John G. Jelesko

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

An effective plant alkaloid chemical defense requires a variety of transport processes, but few alkaloid transporters have been characterized at the molecular level. Previously, a gene fragment encoding a putative plasma membrane proton symporter was isolated, because it was coordinately regulated with several nicotine biosynthetic genes. Here, we show that this gene fragment corresponds to a Nicotiana tabacum gene encoding a nicotine uptake permease (NUP1). NUP1 belongs to a plant-specific class of purine uptake permease-like transporters that originated after the bryophytes but before or within the lycophytes. NUP1 expressed in yeast cells preferentially transported nicotine relative to other pyridine alkaloids, tropane alkaloids, kinetin, and adenine. NUP1-GFP primarily localized to the plasma membrane of tobacco Bright Yellow-2 protoplasts. WT NUP1 transcripts accumulated to high levels in the roots, particularly in root tips. NUP1-RNAi hairy roots had reduced NUP1 mRNA accumulation levels, reduced total nicotine levels, and increased nicotine accumulation in the hairy root culture media. Regenerated NUP1-RNAi plants showed reduced foliar and root nicotine levels as well as increased seedling root elongation rates. Thus, NUP1 affected nicotine metabolism, localization, and root growth.

Glutamate Receptor Homologs in Plants: Functions and Evolutionary Origins

The plant glutamate-like receptor homologs (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) which were discovered more than 10 years ago, and are hypothesized to be potential amino acid sensors in plants. Although initial progress on this gene family has been hampered by gene redundancy and technical issues such as gene toxicity; genetic, pharmacological, and electrophysiological approaches are starting to uncover the functions of this protein family. In parallel, there has been tremendous progress in elucidating the structure of animal glutamate receptors (iGluRs), which in turn will help understanding of the molecular mechanisms of plant GLR functions. In this review, we will summarize recent progress on the plant GLRs. Emerging evidence implicates plant GLRs in various biological processes in and beyond N sensing, and implies that there is some overlap in the signaling mechanisms of amino acids between plants and animals. Phylogenetic analysis using iGluRs from metazoans, plants, and bacteria showed that the plant GLRs are no more closely related to metazoan iGluRs as they are to bacterial iGluRs, indicating the separation of plant, other eukaryotic, and bacterial GLRs might have happened as early on as the last universal common ancestor. Structural similarities and differences with animal iGluRs, and the implication thereof, are also discussed.

Enhanced production of the alkaloid nicotine in hairy root cultures of Nicotiana tabacum L.

The utility of hairy root cultures to produce valuable phytochemicals could be improved by repartitioning more of the desired phytochemical into the spent culture media, thereby simplifying the bioprocess engineering associated with the purification of the desired phytochemical. The majority of nicotine produced by tobacco hairy root cultures is retained within roots, with lesser amounts exuded into the spent culture media. Reduced expression of the tobacco nicotine uptake permease (NUP1) results in significantly more nicotine accumulating in the media. Thus, NUP1-reduced expression lines provide a genetic means to repartition more nicotine into the culture media. The present study examined a wild type and a NUP1-reduced expression hairy root line during a variety of treatments to identify culture conditions that increased nicotine accumulation in the media. The NUP1-reduced expression line grew faster, used less oxygen, and exuded more nicotine into the media. Basification of the culture media associated with root growth resulted in a dramatic reduction in nicotine accumulation levels in the media, which was reversed by decreasing the pH of the media. Kinetic analysis of hairy root growth and nicotine accumulation in the media revealed a potential improvement in nicotine yields in the media by stimulating the branching of tobacco hairy roots.

