The objective of this research is to study the nature of the competition for cysteine (Cys), the ... more The objective of this research is to study the nature of the competition for cysteine (Cys), the first organic sulfur-containing compound, between its two main metabolites, glutathione (GSH) and methionine (Met). GSH plays a central role in protecting plants during various stresses, while Met, an essential amino acid, regulates essential processes and metabolites in plant cells through its metabolite S-adenosyl-Met. Our results, which are based on flux analysis and measurements of Met- metabolites, show that the flux towards Met synthesis is high during non-stress conditions, however the flux is significantly reduced under stress conditions, when there is high synthesis of GSH. Under oxidative stress the expression level of the regulatory enzyme of Met synthesis, cystathionine g-synthase (CGS) was reduced. By using three different systems, we have found that that GSH down regulates the expression level of CGS, thus reducing Met synthesis. We have found that this regulation occurs at...
Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown t... more Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown that both wool quality and milk production can be limited by the post-ruminal supply of sulfur-containing amino acids. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little success. With the objective to increase the S-amino acid content of forage legumes, the goal of this project was to co- express the methionine rich zein genes from corn along with a gene for a key enzyme in methionine biosynthesis, aspartate kinase(AK). The zeins are seed storage proteins from corn and are groupec into four distinct classes based on their amino acid sequence homologies. The b-zein (15kd) and the 6zein (10kD and 18kD) have proportionately high levels of methionine (10%, 22% and 28%, respectively). Initial studies from our lab had shown that while the 15kD zein accumulated to high levels in vegetative tissues of trans...
Additional file 3: Figure S1. Phenotype of calli formed from the outer peels of accession UG28 gr... more Additional file 3: Figure S1. Phenotype of calli formed from the outer peels of accession UG28 grown on MS plates supplemented with different concentrations of sucrose under light (upper panel) and dark (lower panel) conditions. Figure S2. Fresh weight (gr) of pomegranate peel calli grown for 75 days (collected at five time points) on MS plates supplemented with different sucrose concentrations (1, 2.5, 5, 7.5%) under light and dark conditions. The media were replaced every 15 days. The values presented are the average ± SD of four biological replicates. Figure S3. The fresh weight, dry weight and water content in calli formed from the outer peels of accession UG28 grown on MS plates supplemented with different concentrations of sucrose. The plates were placed in the light (upper panel) and the dark (lower panel), and measurements were taken after 75 days. The data presented represent the mean ± SD of four samples taken from four biological replicates. Statistically significant chan...
Methionine is a nutritionally essential sulfur-containing amino acid whose low level in plants di... more Methionine is a nutritionally essential sulfur-containing amino acid whose low level in plants diminishes their value as a source of dietary protein for humans and animals. Methionine is also a fundamental metabolite in plant cells since, through its first metabolite Sadenosylmethionine (SAM), it controls the levels of several key metabolites, such as ethylene, polyamines and biotin. SAM is also the primary methyl group donor that regulates different processes in plants. Despite its nutritional and regulatory significance, the factors regulating its synthesis and catabolism in plants are not fully known. In recent years, genetic molecular biology techniques have been used to increase and decrease the expression levels of several genes encoded to enzymes in the methionine metabolism in order to gain more knowledge about its role in plant metabolism, as well as to increase methionine level and thus improve the nutritional quality of plants. In this review, recent progress made in the ...
Proceedings of the International Plant Sulfur Workshop, 2017
Plant seeds accumulate low contents of methionine in their seeds, limiting their nutritional valu... more Plant seeds accumulate low contents of methionine in their seeds, limiting their nutritional values as a source of proteins. Previous conventional and molecular attempts to increase methionine levels in seeds by classical breeding, selection of mutants or creating ‘additional protein sinks’ for soluble methionine by expressing methionine-rich seed-storage proteins, have yielded only limited success. Here, we summarize our efforts to increase methionine contents in transgenic Arabidopsis, soybean and tobacco seeds by seed-specific expression of feedback-insensitive mutated forms of the Arabidopsis thaliana cystathionine γ-synthase (AtCGS), methionine main regulatory enzyme. Each of these species represents different plant families (Solanaceae, Brassicaceae and Fabaceae). The effects of the manipulations on the levels of soluble and total methionine and on the accumulation of storage compounds in these transgenic seeds are discussed.
