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Demonstrates that the knotted-1 gene encodes a homeodomain-containing protein, the first identified in the plant kingdom. Uses the knotted-1 homeobox to isolate other expressed homeobox genes in maize.

Brassinosteroids (BRs) are important regulators that control myriad aspects of plant growth and development, including biotic and abiotic stress responses, such that modulating BR homeostasis and signaling presents abundant opportunities for plant breeding and crop improvement. Enzymes and other proteins involved in the biosynthesis and signaling of BRs are well understood from molecular genetics and phenotypic analysis in Arabidopsis thaliana; however, knowledge of the molecular functions of these genes in other plant species, especially cereal crop plants, is minimal. In this manuscript, we comprehensively review functional studies of BR genes in Arabidopsis, maize, rice, Setaria, Brachypodium, and soybean to identify conserved and diversified functions across plant species and to highlight cases for which additional research is in order. We performed phylogenetic analysis of gene families involved in the biosynthesis and signaling of BRs and re-analyzed publicly available transcriptomic data. Gene trees coupled with expression data provide a valuable guide to supplement future research on BRs in these important crop species, enabling researchers to identify gene-editing targets for BR-related functional studies., (Published by Elsevier Inc.)

Maize (Zea mays L.) C-type cytoplasmic male sterility (CMS-C) is a highly used CMS system for maize commercial hybrid seed production. Rf4 is the major dominant restorer gene for CMS-C. Inbreds were recently discovered which contain the restoring Rf4 allele yet are unable to restore fertility due to the lack of an additional gene required for Rf4's restoration. To find this additional gene, QTL mapping and positional cloning were performed using an inbred that contained Rf4 but was incapable of restoring CMS-C. The QTL was mapped to a 738-kb interval on chromosome 2, which contains a Pentatricopeptide Repeat (PPR) gene cluster. Allele content comparisons of the inbreds identified three potential candidate genes responsible for fertility restoration in CMS-C. Complementation via transformation of these three candidate genes showed that PPR153 (Zm00001eb114660) is required for Rf4 to restore fertility to tassels. The PPR153 sequence is present in B73 genome, but it is not capable of restoring CMS-C without Rf4. Analysis using NAM lines revealed that Rf4 requires the presence of PPR153 to restore CMS-C in diverse germplasms. This research uncovers a major CMS-C genetic restoration pathway and can be used for identifying inbreds suitable for maize hybrid production with CMS-C cytoplasm., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Jaqueth et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Grass inflorescences support floral structures that each bear a single grain, where variation in branch architecture directly impacts yield. The maize (Zea mays) RAMOSA1 (ZmRA1) transcription factor acts as a key regulator of inflorescence development by imposing branch meristem determinacy. Here, we show RA1 transcripts accumulate in boundary domains adjacent to spikelet meristems in sorghum (Sorghum bicolor, Sb) and green millet (Setaria viridis, Sv) inflorescences similar as in the developing maize tassel and ear. To evaluate the functional conservation of syntenic RA1 orthologs and promoter cis sequences in maize, sorghum, and setaria, we utilized interspecies gene transfer and assayed genetic complementation in a common inbred background by quantifying recovery of normal branching in highly ramified ra1-R mutants. A ZmRA1 transgene that includes endogenous upstream and downstream flanking sequences recovered normal tassel and ear branching in ra1-R. Interspecies expression of two transgene variants of the SbRA1 locus, modeled as the entire endogenous tandem duplication or just the nonframeshifted downstream copy, complemented ra1-R branching defects and induced unusual fasciation and branch patterns. The SvRA1 locus lacks conserved, upstream noncoding cis sequences found in maize and sorghum; interspecies expression of a SvRA1 transgene did not or only partially recovered normal inflorescence forms. Driving expression of the SvRA1 coding region by the ZmRA1 upstream region, however, recovered normal inflorescence morphology in ra1-R. These data leveraging interspecies gene transfer suggest that cis-encoded temporal regulation of RA1 expression is a key factor in modulating branch meristem determinacy that ultimately impacts grass inflorescence architecture., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Suboptimal treatment of hypertension remains a widespread problem, particularly among minorities and socioeconomically disadvantaged groups. We present a health system-based intervention with diverse patient populations using readily available smartphone technology. This intervention is designed to empower patients and create partnerships between patients and their provider team to promote hypertension control., Objective: The mGlide randomized controlled trial is a National Institutes of Health-funded study, evaluating whether a mobile health (mHealth)-based intervention that is an active partnership between interprofessional health care teams and patients results in better hypertension control rates than a state-of-clinical care comparison., Methods: We are recruiting 450 participants including stroke survivors and primary care patients with elevated cardiovascular disease risk from diverse health systems. These systems include an acute stroke service (n=100), an academic medical center (n=150), and community medical centers including Federally Qualified Health Centers serving low-income and minority (Latino, Hmong, African American, Somali) patients (n=200). The primary aim tests the clinical effectiveness of the 6-month mHealth intervention versus standard of care. Secondary aims evaluate sustained hypertension control rates at 12 months; describe provider experiences of system usability and satisfaction; examine patient experiences, including medication adherence and medication use self-efficacy, self-rated health and quality of life, and adverse event rates; and complete a cost-effectiveness analysis., Results: To date, we have randomized 107 participants (54 intervention, 53 control)., Conclusions: This study will provide evidence for whether a readily available mHealth care model is better than state-of-clinical care for bridging the guideline-to-practice gap in hypertension treatment in health systems serving diverse patient populations., Trial Registration: Clinicaltrials.gov NCT03612271; https://clinicaltrials.gov/ct2/show/NCT03612271., International Registered Report Identifier (irrid): DERR1-10.2196/25424., (©Kamakshi Lakshminarayan, Thomas A Murray, Sarah M Westberg, John Connett, Val Overton, John A Nyman, Kathleen A Culhane-Pera, Shannon L Pergament, Paul Drawz, Emily Vollbrecht, Txia Xiong, Susan A Everson-Rose. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 25.01.2021.)

