Gantner, J.; Ordon, J.; Ilse, T.; Kretschmer, C.; Gruetzner, R.; Löfke, C.; Dagdas, Y.; Bürstenbinder, K.; Marillonnet, S.; Stuttmann, J. Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system PLoS ONE 13, e0197185, (2018) DOI: 10.1371/journal.pone.0197185
Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.
Iglesias, M. J.; Terrile, M. C.; Correa-Aragunde, N.; Colman, S. L.; Izquierdo-Álvarez, A.; Fiol, D. F.; París, R.; Sánchez-López, N.; Marina, A.; Calderón Villalobos, L. I. A.; Estelle, M.; Lamattina, L.; Martínez-Ruiz, A.; Casalongué, C. A. Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling Redox Biol 18, 200-210, (2018) DOI: 10.1016/j.redox.2018.07.003
The F-box proteins (FBPs) TIR1/AFBs are the
substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin
ligase complexes and together with Aux/IAAs form the auxin co-receptor.
Although tremendous knowledge on auxin perception and signaling has been
gained in the last years, SCFTIR1/AFBs complex assembly and
stabilization are emerging as new layers of regulation. Here, we
investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is
involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is
S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118
residues in vitro. Both, in vitro and in vivo protein-protein
interaction assays show that NO enhances ASK1 binding to CUL1 and
TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we
demonstrate that Cys37 and Cys118 are essential residues for proper
activation of auxin signaling pathway in planta. Phylogenetic analysis
revealed that Cys37 residue is only conserved in SKP proteins in
Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an
evolutionary adaption for SKP1 function in flowering plants.
Collectively, these findings indicate that multiple events of redox
modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs
for proper auxin signal transduction.
Wasternack, C.; Hause, B. A Bypass in Jasmonate Biosynthesis – the OPR3-independent Formation Trends Plant Sci 23, 276-279, (2018) DOI: 10.1016/j.tplants.2018.02.011
For the first time in 25 years, a new pathway for
biosynthesis of jasmonic acid (JA) has been identified. JA production
takes place via 12-oxo-phytodienoic acid (OPDA) including reduction by
OPDA reductases (OPRs). A loss-of-function allele, opr3-3, revealed an
OPR3-independent pathway converting OPDA to JA.
Bochnia, M.; Scheidemann, W.; Ziegler, J.; Sander, J.; Vollstedt, S.; Glatter, M.; Janzen, N.; Terhardt, M.; Zeyner, A. Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners Equine Vet Educ 30, 24-28, (2018) DOI: 10.1111/eve.12596
Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]-conjugates) increased in body fluids of the AM horse (MCPA-carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA-carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA-conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA-conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA-conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease.
Jablonická, V.; Ziegler, J.; Vatehová, Z.; Lišková, D.; Heilmann, I.; Obložinský, M.; Heilmann, M. Inhibition of phospholipases influences the metabolism of wound-induced benzylisoquinoline alkaloids in Papaver somniferum L. J Plant Physiol 223, 1-8, (2018) DOI: 10.1016/j.jplph.2018.01.007
Benzylisoquinoline alkaloids (BIAs) are important
secondary plant metabolites and include medicinally relevant drugs, such
as morphine or codeine. As the de novo synthesis of BIA backbones is
(still) unfeasible, to date the opium poppy plant Papaver somniferum L.
represents the main source of BIAs. The formation of BIAs is induced in
poppy plants by stress conditions, such as wounding or salt treatment;
however, the details about regulatory processes controlling BIA
formation in opium poppy are not well studied. Environmental stresses,
such as wounding or salinization, are transduced in plants by
phospholipid-based signaling pathways, which involve different classes
of phospholipases. Here we investigate whether pharmacological
inhibition of phospholipase A2 (PLA2, inhibited by aristolochic acid
(AA)) or phospholipase D (PLD; inhibited by 5-fluoro-2-indolyl
des-chlorohalopemide (FIPI)) in poppy plants influences wound-induced
BIA accumulation and the expression of key biosynthetic genes. We show
that inhibition of PLA2 results in increased morphinan biosynthesis
concomitant with reduced production of BIAs of the papaverine branch,
whereas inhibition of PLD results in increased production of BIAs of the
noscapine branch. The data suggest that phospholipid-dependent
signaling pathways contribute to the activation of morphine biosynthesis
at the expense of the production of other BIAs in poppy plants. A
better understanding of the effectors and the principles of regulation
of alkaloid biosynthesis might be the basis for the future genetic
modification of opium poppy to optimize BIA production.
