Sucrose Synthesis Pathway Research Articles

Sucrose-Phosphate Synthase and Sucrose Synthase contribute to refoliation in ryegrass, a grassland fructan-accumulating species.

The perennity of grassland species such as Lolium perenne greatly depends on their ability to regrow after cutting or grazing. Refoliation largely relies on the mobilization of fructans in the remaining tissues and on the associated sucrose synthesis and transport towards the basal leaf meristems. However, nothing is known yet about the sucrose synthesis pathway. Sucrose Phosphate Synthase (SPS) and Sucrose Synthase (SuS) activities, together with their transcripts, were monitored during the first hours after defoliation along the leaf axis of mature leaf sheaths and elongating leaf bases (ELB) where the leaf meristems are located. In leaf sheaths, which undergo a sink-source transition, fructan and sucrose contents declined while SPS and SuS activities increased, along with the expression of LpSPSA, LpSPSD.2, LpSuS1, LpSuS2, and LpSuS4. In ELB, which continue to act as a strong carbon sink, SPS and SuS activities increased to varying degrees while the expression of all the LpSPS and LpSuS genes decreased after defoliation. SPS and SuS both contribute to refoliation but are regulated differently depending on the source or sink status of the tissues. Together with fructan metabolism, they represent key determinants of ryegrass perennity and, more generally, of grassland sustainability.

Engineering Cyanobacterial Cell Factories for Photosynthetic Production of Fructose.

Fructose is an important monosaccharide product widely applied in the food, medicine, and chemical industries. Currently, fructose is mainly manufactured with plant biomass-sourced polysaccharides through multiple steps of digestion, conversion, separation, and purification. The development of cyanobacterial metabolic engineering provides an attractive alternative route for the one-step direct production of fructose utilizing carbon dioxide and solar energy. In this work, we developed a paradigm for engineering cyanobacterial chassis cells into efficient cell factories for the photosynthetic production of fructose. In a representative cyanobacterial strain, Synechococcus elongatus PCC 7942, knockout of fructokinase effectively activated the synthesis and secretion of fructose in hypersaline conditions, independent of any heterologous transporters. The native sucrose synthesis pathway was identified as playing a primary role in fructose synthesis. Through combinatory optimizations on the levels of metabolism, physiology, and cultivation, the fructose yield of the Synechococcus cell factories was stepwise improved to 3.9 g/L. Such a paradigm was also adopted to engineer another Synechococcus strain, the marine species Synechococcus sp. PCC 7002, and facilitated an even higher fructose yield of over 6 g/L. Finally, the fructose synthesized and secreted by the cyanobacterial photosynthetic cell factories was successfully extracted and prepared from the culture broth in the form of products with 86% purity through multistep separation-purification operations. This work demonstrated a paradigm for systematically engineering cyanobacteria for photosynthetic production of desired metabolites, and it also confirmed the feasibility and potential of cyanobacterial photosynthetic biomanufacturing as a simple and efficient route for fructose production.

Genome-Wide Identification of GmSPS Gene Family in Soybean and Expression Analysis in Response to Cold Stress.

Sucrose metabolism plays a critical role in development, stress response, and yield formation of plants. Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in the sucrose synthesis pathway. To date, genome-wide survey and comprehensive analysis of the SPS gene family in soybean (Glycine max) have yet to be performed. In this study, seven genes encoding SPS were identified in soybean genome. The structural characteristics, phylogenetics, tissue expression patterns, and cold stress response of these GmSPSs were investigated. A comparative phylogenetic analysis of SPS proteins in soybean, Medicago truncatula, Medicago sativa, Lotus japonicus, Arabidopsis, and rice revealed four families. GmSPSs were clustered into three families from A to C, and have undergone five segmental duplication events under purifying selection. All GmSPS genes had various expression patterns in different tissues, and family A members GmSPS13/17 were highly expressed in nodules. Remarkably, all GmSPS promoters contain multiple low-temperature-responsive elements such as potential binding sites of inducer of CBF expression 1 (ICE1), the central regulator in cold response. qRT-PCR proved that these GmSPS genes, especially GmSPS8/18, were induced by cold treatment in soybean leaves, and the expression pattern of GmICE1 under cold treatment was similar to that of GmSPS8/18. Further transient expression analysis in Nicotiana benthamiana and electrophoretic mobility shift assay (EMSA) indicated that GmSPS8 and GmSPS18 transcriptions were directly activated by GmICE1. Taken together, our findings may aid in future efforts to clarify the potential roles of GmSPS genes in response to cold stress in soybean.