In flowering plants, gene expression in the haploid male gametophyte (pollen) is essential for sperm delivery and double fertilization. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene expression is not clearly understood. To explore relationships among these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and sperm cells) via RNA-seq. We found that, in contrast with vegetative cell-limited TE expression in Arabidopsis pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinate expression was observed between highly expressed protein-coding genes and their neighboring TEs, specifically in mature pollen and sperm cells. To investigate a potential relationship between elevated gene transcript level and pollen function, we measured the fitness cost (male-specific transmission defect) of GFP-tagged coding sequence insertion mutations in over 50 genes identified as highly expressed in the pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in seedling genes or sperm cell genes (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation when mutant. Insertions in maize gamete expressed2 (Zm gex2), the sole sperm cell gene with measured contributions to fitness, also triggered seed defects when crossed as a male, indicating a conserved role in double fertilization, given the similar phenotype previously demonstrated for the Arabidopsis ortholog GEX2. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes in pollen, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes., Competing Interests: The authors have declared that no competing interests exist.

Type C cytoplasmic male sterility (CMS-C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS-C, is located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non-restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map-based cloning interval of 12 kb. Within this 12-kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix-loop-helix (bHLH) transcription factor with tassel-specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non-restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR-Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Floral morphology is shaped by factors that modulate floral meristem activity and size, and the identity, number and arrangement of the lateral organs they form. We report here that the maize CRABS CLAW co-orthologs drooping leaf1 ( drl1 ) and drl2 are required for development of ear and tassel florets. Pistillate florets of drl1 ears are sterile with unfused carpels that fail to enclose an expanded nucellus-like structure. Staminate florets of drl1 tassels have extra stamens and fertile anthers. Natural variation and transposon alleles of drl2 enhance drl1 mutant phenotypes by reducing floral meristem (FM) determinacy. The drl paralogs are co-expressed in lateral floral primordia, but not within the FM. drl expression together with the more indeterminate mutant FMs suggest that the drl genes regulate FM activity and impose meristem determinacy non-cell-autonomously from differentiating cells in lateral floral organs. We used gene regulatory network inference, genetic interaction and expression analyses to suggest that DRL1 and ZAG1 target each other and a common set of downstream genes that function during floret development, thus defining a regulatory module that fine-tunes floret patterning and FM determinacy., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The maize W22 inbred has served as a platform for maize genetics since the mid twentieth century. To streamline maize genome analyses, we have sequenced and de novo assembled a W22 reference genome using short-read sequencing technologies. We show that significant structural heterogeneity exists in comparison to the B73 reference genome at multiple scales, from transposon composition and copy number variation to single-nucleotide polymorphisms. The generation of this reference genome enables accurate placement of thousands of Mutator (Mu) and Dissociation (Ds) transposable element insertions for reverse and forward genetics studies. Annotation of the genome has been achieved using RNA-seq analysis, differential nuclease sensitivity profiling and bisulfite sequencing to map open reading frames, open chromatin sites and DNA methylation profiles, respectively. Collectively, the resources developed here integrate W22 as a community reference genome for functional genomics and provide a foundation for the maize pan-genome.

