Plant Science 223 (2014) 25–35 Contents lists available at ScienceDirect Plant Science j ourna l ho me pa ge: www.elsevier.com/locate/plantsci The maize pentatricopeptide repeat gene empty pericarp4 (emp4) is required for proper cellular development in vegetative tissues Damiano Gabotti a , Elisabetta Caporali b , Priscilla Manzotti a , Martina Persico a , Gianpiero Vigani a , Gabriella Consonni a,∗ a DISAA - Dipartimento di Scienze Agrarie e Ambientali - Produzione, Territorio, Agroenergia Università degli Studi di Milano - Via Celoria 2, 20133 Milano, Italy b Dipartimento di Bioscienze, Università degli Studi di Milano - Via Celoria 26, 20133 Milano, Italy a r t i c l e i n f o Article history: Received 4 December 2013 Received in revised form 21 February 2014 Accepted 24 February 2014 Available online 3 March 2014 Keywords: Pentatricopeptide repeat proteins Empty pericarp mutants Leaf cell ultra-structure Mitochondria Zea mays a b s t r a c t The empty pericarp4 (emp4) gene encodes a mitochondrion-targeted pentatricopeptide repeat (ppr) protein that is involved in the regulation of mitochondrial gene expression and is required for seed devel- opment. In homozygous mutant emp4-1 kernels the endosperm is drastically reduced and the embryo is retarded in its development and unable to germinate. With the aim of investigating the role of emp4 during post-germinative development, homozygous mutant seedlings were obtained by cultivation of excised immature embryos on a synthetic medium. In the mutants both germination frequency as well as the proportion of seedlings reaching the first and second leaf stages were reduced. The anatomy of the leaf blades and the root cortex was not affected by the mutation, however severe alterations such as the presence of empty cells or cells containing poorly organized organelles, were observed. Moreover both mitochondria and chloroplast functionality was impaired in the mutants. Our hypothesis is that mito- chondrial impairment, the primary effect of the mutation, causes secondary effects on the development of other cellular organelles. Ultra-structural features of mutant leaf blade mesophyll cells are reminiscent of cells undergoing senescence. Interestingly, both structural and functional damage was less severe in seedlings grown in total darkness compared with those exposed to light, thus suggesting that the effects of the mutation are enhanced by the presence of light. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The maize empty pericarp4 (emp4) gene encodes a pro- tein containing nine pentatricopeptide repeat (PPR) motifs, an N-terminal mitochondrion-targeted sequence peptide and a C- terminal domain of unknown function. Its sequence composition allows us to assign EMP4 to the P subfamily of PPR proteins [1]. The emp4 gene is expressed in all maize tissues analyzed, includ- ing embryos and endosperm, leaves, roots, stems and ovaries. Its product is necessary for the correct expression of a small subset of mitochondrial transcripts in the endosperm, as shown through Abbreviations: BETL, basal endosperm transfer layer; ER, embryo-rescue; DAC, day after beginning of culture; DAP, days after pollination; NGE, nuclear gene expression; OGE, organellar gene expression; PPR, pentatricopeptide repeat; WT, wild-type. ∗ Corresponding author at: DISAA - Dipartimento di Scienze Agrarie e Ambientali Produzione, Territorio, Agroenergia, Università degli Studi di Milano - Via Celoria 2, 20133 Milano, Italy. Tel.: +39 02 50316634. E-mail address: gabriella.consonni@unimi.it (G. Consonni). microarray analysis of mitochondrial gene expression in wild-type and emp4-1 mutant endosperms [2]. The PPR family, first identified in the Arabidopsis thaliana genome sequence [3], is typical of terres- trial plant species in which it is present and comprises hundreds of members. The main feature of PPR proteins is a tandem array of 35 amino acids that forms two antiparallel alpha helices; this is known as the PPR motif [3]. PPRs are defined as RNA-interacting proteins regulating post-transcriptional processes in organelles [4]. PPRs can also bind to other proteins as shown in Arabidopsis for the product of DELAYED GREENING1 (DG1), which binds to the SIG6 sigma factor involved in chloroplast transcription, and for pnm1 (a PPR protein localized in the nucleus and chloroplast), which inter- acts with a nucleosome assembly protein in the chloroplast and transcription factor in the nucleus [5,6]. The involvement of specific PPR proteins in processes such as splicing [7–9] and cleavage of plastidial mRNAs [10], the stability of chloroplast transcripts [11–13], RNA editing [14–21] and trans- lation initiation [22] is well documented. In plants most of the PPR genes that have been functionally analyzed are required for embryo and/or kernel development and http://dx.doi.org/10.1016/j.plantsci.2014.02.012 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.