Functional Characterization of Class I Trehalose Biosynthesis Genes in Physcomitrella patens.

The function of trehalose metabolism in plants during growth and development has been extensively studied, mostly in the eudicot Arabidopsis thaliana. So far, however, not much is known about trehalose metabolism in the moss Physcomitrella patens. Here, we show that in P. patens, two active trehalose-6-phosphate synthase enzymes exist, PpTPS1 and PpTPS2. Expression of both enzymes in Saccharomyces cerevisiae can complement the glucose-growth defect of the yeast tps1∆ mutant. Truncation of N-terminal extension in PpTPS1 and PpTPS2 resulted in higher TPS activity and high trehalose levels, upon expression in yeast. Physcomitrella knockout plants were generated and analyzed in various conditions to functionally characterize these proteins. tps1∆ and tps2∆ knockouts displayed a lower amount of caulonema filaments and were significantly reduced in size of gametophores as compared to the wild type. These phenotypes were more pronounced in the tps1∆ tps2∆ mutant. Caulonema formation is induced by factors such as high energy and auxins. Only high amounts of supplied energy were able to induce caulonema filaments in the tps1∆ tps2∆ mutant. Furthermore, this mutant was less sensitive to auxins as NAA-induced caulonema development was arrested in the tps1∆ tps2∆ mutant. In contrast, formation of caulonema filaments is repressed by cytokinins. This effect was more severe in the tps1∆ and tps1∆ tps2∆ mutants. Our results demonstrate that PpTPS1 and PpTPS2 are essential for sensing and signaling sugars and plant hormones to monitor the balance between caulonema and chloronema development.

Heterologous protein production in the moss Physcomitrella patens

Over the last decades plants have been used for the heterologous production of pharmaceuticals, industrial enzymes and edible vaccines. The moss Physcomitrella patens is considered as an experimental model of choice for basic molecular, cytological and developmental questions in plant biology, as well as an outstanding plant model system for heterologous protein production. Here we use P. patens to produce osmotin, a tobacco protein with fungicidal properties. We have generated a transgenic plant able to synthesize and secrete a biologically active osmotin protein(AU)

Durante las últimas décadas las plantas han sido utilizadas para la producción heteróloga de farmacéuticos, enzimas de uso industrial y vacunas. El musgo Physcomitrella patens es considerado como un modelo de experimentación de elección para abordar preguntas en las áreas de biología molecular, citología y de biología del desarrollo en plantas; así como un excelente sistema modelo para la producción de proteínas heterólogas. En este trabajo se utilizó P. patens para la producción de osmotina, una proteína de tabaco con propiedades fungicidas. Se generó una planta transgénica capaz de sintetizar y secretar osmotina biológicamente activa(AU)

Heterologous protein production in the moss Physcomitrella patens

Over the last decades plants have been used for the heterologous production of pharmaceuticals, industrial enzymes and edible vaccines. The moss Physcomitrella patens is considered as an experimental model of choice for basic molecular, cytological and developmental questions in plant biology, as well as an outstanding plant model system for heterologous protein production. Here we use P. patens to produce osmotin, a tobacco protein with fungicidal properties. We have generated a transgenic plant able to synthesize and secrete a biologically active osmotin protein(AU)

Durante las últimas décadas las plantas han sido utilizadas para la producción heteróloga de farmacéuticos, enzimas de uso industrial y vacunas. El musgo Physcomitrella patens es considerado como un modelo de experimentación de elección para abordar preguntas en las áreas de biología molecular, citología y de biología del desarrollo en plantas; así como un excelente sistema modelo para la producción de proteínas heterólogas. En este trabajo se utilizó P. patens para la producción de osmotina, una proteína de tabaco con propiedades fungicidas. Se generó una planta transgénica capaz de sintetizar y secretar osmotina biológicamente activa(AU)

The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules.

Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

Functional screening of a cDNA library from the desiccation-tolerant plant Selaginella lepidophylla in yeast mutants identifies trehalose biosynthesis genes of plant and microbial origin.

