Isolation And Functional Characterization Of Transcription Factor RhERF092 In Rose Flower Opening

Rose is one of the most important ornamental plants around the world with a huge aesthetic and market value. However, improper postharvest handling and transportation often results in great loss to the quality of cut roses. It has been well know that ethylene is a key contributor in affecting the opening quality of rose flower along with some biotic and environmental factors. Ethylene is one of the crucial phytohormone playing its role as potential modulator in growth and development of plant. During the course of its life cycle,a miniature seedling always needs to cope with ethylene during various aspects of nourishment and maturity such as, seed germination, plant organ development, root system proliferation, flower opening & senescence, abscission, and fruit ripening. Ethylene production is sensitively regulated by various internal and external cues in form of biotic(pathogen) and abiotic stresses. And abiotic stress includes drought, freezing, chilling,ozone, wounding, or hypoxia. Here, flowers of cut rose cv. 'Samantha' were used as materials to enlighten the overall roles of ethylene in rose flower opening and senescence.We focused on one of the essential transcription factors family-Ethylene Responsive Factor ERF family which generally encodes transcriptional regulators performing a range of functions and thus regulating the physiological and developmental processes of plants.Based on the microarray analysis, a rose uni-transcript annotated as RU27155 was selected as a candidate RhERF gene for our research work for its high level expression response upon 1 hr ethylene treatment. A total length of 1,289 bp sequence of RU27155 was cloned from rose petal through RACE method including 3' and 5' UTRs and an open frame reading (ORF) region of 810 bp. Phylogenetic analysis of deduced amino acid sequence showed that RU27155 is comprised with a typical AP2/EREBP domain, and belongs to Subgroup IX of ERF family. RU27155 was named as RhERF092 since it is a homolog of AtERF092. Analysis of subcellular localization revealed that RhERF092 is located in the nucleus. Transactivation assay showed that it possesses transactivation activity in its C-terminus. According to qRT-PCR test, the relatively strong expression level of RhERF092 was observed at opening stage-0 of rose flower and the transcript level gradually decreased till the senescence phase. After ethylene treatment, transcript level of ERF092 instantly increases and reached to the highest level at 1 hr and then drops down.Silencing of RhERF092 in rose flower resulted in mild acceleration of flower opening process and also increased the petal width and length,suggesting that RhERF092 maybe a negative regulator for petal cell expansion. Moreover, overexpression of RhERF092 in Arabidopsis resulted in delayed growth, shrink organ size and sterile inflorescence as compared to wild type (WT). Interestingly,the transgenic plants has also been observed with some novel features such as extra branches (lateral outgrowth) developed at every node, and more number of tillers from rosette nodes, indicating that RhERF092 might anticipate in the determination of meristem.Triple responses assay revealed that RhERF092-ox plants are more sensitive to ACC treatment. Transgenic line RhERF092-13ox with highest RhERF092 transcript level shows higher sensitivity and displayed smallest phenotype with shorter hypocotyls and roots system as compare to wild type. Moving Ahead, downstream genes expression analysis using over-expressed lines of RhERF092 revealed that a couple of developmental genes responsible for cell proliferation, cell expansion and cell cycle mitosis such as; ARGOS,ARGOS-LIKE, SIM, and JAGGED,AN3, CYCD3;1 and CYCD3;2 were suppressed in the transgene lines as compare to wild type. Since RhERF092-13ox phenocopied the ethylene hypersensitive mutants (ctrl) and ethylene-treated wild type Arabidopsis, we presumed that RhERF092-13ox is also producing numerous amount of ethylene as compare to wild type and some linkage is present between RhERF092 and ethylene biosynthesis. Therefore,we further focused on RhACSs genes in rose flower, the rate-limiting enzymes during ethylene biosynthesis pathway. RhACS1 and RhACS2 promoter isolated from cut rose genome in our earlier study were used to characterize its activities in Arabidopsis.In transgenic Arabidopsis harboring the RhACS2::GUS constructs, the activities of RhACS2 was found to be developmental-dependent in almost all of the tested organs.Activity of RhACS2 was strong in course of development from the young seedling stage to the mature flowering plant, including hypocotyls, cotyledons, leaves, roots, and floral organs of the inflorescence. Moreover, the RhACSl and RhACS2 promoters are sensitive to all tested hormonal and abiotic stresses including ABA, Mannitol and dehydration. Activity of RhACS2 promoter increased with ABA, Mannitol and dehydration in rosette and the promoter activity declined with rewater treatment in roots. Activity of RhACS2 promoter is more sensitive in roots of ProRhACS2::GUS transgene plants to ABA and drought as compare to rosette.Taken together, RhERF092 is an ethylene sensitive transcription factor gene which may be regulating some cell proliferation and expansion genes and thus modulating the developmental process at the cellular level during cut rose flower opening. Activity of the RhACS1 and RhACS2 promoters are strongly regulated by abiotic and hormonal stresses.We are also presuming that RhERF092 maybe regulator of RhACSs genes in cut rose flower.