Auxin Metabolite Profiling in Isolated and Intact Plant Nuclei.

Vladimír Skalický,Aleš Pěnčík,Veronika Koláčková,Martin F Kubeš,Jan Vrána,Ondřej Novák,Tereza Vojtková,Michaela Sedlářová,Katarzyna Juzoń

doi:10.3390/ijms222212369

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.

Highlights

The processes of plant growth, development, growth, and plasticity are driven mainly by plant hormones
An innovative combination of Flow cytometry (FCM) with ultra-sensitive mass spectrometry analysis has been shown to provide a useful tool for monitoring indole-3-acetic acid (IAA) and other related compounds at the subcellular level
We summarized the main pros and cons of different nuclei isolation approaches, such as conventional biochemical methods of differential centrifugation (DC) or density gradient centrifugation (GC), and modern methods exploiting the technique of FCM or affinity purification (AP)

Summary

Introduction

The processes of plant growth, development, growth, and plasticity are driven mainly by plant hormones. Auxin homeostasis is tightly regulated by the coordination of transport, biosynthesis, and metabolism, which altogether regulate the availability of IAA. It seems that cellular and subcellular compartmentalization of auxin may be functionally important for the control of physiological processes [4]. It was shown that TIR1/AFBs and ARF3, known as ETTIN, are not found exclusively in the nucleus and in the cytosol They may be involved in non-transcriptional responses to auxin input known as non-canonical auxin signaling [7,8,9,10,11]

Methods

Results

Discussion

Conclusion