Low-input transcriptomics of developing pollen isolated from a single anther

Chakkatu SP, Day R, Harrop T and Brownfield L

University of Otago, Dunedin, New Zealand.

Pollen are the male gametophytes of plants that deliver the sperm cells to the female gametes. Development of pollen from a unicellular microspore to a mature triploid pollen grain is a highly complex process involving precise and co-ordinated regulation of cell cycle, specification and differentiation of two different cell types. A key challenge in plant biology is to understand the genes that participate in this complex regulation. However, transcriptomic analysis of developing pollen has been a challenge due to their relative inaccessibility within an anther and also because of a tough outer pollen wall. Due to these challenges, previous studies of pollen transcriptomes have used either whole anthers (comprising parental tissue) or pollen from multiple plants encompassing a broad developmental window. To enable transcriptomic analysis on pollen at a particular developmental stage and from a small number of plants, we have developed a protocol to analyze pollen from a single anther in Arabidopsis thaliana. This protocol involves removal of an anther from a bud. The anther is ruptured and the released pollen is washed before being frozen for storage. The remaining anther material with pollen is fixed and later stained to be viewed under a microscope to determine its developmental stage. The frozen pollen is chemically lysed and used in direct cDNA synthesis. We will report on our development and testing of the method for RT-qPCR, digital droplet PCR and RNA sequencing and its use to study gene expression in pollen.