Conserved signalling components coordinate epidermal patterning and cuticle deposition in barley.

Faced with terrestrial threats, land plants seal their aerial surfaces with a lipid-rich cuticle. To breathe, plants interrupt their cuticles with adjustable epidermal pores, called stomata, that regulate gas exchange, and develop other specialised epidermal cells such as defensive hairs. Mechanisms coordinating epidermal features remain poorly understood. Addressing this, we studied two loci whose allelic variation causes both cuticular wax-deficiency and misarranged stomata in barley, identifying the underlying genes, Cer-g/ HvYDA1, encoding a YODA-like (YDA) MAPKKK, and Cer-s/ HvBRX-Solo, encoding a single BREVIS-RADIX (BRX) domain protein. Both genes control cuticular integrity, the spacing and identity of epidermal cells, and barley's distinctive epicuticular wax blooms, as well as stomatal patterning in elevated CO2 conditions. Genetic analyses revealed epistatic and modifying relationships between HvYDA1 and HvBRX-Solo, intimating that their products participate in interacting pathway(s) linking epidermal patterning with cuticular properties in barley. This may represent a mechanism for coordinating multiple adaptive features of the land plant epidermis in a cultivated cereal.

Sunflower (Helianthus annuus) fatty acid synthase complex: ß-Ketoacyl-[acyl carrier protein] reductase genes.

Fatty acids play many roles in plants, but the function of some key genes involved in fatty acid biosynthesis in plant development are not yet properly understood. Here, we clone two ß-ketoacyl-[ACP] reductase (KAR) genes from sunflower, HaKAR1 and HaKAR2, and characterize their functional roles. The enzymes cloned were the only two copies present in the sunflower genome. Both displayed a high degree of similarity, but their promoters infer different regulation. The two sunflower KAR genes were constitutively expressed in all tissues examined, being maximum in developing cotyledons at the start of oil synthesis. Over-expression of HaKAR1 in E. coli changed the fatty acid composition by promoting the elongation of C16:0 to C18:0 fatty acids. The enzymatic characterization of HaKAR1 revealed similar kinetic parameters to homologues from other oil accumulating species. The results point to a partially functional redundancy between HaKAR1 and HaKAR2. This study clearly revealed that these genes play a prominent role in de novo fatty acids synthesis in sunflower seeds.

Ecophysiology with barley eceriferum (cer) mutants: the effects of humidity and wax crystal structure on yield and vegetative parameters.

BACKGROUND AND AIMS: In addition to preventing water loss, plant cuticles must also regulate nutrient loss via leaching. The eceriferum mutants in Hordeum vulgare (barley) potentially influence these functions by altering epicuticular wax structure and composition. METHODS: Cultivar 'Bonus' and five of its cer mutants were grown under optimal conditions for vegetative growth and maturation, and nine traits were measured. Nutrient and water amounts going through the soil and the amount of simulated rain as deionized water, affecting phyllosphere humidity, delivered during either the vegetative or maturation phase, were varied. Cer leaf genes and three wilty (wlt) mutations were characterized for reaction to toluidine blue and the rate of non-stomatal water loss. KEY RESULTS: Vegetative phase rain on 'Bonus' significantly decreased kernel weight and numbers by 15-30 %, while in cer.j59 and .c36 decreases of up to 42 % occurred. Maturation phase findings corroborated those from the vegetative phase. Significant pleiotropic effects were identified: cer.j59 decreased culm and spike length and 1000-kernel weight, .c36 decreased kernel number and weight, .i16 decreased spike length and .e8 increased culm height. Excepting Cer.zv and .ym mutations, none of the other 27 Cer leaf genes or wlt mutations played significant roles, if any, in preventing water loss. Cer.zv and .ym mutants lost non-stomatal water 13.5 times faster than those of Cer.j, .yi, .ys and .zp and 18.3 times faster than those of four cultivars and the mutants tested here. CONCLUSIONS: Using yield to measure the net effect of phyllosphere humidity and wax crystal structure revealed that the former is far more important than the latter. The amenable experimental setup described here can be used to delve deeper. Significant pleiotropic effects were identified for mutations in four Cer genes, of which one is known to participate in wax biosynthesis. Twenty-seven Cer leaf genes and three wlt mutations have little if any effect on water loss.

