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Soluble sugars such as sucrose, glucose and fructose in plant host cells not only play the role as donors of carbon skeletons, but they may also induce metabolic signals influencing the expression of defense genes. These metabolites function in a complex network with many bioactive molecules, which independently or in dialogue, induce successive defense mechanisms. The aim of this study was to determine the involvement of sucrose and monosaccharides as signaling molecules in the regulation of the levels of phytohormones and hydrogen peroxide participating in the defense responses of Lupinus luteus L. to a hemibiotrophic fungus Fusarium oxysporum Schlecht f. sp. lupini . A positive correlation between the level of sugars and postinfection accumulation of salicylic acid and its glucoside, as well as abscisic acid, was noted. The stimulatory effect of sugars on the production of ethylene was also reported. The protective role of soluble sugars in embryo axes of yellow lupine was seen in the limited development of infection and fusariosis. These results provide evidence for the enhanced generation of signaling molecules both by sugar alone as well as during the crosstalk between sugars and infection caused by F. oxysporum . However, a considerable postinfection increase in the level of these signaling molecules under the influence of sugars was recorded. The duration of the postinfection generation of these molecules in yellow lupine was also variable.

The aim of the study was to examine cross-talk interactions of soluble sugars (sucrose, glucose and fructose) and infection caused by Fusarium oxysporum f.sp. lupini on the synthesis of genistein in embryo axes of Lupinus luteus L.cv. Juno. Genistein is a free aglycone, highly reactive and with the potential to inhibit fungal infection and development of plant diseases. As signal molecules, sugars strongly stimulated accumulation of isoflavones, including genistein, and the expression of the isoflavonoid biosynthetic genes. Infection significantly enhanced the synthesis of genistein and other isoflavone aglycones in cells of embryo axes of yellow lupine with high endogenous sugar levels. The activity of β-glucosidase, the enzyme that releases free aglycones from their glucoside bindings, was higher in the infected tissues than in the control ones. At the same time, a very strong generation of the superoxide anion radical was observed in tissues with high sugar contents already in the initial stage of infection. During later stages after inoculation, a strong generation of semiquinone radicals was observed, which level was relatively higher in tissues deficient in sugars than in those with high sugar levels. Observations of actin and tubulin cytoskeletons in cells of infected embryo axes cultured on the medium with sucrose, as well as the medium without sugar, showed significant differences in their organization.

The aim of the study was to examine cross-talk of exogenous nitric oxide (NO) and sucrose in the mechanisms of synthesis and accumulation of isoflavonoids in embryo axes of Lupinus luteus L. cv. Juno. It was verified whether the interaction of these molecules can modulate the defense response of axes to infection and development of the pathogenic fungus Fusarium oxysporum f. sp. lupini. Sucrose alone strongly stimulated a high level of genistein glucoside in axes pretreated with exogenous nitric oxide (SNP or GSNO) and non-pretreated axes. As a result of amplification of the signal coming from sucrose and GSNO, high isoflavonoids accumulation was observed (+Sn+GSNO). It needs to be stressed that infection in tissues pretreated with SNP/GSNO and cultured on the medium with sucrose (+Si+SNP/+Si+GSNO) very strongly enhances the accumulation of free isoflavone aglycones. In +Si+SNP axes phenylalanine ammonia-lyase activity was high up to 72h. As early as at 12h in +Si+SNP axes an increase was recorded in gene expression level of the specific isoflavonoid synthesis pathway. At 24h in +Si+SNP axes a very high total antioxidant capacity dependent on the pool of fast antioxidants was noted. Post-infection generation of semiquinone radicals was lower in axes with a high level of sucrose than with a deficit., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

This study investigated the effects of cross-talk interactions of sucrose and infection caused by a pathogenic fungus Fusarium oxysporum f.sp. lupini on the regulation of the phenylpropanoid pathway, i.e. the level of expression of genes encoding enzymes participating in flavonoid biosynthesis, as well as cell location and accumulation of these compounds in embryo axes of Lupinus luteus L. cv. Polo. Embryo axes, both non-inoculated and inoculated, were cultured for 96h on Heller medium with 60mM sucrose (+Sn and +Si) or without it (-Sn and -Si). Real-time RT-PCR to assess expression levels of the flavonoid biosynthetic genes, phenylalanine ammonialyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI) and isoflavone synthase (IFS) were used. Sucrose alone strongly stimulated the expression of these genes. There was a very high expression level of these genes in +Si embryo axes in the early phase of infection. Signal amplification by sucrose and the infection was most intense in the 48-h +Si axes, resulting in the highest level of expression of flavonoid biosynthetic genes. In -Si tissues, the expression level of these genes increased at 48 and 72h after inoculation relative to 24h; however, the relative level of expression was much lower than in +Si axes, except at 72h for PAL and CHS.Moreover, at 48h of culture, considerably higher activity of CHI (EC 5.5.1.6) was observed in axes with a high level of sucrose than in those with a sucrose deficit. CHI activity in +Si axes at 48 and 96h post-inoculation was over 1.5 and 2 times higher than that in +Sn axes, as well as higher than in -Si axes.Observations of yellow lupine embryo axes under a confocal microscope showed an increased post-infection accumulation of flavonoids, particularly in cells of embryo axes infected with F. oxysporum and cultured on a medium containing sucrose (+Si). Up to 48h post-infection in +Si axes, a very intensive emission of green fluorescence was observed, indicating high accumulation of these compounds in whole cells. Moreover, a nuclear location of flavonoids was recorded in cells. Strong staining of flavonoid end products in +Si embryo axes was consistent with the expression of PAL, CHS, CHI and IFS.These results indicate that, in the early phase of infection, the flavonoid biosynthesis pathway is considerably enhanced in yellow lupine embryo axes as a strong signal amplification effect of sucrose and the pathogenic fungus F. oxysporum., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

