Characterization of four bifunctional plant IAM/PAM-amidohydrolases capable of contributing to auxin biosynthesis.

Amidases [EC 3.5.1.4] capable of converting indole-3-acetamide (IAM) into the major plant growth hormone indole-3-acetic acid (IAA) are assumed to be involved in auxin de novo biosynthesis. With the emerging amount of genomics data, it was possible to identify over forty proteins with substantial homology to the already characterized amidases from Arabidopsis and tobacco. The observed high conservation of amidase-like proteins throughout the plant kingdom may suggest an important role of theses enzymes in plant development. Here, we report cloning and functional analysis of four, thus far, uncharacterized plant amidases from Oryza sativa, Sorghum bicolor, Medicago truncatula, and Populus trichocarpa. Intriguingly, we were able to demonstrate that the examined amidases are also capable of converting phenyl-2-acetamide (PAM) into phenyl-2-acetic acid (PAA), an auxin endogenous to several plant species including Arabidopsis. Furthermore, we compared the subcellular localization of the enzymes to that of Arabidopsis AMI1, providing further evidence for similar enzymatic functions. Our results point to the presence of a presumably conserved pathway of auxin biosynthesis via IAM, as amidases, both of monocot, and dicot origins, were analyzed

COP1 destabilizes DELLA proteins in Arabidopsis

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana. Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GAinsensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.Fil: Blanco Touriñán, Noel. Universidad Politécnica de Valencia; EspañaFil: Legris, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Minguet, Eugenio G.. Universidad Politécnica de Valencia; EspañaFil: Costigliolo Rojas, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Nohales, María A.. University of Southern California; Estados UnidosFil: Iniesto, Elisa. Consejo Superior de Investigaciones Científicas; EspañaFil: García León, Marta. Consejo Superior de Investigaciones Científicas; EspañaFil: Pacín, Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; ArgentinaFil: Heucken, Nicole. Universitat Dusseldorf; AlemaniaFil: Blomeier, Tim. Universitat Dusseldorf; AlemaniaFil: Locascio, Antonella. Universidad Politécnica de Valencia; EspañaFil: Cerný, Martin. Mendel University in Brno; República ChecaFil: Esteve Bruna, David. Universidad Politécnica de Valencia; EspañaFil: Díez Díaz, Mónica. Univerdiad Catolica de Valencia; EspañaFil: Brzobohatý, Bretislav. Mendel University in Brno; República ChecaFil: Frerigmann, Henning. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Zurbriggen, Matías D.. Universitat Dusseldorf; AlemaniaFil: Kay, Steve A.. University of Southern California; Estados UnidosFil: Rubio, Vicente. Consejo Superior de Investigaciones Científicas; EspañaFil: Blázquez, Miguel A.. Universidad Politécnica de Valencia; EspañaFil: Casal, Jorge José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; ArgentinaFil: Alabadí, David. Universidad Politécnica de Valencia; Españ

COP1 destabilizes DELLA proteins in Arabidopsis

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana. Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness and Agencia Española de Investigación/Fondo Europeo para el Desarrollo Regional/Unión Europea (grants BIO2016-79133-P to D.A. and BIO2013-46539-R and BIO2016-80551-R to V.R.); the European Union SIGNAT-Research and Innovation Staff Exchange (Grant H2020-MSCA-RISE-2014-644435 to M.A.B., D.A., and J.J.C.); the Argentinian Agencia Nacional de Promoción Científica y Tecnológica (Grant Proyectos de Investigación Científica y Tecnológica-2016-1459 to J.J.C.); Universidad de Buenos Aires (grant 20020170100505BA to J.J.C.); the National Institute of General Medical Sciences of the National Institutes of Health (awards R01GM067837 and R01GM056006 to S.A.K.); the German Research Foundation (DFG) under Germany’s Excellence Strategy/Initiative (Cluster of Excellence on Plant Sciences – Excellence Cluster EXC-2048/1, Project ID 390686111 to M.D.Z.); the International Max Planck Research School of the Max Planck Society; the Universities of Düsseldorf and of Cologne to T.B.; Nordrhein Westfalen Bioeconomy Science Center-FocusLabs CombiCom to N.H. and M.D.Z.; and Ministry of Education, Youth and Sports of the Czech Republic (Project LQ1601 Central European Institute of Technology 2020 to B.B. and M.C.). N.B.-T., E.I., and M.G.-L. were supported by Ministerio de Economía y Competitividad-Formación de Personal Investigador Program fellowships

Update on the role of R2R3-MYBs in the regulation of glucosinolates upon sulfur deficiency

