Mechanisms Of N-acylethanolamines Regulation On Senescence In Cut Carnation Flower (Dianthus Caryophyllus L.)

The study of flower senescence should provide not only methods to improve thepostharvest longevity of cut flowers, but insights into the mechanisms underlying thecontrol of plant senescence in general.N-acylethanolamines (NAEs) are fatty-acid amides, which are minor membrane lipidconstituents of plant and animal cells. As a minor membrane lipid constituent of plant cells,NAEs have been shown to be part of the plant defense signaling system, and involve in theregulation of seed development. Recently, NAEs have been used to preserve the freshnessof cut flowers and to delay the ripening of fruit. However, the mechanism of NAEs on theretarding senescence has not yet been studied in cut plants.This research aims at the feeble domain in the research on NAEs. First it wasevaluated the cut carnation (Dianthus earyophyllus L.) and cut rose (Rose hybrida L.)senescence in response to NAEs, and were determined the effects of NAEs on thedevelopment stages of cut flowers. Then it was explored systemically the mechanism ofNAEs on the retarding senescence from the membrane structure and functions level,including the regulation roles of NAEs on enzymatic and non-enzymatic lipid peroxidation,antioxidant defense, the membrane composition and function.The main research results include:1 The effects of N-acylethanolamines on the post-harvest performance of cut flowersThree treatments had been tried to examine the possible involvement ofN-lauroylethanolamine [NAE(12:0)] in the regulation of cut-flower senescence, and theresult showed that only pulse treatment could extend the longevity of cut carnation flowers'Red Barbara'. And similar responses to NAE(12:0) in carnation cultivar 'Snow White' andRose cultivar 'Red Berlin' and 'Avalanche' were observed as well, and the optimumconcentration of NAE(12:0) by pulse treatments to extend the postharvest life of 'RedBarbara', 'Snow White', 'Red Berlin' and 'Avalanche' were 5, 5～25, 25 and 25～50μmol/L, respectively.The effects of another three kinds of NAE species on the cut carnation flowers 'RedBarbara' post-harvest performance were also analyzed in the study, and the vase life of cutcarnation 'Red Barbara' by the pulse treatment with NAEs decreased with increasing acylchain length, such that order of effectiveness rankedNAE(12:0)＞NAE(14:0)＞NAE(16:0)＞NAE(18:0). To evaluate the cut carnation in response to NAE(12:0), 3 stages of development werearbitrarily distinguished, and the results showed that the durations of stage 2 flowerdevelopment were 10.4d and 11.7d for cut carnation flowers 'Red Barbara' and 'SnowWhite', respectively, which were 2.3 and 2.8d longer than that of controls, and theyshowed significantly different compared with the controls(p＜0.05). However, the profiles instage 1 and 3 were not affected by NAE(12:0).2 The role of N-lauroylethanolamine in the regulation of lipid peroxidation andantioxidant defense during the senescence of cut carnations 'Red Barbara'It was conducted the regulation of NAE(12:0) on the lipid peroxidation andantioxidant defense during senescence of cut flowers that were pulse-treated with 5μmol/LNAE(12:0). And the results showed that ion leakage, which is a reliable indicator ofmembrane integrity, was postponed in NAE(12:0)-treated flowers. The lipid peroxidationincreased in carnation petals with time, in parallel to the development in activity oflipoxygenase, superoxide anion and H₂O₂ production rate, and these increases were bothdelayed by NAE(12:0) supplementation. The activities of four enzymes (superoxidedismutase, catalase, glutathione reductase and ascorbate peroxidase) and the contents threenon-enzymatic antioxidants (GSH, ASC andα-tocopherol) that are implicated inantioxidant defense were also up-regulated in the cut carnations that had been treated withNAE(12:0). In addition, significant differences for physiological parameters measured inthe present study between treated samples and controls were only observed during day 12to day 14, which was the stage of end of blooming to onset of wilting.3 N-lauroylethanolamine regulation on membrane composition and function in carnationpetals during senescenceSenescence of flower petals, as of other plant organs, is accompanied by numerouswell-defined compositional, structural and functional changes in their cellular membranes.To assess whether the delay of senescence by NAE(12:0) treatment involved the protectionof petal-membrane structure and function integrity, this work was conducted to examine themodifications occurring in the microsomal fraction, a purified plasma membrane andtonoplast preparation. Microsomal membranes were prepared from petals of carnation usingdifferential centrifugation, and plasma membranes and tonoplast vesicles were isolatedfrom microsomal fraction using discontinuous sucrose gradients partition. The electronmicrograph stained with phosphotungstic acid and marker enzyme activities analysisproved that plasma membrane vesicles had high purity and were not polluted by mictochondria and tonoplast. But the marker enzyme activities analysis showed that thetonoplast vesicles were adulterated by a little plasma membrane vesicles.3.1 The modifications in membrane lipids parameters in the microsomal fractionNAE(12:0) supplementation delayed the decreases in membrane phospholipids and theratio of unsaturation of fatty acids, and the increase in the relative proportion of long-chainfatty acids in the aging carnation petals, which was consistent with the observedimprovements in the lipid fluidity of microsomal fraction isolated from petals ofNAE(12:0)-treated flowers compared to those of controls.3.2 The modifications in membrane protein and membrane enzyme activity in themicrosomal fractionNatural petal senescence of cut carnation flowers is associated with a decline inmembrane protein content and membrane enzyme activity. When the rate of carnationsenescence was modified by exposure to NAE(12:0), the decreases in petal microsomalmembrane proteins and five membrane marker enzymes (vanadate-senesitive ATPase,nitrate-sensitive ATPase, cytochrome c oxidase, cytochrome c reductase and latentIDPase) activitives were delayed accordingly.3.3 The modifications in H⁺-ATPase and Ca²⁺- ATPase of plasma membrane and tonoplastvesicle isolated from carnation petalsThe activities of H⁺-ATPase and Ca²⁺-ATPase located in the plasma membrane andtonoplast vesicles isolated from NAE(12:0)-treated petals were higher than those ofcontrols during the vase-holding, suggest the petal cells treated with NAE(12:0) mightenhance solute transport ability and maintain cytoplasmic Ca²⁺ homeostasis, and hencemaintaining the ongoing metabolism processes and signal transduction events for longertime.As a conclusion, the pulse treatment with NAEs slowed senescence by delaying theonset of initial wilting in cut flowers. NAE(12:0)-induced delays in cut-carnationsenescence involves the regulation of membrane lipid peroxidation and microsomalmembrane lipids, which resulted in the higher lipid fluidity of microsomal fraction, andmaintained the more activities of membrane enzymes; thus preserving the structure andfunction integrity of the membranes. We propose that the stage from the end of blooming tothe onset of wilting is a critical period for NAE(12:0) action.