Identification of the molecular basis for the functional difference between flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase

Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3'- or the 3'- and 5'-position, respectively. Sequence identity between F3'H and F3'5'H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3'5'H from an F3'H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3'H and F3'5'H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5'-hydroxylation activity to recombinant Gerbera hybrida F3'H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3'5'H into an F3'H with low remaining 5'-hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3'- to 3',5'-hydroxylation activity.     

Identification of functional domains in Arabidopsis thaliana mRNA decapping enzyme (AtDcp2)

The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn²⁺- or Mg²⁺-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.     

Hydrogen peroxide-induced gene expression in Arabidopsis thaliana

Hydrogen peroxide (H(2)O(2)) is generated in plants after exposure to a variety of biotic and abiotic stresses, and has been shown to induce a number of cellular responses. Previously, we showed that H(2)O(2) generated during plant-elicitor interactions acts as a signaling molecule to induce the expression of defense genes and initiate programmed cell death in Arabidopsis thaliana suspension cultures. Here, we report for the first time the identification by RNA differential display of four genes whose expression is induced by H(2)O(2). These include genes that have sequence homology to previously identified Arabidopsis genes encoding a late embryogenesis-abundant protein, a DNA-damage repair protein, and a serine/threonine kinase. Their putative roles in H(2)O(2)-induced defense responses are discussed.     

Identification of the gene encoding homoserine kinase from Arabidopsis thaliana and characterization of the recombinant enzyme derived from the gene

Homoserine kinase (EC 2.7.1.39) catalyzes the formation of O-phospho-l-homoserine, a branch point intermediate in the pathways for Met and Thr in plants. A genomic open reading frame located on the top arm of chromosome II and a corresponding cDNA have been identified from Arabidopsis thaliana that encode homoserine kinase. The HSK gene is composed of an 1113-bp continuous open reading frame that could produce a 38-kDa protein. The gene product has homology with homoserine kinase from bacteria and fungi. It contains a conserved motif, known as GHMP, found in a group of ATP-dependent metabolite kinases and thought to comprise the ATP binding site. The amino-terminal 50 amino acids of the HSK protein show features of a transit peptide for localization to plastids. Genomic blot analysis revealed that there is a single locus in A. thaliana to which the HSK cDNA hybridizes. The HSK protein expressed as a His-tagged construct in Escherichia coli shows a specific activity in an l-homoserine-dependent ADP synthesis assay of 3.09 +/- 0.25 micromol min(-1) mg(-1) protein at pH 8.5 and 37 degrees C. The apparent K(m) values are 0.40 mM for l-homoserine and 0.32 mM for Mg-ATP. Other hydroxylated compounds are not used as substrates. The enzyme requires 40 mM K(+) and 3 mM Mg(2+) for activity. It has an unusually high temperature optimum, yet it is very unstable, losing more than 80% of its activity after a single cycle of freeze-thawing. The HSK enzyme shows no significant regulation by amino acids in vitro.     

Characterization of flavonoid 7-O-glucosyltransferase from Arabidopsis thaliana

Most flavonoids found in plants exist as glycosides, and glycosylation status has a wide range of effects on flavonoid solubility, stability, and bioavailability. Glycosylation of flavonoids is mediated by Family 1 glycosyltransferases (UGTs), which use UDP-sugars, such as UDP-glucose, as the glycosyl donor. AtGT-2, a UGT from Arabidopsis thaliana, was cloned and expressed in Escherichia coli as a gluthatione S-transferase fusion protein. Several compounds, including flavonoids, were tested as potential substrates. HPLC analysis of the reaction products indicated that AtGT-2 transfers a glucose molecule into several different kinds of flavonoids, eriodictyol being the most effective substrate, followed by luteolin, kaempferol, and quercetin. Based on comparison of HPLC retention times with authentic flavonoid 7-O-glucosides and nuclear magnetic resonance spectroscopy, the glycosylation position in the reacted flavonoids was determined to be the C-7 hydroxyl group. These results indicate that AtGT-2 encodes a flavonoid 7-O-glucosyltransferase.     

Identification and functional characterization of the BAG protein family in Arabidopsis thaliana

The genes that control mammalian programmed cell death are conserved across wide evolutionary distances. Although plant cells can undergo apoptosis-like cell death, plant homologs of mammalian regulators of apoptosis have, in general, not been found. This is in part due to the lack of primary sequence conservation between animal and putative plant regulators of apoptosis. Thus, alternative approaches beyond sequence similarities are required to find functional plant homologs of apoptosis regulators. Here, we present the results of using advanced bioinformatic tools to uncover the Arabidopsis family of BAG proteins. The mammalian BAG (Bcl-2-associated athanogene) proteins are a family of chaperone regulators that modulate a number of diverse processes ranging from proliferation to growth arrest and cell death. Such proteins are distinguished by a conserved BAG domain that directly interacts with Hsp70 and Hsc70 proteins to regulate their activity. Our searches of the Arabidopsis thaliana genome sequence revealed seven homologs of the BAG protein family. We further show that plant BAG family members are also multifunctional and remarkably similar to their animal counterparts, as they regulate apoptosis-like processes ranging from pathogen attack to abiotic stress and development.     