SlSPS, a Sucrose Phosphate Synthase Gene, Mediates Plant Growth and Thermotolerance in Tomato

Heat stress (HS) has been considered as a severe threat to crop yields in recent years. Sucrose, as a major product of photosynthesis, plays an important role in plant growth and stress response. Sucrose phosphate synthase (SPS) is a key rate-limiting enzyme in the sucrose synthesis pathway in plants. However, its molecular mechanism and signaling pathway remain unclear. In this study, we identified a novel SPS gene (SlSPS) in tomato and generated over-expression and knock-out of SlSPS gene transgenic tomato plants to investigate its biological functions related to the growth and thermotolerance of tomato. Over-expression of SlSPS gene increased the growth and biomass of transgenic tomato plants, such as fresh weight, dry weight, plant height, fruit weight and root length. In contrast, knock-out of SlSPS gene decreased the growth and biomass of transgenic tomato plants. Under heat stress, the survival rates were positively correlated with the expression level of SlSPS gene in different tomato varieties. Furthermore, SlSPS-overexpressing tomato plants showed higher SPS activity and sucrose content and heat stress resistant phenotypes. By comparison, knock-out tomato plants showed lower SPS activity and sucrose content and susceptible to heat stress. The determination of several reference values of oxidative stress parameters were also consistent with their heat resistance of these transgenic plants. In summary, SlSPS gene could positively mediate the growth and thermotolerance in tomato plants.

Disequilibrium evolution of the Fructose-1,6-bisphosphatase gene family leads to their functional biodiversity in Gossypium species

BackgroundFructose-1,6-bisphosphatase (FBP) is a key enzyme in the plant sucrose synthesis pathway, in the Calvin cycle, and plays an important role in photosynthesis regulation in green plants. However, no systemic analysis of FBPs has been reported in Gossypium species.ResultsA total of 41 FBP genes from four Gossypium species were identified and analyzed. These FBP genes were sorted into two groups and seven subgroups. Results revealed that FBP family genes were under purifying selection pressure that rendered FBP family members as being conserved evolutionarily, and there was no tandem or fragmental DNA duplication in FBP family genes. Collinearity analysis revealed that a FBP gene was located in a translocated DNA fragment and the whole FBP gene family was under disequilibrium evolution that led to a faster evolutionary progress of the members in G. barbadense and in At subgenome than those in other Gossypium species and in the Dt subgenome, respectively, in this study. Through RNA-seq analyses and qRT-PCR verification, different FBP genes had diversified biological functions in cotton fiber development (two genes in 0 DPA and 1DPA ovules and four genes in 20–25 DPA fibers), in plant responses to Verticillium wilt onset (two genes) and to salt stress (eight genes).ConclusionThe FBP gene family displayed a disequilibrium evolution pattern in Gossypium species, which led to diversified functions affecting not only fiber development, but also responses to Verticillium wilt and salt stress. All of these findings provide the foundation for further study of the function of FBP genes in cotton fiber development and in environmental adaptability.

Regulation of gene expression involved in the remobilization of rice straw carbon reserves results from moderate soil drying during grain filling.

