Genome sequences of two plant growth-promoting fluorescent Pseudomonas strains, R62 and R81.

Plant growth-promoting rhizobacterial (PGPR) strains R62 and R81 have previously been isolated and characterized as part of the Indo-Swiss Collaboration in Biotechnology. Here we present the draft genome sequences of these two PGPR strains, with the aim of unraveling the mechanisms behind their ability to promote wheat growth.

Redifferentiation of bacteria isolated from Lotus japonicus root nodules colonized by Rhizobium sp. NGR234.

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.

Trehalose metabolism in Arabidopsis: occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues.

Axenically grown Arabidopsis thaliana plants were analysed for the occurrence of trehalose. Using gas chromatography-mass spectrometry (GC-MS) analysis, trehalose was unambiguously identified in extracts from Arabidopsis inflorescences. In a variety of organisms, the synthesis of trehalose is catalysed by trehalose-6-phosphate synthase (TPS; EC 2.4.1.15) and trehalose-6-phosphate phosphatase (TPP; EC 3.1.3.12). Based on EST (expressed sequence tag) sequences, three full-length Arabidopsis cDNAs whose predicted protein sequences show extensive homologies to known TPS and TPP proteins were amplified by RACE-PCR. The expression of the corresponding genes, AtTPSA, AtTPSB and AtTPSC, and of the previously described TPS gene, AtTPS1, was analysed by quantitative RT-PCR. All of the genes were expressed in the rosette leaves, stems and flowers of Arabidopsis plants and, to a lower extent, in the roots. To study the role of the Arabidopsis genes, the AtTPSA and AtTPSC cDNAs were expressed in Saccharomyces cerevisiae mutants deficient in trehalose synthesis. In contrast to AtTPS1, expression of AtTPSA and AtTPSC in the tps1 mutant lacking TPS activity did not complement trehalose formation after heat shock or growth on glucose. In addition, no TPP function could be identified for AtTPSA and AtTPSC in complementation studies with the S. cerevisiae tps2 mutant lacking TPP activity. The results indicate that while AtTPS1 is involved in the formation of trehalose in Arabidopsis, some of the Arabidopsis genes with homologies to known TPS/TPP genes encode proteins lacking catalytic activity in trehalose synthesis.

Trehalose becomes the most abundant non-structural carbohydrate during senescence of soybean nodules.

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.

Induction of ApL3 expression by trehalose complements the starch-deficient Arabidopsis mutant adg2-1 lacking ApL1, the large subunit of ADP-glucose pyrophosphorylase.

The disaccharide trehalose has strong effects on plant metabolism and development. In Arabidopsis seedlings, growth on trehalose-containing medium leads to an inhibition of root elongation, an accumulation of starch in the shoots, an increased activity of ADP-Glc pyrophosphorylase (AGPase), and an induction of the expression of the AGPase gene, ApL3 (A. Wingler, T. Fritzius, A. Wiemken, T. Boller, R.A. Aeschbacher [2000] Plant Physiol 124: 105-114). We used Arabidopsis mutants deficient in starch synthesis to examine whether the primary effect of trehalose was to affect carbohydrate allocation by the induction of AGPase in the photosynthetic tissue. In a mutant lacking the large AGPase subunit, ApL1, (adg2-1 mutant) growth on trehalose restored AGPase activity and led to a strong accumulation of starch in the shoots. In contrast, starch synthesis could not be induced in a mutant lacking the small AGPase subunit, ApS, (adg1-1 mutant) or in a mutant lacking plastidic phosphoglucomutase (pgm1-1 mutant). These results indicate that ApL3 can substitute for ApL1 in the AGPase complex. In addition, root elongation in the mutants, especially in the adg1-1 mutant, was partially resistant to trehalose, suggesting that the induction of ApL3 expression and the resulting accumulation of starch in the shoots were partially responsible for the effects of trehalose on the growth of wild-type plants.

Trehalose and trehalase in Arabidopsis.

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.

Hyphal in vitro growth of the arbuscular mycorrhizal fungus Glomus mosseae is affected by chitinase but not by ß-1,3-glucanase.

