| Oncidium is very popular in the cut flower market because of its beautiful flower and bright color.Oncidium is a respiratory climacteric flower,which is highly sensitive to ethylene.The abscision of the anther cap is an important reason for inducing the production of ethylene.The anther cap of Oncidium is very easy to fall off during harvesting and transportation,thereby releasing endogenous ethylene causes the cut flowers premature aging,which greatly reduces the economic value of Oncidium.In the current study,Onc.hybridum’Lemon Heart’,whose anther cap is easy to fall off and the flower is easy to decay,while Onc.Sweet Sugar ’Million Gold’ Coin ’,whose anther cap is not very easy to fall off and have longevity were used as research materials.The difference in the structure of anther cap and stamen binding site between two varieties of Oncidium,the relationship of abscission of anther cap and release of endogenous ethylene,exogenous ethylene treatment on the flower senescence and its relationship with wax content and structure were investigated.Furthermore,via RNA-seq,RT-qPCR,VIGS,and other biomolecular techniques,the endogenous and exogenous regulation mechanism of ethylene on flower senescence of Oncidium was studied.The main research results are as follows:1.Using the scanning electron microscope the surface structure of the ‘Lemon Green’(the anther cap is easy to fall)and the ‘Million Gold Coin’(the anther cap is not easy to fall off)in the part of the binding part between the stamen and anther cap were observed.There was no matching raised bulk structure on the binding part of ‘Lemon Green’,but on the‘Million Gold Coin’ whose anther cap is not easy to fall off was found that there is a matching raised bulk structure on the edge of the anther cap and the bulk can be combined smoothly with each other,which may effectively reduce abscission of the anther cap.2.In the vase life period,the ‘Lemon Green’ flower began to release endogenous ethylene after 16 hours,and reached its peak on the 6th day,while the ‘Million Gold Coin’ began to release endogenous ethylene after 24 hours and peak on the 9th day.The contents of endogenous ethylene in easy-to-delay ‘Lemon Green’ were 5.74 times higher than that of not-easy-to-delay ‘Million Gold Coin’.After artificial removal of the anther cap,the ‘Lemon Green’ and ‘Million Gold Coins’ released endogenous ethylene 8 hours and 16 hours earlier compared to control(without the removal of the anther cap).On the 6th and 9th days,the endogenous ethylene increased 55.42 times and 8.57 times,respectively compared to the control.After the anther cap fell off,‘Lemon Green’ showed signs of senescence 2 days earlier,while ‘Million Gold Coin’ showed signs of senescence 3 days earlier than control.3.The effect of 10 μl / L of exogenous ethylene treatment on flower senescence and water contents,the malondialdehyde contents were studied.The result revealed that ethylene accelerates the senescence of both varieties of Oncidium.‘Lemon Green’ and ‘Million Gold Coin’ show petal senescence(dehydrating and shrunken)3rd and 4th days after treatment and it were 2 days and 4 days earlier than the control,respectively.It was also observed that after exogenous ethylene treatment,the water content of both varieties of Oncidium significantly decreased by 12.57% and 20.48%,respectively,and the malondialdehyde content also increased significantly by 2.22 and 1.91 times,respectively.The water content of the petals of ‘Lemon Green’ without ethylene treatment for 24 hours is about 3 times lower than that of the petals of ‘Million Gold Coin’.The results indicate that petal water content is one of the main factors cause Oncidium’s flower senescence.4.The flower waxy structure and wax content in Oncidium were investigated after exogenous ethylene treatment.Scanning electron microscopy showed that the wax surface of ‘Lemon Green’ flower change from rough flake to smooth and thin after 3 days of ethylene treatment until no waxy structure can be observed.GC-MS measurement showed that the waxy content of both Oncidium varieties decreased with the flower senescence.Moreover,ethylene treatment significantly accelerated the reduction of the wax content of two varieties of Oncidium.After 3 days of treatment,the surface wax content in ‘Lemon Green’ decreased by 49.98% compared with the control,and the content of alkanes,acids,and esters in the waxy components was significantly reduced by 54.15%,31.75%,and 77.28%,respectively.After 4 days of treatment,the surface wax content in ‘Million Gold Coin’ decreased by 41.70%compared to the control,and the alkanes and acids in the wax component were significantly reduced by 72.80% and 53.36%.The phenolic substance was detected only in the ‘Million Gold Coin’.The wax content of the not-easy-to-delay ‘Million Gold Coin’ was1.64 times higher than that of easy-to-delay ‘Lemon Green’.5.Transcriptome analysis of differentially expressed genes of ‘Lemon Green’ after ethylene treatment was carried out.After 3 days of ethylene treatment,a total of 6190 differentially expressed genes were detected as compared to the control.Among them,4397 were up-regulated and 2417 were down-regulated.Combined with RT-qPCR data analysis of waxy synthesis and transduction pathways,it was found that after ethylene treatment,the expression levels of LACS,KCS,KCR,and other genes which regulate the synthesis of VLCFAs in the waxy component were down-regulated,and OhCER1,OhCER3,and OhCYTB5,which regulate the synthesis of alkane in the waxy component were significantly reduced.Moreover,the expression levels of OhCER4 and OhMAH1 that regulate the synthesis of alcohols in the waxy component were also significantly reduced.A total of 409 differentially expressed transcription factors were screened for 3 days after ethylene treatment,from which 245 TFs were up-regulated and 164 TFs were down-regulated.Among them,NAC,MYB,and WRKY transcription factors were among the highly differentially expressed transcription factors.6.RT-qPCR data showed that the expression levels of OhSAMS1 and OhACS1(key genes of ethylene biosynthesis in ‘Lemon Green’)were down-regulated after ethylene treatment,however exogenous ethylene-induced the expression of OhACO2.After 3 days of treatment,the expression level of OhACO2 was 12.9 times higher than the control.The analysis of expression of key genes in ethylene biosynthesis and waxy biosynthesis pathways in different parts of ‘Lemon Green’ showed that OhSAMS1,OhACS1,and OhACO2 were highly expressed in the pedicel followed by lateral petal,and labella,respectively.The expression level of OhACO2 in the stamen was significantly higher than that of OhSAMS1 and OhACS1,which was 20 times higher of OhSAMS1 and 27.70 times of OhACS1,respectively.It is speculated that OhACO2 is an important gene affecting the senescence of Oncidium.The key genes of waxy biosynthesis were mainly expressed in the lateral petal,sepals,and labella of ‘Lemon Green’.Among them,OhCER1,OhCER3,and OhCYTB5,which control the synthesis of alkanes,were mainly expressed in the lateral petal,and the expression levels of OhCER1 and OhCER3 also higher in the labella.The expression of OhCER4,which controls the synthesis of a primary alcohol,was higher in the lateral petal and labella,and the least in the anther caps and stamens.In addition to the low expression level in the anther cap,OhWSD1 which controls the synthesis of esters had a higher expression level in other parts of ‘Lemon Green’ and the highest expression level in the pedicel was 102.9 times higher than that of OhCER1(the lowest expressed in the pedicel).7.The longevity of the virus silenced OhACO2 in the petal of ‘Lemon Green’ was 15 days longer than that of control.The result indicates that OhACO2 plays a vital role in delaying the senescence of ‘Lemon Green’. |
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