Distinct physiological roles of fructokinase isozymes revealed by gene-specific suppression of Frk1 and Frk2 expression in tomato

There are two divergent fructokinase isozymes, Frk1 and Frk2 in tomato (Lycopersicon esculentum Mill.) plants. To investigate the physiological functions of each isozyme, the expression of each fructokinase mRNA was independently suppressed in transgenic tomato plants, and the respective phenotypes were evaluated. Suppression of Frk1 expression resulted in delayed flowering at the first inflorescence. Suppression of Frk2 did not effect flowering time but resulted in growth inhibition of stems and roots, reduction of flower and fruit number, and reduction of seed number per fruit. Localization of Frk1 and Frk2 mRNA accumulation by in situ hybridization in wild-type tomato fruit tissue indicated that Frk2 is expressed specifically in early tomato seed development. Fruit hexose and starch content were not effected by the suppression of either Frk gene alone. The results collectively indicate that flowering time is specifically promoted by Frk1 and that Frk2 plays specific roles in contributing to stem and root growth and to seed development. Because Frk1 and Frk2 gene expression was suppressed individually in transgenic plants, other significant metabolic roles of fructokinases may not have been observed if Frk1 and Frk2 play, at least partially, redundant metabolic roles.     

Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.

Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.     

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...     

Inhibition of fructokinase and sucrose synthase by cytosolic levels of fructose in young tomato fruit undergoing transient starch synthesis

Immature tomato fruit are characterized by a transient period of starch accumulation. Sucrose synthase (EC 2.4,1.13) and fructokinase (EC 2.7,1.4) are two of the initial enzymes in the sucrose to starch synthetic pathway. Both enzymes in tomato fruit are significantly inhibited by fructose at concentrations physiological to young tomato fruit. Compartmental analysis of immature fruit pericarp indicates that fructose is not specifically compartmentalized in the vacuole and that physiological cytosolic concentrations of fructose in young tomato fruit are above 30 m M . Such physiological levels of fructose significantly inhibit sucrose synthase cleavage activity as well as the activity of a partially purified fructokinase. These data suggest a mechanism of a coordinated, in vivo regulation of tomato sucrose synthase and fructokinase activity, which may be potentially limiting to starch accumulation in young tomato fruit.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Bifidobacterium longum requires a fructokinase (Frk; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) for fructose catabolism

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 0.756 +/- 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.     

A novel gene of tomato preferentially expressed in fruit encodes a protein with a Ca2+-dependent lipid-binding domain

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54 633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca²⁺-dependent lipid-binding domains of cytosolic phospholipase A₂, protein kinase C, Rabphilin-3A, and Synaptotagmin I of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression

Partial amino acid sequences of NAD-dependent sorbitol dehydrogenase (NAD-SDH) were used to identify a full-length cDNA from apple fruit. This clone consisted of 1,433 bp containing an open reading frame of 1,137 bp that could code for a polypeptide with 379 amino acids. To our knowledge, this is the first report about cloning of NAD-SDH cDNA from a plant source. The deduced amino acids from cDNA revealed 43.7% identity to human NAD-SDH. The activity of this enzyme to convert sorbitol to fructose with the reduction of NAD was certified by the fusion protein of this clone expressed in Escherichia coli. Northern blot analysis showed that the mRNA was expressed in matured apple fruit.     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Isolation and characterization of fruit vacuolar invertase genes from two tomato species and temporal differences in mRNA levels during fruit ripening

To determine the relationship between invertase gene expression and glucose and fructose accumulation in ripening tomato fruit, fruit vacuolar invertase cDNA and genomic clones from the cultivated species, Lycopersicon esculentum cv. UC82B, and a wild species, Lycopersicon pimpinellifolium, were isolated and characterized. The coding sequences of all cDNA clones examined are identical. By comparison to the known amino acid sequence of mature L. esculentum fruit vacuolar invertase, a putative signal sequence and putative amino-terminal and carboxy-terminal propeptides were identified in the derived amino acid sequence. Of the residues 42% are identical with those of carrot cell wall invertase. A putative catalytic site and a five-residue motif found in carrot, yeast, and bacterial invertases are also present in the tomato sequence. Minor differences between the nucleotide sequences of the genomic clones from the two tomato species were found in one intron and in the putative regulatory region. The gene appears to be present in one copy per haploid genome. Northern analysis suggests a different temporal pattern of vacuolar invertase mRNA levels during fruit development in the two species, with the invertase mRNA appearing at an earlier stage of fruit development in the wild species. Nucleotide differences found in the putative regulatory regions may be involved in species differences in temporal regulation of this gene, which in turn may contribute to observed differences in hexose accumulation in ripening fruit.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.     

