2016
Walkowiak S., Rowland O., Rodrigue N., and Subramaniam R. (2016). Whole genome sequencing and comparative genomics of closely related Fusarium Head Blight fungi: Fusarium
graminearum, F. meridionale and F. asiaticum. BMC Genomics, 17: 1014Abstract Full text
BACKGROUND: The Fusarium graminearum species complex is composed of many distinct fungal species that cause several diseases in economically important crops, including Fusarium Head Blight of wheat. Despite being closely related, these species and individuals within species have distinct phenotypic differences in toxin production and pathogenicity, with some isolates reported as non-pathogenic on certain hosts. In this report, we compare genomes and gene content of six new isolates from the species complex, including the first available genomes of F. asiaticum and F. meridionale, with four other genomes reported in previous studies. RESULTS: A comparison of genome structure and gene content revealed a 93-99% overlap across all ten genomes. We identified more than 700 k base pairs (kb) of single nucleotide polymorphisms (SNPs), insertions, and deletions (indels) within common regions of the genome, which validated the species and genetic populations reported within species. We constructed a non-redundant pan gene list containing 15,297 genes from the ten genomes and among them 1827 genes or 12% were absent in at least one genome. These genes were co-localized in telomeric regions and select regions within chromosomes with a corresponding increase in SNPs and indels. Many are also predicted to encode for proteins involved in secondary metabolism and other functions associated with disease. Genes that were common between isolates contained high levels of nucleotide variation and may be pseudogenes, allelic, or under diversifying selection. CONCLUSIONS: The genomic resources we have contributed will be useful for the identification of genes that contribute to the phenotypic variation and niche specialization that have been reported among members of the F. graminearum species complex.
Legay S., Guerriero G., André C., Guignard C., Cocco, E., Charton S., Boutry M., Rowland O., and Hausman J.E. (2016). MdMyb93 is a regulator of suberin deposition in russeted apple fruit skins. New Phytologist, 212: 977-991
Abstract Full text
A comparison of the transcriptomes of russeted vs nonrusseted apple skins previously highlighted a tight relationship between a gene encoding an MYB-type transcription factor, MdMYB93, and some key suberin biosynthetic genes. The present work assesses the role of this transcription factor in the suberization process. A phylogenetic analysis of MdMYB93 and Arabidopsis thaliana MYBs was performed and the function of MdMYB93 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight the MdMYB93-regulated genes. Ultraperformance liquid chromatography-triple time-of-flight (UPLC-TripleTOF) and GC-MS were used to investigate alterations in phenylpropanoid, soluble-free lipid and lipid polyester contents. A massive accumulation of suberin and its biosynthetic precursors in MdMYB93 agroinfiltrated leaves was accompanied by a remobilization of phenylpropanoids and an increased amount of lignin precursors. Gene expression profiling displayed a concomitant alteration of lipid and phenylpropanoid metabolism, cell wall development, and extracellular transport, with a large number of induced transcripts predicted to be involved in suberin deposition. The present work supports a major role of MdMYB93 in the regulation of suberin deposition in russeted apple skins, from the synthesis of monomeric precursors, their transport, polymerization, and final deposition as suberin in primary cell wall.
Lukina A.O., Boutin C., Rowland O., and Carpenter D.J. (2016). Evaluating trivalent chromium toxicity on wild terrestrial and wetland plants. Chemosphere, 162: 355-364
Abstract Full text
Elevated chromium levels in soil from mining can impact the environment, including plants. Mining of chromium is concentrated in South Africa, several Asian countries, and potentially in Northern Ontario, Canada, raising concerns since chromium toxicity to wild plants is poorly understood. In the first experiment, concentration-response tests were conducted to evaluate effects of chromium on terrestrial and wetland plants. Following established guidelines using artificial soil, seeds of 32 species were exposed to chromium (Cr(3+)) at concentrations simulating contamination (0-1000 mg kg(-1)). This study found that low levels of chromium (250 mg kg(-1)) adversely affected the germination of 22% of species (33% of all families), while higher levels (500 and 1000 mg kg(-1)) affected 69% and 94% of species, respectively, from 89% of the families. Secondly, effects on seedbanks were studied using soil collected in Northern Ontario and exposed to Cr(3+) at equivalent concentrations (0-1000 mg kg(-1)). Effects were less severe in the seedbank study with significant differences only observed at 1000 mg kg(-1). Seeds exposed to Cr(3+) during stratification were greatly affected. Seed size was a contributing factor as was possibly the seed coat barrier. This study represents an initial step in understanding Cr(3+) toxicity on wild plants and could form the basis for future risk assessments.
Delude C., Fouillen L., Bhar P., Cardinal M.-J., Pascal S., Santos P., Kosma D.K., Joubès J., Rowland O., and Domergue F. (2016). Primary fatty alcohols are major components of suberized root tissues of Arabidopsis in the form of alkyl hydroxycinnamates. Plant Physiology, 171: 1934-1950
Abstract Full text
Suberin is a complex hydrophobic polymer that acts as a barrier controlling water and solute fluxes and restricting pathogen infections. Suberin is deposited immediately outside of the plasmalemma in the cell wall of certain tissues such as endodermis of roots, aerial and underground periderms, and seed coats. Suberin consists of a variety of fatty acid derivatives polymerized with glycerol and phenolics. In this study, we show using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry techniques that most of the fatty alcohols not covalently linked to the suberin polymer are in the form of alkyl hydroxycinnamates (AHCs), with alkyl caffeates predominating. Such compounds are not restricted to the periderm of mature roots but also are present in the endodermis of younger roots, where they are not extracted by rapid dipping in chloroform. Analysis of several mutants affected in key enzymes involved in the biosynthesis and export of suberin monomers suggests that the formation of the suberin polymer and associated waxes involves common pathways and occurs concomitantly in Arabidopsis (Arabidopsis thaliana) roots. Although fatty alcohols represent only minor components of the suberin polymer in Arabidopsis roots, this study demonstrates that they constitute the major aliphatics of suberin-associated waxes in the form of AHCs. Therefore, our results indicate that esterified fatty alcohols, both soluble and polymerized forms, represent major constituents of Arabidopsis root suberized barriers, being as abundant as α,ω-dicarboxylic and unsubstituted fatty acids. In addition, our results show that suberized layers represent a major sink for acyl-lipid metabolism in Arabidopsis roots.
