Nitrogen (N) is an essential macronutrient and its bioavailability plays a major role in tuning plant development to the environmental nutrient status. To find novel factors in early root system architecture responses to N conditions, we performed Arabidopsis thaliana root transcriptome profiling of a short-term time course in limiting and sufficient N conditions. Using this data, we inferred transcriptional regulatory networks in each condition, which revealed the N-condition specific jasmonate responses. We found that the transcription factor (TF) ERF107 plays a generalized role in lateral root development while LBD13 is specific to N-limiting conditions. Further, we used a lateral root single-cell-identity specific transcriptome dataset to model the roles of TFs in early stages of lateral root development. We linked the N time course transcriptomics, lateral root mutant phenotypes and cell type-specific profiling approaches to find and test the roles of TFs that are involved in early root patterning responses to N conditions.

Inflorescences of flowering plants adopt diverse genetically programmed and environmentally tuned architectures. By contrast, continued maintenance of the stem cell pool within the apical meristem is unresponsive to environmental cues. Through a combination of modeling and experimentation in Arabidopsis, we reveal a negative feedback loop that buffers environmental signals. This loop comprises the determinacy-promoting pioneer transcription factor LEAFY (LFY) and the indeterminacy-promoting transcriptional corepressor TERMINAL FLOWER1 (TFL1). At the transition to the flower-producing reproductive phase, LFY directly and quantitatively up-regulates expression of TFL1. TFL1 in turn negatively feeds back on LFY to prevent LFY overaccumulation. This blocks inflorescence termination even under strong florally inductive signals. Our work uncovers a mechanism for robust environmental buffering involving differential responses of two cell populations to the same environmental stimulus.

Morphogen-mediated patterning is a highly dynamic developmental process. To obtain an accurate understanding of morphogen gradients, biophysical parameters such as protein diffusivities must be quantified in vivo . The dorsal-ventral (DV) patterning of early Drosophila embryos by the NF-κB homolog Dorsal (Dl) is an excellent system for understanding morphogen gradient formation. Dl gradient formation is controlled by the inhibitor Cactus/IκB (Cact), which regulates the nuclear import and diffusion of Dl protein. However, quantitative measurements of spatiotemporal Dl movement are currently lacking. Here, we use scanning fluorescence correlation spectroscopy to quantify the mobility of Dl. We find that the diffusivity of Dl varies along the DV axis, with lowest diffusivities on the ventral side, and the DV asymmetry in diffusivity is exclusive to the nuclei. Moreover, we also observe that nuclear export rates are lower in the ventral and lateral regions of the embryo. Both cross correlation spectroscopy measurements and a computational model of Dl/DNA binding suggest that DNA binding of Dl, which is more prevalent on the ventral side of the embryo, is correlated to a lower diffusivity and nuclear export rate. We propose that the variation in Dl/DNA binding along the DV axis is dependent on Cact binding Dl, which prevents Dl from binding DNA in dorsal and lateral regions of the embryo. Thus, our results highlight the complexity of morphogen gradient dynamics and the need for quantitative measurements of biophysical interactions in such systems.

Plant secondary growth is driven by 2 concentric meristems: the inner vascular cambium and outer cork cambium. The periclinal cell divisions of both meristems, providing thickness and protection to plant organs, are activated with a delay after the primary development. Cytokinins and a set of downstream transcription factors are key players in promoting transition from primary to secondary development; however, it is unknown whether other factors play a role in this transition. Here, using time-course transcriptome analysis of cytokinin-treated, cytokinin-deficient isopentenyltransferase1,3,5,7 (ipt1,3,5,7) mutant, we show that during cambium activation cytokinins positively regulate auxin and TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) peptide signaling in Arabidopsis (Arabidopsis thaliana) root. Correspondingly, mutants defective in TDIF peptide signaling displayed reduced cytokinin-induced secondary growth, and auxin signaling was found to be required for proper cytokinin response. Additionally, auxin and cytokinin signaling transiently overlapped in activating procambial cells and acted additively in promoting secondary development. Network analysis revealed that transcription factors belonging to the DNA-BINDING WITH ONE FINGER (DOF) and ETHYLENE RESPONSE FACTOR (ERF) gene families, are regulated by cytokinin during cambium activation and mutant analysis demonstrated delayed cambium activation and xylem formation phenotypes. Overall, we find that cytokinin, auxin, and TDIF form a tightly intertwined network of positive regulators for activation of secondary growth in the Arabidopsis root, indicating extensive redundancy in this process.

