Fluorescence-activated multi-organelle mapping of subcellular plant hormone distribution

Auxins and cytokinins are two major families of phytohormones that control most aspects of plant growth, development and plasticity. Their distribution in plants has been described, but the importance of cell- and subcellular-type specific phytohormone homeostasis remains undefined. Herein, we revealed auxin and cytokinin distribution maps showing their different organelle-specific allocations within the Arabidopsis plant cell. To do so, we have developed Fluorescence-Activated multi-Organelle Sorting (FAmOS), an innovative subcellular fractionation technique based on flow cytometric principles. FAmOS allows the simultaneous sorting of four differently labelled organelles based on their individual light scatter and fluorescence parameters while ensuring hormone metabolic stability. Our data showed different subcellular distribution of auxin and cytokinins, revealing the formation of phytohormone gradients that have been suggested by the subcellular localization of auxin and cytokinin transporters, receptors and metabolic enzymes. Both hormones showed enrichment in vacuoles, while cytokinins were also accumulated in the endoplasmic reticulum

New PEO-IAA-Inspired Anti-Auxins: Synthesis, Biological Activity, and Possible Application in Hemp (Cannabis Sativa L.) Micropropagation

Auxins play an important role in plant physiology and are involved in numerous aspects of plant development, such as cell division, elongation and differentiation, fruit development, and phototropic response. In addition, through their antagonistic interaction with cytokinins, auxins play a key role in the regulation of root growth and apical dominance. Thanks to this capacity to determine plant architecture, natural and synthetic auxins have been successfully employed to obtain more economically advantageous plants. The crosstalk between auxins and cytokinins determines plant development and thus is of particular importance in the field of plant micropropagation, where the ratios between these two phytohormones need to be tightly controlled to achieve proper rooting and shoot generation. Previously reported anti-auxin PEO-IAA, which blocks auxin signalling through binding to TIR1 receptor and inhibiting the expression of auxin-responsive genes, has been successfully used to facilitate hemp micropropagation. Herein, we report a set of new PEO-IAA-inspired anti-auxins capable of antagonizing auxin responses in vivo. The capacity of these compounds to bind to the TIR1 receptor was confirmed in vitro by SPR analysis. Using DESI-MSI analysis, we evaluated the uptake and distribution of the compounds at the whole plant level. Finally, we characterized the effect of the compounds on the organogenesis of hemp explants, where they showed to be able to improve beneficial morphological traits, such as the balanced growth of all the produced shoots and enhanced bud proliferation

Ultra-rapid auxin metabolite profiling for high-throughput mutant screening in Arabidopsis

Auxin (indole-3-acetic acid, IAA) plays fundamental roles as a signalling molecule during numerous plant growth and development processes. The formation of local auxin gradients and auxin maxima/minima, which is very important for these processes, is regulated by auxin metabolism (biosynthesis, degradation, and conjugation) as well as transport. When studying auxin metabolism pathways it is crucial to combine data obtained from genetic investigations with the identification and quantification of individual metabolites. Thus, to facilitate efforts to elucidate auxin metabolism and its roles in plants, we have developed a high-throughput method for simultaneously quantifying IAA and its key metabolites in minute samples (<10 mg FW) of Arabidopsis thaliana tissues by in-tip micro solid-phase extraction and fast LC-tandem MS. As a proof of concept, we applied the method to a collection of Arabidopsis mutant lines and identified lines with altered IAA metabolite profiles using multivariate data analysis. Finally, we explored the correlation between IAA metabolite profiles and IAA-related phenotypes. The developed rapid analysis of large numbers of samples (>100 samples d(-1)) is a valuable tool to screen for novel regulators of auxin metabolism and homeostasis among large collections of genotypes