Sucrose Export Research Articles

PROCERA interacts with JACKDAW in gibberellin-enhanced source-sink sucrose partitioning in tomato.

Proper regulation of the source-sink relationship is an effective way to increase crop yield. Gibberellin (GA) is an important regulator of plant growth and development, and physiological evidence has demonstrated that GA can promote source-sink sucrose partitioning. However, the underlying molecular mechanism remains unclear. Here, we used a combination of physiological and molecular approaches to identify the components involved in GA-enhanced source-sink sucrose partitioning in tomato (Solanum lycopersicum). GA treatment increased the sucrose export rate from source leaves and the sucrose level in young leaves (sink organ). GA-mediated enhancement of source-sink sucrose partitioning depended on SlPROCERA (SlPRO), the DELLA protein in tomato. Sucrose transporter 1 (SlSUT1) was involved in phloem sucrose loading. SlJACKDAW (SlJKD) was identified as an interaction partner of SlPRO. SlJKD negatively regulated the sucrose export rate from source leaves and could directly bind to the promoter of SlSUT1 and repress its expression, while SlPRO enhanced the transcription repression function of SlJKD. This study reveals the molecular mechanism by which GA promotes source-sink sucrose partitioning in tomato and provides potential targets for source-sink relationship optimization.

Fast and simple fluorometric measurement of phloem loading exposes auxin-dependent regulation of Arabidopsis sucrose transporter AtSUC2.

The rate of sucrose export from leaves is a major factor in balancing whole-plant carbon and energy partitioning. A comprehensive study of its dynamics and relationship to photosynthesis, sink demand, and other relevant processes is hampered by the shortcomings of current methods for measuring sucrose phloem loading. We utilize the ability of sucrose transporter proteins, known as SUCs or SUTs, to specifically transport the fluorescent molecule esculin in a novel assay to measure phloem loading rates. Esculin was administered to source leaves and its fluorescence in the leaf extract was measured after 1 or 2 h. Dicot plants with an active phloem loading strategy showed an export-dependent reduction of esculin fluorescence. Relative leaf esculin export rates correlated with leaf export rates of isotopic carbon and phloem exudate sucrose levels. We used esculin experiments to examine the effects of phytohormones on phloem loading in Arabidopsis, showing, for example, that auxin induces phloem loading while cytokinin reduces it. Transcriptional regulation of AtSUC2 by AUXIN RESPONSE FACTOR1 (ARF1) corroborated the link between auxin signaling and phloem loading. Unlike established methods, the esculin assay is rapid and does not require specialized equipment. Potential applications and limitations of the esculin assay are discussed.

Metabolic and transcriptomic analyses of nectaries reveal differences in the mechanism of nectar production between monocots (Ananas comosus) and dicots (Nicotiana tabacum)

BackgroundNectar is offered by numerous flowering plants to attract pollinators. To date, the production and secretion of nectar have been analyzed mainly in eudicots, particularly rosids such as Arabidopsis. However, due to the enormous diversity of flowering plants, further research on other plant species, especially monocots, is needed. Ananas comosus (monocot) is an economically important species that is ideal for such analyses because it produces easily accessible nectar in sufficient quantities. In addition, the analyses were also carried out with Nicotiana tabacum (dicot, asterids) for comparison.ResultsWe performed transcriptome sequencing (RNA-Seq) analyses of the nectaries of Ananas comosus and Nicotiana tabacum, to test whether the mechanisms described for nectar production and secretion in Arabidopsis are also present in these plant species. The focus of these analyses is on carbohydrate metabolism and transport (e.g., sucrose-phosphate synthases, invertases, sucrose synthases, SWEETs and further sugar transporters). In addition, the metabolites were analyzed in the nectar, nectaries and leaves of both plant species to address the question of whether concentration gradients for different metabolites exist between the nectaries and nectar The nectar of N. tabacum contains large amounts of glucose, fructose and sucrose, and the sucrose concentration in the nectar appears to be similar to the sucrose concentration in the nectaries. Nectar production and secretion in this species closely resemble corresponding processes in some other dicots, including sucrose synthesis in nectaries and sucrose secretion by SWEET9. The nectar of A. comosus also contains large amounts of glucose, fructose and sucrose and in this species the sucrose concentration in the nectar appears to be higher than the sucrose concentration in the nectaries. Furthermore, orthologs of SWEET9 generally appear to be absent in A. comosus and other monocots. Therefore, sucrose export by SWEETs from nectaries into nectar can be excluded; rather, other mechanisms, such as active sugar export or exocytosis, are more likely.ConclusionThe mechanisms of nectar production and secretion in N. tabacum appear to be largely similar to those in other dicots, whereas in the monocotyledonous species A. comosus, different synthesis and transport processes are involved.

