A genome-wide identification and analysis of the DYW ... fileRESEARCH ARTICLE A genome-wide...

RESEARCH ARTICLE

A genome-wide identification and analysis

of the DYW-deaminase genes in the

pentatricopeptide repeat gene family in

cotton (Gossypium spp.)

Bingbing Zhang1, Guoyuan Liu1, Xue Li1, Liping Guo1, Xuexian Zhang1, Tingxiang Qi1,

Hailin Wang1, Huini Tang1, Xiuqin Qiao1, Jinfa Zhang2, Chaozhu Xing1*, Jianyong Wu1*

1 State Key Laboratory of Cotton Biology (China)/Institute of Cotton Research of Chinese Academy of

Agricultural Science, Anyang, Henan, China, 2 Department of Plant and Environmental Sciences, New

Mexico State University, Las Cruces, New Mexico, United States of America

* [email protected] (JW); [email protected] (CX)

Abstract

The RNA editing occurring in plant organellar genomes mainly involves the change of cyti-

dine to uridine. This process involves a deamination reaction, with cytidine deaminase as

the catalyst. Pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain are

reportedly associated with cytidine deamination, similar to members of the deaminase

superfamily. PPR genes are involved in many cellular functions and biological processes

including fertility restoration to cytoplasmic male sterility (CMS) in plants. In this study, we

identified 227 and 211 DYW deaminase-coding PPR genes for the cultivated tetraploid cot-

ton species G. hirsutum and G. barbadense (2n = 4x = 52), respectively, as well as 126 and

97 DYW deaminase-coding PPR genes in the ancestral diploid species G. raimondii and

G. arboreum (2n = 26), respectively. The 227 G. hirsutum PPR genes were predicted to

encode 52–2016 amino acids, 203 of which were mapped onto 26 chromosomes. Most

DYW deaminase genes lacked introns, and their proteins were predicted to target the mito-

chondria or chloroplasts. Additionally, the DYW domain differed from the complete DYW

deaminase domain, which contained part of the E domain and the entire E+ domain. The

types and number of DYW tripeptides may have been influenced by evolutionary processes,

with some tripeptides being lost. Furthermore, a gene ontology analysis revealed that DYW

deaminase functions were mainly related to binding as well as hydrolase and transferase

activities. The G. hirsutum DYW deaminase expression profiles varied among different cot-

ton tissues and developmental stages, and no differentially expressed DYW deaminase-

coding PPRs were directly associated with the male sterility and restoration in the CMS-D2

system. Our current study provides an important piece of information regarding the struc-

tural and evolutionary characteristics of Gossypium DYW-containing PPR genes coding for

deaminases and will be useful for characterizing the DYW deaminase gene family in cotton

biology and breeding.

PLOS ONE | https://doi.org/10.1371/journal.pone.0174201 March 24, 2017 1 / 21

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OPENACCESS

Citation: Zhang B, Liu G, Li X, Guo L, Zhang X, Qi

T, et al. (2017) A genome-wide identification and

analysis of the DYW-deaminase genes in the

pentatricopeptide repeat gene family in cotton

(Gossypium spp.). PLoS ONE 12(3): e0174201.

https://doi.org/10.1371/journal.pone.0174201

Editor: David D. Fang, USDA-ARS Southern

Regional Research Center, UNITED STATES

Received: November 30, 2016

Accepted: March 6, 2017

Published: March 24, 2017

Copyright: © 2017 Zhang et al. This is an open

access article distributed under the terms of the

Creative Commons Attribution License, which

permits unrestricted use, distribution, and

reproduction in any medium, provided the original

author and source are credited.

Data Availability Statement: All relevant data are

within the paper and its Supporting Information

files. All of the raw sequence data from the Illumina

sequencing platform can be found in the Short

Read Archive (SRA) database of the National

Center for Biotechnology Information (NCBI) under

accession number SRX2578795.

