A Loss of Function Mutation in Protein phosphatase 1 Regulatory Subunit INH3 Improves Poor hpat1/3 Fertility and Pollen Tube Growth

Abstract

The MacAlister lab previously found that HYDROXYPROLINE O ARABINOSYLTRANSFERASE 1 and 3 (HPAT1 and HPAT3) were required for full pollen fertility in Arabidopsis thaliana by initiating posttranslational protein modification on cell wall structural proteins (Beuder, et al., 2020). The hpat1 hpat3 double knockout mutants (hpat1/3) showed low male fertility and disruption in pollen tube growth, which led to a large decrease in seed set (the average number of seeds per silique) compared to wild-type (WT) (MacAlister et al., 2016). They noticed that with the hpat1/3 mutation, the pollen tube cell wall would frequently rupture, the pollen tube would sometimes initiate a second point of growth leading to pollen tube branching, and the growth rate of the pollen tubes would be slower. To learn more about how HPATs contributed to pollen tube growth, they looked for suppressor genes involved in the HPAT pathway that affects pollen tube growth and fertility and analyzed the role of each gene and looked for genes that were involved with pollen tube cell wall. Finally, they determined the suppressor was in the gene INHIBITOR3 (INH3), which codes for a regulatory subunit of protein phosphatase 1 (PP1), and has been reported to be required for the embryo development and negative phosphoprotein phosphatase regulation (Zhang, et al., 2008). The suppressor mutation in the gene INH3 was caused by a G to A single-nucleotide substitution, which was predicted to cause an early truncation after amino acid W43. This allele was named inh3-3 because inh3-1 (SALK_0445593) and inh3-2 (SAIL_806_C02) T-DNA insertion alleles had been previously characterized (Takemiya et al., 2009). In order to better understand the mechanism behind inh3-1 and inh3-3 mutations and the hpat1/3 pollen fertility pathway, the objectives of the research in my honor's thesis are to study the following: 1) how does inh3-3 mutation suppress hpat1/3 pollen tube fertility defects, 2) if inh3-1 will also suppress the hpat1/3 fertility defect as inh3-3 does, and 3) if inh3-3 and inh3-1 can both lead to the decrease in fertility in the WT background. The experiments in this thesis show that knocking out INH3 with either inh3-1 or the inh3-3 allele will suppress hpat1/3 pollen fertility defects. Both inh3-3 and inh3-1 mutants in pollen could increase the transmission in hpat1/3 pollen and the fertility of hpat1/3 plants. However, the inh3-3 pollen actually decreased the transmission through the pollen in the WT background without hpat1/3 mutants as inh3-1 did. However, the exact mechanism of how the loss-of-function INH3 interacts with the hpat1/3 mutant protein in the pollen tube fertility pathway at the cellular level is still unknown and requires further investigation.