**3. Study of leaf senescence process in sunflower**

**1. Introduction**

30 Senescence - Physiology or Pathology

[15–23].

Leaf senescence is an age-dependent complex process at the cellular, tissue, organ or organ‐ ism level, leading to death at the end of the life span [1]. Annual plants as grain and oil crops undergo a visual process toward the end of the reproductive stage that is accompanied by nutrient remobilization from leaf to developing seeds [2]. Senescence process is controlled by multiple genetic and environmental variables, which has strong impact on crop yield [3].

biotic stresses caused by disease may affect the rate of senescence. In this context, not only environmental conditions but also biotic factors influence senescence, being this an irrevers‐ ible process prematurely induced under these adverse conditions [4]. Moreover, reproductive growth is mentioned as a factor that usually impacts on leaf senescence, and particularly in sunflower, the lack of sinks delays the onset of senescence [5]. During this process, changes in gene expression result in a metabolic shift from anabolism to catabolism, which leads to decreased photosynthetic activity, progressive degradation of cellular structures and oxida‐ tive burst [6–8]. It has been documented that a delay in leaf senescence has a substantial impact on grain yield through the maintenance of the photosynthetic leaf area during the

In sunflower (*Helianthus annuus* L.), the largest important oil crop worldwide, the senescence process reduces the capacity of plants to maintain their green leaf area for longer periods, especially during the grain-filling phase, affecting the yield and thus leading to economic losses [11, 12]. This production constraint has deepened since sunflower crop production has been gradually moved to marginal areas due to the rapid change of agricultural practices in crops such as soybean and maize, which have greatly increased their cultivated areas as a consequence of favorable commodity prices and because farmers found more profitable to

During the last years, many efforts have been achieved to build up useful functional genomics tools for cultivated sunflower involving physiological, transcriptional and metabolic profiles

In crop species, considering the temporal gap between onset and phenotypic detection of senes‐ cence process, the availability of candidate genes and molecular markers to the early detection of senescence is indispensable to discriminate between early-senescing and late-senescing lines to be applied in the different context of breeding activities [24]. For example, the identification of functional stay‐green genotypes for breeding applications and/or for elucidating molecular

Senescence is an essential process for the normal growth and development of plants, being an

important mechanism for the adaptation to several environmental conditions.

sow transgenic crops with resistance to herbicides and insects [13, 14].

**2. Senescence and crop yields: stay‐green genotypes**

, abiotic and

Environmental factors such as light, nutrient availability, concentration of CO<sup>2</sup>

reproductive stage in different crops [3, 9, 10].

mechanisms involved in this complex trait.

Sunflower is the fourth most important oil crop worldwide and the second one in the Argentine. Moreover, Argentina is the third largest exporter of crude oil and the second of protein and pellet flour. The added value of oilseed industrialization contributes in the econ‐ omy with US\$ 1400 million approximately, with a total production between 3.2 and 3.8 million tons of grain annually [34].

Sunflower is an annual monocarpic species in which reproductive phase exerts a strong control on leaf senescence and nutrient remobilization, affecting grain weight [35]. Potential yields of sunflower crop are far from the real ones in all Argentina productive regions. In Balcarce loca‐ tion, for instance (Southeast of Buenos Aires province), one of the best productive regions of Argentina, while the potential yields are estimated in 5000 kg ha−1, those obtained by the best producers only reach 3000 kg ha−1, and the average in the region ranges in 1800 kg ha−1 [36]. Among the factors that contribute to the productivity gap, one of the most important is the inability of current hybrids to keep their green leaf area for long periods, limiting the incident radiation capture during the grain-filling period and impacting negatively on the yield and oil concentration [12, 37].

Sunflower genome complexity characterized by of long and highly similar repeats has slowed the pace of getting a complete genome reference. Recently, a high-quality assembly comprising 3.6 Gigabases has being achieved by means of PACBIO sequencing [38]. The reference genome together with extensive transcriptomic data from vegetative and floral organs is accessible at https://www.heliagene.org/HanXRQ-SUNRISE. In the last years, our group accomplished a series of transcriptional and metabolic profiling studies that were integrated into physiologi‐ cal, molecular and cytological analysis to contribute to the understanding the senescence pro‐ cess in this crop and breeding genotypes against abiotic constraints [19–23, 39–41].

