Characterization of two Arabidopsis thaliana fructokinases

Two different fructokinase isoforms of Arabidopsis thaliana have been identified and characterized by non-denaturing electrophoresis followed by activity-staining. The two fructokinases, fructokinase1 (FRK1) and fructokinase2 (FRK2), showed a high specificity for fructose and did not stain when glucose or mannose were used as substrate. Fructose and ATP at high concentrations (above 5 mM) induced a substrate inhibition of the two enzymatic activities. Arabidopsis FRK1 and FRK2 were capable of employing GTP, CTP, UTP and TTP as phosphate donors, although with a significantly lower efficiency than ATP. The two fructokinase activities were also activated by K⁺, at around 10–20 mM, and inhibited by ADP and AMP at concentrations above 10 mM. Finally, FRK1 and FRK2 showed a different expression pattern in the plant, with FRK1 being more abundant in the roots and FRK2 in the shoots. The results demonstrate a simple technique that provides important information about fructokinase activities in the plants and which can be useful for the analysis of Arabidopsis mutants.     

海巴戟果实发育过程中果糖、果糖激酶及其基因的变化

为揭示海巴戟果实风味形成机制,对果实发育过程的果糖激酶(FRK)活性及其基因的表达模式进行了研究.结果表明,海巴戟果实中的果糖、蔗糖、葡萄糖含量随发育不断积累,均在果实完全成熟时达到最高值,而果糖激酶活性随果实发育不断下降.从果实中克隆了果糖激酶基因TRINITY_DN17192_c0_g1,命名为McFRK2,Gen...     

Characterization of <Emphasis Type="Italic">SWEET</Emphasis> family members from loquat and their responses to exogenous induction

The plant SWEET family is a sugar transporter family that plays a significant role in plant development. Here, seven loquat SWEET family members were identified by RNA-seq. These were designated as EjSWEET1, EjSWEET2a, EjSWEET2b, EjSWEET2c, EjSWEET4, EjSWEET15, and EjSWEET17. Phylogenetic and predictive functional annotation analyses suggest that the loquat SWEETs are classified as having sucrose, glucose and fructose transportation features. The in vivo responses of loquat SWEETs to exogenous sugar or NaCl was investigated by applying high concentrations of sugar or salt to 7-month-old loquat seedlings cultured in a nutrient medium. The results showed that most loquat SWEET genes can respond to exogenous applications of sucrose, glucose, fructose and salt. The response of EjSWEET1 to exogenous fructose was faster than the others, indicating that EjSWEET1 is more sensitive to exogenous fructose compared with other loquat SWEETs. EjSWEET15 can be induced by sucrose, but is suppressed by glucose. This indicates its possible role in sucrose transporting. The response of loquat SWEETs to NaCl showed broadly similar patterns compared to sugars. However, after a longer time of NaCl treatment, most loquat SWEETs are upregulated, especially EjSWEET15. This indicates its long-term response to high salinity.     

Expression of fructokinase isoforms in the sugarcane culm

Two isoforms of fructokinase (FRK), FRK1 and FRK2, are present in sugarcane (Saccharum spp. var N19) internodal tissue. Both isoforms are highly specific for fructose as the hexose substrate. FRK2 is inhibited by fructose concentrations exceeding 0.1 mM and 50% inhibition is attained at 230 μM (K_i (Fru) = 160 μM), while FRK1 activity is not negatively affected even at 1.0 mM fructose. The ratio of FRK2 to FRK1 activity is dependent on the developmental stage of the tissue. FRK1 appears to be the isoform that is preferentially expressed in mature tissue. Total FRK activity decreases during tissue maturation. This is the result of changes in expression of the isoforms and not inactivation of existing protein. A mathematical method that allows accurate estimation of the activities of the two isoforms of FRK in crude sugarcane extracts is presented.     

Control of the hexose content of potato tubers

The amounts of glucose and fructose in a range of harvested tubers of Solanum tuberosum were compared with the labelling of these hexoses by [U-¹⁴C]sucrose supplied to the tubers. Hexose content varied. Fructose was more heavily labelled than glucose. There was no correlation between the amounts of glucose and fructose in the tuber and their labelling. The maximum catalytic activities of α-glucan phosphorylase, acid invertase, alkaline invertase, sucrose synthase, α-amylase and β-amylase in tubers stored for 17 weeks at 5° and at 10° were estimated. The values showed no clear correlation with hexose content, but provided sound evidence that starch breakdown was phosphorolytic. It is suggested that the amounts of glucose and fructose in mature harvested tubers may be determined more by the partitioning of the translocated sucrose during the development of the tubers than by the metabolism of the harvested tuber.     