Carbon reserves in rice straw before flowering contribute greatly to grain filling. Moderate soil drying imposed at the post-anthesis stage significantly promotes carbon reserve remobilization in straws of rice, but the regulation of this process at the proteomic and transcriptomic level remains poorly understood. In this study, we applied moderate soil drying (MD) to rice at the post-anthesis stage, which was followed by dynamic proteomic and transcriptomic studies using SWATH-MS and RNA-seq analysis. MD treatment upregulated the proteins alpha-glucosidase, beta-glucosidase and starch phosphorylase, which are responsible for starch degradation. Furthermore, MD treatment enhanced the expression of proteins involved in the sucrose synthesis pathway, including SPS8 and SPP1. In addition, various monosaccharide transporters (MSTs) and sucrose transporter 2 (SUT2), which are pivotal in carbon reserve remobilization, were also upregulated in straw by MD treatment. Differentially expressed transcription factors, including GRAS, TCP, trihelix, TALE, C3H, and NF-YC, were predicted to interact with other proteins to mediate carbon reserve remobilization in response to MD treatment. Further correlation analysis revealed that the abundances of most of the differentially expressed proteins were not correlated with the corresponding transcript levels, indicating that the carbon reserve remobilization process was probably regulated by posttranscriptional modification. Our results provide insights into the molecular mechanisms underlying the regulation of carbon reserve remobilization from straw to grain in rice under MD conditions.

Transcriptional Analysis of Masson Pine (Pinus massoniana) under High CO2 Stress.

To explore the molecular mechanism of the response of Masson pine (Pinus massoniana), the main coniferous tree in southern China, to high CO2 stress, transcriptome sequencing was carried out to analyze the genome-wide responses of annual seedlings under different durations (0 h, 6 h, 12 h and 24 h) of high CO2 stress. The results showed that a total of 3080/1908, 3110/2115 and 2684/1483 genes were up-/down-regulated after 6 h, 12 h and 24 h of treatment, respectively, compared with control check group (CK, 0 h). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most of these differentially expressed genes (DEGs) were enriched in energy metabolism, carbohydrate synthesis, cell wall precursor synthesis and hormone regulation pathways. For energy metabolism, the expression of most genes involved in photosynthesis (including the light reaction and Calvin cycle) was generally inhibited, while the expression of genes related glycolysis, the tricarboxylic acid (TCA) cycle and PPP pathway was up-regulated. In addition, the increase in the CO2 concentration induced the up-regulation of gene expression in the sucrose synthesis pathway. Among all starch synthesis genes, GBSS (granule-bound starch synthase) had the highest expression level. On the other hand, during the synthesis of hemicellulose and pectin (cell wall precursor substances), the expression levels of GMD (GDP-mannose 4,6-dehydratase), MGP (Mannose-1-phosphate guanylyl transferase) and RHM (Rhamnose biosynthetic enzyme) were the highest, suggesting that the synthesis of the raw materials hemicellulose and pectin in Masson pine under stress were mainly supplied by GDP-Man, GDP-Fuc and UDP-Rha. Finally, stress inhibited gene expression in the ABA (Abscisic Acid) synthesis pathway and induced gene expression in the GA (Gibberellin), SA (Salicylic acid), BR(Brassinolide) and MeJA (Methyl Jasmonate) pathways. Stomatal switches were regulated by hormonal interactions. This experiment elaborated on the response and molecular mechanism of Masson pine to CO2 stress and aided in screening carbon sequestration genes for the corresponding molecular research of Masson pine in the future.

