Presence of inulin in sunflower (Helianthus annuus L.) grown under high irradiance.

Fructans are important polysaccharides synthesized from sucrose which are present in about 12-15% of angiosperms. Sunflower (Helianthus annuus L.) is considered a non-fructan bearing plant even though its close relative, Helianthus tuberosus, accumulates the inulin type of the polymer in large amounts. Previous work suggested that putative fructan-synthesizing enzymes may be expressed in sunflower, but only very limited amounts of the trisaccharide isokestose were found in stems of plants storing high levels of sucrose due to capitulum removal. The present work is aimed at investigating whether intact sunflower plants may indeed synthesize fructans in any of its parts when grown in conditions that favor sucrose availability. Plants were grown in the field at a low density, resulting in a high light availability and low competition for resources, in comparison with controls (usual crop planting density). Plants were harvested at anthesis. Thinned treatment led to an increase in carbohydrates level especially in the capitulum. Carbohydrates analysis of this tissue in thinned plants revealed, for the first time in this species, the presence of inulin-type fructans. The amount of each member of the series appeared to decline starting from isokestose, being DP = 15 the longest fructan detected. Results suggest that, in sunflower, fructans could be synthesized only when sucrose availability exceeds a high threshold, which may not be attained under usual growing conditions. Given the relationship between fructans and tolerance to abiotic stresses including drought, the present finding opens a new perspective for breeding and management of this crop.

Sunflower: a potential fructan-bearing crop?

Grain filling in sunflower (Helianthus annuus L.) mainly depends on actual photosynthesis, being the contribution of stored reserves in stems (sucrose, hexoses, and starch) rather low. Drought periods during grain filling often reduce yield. Increasing the capacity of stem to store reserves could help to increase grain filling and yield stability in dry years. Fructans improve water uptake in soils at low water potential, and allow the storage of large amount of assimilates per unit tissue volume that can be readily remobilized to grains. Sunflower is a close relative to Jerusalem artichoke (H. tuberosus L.), which accumulates large amounts of fructan (inulin) in tubers and true stems. The reason why sunflower does not accumulate fructans is obscure. Through a bioinformatics analysis of a sunflower transcriptome database, we found sequences that are homologous to dicotyledon and monocotyledon fructan synthesis genes. A HPLC analysis of stem sugar composition revealed the presence of low amounts of 1-kestose, while a drastic enhancement of endogenous sucrose levels by capitulum removal did not promote 1-kestose accumulation. This suggests that the regulation of fructan synthesis in this species may differ from the currently best known model, mainly derived from research on Poaceae, where sucrose acts as both a signaling molecule and substrate, in the induction of fructan synthesis. Thus, sunflower might potentially constitute a fructan-bearing species, which could result in an improvement of its performance as a grain crop. However, a large effort is needed to elucidate how this up to now unsuspected potential could be effectively expressed.

Identification of candidate genes associated with leaf senescence in cultivated sunflower (Helianthus annuus L.).

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.

Floret initiation, tissue expansion and carbon availability at the meristem of the sunflower capitulum as affected by water or light deficits.

• The co-ordination between floret initiation and meristem expansion, and their relationships with carbon availability, were studied and quantified in sunflower (Helianthus annuus) plants subjected to light or water shortages. • Meristem size, number of floret primordia, primordium size, rate of plant biomass accumulation, leaf area, photosynthetic rate, and soluble sugar content in the capitulum were measured until completion of floret initiation. • Although treatments differentially affected tissue expansion and biomass acquisition, a common relationship between the final number of florets and the rate and duration of meristem expansion was conserved. In the absence of water deficit, changes in relative expansion rate in the meristem paralleled changes in soluble sugar content. Water deficit reduced tissue expansion both in leaves and in the capitulum, and induced the accumulation of soluble sugars in the meristem. Use of these sugars at re-watering was associated with increased meristem growth and higher floret numbers compared with control plants. • Floret initiation and meristem tissue expansion remained strongly co-ordinated under all studied circumstances, and both depended on local carbon availability when water supply was unlimited. Transient water deficits favoured reproductive meristem growth and floret production. Equations accounting for these results constitute a framework for phenotyping the response to drought.

Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis.

