Accumulation of protein aggregates induces autolytic programmed cell death in hybrid tobacco cells expressing hybrid lethality

Naoya Ueno,Tetsuya Yamada,Motoki Kanekatsu,Wataru Marubashi,Megumi Kashiwagi

doi:10.1038/s41598-019-46619-5

Naoya Ueno, Tetsuya Yamada + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-46619-5

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Abstract

Hybrid cells of Nicotiana suaveolens x N. tabacum grow normally at 36 °C, but immediately express lethality due to probable autoimmune response when transferred from 36 to 28 °C. Our recent study showed that the temperature-sensitive lethality of these hybrid cells occurs through autolytic programmed cell death (PCD). However, what happens in hybrid cells following the induction of autoimmune response to autolytic PCD is unclear. We hypothesized that accumulation of protein aggregates in hybrid cells induces autolytic PCD and examined detergent-insoluble protein (protein aggregates) isolated from hybrid cells expressing lethality. The amount of insoluble proteins increased in hybrid cells. Sodium 4-phenylbutyrate, a chemical chaperone, inhibited both the accumulation of insoluble proteins and irreversible progression of cell death. In contrast, E-64, a cysteine protease inhibitor, accelerated both the accumulation of insoluble proteins and cell death. Moreover, proteome analysis revealed that proteasome-component proteins were accumulated specifically in cells treated with E-64, and proteasome activity of hybrid cells decreased after induction of lethality. These findings demonstrate that accumulation of protein aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This suggests a novel process inducing plant PCD by loss of protein homeostasis and provides clues to future approaches for elucidating the whole process.

Highlights

Hybrid lethality is the phenomenon of death occurring in the hybrids of specific plant crosses[1]
To quantify the progression of cell death in hybrid cell cultures grown at 28 °C, the percentage of trypan www.nature.com/scientificreports for 6 h with or without PBA were transferred to 36 °C for 24 h, and (B) numbers of cells stained with trypan blue were measured
Insoluble protein as a percentage of total proteins in hybrid cells increased in incubation at 28 °C for 3 h before the progression of cell death as indicated by the increase of trypan-blue stained cells (Fig. 1A,B)

Summary

Introduction

Hybrid lethality is the phenomenon of death occurring in the hybrids of specific plant crosses[1]. Hybrid seedlings and suspension cultured cells of Nicotiana suaveolens x N. tabacum are grown normally without any lethal symptoms when they cultured at 36 °C, but immediately express hybrid lethality when transferred from 36 to 28 °C, which is the optimal temperature for growth of the parents of the hybrids[6,7]. Physiological and cell biological features of programmed cell death (PCD) have been observed in these hybrid seedlings and cells expressing temperature-sensitive lethality[7,8,9]. In the cells of N. suaveolens x N. tabacum exhibiting hybrid lethality, autophagy-related features such as the increases of monodansylcadaverine-stained structures and autophagy-related gene transcripts have been observed at early periods of autolytic PCD10. For hybrid cells cultured at 28 and 36 °C for 6 h with

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