Identification of Candidate Genes Associated with Leaf Senescence in Cultivated Sunflower (Helianthus annuus L.) Sebastian Moschen 1,2 , Sofia Bengoa Luoni 3 , Norma B. Paniego 1,2 , H. Esteban Hopp 1,4 , Guillermo A. A. Dosio 2,5 , Paula Fernandez 1,2,3." , Ruth A. Heinz 1,2,4 * ." 1 Instituto de Biotecnologı ´a, Centro de Investigaciones en Ciencias Agrono ´ micas y Veterinarias, Instituto Nacional de Tecnologı ´a Agropecuaria, Hurlingham, Buenos Aires, Argentina, 2 Consejo Nacional de Investigaciones Cientı ´ficas y Te ´cnicas, Ciudad Auto ´ noma de Buenos Aires, Argentina, 3 Escuela de Ciencia y Tecnologı ´a, Universidad Nacional de San Martı ´n, San Martı ´n, Buenos Aires, Argentina, 4 Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Auto ´ noma de Buenos Aires, Argentina, 5 Laboratorio de Fisiologı ´a Vegetal, Unidad Integrada Universidad Nacional de Mar del Plata, Estacio ´ n Experimental Agropecuaria INTA Balcarce, Balcarce, Buenos Aires, Argentina Abstract Cultivated sunflower (Helianthus annuus L.), an important source of edible vegetable oil, shows rapid onset of senescence, which limits production by reducing photosynthetic capacity under specific growing conditions. Carbon for grain filling depends strongly on light interception by green leaf area, which diminishes during grain filling due to leaf senescence. Transcription factors (TFs) regulate the progression of leaf senescence in plants and have been well explored in model systems, but information for many agronomic crops remains limited. Here, we characterize the expression profiles of a set of putative senescence associated genes (SAGs) identified by a candidate gene approach and sunflower microarray expression studies. We examined a time course of sunflower leaves undergoing natural senescence and used quantitative PCR (qPCR) to measure the expression of 11 candidate genes representing the NAC, WRKY, MYB and NF-Y TF families. In addition, we measured physiological parameters such as chlorophyll, total soluble sugars and nitrogen content. The expression of Ha- NAC01, Ha-NAC03, Ha-NAC04, Ha-NAC05 and Ha-MYB01 TFs increased before the remobilization rate increased and therefore, before the appearance of the first physiological symptoms of senescence, whereas Ha-NAC02 expression decreased. In addition, we also examined the trifurcate feed-forward pathway (involving ORE1, miR164, and ETHYLENE INSENSITIVE 2) previously reported for Arabidopsis. We measured transcription of Ha-NAC01 (the sunflower homolog of ORE1) and Ha-EIN2, along with the levels of miR164, in two leaves from different stem positions, and identified differences in transcription between basal and upper leaves. Interestingly, Ha-NAC01 and Ha-EIN2 transcription profiles showed an earlier up-regulation in upper leaves of plants close to maturity, compared with basal leaves of plants at pre-anthesis stages. These results suggest that the H. annuus TFs characterized in this work could play important roles as potential triggers of leaf senescence and thus can be considered putative candidate genes for senescence in sunflower. Citation: Moschen S, Bengoa Luoni S, Paniego NB, Hopp HE, Dosio GAA, et al. (2014) Identification of Candidate Genes Associated with Leaf Senescence in Cultivated Sunflower (Helianthus annuus L.). PLoS ONE 9(8): e104379. doi:10.1371/journal.pone.0104379 Editor: Cynthia Gibas, University of North Carolina at Charlotte, United States of America Received January 17, 2014; Accepted July 13, 2014; Published August 11, 2014 Copyright: ß 2014 Moschen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by ANPCyT/FONCYT, Pre ´stamo BID PICT 15-32905 and PAE 37100-PICT 019, INTA-PE AEBIO 241001 and 245001, INTA-PE AEBIO 245711, INTA-AEBI0 243532, INTA PN CER 1336 and UNMdP, AGR212, AGR260. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: rheinz@cnia.inta.gov.ar . These authors contributed equally to this work. " These authors are joint last authors on this work. Introduction As the last stage of leaf development, the genetically determined and highly ordered process of senescence involves characteristic changes in gene expression that result in decreased photosynthetic activity, active degradation of cellular structures, nutrient recycling, lipid peroxidation and, ultimately, cell death [1,2]. Multiple variables control the complex mechanism of senescence; these genetic and environmental variables have a strong effect on crop yield [3]. Annual plants, such as grain and oil crops, undergo visible senescence towards the end of the reproductive stage, accompanied by nutrient remobilization from leaves to developing seeds [4]. In monocarpic species, the development of the reproductive structure controls leaf senescence [5]. Prematurely induced senescence, caused by biotic or abiotic stress, can reduce crop yield. Thus, leaf senescence has an economic impact, affecting the gap between potential and real yields. Sunflower (Helianthus annuus L.) is the third most important source of edible vegetable oil worldwide, and the second in Argentina. It also provides an important source of biodiesel [6] (Sunflower Statistics NSA 2007–2009, USA) [7]. Recent work has produced some genomic information for this crop [8], but the complete genome sequence remains unavailable. However, functional genomics tools for cultivated sunflower have been PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104379