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Background Gene expression is a key determinant of cellular response. Natural variation in gene expression bridges genetic variation to phenotypic alteration. Identification of the regulatory variants controlling the gene expression in response to drought, a major environmental threat of crop production worldwide, is of great value for drought-tolerant gene identification. Results A total of 627 RNA-seq analyses are performed for 224 maize accessions which represent a wide genetic diversity under three water regimes; 73,573 eQTLs are detected for about 30,000 expressing genes with high-density genome-wide single nucleotide polymorphisms, reflecting a comprehensive and dynamic genetic architecture of gene expression in response to drought. The regulatory variants controlling the gene expression constitutively or drought-dynamically are unraveled. Focusing on dynamic regulatory variants resolved to genes encoding transcription factors, a drought-responsive network reflecting a hierarchy of transcription factors and their target genes is built. Moreover, 97 genes are prioritized to associate with drought tolerance due to their expression variations through the Mendelian randomization analysis. One of the candidate genes, Abscisic acid 8′-hydroxylase , is verified to play a negative role in plant drought tolerance. Conclusions This study unravels the effects of genetic variants on gene expression dynamics in drought response which allows us to better understand the role of distal and proximal genetic effects on gene expression and phenotypic plasticity. The prioritized drought-associated genes may serve as direct targets for functional investigation or allelic mining.

We created waxy corn hybrids by CRISPR–Cas9 editing of a waxy allele in 12 elite inbred maize lines, a process that was more than a year faster than conventional trait introgression using backcrossing and marker-assisted selection. Field trials at 25 locations showed that CRISPR-waxy hybrids were agronomically superior to introgressed hybrids, producing on average 5.5 bushels per acre higher yield.Gene-edited waxy corn lines have higher yields in field trials than hybrids produced by traditional trait introgression.

Alternative splicing enhances transcriptome diversity in all eukaryotes and plays a role in plant tissue identity and stress adaptation. To catalog new maize (Zea mays) transcripts and identify genomic loci that regulate alternative splicing, we analyzed over 90 RNA-seq libraries from maize inbred lines B73 and Mo17, as well as Syn10 doubled haploid lines (progenies from B73 × Mo17). Transcript discovery was augmented with publicly available data from 14 maize tissues, expanding the maize transcriptome by more than 30,000 and increasing the percentage of intron-containing genes that undergo alternative splicing to 40%. These newly identified transcripts greatly increase the diversity of the maize proteome, sometimes coding for entirely different proteins compared with their most similar annotated isoform. In addition to increasing proteome diversity, many genes encoding novel transcripts gained an additional layer of regulation by microRNAs, often in a tissue-specific manner. We also demonstrate that the majority of genotype-specific alternative splicing can be genetically mapped, with cis-acting quantitative trait loci (QTLs) predominating. A large number of trans-acting QTLs were also apparent, with nearly half located in regions not shown to contain genes associated with splicing. Taken together, these results highlight the currently underappreciated role that alternative splicing plays in tissue identity and genotypic variation in maize.

Background Expression QTL analyses have shed light on transcriptional regulation in numerous species of plants, animals, and yeasts. These microarray-based analyses identify regulators of gene expression as either cis-acting factors that regulate proximal genes, or trans-acting factors that function through a variety of mechanisms to affect transcript abundance of unlinked genes. Results A hydroponics-based genetical genomics study in roots of a Zea mays IBM2 Syn10 double haploid population identified tens of thousands of cis-acting and trans-acting eQTL. Cases of false-positive eQTL, which results from the lack of complete genomic sequences from both parental genomes, were described. A candidate gene for a trans-acting regulatory factor was identified through positional cloning. The unexpected regulatory function of a class I glutamine amidotransferase controls the expression of an ABA 8'-hydroxylase pseudogene. Conclusions Identification of a candidate gene underlying a trans-eQTL demonstrated the feasibility of eQTL cloning in maize and could help to understand the mechanism of gene expression regulation. Lack of complete genome sequences from both parents could cause the identification of false-positive cis- and trans-acting eQTL.

Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3′-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well.

DNA polymorphisms such as insertion/deletions and duplications affecting genome segments larger than 1 kb are known as copy-number variations (CNVs) or structural variations (SVs). They have been recently studied in animals and humans by using array-comparative genome hybridization (aCGH), and have been associated with several human diseases. Their presence and phenotypic effects in plants have not been investigated on a genomic scale, although individual structural variations affecting traits have been described. We used aCGH to investigate the presence of CNVs in maize by comparing the genome of 13 maize inbred lines to B73. Analysis of hybridization signal ratios of 60,472 60-mer oligonucleotide probes between inbreds in relation to their location in the reference genome (B73) allowed us to identify clusters of probes that deviated from the ratio expected for equal copy-numbers. We found CNVs distributed along the maize genome in all chromosome arms. They occur with appreciable frequency in different germplasm subgroups, suggesting ancient origin. Validation of several CNV regions showed both insertion/deletions and copy-number differences. The nature of CNVs detected suggests CNVs might have a considerable impact on plant phenotypes, including disease response and heterosis.

