A high-throughput seed germination assay for root parasitic plants.

BACKGROUND: Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope. RESULTS: We developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann's protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose-response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves. CONCLUSION: The developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination.

Germination stimulants of Phelipanche ramosa in the rhizosphere of Brassica napus are derived from the glucosinolate pathway.

Phelipanche ramosa is a major parasitic weed of Brassica napus. The first step in a host-parasitic plant interaction is stimulation of parasite seed germination by compounds released from host roots. However, germination stimulants produced by B. napus have not been identified yet. In this study, we characterized the germination stimulants that accumulate in B. napus roots and are released into the rhizosphere. Eight glucosinolate-breakdown products were identified and quantified in B. napus roots by gas chromatography-mass spectrometry. Two (3-phenylpropanenitrile and 2-phenylethyl isothiocyanate [2-PEITC]) were identified in the B. napus rhizosphere. Among glucosinolate-breakdown products, P. ramosa germination was strongly and specifically triggered by isothiocyanates, indicating that 2-PEITC, in particular, plays a key role in the B. napus-P. ramosa interaction. Known strigolactones were not detected by ultraperformance liquid chromatography-tandem mass spectrometry, and seed of Phelipanche and Orobanche spp. that respond to strigolactones but not to isothiocyanates did not germinate in the rhizosphere of B. napus. Furthermore, both wild-type and strigolactone biosynthesis mutants of Arabidopsis thaliana Atccd7 and Atccd8 induced similar levels of P. ramosa seed germination, suggesting that compounds other than strigolactone function as germination stimulants for P. ramosa in other Brassicaceae spp. Our results open perspectives on the high adaptation potential of root-parasitic plants under host-driven selection pressures.

A detailed survey of seed coat flavonoids in developing seeds of Brassica napus L.

Proanthocyanidins (PAs) are seed coat flavonoids that impair the digestibility of Brassica napus meal. Development of low-PA lines is associated with a high-quality meal and with increased contents in oil and proteins, but requires better knowledge of seed flavonoids. Flavonoids in Brassica mature seed are mostly insoluble so that very few qualitative and quantitative data are available yet. In the present study, the profiling of seed coat flavonoids was established in eight black-seeded B. napus genotypes, during seed development when soluble flavonoids were present and predominated over the insoluble forms. Thirteen different flavonoids including (-)-epicatechin, five procyanidins (PCs which are PAs composed of epicatechin oligomers only) and seven flavonols (quercetin-3-O-glucoside, quercetin-dihexoside, isorhamnetin-3-O-glucoside, isorhamnetin-hexoside-sulfate, isorhamnetin-dihexoside, isorhamnetin-sinapoyl-trihexoside and kaempferol-sinapoyl-trihexoside) were identified and quantified using liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS(n)). These flavonol derivatives were characterized for the first time in the seed coat of B. napus, and isorhamnetin-hexoside-sulfate and isorhamnetin-sinapoyl-trihexoside were newly identified in Brassica spp. High amounts of PCs accumulated in the seed coat, with solvent-soluble polymers of (-)-epicatechin reaching up to 10% of the seed coat weight during seed maturation. In addition, variability for both PC and flavonol contents was observed within the panel of eight black-seeded genotypes. Our results provide new insights into breeding for low-PC B. napus genotypes.

Brassica orthologs from BANYULS belong to a small multigene family, which is involved in procyanidin accumulation in the seed.

As part of a research programme focused on flavonoid biosynthesis in the seed coat of Brassica napus L. (oilseed rape), orthologs of the BANYULS gene that encoded anthocyanidin reductase were cloned in B. napus as well as in the related species Brassica rapa and Brassica oleracea. B. napus genome contained four functional copies of BAN, two originating from each diploid progenitor. Amino acid sequences were highly conserved between the Brassicaceae including B. napus, B. rapa, B. oleracea as well as the model plant Arabidopsis thaliana. Along the 200 bp in 5' of the ATG codon, Bna.BAN promoters (ProBna.BAN) were conserved with AtANR promoter and contained putative cis-acting elements. In addition, transgenic Arabidopsis and oilseed rape plants carrying the first 230 bp of ProBna.BAN fused to the UidA reporter gene were generated. In the two Brassicaceae backgrounds, ProBna.BAN activity was restricted to the seed coat. In B. napus seed, ProBna.BAN was activated in procyanidin-accumulating cells, namely the innermost layer of the inner integument and the micropyle-chalaza area. At the transcriptional level, the four Bna.BAN genes were expressed in the seed. Laser microdissection assays of the seed integuments showed that Bna.BAN expression was restricted to the inner integument, which was consistent with the activation profile of ProBna.BAN. Finally, Bna.BAN genes were mapped onto oilseed rape genetic maps and potential co-localisations with seed colour quantitative trait loci are discussed.

The promoter of the Arabidopsis thaliana BAN gene is active in proanthocyanidin-accumulating cells of the Brassica napus seed coat.

As part of an ongoing research program dedicated to the understanding of proanthocyanidin (PA) accumulation in Brassica napus seed coat, transgenic rapeseed plants carrying a 2.3-kb fragment of the Arabidopsis thaliana BAN promoter (ProAtBAN) fused to the uidA reporter gene (GUS) were generated. Analysis of these plants revealed that ProAtBAN was activated in B. napus seed coat, following a spatio-temporal pattern that was very similar to the PA deposition profile in rapeseed and also to the one previously described in Arabidopsis. ProAtBAN activity occurred as soon as the early stages of embryogenesis and was restricted to the cells where PAs were shown to accumulate. Therefore, the Arabidopsis BAN promoter can be used to trigger gene expression in B. napus seed coat for both genetic engineering and functional validation of candidate genes. In addition, these data strongly suggest that the transcriptional regulatory network of the BAN gene is conserved between Arabidopsis and rapeseed. This is consistent with the fact that similarity searches of the public rapeseed sequence databases allowed recovering the rapeseed homologs for several BAN regulators, namely TT1, TT2, TT8, TT16 and TTG1, which have been previously described in Arabidopsis.