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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.

ABSTRACT Cysteine is the precursor for the biosynthesis of glutathione, a key stress‐protective metabolite, and methionine, which is imperative for cell growth and protein synthesis. The exact mechanism that governs the routing of cysteine toward glutathione or methionine during stresses remains unclear. Our study reveals that under oxidative stress, methionine and glutathione compete for cysteine and that the increased oxidized glutathione (GSSG) levels under stress hinder methionine biosynthesis. Moreover, we find that inhibition occurs as GSSG binds to and accelerates the degradation of cystathionine γ‐synthase, a key enzyme in the methionine synthesis pathway. Consequently, this leads to a reduction in the flux toward methionine‐derived metabolites and redirects cysteine utilization toward glutathione, thereby enhancing plant protection. Our study suggests a novel regulatory feedback loop involving glutathione, methionine, and cysteine, shedding light on the plant stress response and the adaptive rerouting of cysteine. These findings offer new insights into the intricate balance of growth and protection in plants and its impact on their nutritional value due to low methionine levels under stress. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 g-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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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 34SO42– 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. Significance Statement: This study showed that differences in the expression levels of glutathione reductase (GR) in tobacco and Arabidopsis transgenic and mutant plants significantly affect the level of metabolites associated and in the sulfur assimilation pathway. The findings suggest that GR plays an important role in the activity of 5′ phosphosulfate reductase, a key enzyme of the sulfur assimilation pathway and cysteine formation, and thus give substantial evidence of the function of GR in plants. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

It is not clear why herbicides targeting aromatic and branched-chain amino acid biosynthesis successfully control broomrapes--obligate parasitic plants that obtain all of their nutritional requirements, including amino acids, from the host. Our objective was to reveal the mode of action of imazapic and glyphosate in controlling the broomrape Phelipanche aegyptiaca and clarify if this obligatory parasite has its own machinery for the amino acids biosynthesis. P. aegyptiaca callus was studied to exclude the indirect influence of the herbicides on the parasite through the host plant. Using HRT - tomato plants resistant to imidazolinone herbicides, it was shown that imazapic is translocated from the foliage of treated plants to broomrape attachments on its roots and controls the parasite. Both herbicides inhibited P. aegyptiaca callus growth and altered the free amino acid content. Blasting of Arabidopsis thaliana 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and acetolactate synthase (ALS) cDNA against the genomic DNA of P. aegyptiaca yielded a single copy of each homolog in the latter, with about 78 and 75% similarity, respectively, to A. thaliana counterparts at the protein level. We also show for the first time that both EPSPS and ALS are active in P. aegyptiaca callus and flowering shoots and are inhibited by glyphosate and imazapic, respectively. Thus leading to deficiency of those amino acids in the parasite tissues and ultimately, death of the parasite, indicating the ability of P. aegyptiaca to synthesize branched-chain and aromatic amino acids through the activity of ALS and EPSPS, respectively. [ABSTRACT FROM AUTHOR]

Phelipanche aegyptiaca Pers. is a root holoparasitic plant considered to be among the most destructive agricultural weeds worldwide. In order to gain more knowledge about the metabolic profile of the parasite during its developmental stages, we carried out primary metabolic and lipid profiling using GC-MS analysis. In addition, the levels of amino acids that incorporate into proteins, total protein in the albumin fraction, nitrogen, reduced sugars, and phenols were determined. For the assays, the whole plants from the four developmental stages--tubercle, pre-emergent shoot, post-emergent shoot, and mature flowering plants--were taken. Thirty-five metabolites out of 66 differed significantly between the various developmental stages. The results have shown that the first three developmental stages were distinguished in their profiles, but the latter two did not differ from the mature stage. Yet, 46% of the metabolites detected did not change significantly during the developmental stages. This is unlike other studies of non-parasitic plants showing that their metabolic levels tend to alter significantly during development. This implies that the parasite can control the levels of these metabolites. We further studied the metabolic nature of five organs (adventitious roots, lower and upper shoot, floral buds, and flowers) in mature plants. Similar to non-parasitic plants, the parasite exhibited significant differences between the vegetative and reproductive organs. Compared to other organs, floral buds had higher levels of free amino acids and total nitrogen, whereas flowers accumulated higher levels of simple sugars such as sucrose, and the putative precursors for nectar synthesis, color, and volatiles. This suggests that the reproductive organs have the ability to accumulate metabolites that are required for the production of seeds and as a source of energy for the reproductive processes. The data contribute to our knowledge about the metabolic behavior of parasites that rely on their host for its basic nutrients. [ABSTRACT FROM AUTHOR]