This study was designed to investigate whether and to what extent oxidative stress is induced in embryo axes of Lupinus luteus L. cv. Polo inoculated with a necrotrophic fungus, Fusarium oxysporum and cultured on Heller medium for 96h. Four variants were compared: inoculated embryo axes cultured with 60mM sucrose (+Si) or without it (-Si), and non-inoculated embryo axes cultured with 60mM sucrose (+Sn) or without it (-Sn). After inoculation, an accumulation of stable free radicals and Mn2+ ions in +Si and -Si were detected by electron paramagnetic resonance. Concentrations of the radicals with g-values of 2.0052+/-0.0004 and 2.0029+/-0.0003 were generally higher in -Si than in +Si. Beginning at 24h after inoculation, in both +Si and -Si the concentrations of these ions decreased, but more strongly in -Si than in +Si. After inoculation, the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) were higher in -Si than in +Si. SOD and CAT zymograms showed that the synthesis of new isoforms was induced after inoculation. Simultaneously, superoxide anions were assayed in embryo axes by using their specific indicator dihydroethidium (DHE). The DHE-derived fluorescence was stronger and covered a much larger tissue area in +Si than in -Si. The respiration rate was generally much higher in +Si than in -Si. Electron micrographs revealed that, in contrast to -Si cells, +Si cells had numerous mitochondria with less reduced numbers of cristae and long sections of rough endoplasmic reticulum and Golgi bodies. These results indicate that different defensive strategies against F. oxysporum were induced depending on soluble sugar levels in yellow lupine embryo axes.

Peroxidase activity (EC 1.11.1.7) towards phenolic substrates, i.e. pyrogallol, syringaldazine and guaiacol, and ascorbate peroxidase activity (EC 1.11.1.11) were analyzed in embryo axes of Lupinus luteus L. cv. Polo cultured on Heller medium for 96h after inoculation with the necrotrophic fungus Fusarium oxysporum f.sp. Schlecht lupini. Four variants were compared: inoculated embryo axes cultured with 60mM sucrose (+Si) or without it (-Si), and non-inoculated embryo axes cultured with 60mM sucrose (+Sn) or without it (-Sn). Between 0 and 96h of culture, peroxidase activity towards the phenolic substrates increased in all variants except -Si, where a decrease was noted in peroxidase activity towards syringaldazine and guaiacol, but not towards pyrogallol. In +Si tissues, a considerable increase in enzyme activity towards these substrates was recorded starting from 72h of culture. Lignin content of +Si tissues increased already at the first stage of infection, i.e. 24h after inoculation. Additionally, in +Sn tissues, high ascorbate peroxidase activity was observed during the culture. Its activity increased in +Si tissues, beginning at 72h after inoculation. However, this was lower than in +Sn tissues. At 72h after inoculation, a considerably stronger development of the infection was observed in -Si than in +Si tissues during our earlier research [Morkunas, I. et al., 2005. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005; 43: 363-73]. Both peroxidases assayed towards phenolic substrates and ascorbate peroxidase was less active in -Si tissues than in -Sn tissues. Hydrogen peroxide concentration was much higher in -Si than in +Si tissues. These results indicate that peroxidases may be some of the elements of the defense system that are stimulated by sucrose in yellow lupine embryo axes in response to infection caused by F. oxysporum.

