Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice

Accepted Manuscript Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice Hai Zheng, Zhuoran Wang, Yunlu Tian, LingLong Liu, Feng Lv, Weiyi Kong, Wenting Bai, Peiran Wang, Chaolong Wang, Xiaowen Yu, Xi Liu, Ling Jiang, Zhigang Zhao, Jianmin Wan PII:

S0981-9428(19)30142-1

DOI:

https://doi.org/10.1016/j.plaphy.2019.04.008

Reference:

PLAPHY 5660

To appear in:

Plant Physiology and Biochemistry

Received Date: 8 January 2019 Revised Date:

8 April 2019

Accepted Date: 9 April 2019

Please cite this article as: H. Zheng, Z. Wang, Y. Tian, L. Liu, F. Lv, W. Kong, W. Bai, P. Wang, C. Wang, X. Yu, X. Liu, L. Jiang, Z. Zhao, J. Wan, Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast development and establishment of the plastidic ribosome system in rice, Plant Physiology et Biochemistry (2019), doi: https://doi.org/10.1016/j.plaphy.2019.04.008. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Rice albino 1, encoding a glycyl-tRNA synthetase, is involved in chloroplast

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development and establishment of the plastidic ribosome system in rice

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Hai Zhenga,1 • Zhuoran Wanga,1 • Yunlu Tiana • LingLong Liua • Feng Lva • Weiyi

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Konga • Wenting Baia • Peiran Wanga • Chaolong Wanga • Xiaowen Yua • Xi Liua

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• Ling Jianga • Zhigang Zhaoa* • Jianmin Wana, b

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a

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Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing

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210095, China

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b

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National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu

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National Key Facility for Crop Gene Resources and Genetic Improvement, Institute

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of Crop Science, Chinese Academy of Agriculture Sciences, Beijing 100081, China

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1

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*Corresponding author:

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Zhigang Zhao

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Telephone: +86-25-84396516

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Fax: +86-25-84396516

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E-mail: [email protected];

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H.Z and Z.R.W have contributed equally to this work.

ACCEPTED MANUSCRIPT Abstract

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The chloroplast is an important organelle that performs photosynthesis as well as

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biosynthesis and storage of many metabolites. Aminoacyl-tRNA synthetases (aaRSs)

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are key enzymes in protein synthesis. However, the relationship between chloroplast

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development and aaRSs still remains unclear. In this study, we isolated a rice albino 1

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(ra1) mutant through methane sulfonate (EMS) mutagenesis of rice japonica cultivar

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Ningjing 4 (Oryza sativa L.), which displayed albinic leaves in seedling stage due to

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abnormal chloroplast development. Compared with wild type (WT), ra1 showed

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significantly decreased levels of chlorophylls (Chl) and carotenoids (Car) in

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2-week-old seedlings, which also showed obvious plastidic structural defects

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including abnormal thylakoid membrane structures and more osmiophilic particles.

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These defects caused albino phenotypes in seedlings. Map-based cloning revealed that

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RA1 gene encodes a glycyl-tRNA synthetase (GlyRS), which was confirmed by

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genetic complementation and knockout by Crispr/Cas9 technology. Sequence analysis

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showed that a single base mutation (T to A) occurred in the sixth exon of RA1 and

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resulted in a change from Isoleucine (Ile) to Lysine (Lys). Real-time PCR analyses

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showed that RA1 expression levels were constitutive in most tissues, but most

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abundant in the leaves and stems. By transient expression in Nicotiana benthamiana,

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we found that RA1 protein was localized in the chloroplast. Expression levels of

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chlorophyll biosynthesis and plastid development related genes were disordered in the

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ra1 mutant. RNA analysis revealed biogenesis of chloroplast rRNAs was abnormal in

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ra1. Meanwhile, western blotting showed that synthesis of proteins associated with

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plastid development was significantly repressed. These results suggest that RA1 is

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involved in early chloroplast development and establishment of the plastidic ribosome

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system in rice.

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Highlights

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• RA1 is a novel gene controlling chlorophyll synthesis and the ribosome system.

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• Absence of aminoacyl-tRNA synthetases lead to plastid developmental disorders.

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• Biosynthesis of plastidic ribosomes is severely impaired in the ra1 mutant.

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• RA1 protein is targeted to the chloroplast.

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Keywords

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Chloroplast development • Glycyl-tRNA synthetase • Rice • Ribosome

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Abbreviations

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dCAPS

derived cleaved amplified polymorphic sequence

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GluTR

Glutamyl t-RNA reductase

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NEP

Nuclear-encoded RNA polymerase

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PEP

Plastid-encoded RNA polymerase

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psaA1

PS I P700 apoprotein A1

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psaA2

PS I P700 apoprotein A2

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RBCL

Rubisco large subunits

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RBCS

Rubisco small subunit

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RCA

Rubisco activase

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1. Introduction

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Chloroplasts are not only the important site of photosynthesis in higher plants, but

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also responsible for biosynthesis and storage of metabolites (Ruuska et al., 2004;

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Millar et al., 2006). Chloroplasts retain their own genome and translation system,

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nuclear and plastid genomes collectively regulate chloroplast development (Millar et

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al., 2006). In addition, chloroplast development requires cooperation between the

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genes encoded by the nucleus and those encoded by the plastid. In plants, there are

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two types of RNA polymerase in the chloroplast. One type is a nuclear-encoded RNA

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polymerase (NEP), and the other type is a plastid-encoded RNA polymerase (PEP).

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PEP is a multimeric enzyme of the prokaryotic type and NEP is a monomeric enzyme

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of the phage type (Azevedo et al., 2006). PEP catalytic core components are

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composed of four subunits, i.e., α, β, βʹ and βʹʹ, encoded by the plastidic genes RNA

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polymerase α subunit (rpoA), RNA polymerase β subunit (rpoB), RNA polymerase βʹ

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ACCEPTED MANUSCRIPT subunit (rpoC1), and RNA polymerase βʹʹ subunit (rpoC2), respectively. PEP are

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responsible for transcription of photosystem proteins (Shiina et al., 2005). The

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development of chloroplasts can be divided into three periods: plastid replication and

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plastid genome replication; chloroplast genesis and translation system construction

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and the establishment of the chloroplast light-harvesting system (Kusumi et al., 2010).

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Loss of function of proteins involved in the PEP and NEP could lead to the plant

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albinic phenotype (Liu et al., 2016; Steiner et al., 2011; Yagi et al., 2012; Pfalz et al.,

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2013). In Arabidopsis, plastidial thioredoxin z (trx z) had pale yellowish leaves when

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grown on agar plates and subsequently died. TRX z encodes a plastidial thioredoxin,

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which can interact with two fructokinase-like proteins FLN1 and FLN2 by conserved

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Cys residues (Arsova et al., 2010).

