Sourcing DNA parts for synthetic biology applications in plants.

Transgenic approaches are now standard in plant biology research aiming to characterize gene function or improve crops. Recent advances in DNA synthesis and assembly make constructing transgenes a routine task. What remains nontrivial is the selection of the DNA parts and optimization of the transgene design. Early career researchers and seasoned molecular biologists alike often face difficult decisions on what promoter or terminator to use, what tag to include, and where to place it. This review aims to inform about the current approaches being employed to identify and characterize DNA parts with the desired functionalities and give general advice on basic construct design. Furthermore, we hope to share the excitement about new experimental and computational tools being developed in this field.

Arabidopsis as a model for translational research.

Arabidopsis thaliana is currently the most-studied plant species on earth, with an unprecedented number of genetic, genomic, and molecular resources having been generated in this plant model. In the era of translating foundational discoveries to crops and beyond, we aimed to highlight the utility and challenges of using Arabidopsis as a reference for applied plant biology research, agricultural innovation, biotechnology, and medicine. We hope that this review will inspire the next generation of plant biologists to continue leveraging Arabidopsis as a robust and convenient experimental system to address fundamental and applied questions in biology. We aim to encourage lab and field scientists alike to take advantage of the vast Arabidopsis datasets, annotations, germplasm, constructs, methods, molecular and computational tools in our pursuit to advance understanding of plant biology and help feed the world's growing population. We envision that the power of Arabidopsis-inspired biotechnologies and foundational discoveries will continue to fuel the development of resilient, high-yielding, nutritious plants for the betterment of plant and animal health and greater environmental sustainability.

A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters.

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 tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter 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.

Deciphering the molecular basis of tissue-specific gene expression in plants: Can synthetic biology help?

Gene expression differences between distinct cell types are orchestrated by specific sets of transcription factors and epigenetic regulators acting upon the genome. In plants, the mechanisms underlying tissue-specific gene activity remain largely unexplored. Although transcriptional and epigenetic profiling of individual organs, tissues, and more recently, of single cells can easily detect the molecular signatures of different biological samples, how these unique cell identities are established at the mechanistic level is only beginning to be decoded. Computational methods, including machine learning, used in combination with experimental approaches, enable the identification and validation of candidate cis-regulatory elements driving cell-specific expression. Synthetic biology shows great promise not only as a means of testing candidate DNA motifs but also for establishing the general rules of nature driving promoter architecture and for the rational design of genetic circuits in research and agriculture to confer tissue-specific expression to genes or molecular pathways of interest.

A Ribo-Seq Method to Study Genome-Wide Translational Regulation in Plants.

Protein production from mRNA is one of the fundamental molecular processes in a cell. Accurate genome-wide information on the levels of translation and ribosome distribution on mRNA can be gathered by carrying out ribosome footprinting, aka Ribo-seq. Herein, we present a detailed protocol describing the construction of parallel Ribo-seq and RNA-seq libraries from Arabidopsis seedlings treated with the plant hormone auxin. The improved protocol for ribosome footprint library generation can be easily adapted to analyzing the effects on translation of genetic perturbations and various abiotic and biotic factors to shed the much-needed light on translational regulation in plants.

Auxin Interactions with Other Hormones in Plant Development.

Auxin is a crucial growth regulator that governs plant development and responses to environmental perturbations. It functions at the heart of many developmental processes, from embryogenesis to organ senescence, and is key to plant interactions with the environment, including responses to biotic and abiotic stimuli. As remarkable as auxin is, it does not act alone, but rather solicits the help of, or is solicited by, other endogenous signals, including the plant hormones abscisic acid, brassinosteroids, cytokinins, ethylene, gibberellic acid, jasmonates, salicylic acid, and strigolactones. The interactions between auxin and other hormones occur at multiple levels: hormones regulate one another's synthesis, transport, and/or response; hormone-specific transcriptional regulators for different pathways physically interact and/or converge on common target genes; etc. However, our understanding of this crosstalk is still fragmentary, with only a few pieces of the gigantic puzzle firmly established. In this review, we provide a glimpse into the complexity of hormone interactions that involve auxin, underscoring how patchy our current understanding is.

Leveraging synthetic biology approaches in plant hormone research.

The study of plant hormones is critical to understanding development, physiology and interactions of plants with their environment. Synthetic biology holds promise to provide a new perspective and shed fresh light on the molecular mechanisms of plant hormone action and propel the design of novel biotechnologies. With the recent adoption of synthetic biology in plant sciences, exciting first examples of successful tool development and their applications in the area of plant hormone research are emerging, paving the way for new cadres to enter this promising field of science.

To Fight or to Grow: The Balancing Role of Ethylene in Plant Abiotic Stress Responses.

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.

Structure-Function Analysis of Interallelic Complementation in ROOTY Transheterozygotes.

Auxin is a crucial plant growth regulator. Forward genetic screens for auxin-related mutants have led to the identification of key genes involved in auxin biosynthesis, transport, and signaling. Loss-of-function mutations in genes involved in glucosinolate biosynthesis, a metabolically related route that produces defense compounds, result in auxin overproduction. We identified an allelic series of fertile, hypomorphic Arabidopsis (Arabidopsis thaliana) mutants for the essential glucosinolate biosynthetic gene ROOTY (RTY) that exhibit a range of phenotypic defects characteristic of enhanced auxin production. Genetic characterization of these lines uncovered phenotypic suppression by cyp79b2 cyp79b3, wei2, and wei7 mutations and revealed the phenomenon of interallelic complementation in several RTY transheterozygotes. Structural modeling of RTY elucidated the relationships between structure and function in the RTY homo- and heterodimers, and unveiled the likely structural basis of interallelic complementation. This work underscores the importance of employing true null mutants in genetic complementation studies.