Defense responses to inoculation with Fusarium oxysporum SCHLECHT f. sp. lupini were studied in embryo axes of Lupinus luteus L. cv. Polo cultured on a medium with sucrose (60 mM) or without it. Exogenous sucrose caused a marked endogenous increase in concentrations of sucrose, glucose and fructose in embryo axes. In axes cultured with sucrose, high performance liquid chromatography (HPLC) revealed generally higher levels of isoflavone glycosides (particularly until 48 h of culture) and free aglycones (genistein, wighteone, luteone). Inoculation resulted in a considerable decline in soluble carbohydrates between 24 and 72 h of culture. Simultaneously, the infection stimulated an increase in the level of free isoflavone aglycones in inoculated embryo axes, as compared to non-inoculated ones. Concentrations of free aglycones (i.e. genistein, wighteone and luteone) after infection were particularly high in inoculated embryo axes fed with sucrose. Genistein was a better inhibitor to F. oxysporum growth than genistein 7-O-glucoside tested. Exogenous sucrose also stimulated the activity of phenylalanine ammonialyase (PAL, EC 4.3.1.5)--an important enzyme initiating phenylpropanoid metabolism. After infection of tissues, a strong increase was observed in the activity of PAL and beta-glucosidase (EC 3.2.1.21)--an enzyme hydrolyzing isoflavone glycosides. Furthermore, the growth of inoculated embryo axes cultured with sucrose was less inhibited as a result of infection than inoculated axes cultured under carbohydrate deficiency conditions. Additionally, it had been reported previously that disease symptoms of embryo axes growing in the presence of sucrose were less intensive [30]. These results suggest that soluble sugars are involved in the mechanism of resistance, as they can stimulate phenylpropanoid metabolism and contribute to the increase in concentration of isoflavonoids, which are important elements of the defense system of legumes.

Defence responses of embryo axes of Lupinus luteus L. cv. Polo were studied 48-96 h after inoculation with Fusarium oxysporum Schlecht f.sp. lupini. The infection restricted the growth of embryo axes, the lengths of infected embryo axes 72 and 96 h after inoculation were 11 and 12 mm less in the controls, respectively, while their masses c. 0.03 g less than in the controls. The concentration of H2O2 in embryo axes of inoculated germinating seeds was higher than in the control. This was probably a consequence of oxidative burst as well as H2O2 generation by the invading necrotrophic fungal pathogen. EPR-based analyses detected the presence of free radicals with g1 and g2 values of 2.0052 +/- 0.0004 and 2.0031 +/- 0.0005, respectively. Concentrations of the radicals 72 and 96 h after inoculation were 50% higher than in the control. The values of the spectroscopic splitting coefficients suggest that they are quinone radicals. However, inoculated embryo axes possess a number of adaptive mechanisms protecting them from oxidative damage. A twofold increase in catalase (CAT, EC 1.11.1.6) activity was evidenced in embryo axes infected with F. oxysporum Schlecht f. sp. lupini, as compared to the control 48-96 h after inoculation. Superoxide dismutase (SOD, EC 1.15.1.1) activity 96 h after inoculation was 80% higher than in the control. Furthermore, EPR-based analyses revealed a higher concentration of Mn2+ ions after 72 h for inoculated embryo axes, as compared to the control. On the other hand, no increase was detected in the concentration of thiobarbituric acid reactive substances (products of lipid peroxidation) in infected embryo axes. The protective mechanisms induced in lupine embryo axes in response to F. oxysporum Schlecht f.sp. lupini were compared with responses to infections with pathogenic fungi elicited in other plant families.

The level of free radicals and activities of antioxidative enzymes were examined in roots of lupine seedlings (Lupinus luteus L.) that were deprived of oxygen by subjecting them to root hypoxia for 48 and 72 h and then re-aerated for up to 24 h. Using electron paramagnetic resonance (EPR), we found that the exposure of previously hypoxically grown roots to air caused the increase in free radicals level, irrespective of duration of hypoxic pretreatment. Immediately after re-aeration the level of free radicals was two times higher than in aerated control. The EPR signal with the g-values at the maximum absorption of 2.0057 and 2.0040 implied that the paramagnetic radicals are derived from a quinone. Directly after re-aeration of hypoxically pretreated roots, the activity of superoxide dismutase (SOD, EC 1.15.1.1) increased to its highest value, followed by a decline below the initial level, whereas activities of catalase (CAT, EC 1.11.1.6) and peroxidase (POX, EC 1.11.1.7) were diminished or only slightly influenced during re-aeration. The electrophoretic patterns of the soluble extracts show 4 isozymes of SOD, 4 isozymes of POX and 1 isozyme of CAT. The level of H2O2 was enhanced or lowered by re-aeration, depending on the previous duration of hypoxia. At the onset of re-aeration products of lipid peroxidation were present at a three-fourth of the levels found in aerobic control. Their levels increased after prolonged exposure to air but remained lower than those in aerobic control even after 24 h of re-aeration. Re-admission of oxygen resulted in about 20% rise in oxygen uptake by root axes segments immediately after transfer of roots from hypoxia and the high uptake rates were observed over whole re-aeration period. Oxygen consumption by root tips was significantly reduced just after transfer from hypoxic conditions as compared to aerated control but after 24 h of re-aeration even approached the control level. The results are discussed in relation to the ability of lupine roots to cope with oxidative stress caused by re-aeration following hypoxic pretreatment.

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.