The role of methionine recycling for ethylene synthesis in Arabidopsis

The methionine (Met) cycle contributes to sulfur metabolism through the conversion of methylthioadenosine (MTA) to Met at the expense of ATP. MTA is released as a by-product of ethylene synthesis from S-adenosylmethionine (AdoMet). Disruption of the Met cycle in the Arabidopsis mtk mutant resulted in an imbalance of AdoMet homeostasis at sulfur-limiting conditions, irrespective of the sulfur source supplied to the plants. At a low concentration of 100 mum sulfate, the mtk mutant had reduced AdoMet levels and growth was retarded as compared with wild type. An elevated production of ethylene was measured in seedlings of the ethylene-overproducing eto3 mutant. When Met cycle knockout and ethylene overproduction were combined in the mtk/eto3 double mutant, a reduced capacity for ethylene synthesis was observed in seedlings. Even though mature eto3 plants did not produce elevated ethylene levels, and AdoMet homeostasis in eto3 plants did not differ from that in wild type, shoot growth was severely retarded. The mtk/eto3 double mutant displayed a metabolic plant phenotype that was similar to mtk with reduced AdoMet levels at sulfur-limiting conditions. We conclude from our data that the Met cycle contributes to the maintenance of AdoMet homeostasis, especially when de novo AdoMet synthesis is limited. Our data further showed that the Met cycle is required to sustain high rates of ethylene synthesis. Expression of the Met cycle genes AtMTN1, AtMTN2, AtMTK, AtARD1, AtARD2, AtARD3 and AtARD4 was not regulated by ethylene. This result is in contrast to that found in rice where OsARD1 and OsMTK are induced in response to ethylene. We hypothesize that the regulation of the Met cycle by ethylene may be restricted to plants that naturally produce high quantities of ethylene for a prolonged period of time.     

Recycling of methylthioadenosine is essential for normal vascular development and reproduction in Arabidopsis

5'-Methylthioadenosine (MTA) is the common by-product of polyamine (PA), nicotianamine (NA), and ethylene biosynthesis in Arabidopsis (Arabidopsis thaliana). The methylthiol moiety of MTA is salvaged by 5'-methylthioadenosine nucleosidase (MTN) in a reaction producing methylthioribose (MTR) and adenine. The MTN double mutant, mtn1-1mtn2-1, retains approximately 14% of the MTN enzyme activity present in the wild type and displays a pleiotropic phenotype that includes altered vasculature and impaired fertility. These abnormal traits were associated with increased MTA levels, altered PA profiles, and reduced NA content. Exogenous feeding of PAs partially recovered fertility, whereas NA supplementation improved fertility and also reversed interveinal chlorosis. The analysis of PA synthase crystal structures containing bound MTA suggests that the corresponding enzyme activities are sensitive to available MTA. Mutant plants that expressed either MTN or human methylthioadenosine phosphorylase (which metabolizes MTA without producing MTR) appeared wild type, proving that the abnormal traits of the mutant are due to MTA accumulation rather than reduced MTR. Based on our results, we propose that the key targets affected by increased MTA content are thermospermine synthase activity and spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A.     

Functional analysis of methylthioribose kinase genes in plants

Through a biochemical and a genetic approach, we have identified several plant genes encoding methylthioribose (MTR) kinase, an enzyme involved in recycling of methionine through the methylthioadenosine (MTA) cycle. OsMTK1, an MTR kinase from rice (Oryza sativa), is 48.6 kD in size and shows cooperative kinetics with a V(max) of 4.9 pmol/min and a K0.5 of 16.8 microm. MTR kinase genes are the first genes to be identified from the MTA cycle in plants. Insertional mutagenesis of the unique AtMTK gene in Arabidopsis (Arabidopsis thaliana) resulted in an inability of plants to grow on MTA as a supplemental sulfur source. MTK knock-out plants were not impaired in growth under standard conditions, indicating that the MTA cycle is a nonessential metabolic pathway in Arabidopsis when sulfur levels are replete. In rice, OsMTK genes were strongly up-regulated in shoots and roots when plants were exposed to sulfur starvation. Gene expression was largely unaffected by lack of nitrogen or iron in the nutrient solution, indicating that OsMTK regulation was linked specifically to sulfur metabolism.     

The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity

Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.     