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. aegyptiaca to attract nutrients, limiting the translocation to the parasite. By using a glyphosate-resistant host plant expressing the "phloem-mobile" green fluorescent protein (GFP), it was shown that glyphosate interacts specifically with P. aegyptiaca, initiating a deceleration of GFP translocation to the parasite within 24 h of treatment. Additionally, changes in the entire sugars profile (together with that of other metabolites) of P. aegyptiaca were induced by glyphosate. In addition, glyphosate did not impair germination or seedling development of P. aegyptiaca but begun to exert its action only after the parasite has established a connection to the host vascular system and became exposed to the herbicide. Our findings thus indicate that glyphosate does indeed have a secondary effect in P. aegyptiaca, probably as a consequence of its primary target inhibition-via inhibition of the translocation of phloem-mobile solutes to the parasite, as was simulated by the mobile GFP. The observed disruption in the metabolism of major sugars that are abundant in P. aegyptiaca within 48 h after glyphosate treatment provides a possible explanation for this inhibition of translocation and might reflect a critical secondary effect of the herbicide's primary action that results in loss of the parasite's superior sink for solutes. [ABSTRACT FROM AUTHOR]

Broomrape ( Phelipanche aegyptiaca ) is a root holoparasitic plant considered among the most destructive agricultural weeds worldwide. In order to acquire more knowledge about the metabolism of broomrape and its interaction with its tomato host, we performed primary metabolic profiling using GCMS analysis for the early developmental stage of the parasite and of infected and non-infected roots. The analysis revealed that out of 59 metabolites detected, the levels of 37 significantly increased in the parasite while the levels of 10 significantly decreased compared to the infected roots. In addition, the analysis showed that the levels of total protein in the albumin fraction, reducing sugars (representing starch) and total phenols increased by 9.8-, 4.6- and 3.3-fold, respectively, in the parasite compared to the roots. These changes suggest that P. aegyptiaca has its own metabolism that differs significantly in its regulation from those found in their host. In addition, the results have shown that the levels of most of the metabolites in the infected roots were similar to levels detected in the non-infected roots, except for seven metabolites whose levels increased in the infected versus the non-infected roots. This suggests that the parasite did not significantly affect the host primary metabolic pathways. [ABSTRACT FROM AUTHOR]

Glutathione (GSH), a tripeptide thiol compound has multiple functions in plants. Recent works suggested that GSH plays a regulatory role in signaling in plants as part of their adaptation to stress. To better understand the role of GSH as a regulatory molecule, 14 days old Arabidopsis thaliana seedlings were treated with 5 mM of GSH for 4 h. Changes in gene expression patterns were studied by cDNA microarray analysis. The expression of 453 genes was significantly changed compared to the untreated control, of which 261 genes were up-regulated and 192 genes were down-regulated. Genes from several groups were affected, including those of sulfur metabolism, degradation and synthesis of macromolecules and transcription factors. Up-regulation of genes involved in responses to biotic stresses, or in jasmonate or salicylic acid synthesis and their signaling, suggests that GSH triggers genes that help protect the plants during stresses. In addition, GSH down regulated genes involved in plant growth and development, like those involved in cell wall synthesis and its extension, and genes associated with auxin and cytokinins response, which are related to growth and development of the plants. The results suggest that GSH might have a role in response to biotic stress by initiating defense responses and modifying plants’ growth and development in an effort to tune their sessile lifestyle of plants to environmental constraints. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cystathionine γ-synthase (CGS), the first enzyme of methionine biosynthesis in higher plants, plays an important role in the biosynthesis pathway and in regulating methionine metabolism in plant cells. In response to methionine, the expression of this enzyme is regulated via amino acid sequences located in its N-terminal. Here, using reverse transcription PCR and ribonuclease protection analysis, we demonstrate that, in addition to the full-length CGS transcript, a deleted form exists in Arabidopsis. The deleted transcript of CGS that lacks 90 or 87 nt located internally in the regulatory N-terminal region of CGS maintains the reading frame of the protein. Its association with polyribosomes indicates that this deleted form of CGS is translated. In order to study the function of this deleted form of CGS, we overexpressed it in transgenic tobacco plants. We found that the transgenic plants engineered to express only the deleted form of CGS accumulated methionine to a much higher level than those that expressed the full-length CGS. Furthermore, in vitro feeding experiments revealed that the deleted form of CGS is not subject to feedback regulation by methionine, as reported for the full-length transcript. Therefore, although most likely produced from the full-length CGS, the transcript of the deleted form is insensitive to methionine application and its expression may be important for maintaining methionine metabolism even in the presence of a high level of methionine. [ABSTRACT FROM AUTHOR]

