# PLASTID PROTEOSTASIS: RELEVANCE OF TRANSCRIPTION, TRANSLATION AND POST-TRANSLATIONAL MODIFICATIONS

EDITED BY: Fiammetta Alagna, Michele Bellucci, Dario Leister and Andrea Pompa PUBLISHED IN: Frontiers in Plant Science

#### *Frontiers Copyright Statement*

*© Copyright 2007-2017 Frontiers Media SA. All rights reserved. All content included on this site, such as text, graphics, logos, button icons, images, video/audio clips, downloads, data compilations and software, is the property of or is licensed to Frontiers Media SA ("Frontiers") or its licensees and/or subcontractors. The copyright in the text of individual articles is the property of their respective authors, subject to a license granted to Frontiers.*

*The compilation of articles constituting this e-book, wherever published, as well as the compilation of all other content on this site, is the exclusive property of Frontiers. For the conditions for downloading and copying of e-books from Frontiers' website, please see the Terms for Website Use. If purchasing Frontiers e-books from other websites or sources, the conditions of the website concerned apply.*

*Images and graphics not forming part of user-contributed materials may not be downloaded or copied without permission.*

*Individual articles may be downloaded and reproduced in accordance with the principles of the CC-BY licence subject to any copyright or other notices. They may not be re-sold as an e-book.*

*As author or other contributor you grant a CC-BY licence to others to reproduce your articles, including any graphics and third-party materials supplied by you, in accordance with the Conditions for Website Use and subject to any copyright notices which you include in connection with your articles and materials.*

> *All copyright, and all rights therein, are protected by national and international copyright laws.*

*The above represents a summary only. For the full conditions see the Conditions for Authors and the Conditions for Website Use.*

ISSN 1664-8714 ISBN 978-2-88945-343-6 DOI 10.3389/978-2-88945-343-6

### About Frontiers

Frontiers is more than just an open-access publisher of scholarly articles: it is a pioneering approach to the world of academia, radically improving the way scholarly research is managed. The grand vision of Frontiers is a world where all people have an equal opportunity to seek, share and generate knowledge. Frontiers provides immediate and permanent online open access to all its publications, but this alone is not enough to realize our grand goals.

### Frontiers Journal Series

The Frontiers Journal Series is a multi-tier and interdisciplinary set of open-access, online journals, promising a paradigm shift from the current review, selection and dissemination processes in academic publishing. All Frontiers journals are driven by researchers for researchers; therefore, they constitute a service to the scholarly community. At the same time, the Frontiers Journal Series operates on a revolutionary invention, the tiered publishing system, initially addressing specific communities of scholars, and gradually climbing up to broader public understanding, thus serving the interests of the lay society, too.

### Dedication to Quality

Each Frontiers article is a landmark of the highest quality, thanks to genuinely collaborative interactions between authors and review editors, who include some of the world's best academicians. Research must be certified by peers before entering a stream of knowledge that may eventually reach the public - and shape society; therefore, Frontiers only applies the most rigorous and unbiased reviews.

Frontiers revolutionizes research publishing by freely delivering the most outstanding research, evaluated with no bias from both the academic and social point of view. By applying the most advanced information technologies, Frontiers is catapulting scholarly publishing into a new generation.

### What are Frontiers Research Topics?

Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office<EMAIL_ADDRESS>
# **PLASTID PROTEOSTASIS: RELEVANCE OF TRANSCRIPTION, TRANSLATION AND POST-TRANSLATIONAL MODIFICATIONS**

### Topic Editors:

**Fiammetta Alagna,** Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria (CREA), Italy

**Michele Bellucci,** Consiglio Nazionale delle Ricerche (CNR), Italy **Dario Leister**, Ludwig-Maximilians-Universität München, Germany **Andrea Pompa,** Consiglio Nazionale delle Ricerche (CNR), Italy

An image of the adaxial side of a sugar beet leaf taken with a conventional fluorescence microscope. The green color is due to autofluorescence of chlorophyll in the chloroplasts. In the box, the optical section of a tobacco protoplast taken with a confocal microscope (at left) and a representation of the chloroplasts structure based on a transmission electron microscopy (TEM) image (at right) is shown. The drawings inside the TEM image represent translation of chloroplast mRNA by 70S ribosomes.

Image: Michele Bellucci.

Due to their bacterial endosymbiotic origin plastids are organelles with both nuclear-encoded and plastid-encoded proteins. Therefore, a highly integrated modulation of gene expression between the nucleus and the plastome is needed in plant cell development. Plastids have retained for the most part a prokaryotic gene expression machinery but, differently from prokaryotes and eukaryotes, they have largely abandoned transcriptional control and switched to predominantly translational control of their gene expression. Some transcriptional regulation is known to occur, but the coordinate expression between the nucleus and the plastome takes place mainly through translational regulation. However, the regulatory mechanisms of plastid gene expression (PGE) are mediated by intricate plastid-nuclear interactions and are still far from being fully understood. Although, for example, translational autoregulation mechanisms in algae have been described for subunits of heteromeric protein complexes and termed control by epistasy of synthesis (CES), only few autoregulatory proteins have been identified in plant plastids. It should be noted of course that PGE in C. reinhardtii is different from that in plants in many aspects. Another example of investigation in this research area is to understand the interactions that occur during RNA binding between nucleus-encoded RNA-binding proteins and the respective RNA sequences, and how this influences the translation initiation process.

In addition to this, the plastid retains a whole series of mechanisms for the preservation of its protein balance (proteostasis), including specific proteases, as well as molecular chaperones and enzymes useful in protein folding. After synthesis, plastid proteins must rapidly fold into stable three dimensional structures and often undergo co- and posttranslational modifications to perform their biological mission, avoiding aberrant folding, aggregation and targeting with the help of molecular chaperones and proteases.

We believe that this topic is highly interesting for many research areas because the regulation of PGE is not only of wide interest for plant biologists but has also biotechnological implications. Indeed, plastid transformation turns out to be a very promising tool for the production of recombinant proteins in plants, yet some limitations must still be overcome and we believe that this is mainly due to our limited knowledge of the mechanisms in plastids influencing the maintenance of proteostasis.

**Citation:** Alagna, F., Bellucci, M., Leister, D. , Pompa, A., eds. (2017). Plastid Proteostasis: Relevance of Transcription, Translation and Post-Translational Modifications. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-343-6

# Table of Contents

*05 Editorial: Plastid Proteostasis: Relevance of Transcription, Translation, and Post-translational Modifications*

Fiammetta Alagna, Michele Bellucci, Dario Leister and Andrea Pompa

### **Chapter 1. Transcriptional control of gene-expression**

Duorong Xu, Dario Leister and Tatjana Kleine

### **Chapter 2. Post-translational mechanisms affecting plastid proteostasis**

*46 Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms*

Inga Sjuts, Jürgen Soll and Bettina Bölter

*61 Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances*

Magda Grabsztunowicz, Minna M. Koskela and Paula Mulo

### **Chapter 3. Chloroplast-nucleus communication**

Yoshihiro Hirosawa, Yasuko Ito-Inaba and Takehito Inaba

# Editorial: Plastid Proteostasis: Relevance of Transcription, Translation, and Post-translational Modifications

#### Fiammetta Alagna1 \*, Michele Bellucci 2 \*, Dario Leister 3 \* and Andrea Pompa2 \*

1 Viticoltura ed Enologia, Consiglio per la Ricerca in Agricoltura e l'Analisi dell'Economia Agraria (CREA), Turi, Italy, 2 Istituto di Bioscienze e Biorisorse, Consiglio Nazionale delle Ricerche (CNR), Perugia, Italy, 3 Plant Molecular Biology, Department Biology I, Ludwig-Maximilians-Universität, München, Germany

Keywords: plastome, gene expression, protein balance, regulation, nuclear-plastid interactions

### **Editorial on the Research Topic**

### **Plastid Proteostasis: Relevance of Transcription, Translation, and Post-translational Modifications**

Plastids are sites of biochemical and biological processes that are fundamental for plant life. The genome of these endosymbiotic organelles encodes for almost one hundred of the three thousand proteins that make up the chloroplast proteome. Genes coding for plastid multi-subunit protein complexes derive from both nuclear and plastid genomes, so it is clear that there is the need of a highly integrated coordination between this two subcellular compartments.

### Edited and reviewed by:

Julian Eaton-Rye, University of Otago, New Zealand

#### \*Correspondence:

Fiammetta Alagna<EMAIL_ADDRESS>Michele Bellucci<EMAIL_ADDRESS>Dario Leister<EMAIL_ADDRESS>Andrea Pompa<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

Received: 11 August 2017 Accepted: 26 September 2017 Published: 17 October 2017

#### Citation:

Alagna F, Bellucci M, Leister D and Pompa A (2017) Editorial: Plastid Proteostasis: Relevance of Transcription, Translation, and Post-translational Modifications. Front. Plant Sci. 8:1759. doi: 10.3389/fpls.2017.01759

The coordination between the nucleus and the plastome takes place at many different levels, including the modulation of nuclear and plastid transcription, RNA processing and translation, post-translational modifications, and protein targeting. In addition, the plastid retains a whole series of mechanisms for the preservation of its protein balance (proteostasis), including proteases and molecular chaperones (**Figure 1**).

Plastids have largely abandoned transcriptional control switching predominantly to translational and post-translational control of their gene expression, but some transcriptional regulation is known to occur. Transcription of plastid genes is performed by two different types of RNA polymerases: plastid-encoded RNA polymerase (PEP) and a nuclear-encoded RNA polymerases (NEP). Liebers et al. propose that targeted changes in plastid transcription, mostly by controlling the relative activities of NEP and PEP enzymes, impact the establishment of the plastid proteome and these represent key determinants for the transitions between the different plastid types.

The transcriptional regulation mechanisms are still far from being completely elucidated. An unusual light- and stress-responsive promoter (psbD LRP), regulated by a AAG-box immediately upstream of the –35 element, has been recently mapped. Shimmura et al. analyzed psbD LRP promoter regions in 11 embryophytes, at different evolutionary stages, from liverworts to angiosperms. This analysis identified conserved features of the promoter and facilitated study of its emergence and evolution in plant species.

Among the proteins that regulate plastid gene expression, the nucleus-encoded proteins of the mitochondrial transcription termination factor (mTERF) family have been recently identified. Information on their function is only beginning to emerge. Xu et al. investigate the function of the chloroplast-associated mTERF. They report that these proteins are localized to chloroplast nucleoids and identify two of them involved in the salt stress response.

The import of plastid precursor proteins into plastids is another checkpoint affecting plastid proteostasis that is regulated in response to the fluctuating environmental conditions. This fine regulation ensures the optimal functioning of important biological processes

taking place in this cellular compartment. The import of plastid precursor proteins is mediated by two distinct translocation complexes called TOC and TIC, located respectively at the outer and at the inner envelope membrane of chloroplasts. The individual steps involved in protein translocation and the corresponding regulation mechanisms used by plants to modulate protein import are reviewed by Sjuts et al.

Upon transition from an endosymbiont to a plant cell organelle, the plastid retains a set of mechanisms that involves enzymes and proteins of prokaryotic origin which are responsible for protein maturation, post-translational modification, correct folding, protein abundance control, and removal of misfolded or damaged components. These processes ensure that plastid proteins are ready to exert their biological mission and require an intricate system of signals from nucleus to plastid and backwards. Plastid-derived signals can regulate availability of nuclear-encoded plastid precursors controlling their de novo synthesis, and targeting. Recently, an important player in the chloroplast-to-nucleus retrograde communication has been identified: the protein GENOMES UNCOUPLED1 (GUN1). Recent studies indicate that GUN1 might play a role in the coordination of translation, import, and degradation of plastid proteins. The molecular function of different GUN1 partners has been reviewed by Colombo et al., highlighting its potential role in plastid proteostasis. Another important mechanism during nuclear-plastid interaction is the degradation of multiple components through the ubiquitin–proteasome system. It has become increasingly clear that, together with feedback regulation of nuclear gene expression by plastid-derived signals, this mechanism avoids the accumulation of non-imported proteins in the cytosol. In addition to the anterograde signaling pathway, recent studies in A. thaliana demonstrated that also the retrograde signaling pathway can be subjected to ubiquitin– proteasome regulation. Hirosawa et al. review recent advances in understanding how the ubiquitin–proteasome system regulates the nuclear–plastid interaction and plastid biogenesis.

The maturation of plastid proteins is another highly regulated process that in some cases needs complex apparatuses to occur. This is the case of c-type cytochromes that require a multicomponent assembly pathway for their maturation, as reviewed by Gabilly and Hamel. The regulation of this pathway has not yet been clarified. Cline et al. describe the functional characterization of the CCS2 gene of Chlamydomonas reinhardtii required for cytochrome c assembly. The authors discuss the possible functions of CCS2 in the heme attachment reaction.

A wide range of post-translational modifications (PTMs) contribute to finely regulate the biological processes that take place in plastids. PTMs alter the physicochemical properties of the plastidial proteins thus affecting their function. Grabsztunowicz et al. review the current knowledge on the PTMs regulating important metabolic processes in chloroplasts such as DNA replication and gene expression, photosynthetic carbon assimilation, and starch metabolism and report the known physiological effects of these modifications.

Considering that there is limited knowledge of the combined action among the molecular mechanisms that regulate plastid proteostasis, the goal of this Research Topic is to bring together a set of articles that contribute to our understanding of how transcription, translation, and post-translational modifications (including protein targeting) maintain plastid proteostasis. As a consequence, this Research Topic will help us understand more deeply how plastids function.

### AUTHOR CONTRIBUTIONS

MB, DL, and AP prepared the research topic. All the authors edited the manuscripts. FA invited the authors and wrote the editorial. All the authors read and approved the editorial.

