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Abstract Fatty acid amide hydrolase (FAAH) is a conserved amidase that is known to modulate the levels of endogenousN‐acylethanolamines (NAEs) in both plants and animals. The activity of FAAH is enhancedin vitroby synthetic phenoxyacylethanolamides resulting in greater hydrolysis of NAEs. Previously, 3‐n‐pentadecylphenolethanolamide (PDP‐EA) was shown to exert positive effects on the development of Arabidopsis seedlings by enhancing Arabidopsis FAAH (AtFAAH) activity. However, there is little information regarding FAAH activity and the impact of PDP‐EA in the development of seedlings of other plant species. Here, we examined the effects of PDP‐EA on growth of upland cotton (Gossypium hirsutumL. cv Coker 312) seedlings including two lines of transgenic seedlings overexpressingAtFAAH. Independent transgenic events showed accelerated true‐leaf emergence compared with non‐transgenic controls. Exogenous applications of PDP‐EA led to increases in overall seedling growth in AtFAAH transgenic lines. These enhanced‐growth phenotypes coincided with elevated FAAH activities toward NAEs and NAE oxylipins. Conversely, the endogenous contents of NAEs and NAE‐oxylipin species, especially linoleoylethanolamide and 9‐hydroxy linoleoylethanolamide, were lower in PDP‐EA treated seedlings than in controls. Further, transcripts for endogenous cottonFAAHgenes were increased following PDP‐EA exposure. Collectively, our data corroborate that the enhancement of FAAH enzyme activity by PDP‐EA stimulates NAE‐hydrolysis and that this results in enhanced growth in seedlings of a perennial crop species, extending the role of NAE metabolism in seedling development beyond the model annual plant species,Arabidopsis thaliana.
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Critical parameters for robust Agrobacterium ‐mediated transient transformation and quantitative promoter assays in Catharanthus roseus seedlings
Abstract Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect ofAgrobacterium‐infiltration parameters on the transient transformation efficiency ofCatharanthus roseusseedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to theAgrobacteriumgrowth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold.Agrobacteriumin exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in ourAgrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and rawRenillaluciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation ofAgrobacteriuminfiltration inC. roseusseedlings will facilitate the study of this important medicinal plant and will expand the application ofAgrobacterium‐mediated transformation methods in other plant species.
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- Award ID(s):
- 2031237
- PAR ID:
- 10513047
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 8
- Issue:
- 6
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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