γVPE plays an important role in programmed cell death for xylem fiber cells by activating protease CEP1 maturation in Arabidopsis thaliana

https://doi.org/10.1016/j.ijbiomac.2019.07.017Get rights and content

Highlights

  • γVPE was expressed in cambium, phloem and xylem during stem development.

  • γVPE is involved in PCD of xylem fiber cells development.

  • The mature γVPE is directly participates in the maturation of CEP1.

Abstract

The vacuolar processing enzyme (VPE) plays an important role in PCD and was originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants. We found that γVPE is involved in PCD of xylem fiber cells through the activation of CEP1 proproteases into mature protease in Arabidopsis. The γVPE protein was expressed specifically in cambium cells cambium, the primary phloem and the primary xylem during stem development. The recombinant γVPE appearing as a proenzyme at pH 7.0, and then transforming into a 40-kD mature enzyme at pH 5.5 in vitro by self-cleaving. The mature γVPE protein activated CEP1 maturation in vitro, whereas this activity was inhibited in the γvpe mutant. Transmission electron microscopy showed delayed PCD in fiber cells and thickening of secondary fiber cell walls in the γvpe mutant. Transcriptome analysis showed that the expression of 2001 genes was significantly altered expression in the γvpe mutants, and most of them are important for secondary cell wall formation and PCD. Our results demonstrate that γVPE is a crucial processing enzyme for xylem fiber cells PCD during stem development.

Introduction

Xylem is a complex tissue essential for long-distance water transport and mechanical support in plants; thus, it plays key roles in plant growth and development. Xylem cells include tracheary elements (TEs), xylary fibers, and parenchymal cells, which are differentiated from procambial or cambial cells [1]. Xylem development is a typical example of plant cell terminal differentiation, which includes initial cell division, cell expansion, secondary cell wall formation, and programmed cell death (PCD) [2]. TEs and fiber cells undergo PCD during xylem development. The release of hydrolytic enzymes stored in the vacuole leads to the rapid degradation of organelles, nuclear DNA, and part of the cell wall [3]. Many hydrolytic enzymes, including the cysteine proteases XCP1 and XCP2 [4], Tr-cp 14 (homologs of XCP1 and XCP2) [5], metacaspase 9 (AtMC9) [6], and the Zn2+-dependent nuclease ZEN1 [7], are associated with PCD during the development of TEs. Our previous studies demonstrated that CEP1 is involved in PCD and secondary wall thickening during xylem development in Arabidopsis [8]. Although several proteases involved in PCD during xylem development have been characterized, the full extent of the involvement of cysteine proteases remains unknown.

Previous studies have explored vacuole-localized cysteine proteases known as vacuolar processing enzymes (VPEs), which were originally associated with the maturation of seed storage proteins. VPEs are involved in virus-induced hypersensitive cell death in tobacco, exhibiting caspase 1–like activity to substitute orthologous caspases [9,10]. In one previous study, features of fumonisin B1–induced cell death were completely abolished in an Arabidopsis VPE-null mutant whose genome lacked all four VPE genes [11]. Although VPEs and caspase 1 share several structural properties, they share limited sequence identity. Full genome analysis indicated the complete absence of caspase-encoding genes in the Arabidopsis genome. Furthermore, the subcellular localizations of the two proteases differ: VPEs are localized in the vacuole, unlike animal caspases, which are localized in the cytosol [9].

The Arabidopsis genome contains four VPE genes – αVPE, βVPE, γVPE, and δVPE – which can be divided into two subfamilies: vegetative- and seed-type VPEs. αVPE and γVPE are expressed in vegetative organs, whereas βVPE and δVPE are expressed in seeds [12,13]. VPEs are synthesized as larger, inactive pro-protein precursors, from which the C- and N-terminal propeptides are sequentially removed self-catalytically to produce active mature forms under acidic conditions (pH 5.5) [14,15]. For example, γVPE possesses unique autoactivation mechanisms, including a plant-specific two-chain activation state, which remains stable at neutral pH and is a prerequisite for full ligase activity and survival in different cell compartments in Arabidopsis [16].

To investigate the role of γvpe in xylem development, we characterized the expression of γVPE in stems, and detected the maturation of the cysteine proteinase CEP1 by γVPE in vitro and in vivo. Our results indicate that γVPE acts as a trigger during xylem fiber cells PCD by activating cysteine proteinases in acidic vacuolar environments.

Section snippets

Plant materials and growth conditions

Seeds of Arabidopsis T-DNA insertion mutants were obtained from the Arabidopsis Biological Resource Center (https://abrc.osu.edu). The Columbia-0 variety and its mutants were grown in a greenhouse at 23 °C (16 h light, 8 h dark). The stem height of the plant was counted by 30 plants of each lines.

Molecular cloning and plasmid construction

A 1,788-bp promoter of γVPE (γVPEp) and a 2,134-bp γVPE cDNA sequence were amplified using Pro-γVPE-F/R primers (F: 5′-TCATGATTGGAACAAGTGAGG-3′; R: 5′-GAGTCTTTGAAAAGAAAAAAAAG-3′) and γVPE-F/R primers

Expression characterization of γvpe in Arabidopsis

To identify the function of γVPE (AT4G32940) during Arabidopsis stem development, we investigated γVPE expression characteristics. We performed quantitative real-time polymerase chain reaction (qRT-PCR) analysis with total RNA extracted from various organs, including roots, stems, and leaves. γVPE was highly expressed in vegetative tissues (Fig. 1A). During stem development, γVPE expression appeared in stem bolting, reached the maximum level 14–28 days after bolting, and then sharply declined

Discussion

Previous studies have showed the expression patterns of the VPE genes appear to be significantly different from each other in Arabidopsis: αVPE and γVPE are expressed in vegetative organs, whereas βVPE and δVPE are expressed in seeds [12,13]. αVPE and γVPE might play a role in the maturation of some proteins in lytic vacuoles while both βVPE and δVPE might be involved in the maturation of the seed proteins in protein-storage vacuoles [12]. Previous studies have shown that γVPE is specific to

Acknowledgements

This work was funded by the National Natural Science Foundation of China (No.31570582)

Authors contributions

Li H and Lu H designed the experiments; Cheng ZY, Yin B, Zhang JX, Lu YD, and Wang B performed the experiments; Cheng ZY, Yin B, and Zhang JX performed statistical analysis; Li H and Lu H wrote the manuscript. All authors read and approved the final manuscript.

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    These authors contributed equally to this work.

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