Both Met (methionine) and SAM (S-adenosylmethionine), the activated form of Met, participate in a number of essential metabolic pathways in plants. The subcellular compartmentalization of Met fluxes will be discussed in the present review with respect to regulation and communication with the sulfur assimilation pathway, the network of the aspartate-derived amino acids and the demand for production of SAM. SAM enters the ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for the majority of methylation reactions required for plant growth and development. The multiple essential roles of SAM require regulation of its synthesis, recycling and distribution to sustain these different pathways. A particular focus of the present review will be on the function of recently identified genes of the Met salvage cycle or Yang cycle and the importance of the Met salvage cycle in the metabolism of MTA (5′-methylthioadenosine). MTA has the potential for product inhibition of ethylene, nicotianamine and polyamine biosynthesis which provides an additional link between these pathways. Interestingly, regulation of Met cycle genes was found to differ between plant species as shown for Arabidopsis thaliana and Oryza sativa.

INTRODUCTION

Ethylene is a volatile hormone that participates in close-range and long-distance communication within and between plants. It mediates responses to pathogens and abiotic stresses such as the adaptation to soil water logging and submergence. It promotes fruit ripening in climacteric fruits, senescence, and is known as a growth-regulating hormone responsible, for instance, for the triple response of etiolated seedlings which respond to ethylene with reduced shoot elongation, enhanced growth in width and an exaggerated apical hook curvature [1,2]. Ethylene is synthesized from SAM (S-adenosylmethionine) in two enzymatic steps, the first of which releases MTA (5′-methylthioadenosine), a metabolite that is reused in the Met (methionine) salvage cycle, which is also known as the Met or Yang cycle. As MTA is also a product of PA (polyamine) and NA (nicotianamine) synthesis and is present in various microbial synthetic pathways, this suggests that the Met salvage cycle is ubiquitously present in eukaryotic and prokaryotic organisms. The two main roles of the Met salvage cycle are to detoxify MTA and recycle the valuable reduced sulfur group of its degradation product MTR (5′-methylthioribose). The cyclic nature of this pathway also results in the replenishment of the substrate Met and its activated derivative SAM that is consumed by MTA-producing reactions. As such, the Met salvage cycle can be viewed as an elegant way of problem solving. SAM is one of the most abundant co-factors in plant metabolism and is responsible for donation of C1-units to SAM-dependent methyltransferases (EC 2.1.1.x) which target highly abundant cell-wall compounds, mRNAs, promoter regions of genomic DNA, and numerous soluble secondary metabolites. Aside from the Met cycle, two additional salvage pathways contribute to the maintenance of cytosolic SAM levels in higher plants.

In the present review we highlight recent findings on the co-regulation of ethylene and PA synthesis, the three salvage cycles for SAM in the cytosol, and regulation of Met synthesis in plastids to meet demands of cytosolic SAM production for phytohormone synthesis.

BIOSYNTHESIS AND FUNCTION OF ETHYLENE

In plants, ethylene is synthesized from SAM, an activated form of Met produced by SAMS (SAM synthetase; EC 2.5.1.6). The ethylene biosynthetic pathway involves two enzymatic reactions catalysed by ACS [ACC (1-aminocyclopropane-1-carboxylic acid) synthase; EC 4.4.1.14] and ACO (ACC oxidase; EC 1.14.17.4, Figure 1). ACS requires pyridoxal phosphate as a cofactor to metabolize SAM to ACC. The by-product of this reaction, MTA, retains the reduced sulfur and methyl group of Met. ACO is a non-haem iron oxygenase that converts ACC into ethylene, CO2, HCN and H2O in the presence of molecular oxygen and ascorbate. ACS and ACO proteins are encoded by small gene families of up to nine members each in rice, Arabidopsis thaliana and tomato. In most tissues, ACS activity is rate-limiting for ethylene biosynthesis. ACC can be diverted away from ethylene synthesis by conjugation to 1-malonyl-ACC [3] or to γ-glutamyl-ACC [4].

Function of the Met salvage cycle in ethylene synthesis

Figure 1
Function of the Met salvage cycle in ethylene synthesis

The Met salvage cycle recycles Met from MTA that is a by-product of ACS activity with SAM as a substrate. ACC is metabolized to ethylene, water, HCN and CO2 by ACO in the presence of ascorbate. MTN catalyses the depurination of MTA, releasing adenine and MTR. Phosphorylation by MTK consumes ATP and yields MTR-P and ADP. MTI catalyses formation of 5′-methylthioribulose-phosphate (MTRu-P), which is metabolized to DHKMP by a trifunctional DEP complex, releasing Pi and water. An ARD with Fe2+ as a cofactor metabolizes DHKMP to 2-oxo-4-methylthiobutyrate (KMTB) and formate. Transamination of KMTB by an as-yet unknown enzyme and co-substrate results in the formation of Met, which is converted into SAM by SAMS.

Figure 1
Function of the Met salvage cycle in ethylene synthesis

The Met salvage cycle recycles Met from MTA that is a by-product of ACS activity with SAM as a substrate. ACC is metabolized to ethylene, water, HCN and CO2 by ACO in the presence of ascorbate. MTN catalyses the depurination of MTA, releasing adenine and MTR. Phosphorylation by MTK consumes ATP and yields MTR-P and ADP. MTI catalyses formation of 5′-methylthioribulose-phosphate (MTRu-P), which is metabolized to DHKMP by a trifunctional DEP complex, releasing Pi and water. An ARD with Fe2+ as a cofactor metabolizes DHKMP to 2-oxo-4-methylthiobutyrate (KMTB) and formate. Transamination of KMTB by an as-yet unknown enzyme and co-substrate results in the formation of Met, which is converted into SAM by SAMS.