Ascorbate free radical reductase mRNA levels are induced by wounding.

A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization.     

Cloning and characterization of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase in melon (Cucumis melo L. reticulatus)

We have isolated a cDNA for Cm-HMGR, encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in melon (Cucumis melo L. reticulatus; Genbank Accession No. AB021862). Cm-HMGR encodes a polypeptide of 588 amino acids that contains two transmembrane domains and a catalytic domain. Database searches revealed that Cm-HMGR shows homology to HMG1 (63.7%) and HMG2 (70.3%) of tomato, to HMG1 (77.2%) and HMG2 (69.4%) of Arabidopsis thaliana, and to HMGR of tobacco (72.6%). Functional expression in a HMG-CoA reductase-deficient mutant yeast showed that Cm-HMGR products mediate the synthesis of mevalonate. Northern analysis revealed that the level of Cm-HMGR mRNA in the fruit increased after pollination and markedly decreased at the end of fruit enlargement. During ripening, Cm-HMGR mRNA levels increased markedly in the fruit. In parallel with mRNA expression, Cm-HMGR activity increased after pollination, whereas no Cm-HMGR activity was detectable during fruit ripening. Our results suggest that Cm-HMGR is important during early post-pollination development of the fruit in melon.     

Isolation and characterization of a novel, developmentally regulated proteinase inhibitor I protein and cDNA from the fruit of a wild species of tomato

A novel member of the proteinase Inhibitor I family having a trypsin inhibitor specificity was isolated from the fruit of the wild tomato species Lycopersicon peruvianum (L.) Mill. (LA 107) and characterized. The protein is among the isoinhibitors of Inhibitor I that comprise 50% of the soluble proteins in the fruit of this wild species of tomato. A cDNA corresponding to the inhibitor protein and mRNA was isolated and characterized. The Inhibitor I mRNA represented 0.06% of the poly(A) RNA and gene copy number reconstruction experiments gave an estimate of two to four genes/haploid genome. The open reading frame of the cDNA codes for a protein of 111 amino acids having a 42-amino acid prepropolypeptide. The NH2-terminal sequence of the first 21 amino acids of the purified Inhibitor I protein confirmed that the cDNA was identical to the protein. The amino acid sequence of the L. peruvianum fruit Inhibitor I exhibits 74% identity with the wound-inducible Inhibitor I from tomato leaves. Whereas all previously identified members of the Inhibitor I family have either Met, Leu, or Asp at the P1 site and can inhibit enzymes such as chymotrypsin, subtilisin, and elastase, the fruit Inhibitor I possesses Lys at the P1 position. Thus, this is the first member of the extensive Inhibitor I family from plants and animals that exhibits trypsin inhibitory specificity. The presence of this inhibitor in wild tomato fruit may reflect a functional role to protect the tissues against herbivory.     

Iron-superoxide dismutase and monodehydroascorbate reductase transcripts accumulate in response to internode rubbing in tomato

A cDNA encoding an iron-superoxide dismutase (Fe-SOD) was isolated by RACE-PCR from a Lycopersicon esculentum cDNA library. The Fe-SOD cDNA consists of a 746-bp open reading frame and is predicted to encode a protein of 249 amino acids with a calculated molecular mass of 27.9 kDa. The deduced amino acid sequence was very similar to other plant Fe-SODs and a potential chloroplastic targeting was found. To study the induction of oxidative burst in response to mechanical stimulation, the accumulation of Fe-SOD and monodehydroascorbate reductase (MDHAR) mRNAs was analysed in response to young growing internode rubbing in tomato plants. Northern analyses show that Fe-SOD mRNA and MDHAR mRNA accumulated in tomato internodes 10 min after the mechanical stimulation. These results suggest that reactive oxygen species are early involved in the response of a plant to a mechanical stimulation, such as rubbing. The nucleotide sequence data reported in this paper will appear in the NCBI Nucleotide Sequence Databases under the accession number AY262025.     

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.     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.