Diversification and conservation of DNA binding specificities of SPL family of transcription factors.

SQUAMOSA Promoter-Binding Protein-Like (SPL) transcription factors play vital roles in plant development and stress responses. In this study, we report a comprehensive DNA Affinity Purification sequencing (DAP-seq) analysis for 14 of the 16 SPL transcription factors in Arabidopsis thaliana, providing valuable insights into their DNA-binding specificities. We performed Gene Ontology (GO) analysis of the target genes to reveal their convergent and diverse biological functions among SPL family proteins. Comparative analysis between the paralogs AtSPL9 and AtSPL15 revealed differences in their binding motifs, suggesting divergent regulatory functions. Additionally, we expanded our investigation to homologs of AtSPL9/15 in Zea mays (ZmSBP8/30) and Triticum aestivum (TaSPL7/13), identifying conserved and unique DNA-binding patterns across species. These findings provide key resources for understanding the molecular mechanisms of SPL transcription factors in regulating plant development and evolution across different species.

Transcriptional corepressors in maize maintain meristem development.

The formation of the plant body proceeds in a sequential post-embryonic manner through the action of meristems. Tightly coordinated meristem regulation is required for development and reproductive success, eventually determining yield in crop species. In maize (Zea mays), the RAMOSA1 ENHANCER LOCUS2 (REL2) family of transcriptional corepressors includes four members, REL2, RELK1 (REL2-LIKE1), RELK2, and RELK3. In a screen for rel2 enhancers, we identified shorter double mutants with enlarged ear inflorescence meristems (IMs) carrying mutations in RELK1. Expression and genetic analysis indicated that REL2 and RELK1 cooperatively regulate ear IM development by controlling genes involved in redox balance, hormone homeostasis, and differentiation, ultimately tipping the meristem toward an environment favorable to expanded expression of the ZmWUSCHEL1 gene, which encodes a key stem-cell promoting transcription factor. We further demonstrated that RELK genes have partially redundant yet diverse functions in the maintenance of various meristem types during development. By exploiting subtle increases in ear IM size in rel2 heterozygous plants, we also showed that extra rows of kernels are formed across a diverse set of F1 hybrids. Our findings reveal that the REL2 family maintains development from embryonic initiation to reproductive growth and can potentially be harnessed for increasing seed yield in a major crop species.

Transcription factor binding site divergence across maize inbred lines drives transcriptional and phenotypic variation.

Regulatory elements are important constituents of plant genomes that have shaped ancient and modern crops. Their identification, function, and diversity in crop genomes however are poorly characterized, thus limiting our ability to harness their power for further agricultural advances using induced or natural variation. Here, we use DNA affinity purification-sequencing (DAP-seq) to map transcription factor (TF) binding events for 200 maize TFs belonging to 30 distinct families and heterodimer pairs in two distinct inbred lines historically used for maize hybrid plant production, providing empirical binding site annotation for 5.3% of the maize genome. TF binding site comparison in B73 and Mo17 inbreds reveals widespread differences, driven largely by structural variation, that correlate with gene expression changes. TF binding site presence-absence variation helps clarify complex QTL such as vgt1, an important determinant of maize flowering time, and DICE, a distal enhancer involved in herbivore resistance. Modification of TF binding regions via CRISPR-Cas9 mediated editing alters target gene expression and phenotype. Our functional catalog of maize TF binding events enables collective and comparative TF binding analysis, and highlights its value for agricultural improvement.

NAKED ENDOSPERM1, NAKED ENDOSPERM2, and OPAQUE2 interact to regulate gene networks in maize endosperm development.

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.

Double DAP-seq uncovered synergistic DNA binding of interacting bZIP transcription factors.

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.

ZMP recruits and excludes Pol IV-mediated DNA methylation in a site-specific manner.