Revealing the important role of allosteric property in sucrose phosphate synthase from sugarcane with N-terminal domain deletion

Sucrose occupies many essential roles to control regulation of carbon partitioning in plants, including prokaryotic cells. Sucrose phosphate synthase (SPS; EC 2.4.1.14) is a key enzyme to catalyze the form of sucrose in primary sucrose synthesis pathway. Plants SPS has a molecular size around 120 kDa, which consists of N-terminal domain, C-terminal domain, and central domain. We produced the recombinant sugarcane SPS (SoSPS1) in Escherichia coli, however, the expression often appears to be a shorter form with retained enzyme activity. In our result, we reported that the shorter form is suggested to have a truncated N-terminal 20-kDa region. The truncated form of So SPS1 (ΔN-SPS) tends to enhance the specific activity 10-fold compared to full-length SoSPS1. The full-lenght SoSPS1 showed a remarkable allosteric activation by glucose-6-phosphate (G6P), while none of the N-terminal truncated form had such a characteristic. By kinetic analysis of full-length SoSPS1, a higher substrate affinity was shown in the presence of G6P. Conversely, the ΔN-SPS showed a similar substrate affinity whether G6P was added or not. Based on these results, we revealed that N-terminal region of SoSPS1 has essential role for allosteric regulation by G6P and may function like a suppressor domain for the enzyme activity.

Flux control analysis of a lactate and sucrose metabolic network at different storage temperatures for Hami melon (Cucumis melo var. saccharinus)

Cold storage of fruits and vegetables is essentially a respiring and metabolic network flow and control process for which the further study of metabolic control rules are needed in order to improve the shelf-life of these living food products. This study used Hami melon as a test subject to study the respiratory metabolic network and flux control of fruits and vegetables during their storage. Hami melon was stored at different temperatures (0, 5, 10, 15, 20, 25, 30, 35, 40, 45°C), in order to obtain the metabolic flux of the following pathways: Embden–Meyerh of pathway (EMP), pentose phosphate pathway (PPP), sucrose synthesis pathway (SSP) and TCA cycle (TCA). The quantity and activities of primary enzymes (enzymatic expression variate) were measured during storage. In addition, we got the control coefficients of synthesis flux to sucrose and metabolic flux to lactate. The results showed that sucrose had a higher synthesis flux at the temperatures of 5°C and 45°C, and lactate had a higher metabolic flux at the temperature of 5°C. The study on flux control coefficients indicated that sucrose phosphate synthase (SPS, FCC=0.424) produced a larger impact on sucrose flux control in the sucrose synthesis pathway. Lactic dehydrogenase (LDH, FCC=0.143), triosephosphate isomerase (TPI, FCC=0.136) and pyruvate kinase (PK, FCC=0.136) had a large control effect on lactate flux, while glucose-6-phosphate dehydrogenase (G6PDH, FCC=−0.246) had a negative control function. These findings will enable future detailed investigation of metabolic mechanism of fruits and research of new preservatives.

Unravelling the mechanisms that improve photosynthetic performance of N2-fixing pea plants exposed to elevated [CO2

Although the predicted enhanced photosynthetic rates of plants exposed to elevated [CO2] are expected to increase carbohydrate and plant growth, recent findings have shown a complex regulation of these processes. The aim of this study was to determine the effect of elevated [CO2] on pathways leading to the main forms of leaf C storage (starch) and export (sucrose) and the implications of this increased [CO2] on photosynthetic performance of exclusively N2 fixing plants. For this purpose, exclusively N2-fixing pea plants were exposed to elevated [CO2] (1000μmolmol−1versus 360μmolmol−1 CO2). The data obtained highlighted that plants exposed to elevated [CO2] were capable of maintaining hexose levels (involved in Rubisco down regulation) at control levels with the consequent avoidance of photosynthetic acclimation. More specifically, in plants exposed to elevated [CO2] there was an increase in the activity of pathways involved in the main forms of leaf C storage (starch) and export (sucrose). Furthermore, the study highlighted that although starch content increased by up to 40% under elevated [CO2], there was also an increase in the proteins and compounds involved in starch degradation. Such a finding, together with an increase in the activity of proteins involved in sucrose synthesis revealed that these plants up-regulated the sucrose synthesis pathway in order to meet the large nodule photoassimilate requirements. As a consequence, the study highlighted the relevance of controlling the activity of pathways that determine leaf cellular carbohydrate availability and how this is linked with C-demanding organs such as nodules.