The ß-Ketoacyl-CoA Synthase HvKCS1, Encoded by Cer-zh, Plays a Key Role in Synthesis of Barley Leaf Wax and Germination of Barley Powdery Mildew.

The cuticle coats the primary aerial surfaces of land plants. It consists of cutin and waxes, which provide protection against desiccation, pathogens and herbivores. Acyl cuticular waxes are synthesized via elongase complexes that extend fatty acyl precursors up to 38 carbons for downstream modification pathways. The leaves of 21 barley eceriferum (cer) mutants appear to have less or no epicuticular wax crystals, making these mutants excellent tools for identifying elongase and modification pathway biosynthetic genes. Positional cloning of the gene mutated in cer-zh identified an elongase component, ß-ketoacyl-CoA synthase (CER-ZH/HvKCS1) that is one of 34 homologous KCSs encoded by the barley genome. The biochemical function of CER-ZH was deduced from wax and cutin analyses and by heterologous expression in yeast. Combined, these experiments revealed that CER-ZH/HvKCS1 has a substrate specificity for C16-C20, especially unsaturated, acyl chains, thus playing a major role in total acyl chain elongation for wax biosynthesis. The contribution of CER-ZH to water barrier properties of the cuticle and its influence on the germination of barley powdery mildew fungus were also assessed.

The Polyketide Components of Waxes and the Cer-cqu Gene Cluster Encoding a Novel Polyketide Synthase, the ß-Diketone Synthase, DKS.

The primary function of the outermost, lipophilic layer of plant aerial surfaces, called the cuticle, is preventing non-stomatal water loss. Its exterior surface is often decorated with wax crystals, imparting a blue-grey color. Identification of the barley Cer-c, -q and -u genes forming the 101 kb Cer-cqu gene cluster encoding a novel polyketide synthase-the ß-diketone synthase (DKS), a lipase/carboxyl transferase, and a P450 hydroxylase, respectively, establishes a new, major pathway for the synthesis of plant waxes. The major product is a ß-diketone (14,16-hentriacontane) aliphatic that forms long, thin crystalline tubes. A pathway branch leads to the formation of esterified alkan-2-ols.

The Cer-cqu gene cluster determines three key players in a ß-diketone synthase polyketide pathway synthesizing aliphatics in epicuticular waxes.

Aliphatic compounds on plant surfaces, called epicuticular waxes, are the first line of defense against pathogens and pests, contribute to reducing water loss and determine other important phenotypes. Aliphatics can form crystals affecting light refraction, resulting in a color change and allowing identification of mutants in their synthesis or transport. The present study discloses three such Eceriferum (cer) genes in barley - Cer-c, Cer-q and Cer-u - known to be tightly linked and functioning in a biochemical pathway forming dominating amounts of ß-diketone and hydroxy-ß-diketones plus some esterified alkan-2-ols. These aliphatics are present in many Triticeae as well as dicotyledons such as Eucalyptus and Dianthus. Recently developed genomic resources and mapping populations in barley defined these genes to a small region on chromosome arm 2HS. Exploiting Cer-c and -u potential functions pinpointed five candidates, of which three were missing in apparent cer-cqu triple mutants. Sequencing more than 50 independent mutants for each gene confirmed their identification. Cer-c is a chalcone synthase-like polyketide synthase, designated diketone synthase (DKS), Cer-q is a lipase/carboxyl transferase and Cer-u is a P450 enzyme. All were highly expressed in pertinent leaf sheath tissue of wild type. A physical map revealed the order Cer-c, Cer-u, Cer-q with the flanking genes 101kb apart, confirming they are a gene cluster, Cer-cqu. Homology-based modeling suggests that many of the mutant alleles affect overall protein structure or specific active site residues. The rich diversity of identified mutations will facilitate future studies of three key enzymes involved in synthesis of plant apoplast waxes.

Sunflower (Helianthus annuus) fatty acid synthase complex: ß-hydroxyacyl-[acyl carrier protein] dehydratase genes.

