Understanding how to engineer transcriptional regulation in plants is key to advancing both fundamental knowledge and practical applications in plant biology. Native gene promoters, while widely used, are constrained by evolutionary pressures that limit their modularity, tunability, and predictability across genetic backgrounds and species. Synthetic promoters, artificial DNA sequences composed of defined cis-regulatory elements (CREs) for recruitment of gene-specific transcription factors (TFs) and general transcriptional machinery, provide a powerful alternative for achieving fine-tuned transcriptional control. This review examines the design and application of synthetic promoters in plants, emphasizing current strategies, ongoing challenges, and avenues for innovation. We cover the structure of plant promoter architecture, including the contributions of core, proximal, and distal regions, and highlight how promoter grammar (i.e., motif identity, motif distance from transcription start site, spacing between motifs, helical phase of TF binding, motif orientation, and combinatorial interactions between motifs) impacts transcriptional activity. We outline how synthetic promoters are designed and validated via high-throughput reporter assays. Applications of synthetic promoters are discussed across functional genomics studies, biosensor creation, logic gate-based genetic circuits, and practical crop engineering, with examples covering constitutively expressing, hormone-responsive, pathogen-inducible, and abiotic stress-responsive promoter designs. We discuss traditional and emerging computational frameworks that enable CRE identification, novel synthetic promoter generation, and prediction of promoter sequence activity in silico to inform the rational design of promoters with predictable performance and spatiotemporal expression. We emphasize the importance of integrating experimental studies and computational approaches through iterative Design-Build-Test-Learn (DBTL) cycles to standardize and optimize frameworks for synthetic promoter development. By combining insights from plant promoter studies with advances in both plant-specific and non-plant synthetic promoter generation and computational modeling, researchers can expand synthetic promoter libraries to enable complex man-driven transcriptional regulation across various plant systems.

Protein synthesis is an essential process for all living organisms and is tightly regulated to ensure the proper production of proteins needed for growth, development, and stress responses. As sessile organisms, plants have evolved distinct mechanisms to regulate translation, allowing them to adapt to their environment. In this review, we highlight the general translation process, discuss the translational machinery in plants, and examine cis-regulatory elements that influence translation. Additionally, we explore recent studies on how plants regulate translation in response to environmental and developmental cues.

Ethylene is a well-established negative regulator of nodulation, yet how ethylene biosynthesis and perception are spatially coordinated during early symbiotic signaling remains unresolved. Here, we investigate the dynamics of ethylene responses in Medicago (Medicago truncatula) using transcriptomics, promoter-reporter analyses, loss-of-function approaches and a synthetic reporter. We show that the activity of the ethylene-responsive EBSn reporter shifts from inner root tissues under nonsymbiotic conditions to the outer cortex and epidermis following rhizobial inoculation, revealing a spatial reprogramming of ethylene signaling. Among the 8 Medicago 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE (ACS) genes, MtACS3 is induced in outer root cell layers upon rhizobia application, while MtACS10 is repressed in the inner cortex and pericycle, mirroring the shift in ethylene perception. Functional analysis demonstrates that MtACS10 restricts nodule initiation, whereas MtACS3 modulates the number of infection threads, prevents nodule clustering, and contributes to the radial positioning of nodule primordia. Rhizobial induced ectopic ACS expression in the root interior counteracts MtACS10 repression and blocks nodulation, highlighting the requirement for spatially confined downregulation of ethylene biosynthesis. Together, these findings establish a framework in which localized shifts in ethylene biosynthesis, mediated by distinct Medicago ACS genes, balance infection and organogenesis while co-defining the spatial limits of the root susceptible zone.

Ascorbate is the most abundant water-soluble antioxidant in plants and an essential molecule for normal plant development. Although present in all green plants, ascorbate concentrations vary among plant species and tissues. While ascorbate accumulation is a trait of nutritional, and therefore, agronomical interest, the impact of different concentrations on cellular homeostasis remains elusive. To shed light on this question, we compared Arabidopsis (Arabidopsis thaliana) lines with very low (vtc2 mutant, 20% of wild-type (WT) levels), low (vtc4 mutant, 65% of WT levels), and high (vtc2/OE-VTC2, 165% of WT levels) ascorbate concentration in 4-wk-old rosette leaves. An 80% reduction of ascorbate increased the expression of genes implicated in defense against pathogens but repressed genes associated with abiotic stress responses. Unexpectedly, lines with increased (165% of WT) and decreased (65% of WT) ascorbate levels shared 85% of induced transcription factors and the gene ontology terms associated with their transcriptional programs. We identified TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1), a gene encoding the enzyme that catalyzes the first step of auxin biosynthesis, among the group of genes whose expression was positively correlated with ascorbate content. Using a combination of genetic and pharmacological approaches in fluorescent and histochemical reporter lines for auxin biosynthesis and signaling activity, we revealed that TAA1- and TAA1 RELATED 2 (TAR2)-mediated auxin biosynthesis is necessary for plants to cope with increased ascorbate concentration in a light-dependent manner, revealing a layer of complexity in the regulatory landscape of redox homeostasis.

