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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 often thought to play a housekeeping role or have low activity during plant growth and development. 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.

Made available in DSpace on 2022-04-28T19:50:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-12-01 Agricultural Adaptation Council Genome Canada Ontario Genomics Institute Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada Natural Sciences and Engineering Research Council of Canada Grain Farmers of Ontario Background: Symbiotic Methylobacterium strains comprise a significant part of plant microbiomes. Their presence enhances plant productivity and stress resistance, prompting classification of these strains as plant growth-promoting bacteria (PGPB). Methylobacteria can synthesize unusually high levels of plant hormones, called cytokinins (CKs), including the most active form, trans-Zeatin (tZ). Results: This study provides a comprehensive inventory of 46 representatives of Methylobacterium genus with respect to phytohormone production in vitro, including 16 CK forms, abscisic acid (ABA) and indole-3-acetic acid (IAA). High performance-liquid chromatography—tandem mass spectrometry (HPLC–MS/MS) analyses revealed varying abilities of Methylobacterium strains to secrete phytohormones that ranged from 5.09 to 191.47 pmol mL−1 for total CKs, and 0.46 to 82.16 pmol mL−1 for tZ. Results indicate that reduced methanol availability, the sole carbon source for bacteria in the medium, stimulates CK secretion by Methylobacterium. Additionally, select strains were able to transform L-tryptophan into IAA while no ABA production was detected. Conclusions: To better understand features of CKs in plants, this study uncovers CK profiles of Methylobacterium that are instrumental in microbe selection for effective biofertilizer formulations. Environmental and Life Sciences Graduate Program Trent University, 1600 West Bank Drive Department of Biology Trent University, 1600 West Bank Drive Department of Technology Sao Paulo State University Department of Technology Sao Paulo State University Agricultural Adaptation Council: ON-RP3 Genome Canada: ON-RP3 Ontario Genomics Institute: ON-RP3 Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada: RGPIN-2018-05436 Natural Sciences and Engineering Research Council of Canada: RGPIN-2018-05436 Grain Farmers of Ontario: S2013AG04