Sunflower ( Helianthus annuus L . ) leaf senescence analysis by a physiological and molecular approach

 Leaf senescence is a complex mechanism controlled by multiple variables, either from genetic and environmental origin that strongly impacts on crop yield. It has been previously described that water stress accelerates leaf senescence in sunflower while head removal delays the process. Even when senescence has been extensively studied in model plants, the molecular aspects that take place at the onset of leaf senescence in sunflower are still unknown. Visual parameters as yellowing or necrosis of leaves are detectable long after the signaling cascade of senescence process is activated. Recently, different transcription factors with NAC domain have been characterized as early SAGs (Senescence Associated Genes) which might play a key role in leaf senescence and program cell death. The aims of this work were: (i) the evaluation of physiological and biochemical parameters associated to senescence in a time frame, for different growing and leaf-age conditions, (ii) the early detection of leaf senescence by the molecular isolation of a potential SAG, (iii) the study of the differential expression of these candidates along the development of leaf senescence.  Two field experiments were performed at INTA Balcarce, in two growing seasons. Sunflower plants (VDH487, Advanta) were grown under good water and nutrients conditions. Treatments were: (i) head excision, (ii) a moderate water stress during all developmental period, and (iii) control. The evolution along a thermal time scale of physiological (nitrogen, leaf area), biochemical (soluble sugars, chlorophyll) and molecular (NAC transcription factor expression) events related to senescence process were evaluated. Parameters evaluated to detect leaf senescence were 80% (nitrogen, leaf area, soluble sugars and chlorophyll), 20 % (chlorophyll) and 0% (chlorophyll) of the maximal value reached for each variable. Identification of candidate sunflower SAGs was performed by searching tentative orthologous to described SAGs in different plant species. Transcriptional profile of candidate SAGs was done by qPCR in leaves at different ages and growing conditions, using reference genes with stable expression pattern, previously identified and characterized by our group.  The time course of the evaluated parameters showed differences among treatments but also a strong interaction of growing conditions and leaf ages was detected. An early water stress showed a delay in the occurrence of the events in the oldest measured leaf, while in the youngest, the opposite was observed. Nitrogen fall was the very first physiological marker detected, whereas other relevant parameters were detected later in the cascade of events in the senescence process. The expression profile of one of the identified sunflower orthologous SAGs, an ORE1 transcription factor containing a NAC domain, was assessed through qPCR. Expression patterns of ORE1 showed an early activation in relation to physiological and biochemical events, which makes this gene a potential indicator of the triggering of the process.  Knowing the time of the onset of the sequence of events related to senescence could determine the causes of this process and generate physiological and molecular tools to facilitate future interventions on it. This can be useful for application in assisted breeding of this crop with great economic importance. According to the results of this study, the transcription factor ORE1 appears as a potential candidate by qPCR with an early expression pattern, followed by an increase of expression prior to anthesis.  The main contribution of this work was the identification and characterization of genetic sequences involved in the onset and evolution of the leaf senescence process through the study of physiological and transcriptional profiles in sunflower leaves from plants challenged to treatments that alter the senescence rate.