Current status of community resources and priorities for weed genomics research.

Weeds are attractive models for basic and applied research due to their impacts on agricultural systems and capacity to swiftly adapt in response to anthropogenic selection pressures. Currently, a lack of genomic information precludes research to elucidate the genetic basis of rapid adaptation for important traits like herbicide resistance and stress tolerance and the effect of evolutionary mechanisms on wild populations. The International Weed Genomics Consortium is a collaborative group of scientists focused on developing genomic resources to impact research into sustainable, effective weed control methods and to provide insights about stress tolerance and adaptation to assist crop breeding.

Standing genetic variation fuels rapid evolution of herbicide resistance in blackgrass.

Repeated herbicide applications in agricultural fields exert strong selection on weeds such as blackgrass (Alopecurus myosuroides), which is a major threat for temperate climate cereal crops. This inadvertent selection pressure provides an opportunity for investigating the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much target-site resistance (TSR) relies on de novo mutations vs. standing variation is important for developing strategies to manage herbicide resistance. We first generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Next, through empirical data in the form of highly accurate long-read amplicons of alleles encoding acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) variants, we showed that most populations with resistance due to TSR mutations-23 out of 27 and six out of nine populations for ACCase and ALS, respectively-contained at least two TSR haplotypes, indicating that soft sweeps are the norm. Finally, through forward-in-time simulations, we inferred that TSR is likely to mainly result from standing genetic variation, with only a minor role for de novo mutations.

Inhibition profile of trifludimoxazin towards PPO2 target site mutations.

BACKGROUND: Target site resistance to herbicides that inhibit protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) has been described mainly in broadleaf weeds based on mutations in the gene designated protoporphyrinogen oxidase 2 (PPO2) and in one monocot weed species in protoporphyrinogen oxidase 1 (PPO1). To control PPO target site resistant weeds in future it is important to design new PPO-inhibiting herbicides that can control problematic weeds expressing mutant PPO enzymes. In this study, we assessed the efficacy of a new triazinone-type inhibitor, trifludimoxazin, to inhibit PPO2 enzymes carrying target site mutations in comparison with three widely used PPO-inhibiting herbicides. RESULTS: Mutated Amaranthus spp. PPO2 enzymes were expressed in Escherichia coli, purified and measured biochemically for activity and inhibition kinetics, and used for complementation experiments in an E. coli hemG mutant that lacks the corresponding microbial PPO gene function. In addition, we used ectopic expression in Arabidopsis and structural PPO protein modeling to support the enzyme inhibition study. The generated data strongly suggest that trifludimoxazin is a strong inhibitor both at the enzyme level and in transgenics Arabidopsis ectopically expressing PPO2 target site mutations. CONCLUSION: Trifludimoxazin is a potent PPO-inhibiting herbicide that inhibits various PPO2 enzymes carrying target site mutations and could be used as a chemical-based control strategy to mitigate the widespread occurrence of PPO target site resistance as well as weeds that have evolved resistance to other herbicide mode of actions. © 2022 BASF SE and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Survey of ACCase and ALS resistance in winter annual grasses identifies target-site and nontarget-site imazamox resistance in Secale cereale.

BACKGROUND: Early detection of herbicide resistance in weeds is crucial for successful implementation of integrated weed management. We conducted a herbicide resistance survey of the winter annual grasses feral rye (Secale cereale), downy brome (Bromus tectorum), and jointed goatgrass (Aegilops cylindrica) from Colorado winter wheat production areas for resistance to imazamox and quizalofop. RESULTS: All samples were susceptible to quizalofop. All downy brome and jointed goatgrass samples were susceptible to imazamox. Out of 314 field collected samples, we identified three feral rye populations (named A, B, and C) that were imazamox resistant. Populations B and C had a target-site mechanism with mutations in the Ser653 residue of the acetolactate synthase (ALS) gene to Asn in B and to Thr in C. Both populations B and C had greatly reduced ALS in vitro enzyme inhibition by imazamox. ALS feral rye protein modeling showed that steric interactions induced by the amino acid substitutions at Ser653 impaired imazamox binding. Individuals from population A had no mutations in the ALS gene. The ALS enzyme from population A was equally sensitive to imazamox as to known susceptible feral rye populations. Imazamox was degraded two times faster in population A compared with a susceptible control. An oxidized imazamox metabolite formed faster in population A and this detoxification reaction was inhibited by malathion. CONCLUSION: Population A has a nontarget-site mechanism of enhanced imazamox metabolism that may be conferred by cytochrome P450 enzymes. This is the first report of both target-site and metabolism-based imazamox resistance in feral rye. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Involvement of glutamine synthetase 2 (GS2) amplification and overexpression in Amaranthus palmeri resistance to glufosinate.

