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The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.

Description, Analysis of plant protective surface hair chemistry revealed evolutionary mechanisms leading to metabolic innovation., Plants synthesize myriad phylogenetically restricted specialized (aka “secondary”) metabolites with diverse structures. Metabolism of acylated sugar esters in epidermal glandular secreting trichomes across the Solanaceae (nightshade) family is ideal for investigating the mechanisms of evolutionary metabolic diversification. We developed methods to structurally analyze acylhexose mixtures by 2D NMR, which led to the insight that the Old World species black nightshade (Solanum nigrum) accumulates acylglucoses and acylinositols in the same tissue. Detailed in vitro biochemistry, cross-validated by in vivo virus-induced gene silencing, revealed two unique features of the four-step acylglucose biosynthetic pathway: A trichome-expressed, neofunctionalized invertase-like enzyme, SnASFF1, converts BAHD-produced acylsucroses to acylglucoses, which, in turn, are substrates for the acylglucose acyltransferase, SnAGAT1. This biosynthetic pathway evolved independently from that recently described in the wild tomato Solanum pennellii, reinforcing that acylsugar biosynthesis is evolutionarily dynamic with independent examples of primary metabolic enzyme cooption and additional variation in BAHD acyltransferases.

Acylsugars constitute an abundant class of pest- and pathogen-protective Solanaceae family plant-specialized metabolites produced in secretory glandular trichomes. Solanum pennellii produces copious triacylated sucrose and glucose esters, and the core biosynthetic pathway producing these compounds was previously characterized. We performed untargeted metabolomic analysis of S. pennellii surface metabolites from accessions spanning the species range, which indicated geographic trends in the acylsugar profile and revealed two compound classes previously undescribed from this species, tetraacylglucoses and flavonoid aglycones. A combination of ultrahigh-performance liquid chromatography&ndash, high resolution mass spectrometry (UHPLC&ndash, HR-MS) and NMR spectroscopy identified variations in the number, length, and branching pattern of acyl chains, and the proportion of sugar cores in acylsugars among accessions. The new dimensions of acylsugar variation revealed by this analysis further indicate variation in the biosynthetic and degradative pathways responsible for acylsugar accumulation. These findings provide a starting point for deeper investigation of acylsugar biosynthesis, an understanding of which can be exploited through crop breeding or metabolic engineering strategies to improve the endogenous defenses of crop plants.

Acylsugars constitute an abundant class of pest- and pathogen-protective Solanaceae family plant specialized metabolites produced in secretory glandular trichomes. Solanum pennellii produces copious triacylated sucrose and glucose esters, and the core biosynthetic pathway producing these compounds was previously characterized. We performed untargeted metabolomic analysis of S. pennellii surface metabolites from accessions spanning the species range, which indicated geographic trends in acylsugar profile and revealed two compound classes previously undescribed from this species, tetraacylglucoses and flavonoid aglycones. A combination of ultrahigh performance liquid chromatography high resolution mass spectrometry (UHPLC-HR-MS) and NMR spectroscopy identified variations in number, length, and branching pattern of acyl chains, and the proportion of sugar cores in acylsugars among accessions. The new dimensions of acylsugar variation revealed by this analysis further indicate variation in the biosynthetic and degradative pathways responsible for acylsugar accumulation. These findings provide a starting point for deeper investigation of acylsugar biosynthesis, an understanding of which can be exploited through crop breeding or metabolic engineering strategies to improve endogenous defenses of crop plants.

Switchgrass (Panicum virgatum L.) is a bioenergy crop that grows productively on lands not suitable for food production, and is an excellent target for low-pesticide input biomass production. We hypothesize that resistance to insect pests and microbial pathogens is influenced by low molecular weight compounds known as specialized metabolites. We employed untargeted liquid chromatography-mass spectrometry (LC-MS), quantitative gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy to identify differences in switchgrass ecotype metabolomes. This analysis revealed striking differences between upland and lowland switchgrass metabolomes as well as distinct developmental profiles. Terpenoid and polyphenol derived specialized metabolites were identified, including steroidal saponins, di- and sesqui-terpenoids and flavonoids. The saponins are especially abundant in switchgrass extracts and have diverse aglycone cores and sugar moieties. We report seven structurally distinct steroidal saponin classes with unique steroidal cores and glycosylated at one or two positions. Quantitative GC-MS revealed differences in total saponin concentrations in leaf blade, leaf sheath, stem, rhizome and root. The quantitative data also demonstrated that saponin concentrations is higher in roots of lowland than upland ecotype plants, suggesting ecotypic specific biosynthesis and/or biological functions. These results enable future testing of these specialized metabolites on biotic and abiotic stress tolerance and can inform development of low-input bioenergy crops.One sentence summaryIntegrated mass-spectrometry and nuclear magnetic resonance spectroscopy based metabolomics reveal that switchgrass accumulates structurally diverse terpenoids and phenolics, which vary in abundance and structure in a tissue- and ecotype-specific manner.

Natural pyrethrin insecticides produced by Dalmatian pyrethrum (Tanacetum cinerariifolium) have low mammalian toxicity and short environmental persistence, providing an alternative to widely used synthetic agricultural insecticides that pose a threat to human health and the environment. A recent surge of interest in the use of pyrethrins as agricultural insecticides coincides with the discovery of several new genes in the pyrethrin biosynthetic pathway. Elucidation of this pathway facilitates efforts to breed improved pyrethrum varieties and to engineer plants with improved endogenous defenses or hosts for heterologous pyrethrin production. We describe the current state of knowledge related to global pyrethrum production, the pyrethrin biosynthetic pathway and its regulation, and recent efforts to engineer the pyrethrin pathway in diverse plant hosts.

Plants produce phylogenetically and spatially restricted, as well as structurally diverse specialized metabolites via multistep metabolic pathways. Hallmarks of specialized metabolic evolution include enzymatic promiscuity and recruitment of primary metabolic enzymes and examples of genomic clustering of pathway genes. Solanaceae glandular trichomes produce defensive acylsugars, with sidechains that vary in length across the family. We describe a tomato gene cluster on chromosome 7 involved in medium chain acylsugar accumulation due to trichome specific acyl-CoA synthetase and enoyl-CoA hydratase genes. This cluster co-localizes with a tomato steroidal alkaloid gene cluster and is syntenic to a chromosome 12 region containing another acylsugar pathway gene. We reconstructed the evolutionary events leading to this gene cluster and found that its phylogenetic distribution correlates with medium chain acylsugar accumulation across the Solanaceae. This work reveals insights into the dynamics behind gene cluster evolution and cell-type specific metabolite diversity., eLife digest Plants produce a vast variety of different molecules known as secondary or specialized metabolites to attract pollinating insects, such as bees, or protect themselves against herbivores and pests. The secondary metabolites are made from simple building blocks that are readily available in plants, including amino acids, fatty acids and sugars. Different species of plant, and even different parts of the same plant, produce their own sets of secondary metabolites. For example, the hairs on the surface of tomatoes and other members of the nightshade family of plants make metabolites known as acylsugars. These chemicals deter herbivores and pests from damaging the plants. To make acylsugars, the plants attach long chains known as fatty acyl groups to molecules of sugar, such as sucrose. Some members of the nightshade family produce acylsugars with longer chains than others. In particular, acylsugars with long chains are only found in tomatoes and other closely-related species. It remained unclear how the nightshade family evolved to produce acylsugars with chains of different lengths. To address this question, Fan et al. used genetic and biochemical approaches to study tomato plants and other members of the nightshade family. The experiments identified two genes known as AACS and AECH in tomatoes that produce acylsugars with long chains. These two genes originated from the genes of older enzymes that metabolize fatty acids – the building blocks of fats – in plant cells. Unlike the older genes, AACS and AECH were only active at the tips of the hairs on the plant’s surface. Fan et al. then investigated the evolutionary relationship between 11 members of the nightshade family and two other plant species. This revealed that AACS and AECH emerged in the nightshade family around the same time that longer chains of acylsugars started appearing. These findings provide insights into how plants evolved to be able to produce a variety of secondary metabolites that may protect them from a broader range of pests. The gene cluster identified in this work could be used to engineer other species of crop plants to start producing acylsugars as natural pesticides.

