Monique Berger

The isoflavones in the cotyledon of soybean seeds mimic human estrogen in structure, conferring them complex effects on health. Their regulation represents a major challenge for the sustainable breeding of new varieties with lower levels of potential endocrine disruptors. To develop a rapid, nondestructive, and eco-friendly analysis method, this study explores how sample grinding affects the results of near-infrared spectroscopy (NIRS) and the preprocessing methods. The prediction of the daidzein and genistein content would help the specific reduction in isoflavones in the cotyledon without harming seed development. The potential of a nonlinear approach (artificial neural network) is also compared with the more conventional partial least squares (PLS) regression. The isoflavone content of cotyledons from 529 soybean samples (65 genotypes) was quantified by HPLC, and the NIR spectra of these samples were collected using a Brucker multi-purpose analyzer. The spectra of whole and ground cotyledons were also collected for 155 samples. The results show that grain fragmentation improves the model calibration, although spectral preprocessing can harmonize this effect. Although the best PLS regression in cross-validation did not suffice to quantify the daidzein and genistein percentages, the artificial neural network (ANN) approach allowed us to develop much more reliable models than PLS. The performance of ANNs in external validation is remarkable in terms of both precision and applicability (R² = 0.89 and a ratio of prediction to deviation of 2.92), making ANNs suitable in the breeding context for screening soybean grains regarding their isoflavone content.

Glycosylation is a key modification that contributes to determine bioactivity and bioavailability of plant natural products, including that of terpenoids and steviol glycosides (SVglys). It is mediated by uridine-diphosphate glycosyltransferases (UGTs), that achieve their activity by transferring sugars on small molecules. Thus, the diversity of SVglys is due to the number, the position and the nature of glycosylations on the hydroxyl groups in C-13 and C-19 of steviol. Despite the intense sweetener property of SVglys and the numerous studies conducted, the SVglys biosynthetic pathway remains largely unknown. More than 60 SVglys and 68 putative UGTs have been identified in Stevia rebaudiana. This study aims to provide methods to characterize UGTs putatively involved in SVglys biosynthesis. After agroinfiltration-based transient gene expression in Nicotiana benthamiana, functionality of the recombinant UGT can be tested simply and directly in plants expressing it or from a crude extract. The combined use of binary vectors from pGWBs series to produce expression vectors containing the stevia's UGT, enables functionality testing with many substrates as well as other applications for further analysis, including subcellular localization.

OCL, Oilseeds and fats, Crops and Lipids

The need for medicinal and aromatic plants for industrial uses creates an opportunity for farmers to produce alternative crops. Stevia rebaudiana Bertoni, a perennial shrub originating from Paraguay, is of increasing interest as a source of zero-calorie natural sweeteners: the steviol glycosides (SVglys). The aim of this study was to investigate the relevance of nitrogen (N) supply for leaf yield and for SVgly concentrations in leaves, which are the two major components of S. rebaudiana productivity. In this regard, the relationship between leaf N concentration, CO₂ assimilation, leaf production and SVgly accumulation was investigated. The experiments were conducted consecutively in growth-chamber (CC: controlled conditions), in greenhouse (SCC: semi-controlled conditions) and in field conditions (FC) on two genotypes. In CC and SCC, three levels of N fertilization were applied. Plants were grown on four locations in the FC experiment. Both N supply (CC and SCC) and location (FC) had a significant effect on N content in leaves. When light was not limiting (SCC and FC) N content in leaves was positively correlated with CO₂ assimilation rate and biomass accumulation. Irrespective of the growth conditions, N content in leaves was negatively correlated with SVgly content. However, increased SVgly content was correlated with a decreased ratio of rebaudioside A over stevioside. The evidence that the increased SVgly accumulation compensates for the negative effect on biomass production suggests that adequate SVgly productivity per plant may be achieved with relatively low fertilization.