Trehalose is a non-reducing disaccharide that accumulates to large quantities in microbial cells, but in plants it is generally present in very low, barely-detectible levels. A notable exception is the desiccation-tolerant plant Selaginella lepidophylla, which accumulates very high levels of trehalose in both the hydrated and dehydrated state. As trehalose is known to protect membranes, proteins, and whole cells against dehydration stress, we have been interested in the characterization of the trehalose biosynthesis enzymes of S. lepidophylla; they could assist in engineering crop plants towards better stress tolerance. We previously isolated and characterized trehalose-6-phosphate synthases from Arabidopsis thaliana (desiccation sensitive) and S. lepidophylla (desiccation tolerant) and found that they had similar enzymatic characteristics. In this paper, we describe the isolation and characterization of trehalose-6-phosphate phosphatase from S. lepidophylla and show that its catalytic activities are also similar to those of its homolog in A. thaliana. Screening of an S. lepidophylla cDNA library using yeast trehalose biosynthesis mutants resulted in the isolation of a large number of trehalose biosynthesis genes that were of microbial rather than plant origin. Thus, we suggest that the high trehalose levels observed in S. lepidophylla are not the product of the plant but that of endophytes, which are known to be present in this plant. Additionally, the high trehalose levels in S. lepidophylla are unlikely to account for its desiccation tolerance, because its drought-stress-sensitive relative, S. moellendorffii, also accumulated high levels of trehalose.

Metabolic engineering of Kluyveromyces lactis for L-ascorbic acid (vitamin C) biosynthesis.

BACKGROUND: L-ascorbic acid (L-AA) is naturally synthesized in plants from D-glucose by 10 steps pathway. The pathway branch to synthesize L-galactose, the key intermediate for L-ascorbic acid biosynthesis, has been recently elucidated. Budding yeast produces an 5-carbon ascorbic acid analogue Dehydro-D-arabinono 1,4-lactone (D-DAL), which is synthesized from D-arabinose. Yeast is able to synthesize L-ascorbic acid only if it is cultivated in the presence of one of its precursors: L-galactose, L-galactono 1,4-lactone, or L-gulono 1,4-lactone extracted from plants or animals. To avoid feeding the yeast culture with this "L" enantiomer, we engineered Kluyveromyces lactis with L-galactose biosynthesis pathway genes: GDP-mannose 3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2) and L-galactose-1-phosphate phosphatase (VTC4) isolated from Arabidopsis thaliana. RESULTS: Plasmids were constructed and modified such that the cloned plant genes were targeted to the K. lactis LAC4 Locus by homologous recombination and that the expression was associated to the growth on D-galactose or lactose. Upon K. lactis transformation, GME was under the control of the native LAC4 promoter whereas VTC2 and VTC4 were expressed from the S. cerevisiae promoters GPD1 and ADH1 respectively. The expression in K. lactis, of the L-galactose biosynthesis genes was determined by Reverse Transcriptase-PCR and western blotting. The recombinant yeasts were capable to produce about 30 mg.L(-1) of L-ascorbic acid in 48 hours of cultivation when cultured on rich medium with 2% (w/v) D-galactose. We also evaluated the L-AA production culturing recombinant recombinant strains in cheese whey, a waste product during cheese production, as an alternative source of lactose. CONCLUSIONS: This work is the first attempt to engineer K. lactis cells for L-ascorbic acid biosynthesis by a fermentation process without any trace of "L" isomers precursors in the culture medium. We have engineered K. lactis strains capable of converting lactose and D-galactose into L-galactose, by the integration of the genes from the A. thaliana L-galactose pathway. L-galactose is a rare sugar, which is one of the main precursors for L-AA production.

Overexpression of the trehalase gene AtTRE1 leads to increased drought stress tolerance in Arabidopsis and is involved in abscisic acid-induced stomatal closure.

Introduction of microbial trehalose biosynthesis enzymes has been reported to enhance abiotic stress resistance in plants but also resulted in undesirable traits. Here, we present an approach for engineering drought stress tolerance by modifying the endogenous trehalase activity in Arabidopsis (Arabidopsis thaliana). AtTRE1 encodes the Arabidopsis trehalase, the only enzyme known in this species to specifically hydrolyze trehalose into glucose. AtTRE1-overexpressing and Attre1 mutant lines were constructed and tested for their performance in drought stress assays. AtTRE1-overexpressing plants had decreased trehalose levels and recovered better after drought stress, whereas Attre1 mutants had elevated trehalose contents and exhibited a drought-susceptible phenotype. Leaf detachment assays showed that Attre1 mutants lose water faster than wild-type plants, whereas AtTRE1-overexpressing plants have a better water-retaining capacity. In vitro studies revealed that abscisic acid-mediated closure of stomata is impaired in Attre1 lines, whereas the AtTRE1 overexpressors are more sensitive toward abscisic acid-dependent stomatal closure. This observation is further supported by the altered leaf temperatures seen in trehalase-modified plantlets during in vivo drought stress studies. Our results show that overexpression of plant trehalase improves drought stress tolerance in Arabidopsis and that trehalase plays a role in the regulation of stomatal closure in the plant drought stress response.

Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis.

Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-ß-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.

Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome.

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.

The Cytophaga hutchinsonii ChTPSP: First characterized bifunctional TPS-TPP protein as putative ancestor of all eukaryotic trehalose biosynthesis proteins.

The most widely distributed pathway to synthesize trehalose in nature consists of two consecutive enzymatic reactions with a trehalose-6-P (T6P)-synthase (TPS) enzyme, producing the intermediate T6P, and a T6P-phosphatase (TPP) enzyme, which dephosphorylates T6P to produce trehalose and inorganic phosphate. In plants, these enzymes are called Class I and Class II proteins, respectively, with some Class I proteins being active enzymes. The Class II proteins possess both TPS and TPP consensus regions but appear to have lost enzymatic activity during evolution. Plants also contain an extra group of enzymes of small protein size, of which some members have been characterized as functional TPPs. These Class III proteins have less sequence similarity with the Class I and Class II proteins. Here, we characterize for the first time, by using biochemical analysis and yeast growth complementation assays, the existence of a natural TPS-TPP bifunctional enzyme found in the bacterial species Cytophaga hutchinsonii. Through phylogenetic analysis, we show that prokaryotic genes such as ChTPSP might be the ancestor of the eukaryotic trehalose biosynthesis genes. Second, we show that plants have recruited during evolution, possibly by horizontal transfer from bacteria such as Rhodoferax ferrireducens, a new type of small protein, encoding TPP activity, which have been named Class III proteins. RfTPP has very high TPP activity upon expression in yeast. Finally, we demonstrate that TPS gene duplication, the recruitment of the Class III enzymes, and recruitment of an N-terminal regulatory element, which regulates the Class I enzyme activity in higher plants, were initiated very early in eukaryan evolution as the three classes of trehalose biosynthesis genes are already present in the alga Ostreococcus tauri.

A bifunctional TPS-TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis.

Improving stress tolerance is a major goal for agriculture. Trehalose is a key molecule involved in drought tolerance in anhydrobiotic organisms. Here we describe the construction of a chimeric translational fusion of yeast trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase. This construct was overexpressed in yeast cells displaying both TPS and TPP enzyme activities and trehalose biosynthesis capacity. In Arabidopsis thaliana, the gene fusion was overexpressed using either the 35S promoter or the stress-regulated rd29A promoter. Transgene insertion in the genome was checked by PCR and transcript expression by RT-PCR. Several independent homozygous lines were selected in the presence of kanamycin and further analyzed. Trehalose was accumulated in all these lines at low levels. No morphological or growth alterations were observed in lines overexpressing the TPS1-TPS2 construct, whereas plants overexpressing the TPS1 alone under the control of the 35S promoter had aberrant growth, color and shape. TPS1-TPS2 overexpressor lines were glucose insensitive, consistent with a suggested role of trehalose/T6P in modulating sugar sensing and carbohydrate metabolism. Moreover, TPS1-TPS2 lines displayed a significant increase in drought, freezing, salt and heat tolerance. This is the first time that trehalose accumulation in plants is shown to protect against freezing and heat stress. Therefore, these results demonstrate that engineering trehalose metabolism with a yeast TPS-TPP bifunctional enzyme confers multiple stress protection in plants, comprising a potential tool to improve stress-tolerance in crops.

Insights on the evolution of trehalose biosynthesis.

BACKGROUND: The compatible solute trehalose is a non-reducing disaccharide, which accumulates upon heat, cold or osmotic stress. It was commonly accepted that trehalose is only present in extremophiles or cryptobiotic organisms. However, in recent years it has been shown that although higher plants do not accumulate trehalose at significant levels they have actively transcribed genes encoding the corresponding biosynthetic enzymes. RESULTS: In this study we show that trehalose biosynthesis ability is present in eubacteria, archaea, plants, fungi and animals. In bacteria there are five different biosynthetic routes, whereas in fungi, plants and animals there is only one. We present phylogenetic analyses of the trehalose-6-phosphate synthase (TPS) and trehalose-phosphatase (TPP) domains and show that there is a close evolutionary relationship between these domains in proteins from diverse organisms. In bacteria TPS and TPP genes are clustered, whereas in eukaryotes these domains are fused in a single protein. CONCLUSION: We have demonstrated that trehalose biosynthesis pathways are widely distributed in nature. Interestingly, several eubacterial species have multiple pathways, while eukaryotes have only the TPS/TPP pathway. Vertebrates lack trehalose biosynthetic capacity but can catabolise it. TPS and TPP domains have evolved mainly in parallel and it is likely that they have experienced several instances of gene duplication and lateral gene transfer.