MAIN CONCLUSION: Two sunflower hydroxyacyl-[acyl carrier protein] dehydratases evolved into two different isoenzymes showing distinctive expression levels and kinetics' efficiencies. ß-Hydroxyacyl-[acyl carrier protein (ACP)]-dehydratase (HAD) is a component of the type II fatty acid synthase complex involved in 'de novo' fatty acid biosynthesis in plants. This complex, formed by four intraplastidial proteins, is responsible for the sequential condensation of two-carbon units, leading to 16- and 18-C acyl-ACP. HAD dehydrates 3-hydroxyacyl-ACP generating trans-2-enoyl-ACP. With the aim of a further understanding of fatty acid biosynthesis in sunflower (Helianthus annuus) seeds, two ß-hydroxyacyl-[ACP] dehydratase genes have been cloned from developing seeds, HaHAD1 (GenBank HM044767) and HaHAD2 (GenBank GU595454). Genomic DNA gel blot analyses suggest that both are single copy genes. Differences in their expression patterns across plant tissues were detected. Higher levels of HaHAD2 in the initial stages of seed development inferred its key role in seed storage fatty acid synthesis. That HaHAD1 expression levels remained constant across most tissues suggest a housekeeping function. Heterologous expression of these genes in E. coli confirmed both proteins were functional and able to interact with the bacterial complex 'in vivo'. The large increase of saturated fatty acids in cells expressing HaHAD1 and HaHAD2 supports the idea that these HAD genes are closely related to the E. coli FabZ gene. The proposed three-dimensional models of HaHAD1 and HaHAD2 revealed differences at the entrance to the catalytic tunnel attributable to Phe166/Val1159, respectively. HaHAD1 F166V was generated to study the function of this residue. The 'in vitro' enzymatic characterization of the three HAD proteins demonstrated all were active, with the mutant having intermediate K m and V max values to the wild-type proteins.

Sunflower (Helianthus annuus) fatty acid synthase complex: enoyl-[acyl carrier protein]-reductase genes.

MAIN CONCLUSION: Enoyl-[acyl carrier protein]-reductases from sunflower. A major factor contributing to the amount of fatty acids in plant oils are the first steps of their synthesis. The intraplastidic fatty acid biosynthetic pathway in plants is catalysed by type II fatty acid synthase (FAS). The last step in each elongation cycle is carried out by the enoyl-[ACP]-reductase, which reduces the dehydrated product of ß-hydroxyacyl-[ACP] dehydrase using NADPH or NADH. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus) seeds, two enoyl-[ACP]-reductase genes have been identified and cloned from developing seeds with 75 % identity: HaENR1 (GenBank HM021137) and HaENR2 (HM021138). The two genes belong to the ENRA and ENRB families in dicotyledons, respectively. The genetic duplication most likely originated after the separation of di- and monocotyledons. RT-qPCR revealed distinct tissue-specific expression patterns. Highest expression of HaENR1 was in roots, stems and developing cotyledons whereas that of H a ENR2 was in leaves and early stages of seed development. Genomic DNA gel blot analyses suggest that both are single-copy genes. In vivo activity of the ENR enzymes was tested by complementation experiments with the JP1111 fabI(ts) E. coli strain. Both enzymes were functional demonstrating that they interacted with the bacterial FAS components. That different fatty acid profiles resulted infers that the two Helianthus proteins have different structures, substrate specificities and/or reaction rates. The latter possibility was confirmed by in vitro analysis with affinity-purified heterologous-expressed enzymes that reduced the crotonyl-CoA substrate using NADH with different V max.

The inhibitor of wax 1 locus (Iw1) prevents formation of ß- and OH-ß-diketones in wheat cuticular waxes and maps to a sub-cM interval on chromosome arm 2BS.

Glaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non-glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F2 gametes, Iw1 was fine-mapped to a sub-cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, ß-diketones and n-alkanes. Small amounts of C19-C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC2 F3 near-isogenic lines, we show that Iw1 inhibits the formation of ß- and hydroxy-ß-diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n-alkanes and C24 primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near-isogenic lines differing at Iw1.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase.

Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The beta-ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the alpha-methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C(2) fatty acid elongation reaction using either a Cys-His-His or Cys-His-Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C(16) and C(18) products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X-ray crystal structures of the Cys-His-His-containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C(6) and C(10)-C(12)) substrate preferences leading to the C(8) lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl-binding-pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active-site histidines upon primer binding.

Analyses of barley spike mutant waxes identify alkenes, cyclopropanes and internally branched alkanes with dominating isomers at carbon 9.

