Nature Plants

Biological DNA transfer into plant cells mediated by Agrobacterium represents one of the most powerful tools for the engineering and study of plant systems. Transient expression of transfer DNA (T-DNA) in particular enables rapid testing of gene products and has recently been harnessed for facile combinatorial expression of multiple genes. In analogous mammalian cell-based gene expression systems, a clear sense of the multiplicity of infection (MOI) allows users to predict and control viral transfection frequencies for applications requiring single vs. multiple transfection events per cell. Despite the value of Agrobacterium-mediated transient transformation of plants, MOI has not been quantified. Establishing MOI for Agrobacterium T-DNA delivery at the level of single plant cells would allow users to design genomic library delivery conditions (at most 1 event/cell), or maximize co-delivery of T-DNA loads from separate Agrobacterium (>1 event/cell). Here, we analyze the Poisson probability distribution of T-DNA transfer in leaf pavement cells to determine the MOI for the widely used model system Agrobacterium GV3101/Nicotiana benthamiana. These data delineate the relationship between an individual Agrobacterium strain infiltration OD600, plant cell perimeter and leaf age, as well as plant cell co-infection rates. Our analysis also establishes experimental regimes where the probability of near-simultaneous delivery of >20 unique T-DNAs to a given plant cell remains high throughout the leaf. We anticipate that these data will enable users to develop new approaches to in-leaf library development using Agrobacterium transient expression and the reliable combinatorial assaying of multiple heterologous proteins in a single plant cell.

Photosynthesis by which plants convert carbon dioxide to sugars using the energy of light is fundamental to life as it forms the basis of nearly all food chains. Surprisingly, our knowledge about its transcriptional regulation remains incomplete. Effort for its agricultural optimization have mostly focused on post-translational regulatory processes1-3 but photosynthesis is regulated at the post-transcriptional4 and the transcriptional level5. Stacked transcription factor mutations remain photosynthetically active5,6 and additional transcription factors have been difficult to identify possibly due to redundancy6 or lethality. Using a random forest decision tree-based machine learning approach for gene regulatory network calculation7 we determined ranked candidate transcription factors and validated five out of five tested transcription factors as controlling photosynthesis in vivo. The detailed analyses of previously published and newly identified transcription factors suggest that photosynthesis is transcriptionally regulated in a partitioned, non-hierarchical, interlooped network.

Fluctuating bioavailability of inorganic phosphate (Pi), often caused by complex Pi-metal interactions, guide root tip growth and root system architecture for maximizing the foraged soil volume. Two interacting genes in Arabidopsis thaliana, PDR2 (P5-type ATPase) and LPR1 (multicopper oxidase), are central to external Pi monitoring by root tips, which is modified by iron (Fe) co-occurrence. Upon Pi deficiency, the PDR2-LPR1 module facilitates cell type-specific Fe accumulation and cell wall modifications in root meristems, inhibiting intercellular communication and thus root growth. LPR1 executes local Pi sensing, whereas PDR2 restricts LPR1 function. We show that native LPR1 displays specific ferroxidase activity and requires a conserved acidic triad motif for high-affinity Fe2+ binding and root growth inhibition under limiting Pi. Our data indicate that substrate availability tunes LPR1 function and implicate PDR2 in maintaining Fe homeostasis. LPR1 represents the prototype of an ancient ferroxidase family, which evolved very early upon bacterial colonization of land. During plant terrestrialization, horizontal gene transfer transmitted LPR1-type ferroxidase from soil bacteria to the common ancestor of Zygnematophyceae algae and embryophytes, a hypothesis supported by homology modeling, phylogenomics, and activity assays of bacterial LPR1-type multicopper oxidases.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has inherently low catalytic efficiency, making it a key target for photosynthesis and crop improvement. However, introducing mutations to the chloroplast-encoded Rubisco large subunit (rbcL), which contains the enzymes catalytic sites, is technically challenging. In this study, we successfully generated a range of chloroplast-genome-edited Arabidopsis thaliana plants targeting rbcL by a targeted base editor, ptpTALECD. The M309I and D397N substitutions in rbcL resulted in an increased Rubisco catalytic rate (kcat) without any reductions of Rubisco content, thereby enhancing photosynthetic rates and plant growth under both current atmospheric CO2 concentrations (i.e., 381 mol mol-1) and projected future concentrations (i.e., 549 mol mol-1). Cryo-electron microscopy (cryo-EM) structural analysis showed that the M309I and D397N substitutions, although located far from the catalytic site, induce structural alterations in the catalytic (60s) loops. Our findings highlight the potential of Rubisco engineering to improve plant photosynthesis and growth, and underscore the unique opportunities that chloroplast genome editing offers for enhancing photosynthesis and crop productivity and reducing atmospheric CO2 levels in a non-GMO context.

