Manipulation of the Cellular Membrane-Cytoskeleton Network for RNA Virus Replication and Movement in Plants.

Viruses infect all cellular life forms and cause various diseases and significant economic losses worldwide. The majority of viruses are positive-sense RNA viruses. A common feature of infection by diverse RNA viruses is to induce the formation of altered membrane structures in infected host cells. Indeed, upon entry into host cells, plant-infecting RNA viruses target preferred organelles of the cellular endomembrane system and remodel organellar membranes to form organelle-like structures for virus genome replication, termed as the viral replication organelle (VRO) or the viral replication complex (VRC). Different viruses may recruit different host factors for membrane modifications. These membrane-enclosed virus-induced replication factories provide an optimum, protective microenvironment to concentrate viral and host components for robust viral replication. Although different viruses prefer specific organelles to build VROs, at least some of them have the ability to exploit alternative organellar membranes for replication. Besides being responsible for viral replication, VROs of some viruses can be mobile to reach plasmodesmata (PD) via the endomembrane system, as well as the cytoskeleton machinery. Viral movement protein (MP) and/or MP-associated viral movement complexes also exploit the endomembrane-cytoskeleton network for trafficking to PD where progeny viruses pass through the cell-wall barrier to enter neighboring cells.

Transcriptomic analysis of wound-healing in Solanum tuberosum (potato) tubers: Evidence for a stepwise induction of suberin-associated genes.

Suberin deposition involves both phenolic and aliphatic polymer biosynthesis and deposition in the same tissue. Therefore, any consideration of exploiting suberin for crop enhancement (e.g., enhanced storage, soil borne disease resistance) requires knowledge of both phenolic and aliphatic component biosynthesis and their coordinated, temporal deposition. In the present study, we use a wound-healing potato tuber system to explore global transcriptome changes during the early stages of wound-healing. Wounding leads to initial and substantial transcriptional changes that follow distinctive temporal patterns - primary metabolic pathways were already functional, or up-regulated immediately, and maintained at levels that would allow for precursor carbon skeletons and energy to feed into downstream metabolic processes. Genes involved in pathways for phenolic production (i.e., the shikimate pathway and phenylpropanoid metabolism) were up-regulated early while those involved in aliphatic suberin production (i.e., fatty acid biosynthesis and modification) were transcribed later into the time course. The pattern of accumulation of genes associated with ABA biosynthesis and degradation steps support a role for ABA in regulating aliphatic suberin production. Evaluation of putative Casparian strip membrane-like genes pinpointed wound-responsive candidates that may mediate the suberin deposition process.

Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide.

Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact (such as through standard horticultural practices). Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by R genes Tm-1, Tm-2, and Tm-22 in tomato and L1 and L2 alleles in pepper. Currently, no commercial ToBRFV-resistant tomato cultivars are available. Integrated pest management-based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long-term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment-friendly strategy for pathogen control. TAXONOMY: Tomato brown rugose fruit virus belongs to the genus Tobamovirus, in the family Virgaviridae. The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus. GENOME AND VIRION: The ToBRFV genome is a single-stranded, positive-sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod-shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time. DISEASE SYMPTOMS: Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose dots and necrotic blotches on fruits.

ß-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified ß-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and ß-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its ß-cyanoalanine synthase activity. Consistent with the ß-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the ß-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite's ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Suberin Biosynthesis, Assembly, and Regulation.

Suberin is a specialized cell wall modifying polymer comprising both phenolic-derived and fatty acid-derived monomers, which is deposited in below-ground dermal tissues (epidermis, endodermis, periderm) and above-ground periderm (i.e., bark). Suberized cells are largely impermeable to water and provide a critical protective layer preventing water loss and pathogen infection. The deposition of suberin is part of the skin maturation process of important tuber crops such as potato and can affect storage longevity. Historically, the term "suberin" has been used to describe a polyester of largely aliphatic monomers (fatty acids, ω-hydroxy fatty acids, α,ω-dioic acids, 1-alkanols), hydroxycinnamic acids, and glycerol. However, exhaustive alkaline hydrolysis, which removes esterified aliphatics and phenolics from suberized tissue, reveals a core poly(phenolic) macromolecule, the depolymerization of which yields phenolics not found in the aliphatic polyester. Time course analysis of suberin deposition, at both the transcriptional and metabolite levels, supports a temporal regulation of suberin deposition, with phenolics being polymerized into a poly(phenolic) domain in advance of the bulk of the poly(aliphatics) that characterize suberized cells. In the present review, we summarize the literature describing suberin monomer biosynthesis and speculate on aspects of suberin assembly. In addition, we highlight recent advances in our understanding of how suberization may be regulated, including at the phytohormone, transcription factor, and protein scaffold levels.