Purified basic chitinase or ß-1,3-glucanase or a combination of the two enzymes were applied to hyphae of the arbuscular mycorrhizal fungus Glomus mosseae grown in vitro. Chitinase applied to the hyphal tip produced an inhibition of hyphal extension, lysis of the apex and alterations of the growth pattern of the fungus. No effect was observed, however, when chitinase was applied to subapical parts of the hyphae or when glucanase was applied to any part of the hyphae. Application of a combination of the two enzymes to the hyphal tip produced an effect similar to that of chitinase alone.

Cloning and functional analysis of sucrose:sucrose 1-fructosyltransferase from tall fescue.

Enzymes of grasses involved in fructan synthesis are of interest since they play a major role in assimilate partitioning and allocation, for instance in the leaf growth zone. Several fructosyltransferases from tall fescue (Festuca arundinacea) have previously been purified (Lüscher and Nelson, 1995). It is surprising that all of these enzyme preparations appeared to act both as sucrose (Suc):Suc 1-fructosyl transferases (1-SST) and as fructan:fructan 6(G)-fructosyl transferases. Here we report the cloning of a cDNA corresponding to the predominant protein in one of the fructosyl transferase preparations, its transient expression in tobacco protoplasts, and its functional analysis in the methylotrophic yeast, Pichia pastoris. When the cDNA was transiently expressed in tobacco protoplasts, the corresponding enzyme preparations produced 1-kestose from Suc, showing that the cDNA encodes a 1-SST. When the cDNA was expressed in P. pastoris, the recombinant protein had all the properties of known 1-SSTs, namely 1-kestose production, moderate nystose production, lack of 6-kestose production, and fructan exohydrolase activity with 1-kestose as the substrate. The physical properties were similar to those of the previously purified enzyme, except for its apparent lack of fructan:fructan 6(G)-fructosyl transferase activity. The expression pattern of the corresponding mRNA was studied in different zones of the growing leaves, and it was shown that transcript levels matched the 1-SST activity and fructan content.

Trehalose induces the ADP-glucose pyrophosphorylase gene, ApL3, and starch synthesis in Arabidopsis.

In Arabidopsis, genes encoding functional enzymes for the synthesis and degradation of trehalose have been detected recently. In this study we analyzed how trehalose affects the metabolism and development of Arabidopsis seedlings. Exogenously applied trehalose (25 mM) strongly reduced the elongation of the roots and, concomitantly, induced a strong accumulation of starch in the shoots, whereas the contents of soluble sugars were not increased. When Arabidopsis seedlings were grown on trehalose plus sucrose (Suc), root elongation was restored, but starch still accumulated to a much larger extent than during growth on Suc alone. The accumulation of starch in the shoots of trehalose-treated seedlings was accompanied by an increased activity of ADP-glucose pyrophosphorylase and an induction of the expression of the ADP-glucose pyrophosphorylase gene, ApL3. Even in the presence of 50 mM Suc, which itself also slightly induced ApL3, trehalose (5 mM) led to a further increase in ApL3 expression. These results suggest that trehalose interferes with carbon allocation to the sink tissues by inducing starch synthesis in the source tissues. Furthermore, trehalose induced the expression of the beta-amylase gene, AT-beta-Amy, in combination with Suc but not when trehalose was supplied alone, indicating that trehalose can modulate sugar-mediated gene expression.

Differential expression of eight chitinase genes in Medicago truncatula roots during mycorrhiza formation, nodulation, and pathogen infection.