### ACKNOWLEDGMENTS

Research in the lab of Dario Leister is supported by the "Deutsche Forschungsgemeinschaft (DFG)" (grant number TRR175/C05).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Alagna, Bellucci, Leister and Pompa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development

Monique Liebers, Björn Grübler, Fabien Chevalier, Silva Lerbs-Mache, Livia Merendino, Robert Blanvillain and Thomas Pfannschmidt\*

Laboratoire de Physiologie Cellulaire et Végétale, Institut de Biosciences et Biotechnologies de Grenoble, CNRS, CEA, INRA, Université Grenoble Alpes, Grenoble, France

#### Edited by:

Fiammetta Alagna, The Council for Agricultural Research and Economics, Italy

#### Reviewed by:

Takashi Shiina, Kyoto Prefectural University, Japan Pal Maliga, Rutgers University, USA Yan Zubo, Dartmouth college, USA

#### \*Correspondence:

Thomas Pfannschmidt<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

Received: 30 November 2016 Accepted: 05 January 2017 Published: 19 January 2017

#### Citation:

Liebers M, Grübler B, Chevalier F, Lerbs-Mache S, Merendino L, Blanvillain R and Pfannschmidt T (2017) Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development. Front. Plant Sci. 8:23. doi: 10.3389/fpls.2017.00023 Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Keywords: plastids, plastid morphology, photomorphogenesis, plant development, transcription, gene regulation, NEP, PEP

### INTRODUCTION

Plastids are cellular organelles that can be found only in plant and algae cells. They are of endosymbiotic origin that traces back to an evolutionary event in which a mitochondriate eukaryote took up a photosynthetically active cyanobacteria-like bacterium and established it as a permanent component of the cell, likely with the help of Chlamydiae (Ball et al., 2016). The

most prominent benefit for the eukaryotic cell in this process was the gain of photosynthesis and the concomitant switch from a heterotrophic to an autotrophic lifestyle (Hohmann-Marriott and Blankenship, 2011). The establishment of a stable endosymbiosis was, however, not an immediate evolutionary jump but a longongoing adaptation process in which the engulfed cyanobacterialike ancestor has lost slowly most of its genetic information toward the nucleus of the host cell by horizontal gene transfer (Abdallah et al., 2000; Martin et al., 2002; Reyes-Prieto et al., 2007). Only a small, but highly conserved set of genes finally remained encoded in the plastids' own genome of present plants, the plastome (Bock, 2007; Wicke et al., 2011). The vast majority of the proteome of present-day plant plastids is, therefore, encoded in the nucleus and must be imported from the cytosol (Rolland et al., 2012; Demarsy et al., 2014). Nevertheless, the proper expression of plastid genes is absolutely essential for the build-up of protein complexes involved in plastid gene transcription and translation as well as in metabolic processes such as photosynthesis or fatty acid biosynthesis (Jarvis and Lopez-Juez, 2013; Lyska et al., 2013). All major plastid multisubunit protein complexes are composed of a patchwork of nuclear and plastid encoded subunits and can be established only by a tight coordination of gene expression between the two genetic compartments (Pogson et al., 2015).

Alongside with these molecular and sub-cellular constraints, the establishment of plastid proteomes is strongly influenced by tissue-dependent and environmental cues. Multicellular, terrestrial plants are comprised of different organs with very divergent tissue organization and function. Plastids in these different tissues display large morphological and functional variations which are tightly connected to the function of the corresponding tissue (Schnepf, 1980; Lopez-Juez and Pyke, 2005). An individual plant, thus, possesses several different plastid types that represent distinct manifestations of the same cell organelle. Interestingly, most of these plastid types can interconvert upon environmentally induced changes in plant and tissue development. These morphological and functional conversions are only possible by corresponding changes in the plastid proteome composition. In this mini-review we focus on the specific changes in plastid gene expression that occur before or during transitions between different plastid types in the course of plant development.

### The Different Plastid Types of Plant Cells

Plant cells cannot generate plastids de novo but they gain them by inheritance from their progenitor cell. During division of the mother cell plastids are distributed arbitrarily between daughter cells and multiply afterward, by fission using a prokaryotictype division apparatus (Osteryoung and Pyke, 2014). The final number of plastids within a cell is cell-type specific and depends on regulatory mechanisms that are far from being understood yet (Cole, 2016). In addition, an individual cell does typically contain only one specific plastid type indicating that plastid development and cell development are interlinked. The various developmental lines and possible conversions between plastid types are subsequently discussed using the life cycle of the angiosperm Arabidopsis as a model (**Figure 1**). Because of space constraints detailed species-specific differences or special cases will be not considered here.

In Arabidopsis (like in most angiosperms) plastids are inherited maternally as a undifferentiated and small precursor form called proplastid (Pyke, 2007). In other species proplastids might be inherited also by paternal or biparental means. Knowledge in this field is poor and active regulation mechanisms remain to be clarified (Greiner et al., 2015). After fertilization of the egg cell Arabidopsis embryos undergo a morphological program typical for angiosperms that eventually ends with dry seeds (Le et al., 2010; compare **Figure 1**, outer circle). Like in many other oilseed crops Arabidopsis embryogenesis is characterized by an intermediate photosynthetically active period in which proplastids develop into chloroplasts in a stagespecific manner (Tejos et al., 2010). Chloroplast containing cells are already detected at the globular stage, but are most abundant during 6–12 days after fertilization (Allorent et al., 2013). This phase appears to be important for the fitness of the seed (Allorent et al., 2015). In a subsequent desiccation phase these chloroplasts then de-differentiate into non-photosynthetic, colorless leucoplasts, called eoplasts (Mansfield and Briarty, 1991, 1992). After seed imbibition and germination these eoplasts then re-differentiate into various plastid types depending on tissue context and environmental conditions.

In the dark, seedlings follow a developmental program called skotomorphogenesis (Solymosi and Schoefs, 2010). In cotyledons of such seedlings eoplasts develop into etioplasts while those located in hypocotyl and root develop into different types of colorless leucoplasts that are difficult to distinguish at the morphological level. Etioplasts are characteristic for this developmental program and represent an intermediate stand-by state of chloroplast formation. They do not develop a thylakoid membrane system, but a prolamellar body (PLB) that is composed of regular arrangements of NADPH, the enzyme protochlorophyllide-oxido-reductase (POR), the chlorophyll precursor protochlorophyllide and the thylakoid membrane lipids digalactosyl-diacylglycerol (DGDG) and monogalactosyldiacylglycerol (MGDG; Bastien et al., 2016). Upon illumination another developmental program called photomorphogenesis is initiated by the phytochrome-mediated photoreceptor network that triggers the expression of many nucleus located genes coding for chloroplast proteins (Arsovski et al., 2012). In parallel, thylakoid membranes begin to form and the lightdependent POR induces chlorophyll biosynthesis within the PLB. Chloroplast biogenesis then is usually completed after just 6–24 h. If seeds germinate directly in light the skotomorphogenic program is skipped and the eoplasts within the cotyledons differentiate directly into chloroplasts. Whether or not proplastids and eoplasts represent fully equivalent developmental stages remain to be elucidated. Studies on the transition of dedifferentiated desiccoplasts into etio- or chloroplasts after rehydration and illumination in the poikilochlorophyllous plant Xerophyta humilis may provide novel clues for the understanding of proplastid/eoplast-to-chloroplast transitions (Solymosi et al., 2013).

During primary leaf formation chloroplasts originate directly from proplastids present in the shoot apical meristem (SAM;

(indicated by rolling lines). Changes in plastid transcriptional apparatus or activity that occur during these transitions are indicated by symbols NEP and PEP. Size of letters represents the relative activities of the two types of RNA polymerases in the respective plastid type. For details see text.

Charuvi et al., 2012). Fully developed chloroplasts in green parts of plants multiply then by fission until they reach the celltype specific number. This, however, accounts mainly for the mesophyll tissue while in the epidermis a likely tissue-specific program leads to a differential development of chloroplasts. Guard cells were reported to display high numbers of fully developed chloroplasts while pavement cells contain rather low numbers of relatively small chloroplasts (around half the size of those in mesophyll cells) that may contain reduced levels of chlorophyll (Barton et al., 2016). In reproductive organs such as fruits or flowers chloroplasts usually transform into chromoplasts as part of maturation or developmental programs. In senescing tissues the valuable resources of chloroplasts, notably the nitrogen bound in chlorophylls and photosynthesis proteins such as RubisCO, are reallocated and the plastids turn into gerontoplasts, the aging form of plastids.

In hypocotyls and roots of growing seedlings eoplasts usually develop into a number of colorless plastids commonly summarized under the term leucoplasts. This group comprises amyloplasts, statoliths, and elaioplasts (**Figure 1** and **Table 1**) and, in later stages, may develop also in other parts of the plant. These colorless plastids do develop even if the tissues are exposed to light. This strongly suggests that the transition from proplastids/eoplasts into chloroplasts is actively inhibited in these tissues, likely by internal factors. Recent studies have demonstrated that the developmental block of chloroplast development in Arabidopsis roots can be released either genetically or by external hormone treatment (Kobayashi et al., 2012) supporting the view of an active inhibition in chloroplast biogenesis in these tissues. Release of such an inhibition represents not only an artificial effect but does occur also under physiological conditions as some studies reported the presence of fully developed chloroplasts in Arabidopsis hypocotyls (Jin et al., 2001; Hermkes et al., 2011). These chloroplasts were found to be involved in phototropic responses suggesting that they play a defined physiological role (Jin et al., 2001). Studying the mechanisms that control this eoplastchloroplast transition could help to understand principle steps

#### TABLE 1 | Summary of major plastid types in plant cells.

fpls-08-00023 January 17, 2017 Time: 16:47 # 4

Typical plastid types found in vascular plants are listed. Leucoplasts are not included as they represent a group of plastids (summarizing all non-green plastids lacking pigments including amyloplasts) rather than defining a specific plastid type. The group of leucoplasts contains also other less prominent plastid types that are not well investigated and not discussed in this review such as root plastids and proteinoplasts. For more information on these specialized plastid forms readers are referred to corresponding reviews (Schnepf, 1980; Pyke, 2007).

of early chloroplast biogenesis and to identify novel regulatory factors of plastid transitions (Chiang et al., 2012).

### Shifts in Plastid Transcription during Morphological Transitions of Plastids

The different types of plastids mentioned above perform very different functions that are highly specific for the tissue in which they reside (Pyke, 2007) (**Table 1**). Despite their morphological and functional diversity they all contain the same genome (Bock, 2007). However, their strong functional diversity implies a specific enzymatic configuration for each plastid type. This requires a controlled adjustment in the expression of both plastid and nuclear genes encoding the proteins for each of these specific organelle manifestations. Here, we focus on the adjustment of plastid gene expression.

Molecular and genetic studies uncovered that transcription of plastid genes is performed by two different types of RNA polymerases. One type is comprised by two single-subunit phage-type RNA polymerases encoded by two different nuclear genes (nuclear-encoded RNA polymerases, NEP). These proteins are targeted either only to the plastid (RpoTp) or dually to plastids and mitochondria (RpoTmp). The other type of RNA polymerase is a multi-subunit enzyme of prokaryotic type with four basic subunits encoded in the plastid genome (RpoA, RpoB, RpoC1, and RpoC2; plastid-encoded RNA polymerase, PEP). For promoter recognition this enzyme complex is dependent on the interaction with sigma factors (called Sig1 – Sig6 in Arabidopsis) that are encoded in the nucleus (Toyoshima et al., 2005; Schweer et al., 2010; Lerbs-Mache, 2011; Borner et al., 2015; Pfannschmidt et al., 2015). The two types of RNA polymerase activities utilize different promoters and depending on their respective promoter structure the genes on the plastid genome can be categorized into three different classes. Class I comprises genes possessing only PEP promoters (only photosynthesis genes). Class II covers genes that have both NEP and PEP promoters (most other genes including genes for the ATP synthase and many components of the gene expression system). Class III represents genes with NEP promoters only and comprises ycf2 (encoding a still unknown protein), accD (encoding the β-carboxyltransferase subunit of the acetyl CoA carboxylase) and the rpoBC1C2 operon (Liere et al., 2011). This diversity of promoter structures and the multiplicity of transcriptional components (see also below) represent a prerequisite for efficient transcriptional regulation during plastid conversion where plastid housekeeping genes are preferentially transcribed by NEP and photosynthesis related genes are transcribed by PEP (Allison et al., 1996; Hajdukiewicz et al., 1997).

We propose that targeted changes in plastid transcription, mostly by controlling the relative activities of NEP and PEP enzymes, impact the establishment of the plastid proteome and, therefore, represent key determinants for the transitions between the different plastid types.

#### Proplastid/Eoplast-Chloroplast Transition

Proplastids can be found only in meristematic cells of plants and in in vitro cultured cells. Isolation of proplastids from meristematic cells is technically not feasible. However, as meristematic cells give rise to various plant organs, proplastids might be considered as starting point for differentiationdependent plastid conversion. Also, plastid gene expression in proplastids and after controlled conversion of proplastids into amyloplasts has been analyzed using in vitro cultured cells [(Sakai et al., 1992), see below]. These early experiments already showed that such plastid conversion is accompanied by changes in plastid transcriptional activity.

Proplastid/eoplast-chloroplast conversion-associated changes in plastid gene expression patterns have been characterized

in detail during Arabidopsis seed formation and germination (Demarsy et al., 2012; Allorent et al., 2013). Although slight increases of NEP transcribed mRNAs were observed in this transition, the predominant changes concern remarkable increases of mRNAs of photosynthesis related proteins. If proplastid/eoplast-chloroplast conversion is prevented by deletion of plastid rpo genes, colorless plastids of 2–5 µm length are formed that might be considered as a genetically induced type of leucoplasts (Allison et al., 1996; De Santis-MacIossek et al., 1999). Thus, establishment of the correct NEP/PEP configuration and their relative activities at a given developmental stage is absolutely essential for successful chloroplast differentiation.