Kosma D.K., and Rowland O. (2016). Answering a four decade-old question on epicuticular wax biosynthesis. Journal of Experimental Botany, 67: 2538-2540
Abstract Full text
In this issue of Journal of Experimental Botany (pages 2715–2730) Schneider et al. report the identity of three genes from barley described in the 1970s as important for the synthesis of β-diketone cuticular waxes, thereby revealing a novel polyketide synthase pathway responsible for their production. It is a perfect example of how modern sequencing technologies can resolve age-old questions on important food crops.
Monreal C.M., Chahal A., Schnitzer M., and Rowland O. (2016). Chemical characterization of fatty acids, alkanes, n-diols and alkyl esters produced by a mixed culture of Trichoderma koningii and Penicillium janthinellum grown aerobically on undecanoic acid, potato dextrose and their mixture. Journal of Environmental Science and Health, Part B, 51: 326-339
Abstract Full text
Little is known about the mixed fungal synthesis of high-value aliphatics derived from the metabolism of simple and complex carbon substrates. Trichoderma koningii and Penicillium janthinellum were fed with undecanoic acid (UDA), potato dextrose broth (PDB), and their mixture. Pyrolysis Field Ionization Mass Spectrometry (Py-FIMS) together with (1)H and (13)C Nuclear Magnetic Resonance (NMR) characterized CHCl3 soluble aliphatics in the fungal cell culture. Data from NMR and Py-FIMS analysis were complementary to each other. On average, the mixed fungal species produced mostly fatty acids (28% of total ion intensity, TII) > alkanes (2% of TII) > n-diols (2% of TII) > and alkyl esters (0.8% of TII) when fed with UDA, PDB or UDA+PDB. The cell culture accumulated aliphatics extracellularly, although most of the identified compounds accumulated intracellularly. The mixed fungal culture produced high-value chemicals from the metabolic conversion of simple and complex carbon substrates.
2015
Kosma D.K., Molina I., and Rowland O. (2015). GC-MS-based analysis of chloroform extracted suberin-associated root waxes from Arabidopsis and other plant species. Bio-protocol, 5(24): e1679Abstract Full text
The periderm and exodermis of taproots and tuberous taproots contain an extracellular lipid polymer, suberin, deposited in their cell walls. This polymer is intractable in organic solvents, and is co-deposited with chloroform-extractable waxes. These suberin-associated root waxes are typically composed of alkanes, primary alcohols, fatty acids, alkyl ferulates, alkyl caffeates, and alkyl coumarates (Espelie et al., 1980; Li et al., 2007; Kosma et al., 2015). They are believed to contribute to the diffusion barrier properties of suberized cell walls (Soliday et al., 1979), and possibly have other roles yet to be discovered. Here we describe a protocol to extract and analyze waxes associated with root suberin. This fraction of aliphatic components is extracted by whole root immersion in chloroform, and is then chemically modified to prepare samples that are more suitable to gas-chromatography analysis. This protocol is optimized for Arabidopsis thaliana, but can be used with roots of other plants as described herein.
HadiNezhad M., Rowland O., and Hosseinian F. (2015). The fatty acid profile and phenolic composition of Descurainia sophia seeds extracted by supercritical CO2. Journal of the American Oil Chemists' Society, 92: 1379-1390
Abstract Full text
Oil and phenolics were extracted from Descurainia sophia (Sophia) seeds by a supercritical CO2 system. Extractions were conducted in two sequential steps, first using 100 % CO2 and then adding 10 % ethanol as co-solvent. The extracts were collected in each step using two separate collectors operating at different pressures. The extraction run was 3 and 4 h for the first period, and 2 h for the second period. The majority of the oil was collected in the first extraction period while phenolic compounds were obtained in the second extraction period. A combined mode of static/dynamic extraction (3 h running and 1 h soaking in CO2) was also used in the first extraction period, which enhanced the total extraction yield (29.3 ± 0.5 %) and was comparable to the 4 h extraction yield (31.4 ± 0.1 %). The total fatty acid (FA) content of oil in collector 1 (0.94 g) was nearly twice that in collector 2 (0.60 g). The oil contained 14 FAs with α-linolenic being predominant (48.5 %), with a total 91.1 % unsaturated FAs, a ω3/ω6 ratio of 2.7, and an erucic acid content of 6.2 %. More than 10 phenolic compounds were detected by HPLC in the Sophia seed extracts of which sinapic acid was the dominant compound. Sophia seed extracts showed high levels of antioxidant activity. These results suggest that Sophia seed oil and phenolics have the potential for functional food and pharmaceutical applications.
