RNA interference-mediated repression of cell wall invertase impairs defense in source leaves of tobacco

The significance of cell wall invertase (cwINV) for plant defense was investigated by comparing wild-type tobacco (Nicotiana tabacum) Samsun NN (SNN) with plants with RNA interference (RNAi)-mediated repression of cwINV (SNNcwINV). In source leaves of SNNcwINV, the activity of cwINV was repressed by about 90%. Sucrose export and apoplastic carbohydrate levels were significantly reduced, while photosynthesis and dark respiration exhibited little or no change. Activities of sucrose synthase and phosphofructokinase were depressed moderately, while ADP-glucose pyrophosphorylase was diminished greatly. Yet, the content of cytosolic/vacuolar carbohydrates was not significantly lower, which correlated with the absence of phenotypic effects in SNNcwINV under normal growing conditions. By contrast, defense-related processes in primary metabolism and hypersensitive cell death were impaired and delayed in correlation with repression of cwINV. The increase in cwINV observed in source leaves of the resistant wild type following infection with Phytophthora nicotianae was absent in SNNcwINV. Also, defense-related callose deposition at cell-to-cell interfaces, the related decline in sugar export, and accumulation of apoplastic carbohydrates were reduced and delayed. Expression of pathogenesis-related proteins and increase in phenylalanine ammonia-lyase and glucose-6-phosphate dehydrogenase activities were alleviated. Formation of hydrogen peroxide and development of hypersensitive lesions were weak and heterogeneous, and the pathogen was able to sporulate. We conclude that in photosynthetically active leaves of the apoplastic phloem loader, tobacco cwINV plays an essential role for acquisition of carbohydrates during plant-pathogen interactions and that the availability of these carbohydrates supports the onset of the hypersensitive reaction and ensures successful defense.     

Auxin distribution in Lotus japonicus during root nodule development

For this work, Lotus japonicus transgenic plants were constructed expressing a fusion reporter gene consisting of the genes beta-glucuronidase (gus) and green fluorescent protein (gfp) under control of the soybean auxin-responsive promoter GH3. These plants expressed GUS and GFP in the vascular bundle of shoots, roots and leafs. Root sections showed that in mature parts of the roots GUS is mainly expressed in phloem and vascular parenchyma of the vascular cylinder. By detecting GUS activity, we describe the auxin distribution pattern in the root of the determinate nodulating legume L. japonicus during the development of nodulation and also after inoculation with purified Nod factors, N-naphthylphthalamic acid (NPA) and indoleacetic acid (IAA). Differently than white clover, which forms indeterminate nodules, L. japonicus presented a strong GUS activity at the dividing outer cortical cells during the first nodule cell divisions. This suggests different auxin distribution pattern between the determinate and indeterminate nodulating legumes that may be responsible of the differences in nodule development between these groups. By measuring of the GFP fluorescence expressed 21 days after treatment with Nod factors or bacteria we were able to quantify the differences in GH3 expression levels in single living roots. In order to correlate these data with auxin transport capacity we measured the auxin transport levels by a previously described radioactive method. At 48 h after inoculation with Nod factors, auxin transport showed to be increased in the middle root segment. The results obtained indicate that L. japonicus transformed lines expressing the GFP and GUS reporters under the control of the GH3 promoter are suitable for the study of auxin distribution in this legume.     

Chronic rapamycin treatment or lack of S6K1 does not reduce ribosome activity in vivo

Reducing activity of the mTORC1/S6K1 pathway has been shown to extend lifespan in both vertebrate and invertebrate models. For instance, both pharmacological inhibition of mTORC1 with the drug rapamycin or S6K1 knockout extends lifespan in mice. Since studies with invertebrate models suggest that reducing translational activity can increase lifespan, we reasoned that the benefits of decreased mTORC1 or S6K1 activity might be due, at least in part, to a reduction of general translational activity. Here, we report that mice given a single dose of rapamycin have reduced translational activity, while mice receiving multiple injections of rapamycin over 4 weeks show no difference in translational activity compared with vehicle-injected controls. Furthermore, mice lacking S6K1 have no difference in global translational activity compared with wild-type littermates as measured by the percentage of ribosomes that are active in multiple tissues. Translational activity is reduced in S6K1-knockout mice following single injection of rapamycin, demonstrating that rapamycin’s effects on translation can occur independently of S6K1. Taken together, these data suggest that benefits of chronic rapamycin treatment or lack of S6K1 are dissociable from potential benefits of reduced translational activity, instead pointing to a model whereby changes in translation of specific subsets of mRNAs and/or translation-independent effects of reduced mTOR signaling underlie the longevity benefits.     