ACS activity is regulated at the level of gene transcription, as well as by protein dimerization and protein stability. ACS genes are differentially regulated during development, in response to stresses and with high spatial resolution in defined cell and tissue types [5,6]. ACS enzymes are categorized into three groups on the basis of their C-terminal domains [7,8]. Type-1 ACS proteins with a relatively long C-terminus are designated for phosphorylation by mitogen-activated protein kinase (EC 2.7.11.24) and calcium-dependent protein kinase (EC 2.7.11.1). Type-2 ACS proteins have a single predicted calcium-dependent protein kinase phosphorylation site and both type-1 and type-2 ACS proteins are subject to ubiquitination and proteasome degradation depending on their phosphorylation status [9,10]. Type-3 ACS proteins have rudimentary C-termini with no predicted modification. ACS proteins generally have short half-lives; ethylene overproduction in some mutants results from increased ACS protein stability [11]. For example, the ethylene overproducer1 (eto1) mutant of A. thaliana is defective in a BTB (Bric a brac, Tramtrack and Broad Complex) subunit of E3 ubiquitin ligase. Mutation of ETO1 prevents degradation of type-2 ACS, resulting in elevated ACS activity and ethylene overproduction [12,13]. The A. thaliana eto2-1 mutation stabilizes the ACS5 protein and promotes ethylene synthesis as a consequence of phosphorylation at Ser461 of ACS5. eto3 of A. thaliana possesses a single amino acid substitution in the C-terminus of ACS9 that affects its phosphorylation and protein stability [11].

ACS activity is further regulated at the level of dimerization [14]. Most heterodimers of A. thaliana ACS formed within one type of synthase are active, whereas most heterodimers formed between ACS types are inactive [14]. The Km value of ACS isoenzymes from A. thaliana for their substrate SAM varies between 8.3 and 45 μM [15]. Given that ACS genes are differentially expressed, their various enzyme properties may reflect different cellular environments in particular with respect to SAM availability. Expression of SAMS1–SAMS4 genes is up-regulated in tomato fruit in the presence of the ethylene perception inhibitor 1-MCP (1-methylcyclopropene), indicative of negative regulation by ethylene signalling [16]. ACS activity from tomato fruit is inhibited in vitro by Spm (spermine; 1,12-diamino-4,9-diazadodecane) and Spd (spermidine; 1,8-diamino-4-azaoctane) with Ki values of 1.6 mM and 2.9 mM [17]. In A. thaliana, activation of high-affinity sulfate uptake through SULTR1;2 under sulfur deprivation is dependent upon SLIM1 (SULFUR LIMITATION 1) which is identical with EIL3 (ETHYLENE-INSENSITIVE3-LIKE3). Two members of the EI3/EIL transcription factor family, EIN3 and EIL1, are central regulators of ethylene signalling. By contrast, EIL2 and SLIM1/EIL3 are implicated in the regulation of sulfate metabolism [18], but activity of EIL2 and SLIM1/EIL3 has not been linked to ethylene signalling.

FUNCTION OF PAs

As well as being required for ethylene synthesis, SAM, once decarboxylated, provides the aminopropyl group for PA synthesis. PAs are low-molecular-mass organic polycations that are found in all living organisms where they contribute to a myriad of activities, such as protection of photosystem II, pollen and flower development, and stress responses [1921]. PAs have been found in the cytoplasm, chloroplasts, mitochondria and vacuoles of plants [19]. The best studied PAs are the diamine Put (putrescine; 1,4-diaminobutane), the triamine Spd and the tetramine Spm. Plants also produce an isomer of Spm, Tspm (thermospermine), that appears to have an important role in vascular development [22]; Tspm has not been reported in animals.

BIOSYNTHESIS OF PAs

Put is the precursor for higher PAs and can be derived from arginine through two routes (Figure 1), a direct route mediated by ODC (ornithine decarboxylase; EC 4.1.1.17) and an indirect route via agmatine catalysed by ADC (arginine decarboxylase; EC 4.1.1.19). ODC and ADC do not co-exist in all taxa perhaps in association with reduced genome sizes; for example, ODC is not present in some members of the Brassicaceae including A. thaliana [23]. Spd, Spm and Tspm are synthesized sequentially from Put in reactions catalysed by the respective aminopropyl transferases (Figure 2). Complete loss of Spd synthase (EC 2.5.1.16) results in embryo lethality in A. thaliana, whereas Spm synthase (EC 2.5.1.22) mutants have no phenotype, indicating Spd has an essential role in development, but Spm may not [21]. The aminopropyl donor for PA biosynthesis is dcSAM (decarboxylated SAM). The production of dcSAM by SAMDC (SAM decarboxylase; EC 4.1.1.50) allows plants to direct flux from other SAM-dependent reactions such as ethylene synthesis to PA synthesis. A mutation causing reduced Tspm synthase (EC 2.5.1.79) expression (ACAULIS5, acl5) leads to increased Spd and Spm content and an accumulation of acl5 mutant transcripts. Interestingly there is also an increase in SAMDC4 transcript level in Tspm synthase mutants; feeding of Tspm reverses both acl5 and SAMDC4 transcriptional changes, suggesting that these genes are co-ordinately regulated [24]. Although aminopropyl transferase activities limit PA synthesis, the availability of dcSAM and Put appear to be the most critical factors affecting the abundance of PAs.

PA biosynthesis and its relationship with the SAM cycle and the Met salvage cycle

Figure 2
PA biosynthesis and its relationship with the SAM cycle and the Met salvage cycle

Biosynthesis (grey panel, black arrows) of Put from L-arginine follows two routes. Spd is synthesized from Put via Spd synthase. Spm and Tspm are synthesized via their respective synthases. The synthesis of Spd, Spm and Tspm require the addition of an aminopropyl group from dcSAM. dcSAM is generated from SAM by the action of SAMDC. SAM is also required for ethylene and NA biosynthesis (blue arrows). Met is salvaged from MTA produced from PA, ethylene and NA biosynthesis (blue and black broken line). In the SAM cycle (green arrows), SAM is synthesized from Met by SAMS. SAH is generated from SAM through donation of the methyl group catalysed by one of hundreds of methyltransferases. SAH is hydrolysed to homocysteine, which is converted into Met by MS. Enzymes are indicated in dark grey boxes. Negative (−)-feedback loops by metabolites (italic) are highlighted in red.