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Plastid proteins are translated by 70S ribosomes, which consist of 30S rRNA and 50S rRNA complexes. The former is composed of a 16S rRNA molecule and 21

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different proteins and the latter includes a 23S rRNA, a 5S rRNA, a 4.5S rRNA and

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32 different proteins. In rice and Arabidopsis, mutations in Spo0B-associated

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GTP-binding protein C (ObgC) affect ribosomal biosynthesis, leading to chloroplast

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developmental disorders (Bang et al., 2009; Bang et al., 2012). Dysfunction of Albino

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Leaf1 (AL1) resulted in altered abundances of ribosomal proteins and breakdown of

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chloroplast (Zhang et al., 2016).

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The accuracy of mRNA translation in protein synthesis is determined by two factors, the fidelity of the amino acylation reactions binding amino acids to their

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cognate tRNAs, and the accuracy of the binding of the anticodons to the mRNA

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codons exposed at the "A" site of the ribosome. The role of aminoacyl-tRNA

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synthetases (aaRSs) in translation is to define the genetic code by accurately pairing

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cognate tRNAs with their corresponding amino acids (Ling et al., 2009). There are 20

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aaRSs in eukaryotes, corresponding to each amino acid. These aaRSs not only

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catalyze protein synthesis, but also perform translational regulation, RNA splicing,

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apoptosis, and rRNA synthesis (Guo et al., 2010; Wang et al., 2016). For example,

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Methionine-tRNA synthetase (MetRS) is involved in protein synthesis in cytoplasm

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and rRNA synthesis in the nucleus (Ko et al., 2000). Glutamate-tRNA synthetase

ACCEPTED MANUSCRIPT (GluRs) can regulate apoptosis (Ko et al., 2001). In Arabidopsis, EDD1 encodes a

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plastid and mitochondrial localized glycyl-tRNA synthetase and embryo defective

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development 1 (edd1) were characterized by aborted ovules and change in distal

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regions of the leaf. EDD1 control the abaxial fate genes kanadi 1 (KAN1) and auxin

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response factor 3 (ARF3) (Uwer et al., 1998; Moschopoulos et al., 2012). All aaRSs

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are encoded by the nuclear genome and transported to the designated compartments.

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A complete set of 20 aaRSs are present in every cell compartment that requires

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protein synthesis (Duchene et al., 2005). In total, 45 expressed aaRSs genes were

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identified in the Arabidopsis genome. Previous reports on aaRS gene mutants were

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mainly focused on morphology. In rice, white panicle 1 (WP1) encodes a Val-tRNA

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synthetase. Dysfunction of WP1 displayed virescent phenotypes in seedlings and

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white panicles at heading, suggesting WP1 plays an essential role in chloroplast

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development (Wang et al., 2016). However, little is known about aaRSs affecting of

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plant growth and development.

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In this study, we isolated a rice albino 1 (ra1) mutant that displayed albino leaves at the seedling stage and died after 18 days. RA1 encodes a glycyl-tRNA synthetase

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(GlyRS), which is targeted to chloroplasts. Irregularly-shaped membranes with no

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clear thylakoids and vacuole-like structures were observed in ra1 chloroplasts. In

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addition, establishment of the plastidic ribosome system was defective in ra1. We

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demonstrate that RA1 is important for chloroplast development and ribosomal

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biogenesis.

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2. Materials and methods

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2.1. Plant materials and growth conditions

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The ra1 mutant was isolated from an ethyl methane sulfonate (EMS)-mutagenized

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japonica cultivar Ningjing 4 (NJ4) M1 population. WT, ra1 mutant and an F2 mapping

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population were grown in the field or growth chambers. Growth chambers conditions

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were 10 h light (Light intensity was 300 µmol.m-2.s-1) at 30℃ and 14 h dark at 28℃.

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2.2. Measurement of chlorophyll content and observation of chloroplast

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autofluorescence

ACCEPTED MANUSCRIPT Chlorophyll contents of WT and ra1 were measured in vitro as described previously

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(Wang et al., 2016). In brief, approximately 0.5 g fresh 7-day-old WT and ra1

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seedling leaves were cut into 5mm pieces, then transferred to a centrifuge tube, and

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placed in 95% ethanol at 4℃ under dark conditions for 24 h, repeatedly shaking.

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Absorbance of the supernatants was measured at 665, 649, and 470 nm. The equations

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used to calculate the chlorophyll and carotenoid concentrations were: Chlorophyll a:

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Ca = 13.95D665 - 6.88D640, Chlorophyll b: Cb = 24.96D649 - 7.32D665, Carotenoid: Cx =

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(1000D470 - 2.05Ca - 114Cb) / 245. Fluorescence microscopy (NIKON AZ100) was

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used to observe chlorophyll autofluorescence of rice at different stages according to

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the previous method (Itoh et al., 2005). At least 2 biological and 3 technical replicates

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were performed.

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2.3. Transmission electron microscopy (TEM)

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Leaves of WT and ra1 plants grown for 2 weeks were cut into small pieces and

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transferred to 2.5% glutaraldehyde (0.2 M phosphate buffer, pH 7.2). Vacuum was

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applied to infiltrate the solution, resulting in the samples sinking to the bottoms of the

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tubes. Then the samples were incubated at room temperature for 12 h, rinsed and

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incubated overnight in 1% OsO4 at 4℃. After several rounds of rinsing with 0.2 M

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phosphate buffer (PH 7.2), the samples were dehydrated through 30, 50, 70, 80, 90

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and 100% of ethanol solutions for 20 min, and embedded in Spurr’s medium before

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ultrathin sectioning. The samples were stained with uranyl acetate and observed using

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a Hitachi H-7650 transmission electron microscope (Han et al., 2006).

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2.4. Map-based cloning of the RA1 gene

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Since the recessive homozygous ra1 seedlings were albino lethal, crosses were made

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between green heterozygous RA1/ra1 plants and N22 (indica) for construction of the

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F2 mapping population. Within the F2 mapping population, 318 albino plants were

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used for fine mapping and genetic analysis. Based on the published simple sequence

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repeats (SSR) markers, the polymorphic markers between the ra1 mutant and N22

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were used for initial mapping. The DNA sequences in this region of japonica

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(http://www.ncbi.nlm.nih.gov/) and indica (http://www.gramene.org/) were aligned,

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and new genetic markers were designed using Primer Premier 5

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ACCEPTED MANUSCRIPT (http://www.premierbiosoft.com/). All of the molecular markers for genetic mapping

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and gene cloning are listed in Supplementary Table 1.

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2.5. Complementation and Crispr/Cas9 targeting of RA1

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For functional complementation of the ra1 mutant, full length WT cDNA (entire

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coding sequences) and a genomic fragment upstream of native promoter region were

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amplified and inserted into the binary vector pCUbi1390. Fusion vector was

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transformed into the ra1 callus by Agrobacterium-mediated transformation (Hiei et al.,

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1994). The target sequence of RA1 for Crispr/Cas9 deletion was

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“AACAGACCCACTAGACTA”. The sequence were amplified and fused to a binary

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vector pCAMBIA 1305 and transformed into Nipponbare by Agrobacterium-mediated

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transformation as described above.

