Overexpression of S‐adenosyl‐l‐methionine synthetase increased tomato tolerance to alkali stress through polyamine metabolism

S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na⁺ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.     

FERONIA receptor kinase interacts with S‐adenosylmethionine synthetase and suppresses S‐adenosylmethionine production and ethylene biosynthesis in Arabidopsis

Environmental inputs such as stress can modulate plant cell metabolism, but the detailed mechanism remains unclear. We report here that FERONIA (FER), a plasma membrane receptor‐like kinase, may negatively regulate the S‐adenosylmethionine (SAM) synthesis by interacting with two S‐adenosylmethionine synthases (SAM1 and SAM2). SAM participates in ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for protein and DNA methylation reactions. The Arabidopsis fer mutants contained a higher level of SAM and ethylene in plant tissues and displayed a dwarf phenotype. Such phenotype in the fer mutants was mimicked by over‐expressing the S‐adenosylmethionine synthetase in transgenic plants, whereas sam1/2 double mutant showed an opposite phenotype. We propose that FER receptor kinase, in response to environmental stress and plant hormones such as auxin and BR, interacts with SAM synthases and down‐regulates ethylene biosynthesis.     

Reduced ethylene synthesis by transgenic tomatoes expressing S-adenosylmethionine hydrolase

We have utilized a gene from bacteriophage T3 that encodes the enzyme S-adenosylmethionine hydrolase (SAMase) to generate transgenic tomato plants that produce fruit with a reduced capacity to synthesize ethylene. S-adenosylmethionine (SAM) is the metabolic precursor of 1-aminocyclopropane-1-carboxylic acid, the proximal precursor to ethylene. SAMase catalyzes the conversion of SAM to methylthioadenosine and homoserine. To restrict the presence of SAMase to ripening fruit, the promoter from the tomato E8 gene was used to regulate SAMase gene expression. Transgenic tomato plants containing the 1.1 kb E8 promoter bore fruit that expressed SAMase during the breaker and orange stage of fruit ripening and stopped expression after the fruit fully ripened. Plants containing the 2.3 kb E8 promoter expressed SAMase at higher levels during the post-breaker phases of fruit ripening and had a substantially reduced capacity to synthesize ethylene.     

Regulatory Role of Cystathionine-γ-Synthase and de novo Synthesis of Methionine in Ethylene Production during tomato Fruit Ripening

The essential amino acid methionine is a substrate for the synthesis of S-adenosyl-methionine (SAM), that donates its methyl group to numerous methylation reactions, and from which polyamines and ethylene are generated. To study the regulatory role of methionine synthesis in tomato fruit ripening, which requires a sharp increase in ethylene production, we cloned a cDNA encoding cystathionine γ-synthase (CGS) from tomato and analysed its mRNA and protein levels during tomato fruit ripening. CGS mRNA and protein levels peaked at the “turning” stage and declined as the fruit ripened. Notably, the tomato CGS mRNA level in both leaves and fruit was negatively affected by methionine feeding, a regulation that Arabidopsis, but not potato CGS mRNA is subject to. A positive correlation was found between elevated ethylene production and increased CGS mRNA levels during the ethylene burst of the climacteric ripening of tomato fruit. In addition, wounding of pericarp from tomato fruit at the mature green stage stimulated both ethylene production and CGS mRNA level. Application of exogenous methionine to pericarp of mature green fruit increased ethylene evolution, suggesting that soluble methionine may be a rate limiting metabolite for ethylene synthesis. Moreover, treatment of mature green tomato fruit with the ethylene-releasing reagent Ethephon caused an induction of CGS mRNA level, indicating that CGS gene expression is regulated by ethylene. Taken together, these results imply that in addition to recycling of the methionine moieties via the Yang pathway, operating during synthesis of ethylene, de novo synthesis of methionine may be required when high rates of ethylene production are induced.     

Effect of methylglyoxal bis-(guanylhydrazone) on polyamine and ethylene biosynthesis of apple fruit after harvest

Polyamine and ethylene both play important roles in fruit ripening, whose biosynthetic pathways share a common substrate, S-adenosylmethionine (SAM). To unravel the interrelationship between polyamine and ethylene, their metabolism and expression of relevant genes were investigated in apple fruit (Malus domestica Borkh.) treated with methylglyoxal bis-(guanylhydrazone) (MGBG). The MGBG-treated fruit had higher ethylene production until 16 days after treatment (DAT) with preceding accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) than control fruit and then decreased to nearly the same level as control. Ethylene promotion at the early stage by MGBG was accompanied by increased expression of apple ACC synthase (Md-ACS1) and ACC oxidase (MdACO). The expression of apple SAM synthase (MdSAMS) in MGBG-treated fruit was slightly higher than that in control. On the other hand, significant changes in free polyamine titers were observed at some stages, but the changes did not show consistent trends. Based on these observations, possible relationship between polyamine and ethylene pathways was discussed.     

