Qantitative Detection Of Endogenous IAA And ABA In Litchi Fruit And Its Relationship With Fruit Abscission

Lychee?Litchi chinensis Sonn.?is an important fruit tree with high economic value in the south of China,but there is a serious problem that heavy physiological fruit abscission leading to low production and the limitation the deveolpment of litchi industry.The abscission of Litchifruitis a phenomenon controlled by multi-factors mutual coordination and interaction,in which endogenous hormones play an important role,especially IAA and ABA.Liquid-mass spectrometry?LC-MS?technique is currently recognized as a highly accurate endogenous hormone detection method,but it has not been establishedin litchi research.Thisstudy firstexploredandestablisheda UPLC-ESI-MS/MS method for the accurate deter mination of endogenous IAA and ABA content in litchi fruit,and then compared the differences of endogenous IAA and ABA levels in fruits in two independent experiments,including two varieties with having high and low preharvest fruit drop,and carbohydrate stress inducing fruitlet abscission.The purpose of this study was to explore the relationship between endogenous IAA and ABA in the fruit,and to provide theoretical basis for developing measures to alleviate fruit abscission of lychee.The main results are showed as follows:1?A UPLC-ESI-MS/MS method was established for the simultaneous deter mination of endogenous IAA and ABA in litchi fruits using ultra performance liquid chromatography-triple quadrupole mass spectrometry.The three different pretreatment methods of IAA and ABA deter mination including liquid-liquid extraction and external standard method,dispersive liquid-liquid micro-extraction and external standard method,liquid-liquid extraction and internal standard method,were evlauated mainly from the total ion chromatogram peak type,response value and convenience of the operation,the main results showed that:?1?Liquid-liquid extraction and external standard method could not detect any IAA and ABA content in samples.?2?It is not possible to detect IAA by dispersive liquid-liquid micro-extraction and external standard method,but it could detect ABA better.And then,the effects of different extraction time,extractant and NaCl concentration on ABA retention behavior and enrichment effect were compared.The optimization results showed that when the extraction time was 30 min,tributyl phosphate was used as the extractant,and the salt concentration was 0%,the ABA retention behavior and enrichment effect were the best.The correlation coefficient?r²?of ABA in the linear range was 0.99,the limitation of detection was 2.3pg/?L,and the recovery rate was 73.12%¹07.02%,the relative standard deviation was not higher than 4.0%.And we detect the ABA content in the‘Guiwei'and‘Nuomici'samples that was 123.24 ng/g·FW and 358.91 ng/g·FW,respectively.This pre-treatment is suitable for quantification of ABA in litchi samples.?3?Liquid-liquid extraction plus internal standard could detect IAA and ABA content in samples simultaneously;and the effects of different antioxidants,extractants,decolorizers,and extractants on the retention behavior and enrichment effects of the IAA and ABA were compared.The optimization results showed that the litchi samples were precooled 80%acetone leaching,petroleum ether decolorization?pre-adjust pH to 8?,ethyl acetate extraction?adjust pH to 3 with extractant?,mobile phase 0.1%formic acid in XBridgeTM BEHC18 column and the gradient elution of 0.1%formic acid and acetonitrile and the addition of isotopen ¹³C₆-IAA and D₆-ABA as internal standards could quantitatively detect the content of IAA and ABA in the sample.The limitation of detection of IAA and ABA was 0.283 pg/?L and 0.102 pg/?L.The limitation of Quantity of IAA and ABA was 0.339 pg/?L and 0.943 pg/?L.The relative standard deviations of IAA and ABA were 3.75%and 1.38%,respectively.2.The UPLC-ESI-MS/MS method established in this experiment was used to deter mine the contents of IAA and ABA in the pulp and pericarp of the'Nuomici'and'Guiwei'during the period of rapid fruit growth?about 1 month before harvest,from May 23 to June20?.The results showed that the ABA contents in the pericarp and pulp of'Nuomici'were between 397²640 ng/g.FW and 530⁹16ng/g.FW,respectively,‘Guiwei'were 196 to 1805ng/g.FW and 247 to 600 ng/g.FW,respectively.During the pre-harvesting fruit abscission?June 6 to June 20?,the cumulative fruit abscission rates of'Nuomici'and'Guiwei'litchi were 39.43%and 12.86%,respectively,and the ABA content in the pericarp and pulp of'Nuomici'was higher than those of‘Guiwei'except for June 20,but there were no obvious difference in the content of IAA between the two varieties.Based on this,it is believed that the reason of pre-harvest fruit abscission of‘Nuomici'significantly higher than that of‘Guiwei'might be related to the high content of ABA in pulp.3.Using the UPLC-ESI-MS/MS method established in this experiment,the contents of endogenous IAA and ABA in'Huaizhi'fruitlet with GPD treatment and untreated control were analyzed.The results showed that the fruit abscission and the contents of IAA and ABA in'Huaizhi'fruitlet were increased after treatment with GPD.The accumulative fruit abscission rate was nearly to 100%after 5 days after GPD treatment,but there was about8%for the untreated control.The IAA content in control and treatment group was about110ng/g.FW on the 1d and 2d after,but in the 3d suddenly increased,reaching1612ng/g.FW,then slightly decreased on the 4th day,finally reaching the highest value of1732ng/g.FW on the 5th day,while the IAA content in control changed from 125 to441ng/g.FW on the 3rd day to the 5th day.There were no significant difference between the control and treatment group about the ABA content in the three days.On the 4th day,the ABA was rapidly increased and reached to 494 ng/g.FWon the 5th day for GPD treatment,which was significantly higher in the control group.Through the bio-informatics analysis,seven ABA synthesis-related genes NCED were screened from the litchi genome database?http://litchidb.genomics.cn/page/species/index.jsp?.The expression level of the seven NCED genes were quantified by qRT-PCR.The expression levels of'Huaizhi'fruitlet during the shedding process showed that LcNCED1,LcNCED3 and LcNCED5 responded to starvation stress.Based on the above results,GPD treatment promotes the fruitlet abscission.It may be associated with the promotion of ABA accumulation in fruit and inhibition of IAA output from fruit.LcNCED1,LcNCED3 and LcNCED5 may be the key genes that lead to increase ABA content.