Figure 2
PA biosynthesis and its relationship with the SAM cycle and the Met salvage cycle

Biosynthesis (grey panel, black arrows) of Put from L-arginine follows two routes. Spd is synthesized from Put via Spd synthase. Spm and Tspm are synthesized via their respective synthases. The synthesis of Spd, Spm and Tspm require the addition of an aminopropyl group from dcSAM. dcSAM is generated from SAM by the action of SAMDC. SAM is also required for ethylene and NA biosynthesis (blue arrows). Met is salvaged from MTA produced from PA, ethylene and NA biosynthesis (blue and black broken line). In the SAM cycle (green arrows), SAM is synthesized from Met by SAMS. SAH is generated from SAM through donation of the methyl group catalysed by one of hundreds of methyltransferases. SAH is hydrolysed to homocysteine, which is converted into Met by MS. Enzymes are indicated in dark grey boxes. Negative (−)-feedback loops by metabolites (italic) are highlighted in red.

REGULATION OF PA BIOSYNTHESIS

Regulation of PA intracellular homoeostasis involves their transport to vacuoles, and conjugation with small molecules, nucleic acids, membranes or proteins. Owing to limited space, these topics will not be covered in the present review; however, informative reviews are available [19,21]. Catabolism or back-conversion of PAs by diamine or PA oxidases (EC 1.5.3.11) also maintains PA levels and contributes to signalling through the production of H2O2 (for a review, see [25]).

ADC and SAMDC are synthesized as proenzymes that require additional processing to be functional [19]. The regulation of this processing appears to differ between plants and animals; the processing and subsequent enzyme activity of human SAMDC is enhanced by Put, thereby co-ordinating the abundance of the two precursors of Spd synthesis, Put and dcSAM. The plant enzyme lacks a critical amino acid residue essential for this activation, allowing it to be maximally active in the absence of Put [26]. PA homoeostasis is also aided by the irreversible inactivation of SAMDC by its product, dcSAM [19].

There are four genes encoding SAMDC in A. thaliana; all are expressed ubiquitously, to varying degrees and are partially functionally redundant on the basis of double mutant studies. SAMDC4 mutants (bud2-1) have reduced petiole lengths and altered root and shoot vasculature. These changes are associated with an increase in Put and a decrease in Spd and Spm as measured in whole seedlings. SAMDC1 mutants have a similar but more severe phenotype that is accompanied by elevated expression of SAMDC4 [27]. There are no loss-of-function mutants available for SAMDC2 and SAMDC3, so their contributions to plant development have yet to be determined.

PAs exert translational control over the expression of all SAMDC transcripts, except those of SAMDC4. The regulated mRNAs contain two conserved uORFs (upstream open reading frames): the 5′ ‘tiny uORF’ and the 3′ ‘small uORF’. The small uORF peptide represses translation of the downstream SAMDC coding sequence, whereas the tiny uORF is responsive to PA abundance. The data suggest that in low PA conditions the tiny uORF peptide and SAMDC are synthesized; in high PA conditions the small uORF is translated in a PA-mediated mechanism, limiting SAMDC expression [28]. SAMDC4 lacks the two uORFs and instead is sensitive to Tspm, suggesting that this isoform has a distinct role [25]. The similarity in the phenotypes of SAMDC4 and Tspm synthase mutants suggests that this function may be related to Tspm biosynthesis [22].

CO-REGULATION OF PA, IRON AND HORMONE METABOLISM

SAMDC1 expression is strongly repressed by auxin, whereas the other SAMDC genes are induced; bud2 mutants are hyposensitive to auxin and show down-regulation of auxin-inducible genes, suggesting an unidentified role of PAs in auxin-regulated development [29]. A recent report shows that auxin and Tspm may act antagonistically in xylem vessel development: xylem vessel differentiation is enhanced by synthetic auxin analogues in acl5 seedlings, but not in the wild-type [30]. The application of Tspm suppressed the xylem-enhancing effects of auxin in acl5 seedlings. Suppressor mutations that express SAC51 (SUPPRESSOR OF ACAULIS 51) in the absence of Tspm and mask the acl5 phenotype also suppress the effects of auxin, suggesting that Tspm may act through regulation of SAC51 expression rather than directly [30].

SAM is the substrate for PA, NA and ethylene biosynthesis, which consequently compete for their common substrate. Ethylene and PAs have opposing roles. Ethylene is well known as a promoter of senescence, whereas PAs are known to suppress senescence by slowing down membrane deterioration, loss of chlorophyll, and enhancing protease and RNase activities. The relationship between ethylene and PAs is clear in some species, tissues and developmental stages, but not for all. For example, during leaf and flower senescence and fruit ripening, PA synthetic enzymes compete with ACS, which catalyses the rate-limiting step of ethylene biosynthesis [31]. In pea seedlings, ethylene treatment inhibits ADC and SAMDC enzyme activity, resulting in decreased PA levels and inhibited growth [32]. In olive fruit abscission, an event mediated by ethylene and ACC accumulation, OeSAMDC1 transcription is down-regulated [33]. Microarray data of A. thaliana show that the exogenous application of ACC reduces the transcript abundance of AtSAMDC1, AtSPDS1 and AtSPDS2 (https://www.genevestigator.com/gv/), thereby reducing PA synthesis, revealing regulation of ethylene and PA synthesis by a mechanism other than their competition for SAM. Even more so, iron deficiency in rice induces ethylene synthesis and expression of NA synthase genes by ethylene, resulting in joint up-regulation of two SAM-consuming pathways [34]. In contrast with the abundant research on Put synthesis, details about Spd, Spm and Tspm in plants are lacking. This is particularly true for Tspm synthase, which is present in very low abundance relative to the other synthases and has only been recently discovered [22]. Although there have been some recent advances in measuring PA content, such as the improved FMOC-Cl method [35], there are still difficulties to overcome. Also, the majority of studies examine only free PA levels, often ignoring conjugates, in part probably because in the complexity of these measurements. The physiological significance of conjugated PAs cannot not be dismissed as they can make up a large portion of total PAs, as is the case with olive flowers and fruit, where 90% of the PA pool is in the conjugated form [36].

