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NAKED ENDOSPERM1 (NKD1), NKD2, and OPAQUE2 (O2) are transcription factors important for cell patterning and nutrient storage in maize (Zea mays) endosperm. To study the complex regulatory interrelationships among these 3 factors in coregulating gene networks, we developed a set of nkd1, nkd2, and o2 homozygous lines, including all combinations of mutant and wild-type genes. Among the 8 genotypes tested, we observed diverse phenotypes and gene interactions affecting cell patterning, starch content, and storage proteins. From ∼8 to ∼16 d after pollination, maize endosperm undergoes a transition from cellular development to nutrient accumulation for grain filling. Gene network analysis showed that NKD1, NKD2, and O2 dynamically regulate a hierarchical gene network during this period, directing cellular development early and then transitioning to constrain cellular development while promoting the biosynthesis and storage of starch, proteins, and lipids. Genetic interactions regulating this network are also dynamic. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) showed that O2 influences the global regulatory landscape, decreasing NKD1 and NKD2 target site accessibility, while NKD1 and NKD2 increase O2 target site accessibility. In summary, interactions of NKD1, NKD2, and O2 dynamically affect the hierarchical gene network and regulatory landscape during the transition from cellular development to grain filling in maize endosperm.

 

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-versusheterodimers 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 inArabidopsisand 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 GCN4cis-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.

 

ZMP enables RNA-directed DNA methylation at pericentromeric repeats and prevents it from euchromatic regions. 

Machine learning approaches have been applied to identify transcription factor (TF)–DNA interaction important for gene regulation and expression. However, due to the enormous search space of the genome, it is challenging to build models capable of surveying entire reference genomes, especially in species where models were not trained. In this study, we surveyed a variety of methods for classification of epigenomics data in an attempt to improve the detection for 12 members of the auxin response factor (ARF)-binding DNAs from maize and soybean as assessed by DNA Affinity Purification and sequencing (DAP-seq). We used the classification for prediction by minimizing the genome search space by only surveying unmethylated regions (UMRs). For identification of DAP-seq-binding events within the UMRs, we achieved 78.72 % accuracy rate across 12 members of ARFs of maize on average by encoding DNA with count vectorization for k-mer with a logistic regression classifier with up-sampling and feature selection. Importantly, feature selection helps to uncover known and potentially novel ARF-binding motifs. This demonstrates an independent method for identification of TF-binding sites. Finally, we tested the model built with maize DAP-seq data and applied it directly to the soybean genome and found high false-negative rates, which accounted for more than 40 % across the ARF TFs tested. The findings in this study suggest the potential use of various methods to predict TF–DNA interactions within and between species with varying degrees of success.

 

Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinteddosage-effect defective1(ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5–10% seed weight reduction whended1is transmitted through the male, while homozygous mutants are defective with a 70–90% seed weight reduction.Ded1encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting ofDed1causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

 

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 dominantBarren inflorescence3(Bif3) mutant harbors a tandem duplicated copy of theZmWUS1gene,ZmWUS1-B, whose novel promoter enhances transcription in a ring-like pattern. Overexpression ofZmWUS1-Bis 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 ofBif3inflorescence meristems, leading to the formation of ball-shaped ears and severely affecting productivity. These findings establishZmWUS1as an essential meristem size regulator in maize and highlight the striking effect of cis-regulatory variation on a key developmental program.

 

Maintaining sufficient water transport during flowering is essential for proper organ growth, fertilization, and yield. Water deficits that coincide with flowering result in leaf wilting, necrosis, tassel browning, and sterility, a stress condition known as “tassel blasting.” We identified a mutant, necrotic upper tips1 ( nut1 ), that mimics tassel blasting and drought stress and reveals the genetic mechanisms underlying these processes. The nut1 phenotype is evident only after the floral transition, and the mutants have difficulty moving water as shown by dye uptake and movement assays. These defects are correlated with reduced protoxylem vessel thickness that indirectly affects metaxylem cell wall integrity and function in the mutant. nut1 is caused by an Ac transposon insertion into the coding region of a unique NAC transcription factor within the VND clade of Arabidopsis . NUT1 localizes to the developing protoxylem of root, stem, and leaf sheath, but not metaxylem, and its expression is induced by flowering. NUT1 downstream target genes function in cell wall biosynthesis, apoptosis, and maintenance of xylem cell wall thickness and strength. These results show that maintaining protoxylem vessel integrity during periods of high water movement requires the expression of specialized, dynamically regulated transcription factors within the vasculature. 

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 ofcis-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.