An expanding role for purine uptake permease-like transporters in plant secondary metabolism

For the past decade, our understanding of the plant purine uptake permease (PUP) transporter family was primarily oriented on purine nucleobase substrates and their tissue-specific expression patterns in Arabidopsis. However, a tobacco PUP-like homolog demonstrating nicotine uptake permease activity was recently shown to affect both nicotine metabolism and root cell growth. These new findings expand the physiological role for PUP-like transporters to include plant secondary metabolism. Molecular evolution analyses of PUP-like transporters indicate they are distinct group within an ancient super family of drug and metabolite transporters (DMTs). The PUP-like family originated during terrestrial plant evolution sometime between the bryophytes and the lycophytes. A phylogenetic analysis indicates that the PUP-like transporters were likely derived from a pre-existing nucleotide-sugar transporter family within the DMT super family. Within the lycophyte Selaginella, there are three paralogous groups of PUP-like transporters. One of the three PUP-like paralogous groups showed an extensive pattern of gene duplication and diversification within the angiosperm lineage, whereas the more ancestral PUP-like paralogous groups did not. Biochemical characterization of four closely related PUP-like paralogs together with model-based phylogenetic analyses indicate both subfunctionalization and neofunctionalization during the molecular evolution of angiosperm PUP-like transporters. These findings suggest that members of the PUP-like family of DMT transporters are likely involved in diverse primary and secondary plant metabolic pathways.

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

Plants use phenylalanine to produce abundant and diverse phenylpropanoid compounds, such as flavonoids, tannins, and lignin. Through phylogenetic, bioinformatic, and biochemical analyses of prephenate aminotransferase enzymes from deep taxonomic lineages, this study revealed unique evolutionary history and molecular changes of key enzymes responsible for phenylalanine biosynthesis in plants. The aromatic amino acid Phe is required for protein synthesis and serves as the precursor of abundant phenylpropanoid plant natural products. While Phe is synthesized from prephenate exclusively via a phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is predominant in plant Phe biosynthesis. However, the molecular and biochemical evolution of the plant arogenate pathway is currently unknown. Here, we conducted phylogenetically informed biochemical characterization of prephenate aminotransferases (PPA-ATs) that belong to class-Ib aspartate aminotransferases (AspAT Ibs) and catalyze the first committed step of the arogenate pathway in plants. Plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were found to be most closely related to homologs from Chlorobi/Bacteroidetes bacteria. The Chlorobium tepidum PPA-AT and ADT homologs indeed efficiently converted prephenate and arogenate into arogenate and Phe, respectively. A subset of AspAT Ib enzymes exhibiting PPA-AT activity was further identified from both Plantae and prokaryotes and, together with site-directed mutagenesis, showed that Thr-84 and Lys-169 play key roles in specific recognition of dicarboxylic keto (prephenate) and amino (aspartate) acid substrates. The results suggest that, along with ADT, a gene encoding prephenate-specific PPA-AT was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of Plantae, allowing efficient Phe and phenylpropanoid production via arogenate in plants today.

Retrotransposon-based markers from potato monoploids used in somatic hybridization

In an attempt to remove lethal and deleterious genes and enhance the heterozygosity of the potato genome, we developed several diverse somatic hybrids through the electrofusion of selected monoploids. Somatic hybrids and somaclones resulting from fused and unfused protoplasts, respectively, were verified with microsatellites. Molecular markers anchored in the Tst1 retrotransposon were used to examine polymorphisms in the regenerated plants and to reveal any somaclonal variation. Inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon display (sequence-specific amplified polymorphism (S-SAP), anchored in a retransposon) were examined on an ALFexpress DNA sequencer. Because of inconsistencies in the number and quality of bands revealed by the combination of either class of marker in combination with the ALFexpress, we cloned and sequenced 11 S-SAP bands to use as restriction fragment length polymorphism (RFLP) probes in Southern blot analyses of genetic relationships in our potato populations and among related Solanaceae. Readily scorable bands (n = 27) that separated somatic hybrids from parental monoploids and somaclones and grouped monoploids according to known genetic relationships were produced. Some of the probes could be used to differentiate tomato and Datura from potato. Sequence analysis of 5 cloned IRAP and 11 cloned S-SAP markers confirmed that they were anchored in the Tst1 retrotransposon. BLAST searches within GenBank produced 10 highly significant hits (5 nucleotide, 4 expressed sequence tag (EST), and 1 protein) within closely related Solanaceae, suggesting that Tst1 represents an old retroelement that was inserted before the diversion of genera within Solanaceae; however, most sequences were undescribed.