Walkowiak S., Bonner C.T., Wang L., Blackwell B., Rowland O., and Subramaniam R. (2015). Intraspecies interaction of Fusarium graminearum contributes to reduced toxin production and virulence. Molecular Plant Microbe Interactions, 28: 1256-1267
Abstract Full text
Fusarium graminearum is a pathogenic fungus that causes Fusarium Head Blight in wheat and lowers the yield and quality of grains by contamination with the trichothecene mycotoxin, deoxynivalenol. The fungi coexist and interact with several different Fusaria, as well as other plant pathogenic fungi and bacteria in the field. In Canada, F. graminearum exists as two main trichothecene chemotypes: 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol. To understand the potential interactions between two isolates of these chemotypes, we conducted co-inoculation studies both in culture and in planta. The studies showed that intraspecies interaction reduces trichothecene yield in culture and disease symptoms in wheat. To elucidate the genes involved in the intraspecies interaction, expression profiling was performed on RNA samples isolated from co-inoculated cultures and potential genes were identified by using the genome sequences of the respective isolates.
Vishwanath S.J., Delude C., Domergue F., and Rowland O. (2015). Suberin: biosynthesis, regulation, and polymer assembly of a protective extracellular barrier. Plant Cell Reports, 34: 573-586
Abstract Full text
Suberin is a lipid-phenolic biopolyester deposited in the cell walls of certain boundary tissue layers of plants, such as root endodermis, root and tuber peridermis, and seed coats. Suberin serves as a protective barrier in these tissue layers, controlling, for example, water and ion transport. It is also a stress-induced anti-microbial barrier. The suberin polymer contains a variety of C16-C24 chain-length aliphatics, such as ω-hydroxy fatty acids, α,ω-dicarboxylic fatty acids, and primary fatty alcohols. Suberin also contains high amounts of glycerol and phenolics, especially ferulic acid. In addition, non-covalently linked waxes are likely associated with the suberin polymer. This review focusses on the suberin biosynthetic enzymes identified to date, which include β-ketoacyl-CoA synthases, fatty acyl reductases, long-chain acyl-CoA synthetases, cytochrome P450 monooxygenases, glycerol 3-phosphate acyltransferases, and phenolic acyltransferases. We also discuss recent advances in our understanding of the transport of suberin components intracellularly and to the cell wall, polymer assembly, and the regulation of suberin deposition.
2014
Kosma D.K., Murmu J., Razeq F.M., Santos P., Bourgault R., Molina I., and Rowland O. (2014). AtMYB41 activates ectopic suberin synthesis and assembly in multiple plant species and cell types. The Plant Journal, 80: 216-229Abstract Full text
Suberin is a lipid and phenolic cell wall heteropolymer found in roots and other organs of all vascular plants. Suberin plays a critical role in plant water relations and protecting plants from biotic and abiotic stresses. Here we describe a transcription factor AtMYB41 (At4g28110) that can activate the steps necessary for aliphatic suberin synthesis and deposition of cell wall-associated suberin-like lamellae in both Arabidopsis thaliana and Nicotiana benthamiana. Overexpression of AtMYB41 increased the abundance of suberin biosynthetic gene transcripts by orders of magnitude and resulted in the accumulation of up to 22-times more suberin-type than cutin-type aliphatic monomers in leaves. Overexpression of AtMYB41 also resulted in elevated amounts of monolignols in leaves and an increase in the accumulation of phenylpropanoid and lignin biosynthetic gene transcripts. Surprisingly, ultrastructural data indicated that overexpression led to formation of suberin-like lamellae in both epidermal and mesophyll cells of leaves. We further implicate AtMYB41 in the production of aliphatic suberin under abiotic stress conditions. These results provide insight into the molecular-genetic mechanisms of the biosynthesis and deposition of a ubiquitous cell wall-associated plant structure and will serve as a basis for discovering the transcriptional network behind one of the most abundant lipid-based polymers in nature.
Razeq F.M., Kosma D.K., Rowland O., and Molina I. (2014). Extracellular lipids of Camelina sativa: Characterization of chloroform-extractable waxes from aerial and subterranean surfaces. Phytochemistry, 106: 188-196
Abstract Full text
Camelina sativa (L.) Crantz is an emerging low input, stress tolerant crop with seed oil composition suitable for biofuel and bioproduct production. The chemical compositions and ultrastructural features of surface waxes from C. sativa aerial cuticles, seeds, and roots were analyzed using gas chromatography and microscopy. Alkanes, primary fatty alcohols, and free fatty acids were common components of all analyzed organs. A particular feature of leaf waxes was the presence of alkyl esters of long-chain fatty acids and very long-chain fatty alcohols, ranging from C38 to C50 and dominated by C42, C44 and C46 homologues. Stem waxes were mainly composed of non-sterol pentacyclic triterpenes. Flowers accumulated significant amounts of methyl-branched iso-alkanes (C29 and C31 total carbon number) in addition to straight-chain alkanes. Seed waxes were mostly primary fatty alcohols of up to 32 carbons in length and unbranched C29 and C31 alkanes. The total amount of identified wax components extracted by rapid chloroform dipping of roots was 280 μg/g (fresh weight), and included alkyl hydroxycinnamates, predominantly alkyl coumarates and alkyl caffeates. This study provides qualitative and quantitative information on the waxes of C. sativa root, shoot, and seed boundary tissues, allowing the relative activities of wax biosynthetic pathways in each respective plant organ to be assessed. This detailed description of the protective surface waxes of C. sativa may provide insights into its drought-tolerant and pathogen-resistant properties, and also identifies C. sativa as a potential source of renewable high-value natural products.