Through a system biology approach and using a commercial sunflower hybrid, we character‐ ized the leaf senescence process by integrating transcriptomic and metabolomic analyses using both glasshouse and field conditions [22]. Our results revealed early metabolic changes before to anthesis in the absence of the onset of the first visual senescence symptoms, with more pro‐ nounced changes observed when physiological and molecular variables were assessed under field conditions (**Figure 1**). Metabolite remobilization from mature and senescent leaves to the different sinks, particularly into seed development, affects their quality and quantity and is one of the most important aspects of crop improvement [3]. In this study, we showed a decrease of photosynthetic activity and cell growth before anthesis, whereas sucrose, fatty acid, nucleo‐ tide and amino acid metabolisms increased. The role of sugars in senescence has been widely discussed in recent years. Sugars are central elements of the source-sink relationships [42, 43] and have been reported as growth [44] and photosynthetic rate regulators [45]. However, the effect of sugars on senescence is controversial and differs between different species [1, 46, 47]. In sunflower, sugar content decreases during leaf development (**Figure 1**). This finding is in line with previous studies in tomato and higher plants, in which the photosynthetic rate dropped together with sugar levels in a mature leaf [48–50]. Furthermore, sunflower is a plant with a high demand for nutrient, especially sugars as substrate for oil synthesis, during the grain-filling phase. Likewise, low levels of sugars may increase production and/or ethylene sensitivity, which acts as senescence enhancer [51, 52].

Pathways related to nutrient recycling processes were also up-regulated. We found high expression levels of enzymes involved in recycling, such as asparagine synthetase and gluta‐ mine synthase, as well as the associated metabolites, asparagine and glutamine. These amino acids are involved in nitrogen and carbon transport between the different organs and are the most abundant amino acids in the xylem and phloem [53, 54], indicating a high recycling activity at early stages of leaf development.

Transcription factors (TFs) are key proteins involved in the regulation of gene expression and signal transduction networks, regulating different biological processes and their function is crucial for triggering and/or controlling the different aspect of senescence process. Members of the NAC, AP2-EREBP, HB, bZIP and MYB transcription factor families showed high expression levels, and their expression level was highly correlated, suggesting their involve‐ ment in sunflower senescence. These results are in agreement with previous results described for *Arabidopsis thaliana* [55]. Particularly, we found a transcript with high sequence identity Sunflower Leaf Senescence: A Complex Genetic Process with Economic Impact on Crop Production http://dx.doi.org/10.5772/intechopen.68588 33

radiation capture during the grain-filling period and impacting negatively on the yield and

Sunflower genome complexity characterized by of long and highly similar repeats has slowed the pace of getting a complete genome reference. Recently, a high-quality assembly comprising 3.6 Gigabases has being achieved by means of PACBIO sequencing [38]. The reference genome together with extensive transcriptomic data from vegetative and floral organs is accessible at https://www.heliagene.org/HanXRQ-SUNRISE. In the last years, our group accomplished a series of transcriptional and metabolic profiling studies that were integrated into physiologi‐ cal, molecular and cytological analysis to contribute to the understanding the senescence pro‐

Through a system biology approach and using a commercial sunflower hybrid, we character‐ ized the leaf senescence process by integrating transcriptomic and metabolomic analyses using both glasshouse and field conditions [22]. Our results revealed early metabolic changes before to anthesis in the absence of the onset of the first visual senescence symptoms, with more pro‐ nounced changes observed when physiological and molecular variables were assessed under field conditions (**Figure 1**). Metabolite remobilization from mature and senescent leaves to the different sinks, particularly into seed development, affects their quality and quantity and is one of the most important aspects of crop improvement [3]. In this study, we showed a decrease of photosynthetic activity and cell growth before anthesis, whereas sucrose, fatty acid, nucleo‐ tide and amino acid metabolisms increased. The role of sugars in senescence has been widely discussed in recent years. Sugars are central elements of the source-sink relationships [42, 43] and have been reported as growth [44] and photosynthetic rate regulators [45]. However, the effect of sugars on senescence is controversial and differs between different species [1, 46, 47]. In sunflower, sugar content decreases during leaf development (**Figure 1**). This finding is in line with previous studies in tomato and higher plants, in which the photosynthetic rate dropped together with sugar levels in a mature leaf [48–50]. Furthermore, sunflower is a plant with a high demand for nutrient, especially sugars as substrate for oil synthesis, during the grain-filling phase. Likewise, low levels of sugars may increase production and/or ethylene