In plants, RNA-directed DNA methylation (RdDM) uses small interfering RNAs (siRNAs) to target transposable elements (TEs) but usually avoids genes. RNA polymerase IV (Pol IV) shapes the landscape of DNA methylation through its pivotal role in siRNA biogenesis. However, how Pol IV is recruited to specific loci, particularly how it avoids genes, is poorly understood. Here, we identified a Pol IV-interacting protein, ZMP (zinc finger, mouse double-minute/switching complex B, Plus-3 protein), which exerts a dual role in regulating siRNA biogenesis and DNA methylation at specific genomic regions. ZMP is required for siRNA biogenesis at some pericentromeric regions and prevents Pol IV from targeting a subset of TEs and genes at euchromatic loci. As a chromatin-associated protein, ZMP prefers regions with depleted histone H3 lysine 4 (H3K4) methylation abutted by regions with H3K4 methylation, probably monitoring changes in local H3K4 methylation status to regulate Pol IV's chromatin occupancy. Our findings uncover a mechanism governing the specificity of RdDM.

Paternal imprinting of dosage-effect defective1 contributes to seed weight xenia in maize.

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 imprinted dosage-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 when ded1 is transmitted through the male, while homozygous mutants are defective with a 70-90% seed weight reduction. Ded1 encodes 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 of Ded1 causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size.

Mechanisms of temperature-regulated growth and thermotolerance in crop species.

Temperature is a major environmental factor affecting the development and productivity of crop species. The ability to cope with periods of high temperatures, also known as thermotolerance, is becoming an increasingly indispensable trait for the future of agriculture owing to the current trajectory of average global temperatures. From temperature sensing to downstream transcriptional changes, here, we review recent findings involving the thermal regulation of plant growth and the effects of heat on hormonal pathways, reactive oxygen species, and epigenetic regulation. We also highlight recent approaches and strategies that could be integrated to confront the challenges in sustaining crop productivity in future decades.

Structural variation at the maize WUSCHEL1 locus alters stem cell organization in inflorescences.

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.

VviNAC33 promotes organ de-greening and represses vegetative growth during the vegetative-to-mature phase transition in grapevine.

Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green-to-mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood. We investigated the function of the transcriptional regulator VviNAC33 by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP-seq) approach combined with transcriptomic data. We demonstrated that VviNAC33 induces leaf de-greening, inhibits organ growth and directly activates the expression of STAY-GREEN PROTEIN 1 (SGR1), which is involved in Chl and photosystem degradation, and AUTOPHAGY 8f (ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibits AUXIN EFFLUX FACILITATOR PIN1, RopGEF1 and ATP SYNTHASE GAMMA CHAIN 1T (ATPC1), which are involved in photosystem II integrity and activity. Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative-to-mature phase transition.

OsFD4 promotes the rice floral transition via florigen activation complex formation in the shoot apical meristem.

In rice, the florigens Heading Date 3a (Hd3a) and Rice Flowering Locus T 1 (RFT1), OsFD-like basic leucine zipper (bZIP) transcription factors, and Gf14 proteins assemble into florigen activation/repressor complexes (FACs/FRCs), which regulate transition to flowering in leaves and apical meristem. Only OsFD1 has been described as part of complexes promoting flowering at the meristem, and little is known about the role of other bZIP transcription factors, the combinatorial complexity of FAC formation, and their DNA-binding properties. Here, we used mutant analysis, protein-protein interaction assays and DNA affinity purification (DAP) sequencing coupled to in silico prediction of binding syntaxes to study several bZIP proteins that assemble into FACs or FRCs. We identified OsFD4 as a component of a FAC promoting flowering at the shoot apical meristem, downstream of OsFD1. The osfd4 mutants are late flowering and delay expression of genes promoting inflorescence development. Protein-protein interactions indicate an extensive network of contacts between several bZIPs and Gf14 proteins. Finally, we identified genomic regions bound by bZIPs with promotive and repressive effects on flowering. We conclude that distinct bZIPs orchestrate floral induction at the meristem and that FAC formation is largely combinatorial. While binding to the same consensus motif, their DNA-binding syntax is different, suggesting discriminatory functions.

The FUSED LEAVES1-ADHERENT1 regulatory module is required for maize cuticle development and organ separation.

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.

Mapping Regulatory Determinants in Plants.