MAIN CONCLUSION: Amplification and overexpression of the target site glutamine synthetase, specifically the plastid-located isoform, confers resistance to glufosinate in Amaranthus palmeri. This mechanism is novel among glufosinate-resistant weeds. Amaranthus palmeri has recently evolved resistance to glufosinate herbicide. Several A. palmeri populations from Missouri and Mississippi, U.S.A. had survivors when sprayed with glufosinate-ammonium (GFA, 657 g ha-1). One population, MO#2 (fourfold resistant) and its progeny (sixfold resistant), were used to study the resistance mechanism, focusing on the herbicide target glutamine synthetase (GS). We identified four GS genes in A. palmeri; three were transcribed: one coding for the plastidic protein (GS2) and two coding for cytoplasmic isoforms (GS1.1 and GS1.2). These isoforms did not contain mutations associated with resistance. The 17 glufosinate survivors studied showed up to 21-fold increase in GS2 copies. GS2 was expressed up to 190-fold among glufosinate survivors. GS1.1 was overexpressed > twofold in only 3 of 17, and GS1.2 in 2 of 17 survivors. GS inhibition by GFA causes ammonia accumulation in susceptible plants. Ammonia level was analyzed in 12 F1 plants. GS2 expression was negatively correlated with ammonia level (r = - 0.712); therefore, plants with higher GS2 expression are less sensitive to GFA. The operating efficiency of photosystem II (ϕPSII) of Nicotiana benthamiana overexpressing GS2 was four times less inhibited by GFA compared to control plants. Therefore, increased copy and overexpression of GS2 confer resistance to GFA in A. palmeri (or other plants). We present novel understanding of the role of GS2 in resistance evolution to glufosinate.

Field-Evolved ΔG210-ppo2 from Palmer Amaranth Confers Pre-emergence Tolerance to PPO-Inhibitors in Rice and Arabidopsis.

Resistance to protoporphyrinogen IX oxidase (PPO)-inhibitors in Amaranthus palmeri and Amaranthus tuberculatus is mainly contributed by mutations in the PPO enzyme, which renders herbicide molecules ineffective. The deletion of glycine210 (ΔG210) is the most predominant PPO mutation. ΔG210-ppo2 is overexpressed in rice (Oryza sativa c. 'Nipponbare') and Arabidopsis thaliana (Col-0). A foliar assay was conducted on transgenic T1 rice plants with 2× dose of fomesafen (780 g ha−1), showing less injury than the non-transgenic (WT) plants. A soil-based assay conducted with T2 rice seeds confirmed tolerance to fomesafen applied pre-emergence. In agar medium, root growth of WT rice seedlings was inhibited >90% at 5 µM fomesafen, while root growth of T2 seedlings was inhibited by 50% at 45 µM fomesafen. The presence and expression of the transgene were confirmed in the T2 rice survivors of soil-applied fomesafen. A soil-based assay was also conducted with transgenic A. thaliana expressing ΔG210-ppo2 which confirmed tolerance to the pre-emergence application of fomesafen and saflufenacil. The expression of A. palmeri ΔG210-ppo2 successfully conferred tolerance to soil-applied fomesafen in rice and Arabidopsis. This mutant also confers cross-tolerance to saflufenacil in Arabidopsis. This trait could be introduced into high-value crops that lack chemical options for weed management.