### Etioplast-Chloroplast Transition

Etioplasts and their light-induced transition to chloroplasts are well studied in numerous dicotyledonous and monocotyledonous species. Most striking is the very rapid development of thylakoid membranes, increase in chlorophyll content and construction of the photosynthetic apparatus that requires both a massive import of nuclear encoded plastid proteins and high expression of plastid-encoded genes (Lonosky et al., 2004; von Zychlinski et al., 2005; Philippar et al., 2007; Pudelski et al., 2009; Majeran et al., 2010; Ploscher et al., 2011). Etioplasts display just a basic transcriptional activity and accumulate photosynthesis transcripts only to very low levels. Shifting dark-grown seedlings to light, however, rapidly induce a plastome-wide transcript accumulation of photosynthesis genes reaching a maximum level after 10–44 h mRNA levels followed by decrease to approximate pre-illumination levels (Rodermel and Bogorad, 1985). The initial increase in mRNA is followed by subsequent translation of the corresponding proteins (Kanervo et al., 2008). It should be noted that tissue-specific gene expression analyses distinguishing epidermal and mesophyll tissues were never reported and that the results in all studies to date, thus, represent a mixture of both cell types. This is critical with respect to the notion that recent studies suggest a specific sensor function for epidermal chloroplasts (Virdi et al., 2015, 2016). Targeted research on this special type of chloroplasts will be required in order to understand their detailed physiological function.

The light-dependent activation of plastid gene expression during etioplast-chloroplast conversion includes posttranslational modifications such as phosphorylation of PEP subunits and sigma factors (Tiller and Link, 1993) and a restructuring of the PEP complex. While in etiolated mustard seedlings PEP was found to exist in its prokaryotic composition (α2, β, β 0 , β 00 subunits), a much larger PEP complex with many additional subunits was purified from fully developed chloroplasts. Studies on intermediate plastids isolated from seedlings illuminated for just 16 h identified both complexes to around equal activities suggesting a lightinduced conversion between these two plastid RNA polymerase complexes (Pfannschmidt and Link, 1994). Detailed mass spectrometry analyses identified these subunits and a set of conserved PEP-associated proteins (PAPs) could be defined (Pfannschmidt et al., 2000; Suzuki et al., 2004; Pfalz et al., 2006; Steiner et al., 2011).

PEP-associated proteins are all nuclear encoded and are rapidly light-induced during etioplast-chloroplast transition yielding the observed PEP restructuring (Yagi et al., 2012). Genetic inactivation of any of these PAPs in Arabidopsis, maize or rice results in a block of proper chloroplast development and ends up in albinoic phenotypes suggesting that pap gene expression and/or subsequent PEP re-structuring represent essential steps in early chloroplast biogenesis. Evolutionary presence of pap genes appears to be restricted to terrestrial plants and ferns suggesting that their appearance is connected to the conquest of land (Pfalz and Pfannschmidt, 2013). These genes, thus, likely represent an evolutionary indicator for the development of chloroplast-containing multi-cellular plants (de Vries et al., 2016).

Plastid gene expression changes during etioplast to chloroplast conversion were also analyzed in the monocotyledonous plant maize. In monocotyledons, leaf development is initiated at a basal meristem resulting in a gradient of chloroplast development from the bottom to the tip (Baumgartner et al., 1989, 1993; Hess et al., 1993). This gradient has been used extensively as a model for chloroplast biogenesis. About 51 plastid genes were found to be at least two times higher expressed in tips than in the leaf base (Cahoon et al., 2008). It is, however, still debated how far this plastid developmental gradient reflects the corresponding situation (proplastid-to-chloroplast conversion) in dicotyledonous plants.

### Chloroplast-Chromoplast Transition

Chromoplasts mainly develop from chloroplasts in formerly green plant tissues e.g., during fruit ripening or flower development. They can also develop directly from proplastids or amyloplast depending on species and tissue (Egea et al., 2010). Plastid gene expression during conversion of green chloroplasts toward red chromoplasts has been characterized in detail during tomato fruit ripening. In contrast to the rapid etioplast-chloroplast transition in cotyledons, the chloroplastchromoplast transition in tomato fruits requires several days or even weeks allowing transcript analyses of various intermediary stages. These studies uncovered both systemic and gene-specific effects (Kahlau and Bock, 2008). Most important, green tomato fruits displayed a dramatic reduction in chloroplast transcripts compared to green leaves from the same plant. This indicates that the fruit developmental program provides a dominant repressive impact on plastid transcriptional activities even before ripening effects became visible. This may prevent the unnecessary production of photosynthesis proteins in the tomato fruit already in early stages of ripening.

In contrast, changes in plastid gene expression in the subsequent stages (turning, light red, red) remained relatively subtle suggesting that the chloroplast-chromoplast conversion itself is not accompanied by major changes in plastid gene expression. An exception was observed for the accD gene that displayed a targeted accumulation at both, transcript and protein levels. Accumulation of the protein AccD as part of the fatty acid biosynthesis complex may allow the accumulation of lipids necessary for storage of carotenoids produced during fruit ripening. AccD gene expression requires at least a low level of expression of genes involved in transcription/translation. Indeed, the repression by the fruit developmental program was stronger for photosynthesis genes than for genetic system genes (Kahlau and Bock, 2008) suggesting that low levels of plastid gene expression activity may remain. These remaining activities may be directed to the observed targeted accD gene expression.

#### Proplastid-Amyloplast Transition

fpls-08-00023 January 17, 2017 Time: 16:47 # 6

Amyloplasts are the plastids of storage organ tissues and roots and typically contain high amounts of starch. Systematic gene expression studies in this plastid type were done using potato tubers (Brosch et al., 2007; Valkov et al., 2009). When compared to leaf chloroplasts tuber amyloplasts displayed very low levels of gene expression in terms of transcriptional rate, transcript accumulation and maturation as well as ribosome association of mRNAs and translation. Both, NEP and PEP enzymes are present, but run-on transcription experiments revealed very low transcriptional rates of both enzyme activities. Interestingly, like in chromoplasts accD expression appeared to be an exception. It displayed relatively stable transcript levels and ribosome association (Valkov et al., 2009) thus confirming the importance of AccD for the maintenance of plastids regardless of their morphological type. In addition, trans-plastomic inactivation of the plastid accD gene in tobacco revealed to be impossible (Kode et al., 2005).

Tissue cultures of tobacco bright-yellow (BY)-2 cells represent another test system to study amyloplasts (Miyazawa et al., 1999; Enami et al., 2011). In presence of cytokinin these cells develop amyloplasts from proplastids. Microarray analysis of the transcriptome did not reveal specific changes between the two plastid types, including the accD gene. Interestingly, inhibitors of plastid transcription or translation blocked the hormone-induced differentiation of amyloplasts indicating signaling of plastid gene expression to the hormone-induced plastid developmental pathway. This specific retrograde signaling pathway seems to act via intermediates of tetrapyrrole biosynthesis, i.e., haem (Enami et al., 2011).

### REFERENCES

### CONCLUSION AND PERSPECTIVES

In all plastid conversions investigated so far, changes in the plastid transcriptional apparatus and/or transcriptional activity either accompany or even precede the transition. Proper control of plastome transcription, thus, appears to be an important determinant for these developmental steps. Future research will focus on the identification of regulators that may serve as master switches of plastid development in response to internal and external cues (Lopez-Juez, 2007). In addition, more detailed studies on gene expression in proplastids or eoplasts may be highly informative for understanding the molecular regulation of plastid development especially in their initial steps.

### AUTHOR CONTRIBUTIONS

ML contributed a figure, contributed to the manuscript text, read, and approved the final version. BG contributed to the manuscript text, read, and approved the final version. FC contributed to the manuscript text, read, and approved the final version. SL-M contributed to the manuscript text, read, and approved the final version. LM contributed to the manuscript text, read, and approved the final version. RB contributed to the manuscript text, read, and approved the final version. TP developed manuscript idea, wrote the manuscript with the help of all co-authors.

### FUNDING

Work in our team was supported by grants from the Deutsche Forschungsgemeinschaft to TP (PF323-4-2; PF323-5-2) and the DFG research group FOR 804. Our studies received also institutional financial support from the French National Research Agency (ANR-10-LABEX-04 GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology), the University Grenoble-Alpes (AGIR program to RB) and the CNRS (PEPS-ExoMod program to TP).

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Liebers, Grübler, Chevalier, Lerbs-Mache, Merendino, Blanvillain and Pfannschmidt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Comparative Analysis of Chloroplast psbD Promoters in Terrestrial Plants

Shuichi Shimmura1† , Mikio Nozoe1,2† , Shota Kitora1 , Satoko Kin1 , Shigeru Matsutani1,3 , Yoko Ishizaki1 , Yoichi Nakahira1,4 and Takashi Shiina1 \*

1 Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan, 2 AMITA Institute for Sustainable Economies Co., Ltd., Kyoto, Japan, 3 Kyoto Botanical Garden, Kyoto, Japan, 4 College of Agriculture, Ibaraki University, Ibaraki, Japan

The transcription of photosynthesis genes encoded by the plastid genome is mainly mediated by a prokaryotic-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). Standard PEP-dependent promoters resemble bacterial sigma-70-type promoters containing the so-called −10 and −35 elements. On the other hand, an unusual light- and stress-responsive promoter (psbD LRP) that is regulated by a 19-bp AAG-box immediately upstream of the −35 element has been mapped upstream of the psbD-psbC operon in some angiosperms. However, the occurrence of the AAG-box containing psbD LRP in plant evolution remains elusive. We have mapped the psbD promoters in eleven embryophytes at different evolutionary stages from liverworts to angiosperms. The psbD promoters were mostly mapped around 500–900 bp upstream of the psbD translational start sites, indicating that the psbD mRNAs have unusually long 50 -UTR extensions in common. The −10 elements of the psbD promoter are well-conserved in all embryophytes, but not the −35 elements. We found that the AAG-box sequences are highly conserved in angiosperms and gymnosperms except for gnetaceae plants. Furthermore, partial AAG-box-like sequences have been identified in the psbD promoters of some basal embryophytes such as moss, hornwort, and lycophyte, whereas liverwort has the standard PEP promoter without the AAG-box. These results suggest that the AAG-box sequences of the psbD LRP may have evolved from a primitive type of AAG-box of basal embryophytes. On the other hand, monilophytes (ferns) use another type of psbD promoter composed of a distinct cis-element upstream of the potential −35 element. Furthermore, we found that psbD expression is not regulated by light in gymnosperms or basal angiosperms, although they have the well-conserved AAG-box sequences. Thus, it is unlikely that acquisition of the AAG-box containing psbD promoter is directly associated with light-induced transcription of the psbD-psbC operon. Light- and stress-induced transcription may have evolved independently and multiple times during terrestrial plant evolution.

Keywords: psbD LRP, chloroplast, promoter, evolution, stress

### INTRODUCTION

Chloroplasts in plant and algal cells are semiautonomous organelles that have their own genome and gene expression system, reflecting their cyanobacterial origin. Chloroplast transcription is mediated by two distinct RNA polymerase systems, a prokaryotic multi-subunit RNA polymerase (PEP) whose core subunits are encoded by chloroplast genomes and single-subunit

#### Edited by:

Dario Leister, Ludwig-Maximilians-Universität München, Germany

#### Reviewed by:

Karin Krupinska, University of Kiel, Germany Kristina Kuehn, Humboldt University of Berlin, Germany

\*Correspondence: Takashi Shiina<EMAIL_ADDRESS>†These authors have contributed equally to this work.

#### Specialty section:

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

Received: 31 March 2017 Accepted: 21 June 2017 Published: 13 July 2017

#### Citation:

Shimmura S, Nozoe M, Kitora S, Kin S, Matsutani S, Ishizaki Y, Nakahira Y and Shiina T (2017) Comparative Analysis of Chloroplast psbD Promoters in Terrestrial Plants. Front. Plant Sci. 8:1186. doi: 10.3389/fpls.2017.01186

bacteriophage-type RNA polymerases (NEP) that are encoded by the nuclear genome (Hess and Börner, 1999; Liere et al., 2011; Yagi and Shiina, 2012, 2014; Liebers et al., 2017). The PEP core enzyme consists of four major subunits, designated as α, β, β 0 , and β 00" subunits, which are homologous to bacterial subunits. Another dissociable subunit called a sigma factor allows the core enzyme to initiate transcription from the specific promoters. Multiple sigma factor genes have been identified in embryophytes (Tanaka et al., 1997; Morikawa et al., 1999; Fujiwara et al., 2000; Hara et al., 2001; Kasai et al., 2004; Kubota et al., 2007; Kanazawa et al., 2013), and they play specific roles in transcriptional regulation in response to developmental and/or environmental cues (reviewed by Kanamaru and Tanaka, 2004; Shiina et al., 2005; Schweer et al., 2010; Börner et al., 2015; Chi et al., 2015). Standard PEP-dependent promoters resemble bacterial sigma-70 type promoters containing −10 (TATAAT) and −35 (TTGACA) elements, reflecting their bacterial origin. NEP recognizes distinct types of promoters containing a core YRTA motif (Hess and Börner, 1999; Liere and Maliga, 1999; Liere et al., 2004; Börner et al., 2015). PEP transcribes mainly photosynthesis genes in mature chloroplasts while NEP transcribes housekeeping genes in both chloroplasts and non-photosynthetic plastids (Allison et al., 1996; Hajdukiewicz et al., 1997). The functional coordination of PEP and NEP plays a critical role in plastid differentiation in angiosperms.

In contrast to angiosperms, chloroplasts of the green algae C. reinhardtii harbor a simple transcription system, which is dependent on PEP and a single sigma factor SIG1 (Bohne et al., 2006; Yagi and Shiina, 2014). No NEP has been identified in Chlamydomonas. It is considered that embryophytes have developed complex transcription systems to adapt to marked environmental stresses. However, the evolutionary process of chloroplast transcription systems in embryophytes remains largely elusive.

