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The variety of plant architectures observed in nature is predominantly determined by vegetative and reproductive branching patterns, the positioning of lateral organs, and differential stem elongation. Branches, lateral organs, and stems are the final products of the activity of meristems, groups of stem cells whose function is genetically determined and environmentally influenced. Several decades of studies in different plant species have shed light on the essential role of the hormone auxin in plant growth and development. Auxin influences stem elongation and regulates the formation, activity, and fate of meristems, and has therefore been recognized as a major hormone shaping plant architecture. Increasing our knowledge of the molecular mechanisms that regulate auxin function is necessary to understand how different plant species integrate a genetically determined developmental programme, the establishment of a body plan, with constant inputs from the surrounding environment. This information will allow us to develop the molecular tools needed to modify plant architecture in several crop species and in rapidly changing environments.

This protocol describes DAP-seq, a transcription-factor binding site discovery assay that can be used to produce cistrome and epicistrome maps for any organism. To enable low-cost, high-throughput generation of cistrome and epicistrome maps for any organism, we developed DNA affinity purification sequencing (DAP-seq), a transcription factor (TF)-binding site (TFBS) discovery assay that couples affinity-purified TFs with next-generation sequencing of a genomic DNA library. The method is fast, inexpensive, and more easily scaled than chromatin immunoprecipitation sequencing (ChIP-seq). DNA libraries are constructed using native genomic DNA from any source of interest, preserving cell- and tissue-specific chemical modifications that are known to affect TF binding (such as DNA methylation) and providing increased specificity as compared with in silico predictions based on motifs from methods such as protein-binding microarrays (PBMs) and systematic evolution of ligands by exponential enrichment (SELEX). The resulting DNA library is incubated with an affinity-tagged in vitro -expressed TF, and TF–DNA complexes are purified using magnetic separation of the affinity tag. Bound genomic DNA is eluted from the TF and sequenced using next-generation sequencing. Sequence reads are mapped to a reference genome, identifying genome-wide binding locations for each TF assayed, from which sequence motifs can then be derived. A researcher with molecular biology experience should be able to follow this protocol, processing up to 400 samples per week.

Structural variation in plant genomes is a significant driver of phenotypic variability in traits important for the domestication and productivity of crop species. Among these are traits that depend on functional meristems, populations of stem cells maintained by the CLAVATA-WUSCHEL (CLV-WUS) negative feedback-loop that controls the expression of the WUS homeobox transcription factor. WUS function and impact on maize development and yield remain largely unexplored. Here we show that the maize dominant Barren inflorescence3 ( Bif3 ) mutant harbors a tandem duplicated copy of the ZmWUS1 gene, ZmWUS1-B , whose novel promoter enhances transcription in a ring-like pattern. Overexpression of ZmWUS1-B is due to multimerized binding sites for type-B RESPONSE REGULATORs (RRs), key transcription factors in cytokinin signaling. Hypersensitivity to cytokinin causes stem cell overproliferation and major rearrangements of Bif3 inflorescence meristems, leading to the formation of ball-shaped ears and severely affecting productivity. These findings establish ZmWUS1 as an essential meristem size regulator in maize and highlight the striking effect of cis-regulatory variation on a key developmental program. The WUSCHEL transcription factor promotes plant stem cell proliferation. Here the authors show that the maize Bif3 mutant contains a duplication of the ZmWUS1 locus leading to cytokinin hypersensitivity and overproliferation at the shoot meristem demonstrating the role of WUSCHEL in maize and how structural variation can impact plant morphology.

AUXIN RESPONSE FACTORS (ARFs) are plant-specific transcription factors (TFs) that couple perception of the hormone auxin to gene expression programs essential to all land plants. As with many large TF families, a key question is whether individual members determine developmental specificity by binding distinct target genes. We use DAP-seq to generate genome-wide in vitro TF:DNA interaction maps for fourteen maize ARFs from the evolutionarily conserved A and B clades. Comparative analysis reveal a high degree of binding site overlap for ARFs of the same clade, but largely distinct clade A and B binding. Many sites are however co-occupied by ARFs from both clades, suggesting transcriptional coordination for many genes. Among these, we investigate known QTLs and use machine learning to predict the impact of cis -regulatory variation. Overall, large-scale comparative analysis of ARF binding suggests that auxin response specificity may be determined by factors other than individual ARF binding site selection. AUXIN RESPONSE FACTORS (ARFs) are a family of plant-specific transcriptional factors involved in auxin signaling. Here, the authors adapt DAP-seq technology to show the binding landscape of 14 maize ARFs and reveal class-specific binding properties and transcriptional coordination by ARFs from different classes.