Perturbation of tonoplast sucrose transport alters carbohydrate utilization for seasonal growth and defense metabolism in coppiced poplar.

Nonstructural carbohydrate reserves of stems and roots underpin overall tree fitness and productivity under short-rotation management practices such as coppicing for bioenergy. While sucrose and starch comprise the predominant stem carbohydrate reserves of Populus, utilization for fitness and agricultural productivity is understood primarily in terms of starch turnover. The tonoplast sucrose transport protein SUT4 modulates sucrose export from source leaves to distant sinks during photoautotrophic growth, but the possibility of its involvement in remobilizing carbohydrates from storage organs during heterotrophic growth has not been explored. Here, we used PtaSUT4-knockout mutants of Populus tremula × P. alba (INRA 717-1B4) in winter (cool) and summer (warm) glasshouse coppicing experiments to assess SUT4 involvement in reserve utilization. Conditions preceding and supporting summer sprouting were considered favorable for growth, while those preceding and supporting cool temperature sprouting were suboptimal akin to conditions associated with coppicing as generally practiced. Epicormic bud emergence was delayed in sut4 mutants following lower temperature 'winter' but not summer coppicing. Winter xylem hexose increases were observed in control but not in sut4 stumps after coppicing. The magnitude of starch and sucrose reserve depletion was similar in control and sut4 stumps during the winter and did not explain the sprouting and xylem hexose differences. However, winter maintenance costs appeared higher in sut4 based partly on Krebs cycle intermediate levels. In control plants, bark accrual of abundant defense metabolites, including salicinoids and condensed tannins, was higher in summer than in winter, but this increase of summer defense allocations was attenuated in sut4 mutants. Temperature-sensitive trade-offs between growth and other priorities may therefore depend on SUT4 in Populus.

Reduced global plant respiration due to the acclimation of leaf dark respiration coupled with photosynthesis.

Leaf dark respiration (Rd ) acclimates to environmental changes. However, the magnitude, controls and time scales of acclimation remain unclear and are inconsistently treated in ecosystem models. We hypothesized that Rd and Rubisco carboxylation capacity (Vcmax ) at 25°C (Rd,25 , Vcmax,25 ) are coordinated so that Rd,25 variations support Vcmax,25 at a level allowing full light use, with Vcmax,25 reflecting daytime conditions (for photosynthesis), and Rd,25 /Vcmax,25 reflecting night-time conditions (for starch degradation and sucrose export). We tested this hypothesis temporally using a 5-yr warming experiment, and spatially using an extensive field-measurement data set. We compared the results to three published alternatives: Rd,25 declines linearly with daily average prior temperature; Rd at average prior night temperatures tends towards a constant value; and Rd,25 /Vcmax,25 is constant. Our hypothesis accounted for more variation in observed Rd,25 over time (R2 = 0.74) and space (R2 = 0.68) than the alternatives. Night-time temperature dominated the seasonal time-course of Rd , with an apparent response time scale of c. 2 wk. Vcmax dominated the spatial patterns. Our acclimation hypothesis results in a smaller increase in global Rd in response to rising CO2 and warming than is projected by the two of three alternative hypotheses, and by current models.

