Cystathionine γ-synthase expression in seeds alters metabolic and DNA methylation profiles in Arabidopsis.

Arabidopsis (Arabidopsis thaliana) seeds expressing the feedback-insensitive form of cystathionine γ-synthase (AtD-CGS), the key gene of methionine (Met) synthesis, under the control of a seed-specific phaseolin promoter (SSE plants) show a significant increase in Met content. This elevation is accompanied by increased levels of other amino acids (AAs), sugars, total protein, and starch, which are important from a nutritional aspect. Here, we investigated the mechanism behind this phenomenon. Gas chromatography-mass spectrometry (GC-MS) analysis of SSE leaves, siliques, and seeds collected at 3 different developmental stages showed high levels of Met, AAs, and sugars compared to the control plants. A feeding experiment with isotope-labeled AAs showed an increased flux of AAs from nonseed tissues toward the developing seeds of SSE. Transcriptome analysis of leaves and seeds displayed changes in the status of methylation-related genes in SSE plants that were further validated by methylation-sensitive enzymes and colorimetric assay. These results suggest that SSE leaves have higher DNA methylation rates than control plants. This occurrence apparently led to accelerated senescence, together with enhanced monomer synthesis, which further resulted in increased transport of monomers from the leaves toward the seeds. The developing seeds of SSE plants, however, show reduced Met levels and methylation rates. The results provide insights into the role of Met in DNA methylation and gene expression and how Met affects the metabolic profile of the plant.

Conversion of methionine biosynthesis in Escherichia coli from trans- to direct-sulfurylation enhances extracellular methionine levels.

Methionine is an essential amino acid in mammals and a precursor for vital metabolites required for the survival of all organisms. Consequently, its inclusion is required in diverse applications, such as food, feed, and pharmaceuticals. Although amino acids and other metabolites are commonly produced through microbial fermentation, high-yield biosynthesis of L-methionine remains a significant challenge due to the strict cellular regulation of the biosynthesis pathway. As a result, methionine is produced primarily synthetically, resulting in a racemic mixture of D,L-methionine. This study explores methionine bio-production in E. coli by replacing its native trans-sulfurylation pathway with the more common direct-sulfurylation pathway used by other bacteria. To this end, we generated a methionine auxotroph E. coli strain (MG1655) by simultaneously deleting metA and metB genes and complementing them with metX and metY from different bacteria. Complementation of the genetically modified E. coli with metX/metY from Cyclobacterium marinum or Deinococcus geothermalis, together with the deletion of the global repressor metJ and overexpression of the transporter yjeH, resulted in a substantial increase of up to 126 and 160-fold methionine relative to the wild-type strain, respectively, and accumulation of up to 700 mg/L using minimal MOPS medium and 2 ml culture. Our findings provide a method to study methionine biosynthesis and a chassis for enhancing L-methionine production by fermentation.

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway.

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5' phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using 34 SO42- also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non-stress and oxidative stress conditions. However, when germinating on sulfur-deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.

Discovery and characterization of small molecule inhibitors of cystathionine gamma-synthase with in planta activity.

The synthesis of essential amino acids in plants is pivotal for their viability and growth, and these cellular pathways are therefore targeted for the discovery of new molecules for weed control. Herein, we describe the discovery and design of small molecule inhibitors of cystathionine gamma-synthase, a key enzyme in the biosynthesis of methionine. Based on in silico screening and filtering of a large molecular database followed by the in vitro selection of molecules, we identified small molecules capable of binding the target enzyme. Molecular modelling of the interaction and direct biophysical binding enabled us to explore a focussed chemical expansion set of molecules characterized by an active phenyl-benzamide chemical group. These molecules are bio-active and efficiently inhibit the viability of BY-2 tobacco cells and seedlings growth of Arabidopsis thaliana on agar plates.

Elucidating the role of shikimate dehydrogenase in controlling the production of anthocyanins and hydrolysable tannins in the outer peels of pomegranate.