About 15% of the epidermal wax on Hordeum vulgare cv. Bonus barley spikes is n-alkanes. Longer homologues are greatly reduced in the eceriferum mutants, cer-a(6), cer-e(8), cer-n(26), cer-n(53), cer-n(985), cer-x(60), cer-yc(135) and cer-yl(187). Simultaneously hydrocarbons accounting for only traces in the wild-type become prominent in the mutants, although their chain-length distributions remain unchanged. Accordingly several new hydrocarbon series were identified. The two major ones were C(23)-C(35)cis monoenoic alkenes (the major 9-ene isomer was part of a homologous series including 11, 13 and 15-enes), and the novel C(27)-C(31) cyclopropanes (the ring carbons of major isomers were 9,10 and 11,12 with lesser amounts of 13,14). Three minor series included 2- and 3-methylalkanes plus C(25)-C(33) internally branched alkanes (methyls on carbons 9, 11, 13, 15 or 17; shorter homologues dominated by the 9 isomer, longer homologues by 11, 13 or 15 isomers). Acyl chains destined for spike waxes are synthesized via acyl and polyketide elongase systems plus associated reductive and decarbonylative/decarboxylative enzyme systems. Both elongation systems are defective in synthesizing C(32) acyl chains in all nine mutants. The similarities in the position of the chemical groups (primarily on carbon 9, secondarily on carbon 11) of the alkenes, cyclopropanes and internally branched methyl alkanes imply an origin from a common, hitherto unrecognized associated pathway in barley, designated the enoic pathway. The elongation system leading to the enoic derived hydrocarbons differs from the known elongation systems by inclusion of a mechanism for introducing a double bond.

Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases.

Beta-ketoacyl-acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl-ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of beta-ketoacyl-ACP synthases, including mitochondrial beta-ketoacyl-ACP synthase, bacterial plus plastid beta-ketoacyl-ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys-His-His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia colibeta-ketoacyl-ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild-type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6-8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at Nepsilon, directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.

Structure of the mitochondrial beta-ketoacyl-[acyl carrier protein] synthase from Arabidopsis and its role in fatty acid synthesis.

Mitochondrial fatty acid synthesis is catalyzed by a dissociated fatty acid synthase similar to those of plant plastids and bacteria. The crystal structure of a mitochondrial beta-ketoacyl-[acyl carrier protein] synthase (mtKAS), namely that from Arabidopsis thaliana, has been determined for the first time. This enzyme accomplishes the vital condensation steps in constructing fatty acid carbon skeletons. The product profile of mtKAS is unusual in that C8 and C(14-16) fatty acyl chains predominate. An enzyme architecture that likely is the basis for the observed bimodal profile of mtKAS products can be derived from the shape of the acyl binding pocket.

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase.

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.

A KAS2 cDNA complements the phenotypes of the Arabidopsis fab1 mutant that differs in a single residue bordering the substrate binding pocket.

The fab1 mutant of Arabidopsis is partially deficient in activity of beta-ketoacyl-[acyl carrier protein] synthase II (KAS II). This defect results in increased levels of 16:0 fatty acid and is associated with damage and death of the mutants at low temperature. Transformation of fab1 plants with a cDNA from Brassica napus encoding a KAS II enzyme resulted in complementation of both mutant phenotypes. The dual complementation by expression of the single gene proves that low-temperature damage is a consequence of altered membrane unsaturation. The fab1 mutation is a single nucleotide change in Arabidopsis KAS2 that results in a Leu337Phe substitution. The Leu337 residue is conserved among plant and bacterial KAS proteins, and in the crystal structures of E. coli KAS I and KAS II, this leucine abuts a phenylalanine whose imidazole ring extends into the substrate binding cavity causing the fatty acid chain to bend. For functional analysis the equivalent Leu207Phe mutation was introduced into the fabB gene encoding the E. coli KAS I enzyme. Compared to wild-type, the Leu207Phe protein showed a 10-fold decrease in binding affinity for the fatty acid substrate, exhibited a modified behavior during size-exclusion chromatography and was severely impaired in condensation activity. These results suggest that the molecular defect in fab1 plants is a structural instability of the KAS2 gene product induced by insufficient space for the imidazole ring of the mutant phenylalanine residue.