Substrate inhibition of maize endosperm sucrose synthase by fructose and its interaction with glucose inhibition

Sucrose synthase (EC 2.4.1.13) was purified to homogeneity from developing maize (Zea mays L.) endosperm. Substrate saturation and inhibitor kinetics were examined for the sucrose synthase reaction. The K_m-values for fructose and uridine diphosphate glucose (UDPGlc) were estimated to be 7.8 mM and 76 μM, respectively. Fructose concentrations over 20 mM inhibited sucrose synthase in an uncompetitive manner with respect to UDPGlc. Glucose was also found to be an uncompetitive inhibitor with respect to both fructose and UDPGlc. At inhibitory concentrations of fructose, the apparent K_i for glucose increased linearly with increasing fructose concentration. The results suggest an ordered kinetic mechanism for sucrose synthase where UDPGlc binds first and UDP dissociates last. Fructose and glucose both inhibit by binding to the enzyme-UDP complex. Fructose and glucose, which are present in maize endosperm as the products of invertase, could inhibit sucrose synthase, especially in basal regions of the kernel where hexosesmay accumulate.     

The uptake of fructose by Pseudomonas putida

Fructose transport was not apparently affected in a number of Pseudomonas putida strains with deranged activity of a common glucose-gluconate uptake system, indicating the existence of an independent fructose uptake system. Fructose uptake by glucose-gluconate uptake mutants was induced by fructose and obeyed saturation kinetics (apparent K _m =0.3 mM). The fructose uptake system serves to transport glucose in addition to fructose. The entry of fructose into P. putida cells appears to be mediated also by the glucose-gluconate uptake system, as shown by the ability to accumulate fructose of wild type cells grown on glucose, a substrate that induces the glucose-gluconate uptake system but not the fructose uptake system. In addition, fructose was found to be an inducer of the glucose-gluconate uptake system. The physiological significance of these observations is not clear because the fructose uptake system can provide the cell with a high enough internal concentration of fructose to support maximum growth rate on this hexose, as shown by following the growth course of glucose-gluconate uptake mutants on fructose.     

Regulation of Dual Glycolytic Pathways for Fructose Metabolism in Heterofermentative Lactobacillus panis PM1

Lactobacillus panis PM1 belongs to the group III heterofermentative lactobacilli that use the 6-phosphogluconate/phosphoketolase (6-PG/PK) pathway as their central metabolic pathway and are reportedly unable to grow on fructose as a sole carbon source. We isolated a variant PM1 strain capable of sporadic growth on fructose medium and observed its distinctive characteristics of fructose metabolism. The end product pattern was different from what is expected in typical group III lactobacilli using the 6-PG/PK pathway (i.e., more lactate, less acetate, and no mannitol). In addition, in silico analysis revealed the presence of genes encoding most of critical enzymes in the Embden-Meyerhof (EM) pathway. These observations indicated that fructose was metabolized via two pathways. Fructose metabolism in the PM1 strain was influenced by the activities of two enzymes, triosephosphate isomerase (TPI) and glucose 6-phosphate isomerase (PGI). A lack of TPI resulted in the intracellular accumulation of dihydroxyacetone phosphate (DHAP) in PM1, the toxicity of which caused early growth cessation during fructose fermentation. The activity of PGI was enhanced by the presence of glyceraldehyde 3-phosphate (GAP), which allowed additional fructose to enter into the 6-PG/PK pathway to avoid toxicity by DHAP. Exogenous TPI gene expression shifted fructose metabolism from heterolactic to homolactic fermentation, indicating that TPI enabled the PM1 strain to mainly use the EM pathway for fructose fermentation. These findings clearly demonstrate that the balance in the accumulation of GAP and DHAP determines the fate of fructose metabolism and the activity of TPI plays a critical role during fructose fermentation via the EM pathway in L. panis PM1.     