BACKGROUND: The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols that have health beneficial properties: anthocyanins (ATs, which also affect peel color); and hydrolysable tannins (HTs). Their biosynthesis intersects at 3-dehydroshikimate (3-DHS) in the shikimate pathway by the activity of shikimate dehydrogenase (SDH), which converts 3-DHS to shikimate (providing the precursor for AT biosynthesis) or to gallic acid (the precursor for HTs biosynthesis) using NADPH or NADP+ as a cofactor. The aim of this study is to gain more knowledge about the factors that regulate the levels of HTs and ATs, and the role of SDH. RESULTS: The results have shown that the levels of ATs and HTs are negatively correlated in the outer fruit peels of 33 pomegranate accessions, in the outer peels of two fruits exposed to sunlight, and in those covered by paper bags. When calli obtained from the outer fruit peel were subjected to light/dark treatment and osmotic stresses (imposed by different sucrose concentrations), it was shown that light with high sucrose promotes the synthesis of ATs, while dark at the same sucrose concentration promotes the synthesis of HTs. To verify the role of SDH, six PgSDHs (PgSDH1, PgSDH3-1,2, PgSDH3a-1,2 and PgSDH4) were identified in pomegranate. The expression of PgSDH1, which presumably contributes to shikimate biosynthesis, was relatively constant at different sucrose concentrations. However, the transcript levels of PgSDH3s and PgSDH4 increased with the accumulation of gallic acid and HTs under osmotic stress, which apparently accumulates to protect the cells from the stress. CONCLUSIONS: The results strongly suggest that the biosynthesis of HTs and ATs competes for the same substrate, 3-DHS, and that SDH activity is regulated not only by the NADPH/NADP+ ratio, but also by the expression of the PgSDHs. Since the outer peel affects the customer's decision regarding fruit consumption, such knowledge could be utilized for the development of new genetic markers for breeding pomegranates having higher levels of both ATs and HTs.

Revisiting the attempts to fortify methionine content in plant seeds.

The sulfur-containing amino acid methionine belongs to the group of essential amino acids, meaning that humans and animals must consume it in their diets. However, plant seeds have low levels of methionine, limiting their nutritional potential. For this reason, efforts have been made over the years to increase methionine levels in seeds. Here, we summarize these efforts and focus particularly on those utilizing diverse genetic and molecular tools. Four main approaches are described: (i) expression of methionine-rich storage proteins in a seed-specific manner to incorporate more soluble methionine into the protein fraction; (ii) reduction of methionine-poor storage proteins inside the seeds to reinforce the accumulation of methionine-rich proteins; (iii) silencing methionine catabolic enzymes; and (iv) up-regulation of key biosynthetic enzymes participating in methionine synthesis. We focus on the biosynthetic genes that operate de novo in seeds and that belong to the sulfur assimilation and aspartate family pathways, as well as genes from the methionine-specific pathway. We also include those enzymes that operate in non-seed tissues that contribute to the accumulation of methionine in seeds, such as S-methylmethionine enzymes. Finally, we discuss the biotechnological potential of these manipulations to increase methionine content in plant seeds and their effect on seed germination.

Repression of CYSTATHIONINE γ-SYNTHASE in Seeds Recruits the S-Methylmethionine Cycle.

S-Methylmethionine (SMM) was suggested previously to participate in the metabolism of methionine (Met) in seeds. To further reveal its roles, we had previously produced transgenic Arabidopsis (Arabidopsis thaliana) RNA interference (RNAi) seeds with lower transcript expression of CYSTATHIONINE γ-SYNTHASE (AtCGS), Met's main regulatory enzyme. Unexpectedly, these seeds accumulated significantly higher levels of Met compared with control seeds through an as yet unknown mechanism. Here, transcript and metabolic analyses coupled with isotope-labeled [13C]SMM and [13C]Met feeding experiments enabled us to reveal that SMM that was synthesized in rosette leaves of RNAi plants significantly contributed to the accumulation of Met in their seeds at late stages of development. Seed-specific repression of AtCGS in RNAi seeds triggered the induction of genes operating in the SMM cycle of rosette leaves, leading to elevated transport of SMM toward the seeds, where higher reconversion rates of SMM to Met were detected. The metabolic rearrangements in RNAi seeds resulted in an altered sulfur-associated metabolism, such as lower amounts of Cys and glutathione, as well as a differential composition of glucosinolates. Together, the data propose a novel cross talk existing between seeds and rosette leaves along with mutual effects between the Asp family and SMM pathways operating in these tissues. They also shed light on the effects of higher Met levels on seed physiology and behavior.

Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.

The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

Transgenic tobacco plants having a higher level of methionine are more sensitive to oxidative stress.

Methionine is an essential amino acid the low level of which limits the nutritional quality of plants. We formerly produced transgenic tobacco (Nicotiana tabacum) plants overexpressing CYSTATHIONE γ-SYNTHASE (CGS) (FA plants), methionine's main regulatory enzyme. These plants accumulate significantly higher levels of methionine compared with wild-type (WT) plants. The aim of this study was to gain more knowledge about the effect of higher methionine content on the metabolic profile of vegetative tissue and on the morphological and physiological phenotypes. FA plants exhibit slightly reduced growth, and metabolic profiling analysis shows that they have higher contents of stress-related metabolites. Despite this, FA plants were more sensitive to short- and long-term oxidative stresses. In addition, compared with WT plants and transgenic plants expressing an empty vector, the primary metabolic profile of FA was altered less during oxidative stress. Based on morphological and metabolic phenotypes, we strongly proposed that FA plants having higher levels of methionine suffer from stress under non-stress conditions. This might be one of the reasons for their lesser ability to cope with oxidative stress when it appeared. The observation that their metabolic profiling is much less responsive to stress compared with control plants indicates that the delta changes in metabolite contents between non-stress and stress conditions is important for enabling the plants to cope with stress conditions.

The relative contribution of genes operating in the S-methylmethionine cycle to methionine metabolism in Arabidopsis seeds.

KEY MESSAGE: Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine synthesis in seeds. In addition, mutual effects exist between the S -methylmethionine cycle and the aspartate family pathway in seeds. Methionine, a sulfur-containing amino acid, is a key metabolite in plant cells. The previous lines of evidence proposed that the S-methylmethionine (SMM) cycle contributes to methionine synthesis in seeds where methionine that is produced in non-seed tissues is converted to SMM and then transported via the phloem into the seeds. However, the relative regulatory roles of the S-methyltransferases operating within this cycle in seeds are yet to be fully understood. In the current study, we generated transgenic Arabidopsis seeds with altered expression of three HOMOCYSTEINE S-METHYLTRANSFERASEs (HMTs) and METHIONINE S-METHYLTRANSFERASE (MMT), and profiled them for transcript and metabolic changes. The results revealed that AtHMT1 and AtHMT3, but not AtHMT2 and AtMMT, are the predominant enzymes operating in seeds as altered expression of these two genes affected the levels of methionine and SMM in transgenic seeds. Their manipulations resulted in adapted expression level of genes participating in methionine synthesis through the SMM and aspartate family pathways. Taken together, our findings provide new insights into the regulatory roles of the SMM cycle and the mutual effects existing between the two methionine biosynthesis pathways, highlighting the complexity of the metabolism of methionine and SMM in seeds.

ASCORBATE PEROXIDASE6 protects Arabidopsis desiccating and germinating seeds from stress and mediates cross talk between reactive oxygen species, abscisic acid, and auxin.