THE Met SALVAGE CYCLE OR YANG CYCLE

General function and feedback regulation by MTA and adenine

Met is required for protein synthesis, and as a substrate for the synthesis of SAM. As mentioned above, SAM serves as a methyl donor in numerous methylation reactions and is substrate for the synthesis of the ethylene precursor ACC and of the iron chelator NA. A derivate of SAM, dcSAM, is the aminopropyl donor for the synthesis of the PAs: Spm, Tspm and Spd. The common by-product of these reactions, MTA, is subsequently metabolized in a cyclic pathway known as the Met salvage cycle or Yang cycle in plants [1]. In bacteria, MTA is released from SAM during the synthesis of autoinducer quorum signals, which serve in communication within and between species to coordinate bacterial cell density and adaptation of bacteria to their biotic or abiotic environment via transcriptional regulation. The Met salvage cycle is thus a universal metabolic pathway present in bacteria, Archaea, animals and plants. Methylation reactions using SAM as a methyl group donor release SAH (S-adenosylhomocysteine) as a by-product. In plants, MTA and SAH are metabolized by two separate enzymes, MTN (MTA nucleosidase; EC 2.4.2.28) and SAHH (SAH hydrolase; EC 3.3.1.1). In bacteria, a dual-substrate enzyme, MTAN (MTA/SAH nucleosidase), metabolizes MTA and SAH. Hydrolysis of the N-glycosidic bond of MTA by MTN releases MTR and adenine [37]. Adenine acts as a potent competitive inhibitor of MTN with a Ki of 11 μM in lupin. Hence its metabolism by adenine phosphoribosyltransferase (EC 2.4.2.7) is required to sustain MTN activity. By contrast, MTR is a poor inhibitor of MTN with a Ki of 1.06 mM [37]. A. thaliana plants deficient in the MTR-metabolizing enzyme MTK (MTR kinase; EC 2.7.1.100) have a wild-type phenotype [38], supporting the conclusion that MTR levels do not reach cytotoxic levels even when not metabolized to MTR-P (MTR-1-phosphate) by MTK. A. thaliana mtk mutants have a wild-type phenotype when in sulfur-replete conditions, but are severely growth-retarded when grown on MTA as their sulfur source. These phenotypes indicate that metabolism of MTA is dispensable when sulfur is available in sufficient levels, but becomes essential under sulfur-deplete conditions. Accordingly, MTK gene expression is induced in rice by sulfur starvation [38].

Regulation of MTA degradation

Oryza sativa L. (rice) and Solanum lycopersicum (tomato) possess one MTK gene whereas two genes, AtMTN1 and AtMTN2, encode for MTN proteins in A. thaliana [39,40]. MTN from Lupinus luteus (lupin), rice and A. thaliana have a Km between 2.1 μM and 7.1 μM [37,39]. AtMTN1 from A. thaliana and lupin MTN show no activity towards SAH, whereas AtMTN2 and rice OsMTN show a minor rate of SAH metabolism. MTN proteins are structurally conserved not only between plant species but also between bacterial and plant enzymes with conserved adenine- and ribose-binding sites raising the question of how plant MTNs evolved to preferentially utilize MTA as a substrate. Structural analysis of AtMTN1 revealed that it binds SAH through its adenine and ribose groups with an affinity that is comparable with that of Escherichia coli MTAN [41]. However, binding does not occur in a catalytically competent manner, resulting in a lack of SAH hydrolysis. Subtle structural differences at the active site of MTN proteins are held responsible for residual activity of some plant enzymes towards SAH [41]. A. thaliana MTN1 is expressed ectopically in E. coli as a homodimer [42]. It is conceivable that heterodimerization of MTN proteins with differing enzymatic properties provides a mode of enzyme regulation in plants encoding more than one isoenzyme. MTN catalyses the first reaction in the Met salvage cycle and may regulate the flux of Met recycling through this pathway. In A. thaliana the calcium sensor protein calcineurin B-like 3 physically interacts with AtMTN1 in a calcium-dependent manner, which results in inhibition of MTN activity by approximately 65% [43]. In mammals, yeasts, cyanobacteria and archaea, MTA is metabolized to MTR-P by MTI (MTR-P isomerase; EC 5.3.1.23). The different routes for metabolism of MTA in humans compared with bacteria and plants render bacterial MTN a potential target for drug design.

Recycling of MTR to Met

The first step of MTR recycling is the phosphorylation of MTR by MTK (Figure 3). The resulting MTR-P is linearized by MTI. A. thaliana MTI1 complements a Saccharomyces cerevisiae MTI-deficient mutant strain, thus confirming its predicted function [44]. Conversion of MTR into DHKMP (1,2-dihydro-3-oxo-5-methylthiopentene) is catalysed by a DEP (dehydratase–enolase–phosphatase) complex [44]. In animals and yeasts, two distinct enzymes, a dehydratase and an enolase/phosphatase, carry out three catalytic activities. By contrast, these enzyme activities are combined in a single protein in plants. Alignment of the respective protein domains revealed that the N-terminal half of plant DEP aligns with dehydratases from other organisms and the C-terminal portion aligns with dual-function enolase–phosphatase enzymes. DEP1 from A. thaliana was sufficient to complement a dehydratase-deficient yeast mutant as well as an enolase–phosphatase-deficient yeast mutant, confirming trifunctional DEP1 activity [44].

Recycling of cytosolic SAM used by SAM-dependent methyltransferases

Figure 3
Recycling of cytosolic SAM used by SAM-dependent methyltransferases

Schematic presentation of the SAM (green) and SMM (blue) cycles that operate in the cytosol of plant cells to salvage the sulfur of Met used by SAM-dependent methyltransferases. MS re-methylates the HCys generated by SAHH from SAH at the expense of CH3-THG in the SAM cycle. In the SMM cycle, methylation of HCys is catalysed by HMT with SMM as the methyl donor. SMM is produced by MMT. Enzymes are indicated in dark grey boxes. Negative (−)- feedback loops by metabolites (italic) are highlighted in red.

Figure 3
Recycling of cytosolic SAM used by SAM-dependent methyltransferases

Schematic presentation of the SAM (green) and SMM (blue) cycles that operate in the cytosol of plant cells to salvage the sulfur of Met used by SAM-dependent methyltransferases. MS re-methylates the HCys generated by SAHH from SAH at the expense of CH3-THG in the SAM cycle. In the SMM cycle, methylation of HCys is catalysed by HMT with SMM as the methyl donor. SMM is produced by MMT. Enzymes are indicated in dark grey boxes. Negative (−)- feedback loops by metabolites (italic) are highlighted in red.