Funding: This research was financed by funds

from the National Key Research and Development

program of China (2016YFD0101400) and Cotton

Germplasm Innovation and the Molecular breeding


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http://creativecommons.org/licenses/by/4.0/

Introduction

RNA editing is a post-transcriptional process affecting organellar transcripts that results in

nucleotide changes from DNA to RNA. There are various types of RNA editing in different

organisms. In mammals, a zinc-dependent enzyme related to cytidine deaminase is responsi-

ble for converting cytidines (C) to uredines (U) in apoB mRNA [1, 2]. In vascular plants, RNA

editing occurs in chloroplasts and mitochondria, and almost exclusively involves C-to-U

changes [2]. Additionally, nuclear genes encoding proteins involved in RNA editing belong to

the gene subfamily associated with pentatricopeptide repeat (PPR) proteins.

The PPR protein family is one of the largest protein families in higher plants [3]. The PPR

proteins usually have 2–27 repeating motifs consisting of 35 amino acids arranged in clusters

[3, 4]. These motifs are divided into three classes. The canonical PPR motifs (i.e., P motifs) are

common among eukaryotes, while the short PPR-like (i.e., S motifs) and long PPR-like motifs

(i.e., L motifs) are present only in higher plants [4]. The PPR protein family is divided into two

subfamilies (i.e., P and PLS subfamilies) based on the different motifs. The P subfamily mem-

bers contain only the P motif, while the PLS subfamily members usually consist of a tandem

array of degenerated P, L, and S motifs. The PLS subfamily proteins are plant-specific and usu-

ally contain an additional C-terminal extension with E, E+, and DYW domains. According to

the C-terminal extension, this subfamily can be divided into the following four subgroups:

PLS, E, E+, and DYW [4]. Arabidopsis thaliana carries 87 genes encoding PPR proteins with

the DYW domain. The DYW domain is named for the frequent presence of an Asp–Tyr–Trp

tripeptide at the C-terminal, and has been detected only in the proteins of upland plants. The

E and E+ domains are usually highly degraded and difficult to identify. The DYW domain

contains a highly conserved region with α-β-α secondary structure consisting of conserved

amino acids (e.g., Cys and His) [5].

The DYW domain is the candidate catalytic domain for cytidine deaminase. The HxExx. . .

CxxCH motifs in the DYW domain align with the active site of cytidine deaminase (i.e., C/

HxExx. . .xPCxxC). Additionally, the DYW domain is highly evolutionarily correlated with

RNA editing. Thus, proteins containing the DYW domain have been classified as deaminases

involved in RNA editing [6, 7]. As such, proteins are grouped into the nucleic acid deaminase

superfamily (i.e., DYW deaminase family; Pfam: Pf14432) according to DYW domain charac-

teristics. The DYW deaminase domain contains part of the E domain, the whole E+ domain,

and most of the DYW domain [7]. Recently, Hayes et al. (2015) suggested that the conserved

Glu residue of the HXE motif in the DYW domain is necessary for RNA editing. When the

codon for the Glu residue in the DYW domain of OTP84 and CREF7 was mutagenized, the

transgenic plants containing the mutated genes could not efficiently edit the cognate editing

sites [8]. Wagoner et al. (2015) proposed that the DYW domain and the E (or E+) domain are

essential for cytidine deaminase activity because truncating either domain resulted in a QED1

protein that completely lacked any editing activity. In contrast, the DYW domain was revealed

to be unnecessary for four RNA editing factors (i.e., CRR22, CRR28, OTP82, and ELI1) [9].

The DYW deaminase domain is different from the DYW domain in PPR proteins, which is

essential for the RNA editing activity of some enzymes but not for others. Thus, the role of the

DYW domain during RNA editing has not been fully characterized. Boussardon et al. (2012)

suggested that the interaction between CRR4 and DYW1 contributes to the editing of the

ndhD-1 site. The DYW domain, which functioned as the catalytic domain, may have been lost

in CRR4, which now requires the DYW domain from DYW1 to provide catalytic activity. The

DYW1 protein carrying the E+ and DYW domains may interact with CRR4 to edit the ndhD

C2 site [10]. In this case, CRR4 may use the DYW1 as the catalytic domain. In summary, as

A genome-wide identification and analysis of the DYW-deaminase genes in cotton


of High yield varieties in National Natural Science

Foundation of China (31621005).

Competing interests: The authors have declared

that no competing interests exist.


one of the most specific subgroups of the PPR protein family, the DYW deaminase domain-

containing PPR proteins are particularly striking because of their RNA editing activities.