A seed's ability to properly germinate largely depends on its oxidative poise. The level of reactive oxygen species (ROS) in Arabidopsis (Arabidopsis thaliana) is controlled by a large gene network, which includes the gene coding for the hydrogen peroxide-scavenging enzyme, cytosolic ASCORBATE PEROXIDASE6 (APX6), yet its specific function has remained unknown. In this study, we show that seeds lacking APX6 accumulate higher levels of ROS, exhibit increased oxidative damage, and display reduced germination on soil under control conditions and that these effects are further exacerbated under osmotic, salt, or heat stress. In addition, ripening APX6-deficient seeds exposed to heat stress displayed reduced germination vigor. This, together with the increased abundance of APX6 during late stages of maturation, indicates that APX6 activity is critical for the maturation-drying phase. Metabolic profiling revealed an altered activity of the tricarboxylic acid cycle, changes in amino acid levels, and elevated metabolism of abscisic acid (ABA) and auxin in drying apx6 mutant seeds. Further germination assays showed an impaired response of the apx6 mutants to ABA and to indole-3-acetic acid. Relative suppression of abscisic acid insensitive3 (ABI3) and ABI5 expression, two of the major ABA signaling downstream components controlling dormancy, suggested that an alternative signaling route inhibiting germination was activated. Thus, our study uncovered a new role for APX6, in protecting mature desiccating and germinating seeds from excessive oxidative damage, and suggested that APX6 modulate the ROS signal cross talk with hormone signals to properly execute the germination program in Arabidopsis.

Brassinosteroid perception in the epidermis controls root meristem size.

Multiple small molecule hormones contribute to growth promotion or restriction in plants. Brassinosteroids (BRs), acting specifically in the epidermis, can both drive and restrict shoot growth. However, our knowledge of how BRs affect meristem size is scant. Here, we study the root meristem and show that BRs are required to maintain normal cell cycle activity and cell expansion. These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. In addition, BR activity in the meristem is not accompanied by changes in the expression level of the auxin efflux carriers PIN1, PIN3 and PIN7, which are known to control the extent of mitotic activity and differentiation. We further demonstrate that BR signaling in the root epidermis and not in the inner endodermis, quiescent center (QC) cells or stele cell files is sufficient to control root meristem size. Interestingly, expression of the QC and the stele-enriched MADS-BOX gene AGL42 can be modulated by BRI1 activity solely in the epidermis. The signal from the epidermis is probably transmitted by a different component than BES1 and BZR1 transcription factors, as their direct targets, such as DWF4 and BRox2, are regulated in the same cells that express BRI1. Taken together, our study provides novel insights into the role of BRs in controlling meristem size.

New Analytical Approach to Quinolizidine Alkaloids and Their Assumed Biosynthesis Pathways in Lupin Seeds.

Alkaloids play an essential role in protecting plants against herbivores. Humans can also benefit from the pharmacological effects of these compounds. Plants produce an immense variety of structurally different alkaloids, including quinolizidine alkaloids, a group of bi-, tri-, and tetracyclic compounds produced by Lupinus species. Various lupin species produce different alkaloid profiles. To study the composition of quinolizidine alkaloids in lupin seeds, we collected 31 populations of two wild species native to Israel, L. pilosus and L. palaestinus, and analyzed their quinolizidine alkaloid contents. Our goal was to study the alkaloid profiles of these two wild species to better understand the challenges and prospective uses of wild lupins. We compared their profiles with those of other commercial and wild lupin species. To this end, a straightforward method for extracting alkaloids from seeds and determining the quinolizidine alkaloid profile by LC-MS/MS was developed and validated in-house. For the quantification of quinolizidine alkaloids, 15 analytical reference standards were used. We used GC-MS to verify and cross-reference the identity of certain alkaloids for which no analytical standards were available. The results enabled further exploration of quinolizidine alkaloid biosynthesis. We reviewed and re-analyzed the suggested quinolizidine alkaloid biosynthesis pathway, including the relationship between the amino acid precursor l-lysine and the different quinolizidine alkaloids occurring in seeds of lupin species. Revealing alkaloid compositions and highlighting some aspects of their formation pathway are important steps in evaluating the use of wild lupins as a novel legume crop.

Tobacco seeds expressing feedback-insensitive cystathionine gamma-synthase exhibit elevated content of methionine and altered primary metabolic profile.

