Identification and analysis of candidate kinases for a function in gravitropic signalling using a novel phenotyping pipeline

Roots use gravity to guide their growth (gravitropism). Gravitropism involves the establishment of an auxin gradient across the root, which is driven by polar auxin transport (PAT). Auxin acts as a growth inhibitor, triggering a differential growth response that results in root bending towards gravity. Using a three-step process, my aim was to identify and investigate \at kinases likely to function in gravitropic signalling (candidate kinases). First, I utilised my Kinase Subsequence SELECTion (KSselect) algorithm to identify candidate kinases. Second, transcriptional and translational fusions are used to characterise candidate kinases. Third, I used our Quantitative Gravitropic Analysis Pipeline(qGAP) to phenotype candidate kinase mutants. My research focused on the D6PKL3 kinase and the redundant CIPK4 and CIPK7 (CIPK4/7) kinases. D6PKL3 is identified as auxin-inducible and closely related to D6PK, which regulates PAT by targeting PIN proteins. Although KSselect predicts D6PKL3 and D6PK functional divergence, further analysis suggests that both target PINs. D6PKL3 is expressed in columella cells (continuous) and the basal meristem (auxin-induced). In the basal meristem, D6PKL3 does not co-localise with PINs, allowing for the possibility that D6PKL3 targets the ABCB1 transporter. The qGAP results exclude a non-redundant function of D6PKL3 in gravitropism. CIPK4/7 are identified by KSselect as putative orthologs of ZmCIPK15, which is associated with the regulation of crown root angle in \zm. Transcriptional fusions locate CIPK4/7 in the maturation zone and the basal meristem in \at. Although the auxin inducible CIPK7 is expressed predominantly over CIPK4 in the basal meristem, the qGAP results conclude that neither function in gravitropic signalling. I then extended my CIPK4/7 research to the cold stress response and found that the CIPK4/7 mutants did not show a cold-conditional phenotype. My research addresses the challenges faced by gravitropic research, and offers novel tools including KSselect and qGAP. These tools are timely and can be adapted by researchers in a variety of fields