Plants make hundreds of thousands of biologically active specialized metabolites varying widely in structure, biosynthesis and the processes that they influence. An increasing number of these compounds are documented to protect plants from harmful insects, pathogens, or herbivores, or mediate interactions with beneficial organisms including pollinators and nitrogen fixing microbes. Acylsugars – one class of protective compounds – are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop Solanum quitoense (Naranjilla) produces acylsugars that contain a myo-inositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the Solanum lycopersicum acylsucrose biosynthetic pathway. Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin.Material availabilityThe author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Robert L. Last (lastr@msu.edu).One sentence summaryEvidence that the final step in Solanum quitoense acylinositol biosynthesis evolved from an acylsucrose acetyltransferase enzyme.

Plants make many biologically active, specialized metabolites, which vary in structure, biosynthesis, and the processes they influence. An increasing number of these compounds are documented to protect plants from insects, pathogens, or herbivores or to mediate interactions with beneficial organisms, including pollinators and nitrogen-fixing microbes. Acylsugars, one class of protective compounds, are made in glandular trichomes of plants across the Solanaceae family. While most described acylsugars are acylsucroses, published examples also include acylsugars with hexose cores. The South American fruit crop naranjilla (lulo; Solanum quitoense) produces acylsugars containing a myoinositol core. We identified an enzyme that acetylates triacylinositols, a function homologous to the last step in the acylsucrose biosynthetic pathway of tomato (Solanum lycopersicum). Our analysis reveals parallels between S. lycopersicum acylsucrose and S. quitoense acylinositol biosynthesis, suggesting a common evolutionary origin.

The pyrethrum plant, Tanacetum cineramfolium (Asteraceae) synthesizes a class of compounds called pyrethrins that have strong insecticidal properties but are safe to humans. Class I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of three jasmonic-acid derived alcohols. We reconstructed the trans-chry-santhemic acid biosynthetic pathway in tomato fruits, which naturally produce high levels of the tetraterpene pigment lycopene, an isoprenoid which shares a common precursor, dimethylallyl diphosphate (DMAPP), with trans-chrysanthemic acid. trans-Chrysanthemic acid biosynthesis in tomato fruit was achieved by expressing the chrysanthemyl diphosphate synthase gene from T. cinerariifolium, encoding the enzyme that uses DMAPP to make trans-chrysanthemol, under the control of the fruit specific promoter PG, as well as an alcohol dehy-drogenease (ADH) gene and aldehyde dehydrogenase (ALDH) gene from a wild tomato species, also under the control of the PG promoter. Tomato fruits expressing all three genes had a concentration of trans-chrysanthemic acid that was about 1.7-fold higher (by weight) than the levels of lycopene present in non-transgenic fruit, while the level of lycopene in the transgenic plants was reduced by 68%. Ninety seven percent of the diverted DMAPP was converted to trans-chrysanthemic acid, but 62% of this acid was further glycosylated. We conclude that the tomato fruit is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.

Flowers of Tanacetum cinerariifolium produce a set of compounds known collectively as pyrethrins, which are commercially important pesticides that are strongly toxic to flying insects but not to most vertebrates. A pyrethrin molecule is an ester consisting of either trans-chrysanthemic acid or its modified form, pyrethric acid, and one of three alcohols, jasmolone, pyrethrolone, and cinerolone, that appear to be derived from jasmonic acid. Chrysanthemyl diphosphate synthase (CDS), the first enzyme involved in the synthesis of trans-chrysanthemic acid, was characterized previously and its gene isolated. TcCDS produces free trans-chrysanthemol in addition to trans-chrysanthemyl diphosphate, but the enzymes responsible for the conversion of trans-chrysanthemol to the corresponding aldehyde and then to the acid have not been reported. We used an RNA sequencing-based approach and coexpression correlation analysis to identify several candidate genes encoding putative trans-chrysanthemol and trans-chrysanthemal dehydrogenases. We functionally characterized the proteins encoded by these genes using a combination of in vitro biochemical assays and heterologous expression in planta to demonstrate that TcADH2 encodes an enzyme that oxidizes trans-chrysanthemol to trans-chrysanthemal, while TcALDH1 encodes an enzyme that oxidizes trans-chrysanthemal into trans-chrysanthemic acid. Transient coexpression of TcADH2 and TcALDH1 together with TcCDS in Nicotiana benthamiana leaves results in the production of trans-chrysanthemic acid as well as several other side products. The majority (58%) of trans-chrysanthemic acid was glycosylated or otherwise modified. Overall, these data identify key steps in the biosynthesis of pyrethrins and demonstrate the feasibility of metabolic engineering to produce components of these defense compounds in a heterologous host.

The branched-chain amino acids (BCAAs) Ile, Val and Leu are essential nutrients that humans and other animals obtain from plants. However, total and relative amounts of plant BCAAs rarely match animal nutritional needs, and improvement requires a better understanding of the mechanistic basis for BCAA homeostasis. We present an in vivo regulatory model of BCAA homeostasis derived from analysis of feedback-resistant Arabidopsis thaliana mutants for the three allosteric committed enzymes in the biosynthetic network: threonine deaminase (also named L-O-methylthreonine resistant 1, OMR1), acetohydroxyacid synthase small subunit 2 (AHASS2) and isopropylmalate synthase 1 (IPMS1). In this model, OMR1 exerts primary control on Ile accumulation and functions independently of AHAS and IPMS. AHAS and IPMS regulate Val and Leu homeostasis, where AHAS affects total Val+Leu and IPMS controls partitioning between these amino acids. In addition, analysis of feedback-resistant and loss-of-function single and double mutants revealed that each AHAS and IPMS isoenzyme contributes to homeostasis rather than being functionally redundant. The characterized feedback-resistant mutations caused increased free BCAA levels in both seedlings and seeds. These results add to our understanding of the basis of in vivo BCAA homeostasis and inform approaches to improve the amount and balance of these essential nutrients in crops.

