Visualization of symbiotic tissue in intact root nodules of Vicia tetrasperma using GFP-marked Rhizobium leguminosarum bv. viciae.

In rhizobial symbiosis with legume plant hosts, the symbiotic tissue in the root nodules of indeterminate type is localized to the basal part of the nodule where the symbiotic zones contain infected cells (IC) interspersed with uninfected cells (UC) that are devoid of rhizobia. Although IC are easily distinguished in nodule sections using standard histochemical techniques, their observation in intact nodules is hampered by nodule tissue characteristics. Tagging of Rhizobium leguminosarum bv. viciae strain 128C30 with a constitutively expressed gene for green fluorescent protein (nonshifted mutant form cycle3) in combination with the advantages of the tiny nodules formed by Vicia tetrasperma (L.) SCHREB . allowed for vital observation of symbiotic tissue using fluorescence microscopy. Separation of a red-shifted background channel and digital image stacking along z-axis enabled us to construct a nodule image in a classical fluorescence microscopy of nodules exceeding 1 mm in diameter. In parallel, visualization of nodule bacteria inside the symbiotic tissue by confocal microscopy at the excitation wavelength 488 nm clearly distinguished IC/UC pattern in the nodule virtual sections and revealed red-shifted fluorescence of nonrhizobial origin. This signal was located on the periphery of IC and increased with their degradation, thus suggesting accumulation of secondary metabolites, presumably flavonoids. The simultaneous detection of bacteria and secondary metabolites can be used for monitoring changes to intact nodule physiology in the model legumes. The advantage of V. tetrasperma as a suggested laboratory model for pea cross-inoculation group has been demonstrated.

Visualization of nodulation gene activity on the early stages of Rhizobium leguminosarum bv. viciae symbiosis.

A technique was optimized for the in situ detection of nodulation (nod) gene activity in Rhizobium leguminosarum bv. viciae symbiosis with compatible plant hosts Vicia tetrasperma (L.) SCHREB. and Pisum sativum L. The transcription of nodABC-lacZ fusion was visualized as beta-galactosidase (beta-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the background of the indigenous beta-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment. V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model of V. hirsuta (L.) S.F. GRAY: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent of nod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the pea sym8 locus. Judging from the nod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.

Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.).

Selected flavonoids that are known as inducers and a suppressor of nodulation (nod) genes of the symbiotic bacterium Rhizobium leguminosarum bv. viciae were tested for their effect on symbiosis formation with garden pea as the host. A solid substrate was omitted from the hydroponic growing system in order to prevent losses of flavonoids due to adsorption and degradation. The presumed interaction of the tested flavonoids with nod genes has been verified for the genetic background of strain 128C30. A stimulatory effect of a nod gene inducer naringenin on symbiotic nodule number formed per plant 14 d after inoculation was detected at concentrations of 0.1 and 1 micro g ml(-1) nutrient solution. At 10 micro g ml(-1), the highest concentration tested, naringenin was already inhibitory. By contrast, nodulation was negatively affected by a nod gene suppressor, quercetin, at concentrations above 1 micro g ml(-1), as well as by another tested nod gene inducer, hesperetin. The deleterious effect of hesperetin might be due to its toxicity or to the toxicity of its degradation product(s) as indicated by the inhibition of root growth. Both the stimulatory effect of naringenin and the inhibitory effect of quercetin on nodule number were more pronounced at earlier stages of nodule development as revealed with specific staining of initial nodules. The lessening of the flavonoid impact during nodule development was ascribed to the plant autoregulatory mechanisms. Feedback regulation of nodule metabolism might also be responsible for the fact that the naringenin-conditioned increase in nodule number was not accompanied by any increase in nitrogenase activity. By contrast, the inhibitory action of quercetin and hesperetin on nodule number was associated with decreases in total nitrogenase activity. Naringenin also stimulated root hair curling (RHC) as one of the earliest nodulation responses at concentrations of 1 and 10 microg ml(-1), however, the same effect was exerted by the nod gene suppressor, quercetin, suggesting that feedback regulatory mechanisms control RHC in the range of nodulation-inhibiting high flavonoid concentrations. The comparison of the effect of the tested flavonoids in planta with nod gene activity response showed a two orders of magnitude shift to higher concentrations. This shift is explained by the absorption and degradation of flavonoids by both the symbionts during 3 d intervals between hydroponic solution changes. The losses were 99, 96.4, and 90% of the initial concentration of 10 micro g ml(-1) for naringenin, hesperetin, and quercetin, respectively.

Glutamate decarboxylase activity in Trichoderma viride conidia and developing mycelia.

Glutamic acid decarboxylase (GAD) activity was measured in homogenates of conidia and both submerged and aerial mycelia of Trichoderma viride. The GAD activity in conidia had a temperature optimum at 30 degrees C and a pH optimum at pH 4. GAD was stimulated by EDTA (2 mM) and was insensitive to treatment with calmodulin antagonists calmidazolium (10 microM) or phenothiazine neuroleptics (60 microM). Cyclosporin A (up to 300 microM) partially inhibited GAD in the homogenate, but not in the supernatant obtained after centrifuging the homogenate. Attempts to release GAD activity from the homogenate using high ionic strength, detergents, or urea failed. Freezing-thawing led to the partial increase of activity in the conidial homogenate. These results indicate that GAD is a membrane-bound enzyme. The highest specific activity of GAD was present in the mitochondrial/vacuolar organellar fraction. Germination of conidia in the submerged culture led to a temporary decrease in GAD activity. After prolonged cultivation, the activity displayed quasi-oscillatory changes. The stationary state was characterized by a high GAD activity. The presence of gamma-aminobutyric acid in the submerged mycelia was demonstrated. In surface culture in the dark, GAD activity increased in a monophasic manner until conidia formation. The illumination of dark-cultivated mycelia by a white-light pulse caused a dramatic increase in GAD activity. Light-induced changes were not observed in mutants with delayed onset of conidiation. In the dark or upon illumination by light pulse, the increase of GAD activity preceded the appearance of conidia. Thus, GAD activity in T. viride is closely associated with its developmental status and may represent a link between differentiation events and energy metabolism.

Vegetative growth, aging- and light-induced conidiation of Trichoderma viride cultivated on different carbon sources.

The growth and conidiation of the aged Trichoderma viride culture grown in the dark, and after an induction by a light pulse, was examined in the presence of selected mono-, di(tri)saccharides, amino acids and alcohols as sole carbon sources. Hexoses and disaccharides, but not pentoses and amino acids, promoted proportionally both growth and conidiation induced by aging or light. All compounds but pentoses promoted the conidiation in aged cultures and photoconidiation in a close correlation. Ethanol, glycerol and ethylene glycol supported both growth and conidiation but these processes were not supported equally. Conidia formation with hexoses and amino acids as sole carbon sources seems to be a function of growth promotion, rather than of growth restriction (starvation, stress, aging). With glucose as sole carbon source the conidiation was not triggered by nutrient limitation, nor by the accumulation of waste metabolites. The aging-induced conidiation can be considered to be triggered by the genetic program of the microorganism rather than by its nutrient status.