A cis-regulatory atlas in maize at single-cell resolution.

cis-regulatory elements (CREs) encode the genomic blueprints of spatiotemporal gene expression programs enabling highly specialized cell functions. Using single-cell genomics in six maize organs, we determined the cis- and trans-regulatory factors defining diverse cell identities and coordinating chromatin organization by profiling transcription factor (TF) combinatorics, identifying TFs with non-cell-autonomous activity, and uncovering TFs underlying higher-order chromatin interactions. Cell-type-specific CREs were enriched for enhancer activity and within unmethylated long terminal repeat retrotransposons. Moreover, we found cell-type-specific CREs are hotspots for phenotype-associated genetic variants and were targeted by selection during modern maize breeding, highlighting the biological implications of this CRE atlas. Through comparison of maize and Arabidopsis thaliana developmental trajectories, we identified TFs and CREs with conserved and divergent chromatin dynamics, showcasing extensive evolution of gene regulatory networks. In addition to this rich dataset, we developed single-cell analysis software, Socrates, which can be used to understand cis-regulatory variation in any species.

Genetic dissection of cis-regulatory control of ZmWUSCHEL1 expression by type B RESPONSE REGULATORS.

Mutations in cis-regulatory regions play an important role in the domestication and improvement of crops by altering gene expression. However, assessing the in vivo impact of cis-regulatory elements (CREs) on transcriptional regulation and phenotypic outcomes remains challenging. Previously, we showed that the dominant Barren inflorescence3 (Bif3) mutant of maize (Zea mays) contains a duplicated copy of the homeobox transcription factor gene ZmWUSCHEL1 (ZmWUS1), named ZmWUS1-B. ZmWUS1-B is controlled by a spontaneously generated novel promoter region that dramatically increases its expression and alters patterning and development of young ears. Overexpression of ZmWUS1-B is caused by a unique enhancer region containing multimerized binding sites for type B RESPONSE REGULATORs (RRs), key transcription factors in cytokinin signaling. To better understand how the enhancer increases the expression of ZmWUS1 in vivo, we specifically targeted the ZmWUS1-B enhancer region by CRISPR-Cas9-mediated editing. A series of deletion events with different numbers of type B RR DNA binding motifs (AGATAT) enabled us to determine how the number of AGATAT motifs impacts in vivo expression of ZmWUS1-B and consequently ear development. In combination with dual-luciferase assays in maize protoplasts, our analysis reveals that AGATAT motifs have an additive effect on ZmWUS1-B expression, while the distance separating AGATAT motifs does not appear to have a meaningful impact, indicating that the enhancer activity derives from the sum of individual CREs. These results also suggest that in maize inflorescence development, there is a threshold of buffering capacity for ZmWUS1 overexpression.

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.

ZmCTLP1 is required for the maintenance of lipid homeostasis and the basal endosperm transfer layer in maize kernels.

Maize kernel weight is influenced by the unloading of nutrients from the maternal placenta and their passage through the transfer tissue of the basal endosperm transfer layer (BETL) and the basal intermediate zone (BIZ) to the upper part of the endosperm. Here, we show that Small kernel 10 (Smk10) encodes a choline transporter-like protein 1 (ZmCTLP1) that facilitates choline uptake and is located in the trans-Golgi network (TGN). Its loss of function results in reduced choline content, leading to smaller kernels with a lower starch content. Mutation of ZmCTLP1 disrupts membrane lipid homeostasis and the normal development of wall in-growths. Expression levels of Mn1 and ZmSWEET4c, two kernel filling-related genes, are downregulated in the smk10, which is likely to be one of the major causes of incompletely differentiated transfer cells. Mutation of ZmCTLP1 also reduces the number of plasmodesmata (PD) in transfer cells, indicating that the smk10 mutant is impaired in PD formation. Intriguingly, we also observed premature cell death in the BETL and BIZ of the smk10 mutant. Together, our results suggest that ZmCTLP1-mediated choline transport affects kernel development, highlighting its important role in lipid homeostasis, wall in-growth formation and PD development in transfer cells.

A Synthetic Approach Allows Rapid Characterization of the Maize Nuclear Auxin Response Circuit.

Auxin plays a key role across all land plants in growth and developmental processes. Although auxin signaling function has diverged and expanded, differences in the molecular functions of signaling components have largely been characterized in Arabidopsis (Arabidopsis thaliana). Here, we used the nuclear Auxin Response Circuit recapitulated in yeast (Saccharomyces cerevisiae) system to functionally annotate maize (Zea mays) auxin signaling components, focusing on genes expressed during the development of ear and tassel inflorescences. All 16 maize auxin/indole-3-acetic acid repressor proteins were degraded in response to auxin with rates that depended on both receptor and repressor identities. When fused to the maize TOPLESS homolog RAMOSA1 ENHANCER LOCUS2, maize auxin/indole-3-acetic acids were able to repress AUXIN RESPONSE FACTOR transcriptional activity. A complete auxin response circuit comprising all maize components, including the ZmAFB2/3 b1 maize AUXIN SIGNALING F-BOX (AFB) receptor, was fully functional. The ZmAFB2/3 b1 auxin receptor was more sensitive to hormone than AtAFB2 and allowed for rapid circuit activation upon auxin addition. These results validate the conserved role of predicted auxin response genes in maize as well as provide evidence that a synthetic approach can facilitate broader comparative studies across the wide range of species with sequenced genomes.