Identification of a tRNA isopentenyltransferase gene from Arabidopsis thaliana

The tRNA of most organisms contain modified adenines called cytokinins. Situated next to the anticodon, they have been shown to influence translational fidelity and efficiency. The enzyme that synthesizes cytokinins on pre-tRNA, tRNA isopentenyltransferase (EC 2.5.1.8), has been studied in micro-organisms like Escherichia coli and Saccharomyces cerevisiae, and the corresponding genes have been cloned. We here report the first cloning and functional characterization of a homologous gene from a plant, Arabidopsis thaliana. Expression in S. cerevisiae showed that the gene can complement the anti-suppressor phenotype of a mutant that lacks MOD5, the intrinsic tRNA isopentenyltransferase gene. This was accompanied by the reintroduction of isopentenyladenosine in the tRNA. The Arabidopsis gene is constitutively expressed in seedling tissues.     

Implications of long-distance flavonoid movement in Arabidopsis thaliana

Flavonoid synthesis is modulated by developmental and environmental signals that control the amounts and localization of the diverse flavonoids found in plants. Flavonoids are implicated in regulating a number of physiological processes including UV protection, fertilization, auxin transport, plant architecture, gravitropism and pathogenic and symbiotic interactions with other organisms. Recently we showed that flavonoids can move long distances in plants, which may facilitate these molecules reaching positions in the plant where these processes are regulated. The localised application of selective flavonoids to tt4 mutants such as naringenin, dihydrokaempferol and dihydroquercetin showed that they were taken up at the root tip, mid-root or cotyledons and travelled long distances via cell-to-cell movement to distal tissues and converted to quercetin and kaempferol. In contrast, kaempferol and quercetin do not move long distances. They were taken up only at the root tip and did not move from this position. Here we show the movement of endogenous flavonoids by using reciprocal grafting experiments between tt4 and wild-type seedlings. These results demonstrated that to understand the distribution of flavonoids in Arabidopsis, it is necessary to know where the flavonoid biosynthetic enzymes are made and to understand the mechanisms by which certain flavonoids move from their site of synthesis.Key words: flavonoid movement, reciprocal graft, quercetin, kaempferol, Arabidopsis thaliana, fluorescence, aglyconeFlavonoids are plant secondary metabolites made by the phenylpropanoid pathway. The central biosynthetic pathway is known and in Arabidopsis most of the enzymes in flavonoid synthesis are encoded by single copy genes.¹ The isolation of mutants with defects in the genes encoding these flavonoid biosynthetic enzymes has allowed researchers to understand the biochemical complexity of flavonoid synthesis and their biological roles. Flavonoid synthesis is more complex in other species, such as legumes, which produce a greater diversity of flavonoid molecules, and in which gene families encode the key enzymatic branch points of the pathway.^2,3The functions of flavonoids were demonstrated using genetic approaches that blocked flavonoid synthesis in Arabidopsis and other species. In Arabidopsis, flavonoids play important roles in UV protection⁴ and regulate auxin transport and dependent physiological processes, such as gravity responses,^5–7 and lateral root formation.⁸ In petunia, maize and tomato, pollen without flavonoids is infertile and this phenotype is reversed by flavonoid addition.^9–11 However, the enigma of why flavonoid-deficient Arabidopsis seedlings are fertile has not been resolved.¹² Flavonoids appear to interact with Multidrug resistance (MDR)/P-glycoproteins (PGP)/ABC-Type B proteins⁷ that transport auxin, regulate phosphatases and kinases, and may have regulatory roles as scavengers of reactive oxygen species (reviewed in ref. ¹³). These results are consistent with a diversity of important functions for flavonoids in plants that require careful control of flavonoid synthesis and localization.We have explored the possibility that flavonoid accumulation in specific locations is also modulated by movement of early intermediates of the flavonoid pathway. Long-distance movement of secondary metabolites is largely unexplored but potentially has profound developmental effects. Grafting experiments conducted in the early 1900s suggested that alkaloids move from the site of manufacture (the root) to the aerial tissue.¹⁴ More recent grafting experiments showed that root synthesised metabolites, perhaps carotenoids, regulate shoot development,^15,16 flowering inducers travel long distances,¹⁷ and phytohormones are translocated (reviewed in ref. ¹⁸).We recently showed that flavonoids moved long distances in Arabidopsis using several approaches.¹⁹ The roots of Arabidopsis seedlings grown in complete darkness do not accumulate flavonoids⁵ since expression of early genes encoding enzymes of flavonoid biosynthesis are light dependent.