Alteration in the expression of tomato sucrose transporter gene SlSUT4 modulates sucrose subcellular compartmentation and affects responses of plants to drought stress

Sucrose transporters (SUTs) are involved in the transmembrane transport of sucrose. The roles of SUTs in regulating stress tolerance of plants have been reported, but differential results were observed in different plants, implying the need for more studies in different plants. Moreover, the mechanism of SUTs in regulating stress tolerance is still poorly understood. Here, we investigated the role of SlSUT4 from tomato in regulating drought tolerance by using SlSUT4-overexpressed and -RNAi plants. The results showed that SlSUT4 overexpression decreased the drought tolerance of plants, as demonstrated by reduced seed germination, decreased plant growth, photosynthesis and relative water content, and higher oxidative damage under osmotic stress or drought stress; whereas silencing of SlSUT4 increased the stress tolerance. In the control conditions, the leaf sucrose export rate was reduced in the overexpression plants, but promoted in the RNAi plants, and all transgenic lines had higher leaf sucrose concentrations than the wild type. Accumulation of soluble sugars was more obvious in SlSUT4-overexpressed plants and the wild type than the RNAi plants under drought. Overexpressing SlSUT4 decreased the proportion of sucrose in the vacuole in the control conditions, whereas the RNAi plants showed an opposite change. These results suggest that alteration in SlSUT4 expression modulates sucrose vacuole-cytosol compartmentation, thus affecting the responses of tomato plants to drought stress.

Tomato sucrose transporter SlSUT4 participates in flowering regulation by modulating gibberellin biosynthesis.

The functions of sucrose transporters (SUTs) differ among family members. The physiological function of SUT1 has been studied intensively, while that of SUT4 in various plant species including tomato (Solanum lycopersicum) is less well-understood. In this study, we characterized the function of tomato SlSUT4 in the regulation of flowering using a combination of molecular and physiological analyses. SlSUT4 displayed transport activity for sucrose when expressed in yeast (Saccharomyces cerevisiae), and it localized at both the plasma membrane and tonoplast. SlSUT4 interacted with SlSUT1, causing partial internalization of the latter, the main phloem loader of sucrose in tomato. Silencing of SlSUT4 promoted SlSUT1 localization to the plasma membrane, contributing to increased sucrose export and thus increased sucrose level in the shoot apex, which promoted flowering. Both silencing of SlSUT4 and spraying with sucrose suppressed gibberellin biosynthesis through repression of ent-kaurene oxidase and gibberellin 20-oxidase-1 (2 genes encoding key enzymes in gibberellin biosynthesis) expression by SlMYB76, which directly bound to their promoters. Silencing of SlMYB76 promoted gibberellin biosynthesis. Our results suggest that SlSUT4 is a functional SUT in tomato; downregulation of SlSUT4 expression enhances sucrose transport to the shoot apex, which promotes flowering by inhibiting gibberellin biosynthesis.

Physiological implications of SWEETs in plants and their potential applications in improving source-sink relationships for enhanced yield.

The sugars will eventually be exported transporters (SWEET) family of transporters in plants is identified as a novel class of sugar carriers capable of transporting sugars, sugar alcohols and hormones. Functioning in intercellular sugar transport, SWEETs influence a wide range of physiologically important processes. SWEETs regulate the development of sink organs by providing nutritional support from source leaves, responses to abiotic stresses by maintaining intracellular sugar concentrations, and host-pathogen interactions through the modulation of apoplastic sugar levels. Many bacterial and fungal pathogens activate the expression of SWEET genes in species such as rice and Arabidopsis to gain access to the nutrients that support virulence. The genetic manipulation of SWEETs has led to the generation of bacterial blight (BB)-resistant rice varieties. Similarly, while the overexpression of the SWEETs involved in sucrose export from leaves and pathogenesis led to growth retardation and yield penalties, plants overexpressing SWEETs show improved disease resistance. Such findings demonstrate the complex functions of SWEETs in growth and stress tolerance. Here, we review the importance of SWEETs in plant-pathogen and source-sink interactions and abiotic stress resistance. We highlight the possible applications of SWEETs in crop improvement programmes aimed at improving sink and source strengths important for enhancing the sustainability of yield. We discuss how the adverse effects of the overexpression of SWEETs on plant growth may be overcome.

Genetic manipulation of protein phosphatase 2A affects multiple agronomic traits and physiological parameters in potato (Solanum tuberosum).