Simultaneous Determination and Subcellular Localization of Protein-Protein Interactions in Plant Cells Using Bimolecular Fluorescence Complementation Assay.

The bimolecular fluorescence complementation (BiFC) assay allows the visualization of protein-protein interactions in their native state within living systems. The BiFC assay is based on the in vivo complementation of nonfluorescent component parts of a fluorescent protein through the interaction or proximity target proteins, each fused to a different component of the fluorescent protein. Expansion of the BiFC toolkit with an increasing spectrum of fluorescence markers and catalog of Gateway-compatible vectors for high-throughput screening, has made BiFC an exceedingly powerful tool in discovering new protein interactions or providing backup evidence for known ones. Apart from the validation of protein-protein interactions, BiFC offers the additional benefit of providing information on the subcellular localization of protein interaction complexes. Subcellular localization to a specific subcellular compartment or organelle may be further validated by the coexpression of a fluorescence-labeled protein marker. Here we describe an efficient yet simple protocol for simultaneous determination and subcellular localization of protein-protein interactions in plant cells.

Purification of Plasmodesmata-Enriched Fraction for Proteomic Analyses.

In plants, plasmodesmata (PD) are plasmamembrane-lined pores that traverse the cell wall to establish cytoplasmic and endomembrane continuity between neighboring cells. As intercellular channels, PD play pivotal roles in plant growth and development, defense responses, and are also co-opted by viruses to spread cell-to-cell to establish systemic infection. Proteomic analyses of PD-enriched fractions may provide critical insights on plasmodesmal biology and PD-mediated virus-host interactions. However, it is difficult to isolate PD from plant tissues as they are firmly embedded in the cell wall. Here, we describe a protocol for the purification of PD from Nicotiana benthamiana leaves for proteomic analysis.

Multiple indole glucosinolates and myrosinases defend Arabidopsis against Tetranychus urticae herbivory.

Arabidopsis (Arabidopsis thaliana) defenses against herbivores are regulated by the jasmonate (JA) hormonal signaling pathway, which leads to the production of a plethora of defense compounds. Arabidopsis defense compounds include tryptophan-derived metabolites, which limit Arabidopsis infestation by the generalist herbivore two-spotted spider mite, Tetranychus urticae. However, the phytochemicals responsible for Arabidopsis protection against T. urticae are unknown. Here, we used Arabidopsis mutants disrupted in the synthesis of tryptophan-derived secondary metabolites to identify phytochemicals involved in the defense against T. urticae. We show that of the three tryptophan-dependent pathways found in Arabidopsis, the indole glucosinolate (IG) pathway is necessary and sufficient to assure tryptophan-mediated defense against T. urticae. We demonstrate that all three IGs can limit T. urticae herbivory, but that they must be processed by myrosinases to hinder T. urticae oviposition. Putative IG breakdown products were detected in mite-infested leaves, suggesting in planta processing by myrosinases. Finally, we demonstrate that besides IGs, there are additional JA-regulated defenses that control T. urticae herbivory. Together, our results reveal the complexity of Arabidopsis defenses against T. urticae that rely on multiple IGs, specific myrosinases, and additional JA-dependent defenses.

Preen gland microbiota of songbirds differ across populations but not sexes.

Metabolites produced by symbiotic microbes can affect the odour of their hosts, providing olfactory cues of identity, sex or other salient features. In birds, preen oil is a major source of body odour that differs between populations and sexes. We hypothesized that population and sex differences in preen oil chemistry reflect underlying differences in preen gland microbiota, predicting that these microbes also differ among populations and between the sexes. We further predicted that pairwise similarity in the community composition of preen gland microbiota would covary with that of preen oil chemical composition, consistent with the fermentation hypothesis for chemical recognition. We analysed preen oil chemistry and preen gland bacterial communities of song sparrows Melospiza melodia. Birds were sampled at sites for which population and sex differences in preen oil have been reported, and at a third site that has been less studied. Consistent with prior work in this system, we found population and sex differences in preen oil chemistry. By contrast, we found population differences but not sex differences in the community composition of preen gland microbes. Overall similarity in the community composition of preen gland microbiota did not significantly covary with that of preen oil chemistry. However, we identified a subset of six microbial genera that maximally correlated with preen oil composition. Although both preen gland microbiota and preen oil composition differ across populations, we did not observe an overall association between them that would implicate symbiotic microbes in mediating variation in olfactory cues associated with preen oil. Instead, certain subsets of microbes may be involved in mediating olfactory cues in birds, but experiments are required to test this.