Most PEP-dependent genes are actively transcribed in green tissues including the leaves. The chloroplast run-on experiments demonstrated that PEP-dependent transcription is activated by high light compared to normal growth light (Baena-González et al., 2001). However, the accumulation of most PEP-dependent transcripts is not regulated by light/dark transitions or environmental stresses, possibly due to the extraordinary stability of their transcripts (Shiina et al., 1998; Hayes et al., 1999). The only exception is a psbD light-responsive promoter designated psbD LRP, which is located upstream of a psbD-psbC operon encoding D2 (PsbD) and CP47 (PsbC) subunits of the PSII reaction center complex (Christopher et al., 1992; Wada et al., 1994; Allison and Maliga, 1995; To et al., 1996; Hoffer and Christopher, 1997). Transcription from the psbD LRP is activated by not only high-irradiance light, but also various abiotic stresses, including salt, high osmolarity and heat (Nagashima et al., 2004) and circadian rhythm (Noordally et al., 2013). The psbD LRP contains unique signature sequences named the AAG-box, immediately upstream of the -35 element (Allison and Maliga, 1995; Kim and Mullet, 1995; To et al., 1996; Nakahira et al., 1998; Kim et al., 1999). The AAG-box is composed of two different repeat units (AAGT and GACC/T repeats). In vitro transcription assays from the psbD LRP revealed that both repeat motifs are important for transcription, but not the −35 element in barley or wheat (Nakahira et al., 1998; Kim et al., 1999). Furthermore, the AAGT repeat interacts with the sequence-specific DNA-binding protein AGF (Kim and Mullet, 1995; Nakahira et al., 1998; Kim et al., 1999). It has also been shown that the stress-responsive plastid sigma factor SIG5 directs the activation of the psbD LRP in Arabidopsis thaliana (Nagashima et al., 2004; Tsunoyama et al., 2004; Onda et al., 2008).

The psbD promoter mapped in Chlamydomonas has a well-conserved −10 element, but lacks the AAG-box and standard −35 element (Klein et al., 1992; Klinkert et al., 2005). In addition, nucleotide sequence comparison of the upstream regions of the psbD among embryophytes suggests that A. thaliana (angiosperm) and Pinus thunbergii (gymnosperm) have the psbD LRP in their genome, but not the other basal embryophytes Physcomitrella patens (moss) and Marchantia polymorpha (liverwort) (Kanazawa et al., 2013). These findings suggest that the psbD LRP may have emerged during the evolution of embryophytes. However, evolution of the psbD promoter remains elusive. In this study, we mapped the promoter region of the psbD-psbC operon in eleven embryophytes at different evolutionary stages from liverwort to angiosperm. The results suggest that AAG-box sequences of the psbD LRP in angiosperms and gymnosperms may have evolved from the partial AAG-box-like sequences detected in the psbD promoters of basic embryophytes such as moss, hornwort, and lycophyte, while monilophytes (ferns) use a distinct type of psbD promoter lacking the AAG-box. On the other hand, lightdependent psbD expression was not observed in gymnosperms or primitive angiosperms that possess the well-conserved AAGbox, suggesting that the AAG-box containing psbD promoter acquisition is unlikely to be associated with the occurrence of light-dependent psbD expression.

### MATERIALS AND METHODS

### Plant Materials and Growth Condition

For primer extension analysis, A. thaliana, Adiantum capillusveneris, P, patens, and M. polymorpha (Tak-1) were grown in the light in growth chambers at 22◦C under 16-h-light/8-hdark (80–100 µmol photons m−2 s −1 ). Other samples (Laurus nobilis, Ginkgo biloba, P. thunbergii, Equisetum hyemale, Psilotum nudum, and Lycopodium clavatum were collected from plants cultivated at Kyoto Botanical Garden. Leaf samples were collected in the daytime, and immediately frozen in liquid N2. Light-induced gene expression analysis was carried out with plants (A. capillus-veneris, C. revoluta, P. thunbergii, L. nobilis, A. thaliana) grown in the growth chambers at 22◦C under 16 h-light/8-h-dark (80 µmol photons m−2 s −1 ). Plants were darkadapted for 72 h, and then exposed to light of 180 µmol photons m−2 s −1 for 4 h in the growth chamber. Collected leaf samples were immediately frozen in liquid N2.

Light-induced gene expression analysis (**Supplementary Figures S4–S8**) was also carried out with a wide range of embryophytes at different evolutionary stages from moss to

angiosperms grown in the growth chambers at 22◦C under 16 h-light/8-h-dark (80 µmol photons m−2 s −1 ). Plants were darkadapted for 72 h (D), and then illuminated (275 µmolm−2 s −1 ) for up to 12 h (L). Osmotic stress was achieved by 250 mM mannitol treatment for 6 h to the detached leaves. Collected leaf samples were immediately frozen in liquid N2.

Arabidopsis thaliana wild-type Columbia ecotype and AtSIG5 overexpressing plants were germinated and grown on two layers of filter paper on the one-half Murashige and Skoog (MS) medium containing 0.8% (w/v) agar at 22◦C with 16-h light (80 µmol photons m−2 s −1 )/8-h dark cycles for 10 days. For salt and high osmotic stress treatments, the seedlings were transferred to one-half MS medium containing 250 mM NaCl or 250 mM mannitol, and incubated under light (80 µmol photons m−2 s −1 ) for 6–24 h. For low temperature treatment, the seedlings were incubated at 4◦C for 6–24 h under light conditions of 50 µmol photons m−2 s −1 . For light response experiments, the seedlings were dark adapted for 72 h (D) and illuminated for 4 h with white light (80 µmol photons m–2 s–1) or a blue LED light (50 µmol photons m−2 s −1 ) or a red LED light (50 µmol photons m−2 s −1 ).

### Transgenic Plants

First strand cDNA of AtSIG5 was synthesized from total RNA prepared from Arabidopsis seedlings using AMV reverse transcriptase (TaKaRa). cDNA was amplified by PCR using KOD-plus-DNA polymerase (TOYOBO) according to the manufacturer's protocols. To obtain an AtSIG5 overexpression construct under the control of CaMV 35S promoter, the GUS gene of the binary vector pBI121 was replaced with the AtSIG5 cDNA. The resulting constructs were introduced into Agrobacterium tumefaciens and used to transform the wild-type (Col-0) plants.

### Total RNA Isolation, Primer Extension Analysis, and Northern Blot Analysis

Total RNA was extracted from the leaves using the RNeasy Plant Mini kit (Qiagen, United States) or TRIZOL R following the manufacturer's instructions. The primer extension assays were performed on the total RNA using the Primer Extension System (Promega) with the AMV reverse transcriptase following the manufacturer's instructions. Primers used are listed in Supplementary Table S1. Primer extension products were analyzed on a 6% polyacrylamide-7 M urea sequencing gel. For northern blot analysis, total RNA samples (2 µg) were separated by denaturing agarose gelelectrophoresis. After capillary blotting onto Hybond-N nylon membrane, RNA gel blots were hybridized to the randomly primed DNA probes for psbA and psbD of each plant. The psbD UTR probe (−1085 to −726 of the psbD translation start codon) was designed to detect specifically the transcripts from the psbD LRP in Arabidopsis. The psbD and psbA coding region probes were designed to detect transcripts produced from all multiple promoters in the psbD-psbC operon. The AtSIG5 probe was also prepared using specific primers. The psbD UTR probes specific for each plant were also generated by using PCR primers.

### RESULTS

### psbD LRP Transcription Is Dependent on SIG5

The psbD LRP is a unique PEP-dependent chloroplast promoter, which is responsible for the transcription of the psbD-psbC operon. The psbD-psbC operon is well-conserved among plants and cyanobacteria. Unlike standard PEP-dependent promoters composed of sigma-70 type −10 (TATAAT) and −35 (TTGACA) elements, it has been shown that psbD LRP activity is dependent on the upstream AAG-box in tobacco (Allison and Maliga, 1995), barley (Kim and Mullet, 1995), rice (To et al., 1996), and wheat (Nakahira et al., 1998; **Figure 1A**). On the other hand, the −35 element of the psbD LRP is not essential for transcription activity (To et al., 1996; Nakahira et al., 1998; Kim et al., 1999; Thum et al., 2001b). These findings suggest that the upstream AAG-box may take over the role of the pseudo −35 element in the psbD LRP.

Multiple promoters have been identified in the psbD-psbC operon (Hoffer and Christopher, 1997). The most upstream promoter (∼950 of the psbD translation start site) is a so-called psbD LRP that is specifically activated by blue light. To identify specifically mRNAs transcribed from the psbD LRP, we used a psbD UTR probe (−1085 to −726 of the psbD translation start codon) that is designed to be located upstream of the second promoter at −550 (Tsunoyama et al., 2004).

As shown in **Figure 1B**, the 4.5- and 3.7-kb transcripts from the psbD LRP were specifically detected by the psbD LRP UTR probe. As reported by Nagashima et al. (2004), various abiotic stresses including salt, cold, and hyperosmotic stresses induce transcription at the psbD LRP in a time-dependent manner (**Figure 1B**). Similarly, SIG5 transcription is activated by abiotic stresses. Previous reports (Nagashima et al., 2004; Tsunoyama et al., 2004) demonstrated that psbD LRP activity is abolished in AtSIG5-deficient mutants in Arabidopsis. In order to further define the role of AtSIG5 in transcription at the psbD LRP, we developed SIG5 overexpression lines (SIG5oxA and SIG5oxH) and examined transcription activity from the psbD LRP. The accumulation of psbD LRP transcripts was significantly increased by the overexpression of AtSIG5 in illuminated plants irrespective of the presence of white, blue, and red light, but only slightly in the dark (**Figure 1C**). These results clearly demonstrate that SIG5 specifically mediates transcription from the psbD LRP in the light. Photoreceptors including CRY1, CRY2, and PhyA have been shown to be involved in the light-induced expression of the psbD-psbC operon in Arabidopsis (Thum et al., 2001a), while AtSIG5 overexpression cannot activate transcription from the psbD LRP in the dark (**Figure 1C**). Taken together, photoreceptor-mediated signaling may modify SIG5 or SIG5 import in a light-dependent manner and activate transcription at the psbD LRP.

#### psbD Transcripts have Markedly Long 5 0 -UTRs in Common

Transcription initiation sites of the psbD-psbC operon have only been identified in some angiosperm model plants, including barley, wheat, rice, tobacco, and Arabidopsis (Yao et al., 1989;

Christopher et al., 1992; Wada et al., 1994; To et al., 1996; Hoffer and Christopher, 1997), and green algae Chlamydomonas reinhardtii (Klein et al., 1992). In order to address the evolutionary changes of psbD promoter structures, we mapped 5 0 -ends of psbD transcripts of eleven embryophytes at different evolutionary stages from liverworts to angiosperms using primer extension analysis (**Figure 2** and **Supplementary Figure S1**, **S2**). Leaf samples were collected from plants grown in Kyoto Botanical Garden in the daytime, except for A. thaliana, A. capillus-veneris, P. patens, and M. polymorpha that were grown in the light in growth chambers. We estimated the size of primer extension products approximately by comparing their mobility profiles to single-strand DNA ladders. In order to determine the start sites as exactly as possible, we designed appropriate primers that produce primer extension products shorter than 300 bases. Next we searched for sequences similar to the conserved −10 sequences (TATTCT) of the psbD LRP in close proximity to the identified transcription initiation sites. Then, we aligned the deduced psbD promoter sequences with those of other plants using the −10 element as reference. In this study, we considered the psbD transcripts with the most upstream terminus as the primary transcripts, except for P. thunbergii and E. hyemale, which have another promoter upstream of the potential psbD LRP.

Transcription initiation sites from the psbD LRP have been mapped at 572, 610, 566, 905, and 948 bp upstream of the psbD translation start site of barley (Christopher et al., 1992), wheat (Wada et al., 1994), rice (To et al., 1996), tobacco (Yao et al., 1989), and Arabidopsis (Hoffer and Christopher, 1997), respectively. Similarly, 50 -ends of the psbD primary transcripts of the psbD-psbC operon were mapped at 800–900 bp upstream of the psbD gene in the most angiosperms including the basal angiosperm L. nobilis, gymnosperm P. thunbergii, primitive gymnosperm G. biloba, monilophyte (Leptosporangiate fern) A. capillus-veneris, monilophyte (Eusporangiate fern) P. nudum, monilophyte (Eusporangiate fern) E. hyemale, and lycophyte Huperzia lucidula (**Figure 3**). Furthermore, we found sequences similar to the psbD promoter of H. lucidula at the far upstream position (−919) of the psbD translation start site in Anthoceros formosae (hornwort). On the other hand, 50 -ends of the longest psbD transcripts of other bryophytes, P. patens (moss), and M. polymorpha (liverwort) are located at −246 and −243 of the psbD gene, respectively. These results indicate that psbD mRNAs have unusually long 50 -UTR extensions in common, except for mosses and liverworts. It is of note that intergenic distances between psbD and the upstream trnT are much shorter in mosses and liverworts compared with those of other plants.

### AAG-Box of the psbD LRP Is Highly Conserved among Angiosperms and Gymnosperms, and Partially Conserved in Lycophytes and Mosses

Next, we compared sequences immediately upstream of the psbD transcription initiation sites that were mapped in this and previous studies. The typical −10 elements (TATTCT) are wellconserved in all psbD promoters (**Figure 4** and Supplementary Table S2). Conversely, the potential −35 elements are less conserved among terrestrial plants and show weak similarity (less than ∼50%) to the consensus sequences (TTGACA). On the other hand, liverworts possess a typical sigma-70 type promoter with conserved −35 and −10 elements with 18-nt spacing. As expected, the AAG-box is well-conserved among angiosperms and gymnosperms. The consensus sequence of the AAG-box is "RAAGTAAGTRRACCTRACYY," which contains an AAGT

are shown as numbers in parentheses. Lane C shows an experiment with the control RNA and primer provided by the manufacturer that produces an 87-base

repeat and a GACC/T repeat. The AAG-box sequences are almost 80% identical in most gymnosperms and angiosperms, including the primitive gymnosperm G. biloba and basal angiosperm

L. nobilis (Supplementary Table S2). We found that a 13 bp core sequence of the AAG-box is also highly conserved (∼85%) in lycophytes and hornworts, and partially conserved

primer extension product.

arrows. The psbD LRP-related promoters analyzed in this study are indicated by red shadows.

in mosses (69%), but not in liverworts. The AAG-box of lycophytes and hornworts harbors the GACC/T repeat-like sequences, but lacks the AAGT repeat (**Figure 4B**). On the other hand, neither the AAGT repeat nor GACC/T repeat are conserved in the psbD promoter of monilophytes (ferns). Instead, sequences upstream of the −35 element are wellconserved among standard monilophytes and P. nudum, but not in the primitive monilophyte E. hyemale (**Figure 4B** and **Supplementary Figure S3**). These results suggest that the AAGbox was acquired at a very early stage of embryophyte evolution, and is likely conserved in gymnosperms and angiosperms. On the other hand, monilophytes may have acquired another type of psbD promoter with a distinct cis element upstream of the −35 element.