In this study, agronomic and functional characteristics of potato (Solanum tuberosum ) plants constitutively overexpressing the protein phosphatase 2A (PP2A) catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were evaluated. StPP2Ac2b-OE plants display reduced vegetative growth, tuber yield and tuber weight under well-watered and drought conditions. Leaves of StPP2Ac2b-OE plants show an increased rate of water loss, associated with an impaired ability to close stomata in response to abscisic acid. StPP2Ac2b-OE lines exhibit larger stomatal size and reduced stomatal density. These altered stomatal characteristics might be responsible for the impaired stomatal closure and the elevated transpiration rates, ultimately leading to increased sensitivity to water-deficit stress and greater yield loss under drought conditions. Overexpression of StPP2Ac2b accelerates senescence in response to water-deficit stress, which could also contribute to the increased sensitivity to drought. Actively photosynthesising leaves of StPP2Ac2b-OE plants exhibit elevated levels of carbohydrates and a down-regulation of the sucrose transporter StSWEET11 , suggesting a reduced sucrose export from leaves to developing tubers. This effect, combined with the hindered vegetative development, may contribute to the reduced tuber weight and yield in StPP2Ac2b-OE plants. These findings offer novel insights into the physiological functions of PP2A in potato plants and provide valuable information for enhancing potato productivity by modulating the expression of StPP2Ac2b .

Long-distance transport of sucrose in source leaves promotes sink root growth by the EIN3-SUC2 module.

In most plants, sucrose, a major storage sugar, is transported into sink organs to support their growth. This key physiological process is dependent on the function of sucrose transporters. Sucrose export from source tissues is predominantly controlled through the activity of SUCROSE TRANSPORTER 2 (SUC2), required for the loading of sucrose into the phloem of Arabidopsis plants. However, how SUC2 activity is controlled to support root growth remains unclear. Glucose is perceived via the function of HEXOKINASE 1 (HXK1), the only known nuclear glucose sensor. HXK1 negatively regulates the stability of ETHYLENE-INSENSITIVE3 (EIN3), a key ethylene/glucose interaction component. Here we show that HXK1 functions upstream of EIN3 in the regulation of root sink growth mediated by glucose signaling. Furthermore, the transcription factor EIN3 directly inhibits SUC2 activity by binding to the SUC2 promoter, regulating glucose signaling linked to root sink growth. We demonstrate that these molecular components form a HXK1-EIN3-SUC2 module integral to the control of root sink growth. Also, we demonstrate that with increasing age, the HXK1-EIN3-SUC2 module promotes sucrose phloem loading in source tissues thereby elevating sucrose levels in sink roots. As a result, glucose signaling mediated-sink root growth is facilitated. Our findings thus establish a direct molecular link between the HXK1-EIN3-SUC2 module, the source-to sink transport of sucrose and root growth.

Elucidation of the interactome of the sucrose transporter StSUT4: sucrose transport is connected to ethylene and calcium signalling.

Sucrose transporters of the SUT4 clade show dual targeting to both the plasma membrane as well as to the vacuole. Previous investigations revealed a role for the potato sucrose transporter StSUT4 in flowering, tuberization, shade avoidance response, and ethylene production. Down-regulation of StSUT4 expression leads to early flowering, tuberization under long days, far-red light insensitivity, and reduced diurnal ethylene production. Sucrose export from leaves was increased and a phase-shift of soluble sugar accumulation in source leaves was observed, arguing for StSUT4 to be involved in the entrainment of the circadian clock. Here, we show that StSUT4, whose transcripts are highly unstable and tightly controlled at the post-transcriptional level, connects components of the ethylene and calcium signalling pathway. Elucidation of the StSUT4 interactome using the split ubiquitin system helped to prove direct physical interaction between the sucrose transporter and the ethylene receptor ETR2, as well as with the calcium binding potato calmodulin-1 (PCM1) protein, and a calcium-load activated calcium channel. The impact of calcium ions on transport activity and dual targeting of the transporter was investigated in detail. For this purpose, a reliable esculin-based transport assay was established for SUT4-like transporters. Site-directed mutagenesis helped to identify a diacidic motif within the seventh transmembrane spanning domain that is essential for sucrose transport activity and targeting, but not required for calcium-dependent inhibition. A link between sucrose, calcium and ethylene signalling has been previously postulated with respect to pollen tube growth, shade avoidance response, or entrainment of the circadian clock. Here, we provide experimental evidence for the direct interconnection of these signalling pathways at the molecular level by direct physical interaction of the main players.