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2.6. Subcellular localization of RA1

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The full-length RA1 cDNA (with the stop codon deleted) was amplified using the

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primers listed in Supplementary Table 1 and translationally-fused to the N-terminus of

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green fluorescent protein (GFP) in the binary vector pCAMBIA1305. The vectors

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were transiently expressed in Nicotiana benthamiana mesophyll cells by

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Agrobacterium infiltration. The fluorescence was observed using a confocal laser

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scanning microscope (Zeiss LSM 780), and red chlorophyll autofluorescence was

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used as a marker for reference.

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2.7. Real-time PCR

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According to a RNA Prep Pure Plant kit protocol (Tiangen Co, Beijing, China), total

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RNA was extracted from 7-day-old rice seedlings. From each rice seedling RNA

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sample (2 µg) was reverse-transcribed using an oligo-dT or random primer and

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PrimeScript I (Takara Bio, Kusatsu, Japan). Quantitative Real-time PCR (qRT-PCR)

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using a SYBR Premix Ex TaqTM kit (TaKaRa) was executed on an ABI prism 7900

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Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). At least 2

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biological and 4 technical replicates were performed. The primer sequences for the

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genes related to chlorophyll synthesis and plastid development are listed in

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Supplementary Table 1, respectively. The 2-ΔΔCT method was used to calculate relative

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changes in gene expression (Livak et al., 2001).

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ACCEPTED MANUSCRIPT 2.8. rRNA analysis

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The concentrations and purities of 7-day-old rice seedlings RNA extracts were first

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assayed with a NanoDrop Spectrometer (Thermo Fisher Scientific, Waltham, MA,

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USA). RNAs were diluted to ~10 ng.uL-1 and analyzed by an Agilent 2100

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Bioanalyzer. An RNA 6000 Nano Total RNA Analysis Kit (Agilgent) was used for

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analysis following the manufacturer’s instructions. At least 2 biological replicates

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were performed.

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2.9. Western blot analysis

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Total proteins were isolated from 7-day-old WT and ra1 seedlings. In brief, 0.2g fresh

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7-day-old WT and ra1 seedlings were ground into powder in liquid nitrogen and

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transferred to a centrifuge tube. Then 600 µL NBI extraction buffer (50 mM Tris-HCl,

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pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.5 (v/v) β-mercaptoethanol,

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1 mM 4-(2- aminoethyl)-benzenesulfonyl fluoride, 2 µg.mL-1 antipain, 2 µg.mL-1

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leupeptin, 2 µg.mL-1 aprotinin) was added to centrifuge tube and incubated for 30 min

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on ice. The solutions were centrifuged at 12,000 g for 20 min, and then all

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supernatants were aspirated and centrifuged at 12,000 g for 10 min. 80 µL of

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supernatant were transferred to a new centrifuge tube, mixed with 20 µL 5 X protein

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loading buffer and incubated for 5 min at 100℃. 10 µL of this mix were then loaded

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on 8-15% SDS-PAGE gradient gels and electrophoresis using Tris-Glycine

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Electrophoresis Buffer. Gel-membrane Sandwich model was used for transferred to

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polyvinylidene difluoride membrane. The proteins were immunoblotted with various

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specific antibodies, and detected using High-sig ECL Western Blotting Substrate

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(Tanon). Specific antibodies were obtained from Beijing Protein Innovation

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(http://www.proteomics.org.cn/). At least 2 biological replicates were performed.

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2.10. Statistical analysis

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Results were subjected to Student's t-test to compare the statistical significant

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differences of the mean values between WT and ra1 (Microsoft Excel software).

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Statistical significance differences at P < 0.05 and P < 0.01 were indicated by

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asterisks * and **, respectively. The number of replicates (n) for each measured

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parameter refers to the number of repetitions of technical replicates. All values in

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figures were means ± SD (standard deviation).

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3. Results

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3.1. Phenotypic characterization of the ra1 mutant

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The ra1 mutant was derived from the japonica variety NJ4 by EMS mutagenesis.

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Under chamber and paddy field conditions, the ra1 mutant exhibited obvious albinic

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leaf during early leaf development and died 18 days after germination (Fig. 1a). In

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addition, we also observed the chlorophyll auto-fluorescence of 3, 10 and 18-day-old

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seedlings, and found that the auto-fluorescence of chlorophyll was always observed in

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the WT, and showed an increasing trend as the seedlings developed. In contrast, at 3

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day after sowing, only a small amount of auto-fluorescence was detected in ra1

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mutant. At 10 and 18 days after sowing, chloroplast auto-fluorescence was almost

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undetectable with the ra1 mutants (Fig. 1b). Subsequently, leaves of ra1 mutant began

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to wilt and died.

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3.2. Early chloroplast development is affected in the ra1 mutant

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We measured the pigment contents of WT and ra1 seedlings. The various pigment

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content showed a very significant decrease, and Chl a, Chl b and Car did not

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accumulate in the ra1 mutant seedlings as compared to WT (Fig. 2a and 2b). In order

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to determine whether the morphology and development of chloroplasts in ra1 were

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affected, the chloroplast ultrastructures of both WT and ra1 were observed by TEM.

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The results revealed that a large number of lamellar structures in the wild-type

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chloroplasts thylakoid could be observed at 2-weeks-old seedling (Fig. 2c and 2d).

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Instead, only small and irregular lamellas with no clear thylakoid structures were

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shown in ra1 plastids. Besides, a large amount of vacuole-like structures and

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osmiophilic particles appeared in ra1 chloroplasts (Fig. 2e and 2f). Clearly, these

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results emphasized RA1 is essential for chloroplasts development.

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3.3. Map-based cloning of the RA1 gene

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Genetic analysis indicated that selfed F1 plants corresponded to 3: 1 segregation in a

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segregating genetic analysis population (green plants n = 260, albino plants n = 73, χ2

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= 1.68 < χ20.05, 1=3.84). For further gene mapping of the RA1 locus, we performed a F2

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ACCEPTED MANUSCRIPT mapping population from the cross between heterozygous green plant (RA1/ra1) and

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N22. Totally 135 polymorphic markers evenly covering the whole genome were used

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for initial mapping. From the F2 mapping population, 10 green plants and 10 albino

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plants were randomly selected. Preliminary linkage analysis indicated that the ra1

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locus was located near to SSR markers RM1369 on the short arm of chromosome 6.

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To further locate the gene, 318 additional albino plants from the F2 mapping

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population were analyzed. Further dissection delimited the ra1 locus to about 266-kb

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region from InDel markers Bh6-12 to telomere of chromosome 6 including two BACs

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B1460A05 and OSJNBa0075G19 based on the Nipponbare genome sequence (Fig.

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3a). Within the target region, 17 open reading frames (ORFs) were predicted by the

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Rice Expression Profile Database (http://ricexpro.dna.affrc.go.jp) (Supplementary

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Table 2). These ORFs were analyzed by the online gene expression database, and only

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ORF4 and ORF15 showed high expression in leaf tissue. ORF4 and ORF15 were

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annotated for plastocyanin, chloroplast precursor (LOC_Os06g01210) and

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aminoacyl-t-RNA synthetase precursor (LOC_Os06g01400), respectively. No genome

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sequence difference was found for ORF4 between WT and mutant (Date not shown).

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However, a T-to-A transition at nucleotide position 1346 in the 6th exon of ORF15

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was identified, leading to an amino acid change from Ile to Lys at 228th residue (Fig.

