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MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression in many eukaryotes. Next-generation sequencing of small RNAs (small RNA-seq) is central to the discovery and annotation of miRNAs. Newly annotated miRNAs and their longer precursors encoded byMIRNAloci are typically submitted to databases such as the miRBase microRNA registry following the publication of a peer-reviewed study. However, genome-wide scans using small RNA-seq data often yield high rates of false-positiveMIRNAannotations, highlighting the need for more robust validation methods. miRScore was developed as an independent and efficient tool for evaluating newMIRNAannotations using sRNA-seq data. miRScore combines structural and expression-based analyses to provide rapid and reliable validation of newMIRNAannotations. By providing users with detailed metrics and visualization, miRScore enhances the ability to assess confidence inMIRNAannotations. miRScore has the potential to advance the overall quality ofMIRNAannotations by improving accuracy of new submissions to miRNA databases and serving as a resource for re-evaluating existing annotations. 

Three cross-incompatibility loci each control a distinct reproductive barrier in both domesticated maize (Zea mays ssp. mays) and its wild teosinte relatives. These three loci, Teosinte crossing barrier1 (Tcb1), Gametophytic factor1 (Ga1), and Ga2, each play a key role in preventing hybridization between incompatible populations and are proposed to maintain the barrier between domesticated and wild subspecies. Each locus encodes both a silk-active and a matching pollen-active pectin methylesterase (PMEs). To investigate the diversity and molecular evolution of these gametophytic factor loci, we identified existing and improved models of the responsible genes in a new genome assembly of maize line P8860 that contains active versions of all three loci. We then examined fifty-two assembled genomes from seventeen species to classify haplotype diversity and identify sites under diversifying selection during the evolution of these genes. We show that Ga2, the oldest of these three loci, was duplicated to form Ga1 at least 12 million years ago. Tcb1, the youngest locus, arose as a duplicate of Ga1 before or around the time of diversification of the Zea genus. We find evidence of positive selection during evolution of the functional genes at an active site in the pollen-expressed PME and predicted surface sites in both the silk- and pollen-expressed PMEs. The most common allele at the Ga1 locus is a conserved ga1 allele (ga1-Off), which is a specific haplotype containing three full-length PME gene copies, all of which are non-coding due to conserved stop codons and are between 610 thousand and 1.5 million years old. We show that the ga1-Off allele is associated with and likely generates 24-nt siRNAs in developing pollen-producing tissue, and these siRNAs map to functional Ga1 alleles. In previously-published crosses, the ga1-Off allele was associated with reduced function of the typically dominant functional alleles for the Ga1 and Tcb1 barriers. Taken together, this seems to be an example of a type of epigenetic trans-homolog silencing known as paramutation, functioning at a locus controlling a reproductive barrier. 

Abstract The basal endosperm transfer layer (BETL) of the maize (Zea mays L.) kernel is composed of transfer cells for nutrient transport to nourish the developing kernel. To understand the spatiotemporal processes required for BETL development, we characterized 2 unstable factor for orange1 (Zmufo1) mutant alleles. The BETL defects in these mutants were associated with high levels of reactive oxygen species, oxidative DNA damage, and cell death. Interestingly, antioxidant supplementation in in vitro cultured kernels alleviated the cellular defects in mutants. Transcriptome analysis of the loss-of-function Zmufo1 allele showed differential expression of tricarboxylic acid cycle, redox homeostasis, and BETL-related genes. The basal endosperms of the mutant alleles had high levels of acetyl-CoA and elevated histone acetyltransferase activity. The BETL cell nuclei showed reduced electron-dense regions, indicating sparse heterochromatin distribution in the mutants compared with wild-type. Zmufo1 overexpression further reduced histone methylation marks in the enhancer and gene body regions of the pericarp color1 (Zmp1) reporter gene. Zmufo1 encodes an intrinsically disordered nuclear protein with very low sequence similarity to known proteins. Yeast two-hybrid and luciferase complementation assays established that ZmUFO1 interacts with proteins that play a role in chromatin remodeling, nuclear transport, and transcriptional regulation. This study establishes the critical function of Zmufo1 during basal endosperm development in maize kernels. 

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs–named premeiotic 24-nt phasiRNAs–have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediatingPHASprecursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known. 