### The AAG-Box Containing psbD Promoter Is not Associated with Light-Induced psbD Expression

In order to address whether the psbD LRP is responsible for light-induced transcription, we examined the light-induced expression of psbD transcripts in some embryophytes, including monilophytes, gymnosperms, and angiosperms. As shown in **Figure 5A**, psbD expression is clearly induced by light in A. thaliana (angiosperm). However, unexpectedly, no lightinduced psbD expression was detected in L. nobilis (basal angiosperm), P. thunbergii (gymnosperm), or Cycas revoluta (primitive gymnosperm), although they all have a well-conserved AAG-box containing psbD promoter. Similarly, psbD expression

is also not regulated by light in A. capillus-veneris (monilophyte), which does not have the conserved AAG-box in the psbD promoter. We further examined the light-mediated regulation of the AAG-box containing psbD promoter transcripts by primer extension analysis. As shown in **Figure 5B**, the abundance of the transcripts from the AAG-box containing psbD promoter was not regulated by light in L. nobilis or P. thunbergii (**Figure 5B**). These results suggest that the AAG-box containing psbD promoter is not directly associated with light-induced psbD expression.

Moreover, we examined light- and osmotic stress-induced psbD expression in a wide range of plants. In angiosperms, light-induced psbD expression was detected in a number of eudicots (C. sativus, A. thaliana, and L. sativa) and monocots (wheat and maize), whereas psbD expression is not regulated by light in basal angiosperms except for C. glaber (**Supplementary Figures S4–S6**). It is to be noted that psbD expression is activated by long-term illumination (12 h) in C. glaber (**Supplementary Figure S6**). In contrast, the osmotic stress-induced expression of psbD was detected only in eudicots. Expression of psbD was not activated by osmotic stress in monocots and basal angiosperms except for C. glaber (**Supplementary Figure S5**, **S6**). Furthermore, neither light nor osmotic stress-induced psbD expression was detected in gymnosperms, Gingko and Cycas (**Supplementary Figure S6**). Although psbD expression was not activated by moderate light (180 µmolm−2 s −1 ) in Pinus (**Figure 5**), high light exposure (245 µmolm−2 s −1 ) induced transient expression of psbD (**Supplementary Figure S6**). It is of note that gymnosperms and basal angiosperms have a wellconserved AAG-box containing psbD promoter. On the other hand, psbD expression is induced by light and/or osmotic stress in some monilophytes that lack the typical AAG-box containing psbD promoter. In addition, light and salt stress barely affect psbA expression in any of the plants examined (**Supplementary Figures S5–S8**). These results indicate that light and salt stressinduced transcription has evolved independently and multiple times during land plant evolution. Furthermore, the AAG-box in the psbD promoter is unlikely to be directly associated with the occurrence of light and salt stress-induced psbD expression.

### Gnetaceae Plants in Gymnosperms Have Lost the AAG-Box Containing psbD Promoter

Gnetaceae plants are a unique group of gymnosperms that have evolved a morphological system related to that of the angiosperms. The most upstream transcription initiation sites have been mapped at 317 and 114 bp upstream of the psbD translation start site of Gnetum gnemon and Ephedra sinica, respectively (**Figures 6A,B**). The psbD transcripts of gnetaceae have shorter 50 -UTR compared with the standard psbD transcripts in other plants. The upstream sequences of the psbD transcripts are well-conserved among gnetaceae. No AAGbox-like sequences are found upstream of the psbD transcripts in gnetaceae, whereas the gnetaceae psbD promoters possess −35- and −10-like sequences (**Figure 6C**). The similar sequences

are also found upstream of the psbD-C operon of Welwitschia mirabilis. Moreover, AAG-box-like sequences have not been identified in the trnT-psbD intergenic regions of gnetaceae. We suggest that the AAG-box containing psbD promoter was lost in gnetaceae during their evolution. It would be very interesting to determine whether psbD expression is dependent on light in gnetaceaes.

### DISCUSSION

The psbD and psbC genes are organized in a psbD-psbC operon, which is well-conserved among plants and cyanobacteria. Transcription from the psbD-psbC operon is mediated solely by PEP in green tissues. Multiple transcriptional start sites (TSS) generating mRNAs with heterogeneous 50 transcript leaders have been mapped on the psbD-psbC operon in several angiosperm plants, including tobacco (Yao et al., 1989), barley (Christopher et al., 1992), wheat (Wada et al., 1994), rice (To et al., 1996), and Arabidopsis (Hoffer and Christopher, 1997). Light activates the expression of some psbD-psbC mRNAs, whereas the accumulation of other mRNAs is not regulated by light. Analysis of the promoter sequences immediately upstream of the light-induced TSS identified an unusual PEP promoter consisting of a core promoter with a weakly conserved −35 element along with an upstream cis element termed the AAG-box (**Figure 1A**). This promoter is specifically activated by highirradiance blue light and UV-A (Christopher and Mullet, 1994), and is designated as the psbD light-responsive promoter (psbD LRP) or psbD blue light-responsive promoter (psbD BLRP). As shown in **Figure 1B**, the psbD LRP is also activated by various environmental stresses (Nagashima et al., 2004). On the other hand, other light-insensitive promoters mapped on the psbD-psbC operon are standard PEP promoters composed of well-conserved −10 and −35 elements.

In order to investigate psbD LRP evolution, we mapped promoters responsible for the expression of the psbD-psbC operon by primer extension analysis in 11 embryophytes at different evolutionary stages from liverworts to angiosperms. We considered the psbD transcripts with the most upstream terminus as the primary transcripts, except for P. thunbergii and E. hyemale, which have another promoter upstream of the potential AAG-box containing psbD promoter. All psbD promoters identified at the most upstream mRNA terminus

have the well-conserved −10 element (TATTCT) that is similar to the standard −10 element (TATAAT). On the other hand, the potential −35 elements of the psbD promoters are less conserved, suggesting the limited role of the −35 element in psbD promoter activity. In vitro transcription experiments have demonstrated that the −10 element is important for transcription from the AAG-box containing psbD promoter, but the −35 element is not essential for transcription in barley, rice, or wheat (To et al., 1996; Nakahira et al., 1998; Kim et al., 1999). It is considered that the −10 element is important for psbD promoter activity, but not the poorly conserved −35 element.

We found that the 19 bp AAG-box sequences of the psbD promoters are highly conserved among gymnosperms and angiosperms, including the basal angiosperm L. nobilis and primitive gymnosperm G. biloba. The consensus AAGbox sequence is "RAAGTAAGTRRACCTRACY," which is at least 80% identical in gymnosperms and angiosperms. The AAG-box is composed of two repeat sequences: AAGT and GACC/T repeats. Extensive analyses of the AAG-box containing psbD promoter structure using in vitro transcription systems have revealed that both the AAGT and GACC/T repeats are important for AAG-box containing psbD promoter activity (Kim and Mullet, 1995; To et al., 1996; Nakahira et al., 1998). Moreover, the AAG-box is also partially conserved in basal land plants such as lycophytes, hornworts, and mosses. We identified the shorter conserved AAG-box-like sequences in H. lucidula (lycophyte) and A. formosae (hornwort). The AAG-box-like sequences in lycophyta and hornworts retain a partially conserved GACC/T repeat, but lack AAGT repeats. Deletion of the AAGT repeat resulted in only a partial reduction of in vitro transcription activity of the AAG-box containing psbD promoter in wheat, suggesting that the GACC/T repeat is sufficient to mediate AAG-box containing psbD promoter activity (Nakahira et al., 1998). Thus, the AAG-box-like sequences may act as a transcription activation element in the psbD promoters of basal land plants. Considering the highly conserved −10 element among bryophytes and angiosperms, it is likely that the last common ancestor of bryophytes and spermatophytes likely already possessed an AAG box-containing psbD LRP (**Supplementary Figure S9**). The AAG box may have developed to take over the function of the −35 element and support the highlevel transcription activity of the AAG-box containing psbD promoter.

On the other hand, the AAG-box like sequences have not been identified in the psbD promoter of M. polymorpha (liverwort). The liverwort psbD promoter is composed of the typical −35 element (TTGAAA) and the −10 element (TATTCT) with standard spacing, suggesting that psbD is transcribed from a standard PEP promoter in liverworts. It is to be noted that the psbD promoter has a well-conserved −10 element, but lacks the AAG-box and standard −35 element in Chlamydomonas (Klein et al., 1992; Klinkert et al., 2005).

Monilophytes (ferns) have another type of psbD promoter that lacks the conserved AAG-box. Instead, 11-bp sequences upstream of the potential −35 element are well-conserved among standard (A. capillus-veneris) and primitive (P. nudum) monilophytes. However, it remains elusive whether the conserved sequences upstream of the psbD transcription stat site in monilophytes is required for psbD transcription. Interestingly, gnetaceae plants in gymnosperms have lost the AAG-boxcontaining psbD LRP, suggesting that the AAG-box containing psbD promoter is not essential for plant development.

It has also been shown that an AAG-box-binding factor (AGF) specifically binds to the AAGT repeat, that it is partially associated with the GACC/T repeats (Kim and Mullet, 1995; Kim et al., 1999), and that it activates transcription from the AAG-box containing psbD promoter (Wada et al., 1994; Kim and Mullet, 1995; Allison and Maliga, 1995; To et al., 1996; Nakahira et al., 1998; Kim et al., 1999). PTF1 (plastid transcription factor 1) is a basic helix-loop-helix DNA-binding protein, which binds to the AAG box and is involved in transcription from the psbD LRP in Arabidopsis (Baba et al., 2001). However, close orthologs of the Arabidopsis PTF1 have only been found in angiosperms, suggesting that PTF1 is responsible for AAG-box-dependent transcription at the AAG-box containing psbD promoter in angiosperms. However, the role of AGF in the light-dependent transcription remains to be elucidated.

In addition, reverse genetic analysis of sigma factors revealed that the AAG-box containing psbD promoter is specifically recognized by SIG5 in Arabidopsis (Nagashima et al., 2004; Tsunoyama et al., 2004). Transcription from the AAG-box containing psbD promoter is likely to be mediated by SIG5 containing PEP and activated by AGF that binds to the AAG-box. However, overexpression of SIG5 cannot activate transcription from the AAG-box containing psbD promoter in the dark (**Figure 1C**). Furthermore, photoreceptors including CRY1, CRY2, and PhyA are involved in the light-induced expression of the psbD-psbC operon in Arabidopsis (Thum et al., 2001a). Taken together, photoreceptor-mediated signaling may modify SIG5 or SIG5 import into the chloroplasts in a light-dependent manner and activate transcription at the AAG-box containing psbD promoter.

SIG5 orthologs have been identified in a number of angiosperms. Moreover, SIG5 has been identified as a gene that was abundant when water availability was low in gymnosperm Pseudotsuga menziesii (Hess et al., 2016). Furthermore, SIG5 orthologs have been identified in P. patens (Ichikawa et al., 2008) and Selaginella moellendorffii (XP\_002970534). It has been shown that PpSIG5 is involved in high-intensity light and circadian control of psbD expression in the moss P. patens (Ichikawa et al., 2004, 2008). Taken together, these results suggest that SIG5 plays a role in transcription from the psbD promoter consisting of the AAG-box or AAG-like element in embryophytes including mosses. On the other hand, an SIG5 ortholog was also identified in the liverwort M. polymorpha (Kanazawa et al., 2013). MpSIG5 is not necessary for light-dependent psbD expression in M. polymorpha (Kanazawa et al., 2013). MpSIG5 may have another role in chloroplast transcription in M. polymorpha that lacks the AAG-box containing psbD promoter. Furthermore, SIG5 orthologs have been identified in some charophytes such as K. flaccidum, but not in the green alga C. reinhardtii or in the primitive red alga C. merolae. It is likely that SIG5 was acquired in charophytes before the occurrence of AAG-box containing psbD promoter in basal embryophytes.

Transcripts from the AAG-box containing psbD promoter are characterized by a markedly long 50 -UTR. All AAG-box containing psbD promoter transcripts except for P. patens and M. polymorpha have a 50 -UTR longer than 500 nucleotides from the translation start site. Detailed mapping of the psbD transcripts in angiosperm plants revealed that no intron is present in the 5 0 -UTR and the AAG-box containing psbD promoter transcripts actually have a long 50 -UTR (Wada et al., 1994; Hoffer and Christopher, 1997; Kim et al., 1999). The 50 -UTR of chloroplast transcripts may be involved in the stability of the transcripts and/or efficiency of translation (Shiina et al., 1998). However, no conserved sequences have been identified in the 50 -UTR of embryophytes. Further characterization of the 50 -UTR would shed light on the role of the unusually long 50 -UTR of the AAG-box containing psbD promoter in most embryophytes,

This study suggest that the AAG-box containing psbD promoter appeared in basal embryophytes more than 450 million years ago, and the common ancestor of bryophytes and spermatophytes likely possessed an AAG box-containing psbD promoter. On the other hand, the −35 and −10 elements of the psbA and rbcL promoters are almost identical among liverworts and angiosperms (**Supplementary Figure S10**). It is suggested that ecological and/or physiological demands may have accelerated the evolution of the AAG-box containing psbD promoter in embryophytes. One of the unique characteristics of the AAG-box containing psbD promoter is light- and stressinduced transcription. However, extensive expression analysis of psbD in a variety of plants revealed that the light and/or stressinduced expression of the psbD gene developed independently in several plants. Thus, it is unlikely that that light and/or stress responses of the AAG-box containing psbD promoter are directly associated with AAG-box containing psbD promoter evolution. Recent studies demonstrated that ABA and the circadian rhythm regulate chloroplast AAG-box containing psbD promoter activity via the activation of SIG5 (Noordally et al., 2013; Yamburenko et al., 2015; Belbin et al., 2017) Further analysis of the role of the AAG-box containing psbD promoter in chloroplasts may shed light on AAG-box containing psbD promoter evolution.