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3b and 3c). The mutation was the conserved amino acid in α-core domain by amino

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acid sequence alignment (Supplementary Figure 1). Moreover, a dCAPS marker

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confirmed the mutation site in the ra1 mutant (Fig. 3d). ORF15 encodes a GlyRS

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from known gene annotation. This gene is 10,538 bp in length and contains 34 exons

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and 33 introns. Phylogenetic analysis showed that proteins homologous to RA1 exist

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in both monocots and dicots and RA1 was highly conserved in higher plants

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(Supplementary Figure 2). In summary, ORF15 may be as a candidate gene.

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3.4. Confirmation of RA1 function

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To further demonstrate that the mutation in candidate gene ORF15 causes the albino

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leaf phenotype, we conducted a complementation experiment. Because homozygous

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ra1 seedlings couldn’t live to maturity, we selected the phenotype of albino seedlings

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for transgenic complementation experiments in the callus. A 6.2-kb genomic fragment

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ACCEPTED MANUSCRIPT containing its native promoter and complete CDS sequence was introduced into ra1

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callus. Thirty-seven independent positive T0 transgenic lines were obtained and they

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all showed the WT phenotype (Fig. 4a). In order to better confirm complementation

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result, CRISPR/Cas9 technology was used to editing RA1 in the WT plants. As a

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result, knockout lines showed a severe albino plants phenotype and died early (Fig.

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4b). Specifically, there were 29-bp, 10-bp and 1-bp deletion of mutant plants by

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sequencing, respectively (Fig. 4c). These sequencing changes led to frameshift

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mutations. Thus we validated the mutation in ORF15 was the cause of albinic

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phenotype.

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3.5. Tissue analysis of RA1 and subcellular localization

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To detect the tissue expression patterns of RA1, we detected mRNA levels in various

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organs of WT by quantitative RT-PCR. RA1 mainly expressed in stems, leaves, paleas,

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and other organs that contain abundant chloroplasts (Fig. 5a). Previous studies found

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that aaRSs had different subcellular localization results (Duchene et al., 2005). In rice,

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lethal albinic seedling (LAS) was chloroplast localization, while WP1 was

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co-localized in chloroplast and mitochondria (Wang et al., 2016; Zhang et al., 2017).

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In Arabidopsis, aaRSs are not only located in the chloroplast and mitochondria, but

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also synthesized in the cytoplasm (Duchene et al., 2005; Berg et al., 2005). To

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determine the subcellular localization of RA1 protein, a transient expression system

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was performed in Nicotiana benthamiana. We constructed GFP fusion proteins driven

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by the 35S promoter, and transiently expressed them in Nicotiana benthamiana.

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Strong green fluorescence signals were co-localized with the chlorophyll (Fig. 5b).

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3.6. RA1 affects expression levels of chlorophyll biosynthesis and plastid development

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related genes

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Normal plastids development and chlorophyll synthesis are the result of the

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coordination between NEP and PEP (Kusumi et al., 2014). In Arabidopsis,

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Glutamyl-tRNA may mediate the switch of RNA polymerase usage from NEP to PEP

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during chloroplast development (Hanaoka et al., 2005). We hypothesized that the

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expression of chlorophyll biosynthesis and plastid development related genes may be

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affected by defective glycyl-tRNA synthetase in the ra1 mutant. Quantitative analysis

ACCEPTED MANUSCRIPT showed that the expression of genes related to chlorophyll biosynthesis and plastid

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development had significantly changed in WT and ra1. Firstly, expression of related

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genes involved in the chlorophyll biosynthetic pathway, those from the synthesis of

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Glutamyl t-RNA (GluTR) to aminolevulinic acid (ALA) and ALA to Protoporphyrin

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IX in ra1 were increased compared to WT, including HEMA, HEML, HEMB, HEMC,

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HEME and HEMF. However, expression of related genes for the synthesis of

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Protoporphyrin Ⅸ to Chlorophyll a and b in ra1 kept the same as compared to WT,

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including CHLH, CHLI, CHLM, DVR, POR and CAO (Fig. 6a). Secondly, the

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expression levels of plastid development related genes, including class I genes RBCL,

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RCA, PsaA1, PsaA2 and PsbD1, as well as class Ⅸ genes ATP synthase coupling

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factor1 alpha (ATPCFα) and NADH dehydrogenase subunit 2 (NADH2) in the ra1

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mutant, showed a great of reduction compared to wild type. Conversely, expression

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levels of class Ⅸ genes RpoA, RpoB, RpoC1and RpoC2 were increased relative to

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wild type (Fig. 6b). In short, expression levels of chlorophyll biosynthesis and plastid

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development related genes were disordered in the ra1 mutant.

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3.7. Defective plastid ribosomal RNA biogenesis system in ra1

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On account of expression of plastid development related genes were disordered, we

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suspected the changes might affect corresponding protein levels in the ra1 mutant. In

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order to verify this, total proteins from wild type and ra1 seedlings was extracted and

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analyzed by western blotting. Before that, levels of RBCL protein was examined by

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Coomassie blue staining. The results showed that RBCLs were significantly reduced

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in the ra1 mutant (Fig. 7a). Likewise, protein abundance of RBCS, RCA, subunits of

340

photosynthetic complexes (PsbC and PsbD) and ATP synthase CF1 beta (AtpB) were

341

dramatically decreased in ra1 (Fig. 7b). Thus, the decrease of the protein levels may

342

be the reason of the arrest of plastid development.

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Based on the previous reports, ra1 showed similar phenotype to obgc1, wp1,

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temperature-sensitive virescent (tsv), las mutants (Bang et al., 2012; Wang et al., 2016;

345

Sun et al., 2017; Zhang et al., 2017). The chloroplast ribosome is composed of a 50S

346

large subunit and another 30S small subunit, which further include four rRNA (23S,

347

16S, 5S and 4.5S) and ribosomal proteins. We hypothesized that the chloroplast

ACCEPTED MANUSCRIPT ribosomal biosynthesis in the ra1 might be impaired. To prove this hypothesis, we

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used the Agilent 2100 to analyze the composition and amount of rRNA. In the

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seedlings of the ra1mutant, 23S rRNA and 16S rRNA were marginally detected (Fig.

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7c and 7d). Overall, the chloroplast ribosomal biosynthesis in the ra1 mutant is

352

severely impaired.

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4. Discussion

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Chloroplasts are plant organelles for photosynthesis, and are also important places for

356

metabolism. In plants, many mutants are associated with early plastid development

357

(Xu et al., 2013, Dong et al., 2013, Qiao et al., 2013, Motohashi et al., 2007, Pyo et al.,

358

2013). Aminoacyl-tRNA synthetases (aaRSs) are enzymes that catalyze the binding of

359

amino acids and their corresponding tRNAs during protein synthesis, which plays a

360

crucial role in the protein synthesis process and is necessary for plastid development.

361

In this study, we determined that ra1 was a mutation of a glycyl-tRNA synthetase

362

(GlyRS) gene. Our results suggest that RA1 is essential for ribosomal biosynthesis and

363

the absence of this aaRS leads to plastid developmental disorders.