Development of a relative quantification method for infrared matrix-assisted laser desorption electrospray ionization mass spectrometry imaging of Arabidopsis seedlings.

RATIONALE: Mass 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. METHODS: Wild-type Arabidopsis thaliana seedlings, sur2 and wei8 tar2 loss-of-function mutants, and YUC1 gain-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. RESULTS: IAA 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 in sur2 mutants 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. CONCLUSIONS: Agarose was shown to be an appropriate sampling surface for IR-MALDESI mass spectrometry imaging of Arabidopsis seedlings. 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.

An Improved Recombineering Toolset for Plants.

Gene functional studies often rely on the expression of a gene of interest as transcriptional and translational fusions with specialized tags. Ideally, this is done in the native chromosomal contexts to avoid potential misexpression artifacts. Although recent improvements in genome editing have made it possible to directly modify the target genes in their native chromosomal locations, classical transgenesis is still the preferred experimental approach chosen in most gene tagging studies because of its time efficiency and accessibility. We have developed a recombineering-based tagging system that brings together the convenience of the classical transgenic approaches and the high degree of confidence in the results obtained by direct chromosomal tagging using genome-editing strategies. These simple, scalable, customizable recombineering toolsets and protocols allow a variety of genetic modifications to be generated. In addition, we developed a highly efficient recombinase-mediated cassette exchange system to facilitate the transfer of the desired sequences from a bacterial artificial chromosome clone to a transformation-compatible binary vector, expanding the use of the recombineering approaches beyond Arabidopsis (Arabidopsis thaliana). We demonstrated the utility of this system by generating more than 250 whole-gene translational fusions and 123 Arabidopsis transgenic lines corresponding to 62 auxin-related genes and characterizing the translational reporter expression patterns for 14 auxin biosynthesis genes.

Local Auxin Biosynthesis Is a Key Regulator of Plant Development.

Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.

A Plant Biologist's Toolbox to Study Translation.

Across a broad range of species and biological questions, more and more studies are incorporating translation data to better assess how gene regulation occurs at the level of protein synthesis. The inclusion of translation data improves upon, and has been shown to be more accurate than, transcriptional studies alone. However, there are many different techniques available to measure translation and it can be difficult, especially for young or aspiring scientists, to determine which methods are best applied in specific situations. We have assembled this review in order to enhance the understanding and promote the utilization of translational methods in plant biology. We cover a broad range of methods to measure changes in global translation (e.g., radiolabeling, polysome profiling, or puromycylation), translation of single genes (e.g., fluorescent reporter constructs, toeprinting, or ribosome density mapping), sequencing-based methods to uncover the entire translatome (e.g., Ribo-seq or translating ribosome affinity purification), and mass spectrometry-based methods to identify changes in the proteome (e.g., stable isotope labeling by amino acids in cell culture or bioorthogonal noncanonical amino acid tagging). The benefits and limitations of each method are discussed with a particular note of how applications from other model systems might be extended for use in plants. In order to make this burgeoning field more accessible to students and newer scientists, our review includes an extensive glossary to define key terms.

The Triple Response Assay and Its Use to Characterize Ethylene Mutants in Arabidopsis.

Exposure of plants to ethylene results in drastic morphological changes. Seedlings germinated in the dark in the presence of saturating concentrations of ethylene display a characteristic phenotype known as the triple response. This phenotype is robust and easy to score. In Arabidopsis the triple response is usually evaluated at 3 days post germination in seedlings grown in the dark in rich media supplemented with 10 µM of the ethylene precursor ACC in air or in unsupplemented media in the presence of 10 ppm ethylene. The triple response in Arabidopsis consists of shortening and thickening of hypocotyls and roots and exaggeration of the curvature of apical hooks. The search for Arabidopsis mutants that fail to show this phenotype in ethylene or, vice versa, display the triple response in the absence of exogenously supplied hormone has allowed the identification of the key components of the ethylene biosynthesis and signaling pathways. Herein, we describe a simple protocol for assaying the triple response in Arabidopsis. The method can also be employed in many other dicot species, with minor modifications to account for species-specific differences in germination. We also compiled a comprehensive table of ethylene-related mutants of Arabidopsis, including many lines with auxin-related defects, as wild-type levels of auxin biosynthesis, transport, signaling, and response are necessary for the normal response of plants to ethylene.

Translation regulation in plants: an interesting past, an exciting present and a promising future.

Changes in gene expression are at the core of most biological processes, from cell differentiation to organ development, including the adaptation of the whole organism to the ever-changing environment. Although the central role of transcriptional regulation is solidly established and the general mechanisms involved in this type of regulation are relatively well understood, it is clear that regulation at a translational level also plays an essential role in modulating gene expression. Despite the large number of examples illustrating the critical role played by translational regulation in determining the expression levels of a gene, our understanding of the molecular mechanisms behind such types of regulation has been slow to emerge. With the recent development of high-throughput approaches to map and quantify different critical parameters affecting translation, such as RNA structure, protein-RNA interactions and ribosome occupancy at the genome level, a renewed enthusiasm toward studying translation regulation is warranted. The use of these new powerful technologies in well-established and uncharacterized translation-dependent processes holds the promise to decipher the likely complex and diverse, but also fascinating, mechanisms behind the regulation of translation.

Cutting Out the Middle Man in Light-Hormone Interactions.

In this issue of Developmental Cell, Shi et al. (2016a) show that red-light-activated phytochrome B interacts with transcriptional regulators of ethylene signaling, EIN3/EIL1, triggering their degradation by bringing the F-box proteins EBF1 and 2 to the complex. These findings provide a paradigm for crosstalk between light and hormone signaling pathways.