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants.

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1-97), the core (amino acids 98-245), and the C-terminus (amino acids 246-288). We found that deletion of CP or its segments amino acids 51-199, amino acids 200-283, or amino acids 265-274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6-50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.

The genetic basis of female pheromone differences between Drosophila melanogaster and D. simulans.

Chemical signals are one means by which many insect species communicate. Differences in the combination of surface chemicals called cuticular hydrocarbons (CHCs) can influence mating behavior and affect reproductive isolation between species. Genes influencing three CHC compounds have been identified in Drosophila melanogaster. However, the genetic basis of other CHC compounds, whether these genes affect species differences in CHCs, and the genes' resulting effect on interspecies mating, remains unknown. We used fine-scale deficiency mapping of the third chromosome to identify 43 genomic regions that influence production of CHCs in both D. melanogaster and Drosophila simulans females. We identified an additional 23 small genomic regions that affect interspecies divergence in CHCs between females of these two species, one of which spans two genes known to influence the production of multiple CHCs within D. melanogaster. By testing these genes individually, we determined that desat1 also affects interspecific divergence in one CHC compound, while desat2 has no effect on interspecific divergence. Thus, some but not all genes affecting intraspecific amounts of CHCs also affect interspecific divergence, but not all genes or all CHCs. Lastly, we find no evidence of a relationship between the CHC profile and female attractiveness or receptivity towards D. melanogaster males.

Wax Ester Composition of Songbird Preen Oil Varies Seasonally and Differs between Sexes, Ages, and Populations.

Chemical signaling has been well studied in invertebrates and mammals but less so in birds, due to the longstanding misconception that olfaction is unimportant or even non-existent in this taxon. However, recent findings suggest that olfaction plays an important role in avian mate choice and reproductive behavior, similar to other taxa. The leading candidate source for compounds involved in avian chemical communication is preen oil, a complex mixture secreted from the uropygial gland. Preen oil contains volatile compounds and their potential wax ester precursors, and may act as a reproductive chemosignal. Reproductive signals are generally sexually dimorphic, age-specific, seasonally variable, and may also vary geographically. We tested whether preen oil meets these expectations by using gas chromatography to examine the wax ester composition of preen oil in song sparrows (Melospiza melodia). We found that the wax ester composition of preen oil was significantly different between sexes, age classes, seasons, and populations. Collectively, our results suggest that song sparrow preen oil meets the criteria of a chemical cue that may influence mate choice and reproduction. Our findings in song sparrows, which are sexually monomorphic in plumage, also parallel patterns described for dark-eyed juncos (Junco hyemalis), a closely related songbird with sexually dimorphic plumage. Behavioral tests are needed to confirm that song sparrows attend to the cues present in preen oil, but our findings support the increasingly accepted idea that chemical communication is common and widespread in birds as it is in other taxa.

Unraveling inter-species differences in hagfish slime skein deployment.

Hagfishes defend themselves from fish predators by producing defensive slime consisting of mucous and thread components that interact synergistically with seawater to pose a suffocation risk to their attackers. Deployment of the slime occurs in a fraction of a second and involves hydration of mucous vesicles as well as unraveling of the coiled threads to their full length of ∼150 mm. Previous work showed that unraveling of coiled threads (or 'skeins') in Atlantic hagfish requires vigorous mixing with seawater as well as the presence of mucus, whereas skeins from Pacific hagfish tend to unravel spontaneously in seawater. Here, we explored the mechanisms that underlie these different unraveling modes, and focused on the molecules that make up the skein glue, a material that must be disrupted for unraveling to proceed. We found that Atlantic hagfish skeins are also held together with a protein glue, but compared with Pacific hagfish glue, it is less soluble in seawater. Using SDS-PAGE, we identified several soluble proteins and glycoproteins that are liberated from skeins under conditions that drive unraveling in vitro Peptides generated by mass spectrometry of five of these proteins and glycoproteins mapped strongly to 14 sequences assembled from Pacific hagfish slime gland transcriptomes, with all but one of these sequences possessing homologs in the Atlantic hagfish. Two of these sequences encode unusual acidic proteins that we propose are the structural glycoproteins that make up the skein glue. These sequences have no known homologs in other species and are likely to be unique to hagfishes. Although the ecological significance of the two modes of skein unraveling described here are unknown, they may reflect differences in predation pressure, with selection for faster skein unraveling in the Eptatretus lineage leading to the evolution of a glue that is more soluble.