The selection and validation of reference genes constitute a key point for gene expression analysis based on qPCR, requiring efficient normalization approaches. In this work, the expression profiles of eight genes were evaluated to identify novel reference genes for transcriptional studies associated to the senescence process in sunflower. Three alternative strategies were applied for the evaluation of gene expression stability in leaves of different ages and exposed to different treatments affecting the senescence process: algorithms implemented in geNorm, BestKeeper software, and the fitting of a statistical linear mixed model (LMModel). The results show that geNorm suggested the use of all combined genes, although identifying α-TUB1 as the most stable expressing gene. BestKeeper revealed α-TUB and ß-TUB as stable genes, scoring ß-TUB as the most stable one. The statistical LMModel identified α-TUB, actin, PEP, and EF-1α as stable genes in this order. The model-based approximation allows not only the estimation of systematic changes in gene expression, but also the identification of sources of random variation through the estimation of variance components, considering the experimental design applied. Validation of α-TUB and EF-1α as reference genes for expression studies of three sunflower senescence associated genes showed that the first one was more stable for the assayed conditions. We conclude that, when biological replicates are available, LMModel allows a more reliable selection under the assayed conditions. This study represents the first analysis of identification and validation of genuine reference genes for use as internal control in qPCR expression studies in sunflower, experimentally validated throughout six different controlled leaf senescence conditions.

Using a 3-D virtual sunflower to simulate light capture at organ, plant and plot levels: contribution of organ interception, impact of heliotropism and analysis of genotypic differences.

BACKGROUND AND AIMS: Light interception is a critical factor in the production of biomass. The study presented here describes a method used to take account of architectural changes over time in sunflower and to estimate absorbed light at the organ level. METHODS: The amount of photosynthetically active radiation absorbed by a plant is estimated on a daily or hourly basis through precise characterization of the light environment and three-dimensional virtual plants built using AMAP software. Several treatments are performed over four experiments and on two genotypes to test the model, quantify the contribution of different organs to light interception and evaluate the impact of heliotropism. KEY RESULTS: This approach is used to simulate the amount of light absorbed at organ and plant scales from crop emergence to maturity. Blades and capitula were the major contributors to light interception, whereas that by petioles and stem was negligible. Light regimen simulations showed that heliotropism decreased the cumulated light intercepted at the plant scale by close to 2.2% over one day. CONCLUSIONS: The approach is useful in characterizing the light environment of organs and the whole plant, especially for studies on heterogeneous canopies or for quantifying genotypic or environmental impacts on plant architecture, where conventional approaches are ineffective. This model paves the way to analyses of genotype-environment interactions and could help establish new selection criteria based on architectural improvement, enhancing plant light interception.

How does the meristem of sunflower capitulum cope with tissue expansion and floret initiation? A quantitative analysis.

The coordination between floret initiation and tissue expansion has been studied and quantified in the apical meristem of sunflower (Helianthus annuus) plants grown under different light availability. A method was developed to quantify tissue expansion in the meristem during floret initiation from measurements of meristem area, number of florets and primordium size. Initially, floret initiation and tissue expansion occurred simultaneously at the meristem surface. The duration of this phase remained unchanged across environments, whereas the rate of tissue expansion varied greatly. Floret initiation rate depended on meristem initial size and tissue-expansion rate. Thereafter, floret initiation continued without tissue expansion in the meristem, resulting in a rapid decrease of meristem area. A set of equations was proposed to predict floret initiation rate and floret number as a function of the rates of tissue expansion in the meristem before and during floret initiation. This formalism demonstrated the role of tissue expansion in determining the final number of florets, and provided a framework to analyse the response of floret initiation to genotype and environment.

A whole-plant analysis of the dynamics of expansion of individual leaves of two sunflower hybrids.

Common features in the time-course of expansion of leaves which considerably differed in final area, due to phytomer position, growing conditions and genotype, were identified. Leaf development consisted of two phases of exponential growth, followed by a third phase of continuous decrease of the relative expansion rate. The rate and the duration of the first exponential phase were common to all phytomers, growing conditions and genotypes. Leaves differed in the rate and the duration of the second exponential phase. The decrease of the relative expansion rate during the third phase depended on neither genotype nor growing conditions. It was phytomer-dependent and was deduced from the rate of the second phase via a parameter common to all cases studied. Differences in final leaf area among growing conditions were linked to different expansion rates during the second exponential phase. The duration of the phases at any given phytomer position was the same for the two hybrids in different growing conditions. The dates of developmental events (initiation, end of the two exponential phases, full expansion), and the rate of the second exponential phase, were related to phytomer position, defining a strict pattern of leaf development at the whole plant level. Using this framework simplified the analysis of the response of leaf expansion to genotype and environment.