N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. In plants, NAEs are generally elevated in desiccated seeds, suggesting that they may play a role in seed physiology. NAE and abscisic acid (ABA) levels were depleted during seed germination, and both metabolites inhibited the growth of Arabidopsis thaliana seedlings within a similar developmental window. Combined application of low levels of ABA and NAE produced a more dramatic reduction in germination and growth than either compound alone. Transcript profiling and gene expression studies in NAE-treated seedlings revealed elevated transcripts for a number of ABA-responsive genes and genes typically enriched in desiccated seeds. The levels of ABI3 transcripts were inversely associated with NAE-modulated growth. Overexpression of the Arabidopsis NAE degrading enzyme fatty acid amide hydrolase resulted in seedlings that were hypersensitive to ABA, whereas the ABAinsensitive mutants, abi1-1, abi2-1, and abi3-1, exhibited reduced sensitivity to NAE. Collectively, our data indicate that an intact ABA signaling pathway is required for NAE action and that NAE may intersect the ABA pathway downstream from ABA. We propose that NAE metabolism interacts with ABA in the negative regulation of seedling development and that normal seedling establishment depends on the reduction of the endogenous levels of both metabolites.

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca 2+

N-acylethanolamines (NAEs) are bioactive lipids derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NAPE). In animal systems this reaction is part of the “endocannabinoid” signaling pathway, which regulates a variety of physiological processes. The signaling function of NAE is terminated by fatty acid amide hydrolase (FAAH), which hydrolyzes NAE to ethanolamine and free fatty acid. Our previous work in Arabidopsis thaliana showed that overexpression of AtFAAH (At5g64440) lowered endogenous levels of NAEs in seeds, consistent with its role in NAE signal termination. Reduced NAE levels were accompanied by an accelerated growth phenotype, increased sensitivity to abscisic acid (ABA), enhanced susceptibility to bacterial pathogens, and early flowering. Here we investigated the nature of the early flowering phenotype of AtFAAH overexpression. AtFAAH overexpressors flowered several days earlier than wild type and AtFAAH knockouts under both non-inductive short day (SD) and inductive long day (LD) conditions. Microarray analysis revealed that the FLOWERING LOCUS T (FT) gene, which plays a major role in regulating flowering time, and one target MADS box transcription factor, SEPATALLA3 (SEP3), were elevated in AtFAAH overexpressors. Furthermore, AtFAAH overexpressors, with the early flowering phenotype had lower endogenous NAE levels in leaves compared to wild type prior to flowering. Exogenous application of NAE 12:0, which was reduced by up to 30% in AtFAAH overexpressors, delayed the onset of flowering in wild type plants. We conclude that the early flowering phenotype of AtFAAH overexpressors is, in part, explained by elevated FT gene expression resulting from the enhanced NAE hydrolase activity of AtFAAH, suggesting that NAE metabolism may participate in floral signaling pathways.

N-acylethanolamines (Naes) are lipid metabolites derived from the hydrolysis of the membrane phospholipid N-acylphosphatidylethanolamine (NaPe). recent work in Arabidopsis thaliana seedlings showed that combined treatments of Nae 12:0 and aBa inhibited seedling growth synergistically, suggesting low levels of Nae could potentiate the action of aBa. here we examined the interplay between compound concentrations, growth inhibition and mutant genotypes with impaired sensitivities to these regulators. Nae 12:0 and aBa both induced dose-dependent increases in transcript levels of ABI3, and two ABI3 responsive genes, AtHVA22B and RD29B. Interestingly, even in the absence of growth inhibition, RD29B transcripts were elevated by aBa but not Nae treatment outside the sensitive window for aBa/Nae treatment, indicating some differences in the regulation of growth and the modulation of gene expression by these two compounds. also noteworthy, the growth of aBa insensitive mutant (abi 3-1) seedlings was inhibited at higher concentrations of Nae 12:0 but not aBa, suggesting that Nae may act to inhibit early seedling establishment by both aBI3-dependent and aBI3-independent pathways. collectively our results reinforce the concept that Nae12:0 interacts with aBa signaling in seedling establishment, but also points to a complexity in this interaction that modulates the sensitivity of young seedlings to phytohormone-mediated growth arrest. N-Acylethanolamine