### AUTHOR CONTRIBUTIONS

TS, SS, SM, and YN designed research. SS, MN, ShK, SaK, and YI performed research. TS, SS, and MN analyzed data. TS, SS, and MN wrote the paper. SS and MN have contributed equally to this work.

### FUNDING

This work was supported by JSPS and MEXT Grants- in-Aid for Scientific Research to TS (25291065, 15K14553, 15H01236, 17H05728, and 17H03968) and YN (15K14912), a grant from the Mitsubishi Foundation to TS, and a grant for high-priority study from Kyoto Prefectural University.

### ACKNOWLEDGMENTS

fpls-08-01186 July 11, 2017 Time: 15:44 # 11

We acknowledge Kyoto Botanical Garden for co-operation for plant sample collection. We also thank Dr. H. Tobe and Dr. M. H. Sato for helpful discussion.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.01186/ full#supplementary-material

FIGURE S1 | Mapping of the 50 ends of the psbD transcripts psbD transcripts were isolated from liverwort M. polymorpha (A), lycophyte H. lucidula (B), and monilophyte P. nudum (C). Primers used are indicated by numbers on the top of each lane. The size of the extension product is shown on the right. The position of primers and the size of the extension products are shown on the gene map. The deduced sites of the 50 -end of each transcript are shown as numbers in parentheses. Lane Xs show the results of unrelated samples. Lane C shows an experiment with the control RNA and primer provided by the manufacturer that produces an 87-base primer extension product.

FIGURE S2 | Mapping of the 5' ends of the psbD transcripts psbD transcripts were isolated from monilophyte E. hyemale (A), gymnosperm P. thunbergii (B), and angiosperm L. nobilis (C). Primers used are indicated by numbers on the top of each lane. The size of the extension product is shown on the left in (A) and on the right in (B,C). The position of primers and the size of the extension products are shown on the gene map. The deduced sites of the 50 -end of each transcript are shown as numbers in parentheses. Lane C shows an experiment with the control RNA and primer provided by the manufacturer that produces an 87-base primer extension product.

FIGURE S3 | A comparison of the potential psbD promoter sequences in monilophytes DNA sequences upstream of the psbD transcription initiation site of A. capillus-veneris and corresponding sequences of three other leptosporangiate ferns are compared with the psbD promoter sequences of two primitive ferns

### REFERENCES

(P. nudum and E. hyemale). Transcription initiation sites identified in this study are indicated by asterisks. Nucleotides that are identical to the Adiantum sequences are shown in red. The monilophytes-type upstream conserved sequences are indicated by a green box. The −35 and −10 elements are indicated by orange and yellow boxes, respectively.

FIGURE S4 | Northern blot analysis of psbD and psbA transcripts in eudicots. Plants were dark-adapted for 72 h (D) and illuminated for up to12 h (275 µmolm−2 s −1 ; L). Osmotic stress was applied by 250 mM mannitol for 6 h. Light- or salt stress-induced psbD transcripts are indicated by red arrow heads. rRNA stained with EtBr was used as an RNA-loading control for the total RNA sample.

FIGURE S5 | Northern blot analysis of psbD and psbA transcripts in monocots. Plants were dark-adapted for 72 h (D) and illuminated for up to12 h (275 µmolm−2 s −1 ; L). Osmotic stress was applied by 250 mM mannitol for 6 h. Light- or salt stress-induced psbD transcripts are indicated by red arrow heads. rRNA stained with EtBr was used as an RNA-loading control for the total RNA sample.

FIGURE S6 | Northern blot analysis of psbD and psbA transcripts in basal angiosperms. Plants were dark-adapted for 72 h (D) and illuminated for 12 h (275 µmolm−2 s −1 ; L). Osmotic stress was applied by 250 mM mannitol for 6 h. Light- or salt stress-induced psbD transcripts are indicated by red arrow heads. rRNA stained with EtBr was used as an RNA-loading control for the total RNA sample.

FIGURE S7 | Northern blot analysis of psbD and psbA transcripts in gymnosperms. Plants were dark-adapted for 72 h (D) and illuminated for up to 12 h (275 µmolm−2 s −1 ; L). Osmotic stress was applied by 250 mM mannitol for 6 h. Light- or salt stress-induced psbD transcripts are indicated by red arrow heads. rRNA stained with EtBr was used as an RNA-loading control for the total RNA sample.

FIGURE S8 | Northern blot analysis of psbD and psbA transcripts in monilophytes. Plants were dark-adapted for 72 h (D) and illuminated for up to 12 h (275 µmolm−2 s −1 ; L). Osmotic stress was applied by 250 mM mannitol for 6 h. Light- or salt stress-induced psbD transcripts are indicated by red arrow heads. rRNA stained with EtBr was used as an RNA-loading control for the total RNA sample.

FIGURE S9 | Phylogenetic tree of AAG box. Alignments were undertaken with 19 bps sequences (from −54 to −36 of the transcription initiation site) of 18 plants from C. reinhardtii to A. thaliana.

FIGURE S10 | Comparion of psbA and rbcL promoters of terrestrial plants.

chloroplast transcription. Curr. Genet. 49, 333–340. doi: 10.1007/s00294-006- 0060-7

distinct groups of genes in tobacco plastids. EMBO J. 16, 4041–4048. doi: 10.1093/emboj/16.13.4041

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Shimmura, Nozoe, Kitora, Kin, Matsutani, Ishizaki, Nakahira and Shiina. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Arabidopsis thaliana mTERF10 and mTERF11, but Not mTERF12, Are Involved in the Response to Salt Stress

### Duorong Xu, Dario Leister and Tatjana Kleine\*

Plant Molecular Biology, Department Biology I, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany

Plastid gene expression (PGE) is crucial for plant development and acclimation to various environmental stress conditions. Members of the "mitochondrial transcription termination factor" (mTERF) family, which are present in both metazoans and plants, are involved in organellar gene expression. Arabidopsis thaliana contains 35 mTERF proteins, of which mTERF10, mTERF11, and mTERF12 were previously assigned to the "chloroplast-associated" group. Here, we show that all three are localized to chloroplast nucleoids, which are associated with PGE. Knock-down of MTERF10, MTERF11, or MTERF12 has no overt phenotypic effect under normal growth conditions. However, in silico analysis of MTERF10, -11, and -12 expression levels points to a possible involvement of mTERF10 and mTERF11 in responses to abiotic stress. Exposing mutant lines for 7 days to moderate heat (30◦C) or light stress (400 µmol photons m−2 s −1 ) fails to induce a phenotype in mterf mutant lines. However, growth on MS medium supplemented with NaCl reveals that overexpression of MTERF11 results in higher salt tolerance. Conversely, mterf10 mutants are hypersensitive to salt stress, while plants that modestly overexpress MTERF10 are markedly less susceptible. Furthermore, MTERF10 overexpression leads to enhanced germination and growth on MS medium supplemented with ABA.  Thus, characterization of an increasing number of plant mTERF proteins reveals their roles in the response, tolerance and acclimation to different abiotic stresses.

#### Keywords: Arabidopsis, chloroplast, nucleoid, mTERF, acclimation, stress, salt

### INTRODUCTION

Chloroplasts are of cyanobacterial origin (Raven and Allen, 2003) and harbor nowadays a reduced genome that mainly encodes proteins involved in photosynthesis and plastid gene expression (PGE). PGE is crucial for plant development and photosynthesis, but its regulation is only partially understood. This is largely because, although plastids still display characteristics of a prokaryoticlike structure of their genome, their gene expression machinery is much more elaborated compared to that of their cyanobacterial ancestor (reviewed in: Liere et al., 2011). Therefore, PGE requires plenty of proteins encoded in the nucleus that support transcription, splicing, trimming and editing of organellar RNAs, and regulate their translation (Schmitz-Linneweber and Small, 2008; Stern et al., 2010; Hammani et al., 2014; Tiller and Bock, 2014; Börner et al., 2015).

Edited by:

Julian Eaton-Rye, University of Otago, New Zealand

#### Reviewed by:

Victor Quesada, Universidad Miguel Hernández de Elche, Spain Ren Maozhi, Chongqing University, China

> \*Correspondence: Tatjana Kleine<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Plant Cell Biology, a section of the journal Frontiers in Plant Science

Received: 20 March 2017 Accepted: 27 June 2017 Published: 14 July 2017

#### Citation:

Xu D, Leister D and Kleine T (2017) Arabidopsis thaliana mTERF10 and mTERF11, but Not mTERF12, Are Involved in the Response to Salt Stress. Front. Plant Sci. 8:1213. doi: 10.3389/fpls.2017.01213

Also the nucleus-encoded proteins of the mitochondrial transcription termination factor (mTERF) family regulate mitochondrial and PGE at diverse levels (Kleine and Leister, 2015). The mTERF proteins have been identified in both plants and metazoans (Linder et al., 2005). Human mTERF1, which is the first characterized mTERF, is one of four mammalian mTERF proteins, and was identified nearly 30 years ago as a factor that acts on transcription termination in mitochondrial extracts (Kruse et al., 1989). Its presumptive function as a transcription terminator (of heavy-strand transcripts) gave the family its name. More recently however, models have been suggested in which mTERF1 acts chiefly as a terminator of antisense transcription (Terzioglu et al., 2013) or in polar replication fork arrest (Shi et al., 2016). The true molecular function of mouse mTERF2 also remains unclear, with some reports suggesting that it binds to the same mitochondrial DNA region as mTERF1 and mTERF3 (Wenz et al., 2009), while another contends that the DNAbinding activity of mTERF2 is not sequence-specific (Pellegrini et al., 2009). Knock-out of Mterf3 in mice leads to embryonic lethality (Park et al., 2007), and conditional knockout of Mterf3 in the heart has identified a novel role for its protein product in the biogenesis of metazoan mitochondrial ribosomes (Wredenberg et al., 2013). Mterf4 knock-out mice are also embryonic lethal (Camara et al., 2011). Interestingly, human mTERF4 forms a complex with NSUN4, which is required for assembly of the small and large ribosomal subunits of the mitochondrial ribosome (Metodiev et al., 2014). Consequently, while the function for mTERF2 remains to be clarified, the remainder of the mammalian mTERFs do not support transcription termination, as it is suggested by their notation, but seem to take part in antisense transcription termination and ribosome biogenesis.

The number of mTERF family members has increased to approximately 30 throughout the evolution of land plants (Kleine, 2012), but information on their functions is only beginning to emerge. Most of the 35 A. thaliana mTERF proteins (mTERF1-mTERF35; Kleine, 2012) are localized to chloroplasts and/or mitochondria (Babiychuk et al., 2011), and seven of them (mTERF1, -4, -5, -6, -9, -15, and -18) have been functionally investigated in more detail (reviewed in: Kleine and Leister, 2015; Quesada, 2016). Essential functions of mTERF proteins in plant development are revealed by the effects of complete inactivation of three MTERF genes: A. thaliana mutants devoid of SOLDAT10 (SINGLET OXYGEN-LINKED DEATH ACTIVATOR10)/mTERF1 (Meskauskiene et al., 2009) or BSM (BELAYA SMERT)/RUG2 (RUGOSA2)/mTERF4 (Babiychuk et al., 2011; Quesada et al., 2011) are arrested in embryo development, and knock-out mterf6-2 plants are albinotic and stop growing after 2 weeks (Romani et al., 2015). Moreover, the dissection of mterf mutants supports an involvement of plant mTERFs in responses to abiotic stress (reviewed in: Kleine and Leister, 2015; Quesada, 2016). Indeed, SOLDAT10 (Meskauskiene et al., 2009) and SUPPRESSOR OF hot1– 4 1 (SHOT1; Kim et al., 2012) were isolated in forward genetic screens for loci that influence responses to abiotic stress. The hot1-4 mutant is a dominant-negative allele of the heat-shock protein gene HSP101. SHOT1/mTERF18 is a mitochondrial protein and the shot1-1 missense mutant and the shot1-2 T-DNA insertion mutant each suppress the heat hypersensitivity of hot1-4 plants. Moreover, other heat-sensitive mutant phenotypes are also suppressed by shot1-2, and shot1- 2 single mutants display a higher heat tolerance (Kim et al., 2012). SOLDAT10 is localized to chloroplasts, and plants homozygous for a weaker soldat10 allele suffer from mild photooxidative stress already in low-light conditions; this results in turn in a stress acclimation response, which appears to confer improved hardiness against a combination of high-light and low-temperature stress (Meskauskiene et al., 2009). Other mterf mutants are also linked to stress responses. For example, mda1 (mterf5), and mterf9 seedlings are less susceptible to osmotic and salt treatments, which might be linked to their decreased sensibility to ABA (Robles et al., 2012, 2015). Furthermore, the rug2-1 mutant is abnormally sensitive to temperature stress. At 26◦C, rug2-1 homozygotes undergo growth arrest, whereas at 16◦C this growth phenotype is not expressed (Quesada et al., 2011).

A co-expression network for all MTERF genes (26 out of 35) which were present on the Affymetrix ATH1 genome array has been constructed (Kleine, 2012). The resulting clusters and information related to the subcellular locations of the proteins that are encoded by genes co-expressed with each MTERF gene were then used to assign the mTERFs into five groups, referred to as the "chloroplast," "chloroplast-associated," "mitochondrial," "mitochondrion-associated," and the "low expression" clusters.