Soybean seeds accumulate large amounts of isoflavones (genistein, daidzein and glycitein), secondary metabolites known for their phytoestrogenic activities. Isoflavone composition depends on the seed part and glycitein is almost found exclusively in hypocotyls. Moreover, two major phenotypes are encountered in soybean cultivars, with either low (35 %) or high (55 %) levels of glycitein in their hypocotyls. This trait was under a quasi-mendelian heredity, implicating at most one or two genes. A CYP71D9 cDNA displaying a flavonoid 6-hydroxylase (F6H) activity had previously been isolated from elicitor-induced soybean (Glycine max L.) cells. This enzyme allows the synthesis of the glycitein flavanone intermediate (6,7,4′- trihydroxyflavanone) by catalyzing the A-ring hydroxylation of liquiritigenin. In this study, the CYP71D9 gene (F6H1) and two other candidates (F6H2 and F6H3) were studied using contrasted soybean cultivars for glycitein content (0, 35, 55 and 80 %). Their expression was observed in chitosan elicited leaves. They encode P450 proteins of 496, 469 and 481 amino acids respectively and were expressed in leaves with or without elicitation. The expression patterns of these three genes were performed in cotyledons and hypocotyls at different developmental stages. F6H1 and F6H2 were not expressed in the developing seed. F6H3 was only expressed in hypocotyls. Its expression levels did not correlate with hypocotyls glycitein content, but it was not expressed in the null mutant for glycitein. Thus, this F6H3 gene is a good potential candidate for glycitein biosynthesis in soybean seed.

Soybean [Glycine max (L.) Merr.] seed accumulates large amounts of isoflavones, which are known for their phytoestrogenic activities. Three isoflavones (genistein, daidzein, and glycitein) are found in the seed. They are 4 to 10 times more concentrated in hypocotyls than in cotyledons. Isoflavone composition markedly diverges between these two seed parts and their accumulation begins in cotyledons at 40 d after flowering (DAF) once completed in hypocotyls. The relation between the genes of the isoflavonoid pathway and the isoflavone content has been investigated through (i) the variability of isoflavone synthase (IFS) genomic sequences in six cultivars with contrasted isoflavone content and (ii) the expression of key genes in two cultivars at 25, 40, and 60 DAF in hypocotyls and cotyledons. IFS1 and IFS2 polymorphism was not related to the isoflavone content. The expression of chalcone synthases (CHS7 and CHS8) was related to isoflavone accumulation kinetics in the cotyledons but not with the total content at maturity. CHS9 was the most expressed CHS isoform and the only one correlated with the hypocotyl isoflavone accumulation kinetics. The chalcone reductase (CHR) and CHI expression profiles were significantly different between the two seed parts. These results indicate that the regulation of the isoflavone pathway is controlled differently in cotyledons and hypocotyls during seed maturation. This discrepancy should be taken into account in the studies on genetic and environmental effects on isoflavone contents.

Sunflower oil is widely consumed in France and Spain. This oil has a good reputation among consumers, due to its high polyunsaturated fatty acid content. Thirty years after its discovery, the oleic mutation (high C18:1 contents) has now reached agricultural production, with fast increasing surfaces for the last three years. The challenge is now the production of very high oleic (> 90%) sunflowers and new fatty acid profiles, such as high linoleic, palmitic, stearic or stearic + oleic hybrids, which are of high interest for chemistry or food industry. The problem is now the stability of the expression in varying genetic backgrounds, and the understanding of environmental effects. Minor components are also a main strength of sunflower oil: its high tocopherols (Vitamin E) content (0,5 to 1%) is all the more interesting for nutritionists that they be almost exclusively α-tocopherol (> 95%), which is the most vitaminic active component. Nowadays, the unraveling of tocopherols biosynthesis and its key points of regulation is in progress. New tocopherol profiles have been recently obtained. Higher protective effects are attributed to γ- or δ-tocopherols. They could increase the stability of oils or margarines. Genetic progress for total content is possible, as in sunflower like in other oilseed crops, heritability is rather high (> 0.7). Sunflower oil also contains twice as much phystosterols as olive oil (3 vs 1,5%), and mainly β-sitosterol. Major mutations of this pathway can induce penalizing phenotypic modifications: 1) campesterol is the precursor of plant hormones brassinosteroids, and 2) stigmasterol/campesterol ratio is also implied in plant growth regulation. However, hybrids can significantly differ in their total content. Either for fatty acids or minor components, breeding programs for new profiles, or higher contents, need high throughout oil extraction and analysis. Chemometric analysis offer interesting solutions despite their need of large sets of reference analysis