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Summary DNA assembly systems based on the Golden Gate method are popular in synthetic biology but have several limitations: small insert size, incompatibility with other cloning platforms, DNA domestication requirement, generation of fusion scars, and lack of post‐assembly modification. To address these obstacles, we present the DASH assembly toolset, which combines features of Golden Gate‐based cloning, recombineering, and site‐specific recombinase systems. We developed (1) a set of donor vectors based on the GoldenBraid platform, (2) an acceptor vector derived from the plant transformation‐competent artificial chromosome (TAC) vector, pYLTAC17, and (3) a re‐engineered recombineering‐readyE. colistrain, CZ105, based on SW105. The initial assembly steps are carried out using the donor vectors following standard GoldenBraid assembly procedures. Importantly, existing parts and transcriptional units created using compatible Golden Gate‐based systems can be transferred to the DASH donor vectors using standard single‐tube restriction/ligation reactions. The cargo DNA from a DASH donor vector is then efficiently transferredin vivoinE. coliinto the acceptor vector by the sequential action of a rhamnose‐inducible phage‐derived PhiC31 integrase and arabinose‐inducible yeast‐derived Flippase (FLP) recombinase using CZ105. Furthermore, recombineering‐based post‐assembly modification, including the removal of undesirable scars, is greatly simplified. To demonstrate the utility of the DASH system, a 116 kilobase (kb) DNA construct harbouring a 97 kb cargo consisting of 35 transcriptional units was generated. One of the coding DNA sequences (CDSs) in the final assembly was replaced through recombineering, and thein plantafunctionality of the entire construct was tested in both transient and stable transformants. 

Summary Advancement of DNA‐synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high‐complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single‐tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single‐tube digestion–ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone‐responsive, synthetic, repetitive proximal promoters were generated and testedin plantain the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene‐responsive promoter10x2EBS‐S10fused to theGUSreporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution. 

RationaleMass spectrometry imaging of young seedlings is an invaluable tool in understanding how mutations affect metabolite accumulation in plant development. However, due to numerous biological considerations, established methods for the relative quantification of analytes using infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) mass spectrometry imaging are not viable options. In this study, we report a method for the quantification of auxin‐related compounds using stable‐isotope‐labelled (SIL) indole‐3‐acetic acid (IAA) doped into agarose substrate. MethodsWild‐typeArabidopsis thalianaseedlings,sur2andwei8 tar2loss‐of‐function mutants, andYUC1gain‐of‐function line were grown for 3 days in the dark in standard growth medium. SIL‐IAA was doped into a 1% low‐melting‐point agarose gel and seedlings were gently laid on top for IR‐MALDESI imaging with Orbitrap mass spectrometry analysis. Relative quantification was performed post‐acquisition by normalization of auxin‐related compounds to SIL‐IAA in the agarose. Amounts of auxin‐related compounds were compared between genotypes to distinguish the effects of the mutations on the accumulation of indolic metabolites of interest. ResultsIAA added to agarose was found to remain stable, with repeatability and abundance features of IAA comparable with those of other compounds used in other methods for relative quantification in IR‐MALDESI analyses. Indole‐3‐acetaldoxime was increased insur2mutants compared with wild‐type and other mutants. Other auxin‐related metabolites were either below the limits of quantification or successfully quantified but showing little difference among mutants. ConclusionsAgarose was shown to be an appropriate sampling surface for IR‐MALDESI mass spectrometry imaging ofArabidopsisseedlings. SIL‐IAA doping of agarose was demonstrated as a viable technique for relative quantification of metabolites in live seedlings or tissues with similar biological considerations. 

Abstract Phytohormone ethylene regulates numerous aspects of plant physiology, from fruit ripening to pathogen responses. The molecular basis of ethylene biosynthesis and action has been investigated for over 40 years, and a combination of biochemistry, genetics, cell, and molecular biology have proven successful at uncovering the core machinery of the ethylene pathway. A number of molecular tools have been developed over the years that enable visualization of the sites of ethylene production and response in the plant. Genetically encoded biosensors take advantage of reporter proteins, i.e., fluorescent, luminescent, or colorimetric markers, to highlight the tissues that make, perceive, or respond to the hormone. This review describes the different types of biosensors currently available to the ethylene community and discusses potential new strategies for developing the next generation of genetically encoded ethylene reporters. 

Plants often live in adverse environmental conditions and are exposed to various stresses, such as heat, cold, heavy metals, salt, radiation, poor lighting, nutrient deficiency, drought, or flooding. To adapt to unfavorable environments, plants have evolved specialized molecular mechanisms that serve to balance the trade-off between abiotic stress responses and growth. These mechanisms enable plants to continue to develop and reproduce even under adverse conditions. Ethylene, as a key growth regulator, is leveraged by plants to mitigate the negative effects of some of these stresses on plant development and growth. By cooperating with other hormones, such as jasmonic acid (JA), abscisic acid (ABA), brassinosteroids (BR), auxin, gibberellic acid (GA), salicylic acid (SA), and cytokinin (CK), ethylene triggers defense and survival mechanisms thereby coordinating plant growth and development in response to abiotic stresses. This review describes the crosstalk between ethylene and other plant hormones in tipping the balance between plant growth and abiotic stress responses.