DHKMP is subsequently converted into 2-oxo-4-methylthiobutyrate by an ARD (acireductone dioxygenase) complexed with Fe2+ as a cofactor (Fe-ARD). ARDs are unique proteins in that the same apoprotein can acquire two different enzymatic activities which metabolize the same substrate to different products depending on the co-factor bound to it [45,46]. Fe-ARD (EC 1.13.11.54) is the Met cycle enzyme, whereas Ni-ARD (EC 1.13.11.53) catalyses an off-pathway that produces methylthiopropionate, formate and carbon monoxide from DHKMP. ARD isoenzymes are encoded by two genes in rice and tomato, and four genes in A. thaliana [40,44,46]. The final step in Met recycling is catalysed by a transaminase which has yet to be identified in plants. Amino transfer could be accomplished by a specific enzyme or by one or more wide-substrate enzymes not unique to the Met salvage cycle.

Regulation of the Met salvage cycle

The SAM- or dcSAM-consuming enzymes ACS, PA synthases and NA synthase (EC 2.5.1.43) release MTA that is recycled to the SAM precursor Met at the expense of one ATP and of an amino group transfer. In return, the cell disposes of a side-product not otherwise used, salvages the highly reduced sulfur, and replenishes the Met/SAM pool. As MTA is considered a toxic metabolite due to product inhibition, the Met cycle can also be viewed as a detoxifying pathway. In mammals, loss of MTA phosphorylase is associated with malignant cell growth [47]. In extracts of Cucurbita maxima (winter squash), ACS activity was inhibited by MTA with a Ki value of 0.39 mM [17]. MTA levels are approximately 10-fold lower than Met levels and 6-fold lower than SAM levels in rice; similar ratios were reported for A. thaliana, indicating that MTA is rapidly metabolized in plant tissues [38,48]. A. thaliana mtn1-1 mtn2-1 plants with severely reduced MTN activity show pleiotropic phenotypes, including strongly reduced fertility which goes along with elevated MTA levels in inflorescences [49]. A. thaliana plants exposed to MTA show elevated MTN protein levels and elevated MTN activity, suggestive of MTN activation by its substrate MTA. Interestingly, transcript levels of the major transcribed MTN gene in A. thaliana, MTN1, are not elevated, pointing to regulation of MTN activity at the level of protein synthesis and/or stability [50]. Constitutive MTN activity is several-fold higher in A. thaliana seedlings than in mature plants and is not induced by MTA.

An interesting question is how the Met cycle activity is adjusted to varying rates of MTA production when ethylene synthesis rates change. MTN activity is up-regulated in submerged rice, which is known to accumulate ethylene. Elevated MTN activity was accompanied by increased MTN mRNA levels indicative of transcriptional regulation. Treatment with ethylene neither promoted MTN gene expression nor MTN activity. It was therefore hypothesized that MTN is activated by MTA that is released as a result of enhanced ethylene synthesis in submerged plants [39]. Similarly, expression of MTN, MTK and ARD genes was not induced by ethylene in A. thaliana nor were transcript levels altered in ethylene signalling mutants [50]. Current studies hence do not support the idea that the Met cycle is generally controlled by ethylene signalling. Rice ARD1 is an exception in that gene expression is induced as an immediate-early response to ethylene [46].

Solanum lycopersicon (tomato) is a model plant used to study fruit ripening which is controlled by ethylene [51]. During early fruit development ethylene synthesis occurs at basal levels and is autoinhibitory (system 1). At the breaker stage ethylene synthesis switches to a different regulatory mode (system 2) with autocatalytic feedback activation resulting in high rates of ethylene synthesis which in turn promote climacteric fruit ripening. When tomato fruits are ripe ethylene synthesis declines, whereas ACC continues to be produced post-harvest, but is not converted into ethylene due to a low ACO activity. Instead, ACC is conjugated to its inactive storage form malonyl-ACC [40]. Changes in ACS activity with two peaks at the light orange stage and 7 days post-harvest correlated well with elevated transcript levels of MTN, MTK, ARD1 and ARD2. By contrast DEP and MTI transcripts were not elevated at high ACS activity, indicating that some, but not all, Met cycle gene activities were adjusted to different rates of MTA release.

THE SIGNIFICANCE OF SAM SYNTHESIS AND RECYCLING IN THE CYTOSOL

Three independent Met recycling systems exist in the cytosol to cope with the high demand of Met for SAM-dependent reactions: the Met salvage cycle, the SAM cycle and the SMM (S-methylmethionine) cycle. The Met salvage pathway recycles the sulfur of MTA produced during synthesis of ethylene, PAs and NA. The SAM and SMM cycle salvage the bulk of SAM-bound sulfur used in methylation reactions catalysed by methyltransferases (Figure 2). The many substrates for methyltransferases are pervasive throughout all plant cells and compartments. Of the 239 methyltransferase reactions (EC 2.1.1.x) classified in the Brenda database [52], 90% utilize SAM as the methyl donor. These enzymes methylate small molecules, proteins, lipids, cell wall polymers (such as the pectin) as well as nucleic acids, leading to changes in chemical strength, volatility, signalling and intermolecular interactions (Figure 2). Thus the flux through these pathways and the need for SAM is cell specific and very dynamic with respect to development and environment. In rosette leaves of A. thaliana, down-regulation of SAMS activity in the mto3 mutant (SAMS3, At3g17390) causes a 200-fold accumulation of Met, which indicates that a massive flux from Met into SAM occurs in photosynthetically active tissue in wild-type plants (reviewed in [53]). Three additional genes encode for cytosolic SAMS (SAMS1, SAMS2 and SAMS4) in A. thaliana. Nitrosylation of SAMS1 (At1g02500) results in strong inactivation of enzymatic activity. The nitrosylated Cys114 is not conserved in the other SAMS isoforms, which consequently are not targets of this regulation. Application of the NO donor S-nitrosoglutathione to A. thaliana cell cultures causes down-regulation of ethylene production in a time- and concentration-dependent manner and is suggestive of cross-talk between ethylene and NO signalling in plants via regulation of SAM synthesis [54].

