Control of cellularization, nuclear localization, and antipodal cell cluster development in maize embryo sacs.

The maize female gametophyte contains four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In maize, these cells are produced after three rounds of free-nuclear divisions followed by cellularization, differentiation, and proliferation of the antipodal cells. Cellularization of the eight-nucleate syncytium produces seven cells with two polar nuclei in the central cell. Nuclear localization is tightly controlled in the embryo sac. This leads to precise allocation of the nuclei into the cells upon cellularization. Nuclear positioning within the syncytium is highly correlated with their identity after cellularization. Two mutants are described with extra polar nuclei, abnormal antipodal cell morphology, and reduced antipodal cell number, as well as frequent loss of antipodal cell marker expression. Mutations in one of these genes, indeterminate gametophyte2 encoding a MICROTUBULE ASSOCIATED PROTEIN65-3 homolog, shows a requirement for MAP65-3 in cellularization of the syncytial embryo sac as well as for normal seed development. The timing of the effects of ig2 suggests that the identity of the nuclei in the syncytial female gametophyte can be changed very late before cellularization.

High expression in maize pollen correlates with genetic contributions to pollen fitness as well as with coordinated transcription from neighboring transposable elements.

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.

Correction to: Genome-wide discovery and characterization of maize long non-coding RNAs.

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.

Live-Cell Imaging of Auxin and Cytokinin Signaling in Maize Female Gametophytes.

The plant life cycle is characterized by the alternation of generations between genetically active diploid sporophytes and haploid gametophytes. The gametophytes of flowering plants are sexually dimorphic. While the male gametophyte consists of only three cells (two sperm and a vegetative cell) and is released by the parent sporophyte, the female gametophyte (or embryo sac) is more complex and remains imbedded within diploid sporophyte tissues. In maize, the female gametophyte is embedded in a large ovule surrounded with multiple nucellar cell layers impeding live-cell imaging approaches to study embryo sac functions. Here, we describe a simple protocol to visualize embryo sacs with hormonal fluorescent reporters by increasing accessibility of the female gametophyte. The method described is applicable for visualization of any fluorescent embryo sac reporter. The embryo sacs visualization method developed for maize could be extended to facilitate visualization of embryos sac in other important cereals like wheat, rice, and oats.

Maternal Gametophyte Effects on Seed Development in Maize.

Flowering plants, like placental mammals, have an extensive maternal contribution toward progeny development. Plants are distinguished from animals by a genetically active haploid phase of growth and development between meiosis and fertilization, called the gametophyte. Flowering plants are further distinguished by the process of double fertilization that produces sister progeny, the endosperm and the embryo, of the seed. Because of this, there is substantial gene expression in the female gametophyte that contributes to the regulation of growth and development of the seed. A primary function of the endosperm is to provide growth support to its sister embryo. Several mutations in Zea mays subsp. mays have been identified that affect the contribution of the mother gametophyte to the seed. The majority affect both the endosperm and the embryo, although some embryo-specific effects have been observed. Many alter the pattern of expression of a marker for the basal endosperm transfer layer, a tissue that transports nutrients from the mother plant to the developing seed. Many of them cause abnormal development of the female gametophyte prior to fertilization, revealing potential cellular mechanisms of maternal control of seed development. These effects include reduced central cell size, abnormal architecture of the central cell, abnormal numbers and morphology of the antipodal cells, and abnormal egg cell morphology. These mutants provide insight into the logic of seed development, including necessary features of the gametes and supporting cells prior to fertilization, and set up future studies on the mechanisms regulating maternal contributions to the seed.

Correlation between a loss of auxin signaling and a loss of proliferation in maize antipodal cells.

The plant life cycle alternates between two genetically active generations: the diploid sporophyte and the haploid gametophyte. In angiosperms the gametophytes are sexually dimorphic and consist of only a few cells. The female gametophyte, or embryo sac, is comprised of four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In some species the antipodal cells are indistinct and fail to proliferate, so many aspects of antipodal cell function and development have been unclear. In maize and many other grasses, the antipodal cells proliferate to produce a highly distinct cluster at the chalazal end of the embryo sac that persists at the apex of the endosperm after fertilization. The antipodal cells are a site of auxin accumulation in the maize embryo sac. Analysis of different families of genes involved in auxin biosynthesis, distribution, and signaling for expression in the embryo sac demonstrates that all steps are expressed within the embryo sac. In contrast to auxin signaling, cytokinin signaling is absent in the embryo sac and instead occurs adjacent to but outside of the antipodal cells. Mutant analysis shows a correlation between a loss of auxin signaling and a loss of proliferation of the antipodal cells. The leaf polarity mutant Laxmidrib1 causes a lack of antipodal cell proliferation coupled with a loss of DR5 and PIN1a expression in the antipodal cells.

A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function.

Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes.

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.

Genome-wide discovery and characterization of maize long non-coding RNAs.

BACKGROUND: Long non-coding RNAs (lncRNAs) are transcripts that are 200 bp or longer, do not encode proteins, and potentially play important roles in eukaryotic gene regulation. However, the number, characteristics and expression inheritance pattern of lncRNAs in maize are still largely unknown. RESULTS: By exploiting available public EST databases, maize whole genome sequence annotation and RNA-seq datasets from 30 different experiments, we identified 20,163 putative lncRNAs. Of these lncRNAs, more than 90% are predicted to be the precursors of small RNAs, while 1,704 are considered to be high-confidence lncRNAs. High confidence lncRNAs have an average transcript length of 463 bp and genes encoding them contain fewer exons than annotated genes. By analyzing the expression pattern of these lncRNAs in 13 distinct tissues and 105 maize recombinant inbred lines, we show that more than 50% of the high confidence lncRNAs are expressed in a tissue-specific manner, a result that is supported by epigenetic marks. Intriguingly, the inheritance of lncRNA expression patterns in 105 recombinant inbred lines reveals apparent transgressive segregation, and maize lncRNAs are less affected by cis- than by trans-genetic factors. CONCLUSIONS: We integrate all available transcriptomic datasets to identify a comprehensive set of maize lncRNAs, provide a unique annotation resource of the maize genome and a genome-wide characterization of maize lncRNAs, and explore the genetic control of their expression using expression quantitative trait locus mapping.

Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope.

The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells [1] (Kasas and Ikai, 1995), [2] (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.