Adaptive responses of carbon and nitrogen metabolisms to nitrogen-deficiency in Citrus sinensis seedlings

BackgroundIn China, nitrogen (N)-deficiency often occurs in Citrus orchards, which is one of the main causes of yield loss and fruit quality decline. Little information is known about the adaptive responses of Citrus carbon (C) and N metabolisms to N-deficiency. Seedlings of ‘Xuegan’ (Citrus sinensis (L.) Osbeck) were supplied with nutrient solution at an N concentration of 0 (N-deficiency), 5, 10, 15 or 20 mM for 10 weeks. Thereafter, we examined the effects of N supply on the levels of C and N in roots, stems and leaves, and the levels of organic acids, nonstructural carbohydrates, NH4+-N, NO3−-N, total soluble proteins, free amino acids (FAAs) and derivatives (FAADs), and the activities of key enzymes related to N assimilation and organic acid metabolism in roots and leaves.ResultsN-deficiency elevated sucrose export from leaves to roots, C and N distributions in roots and C/N ratio in roots, stems and leaves, thus enhancing root dry weight/shoot dry weight ratio and N use efficiency. N-deficient leaves displayed decreased accumulation of starch and total nonstructural carbohydrates (TNC) and increased sucrose/starch ratio as well as a partitioning trend of assimilated C toward to sucrose, but N-deficient roots displayed elevated accumulation of starch and TNC and reduced sucrose/starch ratio as well as a partitioning trend of assimilated C toward to starch. N-deficiency reduced the concentrations of most FAADs and the ratios of total FAADs (TFAADs)/N in leaves and roots. N-deficiency reduced the demand for C skeleton precursors for amino acid biosynthesis, thus lowering TFAADs/C ratio in leaves and roots. N-deficiency increased (decreased) the relative amounts of C-rich (N-rich) FAADs, thus increasing the molar ratio of C/N in TFAADs in leaves and roots.ConclusionsOur findings corroborated our hypothesis that C and N metabolisms displayed adaptive responses to N-deficiency in C. sinensis seedlings, and that some differences existed between roots and leaves in N-deficiency-induced alterations of and C and N metabolisms.

Regulatory Modules of Metabolites and Protein Phosphorylation in Arabidopsis Genotypes With Altered Sucrose Allocation.

Multi-omics data sets are increasingly being used for the interpretation of cellular processes in response to environmental cues. Especially, the posttranslational modification of proteins by phosphorylation is an important regulatory process affecting protein activity and/or localization, which, in turn, can have effects on metabolic processes and metabolite levels. Despite this importance, relationships between protein phosphorylation status and metabolite abundance remain largely underexplored. Here, we used a phosphoproteomics–metabolomics data set collected at the end of day and night in shoots and roots of Arabidopsis to propose regulatory relationships between protein phosphorylation and accumulation or allocation of metabolites. For this purpose, we introduced a novel, robust co-expression measure suited to the structure of our data sets, and we used this measure to construct metabolite-phosphopeptide networks. These networks were compared between wild type and plants with perturbations in key processes of sugar metabolism, namely, sucrose export (sweet11/12 mutant) and starch synthesis (pgm mutant). The phosphopeptide–metabolite network turned out to be highly sensitive to perturbations in sugar metabolism. Specifically, KING1, the regulatory subunit of SnRK1, was identified as a primary candidate connecting protein phosphorylation status with metabolism. We additionally identified strong changes in the fatty acid network of the sweet11/12 mutant, potentially resulting from a combination of fatty acid signaling and metabolic overflow reactions in response to high internal sucrose concentrations. Our results further suggest novel protein-metabolite relationships as candidates for future targeted research.

Rubisco regulation in response to altered carbon status in the cyanobacterium Synechococcus elongatus PCC 7942.