Expression of eight different chitinase genes, representing members of five chitinase classes, was studied in Medicago truncatula roots during formation of arbuscular mycorrhiza with Glomus intraradices, nodulation with Rhizobium meliloti, and pathogen attack by Phytophthora megasperma f. sp. medicaginis, Fusarium solani f. sp. phaseoli (compatible interactions with root rot symptoms), Ascochyta pisi (compatible, symptomless), and F. solani f. sp. pisi (incompatible, nonhost interaction). In the compatible plant-pathogen interactions, expression of class I, II, and IV chitinase genes was enhanced. The same genes were induced during nodulation. Transcripts of class I and II chitinase genes accumulated transiently during early stages of the interaction, and transcripts of the class IV chitinase gene accumulated in mature nodules. The pattern of chitinase gene expression in mycorrhizal roots was markedly different: Expression of class I, II, and IV chitinase genes was not enhanced, whereas expression of three class III chitinase genes, with almost no basal expression, was strongly induced. Two of these three (Mtchitinase III-2 and Mtchitinase III-3) were not induced at all in interactions with pathogens and rhizobia. Thus, the expression of two mycorrhiza-specific class III chitinase genes can be considered a hallmark for the establishment of arbuscular mycorrhiza in Medicago truncatula.

Disaccharide-mediated regulation of sucrose:fructan-6-fructosyltransferase, a key enzyme of fructan synthesis in barley leaves.

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 Suc, 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 Suc, and also that the structurally related disaccharide Tre can replace Suc as a regulatory compound.

Arbuscular mycorrhizal fungi influence life history traits of a lepidopteran herbivore.

Results from pot and microcosm studies in the greenhouse have shown that plant growth and foliar chemistry is altered by the presence and species composition of arbuscular mycorrhizal fungi (AMF). The growth and survival of herbivores which feed on plants could, as a consequence, also be affected by these mutualistic soil fungi. Consequently, interactions between AMF, plants and herbivores could occur. To test this, larvae of the common blue butterfly, Polyommatus icarus (Lycaenidae), were fed with sprigs of Lotus corniculatus (Fabaceae) plants which were inoculated with one of two different AMF species, with a mixture of these AMF species or with sprigs of plants which were not inoculated with AMF. Survival and larval weight of third instar larvae fed with plants colonised by AMF were greater than those of larvae fed with non-mycorrhizal plants. Survival of larvae feeding on non-mycorrhizal plants was 1.6 times lower than that of larvae feeding on plants inoculated with a mixture of AMF species and 3.8 times lower than that of larvae feeding on plants inoculated with single AMF species. Furthermore, larvae fed with non-mycorrhizal plants attained only about half the weight of larvae fed with mycorrhizal plants after 11 days of growth. These differences in larval performance might be explained by differences in leaf chemistry, since mycorrhizal plants had a 3 times higher leaf P concentration and a higher C/N-ratio. Our results, thus, show that the presence of belowground mutualistic soil fungi influences the performance of aboveground herbivores by altering their food quality. Larval consumption, larval food use and adult lipid concentrations of the common blue butterfly differed between larvae which were fed with plants inoculated with different AMF species. This suggests that the performance of herbivores is not only influenced by the presence of AMF but also depends on the identity of the AMF species colonising the host plants. Moreover, a significant interaction term between AMF species and maternal identity of the larvae occurred for adult dry weight, indicating that the performance of offspring from different females was differently influenced by AMF species composition. To our knowledge, these results show for the first time that the species composition of AMF communities can influence life-history traits of butterfly larvae and possibly herbivores in general.

The thermophilic yeast Hansenula polymorpha does not require trehalose synthesis for growth at high temperatures but does for normal acquisition of thermotolerance.

The TPS1 gene from Hansenula polymorpha, which encodes trehalose-6-phosphate (Tre6P) synthase, has been isolated and characterized. The deletion of TPS1 rendered H. polymorpha cells incapable of trehalose synthesis under conditions where wild-type cells normally accumulate high levels of trehalose. Interestingly, the loss of Tre6P synthase did not cause any obvious growth defects on a glucose-containing medium, even at high temperatures, but seriously compromised the cells' ability to acquire thermotolerance.

Purification of the trehalase GMTRE1 from soybean nodules and cloning of its cDNA. GMTRE1 is expressed at a low level in multiple tissues.

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.