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It is well-known that aaRSs are conserved in function and aaRSs plays an

365

important role in the study of biological evolution. The aaRSs are present in all cell

366

organisms and its catalytic response is mainly divided into two steps. The first step is

367

that the amino acids are catalyzed, under the action of enzymes and ATP, to produce a

368

PPi and aminoacyl adenylate. Generally, aminoacyl adenylate is bound firmly to the

369

enzyme. The second step is that the aminoacyl adenylate-enzyme conjugated complex

370

and tRNA to generate aaRSs, AMP and enzymes and both step are reversible reactions.

371

There are 20 aaRSs in organisms, and class I and class II contain 10 aaRSs,

372

respectively (Sugiura et al., 2000). All class I aaRSs contain a Rossman fold domain

373

in its core site, as well as the conserved HIGH and KMSKS domains. Moreover, the

374

class II aaRSs has an anti-parallel β-sheet structure surrounded by an alpha helix. In

375

this study, GlyRS was classified as class II and consists of four domains

376

(Supplementary Figure 1a). Amino acid at 1-72 is the N-terminal signal peptide. This

377

domain is essential for recognition of chloroplast membrane after protein synthesis in

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ACCEPTED MANUSCRIPT cytoplasm and transport of protein into chloroplast. The 73-351aa region is the α-core

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domain, which has function of glycine-tRNA ligase activity. The 386-933aa region is

380

the β subunit, which was supposed to decompose ATP to provide energy for the

381

normal functioning of GlyRS. The 962-1054aa region of C-terminal is

382

anticodon-binding domain, and its role is likely to act as anticodon to recognize

383

glycine (Hausmann et al., 2008; Ling et al., 2009). By amino acid sequence alignment,

384

it was found that the T to A mutation resided in a highly conserved α-core domain in

385

the ra1 mutant (Supplementary Figure 1b). Thus it is reasonable that the T to A

386

mutation results in pivotal amino acid change from Ile to Lys at 228th residue and loss

387

of enzymatic activity of the RA1, although it causes difference for RA1 transcriptional

388

levels between the wild type and ra1 (Supplementary Figure 3).

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In eukaryotes, cytoplasm, mitochondria and plastids all contain their own protein synthesis systems. In Arabidopsis, some aaRSs are localized in more than one

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organelle. Seventeen aaRSs are localized in both mitochondria and plastids, five

392

aaRSs are localized in both the cytosol and mitochondria, and two aaRSs are only

393

localized to the chloroplast (Duchene et al., 2005). In rice, previous studies have

394

found that WP1 was co-localized in both chloroplasts and mitochondria in rice,

395

whereas OsValRS1 was only localized in the cytoplasm. Three different aaRSs

396

localization are crucial to plants (Wang et al., 2016). In this study, strong green

397

fluorescence signal co-localized with chlorophylls autofluorescence (Fig. 5c). In

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addition, we found two other glycine-tRNA synthetases in rice by database analysis

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(http://ricexpro.dna.affrc.go.jp/). OsGlyRS1 (LOC_Os04g32650) was highly

400

expressed in leaf and inflorescence, while OsGlyRS3 (LOC_Os08g42560) mainly in

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ovary and anther in rice. Similar to WP1, localization of RA1, GlyRS1 and GlyRS3

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may cover the cytoplasm, mitochondria and chloroplast.

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Previous evidence showed that impairment of PEP activity led to a reduction in

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glutamate-tRNA content in the plastid (Hanaoka et al., 2005; Pfalz et al., 2006;

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Arsova et al., 2010; Yagi et al., 2012). The down regulation of PEP expression and the

406

up-regulation of NEP expression in the clb19, obg, cotp70, wp1, las and ys1mutant

407

indicated that their PEP activity was severely impaired (Bang et al., 2009; Bang et al.,

ACCEPTED MANUSCRIPT 2012; Chateigner-Boutin et al., 2008; Chateigner-Boutin et al., 2011; Wang et al.,

409

2016; Zhang et al., 2017). Similarly, expression levels of class I genes in the ra1 were

410

reduced compared to wild type. Moreover, expression levels of class Ⅸ genes were

411

increased (Fig. 6b). We suspect that dysfunction of RA1 may affect the activity of the

412

PEP, which affects the expression level of genes encoded by the plastid. In addition,

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in the seedlings of the ra1, almost no 23S rRNA and 16S rRNA were detected (Fig. 7c

414

and 7d). A significant decrease in the amount of its Gly-tRNA in the mutant may

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disturb the synthesis of early ribosomal proteins and therefore Gly-tRNA is essential

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for the synthesis of ribosomal rRNA.

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In Arabidopsis, EDD1 is a homolog gene of RA1. EDD1 encoding a glycyl-tRNA

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synthetase, an essential enzymes that catalyzes the addition of amino acids to specific

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tRNA (Uwer et al., 1998; Moschopoulos et al., 2012). EDD1 was targeted to plastid

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and mitochondrial, and dysfunction of EDD1 displayed embryo and leaves defective

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development, suggesting it plays an essential role in embryo and leaves development.

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Similar to the phenotype of the edd1 mutant, ra1 show abnormal chloroplast

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developmental. The similar localization and function of RA1 and EDD1 suggested

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that function of glycyl-tRNA synthetase should be conserved function in higher

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plants.

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5.

Conclusion

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We identified a seedling albino mutant, whose chloroplasts showed no intact

429

thylakoid membranes and obvious albinic leaf during early leaf development.

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Map-based cloning revealed that RA1 encodes a glycyl-tRNA synthetase that is

431

essential for plant chloroplast development. RA1 protein was located in the

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chloroplast. Expression levels of genes transcribed by the NEP and PEP were

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disrupted in the mutant and ribosome synthesis was abnormal. Hence, RA1 is crucial

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for chloroplast development and ribosomal synthesis.

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Author contribution statement

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Hai Zheng, Zhuoran Wang and Feng Lv conducted the experiments. Hai Zheng,

ACCEPTED MANUSCRIPT Peiran Wang, Xi Liu. Ling Jiang designed the research. Hai Zheng and Wenting Bai

439

conducted western blot experiments. Yunlu Tian and Weiyi Kong found the mutant

440

and analyzed the data. Chaolong Wang and Xiaowen Yu guide the writing of

441

manuscripts. Hai Zheng wrote the manuscript. Zhigang Zhao and LingLong Liu

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edited and modify the manuscript. Hai Zheng and Zhuoran Wang have contributed

443

equally to this work. All authors read and approved the manuscript.

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Acknowledgements

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This research was supported by the National Transform Science and Technology

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Program (2016ZX08001004-002); the Key Laboratory of Biology, Genetics and

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Breeding of Japonica Rice in the Mid-lower Yangtze River; the Ministry of

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Agriculture, China, Jiangsu Plant Gene Engineering Research Center; the Jiangsu

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Collaborative Innovation Center for Modern Crop Production.

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Confict of interest

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The authors declare that they have no conficts of interest.