BACKGROUND: The essential sulfur-containing amino acid methionine plays a vital role in plant metabolism and human nutrition. In this study, we aimed to elucidate the regulatory role of the first committed enzyme in the methionine biosynthesis pathway, cystathionine γ-synthase (CGS), on methionine accumulation in tobacco seeds. We also studied the effect of this manipulation on the seed's metabolism. RESULTS: Two forms of Arabidopsis CGS (AtCGS) were expressed under the control of the seeds-specific promoter of legumin B4: feedback-sensitive F-AtCGS (LF seeds), and feedback-insensitive T-AtCGS (LT seeds). Unexpectedly, the soluble content of methionine was reduced significantly in both sets of transgenic seeds. Amino acids analysis and feeding experiments indicated that although the level of methionine was reduced, the flux through its synthesis had increased. As a result, the level of protein-incorporated methionine had increased significantly in LT seeds by up to 60%, but this was not observed in LF seeds, whose methionine content is tightly regulated. This increase was accompanied by a higher content of other protein-incorporated amino acids, which led to 27% protein content in the seeds although this was statistically insignificantly. In addition, the levels of reducing sugars (representing starch) were slightly but significantly reduced, while that of oil was insignificantly reduced. To assess the impact of the high expression level of T-AtCGS in seeds on other primary metabolites, metabolic profiling using GC-MS was performed. This revealed significant alterations to the primary seed metabolism manifested by a significant increase in eight annotated metabolites (mostly sugars and their oxidized derivatives), while the levels of 12 other metabolites were reduced significantly in LT compared to wild-type seeds. CONCLUSION: Expression of T-AtCGS leads to an increase in the level of total Met, higher contents of total amino acids, and significant changes in the levels of 20 annotated metabolites. The high level of oxidized metabolites, the two stress-associated amino acids, proline and serine, and low level of glutathione suggest oxidative stress that occurs during LT seed development. This study provides information on the metabolic consequence of increased CGS activity in seeds and how it affects the seed's nutritional quality.

Soybean seeds expressing feedback-insensitive cystathionine γ-synthase exhibit a higher content of methionine.

Soybean seeds provide an excellent source of protein for human and livestock nutrition. However, their nutritional quality is hampered by a low concentration of the essential sulfur amino acid, methionine (Met). In order to study factors that regulate Met synthesis in soybean seeds, this study used the Met-insensitive form of Arabidopsis cystathionine γ-synthase (AtD-CGS), which is the first committed enzyme of Met biosynthesis. This gene was expressed under the control of a seed-specific promoter, legumin B4, and used to transform the soybean cultivar Zigongdongdou (ZD). In three transgenic lines that exhibited the highest expression level of AtD-CGS, the level of soluble Met increased significantly in developing green seeds (3.8-7-fold). These seeds also showed high levels of other amino acids. This phenomenon was more prominent in two transgenic lines, ZD24 and ZD91. The total Met content, which including Met incorporated into proteins, significantly increased in the mature dry seeds of these two transgenic lines by 1.8- and 2.3-fold, respectively. This elevation was accompanied by a higher content of other protein-incorporated amino acids, which led to significantly higher total protein content in the seeds of these two lines. However, in a third transgenic line, ZD01, the level of total Met and the level of other amino acids did not increase significantly in the mature dry seeds. This line also showed no significant change in protein levels. This suggests a positive connection between high Met content and the synthesis of other amino acids that enable the synthesis of more seed proteins.

Light and sucrose up-regulate the expression level of Arabidopsis cystathionine γ-synthase, the key enzyme of methionine biosynthesis pathway.