Cuscuta campestris (dodder) is a stem holoparasitic plant without leaves or roots that parasitize... more Cuscuta campestris (dodder) is a stem holoparasitic plant without leaves or roots that parasitizes various types of host plants and causes damage to certain crops worldwide. This study aimed at gaining more knowledge about the effect of the hosts on the parasite’s levels of primary metabolites. To this end, metabolic profiling analyses were performed on the parasite’s three main organs, haustoria, stem and flowers, which developed on three hosts, Heliotropium hirsutissimum, Polygonum equisetiforme and Amaranthus viridis. The results showed significant differences in the metabolic profiles of C. campestris that developed on the different hosts, suggesting that the parasites rely highly on the host’s metabolites. However, changes in the metabolites’ contents between the organs that developed on the same host suggest that the parasite can also self-regulate its metabolites. Flowers, for example, have significantly higher levels of most of the amino acids and sugar acids, while haustori...
Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precurso... more 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. Meta...
Background The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols ... more 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 (impo...
Plant Cell, Tissue and Organ Culture (PCTOC), 2019
Abstract The pomegranate (Punica granatum L.) fruit harbors remarkable health-beneficial properti... more Abstract The pomegranate (Punica granatum L.) fruit harbors remarkable health-beneficial properties. Most of its healthy secondary metabolites are located in their peels. Therefore, pomegranate peels (PPs) can be used to produce high-value healthy compounds. PP cell cultures may also be an attractive alternative source since it can be established throughout the year regardless of seasonal effects. The aim of this study was to establish a novel PP cell culture for future research and biotechnological applications. We first established procedures to sterilize and control oxidative browning of the peel explants. We found that surface sterilizing with 3% sodium hypochlorite for 15 min, frequent sub-culturing of the explants on media containing polyvinylpyrrolidone plus silver nitrate was the optimal treatment for oxidative browning control and callus initiation. We also found that 6-benzylaminopurine (BAP) induced callus initiation, whereas 3-indoleacetic acid (IAA) repressed it. The callus daily growth rate was maintained at 1% regardless of the various growth media examined. Moreover, our results show that although there was a minor effect of the growth media on polyphenol accumulation and antioxidant activity, the exposure to light and high sucrose levels led to higher levels (up to 205 times than the control) of secondary metabolites such as anthocyanins, ellagic acid, punicalagin and gallic acid. In summary, this work laid the foundations for further research that will be needed to study and commercialize PP cell cultures.Key messageIn this present study, we addresses the establishment of pomegranate peel cell cultures for future research and biotechnological applications, such as production of health-beneficial secondary metabolites.
Galloylated plant specialized metabolites play important roles in plant-environment interactions ... more Galloylated plant specialized metabolites play important roles in plant-environment interactions as well as the promotion of human and animal health. The galloylation reactions are mediated by the formation of galloylglucose esters from gallic acid and UDP-glucose, catalyzed by the plant UGT84 family glycosyltransferases. To explore and exploit the structural determinants of UGT84 activities, we performed homology modeling and substrate docking of PgUGT84A23, a galloylglucose ester-forming family 84 UGT, as well as sequence comparisons of PgUGT84A23 with other functionally characterized plant UGTs. By employing site-directed mutagenesis of candidate amino acids, enzyme assays with analogous substrates, and kinetic analysis, key amino acid sites for PgUGT84A23 substrate binding and reactivity were elucidated. The galloylglucose ester-forming UGT84s have not been shown to glycosylate genistein (an isoflavonoid) in vivo. Unexpectedly, amino acids highly conserved among UGT84s were iden...
Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine... more 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 a...
It is currently held that glyphosate efficiently controls the obligate holoparasite Phelipanche a... more It is currently held that glyphosate efficiently controls the obligate holoparasite Phelipanche aegyptiaca (Egyptian broomrape) by inhibiting its endogenous shikimate pathway, thereby causing a deficiency in aromatic amino acids (AAA). While there is no argument regarding the shikimate pathway being the primary site of the herbicide's action, the fact that the parasite receives a constant supply of nutrients, including proteins and amino acids, from the host does not fit with an AAA deficiency. This apparent contradiction implies that glyphosate mechanism of action in P. aegyptiaca is probably more complex and does not end with the inhibition of the AAA biosynthetic pathway alone. A possible explanation would lie in a limitation of the translocation of solutes from the host as a secondary effect. We examined the following hypotheses: (a) glyphosate does not affects P. aegyptiaca during its independent phase and (b) glyphosate has a secondary effect on the ability of P. aegyptiac...
