Role Of RhWRKY40 In Ethylene-induced Rose Petal Senescence

Rose(Rosa hybrid L.)is one of the most major and economically and commercially important perennials ornamental plants in many countries all around the world,not currently but since early times.It is commonly known as the 'King of Flowers' as well as 'Queen of Flowers' because of unusual quality such as great size or beauty,ablaze and amazing fragrance.Although every process in the life of plant is very important but the opening of flower has a unique value because,the onset of pollination depend on opening of petals too much.For proper pollination the petals opening on right time,when the pollens are mature and the ovary becomes receptacle to the pollen.As insects have their own role in the process of pollination which are attracted by the color and fragrance of the petals,and after the pollination the flowers wilts or abscise.The wilting or senescence is the terminal phase of the flower opening,exhibits many changes which are characteristic of programmed cell death(PCD).More importantly these process are regulated by phytohormones.In which the most important one is ethylene which is a gaseous hormone and is responsible for the many of these process in life cycle of the plants.Such as seed germination,elongation of cell organs,opening of flower,fruit ripening and senescence etc.The process of senescence is age dependent,but it is also dependent on the production of ethylene specifically in fruits and cut flowers.The climacteric upswing of the production of endogenous ethylene flowers is reported to play a regulatory part in the proceedings of senescence of different ornamental plants flowers and its organs.In the count,the flower senescence can be prompted by various exterior and interior cues and phytohormones,which role in regulating,promoting and modulating the senescence is of very important,often through integrative interactions.As in these hormones,ethylene is considered as a crucial accelerator of flower senescence.However,it is still largely unknown about the interacting mechanism among the hormones during senescence.Further,in order to examine the functional characteristics of WRKY family genes regulating the senescence of rose petals,in preliminary stage we had selected several WRKY family members,from our transcriptome data of rose petal treated by ethylene.In total,5 WRKY genes were sorted out including RU58577,RU02827,RU03948,RU25337 and RU01910.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 five genes during flower opening.The results of qRT-PCR showed that the expression levels of RU02827 and RU25337 were significantly increased at early stages of flower opening.In addition,RU58577,RU01910,and RU02827 transcripts were expressed at later stages of flower opening(stage 4-6).Intriguingly,RU01910 avail a tendency of increasing expression and exposed the highest level before petal senescence(stage 4),and decreased in senesced petal(stage 6),and so we focus on this gene for further analysis.In this regard,cDNA sequence of RU01910 about 993bp with open reading frame.The sequence alignment showed that RU01910 has a conserved WRKY protein domain,and had a high degree of sequence homology to AtWRKY40 in Arabidopsis and belongs to the subgroup IIA.Therefore,we named RU01910 as Rh WRKY40.The expression of RhWRKY40 in different organs of rose flower during flower opening was also determined.The expression levels of RhWRKY40 sustained higher and posed peak at the stage-4-5 in most of organs rose flower including sepals,petals,stamens,and pistil.Thereafter,we investigated the expression in response to exogenous ethylene and ethylene actins inhibitor 1-methylcyclopropene(1-MCP).The results showed that the expression level of Rh WRKY40 gene appreciably increased from 12h treatment and reached to the peak at 24 hours ethylene treatment and as well as continual exceptionally higher level during the intact treatment.However,during 1-MCP treatment,its expression level was subdued.Thus,at this stage we had hypothesized that Rh WRKY40 considered as potential member for further experimentations,especially concern to senescence.In order to identify the biological role of Rh WRKY40 gene in senescence of rose flower,we silenced Rh WRKY40 in rose petal disc using virus induced gene silencing(VIGS)technique.We constructed a tobacco rattle virus vector(TRV-RhWRKY40)by the 3' end region of RhWRKY40 to specifically silence Rh WRKY40.The color fading of discs was started promptly in RhWRKY40-silencing compared to that of control(TRV2)after the 7th day of treatment.Similarly,after 15th day,the color of Rh WRKY40-silenced discs,become almost yellowish,whereas,discs of TRV-control showed slightly fading color.Further at 19th day the treatment,all the petals disc completely turn yellow as was compared to TRV-2.The expression of RhWRKY40 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 Rh WRKY40-silenced petal disc than in TRV2(control).We further silenced RhWRKY40 in rose plantlets by VIGS approach,and observed the flower opening pattern and its life durability.The flower life span of RhWRKY40-silencing plants is shorter than control plants.The durability of flower was 9.1±0.0326 days in RhWRKY40-silencing plants,however,in control it was 11.9±0.7382 days in order to investigate the involvement of phytohormones and we observe different kind of phenotype.With treatment with ethylene the color fading in the TRV-Rh WRKY40-silenced petals disc and TRV-2 along with the water treatment starts at 5th day.furthermore the disc start yellowing on the 7th day and on the 15th day all the disc turn yellow that of TRV-RhWRKY40 as compare to the TRV-2(Control).Along with the stress hormone ethylene the disc were also treated with jasmonic acid and the phenotype ware observed.The phenotype shows color was faded at 7th day of the treatment and in control TRV-2 senescence was started at the 10 days.But on later days as the experiment progresses the TRV-2 kept it color as compare to the TRV-RhWRKY40-petals disc.Another hormones were also investigated on the TRV-RhWRKY40-petals which has role in the senescence.GA3 which has an antagonistic effect on senescence to check whether the senescence in the petals disc was because of the silencing of Rh WRKY40.In the treatment with GA3 the senescence was induced in the TRV-RhWRKY40-petals disc as well as in TRV-2 control up to nineteen days there were less number of petals senescence.The expression level SAG 12 and their ion leakage was also investigate which also shows a little differences in TRV-2 control and TRV-Rh WRKY40.RhWRKY40 promote ethylene through ACS,the key enzyme Ethylene biosynthesis pathway,and on the other hand reduce ABA through NCED,the key enzyme ABA biosynthesis pathway.We also studied ABA responsive marker gene RhRD22 in the RhWRKY40-silent sample by RT-PCR.The result shows that there is about two folds increase in the expression of the RhRD22 in silent sample in comparison to the TRV2.Further we investigated a cytokinin pathways marker gene RhIPT9 also with the silenced sample of the RhWRKY40.RhIPT9 shows a significant difference in the expression to that of the TRV2 plants.Which shows that the RhRD22 a stress related gene in the ABA pathway may act as a downregulated gene of RhWRKY40,which promote senescence in the rose flowers.