The sulfur-containing essential amino acid methionine controls the level of important metabolites and processes in plants. In addition, methionine levels limit the nutritional quality of many crop plants. The level of methionine is regulated mainly by cystathionine γ-synthase (CGS), the first enzyme committed to its biosynthesis. Within our efforts to reveal factors that regulate CGS and methionine content in plants, we have analyzed how light regulates the transcript and protein level of Arabidopsis CGS (AtCGS). The expression of AtCGS is up-regulated in the light and reduced in the dark, independent of the diurnal cycle. Using tobacco plants overexpressing AtCGS, we have found that the light sensitive motives of the AtCGS gene are found within the coding sequence of AtCGS and not in its promoter, terminator or the untranslated regions of the gene. Sucrose can partially mimic the effect of light in dark grown plants while the addition of nitrogen and sulfur sources does not have any effect. The kinetics of the changes in the expression level of AtCGS suggest that its level can be maintained during extended darkness, or even when the sucrose content is reduced, such as during abiotic stresses. The up-regulation of AtCGS by light is in agreement with previous studies showing that other enzymes regulating the level of the carbon/amino skeleton and the sulfur group of Met, are up-regulated by light. The results indicate that light and dark participate in the regulation of the carbon/amino skeleton flux in the synthesis of amino acids of the aspartate family.

Elucidating the importance of the catabolic enzyme, methionine-gamma-lyase, in stresses during Arabidopsis seed development and germination.

The sulfur-containing essential amino acid, methionine, is a key metabolite in plant cells since it is used as a precursor for the synthesis of vital metabolites. The transcript level of methionine's catabolic enzyme, methionine γ-lyase (MGL), accumulates in the seeds to a high level compared to other organs. The aim of this study was to reveal the role of MGL during seed development and germination. Using [13C]S-methylmethionine (SMM), the mobile form of methionine that is used to feed flower stalks of wild-type (WT) plants, revealed that the contents of [13C]methionine in seeds were significantly reduced when the plants underwent heat and osmotic stresses. Moreover, the levels of [13C]isoleucine, a product of MGL, significantly increased. Also, using the MGL promoter and gene fused to the GUS reporter gene, it was demonstrated that the heat stress significantly increased the protein level in the seeds. Therefore, we can conclude that MGL became active under stresses apparently to produce isoleucine, which is used as an osmoprotectant and an energy source. Transgenic Arabidopsis thaliana RNAi seeds with targeted repression of AtMGL during the late developmental stages of seeds show that the seeds did not accumulate methionine when they were grown under standard growth conditions, unlike the mgl-2, a knockout mutant, which showed a three-fold higher level of methionine. Also, when the RNAi plants developed under mid-heat stress, the level of methionine significantly increased while the content of isoleucine decreased compared to the control seeds, which strengthened the assumption that MGL is active under stress. The germination efficiency of the RNAi lines and mgl seeds were similar to their controls. However, the seeds that developed during heat or salt stress showed significantly lower germination efficiency compared to the control seeds. This implies that MGL is important to maintain the ability of the seeds to germinate. The RNAi lines and mgl seeds that developed under regular conditions, but germinated during salt or osmotic stress, exhibited a lower germination rate, suggesting an essential role of MGL also during this process. The results of this study show the important role of AtMGL in seeds under stresses.

The Effect of 10 Crop Plants That Served as Hosts on the Primary Metabolic Profile of the Parasitic Plant Phelipanche aegyptiaca.

Phelipanche aegyptiaca Pers. is a holoparasitic plant that parasitizes various types of host plants. Its penetration into host roots causes a massive reduction in the yield of many crop plants worldwide. The nature of the compounds taken by the parasite from its host is still under debate in the scientific literature. To gain more knowledge about the effect of the hosts on the parasite's primary metabolic profile, GC-MS analyses were conducted on the parasites that developed on 10 hosts from four plant families. There are three hosts from each family: Brassicaceae, Apiaceae and Solanaceae and one host from Fabaceae. The results showed significant differences in the metabolic profiles of P. aegyptiaca collected from the different hosts, indicating that the parasites rely strongly on the host's metabolites. Generally, we found that the parasites that developed on Brassicaceae and Fabaceae accumulated more amino acids than those developed on Apiaceae and Solanaceae that accumulated more sugars and organic acids. The contents of amino acids correlated positively with the total soluble proteins. However, the aromatic amino acid, tyrosine, correlated negatively with the accumulation of the total phenolic compounds. This study contributes to our knowledge of the metabolic relationship between host and parasite.