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References

456

Arsova, B., Hoja, U., Wimmelbacher, M., Greiner, E., Ustun, S., Melzer, M., Petersen,

EP

455

K., Lein, W., Bornke, F., 2010. Plastidial thioredoxin z interacts with two

458

fructokinase-like proteins in a thiol-dependent manner: evidence for an essential

459

role in chloroplast development in Arabidopsis and Nicotiana benthamiana. Plant

462

AC C

457

463

Res. 34, 436-444.

460 461

464

Cell. 22, 1498-1515.

Azevedo, J., Courtois, F., Lerbs-Mache, S., 2006. Sub-plastidial localization of two different phage-type RNA polymerases in spinach chloroplasts. Nucleic. Acids.

Bang, W.Y., Chen, J., Jeong, I.S., Kim, S.W., Kim, C.W., Jung, H.S., Lee, K.H.,

465

Kweon, H.S., Yoko, I., Shiina, T., Bahk, J.D., 2012. Functional characterization

466

of ObgC in ribosome biogenesis during chloroplast development. Plant J. 71,

467

122-134.

ACCEPTED MANUSCRIPT 468

Bang, W.Y., Hata, A., Jeong, I.S., Umeda, T., Masuda, T., Chen, J., Yoko, I., Suwastika, I.N., Kim, D.W., Im, C.H., Lee, B.H., Lee, Y., Lee, K.W., Shiina, T.,

470

Bahk, J.D., 2009. AtObgC, a plant ortholog of bacterial Obg, is a

471

chloroplast-targeting GTPase essential for early embryogenesis. Plant Mol. Biol.

472

71, 379-390.

473

RI PT

469

Berg, M., Rogers, R., Muralla, R., Meinke, D., 2005. Requirement of

474

aminoacyl-tRNA synthetases for gametogenesis and embryo development in

475

Arabidopsis. Plant J. 44, 866-878.

Chateigner-Boutin, A.L., Ramos-Vega, M., Guevara-Garcia, A., Andres, C., de la Luz,

477

G.M., Cantero, A., Delannoy, E., Jimenez, L.F., Lurin, C., Small, I., Leon, P.,

478

2008. CLB19, a pentatricopeptide repeat protein required for editing of rpoA and

479

clpP chloroplast transcripts. Plant J. 56, 590-602.

M AN U

480

SC

476

Chateigner-Boutin, A.L., des Francs-Small, C.C., Delannoy, E., Kahlau, S., Tanz, S.K., de Longevialle, A.F., Fujii, S., Small, I., 2011. OTP70 is a

482

pentatricopeptide repeat protein of the E subgroup involved in splicing of the

483

plastid transcript rpoC1. Plant J. 65, 532-542.

484

TE D

481

Dong, H., Fei, G.L., Wu, C.Y., Wu, F.Q., Sun, Y.Y., Chen, M.J., Ren, Y.L., Zhou, K.N., Cheng, Z.J., Wang, J.L., Jiang, L., Zhang, X., Guo, X.P., Lei, C.L., Su, N.,

486

Wang, H., Wan, J.M., 2013. A rice virescent-yellow leaf mutant reveals new

487

insights into the role and assembly of plastid caseinolytic protease in higher

488

plants. Plant Physiol. 162, 1867-1880.

EP

485

Duchene, A.M., Giritch, A., Hoffmann, B., Cognat, V., Lancelin, D., Peeters, N.M.,

490

Zaepfel, M., Marechal-Drouard, L., Small, I.D., 2005. Dual targeting is the rule

491 492 493 494 495

AC C

489

for organellar aminoacyl-tRNA synthetases in Arabidopsis thaliana. Proc. Natl. Acad. Sci. 102, 16484-16489.

Guo, M., Yang, X.L., Schimmel, P., 2010. New functions of aminoacyl-tRNA synthetases beyond translation. Nat. Rev. Mol. Cell. Biol. 11, 668-674. Han, M., Jung, K., Yi, G., Lee, D., An, G., 2006. Rice Immature Pollen 1 (RIP1) is a

496

regulator of late pollen development. Plant Cell Physiol. 47, 1457-1472.

497

Hanaoka, M., Kanamaru, K., Fujiwara, M., Takahashi, H., Tanaka, K., 2005.

ACCEPTED MANUSCRIPT 498

Glutamyl-tRNA mediates a switch in RNA polymerase use during chloroplast

499

biogenesis. EMBO REP. 6, 545-550.

500 501

Hausmann, C.D., Ibba, M., 2008. Aminoacyl-tRNA synthetase complexes: molecular multitasking revealed. FEMS Microbiol Rev. 32, 705-721. Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., 1994. Efficient transformation of rice

503

(Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the

504

boundaries of the T-DNA. Plant J. 6, 271-282.

505

RI PT

502

Itoh, J., Nonomura, K., Ikeda, K., Yamaki, S., Inukai, Y., Yamagishi, H., Kitano, H., Nagato, Y., 2005. Rice plant development: from zygote to spikelet. Plant Cell

507

Physiol. 46, 23-47.

Ko, Y.G., Kang, Y.S., Kim, E.K., Park, S.G., Kim, S., 2000. Nucleolar localization of

M AN U

508

SC

506

509

human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis. J.

510

Cell. Biol. 149, 567-574.

511

Ko, Y.G., Kim, E.Y., Kim, T., Park, H., Park, H.S., Choi, E.J., Kim, S., 2001. Glutamine-dependent antiapoptotic interaction of human glutaminyl-tRNA

513

synthetase with apoptosis signal-regulating kinase 1. J. Biol. Chem. 276,

514

6030-6036.

515

TE D

512

Kusumi, K., Hirotsuka, S., Shimada, H., Chono, Y., Matsuda, O., Iba, K., 2010. Contribution of chloroplast biogenesis to carbon-nitrogen balance during early

517

leaf development in rice. J. Plant. Res. 123, 617-622.

519 520 521

Kusumi, K., Iba, K., 2014. Establishment of the chloroplast genetic system in rice during early leaf development and at low temperatures. Front. Plant. Sci. 5, 386.

AC C

518

EP

516

Ling, J., Reynolds, N., Ibba, M., 2009. Aminoacyl-tRNA synthesis and translational quality control. Annu. Rev. Microbiol. 63, 61-78.

522

Liu, C., Zhu, H., Xing, Y., Tan, J., Chen, X., Zhang, J., Peng, H., Xie, Q., Zhang, Z.,

523

2016. Albino Leaf 2 is involved in the splicing of chloroplast group I and II

524

introns in rice. J. Exp. Bot. 67, 5339-5347.

525

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using

526

real-time quantitative PCR and the 2-∆∆Ct method. Methods. 25, 402-408.

527

Millar, A.H., Whelan, J., Small, I., 2006. Recent surprises in protein targeting to

ACCEPTED MANUSCRIPT 528

mitochondria and plastids. Curr. Opin. Plant. Biol. 9, 610-615.

529

Moschopoulos, A., Derbyshire, P., Byrne, M.E., 2012. The Arabidopsis organelle-localized glycyl-tRNA synthetase encoded by EMBRYO DEFECTIVE

531

DEVELOPMENT1 is required for organ patterning. J. EXP. BOT. 63,

532

5233-5243.

