Metabolism control over growth: a case for trehalose-6-phosphate in plants

How plants relate their requirements for energy with the reducing power necessary to fuel growth is not understood. The activated glucose forms and NADPH are key precursors in pathways yielding, respectively, energy and reducing power for anabolic metabolism. Moreover, they are substrates or allosteric regulators of trehalose-phosphate synthase (TPS1) in fungi and probably also in plants. TPS1 synthesizes the signalling metabolite trehalose-6-phosphate (T6P) and, therefore, has the potential to relate reducing power with energy metabolism to fuel growth. A working model is discussed where trehalose-6-phosphate (T6P) inhibition of SnRK1 is part of a growth-regulating loop in young and metabolically active heterotrophic plant tissues. SnRK1 is the Snf1 Related Kinase 1 and the plant homologue of the AMP-dependent protein kinase of animals, a central energy gauge. T6P accumulation in response to high sucrose levels in a cell inhibits SnRK1 activity, thus promoting anabolic processes and growth. When T6P levels drop due to low glucose-6-phosphate, uridine-diphosphoglucose, and altered NADPH or due to restricted TPS1 activity, active SnRK1 promotes catabolic processes required to respond to energy and carbon deprivation. The model explains why too little or too much T6P has been found to be growth inhibitory: Arabidopsis thaliana embryos and seedlings without TPS1 are growth arrested and Arabidopsis seedlings accumulating T6P on a trehalose medium are growth arrested. Finally, the insight gained with respect to the possible role of T6P metabolism, where it is known to alter developmental and environmental responses of plants, is discussed.     

植物海藻糖代谢及海藻糖-6-磷酸信号研究进展

海藻糖代谢和海藻糖-6-磷酸（T6P）信号途径在植物生长和发育过程中具有重要的调控作用。T6P是海藻糖的代谢前体,是植物响应碳元素可用性、调控生长发育的关键信号分子。植物体中除了自身的海藻糖合成途径外,由病原菌产生的海藻糖或T6P能够导致植物代谢和发育的重新编程。植物不同阶段的生长发育,包括胚胎发育、幼苗生长、成花诱导及叶片衰老等,都受T6P的调控。T6P信号的一个关键互作因子是蔗糖非发酵相关激酶1（SnRKl）,T6P能够抑制SnRK1的催化活性,进而调控植物的生长和发育过程。     

Influence of the StubSNF1 kinase complex and the expression of the yeast TPS1 gene on growth and tuber yield in potato

Expression of the yeast trehalose-6-phosphate synthase-1 (TPS1) gene in potato results in growth aberrations and arrest of development. Recent studies have shown that this phenomenon could be related to the inhibitory effect of trehalose-6-phosphate on SnRK1s, a family of sucrose non-fermenting-1 (SNF1)-related protein kinases that link metabolic and stress signalling in plants. SnRK1s are heterotrimeric enzymes similar to yeast SNF1 and mammalian AMP-activated protein kinases (AMPKs). Previously, we showed that antisense repression of StubGAL83, one of the three subunits of the potato SnRK1 complex, results in a delay in rooting and increases sensitivity to salt stress. Here we report that StubGAL83 is a positive regulator of SNF1 kinase activity in potato and that repression of the kinase subunit of the SnRK1 complex, StubSNF1, reduces growth and tuber yield in potato plants. Co-repression of StubGAL83 and StubSNF1 at a certain level, however, can result in larger plants and increased tuber yield. We found that repression of StubGAL83, but not repression of StubSNF1 attenuated growth aberrations caused by TPS1 expression. We provide evidence that the increased plant size and yield in StubGAL83-StubSNF1 co-repressed plants as well as the attenuation of aberrations caused by TPS1 expression are related to increased nitrate reductase activity.     

