Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for construct engineering for biological research and synthetic biology. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the modular cloning system MoClo. Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level-1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module.

L’Hér. (Myrtaceae) is one of the economically most important and widely cultivated trees for wood crop purposes worldwide. Climatic changes together with the constant need to expand plantations to areas that do not always provide optimal conditions for plant growth highlight the need to assess the impact of abiotic stresses on eucalypt trees. We aimed to unveil the drought effect on the leaf metabolome of commercial clones with differential phenotypic response to this stress. For this, seedlings of 13 clones were grown at well-watered (WW) and water-deficit (WD) conditions and their leaf extracts were subjected to comparative analysis using ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC-MS) and nuclear magnetic resonance spectroscopy (NMR). UPLC-MS and NMR analyses led to the annotation of over 100 molecular features of classes such as cyclitols, phenolics, flavonoids, formylated phloroglucinol compounds (FPCs) and fatty acids. Multivariate data analysis was employed for specimens\' classifications and markers identification from both platforms. The results obtained in this work allowed us to classify clones differing in drought tolerance. Classification models were validated using an extra subset of samples. Tolerant plants exposed to water deficit accumulated arginine, gallic acid derivatives, caffeic acid and tannins at higher levels. In contrast, stressed drought-sensitive clones were characterised by a significant reduction in glucose, inositol and shikimic acid levels. These changes in contrasting drought response eucalypt pave ways for differential outcomes of tolerant and susceptible phenotypes. Under optimal growth conditions, all clones were rich in FPCs. These results can be used for early screening of tolerant clones and to improve our understanding of the role of these biomarkers in Eucalyptus tolerance to drought stress.

Cis-(+)-12-oxophytodienoic acid (cis-(+)-OPDA) is a bioactive jasmonate, a precursor of jasmonic acid, which also displays signaling activity on its own. Modulation of cis-(+)-OPDA actions may be carried out via biotransformation leading to metabolites of various functions. This work introduces a methodology for the synthesis of racemic cis-OPDA conjugates with amino acids (OPDA-aa) and their deuterium-labeled analogs, which enables the unambiguous identification and accurate quantification of these compounds in plants. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry-based method for the reliable determination of seven OPDA-aa (OPDA-Alanine, OPDA-Aspartate, OPDA-Glutamate, OPDA-Glycine, OPDA-Isoleucine, OPDA-Phenylalanine, and OPDA-Valine) from minute amount of plant material. The extraction from 10 mg of fresh plant tissue by 10% aqueous methanol followed by single-step sample clean-up on hydrophilic–lipophilic balanced columns prior to final analysis was optimized. The method was validated in terms of accuracy and precision, and the method parameters such as process efficiency, recovery and matrix effects were evaluated. In mechanically wounded 30-day-old Arabidopsis thaliana leaves, five endogenous (+)-OPDA-aa were identified and their endogenous levels were estimated. The time-course accumulation revealed a peak 60 min after the wounding, roughly corresponding to the accumulation of cis-(+)-OPDA. Our synthetic and analytical methodologies will support studies on cis-(+)-OPDA conjugation with amino acids and research into the biological significance of these metabolites in plants.

Signaling proteins trigger a sequence of molecular switches in the cell, which permit development, growth, and rapid adaptation to changing environmental conditions. SCF-type E3 ubiquitin ligases recognize signaling proteins prompting changes in their fate, one of these being ubiquitylation followed by degradation by the proteasome. SCFs together with their ubiquitylation targets (substrates) often serve as phytohormone receptors, responding and/or assembling in response to fluctuating intracellular hormone concentrations. Tracing and understanding phytohormone perception and SCF-mediated ubiquitylation of proteins could provide powerful clues on the molecular mechanisms utilized for plant adaptation. Here, we describe an adaptable in vitro system that uses recombinant proteins and enables the study of hormone-triggered SCF-substrate interaction and the dynamics of protein ubiquitylation. This system can serve to predict the requirements for protein recognition and to understand how phytohormone levels have the power to control protein fate.

Multicomponent reactions (MCRs) are recently expanding the plethora of solid-phase protocols for the synthesis and derivatization of peptides. Herein, we describe a solid-phase-compatible strategy based on MCRs as a powerful strategy for peptide cyclization and ligation . We illustrate, using Gramicidin S as a model peptide, how the execution of on-resin Ugi reactions enables the simultaneous backbone N-functionalization and cyclization, which are important types of derivatizations in peptide-based drug development or for incorporation of conjugation handles, or labels.

Protein expression in plants by agroinfiltration and subsequent purification is increasingly used for the biochemical characterization of plant proteins. In this chapter we describe the purification of secreted, His-tagged proteases from the apoplast of agroinfiltrated Nicotiana benthamiana using immobilized metal affinity chromatography (IMAC). We show quality checks for the purified protease and discuss potential problems and ways to circumvent them. As a proof of concept, we produce and purify tomato immune protease Pip1 and demonstrate that the protein is active after purification.

Jasmonates are essential engineers of plant defense responses against many pests, including herbivorous insects. Herbivory induces the production of jasmonic acid (JA) and its bioactive conjugate jasmonoyl-l-isoleucine (JA-Ile), which then triggers a large transcriptional reprogramming to promote plant acclimation. The contribution of the JA pathway, including its components and regulators, to defense responses against insect herbivory can be evaluated by conducting bioassays with a wide range of host plants and insect pests. Here, we describe a detailed and reproducible protocol for testing feeding behavior of the generalist herbivore Spodoptera littoralis on the model plant Arabidopsis thaliana and hence infer the contribution of JA-mediated plant defense responses to a chewing insect.

Modular cloning systems that rely on type IIS enzymes for DNA assembly have many advantages for complex pathway engineering. These systems are simple to use, efficient, and allow users to assemble multigene constructs by performing a series of one-pot assembly steps, starting from libraries of cloned and sequenced parts. The efficiency of these systems also facilitates the generation of libraries of construct variants. We describe here a protocol for assembly of multigene constructs using the Modular Cloning system MoClo. Making constructs using the MoClo system requires users to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. The assembly strategy is then defined following a set of standard rules. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Changing environmental conditions greatly affect the accumulation of many proteins; therefore, the analysis of alterations in the proteome is essential to understand the plant response to abiotic stress. Proteomics provides a platform for the identification and quantification of plant proteins responsive to cold stress and taking part in cold acclimation. Here, we describe the preparation of proteins for LC-MS measurement to monitor the changes of protein patterns during cold treatment in Arabidopsis thaliana. In our protocol, proteins are precipitated using TCA/acetone, quantified with 2D Quant Kit and digested with trypsin using a filter-based method and analyzed using an LC-MS approach. The acquired results can be further applied for label-free protein quantification.