Refolding of a fully functional flavivirus methyltransferase revealed that S‐adenosyl methionine but not S‐adenosyl homocysteine is copurified with flavivirus methyltransferase

Methylation of flavivirus RNA is vital for its stability and translation in the infected host cell. This methylation is mediated by the flavivirus methyltransferase (MTase), which methylates the N7 and 2′‐O positions of the viral RNA cap by using S‐adenosyl‐l ‐methionine (SAM) as a methyl donor. In this report, we demonstrate that SAM, in contrast to the reaction by‐product S‐adenosyl‐l ‐homocysteine, which was assumed previously, is copurified with the Dengue (DNV) and West Nile virus MTases produced in Escherichia coli (E. coli). This endogenous SAM can be removed by denaturation and refolding of the MTase protein. The refolded MTase of DNV serotype 3 (DNV3) displays methylation activity comparable to native enzyme, and its crystal structure at 2.1 Å is almost identical to that of native MTase. We characterized the binding of Sinefungin (SIN), a previously described SAM‐analog inhibitor of MTase function, to the native and refolded DNV3 MTase by isothermal titration calorimetry, and found that SIN binds to refolded MTase with more than 16 times the affinity of SIN binding to the MTase purified natively. Moreover, we show that SAM is also copurified with other flavivirus MTases, indicating that purification by refolding may be a generally applicable tool for studying flavivirus MTase inhibition.     

Crystal structure of norcoclaurine‐6‐O‐methyltransferase,a key rate‐limiting step in the synthesis of benzylisoquinoline alkaloids

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate‐limiting enzymes and negative feedback inhibition by end‐products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)‐norcoclaurine‐6‐O‐methyltransferase (6OMT), a key rate‐limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end‐products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S‐adenosyl‐l ‐homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time‐dependent sensitivity toward sanguinarine.     

A novel tomato F‐box protein,SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F‐BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene‐dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening‐associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening‐related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene‐related alterations, including inhibition of fruit ripening, attenuated triple‐response and delayed petal abscission. Yeast‐two‐hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3‐mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.     

Inhibition of the ethylene response by 1-MCP in tomato suggests that polyamines are not involved in delaying ripening, but may moderate the rate of ripening or over-ripening

Ethylene initiates the ripening and senescence of climacteric fruit, whereas polyamines have been considered as senescence inhibitors. Ethylene and polyamine biosynthetic pathways share S-adenosylmethionine as a common intermediate. The effects of 1-methylcyclopropene (1-MCP), an inhibitor of ethylene perception, on ethylene and polyamine metabolism and associated gene expression was investigated during ripening of the model climacteric fruit, tomato (Solanum lycopersicum L.), to determine whether its effect could be via polyamines as well as through a direct effect on ethylene. 1-MCP delayed ripening for 8 d compared with control fruit, similarly delaying ethylene production and the expression of 1-aminocyclopropane-1-carboxylic acid (ACC)-synthase and some ethylene receptor genes, but not that of ACC oxidase. The expression of ethylene receptor genes returned as ripening was reinitiated. Free putrescine contents remained low while ripening was inhibited by 1-MCP, but increased when the fruit started to ripen; bound putrescine contents were lower. The activity of the putrescine biosynthetic enzyme, arginine decarboxylase, was higher in 1-MCP-treated fruit. Activity of S-adenosylmethionine-decarboxylase peaked at the same time as putrescine levels in control and treated fruit. Gene expression for arginine decarboxylase peaked early in non-treated fruit and coincident with the delayed peak in putrescine in treated fruit. A coincident peak in the gene expression for arginase, S-adenosylmethionine-decarboxylase, and spermidine and spermine synthases was also seen in treated fruit. No effect of treatment on ornithine decarboxylase activity was detected. Polyamines are thus not directly associated with a delay in tomato fruit ripening, but may prolong the fully-ripe stage before the fruit tissues undergo senescence.     