The plant pyrethrum (Tanacetum cinerariifolium) synthesizes highly effective natural pesticides known as pyrethrins. Pyrethrins are esters consisting of an irregular monoterpenoid acid and an alcohol derived from jasmonic acid (JA). These alcohols, referred to as rethrolones, can be jasmolone, pyrethrolone, or cinerolone. We recently showed that jasmolone is synthesized from jasmone, a degradation product of JA, in a single hydroxylation step catalyzed by jasmone hydroxylase (TcJMH). TcJMH belongs to the CYP71 clade of the cytochrome P450 oxidoreductase family. Here, we used coexpression analysis, heterologous gene expression, and in vitro biochemical assays to identify the enzyme responsible for conversion of jasmolone to pyrethrolone. A further T. cinerariifolium cytochrome P450 family member, CYP82Q3 (designated Pyrethrolone Synthase; TcPYS), appeared to catalyze the direct desaturation of the C1–C2 bond in the pentyl side chain of jasmolone to produce pyrethrolone. TcPYS is highly expressed in the trichomes of the ovaries in pyrethrum flowers, similar to TcJMH and other T. cinerariifolium genes involved in JA biosynthesis. Thus, as previously shown for biosynthesis of the monoterpenoid acid moiety of pyrethrins, rethrolones are synthesized in the trichomes. However, the final assembly of pyrethrins occurs in the developing achenes. Our data provide further insight into pyrethrin biosynthesis, which could ultimately be harnessed to produce this natural pesticide in a heterologous system.

Evolution of a cell specific invertase enzyme creates phenotypic diversity in wild tomato insecticidal metabolite production., Plants produce a myriad of taxonomically restricted specialized metabolites. This diversity—and our ability to correlate genotype with phenotype—makes the evolution of these ecologically and medicinally important compounds interesting and experimentally tractable. Trichomes of tomato and other nightshade family plants produce structurally diverse protective compounds termed acylsugars. While cultivated tomato (Solanum lycopersicum) strictly accumulates acylsucroses, the South American wild relative Solanum pennellii produces copious amounts of acylglucoses. Genetic, transgenic, and biochemical dissection of the S. pennellii acylglucose biosynthetic pathway identified a trichome gland cell–expressed invertase-like enzyme that hydrolyzes acylsucroses (Sopen03g040490). This enzyme acts on the pyranose ring–acylated acylsucroses found in the wild tomato but not on the furanose ring–decorated acylsucroses of cultivated tomato. These results show that modification of the core acylsucrose biosynthetic pathway leading to loss of furanose ring acylation set the stage for co-option of a general metabolic enzyme to produce a new class of protective compounds.

Glandular trichomes of cultivated tomato (Solanum lycopersicum) and many other species throughout the Solanaceae produce and secrete mixtures of sugar esters (acylsugars) on the plant aerial surfaces. In wild and cultivated tomato, these metabolites consist of a sugar backbone, typically glucose or sucrose, and two to five acyl chains esterified to various positions on the sugar core. The aliphatic acyl chains vary in length and branching and are transferred to the sugar by a series of reactions catalyzed by acylsugar acyltransferases. A phenotypic screen of a set of S. lycopersicum M82 × Solanum pennellii LA0716 introgression lines identified a dominant genetic locus on chromosome 5 from the wild relative that affected total acylsugar levels. Genetic mapping revealed that the reduction in acylsugar levels was consistent with the presence and increased expression of two S. pennellii genes (Sopen05g030120 and Sopen05g030130) encoding putative carboxylesterase enzymes of the α/β-hydrolase superfamily. These two enzymes, named ACYLSUGAR ACYLHYDROLASE1 (ASH1) and ASH2, were shown to remove acyl chains from specific positions of certain types of acylsugars in vitro. A survey of related genes in M82 and LA0716 identified another trichome-expressed ASH gene on chromosome 9 (M82, Solyc09g075710; LA0716, Sopen09g030520) encoding a protein with similar activity. Characterization of the in vitro activities of the SpASH enzymes showed reduced activities with acylsugars produced by LA0716, presumably contributing to the high-level production of acylsugars in the presence of highly expressed SpASH genes.

Flavonoids are ubiquitous plant aromatic specialized metabolites found in a variety of cell types and organs. Methylated flavonoids are detected in secreting glandular trichomes of various Solanum species, including the cultivated tomato (Solanum lycopersicum). Inspection of the sequenced S. lycopersicum Heinz 1706 reference genome revealed a close homolog of Solanum habrochaites MOMT1 3'/5' myricetin O-methyltransferase gene, but this gene (Solyc06g083450) is missing the first exon, raising the question of whether cultivated tomato has a distinct 3' or 3'/5' O-methyltransferase. A combination of mining genome and cDNA sequences from wild tomato species and S. lycopersicum cultivar M82 led to the identification of Sl-MOMT4 as a 3' O-methyltransferase. In parallel, three independent ethyl methanesulfonate mutants in the S. lycopersicum cultivar M82 background were identified as having reduced amounts of di- and trimethylated myricetins and increased monomethylated myricetin. Consistent with the hypothesis that Sl-MOMT4 is a 3' O-methyltransferase gene, all three myricetin methylation defective mutants were found to have defects in MOMT4 sequence, transcript accumulation, or 3'-O-methyltransferase enzyme activity. Surprisingly, no MOMT4 sequence is found in the Heinz 1706 reference genome sequence, and this cultivar accumulates 3-methyl myricetin and is deficient in 3'-methyl myricetins, demonstrating variation in this gene among cultivated tomato varieties.

A systematic screen of volatile terpene production in the glandular trichomes of 79 accessions of Solanum habrochaites was conducted and revealed the presence of 21 mono- and sesquiterpenes that exhibit a range of qualitative and quantitative variation. Hierarchical clustering identified distinct terpene phenotypic modules with shared patterns of terpene accumulation across accessions. Several terpene modules could be assigned to previously identified terpene synthase (TPS) activities that included members of the TPS-e/f subfamily that utilize the unusual cis-prenyl diphosphate substrates neryl diphosphate and 2z,6z-farnesyl diphosphate. DNA sequencing and in vitro enzyme activity analysis of TPS-e/f members from S. habrochaites identified three previously unassigned enzyme activities that utilize these cisoid substrates. These produce either the monoterpenes α-pinene and limonene, or the sesquiterpene 7-epizingiberene, with the in vitro analyses that recapitulated the trichome chemistry found in planta. Comparison of the distribution of S. habrochaites accessions with terpene content revealed a strong preference for the presence of particular TPS20 alleles at distinct geographic locations. This study reveals that the unusually high intra-specific variation of volatile terpene synthesis in glandular trichomes of S. habrochaites is due at least in part to evolution at the TPS20 locus.