Can double PPO mutations exist in the same allele and are such mutants functional?

BACKGROUND: Resistance to protoporphyrinogen oxidase (PPO)-inhibiting herbicides is endowed primarily by target-site mutations at the PPX2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus spp. PPX2, the most prevalent target mutations are deletion of the G210 codon, and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket, resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same PPX2 allele. RESULTS: Plants carrying R128G+/+ ΔG210+/-, where + indicates presence of the mutation, were crossed with each other. The PPX2 of the offspring was subjected to pyrosequencing and E. coli-based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G ΔG210 can occur in one allele only; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying ΔG210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions. CONCLUSIONS: Homozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Functional PPO2 mutations: co-occurrence in one plant or the same ppo2 allele of herbicide-resistant Amaranthus palmeri in the US mid-south.

BACKGROUND: Protoporphyrinogen IX oxidase 2 (PPO2) inhibitors are important for the management of glyphosate- and acetolactate synthase-resistant Palmer amaranth [Amaranthus palmeri (S.) Wats.]. The evolving resistance to PPO inhibitors is of great concern. We surveyed the evolution of resistance to fomesafen in the US Mid-south and determined its correlation with the known functional PPO2 target-site mutations (TSM). RESULTS: The 167 accessions analyzed were grouped into five categories, four resistant (147) and one susceptible (20). Arkansas accessions constituted 100% of the susceptible group while the Missouri accessions comprised 60% of the most resistant category. The majority of Mississippi accessions (88%) clustered in the high-survival-high-injury category, manifesting an early-stage resistance evolution. One hundred and fifteen accessions were genotyped for four known TSMs; 74% of accessions carried at least one TSM. The most common single TSM was ΔG210 (18% of accessions) and the predominant double mutation was ΔG210 + G399A (17%). Other mutations are likely less favorable, hence are rare. All TSMs were detected in three accessions. Further examination revealed that 9 and two individuals carried G399A + G210 and G399A + R128G TSM in the same allele, respectively. The existence of these combinations is supported by molecular modeling. CONCLUSIONS: Resistance to PPO inhibitors is widespread across the Mid-southern USA. Highly resistant field populations have plants with multiple mutations. G399A is the most prone to co-occur with other ppo2 mutations in the same allele. Mutation at R128 in the configuration of the PPO2 catalytic domain restrains the co-occurrence of R128G with ΔG210, making ΔG210 + G399A the most plausible, tolerable functional mutation combination to co-occur in the same ppo2 allele.

Prohexadione calcium is herbicidal to the sunflower root parasite Orobanche cumana.

BACKGROUND: The obligatory sunflower root parasite Orobanche cumana Wallr. deprives its host of essential nutrients, resulting in a dramatic reduction in yield and biomass. A post-emergence application with an imidazolinone herbicide on an imidazolinone-tolerant sunflower is highly effective against O. cumana. The herbicide inhibits the enzyme acetohydroxy acid synthase and consequently, growth of the parasite is inhibited, although the sunflower survives the treatment through mutations in the target enzyme. Interestingly, field studies have shown that a combined application of an imidazolinone herbicide with prohexadione resulted in reduced emergence of O. cumana compared with the sole application of the herbicide. The aim of this study was to investigate whether prohexadione is herbicidal to O. cumana. RESULTS: Prohexadione was rapidly distributed within the sunflower, reaching the roots, the site of O. cumana attack, as early as 6 h after application (HAA) on sunflower leaves. A direct impact of prohexadione on O. cumana germination was investigated and a half-maximal inhibitory concentration (IC50 ) of 84 µm prohexadione was found. In addition, the inhibition of germination by prohexadione was terminal, meaning that O. cumana seeds died after prohexadione contact as soon as they were primed for germination. Additionally, excretion studies showed that a small proportion of the applied prohexadione was excreted by sunflower roots. CONCLUSION: We show that prohexadione is an inhibitor of O. cumana germination and that the growth regulator is found in sunflower roots shortly after application. We hypothesize that prohexadione is excreted in sufficient amounts from the sunflower roots, therefore having a direct impact on O. cumana germination. © 2020 Society of Chemical Industry.