²⁰ Yet, flavonoids accumulate in root tips of seedlings with light-grown shoots and light-shielded roots, consistent with shoot-to-root flavonoid movement. Using fluorescence microscopy, a selective flavonoid stain (diphenyl boric acid 2-amino ethyl ester [DPBA]), and localised aglycone application to transparent testa mutants, we showed that flavonoids accumulated in tissues distal to the application site, indicating that early intermediates in the flavonoid pathway can move long distances. This was confirmed by time-course fluorescence experiments and HPLC. Flavonoid applications to root tips resulted in basipetal movement in epidermal layers, with subsequent fluorescence detected 1 cm from application sites after 1 h. Flavonoid application mid-root or to cotyledons showed movement of flavonoids toward the root tip mainly in vascular tissue. Naringenin, dihydrokaempferol and dihydroquercetin were taken up at the root tip, mid-root or through cotyledons and travelled long distances via cell-to-cell movement to distal tissues followed by conversion to quercetin and kaempferol. In contrast, kaempferol and quercetin were only taken up at the root tip. Uptake of flavonoids at the root tip was inhibited by glybenclamide, a specific inhibitor of ABC type transporters²¹ suggesting a possible role for transporters of this class in the movement of flavonoids.To show that endogenous flavonoids are capable of long distance movement, we performed reciprocal butt grafting between tt4 and wild-type seedlings.²² In these experiments we asked whether flavonoids moved from wild-type tissues to flavonoid-deficient tissues of tt4. DPBA fluorescence detection was used to detect flavonoid movement into tt4 tissues.¹⁹ Seedlings were grafted and grown on filter paper in Petri dishes for 8 d. The seedlings were then transferred to equal parts sand, perlite and vermiculite to avoid the possibility of uptake of pre-existing flavonoids that may be natural soil components. After 14 d, the seedlings were stained with DPBA. When tt4 roots were grafted to tt4 shoots, the samples showed dim greenish autofluorescence in roots and only red chlorophyll fluorescence in the shoot. When either tt4 roots or shoots were grafted onto wild-type shoots or roots, respectively, the tt4 tissues showed bright yellow DPBA fluorescence (Fig. 1). The results of these experiments clearly showed that endogenous flavonoids moved across the graft to the reciprocal tissue. The flavonoid movement is specific to certain tissues, as flavonoids are not transported into the seeds developing on tt4 shoots grafted on wild-type roots, which retain the transparent testa phenotype. Although flavonoids clearly travelled from wild-type root tissue to mutant shoots, they were not capable of complementing the seed colour defect of tt4. In addition, adding naringenin to tt4 plants either to the media, or to the soil, also did not complement the seed colour phenotype in tt4 (data not shown). Recent research by Hsieh and Huang²³ may account for this inability to complement seed colouration, as flavonoids in the Brassicaceae end up in the pollen coat rather than the testa. The testa tissue derives from ovular tissue,²⁴ and thus is maternal in origin.Open in a separate windowFigure 1Grafting shows flavonoid movement occurs across grafts. Reciprocal grafting between wild type and tt4 indicated flavonoid movement from the flavonoid producing tissue to the chalcone synthase-deficient tissue. The order of the graft is indicated by the left legend as aerial tissue over root tissue in the graft. Micrographs are DPBA stained tissue excited with 488 nm wavelength. The tt4/tt4 control graft shows no flavonoids are present, even though a wound has occurred on the leaf which generally exacerbates flavonoid fluorescence. Scale bar = 100 µm. Green fluorescence is from kaempferol and gold from quercetin. The red fluorescence is from chlorophyll.The complexity of the flavonoid biosynthetic pathway and the large number of modified flavonoids that can be made through the complex series of glycosylation reactions suggests that distinct flavonoid molecules may have unique function. To fully understand these molecules, it is necessary to dissect the synthesis pathways for these glycosylated flavonoids. Two unnamed flavonoid glycoside mutants isolated in 1998,²⁵ have profound developmental phenotypes, supporting this hypothesis. These mutations resulted in whorled cauline leaves on inflorescences and double the number of rosette leaves. Our lab is in the process of determining if other phenotypes exist in flavonoid mutants.A critical feature of the observations of flavonoid movement is understanding the biological context of this movement. First, a number of recent studies reported physiological functions of flavonoids in roots, ranging from modulation of auxin transport and root gravitropism,^5–8 to nodulation³ and root branching,⁸ while it is clear that flavonoid synthesis is absent in dark-grown seedlings.⁵ Yet, for flavonoids to function in roots of plants grown in soil, the light signal and/or flavonoid precursors must travel to the roots. Additionally, transient flavonoid accumulation has been reported in roots reoriented relative to the gravity vector.⁶ For flavonoids to transiently accumulate at the root tip (at 2 hours after reorientation) and to return to lower levels (within 2 additional hours), suggests that more than flavonoid synthesis is regulated. Perhaps this transient flavonoid accumulation requires localized enzyme activation and transport mechanisms. As flavonoid transport is inhibited by a compound that blocks ABC transporters, which include the newly identified auxin transporters of the MDR/PGP class, perhaps there are connections between flavonoid and auxin transport that allow this transient accumulation. A more detailed understanding of this role of flavonoid movement in controlling plant development awaits additional experimentation.     