SAH is a by-product of all methyltransferase reactions that use SAM as the methyl donor. In plants SAHH catalyses the reversible metabolism of SAH with an equilibrium that lies far towards the synthesis of SAH (reviewed in [55]). This reaction must be drawn in the hydrolysis direction by the removal of the products adenosine and HCys (homocysteine), in order to prevent competitive inhibition of methyltransferase activities by SAH. The HCys is re-methlyated to Met by MS (Met synthase; EC 2.1.1.14) within the SAM cycle (Figure 2). In plants, adenosine is metabolized by ADK (adenosine kinase; EC 2.7.1.20); A. thaliana lines deficient in ADK activity have increased SAH content and reduced DNA and pectin methylation. Reduction of ADK activity to less than 4–5% of that present in wild-type plants is lethal [56]. In humans the SAM to SAH ratio is termed the MP (methylation potential) and is a metabolic indicator for the methylation status of the cell. A decrease in MP is often associated with induction of specific genes due to lowered methylation degree of their promoters and may result, in plants, in inefficient capping of mRNAs (see below).

SAHH and ADK are cytosolic enzymes lacking subcellular targeting signals [57]. Specific SAM/SAH exchangers exist on the outer membranes of plastids [58] and mitochondria [59] to maintain the MT activities within these organelles. In A. thaliana, SAHH and ADK form a complex with the methyltransferase required for capping of mRNAs (EC 2.1.1.56), thereby directing both enzymes to the nucleus to maintain this methylation activity [57]. It is possible that targeting signals on other methyltransferases aid the subcellular localization of SAH and ADK.

CONTRIBUTION OF THE SMM CYCLE TO SUSTAIN CYTOSOLIC Met LEVELS

The tandem action of MMT (Met S-methyltransferase; EC 2.1.1.12) and HMT (HCys S-methyltransferase; EC 2.1.1.10), plus that of SAHH, constitutes the SMM cycle (Figure 2). The entry reaction of the SMM cycle is catalysed by MMT and is unique for plants. SMM is highly abundant in the leaves of plants (low millimolar range) when compared with Met (low micromolar range). It seems to serve as a storage of sulfur and methyl groups in leaves and a transport form of reduced sulfur in the phloem [60,61]. It has been suggested that phloem loading and source-sink partitioning of SMM are important for co-ordination of sulfur and nitrogen metabolism and yield in legumes [61]. Loss-of-function MMT mutants of A. thaliana and maize (Zea mays) lacked the capacity to produce SMM, but grew and reproduced normally ruling out an essential role of MMT in these plant species [62]. SMM serves also as a substrate for conversion of HCys into Met by HMT. In contrast with Met synthesis by MS in the SAM cycle, the HMT reaction is independent of CH3-THG (5-methyl-tetra-hydropteryl-tri-glutamate) and could provide a temporal buffer for HCys salvation in the cytosol, e.g. during CH3-THG limitation (SMM cycle, Figure 2). Two HMTs are present in the cytosol of A. thaliana, one of which is inhibited by Met [63]. If the cytosolic Met level drops due to limitation of CH3-THG, HMT will maintain Met synthesis from HCys in the cytosol at the expense of SMM. Thus accumulation of HCys and consequently SAH could be dependent on a functional SMM cycle during CH3-THG limitation. Indeed, A. thaliana and maize plants that lack MMT activity, have a higher MP, demonstrating that the SMM cycle contributes to maintenance of the MP in plants [62].

CONTRIBUTION OF Met METABOLISM TO HORMONE AND PA SYNTHESIS

The precursor of ethylene and PA biosynthesis, Met, is produced in a three-step pathway (Figure 4). In higher plants the first step is the formation of cystathionine by merging of cysteine and OPH (O-phosphohomoserine). This reaction is catalysed by CGS [cystathionine γ-synthase; EC 2.5.1.48 (formerly 4.2.99.9)], which is rate-limiting for Met de novo biosynthesis (reviewed in [53]). Next, CBL (cystathionine β-lyase; EC 4.4.1.8) decomposes the highly reactive thioether cystathionine into ammonium, pyruvate and HCys [64]. The HCys is methylated to Met by MS that accepts the methyl group from CH3-THG. The by-products of HCys formation, ammonium and pyruvate, are metabolized in the GOGAT (glutamate synthase) pathway and glycolysis respectively. In contrast with exclusively plastid-localized CGS and CBL, MS in A. thaliana is present in plastids and the cytosol [58]. This is because HCys methylation is required not only for de novo Met synthesis but also in the recycling of SAM in the cytosol (see Figure 2 and below). The exclusive cytosolic SAMS localization, the absence of SAHH in plastids and the identification of a plastidic SAM/SAH antiport system indicate that production and recycling of the methyl donor SAM is separated from de novo Met synthesis (reviewed in [65]).

REGULATION OF CARBON FLUX INTO Met

In contrast with cyanobacteria and fungi, OPH is the sole carbon backbone for Met synthesis in plants (Figure 2). A comparison of kinetic parameters of A. thaliana CGSs with steady-state levels of both substrates suggests that plastidic CGS activity is only approximately 2% of its maximal rate [66]. Consequently, regulation of OPH synthesis within the plastid-localized ADAAP (aspartate-derived amino acid pathway) is a major determinant for Met production. Besides its role in Met synthesis, ADAAP provides the plant cell with Lys (lysine), Ile (isoleucine) and Thr (threonine). The entry step of this highly controlled pathway is the phosphorylation of aspartate by AK (aspartate kinase; EC 2.7.2.4). In A. thaliana five genes encode for proteins having AK activity, of which two genes encode for bi-functional enzymes that also possess homoserine dehydrogenase (AK-HSDH) activity (3rd step of ADAAP, Figure 4). The high complexity of the AK gene family in A. thaliana is thought to reflect its importance for regulation of the entire pathway. Indeed, SAM inhibits specifically AK1 (At5g14060), but not AK2 and AK3 or AK-HSDH activity. In contrast, cysteine stimulates AK-HSDH1 activity, but has no impact on any AK or AK-HSDH2 (reviewed in [53]). Stimulation of AK-HSDH and not AK by cysteine is advantageous for the plant, since it directs the flux from aspartate into the Thr/Ile/Met branch of ADAAP and avoids Lys accumulation (Figure 4). For a detailed regulation of all steps of ADAAP by Lys, Thr and Ile that also affect flux of carbon into the Met branch (Figure 4), the reader is referred to [53]. In general, Met synthesis seems to be regulated more by SAM than by Met in higher plants (Figure 4), which reflects the high rate of Met into SAM conversion by SAMS. SAMS direct approximately 80% of the flux of Met into SAM, whereas only 20% of Met ends up in the protein fraction [67]. High SAM levels activate dihydrodipicolinate synthase (EC 4.2.1.52) and TS (Thr synthase; EC 4.2.3.4), which direct the flux of carbon away from de novo Met synthesis in plastids. Competition between CGS and TS for their common substrate OPH is especially important for metabolite partitioning within the ADAAP, as demonstrated by the more than 20-fold accumulation of Met in leaves of the A. thaliana mto2-1 mutant with decreased TS activity [68].