Chahal A., Monreal C.M., Bissett J., Rowland O., Smith M.L., and S. Miller (2014). Metabolism of n-C10:0 and n-C11:0 fatty acids by Trichoderma koningii, Penicillium janthinellum and their mixed culture: I. Biomass and CO2 production, and allocation of intracellular lipids. Journal of Environmental Science and Health, Part B, 49: 945-954
Abstract Full text
The capacity of two soil fungi, Trichoderma koningii and Penicillium janthinellum, to oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and store intracellular lipids during growth is unknown. This article reports for the first time the metabolism of decanoic acid (DA, C10:0), undecanoic acid (UDA, n-C11:0), a mixture of the acids (UDA+DA) and a mixture of UDA+ potato dextrose broth (PDB) by T. koningii and P. janthinellum and their mixed culture. A control PDB complex substrate was used as a substrate control treatment. The fungal cultures were assayed for their capacity to: (1) oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and (2) store lipids intracellularly during growth. On all four fatty acid substrates, the mixed T. koningii and P. janthinellum culture produced more biomass and CO2 than the individual fungal cultures. Per 150 mL culture, the mixed species culture grown on UDA+PDB and on PDB alone produced the most biomass (7,567 mg and 11,425 mg, respectively). When grown in DA, the mixed species culture produced the least amount of biomass (6,400 mg), a quantity that was lower than those obtained in UDA (7,550 mg) or UDA+DA (7,270 mg). Amounts of CO2 produced ranged from 210 mg under DA to 618 mg under PDB, and these amounts were highly correlated with biomass (r(2) = 0.99). Fluorescence microscopy of stained lipids in the mixed fungal cell cultures growing during the exponential phase demonstrated larger fungal cells and higher accumulation of lipids in membranes and storage bodies than those observed during the lag and stationary phases. T. koningii and P. janthinellum grown on n-C10:0 and n-C11:0 fatty acids produced lower amounts of biomass and CO2, but stored higher amounts of intracellular lipids, than when grown on PDB alone.
Monreal C.M., Chahal A., Rowland O., Smith M., and Schnitzer M. (2014). Metabolism of nC11 fatty acid fed to Trichoderma koningii and Penicillium janthinellum II: Production of intracellular and extracellular lipids. Journal of Environmental Science and Health, Part B, 49: 955-965
Abstract Full text
Little is known about the fungal metabolism of nC10 and nC11 fatty acids and their conversion into lipids. A mixed batch culture of soil fungi, T. koningii and P. janthinellum, was grown on undecanoic acid (UDA), a mixture of UDA and potato dextrose broth (UDA+PDB), and PDB alone to examine their metabolic conversion during growth. We quantified seven intracellular and extracellular lipid classes using Iatroscan thin-layer chromatography with flame ionization detection (TLC-FID). Gas chromatography with flame ionization detection (GC-FID) was used to quantify 42 individual fatty acids. Per 150 mL culture, the mixed fungal culture grown on UDA+PDB produced the highest amount of intracellular (531 mg) and extracellular (14.7 mg) lipids during the exponential phase. The content of total intracellular lipids represented 25% of the total biomass-carbon, or 10% of the total biomass dry weight produced. Fatty acids made up the largest class of intracellular lipids (457 mg/150 mL culture) and they were synthesized at a rate of 2.4 mg/h during the exponential phase, and decomposed at a rate of 1.8 mg/h during the stationary phase, when UDA+PDB was the carbon source. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and vaccenic acid (C18:1) accounted for >80% of the total intracellular fatty acids. During exponential growth on UDA+PDB, hydrocarbons were the largest pool of all extracellular lipids (6.5 mg), and intracellularly they were synthesized at a rate of 64 μg/h. The mixed fungal species culture of T. koningii and P. janthinellum produced many lipids for potential use as industrial feedstocks or bioproducts in biorefineries.
Vishwanath S.J., Domergue F., and Rowland O. (2014). Seed coat permeability test: Tetrazolium penetration assay. Bio-protocol, 4(13): e1173
Abstract Full text
Seed coat permeability is important to study as it plays significant roles in seed dormancy, germination, and protection from pathogens. Here we describe a commonly used seed coat permeability test known as the tetrazolium penetration assay with a method to quantify the levels of permeability. Tetrazolium red is a cationic dye that is widely used in seed viability testing. Tetrazolium salts are amphipathic cations, which, after penetrating the dead cells of the seed coat, are reduced to red-colored insoluble precipitates made up of formazans by active dehydrogenases (NADH-dependent reductases) in the embryo of seeds (Berridge et al., 1996). The intensity of red coloration is directly proportional to the permeability of the seeds. The quantification involves extraction of formazans from the incubated seeds and spectrophotometric determination of absorbance of formazan extracts at 485 nm.