The integration of 3D bioprinting into plant science and biotechnology is revolutionizing research and applications. While many high-throughput techniques have advanced plant biology, replicating the complex 3D organization and cellular environments of plant tissues remains a significant challenge. Traditional 2D culture systems fall short of capturing the necessary spatial context for accurate studies of cell behavior, gene expression, and tissue development. Additionally, the lack of precise simulation of plant microenvironments limits control over cellular interactions and responses to external stimuli. Recent advancements in 3D bioprinting address these limitations by allowing precise control over cell positioning and biomaterial arrangement, thereby better replicating natural plant environments. This enables more accurate studies of gene expression, developmental processes, and stress responses. The technology also enhances our ability to test genetic modifications and biotechnological interventions, advancing crop improvement, sustainable agriculture, and precision breeding. This review examines the current state of 3D bioprinting in plant science, discusses its limitations, and explores its potential to transform research and applications in the field.

Plant root development is tightly regulated and depends on the coordination among different cell types located in the root stem cell niche (SCN). A complex network of regulators includes the gasotransmitter nitric oxide (NO) known to inhibit primary root growth, modify root architecture and alter meristem organisation. However, little is known about the molecular targets and the mechanistic insights of this signalling molecule during root SCN homeostasis. Here, genetic, molecular and physiological evidence support that TGA transcription factors of the bZIP family, and specifically PERIANTHIA (PAN), contribute to NO-mediated root stem cell regulation. NO induces reversible S-nitrosylation of PAN, thereby inhibiting its binding to DNA and associated transcriptional activation. In turn, the protein-SNO reductase activity of TRXh5 is able to denitrosylate PAN. This switch on and off mechanism of PAN activation modulates WOX5 expression and quiescent centre activity, which alters columella stem cell maintenance through a NO-dependent mechanism. Different TGA members interact with PAN and are also S-nitrosylated, whose expression in the root SCN together with their accumulated mutations contribute to NO sensing during root apical meristem development. Collectively, these findings reveal a molecular framework where NO controls the ability of PAN to bind to its targets as a master regulator of the root SCN. ### Competing Interest Statement The authors have declared no competing interest.

Feeding the growing human population sustainably amidst climate change is one of the most important challenges in the 21st century. Current practices often lead to the overuse of agronomic inputs, such as synthetic fertilizers and water, resulting in environmental contamination and diminishing returns on crop productivity. The complexity of agricultural systems, involving plant-environment interactions and human management, presents significant scientific and technical challenges for developing sustainable practices. Addressing these challenges necessitates transdisciplinary research, involving intense collaboration among fields such as plant science, engineering, computer science, and social sciences. Five case studies are presented here demonstrating successful transdisciplinary approaches toward more sustainable water and fertilizer use. These case studies span multiple scales. By leveraging whole-plant signaling, reporter plants can transform our understanding of plant communication and enable efficient application of water and fertilizers. The use of new fertilizer technologies could increase the availability of phosphorus in the soil. To accelerate advancements in breeding new cultivars, robotic technologies for high-throughput plant screening in different environments at a population scale are discussed. At the ecosystem scale, phosphorus recovery from aquatic systems and methods to minimize phosphorus leaching are described. Finally, as agricultural outputs affect all people, integration of stakeholder perspectives and needs into research is outlined. These case studies highlight how transdisciplinary research and cross-training among biologists, engineers, and social scientists bring diverse expertise to tackling grand challenges in sustainable agriculture, driving discovery and innovation.

Understanding root development is critical for enhancing plant growth and health, and advanced technologies are essential for unraveling the complexities of these processes. In this review, we highlight select technological innovations in the study of root development, with a focus on the transformative impact of single-cell gene expression analysis. We provide a high-level overview of recent advancements, illustrating how single-cell RNA sequencing (scRNA-seq) has become a pivotal tool in plant biology. scRNA-seq has revolutionized root biology by enabling detailed, cell-specific analysis of gene expression. This has allowed researchers to create comprehensive root atlases, predict cell development, and map gene regulatory networks (GRNs) with unprecedented precision. Complementary technologies, such as multimodal profiling and bioinformatics, further enrich our understanding of cellular dynamics and gene interactions. Innovations in imaging and modeling, combined with genetic tools like CRISPR, continue to deepen our knowledge of root formation and function. Moreover, the integration of these technologies with advanced biosensors and microfluidic devices has advanced our ability to study plant-microbe interactions and phytohormone signaling at high resolution. These tools collectively provide a more comprehensive understanding of root system architecture and its regulation by environmental factors. As these technologies evolve, they promise to drive further breakthroughs in plant science, with substantial implications for agriculture and sustainability.

