Carbohydrate metabolism and symbiont survival were studied in nodules of soybean (G. max [L.] Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101), induced to senesce simultaneously by application of the photosynthesis inhibitor dichloromethyl urea (DCMU). The plant-borne carbohydrates sucrose and starch started to decline after 2 d and reached background levels after 8 d, in parallel with the decline of nitrogenase. However, the microsymbiont-borne disaccharide trehalose declined only by about 40% and subsequently remained at a constant level of c. 6 mg x g(-1) dry weight up to 14 d, when nodules softened and decayed. The number of re-isolated viable bacteria was not significantly decreased in senescent nodules as compared to control nodules. These results indicate that during terminal senescence of nodules an appreciable part of the bacteria conserve their trehalose pools and survive.

In most studies concerning legume root nodules, the question to what extent the nodule-borne bacteroids survive nodule senescence has not been properly addressed. At present, there is no "model system" to study these aspects in detail. Such a system with Lotus japonicus and the broad host range Rhizobium sp. NGR234 has been developed. L. japonicus L. cv. Gifu was inoculated with Rhizobium sp. NGR234 and grown over a 12 week time period. The first nodules could be harvested after 3 weeks. Nodulation reached a plateau after 11 weeks with a mean of 64 nodules having a biomass of nearly 100 mg FW per plant. Nodules were harvested and homogenized at different stages of plant development. Microscopic inspection of the extracts revealed that, typically, nodules contained c. 15x10(9) bacteroids g(-1) FW, and that about 60% of the bacteroids were viable as judged by vital staining. When aliquots of the extracts were plated on selective media, a substantial number of "colony-forming units" was observed in all cases, indicating that a considerable fraction of the bacteroids had the potential to redifferentiate into growing bacteria. In nodules from the early developmental stages, the fraction of total bacteroids yielding CFUs amounted to about 20%, or one-third of the bacteroids judged to be viable after extraction, and it increased slightly when the plants started to flower. In order to see how nodule senescence affected the survival and redifferentiation potential of bacteroids, some plants were placed in the dark for 1 week. This led to typical symptoms of senescence in the nodules such as an almost complete loss of nitrogenase activity and a considerable decrease in soluble proteins. However, surprisingly, the number of total and viable bacteroids g(-1) nodule FW remained virtually constant, and the fraction of total bacteroids yielding CFUs did not decrease but significantly increased up to 75% of the bacteroids judged to be viable after extraction. This result indicates that during nodule senescence bacteroids might be induced to redifferentiate into the state of free-living, growing bacteria.

Trehalase is ubiquitous in higher plants. So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose. A putative trehalase gene, T19F6.15, has been identified in the genome sequencing effort in Arabidopsis. Here we show that this gene encodes a functional trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs. Furthermore, we present results on the distribution and activity of trehalase in Arabidopsis and we describe how inhibition of trehalase by validamycin A affects the plants response to exogenous trehalose (alpha-D-glucopyranosyl-[1, 1]-alpha-D-glucopyranoside). Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g(-1) protein) and maturing siliques (approximately 250 nkat g(-1) protein) and much lower in leaves, stems, and roots (less than 50 nkat g(-1) protein). Inhibition of trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems. Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and trehalase may play a role in regulating the carbohydrate allocation in plants.

Previous work has indicated that sugar sensing may be important in the regulation of fructan biosynthesis in grasses. We used primary leaves of barley (Hordeum vulgare cv Baraka) to study the mechanisms involved. Excised leaf blades were supplied in the dark with various carbohydrates. Fructan pool sizes and two key enzymes of fructan biosynthesis, sucrose (Suc):Suc-1-fructosyltransferase (1-SST; EC 2.4.1.99) and Suc:fructan-6-fructosyltransferase (6-SFT; EC 2.4.1.10) were analyzed. Upon supply of Sue, fructan pool sizes increased markedly. Within 24 h, 1-SST activity was stimulated by a factor of three and 6-SFT-activity by a factor of more than 20, compared with control leaves supplemented with mannitol (Mit). At the same time, the level of mRNA encoding 6-SFT increased conspicuously. These effects were increased in the presence of the invertase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol. Compared with equimolar solutions of Suc, glucose (Glu) and fructose stimulated 6-SFT activity to a lesser extent. Remarkably, trehalose (Tre; Glc-alpha-1 and 1-alpha-Glc) had stimulatory effects on 6-SFT activity and, to a somewhat lesser extent, on 6-SFT mRNA, even in the presence of validoxylamine A, a potent trehalase inhibitor. Tre by itself, however, in the presence or absence of validoxylamine A, did not stimulate fructan accumulation. Monosaccharides phosphorylated by hexokinase but not or weakly metabolized, such as mannose (Man) or 2-deoxy-Glc, had no stimulatory effects on fructan synthesis. When fructose or Man were supplied together with Tre, fructan and starch biosynthesis were strongly stimulated. Concomitantly, phospho-Man isomerase (EC 5.3.1.8) activity was detected. These results indicate that the regulation of fructan synthesis in barley leaves occurs independently of hexokinase and is probably based on the sensing of Sue, and also that the structurally related disaccharide Tre can replace Suc as a regulatory compound.