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge. [ABSTRACT FROM AUTHOR]

The sulfur-containing amino acid methionine is a nutritionally important essential amino acid and is the precursor of several metabolites that regulate plant growth and responses to the environment. Methionine production is largely regulated by cystathionine γ-synthase, the first specific enzyme for its synthesis. This enzyme competes in a complex manner with threonine synthase, the last enzyme in threonine biosynthesis, for their common substrate O-phosphohomoserine. New genetic and molecular data suggest that methionine synthesis and catabolism are coordinately regulated by novel post-transcriptional and post-translational mechanisms that are associated with a regulatory part within the N-terminal part of cystathionine γ-synthase. [ABSTRACT FROM AUTHOR]

A comparison of the three-dimensional structures of the closely related mesophilic Clostridium beijerinckii alcohol dehydrogenase (CBADH) and the hyperthermophilic Thermoanaerobacter brockii alcohol dehydrogenase (TBADH) suggested that extra proline residues in TBADH located in strategically important positions might contribute to the extreme thermal stability of TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline. All eight single-proline mutants and a double-proline mutant of CBADH were enzymatically active. The critical sites for increasing thermostability parameters in CBADH were Leu-316 and Ser-24, and to a lesser degree, Ala-347. Substituting proline for His-222, Leu-275, and Thr-149, however, reduced thermal stability parameters. Our results show that the thermal stability of the mesophilic CBADH can be moderately enhanced by substituting proline at strategic positions analogous to nonconserved prolines in the homologous thermophilic TBADH. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a β-turn and a terminating external loop in the polypeptide chain. Positioning proline at the N-caps of α-helices in CBADH led to adverse effects on thermostability, whereas single-proline mutations in other positions in the polypeptide had varying effects on thermal parameters. The finding presented here support the idea that at least two of the eight extra prolines in TBADH contribute to its thermal stability. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Methionine and threonine are two essential amino acids, the levels of which limit the nutritional quality of plants. Both amino acids diverge from the same branch of the aspartate family biosynthesis pathway; therefore, their biosynthesis pathways compete for the same carbon/amino substrate. To further elucidate the regulation of methionine biosynthesis and seek ways of increasing the levels of these two amino acids, we crossed transgenic tobacco plants overexpressing the bacterial feedback-insensitive aspartate kinase (bAK), containing a significantly higher threonine level, with plants overexpressing Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Plants co-expressing bAK and the full-length AtCGS (F-AtCGS) have significantly higher methionine and threonine levels compared with the levels found in wild-type plants, but the methionine level does not increase beyond that found in plants expressing F-AtCGS alone. This finding can be explained through the feedback inhibition regulation mediated by the methionine metabolite on the transcript level of AtCGS. To test this assumption, plants expressing bAK were crossed with plants expressing two mutated forms of AtCGS in which the domains responsible for the feedback regulation have been deleted. Indeed, significantly higher methionine contents and its metabolites levels accumulated in the newly produced plants, and the levels of threonine were also significantly higher than in the wild-type plants. The transcript level of the two mutated forms of AtCGS significantly increased when there was a high content of threonine in the plants, suggesting that threonine modulates, probably indirectly, the transcript level of AtCGS.

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.