Sink Metabolism in Tomato Fruit : III. Analysis of Carbohydrate Assimilation in a Wild Species

Carbohydrate composition and key enzymes involved in carbohydrate metabolism were assayed throughout development of Lycopersicon esculentum and L. chmielewskii fruit. Translocation and assimilation of asymmetric sucrose and total soluble solids content was also determined in both species. The data showed that L. chmielewskii accumulated less starch than L. esculentum, and this was related to a lower level of ADPglucose pyrophosphorylase and a higher level of phosphorylase in L. chmielewskii. L. chmielewskii accumulated sucrose throughout fruit development rather than glucose and fructose which were accumulated by L. esculentum. A low level of invertase and nondetectable levels of sucrose synthase were associated with the high level of sucrose in L. chmielewskii. Translocation and assimilation of asymmetrically labeled sucrose indicated that sucrose accumulated in L. chmielewskii fruit was imported and stored directly in the fruit without intervening metabolism along the translocation path. In contrast, the relatively low level of radioactive sucrose found in L. esculentum fruit appeared to arise from hydrolysis and resynthesis of sucrose. The possible relationship between the level of soluble solids and differences in carbohydrate metabolism in sink tissue of the two species is discussed.     

In vitro activation of phosphoglucomutase by fructose 2,6-bisphosphate

The hexose bisphosphate activation of phosphoglucomutase was investigated with both plant (pea and mung bean) and animal (rabbit muscle) sources of the enzyme. Plant phosphoglucomutase was purified about 50-fold from seeds, and to a lesser extent, from seedlings of Pisum sativum L. cv Grenadier and seedlings of Phaseolus aureus. It was found that the plant enzyme was isolated in a mostly dephosphorylated form while commercial rabbit muscle phosphoglucomutase was predominantly in the phosphorylated form. Activation studies were done using the dephosphorylated enzymes. The range of activation constant (K_a) values were obtained for each bisphosphate were: for glucose 1-6-P₂, 0.5 to 1.8; fructose 2,6-P₂, 6 to 11.7; and fructose 1,6-P₂, 7 micromolar, respectively. Fructose 2,6-P₂ is known to occur in both plant and animal tissues at changing levels encompassing the K_a values found in this study; hence, these results implicate fructose 2,6-P₂ as a natural activator of phosphoglucomutase, particularly in plants. Also, glucose 1,6-P₂ has not been found in plants, and the method for measuring glucose 1,6-P₂ by monitoring the activation of phosphoglucomutase is not specific.     

Synthesis and degradation of fructose 2,6-bisphosphate in endosperm of castor bean seedlings

The aim of this work was to examine the possibility that fructose 2,6-bisphosphate (Fru-2,6-P₂) plays a role in the regulation of gluconeogenesis from fat. Fru-2,6-P₂ is known to inhibit cytoplasmic fructose 1,6-bisphosphatase and stimulate pyrophosphate:fructose 6-phosphate phosphotransferase from the endosperm of seedlings of castor bean (Ricinus communis). Fru-2,6-P₂ was present throughout the seven-day period in amounts from 30 to 200 picomoles per endosperm. Inhibition of gluconeogenesis by anoxia or treatment with 3-mercaptopicolinic acid doubled the amount of Fru-2,6-P₂ in detached endosperm. The maximum activities of fructose 6-phosphate,2-kinase and fructose 2,6-bisphosphatase (enzymes that synthesize and degrade Fru-2,6-P₂, respectively) were sufficient to account for the highest observed rates of Fru-2,6-P₂ metabolism. Fructose 6-phosphate,2-kinase exhibited sigmoid kinetics with respect to fructose 6-phosphate. These kinetics became hyperbolic in the presence of inorganic phosphate, which also relieved a strong inhibition of the enzyme by 3-phosphoglycerate. Fructose 2,6-bisphosphatase was inhibited by both phosphate and fructose 6-phosphate, the products of the reaction. The properties of the two enzymes suggest that in vivo the amounts of fructose-6-phosphate, 3-phosphoglycerate, and phosphate could each contribute to the control of Fru-2,6-P₂ level. Variation in the level of Fru-2,6-P₂ in response to changes in the levels of these metabolites is considered to be important in regulating flux between fructose 1,6-bisphosphate and fructose 6-phosphate during germination.     

Uptake and metabolism of fructose by rat neocortical cells in vivo and by isolated nerve terminals in vitro

Fructose reacts spontaneously with proteins in the brain to form advanced glycation end products (AGE) that may elicit neuroinflammation and cause brain pathology, including Alzheimer's disease. We investigated whether fructose is eliminated by oxidative metabolism in neocortex. Injection of [¹⁴C]fructose or its AGE‐prone metabolite [¹⁴C]glyceraldehyde into rat neocortex in vivo led to formation of ¹⁴C‐labeled alanine, glutamate, aspartate, GABA, and glutamine. In isolated neocortical nerve terminals, [¹⁴C]fructose‐labeled glutamate, GABA, and aspartate, indicating uptake of fructose into nerve terminals and oxidative fructose metabolism in these structures. This was supported by high expression of hexokinase 1, which channels fructose into glycolysis, and whose activity was similar with fructose or glucose as substrates. By contrast, the fructose‐specific ketohexokinase was weakly expressed. The fructose transporter Glut5 was expressed at only 4% of the level of neuronal glucose transporter Glut3, suggesting transport across plasma membranes of brain cells as the limiting factor in removal of extracellular fructose. The genes encoding aldose reductase and sorbitol dehydrogenase, enzymes of the polyol pathway that forms glucose from fructose, were expressed in rat neocortex. These results point to fructose being transported into neocortical cells, including nerve terminals, and that it is metabolized and thereby detoxified primarily through hexokinase activity.

