Eve Shaw

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

ABSTRACT Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kbEcoRI fragment of plasmid pHRIM620 was expressed inEscherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. colicoded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhAgene).

Seed Storage Oil Mobilization Is Important But Not Essential for Germination or Seedling Establishment in Arabidopsis

Triacylglycerol (TAG) is a major storage reserve in many plant seeds. We previously identified a TAG lipase mutant called sugar-dependent1 (sdp1) that is impaired in TAG hydrolysis following Arabidopsis (Arabidopsis thaliana) seed germination (Eastmond, 2006). The aim of this study was to identify additional lipases that account for the residual TAG hydrolysis observed in sdp1. Mutants were isolated in three candidate genes (SDP1-LIKE [SDP1L], ADIPOSE TRIGLYCERIDE LIPASE-LIKE, and COMPARATIVE GENE IDENTIFIER-58-LIKE). Analysis of double, triple, and quadruple mutants showed that SDP1L is responsible for virtually all of the residual TAG hydrolysis present in sdp1 seedlings. Oil body membranes purified from sdp1 sdp1L seedlings were deficient in TAG lipase activity but could still hydrolyze di- and monoacylglycerol. SDP1L is expressed less strongly than SDP1 in seedlings. However, SDP1L could partially rescue TAG breakdown in sdp1 seedlings when expressed under the control of the SDP1 or 35S promoters and in vitro assays showed that both SDP1 and SDP1L can hydrolyze TAG, in preference to diacylglycerol or monoacylglycerol. Seed germination was slowed in sdp1 sdp1L and postgerminative seedling growth was severely retarded. The frequency of seedling establishment was also reduced, but sdp1 sdp1L was not seedling lethal under normal laboratory growth conditions. Our data show that together SDP1 and SDP1L account for at least 95% of the rate of TAG hydrolysis in Arabidopsis seeds, and that this hydrolysis is important but not essential for seed germination or seedling establishment.

The SUGAR-DEPENDENT1 Lipase Limits Triacylglycerol Accumulation in Vegetative Tissues of Arabidopsis

A triacylglycerol lipase knockout boosts the oil content of wild-type plants and transgenic plants genetically engineered to make more oil. There has been considerable interest recently in the prospect of engineering crops to produce triacylglycerol (TAG) in their vegetative tissues as a means to achieve a step change in oil yield. Here, we show that disruption of TAG hydrolysis in the Arabidopsis (Arabidopsis thaliana) lipase mutant sugar-dependent1 (sdp1) leads to a substantial accumulation of TAG in roots and stems but comparatively much lower TAG accumulation in leaves. TAG content in sdp1 roots increases with the age of the plant and can reach more than 1% of dry weight at maturity, a 50-fold increase over the wild type. TAG accumulation in sdp1 roots requires both ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL ACYLTRANSFERASE1 and can also be strongly stimulated by the provision of exogenous sugar. In transgenic plants constitutively coexpressing WRINKLED1 and DGAT1, sdp1 also doubles the accumulation of TAG in roots, stems, and leaves, with levels ranging from 5% to 8% of dry weight. Finally, provision of 3% (w/v) exogenous Suc can further boost root TAG content in these transgenic plants to 17% of dry weight. This level of TAG is similar to seed tissues in many plant species and establishes the efficacy of an engineering strategy to produce oil in vegetative tissues that involves simultaneous manipulation of carbohydrate supply, fatty acid synthesis, TAG synthesis, and also TAG breakdown.

Suppression of the SUGAR‐DEPENDENT1 triacylglycerol lipase family during seed development enhances oil yield in oilseed rape (Brassica napus L.)

Increasing the productivity of oilseed crops is an important challenge for plant breeders and biotechnologists. To date, attempts to increase oil production in seeds via metabolic pathway engineering have focused on boosting synthetic capacity. However, in the tissues of many organisms, it is well established that oil levels are determined by both anabolism and catabolism. Indeed, the oil content of rapeseed (Brassica napus L.) has been reported to decline by approximately 10% in the final stage of development, as the seeds desiccate. Here, we show that RNAi suppression of the SUGAR-DEPENDENT1 triacylglycerol lipase gene family during seed development results in up to an 8% gain in oil yield on either a seed, plant or unit area basis in the greenhouse, with very little adverse impact on seed vigour. Suppression of lipolysis could therefore constitute a new method for enhancing oil yield in oilseed crops.

bZIP67 Regulates the Omega-3 Fatty Acid Content of Arabidopsis Seed Oil by Activating FATTY ACID DESATURASE3

This work explores the regulation of polyunsaturated fatty acid content in Arabidopsis seed oil. It shows that a transcriptional complex containing the basic Leu zipper protein bZIP67 and LEAFY COTYLEDON1-LIKE is responsible for activation of FATTY ACID DESATURASE3, which encodes an enzyme whose activity determines the level of omega-3 polyunsaturated fatty acids. Arabidopsis thaliana seed maturation is accompanied by the deposition of storage oil, rich in the essential ω-3 polyunsaturated fatty acid α-linolenic acid (ALA). The synthesis of ALA is highly responsive to the level of FATTY ACID DESATURASE3 (FAD3) expression, which is strongly upregulated during embryogenesis. By screening mutants in LEAFY COTYLEDON1 (LEC1)–inducible transcription factors using fatty acid profiling, we identified two mutants (lec1-like and bzip67) with a seed lipid phenotype. Both mutants share a substantial reduction in seed ALA content. Using a combination of in vivo and in vitro assays, we show that bZIP67 binds G-boxes in the FAD3 promoter and enhances FAD3 expression but that activation is conditional on bZIP67 association with LEC1-LIKE (L1L) and NUCLEAR FACTOR-YC2 (NF-YC2). Although FUSCA3 and ABSCISIC ACID INSENSITIVE3 are required for L1L and bZIP67 expression, neither protein is necessary for [bZIP67:L1L:NF-YC2] to activate FAD3. We conclude that a transcriptional complex containing L1L, NF-YC2, and bZIP67 is induced by LEC1 during embryogenesis and specifies high levels of ALA production for storage oil by activating FAD3 expression.