The role of ethylene in the growth response of submerged deep water rice

We investigated the effect of partial submergence on internode elongation in a Bangladesh variety of floating or deep water rice (Oryza sativa L., cv. Habiganj Aman II). In plants which were at least 21 days old, 7 days of submergence led to a 3- to 5-fold increase in internodal length. During submergence, the ethylene concentration in the internodes increased from about 0.02 to 1 microliters per liter. Treatment of nonsubmerged plants with ethylene also stimulated internode elongation. When ethylene synthesis in partially submerged plants was blocked with aminooxyacetic acid and aminoethoxyvinylglycine, internode elongation was inhibited. This growth inhibition was reversed when ethylene biosynthesis was restored with 1-aminocyclopropane-1-carboxylic acid (ACC). Radio-labeling studies showed that ethylene in floating rice was synthesized from methionine via ACC. Internodal tissue from submerged plants had a much higher capacity to form ethylene than did internodal tissue from nonsubmerged plants. This increase in ethylene synthesis appeared to be due to enhanced ACC formation rather than to increased conversion of ACC to ethylene. Our results indicate that ethylene produced during submergence is required for the stimulation of growth in submerged floating rice plants.     

Characterization and functional expression of cDNAs encoding methionine-sensitive and -insensitive homocysteine S-methyltransferases from Arabidopsis

Plants synthesize S-methylmethionine (SMM) from S-adenosylmethionine (AdoMet), and methionine (Met) by a unique reaction and, like other organisms, use SMM as a methyl donor for Met synthesis from homocysteine (Hcy). These reactions comprise the SMM cycle. Two Arabidopsis cDNAs specifying enzymes that mediate the SMM --> Met reaction (SMM:Hcy S-methyltransferase, HMT) were identified by homology and authenticated by complementing an Escherichia coli yagD mutant and by detecting HMT activity in complemented cells. Gel blot analyses indicate that these enzymes, AtHMT-1 and -2, are encoded by single copy genes. The deduced polypeptides are similar in size (36 kDa), share a zinc-binding motif, lack obvious targeting sequences, and are 55% identical to each other. The recombinant enzymes exist as monomers. AtHMT-1 and -2 both utilize l-SMM or (S,S)-AdoMet as a methyl donor in vitro and have higher affinities for SMM. Both enzymes also use either methyl donor in vivo because both restore the ability to utilize AdoMet or SMM to a yeast HMT mutant. However, AtHMT-1 is strongly inhibited by Met, whereas AtHMT-2 is not, a difference that could be crucial to the control of flux through the HMT reaction and the SMM cycle. Plant HMT is known to transfer the pro-R methyl group of SMM. This enabled us to use recombinant AtHMT-1 to establish that the other enzyme of the SMM cycle, AdoMet:Met S-methyltransferase, introduces the pro-S methyl group. These opposing stereoselectivities suggest a way to measure in vivo flux through the SMM cycle.     

Regulation of methionine synthesis in Escherichia coli: effect of metJ gene product and S-adenosylmethionine on the in vitro expression of the metB, metL and metJ genes

The regulation of the expression of three Escherichia coli met genes, metB, which codes for cystathionine gamma-synthetase (EC 4.2.99.9), metL, which codes for aspartokinase II-homoserine dehydrogenase II (EC 2.7.2.4-EC 1.1.1.3) and metJ, which codes for the methionine regulon aporepressor, has been studied using highly purified DNA-directed in vitro protein synthesis systems. In a system where the entire gene product is synthesized, the expression of the metB and metL genes is specifically inhibited by MetJ protein (repressor protein) and S-adenosylmethionine (AdoMet). In a simplified system that measures the formation of the first dipeptide of the gene product (fMet-Ala for the metJ gene), MetJ protein and AdoMet partially repress (approximately 40-60%) metJ gene expression. Thus, the metJ gene can be partially autoregulated by its gene product.     