De novo biosynthesis of Met

Figure 4
De novo biosynthesis of Met

Presentation of the three-step Met biosynthesis pathway and its regulation in higher plants. The biosynthesis of the carbon backbone (O-phosphomoserine) and the sulfur donor (cysteine) for Met biosynthesis by the aspartate-derived amino acid (grey panel, blue arrows) and the sulfur assimilation pathway (grey arrows) are shown schematically in the upper part of the Figure to indicate regulatory feedback loops by SAM and cysteine. Positive (+)- and negative (−)-feedback loops by these metabolites are highlighted in green and red respectively. Arrows indicate enzymatic steps in ADAAP (blue), cysteine (grey) and Met (black) biosynthesis. Enzymes are indicated in dark grey boxes. Key intermediates are italicized. For a better overview the sulfur is highlighted in yellow in the SAM synthesis pathway.

Figure 4
De novo biosynthesis of Met

Presentation of the three-step Met biosynthesis pathway and its regulation in higher plants. The biosynthesis of the carbon backbone (O-phosphomoserine) and the sulfur donor (cysteine) for Met biosynthesis by the aspartate-derived amino acid (grey panel, blue arrows) and the sulfur assimilation pathway (grey arrows) are shown schematically in the upper part of the Figure to indicate regulatory feedback loops by SAM and cysteine. Positive (+)- and negative (−)-feedback loops by these metabolites are highlighted in green and red respectively. Arrows indicate enzymatic steps in ADAAP (blue), cysteine (grey) and Met (black) biosynthesis. Enzymes are indicated in dark grey boxes. Key intermediates are italicized. For a better overview the sulfur is highlighted in yellow in the SAM synthesis pathway.

REGULATION OF S-DONOR SYNTHESIS FOR Met PRODUCTION

CGS catalyses the γ-replacement of the phosphoryl substituent of OPH by cysteine, which is the common S-donor for all metabolites containing reduced sulfur in plants (e.g. biotin, iron–sulfur clusters, glucosinolates, etc.). The Km value of CGS for cysteine is approximately 45-fold higher than the determined concentration of cysteine in plastids (9±1 μM) [66,69]. In plants, cysteine is synthesized from OAS (O-acetylserine) and sulfide by OASTL [OAS-(thiol)-lyase; EC 2.5.1.47]. The synthesis of a bulk amount of OAS is catalysed by SAT (serine acetyltransferase; EC 2.3.1.30) from serine and acetyl-CoA in mitochondria and limits cysteine production in leaves of A. thaliana [70]. In contrast, reduction of sulfate to sulfide takes place exclusively in plastids and is limited by adenosine-5-phospho-sulfate reductase and sulfite reductase activity [71]. Mitochondrial OAS and plastidial sulfide meet high OASTL activity in the cytosol, where the bulk of foliar cysteine is formed [72]. The unusual subcellular compartmentalization of cysteine biosynthesis emerged during evolution of vascular plants, probably to allow efficient regulation (reviewed in [73]) and to avoid the toxic effect of sulfide in mitochondria [74]. However, SAT and OASTL are present in mitochondria, plastids and the cytosol, where they form the cysteine synthase complex, which senses OAS and sulfide supply and regulates SAT activity to meet the demand for cysteine [75].

Limited cysteine supply in plastids due to sulfur deprivation does not cause a significant alteration of the Met steady-state pool, but results in a 10-fold decrease in SAM content [76]. The decreased SAM level will push carbon flux into Met synthesis and activate CGS to counteract the decreased Met synthetic capacity due to cysteine limitation (Figure 4). Interestingly, sulfur limitation results in a specific decrease in SAM and does not affect the SAH level. This causes a significant decrease in the methylation potential of the plant cell and may affect ethylene and PA synthesis capacity [76]. The regulatory function of SAM on Met steady-state levels may also explain why overexpression of SAT results in significant accumulation of cysteine and glutathione, a storage form of cysteine, but does not affect Met levels [77].

REGULATION OF A METHYL-GROUP DONOR FOR Met PRODUCTION

Folate metabolism provides the methyl donor for Met synthesis. Polyglutamylation of the folate backbone was identified as an important trigger for folate compartmentation and homoeostasis and thus folate-dependent metabolic processes [78]. MS accepts the methyl group of CH3-THG with 90–220-fold higher affinity than from methyl-tetra-hydrofolate (the non-polyglutamylated form) [79]. Biosynthesis of the folate backbone takes place exclusively in the mitochondria of plants. The cytosol and plastids are folate-auxotrophic and require import of folate, which is subject to polyglutamylation in all compartments [80]. For these reasons regulation of mitochondrial folate biosynthesis and plastidic FPGS (folylpolyglutamate synthase) activity could be potential targets for regulation of Met synthesis. However, the co-regulation of CH3-THG production by folate metabolism with plastid Met synthesis and cytosolic Met recycling is barely understood [80].