In the present study, we characterized the members of the "chloroplast-associated" group, which comprises mTERF10 (AT2G34620), mTERF11 (AT3G18870), and mTERF12 (AT4G09620). The sub-chloroplast localization of mTERF10, -11, and -12 was defined by fluorescence microscopy of mTERF-GFP fusions and an RFP fusion protein (as a control for nucleoid localization). Lines with altered MTERF10, MTERF11, and MTERF12 levels did not display phenotypes under normal growth conditions. In silico analyses with the eFP browser and Genevestigator were conducted, which pointed to an involvement of these three mTERFs in abiotic stress responses. To follow this up, the mutant lines were exposed to moderate heat (30◦C), high light (400 µmol photons m−2 s −1 ), or salt (175 mM NaCl) stress, and subjected to ABA treatment. The mterf10, -11, and -12 mutant lines responded to heat and high light stress like the wild type (WT). However, lack of mTERF10 or mTERF11 led to enhanced or reduced sensitivity to salt, respectively, while overexpression of MTERF10 rendered seedlings more tolerant than WT to both salt and ABA.

### MATERIALS AND METHODS

### Plant Material and Growth Conditions

The mutants mterf10-1 (SAIL\_12A03), mterf10-2 (SALK\_097699), mterf11-1 (FLAG\_357F09), mterf11-2 (GABI\_211D05), and mterf12-1 (GABI\_407E04) were identified in the SIGnAL database (Alonso et al., 2003), the abi4-1 mutant was ordered from The European Arabidopsis Stock Centre (NASC; ID N8104). All mutants are in the Col-0 background except of mterf11-1 which is a WS line.

Arabidopsis thaliana plants were grown in long-day conditions (16 h light/8 h dark) on potting soil (Stender). Plants were illuminated with HQI Powerstar 400 W/D lamps and a fluence rate of approximately 100 µmol photons m−2 s −1 . To accomplish salt and ABA stress experiments, seedlings were grown on plant agar (Sigma-Aldrich) containing half-strength MS medium and 1.5% (w/v) sucrose at 22◦C under 100 µmol photons m−2 s −1 provided by white fluorescent lamps under continuous light or long-day conditions. For salt stress experiments, MS medium was supplemented with 125 mM or 175 mM NaCl as indicated. For ABA experiments, MS medium was supplemented with 1 µM ABA.

### Nucleic Acid Extraction

For DNA isolation, leaf tissue was homogenized in extraction buffer containing 200 mM Tris/HCl (pH 7.5), 25 mM NaCl, 25 mM EDTA, and 0.5% (w/v) SDS. After centrifugation, DNA was precipitated by adding isopropyl alcohol. After washing with 70% (v/v) ethanol, the DNA was dissolved in distilled water.

For RNA isolation, frozen tissue was ground in liquid nitrogen. Following the addition of TRIZOL (Invitrogen) and chloroform according to the manufacturer's instructions, RNA was precipitated from the aqueous phase with isopropyl alcohol, then washed with 70% (v/v) ethanol, and dissolved in RNasefree water. Concentration and purity of RNA samples were determined spectroscopically in a GeneQuant pro RNA/DNA Calculator (GE Healthcare Europe GmbH). Isolated RNA was stored at −80◦C until further use.

### cDNA Synthesis and Real-Time PCR Analysis

cDNA synthesis and real-time PCR analysis were performed as outlined before (Voigt et al., 2010). All reactions were done in triplicate on three biological replicates. The target genes and the respective primers, are listed in **Supplementary Table S1**. The RCE1 gene was used as an internal reference in other studies (Voigt et al., 2010; Romani et al., 2015). RCE1 transcript levels are not changed upon diverse conditions, especially not under diverse stress conditions including lincomycin and norflurazon treatment which affect organellar gene expression.

### RNAi, Overexpression and Intracellular Protein Localization

To reduce MTERF12 mRNA levels by RNAi, a 145 bp fragment was amplified from genomic DNA with the primer pair AT4G09620-GST-attB1 and –attB2 (see **Supplementary Table S1**). The gel-purified PCR product was used for BP and LR Clonase reactions (GATEWAY Cloning; Invitrogen) which led to the final construct pB7GWIWG2/MTERF12 (for pB7GWIWG2, see Karimi et al., 2002). For overexpression and localization studies of mTERF10, mTERF11 and mTERF12, cDNAs encompassing the coding regions were amplified by PCR (see **Supplementary Table S1** for primer information). Notably, in our Col-0 strain, MTERF11 has an additional triplet (CAT; coding for histidine) inserted after nucleotide 27 (relative to the start codon) compared with the coding sequence from The Arabidopsis Information Resource (TAIR; www. arabidopsis.org). MTERF10, MTERF11, and MTERF12 were cloned by GATEWAY technology (see above) into pB7FWG2 to generate fusions with enhanced GFP (eGFP), expression of which is controlled by the Cauliflower mosaic virus 35S promoter. For RAP-RFP fusions, the pENTR/RAP plasmid (Prof. Jörg Nickelsen, LMU Munich) was introduced into p2GWR7 by GATEWAY cloning. For overexpression of mTERF10, MTERF10 was introduced by classical cloning with the NcoI restriction enzyme into pCAMBIA1302. For RNAi experiments with MTERF12 and overexpression of mTERF10 and mTERF11, the plasmids pB7GWIWG2/MTERF12, pCAMBIA1302/MTERF10, and pB7FWG2/MTERF11 were independently transferred into Agrobacterium tumefaciens, and A. thaliana (ecotype Col-0 for MTERF10 overexpression and MTERF12 RNAi; ecotype WS for MTERF11 overexpression) plants were transformed by the floral-dip method (Clough and Bent, 1998). After seed set, transgenic plants were selected on the basis of their resistance to BASTA (pB7GWIWG2/MTERF12 and pB7FWG2/MTERF11) or hygromycin (pCAMBIA1302/MTERF10), respectively.

For fluorescence visualization, leaves of 3-week-old Col-0 plants grown on MS medium were cut into small pieces and incubated for 16 h at 24◦C in the dark in a protoplasting solution (10 mM MES, 20 mM CaCl2, 0.5 M mannitol (pH 5.8), 0.1 g ml−1 macerozyme (Duchefa), 0.1 g ml−1 cellulase (Duchefa). After isolation and transformation of protoplasts as described (Dovzhenko et al., 2003), preparations were examined with a Fluorescence Axio Imager microscope (Zeiss). Fluorescence was excited with the X-Cite Series 120 fluorescence lamp (EXFO) and images were collected at 500–550 nm (eGFP fluorescence), 570– 640 nm (RFP fluorescence) and 670–750 nm (chlorophyll autofluorescence).

### Chlorophyll a Fluorescence Measurements

In vivo chlorophyll a fluorescence of whole plants was recorded using an imaging chlorophyll fluorometer (ImagingPAM, Walz GmbH, Effeltrich, Germany). Plants were dark adapted for 15 min and then exposed to a pulsed, blue measuring light (1 Hz, intensity 4) and a saturating light flash (intensity 5) to determine the maximum fluorescence Fm and the ratio (Fm-F0)/Fm = Fv/Fm.

### Computational Analyses

Protein sequences were retrieved from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih. gov/) and The Arabidopsis Information Resource (TAIR; http:// www.arabidopsis.org). Amino acid sequences were aligned using the ClustalW program (http://www.ebi.ac.uk/clustalw; Chenna et al., 2003). The unrooted tree was constructed with the Phylip server Mobyle at the Pasteur Institute (http://mobyle.pasteur.fr/ cgi-bin/portal.py#welcome).

### RESULTS

### All Members of the Chloroplast-Associated mTERF Cluster Are Localized to Nucleoids

The localizations of almost all A. thaliana mTERF proteins have been investigated by fluorescence microscopy of mTERF-GFP fusions transiently expressed in isolated protoplasts, and in guard cells of transgenic plants (Babiychuk et al., 2011). These data indicated that mTERF10, -11, and -12 are targeted to chloroplasts. To confirm these results and if possible define the precise locations of the proteins within the chloroplast, the eGFP fluorescence of mTERF10-, mTERF11-, or mTERF12-eGFP fusions, transiently overexpressed in Col-0 protoplasts, was monitored. Localization of all three fusion proteins to chloroplasts was confirmed (**Figure 1A**). However, the fluorescence signals were not uniformly distributed, but

FIGURE 1 | Localization of mTERF10, mTERF11 and mTERF12. (A) Fluorescence microscopy of A. thaliana protoplasts transiently expressing mTERF10, mTERF11 or mTERF12 fused to eGFP (mTERF10-eGFP, mTERF11-eGFP and mTERF12-eGFP). (B) To visualize nucleoids, protoplasts were co-transformed with a RAP-RFP fusion in combination with mTERF10-eGFP, mTERF11-eGFP, or mTERF12-eGFP. The eGFP fluorescence is shown in green (GFP), RFP fluorescence in cyan (RFP), autofluorescence of chloroplasts in red (Auto). The scale bars correspond to 10 µm.

appeared as small dots in the chloroplasts. The size and distribution of these dots were suggestive of nucleoids, which are associated with PGE (Majeran et al., 2012). Localization of the A. thaliana RNA-binding protein RAP to nucleoids was previously established using a transiently expressed RAP-eGFP fusion (Kleinknecht et al., 2014). Therefore, Col-0 protoplasts were co-transformed with a RAP-RFP fusion in combination with mTERF10-eGFP, mTERF11-eGFP, or mTERF12-eGFP. Indeed, for each mTERF-eGFP construct, signals were found in dots together with the RFP signal. Merging of both signals confirmed colocalization of the mTERF10, -11, and -12 fusions with RAP, and therefore localization of all three mTERFs to nucleoids (**Figure 1B**). It is noteworthy, that especially mTERF12—and a minor fraction of RAP—tend to be localized in the chloroplast stroma when both mTERF12-eGFP and RAP-RFP are expressed together in protoplasts.

### Identification and Phenotypic Analysis of Mutants for the MTERF10, MTERF11, MTERF12 Loci

To obtain insight into the physiological functions of mTERFs 10, 11, and 12, T-DNA insertion lines were identified in the SIGnAL database (Alonso et al., 2003). The insertions were confirmed by PCR (**Figure 2A**) and homozygous lines were selected. In the mutants mterf10-1 (SAIL\_12A03) and mterf10- 2 (SALK\_097699) the T-DNAs are inserted in the 5′ UTR and the second exon, respectively (**Figure 2B**). The mterf11-1 (FLAG\_357F09) and mterf11-2 (GABI\_211D05) mutants both have their T-DNA insertion in the gene's single exon. For MTERF12, only one insertion line could be identified (mterf12- 1, GABI\_407E04), which contains a T-DNA in the promoter region (**Figure 2B**). To repress the MTERF12 gene by RNAi, Col-0 lines were generated that contained constructs with an inverted repeat of a fragment spanning the first exon and a part of exon 2 of MTERF12 (**Figure 2A**) which was under control of the constitutive Cauliflower mosaic virus 35S promoter.

**Figure 2C** shows the numbers and distributions of mTERF domains in the mTERF10, -11, and -12 proteins. In mTERF10 and mTERF11, six and five mTERF motifs are predicted by the Simple Modular Architecture Research Tool SMART (http://smart.embl-heidelberg.de/). One mTERF motif has been predicted for mTERF12 (our previous results, and see also Supporting Information of Babiychuk et al., 2011), but this domain is not annotated anymore with confidence by the SMART tool (http://smart.embl-heidelberg.de/smart/ show\_motifs.pl?ID=Q93ZZ2\_ARATH). Thus, the classification of mTERF12 as an mTERF protein must be regarded as uncertain.

All mutants are in the Col-0 background except of mterf11- 1 which is a WS line. Hence, in all following experiments, mterf11-1 was compared with WS, while Col-0 was used as the WT standard for the other lines. Real-time PCR analysis was employed to determine the extent of repression of MTERF transcripts in the different mutant lines (**Figure 3A**). In 3 week-old mterf10-1 and mterf10-2 plants, MTERF10 transcript levels were reduced to 29 and 4% of WT, respectively. To determine MTERF11 transcript levels, primer pair A was chosen to detect transcripts initiated 5′ of the T-DNA insertions (**Figure 2A**). Using this set-up, MTERF11 transcript levels were found to be unchanged (mterf11-1) and nearly 6-fold induced (mterf11-2) relative to their WT (**Figure 3A**). In the mterf11- 1 allele (which is FLAG\_357F09), the T-DNA of the pGKB5 vector integrated in the 5′LB–T-DNA–RB3′ direction. It is of note here that the pGKB5 vector used to generate the FLAGdb T-DNA insertion line collection contains the 35S promoter on the LB side (Samson et al., 2002). The 35S promoter drives the expression of PHOSPHINOTRICIN ACETYL TRANSFERASE (PAT) used to select transgenic plants, and the PAT transcripts are terminated by the G7 terminator. It was already shown with two independent FLAG lines as an example that the G7 terminator can be an inefficient terminator in the context of the pGKB5 vector, allowing transcription to continue through and beyond the terminator sequence (Ulker et al., 2008). However, real-time PCR carried out with a primer pair covering the region 3′ of the T-DNA insertion detected greatly reduced MTERF11 transcript levels in the mterf11 mutants: 0.09% of WT in mterf11-1 and 0.01% in mterf11-2 (**Figure 3A**). MTERF12 transcript levels were not affected in the mterf12- 1 mutant (**Figure 3A**). Therefore, MTERF12 RNAi lines were tested for their ability to repress MTERF12 gene expression. Six independent lines were screened, but the most effectively repressed lines, mterf12i-1 and mterf12i-2, still retained 32% and 59% of WT (Col-0) amounts of MTERF12 transcripts, respectively (**Figure 3A**). Under normal growth conditions, all identified mutant lines were phenotypically indistinguishable from WT (**Figure 3B**). To look for subtle photosynthetic phenotypes, the maximum quantum yield of photosystem II (Fv/Fm) was measured in Col-0, WS and all mterf mutants (**Figure 3B**), but no deviations in this parameter were detected in the mutants.

