Although DNA methylation primarily represses TEs, it also represses select genes that are methylated in plant body tissues but demethylated by DNA glycosylases (DNGs) in endosperm or pollen. Either one of two DNGs, MATERNAL DEREPRESSION OF R1 (MDR1) or DNG102, is essential for pollen viability in maize. Using single-pollen mRNA sequencing on pollen-segregating mutations in both genes, we identify 58 candidate DNG target genes that account for 11.1% of the wild-type transcriptome but are silent or barely detectable in other tissues. They are unusual in their tendency to lack introns but even more so in their TE-like methylation (teM) in coding DNA. The majority have predicted functions in cell wall modification, and they likely support the rapid tip growth characteristic of pollen tubes. These results suggest a critical role for DNA methylation and demethylation in regulating maize genes with the potential for extremely high expression in pollen but constitutive silencing elsewhere.

BACKGROUND:The La-related proteins (LARPs) are a superfamily of RNA-binding proteins associated with regulation of gene expression. Evidence points to an important role for post-transcriptional control of gene expression in germinating pollen tubes, which could be aided by RNA-binding proteins.RESULTS:In this study, a genome-wide investigation of the LARP proteins in eight plant species was performed. The LARP proteins were classified into three families based on a phylogenetic analysis. The gene structure, conserved motifs, cis-acting elements in the promoter, and gene expression profiles were investigated to provide a comprehensive overview of the evolutionary history and potential functions of ZmLARP genes in maize. Moreover, ZmLARP6c1 was specifically expressed in pollen and ZmLARP6c1 was localized to the nucleus and cytoplasm in maize protoplasts. Overexpression of ZmLARP6c1 enhanced the percentage pollen germination compared with that of wild-type pollen. In addition, transcriptome profiling analysis revealed that differentially expressed genes included PABP homologous genes and genes involved in jasmonic acid and abscisic acid biosynthesis, metabolism, signaling pathways and response in a Zmlarp6c1::Ds mutant and ZmLARP6c1-overexpression line compared with the corresponding wild type.CONCLUSIONS:The findings provide a basis for further evolutionary and functional analyses, and provide insight into the critical regulatory function of ZmLARP6c1 in maize pollen germination.

Members of the La-related protein family (LARPs) contain a conserved La module, which has been associated with RNA-binding activity. Expression of the maize gene GRMZM2G323499/Zm00001d018613, a member of the LARP family, is highly specific to pollen, based on both transcriptomic and proteomic assays. This suggests a pollen-specific RNA regulatory function for the protein, designated ZmLARP6c1 based on sequence similarity to the LARP6 subfamily in Arabidopsis. To test this hypothesis, a Ds-GFP transposable element insertion in the ZmLarp6c1 gene (tdsgR82C05) was obtained from the Dooner/Du mutant collection. Sequencing confirmed that the Ds-GFP insertion is in an exon, and thus likely interferes with ZmLARP6c1 function. Tracking inheritance of the insertion via its endosperm-expressed GFP indicated that the mutation was associated with reduced transmission from a heterozygous plant when crossed as a male (ranging from 0.5 to 26.5% transmission), but not as a female. Furthermore, this transmission defect was significantly alleviated when less pollen was applied to the silk, reducing competition between mutant and wild-type pollen. Pollen grain diameter measurements and nuclei counts showed no significant differences between wild-type and mutant pollen. However, in vitro, mutant pollen tubes were significantly shorter than those from sibling wild-type plants, and also displayed altered germination dynamics. These results are consistent with the idea that ZmLARP6c1 provides an important regulatory function during the highly competitive progamic phase of male gametophyte development following arrival of the pollen grain on the silk. The conditional, competitive nature of the Zmlarp6c1::Ds male sterility phenotype (i.e., reduced ability to produce progeny seed) points toward new possibilities for genetic control of parentage in crop production.

KEY MESSAGE:Advances in deep learning are providing a powerful set of image analysis tools that are readily accessible for high-throughput phenotyping applications in plant reproductive biology. High-throughput phenotyping systems are becoming critical for answering biological questions on a large scale. These systems have historically relied on traditional computer vision techniques. However, neural networks and specifically deep learning are rapidly becoming more powerful and easier to implement. Here, we examine how deep learning can drive phenotyping systems and be used to answer fundamental questions in reproductive biology. We describe previous applications of deep learning in the plant sciences, provide general recommendations for applying these methods to the study of plant reproduction, and present a case study in maize ear phenotyping. Finally, we highlight several examples where deep learning has enabled research that was previously out of reach and discuss the future outlook of these methods.

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.