Differential induction of polar and non-polar metabolism during wound-induced suberization in potato (Solanum tuberosum L.) tubers.

Wound-induced suberin deposition involves the temporal and spatial coordination of phenolic and fatty acid metabolism. Phenolic metabolism leads to both soluble metabolites that accumulate as defense compounds as well as hydroxycinnamoyl derivatives that form the basis of the poly(phenolic) domain found in suberized tissue. Fatty acid metabolism involves the biosynthesis of very-long-chain fatty acids, 1-alkanols, ω-hydroxy fatty acids and α,ω-dioic acids that form a poly(aliphatic) domain, commonly referred to as suberin. Using the abscisic acid (ABA) biosynthesis inhibitor fluridone (FD), we reduced wound-induced de novo biosynthesis of ABA in potato tubers, and measured the impact on the expression of genes involved in phenolic metabolism (StPAL1, StC4H, StCCR, StTHT), aliphatic metabolism (StCYP86A33, StCYP86B12, StFAR3, StKCS6), metabolism linking phenolics and aliphatics (StFHT) or acyl chains and glycerol (StGPAT5, StGPAT6), and in the delivery of aliphatic monomers to the site of suberization (StABCG1). In FD-treated tissue, both aliphatic gene expression and accumulation of aliphatic suberin monomers were delayed. Exogenous ABA restored normal aliphatic suberin deposition in FD-treated tissue, and enhanced aliphatic gene expression and poly(aliphatic) domain deposition when applied alone. By contrast, phenolic metabolism genes were not affected by FD treatment, while FD + ABA and ABA treatments slightly enhanced the accumulation of polar metabolites. These data support a role for ABA in the differential induction of phenolic and aliphatic metabolism during wound-induced suberization in potato.

Dietary Fatty Acids Alter Lipid Profiles and Induce Myocardial Dysfunction without Causing Metabolic Disorders in Mice.

Oversupply of bulk saturated fatty acids (SFA) induces metabolic disorders and myocardial dysfunction. We investigated whether, without causing metabolic disorders, the uptake of individual dietary SFA species alters lipid profiles and induces myocardial dysfunction. C57BL/6 mice were fed various customized long-chain SFA diets (40% caloric intake from SFA), including a beef tallow (HBD), cocoa butter (HCD), milk fat (HMD) and palm oil diet (HPD), for 6 months. An isocaloric fat diet, containing medium-chain triglycerides, served as a control (CHD). Long-term intake of dietary long-chain SFA differentially affected the fatty acid composition in cardiac phospholipids. All long-chain SFA diets increased the levels of arachidonic acid and total SFA in cardiac phospholipids. The preferential incorporation of individual SFA into the cardiac phospholipid fraction was dependent on the dietary SFA species. Cardiac ceramide content was elevated in all mice fed long-chain SFA diets, while cardiac hypertrophy was only presented in mice fed HMD or HPD. We have demonstrated that the intake of long-chain SFA species differentially alters cardiac lipid profiles and induces cardiac dysfunction, without causing remarkable metabolic disorders.

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling.