Regulation of Arabidopsis thaliana 14-3-3 gene expression by gamma-aminobutyric acid

The function in plants of the non-protein amino acid, gamma-aminobutyric acid (GABA) is poorly understood. In this study, we show that GABA down-regulates the expression of a large subset of 14-3-3 gene family members in Arabidopsis thaliana seedlings in a calcium, ethylene and abscisic acid (ABA)-dependent manner. Gene expression is not affected when seedlings are supplied with glutamate (GLU), a precursor of GABA. The repression of 14-3-3 gene expression by GABA is dependent on functional ethylene and ABA signalling pathways, because the response is lost in the etr1-1, abi1-1 and abi2-1 mutants. Calcium measurements show that in contrast to GLU, GABA does not elicit a cytoplasmic calcium elevation, suggesting that the GABA response is unlikely to be mediated by GLU receptors (GLRs), as has been suggested previously. We suggest that in addition to its role as a stress-related metabolite, GABA may regulate gene expression in A. thaliana, including members of the 14-3-3 gene family.     

P1-450 and P3-450 gene expression and maximum life span in mice

The effects of beta-naphthoflavone on the inducibility of hepatic P1-450 and P3-450 mRNA were investigated in male B10.RIII/Sn, C57BL/10Sn, C3H/HeSnJ, and A/WYSn mice. Previous work has shown that the maximum level of aryl hydrocarbon hydroxylase induction in these strains correlates with maximum life span. In this study we found that the maximum inducible levels of P1- and P3-450 RNA were significantly different among the strains, and these levels also correlate with life span. The differences were not due to strain-specific differences in the kinetics of P1- or P3-450 RNA induction. The differences were specific to expression of the P-450 genes, since the levels of hepatic alpha-actin and albumin RNA were not significantly different among the strains, and specific RNA levels were normalized to the level of total polyadenylated RNA. beta-Naphthoflavone was found to induce alpha-actin mRNA approximately 2-fold and to transiently repress albumin RNA about 50% in all mouse strains. Maximum P1- and P3-450 gene expression correlated directly with the 10th deciles of survival of the mouse strains. Longer-lived strains expressed higher combined levels of P1- and P3-450 RNAs. Maximum P1- and P3-450 gene expression also correlated generally with the reported aryl hydrocarbon hydroxylase receptor levels of each strain. It is unlikely that the hepatic P1- and P3-450 genes are ever maximally induced under the sheltered laboratory conditions used to determine maximum life span, as we consistently find very low levels of P-450 expression in uninduced animals. These uninduced levels were not statistically different between the strains. Therefore, the reason for the relationship between maximum life span and maximum P1- and P3-450 inducibility is unclear at present.     

Identification of a monofunctional aspartate kinase gene of Arabidopsis thaliana with spatially and temporally regulated expression

We screened a gene trap library of Arabidopsis thaliana and isolated a line in which a gene encoding a homologue of monofunctional aspartate kinase was trapped by the reporter gene. Aspartate kinase (AK) is a key enzyme in the biosynthsis of aspartate family amino acids such as lysine, threonine, isoleucine, and methionine. In plants, two types of AK are known: one is AK which is sensitive to feedback inhibition by threonine and carries both AK and homoserine dehydrogenase (HSD) activities. The other one is monofunctional, sensitive to lysine and synergistically S-adenosylmethionine, and has only AK activity. We concluded that the trapped gene encoded a monofunctional aspartate kinase and designated as AK-lys3, because it lacked the HSD domain and had an amino acid sequence highly similar to those of the monofunctional aspartate kinases ofA. thaliana. AK-lys3 was highly expressed in xylem of leaves and hypocotyls and stele of roots. Significant expression of this gene was also observed in trichomes after bolting. Slight expression of AK-lys3 was detected in vascular bundles and mesophyll cells of cauline leaves, inflorescence stems, sepals, petals, and stigmas. These results indicated that this aspartate kinase gene was not expressed uniformly but in a spatially specific manner.