A peptide of 17 aminoacids from the N-terminal region of maize plastidial transglutaminase is essential for chloroplast targeting

Nefertiti Campos,Josep M Torné,Pilar Fontanet,Mireya Santos,Enrique Villalobos

doi:10.4236/ajmb.2012.23026

Abstract

Transglutaminases (TGases, EC 2.3.2.13) catalyse posttranslational modification of proteins by establishing ε-(γ-glutamyl) links and covalent conjugation of polyamines. In plants, the functionality of these enzymes is scarcely known. The maize transglutaminase gene (tgz), the only cloned plant TGase, produces major alterations in thylakoid membrane architecture when the transglutaminase (chlTGZ) protein was over-expressed in tobacco chloroplasts, significantly increasing the number of grana stacked layers. Here we demonstrate that nuclear transformation of rice plants starting from a tgz gene truncated in 17 N-terminal aas (tgzt) non altered chloroplast thylakoid structures. F3 transformed plants were analysed for TGase activity, chlTGZ presence and tgzt transcription levels. Transformed plants exhibited double the in vitro TGase activity of the non-transformed plants. Immunoblot and quantitative RT-PCR analysis results of tgzt-rice plants grown under different illumination periods revealed that chlTGZ maintains its differential expression depending on the light regime. Nevertheless, the maize protein was localised by confocal microscopy in the cell wall of transformed rice cells. TEM analyses of the transformed cells showed normal, non-altered chloroplast thylakoid structures with the maize protein preferentially located in the cell walls. The results confirmed that the tgz eliminated sequence is essential for chloroplast targeting, being its absence sufficient to the lack of protein expression in its original plastidal compartment. Interestingly, the immunolocalization of a putative endogenous rice TGase protein is also showed. These data give further information on plant TGase functionality and its relationship to photosynthetic membranes.

Highlights

Transglutaminases (TGases) are enzymes that catalyse posttranslational modification of proteins by inter or intra-molecular cross-linking through acyl-transfer between -carboxyamide groups of glutamine residues and -amino groups of lysine
By inducing over-expression of maize chlTGZ in Tobacco chloroplasts, using transformation by a specific chloroplast promoter [12], we found that TGase activity in the over-expressers increased 4 times in comparison to the wild-type
In contrast to results with tgz-transplastomic tobacco plants [12], in the rice transformed plants TGase activity was higher and light dependent, we found that the eliminated peptide demonstrated to be sufficient for chlTGZ not to be expressed within the rice chloroplasts, which maintained a normal appearance and grana size

Summary

Introduction

Transglutaminases (glutamine:amine -glutamyl-transferase, E.C. 2.3.2.13) (TGases) are enzymes that catalyse posttranslational modification of proteins by inter or intra-molecular cross-linking through acyl-transfer between -carboxyamide groups of glutamine residues and -amino groups of lysine. This activity results in the modification of the protein conformation and more extensive conformation changes due to bonding between the same and between different proteins, forming high molecular weight conjugates [1]. In plants, using Helianthus tuberosus isolated leaf chloroplasts it was shown that some antenna proteins of the phothosystems (LHCII, CP29, CP26 and CP24) were substrates of TGase activity [2]. The maize cDNA clones exhibit TGase activity and the general biochemical characteris-

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Conclusion