Sucrose phosphate phosphatase in the green alga Klebsormidium flaccidum (Streptophyta) lacks an extensive C-terminal domain and differs from that of land plants

Previously, it was reported that like land plants, the green alga Klebsormidium flaccidum (Streptophyta) accumulates sucrose during cold acclimation (Nagao et al. Plant Cell Environ 31:872-885, 2008), suggesting that synthesis of sucrose could enhance the freezing tolerance of this alga. Because sucrose phosphate phosphatase (SPP; EC 3.1.3.24) is a key enzyme in the sucrose synthesis pathway in plants, we analyzed the SPP gene in K. flaccidum (KfSPP, GenBank accession number AB669024) to clarify its role in sucrose accumulation. As determined from its deduced amino acid sequence, KfSPP contains the N-terminal domain that is characteristic of the L-2-haloacid-dehalogenase family of phosphatases/hydrolases (the HAD phosphatase domain). However, it lacks the extensive C-terminal domain found in SPPs of land plants. Database searches revealed that the SPPs in cyanobacteria also lack the C-terminal domain. In addition, the green alga Coccomyxa (Chlorophyta) and K. flaccidum, which are closely related to land plants, have cyanobacterial-type SPPs, while Chlorella (Chlorophyta) has a land plant-type SPP. These results demonstrate that even K. flaccidum (Streptophyta), as a recent ancestor of land plants, has the cyanobacterial-type SPP lacking the C-terminal domain. Because SPP and sucrose phosphate synthase (SPS) catalyze sequential reactions in sucrose synthesis in green plant cells and the lack of the C-terminal domain in KfSPP is predicted to decrease its activity, the interaction between decreased KfSPP activity and SPS activity may alter sucrose synthesis during cold acclimation in K. flaccidum.

Identification and characterization of a null-activity mutant containing a cryptic pre-mRNA splice site for cytosolic fructose-1,6-bisphosphatase in Flaveria linearis

Cytosolic fructose-1,6-bisphosphatase (cytFBPase) (E.C. 3.1.3.11) catalyzes the first irreversible reaction of daytime sucrose synthesis. A Flaveria linearis (F. linearis) mutant (line 84-9) previously shown to have ~10% wildtype cytFBPase activity contains no cytFBPase activity based on enzymatic and immunoprecipitation analysis. Genetic segregation and Southern analysis of an F2 population shows one gene copy of cytFBPase in F. linearis and linkage of null cytFBPase activity to the cytFBPase structural gene. A point mutation is present in the structural gene coding for cytFBPase in the mutant, causing a cryptic pre-mRNA splice site and a corresponding 24 amino acid deletion spanning the active site of the enzyme. Collectively, these data support the identification of a null-activity mutant for cytFBPase in F. linearis. This is the first report of a null mutant in the daytime sucrose synthesis pathway confirmed by both enzymatic and molecular analysis. Null cytFBPase in F. linearis does not predispose all lines to high starch accumulation due to an epistatic gene interaction; low starch accumulation in null cytFBPase lines segregates with elevated pyrophosphate-dependent phosphofructokinase (PFP) activity when grown in a 16 h photoperiod. Surprisingly, growth of parental lines and F2 progeny having null cytFBPase in continuous light rescued the wildtype growth phenotype. All null cytFBPase lines showed CO(2)-insensitivity/reversed sensitivity of photosynthesis, indicating that null cytFBPase causes a reduced total capacity for both photosynthesis and end-product synthesis regardless of starch and PFP phenotype. Collectively, the data indicate that F. linearis, a C3-C4 photosynthetic intermediate, has alternative cytFBPase-independent pathways for daytime sucrose synthesis.

TRANSFORMASI GEN SUCROSE PHOSPHATE SYNTHASE (SoSPS1) MENGGUNAKAN Agrobacterium tumefaciens UNTUK MENINGKATKAN SINTESIS SUKROSA PADA TANAMAN TEBU (Saccharum officinarum L.)