Metabolic profiling reveals ethylene mediated metabolic changes and a coordinated adaptive mechanism of ‘Jonagold’ apple to low oxygen stress

Apples are predominantly stored in controlled atmosphere (CA) storage to delay ripening and prolong their storage life. Profiling the dynamics of metabolic changes during ripening and CA storage is vital for understanding the governing molecular mechanism. In this study, the dynamics of the primary metabolism of ‘Jonagold’ apples during ripening in regular air (RA) storage and initiation of CA storage was profiled. 1‐Methylcyclopropene (1‐MCP) was exploited to block ethylene receptors and to get insight into ethylene mediated metabolic changes during ripening of the fruit and in response to hypoxic stress. Metabolic changes were quantified in glycolysis, the tricarboxylic acid (TCA) cycle, the Yang cycle and synthesis of the main amino acids branching from these metabolic pathways. Partial least square discriminant analysis of the metabolic profiles of 1‐MCP treated and control apples revealed a metabolic divergence in ethylene, organic acid, sugar and amino acid metabolism. During RA storage at 18°C, most amino acids were higher in 1‐MCP treated apples, whereas 1‐aminocyclopropane‐1‐carboxylic acid (ACC) was higher in the control apples. The initial response of the fruit to CA initiation was accompanied by an increase of alanine, succinate and glutamate, but a decline in aspartate. Furthermore, alanine and succinate accumulated to higher levels in control apples than 1‐MCP treated apples. The observed metabolic changes in these interlinked metabolites may indicate a coordinated adaptive strategy to maximize energy production.     

Förster resonance energy transfer measurements of cofactor‐dependent effects on protein arginine N‐methyltransferase homodimerization

Protein arginine N‐methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S‐adenosyl‐L ‐methionine (AdoMet) to arginine residues in protein substrates, forming S‐adenosyl‐L ‐homocysteine (AdoHcy) and methylarginine residues. In this study, we use Förster resonance energy transfer (FRET) to determine dissociation constant (K_D) values for dimerization of PRMT1 and PRMT6. By attaching monomeric Cerulean and Citrine fluorescent proteins to their N‐termini, fluorescent PRMTs are formed that exhibit similar enzyme kinetics to unconjugated PRMTs. These fluorescent proteins are used in FRET‐based binding studies in a multi‐well format. In the presence of AdoMet, fluorescent PRMT1 and PRMT6 exhibit 4‐ and 6‐fold lower dimerization K_D values, respectively, than in the presence of AdoHcy, suggesting that AdoMet promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization K_D values for PRMT1 in the presence of AdoMet or AdoHcy are, respectively, 6‐ and 10‐fold lower than the corresponding values for PRMT6. Considering that the affinity of PRMT6 for AdoHcy is 10‐fold higher than for AdoMet, PRMT6 function may be subject to cofactor‐dependent regulation in cells where the methylation potential (i.e., ratio of AdoMet to AdoHcy) is low. Since PRMT1 affinity for AdoMet and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.     

Enhanced flux of substrates into polyamine biosynthesis but not ethylene in tomato fruit engineered with yeast S-adenosylmethionine decarboxylase gene

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled ¹⁴C-Arg, ¹⁴C-Orn, L-[U-¹⁴C]Met, ¹⁴C-SAM and ¹⁴C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-¹⁴C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of ¹⁴C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of ¹⁴C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.     

Viperin is an iron‐sulfur protein that inhibits genome synthesis of tick‐borne encephalitis virus via radical SAM domain activity

Viperin is an interferon‐induced protein with a broad antiviral activity. This evolutionary conserved protein contains a radical S‐adenosyl‐l ‐methionine (SAM) domain which has been shown in vitro to hold a [4Fe‐4S] cluster. We identified tick‐borne encephalitis virus (TBEV) as a novel target for which human viperin inhibits productionof the viral genome RNA. Wt viperin was found to require ER localization for full antiviral activity and to interact with the cytosolic Fe/S protein assembly factor CIAO1. Radiolabelling in vivo revealed incorporation of ⁵⁵Fe, indicative for the presence of an Fe‐S cluster. Mutation of the cysteine residues ligating the Fe‐S cluster in the central radical SAM domain entirely abolished both antiviral activity and incorporation of ⁵⁵Fe. Mutants lacking the extreme C‐terminal W361 did not interact with CIAO1, were not matured, and were antivirally inactive. Moreover, intracellular removal of SAM by ectopic expression of the bacteriophage T3 SAMase abolished antiviral activity. Collectively, our data suggest that viperin requires CIAO1 for [4Fe‐4S] cluster assembly, and acts through an enzymatic, Fe‐S cluster‐ and SAM‐dependent mechanism to inhibit viral RNA synthesis.