ABSTRACT The development of rapid and efficient transformation methods for many plant species remains an obstacle in both the basic and applied plant sciences. A novel method described by Zhao et al. (2017) used magnetic nanoparticles to deliver DNA into pollen grains of several dicot species, and one monocot (lily), to achieve transformation (“pollen magnetofection”). Using the published protocol, extensive trials by two independent research groups showed no indication of transient transformation success with pollen from two monocots, maize and sorghum. To further address the feasibility of magnetofection, lily pollen was used for side-by-side trials of magnetofection with a proven methodology for transient transformation, biolistics. Using a Green Fluorescent Protein reporter plasmid, transformation efficiency with the biolistic approach averaged 0.7% over three trials. However, the same plasmid produced no recognizable transformants via magnetofection, despite screening >3500 individual pollen grains. We conclude that pollen magnetofection is not effective for transient transformation of pollen for at least three species of monocots, and suggest that efforts to replicate the magnetofection protocol in dicot species would be useful to fully assess its potential. ARISING FROM Zhao et al. Nature Plants https://doi.org/10.1038/s41477-017-0063-z (2017)

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.

High-throughput phenotyping systems are becoming increasingly powerful, dramatically changing our ability to document, measure, and detect phenomena. Unfortunately, taking advantage of these trends can be difficult for scientists with few resources, particularly when studying nonstandard biological systems. Here, we describe a powerful, cost-effective combination of a custom-built imaging platform and open-source image processing pipeline. Our maize ear scanner was built with off-the-shelf parts for <$80. When combined with a cellphone or digital camera, videos of rotating maize ears were captured and digitally flattened into projections covering the entire surface of the ear. Segregating GFP and anthocyanin seed markers were clearly distinguishable in ear projections, allowing manual annotation using ImageJ. Using this method, statistically powerful transmission data can be collected for hundreds of maize ears, accelerating the phenotyping process.

The explosion of RNA-Seq data has enabled the identification of expressed genes without relying on gene models with biases toward open reading frames, allowing the identification of many more long noncoding RNAs (lncRNAs) in eukaryotes. Various tissue enrichment strategies and deep sequencing have also enabled the identification of an extensive list of genes expressed in maize gametophytes, tissues that are intractable to both traditional genetic and gene expression analyses. However, the function of very few genes from the lncRNA and gametophyte sets (or from their intersection) has been tested. Methods for isolating and identifying lncRNAs from gametophyte samples of maize are described here. This method is transferable to any maize gametophyte mutant enabling the development of gene networks involving both protein-coding genes and lncRNAs. Additionally, these methods can be adapted to apply to other grass model systems to test for evolutionary conservation of lncRNA expression patterns.

In flowering plants, the haploid male gametophyte (pollen) is essential for sperm delivery, double fertilization, and subsequent initiation of seed development. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene regulation and function is not clearly understood. To identify components of these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and isolated sperm cells) via RNA-seq. We found that, in contrast to Arabidopsis TE expression in pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinated expression was observed between the most highly expressed protein-coding genes and neighboring TEs, specifically in both mature pollen and sperm cells. To test the hypothesis that such elevated expression correlates with functional relevance, we measured the fitness cost (male-specific transmission defect) of GFP-tagged exon insertion mutations in over 50 genes highly expressed in pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in genes highly expressed only in seedling or primarily in sperm cells (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. The gamete expressed2 ( gex2 ) gene was the single sperm cell gene associated with reduced transmission when mutant (<35% for two independent insertions), and also triggered seed defects when crossed as a male, supporting a role for gex2 in double fertilization. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes. Author Summary In flowering plants, pollen is essential for delivering sperm cells to the egg and central cell for double fertilization, initiating the process of seed development. In plants with abundant pollen like maize, this process can be highly competitive. In an added layer of complexity, growing evidence indicates expression of transposable elements (TEs) is more dynamic in pollen than in other plant tissues. How these elements impact pollen function and gene regulation is not well understood. We used transcriptional profiling to generate a framework for both detailed analysis of TE expression and quantitative assessment of gene function during maize pollen development. TEs are expressed early and persist, many showing coordinate activation with highly-expressed neighboring genes in the pollen vegetative cell and sperm cells. Measuring fitness costs for a set of over 50 mutations indicates a correlation between elevated transcript level and gene function in the vegetative cell. Finally, we establish a role in fertilization for the gamete expressed2 ( gex2 ) gene, identified based on its specific expression in sperm cells. These results highlight maize pollen as a powerful model for investigating the developmental interplay of TEs and genes, as well as for measuring fitness contributions of specific genes.

