Examination of the functions of Clade III Cytokinin Response Factors in relation to Cytokinin and the Oxidative Stress Response

Abstract

Cytokinin response factors (CRFs) are a family of transcription factors belonging to the AP2/ERF transcription factor family. A subset of these CRFs have been shown to play a role in both the cytokinin (CK) signal cascade and the response to oxidative stress across several species of angiosperms. In order to investigate the roles Clade III CRFs play in responses to CK and oxidative stress, members of this group of CRFs were examined in both Arabidopsis thaliana and Solanum lycopersicum (tomato). AtCRF6 of Arabidopsis has already been shown to be involved in the oxidative stress response in Zwack et al 2016, but its function in the CK signal cascade remained mostly unstudied. By analyzing the transcriptome of CK-treated Atcrf6 mutant plant, several genes involved in potassium (K+) transport were found to be AtCRF6-dependantly CK-regulated. To further explore the possible connection by AtCRF6 between CK and K+ uptake and transport, mutants of the AtCRF6-depentanly CK-regulated K+ transporters, hak5, skor and nrt1.5, alongside mutant Atrf6, were grown in the presence of CK and reduced K+ levels, and their shoot and root growth, FV/FM levels, and the ionomic content of their roots and shoots were analyzed and compared. After investigating the role of AtCRF6 in CK signaling, the roles of Clade III CRFs in oxidative stress was explored in both Arabidopsis and tomato. While Arabidopsis has two Clade III CRFs, AtCRF5 and AtCRF6, tomato only has one; SlCRF5. Both AtCRF6 and SlCRF5 have been linked to the oxidative stress response, but remaining homolog, AtCRF5 in Arabidopsis, had not been linked. To better understand how these Clade III CRFs are connected to oxidative stress, the transcriptome of oxidative stress-treated Atcrf5 was analyzed and compared to those of oxidative stress-treated Atcrf6 and SlCRF5AS. Genes that were differentially regulated in the presence of oxidative stress were identified in each line, and those that diverged from the regulation observes in wildtype were deemed Clade III CRF-dependently oxidative stress-regulated genes (CDRGs). The functions of the CDRGs of each line were investigated and then compared using GO enrichment analysis. From these genes, several tomato CDRGs were found to be involved in the regulation of CK biosynthesis. To investigate the potential link formed by Clade III CRFs between oxidative stress and CK biosynthesis, CK measurements were taken from the oxidative stress-treated and untreated Atcrf5, Atcrf6, and SlCRF5AS lines, and altered CK levels analyzed.