The objective of this research is to study the nature of the competition for cysteine (Cys), the ... more The objective of this research is to study the nature of the competition for cysteine (Cys), the first organic sulfur-containing compound, between its two main metabolites, glutathione (GSH) and methionine (Met). GSH plays a central role in protecting plants during various stresses, while Met, an essential amino acid, regulates essential processes and metabolites in plant cells through its metabolite S-adenosyl-Met. Our results, which are based on flux analysis and measurements of Met- metabolites, show that the flux towards Met synthesis is high during non-stress conditions, however the flux is significantly reduced under stress conditions, when there is high synthesis of GSH. Under oxidative stress the expression level of the regulatory enzyme of Met synthesis, cystathionine g-synthase (CGS) was reduced. By using three different systems, we have found that that GSH down regulates the expression level of CGS, thus reducing Met synthesis. We have found that this regulation occurs at...
Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown t... more Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown that both wool quality and milk production can be limited by the post-ruminal supply of sulfur-containing amino acids. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little success. With the objective to increase the S-amino acid content of forage legumes, the goal of this project was to co- express the methionine rich zein genes from corn along with a gene for a key enzyme in methionine biosynthesis, aspartate kinase(AK). The zeins are seed storage proteins from corn and are groupec into four distinct classes based on their amino acid sequence homologies. The b-zein (15kd) and the 6zein (10kD and 18kD) have proportionately high levels of methionine (10%, 22% and 28%, respectively). Initial studies from our lab had shown that while the 15kD zein accumulated to high levels in vegetative tissues of trans...
Additional file 3: Figure S1. Phenotype of calli formed from the outer peels of accession UG28 gr... more Additional file 3: Figure S1. Phenotype of calli formed from the outer peels of accession UG28 grown on MS plates supplemented with different concentrations of sucrose under light (upper panel) and dark (lower panel) conditions. Figure S2. Fresh weight (gr) of pomegranate peel calli grown for 75 days (collected at five time points) on MS plates supplemented with different sucrose concentrations (1, 2.5, 5, 7.5%) under light and dark conditions. The media were replaced every 15 days. The values presented are the average ± SD of four biological replicates. Figure S3. The fresh weight, dry weight and water content in calli formed from the outer peels of accession UG28 grown on MS plates supplemented with different concentrations of sucrose. The plates were placed in the light (upper panel) and the dark (lower panel), and measurements were taken after 75 days. The data presented represent the mean ± SD of four samples taken from four biological replicates. Statistically significant chan...
Methionine is a nutritionally essential sulfur-containing amino acid whose low level in plants di... more Methionine is a nutritionally essential sulfur-containing amino acid whose low level in plants diminishes their value as a source of dietary protein for humans and animals. Methionine is also a fundamental metabolite in plant cells since, through its first metabolite Sadenosylmethionine (SAM), it controls the levels of several key metabolites, such as ethylene, polyamines and biotin. SAM is also the primary methyl group donor that regulates different processes in plants. Despite its nutritional and regulatory significance, the factors regulating its synthesis and catabolism in plants are not fully known. In recent years, genetic molecular biology techniques have been used to increase and decrease the expression levels of several genes encoded to enzymes in the methionine metabolism in order to gain more knowledge about its role in plant metabolism, as well as to increase methionine level and thus improve the nutritional quality of plants. In this review, recent progress made in the ...
Proceedings of the International Plant Sulfur Workshop, 2017
Plant seeds accumulate low contents of methionine in their seeds, limiting their nutritional valu... more Plant seeds accumulate low contents of methionine in their seeds, limiting their nutritional values as a source of proteins. Previous conventional and molecular attempts to increase methionine levels in seeds by classical breeding, selection of mutants or creating ‘additional protein sinks’ for soluble methionine by expressing methionine-rich seed-storage proteins, have yielded only limited success. Here, we summarize our efforts to increase methionine contents in transgenic Arabidopsis, soybean and tobacco seeds by seed-specific expression of feedback-insensitive mutated forms of the Arabidopsis thaliana cystathionine γ-synthase (AtCGS), methionine main regulatory enzyme. Each of these species represents different plant families (Solanaceae, Brassicaceae and Fabaceae). The effects of the manipulations on the levels of soluble and total methionine and on the accumulation of storage compounds in these transgenic seeds are discussed.