A secondary function of trehalose-6-phosphate synthase is required for resistance to oxidative and desiccation stress in Fusarium verticillioides

The disaccharide trehalose has long been recognized for its role as a stress solute, but in recent years some of the protective effects previously ascribed to trehalose have been suggested to arise from a function of the trehalose biosynthesis enzyme trehalose-6-phosphate (T6P) synthase that is distinct from its catalytic activity. In this study, we use the maize pathogenic fungus Fusarium verticillioides as a model to explore the relative contributions of trehalose itself and a putative secondary function of T6P synthase in protection against stress as well as to understand why, as shown in a previous study, deletion of the TPS1 gene coding for T6P synthase reduces pathogenicity against maize. We report that a TPS1-deletion mutant of F. verticillioides is compromised in its ability to withstand exposure to oxidative stress meant to simulate the oxidative burst phase of maize defense and experiences more ROS-induced lipid damage than the wild-type strain. Eliminating T6P synthase expression also reduces resistance to desiccation, but not resistance to phenolic acids. Expression of catalytically-inactive T6P synthase in the TPS1-deletion mutant leads to a partial rescue of the oxidative and desiccation stress-sensitive phenotypes, suggesting the importance of a T6P synthase function that is independent of its role in trehalose synthesis.     

HSPRO acts via SnRK1-mediated signaling in the regulation of Nicotiana attenuata seedling growth promoted by Piriformospora indica

Nicotiana attenuata HSPRO (NaHSPRO) is a negative regulator of seedling growth promoted by the fungus Piriformospora indica. Homologs of NaHSPRO in Arabidopsis thaliana (i.e., AtHSPRO1 and AtHSPRO2) are known to physically interact with the AKINβγ subunit of the SnRK1 complex.² To investigate whether NaHSPRO is associated with SnRK1 function during the stimulation of seedling growth by P. indica, we studied N. attenuata plants silenced in the expression of NaGAL83 (as-gal83 plants)—a gene that encodes for the regulatory β-subunit of SnRK1—and plants silenced in the expression of both NaHSPRO and NaGAL83 (ir-hspro/as-gal83 plants). The results showed that P. indica differentially stimulated the growth of both as-gal83 and ir-hspro/as-gal83 seedlings compared with control seedlings, with a magnitude similar to that observed in ir-hspro seedlings. Thus, we showed that, similar to NaHSPRO, NaGAL83 is a negative regulator of seedling growth stimulated by P. indica. We propose that the effect of NaHSPRO on seedling growth is associated with SnRK1 signaling.     

The Trehalose 6-Phosphate/SnRK1 Signaling Pathway Primes Growth Recovery following Relief of Sink Limitation

Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g⁻¹ fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.The nonreducing Glc disaccharide, trehalose [α-d-glucopyranosyl-(1→1)-α-d-glucopyranoside] is widespread in nature. In resurrection plants, fungi, bacteria, and nonvertebrate animals, it performs a role as a carbon source and stress protection compound (Elbein et al., 2003; Paul et al., 2008). In the majority of plants, however, amounts of trehalose are too low to perform this function. Instead, the pathway has developed into a specialized system that regulates and integrates metabolism with growth and development (Schluepmann et al., 2003; Lunn et al., 2006; Ramon and Rolland, 2007; Gómez et al., 2010). This system is indispensible throughout seed and vegetative development (Eastmond et al., 2002; van Dijken et al., 2004; Gómez et al., 2010), and evidence suggests that the critical function is performed by the precursor of trehalose, trehalose 6-P (T6P). There is one known trehalose biosynthesis pathway in plants from the intermediates Glc 6-P and UDP-Glc catalyzed by trehalose phosphate synthase (TPS), which synthesizes T6P. T6P is then converted to trehalose by trehalose phosphate phosphatase (TPP). The regulation of T6P content in plants by TPSs and TPPs is not well understood. TPS1 is thought to account for most TPS catalytic activity in plants (Vandesteene et al., 2010). All 10 TPPs are now known to be catalytically active (Vandesteene et al., 2012); however, their specific contribution to T6P homeostasis is not known. Evidence suggests that T6P is a sugar signal in plants. T6P responds strongly to Suc supply when Suc is fed to seedlings grown in culture and in response to an increase in Suc in illuminated leaves (Lunn et al., 2006). Biosynthetic pathways for cell wall (Gómez et al., 2006) and starch synthesis (Kolbe et al., 2005) are regulated by T6P, supporting the observation that T6P promotes carbon utilization and growth of seedlings at high sugar levels when its content is increased through expression of otsA, a TPS-encoding gene from Escherichia coli (Schluepmann et al., 2003; Paul et al., 2010). In contrast, expression of otsB, a corresponding TPP-encoding gene from E. coli, decreases T6P content and inhibits growth in the presence of high sugar (Schluepmann et al., 2003; Paul et al., 2010). Given the importance of T6P in the regulation of growth and end-product synthesis, targets for its interaction have been eagerly sought.Recently, it was found that T6P inhibits the protein kinase SnRK1 in growing tissues of plants (Zhang et al., 2009; Debast et al., 2011; Delatte et al., 2011; Martínez-Barajas et al., 2011) through an intermediary factor. SnRK1 (AKIN10/AKIN11) is a member of the SNF1-related AMPK group of protein kinases that perform central functions in the regulation of responses of cells to endogenous energy and carbon status (Hardie, 2007). Baena-González et al. (2007) established that over 1000 genes are regulated by SnRK1 involved in biosynthetic, growth, and stress responses. It was observed that, in addition to cell wall and starch synthesis, T6P could regulate amino acid metabolism, protein, and nucleotide synthesis (Zhang et al., 2009) and is most likely connected to hormone signaling (Zhang et al., 2009; Paul et al., 2010). A model is proposed where SnRK1 inhibits growth processes when sugar and energy supplies are scarce, thus enabling survival under starvation stress conditions. When sugar supply is plentiful, T6P accumulates and inhibits SnRK1 blocking expression of genes involved in the stress survival response and inducing genes involved in the feast response, including growth processes. Interestingly, plants with altered SnRK1 activity display similar phenotypes to plants with altered T6P in both growth and developmental processes such that plants with genetically decreased T6P content resemble those with overexpressed SnRK1 and vice versa (Schluepmann et al., 2003; Baena-González et al., 2007; Wingler et al., 2012).Sugars fluctuate widely in plants in response to changes in photosynthesis and in response to environmental variables. Sugar starvation conditions, such as those induced by deep shade, limit growth through lack of sugar availability; SnRK1 would be active under such conditions. High sugar availability, however, does not necessarily indicate good conditions for growth and high growth rates. For example, under low-temperature and limiting nutrient supply, growth is limited in spite of abundant sugar availability (Paul and Stitt, 1993; Usadel et al., 2008). This is termed sink-limited growth, when growth is limited by capacity of sinks, i.e. growing regions to use assimilate. It departs from the famine model of growth regulation by SnRK1. The interrelationship between T6P, SnRK1, and growth is not known under such conditions. Here, we vary growth conditions by temperature and nutrient supply to induce sink-limited growth and feed Suc and Glc at physiological levels (15 mm). We show a strong specific interrelationship between T6P and Suc and SnRK1-regulated gene expression under all conditions irrespective of growth rate. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth. By priming, we mean being in a prepared state with an advanced capacity to activate growth following relief of a growth limitation, such as low temperature. To test that T6P/SnRK1 enable growth recovery following relief from sink limitation, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that T6P responds to Suc induced by growth restriction. This enables growth recovery following relief of limitations downstream of T6P/SnRK1, such as low temperature. Our findings are included in a model for the regulation of growth by the T6P/SnRK1 signaling pathway.     

Cloning and truncation modification of trehalose-6-phosphate synthase gene from Selaginella pulvinata

A homologous sequence was amplified from resurrection plant Selaginella pulvinta by RACE technique, proved to be the full-length cDNA of trehalose-6-phosphate synthase gene by homologous alignment and yeast complementation assay, and nominated as SpTPS1 gene. The open reading frame of this gene was truncated 225 bp at the 5′-end, resulting the N-terminal truncation modification of 75 amino acids for its encoding protein. The TPS1 deletion mutant strain YSH290 of the brewer's yeast transformed by the truncated gene SpTPS1Δ and its original full-length version restored growth on the medium with glucose as a sole carbon source and displayed growth curves with no significant difference, indicating their encoding proteins functioning as TPS enzyme. The TPS activity of the mutant strain transformed by the truncated gene SpTPS1Δ was about six fold higher than that transformed by its original version, reasoning that the extra N-terminal extension of the full-length amino acid sequence acts as an inhibitory domain to trehalose synthesis. However, the trehalose accumulation of the mutant strain transformed by the truncated gene SpTPS1Δ was only 8% higher than that transformed by its original version. This result is explained by the feedback balance of trehalose content coordinated by the comparative activities between trehalose synthase and trehalase. The truncated gene SpTPS1Δ is suggested to be used in transgenic operation, together with the inhibition of trehalase activity by the application of validamycin A or genetic deficiency of the endogenous trehalase gene, for the enhancement of trehalose accumulation and improvement of abiotic tolerance in transgenic plants.     

