Regulation of the methionine cycle through polyamine biosynthesis and ethylene signaling in Arabidopsis thaliana

Im Methioninzyklus wird Methylthioadenosin (MTA), das Nebenprodukt der Polyamin-, Ethylen- und Nicotianaminbiosynthese, zu Methionin recycelt. In dieser Arbeit wurden zum Einen mögliche Regulationen des Methioninzyklus durch die Polyaminbiosynthese in Arabidopsis thaliana untersucht. Arabidopsis besitzt zwei Gene für die MTA-Nukleosidase (MTN; At4g38800, At4g34840), welche den Eingangsschritt des Methioninzyklus katalysiert. T-DNA-Insertionsmutanten von MTN1 zeigten bei Anzucht auf MTA als Schwefelquelle ein stark reduziertes Wachstum. mtn1-1/mtn2-1 Doppelmutanten besaßen eine verlängerte vegetative Wachstumsphase und waren steril. Es wurde bereits gezeigt, dass Spermidin in der Lage ist das reduzierte Keimlingswachstum und die Infertilität der Doppelmutante zu komplementieren. In dieser Arbeit konnte das reduzierte Wurzelwachstum der mtn1 Mutanten durch Spermin komplementiert werden, obwohl die internen Spermingehalte in den Wurzeln auf MTA als Schwefelquelle erhöht waren. Es ist möglich, dass die Inhibierung von Enzymen durch MTA bei dem reduzierten Wurzelwachstum der mtn1 Mutanten eine Rolle spielt. MTA ist in der Lage Enzyme wie die Spermidin-Synthase in vitro zu inhibieren. Um den Zusammenhang von Wurzelwachstum und Polyaminen weiter zu untersuchen, wurden Loss-of-function-Mutanten verwendet. Mutanten der Spermidin-Synthase (At1g23820, At1g70310), Spermin-Synthase (At5g53120), Thermospermin-Synthase (At5g19530) und zwei Acyl-Transferasen (At2g23510, At2g25150) zeigten keinen, den mtn1 Mutanten ähnlichen Phänotyp. Die Inhibierung dieser Enzyme der Polyaminbiosynthese scheint nicht für das reduzierte Wachstum der mtn1 Mutanten verantwortlich zu sein. Über welche Mechanismen Spermin das Wurzelwachstum verändert ist noch nicht verstanden. In dieser Arbeit wurden außerdem die Regulation des Methioninzyklus durch die Ethylenbiosynthese und den Ethylensignalweg untersucht. Es wurde gezeigt, dass Ethylen zu einer Reduktion der MTN-Aktivität über die Reduktion der MTN1 Proteinmenge führt. Die Reduktion von MTN1 scheint weder transkriptionell noch über den Proteinabbau reguliert zu sein. Analysen der Ethylenmutanten ethylene insensitive2 (ein2), ethylene overproducer3 (eto3) und constitutive triple response1 (ctr1) zeigten, dass die Reduktion der MTN1 Proteinmenge nicht über die Ethylenproduktion, sondern über den Ethylensignalweg reguliert wird. Es wäre denkbar, dass der Ethylensignalweg ein Protein reguliert, welches direkt mit MTN1 interagiert um die Proteinmenge zu reduzieren. Protein Interaktion mit MTN1 wurde bereits für CALCINEURIN B-LIKE3 (CBL3) gezeigt. Diese Interaktion ist Kalzium-abhängig und führt zu einer Reduktion der MTN1-Aktivität. In dieser Arbeit wurde untersucht, ob die Reduktion der MTN-Aktivität durch Ethylen über Kalzium-abhängige Interaktion CBL3 reguliert wird. Kalzium führte zu einer Reduktion der MTN-Aktivität, unabhängig von Ethylen. Kalzium-abhängige Interaktion mit CBL3 ist nicht der Grund für die reduzierte MTN-Aktivität in Anwesenheit von Ethylen. Der genaue Mechanismus zur Regulation von MTN1 über den Ethylensignalweg muss noch weiter analysiert werden.The Met cycle salvages the reduced sulfur group from methylthioadenosine (MTA) the by-product of polyamine, ethylene and nicotianamine synthesis. In one part of this thesis Arabidopsis thaliana was employed to study regulation of the Met cycle by the polyamine biosynthesis. In Arabidopsis two genes encode MTA nucleosidase (MTN; At4g38800, At4g34840), which catalyzes the first committed step in the Met cycle. T-DNA-Insertion mutants of MTN1 grown on MTA as sulfur source showed severely reduced growth. mtn1-1/mtn2-1 double mutants had a prolonged vegetative stage and were sterile. It was already shown that spermidine complements retarded growth of mtn1 seedlings and infertility of mtn12/mtn2-1 double mutants. In this thesis reduced root growth of mtn1 plants was complemented by spermine, although internal spermine levels were elevated in roots grown on MTA as sulfur source. It is possible that enzyme inhibition by MTA plays a role in reduced root growth of mtn1. MTA is able to inhibit enzymes like spermidine synthase in vitro. To further study root growth in relation to polyamines, loss-of-function mutants were employed. Mutants of spermidine synthase (At1g23820, At1g70310), spermine synthase (At5g53120), thermospermine synthase (At5g19530) and two acyl transferases (At2g23510, At2g25150) did not have a phenotype comparable to mtn1 grown on MTA. Disrupting activity of these enzymes does not seem to be responsible for reduced mtn1 growth. It is not yet understood how spermine affects root growth of mtn1 mutants. Furthermore this study was employed to analyze regulation of the Met cycle by the ethylene biosynthesis and the ethylene signaling pathway. In this thesis it was shown that ethylene leads to a reduction of MTN activity by reducing the abundance of MTN1 protein. Neither changes in MTN transcripts nor changes in protein degradation were responsible for reduced MTN1 protein abundance. Studying the ethylene mutants ethylene insensitive2 (ein2), ethylene overproducer3 (eto3) and constitutive triple response1 (ctr1) pointed to the ethylene signaling rather than ethylene synthesis as a cause for MTN1 reduction. It is possible that ethylene signaling regulates a protein which interacts with MTN1 to reduce protein abundance. Protein interaction with MTN1 was shown for CALCINEURIN B-LIKE3 (CBL3). It is a calcium-dependent interaction and leads to reduced MTN1 activity. The probability of calcium-dependent interaction with CBL3 as the cause of ethylene-induced reduction of MTN activity was analyzed. Calcium led to a reduction of MTN activity independent of the presence of ethylene. Calcium-dependent interaction with CBL3 is hence not the cause for reduced MTN-activity in the presence of ethylene. Further studies will have to be done to reveal the mechanism of regulation