Axillary branch suppression is a favorable trait bred into many domesticated crop plants including maize compared with its highly branched wild ancestor teosinte. Branch suppression in maize was achieved through selection of a gain of function allele of the teosinte branched1 (tb1) transcription factor that acts as a repressor of axillary bud growth. Previous work indicated that other loci may function epistatically with tb1 and may be responsible for some of its phenotypic effects. Here, we show that tb1 mediates axillary branch suppression through direct activation of the tassels replace upper ears1 ( tru1 ) gene that encodes an ankyrin repeat domain protein containing a BTB/POZ motif necessary for protein-protein interactions. The expression of TRU1 and TB1 overlap in axillary buds, and TB1 binds to two locations in the tru1 gene as shown by chromatin immunoprecipitation and gel shifts. In addition, nucleotide diversity surveys indicate that tru1 , like tb1 , was a target of selection. In modern maize, TRU1 is highly expressed in the leaf trace vasculature of axillary internodes, while in teosinte, this expression is highly reduced or absent. This increase in TRU1 expression levels in modern maize is supported by comparisons of relative protein levels with teosinte as well as by quantitative measurements of mRNA levels. Hence, a major innovation in creating ideal maize plant architecture originated from ectopic overexpression of tru1 in axillary branches, a critical step in mediating the effects of domestication by tb1., Competing Interests: The authors declare no conflict of interest.

Leaf architecture directly influences canopy structure, consequentially affecting yield. We discovered a maize ( Zea mays ) mutant with aberrant leaf architecture, which we named drooping leaf1 ( drl1 ). Pleiotropic mutations in drl1 affect leaf length and width, leaf angle, and internode length and diameter. These phenotypes are enhanced by natural variation at the drl2 enhancer locus, including reduced expression of the drl2-Mo17 allele in the Mo17 inbred. A second drl2 allele, produced by transposon mutagenesis, interacted synergistically with drl1 mutants and reduced drl2 transcript levels. The drl genes are required for proper leaf patterning, development and cell proliferation of leaf support tissues, and for restricting auricle expansion at the midrib. The paralogous loci encode maize CRABS CLAW co-orthologs in the YABBY family of transcriptional regulators. The drl genes are coexpressed in incipient and emergent leaf primordia at the shoot apex, but not in the vegetative meristem or stem. Genome-wide association studies using maize NAM-RIL (nested association mapping-recombinant inbred line) populations indicated that the drl loci reside within quantitative trait locus regions for leaf angle, leaf width, and internode length and identified rare single nucleotide polymorphisms with large phenotypic effects for the latter two traits. This study demonstrates that drl genes control the development of key agronomic traits in maize., (© 2017 American Society of Plant Biologists. All rights reserved.)

Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

The aleurone is the outermost layer of cereal endosperm and functions to digest storage products accumulated in starchy endosperm cells as well as to confer important dietary health benefits. Whereas normal maize (Zea mays [Zm]) has a single aleurone layer, naked endosperm (nkd) mutants produce multiple outer cell layers of partially differentiated cells that show sporadic expression of aleurone identity markers such as a viviparous1 promoter-β-glucuronidase transgene. The 15:1 F2 segregation ratio suggested that two recessive genes were involved, and map-based cloning identified two homologous genes in duplicated regions of the genome. The nkd1 and nkd2 genes encode the INDETERMINATE1 domain (IDD) containing transcription factors ZmIDDveg9 and ZmIDD9 on chromosomes 2 and 10, respectively. Independent mutant alleles of nkd1 and nkd2, as well as nkd2-RNA interference lines in which both nkd genes were knocked down, also showed the nkd mutant phenotype, confirming the gene identities. In wild-type kernels, the nkd transcripts were most abundant around 11 to 16 d after pollination. The NKD proteins have putative nuclear localization signals, and green fluorescent protein fusion proteins showed nuclear localization. The mutant phenotype and gene identities suggest that NKD controls a gene regulatory network involved in aleurone cell fate specification and cell differentiation., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Plant architecture is dictated by precise control of meristematic activity. In the shoot, an imbalance in positive or negative maintenance signals can result in a fasciated or enlarged meristem phenotype. fasciated ear4 (fea4) is a semidwarfed mutant with fasciated ears and tassels as well as greatly enlarged vegetative and inflorescence meristems. We identified FEA4 as a bZIP transcription factor, orthologous to Arabidopsis thaliana PERIANTHIA. FEA4 was expressed in the peripheral zone of the vegetative shoot apical meristem and in the vasculature of immature leaves and conspicuously excluded from the stem cell niche at the tip of the shoot apical meristem and from incipient leaf primordia. Following the transition to reproductive fate, FEA4 was expressed throughout the entire inflorescence and floral meristems. Native expression of a functional YFP:FEA4 fusion recapitulated this pattern of expression. We used chromatin immunoprecipitation-sequencing to identify 4060 genes proximal to FEA4 binding sites, including ones that were potentially bound and modulated by FEA4 based on transcriptional changes in fea4 mutant ears. Our results suggest that FEA4 promotes differentiation in the meristem periphery by regulating auxin-based responses and genes associated with leaf differentiation and polarity, potentially in opposition to factors such as KNOTTED1 and WUSCHEL., (© 2015 American Society of Plant Biologists. All rights reserved.)

Background: Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life cycle is that gene expression is required in the haploid gametophytes. Consequently, many mutant phenotypes are expressed in this phase., Results: We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. Transposon-related transcripts are present in high levels in both gametophytes, suggesting a connection between gamete production and transposon expression in maize not previously identified in any female gametophytes. Two classes of small signaling proteins and several transcription factor gene families are enriched in gametophyte transcriptomes. Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2 indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than subgenome 2 genes with other expression patterns. Analysis of available insertion mutant collections shows a statistically significant deficit in insertions in gametophyte-expressed genes., Conclusions: This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies maize gametophyte functions, gametophyte expression of transposon-related sequences, and unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed genes is supporting evidence for their function in the gametophytes. Expression patterns of extant, duplicated maize genes reveals that selective pressures based on male gametophytic function have likely had a disproportionate effect on plant genomes.

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

Plant axillary meristems are composed of highly organized, self-renewing stem cells that produce indeterminate branches or terminate in differentiated structures, such as the flowers. These opposite fates, dictated by both genetic and environmental factors, determine interspecific differences in the architecture of plants. The Cys(2)-His(2) zinc-finger transcription factor RAMOSA1 (RA1) regulates the fate of most axillary meristems during the early development of maize inflorescences, the tassel and the ear, and has been implicated in the evolution of grass architecture. Mutations in RA1 or any other known members of the ramosa pathway, RAMOSA2 and RAMOSA3, generate highly branched inflorescences. Here, we report a genetic screen for the enhancement of maize inflorescence branching and the discovery of a new regulator of meristem fate: the RAMOSA1 ENHANCER LOCUS2 (REL2) gene. rel2 mutants dramatically increase the formation of long branches in ears of both ra1 and ra2 mutants. REL2 encodes a transcriptional co-repressor similar to the TOPLESS protein of Arabidopsis, which is known to maintain apical-basal polarity during embryogenesis. REL2 is capable of rescuing the embryonic defects of the Arabidopsis topless-1 mutant, suggesting that REL2 also functions as a transcriptional co-repressor throughout development. We show by genetic and molecular analyses that REL2 physically interacts with RA1, indicating that the REL2/RA1 transcriptional repressor complex antagonizes the formation of indeterminate branches during maize inflorescence development. Our results reveal a novel mechanism for the control of meristem fate and the architecture of plants.