     

Assessment of a 1H high-resolution magic angle spinning NMR spectroscopy procedure for free sugars quantification in intact plant tissue

In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4–2.5 g of glucose; and 0.73–2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.     

温州蜜柑果实发育期间果糖激酶与糖积累的关系

研究了温州蜜柑果实发育进程中糖含量变化与果糖激酶活性变化的关系及增施氮肥对果实果糖激酶活性和基因表达的影响.结果表明,随着果实的发育,可食组织果糖激酶活性逐渐降低,糖含量不断增加,果皮中蔗糖和葡萄糖含量在成熟期略有下降,果糖激酶活性略有升高.果实膨大期后增施氮肥的果实在成熟期可食组织及果皮中蔗糖和果糖所占比例均有所下降,葡萄糖比例升高,以单位蛋白质表示的果糖激酶活性也明显高于对照果实.Northern分析表明,增施氮肥能促进发育后期果实可食组织中Cufrkl基因的表达,但对Cufrk2的表达无明显作用.     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

长双歧杆菌NCC2705葡萄糖与果糖代谢的比较蛋白质组学

为揭示长双歧杆菌NCC2705 (Bifidobacterium longum NCC2705)果糖代谢途径, 建立其果糖发酵模型。以本实验室前期构建的长双歧杆菌NCC2705菌株蛋白质参考图谱为基础, 进行了果糖和葡萄糖生长的菌体比较蛋白质组学研究, 利用MALDI-TOF和ESI-MS/MS鉴定差异蛋白, 进一步通过半定量RT-PCR验证二者显著差异表达蛋白。果糖生长的菌体蛋白中鉴定到了所有葡萄糖降解途径中的酶和蛋白质, 另外鉴定到3倍以上差异蛋白点9个, 其对应的5个蛋白在果糖发酵中上调。半定量RT-PCR验证显著差异蛋白, 显示在果糖发酵中具有高水平表达是ABC 转运系统的果糖特异性-结合蛋白BL0033和ATP结合蛋白BL0034。果糖的发酵时间和浓度梯度试验显示诱导时间越长、浓度越高, BL0033的表达量越高。第一, 比较蛋白谱证明果糖和葡萄糖以相同途径降解。第二, BL0033的表达是受果糖诱导的, 果糖的吸收可能是通过一个特殊的转运系统, 即ABC转运系统将果糖从胞外转运到胞内, 其中BL0033和BL0034共同作为系统元件扮演了重要角色。     

Abscisic acid,sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape

The aim of this study was to examine the effect of abscisic acid (ABA), sucrose, and auxin on grape fruit development and to assess the mechanism of these three factors on the grape fruit ripening process. Different concentrations of ABA, sucrose, and auxin were used to treat the grape fruit, and the ripening-related indices, such as physiological and molecular level parameters, were analyzed. The activity of BG protein activity was analyzed during the fruit development. Sucrose, ABA, and auxin influenced the grape fruit sugar accumulation in different ways, as well as the volatile compounds, anthocyanin content, and fruit firmness. ABA and sucrose induced, but auxin blocked, the ripening-related gene expression levels, such as softening genes PE, PG, PL, and CELL, anthocyanin genes DFR, CHI, F3H, GST, CHS, and UFGT, and aroma genes Ecar, QR, and EGS. ABA, sucrose, and glucose induced the fruit dry weight accumulation, and auxin mainly enhanced fruit dry weight through seed weight accumulation. In the early development of grape, starch was the main energy storage; in the later, it was glucose and fructose. Sucrose metabolism pathway-related gene expression levels were significant for glucose and fructose accumulation. BG protein activity was important in the regulation of grape ABA content levels. ABA plays a core role in the grape fruit development; sucrose functions in fruit development through two pathways: one was ABA dependent, the other ABA independent. Auxin blocked ABA accumulation to regulate the fruit development process.