An experimental design mimicking natural plant-microbe interactions is very important to delineate the complex plant-microbe signaling processes. Arabidopsis thaliana-Agrobacterium tumefaciens provides an excellent model system to study bacterial pathogenesis and plant interactions. Previous studies of plant-Agrobacterium interactions have largely relied on plant cell suspension cultures, the artificial wounding of plants, or the artificial induction of microbial virulence factors or plant defenses by synthetic chemicals. However, these methods are distinct from the natural signaling in planta, where plants and microbes recognize and respond in spatial and temporal manners. This work presents a hydroponic cocultivation system where intact plants are supported by metal mesh screens and cocultivated with Agrobacterium. In this cocultivation system, no synthetic phytohormone or chemical that induces microbial virulence or plant defense is supplemented. The hydroponic cocultivation system closely resembles natural plant-microbe interactions and signaling homeostasis in planta. Plant roots can be separated from the medium containing Agrobacterium, and the signaling and responses of both the plant hosts and the interacting microbes can be investigated simultaneously and systematically. At any given timepoint/interval, plant tissues or bacteria can be harvested separately for various "omics" analyses, demonstrating the power and efficacy of this system. The hydroponic cocultivation system can be easily adapted to study: 1) the reciprocal signaling of diverse plant-microbe systems, 2) signaling between a plant host and multiple microbial species (i.e. microbial consortia or microbiomes), 3) how nutrients and chemicals are implicated in plant-microbe signaling, and 4) how microbes interact with plant hosts and contribute to plant tolerance to biotic or abiotic stresses.

Physical, Chemical and Proteomic Evidence of Potato Suberin Degradation by the Plant Pathogenic Bacterium Streptomyces scabiei.

Potato peels consist of a tissue called phellem, which is formed by suberized cell layers. The degradation of suberin, a lipidic and recalcitrant polymer, is an ecological process attributed to soil fungal populations; however, previous studies have suggested that Streptomyces scabiei, the causal agent of potato common scab, possesses the ability to degrade suberin. In the present study, S. scabiei was grown in medium containing suberin-enriched potato phellem as the sole carbon source and its secretome was analyzed periodically (10- to 60-d-old cultures) with a special focus on proteins potentially involved in cell wall degradation. Although the amount and diversity of proteins linked to polysaccharide degradation remained high throughout the experiment, their abundance decreased over time. In contrast, proteins dedicated to lipid metabolism represented a small fraction of the secretome; however, their abundance increased during the experiment. The lipolytic enzymes detected may be involved in the degradation of the aliphatic fraction of suberin because the results of optical and transmission electron microscopy examinations revealed a loss in the integrity of suberized tissues exposed to S. scabiei cells. Chemical analyses identified a time period in which the concentration of aliphatic compounds in potato phellem decreased and the sugar concentration increased; at the end of the 60-d incubation period, the sugar concentration in potato phellem was significantly reduced. This study demonstrated the ability of S. scabiei to degrade the aliphatic portion of suberin.

Fatty acid ω-hydroxylases from Solanum tuberosum.

KEY MESSAGE: Potato StCYP86A33 complements the Arabidopsis AtCYP86A1 mutant, horst - 1. Suberin is a cell-wall polymer that comprises both phenolic and aliphatic components found in specialized plant cells. Aliphatic suberin is characterized by bi-functional fatty acids, typically ω-hydroxy fatty acids and α,ω-dioic acids, which are linked via glycerol to form a three-dimensional polymer network. In potato (Solanum tuberosum L.), over 65 % of aliphatics are either ω-hydroxy fatty acids or α,ω-dioic acids. Since the biosynthesis of α,ω-dioic acids proceeds sequentially through ω-hydroxy fatty acids, the formation of ω-hydroxy fatty acids represents a significant metabolic commitment during suberin deposition. Four different plant cytochrome P450 subfamilies catalyze ω-hydroxylation, namely, 86A, 86B, 94A, and 704B; though to date, only a few members have been functionally characterized. In potato, CYP86A33 has been identified and implicated in suberin biosynthesis through reverse genetics (RNAi); however, attempts to express the CYP86A33 protein and characterize its catalytic function have been unsuccessful. Herein, we describe eight fatty acid ω-hydroxylase genes (three CYP86As, one CYP86B, three CYP94As, and a CYP704B) from potato and demonstrate their tissue expression. We also complement the Arabidopsis cyp86A1 mutant horst-1 using StCYP86A33 under the control of the Arabidopsis AtCYP86A1 promoter. Furthermore, we provide preliminary analysis of the StCYP86A33 promoter using a hairy root transformation system to monitor pStCYP86A33::GUS expression constructs. These data confirm the functional role of StCYP86A33 as a fatty acid ω-hydroxylase, and demonstrate the utility of hairy roots in the study of root-specific genes.

Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination.

During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis.

Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H.

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or 'metabolons', which represent the molecular level of organization. Here, we have confirmed the existence of the long-postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4-hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non-consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.