Use of an antisense gene to characterize glutathione S-transferase functions in transformed suspension-cultured rice cells and calli☆

Abstract Rice glutathione S -transferases (GSTs) conjugate the herbicide pretilachlor with glutathione and this reaction is induced by the safener fenclorim. A cDNA construct of the gene coding for the Os GST III subunit of rice GSTs was inserted in antisense orientation into the vector pBC302 under the control of an enhanced p CaMV 35S promoter. The final clone containing the bar gene as marker was used to transform suspension-cultured cells and calli of Lemont rice ( indica × japonica ) via the Agrobacterium Ti vector pTOK233. Five lines of suspension-cultured rice cells transformed with the antisense GST gene (α-3, α-5, α-8, α-12, and α-15) were selected and analyzed by Southern and Northern blot analyses. Transformed suspension-culture cells and calli of rice exhibited resistance to the herbicide glufosinate because of the bar gene, but were sensitive to pretilachlor. The α-5 and α-8 rice cell lines contained a single copy of the Os GST III gene, while the α-3 line contained two copies of this gene. Os GST III mRNA levels present in wild type rice cells were induced by fenclorim. Os GST III mRNA levels were undetectable in the transformed cell lines of rice and were not induced by fenclorim treatments, except for line α-5. GST activity utilizing cinnamic acid, CDNB, and pretilachlor as substrates was reduced in all cell lines of transformed rice. SDS–PAGE analysis showed that the band of the Os GST III subunit was very faint in α-3 rice cells. HPLC analysis showed that the levels of cinnamic acid, coumaric acid, and phenylalanine were significantly higher in transformed cells than in wild type cells of rice. These results suggest that the Os GST III gene plays an important role in the detoxification of the herbicide pretilachlor and the metabolism of phenolic compounds in suspension-cultured rice cells and calli.

MALDI-MS Imaging of Urushiols in Poison Ivy Stem

Urushiols are the allergenic components of Toxicodendron radicans (poison ivy) as well as other Toxicodendron species. They are alk-(en)-yl catechol derivatives with a 15- or 17-carbon side chain having different degrees of unsaturation. Although several methods have been developed for analysis of urushiols in plant tissues, the in situ localization of the different urushiol congeners has not been reported. Here, we report on the first analysis of urushiols in poison ivy stems by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Our results show that the urushiol congeners with 15-carbon side chains are mainly localized to the resin ducts, while those with 17-carbon side chains are widely distributed in cortex and vascular tissues. The presence of these urushiols in stem extracts of poison ivy seedlings was confirmed by GC-MS. These novel findings provide new insights into the spatial tissue distribution of urushiols that might be biosynthetically or functionally relevant.

First report of seedling blight of Eastern poison ivy (Toxicodendron radicans) by Colletotrichum fioriniae in Virginia.