RNA degradation inside the plant vacuole by the ribonuclease RNS2 is essential for maintaining nucleotide concentrations and cellular homeostasis via the nucleotide salvage pathway. However, the mechanisms by which RNA is transported into the vacuole are not well understood. While selective macroautophagy may contribute to this transport, macroautophagy-independent transport pathways also exist. Here we demonstrate a mechanism for direct RNA transport into vacuoles that is active in purified vacuoles and is ATP hydrolysis-dependent. We identify the RNA helicase SKI2 as a factor required for this transport pathway, as ski2 mutant vacuoles are defective in transport. ski2 mutants have an increased autophagy phenotype that can be rescued by exogenous addition of inosine, consistent with a function in nucleotide salvage. This newly-described transport mechanism is therefore critical for RNA degradation, recycling and cytoplasmic nucleotide homeostasis.

Genome editing allows precise DNA manipulation, but its potential is limited in many crops by low regeneration efficiencies and few transformable genotypes. Here, we show that expression of a chimeric protein including wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) dramatically increases the efficiency and speed of regeneration in wheat, triticale and rice and expands the number of transformable wheat genotypes. Moreover, GRF4-GIF1 induces efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. By combining GRF4-GIF1 and CRISPR-Cas9 technologies, we were able to generate large numbers of edited wheat plants. The GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects, likely due to post-transcriptional regulatory mechanisms operating on GRF4 in adult tissues. Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus suggesting that this strategy can be expanded to dicot crops.

Plant cell wall enlargement is fundamental to crop productivity and its sensitivity to drought1. Tip growth and diffuse growth are contrasting wall enlargement patterns often proposed to be limited by different processes: localized secretion and remodeling of pectins for tip growth versus loosening and sliding of cellulosic networks by -expansins (EXPAs) for diffuse growth2,3. Here, we knocked out root-hair specific EXPA7 and EXPA18 in Arabidopsis, abolishing root-hair tip growth which was restored by complementation with genes from some, but not all, expansin clades. Notably, EXPA13 and EXPA20 failed to complement; they belong to two ancient clades lacking a highly conserved Asp considered essential for expansin activity. Mutation of this Asp in EXPA7 confirmed its requirement for wall enlargement. EXPA-mCherry fusions revealed widely contrasting patterns of subcellular trafficking and wall-binding for different EXPAs. The results demonstrate an essential EXPA requirement for root-hair tip growth and uncover a greater diversity of expansin functions than previously recognized.

Coumarins are phenylpropanoid-derived specialized metabolites that contribute to plant defence, shape plant-microbe interactions in the rhizosphere, and promote iron acquisition. In Arabidopsis thaliana, a model plant for iron-responsive coumarin metabolism, the enzymatic origin of the catecholic coumarin esculetin has long remained unresolved. Here we identify the first O-demethylation reaction in Arabidopsis specialized metabolism and show that 2-oxoglutarate- and Fe(II)-dependent dioxygenases catalyze scopoletin 6-O-demethylation to form esculetin. We designate these enzymes scopoletin 6-O-demethylases (S6ODs) and validate their activity through biochemical characterization, together with metabolomic profiling and independent loss-of-function mutant lines providing genetic evidence in planta. Disruption of S6OD activity remodels coumarin profiles and alters plant performance under limited iron availability, indicating that esculetin biosynthesis contributes to plant responses under these conditions. Our findings resolve the long-sought missing step in esculetin biosynthesis. It establishes O-demethylation as a previously unrecognized reaction in Arabidopsis specialized metabolism and suggest that 2OGD-mediated O-demethylation is recurrently recruited during evolution of plant metabolism, with implications for metabolic engineering and improvement of iron acquisition traits in crops.

