Periodic Reporting for period 1 - LONGFLOW (Role of CONSTANS in flower longevity) Reporting period: 2019-09-01 to 2021-08-31 Summary of the context and overall objectives of the project The overall aim of LONGFLOW is to find a new expression pattern for the key photoperiodic gene CONSTANS (CO) in flowers, discover its role in autophagy and flower senescence, and modify flower longevity by modifying CO expression. The hypothesis is that CO, besides inducing flowering, could also induce autophagy and flower senescence. Results obtained in LONGFLOW are crucial in the flower farming industry, because by decreasing CO levels specifically in flowers, it could delay autophagy and flower senescence and obtain longer-lived flowers. The overall objectives of LONGFLOW are:Objective 1. Identify the role of CONSTANS in autophagy and flower senescence by Systems Biology approaches.Task 1.1. Characterization of CO expression pattern in Arabidopsis flowers.Task 1.2. Demonstrate CO activation of ATG and SAG genes in Arabidopsis flowers by RNAseq analysis.Task 1.3. Identify autophagy and senescence targets of CO binding in Arabidopsis flowers by Chromatin immunoprecipitation (ChIP) experiments.Objective 2. Modify of flower longevity by altering CONSTANS levels. Task 2.1. Demonstrate CO autophagy activation in Arabidopsis flowers.Task 2.2. Control of Arabidopsis flower senescence/longevity by CO.The main conclusions of LONGFLOW are:-CO protein is maintenance in Arabidopsis floral organs with a novel pattern of expression.-Changes in CO expression alter flower senescence and abscission.-CO participates in the control of flower senescence and abscission mediated by jasmonate.-The coordinated recruitment of photoperiodic and jasmonate signaling would be an efficient way for plants to chronologically order the floral process and ensure the success of offspring production. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During LONGFLOW period, CO temporal expression in all Arabidopsis floral organs was identified. Remarkably, the promoter activity of CO was extremely high in all floral organs compared with leaf organs. Circadian expression patterns and CO protein stability were also analysed, differences in CO expression and protein turn-over in leaves and flowers hinted to a different regulation of CO in the flower. From transcriptomic analysis of wild type, CO overexpresor and mutant lines by RNAseq, a significant effect of CO overexpression over senescence and Jasmonate (JA) response, a well-known senescence-inducing agent, were observed in Arabidopsis petals.Concerning objective 2, transgenic lines expressing a conventional autophagy marker and different levels of CO were generated and autophagy was monitored. The results suggest a role for CO in the inhibition of autophagy in Arabidopsis flowers. Concerning CO control over of Arabidopsis flower senescence/longevity, these processes were compared in wild type, CO overexpression and mutant lines. The results suggest a role for CO in the induction of flower senescence and abscission. Due to the possible implication of JA in CO control over flower senescence observed by RNAseq, mutant plants in genes involved in jasmonate synthesis and signalling, and different levels of CO were generated. Flower senescence analysed in these transgenic lines support the effect of CO in JA-mediated flower senescence. It has been documented that red light (RL) mediates CO degradation in Arabidopsis plants. With an applied perspective in ornamental industry and avoiding the use of transgenic plants, RL treatment, aimed to reduce CO levels, was used. The results support this treatment to obtain longer-lived flowers by the reduction of CO levels.Among highlights of the results of LONGFLOW are these peer-reviewed publications in high-impact journals:• Gloria Serrano-Bueno, Fatima E Said, Pedro de Los Reyes, Eva I Lucas-Reina, M Isabel Ortiz-Marchena, José M Romero, Federico Valverde. 2020. CONSTANS-FKBP12 interaction contributes to modulation of photoperiodic flowering in Arabidopsis. Plant J. 101(6):1287-1302. https://doi.org/10.1111/tpj.14590.• Gloria Serrano-Bueno, Víctor Sánchez de Medina Hernández, and Federico Valverde. 2021. Photoperiodic Signaling and Senescence, an Ancient Solution to a Modern Problem? Front Plant Sci. 12:634393. https://doi.org/10.3389/fpls.2021.634393.• Gloria Serrano-Bueno, Andrea Chini, Gabriel Ferreras-Garrucho, Pedro de los Reyes, Roberto Solano, and Federico Valverde. CONSTANS and jasmonate signaling coordinate floral senescence in Arabidopsis. Manuscript Submitted to Mol Plants.And these Seminar/Conference/Meeting participations:• Institute of Plant Biochemistry and Photosynthesis symposium. 2020, “Role of CONSTANS in flower longevity”. https://t.co/tyOFGbDjwB https://t.co/kYEvDknw39" / Twitter.• XV Meeting of Plant Molecular Biology. Online. 2020, “Flower senescence promotion by CONSTANS through Jasmonate pathway in Arabidopsis thaliana” https://www.xvrbmp.com/• SevinOmics Spring Meeting. Online. 2021, “Transcriptional effects of CONSTANS altered expression in Arabidopsis floral organs”.• Plant Biology Worldwide Summit. Online. 2021, “CONSTANS interaction with jasmonate related proteins controls flower senescence in plants”. https://www.eventscribe.net/2021/ASPB/fsPopup.asp?Mode=presInfo&PresentationID=889524. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Understanding the way plants respond to external stimuli is of general interest to secure a sustainable food production in the current scenario of global warming. Coordination of floral development and senescence is crucial for crop yield. In LONGFLOW, a molecular mechanism that aims to explain how plants integrate endogenous and exogenous signals to promote floral senescence by coordinating a hormonal and a temporal response has been described.How jasmonates function in flower senescence can be modified by external cues is still unexplored. In this project, we identified a previously unnoticed role in the flower for the central hub of the photoperiod response, CO, incorporating the robust seasonal regulatory input of the photoperiod pathway into the senescence program.LONGFLOW results could have deep implications in many fields of plant science and can be of interest to improve crop yield and for the industry of cut flower. The manipulation through light quality, day length or genetic approaches can be used to modulate flower senescence, changing flower longevity or delaying fruit production, thus making plants more robust against the deleterious effects of global warming. The proof of this is the project “Role of CONSTANS-homologue (PhCO) in Petunia flower senescence” that we have just gain to apply this research in the ornamental plant Petunia.