Draft Genomes of Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri.

Amaranthus tuberculatus, Amaranthus hybridus, and Amaranthus palmeri are agronomically important weed species. Here, we present the most contiguous draft assemblies of these three species to date. We utilized a combination of Pacific Biosciences long-read sequencing and chromatin contact mapping information to assemble and order sequences of A. palmeri to near-chromosome-level resolution, with scaffold N50 of 20.1 Mb. To resolve the issues of heterozygosity and coassembly of alleles in diploid species, we adapted the trio binning approach to produce haplotype assemblies of A. tuberculatus and A. hybridus. This approach resulted in an improved assembly of A. tuberculatus, and the first genome assembly for A. hybridus, with contig N50s of 2.58 and 2.26 Mb, respectively. Species-specific transcriptomes and information from related species were used to predict transcripts within each assembly. Syntenic comparisons of these species and Amaranthus hypochondriacus identified sites of genomic rearrangement, including duplication and translocation, whereas genetic map construction within A. tuberculatus highlighted the need for further ordering of the A. hybridus and A. tuberculatus contigs. These multiple reference genomes will accelerate genomic studies in these species to further our understanding of weedy evolution within Amaranthus.

Cinmethylin controls multiple herbicide-resistant Lolium rigidum and its wheat selectivity is P450-based.

BACKGROUND: Multiple-herbicide resistance in Lolium rigidum and other weed species is increasingly exerting pressure on herbicide discovery research for solutions against resistance-prone weeds. In this study we investigate: (i) the responses of L. rigidum populations and wheat to the new herbicide cinmethylin in comparison with other pre-emergence herbicides, (ii) the effect of seed burial depths on cinmethylin efficacy and crop selectivity, and (iii) the basis of cinmethylin selectivity in wheat. RESULTS: Cinmethylin at 400 g ha-1 controls herbicide-susceptible and multiple-resistant L. rigidum, with a reduction of >85% in plant emergence and 90% in aboveground biomass. Cinmethylin provides effective control of a large number of field populations of L. rigidum with evident resistance to trifluralin. When the wheat seed is buried ≥1 cm below the cinmethylin-treated soil surface, the emergence of crop seedlings is not different from the untreated control. The organophosphate insecticide phorate synergizes cinmethylin toxicity in wheat, with an LD50 of 682 g ha-1 in the absence of phorate versus 109 g ha-1 in the presence of phorate (84% reduction). The synergistic effect of phorate with cinmethylin on herbicide-susceptible L. rigidum appears smaller (a 44% reduction in the LD50 of cinmethylin). CONCLUSIONS: Cinmethylin is effective in controlling multiple-resistant L. rigidum and appears safe for wheat when the seed is separated at depth from the herbicide applied to the soil surface. The basis of this metabolism-based selectivity is likely regulated by cytochrome P450 monooxygenases. © 2020 Society of Chemical Industry.

A novel mutation A212T in chloroplast Protoporphyrinogen oxidase (PPO1) confers resistance to PPO inhibitor Oxadiazon in Eleusine indica.

BACKGROUND: Protoporphyrinogen oxidase (PPO) with two isoforms, chloroplast-targeted (PPO1) and mitochondrial-targeted (PPO2), catalyzes a step in the biosynthesis of chlorophyll and heme. PPO1 and PPO2 are herbicide target sites of PPO-inhibiting herbicides. Target-site mutations conferring resistance to PPO inhibitors have all thus far been in PPO2. Oxadiazon is a unique PPO inhibitor utilized for preemergence Eleusine indica control. In this research, we evaluated the response of two previously confirmed oxadiazon-resistant and susceptible E. indica biotypes to other PPO inhibitors and identified the resistance mechanism in two oxadiazon-resistant E. indica biotypes. RESULTS: Two E. indica biotypes were resistant to oxadiazon, but not to other structurally unrelated PPO inhibitors, such as lactofen, flumioxazin and sulfentrazone. A novel mutation A212T was identified in the chloroplast-targeted PPO1, conferring resistance to oxadiazon in a heterologous expression system. Computational structural modeling provided a mechanistic explanation for reduced herbicide binding to the variant protein: the presence of a methyl group of threonine 212 changes the PPO1 active site and produces repulsive electrostatic interactions that repel oxadiazon from the binding pocket. CONCLUSION: The novel A212T mutation in PPO1 conferring resistance specifically to PPO inhibitor oxadiazon was characterized. This is the first evidence of the direct role of PPO1 in the PPO mode of action, and the first evidence of evolved resistance in PPO1. © 2019 Society of Chemical Industry.