Sustainable phosphorus fertilization is a growing challenge in agriculture. Phosphorus is necessary for plant growth, but it is typically only bioavailable in its orthophosphate form. Phosphate fertilizers contribute to environmental damage as they leach into aquatic ecosystems. Therefore, it is imperative to develop new fertilization techniques such as controlled-release small-scale phosphate fertilizers. However, iteratively optimizing various new fertilizers using a comparable method is difficult. Here, we use three-dimensional bioprinting as a high-throughput screening platform to evaluate cellular phosphate uptake of various phosphate sources, including triple super phosphate, diammonium phosphate and struvite, which are composed of different chemistries and scales. As a result, we identified ideal phosphate fertilizer sources for the development of controlled-release phosphate fertilizers. Then, we evaluated whether plant growth and root architecture responded differently to the ideal controlled-release fertilizers. This study demonstrates the utility of this screening platform in developing a controlled-release phosphate fertilizer that effectively provides phosphate to plants at the microparticle scale.

Elevated temperatures resulting from climate change adversely affect natural and crop ecosystems, necessitating the development of heat-tolerant crops. Here, we established a framework to precisely identify protein phosphorylation sites associated with varying temperature sensitivities in wheat (Triticum aestivum). We identified specific kinases primarily associated with particular temperatures, but our results also suggest a striking overlap between cold and heat signaling. Furthermore, we propose that the phosphorylation state of a specific set of proteins may represent a signature for heat stress tolerance. These findings can potentially aid in the identification of targets for breeding or genome editing to enhance the sub- and supra-optimal temperature tolerance of crops.

Plant cell differentiation incorporates environmental cues over time to optimize overall growth. Iron deficiency influences development, such as root hair, cortical, and endodermal cell differentiation. However, the mechanisms by which iron deficiency regulates cell differentiation are not well characterized. Root sieve elements serve as an excellent model for cell differentiation since all cells, from undifferentiated to differentiated, are present in a distinct cell file. Here, we use semi-automated image analysis to show that iron deficiency delays sieve element differentiation, particularly enucleation and cell wall thickening, and consequently delays phloem sap unloading to roots. Using Dynamic Bayesian modeling we also characterize how iron deficiency changes the fundamental structure of the gene regulatory network associated with sieve element differentiation. We identify DOF1.5 as a positive regulator of sieve element enucleation and, consequently, of root sap translocation. These results clarify how abiotic stress can influence overall plant growth as a consequence of negatively influencing vascular differentiation.

The emerging bioeconomy presents a promising solution to both economic and environmental challenges. Within the bioeconomy, plants serve as a renewable, sustainable, and cost-effective source of foods, fuels, chemicals, and materials. However, traditional breeding and single-gene engineering approaches fall short in addressing complex traits (e.g., drought tolerance, disease resistance, yield, nutrient use efficiency) which are controlled by multiple genes. The complexity of plant biology often necessitates the use of multigene engineering (MGE), which involves simultaneous ectopic expression, up/down-regulation, or editing of multiple genes, to enhance plant traits relevant to the bioeconomy. These genes may be associated with distinct traits or function as components of specific metabolic and regulatory pathways. This review summarizes current technologies for MGE within the synthetic biology-driven Design-Build-Test-Learn (DBTL) framework, detailing its four key stages: Design - gene construct development; Build - DNA assembly and plant transformation; Test - the molecular, biochemical, and physiological characterization of engineered plants; and Learn - computational modeling to refine, multiplex and iterate the process. Despite good progress in the applications of MGE in biofortification, metabolic engineering, and stress resilience, challenges remain in construct stability, coordinated gene expression, and regulatory predictability. We identified optimization paths and future directions to accelerate MGE deployment in sustainable agriculture, with possible societal benefits including reduced production costs, increased yield, and improved food and nutritional security.

Uneven global distribution of phosphate rock deposits and the supply chains to transport phosphorus (P) make P fertilizers vulnerable to exogenous shocks, including commodity market shocks; extreme weather events or natural disasters; and geopolitical instability, such as trade disputes, disruption of shipping routes, and war. Understanding bidirectional risk transmission (global-to-local and local-to-global) in P supply and consumption chains is thus essential. Ignoring P system interdependencies and associated risks could have major impacts on critical infrastructure operations and increase the vulnerability of global food systems. We highlight recent unanticipated events and cascading effects that have impacted P markets globally. We discuss the need to account for exogenous shocks in local assessments of P flows, policies, and infrastructure design choices. We also provide examples of how accounting for undervalued global risks to the P industry can hasten the transition to a sustainable P future. For example, leveraging internal P recycling loops, improving plant P use efficiency, and utilizing legacy soil P all enhance system resiliency in the face of exogenous shocks and long-term anticipated threats. Strategies applied at the local level, which are embedded within national and global policy systems, can have global-scale impacts in derisking the P supply chain.