This work identifies LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), a Zn finger protein that shows disulfide isomerase activity, interacts with the photosystem II (PSII) core complex, and may act in repair of photodamaged PSII complexes. Two mutants of an unannotated small Zn finger containing a thylakoid membrane protein of Arabidopsis thaliana (At1g75690; LQY1) were found to have a lower quantum yield of PSII photochemistry and reduced PSII electron transport rate following high-light treatment. The mutants dissipate more excess excitation energy via nonphotochemical pathways than wild type, and they also display elevated accumulation of reactive oxygen species under high light. After high-light treatment, the mutants have less PSII–light-harvesting complex II supercomplex than wild-type plants. Analysis of thylakoid membrane protein complexes showed that wild-type LQY1 protein comigrates with the PSII core monomer and the CP43-less PSII monomer (a marker for ongoing PSII repair and reassembly). PSII repair and reassembly involve the breakage and formation of disulfide bonds among PSII proteins. Interestingly, the recombinant LQY1 protein demonstrates a protein disulfide isomerase activity. LQY1 is more abundant in stroma-exposed thylakoids, where key steps of PSII repair and reassembly take place. The absence of the LQY1 protein accelerates turnover and synthesis of PSII reaction center protein D1. These results suggest that the LQY1 protein may be involved in maintaining PSII activity under high light by regulating repair and reassembly of PSII complexes.

Large-scale phenotypic screening presents challenges and opportunities not encountered in typical forward or reverse genetics projects. We describe a modular database and laboratory information management system that was implemented in support of the Chloroplast 2010 Project, an Arabidopsis (Arabidopsis thaliana) reverse genetics phenotypic screen of more than 5,000 mutants (http://bioinfo.bch.msu.edu/2010_LIMS; www.plastid.msu.edu). The software and laboratory work environment were designed to minimize operator error and detect systematic process errors. The database uses Ruby on Rails and Flash technologies to present complex quantitative and qualitative data and pedigree information in a flexible user interface. Examples are presented where the database was used to find opportunities for process changes that improved data quality. We also describe the use of the data-analysis tools to discover mutants defective in enzymes of leucine catabolism (heteromeric mitochondrial 3-methylcrotonyl-coenzyme A carboxylase [At1g03090 and At4g34030] and putative hydroxymethylglutaryl-coenzyme A lyase [At2g26800]) based upon a syndrome of pleiotropic seed amino acid phenotypes that resembles previously described isovaleryl coenzyme A dehydrogenase (At3g45300) mutants. In vitro assay results support the computational annotation of At2g26800 as hydroxymethylglutaryl-coenzyme A lyase.

Summary Glandular secreting trichomes of cultivated tomato (Solanum lycopersicum) and close relatives produce a variety of structurally diverse volatile and non-volatile specialized (‘secondary’) metabolites, including terpenes, flavonoids and acyl sugars. A genetic screen is described here to profile leaf trichome and surface metabolite extracts of nearly isogenic chromosomal substitution lines covering the tomato genome. These lines contain specific regions of the Solanum pennellii LA0716 genome in an otherwise ‘wild-type’ M82 tomato genetic background. Regions that have an impact on the total amount of extractable mono- and sesquiterpenes (IL2-2) or only sesquiterpenes (IL10-3) or specifically influence accumulation of the monoterpene α-thujene (IL1-3 and IL1-4) were identified using GC-MS. A rapid LC-TOF-MS method was developed and used to identify changes in non-volatile metabolites through non-targeted analysis. Metabolite profiles generated using this approach led to the discovery of introgression lines producing different acyl chain substitutions on acyl sugar metabolites (IL1-3/1-4 and IL8-1/8-1-1), as well as two regions that influence the quantity of acyl sugars (IL5-3 and IL11-3). Chromosomal region 1-1/1-1-3 was found to influence the types of glycoalkaloids that are detected in leaf surface extracts. These results show that direct chemical screening is a powerful way to characterize genetic diversity in trichome specialized metabolism.

Traditionally, phenotype-driven forward genetic plant mutant studies have been among the most successful approaches to revealing the roles of genes and their products and elucidating biochemical, developmental, and signaling pathways. A limitation is that it is time consuming, and sometimes technically challenging, to discover the gene responsible for a phenotype by map-based cloning or discovery of the insertion element. Reverse genetics is also an excellent way to associate genes with phenotypes, although an absence of detectable phenotypes often results when screening a small number of mutants with a limited range of phenotypic assays. The Arabidopsis Chloroplast 2010 Project (www.plastid.msu.edu) seeks synergy between forward and reverse genetics by screening thousands of sequence-indexed Arabidopsis (Arabidopsis thaliana) T-DNA insertion mutants for a diverse set of phenotypes. Results from this project are discussed that highlight the strengths and limitations of the approach. We describe the discovery of altered fatty acid desaturation phenotypes associated with mutants of At1g10310, previously described as a pterin aldehyde reductase in folate metabolism. Data are presented to show that growth, fatty acid, and chlorophyll fluorescence defects previously associated with antisense inhibition of synthesis of the family of acyl carrier proteins can be attributed to a single gene insertion in Acyl Carrier Protein4 (At4g25050). A variety of cautionary examples associated with the use of sequence-indexed T-DNA mutants are described, including the need to genotype all lines chosen for analysis (even when they number in the thousands) and the presence of tagged and untagged secondary mutations that can lead to the observed phenotypes.

In traditional mutant screening approaches, genetic variants are tested for one or a small number of phenotypes. Once bona fide variants are identified, they are typically subjected to a limited number of secondary phenotypic screens. Although this approach is excellent at finding genes involved in specific biological processes, the lack of wide and systematic interrogation of phenotype limits the ability to detect broader syndromes and connections between genes and phenotypes. It could also prevent detection of the primary phenotype of a mutant. As part of a systems biology approach to understand plastid function, large numbers of Arabidopsis thaliana homozygous T-DNA lines are being screened with parallel morphological, physiological, and chemical phenotypic assays (www.plastid.msu.edu). To refine our approaches and validate the use of this high-throughput screening approach for understanding gene function and functional networks, approximately 100 wild-type plants and 13 known mutants representing a variety of phenotypes were analyzed by a broad range of assays including metabolite profiling, morphological analysis, and chlorophyll fluorescence kinetics. Data analysis using a variety of statistical approaches showed that such industrial approaches can reliably identify plant mutant phenotypes. More significantly, the study uncovered previously unreported phenotypes for these well-characterized mutants and unexpected associations between different physiological processes, demonstrating that this approach has strong advantages over traditional mutant screening approaches. Analysis of wild-type plants revealed hundreds of statistically robust phenotypic correlations, including metabolites that are not known to share direct biosynthetic origins, raising the possibility that these metabolic pathways have closer relationships than is commonly suspected.