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca2+] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca2+] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca2+] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs fromRhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.

Roots of pea (Pisum sativum L. cv. Frisson and Finale) wildtype and mycorrhiza-resistant mutant plants (P2, P6, DK 10) were treated with the auxin transport inhibitor tri-iodo-benzoic acid (TIBA) and grown in the presence of mycorrhizal inocula. In wildtype plants, mycorrhizal infection was considerably enhanced upon treatment with TIBA. In roots of mycorrhiza-resistant pea mutants, a two-(DK 10) to tenfold (P2, P6) increase of appressoria formation could be observed upon addition of TIBA. In roots of P2 and P6 treated with TIBA, length of intraradical hyphae was nearly 1 cm m(-1) root length as compared to less than 1 mm m(-1) in control roots. In mutant P2, this enhancement of mycorrhiza formation was correlated to an enhancement of growth. These results indicate that the mycorrhiza-resistant phenotype of these mutants can be partially reverted by alterations of the phytohormonal balance of the root.

Trehalose (α-D-glucopyranosyl-[1,1]-α-D-glucopyranoside), a non-reducing disaccharide, is common in lower organisms such as bacteria, fungi and invertebrates. In higher plants, trehalose is generally not accumulated and is even considered to be toxic. Recently, genes for trehalose metabolism have, however, been identified in higher plants and characterized by expression studies and functional complementations of corresponding yeast mutants. Interestingly, data obtained from plants externally supplied with trehalose or from transgenic plants expressing trehalose biosynthesis genes from microorganisms point towards a role of trehalose, or intermediates of its metabolism, in sugar sensing and development. These findings may open up new perspectives for engineering the sugar sensing system in plants. Here, we discuss results concerning trehalose as a regulator of the carbohydrate allocation system of the plant and possible mechanisms by which trehalose might interfere with the sugar sensing mechanisms and other signal transduction pathways.

Trehalose (alpha-D-glucopyranosyl-1,1-alpha-D-glucopyranoside), a disaccharide widespread among microbes and lower invertebrates, is generally believed to be nonexistent in higher plants. However, the recent discovery of Arabidopsis genes whose products are involved in trehalose synthesis has renewed interest in the possibility of a function of trehalose in higher plants. We previously showed that trehalase, the enzyme that degrades trehalose, is present in nodules of soybean (Glycine max [L.] Merr.), and we characterized the enzyme as an apoplastic glycoprotein. Here we describe the purification of this trehalase to homogeneity and the cloning of a full-length cDNA encoding this enzyme, named GMTRE1 (G. max trehalase 1). The amino acid sequence derived from the open reading frame of GMTRE1 shows strong homology to known trehalases from bacteria, fungi, and animals. GMTRE1 is a single-copy gene and is expressed at a low but constant level in many tissues.