The domestication and improvement of many plant species have frequently involved modulation of transcriptional outputs and continue to offer much promise for targeted trait engineering. The cis-regulatory elements (CREs) controlling these trait-associated transcriptional variants however reside within non-coding regions that are currently poorly annotated in most plant species. This is particularly true in large crop genomes where regulatory regions constitute only a small fraction of the total genomic space. Furthermore, relatively little is known about how CREs function to modulate transcription in plants. Therefore understanding where regulatory regions are located within a genome, what genes they control, and how they are structured are important factors that could be used to guide both traditional and synthetic plant breeding efforts. Here, we describe classic examples of regulatory instances as well as recent advances in plant regulatory genomics. We highlight valuable molecular tools that are enabling large-scale identification of CREs and offering unprecedented insight into how genes are regulated in diverse plant species. We focus on chromatin environment, transcription factor (TF) binding, the role of transposable elements, and the association between regulatory regions and target genes.

Necrotic upper tips1 mimics heat and drought stress and encodes a protoxylem-specific transcription factor in maize.

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.

Author Correction: Widespread long-range cis-regulatory elements in the maize genome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Widespread long-range cis-regulatory elements in the maize genome.

Genetic mapping studies on crops suggest that agronomic traits can be controlled by gene-distal intergenic loci. Despite the biological importance and the potential agronomic utility of these loci, they remain virtually uncharacterized in all crop species to date. Here, we provide genetic, epigenomic and functional molecular evidence to support the widespread existence of gene-distal (hereafter, distal) loci that act as long-range transcriptional cis-regulatory elements (CREs) in the maize genome. Such loci are enriched for euchromatic features that suggest their regulatory functions. Chromatin loops link together putative CREs with genes and recapitulate genetic interactions. Putative CREs also display elevated transcriptional enhancer activities, as measured by self-transcribing active regulatory region sequencing. These results provide functional support for the widespread existence of CREs that act over large genomic distances to control gene expression.

NEEDLE1 encodes a mitochondria localized ATP-dependent metalloprotease required for thermotolerant maize growth.

Meristems are highly regulated structures ultimately responsible for the formation of branches, lateral organs, and stems, and thus directly affect plant architecture and crop yield. In meristems, genetic networks, hormones, and signaling molecules are tightly integrated to establish robust systems that can adapt growth to continuous inputs from the environment. Here we characterized needle1 (ndl1), a temperature-sensitive maize mutant that displays severe reproductive defects and strong genetic interactions with known mutants affected in the regulation of the plant hormone auxin. NDL1 encodes a mitochondria-localized ATP-dependent metalloprotease belonging to the FILAMENTATION TEMPERATURE-SENSITIVE H (FTSH) family. Together with the hyperaccumulation of reactive oxygen species (ROS), ndl1 inflorescences show up-regulation of a plethora of stress-response genes. We provide evidence that these conditions alter endogenous auxin levels and disrupt primordia initiation in meristems. These findings connect meristem redox status and auxin in the control of maize growth.

RAMOSA1 ENHANCER LOCUS2-Mediated Transcriptional Repression Regulates Vegetative and Reproductive Architecture.

Transcriptional repression in multicellular organisms orchestrates dynamic and precise gene expression changes that enable complex developmental patterns. Here, we present phenotypic and molecular characterization of the maize (Zea mays) transcriptional corepressor RAMOSA1 ENHANCER LOCUS2 (REL2), a unique member of the highly conserved TOPLESS (TPL) family. Analysis of single recessive mutations in rel2 revealed an array of vegetative and reproductive phenotypes, many related to defects in meristem initiation and maintenance. To better understand how REL2-mediated transcriptional complexes relate to rel2 phenotypes, we performed protein interaction assays and transcriptional profiling of mutant inflorescences, leading to the identification of different maize transcription factors and regulatory pathways that employ REL2 repression to control traits directly impacting maize yields. In addition, we used our REL2 interaction data to catalog conserved repression motifs present on REL2 interactors and showed that two of these, RLFGV- and DLN-type motifs, interact with the C-terminal WD40 domain of REL2 rather than the N terminus, which is known to bind LxLxL EAR motifs. These findings establish that the WD40 domain of TPL family proteins is an independent protein interaction surface that may work together with the N-terminal domain to allow the formation of large macromolecular complexes of functionally related transcription factors.

The DNA binding landscape of the maize AUXIN RESPONSE FACTOR family.

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.