Pathways related to nutrient recycling processes were also up-regulated. We found high expression levels of enzymes involved in recycling, such as asparagine synthetase and gluta‐ mine synthase, as well as the associated metabolites, asparagine and glutamine. These amino acids are involved in nitrogen and carbon transport between the different organs and are the most abundant amino acids in the xylem and phloem [53, 54], indicating a high recycling

Transcription factors (TFs) are key proteins involved in the regulation of gene expression and signal transduction networks, regulating different biological processes and their function is crucial for triggering and/or controlling the different aspect of senescence process. Members of the NAC, AP2-EREBP, HB, bZIP and MYB transcription factor families showed high expression levels, and their expression level was highly correlated, suggesting their involve‐ ment in sunflower senescence. These results are in agreement with previous results described for *Arabidopsis thaliana* [55]. Particularly, we found a transcript with high sequence identity

cess in this crop and breeding genotypes against abiotic constraints [19–23, 39–41].

sensitivity, which acts as senescence enhancer [51, 52].

activity at early stages of leaf development.

oil concentration [12, 37].

32 Senescence - Physiology or Pathology

**Figure 1.** Metabolism overview in the field experiment at pre-anthesis time. Genes and metabolites are represented by squares and circles, respectively. Color intensity corresponds to the expression ratio at logarithmic scale (red: upregulated, blue: down-regulated) [22].

to *ANAC02* or *ATAF1*. This transcript might be associated with an upstream regulation of the signaling pathway involving ORE1 and EIN2 [56], thus activating their expression and inhibiting the expression of Golden2-like (GLKs) genes, which are necessary for chloroplast development and maintenance [57]. ORE1 also acts as an antagonist of GLK protein, adding more complexity to this regulation pathway [57]. In *A. thaliana*, ORE1 TF induces leaf senes‐ cence [58]. In addition, the micro-RNA *miR164* 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* [59]. In a previous work conducted in sun‐ flower, expression profiles of candidate genes *Ha‐EIN2* and *Ha‐NAC01* (with high sequence identity to *ORE1*) were evaluated together with *miR164* levels [21] showing similar expression patterns to Arabidopsis and in line with the increase in the nutrient remobilization rate.

Moreover, using bioinformatic approaches and evaluating two different approaches for gene expression correlation analysis: Weighted Gene Correlation Network Analysis (WGCNA) and BioSignature Discoverer (BioSD, Gnosis Data Analysis, Heraklion, Greece), we integrated tran‐ scriptomic and metabolomic data [39]. WGCNA allowed the detection of 10 metabolites and 13 TFs (**Figure 2**), whereas BioSD allowed the detection of one metabolite and six TFs as potential biomarkers. Comparative analysis demonstrated that three transcription factors were detected

**Figure 2.** Integrated network of hubs metabolites and transcription factors detected by WGCNA [39]. 24 selected hubs metabolites and 82 TFs statistically significant during senescence were correlated (p-value < 0.0001) and visualized in Cytoscape [60] by degree (node size and color) and edge weight (edge size and color).

by both approaches, highlighting them as potentially robust biomarkers associated with leaf senescence in sunflower. This complementary use of network and BioSignature Discoverer analysis of transcriptomic and metabolomic data provided a useful tool for identifying candi‐ date genes and metabolites, which may have a role in the triggering and development of the leaf senescence process.

Transcriptomic analysis in combination with metabolic profile is a comprenhensive tool for the study of leaf senescence. These results suggest a complex regulatory network underlying this process. Thus, the identification of regulatory networks based on expression profiling is an important starting point for the detection of new key genes involved in the triggering of the senescence process in this crop.