5'-Methylthioadenosine nucleosidase and 5-methylthioribose kinase activities in relation to stress-induced ethylene biosynthesis

The activities of 5'-methylthioadenosine (MTA) nucleosidase (EC 2.2.2.28) and 5-methylthioribose (MTR) kinase (EC 2.7.1.100) were related to changes in ethylene biosynthesis in tomato ( Lycopersicon esculentum Mill. cv. Rutgers) and cucumber ( Cucumis sativus Mill. cv. Poinsett 76) fruit following wounding and chemically induced stresses. Stress ethylene formation in wounded tomato and cucumber tissue continued to increase after wounding, reached its peak by 3h, and then declined. The activities of MTA nucleosidase and MTR kinase increased parallel to stress ethylene in both tissues. At peak ethylene formation, MTA and MTR kinase activities were 2- to 4-fold higher in wounded than in intact tissue. Wounded, mature-green tomato tissue treated with specific inhibitors of MTA nucleosidase and MTR kinase showed a significant reduction in the activities of these enzymes, which was concomitant with a decline in stress ethylene biosynthesis. When mature-green tomato discs were infiltrated with [¹⁴CH₃] MTA and wounded, radioactive MTR and methionine were formed. Incubation of mature-green tomato discs with Cu²⁺ and Li⁺ in the presence of kinetin increased ethylene biosynthesis. MTA nucleosidase activity was higher than that of the control in the presence of Cu²⁺ but not in the presence of Li⁺, while MTR kinase activity was lower than that of the control in both Cu²⁺ and Li⁺ treatments. Data indicate that MTA nucleosidase and MTR kinase are required for wound-induced ethylene biosynthesis but not for chemical stress-induced ethylene by Cu²⁺ or Li⁺ treatments.     

Examination of two lowland rice cultivars reveals that gibberellin-dependent early response to submergence is not necessarily mediated by ethylene

Using two lowland rice (Oryza sativa L.) cultivars we found that in both cases submerged-induced elongation early after germination depends on gibberellins (GAs). Submergence increases the content of the active GA₁ by enhancing the expression of GA biosynthesis genes, thus facilitating the seedlings to escape from the water and preventing asphyxiation. However, the two cultivars differ in their response to ethylene. The cultivar Senia (short), by contrast to cultivar Bomba (tall), does not elongate after ethylene application, and submerged-induced elongation is not negated by an inhibitor of ethylene perception. Also, while ethylene emanation in Senia is not altered by submergence, Bomba seedlings emanate more ethylene upon desubmergence, associated with enhanced expression of the ethylene biosynthesis gene OsACS5. The cultivar Senia thus allows the possibility of clarifying the role of ethylene and other factors as triggers of GA biosynthesis enhancement in rice seedlings under submergence.Key words: gibberellins, ethylene, submergence, lowland rice, Oryza sativa     

Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene

If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5′-S-methyl-5′-thioadenosine (MTA) would be the fragment nucleoside. When [Me-¹⁴C] or [³⁵S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by _l-2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid, the conversion of [³⁵S] or [Me¹⁴C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [³⁵S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.     

Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol

The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B(12)-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.     

Biotin synthase exhibits burst kinetics and multiple turnovers in the absence of inhibition by products and product-related biomolecules

Biotin synthase (BS) is a member of the "SAM radical" superfamily of enzymes, which catalyze reactions in which the reversible or irreversible oxidation of various substrates is coupled to the reduction of the S-adenosyl-l-methionine (AdoMet) sulfonium to generate methionine and 5'-deoxyadenosine (dAH). Prior studies have demonstrated that these products are modest inhibitors of BS and other members of this enzyme family. In addition, the in vivo catalytic activity of Escherichia coli BS requires expression of 5'-methylthioadenosine/S-adenosyl-l-homocysteine nucleosidase, which hydrolyzes 5'-methylthioadenosine (MTA), S-adenosyl-l-homocysteine (AdoHcy), and dAH. In the present work, we confirm that dAH is a modest inhibitor of BS (K(i) = 20 μM) and show that cooperative binding of dAH with excess methionine results in a 3-fold enhancement of this inhibition. However, with regard to the other substrates of MTA/AdoHcy nucleosidase, we demonstrate that AdoHcy is a potent inhibitor of BS (K(i) ≤ 650 nM) while MTA is not an inhibitor. Inhibition by both dAH and AdoHcy likely accounts for the in vivo requirement for MTA/AdoHcy nucleosidase and may help to explain some of the experimental disparities between various laboratories studying BS. In addition, we examine possible inhibition by other AdoMet-related biomolecules present as common contaminants in commercial AdoMet preparations and/or generated during an assay, as well as by sinefungin, a natural product that is a known inhibitor of several AdoMet-dependent enzymes. Finally, we examine the catalytic activity of BS with highly purified AdoMet in the presence of MTAN to relieve product inhibition and present evidence suggesting that the enzyme is half-site active and capable of undergoing multiple turnovers in vitro.