The Level of Methionine Residues in Storage Proteins Is the Main Limiting Factor of Protein-Bound-Methionine Accumulation in Arabidopsis Seeds.

The low level of methionine, an essential sulfur-containing amino acid, limits the nutritional quality of seeds. Two main factors can control the level of protein-bound methionine: the level of free methionine that limits protein accumulation and the methionine residues inside the storage proteins. To reveal the main limiting factor, we generated transgenic Arabidopsis thaliana seed-specific plants expressing the methionine-rich sunflower seed storage (SSA) protein (A1/A2). The contents of protein-bound methionine in the water-soluble protein fraction that includes the SSA in A1/A2 were 5.3- and 10.5-fold, respectively, compared to control, an empty vector (EV). This suggests that free methionine can support this accumulation. To elucidate if the level of free methionine could be increased further in the protein-bound methionine, these lines were crossed with previously characterized plants having higher levels of free methionine in seeds (called SSE). The progenies of the crosses (A1S, A2S) exhibited the highest level of protein-bound methionine, but this level did not differ significantly from A2, suggesting that all the methionine residues of A2 were filled with methionine. It also suggests that the content of methionine residues in the storage proteins is the main limiting factor. The results also proposed that the storage proteins can change their content in response to high levels of free methionine or SSA. This was assumed since the water-soluble protein fraction was highest in A1S/A2S as well as in SSE compared to EV and A1/A2. By using these seeds, we also aimed at gaining more knowledge about the link between high free methionine and the levels of metabolites that usually accumulate during abiotic stresses. This putative connection was derived from a previous analysis of SSE. The results of metabolic profiling showed that the levels of 29 and 20 out of the 56 metabolites were significantly higher in SSE and A1, respectively, that had higher level of free methionine, compared A1S/A2S, which had lower free methionine levels. This suggests a strong link between high free methionine and the accumulation of stress-associated metabolites.

Enhanced Production of Aromatic Amino Acids in Tobacco Plants Leads to Increased Phenylpropanoid Metabolites and Tolerance to Stresses.

Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precursors for a wide range of secondary metabolites that are important for plant defense. The goals of the current study were to test the effect of increased AAAs on primary and secondary metabolic profiles and to reveal whether these plants are more tolerant to abiotic stresses (oxidative, drought and salt) and to Phelipanche egyptiaca (Egyptian broomrape), an obligate parasitic plant. To this end, tobacco (Nicotiana tabacum) plants were transformed with a bacterial gene (AroG) encode to feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. Two sets of transgenic plants were obtained: the first had low expression of the AroG protein, a normal phenotype and minor metabolic changes; the second had high accumulation of the AroG protein with normal, or deleterious morphological changes having a dramatic shift in plant metabolism. Metabolic profiling analysis revealed that the leaves of the transgenic plants had increased levels of phenylalanine (up to 43-fold), tyrosine (up to 24-fold) and tryptophan (up to 10-fold) compared to control plants having an empty vector (EV) and wild type (WT) plants. The significant increase in phenylalanine was accompanied by higher levels of metabolites that belong to the phenylpropanoid pathway. AroG plants showed improved tolerance to salt stress but not to oxidative or drought stress. The most significant improved tolerance was to P. aegyptiaca. Unlike WT/EV plants that were heavily infected by the parasite, the transgenic AroG plants strongly inhibited P. aegyptiaca development, and only a few stems of the parasite appeared above the soil. This delayed development of P. aegyptiaca could be the result of higher accumulation of several phenylpropanoids in the transgenic AroG plants and in P. aegyptiaca, that apparently affected its growth. These findings indicate that high levels of AAAs and their related metabolites have the potential of controlling the development of parasitic plants.