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

Abstract

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, sperm cell delivery 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 detailed analysis of TE expression, as well as for quantitative assessment of gene function during maize pollen development. TEs are expressed early and persist, many showing coordinated 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. We also establish a role in fertilization for the maize gamete expressed2 (Zm 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. 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.