The maize (Zea mays) transposable element Dissociation (Ds) was mobilized for large-scale genome mutagenesis and to study its endogenous biology. Starting from a single donor locus on chromosome 10, over 1500 elements were distributed throughout the genome and positioned on the maize physical map. Genetic strategies to enrich for both local and unlinked insertions were used to distribute Ds insertions. Global, regional, and local insertion site trends were examined. We show that Ds transposed to both linked and unlinked sites and displayed a nonuniform distribution on the genetic map around the donor r1-sc:m3 locus. Comparison of Ds and Mutator insertions reveals distinct target preferences, which provide functional complementarity of the two elements for gene tagging in maize. In particular, Ds displays a stronger preference for insertions within exons and introns, whereas Mutator insertions are more enriched in promoters and 5'-untranslated regions. Ds has no strong target site consensus sequence, but we identified properties of the DNA molecule inherent to its local structure that may influence Ds target site selection. We discuss the utility of Ds for forward and reverse genetics in maize and provide evidence that genes within a 2- to 3-centimorgan region flanking Ds insertions will serve as optimal targets for regional mutagenesis.

Modern maize was domesticated from Zea mays parviglumis, a teosinte, about 9000 years ago in Mexico. Genes thought to have been selected upon during the domestication of crops are commonly known as domestication loci. The ramosa1 (ra1) gene encodes a putative transcription factor that controls branching architecture in the maize tassel and ear. Previous work demonstrated reduced nucleotide diversity in a segment of the ra1 gene in a survey of modern maize inbreds, indicating that positive selection occurred at some point in time since maize diverged from its common ancestor with the sister species Tripsacum dactyloides and prompting the hypothesis that ra1 may be a domestication gene. To investigate this hypothesis, we examined ear phenotypes resulting from minor changes in ra1 activity and sampled nucleotide diversity of ra1 across the phylogenetic spectrum between tripsacum and maize, including a broad panel of teosintes and unimproved maize landraces. Weak mutant alleles of ra1 showed subtle effects in the ear, including crooked rows of kernels due to the occasional formation of extra spikelets, correlating a plausible, selected trait with subtle variations in gene activity. Nucleotide diversity was significantly reduced for maize landraces but not for teosintes, and statistical tests implied directional selection on ra1 consistent with the hypothesis that ra1 is a domestication locus. In maize landraces, a noncoding 3'-segment contained almost no genetic diversity and 5'-flanking diversity was greatly reduced, suggesting that a regulatory element may have been a target of selection.

We describe genetic screens, molecular methods and web resources newly available to utilize Dissociation (Ds) as an insertional mutagen in maize. Over 1700 Ds elements have been distributed throughout the maize genome to serve as donor elements for local or regional mutagenesis. Two genetic screens are described to identify Ds insertions in genes-of-interest (goi). In scheme I, Ds is used to generate insertion alleles when a recessive reference allele is available. A Ds insertion will enable the cloning of the target gene and can be used to create an allelic series. In scheme II, Ds insertions in a goi are identified using a PCR-based screen to identify the rare insertion alleles among a population of testcross progeny. We detail an inverse PCR protocol to rapidly amplify sequences flanking Ds insertion alleles and describe a high-throughput 96-well plate-based DNA extraction method for the recovery of high-quality genomic DNA from seedling tissues. We also describe several web-based tools for browsing, searching and accessing the genetic materials described. The development of these Ds insertion lines promises to greatly accelerate functional genomics studies in maize.

Genetic control of grass inflorescence architecture is critical given that cereal seeds provide most of the world's food. Seeds are borne on axillary branches, which arise from groups of stem cells in axils of leaves and whose branching patterns dictate most of the variation in plant form. Normal maize (Zea mays) ears are unbranched, and tassels have long branches only at their base. The ramosa2 (ra2) mutant of maize has increased branching with short branches replaced by long, indeterminate ones. ra2 was cloned by chromosome walking and shown to encode a LATERAL ORGAN BOUNDARY domain transcription factor. ra2 is transiently expressed in a group of cells that predicts the position of axillary meristem formation in inflorescences. Expression in different mutant backgrounds places ra2 upstream of other genes that regulate branch formation. The early expression of ra2 suggests that it functions in the patterning of stem cells in axillary meristems. Alignment of ra2-like sequences reveals a grass-specific domain in the C terminus that is not found in Arabidopsis thaliana. The ra2-dm allele suggests this domain is required for transcriptional activation of ra1. The ra2 expression pattern is conserved in rice (Oryza sativa), barley (Hordeum vulgare), sorghum (Sorghum bicolor), and maize, suggesting that ra2 is critical for shaping the initial steps of grass inflorescence architecture.

