Metabolic regulation of quiescence in plants.

Quiescence is a crucial survival attribute in which cell division is repressed in a reversible manner. Although quiescence has long been viewed as an inactive state, recent studies have shown that it is an actively monitored process that is influenced by environmental stimuli. Here, we provide a perspective of the quiescent state and discuss how this process is tuned by energy, nutrient and oxygen status, and the pathways that sense and transmit these signals. We not only highlight the governance of canonical regulators and signalling mechanisms that respond to changes in nutrient and energy status, but also consider the central significance of mitochondrial functions and cues as key regulators of nuclear gene expression. Furthermore, we discuss how reactive oxygen species and the associated redox processes, which are intrinsically linked to energy carbohydrate metabolism, also play a key role in the orchestration of quiescence.

TaTPP-7A positively feedback regulates grain filling and wheat grain yield through T6P-SnRK1 signalling pathway and sugar-ABA interaction.

Grain size and filling are two key determinants of grain thousand-kernel weight (TKW) and crop yield, therefore they have undergone strong selection since cereal was domesticated. Genetic dissection of the two traits will improve yield potential in crops. A quantitative trait locus significantly associated with wheat grain TKW was detected on chromosome 7AS flanked by a simple sequence repeat marker of Wmc17 in Chinese wheat 262 mini-core collection by genome-wide association study. Combined with the bulked segregant RNA-sequencing (BSR-seq) analysis of an F2 genetic segregation population with extremely different TKW traits, a candidate trehalose-6-phosphate phosphatase gene located at 135.0 Mb (CS V1.0), designated as TaTPP-7A, was identified. This gene was specifically expressed in developing grains and strongly influenced grain filling and size. Overexpression (OE) of TaTPP-7A in wheat enhanced grain TKW and wheat yield greatly. Detailed analysis revealed that OE of TaTPP-7A significantly increased the expression levels of starch synthesis- and senescence-related genes involved in abscisic acid (ABA) and ethylene pathways. Moreover, most of the sucrose metabolism and starch regulation-related genes were potentially regulated by SnRK1. In addition, TaTPP-7A is a crucial domestication- and breeding-targeted gene and it feedback regulates sucrose lysis, flux, and utilization in the grain endosperm mainly through the T6P-SnRK1 pathway and sugar-ABA interaction. Thus, we confirmed the T6P signalling pathway as the central regulatory system for sucrose allocation and source-sink interactions in wheat grains and propose that the trehalose pathway components have great potential to increase yields in cereal crops.

Spatial expression patterns of genes encoding sugar sensors in leaves of C4 and C3 grasses.

BACKGROUND AND AIMS: The mechanisms of sugar sensing in grasses remain elusive, especially those using C4 photosynthesis even though a large proportion of the world's agricultural crops utilize this pathway. We addressed this gap by comparing the expression of genes encoding components of sugar sensors in C3 and C4 grasses, with a focus on source tissues of C4 grasses. Given C4 plants evolved into a two-cell carbon fixation system, it was hypothesized this may have also changed how sugars were sensed. METHODS: For six C3 and eight C4 grasses, putative sugar sensor genes were identified for target of rapamycin (TOR), SNF1-related kinase 1 (SnRK1), hexokinase (HXK) and those involved in the metabolism of the sugar sensing metabolite trehalose-6-phosphate (T6P) using publicly available RNA deep sequencing data. For several of these grasses, expression was compared in three ways: source (leaf) versus sink (seed), along the gradient of the leaf, and bundle sheath versus mesophyll cells. KEY RESULTS: No positive selection of codons associated with the evolution of C4 photosynthesis was identified in sugar sensor proteins here. Expressions of genes encoding sugar sensors were relatively ubiquitous between source and sink tissues as well as along the leaf gradient of both C4 and C3 grasses. Across C4 grasses, SnRK1ß1 and TPS1 were preferentially expressed in the mesophyll and bundle sheath cells, respectively. Species-specific differences of gene expression between the two cell types were also apparent. CONCLUSIONS: This comprehensive transcriptomic study provides an initial foundation for elucidating sugar-sensing genes within major C4 and C3 crops. This study provides some evidence that C4 and C3 grasses do not differ in how sugars are sensed. While sugar sensor gene expression has a degree of stability along the leaf, there are some contrasts between the mesophyll and bundle sheath cells.

Impaired KIN10 function restores developmental defects in the Arabidopsis trehalose 6-phosphate synthase1 (tps1) mutant.

Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6-phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo-lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1). Here, we explored the role of SnRK1 in T6P-mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches. We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10. Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss-of-function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.

The trehalose 6-phosphate pathway impacts vegetative phase change in Arabidopsis thaliana.

The vegetative phase change marks the beginning of the adult phase in the life cycle of plants and is associated with a gradual decline in the microRNA miR156, in response to sucrose status. Trehalose 6-phosphate (T6P) is a sugar molecule with signaling function reporting the current sucrose state. To elucidate the role of T6P signaling in vegetative phase change, molecular, genetic, and metabolic analyses were performed using Arabidopsis thaliana loss-of-function lines in TREHALOSE PHOSPHATE SYNTHASE1 (TPS1), a gene coding for an enzyme that catalyzes the production of T6P. These lines show a significant delay in vegetative phase change, under both short and long day conditions. Induced expression of TPS1 complements this delay in the TPS1 knockout mutant (tps1-2 GVG::TPS1). Further analyses indicate that the T6P pathway promotes vegetative phase transition by suppressing miR156 expression and thereby modulating the levels of its target transcripts, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes. TPS1 knockdown plants, with a delayed vegetative phase change phenotype, accumulate significantly more sucrose than wild-type plants as a result of a feedback mechanism. In summary, we conclude that the T6P pathway forms an integral part of an endogenous mechanism that influences phase transitions dependent on the metabolic state.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1.

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (ß3-ß4 loop to α0 helix) and movement of a 'shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a 'closed' state compared with its 'open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Nitrate acts at the Arabidopsis thaliana shoot apical meristem to regulate flowering time.

Optimal timing of flowering, a major determinant for crop productivity, is controlled by environmental and endogenous cues. Nutrients are known to modify flowering time; however, our understanding of how nutrients interact with the known pathways, especially at the shoot apical meristem (SAM), is still incomplete. Given the negative side-effects of nitrogen fertilization, it is essential to understand its mode of action for sustainable crop production. We investigated how a moderate restriction by nitrate is integrated into the flowering network at the SAM, to which plants can adapt without stress symptoms. This condition delays flowering by decreasing expression of SUPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) at the SAM. Measurements of nitrate and the responses of nitrate-responsive genes suggest that nitrate functions as a signal at the SAM. The transcription factors NIN-LIKE PROTEIN 7 (NLP7) and NLP6, which act as master regulators of nitrate signaling by binding to nitrate-responsive elements (NREs), are expressed at the SAM and flowering is delayed in single and double mutants. Two upstream regulators of SOC1 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (SPL3) and SPL5) contain functional NREs in their promoters. Our results point at a tissue-specific, nitrate-mediated flowering time control in Arabidopsis thaliana.

SnRK1/TOR/T6P: three musketeers guarding energy for root growth.

Sugars derived from photosynthesis, specifically sucrose, are the primary source of plant energy. Sucrose is produced in leaves and transported to the roots through the phloem, serving as a vital energy source. Environmental conditions can result in higher or lower photosynthesis, promoting anabolism or catabolism, respectively, thereby influencing the sucrose budget available for roots. Plants can adjust their root system to optimize the search for soil resources and to ensure the plant's adaptability to diverse environmental conditions. Recently, emerging research indicates that SNF1-RELATED PROTEIN KINASE 1 (SnRK1), trehalose 6-phosphate (T6P), and TARGET OF RAPAMYCIN (TOR) collectively serve as fundamental regulators of root development, together forming a signaling module to interpret the nutritional status of the plant and translate this to growth adjustments in the below ground parts.

Sucrose homeostasis: Mechanisms and opportunity in crop yield improvement.

Sugar homeostasis is a critical feature of biological systems. In humans, raised and dysregulated blood sugar is a serious health issue. In plants, directed changes in sucrose homeostasis and allocation represent opportunities in crop improvement. Plant tissue sucrose varies more than blood glucose and is found at higher concentrations (cytosol and phloem ca. 100 mM v 3.9-6.9 mM for blood glucose). Tissue sucrose varies with developmental stage and environment, but cytosol and phloem exhibit tight sucrose control. Sucrose homeostasis is a consequence of the integration of photosynthesis, synthesis of storage end-products such as starch, transport of sucrose to sinks and sink metabolism. Trehalose 6-phosphate (T6P)-SnRK1 and TOR play central, still emerging roles in regulating and coordinating these processes. Overall, tissue sucrose levels are more strongly related to growth than to photosynthesis. As a key sucrose signal, T6P regulates sucrose levels, transport and metabolic pathways to coordinate source and sink at a whole plant level. Emerging evidence shows that T6P interacts with meristems. With careful targeting, T6P manipulation through exploiting natural variation, chemical intervention and genetic modification is delivering benefits for crop yields. Regulation of cereal grain set, filling and retention may be the most strategically important aspect of sucrose allocation and homeostasis for food security.