Interaction between the endophytic fungus Epichloe bromicola and the grass bromus erectus: effects of endophyte infection, fungal concentration and environment on grass growth and flowering

Epichloe bromicola is an endophytic fungal species that systemically and perennially colonizes intercellular spaces of leaf blades, leaf sheaths and culms of Bromus grass species. E. bromicola causes choke disease in B. erectus, suppressing maturation of most, if not all, host inflorescences. In an investigation of the interaction between fungus and host, we used a quantitative polymerase chain reaction technique to estimate the amount of fungal DNA, and thereby fungal concentration, in host plants. Fungal concentration was directly correlated with vegetative vigour of the plant, as measured by longest leaf length, number of tillers and vegetative above-ground biomass, suggesting that, during vegetative growth, the endophytic fungus is most beneficial for the plant when present in high concentrations. In contrast, the reproduction of the plant, as measured by the number of functional inflorescences, was inversely correlated with fungal concentration: the majority of infected plants, and all that were associated with high concentrations of fungi, were diseased. Thus, the benefit of endophyte infection for the plant is coupled with the disadvantages of infertility. Fungal concentration was shown to be at least in part genetically determined because fungal concentration differed significantly in different plant-endophyte genotype combinations (symbiotum). In a field experiment with normal and CO2-enriched environments, elevated CO2 levels favoured fungal reproductive vigour over host reproductive vigour, suggesting that these plant endophytes would be at a selective advantage in a corresponding environmental-change scenario. We conclude that a dynamic and complex relationship between fungal endophyte infection, fungal concentration, genotype and environment affects growth and fecundity of B. erectus and should contribute to the evolution of these plant-fungal interactions.

Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex.

In the yeast Saccharomyces cerevisiae, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which convert glucose 6-phosphate plus UDP-glucose to trehalose, are part of the trehalose synthase complex. In addition to the TPS1 (previously also called GGS1, CIF1, BYP1, FDP1, GLC6, and TSS1) and TPS2 (also described as HOG2 and PFK3) gene products, this complex also contains a regulatory subunit encoded by TSL1. We have constructed a set of isogenic strains carrying all possible combinations of deletions of these three genes and of TPS3, a homologue of TSL1 identified by systematic sequencing. Deletion of TPS1 totally abolished TPS activity and measurable trehalose, whereas deletion of any of the other genes in most cases reduced both. Similarly, deletion of TPS2 completely abolished TPP activity, and deletion of any of the other genes resulted in a reduction of this activity. Therefore, it appears that all subunits are required for optimal enzymatic activity. Since we observed measurable trehalose in strains lacking all but the TPS1 gene, some phosphatase activity in addition to Tps2 can hydrolyze trehalose 6-phosphate. Deletion of TPS3, in particular in a tsl1Delta background, reduced both TPS and TPP activities and trehalose content. Deletion of TPS2, TSL1, or TPS3 and, in particular, of TSL1 plus TPS3 destabilized the trehalose synthase complex. We conclude that Tps3 is a fourth subunit of the complex with functions partially redundant to those of Tsl1. Among the four genes studied, TPS1 is necessary and sufficient for growth on glucose and fructose. Even when overproduced, none of the other subunits could take over this function of Tps1 despite the homology shared by all four proteins. A portion of Tps1 appears to occur in a form not bound by the complex. Whereas TPS activity in the complex is inhibited by Pi, Pi stimulates the monomeric form of Tps1. We discuss the possible role of differentially regulated Tps1 in a complex-bound or monomeric form in light of the requirement of Tps1 for trehalose production and for growth on glucose and fructose.

Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase.

The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, and SSA3, in induction of thermotolerance and starvation resistance, and in proper G1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase.

Cloning of sucrose:sucrose 1-fructosyltransferase from onion and synthesis of structurally defined fructan molecules from sucrose.

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Trehalose-6-phosphate phosphatases from Arabidopsis thaliana: identification by functional complementation of the yeast tps2 mutant.

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.

Expression of a functional barley sucrose-fructan 6-fructosyltransferase in the methylotrophic yeast Pichia pastoris.

The cDNA encoding sucrose-fructan 6-fructosyltransferase (6-SFT) from barley (Hordeum vulgare) has been expressed in the methylotrophic yeast Pichia pastoris, using a translational fusion into vector pPICZ alphaC, containing the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. Transformed Pichia produced and secreted a functional 6-SFT which had characteristics similar to the barley enzyme, but had a pronounced additional 1-SST activity when incubated with sucrose.