Seryl-phosphorylation of soybean nodule sucrose synthase (nodulin-100) by a Ca-dependent protein kinase

Sucrose synthase (SS; EC 2.4.1.13) was radiolabeled in situ by incubating detached soybean nodules with ³²Pi. Phosphoamino acid analysis indicated that SS was phosphorylated on a serine residue(s). In-vitro phosphorylation of purified nodule SS by desalted nodule extracts was Ca²⁺-dependent. This SS-kinase was partially purified (2200-fold) from nodules harvested from illuminated plants. The molecular mass of the SS-kinase was about 55 000 on a Superdex 75 size-exclusion column or in a denaturing autophosphorylation gel. With either purified nodule SS or Syntide 2 as substrate, exogenous calmodulin and phosphatidylserine showed little or no effect on the in-vitro activity of this partially purified protein kinase. However, its activity was inhibited by W-7. The purified nodule SS-kinase (or CDPK) phosphorylated nodule PEP carboxylase (PEPC; EC 4.1.1.31) in the presence of Ca²⁺. In contrast, a partially purified nodule PEPC-kinase preparation was incapable of phosphorylating nodule SS. Unlike nodule PEPC [Zhang et al. (1995) Plant Physiol. 108, 1561–1568], the phosphorylation state of SS is not likely modulated in planta by photosynthate supply from the shoots.     

Autoregulation of soybean nodulation: Delayed inoculation increases nodule number

The influence of seedling age at the time of inoculation on the regulation of nodule number in soybean (Glycine max [L.] Merr.) was examined in cv. Williams 82 and its hypernodulating mutant NOD1-3. Nodulation was evaluated on plants grown in plastic growth pouches or in vermiculite in 50- or 500-ml glass containers in growth chamber studies. Seeds or seedlings were inoculated once with Bradyrhizobium japonicum strain USDA 110 (10^k cells seedling^?1) between 0 and 15 days after sowing at 3- or 5-day intervals and were grown for 21 days after inoculation. Nodule number plant^?1 was similar across inoculation times in plants grown in growth pouches, but was significantly greater when inoculation was delayed and plants were grown in vermiculite in 500-ml containers. Plant culture in vermiculite in 50- or 500-ml containers confirmed the suppressive effect of restricted space for root growth on nodulation. Inoculation with 10⁵ or 10⁹ USDA 110 cells revealed that nodulation was inhibited by a high inoculum dose. There was a large increase in nodule number plant^?1 when plants were transferred from a restricted rooting environment (growth pouch culture) to a nonrestricted rooting environment (2-1 hydroponic pots). Autoregulation was also examined in split-root assemblies of plants in 500-ml containers of vermiculite. Controls involved concurrent inoculation of both root halves at 0. 4 or 8 days after transplant. Treatments involved time-separated inoculations of root halves with the primary and secondary inoculations being separated by 4 days. Plants were harvested at 21 days after inoculation. Williams 82 exhibited autoregulation of nodule number on the root half receiving delayed inoculation, regardless of plant age at the time of primary inoculation. Total nodule number plant^?1 invariably increased with later inoculation times. In contrast. NOD1 - 3 exhibited little, if any, autoregulation of nodule number. It was concluded that although Williams 82 exhibits autoregulation of nodule number and NODI - 3 does not, there was no finite limit to nodule number in either line since any delay in inoculation resulted in formation of a greater nodule number on both lines if root growth was not restricted. Nodule number in Williams 82 and NODI - 3 appears to be a function of infection sites (root size) at the time of inoculation and of subsequent plant growth.     

Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.     

Low concentrations of ammonium inhibit specific nodulation (nodule number g-1 root DW) in soybean (Glycine max [L.] Merr.)