Distinct ancient structural polymorphisms control heterodichogamy in walnuts and hickories.

The maintenance of stable mating type polymorphisms is a classic example of balancing selection, underlying the nearly ubiquitous 50/50 sex ratio in species with separate sexes. One lesser known but intriguing example of a balanced mating polymorphism in angiosperms is heterodichogamy - polymorphism for opposing directions of dichogamy (temporal separation of male and female function in hermaphrodites) within a flowering season. This mating system is common throughout Juglandaceae, the family that includes globally important and iconic nut and timber crops - walnuts (Juglans), as well as pecan and other hickories (Carya). In both genera, heterodichogamy is controlled by a single dominant allele. We fine-map the locus in each genus, and find two ancient (>50 Mya) structural variants involving different genes that both segregate as genus-wide trans-species polymorphisms. The Juglans locus maps to a ca. 20 kb structural variant adjacent to a probable trehalose phosphate phosphatase (TPPD-1), homologs of which regulate floral development in model systems. TPPD-1 is differentially expressed between morphs in developing male flowers, with increased allele-specific expression of the dominant haplotype copy. Across species, the dominant haplotype contains a tandem array of duplicated sequence motifs, part of which is an inverted copy of the TPPD-1 3' UTR. These repeats generate various distinct small RNAs matching sequences within the 3' UTR and further downstream. In contrast to the single-gene Juglans locus, the Carya heterodichogamy locus maps to a ca. 200-450 kb cluster of tightly linked polymorphisms across 20 genes, some of which have known roles in flowering and are differentially expressed between morphs in developing flowers. The dominant haplotype in pecan, which is nearly always heterozygous and appears to rarely recombine, shows markedly reduced genetic diversity and is over twice as long as its recessive counterpart due to accumulation of various types of transposable elements. We did not detect either genetic system in other heterodichogamous genera within Juglandaceae, suggesting that additional genetic systems for heterodichogamy may yet remain undiscovered.

Genome-Wide Identification of Trehalose-6-phosphate Synthase (TPS) Gene Family Reveals the Potential Role in Carbohydrate Metabolism in Peach.

Trehalose-6-phosphate synthase (TPS) is essential for plant growth and development, linking trehalose-6-phosphate (T6P) to carbon metabolism. However, little is known about the TPS gene family in peaches and their potential roles in regulating carbohydrates in peach fruit. In this study, nine TPS genes were identified in the peach genome and named according to the homologous genes in Arabidopsis. Phylogenetic analysis showed that three subfamilies were identified, including TPSI, TPSII-1, and TPSII-2, which were also consistent with gene structure analysis. Considerable cis-elements were enriched in the promoters, including plant hormone-related elements. Tissue-specific analysis showed that these TPS genes were mainly expressed in leaves, stems, and fruit, showing different expression patterns for each gene. In addition, during fruit development, the content of trehalose-6-phosphate (T6P) was positively correlated with the expression of PpTPS7a and negatively with sucrose non-fermenting-1-related kinase 1 (SnRK1) activity. Transient overexpression and silencing of PpTPS7a in peach fruit validated its function in regulating T6P content and SnRK1 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.

Trehalose 6-Phosphate/SnRK1 Signaling Participates in Harvesting-Stimulated Rubber Production in the Hevea Tree.

Trehalose 6-phosphate (T6P), the intermediate of trehalose biosynthesis and a signaling molecule, affects crop yield via targeting sucrose allocation and utilization. As there have been no reports of T6P signaling affecting secondary metabolism in a crop plant, the rubber tree Hevea brasiliensis serves as an ideal model in this regard. Sucrose metabolism critically influences the productivity of natural rubber, a secondary metabolite of industrial importance. Here, we report on the characterization of the T6P synthase (TPS) gene family and the T6P/SNF1-related protein kinase1 (T6P/SnRK1) signaling components in Hevea laticifers under tapping (rubber harvesting), an agronomic manipulation that itself stimulates rubber production. A total of fourteen TPS genes were identified, among which a class II TPS gene, HbTPS5, seemed to have evolved with a function specialized in laticifers. T6P and trehalose increased when the trees were tapped, this being consistent with the observed enhanced activities of TPS and T6P phosphatase (TPP) and expression of an active TPS-encoding gene, HbTPS1. On the other hand, SnRK1 activities decreased, suggesting the inhibition of elevated T6P on SnRK1. Expression profiles of the SnRK1 marker genes coincided with elevated T6P and depressed SnRK1. Interestingly, HbTPS5 expression decreased significantly with the onset of tapping, suggesting a regulatory function in the T6P pathway associated with latex production in laticifers. In brief, transcriptional, enzymatic, and metabolic evidence supports the participation of T6P/SnRK1 signaling in rubber formation, thus providing a possible avenue to increasing the yield of a valuable secondary metabolite by targeting T6P in specific cells.