Abstract Jasmonic and salicylic acids are key hormones involved in plant responses to pests and pathogens. Existing fluorescence-based approaches to imaging plant defence hormones are constrained by the need for external illumination and by autofluorescence of plant tissues, while luminescence-based ones require exogenous substrates. Here, we use jasmonate- and salicylate-responsive promoters to engineer autoluminescent plants that report hormone signalling activity with up to a 53-fold contrast. Using consumer-grade cameras, we image reporter Arabidopsis thaliana and Nicotiana benthamiana plants throughout normal development and in response to pest and pathogen attacks, visualising local and systemic responses. Because the luminescence is self-sustained, these reporters enable non-invasive, substrate-free imaging of defence signalling over extended time courses without specialised equipment.

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here we harness the bacterial antiphage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives homologous recombination, offering flexible and scar-free genome editing without base replacement or counterselection. In tested yeast, plant and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of chemical moieties to DNA to expand current modalities for precision gene editing.

Viral vectors can be useful tools for expressing recombinant proteins as well as delivering gene-editing machinery. Despite their utility, the development and subsequent optimisation of these tools is often a difficult and tedious process. Thus, although considerable work has been done to create useful viral vectors for gene editing and protein expression, there is a lack of understanding of how best to design these vectors for specific applications. For instance, it is often unclear whether the inclusion of heterologous promoter sequences or different viral components will improve cargo expression or replicon accumulation. To address some of these hurdles, we designed a GoldenBraid (GB)-compatible viral vector system based on the geminivirus-beet curly top virus (BCTV). This system allows for simple, modular cloning of a variety of reporter constructs. Making use of this modular cloning strategy, we compared a variety of alternative viral vector architectures. Interestingly, native BCTV promoters outperformed the constitutive 35S promoter, while the removal of the BCTV virion-sense genes promoted reporter expression. Intriguingly, these modifications had no effect on total replicon accumulation. These results show the utility of the new modular BCTV-based vectors for protein expression and gene targeting applications, as well as uncover design principles that may inform future geminivirus-based viral vector architectures. We anticipate that the availability of this new modular system will spark the broad adoption of replicon-based strategies in protein expression and gene editing experiments in plants.

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-ready E. coli strain, 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 transferred in vivo in E. coli into 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 the in planta functionality of the entire construct was tested in both transient and stable transformants.

Plant cells were one of the first cell types to be imaged by biologists seeking to understand the organizational properties of organisms and to develop new approaches for probing the inner workings of life. Since then, a myriad of new technologies have further enhanced our ability to define and understand the cell biology of plants. Plant cells display a diversity of highly organized and complex forms that are shaped dynamically by developmental programming and external factors. The beauty and wonder of the plant subcellular world continue to fascinate researchers and inspire new questions and technological innovation. This Special Issue aims to highlight recent advances in understanding the structural organization of plant cells, the function of subcellular organelles, and their dynamic relationship with each other and in response to endogenous and exogenous inputs. Additionally, this Special Issue features a sample of new technologies that are enabling cell biologists to probe plant cells with unparalleled precision, resolution, and scale. From these reviews, several themes emerge, as elaborated below.