To study the response of non-mycorrhizal and mycorrhizal maize plants to drought, the changes in the pools of non-structural carbohydrates and amino acids were analysed in leaves and roots of two maize cvs. Plants well colonized by the arbuscular mycorrhizal fungus Glomus mosseae (Nicol. & Gerd.) (60% of root length infected) and comparable non-mycorrhizal plants were subjected to moderate drought stress by reducing the water supply. This stress induced a conspicuous increase in the trehalose pool in the mycorrhizal roots, probably because it was accumulated by the fungal symbiont. Furthermore, glucose and fructose were accumulated in leaves and roots of non-mycorrhizal plants but not in the mycorrhizal ones. Starch disappeared completely from the leaves of both mycorrhizal and non-mycorrhizal plants in response to drought. Activities of soluble acid invertase and trehalase were also measured. Acid invertase activity increased during drought in the leaves of both non-mycorrhizal and mycorrhizal plants whilst in the roots it was unaffected in non-mycorrhizal plants and decreased in the mycorrhizal ones. Without drought stress, trehalase activity was considerably higher in the leaves and roots of mycorrhizal plants than in those of non-mycorrhizal plants. It increased conspicuously during drought, primarily in the leaves of non-mycorrhizal plants. A drought-induced accumulation of amino acids as well as imino acids was found in roots and leaves of both mycorrhizal and non-mycorrhizal plants; leaves of mycorrhizal plants accumulated more imino acids than those of non-mycorrhizal ones. Our results show that drought stress and the presence of a mycorrhizal fungus have a considerable effect on carbon partitioning, imino acid and amino acid accumulation in maize plants.

Roots of Lablab purpureus (L.) Sweet were treated with tri‐iodobenzoic acid (TIBA), kinetin or with nodulation factors (Nod factors) purified from Rhizobium sp. NGR234 and grown in the presence of a mycorrhizal inoculum (Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe. Colonization by the mycorrhizal fungus was increased from <30% to c. 65% of root length when roots were treated with these growth regulators. Moreover, treatment of mycorrhizal L. purpureus roots with Nod factors or TIBA strongly induced sporocarp formation of Glomus mosseae. In parallel, the pool size of the fungal disaccharide trehalose was significantly affected in roots treated with TIBA and Nod factors alone, and with TIBA combined with all effectors, and increased from 0·06 mg g−1 d. wt in control roots to up to 1·7 mg g−1 d. wt (TIBA+kinetin). Conversely, the sucrose pool decreased from 5% d. wt to less than a half in roots treated with Nod factors. Activities of trehalase were significantly enhanced in mycorrhizal roots by the treatment with Nod factors or TIBA. When Nod factors and TIBA were added in combination, these activities were strongly enhanced suggesting synergism between these growth regulators.

It is currently thought that most flowering plants lack the capacity to synthesize trehalose, a common disaccharide of bacteria, fungi and invertebrates that appears to play a major role in desiccation tolerance. Attempts have therefore been made to render plants more drought-resistant by the expression of microbial genes for trehalose synthesis. It is demonstrated here that Arabidopsis thaliana itself possesses genes for at least one of the enzymes required for trehalose synthesis, trehalose-6-phosphate phosphatase. The yeast tps2 mutant, which lacks this enzyme, is heat-sensitive, and Arabidopsis cDNA able to complement this effect has been screened for. Half of the yeast transformants that grew at 38.6 degrees C were also able to produce trehalose. All of these expressed one of two Arabidopsis cDNA, either AtTPPA or AtTPPB, which are both homologous to the C-terminal part of the yeast TPS2 gene and other microbial trehalose-6-phosphate phosphatases. Yeast tps2 mutants expressing AtTPPA or AtTPPB contained trehalose-6-phosphate phosphatase activity that could be measured both in vivo and in vitro. The enzyme dephosphorylated trehalose-6-phosphate but not glucose-6-phosphate or sucrose-6-phosphate. Both genes are expressed in flowers and young developing tissue of Arabidopsis. The finding of these novel Arabidopsis genes for trehalose-6-phosphate phosphatase strongly indicates that a pathway for trehalose biosynthesis exists in plants.

A few genera of angiosperms are known as ‘resurrection plants’ since their leaves withstand complete desiccation. In many organisms, including some resurrection plants, desiccation tolerance is associated with the accumulation of special carbohydrates. We examined whether this is also true for the two European angiosperm genera of resurrection plants, Ramonda and Haberlea in the Gesneriaceae. Using gas chromatography, non‐structural carbohydrates were determined as a percentage of the dry weight in leaves of Ramonda nathaliae subjected to various desiccation regimes. Sucrose was the predominant soluble carbohydrate in all samples, and its level steadily increased from 2 to 10% during desiccation. Starch amounted to ca 2% in control leaves and disappeared completely within 8 days of desiccation. Considerable amounts (1–2.5%) of raffinose and smaller amounts of its precursor galactinol (1‐a‐galactosyl‐myo‐inositol) were present in control leaves; these carbohydrates showed only minor changes upon desiccation. Similar results were obtained when excised leaves of Ramonda nathaliae, Ramonda myconi and Haberlea rhodopensis were subjected to desiccation. These data indicate that sucrose accumulation is connected to desiccation tolerance in Gesneriaceae; the presence of raffinose may be a pre‐adaptation since this sugar prevents crystallization of sucrose during drying.

