Changes in carbohydrate and isoflavonoid metabolism in yellow lupine in response to infection by Fusarium oxysporum during the stages of seed germination and early seedling growth

Little biochemical information is available regarding the responses of embryo axes to Fusarium oxysporum during the stages of seed germination and early seedling growth of yellow lupine, i.e. in the heterotrophic phase and during the transition from the heterotrophic to the autotrophic phase [1]. In the experimental protocol used in this study germinated yellow lupine seeds, both non-inoculated and inoculated with F. oxysporum, were cultured for 96 h in perlite.An analysis of soluble carbohydrates, between 48 and 96 h of culture, shows a decrease in sugar content, i.e. that of sucrose and fructose in infected embryo axes of germinated seeds and that of sucrose and glucose in the controls at 72 h. After infection, lower levels of sucrose, glucose and fructose were observed in infected axes than in controls. Invertase activity (EC 3.2.1.26) in infected tissues was higher than in the controls with the exception of acid invertase activity at 96 h. The highest post-infection alkaline and acid invertase activity was recorded in 48 h embryo axes of germinated seeds. An HPLC analysis in the period between 48 and 96 h after infection showed a decrease in the level of glycosylated isoflavonoids, i.e. genistein 7-O-glucoside, 2′OH genistein 7-O-glucoside, genistein 4′,7-O-diglucoside, 2′OH genistein malonyl glucoside and 2′OH genistein malonyl glucoside, which was particularly marked at 72 h after infection. Concentrations of these glucosides in 72 h and 96 h infected tissues were lower than in controls. We need to stress here a very high isoflavonoid content observed in intact embryo axes of germinating seeds at 48 h after inoculation, i.e. shortly after the embryo axes penetrated the seed coat. At this time point in infected tissues the concentrations of genistein glucosides and that of a free aglycone, genistein, were many times higher than in the controls. At later time points after inoculation genistein content was also higher than in the controls. A different tendency to that observed in infected axes was seen in the control specimens, where the level of flavonoid glucosides rose in the period between 48 and 72 h of culture, followed by a subsequent decrease. The activity of phenylalanine ammonialyase (PAL, EC. 4.3.1.5) in infected embryo axes of germinating seeds was higher than in the controls and the highest post-infection activity of this enzyme was observed in 48 h axes. At this time point, a considerable post-infection accumulation was observed using semi-quantitative RT-PCR for transcripts of phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS), which was consistent with the high level of isoflavonoids.These results indicate that in yellow lupine during the stages of seed germination and early seedling growth changes in carbohydrate and isoflavonoid metabolism occur in embryo axes in response to infection with F. oxysporum f.sp. lupini. These changes may be included in the active protection mechanism in these axes.

Research highlights
► This paper regarding the responses of embryo axes to Fusarium oxysporum during the stages of seed germination and early seedling growth of yellow lupine, that is, in the heterotrophic phase and during the change from the heterotrophic to the autotrophic phase.
► There is very little in the published literature concerning the responses of embryo axes to infection caused pathogenic fungi during stages of seed germination and early seedling growth.
► This stage is unusually significant to the ontogenetic development of plants because, during this phase, before the developing seeds are above the soil surface, the plants are carbon heterotrophs.
► This study supplies additional new information about the responses of seed germination to the pathogenic fungus F. oxysporum, which is a facultative parasite.
► The presented results suggest that, in germinating seeds of the Polo variety of yellow lupine, the metabolism of isoflavonoids is stimulated within 48 h of inoculation, that is, by the time the seed coat is breached and also that a high level of phenylalanine ammonialyase (PAL) may be noted along with accumulation of genistein glucoside, genistein itself and the transcription products of the genes for phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerasae (CHI) and isoflavone synthase (IFS).
► Results of analyses of free aglycones in this study suggest that the profile of these compounds is affected by the presence of cotyledons during embryo axe growth, i.e. close contact between the sink and donor tissues, nutrients and the manner of inoculation.
► Besides these results this study also suggests that the level of soluble carbohydates in germinating embryo axes is not without significance, in particular during the heterotrophic phase, because it may have effects on the mobilization of defence mechanisms aimed at reducing the effects of pathogenic fungi and on the retardation their development.
► A comparison of post-infection changes in carbohydrate and isoflavonoid metabolism in the model system, i.e. in embryo axes, which were cut off from the natural source of carbon, i.e. cotyledons, and they were supplied exogenous sucrose (Morkunas et al. 2005), as well changes occurring in intact embryo axes of germinating yellow lupine embryo axes growing under conditions resembling natural conditions, thus the experimental design applied in the presented study will facilitate better understanding of how significant is carbohydrate level in sink tissues, i.e. the embryo axe of the germinating seed, for defense response triggered against a pathogenic fungus and will make it possible to follow changes in the above-mentioned biochemical indicators during heterotrophic and autotrophic phases of seed germination.