Plant diurnal rhythms are vital environmental adaptations to coordinate internal physiological responses to alternating day-night cycles. A comprehensive view of diurnal biology has been lacking for maize (Zea mays), a major world crop. A photosynthetic tissue, the leaf, and a non-photosynthetic tissue, the developing ear, were sampled under natural field conditions. Genome-wide transcript profiling was conducted on a high-density 105 K Agilent microarray to investigate diurnal rhythms. In both leaves and ears, the core oscillators were intact and diurnally cycling. Maize core oscillator genes are found to be largely conserved with their Arabidopsis counterparts. Diurnal gene regulation occurs in leaves, with some 23% of expressed transcripts exhibiting a diurnal cycling pattern. These transcripts can be assigned to over 1700 gene ontology functional terms, underscoring the pervasive impact of diurnal rhythms on plant biology. Considering the peak expression time for each diurnally regulated gene, and its corresponding functional assignment, most gene functions display temporal enrichment in the day, often with distinct patterns, such as dawn or midday preferred, indicating that there is a staged procession of biological events undulating with the diurnal cycle. Notably, many gene functions display a bimodal enrichment flanking the midday photosynthetic maximum, with an initial peak in mid-morning followed by another peak during the afternoon/evening. In contrast to leaves, in developing ears as few as 47 gene transcripts are diurnally regulated, and this set of transcripts includes primarily the core oscillators. In developing ears, which are largely shielded from light, the core oscillator therefore is intact with little outward effect on transcription.

Maize starchy endosperm mutants have kernel phenotypes that include a brittle texture, susceptibility to insect pests, and inferior functional characteristics of products made from their flour. At least 18 such mutants have been identified, but only in the cases of opaque2 (o2) and floury2 (fl2), which affect different aspects of storage protein synthesis, is the molecular basis of the mutation known. To better understand the relationship between the phenotypes of these mutants and their biochemical bases, we characterized the protein and amino acid composition, as well as the mRNA transcript profiles, of nearly isogenic inbred lines of W64A o1, o2, o5, o9, o11, Mucuronate (Mc), Defective endosperm B30 (DeB30), and fl2. The largest reductions in zein protein synthesis occur in the W64A o2, DeB30, and fl2 mutants, which have ∼35 to 55% of the wild-type level of storage proteins. Zeins in W64A o5, o9, o11, and Mc are within 80 to 90% of the amount found in the wild type. Only in the cases of o5 and Mc were significant qualitative changes in zein synthesis observed. The pattern of gene expression in normal and mutant genotypes was assayed by profiling endosperm mRNA transcripts at 18 days after pollination with an Affymetrix GeneChip containing >1400 selected maize gene sequences. Compared with W64A sugary1, a mutant defective in starch synthesis, alterations in the gene expression patterns of the opaque mutants are very pleiotropic. Increased expression of genes associated with physiological stress, and the unfolded protein response, are common features of the opaque mutants. Based on global patterns of gene expression, these mutants were categorized in four phenotypic groups as follows: W64A + and o1; o2; o5/o9/o11; and Mc and fl2.

Maize (Zea mays) develops an extensive shoot-borne root system to secure water and nutrient uptake and to provide anchorage in the soil. In this study, early coleoptilar node (first shoot node) development was subjected to a detailed morphological and histological analysis. Subsequently, microarray profiling via hybridization of oligonucleotide microarrays representing transcripts of 31,355 unique maize genes at three early stages of coleoptilar node development was performed. These pairwise comparisons of wild-type versus mutant rootless concerning crown and seminal roots (rtcs) coleoptilar nodes that do not initiate shoot-borne roots revealed 828 unique transcripts that displayed RTCS-dependent expression. A stage-specific functional analysis revealed overrepresentation of \"cell wall,\" \"stress,\" and \"development\"-related transcripts among the differentially expressed genes. Differential expression of a subset of 15 of 828 genes identified by these microarray experiments was independently confirmed by quantitative real-time-polymerase chain reaction. In silico promoter analyses revealed that 100 differentially expressed genes contained at least one LATERAL ORGAN BOUNDARIES domain (LBD) motif within 1 kb upstream of the ATG start codon. Electrophoretic mobility shift assay experiments demonstrated RTCS binding for four of these promoter sequences, supporting the notion that differentially accumulated genes containing LBD motifs are likely direct downstream targets of RTCS.