Heat stress (HS) is a major abiotic stress that negatively impacts crop yields across the globe. Plants respond to elevated temperatures by changing gene expression, mediated by transcription factors (TFs) functioning to enhance HS tolerance. The involvement of Group I bZIP TFs in the heat stress response (HSR) is not known. In this study, bZIP18 and bZIP52 were investigated for their possible role in the HSR. Localization experiments revealed their nuclear accumulation following heat stress, which was found to be triggered by dephosphorylation. Both TFs were found to possess two motifs containing serine residues that are candidates for phosphorylation. These motifs are recognized by 14–3–3 proteins, and bZIP18 and bZIP52 were found to bind 14–3–3 ε, the interaction of which sequesters them to the cytoplasm. Mutation of both residues abolished 14–3–3 ε interaction and led to a strict nuclear localization for both TFs. RNA-seq analysis revealed coordinated downregulation of several metabolic pathways including energy metabolism and translation, and upregulation of numerous lncRNAs in particular. These results support the idea that bZIP18 and bZIP52 are sequestered to the cytoplasm under control conditions, and that heat stress leads to their re-localization to nuclei, where they jointly regulate gene expression.

Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

Precise guided pollen tube growth by the female gametophyte is a pre-requisite for successful sexual reproduction in flowering plants. Cysteine-rich proteins (CRPs) secreted from the embryo sac are known pollen tube attractants perceived by pollen tube receptor-like kinases (RLK's). How pre-mRNA splicing facilitates this cell-to-cell communication is not understood. Here, we report novel function of Pre-mRNA PROCESSING factor 8 paralogs, PRP8A and PRP8B, as regulators of pollen tube attraction. Double mutant prp8a prp8b ovules cannot attract pollen tubes, and prp8a prp8b pollen tubes fail in sensing ovules attraction signals. Only 3% of ovule-expressed genes were misregulated in prp8a prp8b. Combination of RNA-seq and MYB98/LURE1.2-YFP reporter revealed the expression of MYB98, LUREs and 49 other CRPs were downregulated suggesting loss of synergid cell fate. Differential Exon usage (DEU) and Intron-retention (IR) analysis revealed autoregulation of PPR8A/PRP8B splicing. In vivo, PRP8A coimmunoprecipitates with splicing enhancer AtSF3A1, suggesting involvement of PRP8A in 3′-splice site selection. Our data hint that PRP8A/PRP8B module exhibit spliceosome-autoregulation to facilitate pollen tube attraction via transcriptional regulation of MYB98, CRPs and LURE pollen tube attractants.