Functional characterization of 5′-regulatory region of flavonoid 3′,5′-hydroxylase-1 gene of banana plants

Protoplasma - Generation of crops with broad-spectrum tolerance to biotic and abiotic stress conditions depends upon availability of genetic elements suitable for varied situations and diverse...     

Gene-dependent flavonoid 3′-hydroxylation in maize

The influence of the gene Pr on flavonoid 3-hydroxylase activity in maize is described. Specific activities are presented for the hydroxylase in seedlings and aleurone tissue homozygous dominant and recessive and heterozygous for Pr. Specific activity levels in both tissues increased in a nearly direct proportion with the increase in Pr dosage, which is consistent with Pr being the structural gene for the hydroxylase. Regression analysis of the gene dosage:enzyme activity comparison yielded correlation coefficients of 0.979 and 0.959 for the seedlings and aleurone, respectively. Quantitative identification of the cyanidin and pelargonidin in the aleurone indicated that cyanidin increased with an increase in dominant Pr, while pelargonidin decreased, although the increases and decreases observed were not directly proportional to the gene dosage. Comparison of the cyanidin/pelargonidin ratio to the gene dosage ratio in the different tissues showed a strong correlation (0.998), which demonstrates that the dosage of Pr controls the ratio of cyanidin to pelargonidin. Cyanidin was found at a low concentration in aleurone homozygous for pr. Hydroxylase activity in maturing field plants reaches its peak concentration near anthesis and is present at an appreciable concentration in mature plant tissue homozygous for pr, as well as in seedlings homozygous for pr. Suggestion is made that pr could be a hypomorphic allele or that a duplicate gene for Pr could exist to account for the hydroxylase activity in homozygous pr tissue. Evidence for the hydroxylase in the aleurone and the seedlings and the pigment ratio data from the aleurone suggest that Pr is indeed a structural gene for NADPH:flavonoid 3-hydroxylase.Cooperative Investigations, Agricultural Research Service, United States Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia, Missouri 65211. Journal Series No. 9958.     

The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3′-hydroxylase on leucoanthocyanidins

Main conclusion

In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3′ position by flavonoid 3’-hydroxylase.

Abstract

The cytochrome P450-dependent monooxygenases flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3′ or 3′ and 5′, respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3′H and F3′5′H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3′H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3′-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3′,5′-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3′Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.     

Modification of the B-ring during flavonoid synthesis in Petunia hybrida: Introduction of the 3′-hydroxyl group regulated by the gene Ht1

The flower-color mutants of Petunia hybrida W37 and W18, which are homozygous recessive for the anthocyanin gene An3, accumulate flavanone glycosides in the flowers. It is concluded that the gene An3 is not directly involved in the synthesis of the C₁₅ skeleton, but that it probably takes part in modifying the skeleton. Complementation experiments with the mutants W18 and M5 show that the hydroxylating gene Ht1, which is reponsible for the introduction of the second hydroxyl group in the B-ring at position 3, is expressed after gene An3. In P. hybrida introduction of the 3-hydroxyl group is therefore not achieved by specific incorporation of caffeic acid during synthesis of the C₁₅ skeleton, but by hydroxylation of a C₁₅ skeleton. When anthocyanin synthesis is blocked by homozygous recessive hydroxylating genes Ht1 and Hf1, as in the mutant M5, dihydrokaempferol-7-glucoside is accumulated. This intermediate is discussed as a possible substrate for B-ring hydroxylation.     

An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3′-hydroxylase gene

The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3′H, the gene for flavonoid 3′-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.     

Flower-specific expression of the Phalaenopsis flavonoid 3′, 5′-hydoxylase modifies flower color pigmentation in Petunia and Lilium

Flavonoid 3′, 5′-hydoxylase (F3′5′H) is a key enzyme for biosynthesis of the blue anthocyanin pigment delphinidin. A number of F3′5′H genes from dicots have been tested for their effects on flower pigmentation; here F3′5′H from a monocot was tested for its effect on delphinidin accumulation in petals. To this end, F3′5′H (PhF3′5′H) from the orchid Phalaenopsis was expressed under the control of the chalcone synthase promoter in petunia flowers. Quantitative RT-PCR showed that PhF3′5′H was expressed mainly in the petal limb; this expression produced an increase in dihydromyricetin and delphinidin and a change in petal color from pink to deeper pink. To increase the accumulation of delphinidin, Hyacinth HyDFR, which encodes dihydroflavonol 4-reductase, and petunia DifF, which encodes a cytochrome b ₅ that is required for full activity of F3′5′H were overexpressed. The HyDFR petunia transformants had a deeper color petal limb, increased dihydromyricetin and delphinidin contents and adaxial petals with a number of blue cells. The flowers of the DifF petunia transformants also showed a slight color change. We also tested PhF3′5′H in Lilium oriental ‘Sorbonne’, where transient PhF3′5′H expression by particle bombardment resulted in purple cells in the petals. Production of blue flowers by Phalaenopsis F3′5′H and hyacinth DFR potentially enables manipulation of flower color in ornamental plants, including production of blue flowers.     