S-adenosyl-L-methionine (SAM) usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S-adenosyl-L-methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum 'Bonaparte'), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1-MCP treatment affects SAM usage by the three major SAM-associated pathways. The 1-MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1-(malonylamino)cyclopropane-1-carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1-MCP-treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1-MCP-treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S-adenosyl-L-homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1-MCP-treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.status: publishe

S-Adenosyl-l-methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S-adenosyl-l-methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum Bonaparte'), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1-MCP treatment affects SAM usage by the three major SAM-associated pathways. The 1-MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1-(malonylamino)cyclopropane-1-carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1-MCP-treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1-MCP-treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S-adenosyl-l-homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1-MCP-treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels

Inhibition of 5 \u27-methylthioadenosine metabolism in the Yang cycle alters polyamine levels, and impairs seedling growth and reproduction in Arabidopsis

The methionine or Yang cycle recycles Met from 5\u27-methylthioadenosine (MTA) which is produced from S-adenosyl-L-methionine (SAM) as a by-product of ethylene, polyamines, and nicotianamine (NA) synthesis. MTA nucleosidase is encoded by two genes in Arabidopsis thaliana, MTN1 and MTN2. Analysis of T-DNA insertion mutants and of wt revealed that MTN1 provides approximately 80% of the total MTN activity. Severe knock down of MTN enzyme activity in the mtn1-1 and mtn1-2 allelic lines resulted in accumulation of SAM/dSAM (decarboxylated SAM) and of MTA in seedlings grown on MTA as sulfur source. While ethylene and NA synthesis were not altered in mtn1-1 and mtn1-2 seedlings grown on MTA, putrescine and spermine were elevated. By contrast, mtn2-1 and mtn2-2 seedlings with near wt enzyme activity had wt levels of SAM/dSAM, MTA, and polyamines. In addition to the metabolic phenotypes, mtn1-1 and mtn1-2 seedlings were growth retarded, while seedlings of wt, mtn2-1, and mtn2-2 showed normal growth on 500 mu m MTA. The double knock down mutant mtn1-1/mtn2-1 was sterile. In conclusion, the data presented identify MTA as a crucial metabolite that acts as a regulatory link between the Yang cycle and polyamine biosynthesis and identifies MTA nucleosidase as a crucial enzyme of the Yang cycle

Recycling of Methylthioadenosine Is Essential for Normal Vascular Development and Reproduction in Arabidopsis1[W][OA]

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

Targeted systems biology profiling of tomato fruit reveals coordination of the Yang cycle and a distinct regulation of ethylene biosynthesis during post-climacteric ripening

The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the postclimacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid oxidase. At the same time, 1-aminocyclopropane-1-carboxylic acid synthase activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during postclimacteric ripening. Postclimacteric red tomatoes on the plant showed only a moderate regulation of 1-aminocyclopropane-1-carboxylic acid synthase and Yang cycle genes compared with the regulation in detached fruit. Treatment of red fruit with 1-methylcyclopropane and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene biosynthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal.status: publishe

Targeted systems biology profiling of tomato fruit reveals coordination of the Yang cycle and a distinct regulation of ethylene biosynthesis during postclimacteric ripening

The concept of system 1 and system 2 ethylene biosynthesis during climacteric fruit ripening was initially described four decades ago. Although much is known about fruit development and climacteric ripening, little information is available about how ethylene biosynthesis is regulated during the postclimacteric phase. A targeted systems biology approach revealed a novel regulatory mechanism of ethylene biosynthesis of tomato (Solanum lycopersicum) when fruit have reached their maximal ethylene production level and which is characterized by a decline in ethylene biosynthesis. Ethylene production is shut down at the level of 1-aminocyclopropane-1-carboxylic acid oxidase. At the same time, 1-aminocyclopropane-1-carboxylic acid synthase activity increases. Analysis of the Yang cycle showed that the Yang cycle genes are regulated in a coordinated way and are highly expressed during postclimacteric ripening. Postclimacteric red tomatoes on the plant showed only a moderate regulation of 1-aminocyclopropane-1-carboxylic acid synthase and Yang cycle genes compared with the regulation in detached fruit. Treatment of red fruit with 1-methylcyclopropane and ethephon revealed that the shut-down mechanism in ethylene biosynthesis is developmentally programmed and only moderately ethylene sensitive. We propose that the termination of autocatalytic ethylene biosynthesis of system 2 in ripe fruit delays senescence and preserves the fruit until seed dispersal