White lupin has developed a complex strategy to limit microbial degradation of secreted citrate required for phosphate acquisition.

White lupins (Lupinus albus L.) respond to phosphate deficiency by producing special root structures called cluster roots. These cluster roots secrete large amounts of carboxylates into the rhizosphere, mostly citrate and malate, which act as phosphate solubilizers and enable the plant to grow in soils with sparingly available phosphate. The success and efficiency of such a P-acquisition strategy strongly depends on the persistence and stability of the carboxylates in the soil, a parameter that is influenced to a large extent by biodegradation through rhizosphere bacteria and fungi. In this study, we show that white lupin roots use several mechanisms to reduce microbial growth. The abundance of bacteria associated with cluster roots was decreased at the mature state of the cluster roots, where a burst of organic acid excretion and a drastic pH decrease is observed. Excretion of phenolic compounds, mainly isoflavonoids, induced fungal sporulation, indicating that vegetative growth, and thus potential citrate consumption, is reduced. In addition, the activity of two antifungal cell wall-degrading enzymes, chitinase and glucanase, were highest at the stage preceding the citrate excretion. Therefore, our results suggest that white lupin has developed a complex strategy to reduce microbial degradation of the phosphate-solubilizing agents.

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage.

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage. To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS). The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.

Rapid analysis of stilbenes and derivatives from downy mildew-infected grapevine leaves by liquid chromatography-atmospheric pressure photoionisation mass spectrometry.

Resveratrol, trans-epsilon-viniferin and trans-delta-viniferin are the major stilbenes induced in downy mildew infected grapevine leaves. In addition, nine minor polyphenolic compounds, described as stilbenes derivatives, have been separated and detected among known stilbenes after a methanolic microextraction of small pieces (1-2 mg) from infected grapevine leaves with a rapid, qualitative and optimized HPLC method coupled to mass spectrometry using atmospheric pressure photoionisation (APPI-MS(n)). The characterization of unknown stilbenic derivatives as six resveratrol dimers, two dimethylated resveratrol dimers and a resveratrol trimer are reported. Therefore, structures have been proposed for the dimethylated resveratrol dimers. Use of an easy sample treatment and the LC-APPI-MS(n) method results in spectral data of these minor naturally occurring viniferin analogues.

Delta-viniferin, a resveratrol dehydrodimer: one of the major stilbenes synthesized by stressed grapevine leaves.

Delta-viniferin is a resveratrol dehydrodimer, an isomer of epsilon-viniferin. This compound has been reported as a molecule produced in vitro by the oxidative dimerization of resveratrol by plant peroxidases or fungal laccases. It was also recently identified in wines and in grape cell cultures. We have now identified this dimer by NMR, high-performance liquid chromatography-diode array detection (HPLC-DAD), and HPLC-mass spectrometry (MS) in grapevine leaves infected by Plasmopara viticola (downy mildew) or UV-C irradiated. Its concentration was higher than that of epsilon-viniferin and constitutes one of the most important phytoalexins derived from resveratrol.

Differentiation of isomeric flavone/isoflavone aglycones by MS2 ion trap mass spectrometry and a double neutral loss of CO.

The fragmentation behaviour of seven pairs of isomeric flavone/isoflavone aglycones (solely hydroxylated and/or methoxylated) was studied using ion trap mass spectrometry with atmospheric pressure ionisation (API, both electrospray and APCI) in the positive and negative ion modes. A major difference was found in the neutral loss of 56 u, which was a common feature of all isoflavones in API(+). It was identified as a double loss of CO by accurate mass tandem mass spectrometric (MS/MS) measurements using a hybrid quadrupole time-of-flight (Q-TOF) instrument. Fragmentation of daidzein with (13)C-isotope labelling of the carbon C2 showed that this double loss occurred from the central ring of the molecule. A mechanism for this selective fragmentation is given. Further isoflavone-specific fragmentations were used to develop a guideline for the identification of isoflavone structures. A software-based neutral loss scan of 56 u in the API(+)-MS(2) mode was applied to extracts of leaves of Lupinus albus and to soy flour. The structure elucidation guideline allowed identification of hydroxy and/or methoxy isoflavones. Structures could be confirmed for those available as reference compounds.