| Rose(Rosa hybrid L.)is one of the most significant and economically important ornamental plants worldwide since ancient time.It is commonly known as the 'King of Flowers' as well as 'Queen of Flowers' due to its magnificent beauty,attractive colour and marvelous fragrance.Flower opening is a natural and programmed plant process,during this process petals unfold for flower pollination,and then wilt or abscise.Flower senescence as the terminal phase of flower opening,exhibits many changes that are hallmarks of programmed cell death.Moreover,ethylene is considered as an important plant gas hormone in regulating numerous developmental processes in plants such as seed germination,elongation of cell organs,flower opening,fruit ripening,organ senescence etc.It is also well-known that flower senescence is associated with increased ethylene production in many flowers.The climacteric rise of endogenous ethylene in these flowers has been shown to play a regulatory role in the events leading to the senescence of flower organs.In addition,flower senescence can be prompted by various exterior or interior cues and phytohormones,which play a pivotal role in triggering and modulating the progression of senescence,often through combinatorial interactions.Amongst these hormones,ethylene is considered as a key accelerator of flower senescence.However,it is still largely unknown about the interacting mechanism among the hormones during senescence.Further,in order to investigate the functional role of AP2/ERF family genes regulating the senescence of rose petals,in preliminary stage we had screened out various AP2/ERF family members from our transcriptome data of rose petal treated by ethylene.In total,six AP2/ERF genes were sorted out including RU03641,RU53500,RU03393,RU06277,RU22804,and RU56654.These genes were selected on the basis of showing maximum amount of fold change in the gene expression level while confronted to ethylene treatment.Furthermore,we evaluated the expression patterns of those six genes during flower opening.The results of qRT-PCR showed that the expression levels of RU06277 and RU56654 were significantly increased at early stages of flower opening.In addition,RU53500,RURU22804,and RU03393 transcripts were accumulated at later stages(stage 4-6)of flower opening.Interestingly,RU03641 exhibited a trend of increasing expression and showed the highest level before petal senescence at stage-5,and decreased in senesced petal at stage-6;thus,we targeted this gene for further analysis.In this regard,we identified cDNA sequence of RU0364 1 which is about 1224bp with a 721bp open reading frame.The sequence alignment showed that RU03641 has a conserved AP2/EREBP domain,and had a high degree of sequence homology to ERF113 in Arabidopsis;therefore,we denominated RU03641 as RhERF113.The expression of RhERF113 in different organs of rose flower during flower opening was also determined.The expression levels of RhERF113 sustained higher and posed peak at the stage-5 in most of organs rose flower including sepals,petals,stamens,and pistil.Thereafter,we tested the expression of RhERF113 in response to exogenous ethylene and ethylene actins inhibitorl-methylcyclopropene(1-MCP).The results showed that the expression level of RhERF113 gene appreciably reached a peak at the 18h ethylene treatment and as well as sustained outstandingly higher level during the intact treatment.However,during 1-MCP treatment,its expression level was inhibited.Thus,at this stage we had hypothesized that RhERF113 considered as potential member for further experimentations,especially concern to senescence.In order to identify the biological role of RhERF113 gene in senescence of rose flower,we silenced RhERF113 in rose petal disc using virus induced gene silencing(VIGS)technique.We constructed a tobacco rattle virus vector(TRV-RhERF113)by the 3' end region of RhERF113 to specifically silence RhERF113.The color fading of discs was started swiftly in RhERF113-silencing compared to that of control(TRV2)after the 7th day of treatment.Similarly,after 15th day,the color of RhERF113-silenced discs was become almost yellowish,whereas,discs of TRV-control showed slightly fading color.The expression of RhERF113 in silenced petal discs was significantly reduced compared to the TRV2(control),confirming the silencing efficiency.In addition,ion leakage rate and RhSAG12,senescence associated gene,expression as markers for senescence progression were significantly higher in RhERF113-silenced petal disc than in TRV2(control).We further silenced RhERF113 in rose plantlets by VIGS approach,and observed the flower opening pattern and its life durability.The flower life span of RhERF113-silencing plants is shorter than control plants.The durability of flower was 9.1±0.0326 days in RhERF113-silencing plants,however,in control it was 11.9±0.7382 days.In order to investigate the involvement of cytokinin(CTK)in RhERF113 silencing-induced flower senescence,we first tested the effect of treating RhERFl 