Ethylene is a gaseous plant hormone that controls a wide array of physiologically relevant processes, including plant responses to biotic and abiotic stress, and induces ripening in climacteric fruits. To monitor ethylene in plants, analytical methods, phenotypic assays, gene expression analysis and transcriptional or translational reporters are typically employed. In the model plant Arabidopsis, two ethylene-sensitive synthetic transcriptional reporters have been described, 5xEBS:GUS and 10x2EBS-S10:GUS. These reporters harbour a different type, arrangement and number of homotypic cis-elements in their promoters and thus may recruit the ethylene master regulator EIN3 in the context of alternative transcriptional complexes. Accordingly, the patterns of GUS activity in these transgenic lines differ and neither of them encompasses all plant tissues even in the presence of saturating levels of exogenous ethylene. Herein, we set out to develop and test a more sensitive version of the ethylene-inducible promoter that we refer to as EBSnew (abbreviated as EBSn). EBSn leverages a tandem of 10 non-identical, natural copies of a novel, dual, everted, 11 bp-long EIN3-binding site, 2EBS(-1). We show that in Arabidopsis, EBSn outperforms its predecessors in terms of its ethylene sensitivity, having the capacity to monitor endogenous levels of ethylene and displaying more ubiquitous expression in response to the exogenous hormone. We demonstrate that the EBSn promoter is also functional in tomato, opening new avenues to manipulating ethylene-regulated processes, such as ripening and senescence, in crops.

Plants are essential for human survival. Over the past three decades, work with the reference plant Arabidopsis thaliana has significantly advanced plant biology research. One key event was the sequencing of its genome 25 years ago, which fostered many subsequent research technologies and datasets. Arabidopsis has been instrumental in elucidating plant-specific aspects of biology, developing research tools, and translating findings to crop improvement. It not only serves as a model for understanding plant biology and but also biology in other fields, with discoveries in Arabidopsis also having led to applications in human health, including insights into immunity, protein degradation, and circadian rhythms. Arabidopsis research has also fostered the development of tools useful for the wider biological research community, such as optogenetic systems and auxin-based degrons. This 4th Multinational Arabidopsis Steering Committee Roadmap outlines future directions, with emphasis on computational approaches, research support, translation to crops, conference accessibility, coordinated research efforts, climate change mitigation, sustainable production, and fundamental research. Arabidopsis will remain a nexus for discovery, innovation, and application, driving advances in both plant and human biology to the year 2030, and beyond.

Genome editing technologies like CRISPR/Cas have greatly accelerated the pace of both fundamental research and translational applications in agriculture. However, many plant biologists are functionally limited to creating small, targeted DNA changes or large, random DNA insertions. The ability to efficiently generate large, yet precise, DNA changes will massively accelerate crop breeding cycles, enabling researchers to more efficiently engineer crops amidst a rapidly changing agricultural landscape. This review provides an overview of existing technologies that allow plant biologists to integrate large DNA sequences within a plant host and some associated technical bottlenecks. Additionally, this review explores a selection of emerging techniques in other host systems to inspire tool development in plants.

The auxin indole-3-acetic acid (IAA) governs plant development and environmental responses. Although the indole-3-pyruvic acid (IPyA) pathway is the predominant route for IAA biosynthesis, other pathways have been proposed, such as the indole-3-acetaldoxime (IAOx) pathway. The IAOx pathway has garnered attention due to its supposed activation in auxin-overproducing mutants (e.g. sur1, sur2, ugt74b1) and the auxin-like responses triggered by exogenous application of its proposed intermediates IAOx, indole-3-acetonitrile (IAN), and indole-3-acetamide (IAM). However, despite the supporting evidence for individual steps of the IAOx pathway, its overall physiological relevance remains inconclusive. Here, using a comprehensive genetic approach combined with metabolic and phenotypic profiling, we demonstrate that mutating gene families proposed to function in the IAOx pathway in Arabidopsis (Arabidopsis thaliana) does not result in prominent auxin-deficient phenotypes, nor are these genes required for the high auxin production in the sur2 mutant. Our findings also challenge the previously postulated linear IAOx pathway. Exogenously provided IAOx, IAN, and IAM can be converted to IAA in vivo, but they do not act as precursors for each other. Finally, our findings question the physiological relevance of IAM and IAN as IAA precursors in plants and suggest the existence of a yet-uncharacterized route for IAA production in the sur2 mutant, likely involving IAOx as an intermediate. The identification of the metabolic steps and the corresponding genes in this pathway may uncover another IAA biosynthesis route in plants.