Krägeloh, T.; Cavalleri, J. M. V.; Ziegler, J.; Sander, J.; Terhardt, M.; Breves, G.; Cehak, A. Identification of hypoglycin A binding adsorbents
as potential preventive measures in co-grazers of atypical myopathy
affected horses Equine Vet J 50, 220-227, (2018) DOI: 10.1111/evj.12723
BackgroundIntestinal absorption of hypoglycin A
(HGA) and its metabolism are considered major prerequisites for atypical
myopathy (AM). The increasing incidence and the high mortality rate of
AM urgently necessitate new therapeutic and/or preventative
approaches.ObjectivesTo identify a substance for oral administration
capable of binding HGA in the intestinal lumen and effectively reducing
the intestinal absorption of the toxin.Study designExperimental in vitro
study.MethodsSubstances commonly used in equine practice (activated
charcoal composition, di‐tri‐octahedral smectite, mineral oil and
activated charcoal) were tested for their binding capacity for HGA using
an in vitro incubation method. The substance most effective in binding
HGA was subsequently tested for its potential to reduce intestinal HGA
absorption. Jejunal tissues of 6 horses were incubated in Ussing
chambers to determine mucosal uptake, tissue accumulation, and serosal
release of HGA in the presence and absence of the target substance.
Potential intestinal metabolism in methylenecyclopropyl acetic acid
(MCPA)‐conjugates was investigated by analysing their concentrations in
samples from the Ussing chambers.ResultsActivated charcoal composition
and activated charcoal were identified as potent HGA binding substances
with dose and pH dependent binding capacity. There was no evidence of
intestinal HGA metabolism.Main limitationsBinding capacity of adsorbents
was tested in vitro using aqueous solutions, and in vivo factors such
as transit time and composition of intestinal content, may affect
adsorption capacity after oral administration.ConclusionsFor the first
time, this study identifies substances capable of reducing HGA
intestinal absorption. This might have major implications as a
preventive measure in cograzers of AM affected horses but also in horses
at an early stage of intoxication.
Ibañez, C.; Delker, C.; Martinez, C.; Bürstenbinder, K.; Janitza, P.; Lippmann, R.; Ludwig, W.; Sun, H.; James, G. V.; Klecker, M.; Grossjohann, A.; Schneeberger, K.; Prat, S.; Quint, M. Brassinosteroids Dominate Hormonal Regulation of Plant Thermomorphogenesis via BZR1 Curr Biol 28, 303-310.e3, (2018) DOI: 10.1016/j.cub.2017.11.077
Thermomorphogenesis is defined as the suite of morphological changes that together are likely to contribute to adaptive growth acclimation to usually elevated ambient temperature [ 1, 2 ]. While many details of warmth-induced signal transduction are still elusive, parallels to light signaling recently became obvious (reviewed in [ 3 ]). It involves photoreceptors that can also sense changes in ambient temperature [ 3–5 ] and act, for example, by repressing protein activity of the central integrator of temperature information PHYTOCHROME-INTERACTING FACTOR 4 (PIF4 [ 6 ]). In addition, PIF4 transcript accumulation is tightly controlled by the evening complex member EARLY FLOWERING 3 [ 7, 8 ]. According to the current understanding, PIF4 activates growth-promoting genes directly but also via inducing auxin biosynthesis and signaling, resulting in cell elongation. Based on a mutagenesis screen in the model plant Arabidopsis thaliana for mutants with defects in temperature-induced hypocotyl elongation, we show here that both PIF4 and auxin function depend on brassinosteroids. Genetic and pharmacological analyses place brassinosteroids downstream of PIF4 and auxin. We found that brassinosteroids act via the transcription factor BRASSINAZOLE RESISTANT 1 (BZR1), which accumulates in the nucleus at high temperature, where it induces expression of growth-promoting genes. Furthermore, we show that at elevated temperature BZR1 binds to the promoter of PIF4, inducing its expression. These findings suggest that BZR1 functions in an amplifying feedforward loop involved in PIF4 activation. Although numerous negative regulators of PIF4 have been described, we identify BZR1 here as a true temperature-dependent positive regulator of PIF4, acting as a major growth coordinator.