Improving architectural traits of maize inflorescences.

The domestication and improvement of maize resulted in radical changes in shoot architecture relative to its wild progenitor teosinte. In particular, critical modifications involved a reduction of branching and an increase in inflorescence size to meet the needs for human consumption and modern agricultural practices. Maize is a major contributor to global agricultural production by providing large and inexpensive quantities of food, animal feed, and ethanol. Maize is also a classic system for studying the genetic regulation of inflorescence formation and its enlarged female inflorescences directly influence seed production and yield. Studies on the molecular and genetic networks regulating meristem proliferation and maintenance, including receptor-ligand interactions, transcription factor regulation, and hormonal control, provide important insights into maize inflorescence development and reveal potential avenues for the targeted modification of specific architectural traits. In this review, we summarize recent findings on the molecular mechanisms controlling inflorescence formation and discuss how this knowledge can be applied to improve maize productivity in the face of present and future environmental challenges.

PPR-SMR1 is required for the splicing of multiple mitochondrial introns, interacts with Zm-mCSF1, and is essential for seed development in maize.

Group II introns are ribozymes that can excise themselves from precursor-RNA transcripts, but plant organellar group II introns have structural deviations that inhibit ribozyme activity. Therefore, splicing of these introns requires the assistance of nuclear- and/or organellar-encoded splicing factors; however, how these splicing factors function remains unclear. In this study, we report the functions and interactions of two splicing factors, PPR-SMR1 and Zm-mCSF1, in intron splicing in maize mitochondria. PPR-SMR1 is a SMR domain-containing pentatricopeptide repeat (PPR) protein and Zm-mCSF1 is a CRM domain-containing protein, and both are targeted to mitochondria. Loss-of-function mutations in each of them severely arrests embryogenesis and endosperm development in maize. Functional analyses indicate that PPR-SMR1 and Zm-mCSF1 are required for the splicing of most mitochondrial group II introns. Among them, nad2-intron 2 and 3, and nad5-intron 1 are PPR-SMR1/Zm-mCSF1-dependent introns. Protein interaction assays suggest that PPR-SMR1 can interact with Zm-mCSF1 through its N-terminus, and that Zm-mCSF1 is self-interacting. Our findings suggest that PPR-SMR1, a novel splicing factor, acts in the splicing of multiple group II introns in maize mitochondria, and the protein-protein interaction between it and Zm-mCSF1 might allow the formation of large macromolecular splicing complexes.

Identification of minor effect QTLs for plant architecture related traits using super high density genotyping and large recombinant inbred population in maize (Zea mays).

BACKGROUND: Plant Architecture Related Traits (PATs) are of great importance for maize breeding, and mainly controlled by minor effect quantitative trait loci (QTLs). However, cloning or even fine-mapping of minor effect QTLs is very difficult in maize. Theoretically, large population and high density genetic map can be helpful for increasing QTL mapping resolution and accuracy, but such a possibility have not been actually tested. RESULTS: Here, we employed a genotyping-by-sequencing (GBS) strategy to construct a linkage map with 16,769 marker bins for 1021 recombinant inbred lines (RILs). Accurately mapping of well studied genes P1, pl1 and r1 underlying silk color demonstrated the map quality. After QTL analysis, a total of 51 loci were mapped for six PATs. Although all of them belong to minor effect alleles, the lengths of the QTL intervals, with a minimum and median of 1.03 and 3.40 Mb respectively, were remarkably reduced as compared with previous reports using smaller size of population or small number of markers. Several genes with known function in maize were shown to be overlapping with or close neighboring to these QTL peaks, including na1, td1, d3 for plant height, ra1 for tassel branch number, and zfl2 for tassel length. To further confirm our mapping results, a plant height QTL, qPH1a, was verified by an introgression lines (ILs). CONCLUSIONS: We demonstrated a method for high resolution mapping of minor effect QTLs in maize, and the resulted comprehensive QTLs for PATs are valuable for maize molecular breeding in the future.

Defective Kernel 39 encodes a PPR protein required for seed development in maize.