The endosperm is a seed tissue supporting embryo growth, similar to the placenta in mammals. It originates after fertilization of the maternal central cell by one of the paternal sperm cells. In the early stages of Arabidopsis thaliana seed development, nuclei divisions in the endosperm are not followed by cellularization. After a defined number of mitotic cycles, the endosperm cellularizes and stops dividing. The timing of endosperm cellularization impacts on final seed size and is differentially controlled by maternal and paternal genome contributions. While increased maternal genome dosage causes early endosperm cellularization and the formation of small seeds, the opposite is caused by increased paternal genome dosage. The parental factors controlling the differential timing of endosperm cellularization remain largely unexplored. Here, we show that a family of maternally expressed auxin response factors (ARFs) promotes endosperm cellularization and regulates final seed size. One-Sentence SummaryEndosperm cellularization is under antagonistic parental control that converges on Auxin Response Factors.

Precise cis-regulatory control of gene expression is essential for plant growth. In Arabidopsis thaliana, PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY) peptides and their receptors (PSYRs) mediate growth-stress trade-offs, yet the transcriptional regulation of these genes remains poorly understood. Here, we mapped transcription factor (TF)-promoter interactions for nine PSY and three PSYR genes by combining high-throughput enhanced yeast one-hybrid screening with DNA Affinity Purification sequencing (DAP-seq) data, uncovering 1,207 interactions that reveal both shared and gene-specific regulatory relationships, defining the global TF-promoter interaction network of the PSY/PSYR pathway. Functional analysis of 25 TF mutants identified 12 regulators that significantly influence root growth, most acting as repressors. Of these, CYTOKININ RESPONSE FACTOR 10 (CRF10) emerged as a strong growth inhibitor. We identified a CRF10-binding motif in the PSYR3 promoter using DAP-seq data and validated it using eY1H. This motif is also located in the last 3' terminal exon of Topoisomerase 3A (TOP3A). Guided by these insights, we used CRISPR/Cas9-mediated promoter editing to delete a small region encompassing or flanking a functional TF-binding site (TFBS). Removal of this motif, or of its surrounding region, enhanced root growth, yielding variants that retained root length comparable to the crf10 mutant. Our results suggest that the observed root growth phenotype results either from disruption of the CRF10 binding motif or from the mutation in the TOP3A exon.

A major bottleneck in plant biotechnology is the inefficient and genotype-dependent regeneration of callus, which severely limits genetic transformation and functional studies across many species. This barrier is acutely exemplified in the study of beneficial plant-microbe interactions, such as the Epichloe-grass symbiosis--a system conferring remarkable stress tolerance to its host but hindered by a lack of efficient genetic tools. To address this, we established a chromosome-scale genome for an Epichloe native host grass Achnatherum inebrians. We discovered that the expression dynamics of evolutionarily conserved cell pluripotency regulators (CPRs) including ARF5/7/19, BBM, WUS/WOX5 and CUC1/2 serve as a precise molecular predictor for callus regenerative capacity, revealing that pluripotency is dynamic and peaks within a narrow, definable time window. Harnessing this predictable window enabled the development of a highly efficient transformation system for A. inebrians (49.4% efficiency). Crucially, this CPR-based strategy proved generalizable: applied to wheat and the legume sainfoin, it pinpointed species-specific optimal regeneration windows, boosting shoot regeneration rates to 65.7% and 87.5%, respectively. Collectively, our work provides an integrated research system and a rational design principle that removes a key barrier to uncovering molecular mechanisms in plant systems, particularly the Epichloe-enhanced stress tolerance symbiosis.