Elevated temperatures resulting from climate change adversely affect natural and crop ecosystems, necessitating the development of heat-tolerant crops. We established a framework to precisely identify wheat protein-phosphorylated sites associated with varying temperature sensitivities. We identified specific kinases primarily associated with a specific temperature, but our results also suggest a striking overlap between cold and heat signaling. We furthermore demonstrated that the phosphorylation state of a specific set of proteins creates a unique signature for heat stress tolerance. These findings can potentially aid in the identification of targets for breeding or genome editing to enhance sub/supra optimal temperature tolerance. ### Competing Interest Statement The authors have declared no competing interest.

Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs(1). To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs.

Today’s challenges with sustainability are driven by complexity, lack necessary information, resist straightforward solutions, span multiple scales, and encompass diverse or sometimes conflicting perspectives. To tackle these issues effectively, research organizations need tools that support and accelerate the integration of disciplinary knowledge across both natural and social sciences so that they can explore and execute workable solutions. Boundary objects are tools that can bring diverse perspectives together through a shared point of focus that is meaningful across different groups and perspectives, enhancing communication between them. Here, we introduce a framework to develop Triple Bottom Line Scenario Sites (TBL Sites) as “convergence” boundary objects and intervention testbeds to support a holistic approach to sustainability research within multidisciplinary and multi-institutional organizations. We describe four key criteria used to identify a potential TBL Site: (1) proximity to researchers, (2) a bounded geographic location encompassing a particular ecosystem, (3) an integrated stakeholder network, and (4) access to existing resources. We explain how TBL Sites may be used to assess research impacts on environmental, economic, and social sustainability goals. Finally, we provide examples of aquatic, agricultural, and urban TBL Sites used by the Science and Technologies for Phosphorus Sustainability (STEPS) Center, demonstrating how these boundary objects facilitate convergence across a large multidisciplinary research team to tackle sustainable phosphorus management.

All differentiated root cells derive from stem cells spatially organized within the stem cell niche (SCN), a microenvironment located within the root tip. Here, we compiled recent advances in the understanding of how the SCN drives the establishment and maintenance of cell types. The quiescent center (QC) is widely recognized as the primary driver of cell fate determination, but it is recently considered a convergence center of multiple signals. Cell identity of the cortex endodermis initials is mainly driven by the regulatory feedback loops between transcription factors (TFs), acting as mobile signals between neighboring cells, including the QC. As exemplified in the vascular initials, the precise spatial expression of these regulatory TFs is connected with a dynamic hormonal interplay. Thus, stem cell maintenance and cell differentiation are regulated by a plethora of signals forming a complex, multilevel regulatory network. Integrating the transcriptional and post-translational regulations, protein-protein interactions, and mobile signals into models will be fundamental for the comprehensive understanding of SCN maintenance and differentiation.

3D bioprinting is an additive manufacturing technology with promise towards facilitating tissue engineering and single-cell investigations of cellular development and microenvironment responses. 3D bioprinting is still a new technology in the field of plant biology so its optimization with plant cells is still widely needed. Here, we present a study in which 3D bioprinting parameters, such as needle gauge, extrusion pressure, and scaffold type, were all tested in 3D bioprinted Tobacco BY-2 cells to evaluate how cell viability is responsive to each parameter. As a result, this study revealed an optimal range of extrusion pressures and needle gauges that resulted in an optimum cell viability. Furthermore, this study applied the identified optimal 3D bioprinting parameters to a different cell line, Arabidopsis root protoplasts, and stress condition, phosphate starvation, to confirm that the identified parameters were optimal in a different species, cell type, and cellular microenvironment. This suggested that phosphate-starved bioprinted Arabidopsis cells were less viable by 7 days, which was consistent with whole root phosphate starvation responses. As a result, the 3D bioprinter optimization yielded optimal cell viabilities in both BY-2 and Arabidopsis cells and facilitated an applied investigation into phosphate starvation stress.### Competing Interest StatementThe authors have declared no competing interest.