To summarize, the expression of all mTERF motifs should be strongly reduced in the mterf10 mutants (particularly mterf10- 2), while the mterf11 mutants produce truncated transcripts. Assuming the latter are translated, the protein products would lack the last two mTERF domains (mterf11-1) or mTERF domain 5 only (mterf11-2) (**Figure 2C**). In the mterf12i lines, transcripts including the single putative mTERF domain were—at best reduced to one-third of Col-0 levels. At all events, none of the mterf mutant lines display any obvious phenotype under normal growth conditions.

### Phylogenetic Position of the mTERF10, -11, and -12 Proteins

Because the mterf10, -11, and -12 mutant lines lacked a clear phenotype under normal growth conditions (**Figure 3**), we asked whether this might be attributable to functional redundancy within the mTERF family. Several MTERF genes have undergone tandem duplications (on chromosome 1) and one block duplication (AT4G19650 and AT5G45113; Kleine, 2012). But neither MTERF12 nor MTERF10 or MTERF11 originated from a duplication event, so we can exclude the possibility of

(Continued)

### FIGURE 2 | Continued

sequences around the T-DNA insertion. The combination of RP and T-DNA left border primer (LB) was used for the verification of the T-DNA insertion. (B) Schematic representation and T-DNA tagging of the MTERF10 (AT2G34620), MTERF11 (AT3G18870), and MTERF12 (AT4G09620) loci. Exons (black boxes), introns (black lines) and the 5′ and 3′ UTRs (gray boxes) are shown. Numbers are given relative to the transcription start site of the gene loci. Locations and orientation of T-DNA insertions are indicated, as deduced from RP + LB PCR products shown in (A) which were subsequently sequenced. Note that the insertions are not drawn to scale. Furthermore, the location of the MTERF12 RNAi-directed sequence is indicated as a dashed line. (C) Schematic representation of mTERF10, mMTERF11, and mTERF12 proteins. The numbers and locations of mTERF domains are shown as white boxes. The relative positions of T-DNA and RNAi tagging are indicated.

mutant plants grown under long-day (16/8 h) light conditions. The maximum quantum yield of PSII (Fv/Fm) was measured with an ImagingPAM fluorometer.

protein redundancy arising from gene duplication. To obtain an impression of the overall degree of sequence similarity within the mTERF protein family, we constructed a phylogenetic tree which included in addition to A. thaliana mTERFs, mTERF members from the green alga Chlamydomonas reinhardtii, Homo sapiens, Mus musculus, Drosophila melanogaster, and other organisms (**Figure 4**). The tree reveals four main clades. The mTERF members of C. reinhardtii, H. sapiens and M. musculus are all in the same clade. The majority of A. thaliana mTERFs form clade I which includes mTERF10 and -11, while mTERF12 along with five other A. thaliana mTERFs and one D. melanogaster mTERF

constitute clade II. The mTERF10, -11, and -12 proteins are most closely related to mTERF1/SOLDAT10 (Meskauskiene et al., 2009), mTERF4/BSM/RUG (Babiychuk et al., 2011; Quesada et al., 2011) and mTERF15 (Hsu et al., 2014) proteins. Mutants for each of these three display phenotypes under normal growth conditions and have been shown to be involved in PGE or mitochondrial gene expression. Moreover, levels of sequence identity/similarity between mTERF10 and mTERF1 (38/68% over a stretch of 240 amino acids), mTERF11 and mTERF4 (26/41% over a stretch of 222 amino acids) and mTERF12 and mTERF15 (29/53% over a stretch of 77 amino acids), respectively, are

### FIGURE 4 | Continued

of four main clades (I–IV). Clade I encompasses proteins encoded by a tandem gene cluster on A. thaliana chromosome 1 and several other A. thaliana mTERF proteins. In clade III, C. reinhardtii mTERFs are grouped together with animal mTERF proteins. Clade IV comprises mTERF proteins from diverse species including paramecium, sea urchin (P. livides), parasites (P. falciparum and B. bovis) together with mTERFs from Drosophila. The mTERF proteins mTERF10 and -11 (highlighted in large, bold letters) form clade I together with 25 other mTERF proteins, while mTERF12 (also highlighted in big, bold letters) is assigned to clade II, together with five other A. thaliana mTERFs and one Drosophila mTERF. C, chloroplast; M, mitochondrion; N, nucleus; Y, cytosol; O, other.

noteworthy for the mTERF10/mTERF1 pair, but negligible for the other two.

### Changes in MTERF Transcript Levels in Response to Abiotic Stresses

To gain deeper insights into the functions of mTERF10, -11, and -12, their mRNA expression patterns were analyzed. Coexpression analysis of 26 MTERF genes and their corresponding gene ontology annotations have already been reported (Kleine, 2012). However, that study was designed to provide a global classification. Hence subsequent Genevestigator analyses only dealt with the numbers of conditions/treatments that altered MTERF gene expression. In the present study, we extracted MTERF transcript levels from the Arabidopsis eFP browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) with "Abiotic Stress" as a data source (Winter et al., 2007). In these experiments, 18-day-old plants were subjected to different stresses, and samples were taken over a time course of 24 h from stress-treated and control plants. We calculated the relative changes in MTERF10, -11, and -12 transcript levels from plants exposed to drought, high salt, heat, or cold compared to control conditions (**Figure 5A**). Because mda1 (mterf5) and mterf9 mutants are known to exhibit altered stress responses (Robles et al., 2012, 2015), MTERF5 and -9 were included for reference. Under drought and heat stress, transcript levels of all investigated MTERF genes were only moderately changed (**Figure 5A**). With a 3-fold rise after 1 h of heat stress (MTERF10) and an approximately 0.3-fold change (MTERF5 and 9), those transcripts were the most responsive. Under salt and cold treatment, MTERF transcript levels tended to be reduced. Under both salt and cold stress, MTERF10 and MTERF11 levels were most responsive, and especially after 24 h of cold treatment MTERF5, 10, 11, and 12 transcript levels were reduced (**Figure 5A**). To confirm these data and to find other conditions under which the MTERFs of interest might be regulated, the Genevestigator Perturbations Tool (https:// genevestigator.com/gv; Hruz et al., 2008) was employed on all deposited A. thaliana ATH1 arrays together with a 2-fold change filter and a p-value of < 0.05. An overview of all changes in MTERF10, MTERF11, and MTERF12 mRNA levels in response to perturbations (relative to untreated controls) can be found in **Supplementary Figures S1**–**S3**. In **Figure 5B** selected conditions are shown which are associated with changes in temperature and light, and with salt and drought stress conditions. Levels of MTERF10 mRNA were most susceptible to perturbation, being induced by light, raised after germination, and strongly reduced under drought conditions and various cold and high-light regimes, and on exposure to salt or ABA (**Figure 5B**).

### Knockdown of MTERF10 or MTERF11 Alters Sensitivity to Salt Stress

To experimentally probe the involvement of mTERFs in stress responses, 3-week-old WT and mterf10, -11, and - 12 mutant plants grown under standard conditions (22◦C at 100 µmol photons m−2 s −1 ) were exposed for 7 days to moderate temperature stress (30◦C, at a fluence rate of 100 µmol photons m−2 s −1 ) or moderate light stress (400 µmol photons m−2 s −1 at a temperature of 22◦C). After 7 days of moderate temperature stress, the leaf petioles of WT and all mterf mutants were shortened, but otherwise all plants looked healthy (**Figure 6A**). After 3 days, Fv/Fm was slightly reduced in all mterf11 and mterf12 mutant lines, but was restored to normal levels after 7 days (**Figures 6A,B**). Also after 7 days of moderate light stress, the leaf petioles of WT and all mterf mutants were shortened—albeit to a lesser extent. Furthermore, the edges of older leaves in all lines began to show signs of necrosis (**Supplementary Figure S4A**). After 1 h of moderate light stress, Fv/Fm was slightly reduced in all lines (**Supplementary Figure S4B**). This reduction continued in the mterf11-1 mutant after 2 and 4 h, but all lines recovered to the initial Fv/Fm values after 96 h (**Supplementary Figure S4B**).

After 3 and 24 h of salt stress, MTERF5 and MTERF9 transcript levels were reduced to half of those in control conditions (**Figure 5A**), and indeed, mda1 (mterf5) and mterf9 seedlings are less sensitive to salt and osmotic stresses (Robles et al., 2012, 2015). Because MTERF10 and MTERF11 transcripts were reduced to an even larger extent than MTERF5 and MTERF9 RNAs following exposure to salt stress for 6 and 24 h (**Figure 5A**), we asked whether inactivation of MTERF10 or MTERF11 might enable the mutant plants to better tolerate salt stress. To this end, WT and mterf mutant lines were germinated on MS medium (control) and MS medium supplemented with 125 mM or 175 mM NaCl, and germination rates were scored after different time points (**Figure 7A**). All lines germinated to nearly 100% on the control MS medium. Germination rates of all lines with a Col-0 background grown for 48 h on MS medium supplemented with 125 mM NaCl or for 72 h on medium supplemented with 175 mM NaCl were very similar (**Figure 7A**). In the aforementioned conditions, germination rates of Col-0 seeds were approximately 60% (**Figure 7A**). The germination rates of mterf10-1 and -2, mterf11-2 and all mterf12 seeds were all lower (ranging from 26 to 47%) than those of Col-0 seeds. However, after 96 h on MS medium supplemented with 175 mM NaCl the germination rates of mterf12 seeds (76 to 87%) were comparable to that of Col-0 seeds (84%). Interestingly, mterf10-1, mterf10-2, and mterf11-2 still displayed enhanced sensitivity to salt inhibition, with germination rates of 54, 55, and 61%, respectively (**Figure 7A**). WS seeds were

FIGURE 5 | In silico analyses of changes in levels of MTERF transcripts in response to abiotic stresses. (A) MTERF transcript levels were extracted from the Arabidopsis eFP Browser (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi) with "Abiotic Stress" as a data source (Winter et al., 2007). Plant material from stress-treated and control plants was analyzed over a time course of 24 h. Here, the expression values are reported relative to the corresponding transcript levels in control conditions. (B) The Genevestigator Perturbations Tool (https://genevestigator.com/gv; Hruz et al., 2008) was applied to all available A.thaliana microarrays in combination with the 2-fold change filter and a p-value of < 0.05. Shown here is a selection of conditions related to abiotic stresses. Conditions were ordered according to the magnitude of the relative change in MTERF10 mRNA (from high to low). An overview of all transcriptional responses of MTERF10, MTERF11, and MTERF12 to perturbations can be found in Supplementary Figures S1–S3.

very susceptible to salt stress and failed to germinate under the conditions used to investigate the Col-0 descendant lines. For this reason, a milder salt stress treatment was applied to all lines with a WS background. Still, after 72 h growth on MS medium supplemented with 125 mM, the germination rate of WS was only 7% and raised to 48% after 96 h (**Figure 7A**). Although the mterf11-2 mutant was more sensitive to salt stress compared to Col-0, the germination rates of mterf11-1 seeds were comparable to their corresponding WT (WS; **Figure 7A**).

To ascertain whether the altered activity of MTERF10 was indeed responsible for the salt-stress phenotypes and whether overexpression of MTERF11 might lead to enhanced saltstress tolerance, 35S:MTERF10:MGFP5 and 35S:MTERF11:EGFP constructs were introduced into Col-0 and WS, respectively, to generate oe-mTERF10 and oe-mTERF11 lines. Although MTERF10 mRNA levels were only approximately 2.3-fold higher in oe-mTERF10-1 and oe-mTERF10-2 lines than in Col-0 (**Figure 8A**), these lines—with germination rates of approximately 85 and 95%, respectively, after 48 h on MS medium with 125 mM NaCl and 72 h on MS medium with 175 mM NaCl—were nevertheless resistant to the deleterious effect of salt (**Figure 7A**). This confirms that the salt sensitivity of mterf10-1 and mterf10-2 mutants is indeed caused by knockdown of the MTERF10 gene. Moreover, we identified three oemTERF11 lines that displayed a high diversity of MTERF11 transcript overaccumulation which ranged from 12- to 117 fold (**Figure 8B**). Two of these lines were challenged with salt stress, and actually displayed much higher germination rates than WS and therefore, enhanced salt stress tolerance (**Figure 7A**).

#### FIGURE 7 | Continued

Seed germination was investigated on MS medium in the absence and presence of 125 and 175 mM NaCl. Radicle emergence was scored after indicated time points. Germination rates were calculated relative to the number of total seeds. (B) Phenotypes of 7-day-old WT and mutant seedlings were germinated on either MS medium supplemented with 125 mM NaCl (grown for 7 days on 125 mM NaCl), or on MS medium and transferred directly after radicle emergence for 12 days to MS medium supplemented with 125 mM NaCl (transferred for 12 days to 125 mM Nacl) or 175 mM NaCl (transferred for 12 days to 175 mM NaCl), respectively. The root lengths (C) and cotyledon greening rates of seedlings grown on 125 mM NaCl—displayed as the ratio of the number of green cotyledons to the total number of cotyledons (D) were determined after 7 and 5 days, respectively. The data in (A,C,D) represent mean values ± SD of three independent experiments, each performed with at least 100 seeds per treatment and genotype. Statistically significant differences (t-test; p < 0.05) between WT (Col-0 or WS) and corresponding mutant lines are indicated by an asterisk (black for Col-0; red for WS).

To investigate this further, the performance of mterf mutants and mTERF overexpression lines was investigated during postgermination development. As shown in **Figures 7B,C**, the root lengths of mterf10-1 and -2 seedlings challenged with 125 mM NaCl were significantly shorter compared to Col-0, while the root lengths of oe-mTERF10 seedlings were longer (**Figure 7C**). Moreover, compared to WS, overexpression of MTERF11 results in longer root lengths, reflecting findings of the germination rates (**Figure 7A**). In addition, cotyledon greening—displayed as the ratio of the number of green cotyledons to the total number of cotyledons—of mterf10-1 and -2 seedlings was delayed by salt stress, while in contrast, overexpression of MTERF10 or MTERF11 enabled seedlings to display higher cotyledon greening rates than their corresponding wild types (**Figure 7D**).