Pulsifer I.P., Lowe C., Narayaran S.A., Busuttil A.S., Vishwanath S.J., Domergue F., and Rowland O. (2014). Acyl-lipid thioesterase1-4 from Arabidopsis thaliana form a novel family of fatty acyl-acyl carrier protein thioesterases with divergent expression patterns and substrate specificities. Plant Molecular Biology, 84: 549-563
Abstract Full text
Hydrolysis of fatty acyl thioester bonds by thioesterases to produce free fatty acids is important for dictating the diversity of lipid metabolites produced in plants. We have characterized a four-member family of fatty acyl thioesterases from Arabidopsis thaliana, which we have called acyl-lipid thioesterase1 (ALT1), ALT2, ALT3, and ALT4. The ALTs belong to the Hotdog fold superfamily of thioesterases. ALT-like genes are present in diverse plant taxa, including dicots, monocots, lycophytes, and microalgae. The four Arabidopsis ALT genes were found to have distinct gene expression profiles with respect to each other. ALT1 was expressed specifically in stem epidermal cells and flower petals. ALT2 was expressed specifically in root endodermal and peridermal cells as well as in stem lateral organ boundary cells. ALT3 was ubiquitously expressed in aerial and root tissues and at much higher levels than the other ALTs. ALT4 expression was restricted to anthers. All four proteins were localized in plastids via an N-terminal targeting sequence of about 48 amino acids. When expressed in Escherichia coli, the ALT proteins used endogenous fatty acyl-acyl carrier protein substrates to generate fatty acids that varied in chain length (C6-C18), degree of saturation (saturated and monounsaturated), and oxidation state (fully reduced and β-ketofatty acids). Despite their high amino acid sequence identities, each enzyme produced a different profile of lipids in E. coli. The biological roles of these proteins are unknown, but they potentially generate volatile lipid metabolites that have previously not been reported in Arabidopsis.
2013
Chacón M.G., Fournier A.E., Tran F., Dittrich-Domergue F., Pulsifer I.P., Domergue F., and Rowland O. (2013). Identification of Amino Acids Conferring Chain-Length Substrate Specificities on Fatty Alcohol-Forming Reductases FAR5 and FAR8 from Arabidopsis thaliana. Journal of Biological Chemistry, 288: 30345-30355 Abstract Full text
Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast, and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine-355 and valine-377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value.
Vishwanath S.J., Kosma D.K., Pulsifer I.P., Scandola S., Pascal S., Joubès J., Dittrich-Domergue F., Lessire R., Rowland O., and Domergue F. (2013). Suberin-Associated Fatty Alcohols in Arabidopsis thaliana: Distributions in Roots and Contributions to Seed Coat Barrier Properties. Plant Physiology, 163: 1118-1132
Abstract Full text
Suberin is found in a variety of tissue such as root endoderms and periderms, storage tuber periderms, trees cork layer, and seed coats. It acts as a hydrophobic barrier to control the movement of water, gases and solutes as well as an anti-microbial barrier. Suberin consists of polymerized phenolics, glycerol, and a variety of fatty acid derivatives, including primary fatty alcohols. We have conducted an in-depth analysis of the distribution of the C18:0-C22:0 fatty alcohols in Arabidopsis roots and found that only 20% are part of the root suberin polymer, together representing about 5% of its aliphatic monomer composition, while the remaining 80% are found in the non-polymeric (soluble) fraction and in suberin-associated root waxes. Down regulation of Arabidopsis FAR1, FAR4 and FAR5, which collectively produce the fatty alcohols found in suberin, reduced their levels by 70 to 80% in: (1) the polymeric and non-polymeric fractions from roots of tissue culture-grown plants, (2) the suberin-associated root waxes from six-week old soil-grown plants, and (3) the seed coat suberin polymer. In contrast, the other main monomers of the suberin were not altered, indicating that reduced levels of fatty alcohols did not influence the suberin polymerization process. Nevertheless, the 75% reduction in total fatty alcohol and diol loads in the seed coat resulted in increased permeability to tetrazolium salts and a higher sensitivity to abscisic acid (ABA). These results suggest that fatty alcohols and diols play an important role in determining the functional properties of the suberin barrier.
Tran F., Penniket C., Patel R.V., Provart N.J., Laroche A., Rowland O., and Robert L.S. (2013). Developmental transcriptional profiling reveals key insights into Triticeae reproductive development. The Plant Journal 74: 971-988
Abstract Full text
Despite their importance, there remains a paucity of large scale gene expression-based studies of reproductive development in the species belonging to the Triticeae. As a first step to address this deficiency, a gene expression atlas of triticale reproductive development was generated using the 55K Affymetrix GeneChip® Wheat Genome Array. The global transcriptional profiles of the anther/pollen, ovary and stigma were analyzed at concurrent developmental stages and co-expressed as well as preferentially expressed genes were identified. Data analysis revealed both novel and conserved regulatory factors underlying Triticeae floral development and function. This comprehensive resource rests upon detailed gene annotations and the expression profiles are readily accessible via a web browser.
Bird D., and Rowland O. (2013). Cuticular Waxes. In “Acyl-Lipid Metabolism” by Li-Beisson et al. The Arabidopsis Book, 11: e0161
Abstract Full text
Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.
Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffusion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http://aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables.
2012
Lü S., Zhao H., Des Marais D.L., Parsons E.P., Wen X., Xu X., Bangarusamy D.K., Wang G., Rowland O., Juenger T., Bressan R.A., and Jenks M.A. (2012). Mutation of Arabidopsis ECERIFERUM9 alters cuticle metabolism and improves tolerance to water deficit. Plant Physiology 159: 930-944
Abstract Full text
Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C24) and hexacosanoic acid (C26). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.
Rowland O., and Domergue F. (2012). Plant Fatty Acyl Reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications. Plant Science 193-194: 28-38
Abstract Full text
Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols.