533

RI PT

530

Motohashi, R., Yamazaki, T., Myouga, F., Ito, T., Ito, K., Satou, M., Kobayashi, M., Nagata, N., Yoshida, S., Nagashima, A., Tanaka, K., Takahashi, S., Shinozaki,

535

K., 2007. Chloroplast ribosome release factor 1 (AtcpRF1) is essential for

536

chloroplast development. Plant Mol. Biol. 64, 481-497.

537

SC

534

Pfalz, J., Liere, K., Kandlbinder, A., Dietz, K.J., Oelmuller, R., 2006. pTAC2, -6, and -12 are components of the transcriptionally active plastid chromosome that are

539

required for plastid gene expression. Plant Cell. 18, 176-197.

540 541 542

M AN U

538

Pfalz, J., Pfannschmidt, T., 2013. Essential nucleoid proteins in early chloroplast development. Trends Plant Sci. 18, 186-194.

Pyo, Y.J., Kwon, K.C., Kim, A., Cho, M.H., 2013. Seedling Lethal1, a pentatricopeptide repeat protein lacking an E/E+ or DYW domain in Arabidopsis,

544

is involved in plastid gene expression and early chloroplast development. Plant

545

Physiol. 163, 1844-1858.

TE D

543

Qiao, J., Li, J., Chu, W., Luo, M., 2013. PRDA1, a novel chloroplast nucleoid protein,

547

is required for early chloroplast development and is involved in the regulation of

548

plastid gene expression in Arabidopsis. Plant Cell Physiol. 54, 2071-2084.

549

Ruuska, S.A., Schwender, J., Ohlrogge, J.B., 2004. The capacity of green oilseeds to

551 552

AC C

550

EP

546

utilize photosynthesis to drive biosynthetic processes. Plant Physiol. 136, 2700-2709.

Shiina, T., Tsunoyama, Y., Nakahira, Y., Khan, M.S., 2005. Plastid RNA

553

polymerases, promoters, and transcription regulators in higher plants. Int. Rev.

554

Cytol. 244, 1-68.

555

Steiner, S., Schroter, Y., Pfalz, J., Pfannschmidt, T., 2011. Identification of essential

556

subunits in the plastid-encoded RNA polymerase complex reveals building

557

blocks for proper plastid development. Plant Physiol. 157, 1043-1055.

ACCEPTED MANUSCRIPT 558

Sugiura, I., Nureki, O., Ugaji-Yoshikawa, Y., Kuwabara, S., Shimada, A., Tateno, M.,

559

Lorber, B., Giege, R., Moras, D., Yokoyama, S., Konno, M., 2000. The 2.0 A

560

crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals

561

two RNA-binding modules. Structure. 8, 197-208. Sun, J., Zheng, T., Yu, J., Wu, T., Wang, X., Chen, G., Tian, Y., Zhang, H., Wang, Y.,

RI PT

562 563

Terzaghi, W., Wang, C., Wan, J., 2017. TSV, a putative plastidic oxidoreductase,

564

protects rice chloroplasts from cold stress during development by interacting

565

with plastidic thioredoxin Z. New Phytol. 215, 240-255.

Uwer, U., Willmitzer, L., Altmann, T., 1998. Inactivation of a glycyl-tRNA

SC

566

synthetase leads to an arrest in plant embryo development. Plant Cell. 10,

568

1277-1294.

569

M AN U

567

Wang, Y., Wang, C., Zheng, M., Lyu, J., Xu, Y., Li, X., Niu, M., Long, W., Wang, D.,

570

Wang, H., Terzaghi, W., Wang, Y., Wan, J., 2016. WHITE PANICLE1, a

571

Val-tRNA Synthetase Regulating Chloroplast Ribosome Biogenesis in Rice, Is

572

Essential for Early Chloroplast Development. Plant Physiol. 170, 2110-2123. Xu, J., Yang, J., Wu, Z., Liu, H., Huang, F., Wu, Y., Carrie, C., Narsai, R., Murcha,

TE D

573

M., Whelan, J., Wu, P., 2013. Identification of a dual-targeted protein belonging

575

to the mitochondrial carrier family that is required for early leaf development in

576

rice. Plant Physiol. 161, 2036-2048.

577

EP

574

Yagi, Y., Ishizaki, Y., Nakahira, Y., Tozawa, Y., Shiina, T., 2012. Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type

579

plastid RNA polymerase. Proc. Natl. Acad. Sci. 109, 7541-7546.

580 581 582 583 584

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Zhang, Y., Hao, Y., Wang, Y., Wang, C., Wang, Y., Long, W., Wang, D., Liu, X., Jiang, L., Wan, J., 2017. Lethal albinic seedling, encoding a threonyl-tRNA synthetase, is involved in development of plastid protein synthesis system in rice. Plant Cell Rep. 36, 1053-1064. Zhang, Z., Tan, J., Shi, Z., Xie, Q., Xing, Y., Liu, C., Chen, Q., Zhu, H., Wang, J.,

585

Zhang, J., Zhang, G., 2016. Albino Leaf 1 that Encodes the Sole Octotricopeptide

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Repeat Protein Is Responsible for Chloroplast Development. Plant Physiol. 171,

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1182-1191.

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Figure Legends

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Figure 1. Phenotypic characteristics of wild type and ra1 mutant

591

a Phenotypes of 3, 10 and 18 day old seedlings. Scale bars = 3 cm. b Chlorophyll

592

autofluorescence of 3, 10 and 18 day old seedlings. Scale bars = 5 mm.

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Figure 2. Phenotypic characteristics and ultrastructure of chloroplasts of

595

wild-type and ra1 mutant in second leaf

596

a Phenotypes of wild type and ra1 mutants. Scale bars = 5 cm. b pigment

597

concentration in wild type and ra1 seedlings. Error bars, ± SD (n = 3). ** indicate

598

statistical significance at P < 0.01 by t-test. c Leaf cells of wild type. d Ultrastructures

599

of chloroplasts of wild type. e Leaf cells of ra1 mutant. f Ultrastructures of

600

chloroplasts of ra1 mutant. Cp, chloroplast; CW, cell wall; Thy, Thylakoid; OG,

601

osmiophilic plastoglobuli.

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Figure 3. Map-based cloning of the RA1 gene

604

a. The RA1 gene was located near to SSR markers RM1369 on the short arm of

605

chromosome 6. Fine mapping, RA1 gene was located between RM1369 and the

606

telomere of chromosome 6. The physical distance of this interval is 266-kb. b.

607

Structure of LOC_Os06g01400. ATG and TAG, represent the start codon and the stop

608

codons, respectively. Black boxes represent exons and lines between black boxes

609

represent introns. Empty boxes at the top and bottom represent the 5 'UTR and the 3'

610

UTR, respectively. On the sixth exon, the T to A mutation of the mutant is indicated

611

by a red box. c. Sequencing peak pattern of mutation site. d. The dCAPs markers

612

were used to verify the mutation sites.