Glucosinolates are secondary metabolites involved in pathogen and insect defense of cruciferous plants. Although seeds and vegetative tissue often have very different glucosinolate profiles, few genetic factors that determine seed glucosinolate accumulation have been identified. An HPLC-based screen of 5500 mutagenized Arabidopsis thaliana lines produced 33 glucosinolate mutants, of which 21 have seed-specific changes. Five of these mutant lines, representing three genetic loci, are compromised in the biosynthesis of benzoyloxyglucosinolates, which are only found in seeds and young seedlings of A. thaliana. Genetic mapping and analysis of T-DNA insertions in candidate genes identified BZO1 (At1g65880), which encodes an enzyme with benzoyl-CoA ligase activity, as being required for the accumulation of benzoyloxyglucosinolates. Long-chain aliphatic glucosinolates are elevated in bzo1 mutants, suggesting substrate competition for the common short-chain aliphatic glucosinolate precursors. Whereas bzo1 mutations have seed-specific effects on benzoyloxyglucosinolate accumulation, the relative abundance of 3-benzoyloxypropyl- and 4-benzoyloxybutylglucosinolates depends on the maternal genotype.

The eskimo1 (esk1) mutation of Arabidopsis resulted in a 5.5 degrees C improvement in freezing tolerance in the absence of cold acclimation. Here we show that the increase in freezing tolerance is not associated with any increase in the ability to survive drought or salt stresses, which are similar to freezing in their induction of cellular dehydration. Genome-wide comparisons of gene expression between esk1-1 and wild type indicate that mutations at esk1 result in altered expression of transcription factors and signaling components and of a set of stress-responsive genes. Interestingly, the list of 312 genes regulated by ESK1 shows greater overlap with sets of genes regulated by salt, osmotic and abscisic acid treatments than with genes regulated by cold acclimation or by the transcription factors CBF3 and ICE1, which have been shown to control genetic pathways for freezing tolerance. Map-based cloning identified the esk1 locus as At3g55990. The wild-type ESK1 gene encodes a 57-kDa protein and is a member of a large gene family of DUF231 domain proteins whose members encode a total of 45 proteins of unknown function. Our results indicate that ESK1 is a novel negative regulator of cold acclimation. Mutations in the ESK1 gene provide strong freezing tolerance through genetic regulation that is apparently very different from previously described genetic mechanisms of cold acclimation.

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330--or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)--and Solyc04g012020 (Sl-ASAT2). These enzymes were used--in concert with two previously identified BAHD acyltransferases--to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.

Plants contain many unique biosynthetic pathways producing a diverse array of natural products that are important for plant function, agriculture, and human nutrition. The tocochromanols define one such class of compounds, comprised of four tocopherols and four tocotrienols that are collectively termed vitamin E. Tocochromanols are synthesized only by plants and other oxygenic, photosynthetic organisms, and the eight individual compounds vary widely in their vitamin E activities. Vitamin E was recognized as an essential component in mammalian diets in the 1920s and the tocochromanol biosynthetic pathway elucidated from radiotracer studies in the mid 1980s. However, it is only recently that genetic and genomics-based approaches in model photosynthetic organisms have allowed the genes and proteins for tocochromanol synthesis to be isolated, setting the stage for targeted manipulation of tocochromanol levels and types in various crops. This article reviews advancements in our molecular and genetic understanding of the tocochromanol biosynthetic pathway in the model photosynthetic organisms Arabidopsis thaliana and Synechocystis sp. PCC6803 and highlights ongoing efforts to use this knowledge to manipulate the levels of this essential nutrient in food crops.

We report the identification and characterization of a low tocopherol Arabidopsis thaliana mutant, vitamin E pathway gene5-1 (vte5-1), with seed tocopherol levels reduced to 20% of the wild type. Map-based identification of the responsible mutation identified a G→A transition, resulting in the introduction of a stop codon in At5g04490, a previously unannotated gene, which we named VTE5. Complementation of the mutation with the wild-type transgene largely restored the wild-type tocopherol phenotype. A knockout mutation of the Synechocystis sp PCC 6803 VTE5 homolog slr1652 reduced Synechocystis tocopherol levels by 50% or more. Bioinformatic analysis of VTE5 and slr1652 indicated modest similarity to dolichol kinase. Analysis of extracts from Arabidopsis and Synechocystis mutants revealed increased accumulation of free phytol. Heterologous expression of these genes in Escherichia coli supplemented with free phytol and in vitro assays of recombinant protein produced phytylmonophosphate, suggesting that VTE5 and slr1652 encode phytol kinases. The phenotype of the vte5-1 mutant is consistent with the hypothesis that chlorophyll degradation-derived phytol serves as an important intermediate in seed tocopherol synthesis and forces reevaluation of the role of geranylgeranyl diphosphate reductase in tocopherol biosynthesis.

We report the characterization of an Arabidopsis thaliana mutant, ups1, isolated on the basis of reduced expression of phosphoribosylanthranilate transferase, a tryptophan biosynthetic enzyme. ups1 also exhibits defects in a wide range of defence responses. After infection with Pseudomonas syringae or Botrytis cinerea, the expression of genes regulated by both the salicylic acid and jasmonic acid/ethylene pathways is reduced in ups1 compared with wild type. Camalexin accumulation in ups1 is greatly reduced after infection with these two pathogens, as well as after amino acid starvation or oxidative stress. Reactive oxygen species (ROS)-mediated gene expression is also compromised in ups1 indicating that this mutant is defective in signalling pathways activated in response to both biotic and abiotic stress. The fact that all three major defence signalling pathways are disrupted in ups1, together with the oxidative stress phenotype, leads us to suggest that UPS1 is involved in ROS signal transduction.

SummaryBeyond their essential function as the building blocks of proteins, amino acids contribute to many aspects ofplant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of thebiochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20–90 min forstandard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels offree amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one ormore amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines withincreased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNAsequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause ofincreased seed Thr levels in one mutant. The assay that was developed for this project has broad applicabilityto Arabidopsis and other plant species.Keywords: amino acid, threonine, Arabidopsis, HPLC-MS/MS, isoleucine, mutant screen.IntroductionAmino acids play many important roles beyond incorpor-ation into proteins in flowering plants (Coruzzi and Last,2000). Glu, Gln, Asp and Asn are sources of nitrogen andprecursors for a wide variety of metabolic pathways. Theseare also major nitrogen transport molecules that are regula-ted in response to environmental signals such as light, andmay in fact act as signaling molecules in the plant (Brenneret al., 2000; Lam et al., 1998). In addition, the role of aminoacids in plant morphogenesis and stress adaptation is wide-spread, witha varietyof aminoacidsacting as precursorsforthe synthesis of hormones and secondary metabolites.The synthesis of amino acids by plants is also agricul-turally important. Animals, including humans, rely onplant foods as dietary sources of essential amino acidsthat they cannot synthesize themselves. However, Lys,Thr, Met, Trp, and Ile are present in lower than optimalamounts in seeds of plants such as corn, soy and rice.These crops are widely used as feed for farm animals andalso make up the majority of the diet of some humanpopulations (Coruzzi and Last, 2000). Currently, animalfeed is supplemented with chemically synthesized ver-sions of these amino acids and, as such, they are

Glucosinolates are a group of defense-related secondary metabolites found in Arabidopsis and other cruciferous plants. Levels of leaf glucosinolates are regulated during plant development and increase in response to mechanical damage or insect feeding. The Arabidopsis TU8 mutant has a developmentally altered leaf glucosinolate profile: aliphatic glucosinolate levels drop off more rapidly, consistent with the early senescence of the mutant, and the levels of two indole glucosinolates are uniformly low. In TU8 seeds, four long-chain aliphatic glucosinolates have significantly increased levels, whereas the indolyl-3-methyl glucosinolate level is significantly reduced relative to wild type. Genetic mapping and DNA sequencing identified the TU8 mutation as tfl2-6, a new allele of TERMINAL FLOWER2 (TFL2), the only Arabidopsis homolog of animal HETEROCHROMATIN PROTEIN1 (HP1). TU8 (tfl2-6) has other previously identified tfl2 phenotypes, including an early transition to flowering, altered meristem structure, and stunted leaves. Analysis of two additional alleles, tfl2-1 and tfl2-2, showed glucosinolate profiles similar to those of line TU8 (tfl2-6).

