The tRNA-degradation pathway impacts the phenotype and metabolome of Arabidopsis thaliana: evidence from atipt2 and atipt9 knockout mutants

Abstract

Isopentenyltransferases (IPTs), including adenosine phosphate-isopentenyltransferases (ATP/ADP-IPTs and AMP-IPTs) and tRNA‐isopentenyltransferases (tRNA-IPTs), are responsible for a rate-limiting step of cytokinin (CK) biosynthesis. tRNA-IPTs, which account for the synthesis of cis-Zeatin (cZ)-type CKs, are less understood and have often been thought to play a housekeeping role or have low activity during plant growth and development. Even with increasing evidence supporting the tRNA-degradation pathway and the role of cis-CK metabolites to perform important functions, a fulsome examination at the metabolome level where critical biological effects are normally absent of phenotypic expression, remains unpublished. Here, two Arabidopsis tRNA-IPT knockout mutants, atipt2 and atipt9, with independent disturbance of the pathway leading to cisCKs were investigated at the phenotype and metabolite levels at four stages of plant development: first leaf, inflorescence, siliques, and mature seed. Phenotypic deviations were noted in rosette diameter, number of non-rosette leaves, shoot height, flowering time, flower number, carotenoid content, trichome development, and above-ground fresh mass. Hormone profiling by high-performance liquid chromatography-high resolution tandem mass spectrometry (HPLC-HRMS/MS) showed that the atipt2 mutant accumulates lower total cisCKs in the first leaves and in siliques. The atipt9 mutant showed reduced total cisCKs in first leaves, but, during silique development, it had higher levels of cisCKs in than those of the wild type (WT) plants. Additionally, metabolite detection was performed via an untargeted approach using HPLC-HRMS. A total of 33 significant features differing in abundance between ipt mutants and the WT were putatively identified based on database search. Matched metabolites included those that participate in hormone cross-talk, fatty acid synthesis, seed set and germination, and in stress acclimation. Evidence indicates that cisCK production is important for plant growth and development, in ways distinct from CKs produced from de novo pathway.