Low-input transcriptomics of developing pollen in Arabidopsis thaliana

Pollen is the male gametophyte of flowering plants that deliver sperm cells to the female gametophyte during fertilization. Pollen development begins with the reduction division (meiosis) of a single pollen mother cell to produce four microspores in a process called microsporogenesis. The microspores undergo two rounds of mitotic divisions to eventually produce a mature three-celled pollen composed of a large vegetative cell and two small sperm cells. A key challenge in plant reproductive biology is to understand the molecular and genetic mechanisms regulating these complex processes. Efforts to analyse the gene expression during pollen development by generating its transcriptome has been limited due to challenges associated with generating a pure and homogeneous developing pollen sample. To facilitate gene expression and transcriptomic analysis of developing pollen, I have developed a novel Low-input Developing Pollen (LiDP) method that allows isolation of pure and homogeneous Arabidopsis thaliana (Arabidopsis) pollen samples belonging to a narrow developmental window. The LiDP method involves the separation of up to three anthers from a bud that is nicked to release their pollen. The pollen is washed to remove non-pollen transcripts and frozen for future use whereas the nicked anther together with remaining pollen is stored in a fixative. The fixed pollen is later staged by DAPI-staining and microscopic analysis to determine its developmental stage. Appropriate pollen samples stored in the freezer are then selected based on their stages and eventually lysed using a chemical called MMNO. Finally, the lysed pollen extract is directly used for RT-qPCR, digital droplet PCR or RNA sequencing (RNA-Seq). The ‘pollen lysis condition’ had minimal effect on RNA quality but showed partial degradation of mRNA 5`-ends. The lysis condition showed negligible effect on RT-qPCR amplification efficiency but reduced the mapping quality of RNA-Seq reads evidenced by increasing number of reads mapping to intergenic regions and reads that mapped to more than one position on the genome. The LiDP method was used to generate the first RNA-Seq-based transcriptome of developing pollen from four discreet stages- uninucleate microspore (UNM), polarised microspore (PUNM), late bicellular pollen (BCP) and late tricellular pollen (TCP) stages. Analysis of the RNA-Seq data revealed global gene expression patterns that coincided with the molecular classification of pollen development into early and late developmental phases. Differential expression analysis revealed novel pollen-expressed genes that may play critical roles in pollen development and can now be tested in planta. Several pollen-specific genes were identified for the future development of cell-type-specific markers. Overall, the LiDP transcriptome offers a roadmap for further investigation of gene regulation in developing pollen , especially from the PUNM stage that was never studied before. Finally, to assess the LiDP method’s usefulness the method was utilized to address a duo4 pollen development mutant and to test the hypothesis that the misexpression of DUO4 may lead to failed germ cell division in duo4 pollen through early degradation of mitotic CYCB1;1 (CYCLINB1;1). Investigation of DUO4 expression using LiDP RT-qPCR revealed considerable DUO4 expression at BCP stages of +/duo4 pollen but not in WT which supports the hypothesis that DUO4 is misexpressed at BCP in +/duo4 pollen. However, targeted amplicon sequencing of WT and duo4 transcripts in +/duo4 plants close to PM I and PM II revealed the typical 1:1 ratio between the two transcripts. These results demonstrated the utility of the LiDP method to address gene expression in specific stages of pollen development mutant