Sucrose phosphate synthase (SPS EC 2.3.1.14) plays an important role in partition of assimilated carbon in most plants. SPS catalyses the penultimate reaction in the pathway of sucrose synthesis, in which sucrose-6-phosphate (Suc6P) is synthesized from UDPglucose (UDPG) and fructose-6-P (Fru6P). To increase the capacity of sugarcane in sucrose synthesis, spindle leaves of sugarcane cv R579 were transformed with cDNA SoSPS1 from sugarcane under the control of constitutive promoter (35S CaMV) that constructed in pBI 121 (pKYS) using Agrobacterium tumefaciens. Based on PCR analysis, we have detected the existence of SPS transgene in some lines of transformed sugarcane, called line 4, 5, 6, and 7. The SPS transgene in transformed sugarcane could be expressed into translation level and increased the amount of leaves SPS protein, so the activity of leaves SPS was higher than wild type sugarcane as control. The transformed sugarcane line 4, 5, 6, and 7 showed 1.4–2.9 fold increases in SPS activity and 1,76–2,2 fold increases in leaves sucrose content. Increasing in SPS activity in transgenic sugarcane was coupled by the increase in invertase activity and ratio between sucrose and starch content.

Research Note: The five families of sucrose-phosphate synthase genes in Saccharum spp. are differentially expressed in leaves and stem.

Sucrose-phosphate synthase (SPS) is a key enzyme in the pathway of sucrose synthesis. Five different gene families encoding SPS have been reported in the Poaceae [Castleden CK, Aoki N, Gillespie VJ, MacRae EA, Quick WP, Buchner P, Foyer CH, Furbank RT, Lunn JE (2004) Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses. Plant Physiology 135, 1753-1764]. Expression of the five families in leaf and stem tissues of Saccharum spp. at different stages of development was determined by quantitative real-time PCR. The type B and C families of SPS genes were predominantly expressed in both immature and mature leaves, whereas the two subfamilies making up the type D family were expressed at similar levels in all tissues examined. In the type A family, expression was lowest in leaves and increased from the meristem region down to internode 7 of the stem.

The Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell

Sucrose-phosphatase (SPP) catalyzes the final step in the pathway of sucrose biosynthesis in both plants and cyanobacteria, and the SPPs from these two groups of organisms are closely related. We have crystallized the enzyme from the cyanobacterium Synechocystis sp PCC 6803 and determined its crystal structure alone and in complex with various ligands. The protein consists of a core domain containing the catalytic site and a smaller cap domain that contains a glucose binding site. Two flexible hinge loops link the two domains, forming a structure that resembles a pair of sugar tongs. The glucose binding site plays a major role in determining the enzyme's remarkable substrate specificity and is also important for its inhibition by sucrose and glucose. It is proposed that the catalytic reaction is initiated by nucleophilic attack on the substrate by Asp9 and involves formation of a covalent phospho-Asp9-enzyme intermediate. From modeling based on the SPP structure, we predict that the noncatalytic SPP-like domain of the Synechocystis sucrose-phosphate synthase could bind sucrose-6(F)-phosphate and propose that this domain might be involved in metabolite channeling between the last two enzymes in the pathway of sucrose synthesis.

Sucrose may play an additional role to that of an osmolyte in Synechocystis sp. PCC 6803 salt-shocked cells

The role of sucrose in cyanobacteria is still not fully understood. It is generally considered a salt-response molecule, and particularly, in Synechocystis sp. strain PCC 6803, it is referred as a secondary osmolyte. We showed that sucrose accumulates transiently in Synechocystis cells at early stages of a salt shock, which could be ascribed to salt activation of sucrose-phosphate synthase (SPS, UDP-glucose: d-fructose-6-phosphate 2-α- d-glucosyltransferase; EC 2.4.1.14), the key enzyme in sucrose synthesis pathway, and to an increase of the expression of the SPS encoding gene. Experiments with a mutant strain impaired in sucrose biosynthesis showed that sucrose is essential in stationary phase cells to overcome a later salt stress. Taken together, these results led us to suggest a more intricate function for sucrose than to be an osmoprotectant compound.

Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis, alters carbohydrate partitioning, and reduces growth

The aim of this work was to examine the role of sucrose-6-phosphate phosphatase (SPP; EC 3.1.3.24) in photosynthetic carbon partitioning. SPP catalyzes the final step in the pathway of sucrose synthesis; however, until now the importance of this enzyme in plants has not been studied by reversed-genetics approaches. With the intention of conducting such a study, transgenic tobacco plants with reduced SPP levels were produced using an RNA interference (RNAi) strategy. Transformants with less than 10% of wild-type SPP activity displayed a range of phenotypes, including those that showed inhibition of photosynthesis, chlorosis, and reduced growth rates. These plants had strongly reduced levels of sucrose and hexoses but contained 3-5 times more starch than the control specimens. The leaves were unable to export transient starch during extended periods of darkness and as consequence showed a starch- and maltose-excess phenotype. This indicates that no alternative mechanism for carbon export was activated. Inhibition of SPP resulted in an approximately 1,000-fold higher accumulation of sucrose-6-phosphate (Suc6P) compared to wild-type leaves, whereas the content of hexose-phosphates was reduced. Although the massive accumulation of Suc6P in the cytosol of transgenic leaves was assumed to impair phosphate-recycling into the chloroplast, no obvious signs of phosphate-limitation of photosynthesis became apparent. 3-Phosphoglycerate (3-PGA) levels dropped slightly and the ATP/ADP ratio was not reduced in the transgenic lines under investigation. It is proposed that in SPP-deficient plants, long-term compensatory responses give rise to the observed acceleration of starch synthesis, increase in total cellular Pi content, decrease in protein content, and related reduction in photosynthetic activity.

Frost resistance and biochemical changes during cold acclimation in different annual legumes

In Mediterranean areas of southern Europe growth of forages is seriously limited by the ability of each species to growth during cold winter. Therefore, the objective of this work was to prove the capacity for cold acclimation and frost resistance in four annual legumes native of Mediterranean region under laboratory conditions. Plants from subterranean clover ( Trifolium subterraneum L. ssp. brachycalycinum cv. Clare), balansa clover ( T. michelianum Savi cv. Giorgia), burr medic ( Medicago polymorpha L. cv. Anglona) and barrel medic ( M. truncatula Gaertn. cv. Paraggio) were cold acclimated by growing them at 10/5 °C. Non-acclimated plants developed at 20/15 °C. Dry matter partition, root solute content and enzymes for sucrose synthesis were determined during cold acclimation period. Freezing tolerance was determined by measuring the lethal temperature of 50% of the population (LT 50) and a thermal analysis was performed to study the dynamics for ice formation in plant tissues. Finally, by measuring forage production after 4 weeks at 20/15 °C it was assessed regrowth capacity. Cold-acclimated plants have increased root solute concentrations coupled with changes in the activity of some enzymes involved in sucrose synthesis in leaves. However, activities of sucrose synthesis pathway are not related with freezing tolerance in a simple way. Lower LT 50 was related to lower specific leaf area (SLA), higher root to shoot ratio and higher root solutes. Non-acclimated plants have LT 50 similar or higher than ice nucleation temperature indicating that these species cannot tolerate freezing. Cold acclimation decreased LT 50 in all species, being lower in Giorgia and Anglona; in Anglona did not change nucleation temperature, resulting values significantly higher than corresponding LT 50. This suggests that this cultivar was able to tolerate freezing. By contrast, in Giorgia, nucleation temperature decreased, which suggests that changes occurring during cold-acclimation might contribute to increase their capacity for avoidance by moderate supercooling. Cold-acclimated plants have higher regrowth capacity than non-acclimated ones except in Paraggio, which exhibited a poor regrowth. This might be due to an ineffective cold acclimation process related to impaired SPS activity and low starch reserves observed in this cultivar. On the other hand, Anglona and Giorgia have the better frost tolerance and higher potential for production of forage mass after freezing, indicating that they may be suitable cultivars for sow under cold winter conditions predominant in some Mediterranean zones.

