| Description |
Plants use root-to-shoot signaling to coordinate shoot development with the conditions experienced by the roots. A root-to-shoot signaling molecule had been implicated in the Arabidopsis bypass 1 (bps1) mutant. The bps1 mutant exhibits defective shoot and root growth that is associated with over-production of a root-derived signal, the bps signal. Our main goal is to characterize the bps signal chemically and work on purification steps for the identification of the bps signal. Our strategy was to create several mutants with altered levels of bps signal, fractionate extracts, test fractions for activity using a bioassay, and analyze the active the fraction using mass spectrometer. I developed a bioassay to follow the bps signal, which is based on the growth-reducing activity using the pCYCB1;1::GUS cell cycle marker. Using the bioassay, we revealed that the bps signal is neither a protein nor RNA but it is a small metabolite. Using the bioassay and several SPE fractionation procedures, including C-18, HILIC, and MCX we showed that the bps signal is a polar, positively charged metabolite. We used genetic and chemical inhibitor approaches to characterize the biosynthetic pathway of the bps signal. We showed that bps1 mutants were resistant to 5-MT, an analog of Tryptophan (Trp). When Trp biosynthesis was limited in bps1 mutants, by creating double mutants with trp2 and trp3 mutants, leaf development was partially rescued. The rescued phenotype was restored when trp2 bps1 double mutants were grown on media containing Trp. Using the bioassay, we further showed that trp2 bps1 double mutants have a reduced level of the bps signal. To characterize the bps signal chemically, we analyzed the numbers and level of compounds in bps1, trp2 bps1, and cyp79B2 cyp79B3 bps1 mutants by HPLC using pHILIC analytical column. Analysis using negative and positive mode MS revealed that there were one and two potential bps signal candidates. Further fractionation of the extracts using a pHILIC semipreparative column and testing the fractions for activity revealed that a single 30-second fraction showed the bps signal activity. However, the compounds in the active 30-second fraction were different than the putative bps signal candidates obtained from pHILIC analytical column. Further fractionating the active 30-second fraction using cHILIC (pH 3.2) chromatography revealed that there were many more compounds in that fraction. Much additional work is required before we can clearly identify the bps signal. |
