PP2A-B’γ modulates foliar trans-methylation capacity and the formation of 4-methoxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves

Glucosinolates (GSL) of cruciferous plants comprise a major group of structurally diverse secondary compounds which act as deterrents against aphids and microbial pathogens and have large commercial and ecological impacts. While the transcriptional regulation governing the biosynthesis and modification of GSL is now relatively well understood, post-translational regulatory components that specifically determine the structural variation of indole glucosinolates have not been reported. We show that the cytoplasmic protein phosphatase 2A regulatory subunit B'gamma (PP2A-B'gamma) physically interacts with indole glucosinolate methyltransferases and controls the methoxylation of indole glucosinolates and the formation of 4-meth-oxy-indol-3-yl-methyl glucosinolate in Arabidopsis leaves. By taking advantage of proteomic approaches and metabolic analysis we further demonstrate that PP2A-B'gamma is required to control the abundance of oligomeric protein complexes functionally linked with the activated methyl cycle and the trans-methylation capacity of leaf cells. These findings highlight the key regulatory role of PP2A-B'gamma in methionine metabolism and provide a previously unrecognized perspective for metabolic engineering of glucosinolate metabolism in cruciferous plants.Peer reviewe

PROTEIN PHOSPHATASE 2A-B 'gamma Controls Botrytis cinerea Resistance and Developmental Leaf Senescence

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'gamma is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'gamma regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'gamma to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'gamma is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'gamma depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'gamma age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.Peer reviewe

The formation of a camalexin-biosynthetic metabolon

Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway by formation of an enzyme complex. To identify such protein interactions, two independent untargeted co49 immunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B1 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were shown to co-purify. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, interaction of CYP71A13 and Arabidopsis P450 Reductase 1 (ATR1) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating allosteric interaction. Camalexin biosynthesis involves glutathionylation of an intermediary indole-3-cyanohydrin, synthesized by CYP71A12 and especially CYP71A13. It was demonstrated by FRET-FLIM and co-IP, that the glutathione transferase GSTU4, which is co-expressed with tryptophan- and camalexin-specific enzymes, was physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knock-out and reduced in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a role in a competing mechanism

PROTEIN PHOSPHATASE 2A-B'γ controls Botrytis cinerea resistance and developmental leaf senescence

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In pre-senescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.</p

The role of MYB34, MYB51 and MYB122 in the regulation of camalexin biosynthesis in Arabidopsis thaliana

The phytoalexin camalexin and indolic glucosinolates share not only a common evolutionary origin and a tightly interconnected biosynthetic pathway, but regulatory proteins controlling the shared enzymatic steps are also modulated by the same R2R3-MYB transcription factors. The indolic phytoalexin camalexin is a crucial defense metabolite in the model plant Arabidopsis. indolic phytoalexins and glucosinolates appear to have a common evolutionary origin and are interconnected on the biosynthetic level: a key intermediate in the biosynthesis of camalexin, indole-3-acetaldoxime (IA0x), is also required for the biosynthesis of indolic glucosinolates and is under tight control by the transcription factors MYB34, MYB51, and MYB122. The abundance of camalexin was strongly reduced in myb34/51 and myb51/122 double and in triple myb mutant, suggesting that these transcription factors are important in camalexin biosynthesis. Furthermore, expression of MYB51 and MYB122 was significantly increased by biotic and abiotic camalexin-inducing agents. Feeding of the triple myb34/51/122 mutant with IA0x or indole-3-acetonitrile largely restored camalexin biosynthesis. Conversely, tryptophan could not complement the low camalexin phenotype of this mutant, which supports a role for the three MYB factors in camalexin biosynthesis upstream of IA0x. Consistently expression of the camalexin biosynthesis genes CYP71815/PAD3 and CYP71A13 was not negatively affected in the triple myb mutant and the MYBs could not activate pCYP71815::uidA expression in trans-activation assays with cultured Arabidopsis cells. In conclusion, this study reveals the importance of MYB factors regulating the generation of IA0x as precursor of camalexin

Characterisation of the tryptophan synthase alpha subunit in maize

<p>Abstract</p> <p>Background</p> <p>In bacteria, such as <it>Salmonella typhimurium</it>, tryptophan is synthesized from indole-3-glycerole phosphate (IGP) by a tryptophan synthase αββα heterotetramer. Plants have evolved multiple α (TSA) and β (TSB) homologs, which have probably diverged in biological function and their ability of subunit interaction. There is some evidence for a tryptophan synthase (TS) complex in Arabidopsis. On the other hand maize (<it>Zea mays</it>) expresses the TSA-homologs BX1 and IGL that efficiently cleave IGP, independent of interaction with TSB.</p> <p>Results</p> <p>In order to clarify, how tryptophan is synthesized in maize, two TSA homologs, hitherto uncharacterized <it>Zm</it>TSA and <it>Zm</it>TSAlike, were functionally analyzed. <it>Zm</it>TSA is localized in plastids, the major site of tryptophan biosynthesis in plants. It catalyzes the tryptophan synthase α-reaction (cleavage of IGP), and forms a tryptophan synthase complex with <it>Zm</it>TSB1 <it>in vitro</it>. The catalytic efficiency of the α-reaction is strongly enhanced upon complex formation. A 160 kD tryptophan synthase complex was partially purified from maize leaves and <it>Zm</it>TSA was identified as native α-subunit of this complex by mass spectrometry. <it>Zm</it>TSAlike, for which no <it>in vitro </it>activity was detected, is localized in the cytosol. <it>Zm</it>TSAlike, BX1, and IGL were not detectable in the native tryptophan synthase complex in leaves.</p> <p>Conclusion</p> <p>It was demonstrated <it>in vivo </it>and <it>in vitro </it>that maize forms a tryptophan synthase complex and <it>Zm</it>TSA functions as α-subunit in this complex.</p