Regulation of genes encoding subunits of the trehalose synthase complex inSaccharomyces cerevisiae: novel variations of STRE-mediated transcription control?

Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress. This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters. In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose. In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex,GGS1/TPS1, TPS2, TPS3 andTSL1. We show that expression of these genes is coregulated under stress conditions. Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway. However, during fermentative growth onlyTSL1 shows an expression pattern like that of the STRE-controlled genesCTT1 andSSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated. This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth. We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression. Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements.     

Chlorophyll fluorescence responses to temperature and water availability in two co-dominant Mediterranean shrub and tree species in a long-term field experiment simulating climate change

A rain exclusion experiment simulating drought conditions expected in Mediterranean areas for the following decades (15% decrease in soil moisture) is being conducted since 1999 in a Mediterranean holm oak forest to study its response to the forecasted climatic changes for the coming decades. The maximum PSII quantum yield of primary photochemistry (Fv/Fm) was measured in Quercus ilex, and Phillyrea latifolia, the co-dominant species of the studied forest, from 1999 to 2009 in four plots: two of them were control plots and the other two plots received the rain exclusion treatment. In both species, the Fv/Fm values were highly dependent on air temperatures, and in a second term, in water availability. P. latifolia was the species with the larger decrease in Fv/Fm values induced by low air temperatures, while in hot seasons, the Fv/Fm values in P. latifolia were even higher than in Q. ilex. Rainfall exclusion decrease Fv/Fm values significantly only in few monitoring dates. The most drought resistant species P. latifolia was more affected by the experimental rainfall exclusion than Q. ilex that instead lost number of leaves per tree. There was a synergic effect of drought stress and winter cold in P. latifolia not observed in Q. ilex, but a more conservative strategy in P. latifolia maintaining leaves with a down-regulation of the linear photosynthetic electron transport. These results indicate that, although other physiological and reproductive strategies at whole plant level must be also taken into account, the warmer and drier environment expected for the following decades could favour the species more sensitive to cold and more resistant to drought, the shrub P. latifolia, in detriment of the tree Q. ilex as already observed in the field after severe heat-drought episodes.     

Enhanced thermotolerance for ethanol fermentation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by overexpression of the gene coding for trehalose-6-phosphate synthase

The effect of overexpression of the trehalose-6-phosphate (T6P) synthase gene (TPS1) on ethanol fermentation of Saccharomyces cerevisiae has been studied at 30 and 38°C. The activity of T6P synthase and the accumulation of trehalose during ethanol fermentation were significantly improved by overexpression of TPS1, and especially at 38°C. Ethanol produced by transformants with and without TPS1 gene overexpression at 38°C was approx. 60 and 37 g/l, respectively. The fermentation efficiency of transformants with TPS1 gene overexpression at 38°C was similar to that at 30°C. The critical growth temperature was increased from 36 to 42°C by TPS1 gene overexpression. These results indicated that overexpression of the TPS1 gene had a beneficial effect on the fermentation capacity of the title yeast strain at high temperatures.     

Nondestructive evaluation of the photosynthetic properties of micropropagated plantlets by imaging photochemical reflectance index under low light intensity

The photochemical reflectance index (PRI) of micropropagated potato leaves was estimated nondestructively from outside the culture vessel using a PRI imaging system developed by the present group. The PRI was determined under low light intensity conditions after dark treatment and compared with the chlorophyll fluorescence parameter Fv/Fm, which denotes photosystem II maximum quantum yield. Short-term high-light treatment decreased Fv/Fm of the plantlets. Culture conditions such as temperature and sucrose concentration also affected Fv/Fm. A linear relationship between the PRI and Fv/Fm was observed in both cases of high-light treatment and different culture conditions, suggesting the potential of the PRI to be used as a substitute for Fv/Fm. PRI estimated from reflection images under low light intensity conditions may be used for rapid and noninvasive evaluation of photosynthetic properties of micropropagated plantlets in a similar manner to Fv/Fm.     

Regulation of the yeast trehalose–synthase complex by cyclic AMP-dependent phosphorylation

Background

Trehalose is an important protectant in several microorganisms. In Saccharomyces cerevisiae, it is synthesized by a large complex comprising the enzymes Tps1 and Tps2 and the subunits Tps3 and Tsl1, showing an intricate metabolic control.

Methods

To investigate how the trehalose biosynthesis pathway is regulated, we analyzed Tps1 and Tps2 activities as well as trehalose and trehalose-6-phosphate (T6P) contents by mass spectrometry.