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Figure 4. Genetic complementation and CRISPR/Cas9 validate the RA1 gene

615

a Complementation experiments validated the seedling phenotype of RA1 gene. Scale

ACCEPTED MANUSCRIPT 616

bars = 5 cm. b The RA1 gene was edited using CRISPR/Cas9 technology and the

617

phenotype of seedlings. c Comparisons of transgene positive and wild type sequences

618

using CRISPR/Cas9. Scale bars = 5 cm.

619

Figure 5. Tissue analysis of RA1 gene and subcellular localization

621

a Expression analysis of the RA1 gene in various tissues. The UBQ gene was used as

622

a control. Error bars, ± SD (n = 4). b RA1 subcellular localization signals coincide

623

with chlorophyll autofluorescence. Scale bars = 20 µm.

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Figure 6. Quantitative RT-PCR analysis of chlorophyll biosynthesis related gene

626

expression levels

627

a Expression level of chlorophyll biosynthesis related gene, and UBQ was as a control.

628

Error bars, ± SD (n = 4). * and ** indicate statistical significance at P < 0.05 and P <

629

0.01 by t-test. b Expression level of plastid development related gene, and UBQ was

630

as a control. Error bars, ± SD (n = 4). * and ** indicate statistical significance at P <

631

0.05 and P < 0.01 by t-test.

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Figure 7. Transcript and immunoblots analysis of plastid development related

634

genes and rRNA analysis of wild type and ra1 mutant

635

a RBCL was detected by Coomassie blue staining. b The protein level of chloroplast

636

related genes was detected by western blot. Hsp90 was used as an internal control. c

637

Wild type and ra1 were analyzed with Agilent 2100. The red boxes indicate the

638

absence of 16S and 23S in ra1, respectively. d rRNA analysis results using Agilent

639

2100. The rRNAs isolated from 7 day old WT and ra1 seedlings.

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Supplementary Figure 1 Protein structure and sequence analysis of RA1. a Protein

642

structure of RA1. b Conserved residues or similarity of the aligned sequences are

643

depicted in the consensus sequence with lowercase. The mutation site of amino acid

644

in RA1 (OsGRS2) is represented by a triangle. Abbreviation: GRS, glycyl-tRNA

645

synthetase; At, Arabidopsis thaliana; Bd, Brachypodium distachyon; Zm, Zea mays;

ACCEPTED MANUSCRIPT 646

Sb, Sorghum bicolor; Tu, Triticum urartu; Gm, Glycine max; Vv, Vitis vinifera; Pt,

647

Populus trichocarpa; Nt, Nicotiana tomentosiformis; Sl, Solanum lycopersicum.

648

Supplementary Figure 2 The phylogenetic tree of RA1 protein and its homologous

650

proteins in different species. Numbers above the branch sites indicate bootstrapping

651

values. RA1 is shown in a red box.

652

Supplementary Figure 3 Quantitative RT-PCR analysis of the RA1 expression levels

653

of wild type and ra1. The sample is young leaves taken from 7-day-old seedlings.

654

Error bars, ± SD (n = 4). * indicate statistical significance at P < 0.05 by t-test.

655

Supplementary Table 1 Primer sequences used in this study.

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Supplementary Table 2 The predicated genes within the 266-kb region.

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Figure 1. Phenotypic characteristics of wild type and ra1 mutant a Phenotypes of 3, 10 and 18 day old seedlings. Scale bars = 3 cm. b Chlorophyll

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autofluorescence of 3, 10 and 18 day old seedlings. Scale bars = 5 mm.

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Figure 2. Phenotypic characteristics and ultrastructure of chloroplasts of wild-type and ra1 mutant in second leaf a Phenotypes of wild type and ra1 mutants. Scale bars = 5 cm. b pigment concentration in wild type and ra1 seedlings. Error bars, ± SD (n = 3). ** indicate statistical significance at P < 0.01 by t-test. c Leaf cells of wild type. d Ultrastructures of chloroplasts of wild type. e Leaf cells of ra1 mutant. f Ultrastructures of chloroplasts of ra1 mutant. Cp, chloroplast; CW, cell wall; Thy, Thylakoid; OG, osmiophilic plastoglobuli.

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Figure 3. Map-based cloning of the RA1 gene a. The RA1 gene was located near to SSR markers RM1369 on the short arm of chromosome 6. Fine mapping, RA1 gene was located between RM1369 and the telomere of chromosome 6. The physical distance of this interval is 266-kb. b. Structure of LOC_Os06g01400. ATG and TAG, represent the start codon and the stop codons, respectively. Black boxes represent exons and lines between black boxes represent introns. Empty boxes at the top and bottom represent the 5 'UTR and the 3' UTR, respectively. On the sixth exon, the T to A mutation of the mutant is indicated by a red box. c. Sequencing peak pattern of mutation site. d. The dCAPs markers were used to verify the mutation sites.

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Figure 4. Genetic complementation and CRISPR/Cas9 validate the RA1 gene a Complementation experiments validated the seedling phenotype of RA1 gene. Scale bars = 5 cm. b The RA1 gene was edited using CRISPR/Cas9 technology and the

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phenotype of seedlings. c Comparisons of transgene positive and wild type sequences

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using CRISPR/Cas9. Scale bars = 5 cm.

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Figure 5. Tissue analysis of RA1 gene and subcellular localization

a Expression analysis of the RA1 gene in various tissues. The UBQ gene was used as a control. Error

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Scale bars = 20 µm.

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bars, ± SD (n = 4). b RA1 subcellular localization signals coincide with chlorophyll autofluorescence.

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Figure 6. Quantitative RT-PCR analysis of chlorophyll biosynthesis related gene expression levels

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a Expression level of chlorophyll biosynthesis related gene, and UBQ was as a control. Error bars, ± SD (n = 4). * and ** indicate statistical significance at P < 0.05 and P < 0.01 by t-test. b Expression level of plastid development related gene, and UBQ was

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as a control. Error bars, ± SD (n = 4). * and ** indicate statistical significance at P < 0.05 and P < 0.01 by t-test.

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Figure 7. Transcript and immunoblots analysis of plastid development related genes and rRNA

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analysis of wild type and ra1 mutant

a RBCL was detected by Coomassie blue staining. b The protein level of chloroplast related genes was

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detected by western blot. Hsp90 was used as an internal control. c Wild type and ra1 were analyzed with Agilent 2100. The red boxes indicate the absence of 16S and 23S in ra1, respectively. d rRNA analysis results using Agilent 2100. The rRNAs isolated from 7 day old WT and ra1 seedlings.

ACCEPTED MANUSCRIPT Author contribution statement Hai Zheng, Zhuoran Wang and Feng Lv conducted the experiments. Hai Zheng, Peiran Wang, Xi Liu. Ling Jiang designed the research. Hai Zheng and Wenting Bai conducted western blot experiments. Yunlu Tian and Weiyi Kong found the mutant

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and analyzed the data. Chaolong Wang and Xiaowen Yu guide the writing of manuscripts. Hai Zheng wrote the manuscript. Zhigang Zhao and LingLong Liu

edited and modify the manuscript. Hai Zheng and Zhuoran Wang have contributed

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equally to this work. All authors read and approved the manuscript.