Experiments on peas (Gulden and Vessey, 1997) have indicated that NH ₄ ⁺ stimulates both whole plant (nodules plant^-1) and specific nodulation (nodules g^-1 root DW). The effect of low concentrations of NH ₄ ⁺ on the soybean/Bradyrhizobium symbiosis is unknown. The objectives of the current study were to determine the immediate and residual effects of NH ₄ ⁺ on nodulation and N₂ fixation in soybean (Glycine max [L.] Merr.) in sand culture. Soybean (cv. Maple Ridge) were exposed to 0.0, 0.5, 1.0 and 2.0 mM of ¹⁵N-labelled (NH₄)₂SO₄ for 28 days after inoculation (DAI). From 29 to 56 DAI the plants were grown on NH ₄ ⁺ -free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI for root, shoot and nodule dry weight (DW), total N content, nodule counts and ¹⁵N enrichment of plant composites. Nitrogenase activity was measured by gas exchange at 28 DAI. The plants in the control (0.0 mM NH ₄ ⁺ ) treatment had consistently lower relative growth rates than the plants in the NH ₄ ⁺ treatments during the first 28 DAI. Plant growth was also less at 2.0 mM NH ₄ ⁺ compared to growth at 0.5 and 1.0 mM NH ₄ ⁺ . At 28 DAI, plants exposed to 0.5 and 1.0 mM NH ₄ ⁺ had significantly more nodules per plant and larger individual nodules than either the NH ₄ ⁺ -free controls or the 2.0 mM NH ₄ ⁺ -supplied plants. However, specific nodulation (nodule number g^-1 root DW) and specific nitrogenase activity (nitrogenase activity g^-1 nodule DW) were on average approximately 286% and 60% higher in the control plants, respectively, than for plants in the NH ₄ ⁺ treatments at 28 DAI. Also at 28 DAI, specific nodule DW (nodule DW g^-1 root DW) were 17, 44 and 53% higher in control plants than plants that had been exposed to 0.5, 1.0 and 2.0 mM NH ₄ ⁺ . At 56 DAI, after an additional 4 weeks of NH ₄ ⁺ -free nutrition, the plants which had previously received 0.5 and 1.0 mM NH ₄ ⁺ still maintained the highest plant DW and N contents, however, specific nodule DW had become similar at 600 mg nodule DW g^-1 root DW among all treatments. It is concluded that NH ₄ ⁺ has a negative effect on the nodulation process in the soybean/Bradyrhizobium symbiosis (as best indicated by the negative effect of NH ₄ ⁺ on specific nodulation). Despite this negative effect on specific nodulation, 0.5 and 1.0 mM NH ₄ ⁺ resulted in higher whole plant nodulation and N₂ fixation due to a compensating, positive effect on overall plant growth (i.e. fewer nodules g^-1 root DW, but much larger roots). Once NH ₄ ⁺ was removed from all treatments, the soybean plants appeared to move toward a consistent level of nodule DW relative to root DW.     

Overexpression of <Emphasis Type="Italic">GmAKR1</Emphasis>, a stress-induced aldo/keto reductase from soybean,retards nodule development

Development of symbiotic root nodules in legumes involves the induction and repression of numerous genes in conjunction with changes in the level of phytohormones. We have isolated several genes that exhibit differential expression patterns during the development of soybean nodules. One of such genes, which were repressed in mature nodules, was identified as a putative aldo/keto reductase and thus named Glycine max aldo/keto reductase 1 (GmAKR1). GmAKR1 appears to be a close relative of a yeast aldo/keto reductase YakC whose in vivo substrate has not been identified yet. The expression of GmAKR1 in soybean showed a root-specific expression pattern and inducibility by a synthetic auxin analogue 2,4-D, which appeared to be corroborated by presence of the root-specific element and the stress-response element in the promoter region. In addition, constitutive overexpression of GmAKR1 in transgenic soybean hairy roots inhibited nodule development, which suggests that it plays a negative role in the regulation of nodule development. One of the Arabidopsis orthologues of GmAKR1 is the ARF-GAP domain 2 protein, which is a potential negative regulator of vesicle trafficking; therefore GmAKR1 may have a similar function in the roots and nodules of legume plants. These authors contributed equally to this work.     