Plants interact with structured microbial communities called the microbiota, which can have a profound impact on plant growth and health. However, how plants perceive and respond to specific core microbiota members at a molecular level is still unclear. We identified natural variation in Arabidopsis thaliana root responses to bacterial strains of the genus Pseudomonas, a core genus of the plant microbiota. Some A. thaliana accessions such as Van-0 show strong root responses to Pseudomonas strains, including changes in root system architecture and transcriptional reprogramming. Through a forward genetic screen using Pseudomonas isolate R569, we found that the nuo NADH dehydrogenase complex, part of the bacterial electron transport chain, contributes to the bacterial activity on Van-0 roots. Using recombinant inbred lines, we further mapped a multigenic quantitative trait locus in the host that is associated with the root responses. In Van-0, the exocyst subunit EXO70E2 positively contributes to the response, while Col-0 haplotypes of malectin-like and leucine-rich-repeat domain-containing receptor-like kinases play an inhibitory role. The identification of these components in the bacteria and the host establishes a genetic framework for how root developmental plasticity is integrated with the microbe-rich soil environment and is subject to intraspecific natural variation.

The REDUCED HEIGHT (RHT) dwarfing alleles Rht-B1b and Rht-D1b were essential in the "Green Revolution" to optimize wheat plant height and increase grain yield. However, those alleles reduce coleoptile length limiting sowing depth, which triggered the search for alternative dwarfing genes. In this study, we engineered the interaction between miR172 and AP2L2 genes to fine-tune wheat and triticale plant height without affecting coleoptile and first-leaf length.

Secondary root emergence is a crucial trait that shapes the plants underground system. Virtually every developmental step of root primordium morphogenesis is controlled by auxin. However, how the hormone controls cell separation in primordium-overlaying tissues through wall loosening is poorly understood. Here, we took advantage of white lupin and its spectacular cluster root development to assess the contribution of auxin to this process. We show that auxins positive role on rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes. Downregulation of a pectinolytic enzyme gene expressed in cells surrounding the primordium resulted in delayed emergence. Pectins were demethylesterified in the emergence zone and auxin treatment further enhanced this effect. Additionally, we report specific rhamnogalacturonan-I modifications during cortical cell separation. In conclusion, we propose a model in which auxin has a dual role during rootlet emergence: Firstly, through active pectin demethylesterification and secondly by regulating the expression of cell wall remodeling enzymes.

Gibberellins (GAs) influence cell division and elongation, profoundly shaping plant architecture and yield. GA perception occurs when bioactive GAs bind the receptor GID1, promoting DELLA degradation and activating transcriptional programs. While GA signaling in the root endodermis is essential for promoting root elongation, functions of other layers in spatial control of GA responses have not been explored. Here, we developed a synthetic GA (sGA) that does not bind endogenous GID1, together with a modified GID1 (mGID1) engineered to selectively recognize sGA, enabling cell-specific activation of GA signaling in vivo. Using this system in Arabidopsis, we demonstrate that coordinated action of GA signaling in the endodermis, epidermis, and other layers is required for full root elongation. Moreover, cell type-specific expression of GA biosynthetic enzymes indicates the existence of intercellular GA transport. The sGA-mGID1 system provides a versatile platform for spatially precise reprogramming of hormone signaling, enabling synthetic control of developmental processes such as root-shoot growth balance, thereby advancing applications in plant synthetic biology and sustainable crop improvement.