Wasternack, C.; Strnad, M. Jasmonates: News on Occurrence, Biosynthesis, Metabolism and Action of an Ancient Group of Signaling Compounds Int J Mol Sci 19, 2539, (2018) DOI: 10.3390/ijms19092539
Jasmonic acid (JA) and its related derivatives are
ubiquitously occurring compounds of land plants acting in numerous
stress responses and development. Recent studies on evolution of JA and
other oxylipins indicated conserved biosynthesis. JA formation is
initiated by oxygenation of α-linolenic acid (α-LeA, 18:3) or 16:3 fatty
acid of chloroplast membranes leading to 12-oxo-phytodienoic acid
(OPDA) as intermediate compound, but in Marchantiapolymorpha and
Physcomitrellapatens, OPDA and some of its derivatives are final
products active in a conserved signaling pathway. JA formation and its
metabolic conversion take place in chloroplasts, peroxisomes and
cytosol, respectively. Metabolites of JA are formed in 12 different
pathways leading to active, inactive and partially active compounds. The
isoleucine conjugate of JA (JA-Ile) is the ligand of the receptor
component COI1 in vascular plants, whereas in the bryophyte M.
polymorpha COI1 perceives an OPDA derivative indicating its functionally
conserved activity. JA-induced gene expressions in the numerous biotic
and abiotic stress responses and development are initiated in a
well-studied complex regulation by homeostasis of transcription factors
functioning as repressors and activators.
Mitra, D.; Kumari, P.; Quegwer, J.; Klemm, S.; Moeller, B.; Poeschl, Y.; Pflug, P.; Stamm, G.; Abel, S.; Bürstenbinder, K. Microtubule-associated protein IQ67 DOMAIN5 regulates interdigitation of leaf pavement cells in Arabidopsis thaliana bioRxiv (2018) DOI: 10.1101/268466
Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis, as indicated by reduced interdigitation of neighboring cells in the leaf epidermis of iqd5 mutants. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced asymmetric expansion. Lastly, we provide evidence for IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate multi-polar growth in PCs.
Bagchi, R.; Melnyk, C. W.; Christ, G.; Winkler, M.; Kirchsteiner, K.; Salehin, M.; Mergner, J.; Niemeyer, M.; Schwechheimer, C.; Calderón Villalobos, L. I. A.; Estelle, M. The Arabidopsis ALF4 protein is a regulator of SCF E3 ligases. EMBO J 37, 255-268, (2018) DOI: 10.15252/embj.201797159
The cullin-RING E3 ligases (CRLs) regulate diverse cellular processes in all eukaryotes. CRL activity is controlled by several proteins or protein complexes, including NEDD8, CAND1, and the CSN. Recently, a mammalian protein called Glomulin (GLMN) was shown to inhibit CRLs by binding to the RING BOX (RBX1) subunit and preventing binding to the ubiquitin-conjugating enzyme. Here, we show that Arabidopsis ABERRANT LATERAL ROOT FORMATION4 (ALF4) is an ortholog of GLMN. The alf4 mutant exhibits a phenotype that suggests defects in plant hormone response. We show that ALF4 binds to RBX1 and inhibits the activity of SCFTIR1, an E3 ligase responsible for degradation of the Aux/IAA transcriptional repressors. In vivo, the alf4 mutation destabilizes the CUL1 subunit of the SCF. Reduced CUL1 levels are associated with increased levels of the Aux/IAA proteins as well as the DELLA repressors, substrate of SCFSLY1. We propose that the alf4 phenotype is partly due to increased levels of the Aux/IAA and DELLA proteins.