REGULATION OF CGS, CBL AND MS ACTIVITIES

Interestingly, none of the enzymes in the trans-sulfuration pathway, CGS, CBL and MS, are allosterically regulated by Met (reviewed in [65]), pointing again to a considerable regulation of Met production by precursor availability (see previous sections). Genes for de novo synthesis of Met are more or less constitutively expressed in all organs of A. thaliana. Nevertheless, several stresses can result in a significant accumulation of Met biosynthesis-related transcripts. In flowers of Nicotiana suaveolens, MS and SAMS transcripts oscillate during day/night transitions, which contributes significantly to the demand for SAM used for methylation of volatile organic compounds and in later stages of flower development for ethylene synthesis. Specifically in these late stages ACO transcript levels fluctuate in a synchronized manner with transcript levels of MS and SAMS. Interestingly, the frequency of this fluctuation negatively correlates with fluctuation in the SAM steady-state level [81]. Regulation of transcript steady-state level by SAM is evident for CGS via a post-transcriptional mechanism. In this case the protein sequence encoded by the first exon of CGS (MTO-1 domain) acts in cis to destabilize its own mRNA after binding of SAM, which acts as the effector [82,83]. The MTO1 domain has no features in common with known SAM-binding domains. It may hence be speculated that SAM regulation involves an additional protein [83]. The importance of the mRNA decay-based regulation of CGS via the MTO-1 domain is underpinned by the identification of a natural CGS transcript with a 90 bp deletion in this domain. Overexpression of this naturally stable transcript results in significantly higher concentrations of Met than the ectopic expression of the full-length mRNA [84].

CATABOLISM OF Met

Metabolism of Met into SAM for methylation reactions in the cytosol is a massive sink (80% of flux [67]). Moreover, Met can be degraded by cytosolic Met γ-lyase (EC 4.4.1.11, At1g64660) to ammonia, methanethiol and 2-oxobutanoate [85]. The latter is also a product of Thr catabolism and can be re-incorporated in Ile (reviewed in [53]). Labelling studies demonstrated the re-fixation of methanethiol in S-methylcysteine, but the mechanism is unclear. A loss-of-function Met γ-lyase mutant displays normal growth, but >9-fold higher Met levels when grown under sulfur-limiting conditions. The Met level of the mutant is wild-type-like under normal growth conditions, indicating that recycling of Met in the cytosol is particularly important during sulfur starvation [86].

CONCLUDING REMARKS

The synthesis of the phytohormone ethylene, the growth-stimulating polyamines, the iron-chelating NA, and the primary metabolite Met is linked by SAM, which also is the predominant methyl donor in eukaryotic cells. Three salvage cycles for Met exist in the cytosol of plant cells to sustain SAM levels. The Met salvage cycle is specific for recycling of SAM consumed in ethylene, NA and PA biosynthesis. SAM metabolism is highly compartmentalized and regulated by various feedback loops that also control synthesis of Met precursors in plastids. The diverse regulatory mechanisms employed by plants to mediate SAM-dependent activities challenges our current methods for dissecting metabolic flux and demand for SAM. The application of improved analysis methods for SAM-related metabolites in the model plants A. thaliana, Plantago major and O. sativa revealed the importance of the Met salvage cycle for phytohormone synthesis in the vasculature, during determination of plant development and in response to abiotic stresses. The identification of Met cycle genes from plants by reverse genetics and improved methods for metabolite analysis will open new avenues to study the complex regulatory circuits between PA, NA, ethylene and Met metabolism.

Abbreviations

     
  • ACC

    1-aminocyclo-propane-1-carboxylic acid

  •  
  • ACO

    ACC oxidase

  •  
  • ACS

    ACC synthase

  •  
  • ADC

    arginine decarboxylase

  •  
  • ADK

    adenosine kinase

  •  
  • ADAAP

    aspartate-derived amino acid pathway

  •  
  • AK

    aspartate kinase

  •  
  • AK-HSDH

    bi-functional AK homoserine dehydrogenase

  •  
  • ARD

    acireductone dioxygenase

  •  
  • CBL

    cystathionine β-lyase

  •  
  • CGS

    cystathionine γ-synthase

  •  
  • CH3-THG

    5-methyl-tetra-hydropteryl-tri-glutamate

  •  
  • dcSAM

    decarboxylated S-adenosylmethionine

  •  
  • DEP

    dehydratase–enolase–phosphatase

  •  
  • DHKMP

    1,2-dihydro-3-keto-5-methylthiopentene

  •  
  • EIL

    ETHYLENE-INSENSITIVE3-LIKE

  •  
  • HCys

    homocysteine

  •  
  • HMT

    HCys S-methyltransferase

  •  
  • Ile

    isoleucine

  •  
  • Lys

    lysine

  •  
  • Met

    methionine

  •  
  • MMT

    Met S-methyltransferase

  •  
  • MP

    methylation potential

  •  
  • MS

    Met synthase

  •  
  • MTA

    5′-methylthioadenosine

  •  
  • MTAN

    MTA/S-adenosylhomocysteine nucleosidase

  •  
  • MTI

    5′-methylthioribose-1-phosphate isomerase

  •  
  • MTK

    5′-methylthioribose kinase

  •  
  • MTN

    MTA nucleosidase

  •  
  • MTR

    5′-methylthioribose

  •  
  • MTR-P

    5′-methylthioribose-1-phosphate

  •  
  • NA

    nicotianamine

  •  
  • OAS

    O-acetylserine

  •  
  • OASTL

    OAS-(thiol)-lyase

  •  
  • ODC

    ornithine decarboxylase

  •  
  • OPH

    O-phosphohomoserine

  •  
  • PA

    polyamine

  •  
  • Put

    putrescine

  •  
  • SAH

    S-adenosylhomocysteine

  •  
  • SAC51

    SUPPRESSOR OF ACAULIS 51

  •  
  • SAHH

    SAH hydrolase

  •  
  • SAM

    S-adenosylmethionine

  •  
  • SAMS

    SAM synthetase, SAMDC, SAM decarboxylase

  •  
  • SAT

    serine acetyltransferase

  •  
  • SLIM1

    SULFUR LIMITATION 1

  •  
  • SMM

    S-methylmethionine

  •  
  • Spd

    spermidine

  •  
  • Spm

    spermine

  •  
  • Thr

    threonine

  •  
  • TS

    Thr synthase

  •  
  • Tspm

    thermospermine

  •  
  • uORF

    upstream open reading frame

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