Identification, Typing, and Insecticidal Activity of Xenorhabdus Isolates from Entomopathogenic Nematodes in United Kingdom Soil and Characterization of the xpt Toxin Loci

ABSTRACT Xenorhabdus strains from entomopathogenic nematodes isolated from United Kingdom soils by using the insect bait entrapment method were characterized by partial sequencing of the 16S rRNA gene, four housekeeping genes (asd, ompR, recA, and serC) and the flagellin gene (fliC). Most strains (191/197) were found to have genes with greatest similarity to those of Xenorhabdus bovienii, and the remaining six strains had genes most similar to those of Xenorhabdus nematophila. Generally, 16S rRNA sequences and the sequence types based on housekeeping genes were in agreement, with a few notable exceptions. Statistical analysis implied that recombination had occurred at the serC locus and that moderate amounts of interallele recombination had also taken place. Surprisingly, the fliC locus contained a highly variable central region, even though insects lack an adaptive immune response, which is thought to drive flagellar variation in pathogens of higher organisms. All the X. nematophila strains exhibited a consistent pattern of insecticidal activity, and all contained the insecticidal toxin genes xptA1A2B1C1, which were present on a pathogenicity island (PAI). The PAIs were similar among the X. nematophila strains, except for partial deletions of a peptide synthetase gene and the presence of insertion sequences. Comparison of the PAI locus with that of X. bovienii suggested that the PAI integrated into the genome first and then acquired the xpt genes. The independent mobility of xpt genes was further supported by the presence of xpt genes in X. bovienii strain I73 on a type 2 transposon structure and by the variable patterns of insecticidal activity in X. bovienii isolates, even among closely related strains.

SUGAR-DEPENDENT6 encodes a mitochondrial flavin adenine dinucleotide-dependent glycerol-3-P dehydrogenase, which is required for glycerol catabolism and postgerminative seedling growth in Arabidopsis

The aim of this study was to clone and characterize the SUGAR-DEPENDENT6 (SDP6) gene, which is essential for postgerminative growth in Arabidopsis (Arabidopsis thaliana). Mutant alleles of sdp6 were able to break down triacylglycerol following seed germination but failed to accumulate soluble sugars, suggesting that they had a defect in gluconeogenesis. Map-based cloning of SDP6 revealed that it encodes a mitochondrial flavin adenine dinucleotide (FAD)-dependent glycerol-3-P (G3P) dehydrogenase:ubiquinone oxidoreductase called FAD-GPDH. This gene has previously been proposed to play a role both in the break down of glycerol (derived from triacylglycerol) and in NAD+/NADH homeostasis. Germinated seeds of sdp6 were severely impaired in the metabolism of [U-14C]glycerol to CO2 and accumulated high levels of G3P. These data suggest that SDP6 is essential for glycerol catabolism. The activity of the glycolytic enzyme phosphoglucose isomerase is competitively inhibited by G3P in vitro. We show that phosphoglucose isomerase is likely to be inhibited in vivo because there is a 6-fold reduction in the transfer of 14C-label into the opposing hexosyl moiety of sucrose when [U-14C]glucose or [U-14C]fructose is fed to sdp6 seedlings. A block in gluconeogenesis, at the level of hexose phosphate isomerization, would account for the arrested seedling growth phenotype of sdp6 and explain its rescue by sucrose and glucose but not by fructose. Measurements of NAD+ and NADH levels in sdp6 seedlings also suggest that NAD+/NADH homeostasis is altered, and this observation is consistent with the hypothesis that SDP6 participates in a mitochondrial G3P shuttle by cooperating with the cytosolic NAD-dependent GPDH protein GPDHC1.

Variation in Microbial Communities Colonizing Horticultural Slow Sand Filter Beds: Implications for Filter Function

The effect of microbial colonization on the function and rejuvenation of slow sand filters was investigated using culture-independent profiling. Colonization resulted in significant reduction in filter pore size, which may be important in order to fully remove pathogens, but was not associated with a specific microbial component. Communities were highly variable, and no common microbial groups were found in effective filters. Bacterial community composition was affected by sand particle size, although high levels of microbial turnover during filter maturation suggested that this was unlikely to have a major influence on community composition. The composition of microbial inoculum from a previous filter could not be maintained through a cycle of culture, storage and re-culture. Furthermore, no significant proportion of the inoculum persisted in filter maturity, and no advantages in terms of time to filter maturation or final filter efficiency were evident. These results may explain why filtration is such an effective and robust water treatment and emphasize the need for further research on the mechanisms involved in pathogen elimination.