Plant resistance to glyphosate has been reported far less frequently than resistance to sulfonylurea and imidazolinone herbicides. However, these studies tend to be anecdotal, without side by side comparisons for a single species or natural isolate. In this study, we tested the frequencies of resistance of three herbicides in a controlled ethylmethanesulfonate (EMS) saturation mutagenesis experiment, allowing a direct comparison of the frequencies at which resistant mutant plants arise. The 100% growth inhibition dose rates of glyphosate, chlorsulfuron (a sulfonylurea herbicide), and imazethapyr (an imidazolinone herbicide) were determined for Arabidopsis. Populations of EMS-mutagenized M(2) seedlings were sprayed with twice the 100% growth inhibition dose of glyphosate, chlorsulfuron, or imazethapyr, and herbicide-resistant mutants were identified. Although there were no glyphosate-resistant mutants among M(2) progeny of 125,000 Columbia and 125,000 Landsberg erecta M(1) lines, chlorsulfuron resistance and imazethapyr resistance each appeared at frequencies of 3.2 x 10(-5). Given the observed frequency of herbicide resistance mutations, we calculate that there are at least 700 mutations in each EMS-mutagenized Arabidopsis line and that fewer than 50,000 M(1) lines are needed to have a 95% chance of finding a mutation in any given G:C base pair in the genome. As part of this study, two previously unreported Arabidopsis mutations conferring resistance to imidazolinone herbicides, csr1-5 (Ala-122-Thr) and csr1-6 (Ala-205-Val), were discovered. Neither of these mutations caused enhanced resistance to chlorsulfuron in Arabidopsis.

To further our understanding of how plants defend against the harmful effects of ultraviolet (UV) light, we characterized an Arabidopsis mutant hypersensitive to UV-B. This mutant, UV resistance locus 8-1 (uvr8-1), contains a single recessive mutation at the bottom of chromosome 5. Fine-scale mapping localized uvr8-1 to a 21-kb locus containing five predicted open reading frames. Sequencing of this entire region revealed that the uvr8-1 allele contains a 15-nucleotide deletion in a gene similar to the human guanine nucleotide exchange factor regulator of chromatin condensation 1. This mutation reduces the UV-B-mediated induction of flavonoids and blocks chalcone synthase mRNA and protein induction. In contrast, uvr8-1 has enhanced induction of PR1 and PR5 proteins in response to UV-B, an indication of increased UV-B injury. These results suggest that UVR8acts in a UV-B signal transduction pathway leading to induction of flavonoid biosynthesis.

Photosystem II (PSII) is a multiprotein complex that catalyzes the light-driven water-splitting reactions of oxygenic photosynthesis. Light absorption by PSII leads to the production of excited states and reactive oxygen species that can cause damage to this complex. Here, we describe Arabidopsis (Arabidopsis thaliana) At1g71500, which encodes a previously uncharacterized protein that is a PSII auxiliary core protein and hence is named PHOTOSYSTEM II PROTEIN33 (PSB33). We present evidence that PSB33 functions in the maintenance of PSII-light-harvesting complex II (LHCII) supercomplex organization. PSB33 encodes a protein with a chloroplast transit peptide and one transmembrane segment. In silico analysis of PSB33 revealed a light-harvesting complex-binding motif within the transmembrane segment and a large surface-exposed head domain. Biochemical analysis of PSII complexes further indicates that PSB33 is an integral membrane protein located in the vicinity of LHCII and the PSII CP43 reaction center protein. Phenotypic characterization of mutants lacking PSB33 revealed reduced amounts of PSII-LHCII supercomplexes, very low state transition, and a lower capacity for nonphotochemical quenching, leading to increased photosensitivity in the mutant plants under light stress. Taken together, these results suggest a role for PSB33 in regulating and optimizing photosynthesis in response to changing light levels.

Vitamin C ( l -ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and l -galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants ( vtc1 ) contains ≈25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Relatively little is known about the types of photomorphogenic responses and signal transduction pathways that plants employ in response to ultraviolet-B (UV-B, 290-320 nm) radiation. In wild-type Arabidopsis seedlings, hypocotyl growth inhibition and cotyledon expansion were both reproducibly promoted by continuous UV-B. The fluence rate response of hypocotyl elongation was examined and showed a biphasic response. Whereas photomorphogenic responses were observed at low doses, higher fluences resulted in damage symptoms. In support of our theory that photomorphogenesis, but not damage, occurs at low doses of UV-B, photomorphogenic responses of UV-B sensitive mutants were indistinguishable from wild-type plants at the low dose. This allowed us to examine UV-B-induced photomorphogenesis in photoreceptor deficient plants and constitutive photomorphogenic mutants. The cry1 cryptochrome structural gene mutant, and phytochrome deficient hy1, phyA and phyB mutant seedlings resembled wild-type seedlings, while phyA/phyB double mutants were less sensitive to the photomorphogenic effects of UV-B. These results suggest that either phyA or phyB is required for UV-B-induced photomorphogenesis. The constitutive photomorphogenic mutants cop1 and det1 did not show significant inhibition of hypocotyl growth in response to UV-B, while det2 was strongly affected by UV-B irradiation. This suggests that COP1 and DET1 work downstream of the UV-B signaling pathway.

Nine blue fluorescent mutants of the flowering plant Arabidopsis thaliana were isolated by genetic selections and fluorescence screens. Each was shown to contain a recessive allele of trp1, a previously described locus that encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase (PAT, called trpD in bacteria). The trp1 mutants consist of two groups, tryptophan auxotrophs and prototrophs, that differ significantly in growth rate, morphology, and fertility. The trp1 alleles cause plants to accumulate varying amounts of blue fluorescent anthranilate compounds, and only the two least severely affected of the prototrophs have any detectable PAT enzyme activity. All four of the trp1 mutations that were sequenced are G to A or C to T transitions that cause an amino acid change, but in only three of these is the affected residue phylogenetically conserved. There is an unusually high degree of sequence divergence in the single-copy gene encoding PAT from the wild-type Columbia and Landsberg erecta ecotypes of Arabidopsis.