Biochemical Characterization of Rice Sucrose Phosphate Synthase under Illumination and Osmotic Stress

Sucrose phosphate synthase (SPS) is one of a number of sucrose-metabolizing enzymes that regulates the sucrose synthesis pathway. SPSs were purified from etiolated rice seedlings (ERS), green rice seedlings (GRS), rice grain suspension cells under osmotic stress (RGSO), and rice grain suspension cells under illumination (RGSI). A native molecular mass of ca. 420 and 520 kDa was found using native-PAGE. The SDS-PAGE analyses revealed SPSs to be homotetramers composed of subunits with a mass of 116-120 kDa. The maximum activity for SPSs was observed on the third day. As far as their biochemical characterization was concerned, the optimum pH of the enzyme reactions lay generally between 6-8, the optimum temperatures between 35-40°C. The ERS and RGSO SPS Km values for Fm 6-P and UDPG were 1.8 and 35mM, respectively. However, the GRS and RGSI SPS had similar Km values for Fm 6-P and UDPG of 1.5 and 28mM, respectively. GRS and RGSI SPS activities were allosterically regulated by Glc 6-P (activator) or Pi (inhibitor), but ERS and RGSO SPS had no effect. From their regulations and Km values two enzyme forms (SPS-I and SPS-II) could be discriminated in the rice. SPS-II was induced by illumination, but SPS-I by osmotic stress. All SPSs were activated by Mg(superscript 2+). The nucleotides AMP, ADP, ATP, UMP, UDP, GDP and UTP inhibited enzyme activity by about 25-50%. Thiol reagents became sensitized to the enzyme activity, but could be restored with DTT or β-ME. Glucose, galactose, glucosamine, maltose, and lactose activated the enzymes and were inhibited by δ-gluconolactone and mannose. SPSs were also inhibited by PCMBS, cibacron blue F3G-A, and DEP.

P4-239 Region specific, age-related, alterations in mitochondrial responses to increasing calcium

Cold storage of fruits and vegetables is essentially a respiring and metabolic network flow and control process for which the further study of metabolic control rules are needed in order to improve the shelf-life of these living food products. This study used Hami melon as a test subject to study the respiratory metabolic network and flux control of fruits and vegetables during their storage. Hami melon was stored at different temperatures (0, 5, 10, 15, 20, 25, 30, 35, 40, 45 °C), in order to obtain the metabolic flux of the following pathways: Embden–Meyerh of pathway (EMP), pentose phosphate pathway (PPP), sucrose synthesis pathway (SSP) and TCA cycle (TCA). The quantity and activities of primary enzymes (enzymatic expression variate) were measured during storage. In addition, we got the control coefficients of synthesis flux to sucrose and metabolic flux to lactate. The results showed that sucrose had a higher synthesis flux at the temperatures of 5 °C and 45 °C, and lactate had a higher metabolic flux at the temperature of 5 °C. The study on flux control coefficients indicated that sucrose phosphate synthase (SPS, FCC = 0.424) produced a larger impact on sucrose flux control in the sucrose synthesis pathway. Lactic dehydrogenase (LDH, FCC = 0.143), triosephosphate isomerase (TPI, FCC = 0.136) and pyruvate kinase (PK, FCC = 0.136) had a large control effect on lactate flux, while glucose-6-phosphate dehydrogenase (G6PDH, FCC = −0.246) had a negative control function. These findings will enable future detailed investigation of metabolic mechanism of fruits and research of new preservatives.