A new herbicidal site of action: Cinmethylin binds to acyl-ACP thioesterase and inhibits plant fatty acid biosynthesis.

The prevalent occurrence of herbicide resistant weeds increases the necessity for new site of action herbicides for effective control as well as to relax selection pressure on the known sites of action. As a consequence, interest increased in the unexploited molecule cinmethylin as a new solution for the control of weedy grasses in cereals. Therefore, the mechanism of action of cinmethylin was reevaluated. We applied the chemoproteomic approach cellular Target Profiling™ from Evotec to identify the cinmethylin target in Lemna paucicostata protein extracts. We found three potential targets belonging to the same protein family of fatty acid thioesterases (FAT) to bind to cinmethylin with high affinity. Binding of cinmethylin to FAT proteins from Lemna and Arabidopsis was confirmed by fluorescence-based thermal shift assay. The plastid localized enzyme FAT plays a crucial role in plant lipid biosynthesis, by mediating the release of fatty acids (FA) from its acyl carrier protein (ACP) which is necessary for FA export to the endoplasmic reticulum. GC-MS analysis of free FA composition in Lemna extracts revealed strong reduction of unsaturated C18 as well as saturated C14, and C16 FAs upon treatment with cinmethylin, indicating that FA release for subsequent lipid biosynthesis is the primary target of cinmethylin. Lipid biosynthesis is a prominent target of different herbicide classes. To assess whether FAT inhibition constitutes a new mechanism of action within this complex pathway, we compared physiological effects of cinmethylin to different ACCase and VLCFA synthesis inhibitors and identified characteristic differences in plant symptomology and free FA composition upon treatment with the three herbicide classes. Also, principal component analysis of total metabolic profiling of treated Lemna plants showed strong differences in overall metabolic changes after cinmethylin, ACCase or VLCFA inhibitor treatments. Our results identified and confirmed FAT as the cinmethylin target and validate FAT inhibition as a new site of action different from other lipid biosynthesis inhibitor classes.

A multifunctional lipoxygenase with fatty acid hydroperoxide cleaving activity from the moss Physcomitrella patens.

A complex mixture of fatty acid-derived aldehydes, ketones, and alcohols is released upon wounding of the moss Physcomitrella patens. To investigate the formation of these oxylipins at the molecular level we isolated a lipoxygenase from P. patens, which was identified in an EST library by sequence homology to lipoxygenases from plants. Sequence analysis of the cDNA showed that it exhibits a domain structure similar to that of type2 lipoxygenases from plants, harboring an N-terminal import signal for chloroplasts. The recombinant protein was identified as arachidonate 12-lipoxygenase and linoleate 13-lipoxygenase with a preference for arachidonic acid and eicosapentaenoic acid. In contrast to any other lipoxygenase cloned so far, this enzyme exhibited in addition an unusual high hydroperoxidase and also a fatty acid chain-cleaving lyase activity. Because of these unique features the pronounced formation of (2Z)-octen-1-ol, 1-octen-3-ol, the dienal (5Z,8Z,10E)-12-oxo-dodecatrienoic acid and 12-keto eicosatetraenoic acid was observed when arachidonic acid was administered as substrate. 12-Hydroperoxy eicosatetraenoic acid was found to be only a minor product. Moreover, the P. patens LOX has a relaxed substrate tolerance accepting C(18)-C(22) fatty acids giving rise to even more LOX-derived products. In contrast to other lipoxygenases a highly diverse product spectrum is formed by a single enzyme accounting for most of the observed oxylipins produced by the moss. This single enzyme might, in a fast and effective way, be involved in the formation of signal and/or defense molecules thus contributing to the broad resistance of mosses against pathogens.

