Histone variants and post-translational modifications in the regulation of plant callus formation

Introduction

The ability of plants to regenerate from dedifferentiated cells opens up many possibilities for molecular bioengineering. By manipulating gene expression in calli, we can derive crops with specific traits. Epigenetically active compounds are frequently used to impact callus formation and subsequent processes of plant organ regeneration. However, little is known about epigenetic changes during callus formation and epigenetic imprint in long-term callus propagation.

Objectives

Comparison of histone variant composition and post-translational modification status in calli induced from the shoot and root parts of Arabidopsis thaliana seedlings in the presence / absence of histone deacetylase inhibitors (HDACi), 0.5 mM sodium butyrate (NaB) or 0.5 µM trichostatin A (TSA).

Materials and methods

Callus cultures originated from shoot or root parts of 7-day-old seedlings were collected for MS analysis after 20 days of cultivation or 1-year propagation. Nuclei from seedlings and calli were isolated using Percoll gradient followed by histone extraction into sulfuric acid. Chemical derivatization of amine residues was performed prior to LC-MS/MS on Orbitrap Fusion Lumos Tribrid. Peaks of histone peptide precursors were quantified in Skyline software.

Results

Increased levels of the histone H3.3 variant were prominent in 20-day calli. The methylation status in root- and shoot-derived calli reached the same level during long-term propagation, while differences in acetylation levels provided a lasting imprint of their origin. Epigenetic markers disappeared after 20 days of callus propagation in the presence of HDACi, with NaB-treated calli resembling root-derived calli and TSA-treated calli resembling shoot-derived calli.

Conclusion

Understanding the factors that control epigenetic changes during callus formation is a prerequisite for targeted plant regeneration and the development of crop manipulation technologies. Here we show that histone variant replacement plays a substantial role in chromatin remodeling during callus induction and propagation. Changes in histone acetylations and methylations represent an additional fine-tuning mechanism that is important for activation/repression of specific genes. Interestingly, signs of calli origins (shoot vs. root) seem to disappear during long-term passaging and in the 20-day cultivation in the presence of HDACi.

Funding

This research was supported by the European Regional Development Fund project "SINGING PLANT" (No. CZ.02.1.01/0.0/0.0/16_026/0008446) and Czech Science Foundation (project No. 22-28190S). CIISB: Instruct-CZ Centre of Instruct-ERIC EU consortium, funded by MEYS CR infrastructure project LM2023042, is gratefully acknowledged for the financial support of the measurements at the CEITEC Proteomics Core Facility. Computational resources were provided by the e-INFRA CZ project (ID:90254), supported by MEYS CR.