Pulsifer I.P., Kluge S., and Rowland O. (2012). Arabidopsis LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiology and Biochemistry 51: 31-39
Abstract Full text
The plant cuticle is a lipid-based barrier on the aerial surfaces of plants that plays a variety of protective roles. The cuticle is comprised largely of long-chain and very-long-chain fatty acids and their derivatives. In Arabidopsis, LONG-CHAIN ACYL-COA SYNTHETASE1 (LACS1), LACS2, and LACS3 are known or suspected cuticle biosynthetic genes. Very-long-chain acyl-coenzyme A (CoA) synthetase activity has been demonstrated for LACS1 and LACS2, although the role for such an activity in cuticle biosynthesis is currently unclear. In yeast and mammalian systems, some very-long-chain acyl-CoA synthetases are also called fatty acid transport proteins (FATPs) due to a second function of mediating transmembrane movement of fatty acids. We sought to determine if LACS1-3 also have this dual functionality. A yeast fat1Δ mutant is deficient in both very-long-chain acyl-CoA synthetase activity and exogenous fatty acid uptake. We demonstrate that heterologous expression of LACS1, 2, or 3 is able to complement both of these deficiencies. Furthermore, expression of each LACS enzyme in yeast resulted in uptake of the long-chain fatty acid analogue, C(1)-BODIPY-C(12). Only expression of LACS1 resulted in uptake of the very-long-chain fatty acid analogue, BODIPY-C(16). These results demonstrate that LACS1, LACS2, and LACS3 have the dual functionality of yeast and mammalian FATP enzymes. These findings have implications in the transmembrane transport and intracellular trafficking of plant lipids destined for export to the cuticle.
Boutin C., Aya K.L., Carpenter D., Thomas P.J., and Rowland O. (2012). Phytotoxicity testing for pesticide regulation: shortcomings in relation to biodiversity and ecosystem services in agrarian systems.Science of the Total Environment 415: 79-92
Abstract Full text
The purpose of this paper is to present current knowledge on methods employed to perform phytotoxicity tests and risk assessments and to highlight shortcomings in relation to biodiversity and ecosystem services. Ecosystem services are benefits provided to humankind by a multitude of organisms present in natural ecosystems. Several studies were conducted between 2001 and 2010 aimed at investigating some of the deficiencies in phytotoxicity testing (new and existing data are presented). Herbicide toxicity responses were similar when comparing a suite of crop versus wild species. However, the validity of the evaluation was limited because of the narrow types of species tested. The number of species tested, currently set between six and ten, appears insufficient. The trait-based approach (i.e. the use of plant attributes to predict species sensitivity to toxicants) can be used to improve species selection. This approach puts more emphasis on the shared biological characteristics that affect plant function within ecological communities rather than on plant phylogeny. Results presented showed that further studies are needed. In test guidelines, protocols require that crop species be sprayed as young vegetative plants, which is assumed to be the most sensitive growth stage to herbicides. In contrast, during herbicide spray, herbicides may reach non-target plants that are at various phenological stages. Several studies demonstrated that plants may be at greater risk when contamination occurs at the reproductive stage. No data on long-term effects, plant recovery or on effects on reproductive stages are requested in current guidelines. Preliminary evidence suggests that this may be an important aspect to consider in risk assessment. In addition, herbicide impacts on plant community diversity as well as biodiversity at other trophic levels have been demonstrated in only a limited number of studies and therefore should warrant more attention in risk assessment.
Abstract Full text
Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C24) and hexacosanoic acid (C26). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.
Rowland O., and Domergue F. (2012). Plant Fatty Acyl Reductases: enzymes generating fatty alcohols for protective layers with potential for industrial applications. Plant Science 193-194: 28-38
Abstract Full text
Primary fatty alcohols are found throughout the biological world, either in free form or in a combined state. They are common components of plant surface lipids (i.e. cutin, suberin, sporopollenin, and associated waxes) and their absence can significantly perturb these essential barriers. Fatty alcohols and/or derived compounds are also likely to have direct functions in plant biotic and abiotic interactions. An evolutionarily related set of alcohol-forming fatty acyl reductases (FARs) is present in all kingdoms of life. Plant microsomal and plastid-associated FAR enzymes have been characterized, acting on acyl-coenzymeA (acyl-CoA) or acyl-acyl carrier protein (acyl-ACP) substrates, respectively. FARs have distinct substrate specificities both with regard to chain length and chain saturation. Fatty alcohols and wax esters, which are a combination of fatty alcohol and fatty acid, have a variety of commercial applications. The expression of FARs with desired specificities in transgenic microbes or oilseed crops would provide a novel means of obtaining these valuable compounds. In the present review, we report on recent progress in characterizing plant FAR enzymes and in understanding the biological roles of primary fatty alcohols, as well as describe the biotechnological production and industrial uses of fatty alcohols.
Pulsifer I.P., Kluge S., and Rowland O. (2012). Arabidopsis LONG-CHAIN ACYL-COA SYNTHETASE 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiology and Biochemistry 51: 31-39
Abstract Full text
The plant cuticle is a lipid-based barrier on the aerial surfaces of plants that plays a variety of protective roles. The cuticle is comprised largely of long-chain and very-long-chain fatty acids and their derivatives. In Arabidopsis, LONG-CHAIN ACYL-COA SYNTHETASE1 (LACS1), LACS2, and LACS3 are known or suspected cuticle biosynthetic genes. Very-long-chain acyl-coenzyme A (CoA) synthetase activity has been demonstrated for LACS1 and LACS2, although the role for such an activity in cuticle biosynthesis is currently unclear. In yeast and mammalian systems, some very-long-chain acyl-CoA synthetases are also called fatty acid transport proteins (FATPs) due to a second function of mediating transmembrane movement of fatty acids. We sought to determine if LACS1-3 also have this dual functionality. A yeast fat1Δ mutant is deficient in both very-long-chain acyl-CoA synthetase activity and exogenous fatty acid uptake. We demonstrate that heterologous expression of LACS1, 2, or 3 is able to complement both of these deficiencies. Furthermore, expression of each LACS enzyme in yeast resulted in uptake of the long-chain fatty acid analogue, C(1)-BODIPY-C(12). Only expression of LACS1 resulted in uptake of the very-long-chain fatty acid analogue, BODIPY-C(16). These results demonstrate that LACS1, LACS2, and LACS3 have the dual functionality of yeast and mammalian FATP enzymes. These findings have implications in the transmembrane transport and intracellular trafficking of plant lipids destined for export to the cuticle.