The Chloroplast 2010 Project (http://www.plastid.msu.edu/) identified and phenotypically characterized homozygous mutants in over three thousand genes, the majority of which encode plastid-targeted proteins. Despite extensive screening by the community, no homozygous mutant alleles were available for several hundred genes, suggesting that these might be enriched for genes of essential function. Attempts were made to generate homozygotes in ∼1200 of these lines and 521 of the homozygous viable lines obtained were deposited in the Arabidopsis Biological Resource Center (http://abrc.osu.edu/). Lines that did not yield a homozygote in soil were tested as potentially homozygous lethal due to defects either in seed or seedling development. Mutants were characterized at four stages of development: developing seed, mature seed, at germination, and developing seedlings. To distinguish seed development or seed pigment-defective mutants from seedling development mutants, development of seeds was assayed in siliques from heterozygous plants. Segregating seeds from heterozygous parents were sown on supplemented media in an attempt to rescue homozygous seedlings that could not germinate or survive in soil. Growth of segregating seeds in air and air enriched to 0.3% carbon dioxide was compared to discover mutants potentially impaired in photorespiration or otherwise responsive to CO2 supplementation. Chlorophyll fluorescence measurements identified CO2-responsive mutants with altered photosynthetic parameters. Examples of genes with a viable mutant allele and one or more putative homozygous-lethal alleles were documented. RT-PCR of homozygotes for potentially weak alleles revealed that essential genes may remain undiscovered because of the lack of a true null mutant allele. This work revealed 33 genes with two or more lethal alleles and 73 genes whose essentiality was not confirmed with an independent lethal mutation, although in some cases second leaky alleles were identified.

Acylsugars are polyesters of short- to medium-length acyl chains on sucrose or glucose backbones that are produced in secretory glandular trichomes of many solanaceous plants, including cultivated tomato (Solanum lycopersicum). Despite their roles in biotic stress adaptation and their wide taxonomic distribution, there is relatively little information about the diversity of these compounds and the genes responsible for their biosynthesis. In this study, acylsugar diversity was assessed for 80 accessions of the wild tomato species Solanum habrochaites from throughout the Andes Mountains. Trichome metabolites were analyzed by liquid chromatography-time of flight-mass spectrometry, revealing the presence of at least 34 structurally diverse acylsucroses and two acylglucoses. Distinct phenotypic classes were discovered that varied based on the presence of glucose or sucrose, the numbers and lengths of acyl chains, and the relative total amounts of acylsugars. The presence or absence of an acetyl chain on the acylsucrose hexose ring caused clustering of the accessions into two main groups. Analysis of the Acyltransferase2 gene (the apparent ortholog of Solyc01g105580) revealed differences in enzyme activity and gene expression correlated with polymorphism in S. habrochaites accessions that varied in acylsucrose acetylation. These results are consistent with the hypothesis that glandular trichome acylsugar acetylation is under selective pressure in some populations of S. habrochaites and that the gene mutates to inactivity in the absence of selection.

A comprehensive understanding of the lipid and fatty acid metabolic machinery is needed for optimizing production of oils and fatty acids for fuel, industrial feedstocks and nutritional improvement in plants. T-DNA mutants in the poorly annotated Arabidopsis thaliana gene At1g08640 were identified as containing moderately high levels (50-100%) of 16∶1Δ7 and 18∶1Δ9 leaf fatty acids and subtle decreases (5-30%) of 16∶3 and 18∶3 (http://www.plastid.msu.edu/). TLC separation of fatty acids in the leaf polar lipids revealed that the chloroplastic galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were the main lipid types affected by this mutation. Analysis of the inferred amino acid sequence of At1g08640 predicted the presence of a transit peptide, three transmembrane domains and an N-terminal J-like domain, and the gene was named CJD1 for Chloroplast J-like Domain 1. GFP reporter experiments and in vitro chloroplast import assays demonstrated CJD1 is a chloroplast membrane protein. Screening of an Arabidopsis cDNA library by yeast-2-hybrid (Y2H) using the J-like domain of CJD1 as bait identified a plastidial inner envelope protein (Accumulation and Replication of Chloroplasts 6, ARC6) as the primary interacting partner in the Y2H assay. ARC6 plays a central role in chloroplast division and binds CJD1 via its own J-like domain along with an adjacent conserved region whose function is not fully known. These results provide a starting point for future investigations of how mutations in CJD1 affect lipid composition.

As part of a project to analyze chloroplast functional networks systematically, we have subjected mutants in >3,200 nuclear genes predicted to encode chloroplast-targeted proteins in Arabidopsis thaliana (http://www.plastid.msu.edu) to parallel phenotypic assays. Detailed methods are presented for the various assays being used in this project to study chloroplast biology. These include morphological analysis of plants, chloroplasts, and seeds using controlled vocabulary. Metabolites synthesized in the chloroplast such as starch, amino acids, and fatty acids are analyzed in groups according to their chemical properties. As an indicator for the relative composition of seed storage oil and proteins, the carbon and nitrogen contents are determined by an elemental analyzer. The methods in this chapter describe how the assays are configured to run in relatively high throughput, maximizing data quality.

We identified a cis -prenyltransferase gene, neryl diphosphate synthase 1 ( NDPS1 ), that is expressed in cultivated tomato ( Solanum lycopersicum ) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 ( PHS1 ), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce β-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the “universal” substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

Photosystem II (PSII) is highly susceptible to photoinhibition caused by environmental stimuli such as high light; therefore plants have evolved multifaceted mechanisms to efficiently protect PSII from photodamage. We previously published data suggesting that Maintenance of PSII under High light 1 (MPH1, encoded by AT5G07020), a PSII-associated proline-rich protein found in land plants, participates in the maintenance of normal PSII activity under photoinhibitory stress. Here we provide additional evidence for the role of MPH1 in protecting PSII against photooxidative damage. Two Arabidopsis thaliana mutants lacking a functional MPH1 gene suffer from severe photoinhibition relative to the wild-type plants under high irradiance light. The mph1 mutants exhibit significantly decreased PSII quantum yield and electron transport rate after exposure to photoinhibitory light. The mutants also display drastically elevated photodamage to PSII reaction center proteins after high-light treatment. These data add further evidence that MPH1 is involved in PSII photoprotection in Arabidopsis. MPH1 homologs are found across phylogenetically diverse land plants but are not detected in algae or prokaryotes. Taken together, these results suggest that MPH1 protein began to play a role in protecting PSII against excess light following the transition from aquatic to terrestrial conditions.