The protein concentration gradients of the master regulators of the root meristem, named the PLETHORA proteins, modulate the root meristem size. Root meristem growth factor 1 (RGF1) peptide extends the PLETHORA2 (PLT2) protein gradients by altering reactive oxygen species (ROS) distributions. However, the underlying mechanism through which the ROS alterations regulate PLT2 remains unknown. Here, we demonstrate that the 212th cysteine of the PLT2 protein plays a pivotal role in modulating PLT2 stability through the ROS altered by RGF1. The substitution of the 212th cysteine of PLT2 with serine (PLT2C212S) enhanced the PLT2 protein stability upon RGF1 and resulted in robust resistance to ROS relative to the native PLT2. Accordingly, PLT2C212S modulated expressions of certain specific root development-related genes to a greater extent than native PLT2. In summary, these findings show that the PLT2 concentration gradient formation through ROS, modulated by RGF1, is dependent on a mechanism involving the 212th cysteine of PLT2.

Synthetic transcription factors have great promise as tools to explore biological processes. By allowing precise alterations in gene expression, they can help elucidate relationships between gene expression and plant morphology or metabolism. However, the years-long timescales, high cost, and technical skill associated with plant transformation have dramatically slowed their use. In this work, we developed a new platform technology called VipariNama (ViN) in which RNA vectors are used to rapidly deploy synthetic transcription factors and reprogram gene expression in planta. We demonstrate how ViN vectors can direct activation or repression of multiple genes, systemically and persistently over several weeks, and in multiple plant species. We also show how this transcriptional reprogramming can create predictable changes to metabolic and morphological phenotypes in the model plants Nicotiana benthamiana and Arabidopsis thaliana in a matter of weeks. Finally, we show how a model of gibberellin signaling can guide ViN vector-based reprogramming to rapidly engineer plant size in both model species as well as the crop Solanum lycopersicum (tomato). In summary, using VipariNama accelerates the timeline for generating phenotypes from over a year to just a few weeks, providing an attractive alternative to transgenesis for synthetic transcription factor-enabled hypothesis testing and crop engineering.

Root branching is an important strategy to explore efficiently large volumes of soil. To economize this process, lateral roots (LR) are formed along the growing root at discrete positions that are instructed by oscillating auxin signals derived from the lateral root cap (LRC). This assumes that auxin moves from the LRC across multiple layers to accumulate in the pericycle. Here, we identified, using gene silencing and CRISPR based approaches, a group of five genetically linked, closely related ABCBs that control LR spacing by modulating the amplitude of the auxin oscillation. The transporters localize to the plasma membrane and reveal significant auxin export activity. These ABCBs are mainly expressed in the LRC and epidermis where they contribute to auxin transport towards the root oscillation zone. Our findings highlight the importance of auxin transport in the outer tissues of the root meristem to regulate LR spacing.

In Arabidopsis thaliana, the negative brassinosteroid (BR) signalling regulator, BR INSENSITIVE2 (BIN2) promotes and restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffolding protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages with either exogenous BRs or the specific BIN2 inhibitor revealed that the two modes of BR activation triggered spatiotemporally distinct transcriptional responses. We established that when in a complex with POLAR and its closest homolog POLAR-LIKE1, BIN2 is insulated from BR-mediated inactivation, nevertheless, it remains sensitive to the inhibitor. Subsequently, BR signalling is attenuated in ACD precursors, whereas it remains active in epidermal cells that would undergo differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

Auxin signaling through the Nuclear Auxin Pathway is essential for plant development and is mediated by competing A-class and B-class Auxin Response Factor (ARF) transcription factors. Recently, proteasomal ARF degradation through a degradation signal embedded within the DNA-Binding Domain, was identified as a key component of auxin response. Here, we investigate the structural requirements and biological relevance of ARF degradation in the bryophyte Marchantia polymorpha. We identify a critical residue for proteolysis of the repressive, B-class MpARF2, and find it to be functionally conserved in degradation of the activating, A-class MpARF1. Unlike MpARF2, however, impaired MpARF1 degradation had little effect on auxin response and development, suggesting differential integration in biological function. We find that MpARF2 degradation occurs across all developmental stages of the life cycle, and is required for MpARF2 function during development. Our findings reveal a degradation mechanism targeting A- and B-class ARFs which shares the same origin, but has evolved along unique evolutionary trajectories.