Co-cultivations of plant suspension cultures of soybean (Glycine max) with compatible phytopathogenic (Pseudomonas syringae pv. glycinea), incompatible phytopathogenic (Pseudomonas syringae pv. tomato), and different non-pathogenic (Erwinia herbicola, Escherichia coli) bacteria were carried out. Growth and viability (triphenyltetrazolium chloride activity) of plant cells and bacteria as well as enzyme activities of peroxidase (PO), polyphenoloxidase (PPO), and phenylalanine ammonialyase (PAL) within the plant cells were investigated over an incubation period of 7 days. The compatible pathogen inhibited growth and viability of the plant cells after 1 day and led to the death of the majority of the plant cells by the seventh day. In contrast, the incompatible pathogen directly reduced growth and viability of the soybean cells and caused a strong induction of enzyme activities of PO and PAL to more than 4 times of the untreated control by the seventh day. The epiphytic bacterium Erwinia herbicola caused a slight inhibition of growth and viability of the plant cells after the second day of co-cultivation. The PO activity increased in the same manner as in the incompatible interaction. The saprophytic Escherichia coli strain had a negligible influence on soybean suspension cells. All the bacteria tested except for Escherichia coli multiplied rapidly during cocultivation and reached populations of 10(8)-10(9) colony-forming units/ml in the stationary phase. The results from this study demonstrate that the soybean suspension cells react differently to compatible, incompatible and saprophytic bacteria.

The aim of this study was to examine how the pools of non‐structural carbohydrates in soybean nodules are affected under water stress conditions depending on the nature of the symbiont strains with particular emphasis on the plant‐borne carbohydrates sucrose and pinitol, and on trehalose, a compatible solute synthesized by the bacteroids. Soybean (Glycine max [L.] Merr. cv. Maple Arrow) plants were inoculated with the nitrogen‐fixing strains Bradyrhizobium japonicum 61‐A‐101 or USDA 110 spc4 and cultivated axenically under conditions in which nodules formed in an upper soil compartment while roots for water supply grew into a compartment filled with nutrient solution. When the nodules were well established (1 month post inoculation), 10% (w/v) PEG 6000 was added to the nutrient solution. This led to a slowly progressing, moderate water stress, as determined by measuring the decrease of transpiration, and to a decrease in nitrogen fixation. The pool sizes of the major non‐structural nodule carbohydrates changed during progression of water stress. Sucrose, the major soluble carbohydrate in nodules of unstressed plants (2 and 4%, respectively of nodule dry weight depending on symbiont strain), strongly increased in nodules of stressed plants, reaching nearly 10% of dry weight. The activities of two major sucrose‐consuming enzymes, sucrose synthase and alkaline invertase, decreased markedly in nodules of stressed plants. Starch decreased only transiently upon water stress. Pinitol, a cyclitol serving as compatible solute in many plants, increased more than 4 times, reaching about 1% of nodule dry weight during the stress. Trehalose, the major soluble carbohydrate synthesized by the bacteroids, increased in nodules colonized by USDA 110 spc4 from about 0.2 to 0.8% of nodule dry weight, while in nodules colonized by 61‐A‐101 it amounted to more than 1.5% of dry weight both with and without stress.

Nodulation mutants of soybean (G. max L. Merr.) forming less (nod 49, nod 139) or more (nts 382, nts 1116) nodules than the corresponding wildtype (cv. Bragg) were infected with Bradyrhizobium japonicum 61-A-101 and grown in sterilized Leonard jars under optimal phytotron conditions. After 1 month, the nodules were harvested and their carbohydrate pools were analyzed. The pools of pinitol, sucrose and starch were greatly increased in the nodules of the weakly nodulating mutants nod 49 and nod 139 as compared with the wildtype. The amount of the disaccharid trehalose, produced by the microsymbiont, was more than 50% lower in nodules from both weakly nodulating and supernodulating plants in comparison to the wildtype, The activity of trehalase, an enzyme stimulated in nodules, was about 60% lower in nodules of the supernodulating mutant nts 382 than in nodules of the isogenic wildtype.

