Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei

Abstract

The plant nucleus plays an irreplaceable role in cellular control and regulation by auxin (indole-3-acetic acid, IAA) mainly because canonical auxin signaling takes place here. Auxin can enter the nucleus from either the endoplasmic reticulum or cytosol. Therefore, new information about the auxin metabolome (auxinome) in the nucleus can illuminate our understanding of subcellular auxin homeostasis. Different methods of nuclear isolation from various plant tissues have been described previously, but information about auxin metabolite levels in nuclei is still fragmented and insufficient. Herein, we tested several published nucleus isolation protocols based on differential centrifugation or flow cytometry. The optimized sorting protocol leading to promising yield, intactness, and purity was then combined with an ultra-sensitive mass spectrometry analysis. Using this approach, we can present the first complex report on the auxinome of isolated nuclei from cell cultures of Arabidopsis and tobacco. Moreover, our results show dynamic changes in auxin homeostasis at the intranuclear level after treatment of protoplasts with free IAA, or indole as a precursor of auxin biosynthesis. Finally, we can conclude that the methodological procedure combining flow cytometry and mass spectrometry offers new horizons for the study of auxin homeostasis at the subcellular level.

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

“…However, the applied formaldehyde fixation resulted in phytohormone analysis deterioration (Figure S5b) as was similarly found in Skalický et al. (2021). Possible explanation might be cross‐linking of some analytes especially IAA and CK ribosides with cellular structures (Friml et al., 2003).…”

“…Initially, we attempted mild fixation with formaldehyde to stop all metabolic processes (Petrovská et al., 2014). However, this step was excluded as formaldehyde was detrimental to detecting phytohormones by LC–MS/MS (Skalický et al., 2021) (Figure S5b). Next, we applied sodium azide as an ATP‐dependent processes inhibitor (Tucker, 1993) or 1‐ N ‐naphtylphthalamic acid (NPA) as auxin transport inhibitor (Petrášek et al., 2006).…”

“…The time would increase both due to the longer isolation process that vacuoles require and to longer sorting time. While auxin and/or CK levels have been previously measured in chloroplasts (Polanská et al., 2007), vacuoles (Jiskrová et al., 2016; Ranocha et al., 2013), ER (Včelařová et al., 2021) and nuclei (Skalický et al., 2021), sample preparation processes and the divergent normalization methods used did not allow comparison of the data between the organelles. Due to the different sizes, shapes and abundance of organelles per plant cell, data normalization was essential for comparison of auxin and CK levels between organelles.…”

“…Flow cytometry has been earlier used for cell sorting in human medicine (Adan et al., 2017) as well as in plant sciences (Carter et al., 2013; Galbraith et al., 2021). This technique has been also utilized for sorting of nuclei (Petrovská et al., 2014; Skalický et al., 2021), chloroplasts (Wolf et al., 2005) or even smaller particles like chromosomes (Thind et al., 2017). Here we used the FACS instrument and achieved a 4‐way simultaneous and efficient sorting of chloroplasts, nuclei, ER and mitochondria from a single sample.…”

“…In Table S3, we show that IAA‐glc and oxIAA‐glc were below the limit of detection at all in examined Arabidopsis cell culture. Similarly, the presence of IAA‐glc and oxIAA‐glc metabolites in Arabidopsis ecotype Landsberg erecta cell culture has not been proven (Skalický et al., 2021). At this point, it should be noted that although we showed that IAA‐amino acid conjugates were present only in vacuoles, their presence cannot be excluded from other organelles as these were not truly absent but just below the limit of detection, and this is the case for all compounds.…”