RNA editing is a posttranscriptional process that is important in mitochondria and plastids of higher plants. All RNA editing-specific trans-factors reported so far belong to PLS-class of pentatricopeptide repeat (PPR) proteins. Here, we report the map-based cloning and molecular characterization of a defective kernel mutant dek39 in maize. Loss of Dek39 function leads to delayed embryogenesis and endosperm development, reduced kernel size, and seedling lethality. Dek39 encodes an E sub-class PPR protein that targets to both mitochondria and chloroplasts, and is involved in RNA editing in mitochondrial NADH dehydrogenase3 (nad3) at nad3-247 and nad3-275. C-to-U editing of nad3-275 is not conserved and even lost in Arabidopsis, consistent with the idea that no close DEK39 homologs are present in Arabidopsis. However, the amino acids generated by editing nad3-247 and nad3-275 are highly conserved in many other plant species, and the reductions of editing at these two sites decrease the activity of mitochondria NADH dehydrogenase complex I, indicating that the alteration of amino acid sequence is necessary for Nad3 function. Our results indicate that Dek39 encodes an E sub-class PPR protein that is involved in RNA editing of multiple sites and is necessary for seed development of maize.

Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize.

Genetic imprinting is a specific epigenetic phenomenon in which a subset of genes is expressed depending on their parent-of-origin. Two types of chromatin modifications, DNA methylation and histone modification, are generally believed to be involved in the regulation of imprinting. However, the genome-wide correlation between allele-specific chromatin modifications and imprinted gene expression in maize remains elusive. Here we report genome-wide high resolution allele-specific maps of DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) in maize endosperm. For DNA methylation, thousands of parent-of-origin dependent differentially methylated regions (pDMRs) were identified. All pDMRs were uniformly paternally hypermethylated and maternally hypomethylated. We also identified 1131 allele-specific H3K27me3 peaks that are preferentially present in the maternal alleles. Maternally expressed imprinted genes (MEGs) and paternally expressed imprinted genes (PEGs) had different patterns of allele-specific DNA methylation and H3K27me3. Allele-specific expression of MEGs was not directly related to allele-specific H3K27me3, and only a subset of MEGs was associated with maternal-specific DNA demethylation, which was primarily located in the upstream and 5' portion of gene body regions. In contrast, allele-specific expression of a majority of PEGs was related to maternal-specific H3K27me3, with a subgroup of PEGs also associated with maternal-specific DNA demethylation. Both pDMRs and maternal H3K27me3 peaks associated with PEGs are enriched in gene body regions. Our results indicate highly complex patterns of regulation on genetic imprinting in maize endosperm.

Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa).

RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, altering the amino acid specified by the DNA sequence. Here we report the identification of a critical editing factor of mitochondrial nad7 transcript via molecular characterization of a small kernel 1 (smk1) mutant in Zea mays (maize). Mutations in Smk1 arrest both the embryo and endosperm development. Cloning of Smk1 indicates that it encodes an E-subclass pentatricopeptide repeat (PPR) protein that is targeted to mitochondria. Loss of SMK1 function abolishes the C â†’ U editing at the nad7-836 site, leading to the retention of a proline codon that is edited to encode leucine in the wild type. The smk1 mutant showed dramatically reduced complex-I assembly and NADH dehydrogenase activity, and abnormal biogenesis of the mitochondria. Analysis of the ortholog in Oryza sativa (rice) reveals that rice SMK1 has a conserved function in C â†’ U editing of the mitochondrial nad7-836 site. T-DNA knock-out mutants showed abnormal embryo and endosperm development, resulting in embryo or seedling lethality. The leucine at NAD7-279 is highly conserved from bacteria to flowering plants, and analysis of genome sequences from many plants revealed a molecular coevolution between the requirement for C â†’ U editing at this site and the existence of an SMK1 homolog. These results demonstrate that Smk1 encodes a PPR-E protein that is required for nad7-836 editing, and this editing is critical to NAD7 function in complex-I assembly in mitochondria, and hence to embryo and endosperm development in maize and rice.

An ultra-high density bin-map for rapid QTL mapping for tassel and ear architecture in a large F2 maize population.