Plant cells accumulate small RNA molecules that regulate plant development, genome stability, and environmental responses. These small RNAs fall into three major classes based on their function and mechanisms of biogenesis—microRNAs, heterochromatic small interfering RNAs, and secondary small interfering RNAs—plus several other less well-characterized categories. Biogenesis of each small RNA class requires a pathway of factors, some specific to each pathway and others involved in multiple pathways. Diverse sequenced plant genomes, along with rapid developments in sequencing, imaging, and genetic transformation techniques, have enabled significant progress in understanding the biogenesis, functions, and evolution of plant small RNAs, including those that had been poorly characterized because they were absent or had low representation in Arabidopsis ( Arabidopsis thaliana). Here, we review recent findings about plant small RNAs and discuss our current understanding of their biogenesis mechanisms, targets, modes of action, mobility, and functions in Arabidopsis and other plant species, including economically important crops. Expected final online publication date for the Annual Review of Plant Biology, Volume 74 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates. 

Abstract Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways. 

Abstract Plant microRNAs (miRNAs) are short, noncoding RNA molecules that restrict gene expression via posttranscriptional regulation and function in several essential pathways, including development, growth, and stress responses. Accurately identifying miRNAs in populations of small RNA sequencing libraries is a computationally intensive process that has resulted in the misidentification of inaccurately annotated miRNA sequences. In recent years, criteria for miRNA annotation have been refined with the aim to reduce these misannotations. Here, we describe miRador, a miRNA identification tool that utilizes the most up-to-date, community-established criteria for accurate identification of miRNAs in plants. We combined target prediction and Parallel Analysis of RNA Ends (PARE) data to assess the precision of the miRNAs identified by miRador. We compared miRador to other commonly used miRNA prediction tools and found that miRador is at least as precise as other prediction tools while being substantially faster than other tools. miRador should be broadly useful for the plant community to identify and annotate miRNAs in plant genomes. 

Teachers’ integration of the Next Generation Science Standards and corresponding Science and Engineering Practices (SEPs) illustrate current science education reform in the United States. Effective teacher professional development (PD) on SEPs is essential for reform success. In this study, we evaluated the Nebraska STEM Education Conference, a PD program for middle school, high school, and first- and second-year post-secondary STEM teachers. This SEP-oriented PD program focused predominantly on the SEPs ‘developing and using models’ and ‘using mathematics and computational thinking.’ An electronic survey was used to measure participants’ (n = 45) prior integration of SEPs, influential factors and barriers to using SEPs, and changes to interest and confidence in using SEPs as a result of attending the PD program. Our results showed that teachers had limited prior use of SEPs in their teaching. Student interest and learning outcomes were the factors found to be most influential to teachers’ use of SEPs, while limited knowledge, confidence, and resources were the most commonly identified barriers. As a result of attending the PD program, participants significantly improved their confidence and interest to incorporate SEPs. We recommend continued SEP-oriented PD to foster successful NGSS integration and to advance reforms in science education. 

Abstract The spatiotemporal development of somatic tissues of the anther lobe is necessary for successful fertile pollen production. This process is mediated by many transcription factors acting through complex, multi-layered networks. Here, our analysis of functional knockout mutants of interacting basic helix–loop–helix genes Ms23, Ms32, basic helix–loop–helix 122 (bHLH122), and bHLH51 in maize (Zea mays) established that male fertility requires all four genes, expressed sequentially in the tapetum (TP). Not only do they regulate each other, but also they encode proteins that form heterodimers that act collaboratively to guide many cellular processes at specific developmental stages. MS23 is confirmed to be the master factor, as the ms23 mutant showed the earliest developmental defect, cytologically visible in the TP, with the most drastic alterations in premeiotic gene expression observed in ms23 anthers. Notably, the male-sterile ms23, ms32, and bhlh122-1 mutants lack 24-nt phased secondary small interfering RNAs (phasiRNAs) and the precursor transcripts from the corresponding 24-PHAS loci, while the bhlh51-1 mutant has wild-type levels of both precursors and small RNA products. Multiple lines of evidence suggest that 24-nt phasiRNA biogenesis primarily occurs downstream of MS23 and MS32, both of which directly activate Dcl5 and are required for most 24-PHAS transcription, with bHLH122 playing a distinct role in 24-PHAS transcription.