Many eukaryotic transcription factors (TF) form homodimer or heterodimer complexes to regulate gene expression. Dimerization of BASIC LEUCINE ZIPPER (bZIP) TFs are critical for their functions, but the molecular mechanism underlying the DNA binding and functional specificity of homo- versus heterodimers remains elusive. To address this gap, we present the double DNA Affinity Purification-sequencing (dDAP-seq) technique that maps heterodimer binding sites on endogenous genomic DNA. Using dDAP-seq we profile twenty pairs of C/S1 bZIP heterodimers and S1 homodimers in Arabidopsis and show that heterodimerization significantly expands the DNA binding preferences of these TFs. Analysis of dDAP-seq binding sites reveals the function of bZIP9 in abscisic acid response and the role of bZIP53 heterodimer-specific binding in seed maturation. The C/S1 heterodimers show distinct preferences for the ACGT elements recognized by plant bZIPs and motifs resembling the yeast GCN4 cis -elements. This study demonstrates the potential of dDAP-seq in deciphering the DNA binding specificities of interacting TFs that are key for combinatorial gene regulation. Here, the authors describe a new method to study how some proteins work together to control gene activity. They show that certain protein pairs can recognize new DNA sequences that they can’t recognize individually and control a wider range of genes.

Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear [rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues, rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.

In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes,BARREN INFLORESCENCE1andBARREN INFLORESCENCE4(BIF1andBIF4), that regulate the early steps required for inflorescence formation.BIF1andBIF4encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.

• All aerial epidermal cells in land plants are covered by the cuticle, an extracellular hydrophobic layer that provides protection against abiotic and biotic stresses and prevents organ fusion during development. • Genetic and morphological analysis of the classic maize adherent1 (ad1) mutant was combined with genome-wide binding analysis of the maize MYB transcription factor FUSED LEAVES1 (FDL1), coupled with transcriptional profiling of fdl1 mutants. • We show that AD1 encodes an epidermally-expressed 3-KETOACYL-CoA SYNTHASE (KCS) belonging to a functionally uncharacterized clade of KCS enzymes involved in cuticular wax biosynthesis. Wax analysis in ad1 mutants indicates that AD1 functions in the formation of very-long-chain wax components. We demonstrate that FDL1 directly binds to CCAACC core motifs present in AD1 regulatory regions to activate its expression. Over 2000 additional target genes of FDL1, including many involved in cuticle formation, drought response and cell wall organization, were also identified. • Our results identify a regulatory module of cuticle biosynthesis in maize that is conserved across monocots and eudicots, and highlight previously undescribed factors in lipid metabolism, transport and signaling that coordinate organ development and cuticle formation.

The micronutrient boron is essential in maintaining the structure of plant cell walls and is critical for high yields in crop species. Boron can move into plants by diffusion or by active and facilitated transport mechanisms. We recently showed that mutations in the maize boron efflux transporter ROTTEN EAR (RTE) cause severe developmental defects and sterility. RTE is part of a small gene family containing five additional members (RTE2–RTE6) that show tissue-specific expression. The close paralogous gene RTE2 encodes a protein with 95% amino acid identity with RTE and is similarly expressed in shoot and root cells surrounding the vasculature. Despite sharing a similar function with RTE, mutations in the RTE2 gene do not cause growth defects in the shoot, even in boron-deficient conditions. However, rte2 mutants strongly enhance the rte phenotype in soils with low boron content, producing shorter plants that fail to form all reproductive structures. The joint action of RTE and RTE2 is also required in root development. These defects can be fully complemented by supplying boric acid, suggesting that diffusion or additional transport mechanisms overcome active boron transport deficiencies in the presence of an excess of boron. Overall, these results suggest that RTE2 and RTE function are essential for maize shoot and root growth in boron-deficient conditions.

Background WUSCHEL (WUS) is a homeodomain transcription factor vital for stem cell proliferation in plant meristems. In maize, ZmWUS1 is expressed in the inflorescence meristem, including the central zone reservoir of stem cells. ZmWUS1 overexpression in the Barren inflorescence3 ( Bif3 ) mutant perturbs inflorescence development due to stem cell over-proliferation. Results Single-cell Assay for Transposase Accessible Chromatin sequencing (scATAC-seq) shows that Bif3 alters central zone chromatin accessibility compared to normal inflorescence meristems. The CAATAATGC motif, a known homeodomain recognition site, is enriched within regions with increased chromatin accessibility in Bif3, suggesting ZmWUS1 could function as a transcriptional activator in the central zone. This motif differs from the TGAATGAA motif identified by DNA Affinity Purification sequencing (DAP-seq) of ZmWUS1, which showed low enrichment in the central zone. Conversely, regions with decreased chromatin accessibility in Bif3 are instead adjacent to AUXIN RESPONSE FACTOR genes, suggesting possible reduced auxin signaling in the Bif3 central zone. Conclusions This study characterized how Bif3 overexpression of ZmWUS1 influences chromatin accessibility in the central zone, reducing auxin signaling, while raising questions about differential ZmWUS1 motif usage in distinct cellular contexts.