Identification of Candidate Genes Associated with Leaf Senescence in Cultivated Sunflower (Helianthus annuus L.)

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.

Integrating transcriptomic and metabolomic analysis to understand natural leaf senescence in sunflower

“…In addition, the micro-RNA miR164 may suppress ORE1 transcript levels; miR164 and ORE1 may be regulated in a loop that would also involve EIN2, where EIN2 would promote the expression of ORE1 and would inhibit miR164 (Kim et al, 2009). In a previous work conducted in sunflower, the expression profiles of candidate genes Ha-EIN2 and Ha-NAC01 (with high sequence identity to ORE1) were evaluated together with miR164 levels (Moschen et al, 2014). In that study, the candidate genes and miR164 displayed high expression levels prior to anthesis and ahead of the first senescence symptoms.…”

“…The sunflower leaf senescence assays were conducted under field and glasshouse conditions. The field experiment was carried out at the INTA Balcarce Experimental Station (37°45 0 S, 58°18 0 W) as previously described (Moschen et al, 2014). The glasshouse experiment was performed at the Biotechnology Institute, INTA Castelar (34°36 0 S, 58°40 0 W).…”

“…The physiological measurements were assessed as previously described (Moschen et al, 2014) including chlorophyll, TSC and nitrogen determinations.…”

“…At the transcriptional level, the regulation of senescence has been well studied in model species such as A. thaliana (Gepstein et al, 2003;Lim et al, 2003;Lin and Wu, 2004). Furthermore, this process has been partially assessed in other species such as rice (Lee et al, 2001), tobacco (Pageau et al, 2006), pea (Pic et al, 2002), wheat (Uauy et al, 2006), cotton (Kong et al, 2013), maize (Sekhon et al, 2012;Zhang et al, 2014), turnip (Gombert et al, 2006), barley (Hollmann et al, 2014;Jukanti et al, 2008) and sunflower (Cabello et al, 2006;Fernandez et al, 2012a;Moschen et al, 2014). Transcription factors (TFs) are key proteins in the regulation of gene expression and signal transduction networks regulating different biological processes.…”

Identification of suitable reference genes by quantitative real-time PCR for gene expression normalization in sunflower

“…As in other plant species, TUB, ACT, and EF1 have been the most frequently employed as reference genes in sunflower (Breccia et al, 2013;Moschen et al, 2014;Ochogavia et al, 2014;Ungerer and Kawakami 2013). However, no detailed validation has been carried out to test the suitability of these genes and novel genes in different sunflower tissues.…”