An ethylene biosynthesis enzyme controls quantitative variation in maize ear length and kernel yield

Maize ear size and kernel number differ among lines, however, little is known about the molecular basis of ear length and its impact on kernel number. Here, we characterize a quantitative trait locus, qEL7, to identify a maize gene controlling ear length, flower number and fertility. qEL7 encodes 1-aminocyclopropane-1- carboxylate oxidase2 (ACO2), a gene that functions in the final step of ethylene biosynthesis and is expressed in specific domains in developing inflorescences. Confirmation of qEL7 by gene editing of ZmACO2 leads to a reduction in ethylene production in developing ears, and promotes meristem and flower development, resulting in a ~13.4% increase in grain yield per ear in hybrids lines. Our findings suggest that ethylene serves as a key signal in inflorescence development, affecting spikelet number, floral fertility, ear length and kernel number, and also provide a tool to improve grain productivity by optimizing ethylene levels in maize or in other cereals.

The maize gene maternal depression of r1 (mdr1) encodes a DNA glycosylase with maternal and paternal fertility functions

Demethylation of transposons can activate expression of nearby genes and cause imprinted gene expression in endosperm, and it is hypothesized to lead to expression of transposon siRNAs that reinforce silencing in the next generation through transfer either into egg or embryo. Here we describe maternal derepression of R1 (mdr1), a DNA glycosylase with homology to Arabidopsis DEMETER that is partially responsible for demethylation of thousands of regions in endosperm. Maternally-expressed imprinted genes were enriched strongly enriched for overlap with demethylated regions, but the majority of genes that overlapped demethylated regions were not imprinted. Demethylated regions were depleted from the majority of repetitive DNA in the genome but enriched in a set of transposon families accounting for about a tenth of the total demethylated regions. Demethylated regions produced few siRNAs and were not associated with excess CHH methylation in endosperm or other tissues. mdr1 and its close homolog dng102 are essential factors in maternal and paternal fertility in maize, as neither double mutant microgametophytes nor megagametophytes gave rise to seeds. These data establish DNA demethylation by glycosylases as essential in maize endosperm and pollen and suggest that neither transposon regulation nor genomic imprinting are its main function.

Maize Plants Chimeric for an Autoactive Resistance Gene Display a Cell Autonomous Hypersensitive Response but Non-Cell Autonomous Defense Signaling.

The maize gene Rp1-D21 is a mutant form of the gene Rp1-D that confers resistance to common rust. Rp1-D21 triggers a spontaneous defense response that occurs in the absence of the pathogen and includes a programed cell death called the hypersensitive response (HR). Eleven plants heterozygous for Rp1-D21, in four different genetic backgrounds, were identified that had chimeric leaves with lesioned sectors showing HR abutting green non-lesioned sectors lacking HR. The Rp1-D21 sequence derived from each of the lesioned portions of leaves was unaltered from the expected sequence whereas the Rp1-D21 sequences from nine of the non-lesioned sectors displayed various mutations and we were unable to amplify Rp1-D21 from the other two non-lesioned sectors. In every case, the borders between the sectors were sharp with no transition zone, suggesting that HR and chlorosis associated with Rp1-D21 activity was cell-autonomous. Expression of defense response marker genes was assessed in the lesioned and non-lesioned sectors as well as in near-isogenic plants lacking and carrying Rp1-D21. Defense gene expression was somewhat elevated in non-lesioned sectors abutting sectors carrying Rp1-D21 compared to near-isogenic plants lacking Rp1-D21. This suggests that while the HR itself was cell autonomous, other aspects of the defense response initiated by Rp1-D21 were not.

A CRISPR/dCas9 toolkit for functional analysis of maize genes

Background The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

Plant homeodomain proteins provide a mechanism for how leaves grow wide

ABSTRACTThe mechanisms whereby leaf anlagen undergo proliferative growth and expansion to form wide, flat leaves are unclear. The maize gene NARROWSHEATH1 (NS1) is a WUSCHEL-related homeobox3 (WOX3) homolog expressed at the margins of leaf primordia, and is required for mediolateral outgrowth. To investigate the mechanisms of NS1 function, we used chromatin immunoprecipitation and laser-microdissection RNA-seq of leaf primordial margins to identify gene targets bound and modulated by NS1. Microscopic analyses of cell division and gene expression in expanding leaves, and reverse genetic analyses of homologous NS1 target genes in Arabidopsis, reveal that NS1 controls mediolateral outgrowth by repression of a growth inhibitor and promotion of cell division at primordial leaf margins. Intriguingly, homologous WOX gene products are expressed in stem cell-organizing centers and traffic to adjoining cells to activate stem-cell identity non-autonomously. In contrast, WOX3/NS1 does not traffic, and stimulates cell divisions in the same cells in which it is transcribed.

A CRISPR/dCas9 toolkit for functional analysis of maize genes

Background: The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

A CRISPR/dCas9 toolkit for functional analysis of maize genes

Background: The C lustered R egularly I nterspaced S hort P alindromic R epeats (CRISPR)/Cas9 system has become a powerful tool for functional genomics in plants. The RNA-guided nuclease can be used to not only generate precise genomic mutations, but also to manipulate gene expression when present as a deactivated protein (dCas9). Results: In this study, we describe a vector toolkit for analyzing dCas9-mediated activation (CRISPRa) or inactivation (CRISPRi) of gene expression in maize protoplasts. An improved maize protoplast isolation and transfection method is presented, as well as a description of dCas9 vectors to enhance or repress maize gene expression. Additionally, we describe the utility of Foxtail Mosaic Virus (FoMV), a positive-sense RNA monocot virus, as a vector for delivering guide RNAs (gRNAs) to maize protoplasts in addition to whole plants. Conclusions: We anticipate that this maize protoplast toolkit will streamline the analysis of gRNA candidates and facilitate genetic studies of important trait genes in this transformation-recalcitrant plant.

Using single-plant -omics in the field to link maize genes to functions and phenotypes

ABSTRACTMost of our current knowledge on plant molecular biology is based on experiments in controlled lab environments. Over the years, lab experiments have generated substantial insights in the molecular wiring of plant developmental processes, stress responses and phenotypes. However, translating these insights from the lab to the field is often not straightforward, in part because field growth conditions are very different from lab conditions. Here, we test a new experimental design to unravel the molecular wiring of plants and study gene-phenotype relationships directly in the field. We molecularly profiled a set of individual maize plants of the same inbred background grown in the same field, and used the resulting data to predict the phenotypes of individual plants and the function of maize genes. We show that the field transcriptomes of individual plants contain as much information on maize gene function as traditional lab-generated transcriptomes of pooled plant samples subject to controlled perturbations. Moreover, we show that field-generated transcriptome and metabolome data can be used to quantitatively predict at least some individual plant phenotypes. Our results show that profiling individual plants in the field is a promising experimental design that could help narrow the lab-field gap.