13-silenced petal discs with 6-benzyl aminopurine(6-BA,synthetic CTK).The slight color fading after 10d in WT,TRV2,and RhERF113-silencing petal discs was noticed.In addition,expression levels of RhSAG12 and ion leakage rates were similar between TRV2 and RhERF113-silencing petal discs.Thus,CTK content in TRV2 and RhERF113-silencing petal discs was measured.The results indicated that RhERF113-silencing induced senescence is associated with CTK content.The results of qRT-PCR showed that the expression levels of RhHB6 and RhPR10.1 were significantly reduced in RhERF113-silencing flowers.Thereafter,we identified a 1500bp region of RhHB6 promoter,which is upstream of RhHB6 coding sequences.Furthermore,we had also assessed the significant role of RhERF113-silencing in leaves senescence of rose.In this part,Virus induced gene silencing(VIGS)approach and functional analysis was used for a potentially novel transcription factor RhERF113 regulation of leave senescence in rose.Silencing approach by virus induced gene silencing(VIGS)study of RhERF113 into rose has resulted in a narrative phenotype.In relating to rose,the phenotype was characterized with yellow fades of leaves which showed in RhERF113-silencing leaves.In order to confirm the efficiency and effectiveness of RhERF113-silencing,we had assessed the changes happened via ion leakage and chlorophyll content,expression of SAG 12(a senescence associated gene)and(PAG)photosynthetic associated genes expression as markers for senescence progression proved that RhERF113-silecing played a critical role in promoting the leaves senescence.The effect of RhERF113-silenicng on leaf senescence was determined by comparing the level of yellowing in the RhERF113-silenced,non-si lenced TRV(control)and wild type during age-dependent leaf senescence and as well as of discs of leaves,in order to know the role of RhERF113-silencing in stimulating the instigation of detached leaf senescence.At 15d after inflation,the leaves were initiated in RhERF113-silenced and started to turn yellow;however,TRV(control)and wild-type leaves remained green.At 25-30d after infiltration,the RhERF113-silenced leaves had turned completely yellow and showed signs of drying to death.Whereas,TRV(control)and wild-type leaves retained their integrity and showed only partial yellowing.In order to retain its substantiation,the leaf senescence symptoms were also analyzed by measuring typical senescence-associated physiological markers,such as chlorophyll content,expression of Photosynthetic related and senescence associated genes(SAG12).After 3 weeks of infiltration,the chlorophyll content of TRV(control)and wild-type leaves started to decline;whereas,after 5 weeks,silenced leaves had already lost 45-50%of their chlorophyll content.Another member of ERF family was also investigated,which played a significant role in accelerating the senescence of rose petals and possesses up regulation in response to ethylene treatment.The expression of RhERF003 was determined during different rose flower opening stages.The expression levels of RhERF003 sustained higher and posed peak at the stage-4 to stage-6.Subsequently,after that we tested the expression of RhERF003 in response to exogenous ethylene and ethylene actins 1-MCP.The results showed that the expression level of RhERF003 gene appreciably reached a peak at the 24h ethylene treatment and as well as sustained outstandingly higher level during the intact treatment,instead of 1-MCP treatment,its expression level was inhibited.Thus,at this stage we had hypothesized that RhERF003 considered as potential member for further experimentations,especially concern to senescence.In order to identify the prospective role of RhERF003 gene in senescence of rose flower,we silenced RhERF003 in rose petal using virus induced gene silencing(VIGS)technique.The results further elucidated,by applying exogenous ethylene on RhERF003-silenced petals,the senescence was highly delayed.Furthermore,the ABA competency on RhERF003-silenced petals senescence was also assessed and slight yellowing of petals was observed at the beginning days of infiltrations.Furthermore,we also had investigated efficiency of inhibitor of senescence such as Auxin and sucrose on RhERF003-silencing.Interestingly,in contrast to other hormones we found,the senescence start slightly in the entire experiment.Slight yellowing of petals was observed from the beginning days till end of experiments for both TRV2 and RhERF003.In order to investigate the involvement of sucrose RhERF003 silencing-induced senescence,we first tested the effect of treating RhERF003-silenced petal with sucrose.The slight color fading after 10d in TRV2,and RhERF003-silencing petal was noticed.In addition,expression levels of RhSAG12 and ion leakage rates were similar between TRV2 and RhERF003-silencing petal.Take all results together;we concluded that RhERF003 play a role in senescence of rose petals by modulating sucrose contents. |
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