Genome-Wide Identification of Cotton (Gossypium spp.) Trehalose-6-Phosphate Phosphatase (TPP) Gene Family Members and the Role of GhTPP22 in the Response to Drought Stress.

Trehalose-6-phosphate phosphatase (TPP) is a key enzyme involved in trehalose synthesis in higher plants. Previous studies have shown that TPP family genes increase yields without affecting plant growth under drought conditions, but their functions in cotton have not been reported. In this study, 17, 12, 26 and 24 TPP family genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. The 79 TPP family genes were divided into three subgroups by phylogenetic analysis. Virus-induced gene silencing (VIGS) of GhTPP22 produced TRV::GhTPP22 plants that were more sensitive to drought stress than the control plants, and the relative expression of GhTPP22 was decreased, as shown by qRT-PCR. Moreover, we analysed the gene structure, targeted small RNAs, and gene expression patterns of TPP family members and the physicochemical properties of their encoded proteins. Overall, members of the TPP gene family in cotton were systematically identified, and the function of GhTPP22 under drought stress conditions was preliminarily verified. These findings provide new information for improving drought resistance for cotton breeding in the future.

Metabolic mechanism of nitrogen modified atmosphere storage on delaying quality deterioration of rice grains.

Nitrogen modified atmosphere was an effective way to control pest infestation in grains. In this study, the quality changes of rice during nitrogen modified atmosphere packaging storage (N2-MAPS) were monitored. An un-targeted metabolomics method was used to detect the rice metabolites and explore the mechanism of N2-MAPS for delaying rice deterioration. In this study, two rice species were studied under N2-MAPS and conventional storage at 30 °C for 150 days. The quality changes of rice during storage were monitored. The results showed that N2-MAPS could retard the increase of fatty acid value and amylose content, and defer the decrease of enzyme activities. And N2-MAPS had no significant influence on texture characteristics of rice. The metabolomics results suggested some metabolites and pathways were affected by N2-MAPS and revealed that N2-MAPS could protect rice cells from oxidative damage, maintain cell integrity and stability by regulating the metabolism to delay the rice deterioration.

What are the regulatory targets for intervention in assimilate partitioning to improve crop yield and resilience?

Sucrose utilisation for the synthesis of cellular components involved in growth and development and the accumulation of biomass determines diversity in the plant kingdom; sucrose utilisation and partitioning also underpin crop yields. As a complex process the use of sucrose for the partitioning of plant products for yield is decided by the interaction of several regulatory hubs and the integration of metabolism and development. Understanding the regulation of assimilate partitioning has been a grand challenge in plant and crop science. There are emerging examples of genes and processes that appear important for assimilate partitioning that underpin yield in crops and which are amenable to intervention. Enzymes of carbon metabolism were some of the first targets in attempts to modify assimilate partitioning at the beginning (source) and end (sink) of the whole plant assimilate partitioning process. Metabolic enzymes are subject to regulatory and homeostatic mechanisms, a key factor to consider in modifying assimilate partitioning. Trehalose 6-phosphate, as a sucrose signal, may represent a special case in its ability to regulate and coordinate source and sink processes. This review summarises recent progress in understanding the underlying regulators of assimilate partitioning and the current and potentially most promising routes to crop yield enhancement with a main focus on cereals. A framework for how source-sink may regulate whole plant assimilate partitioning involving a few key elements and the central importance of reproductive development is presented.

Roots of the Resurrection Plant Tripogon loliiformis Survive Desiccation Without the Activation of Autophagy Pathways by Maintaining Energy Reserves.