Bradyrhizobium strains and the nodulation,nodule efficiency and growth of soybean (Glycine max L.) in Egyptian soils

Six strains and a commercial inoculant ofBradyrhizobium japonicum were evaluated in association withGlycine max (L.) cultivar Clark. Inoculated and uninoculated plants were grown in pot and field experiments. Nodules were counted and weighed and roots and shoots were separated and analysed for total nitrogen. In pot experiments, two of six bacterial strains were superior to the other four, and to the commercial inoculant (Nitragin) in promoting greater root and top growth and plant nitrogen accumulation. In the field experiment, there were indications that environmental conditions may have affected nodulation by the bacteria. The strains could be divided into three groups according to nodule efficiencies, accumulation of plant dry matter, and total nitrogen content. The greater variations in nodule efficiencies of the tested strains could be attributed to the quantities of bacteroid, cytosol protein and leghaemoglobin in the nodules.     

Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6

Ribosomal protein S6 is phosphorylated in response to mitogens by activation of one or more protein kinase cascades. Phosphorylation of S6 in vivo is catalyzed by (at least) two distinct mitogen-activated S6 kinase families distinguishable by size, the 70 kDa and 90 kDa S6 kinases. Both S6 kinases are activated by serine/threonine phosphorylation. Members of each family have been cloned. The 90 kDa S6 kinases are activated more rapidly than the 80 kDa S6 kinase, and may have other intracellular targets. The 70 kDa S6 kinase is relatively specific for 40 S ribosomal subunits. No kinase capable of activating the 70 kDa S6 kinase has been identified. Members of the 90 kDa S6 kinases are activated in vitro by 42 kDa and 44 kDa MAP kinases, which are in turn activated by mitogen-dependent activators. The pathways for mitogen-stimulated S6 phosphorylation are discussed.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.     

Identification of S6K2 as a centrosome-located kinase

Ribosomal S6 kinase 2 (S6K2) acts downstream of the mammalian target of rapamycin (mTOR). Here, we show that some S6K2 localize at the centrosome throughout the cell cycle. S6K2 is found in the pericentriolar area of the centrosome. S6K2 centrosomal localization is unaffected by serum withdrawal or treatment with rapamycin, wortmannin, U0126, or phorbol-12-myristate-13-acetate (PMA). Unlike S6K2, S6 kinase 1 (S6K1) does not localize at the centrosome, suggesting the two kinases may also have nonoverlapping functions. Our data suggest that centrosomal S6K2 may have a role in the phosphoinositide-3-kinase (PI3K)/Akt/mTOR signaling pathway that has also been detected in the centrosome.     

Utilization of nitrate by bacteroids of Bradyrhizobium japonicum in the soybean root nodule

Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O₂ (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 g· ml^-1) or chloramphenicol (50 g·ml^-1). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N₂ fixation.Abbreviations NR nitrate reductase - NiR nitrite reductase - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Ribosomal protein S6 kinase 1 interacts with and is ubiquitinated by ubiquitin ligase ROC1

Ribosomal protein S6 kinase (S6K) is involved in the regulation of cell growth and cellular metabolism. The activation of S6K in response to diverse extracellular stimuli is mediated by multiple phosphorylations coordinated by the mTOR and PI3K signaling pathways. We have recently found that both forms of S6K are modified by ubiquitination. Following these findings, we demonstrate here for the first time that S6K1 associates specifically with ubiquitin ligase ROC1 in vitro and in vivo. The interaction was initially identified in the yeast two-hybrid screening and further confirmed by pull-down and co-immunoprecipitation assays. Furthermore, the overexpression of ROC1 leads to an increase in S6K1 ubiquitination. Consistent with this observation, we showed that the steady-state level of S6K1 is regulated by ROC1, since downregulation of ROC1 by specific siRNA promotes stabilization of S6K1 protein. The results suggest the involvement of ROC1 in S6K1 ubiquitination and subsequent proteasomal degradation.     

Regulation of catalytic activity of S6 kinase 2 during cell cycle

Ribosomal S6 kinase 2 (S6K2) is one of the kinases regulated by the mammalian target of rapamycin (mTOR) signaling pathway. Although it has been identified as a kinase homologous to S6K1, evidence suggests that the two kinases have non-overlapping functions, and the biological function of S6K2 still remains unknown. In order to identify the cell cycle stage(s) during which S6K2 plays a role, we assessed changes in the catalytic activity of S6K2 throughout the cell cycle. Our data show that S6K2 is active throughout the cell cycle with higher activity in G2 and M phases. We also show that S6K1 activity peaks sharply during M phase. Our data suggest that S6K1 and S6K2 likely play yet-unknown roles in G2 and M phases.