Trehalose-6-phosphate synthase as an intrinsic selection marker for plant transformation.

Insertion of foreign DNA into plant genomes occurs randomly and with low frequency. Hence, a selectable marker is generally required to identify transgenic plants. Until now, all selection systems have been based on the use of non-plant genes, derived from microorganisms and usually conferring antibiotic or herbicide resistance. The use of microorganism-derived genes however has raised biosafety concerns. We have developed a novel selection system based on enhancing the expression of a plant-intrinsic gene and the use of a harmless selection agent. Selection takes advantage of the reduced glucose sensitivity of seedlings with enhanced expression of AtTPS1, a gene encoding trehalose-6-P synthase. As a result, transformants can be identified as developing green seedlings amongst the background of small, pale non-transformed plantlets on high glucose medium. In addition, vegetative regeneration of tobacco leaf explants is very sensitive to high external glucose. Overexpression of AtTPS1 in tobacco allows selecting glucose insensitive transgenic shoots.

The Arabidopsis trehalose-6-P synthase AtTPS1 gene is a regulator of glucose, abscisic acid, and stress signaling.

In Arabidopsis (Arabidopsis thaliana), trehalose is present at almost undetectable levels, excluding its role as an osmoprotectant. Here, we report that overexpression of AtTPS1 in Arabidopsis using the 35S promoter led to a small increase in trehalose and trehalose-6-P levels. In spite of this, transgenic plants displayed a dehydration tolerance phenotype without any visible morphological alterations, except for delayed flowering. Moreover, seedlings overexpressing AtTPS1 exhibited glucose (Glc)- and abscisic acid (ABA)-insensitive phenotypes. Transgenic seedlings germinated on Glc were visibly larger with green well-expanded cotyledonary leaves and fully developed roots, in contrast with wild-type seedlings showing growth retardation and absence of photosynthetic tissue. An ABA dose-response experiment revealed a higher germination rate for transgenic plants overexpressing AtTPS1 showing insensitive germination kinetics at 2.5 mum ABA. Interestingly, germination in the presence of Glc did not trigger an increase in ABA content in plants overexpressing AtTPS1. Expression analysis by quantitative reverse transcription-PCR in transgenic plants showed up-regulation of the ABI4 and CAB1 genes. In the presence of Glc, CAB1 expression remained high, whereas ABI4, HXK1, and ApL3 levels were down-regulated in the AtTPS1-overexpressing lines. Analysis of AtTPS1 expression in HXK1-antisense or HXK1-sense transgenic lines suggests the possible involvement of AtTPS1 in the hexokinase-dependent Glc-signaling pathway. These data strongly suggest that AtTPS1 has a pivotal role in the regulation of Glc and ABA signaling during vegetative development.

New selection marker for plant transformation.

A number of systems to insert foreign DNA into a plant genome have been developed so far. However, only a small percentage of transgenic plants are obtained using any of these methods. Stable transgenic plants are selected by co-introduction of a selectable marker gene, which in most cases are genes that confer resistance against antibiotics or herbicides. In this chapter we describe a new method for selection of transgenic plants after transformation. The selection agent used is the nontoxic and common sugar glucose. Wild-type Arabidopsis thaliana plantlets that have been germinated on glucose have small white cotyledons and remain petite because the external sugar switches off the photosynthetic mechanism. The selectable marker gene encodes the essential trehalose-6-phophate synthase, AtTPS1, that catalyzes the first reaction of the two-step trehalose synthesis. Upon ectopic expression of AtTPS1 driven by the 35S promoter, transformed Arabidopsis thaliana plants became insensitive to glucose in comparison to wild-type plants. After transformation using AtTPS1 as a selection marker and 6% glucose as selection agent it is possible to single out the green and normal sized transgenic plants amid the nontransformed plantlets.