synthesis, such compounds are transported and accumulated in apoplastic cell walls or central vacuoles in plant cells. Membrane transporters actively catalyze translocation of a diverse range of these compounds across various membranes within cells. Biochemical, molecular, and genetic studies have begun to reveal functions of a handful of ATP-binding cassette and multidrug and toxic compound extrusion family transporters engaged in transport of organic xenobiotics, heavy metals, metalloids, aluminum, alkaloids, flavonoids, terpenoids, terpenoid-derived phytohormones, cuticle lipids, and monolignols in plants. This detoxification versatility and metabolic diversity may underlie the functional diversification in plants of these families of transporters, which are largely involved in multidrug resistance in microorganisms and animals.

Nicotine alkaloids are synthesized in the root of Nicotiana species, and their synthesis increases after insect attack, wounding and jasmonate treatment of the leaf. Putrescine N-methyltransferase (PMT) catalyzes the first committed step in nicotine biosynthesis. The expression patterns of the three Nicotiana sylvestris PMT genes (NsPMT1, NsPMT2, and NsPMT3) are reported in this study. Transcripts of the NsPMT genes were detected only in the root, and were up-regulated by methyl jasmonate treatment. When the 5'-flanking regions of NsPMT1, NsPMT2, and NsPMT3 were fused independently to beta-glucuronidase reporter gene and introduced into N. sylvestris by Agrobacterium-mediated transformation, all introduced transgenes were expressed in the cortex, endodermis, and xylem in the root, as well as upregulated by methyl jasmonate treatment. These qualitatively similar patterns of expression for the NsPMT genes are achieved with only 0.25 kb of their conserved 5'-flanking regions, which contained no known jasmonate-responsive elements.

The putrescine N-methyltransferase (PMT) cDNA clone previously isolated from tobacco encodes a spermidine synthase-like protein with an 11 amino acid element repeated four times in tandem at the amino terminus. Genomic Southern blot analyses indicated that this N-terminal repeat array is found in tobacco PMTs but absent in Hyoscyamus and Atropa PMTs. A truncated tobacco PMT in which this repeat array was entirely removed still retained full enzymatic activity when expressed in Escherichia coli. Three PMT genes (NsPMT1, NsPMT2, NsPMT3) isolated from Nicotiana sylvestris encode two, five, and nine tandem repeats, respectively, in the first exon, but otherwise encode highly conserved proteins. Analysis of PCR fragments amplified from the genomes of N. tabacum and its two probable progenitors shows that one of the nine repeat elements in NsPMT3 was precisely deleted in the corresponding N. tabacum gene. These results indicate that direct tandem repeats of a 33 bp sequence that encodes 11 amino acids of no obvious function were added to the ancestral Nicotiana PMT gene, and that the tandem repetition was genetically very unstable, contracting or expanding during evolution of the Nicotiana species.

A 41-kD DNA binding protein with a basic pl was purified from chloroplast nucleoids in photomixotrophically cultured tobacco cells, and its amino acid sequence was determined. Using this sequence information, its cDNA (CND41) was isolated, and its nucleotide sequence was determined. The predicted amino acid sequence of CND41 has a transit peptide of 120 amino acids and a mature protein of 382 amino acids. A distinctive helix-turn-helix motif in the lysine-rich N-terminal region of the mature protein and an aspartyl protease active site motif were predicted. Expression of a series of truncated CND41 proteins in Escherichia coli indicated that the lysine-rich region is essential for DNA binding and that CND41 nonspecifically binds chloroplast DNA. Protein gel blot analyses showed CND41 mainly in cells and/or tissues containing nonphotosynthesizing, actively growing plastids. In addition, the accumulation of chloroplast transcripts in these cells and/or tissues (e.g., transcripts for QB binding protein of photosystem II [psbA] and large subunit of ribulose bisphosphate carboxylase [rbcL]) was negatively correlated with the accumulation of CND41. Analyses of cultured cells of transgenic tobacco with reduced CND41 levels showed a higher level of expression of chloroplast genes compared with that of the wild type. We discuss the possible function of CND41 as a negative regulator of chloroplast gene expression.

Biosynthesis of nicotine in tobacco requires N-methylputrescine oxidase (MPO), which belongs to the copper-containing amine oxidase superfamily. Previous studies identified tobacco MPO1 and its close homolog NtDAO1 (formerly called MPO2), of which MPO1 has been shown preferentially to oxidize N-methylated amines. We show here that NtDAO1, as well as a homologous Arabidopsis diamine oxidase (DAO), accept non-N-methylated amines more efficiently than their corresponding N-methylated amines. MPO1 is coordinately regulated with other nicotine biosynthesis genes with regard to COI1-MYC2-dependent jasmonate induction and its dependence on nicotine-specific ERF transcription factors, whereas NtDAO1 is constitutively expressed at low basal levels in tobacco plants. Both MPO1 and NtDAO1 are targeted to peroxisomes by their C-terminal motifs, and the peroxisomal localization of MPO1 is required for it to function in nicotine biosynthesis in jasmonate-elicited cultured tobacco cells. Restricted occurrence of the MPO subfamily in Nicotiana and Solanum indicates that, during the formation of the Solanaceae, MPO has evolved from a DAO, which functions in polyamine catabolism within peroxisomes, by optimizing substrate preference and gene expression patterns to be suitable for alkaloid formation.

In tobacco (Nicotiana tabacum), nicotine and related pyridine alkaloids are produced in the root, and then transported to the aerial parts where these toxic chemicals function as part of chemical defense against insect herbivory. Although a few tobacco transporters have been recently reported to take up nicotine into the vacuole from the cytoplasm or into the cytoplasm from the apoplast, it is not known how the long-range translocation of tobacco alkaloids between organs is controlled. Nicotiana langsdorffii and N. alata are closely related species of diploid Nicotiana section Alatae, but the latter does not accumulate tobacco alkaloids in the leaf. We show here that N. alata does synthesize alkaloids in the root, but lacks the capacity to mobilize the root-borne alkaloids to the aerial parts. Interspecific grafting experiments between N. alata and N. langsdorffii indicate that roots of N. alata are unable to translocate alkaloids to their shoot system. Interestingly, genetic studies involving interspecific hybrids between N. alata and N. langsdorffii and their self-crossed or back-crossed progeny showed that the non-translocation phenotype is dominant over the translocation phenotype. These results indicate that a mechanism to retain tobacco alkaloids within the root organ has evolved in N. alata, which may represent an interesting strategy to control the distribution of secondary products within a whole plant.