To balance the flux of sulfur (S) into glucosinolates (GSL) and primary metabolites plants exploit various regulatory mechanisms particularly important upon S deficiency (S). The role of MYB34, MYB51 and MYB122 controlling the production of indolic glucosinolates (IGs) and MYB28, MYB29, and MYB76 regulating the biosynthesis of aliphatic glucosinolates (AGs) in Arabidopsis thaliana has not been fully addressed at S conditions yet. We show that the decline in the concentrations of GSL during S depletion does not coincide with the globally decreased transcription of R2R3-MYBs. Whereas the levels of GSL are diminished, the expression of MYB34, MYB51, MYB122, and MYB28 is hardly changed in early phase of S limitation. Furthermore, the mRNA levels of these MYBs start to raise under prolonged S starvation. In parallel, we found that SLIM1 can downregulate the MYBs in vitro as demonstrated in trans-activation assays in cultured Arabidopsis cells with SLIM1 as effector and ProMYB51:uidA as a reporter construct. However, in vivo, only the mRNA of MYB29 and MYB76 correlated with the levels of GSL at S. We propose that the negative effect of SLIM1 on GSL regulatory genes can be overridden by a low GSL signal inducing the transcription of MYBs in a feedback regulatory loop. In accordance with this hypothesis, the expression of MYB34, MYB51, MYB122, and CYP83B1 was further induced in cyp79b2 cyp79b3 mutant exposed to S conditions vs. cyp79b2 cyp79b3 plants grown on control medium. In addition, the possible role of MYBs in the regulation of essential S assimilation enzymes, in the regulation of GSL biosynthesis upon accelerated termination of life cycles, in the mobilization of auxin and lateral root formation at S deficiency is discussed

MYB34, MYB51, and MYB122 Distinctly Regulate Indolic Glucosinolate Biosynthesis in Arabidopsis thaliana

The MYB34, MYB51, and MYB122 transcription factors are known to regulate indolic glucosinolate (IG) biosynthesis in Arabidopsis thaliana. To determine the distinct regulatory potential of MYB34, MYB51, and MYB122, the accumulation of IGs in different parts of plants and upon treatment with plant hormones were analyzed in A. thaliana seedlings. It was shown that MYB34, MYB51, and MYB122 act together to control the biosynthesis of I3M in shoots and roots, with MYB34 controlling biosynthesis of IGs mainly in the roots, MYB51 regulating biosynthesis in shoots, and MYB122 having an accessory role in the biosynthesis of IGs. Analysis of glucosinolate levels in seedlings of myb34, myb51, myb122, myb34 myb51 double, and myb34 myb51 myb122 triple knockout mutants grown in the presence of abscisic acid (ABA), salicylic acid (SA), jasmonate (JA), or ethylene (ET) revealed that: (1) MYB51 is the central regulator of IG synthesis upon SA and ET signaling, (2) MYB34 is the key regulator upon ABA and JA signaling, and (3) MYB122 plays only a minor role in JA/ET-induced glucosinolate biosynthesis. The myb34 myb51 myb122 triple mutant is devoid of IGs, indicating that these three MYB factors are indispensable for IG production under standard growth conditions

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

bHLH05 Is an Interaction Partner of MYB51 and a Novel Regulator of Glucosinolate Biosynthesis in Arabidopsis

By means of yeast (Saccharomyces cerevisiae) two-hybrid screening, we identified basic helix-loop-helix transcription factor05 (bHLH05) as an interacting partner of MYB51, the key regulator of indolic glucosinolates (GSLs) in Arabidopsis (Arabidopsis thaliana). Furthermore, we show that bHLH04, bHLH05, and bHLH06/MYC2 also interact with other R2R3-MYBs regulating GSL biosynthesis. Analysis of bhlh loss-of-function mutants revealed that the single bhlh mutants retained GSL levels that were similar to those in wild-type plants, whereas the triple bhlh04/05/06 mutant was depleted in the production of GSL. Unlike bhlh04/06 and bhlh05/06 mutants, the double bhlh04/05 mutant was strongly affected in the production of GSL, pointing to a special role of bHLH04 and bHLH05 in the control of GSL levels in the absence of jasmonic acid. The combination of two specific gain-of-function alleles of MYB and bHLH proteins had an additive effect on GSL levels, as demonstrated by the analysis of the double MYB34-1D bHLH05D94N mutant, which produces 20-fold more indolic GSLs than bHLH05D94N and ecotype Columbia-0 of Arabidopsis. The amino acid substitution D94N in bHLH05D94N negatively affects the interaction with JASMONATE-ZIM DOMAIN protein, thereby resulting in constitutive activation of bHLH05 and mimicking jasmonic acid treatment. Our study revealed the bHLH04, bHLH05, and bHLH06/MYC2 factors as novel regulators of GSL biosynthesis in Arabidopsis