Photosynthetic organisms possess a variety of mechanisms to achieve balance between absorbed light (source) and the capacity to metabolically utilize or dissipate this energy (sink). While regulatory processes that detect changes in metabolic status/balance are relatively well studied in plants, analogous pathways remain poorly characterized in photosynthetic microbes. Here, we explored systemic changes that result from alterations in carbon availability in the model cyanobacterium Synechococcus elongatus PCC 7942 by taking advantage of an engineered strain where influx/efflux of a central carbon metabolite, sucrose, can be regulated experimentally. We observed that induction of a high-flux sucrose export pathway leads to depletion of internal carbon storage pools (glycogen) and concurrent increases in estimates of photosynthetic activity. Further, a proteome-wide analysis and fluorescence reporter-based analysis revealed that upregulated factors following the activation of the metabolic sink are concentrated on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and auxiliary modules involved in Rubisco maturation. Carboxysome number and Rubisco activity also increased following engagement of sucrose secretion. Conversely, reversing the flux of sucrose by feeding exogenous sucrose through the heterologous transporter resulted in increased glycogen pools, decreased Rubisco abundance, and carboxysome reorganization. Our data suggest that Rubisco activity and organization are key variables connected to regulatory pathways involved in metabolic balancing in cyanobacteria.

Coordination Between Phloem Loading and Structure Maintains Carbon Transport Under Drought.

Maintaining phloem transport under water stress is expected to be crucial to whole-plant drought tolerance, but the traits that benefit phloem function under drought are poorly understood. Nearly half of surveyed angiosperm species, including important crops, use sucrose transporter proteins to actively load sugar into the phloem. Plants can alter transporter abundance in response to stress, providing a potential mechanism for active-loading species to closely regulate phloem loading rates to avoid drought-induced reductions or failures in phloem transport. We developed an integrated xylem-phloem-stomatal model to test this hypothesis by quantifying the joint impacts of transporter kinetics, phloem anatomy, and plant water status on sucrose export to sinks. We parameterized the model with phloem hydraulic resistances and sucrose transporter kinetic parameters compiled from the literature, and simulated loading regulation by allowing loading rates to decline exponentially with phloem pressure to prevent excessive sucrose concentrations from inducing viscosity limitations. In the absence of loading regulation, where loading rates were independent of phloem pressure, most resistance values produced unrealistic phloem pressures owing to viscosity effects, even under well-watered conditions. Conversely, pressure-regulated loading helped to control viscosity buildup and improved export to sinks for both lower and higher resistant phloem pathways, while maintaining realistic phloem pressures. Regulation also allowed for rapid loading and export in wet conditions while maintaining export and viable phloem pressures during drought. Therefore, we expect feedbacks between phloem pressure and loading to be critical to carbon transport in active-loading species, especially under drought, and for transporter kinetics to be strongly coordinated with phloem architecture and plant water status. This work provides an important and underexplored physiological framework to understand the ecophysiology of phloem transport under drought and to enhance the genetic engineering of crop plants.

Plasma membrane-localized SEM1 protein mediates sugar movement to sink rice tissues.

The translocation of photosynthate carbohydrates, such as sucrose, is critical for plant growth and crop yield. Previous studies have revealed that sugar transporters, plasmodesmata and sieve plates act as important controllers in sucrose loading into and unloading from phloem in the vascular system. However, other pivotal steps for the regulation of sucrose movement remain largely elusive. In this study, characterization of two starch excesses in mesophyll (sem) mutants and dye and sucrose export assays were performed to provide insights into the regulatory networks that drive source-sink relations in rice. Map-based cloning identified two allelic mutations in a gene encoding a GLUCAN SYNTHASE-LIKE (GSL) protein, thus indicating a role for SEM1 in callose biosynthesis. Subcellular localization in rice showed that SEM1 localized to the plasma membrane. In situ expression analysis and GUS staining showed that SEM1 was mainly expressed in vascular phloem cells. Reduced sucrose transport was found in the sem1-1/1-2 mutant, which led to excessive starch accumulation in source leaves and inhibited photosynthesis. Paraffin section and transmission electron microscopy experiments revealed that less-developed vascular cells (VCs) in sem1-1/1-2 potentially disturbed sugar movement. Moreover, dye and sugar trafficking experiments revealed that aberrant VC development was the main reason for the pleiotropic phenotype of sem1-1/1-2. In total, efficient sucrose loading into the phloem benefits from an optional number of VCs with a large vacuole that could act as a buffer holding tank for sucrose passing from the vascular bundle sheath.

A hybrid kinetic and constraint-based model of leaf metabolism allows predictions of metabolic fluxes in different environments.