The wide distribution of Palmer amaranth (Amaranthus palmeri) in the southern US became a serious weed control problem prior to the extensive use of glyphosate-resistant crops. Currently glyphosate-resistant populations of Palmer amaranth occur in many areas of this geographic region creating an even more serious threat to crop production. Investigations were undertaken using four biotypes (one glyphosate-sensitive, one resistant from Georgia and two of unknown tolerance from Mississippi) of Palmer amaranth to assess bioassay techniques for the rapid detection and level of resistance in populations of this weed. These plants were characterized with respect to chlorophyll, betalain, and protein levels and immunological responses to an antibody of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) the target site of glyphosate. Only slight differences were found in four biotypes grown under greenhouse conditions regarding extractable soluble protein and chlorophyll content, but one biotype was found to be devoid of the red pigment, betalain. Measurement of early growth (seedling shoot elongation) of seedlings was a useful detection tool to determine glyphosate resistance. A leaf disc bioassay (using visual ratings and/or chlorophyll analysis) and an assay for shikimate accumulation were effective methods for determining herbicide resistance levels. The two unknown biotypes were found to be resistant to this herbicide. Some differences were found in the protein profiles of the biotypes, and western blots demonstrated a weak labeling of antibody in the glyphosate-sensitive biotype, whereas strong labeling occurred in the resistant plants. This latter point supports research by others, that increased copy number of the EPSPS gene (and increased EPSPS protein levels) is the resistance mechanism in this species. Results indicate the utility of certain bioassays for the determination of resistance and provide useful comparative information on the levels of inherent constituents among closely related plants.

Glyphosate‐resistant Palmer amaranth from Georgia (GA), USA, possesses multiple copies of the gene that encodes 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), the enzyme target site of this herbicide. The cloned plants of glyphosate‐resistant and glyphosate‐susceptible Palmer amaranth biotypes from Mississippi (MS), USA, and GA were evaluated for glyphosate injury (digital imaging) in leaf disc bioassays. Four groups (three resistant groups: two from MS [G and R] and one from GA [C7]; one susceptible group from GA [C3]) were chosen for cloning to facilitate long‐term studies. After exposure to glyphosate (1.0 mmol L–1, 144 h), the level of injury (mean value) was low in the resistant groups, while a higher level of injury was found in the susceptible group. However, the individual injury values within all groups varied widely. The mean EPSPS gene copy number of these groups was G ≥ R > C7 >>> C3. However, a higher copy number did not always convey increased resistance in these bioassays. When the copy number was high (>20), 81.5% of the bioassayed plants exhibited little or no injury and only ∼20% were significantly injured, while 50% of the plants with a low copy number (<20) remained healthy. Overall, no strong statistical correlation of the copy number versus injury occurred in these cloned plants and no statistical relationship of resistance and copy number with the sex of the MS plants was observed. The results suggest that although an elevated copy number of the EPSPS gene can instill resistance, other mechanisms might contribute to the overall glyphosate resistance of Palmer amaranth in these plants.

The fungus Myrothecium verrucaria (Alb. & Schwein.) (MV), originally isolated from diseased sicklepod (Senna obtusifolia L.), has bioherbicial activity against kudzu and several other weeds when applied with low concentrations of the surfactant Silwet L-77. To more fully understand the initial events of MV infection or disease progression, and to improve knowledge related to its mechanism of action, the effects of MV and its product (roridin A) on kudzu seedlings were examined at the ultrastructural level. Ultrastructural analysis of MV effects on kudzu seedlings revealed a rapid (~1 h after treatment) detachment of the protoplast from the cell wall and plasmodesmata appeared to be broken off and retained in the wall. These symptoms occurred well in advance of the appearance of any fungal growth structures. Some fungal growth was observed after severe tissue degeneration (24 to 48 h after treatment), but this occurred primarily at the extra-cellular location with respect to the kudzu tissues. Kudzu seedlings treated with roridin A, a trichothecene produced by the fungus, exhibited some symptoms similar to those induced by the fungus applied in spore formulations with surfactant. The overall results are the first to report the ultrastructural effects of this bioherbicide on plants and suggest that penetration of a phytotoxic substance(s) in the fungal formulation was facilitated by the surfactant, and that roridin A exerts phytotoxicity toward kudzu.

A Palmer amaranth population (seeds collected in the year 2000; Washington Co., MS) suspected to be susceptible to glyphosate was examined as a population and as individual plants and found to exhibit varying tolerance or resistance to glyphosate. Whole plant spraying of glyphosate (0.84 kg·ha ) to the population revealed that approximately 40% of this population were resistant to glyphosate and an LD 50 of 0.75 kg·ha −1 was determined. Spray application of glyphosate indicated that some plants displayed varying degrees of resistance 14 days after treatment. Initial tests using leaf disc bioassays on 10 individual plants selected randomly from the population, allowed characterization of glyphosate resistance using both visual ratings of injury and quantitative measurement via chlorophyll content analysis. After initial bioassays and spray application, five plants with a range of tolerance to glyphosate were selected for cloning so that further studies could be accomplished on these individuals. Q-PCR analysis of these clones showed that resistance was not due to elevated EPSPS gene copy number. Shikimate levels were lower in the resistant and higher in the susceptible clones which correlated with varying degrees of resistance demonstrated in bioassays and spray application of glyphosate of these clones. Results demonstrate that individuals in a population can vary widely with respect to herbicide resistance and suggest that uptake, translocation, sequestration, metabolism or altered target site may contribute to the resistance in some individuals of this population.