Boutin C., Aya K.L., Carpenter D., Thomas P.J., and Rowland O. (2012). Phytotoxicity testing for pesticide regulation: shortcomings in relation to biodiversity and ecosystem services in agrarian systems.Science of the Total Environment 415: 79-92
Abstract Full text
The purpose of this paper is to present current knowledge on methods employed to perform phytotoxicity tests and risk assessments and to highlight shortcomings in relation to biodiversity and ecosystem services. Ecosystem services are benefits provided to humankind by a multitude of organisms present in natural ecosystems. Several studies were conducted between 2001 and 2010 aimed at investigating some of the deficiencies in phytotoxicity testing (new and existing data are presented). Herbicide toxicity responses were similar when comparing a suite of crop versus wild species. However, the validity of the evaluation was limited because of the narrow types of species tested. The number of species tested, currently set between six and ten, appears insufficient. The trait-based approach (i.e. the use of plant attributes to predict species sensitivity to toxicants) can be used to improve species selection. This approach puts more emphasis on the shared biological characteristics that affect plant function within ecological communities rather than on plant phylogeny. Results presented showed that further studies are needed. In test guidelines, protocols require that crop species be sprayed as young vegetative plants, which is assumed to be the most sensitive growth stage to herbicides. In contrast, during herbicide spray, herbicides may reach non-target plants that are at various phenological stages. Several studies demonstrated that plants may be at greater risk when contamination occurs at the reproductive stage. No data on long-term effects, plant recovery or on effects on reproductive stages are requested in current guidelines. Preliminary evidence suggests that this may be an important aspect to consider in risk assessment. In addition, herbicide impacts on plant community diversity as well as biodiversity at other trophic levels have been demonstrated in only a limited number of studies and therefore should warrant more attention in risk assessment.
2011
Doan T.T.P., Domergue F., Fournier A.E., Vishwanath S.J., Rowland O., Moreau P., Wood C.C., Carlsson A.S., Hamberg M., and Hofvander P. (2011). Biochemical characterization of a chloroplast localized fatty acid reductase from Arabidopsis thaliana. Biochimica et Biophysica Acta 1821: 1244-1255
Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty alcohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta.
2010
Domergue F., Vishwanath S.J., Joubès J., Ono J., Lee J.A., Alhattab R., Lowe C., Pascal S., Bourdon M., Lessire R., and Rowland O. (2010). Three Arabidopsis Fatty Acyl-CoA Reductases, FAR1, FAR4, and FAR5, Generate Primary Fatty Alcohols Associated with Suberin Deposition. Plant Physiology 153: 1539-1554
Suberin is a protective hydrophobic barrier consisting of phenolics, glycerol, and a variety of fatty acid derivatives, including C18:0-C22:0 primary fatty alcohols. An eight-member gene family encoding alcohol-forming fatty acyl-coenzyme A reductases (FARs) has been identified in Arabidopsis (Arabidopsis thaliana). Promoter-driven expression of the β-glucuronidase reporter gene indicated that three of these genes, FAR1(At5g22500), FAR4(At3g44540), and FAR5(At3g44550), are expressed in root endodermal cells. The three genes were transcriptionally induced by wounding and salt stress. These patterns of gene expression coincide with known sites of suberin deposition. We then characterized a set of mutants with T-DNA insertions in FAR1, FAR4, or FAR5 and found that the suberin compositions of roots and seed coats were modified in each far mutant. Specifically, C18:0-OH was reduced in far5-1, C20:0-OH was reduced in far4-1, and C22:0-OH was reduced in far1-1. We also analyzed the composition of polymer-bound lipids of leaves before and after wounding and found that the basal levels of C18:0-C22:0 primary alcohols in wild-type leaves were increased by wounding. In contrast, C18:0-OH and C22:0-OH were not increased by wounding in far5-1 and far1-1 mutants, respectively. Heterologous expression of FAR1, FAR4, and FAR5 in yeast confirmed that they are indeed active alcohol-forming FARs with distinct, but overlapping, chain length specificities ranging from C18:0 to C24:0. Altogether, these results indicate that Arabidopsis FAR1, FAR4, and FAR5 generate the fatty alcohols found in root, seed coat, and wound-induced leaf tissue.