While flux balance analysis (FBA) provides a framework for predicting steady-state leaf metabolic network fluxes, it does not readily capture the response to environmental variables without being coupled to other modelling formulations. To address this, we coupled an FBA model of 903 reactions of soybean (Glycine max) leaf metabolism with e-photosynthesis, a dynamic model that captures the kinetics of 126 reactions of photosynthesis and associated chloroplast carbon metabolism. Successful coupling was achieved in an iterative formulation in which fluxes from e-photosynthesis were used to constrain the FBA model and then, in turn, fluxes computed from the FBA model used to update parameters in e-photosynthesis. This process was repeated until common fluxes in the two models converged. Coupling did not hamper the ability of the kinetic module to accurately predict the carbon assimilation rate, photosystem II electron flux, and starch accumulation of field-grown soybean at two CO2 concentrations. The coupled model also allowed accurate predictions of additional parameters such as nocturnal respiration, as well as analysis of the effect of light intensity and elevated CO2 on leaf metabolism. Predictions included an unexpected decrease in the rate of export of sucrose from the leaf at high light, due to altered starch-sucrose partitioning, and altered daytime flux modes in the tricarboxylic acid cycle at elevated CO2 . Mitochondrial fluxes were notably different between growing and mature leaves, with greater anaplerotic, tricarboxylic acid cycle and mitochondrial ATP synthase fluxes predicted in the former, primarily to provide carbon skeletons and energy for protein synthesis.

A cell wall invertase controls nectar volume and sugar composition.

Nectar volume and sugar composition are key determinants of the strength of plant-pollinator mutualisms. The main nectar sugars are sucrose, glucose and fructose, which can vary widely in ratio and concentration across species. Brassicaspp. produce a hexose-dominant nectar (high in the monosaccharides glucose and fructose) with very low levels of the disaccharide sucrose. Cell wall invertases (CWINVs) catalyze the irreversible hydrolysis of sucrose into glucose and fructose in the apoplast. We found that BrCWINV4A is highly expressed in the nectaries of Brassica rapa. Moreover, a brcwinv4a null mutant: (i) has greatly reduced CWINV activity in the nectaries; (ii) produces a sucrose-rich nectar; but (iii) with significantly less volume. These results definitively demonstrate that CWINV activity is not only essential for the production of a hexose-rich nectar, but also support a hypothetical model of nectar secretion in which its hydrolase activity is required for maintaining a high intracellular-to-extracellular sucrose ratio that facilitates the continuous export of sucrose into the nectary apoplast. The extracellular hydrolysis of each sucrose into two hexoses by BrCWINV4A also likely creates the osmotic potential required for nectar droplet formation. These results cumulatively indicate that modulation of CWINV activity can at least partially account for naturally occurring differences in nectar volume and sugar composition. Finally, honeybees prefer nectars with some sucrose, but wild-type B.rapa flowers were much more heavily visited than flowers of brcwinv4a, suggesting that the potentially attractive sucrose-rich nectar of brcwinv4a could not compensate for its low volume.

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria

Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full "overcapacity" of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be "lost."

Expression Level of Mature miR172 in Wild Type and StSUT4-Silenced Plants of Solanum tuberosum Is Sucrose-Dependent.

In potato plants, the phloem-mobile miR172 is involved in the sugar-dependent transmission of flower and tuber inducing signal transduction pathways and a clear link between solute transport and the induction of flowering and tuberization was demonstrated. The sucrose transporter StSUT4 seems to play an important role in the photoperiod-dependent triggering of both developmental processes, flowering and tuberization, and the phenotype of StSUT4-inhibited potato plants is reminiscent to miR172 overexpressing plants. The first aim of this study was the determination of the level of miR172 in sink and source leaves of StSUT4-silenced as well as StSUT4-overexpressing plants in comparison to Solanum tuberosum ssp. Andigena wild type plants. The second aim was to investigate the effect of sugars on the level of miRNA172 in whole cut leaves, as well as in whole in vitro plantlets that were supplemented with exogenous sugars. Experiments clearly show a sucrose-dependent induction of the level of mature miR172 in short time as well as long time experiments. A sucrose-dependent accumulation of miR172 was also measured in mature leaves of StSUT4-silenced plants where sucrose export is delayed and sucrose accumulates at the end of the light period.