Being sessile, plants must regulate energy balance, potentially via source-sink relations, to compromise growth with survival in stressful conditions. Crops are sensitive, possibly because they allocate their energy resources toward growth and yield rather than stress tolerance. In contrast, resurrection plants tightly regulate sugar metabolism and use a series of physiological adaptations to suppress cell death in their vegetative tissue to regain full metabolic capacity from a desiccated state within 72 h of watering. Previously, we showed that shoots of the resurrection plant Tripogon loliiformis, initiate autophagy upon dehydration as one strategy to reinstate homeostasis and suppress cell death. Here, we describe the relationship between energy status, sugar metabolism, trehalose-mediated activation of autophagy pathways and investigate whether shoots and roots utilize similar desiccation tolerance strategies. We show that despite containing high levels of trehalose, dehydrated Tripogon roots do not display elevated activation of autophagy pathways. Using targeted and non-targeted metabolomics, transmission electron microscopy (TEM) and transcriptomics we show that T. loliiformis engages a strategy similar to the long-term drought responses of sensitive plants and continues to use the roots as a sink even during sustained stress. Dehydrating T. loliiformis roots contained more sucrose and trehalose-6-phosphate compared to shoots at an equivalent water content. The increased resources in the roots provides sufficient energy to cope with stress and thus autophagy is not required. These results were confirmed by the absence of autophagosomes in roots by TEM. Upregulation of sweet genes in both shoots and roots show transcriptional regulation of sucrose translocation from leaves to roots and within roots during dehydration. Differences in the cell's metabolic status caused starkly different cell death responses between shoots and roots. These findings show how shoots and roots utilize different stress response strategies and may provide candidate targets that can be used as tools for the improvement of stress tolerance in crops.

Dynamic changes in the starch-sugar interconversion within plant source and sink tissues promote a better abiotic stress response.

Starch is a significant store of sugars, and the starch-sugar interconversion in source and sink tissues plays a profound physiological role in all plants. In this review, we discuss how changes in starch metabolism can facilitate adaptive changes in source-sink carbon allocation for protection against environmental stresses. The stress-related roles of starch are described, and published mechanisms by which starch metabolism responds to short- or long-term water deficit, salinity, or extreme temperatures are discussed. Numerous examples of starch metabolism as a stress response are also provided, focusing on studies where carbohydrates and cognate enzymes were assayed in source, sink, or both. We develop a model that integrates these findings with the theoretical and known roles of sugars and starch in various species, tissues, and developmental stages. In this model, localized starch degradation into sugars is vital to the plant cold stress response, with the sugars produced providing osmoprotection. In contrast, high starch accumulation is prominent under salinity stress, and is associated with higher assimilate allocation from source to sink. Our model explains how starch-sugar interconversion can be a convergent point for regulating carbon use in stress tolerance at the whole-plant level.

Molecular Insights into the Enigmatic Metabolic Regulator, SnRK1.

Sucrose non-fermenting-1 (SNF1)-related kinase 1 (SnRK1) lies at the heart of metabolic homeostasis in plants and is crucial for normal development and response to stress. Evolutionarily related to SNF1 in yeast and AMP-activated kinase (AMPK) in mammals, SnRK1 acts protectively to maintain homeostasis in the face of fluctuations in energy status. Despite a conserved function, the structure and regulation of the plant kinase differ considerably from its relatively well-understood opisthokont orthologues. In this review, we highlight the known plant-specific modes of regulation involving SnRK1 together with new insights based on a 3D molecular model of the kinase. We also summarise how these differences from other orthologues may be specific adaptations to plant metabolism, and offer insights into possible avenues of future inquiry into this enigmatic enzyme.

Interaction of storage carbohydrates and other cyclic fluxes with central metabolism: A quantitative approach by non-stationary 13C metabolic flux analysis.

13C labeling experiments in aerobic glucose limited cultures of Saccharomyces cerevisiae at four different growth rates (0.054; 0.101, 0.207, 0.307 h-1) are used for calculating fluxes that include intracellular cycles (e.g., storage carbohydrate cycles, exchange fluxes with amino acids), which are rearranged depending on the growth rate. At low growth rates the impact of the storage carbohydrate recycle is relatively more significant than at high growth rates due to a higher concentration of these materials in the cell (up to 560-fold) and higher fluxes relative to the glucose uptake rate (up to 16%). Experimental observations suggest that glucose can be exported to the extracellular space, and that its source is related to storage carbohydrates, most likely via the export and subsequent extracellular breakdown of trehalose. This hypothesis is strongly supported by 13C-labeling experimental data, measured extracellular trehalose, and the corresponding flux estimations.