Molecular analysis of "de novo" purine biosynthesis in solanaceous species and in Arabidopsis thaliana.

Purine nucleotides are essential components to sustain plant growth and development. In plants they are either synthesized "de novo" during the process of purine biosynthesis or are recycled from purine bases and purine nucleosides throughout the salvage pathway. Comparison between animals, microorganisms and Arabidopsis, the first plant species with a completely sequenced genome, shows that plants principally use the same biochemical steps to synthesize purine nucleotides and possess all the essential genes and enzymes. Here we report on the cloning and molecular analysis of the complete purine biosynthesis pathway in plants, and the in planta functional analysis of PRPP (5-phosphoribosyl-1-pyrophoshate) amidotransferase (ATase), catalyzing the first committed step of the "de novo" purine biosynthesis. The cloning of the genes involved in the purine biosynthesis pathway was attained by a screening strategy with heterologous cDNA probes and by using S. cerevisiae mutants for complementation. Southern hybridization showed a complex genomic organization for these genes in solanaceous species and their organ- and developmental specific expression was analyzed by Northern hybridization. The specific role of ATase for plant growth and development was analyzed in transgenic tobacco plants exhibiting a reduced ATase activity and in an Arabidopsis T-DNA mutant (atd2) deficient for ATase2. The transgenic tobacco plants as well as the Arabidopsis mutant exhibit a specific and comparable phenotype, which is characterized by strong growth retardation and severe chlorosis in leaves. The formation of white leaves, but green cotyledons is a characteristic trait of the Arabidopsis atd2 mutant.

New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases.

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.

Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum).

To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv. Desirée) plants under control of the rolC promoter. Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections. Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants. However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud. Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls. During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged. A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly. Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration. Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells. To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase. Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells. Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose. In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls. Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers. In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed. Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.

Cloning and functional characterisation of an enzyme involved in the elongation of Delta6-polyunsaturated fatty acids from the moss Physcomitrella patens.

The moss Physcomitrella patens contains high proportions of polyunsaturated very-long-chain fatty acids with up to 20 carbon atoms. Starting from preformed C18 polyunsaturated fatty acids, their biosynthesis involves a sequence of Delta6-desaturation, Delta6-elongation and Delta5-desaturation. In this report we describe for the first time the characterisation of a cDNA (PSE1) of plant origin with homology to the ELO-genes from Saccharomyces cerevisiae, encoding a component of the Delta6-elongase. Functional expression of PSE1 in S. cerevisiae led to the elongation of exogenously supplied Delta6-polyunsaturated fatty acids. By feeding experiments with different trienoic fatty acids of natural and synthetic origin, both substrate specificity and substrate selectivity of the enzyme were investigated. The activity of Pse1, when expressed in yeast, was not sensitive to the antibiotic cerulenin, which is an effective inhibitor of fatty acid synthesis and elongation. Furthermore, the PSE1 gene was disrupted in the moss by homologous recombination. This led to a complete loss of all C20 polyunsaturated fatty acids providing additional evidence for the function of the cDNA as coding for a component of the Delta6-elongase. The elimination of the elongase was not accompanied by a visible alteration in the phenotype, indicating that C20-PUFAs are not essential for viability of the moss under phytotron conditions.

Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.

Phaeodactylum tricornutum is an unicellular silica-less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N-terminus and the three histidine clusters common to all front-end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Delta5- and Delta6-fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Delta6-specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the omega3- and omega6-pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the omega3- and omega6-fatty acids with similar efficiencies, indicating that none of them was specific for either the omega3- or the omega6-pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.