Colletotrichum fioriniae is a member of the large cosmopolitan C. acutatum species complex (2). Known agricultural hosts of C. acutatum include apple, European blueberry, grape, olive, papaya, and strawberry (2). In contrast, the life history of C. fioriniae ranges from an epizootic of certain scale insect populations to an endophyte of plants (3,4). The present study extends the phytopathology of C. fioriniae to include poison ivy seedlings. Poison ivy (Toxicodendron radicans) drupes were collected from solitary lianas in Roanoke and Montgomery counties, Virginia. These drupes were subjected to experiments aimed at producing sterile seedlings (1); however, there was extensive blighting and wilting in the germinated seedlings. Associated with the drupes and seedlings was a fungus with white to pale olivaceous grey mycelium with orange blister-like conidiomata and sclerotial masses enclosing the drupe mesocarp as well as conidiomata emerging from blighted, necrotic leaves. Condiomata were plated onto acidified potato dextrose agar (APDA) and oatmeal agar (OA). This consistently yielded colonies identical to those described from diseased tissues and were putatively identified as C. acutatum based on the presence of acervuli containing hyaline, smooth-walled, aseptate conidia with acute ends, the absence of setae, and formation of red pigments in culture (2). Conidial dimensions of four isolates most closely aligned with reported measurements for C. fioriniae (4): mean length ± SD × width ± SD = 15.1 ± 1.7 × 4.9 ± 0.3 μm, L/W ratio = 3.04 on OA. Fungal DNA was isolated and used as template in PCR reactions using oligonucleotide primer pairs corresponding to the internal transcribed spacer (ITS) region, and a portion of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. The resulting PCR fragments were sequenced and used as queries in BLASTN searches of the GenBank NR database. All of the amplified ITS DNA sequences (497 bp KF944356 and KF944357) were identical to Glomerella/Colletotrichum fioriniae (JN121190 and KF278459). Similarly, the amplified (672 bp) GAPDH sequences (KF944354 and KF944355) were 99.6% similar over the 254 bp overlapping with C. fioriniae (JQ948622). Pathogenicity of two randomly chosen C. fioriniae isolates, TR-123 and TR-126, was confirmed by placing 4.75 mm diam. inoculated agar plugs from 8-day-old fungal cultures or a sterile plug (negative control) at the base of an axenic young seedling ~1.5 to 6.5 cm in height with at least one set of true leaves (1). Each treatment was replicated five times. Acute wilt and blighting of leaves and production of orange acervuli on cotyledons disease symptoms developed by 3 weeks post inoculation (WPI). By 7 WPI all but one of the Colletotrichum-inoculated plants were dead, whereas all of the control plants were healthy with significantly lower area under the disease progress curve values. Colletotrichum was consistently re-isolated, and confirmed morphologically and molecularly, from six of seven diseased seedlings, whereas two of two randomly chosen control seedlings remained asymptomatic and did not yield Colletotrichum. In summary, C. fioriniae may represent a natural biocontrol agent against poison ivy and scale insect herbivores thereof. References: (1) E. Benhase and J. Jelesko. HortScience 48:1, 2013. (2) U. Damm et al. Stud. Mycol. 73:37, 2012. (3) J. Marcelino et al. J. Insect Sci. 9:25, 2009. (4) R. Shivas et al. Fungal Divers. 39:111.

Differential Responses to Light and Nutrient Availability by Geographically Isolated Poison Ivy Accessions

Abstract The morphology and growth habit of Toxicodendron radicans (Poison Ivy) varies widely across North America. In this study, we evaluated the role of Poison Ivy accessionlevel diversity on growth habit and performance responses to light and nutrient stress in a common-garden greenhouse environment. We grew Poison Ivy seedlings derived from drupes collected from Iowa (IA), Michigan (MI), Texas (TX), and Virginia (VA) in a glasshouse with factorial arrangements of 2 light treatments (full sun and deep shade) and 3 nutrient treatments (full nutrients, full nutrients minus nitrogen, no nutrients). Seedlings from Iowa grew very poorly across all treatments, whereas plants from MI, TX, and VA showed accessional variation, though treatment effects were stronger than accession-level effects. We observed significant accession-level differences in height, branch number, leaf area, total biomass, chlorophyll, and root:shoot ratio biometrics; the Texas accession generally outperformed the MI and VA accessions across all treatments. Overall, light availability was a much stronger driver of overall performance than nutrient availability; plants grown in shade were smaller, had fewer branches and produced ∼20% of the biomass compared to those grown in full-sun. Poison Ivy shows accession-level variation in plastic responses to light and nutrient availability, which partially explains the breadth of habitats the species colonizes.