The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.

Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism.

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Plants respond to both abiotic and biotic stresses with alterations in the expression of genes required to produce protective metabolites. Sometimes plants can be challenged with different stresses simultaneously and as they cannot evade from this situation, priorities have to be set to deal with the most urgent threat. The abiotic stress ultraviolet‐B (UV‐B) light induces the production of UV‐protective flavonols in Arabidopsis Col‐0 cell suspension cultures and this accumulation is attenuated by concurrent application of the bacterial elicitor flg22 (simulating biotic stress). This inhibition correlates with strong suppression of the flavonol biosynthesis genes. In parallel, flg22 induces the production of defence‐related compounds, such as the phytoalexins, camalexin and scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread. This correlated positively with flg22‐mediated expression of enzymes for lignin, scopoletin and camalexin production. As flavonols, lignin and scopoletin are all derived from phenylalanine, it appears that the plant focuses the metabolism on production of scopoletin and lignin at the expense of flavonol production. Furthermore, it appears that this crosstalk involves antagonistic regulation of two opposing MYB transcription factors, the positive regulator of the flavonol pathway MYB12 (UV‐B‐induced and flg22‐suppressed) and the negative regulator MYB4 (UV‐B‐ and flg22‐induced).

While diverse microbe‐ or damage‐associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP‐specific differences in Ca2+ signalling, defence gene expression and MAMP‐mediated growth arrest in Arabidopsis thaliana. Such MAMP‐specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca2+ elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1‐mediated signal amplification to attain MAMP/DAMP‐specific Ca2+ amplitudes in wild‐type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca2+ peak, demonstrating a feedback effect of ROS on Ca2+ signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca2+ response. Thus, fine‐tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up‐ or downstream of Ca2+ signalling.

Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism.

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Plants respond to both abiotic and biotic stresses with alterations in the expression of genes required to produce protective metabolites. Sometimes plants can be challenged with different stresses simultaneously and as they cannot evade from this situation, priorities have to be set to deal with the most urgent threat. The abiotic stress ultraviolet‐B (UV‐B) light induces the production of UV‐protective flavonols in Arabidopsis Col‐0 cell suspension cultures and this accumulation is attenuated by concurrent application of the bacterial elicitor flg22 (simulating biotic stress). This inhibition correlates with strong suppression of the flavonol biosynthesis genes. In parallel, flg22 induces the production of defence‐related compounds, such as the phytoalexins, camalexin and scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread. This correlated positively with flg22‐mediated expression of enzymes for lignin, scopoletin and camalexin production. As flavonols, lignin and scopoletin are all derived from phenylalanine, it appears that the plant focuses the metabolism on production of scopoletin and lignin at the expense of flavonol production. Furthermore, it appears that this crosstalk involves antagonistic regulation of two opposing MYB transcription factors, the positive regulator of the flavonol pathway MYB12 (UV‐B‐induced and flg22‐suppressed) and the negative regulator MYB4 (UV‐B‐ and flg22‐induced).

While diverse microbe‐ or damage‐associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP‐specific differences in Ca2+ signalling, defence gene expression and MAMP‐mediated growth arrest in Arabidopsis thaliana. Such MAMP‐specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca2+ elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1‐mediated signal amplification to attain MAMP/DAMP‐specific Ca2+ amplitudes in wild‐type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca2+ peak, demonstrating a feedback effect of ROS on Ca2+ signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca2+ response. Thus, fine‐tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up‐ or downstream of Ca2+ signalling.

The defense-related plant metabolites known as glucosinolates play important roles in agriculture, ecology, and human health. Despite an advanced biochemical understanding of the glucosinolate pathway, the source of the reduced sulfur atom in the core glucosinolate structure remains unknown. Recent evidence has pointed toward GSH, which would require further involvement of a GSH conjugate processing enzyme. In this article, we show that an Arabidopsis thaliana mutant impaired in the production of the γ-glutamyl peptidases GGP1 and GGP3 has altered glucosinolate levels and accumulates up to 10 related GSH conjugates. We also show that the double mutant is impaired in the production of camalexin and accumulates high amounts of the camalexin intermediate GS-IAN upon induction. In addition, we demonstrate that the cellular and subcellular localization of GGP1 and GGP3 matches that of known glucosinolate and camalexin enzymes. Finally, we show that the purified recombinant GGPs can metabolize at least nine of the 10 glucosinolate-related GSH conjugates as well as GS-IAN. Our results demonstrate that GSH is the sulfur donor in the biosynthesis of glucosinolates and establish an in vivo function for the only known cytosolic plant γ-glutamyl peptidases, namely, the processing of GSH conjugates in the glucosinolate and camalexin pathways.