BACKGROUND: Understanding genetic control of tassel and ear architecture in maize (Zea mays L. ssp. mays) is important due to their relationship with grain yield. High resolution QTL mapping is critical for understanding the underlying molecular basis of phenotypic variation. Advanced populations, such as recombinant inbred lines, have been broadly adopted for QTL mapping; however, construction of large advanced generation crop populations is time-consuming and costly. The rapidly declining cost of genotyping due to recent advances in next-generation sequencing technologies has generated new possibilities for QTL mapping using large early generation populations. RESULTS: A set of 708 F2 progeny derived from inbreds Chang7-2 and 787 were generated and genotyped by whole genome low-coverage genotyping-by-sequencing method (average 0.04×). A genetic map containing 6,533 bin-markers was constructed based on the parental SNPs and a sliding-window method, spanning a total genetic distance of 1,396 cM. The high quality and accuracy of this map was validated by the identification of two well-studied genes, r1, a qualitative trait locus for color of silk (chromosome 10) and ba1 for tassel branch number (chromosome 3). Three traits of tassel and ear architecture were evaluated in this population, a total of 10 QTL were detected using a permutation-based-significance threshold, seven of which overlapped with reported QTL. Three genes (GRMZM2G316366, GRMZM2G492156 and GRMZM5G805008) encoding MADS-box domain proteins and a BTB/POZ domain protein were located in the small intervals of qTBN5 and qTBN7 (~800 Kb and 1.6 Mb in length, respectively) and may be involved in patterning of tassel architecture. The small physical intervals of most QTL indicate high-resolution mapping is obtainable with this method. CONCLUSIONS: We constructed an ultra-high-dentisy linkage map for the large early generation population in maize. Our study provides an efficient approach for fast detection of quantitative loci responsible for complex trait variation with high accuracy, thus helping to dissect the underlying molecular basis of phenotypic variation and accelerate improvement of crop breeding in a cost-effective fashion.

Closed floral structure for self-pollination in cultivated tomato.

Cultivated tomatoes exhibit cleistogamy - self-pollination within closed flowers. Wu et al. report that three HD-Zip IV genes and Style2.1 coordinately control anther trichome formation and style length to form closed anther cones that underpin the development of cleistogamy. Further exploration of causal variation and regulatory elements could provide targets for plant breeding.

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.

Recent advances in crop transformation technologies.

Agriculture is experiencing a technological inflection point in its history, while also facing unprecedented challenges posed by human population growth and global climate changes. Key advancements in precise genome editing and new methods for rapid generation of bioengineered crops promise to both revolutionize the speed and breadth of breeding programmes and increase our ability to feed and sustain human population growth. Although genome editing enables targeted and specific modifications of DNA sequences, several existing barriers prevent the widespread adoption of editing technologies for basic and applied research in established and emerging crop species. Inefficient methods for the transformation and regeneration of recalcitrant species and the genotype dependency of the transformation process remain major hurdles. These limitations are frequent in monocotyledonous crops, which alone provide most of the calories consumed by human populations. Somatic embryogenesis and de novo induction of meristems - pluripotent groups of stem cells responsible for plant developmental plasticity - are essential strategies to quickly generate transformed plants. Here we review recent discoveries that are rapidly advancing nuclear transformation technologies and promise to overcome the obstacles that have so far impeded the widespread adoption of genome editing in crop species.

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.

Auxin boosts energy generation pathways to fuel pollen maturation in barley.

Pollen grains become increasingly independent of the mother plant as they reach maturity through poorly understood developmental programs. We report that the hormone auxin is essential during barley pollen maturation to boost the expression of genes encoding almost every step of heterotrophic energy production pathways. Accordingly, auxin is necessary for the flux of sucrose and hexoses into glycolysis and to increase the levels of pyruvate and two tricarboxylic (TCA) cycle metabolites (citrate and succinate). Moreover, bioactive auxin is synthesized by the pollen-localized enzyme HvYUCCA4, supporting that pollen grains autonomously produce auxin to stimulate a specific cellular output, energy generation, that fuels maturation processes such as starch accumulation. Our results demonstrate that auxin can shift central carbon metabolism to drive plant cell development, which suggests a direct mechanism for auxin's ability to promote growth and differentiation.

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.

PPR14 Interacts With PPR-SMR1 and CRM Protein Zm-mCSF1 to Facilitate Mitochondrial Intron Splicing in Maize.

In plants, splicing of organellar group II introns involves numerous nucleus-encoded trans-factors. But, how these trans-factors function and interact is not well understood. Here we report the function of a pentatricopeptide repeat (PPR) protein PPR14 and its physical relationship with other splicing factors in mitochondria. Null mutations of PPR14 severely arrest the embryo and endosperm development, causing an empty pericarp phenotype. PPR14 is required for the splicing of NADH dehydrogenase 2 (nad2) intron 3 and nad7 introns 1 and 2 in mitochondria. The absence of nad2 and nad7 transcripts leads to disruption of the mitochondrial complex I assembly and abolishes its NADH dehydrogenase activity. This is accompanied with increased levels of other mitochondrial complexes and elevated expression of the alternative oxidase proteins. As the function of PPR14 overlaps with PPR-SMR1 and the CRM-domain containing protein Zm-mCSF1, we tested their interactions. Protein-protein interaction analysis indicated that PPR14 interacts with PPR-SMR1 and Zm-mCSF1, suggesting that these three proteins may form a complex. As PPR proteins and CRM-domain containing proteins have many members in mitochondria and chloroplasts, we propose that organellar group II intron splicing is probably mediated by a dynamic complex that includes different PPR and CRM proteins in plants.

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.