The exocyst, a conserved, octameric protein complex, helps mediate secretion at the plasma membrane, facilitating specific developmental processes that include control of root meristem size, cell elongation, and tip growth. A genetic screen for second-site enhancers in Arabidopsis identified NEW ENHANCER of ROOT DWARFISM1 (NERD1) as an exocyst interactor. Mutations in NERD1 combined with weak exocyst mutations in SEC8 and EXO70A1 result in a synergistic reduction in root growth. Alone, nerd1 alleles modestly reduce primary root growth, both by shortening the root meristem and by reducing cell elongation, but also result in a slight increase in root hair length, bulging, and rupture. NERD1 was identified molecularly as At3g51050, which encodes a transmembrane protein of unknown function that is broadly conserved throughout the Archaeplastida. A functional NERD1-GFP fusion localizes to the Golgi, in a pattern distinct from the plasma membrane-localized exocyst, arguing against a direct NERD1-exocyst interaction. Structural modeling suggests the majority of the protein is positioned in the lumen, in a beta-propeller-like structure that has some similarity to proteins that bind polysaccharides. We suggest that NERD1 interacts with the exocyst indirectly, possibly affecting polysaccharides destined for the cell wall, and influencing cell wall characteristics in a developmentally distinct manner.

A gene's duplication relaxes selection. Loss of duplicate, low-function DNA (fractionation) sometimes follows, mostly by deletion in plants, but mostly via the pseudogene pathway in fish and other clades with smaller population sizes. Subfunctionalization — the founding term of the Xfunctionalization lexicon — while not the general cause of differences in duplicate gene retention, becomes primary as the number of a gene's cis — regulatory sites increases. Balanced gene drive explains retention for the average gene. Both maintenance-of-balance and subfunctionalization drive gene content nonrandomly, and currently fall outside of our accepted Theory of Evolution. The ‘typical’ mutation encountered by a gene duplicate is not a neutral loss-of-function; dominant mutations (Muller's lexicon; these are not neutral) abound, and confound X functionalization terms like ‘neofunctionalization’. Confusion of words may cause confusion of thought. As with many plants, fish tetraploidies provide a higher throughput surrogate-genetic method to infer function from human and other vertebrate ENCODE-like regulatory sites.

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

BACKGROUND:Exocytosis is integral to root growth: trafficking components of systems that control growth (e.g., PIN auxin transport proteins) to the plasma membrane, and secreting materials that expand the cell wall to the apoplast. Spatiotemporal regulation of exocytosis in eukaryotes often involves the exocyst, an octameric complex that tethers selected secretory vesicles to specific sites on the plasma membrane and facilitates their exocytosis. We evaluated Arabidopsis lines with mutations in four exocyst components (SEC5, SEC8, EXO70A1 and EXO84B) to explore exocyst function in primary root growth.RESULTS:The mutants have root growth rates that are 82% to 11% of wild-type. Even in lines with the most severe defects, the organization of the quiescent center and tissue layers at the root tips appears similar to wild-type, although meristematic, transition, and elongation zones are shorter. Reduced cell production rates in the mutants are due to the shorter meristems, but not to lengthened cell cycles. Additionally, mutants demonstrate reduced anisotropic cell expansion in the elongation zone, but not the meristematic zone, resulting in shorter mature cells that are similar in shape to wild-type. As expected, hypersensitivity to brefeldin A links the mutant root growth defect to altered vesicular trafficking. Several experimental approaches (e.g., dose-response measurements, localization of signaling components) failed to identify aberrant auxin or brassinosteroid signaling as a primary driver for reduced root growth in exocyst mutants.CONCLUSIONS:The exocyst participates in two spatially distinct developmental processes, apparently by mechanisms not directly linked to auxin or brassinosteroid signaling pathways, to help establish root meristem size, and to facilitate rapid cell expansion in the elongation zone.

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.

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation.

The original version [1] of this article unfortunately contained a mistake. The additive effects of the eQTLs of lncRNAs were flipped, meaning that the base allele in the contrast to derive the additive effects should have been B73, rather than Mo17, due to the original coding of biallele SNPs as "0s" and "1s". Going through the entire analysis procedure, it was determined that the mistake was made while tabulating the eQTL results from QTL Cartographer.

Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells. This myosin was found to associate primarily not with larger organelles (e.g., Golgi) as was broadly assumed, but with endomembrane vesicles trafficking along F-actin. Subcellular localization and fractionation experiments indicated that the nature of myosin-associated vesicles is organ- and cell type-specific. In leaves, a large proportion of these vesicles aligned and co-fractionated with a motile endoplasmic reticulum (ER) subdomain. In roots, non-ER vesicles were a dominant myosin cargo. Myosin XI-K showed a striking polar localization at the tips of growing, but not mature, root hairs. These results strongly suggest that a major mechanism whereby myosins contribute to plant cell physiology is vesicle transport, and that this activity can be regulated depending on the growth phase of a cell.

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.