The external appearance of flowering plants is determined to a large extent by the forms of flower-bearing branch systems, known as inflorescences, and their position in the overall structure of the plant. Branches and branching patterns are produced by tissues called shoot apical meristems. Thus, inflorescence architecture reflects meristem number, arrangement and activity, and the duration of meristem activity correlates with branch length. The inflorescences of maize, unlike those of related grasses such as rice and sorghum, predominantly lack long branches, giving rise to the tassel and familiar corncob. Here we report the isolation of the maize ramosa1 gene and show that it controls inflorescence architecture. Through its expression in a boundary domain near the nascent meristem base, ramosa1 imposes short branch identity as branch meristems are initiated. A second gene, ramosa2, acts through ramosa1 by regulating ramosa1 gene expression levels. ramosa1 encodes a transcription factor that appears to be absent in rice, is heterochronically expressed in sorghum, and may have played an important role in maize domestication and grass evolution.

Development in higher plants depends on the activity of meristems, formative regions that continuously initiate new organs at their flanks. Meristems must maintain a balance between stem cell renewal and organ initiation. In fasciated mutants, organ initiation fails to keep pace with meristem proliferation. The thick tassel dwarf1 (td1) mutation of maize affects both male and female inflorescence development. The female inflorescence, which results in the ear, is fasciated, with extra rows of kernels. The male inflorescence, or tassel, shows an increase in spikelet density. Floral meristems are also affected in td1 mutants; for example, male florets have an increase in stamen number. These results suggest that td1 functions in the inflorescence to limit meristem size. In addition, td1 mutants are slightly shorter than normal siblings, indicating that td1 also plays a role in vegetative development. td1 encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) that is a putative ortholog of the Arabidopsis CLAVATA1 protein. These results complement previous work showing that fasciated ear2 encodes a CLAVATA2-like protein, and suggest that the CLAVATA signaling pathway is conserved in monocots. td1 maps in the vicinity of quantitative trait loci that affect seed row number, spikelet density and plant height. We discuss the possible selection pressures on td1 during maize domestication.

We describe an efficient system for site-selected transposon mutagenesis in maize. A total of 43,776 F1 plants were generated by using Robertson's Mutator (Mu) pollen parents and self-pollinated to establish a library of transposon-mutagenized seed. The frequency of new seed mutants was between 10-4 and 10-5 per F1 plant. As a service to the maize community, maize-targeted mutagenesis selects insertions in genes of interest from this library by using the PCR. Pedigree, knockout, sequence, phenotype, and other information is stored in a powerful interactive database (maize-targeted mutagenesis database) that enables analysis of the entire population and the handling of knockout requests. By inhibiting Mu activity in most F1 plants, we sought to reduce somatic insertions that may cause false positives selected from pooled tissue. By monitoring the remaining Mu activity in the F2, however, we demonstrate that seed phenotypes depend on it, and false positives occur in lines that appear to lack it. We conclude that more than half of all mutations arising in this population are suppressed on losing Mu activity. These results have implications for epigenetic models of inbreeding and for functional genomics.

The Kn1-2F11 mutation causes protrusions or knots along the lateral veins of the first few leaves of the maize plant. The phenotype is visible when an unlinked gene, presumably Ac, is present in the genome. The mutation is closely linked to a genetically unstable Adh1 mutation that resulted from the insertion of a Ds2 element (Döring et al., 1984; Chen et al., 1986). Using a unique sequence from the Ds2 element as a hybridization probe, a genomic restriction fragment that cosegregated with the knotted phenotype was cloned. It carries the Kn1-2F11 locus by the following criteria. (i) Cosegregation of the fragment is tightly linked to the phenotype. (ii) Somatic and germinal excision produce a fragment which is the expected size of a revertant fragment; progeny containing the revertant size fragment are normal. (iii) The sequences that hybridize to this fragment are significantly altered in the chromosome containing the original knotted mutation, Kn1-O, (iv) The cloned fragment does not hybridize to a chromosome that contains a deletion of Kn1-O.