Nitrogen‐fixing (effective) nodules from various legume‐ Rhizobium combinations were analyzed for trehalose and other soluble carbohydrates using gas chromatography and for trehalase activity using biochemical assays. Whereas the bacterial disaccharide trehalose was present only in the minority of the nodules, trehalase activity was found in all of them. Extracts from determinate nodules had a higher trehalase activity than extracts from indeterminate nodules. More detailed studies were done on soybean nodules formed in interactions with two effective and 5 ineffective Bradyrhizobium japonicum strains. Only in effective soybean nodules colonized by the strain 61‐A‐101 was trehalose a major soluble carbohydrate. Irrespective of the wildtype strains used. effective soybean nodules contained about 10 nkat trehalase g −1 fresh weight, whereas the ineffective nodules colonized by mutant strains derived from these wildtype strains contained 2 to 30 times less trehalase. However, a clear correlation between trehalose content and trehalase activity could not be established.

The bacterial genera Rhizobium and Bradyrhizobium, nitrogen-fixing symbionts of legumes, secrete specific lipo chitooligosaccharides that induce the formation of nodules on their host plants. When preparations of such nodulation-inducing factors (Nod factors) were added to suspension-cultured tomato cells, a rapid and transient alkalinization of the culture medium occurred. Lipo oligosaccharide preparations from Rhizobium or Bradyrhizobium treated with flavonoids, known inducers of Nod factor synthesis, were up to 100 times more potent in inducing alkalinization than the ones from untreated bacteria. The activity was absent from preparations of the mutant strain Rhizobium sp. NGR234 Delta nodABC, unable to produce any Nod factors. Preparations of Nod factors from various bacteria as well as individual, highly purified Nod factors from Rhizobium sp. NGR(pA28) induced alkalinization in the tomato cell cultures at nanomolar concentrations. This demonstrates that Nod factors can be perceived by tomato; a nonhost of rhizobia. Using the alkalinization response as a sensitive bioassay, Nod factors were found to be inactivated by plant chitinases. Root chitinases purified from different legumes differed in their potential to inactivate differently substituted Nod factors produced by Rhizobium sp. NGR(pA28). This indicates that the specificity of the bacterium-host plant interaction may be due, at least in part, to differential inactivation of Nod factors by root chitinases.

Soybean (Glycine max cv. Maple Arrow) was infected with Bradyrhizobium japonicum (strain 61-A-101) and grown under semi-sterile conditions. Trehalase was extracted from nodules under acidic conditions (pH 3.7) and purified 530-fold by chromatography on concanavalin A-agarose, anion exchange chromatography and gel filtration. Its native molecular weight was close to 54 kDa. Activity staining after electrophoresis yielded only one band. Isoelectric focussing under non-denaturing conditions indicated an acidic isoelectric point (ca. pH 5.2). The enzyme had a broad pH-optimum (pH 3.5-7) and a temperature optimum of 59 °C. The Km value for trehalose was 0.28 mM, close to the data given previously for crude nodule extracts. Trehalase was found to be relatively insensitive to chelators and divalent cations, indicating that it does not require a metal cofactor. Validamycin A, a fungitoxic antibioticum isolated from Streptomyces, competitively inhibited trehalase with a Ki value of 2 nM.

The fission yeast Schizosaccharomyces pombe was found to accumulate large amounts of polyphosphate, particularly when grown on arginine as the nitrogen source. Upon transfer to a medium without phosphate, polyphosphate was degraded and served as an endogenous phosphate reserve. When phosphate was added again after a prolonged period of phosphate starvation, fission yeast cells synthesized more polyphosphate than they had contained before starvation, a phenomenon known as over-compensation. Strains carrying mutated structural genes for three different phosphatases, pho1, pho2 or pho3, degraded polyphosphate at the same rate as the wild-type strain during phosphate starvation and showed the same type of over-compensation when phosphate was added again.