Results

Tsl1 deficiency totally abolished the increase in Tps1 activity and accumulation of trehalose in response to a heat stress, whereas absence of Tps3 only reduced Tps1 activity and trehalose synthesis. In extracts of heat stressed cells, Tps1 was inhibited by T6P and by ATP. Mg^2 + in the presence of cAMP. In contrast, cAMP-dependent phosphorylation did not inhibit Tps1 in tps3 cells, which accumulated a higher proportion of T6P after stress. Tps2 activity was not induced in a tps3 mutant.

Conclusion

Taken together these results suggest that Tsl1 is a decisive subunit for activity of the TPS complex since in its absence no trehalose synthesis occurred. On the other hand, Tps3 seems to be an activator of Tps2. To perform this task, Tps3 must be non-phosphorylated. To readily stop trehalose synthesis during stress recovery, Tps3 must be phosphorylated by cAMP-dependent protein kinase, decreasing Tps2 activity and, consequently, increasing the concentration of T6P which would inhibit Tps1.

General significance

A better understanding of TPS complex regulation is essential for understanding how yeast deals with stress situations and how it is able to recover when the stress is over.     

Differential Expression of Genes in the Leaves of Sugarcane in Response to Sugar Accumulation

In C₄ sugarcane (Saccharum spp. hybrids), photosynthetic activity has been shown to be regulated by the demand for carbon from sink tissues. There is evidence, from other plant species, that sink-limitation of photosynthesis is facilitated by sugar-signaling mechanisms in the leaf that affect photosynthesis through regulation of gene expression. In this work, we manipulated leaf sugar levels by cold-girdling leaves (5°C) for 80 h to examine the mechanisms whereby leaf sugar accumulation affects photosynthetic activity and assess whether signaling mechanisms reported for other species operate in sugarcane. During this time, sucrose and hexose concentrations above the girdle increased by 77% and 81%, respectively. Conversely, leaf photosynthetic activity (A) and electron transport rates (ETR) decreased by 66% and 54%, respectively. Quantitative expression profiling by means of an Affymetrix GeneChip Sugarcane Genome Array was used to identify genes responsive to cold-girdling (56 h). A number of genes (74) involved in primary and secondary metabolic pathways were identified as being differentially expressed. Decreased expression of genes related to photosynthesis and increased expression of genes involved in assimilate partitioning, cell wall synthesis, phosphate metabolism and stress were observed. Furthermore four probe sets homologous to trehalose 6-phosphate phosphatase (TPP; EC 5.3.1.1) and trehalose 6-phosphate synthase (TPS; EC 2.4.1.15) were up- and down-regulated, respectively, indicating a possible role for trehalose 6-phosphate (T6P) as a putative sugar-sensor in sugarcane leaves.     

Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales

We investigated the mechanisms of osmoadaptation in the order Halobacteriales, with special emphasis on Haladaptatus paucihalophilus, known for its ability to survive in low salinities. H. paucihalophilus genome contained genes for trehalose synthesis (trehalose-6-phosphate synthase/trehalose-6-phosphatase (OtsAB pathway) and trehalose glycosyl-transferring synthase pathway), as well as for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). H. paucihalophilus cells synthesized and accumulated ∼1.97–3.72 μmol per mg protein of trehalose in a defined medium, with its levels decreasing with increasing salinities. When exogenously supplied, glycine betaine accumulated intracellularly with its levels increasing at higher salinities. RT-PCR analysis strongly suggested that H. paucihalophilus utilizes the OtsAB pathway for trehalose synthesis. Out of 83 Halobacteriales genomes publicly available, genes encoding the OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively. Trehalose (or its sulfonated derivative) production and glycine betaine uptake, or lack thereof, were experimentally verified in 17 different Halobacteriales species. Phylogenetic analysis suggested that trehalose synthesis is an ancestral trait within the Halobacteriales, with its absence in specific lineages reflecting the occurrence of gene loss events during Halobacteriales evolution. Analysis of multiple culture-independent survey data sets demonstrated the preference of trehalose-producing genera to saline and low salinity habitats, and the dominance of genera lacking trehalose production capabilities in permanently hypersaline habitats. This study demonstrates that, contrary to current assumptions, compatible solutes production and uptake represent a common mechanism of osmoadaptation within the Halobacteriales.