Reconstructing interglacial marine environments helps us understand the climate change mechanisms of the past. To contribute to this body of knowledge, we studied a high-resolution 455 cm-thick sediment sequence of the Boreal (Eemian) marine beds directly overlying Moscovian (Saalian) moraine in the Bychye-2 section on the Pyoza River. We analyzed lithological and microfossil (foraminifers, ostracods, pollen, aquatic palynomorphs) variations at the studied site. Stratigraphical zonation is based on the local and well-established regional pollen zones, correlated with the western European pollen zones. The studied marine beds accumulated from the end of the Moscovian glacial (>131 ka) until ca. 119.5 ka. We distinguished three successive phases: a seasonally sea-ice-covered, relatively deep, freshened basin in the initial rapid flooding stage (>131–130.5 ka); a deep basin in the maximum flooding phase with less extensive sea ice cover (130.5–130.25 ka); and a shallow basin with reduced sea ice cover (130.25–119.5 ka). According to a pollen zone comparison with other sites, the regional glacioisostatic rebound started ca. 130 ka. The diverse warm-water assemblages of benthic foraminifers and ostracods containing typical Baltic Sea species occurred during the regression, mainly 128–124 ka, thus giving evidence for a relatively long-lasting connection between the White and Baltic Seas.

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 laboratory and field scientists alike to take advantage of the vast Arabidopsis datasets, annotations, germplasm, constructs, methods, and 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.

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 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.

Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here, we harness the bacterial anti-phage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives efficient homologous recombination in tested bacteria, offering flexible and scar-free genome editing without base replacement nor counterselection. In tested eukaryotes including yeast, plants and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of a chemical moiety to DNA to expand current modalities for precision gene editing.

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.

Contribution: In this work-in-progress paper we describe the process of creating and validating a conceptual assessment in the field of sedimentology for undergraduate geoscience courses. The mechanism can aid future geoscience educators and researchers in the process of academic assessment development aligned with learning objectives in these courses. Background: Prior literature review supports the benefits of using active learning tools in STEM (Science, Technology, Engineering, and Mathematics) courses. This paper is part of a larger project to develop and incorporate research-based active learning software in sedimentology and other geoscience courses to improve grade point average (GPA) and time to graduation for Hispanic students at Texas A&M University. To evaluate the novel tool, we designed and validated the conceptual assessment instrument presented in this work. Research Question: What is the process to develop and validate a conceptual assessment for sedimentology? Methodology: This paper follows quantitative analysis and the assessment triangle approach and focuses on cognition, observation, and interpretation to design and evaluate the conceptual assessment. In the cognition element of the triangle, we explain the mechanism for creating the assessment instrument using students' learning objectives. The observation element explains the mechanism of data collection and the instrument revision. The interpretation element explains the results of the validation process using item response theory and reliability measures. We collected the conceptual assessment data from 17 participants enrolled in two courses where sedimentology topics are taught. Participants were geology majors in one of the courses and engineering majors in the other. Findings: The team developed a conceptual assessment that included eight multiple-choice (MCQ) and four open-ended response questions. The results of the design process described the conceptualization of questions and their validation. Also, the validity of created rubrics was established using inter-rater reliability measures, which showed good agreement between raters. Additionally, the results of the validation process indicated that the conceptual assessment was designed for students with average abilities.

Plant organ phenotyping represents a powerful tool to investigate the effects of biotic and abiotic factors on plant growth and development. Phenotyping typically involves the initial capture of high-resolution images of the plant of interest and the subsequent measurement of different morphometric parameters of specific plant organs. This second step is typically very time consuming and difficult to automate. To deal with this bottleneck, we developed a multi-class segmentation model based on U-shape network to identify hypocotyls and roots of young seedlings of the reference plant Arabidopsis thaliana. We applied a balanced cross entropy loss function to learn an alternative optimal network structure for this multi-class segmentation task. We evaluated our segmentation machine using 66 images of the wild-type Arabidopsis strain Col-0, as well as 34 images of the highly agravitropic and morphologically distinct mutant strain, aux1-7. Our model achieved a mean BFscore of 0.81 for Col-0 seedlings and 0.75 for aux1-7 mutant seedlings on the test dataset. Our model was also able to maintain accuracy in these two morphologically different genotypes suggesting that our segmentation procedure could be successfully applied to Arabidopsis seedlings showing broad morphological differences due to their genotype or treatment conditions. Appropriate segmentation is the first step in identifying phenotypic changes under hormone-mediated stress response. The identified growth parameters will be useful to identify the response associated with both abiotic and biotic stresses, which include but not limited to drought stress, heat stress, and the presence of pests or pathogens. The quantification of these parameters will aid assessment of genetic factors that contribute to the stress response.