Induction of micronuclei in mouse bone-marrow cells by the flavonoid 5,3′, 4′-trihydroxy-3,6,7,8-tetramethoxy-flavone (THTMF)

Flavonoids obtained from various plant species are an important source of food additives and drugs. The finding that several compounds of this group had mutagenic activity in microbial tests (Hardigree and Epler, 1978; MacGregor and Jurd, 1978; Seino et al., 1978; Takahashi et al., 1979), induced cytological alterations in plant cells (Segawa and Kondo, 1978) and increased the frequency of micronuclei in mice (Sahu et al., 1981) revealed the importance of flavonoids as environmental mutagens.THTMF was isolated from the Compositae Gutierrezia resinosa (H. et A.) Blake as part of a screening of Chilean plants for anti-cancer activity, and it exhibited an anti-tumor effect in KB cells (Bhakuni et al., 1976; Bittner et al., 1982). Because a large number of anti-cancer compounds are well-known chromosome-breaking agents, we assayed THTMF with the micronucleus test as a prelilminary study on the biological properties of the drug.     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

The expression of petunia flavonoid 3′ and 3′5′ hydroxylase genes in potatoes (Solanum tuberosum cv. Jopung)

Flavonoid 3′ (F3′OH) and 3′5′ hydroxylase (F3′5′OH) play a major role in the synthesis of flavonoids. They are involved in the flavonoid modification and the B-ring hydroxylation produces quercetin and myricetin, respectively. We introduced the petunia F3′OH and F3′5′OH genes in potato and expression of these enzyme was confirmed by Southern and Northern blot analyses in these transgenic plants. In the flavonoid, staining experiment, all transgenic plants with petunia F3′OH and F3′5′OH genes were successfully changed with their green color to orange, confirming that quercetin was synthesized in those plants. Especially, the F3′5′OH transgenic potatoes showed the strongest orange color, and it was revealed by capillary electrophoresis that they produce quercetin one and a half times as much as the untransformed potatoes.     

Viscosol,a c-3′ prenylated flavonoid from dodonaea viscosa

The structure of viscosol, a new prenylated flavonoid isolated from the aerial parts of Dodonaea viscosa, was established on the basis of spectral studies as well as by the conversion of the flavonol penduletin into permethyl viscosol.     

Allele-specific marker development and selection efficiencies for both flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes in soybean subgenus soja

Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3′-hydroxylase (F3′H; EC1.14.3.21) and flavonoid 3′,5′-hydroxylase (F3′5′H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3′H and the W1 locus co-segregated with a gene encoding F3′5′H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3′H and F3′5′H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3′H and one of three alleles for GmF3′5′H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3′H and F3′5′H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3′H allele explained 92.2 % of the variation in tawny and two gmf3′h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3′5′H alleles and one gmF3′5′h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3′h-b and gmf3′5′h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.     

Catalytic roles of the AMP at the 3′ end of tRNAs

Recent reports suggest that the ribosome retains considerable peptidyl transferase activity even when much of the protein of the ribosome is removed and further suggests that rRNA may be the peptidyl transferase. The work here suggests that the AMP residue at the 3 terminus of each tRNA has some catalytic activity both in the esterification reaction and in forming a pseudopeptide, AcGly, and further suggests that whatever peptidyl transferase is, it finds a cooperative substrate in the aminoacyl-AMP at the 3 terminus of tRNA.     

Distribution of 5′-nucleotidase in the renal interstitium of the rat

Summary The hydrolysis of 5-AMP by 5-nucleotidase is the main source of adenosine. In various tissues adenosine is a local mediator adjusting the organ work to the available energy. In the kidney it regulates renal hemodynamics, glomerular filtration rate and renin release via specific receptors of the arteriolar walls. By immunocytochemistry we identified interstitial and tubular sites of 5-nucleotidase in the rat kidney. In the interstitium the enzyme was detected only in the cortical labyrinth, the compartment that comprises all arteriolar vessels besides other putative targets of adenosine. The 5-nucleotidase-positive cells of the interstitium were identified as fibroblasts. The fibroblasts are in close contact with the tubules as well as with the vessels. Thus, any 5-AMP released by the tubules into the interstitial space would be converted to adenosine in the direct vicinity of its assumed targets. Adenosine produced by tubular cells would hardly have access to its known targets, since 5-nucleotidase is restricted to the luminal cell surface. Pathological events affecting the fibroblasts might influence renal function by modifying the interstitial adenosine production.     

The kinetics and mechanism of the reaction of 2′-deoxy-5′-guanosinemonophosphoric acid and the diaqua form of cis-platin

2′-Deoxy-5′-guanosinemonosphoric acid (B) reacts with cis-[Pt(NH₃)₂(OH₂)₂]²⁺ in two steps to form the cis-[Pt(NH₃)₂B₂]^y+ ion. In the first step 2′-d-5′- GMPH₂ reacts some ten times faster than 5′-GMPH₂ does. Rate constants, ΔH^#, ΔS^# and ΔV^# are very similar for the two bases in the second reaction. It is proposed that the product in the first step contains no water and is cis-[Pt(NH₃)₂B]^x+ in which the nucleobase is bidentate bonding through both N(7) of guanine and an oxygen atom of the phosphate group.     

The Crystal and Molecular Structure of the Complex of 2′3′-Didehydro-2′3′-Dideoxyguanosine with Pyridine

Abstract

The structure of 2′,3′-didehydro-2′,3′-dideoxyguanosine was determined by X-ray crystallographic analysis of the complex with pyridine. The two independent nucleoside molecules have similar, commonly observed glycosyl link (x = -102.3° and -94.2°) and 5′-hydroxyl (y = 54.0° and 47.6°) conformations. The five-membered rings are very planar with r.m.s. deviations from planarity of less than 0.015 A. 2′,3′-Didehydro-2′,3′-dideoxyadenosine has a similar glycosyl link conformation but a different 5′-hydroxyl group orientation and a slightly less planar 5-membered ring.