Cuscuta campestris (dodder) is a stem holoparasitic plant without leaves or roots that parasitize... more Cuscuta campestris (dodder) is a stem holoparasitic plant without leaves or roots that parasitizes various types of host plants and causes damage to certain crops worldwide. This study aimed at gaining more knowledge about the effect of the hosts on the parasite’s levels of primary metabolites. To this end, metabolic profiling analyses were performed on the parasite’s three main organs, haustoria, stem and flowers, which developed on three hosts, Heliotropium hirsutissimum, Polygonum equisetiforme and Amaranthus viridis. The results showed significant differences in the metabolic profiles of C. campestris that developed on the different hosts, suggesting that the parasites rely highly on the host’s metabolites. However, changes in the metabolites’ contents between the organs that developed on the same host suggest that the parasite can also self-regulate its metabolites. Flowers, for example, have significantly higher levels of most of the amino acids and sugar acids, while haustori...
Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precurso... more 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. Meta...
Background The outer peels of pomegranate (Punica granatum L.) possess two groups of polyphenols ... more 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 (impo...
Plant Cell, Tissue and Organ Culture (PCTOC), 2019
Abstract The pomegranate (Punica granatum L.) fruit harbors remarkable health-beneficial properti... more Abstract The pomegranate (Punica granatum L.) fruit harbors remarkable health-beneficial properties. Most of its healthy secondary metabolites are located in their peels. Therefore, pomegranate peels (PPs) can be used to produce high-value healthy compounds. PP cell cultures may also be an attractive alternative source since it can be established throughout the year regardless of seasonal effects. The aim of this study was to establish a novel PP cell culture for future research and biotechnological applications. We first established procedures to sterilize and control oxidative browning of the peel explants. We found that surface sterilizing with 3% sodium hypochlorite for 15 min, frequent sub-culturing of the explants on media containing polyvinylpyrrolidone plus silver nitrate was the optimal treatment for oxidative browning control and callus initiation. We also found that 6-benzylaminopurine (BAP) induced callus initiation, whereas 3-indoleacetic acid (IAA) repressed it. The callus daily growth rate was maintained at 1% regardless of the various growth media examined. Moreover, our results show that although there was a minor effect of the growth media on polyphenol accumulation and antioxidant activity, the exposure to light and high sucrose levels led to higher levels (up to 205 times than the control) of secondary metabolites such as anthocyanins, ellagic acid, punicalagin and gallic acid. In summary, this work laid the foundations for further research that will be needed to study and commercialize PP cell cultures.Key messageIn this present study, we addresses the establishment of pomegranate peel cell cultures for future research and biotechnological applications, such as production of health-beneficial secondary metabolites.
Galloylated plant specialized metabolites play important roles in plant-environment interactions ... more Galloylated plant specialized metabolites play important roles in plant-environment interactions as well as the promotion of human and animal health. The galloylation reactions are mediated by the formation of galloylglucose esters from gallic acid and UDP-glucose, catalyzed by the plant UGT84 family glycosyltransferases. To explore and exploit the structural determinants of UGT84 activities, we performed homology modeling and substrate docking of PgUGT84A23, a galloylglucose ester-forming family 84 UGT, as well as sequence comparisons of PgUGT84A23 with other functionally characterized plant UGTs. By employing site-directed mutagenesis of candidate amino acids, enzyme assays with analogous substrates, and kinetic analysis, key amino acid sites for PgUGT84A23 substrate binding and reactivity were elucidated. The galloylglucose ester-forming UGT84s have not been shown to glycosylate genistein (an isoflavonoid) in vivo. Unexpectedly, amino acids highly conserved among UGT84s were iden...
Enzymes operating in the S -methylmethionine cycle make a differential contribution to methionine... more 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 a...
It is currently held that glyphosate efficiently controls the obligate holoparasite Phelipanche a... more It is currently held that glyphosate efficiently controls the obligate holoparasite Phelipanche aegyptiaca (Egyptian broomrape) by inhibiting its endogenous shikimate pathway, thereby causing a deficiency in aromatic amino acids (AAA). While there is no argument regarding the shikimate pathway being the primary site of the herbicide's action, the fact that the parasite receives a constant supply of nutrients, including proteins and amino acids, from the host does not fit with an AAA deficiency. This apparent contradiction implies that glyphosate mechanism of action in P. aegyptiaca is probably more complex and does not end with the inhibition of the AAA biosynthetic pathway alone. A possible explanation would lie in a limitation of the translocation of solutes from the host as a secondary effect. We examined the following hypotheses: (a) glyphosate does not affects P. aegyptiaca during its independent phase and (b) glyphosate has a secondary effect on the ability of P. aegyptiac...
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Papers by Rachel Amir