The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine essential amino acids that must be obtained from the diet of humans and other animals, and can be nutritionally limiting in plant foods. Despite genetic evidence of its importance in regulating seed amino acid levels, the full BCAA catabolic network is not completely understood in plants, and limited information is available regarding its regulation. In this study, transcript coexpression analyses revealed positive correlations among BCAA catabolism genes in stress, development, diurnal/circadian, and light data sets. A core subset of BCAA catabolism genes, including those encoding putative branched-chain ketoacid dehydrogenase subunits, is highly expressed during the night in plants on a diel cycle and in prolonged darkness. Mutants defective in these subunits accumulate higher levels of BCAAs in mature seeds, providing genetic evidence for their function in BCAA catabolism. In addition, prolonged dark treatment caused the mutants to undergo senescence early and overaccumulate leaf BCAAs. These results extend the previous evidence that BCAAs can be catabolized and serve as respiratory substrates at multiple steps. Moreover, comparison of amino acid profiles between mature seeds and dark-treated leaves revealed differences in amino acid accumulation when BCAA catabolism is perturbed. Together, these results demonstrate the consequences of blocking BCAA catabolism during both normal growth conditions and under energy-limited conditions.

Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

We report the identification and biotechnological utility of a plant gene encoding the tocopherol (vitamin E) biosynthetic enzyme 2-methyl-6-phytylbenzoquinol methyltransferase. This gene was identified by map-based cloning of the Arabidopsis mutation vitamin E pathway gene3-1 (vte3-1), which causes increased accumulation of delta-tocopherol and decreased gamma-tocopherol in the seed. Enzyme assays of recombinant protein supported the hypothesis that At-VTE3 encodes a 2-methyl-6-phytylbenzoquinol methyltransferase. Seed-specific expression of At-VTE3 in transgenic soybean reduced seed delta-tocopherol from 20 to 2%. These results confirm that At-VTE3 protein catalyzes the methylation of 2-methyl-6-phytylbenzoquinol in planta and show the utility of this gene in altering soybean tocopherol composition. When At-VTE3 was coexpressed with At-VTE4 (gamma-tocopherol methyltransferase) in soybean, the seed accumulated to95% alpha-tocopherol, a dramatic change from the normal 10%, resulting in a greater than eightfold increase of alpha-tocopherol and an up to fivefold increase in seed vitamin E activity. These findings demonstrate the utility of a gene identified in Arabidopsis to alter the tocopherol composition of commercial seed oils, a result with both nutritional and food quality implications.

Map-based cloning is an iterative approach that identifies the underlying genetic cause of a mutant phenotype. The major strength of this approach is the ability to tap into a nearly unlimited resource of natural and induced genetic variation without prior assumptions or knowledge of specific genes. One begins with an interesting mutant and allows plant biology to reveal what gene or genes are involved. Three major advances in the past 2 years have made map-based cloning in Arabidopsis fairly routine: sequencing of the Arabidopsis genome, the availability of more than 50,000 markers in the Cereon Arabidopsis Polymorphism Collection, and improvements in the methods used for detecting DNA polymorphisms. Here, we describe the Cereon Collection and show how it can be used in a generic approach to mutation mapping in Arabidopsis. We present the map-based cloning of theVTC2 gene as a specific example of this approach.

Arabidopsis cyt1 mutants have a complex phenotype indicative of a severe defect in cell wall biogenesis. Mutant embryos arrest as wide, heart-shaped structures characterized by ectopic accumulation of callose and the occurrence of incomplete cell walls. Texture and thickness of the cell walls are irregular, and unesterified pectins show an abnormally diffuse distribution. To determine the molecular basis of these defects, we have cloned the CYT1 gene by a map-based approach and found that it encodes mannose-1-phosphate guanylyltransferase. A weak mutation in the same gene, called vtc1 , has previously been identified on the basis of ozone sensitivity due to reduced levels of ascorbic acid. Mutant cyt1 embryos are deficient in N-glycosylation and have an altered composition of cell wall polysaccharides. Most notably, they show a 5-fold decrease in cellulose content. Characteristic aspects of the cyt1 phenotype, including radial swelling and accumulation of callose, can be mimicked with the inhibitor of N-glycosylation, tunicamycin. Our results suggest that N-glycosylation is required for cellulose biosynthesis and that a deficiency in this process can account for most phenotypic features of cyt1 embryos.

Vitamin C (l-ascorbic acid) is a potent antioxidant and cellular reductant present at millimolar concentrations in plants. This small molecule has roles in the reduction of prosthetic metal ions, cell wall expansion, cell division, and in the detoxification of reactive oxygen generated by photosynthesis and adverse environmental conditions. However, unlike in animals, the biosynthesis of ascorbic acid (AsA) in plants is only beginning to be unraveled. The previously described AsA-deficient Arabidopsis mutant vtc1 (vitamin c-1) was recently shown to have a defect in GDP-mannose pyrophosphorylase, providing strong evidence for the recently proposed role of GDP-mannose in AsA biosynthesis. To genetically define other AsA biosynthetic loci, we have used a novel AsA assay to isolate four vtc mutants that define three additional VTC loci. We have also isolated a second mutant allele of VTC1. The four loci represented by the vtc mutant collection have been genetically characterized and mapped onto the Arabidopsis genome. The vtc mutants have differing ozone sensitivities. In addition, two of the mutants, vtc2-1 and vtc2-2, have unusually low levels of AsA in the leaf tissue of mature plants.

We characterized the accumulation patterns of Arabidopsis thaliana proteins, two CuZnSODs, FeSOD, MnSOD, PR1, PR5, and GST1, in response to various pathogen-associated treatments. These treatments included inoculation with virulent and avirulent Pseudomonas syringae strains, spontaneous lesion formation in the lsd1 mutant, and treatment with the salicylic acid (SA) analogs INA (2,6-dichloroisonicotinic acid) and BTH (benzothiadia-zole). The PR1, PR5, and GST1 proteins were inducible by all treatments tested, as expected from previous mRNA blot analysis. The two CuZnSOD proteins were induced by SA analogs and in conjunction with lsd1-mediated spreading cell death. Additionally, LSD1 is a part of a signaling pathway for the induction of the CuZnSOD proteins in response to SA but not in lsd1-mediated cell death. We suggest that the spreading lesion phenotype of lsd1 results from a lack of up-regulation of a CuZnSOD responsible for detoxification of accumulating superoxide before the reactive oxygen species can trigger a cell death cascade.

Little is known about how amino acid metabolism is regulated in higher plants despite our extensive knowledge of the same processes in prokaryotes and fungi (see reviews by Galili, 1995; Herrmann, 1995; Lam et al., 1995; Radwanski and Last, 1995). The physiological roles of different isozymes is not clear, regulatory elements in amino acid biosynthetic gene promoters have not been defined and, in contrast to microorganisms, nothing is known about factors interacting with these promoters to affect gene expression. However, genes have been cloned for all of the enzymes in the Arabidopsis tryptophan biosynthetic pathway, antibodies were produced against six out of the seven proteins, and mutants obtained or antisense plants constructed for six out of the seven proteins (Bender and Fink, 1995; Radwanski and Last, 1995; Li and Last, 1996). In addition, the induction of the tryptophan pathway in response to a variety of environmental conditions (as discussed below) provides an excellent opportunity to pursue genetic approaches to understanding the regulation of this important pathway.