2009
Rowland O. (2009). Green Biotechnology: Harnessing plant biomass for biofuels and biomaterials. Ottawa Life Magazine June 2009
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Lü S., Song T., Kosma D., Parson E., Rowland O.*, and Jenks M.A.* (2009). Arabidopsis CER8 encodes Long-Chain Acyl CoA Synthetase 1 (LACS1) and has overlapping functions with LACS2 in plant cutin and wax biosynthesis. The Plant Journal 59: 553-564
*co-corresponding authors
Plant cuticle is an extracellular lipid-based matrix of cutin and waxes, which covers aerial organs and protects them from many forms of environmental stress. We report here the characterization of CER8/LACS1, one of nine Arabidopsis long-chain acyl-CoA synthetases thought to activate acyl chains. Mutations in LACS1 reduced the amount of wax in all chemical classes on the stem and leaf, except in the very long-chain fatty acid (VLCFA) class wherein acids longer than 24 carbons (C(24)) were elevated more than 155%. The C(16) cutin monomers on lacs1 were reduced by 37% and 22%, whereas the C(18) monomers were increased by 28% and 20% on stem and leaf, respectively. Amounts of wax and cutin on a lacs1-1 lacs2-3 double mutant were much lower than on either parent, and lacs1-1 lacs2-3 had much higher cuticular permeability than either parent. These additive effects indicate that LACS1 and LACS2 have overlapping functions in both wax and cutin synthesis. We demonstrated that LACS1 has synthetase activity for VLCFAs C(20)-C(30), with highest activity for C(30) acids. LACS1 thus appears to function as a very long-chain acyl-CoA synthetase in wax metabolism. Since C(16) but not C(18) cutin monomers are reduced in lacs1, and C(16) acids are the next most preferred acid (behind C(30)) by LACS1 in our assays, LACS1 also appears to be important for the incorporation of C(16) monomers into cutin polyester. As such, LACS1 defines a functionally novel acyl-CoA synthetase that preferentially modifies both VLCFAs for wax synthesis and long-chain (C(16)) fatty acids for cutin synthesis.
Arsovski A.A., Villota M., Rowland O., Subramaniam R., and Western T.L. (2009). MUM ENHANCERS are required for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana. Journal of Experimental Botany 60: 2601-2612
Arsovski A.A., Villota M., Rowland O., Subramaniam R., and Western T.L. (2009). MUM ENHANCERS are required for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana. Journal of Experimental Botany 60: 2601-2612
Abstract Full text
Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.
Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.
Older publications
van den Burg H.A., Tsitsigiannis D.I., Rowland O., Lo J., Rallapalli G., Maclean D., Takken F.L.W, and Jones J.D.G. (2008). The F-box protein ACRE189/ACIF1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato. Plant Cell 20: 697-719
Rowland O., Lee R., Franke R., Schreiber L., and Kunst L. (2007). The CER3 wax biosynthetic gene from Arabidopsis thaliana is allelic to WAX2/YRE/FLP1. FEBS Letters 581: 3538-3544
Rothfels K., Rowland O., and Segall J. (2007). Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene. Nucleic Acids Research 35: 4869-4881
Rowland O., Zheng H., Hepworth S.R., Lam P., Jetter R., and Kunst L. (2006). CER4 encodes an alcohol-forming fatty acyl-coenzyme A reductase involved in cuticular wax production in Arabidopsis. Plant Physiology 142: 866-877
Yang C.W., Gonzalez-Lamothe R., Ewan R.A., Rowland O., Yoshioka H., Shenton M., Ye H., O’Donnell E., Jones J.D.G., and Sadanandom A. (2006). The E3 ubiquitin ligase activity of Arabidosis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense. Plant Cell 18: 1084-1098
Zheng H., Rowland O., and Kunst L. (2005). Disruptions of the Arabidopsis enoyl-CoA reductase gene reveal an essential role for very-long-chain fatty acid synthesis in cell expansion during plant morphogenesis. Plant Cell 17: 1467-2481
Rowland O., Ludwig A.A., Merrick C.J., Baillieul F., Tracy F.E., Durrant W.E., Fritz-Laylin L., Nekrasov V., Sjolander K., Yoshioka H., and Jones J.D.G. (2005). Functional analysis of Avr9/Cf-9 rapidly elicited genes identifies a protein kinase, ACIK1, that is essential for full Cf-9-dependent disease resistance in tomato. Plant Cell 17: 295-310
Katou S., Yoshioka H., Kawakita K., Rowland O., Jones J.D.G., Mori H., and Doke N. (2005). Involvement of PPS3 phosphorylated by elicitor-responsive MAPKs in the regulation of plant cell death. Plant Physiology 139: 1914-1926
Moon H.*, Chowrira G.*, Rowland O.*, Blacklock B.J., Smith M.A., and Kunst L. (2004). A root-specific condensing enzyme from Lesquerella fendleri that elongates very-long-chain saturated fatty acids. Plant Molecular Biology 56: 917-927 *co-first authors
Navarro L., Zipfel C., Rowland O., Keller I., Robatzek S., Boller T., and Jones J.D.G. (2004). The transcriptional innate immune response to flg22: interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis. Plant Physiology 135: 1113-1128
Yoshioka H., Numata N., Nakajima K., Katou S., Kawakita K., Rowland O., Jones J.D.G., and Doke N. (2003). Nicotiana benthamiana gp91phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell 15: 706-718
Rowland O., and Jones J.D.G. (2001). Unraveling regulatory networks in plant defense using microarrays. Genome Biology 2:reviews1001.1-1001.3
Durrant W.E., Rowland O., Piedras P., Hammond-Kosack K.E., and Jones J.D.G. (2000). cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. Plant Cell 12: 963-977
Rowland O., and Segall J. (1998). A hydrophobic segment within the 81-amino-acid domain of TFIIIA from Saccharomyces cerevisiae is essential for its transcription factor activity. Molecular and Cellular Biology 18: 420-432
Rowland O., and Segall J. (1996). Interaction of wild-type and truncated forms of transcription factor IIIA from Saccharomyces cerevisiae with the 5S RNA gene. Journal of Biological Chemistry 271: 12103-12110