Cotton is an economically important fiber crop as the source of the most important natural

textile fiber. The genus Gossypium contains approximately 45 diploid species and five poly-

ploid species. Crossing between the extant of diploid species G. arboreum and G. raimondiiand chromosome doubling 1–2 million years ago resulted in the emergence of allotetraploid

cotton species, including cultivated G. hirsutum (accounting for ca. 95% world cotton produc-

tion) and G. barbadense [11]. The genomes of the two ancestral diploids (i.e., G. arboreum and

G. raimondii) and the two tetraploids (i.e., G. hirsutum and G. barbadense) have recently been

sequenced, providing an opportunity to study cotton gene families. PPR genes including these

responsible for fertility restoration to cytoplasmic male sterility (CMS) have been cloned with

functions analyzed in several plant species. For instance, Rf4 encoded a PPR protein for wild

abortive-type CMS of rice, was confirmed to reduce the orf352-containing transcripts to

restore pollen fertility [12], and a PPR protein Rf6 function with hexokinase 6 (OsHXK6) to

promote the processing of the aberrant CMS-associated transcript atp6-orfH79 to rescue

HL-CMS of rice [13]. In sorghum, two PPR genes were identified for fertility restoration of A1

cytoplasm [14, 15]. However, no such information is currently available in cotton. In this

study, we performed an in silico analysis of the four Gossypium genome sequences to identify

PPR genes encoding for the DYW-containing deaminases. We focused on gene structure and

expression with a specific focus on CMS and fertility restoration in G. hirsutum. The results

of this study will contribute to the understanding of the distribution and functions of DYW

deaminase genes in cotton.

Materials and methods

Materials

The Gossypium hirsutum variety Zhong10 was used for different tissue expression analysis.

Materials were planted under greenhouse. Roots, stems and leaves were collected in one-

month-old seedlings, flowers were collected from plants grown two-month-old on the day of

flowering, all tissue samples were obtained from 3 individual plants. Cytoplasm male sterility

(CMS-D2) A S(rf1rf1) with cytoplasm from Gossypium harknessii and its isogenic restorer line

R S(Rf1rf1) were planted under normal conditions in the farm. Floral buds about 3 mm in

length (nearly represent the stage of male meiosis) were collected from the near isogenic line

with three biological replicates as previous studies, respectively [16, 17]. All collected samples

were immediately frozen in liquid nitrogen and stored at -76˚C before use. Above the materials

were provided by Cotton Research Institute (CRI), Chinese Academy of Agricultural Science

(CAAS), Anyang, Henan, China.

Local databases of sequence data and RNA-seq

Local databases including genome sequence and annotation information files of Gossypiumspecies (G. raimondii, G. arboreum, G. hirsutum and G. barbadense (xinhai)) and other organ-

isms (Cyanidioschyzon merolae, Physcomitrella patens, Selaginella moellendorffii, Sorghumbicolor, Oryza sativa; Japonica Group) were established. The genome sequence and annota-

tion information of Gossypium species was downloaded from the Cottongen (https://www.

cottongen.org/). Other organism genome information was downloaded from the NCBI. Arabi-

dopsis thaliana genome sequencewere downloaded from TAIR (http://www.arabidopsis.org/).

The extraction of total RNA from flower buds about 3 mm in length (at roughly the stage of male

meiosis) of CMS cotton (CMS-D2) A and its isogenic restorer line R was performed by using

Spectrum™ Plant Total RNA Kit according to the manufacturer’s guide. RNA concentration was



https://www.cottongen.org/

https://www.cottongen.org/

http://www.arabidopsis.org/


measured using NANODROP 2000. Six transcriptome libraries (A1-3, R1-3) were con-

structed by using equal amounts of RNA from the three biological replicates. The libraries

preparation and sequencing were performed following standard Illumina methods on the

Illumina sequencing platform HiSeq™ 2500 system (2×150bp read length). All of the raw

sequence data from the Illumina sequencing platform can be found in the Short Read

Archive (SRA) database of the National Center for Biotechnology Information (NCBI)

under accession number SRX2578795. The FPKMs (mean value of three biological repli-

cates) of gene was further used to perform a differential expression analysis. The criterion

was set as: fold change = |log2 (sample1/sample2) |� 1 and P� 0.05. The differentially

expressed genes between CMS cotton (CMS-D2) A and its isogenic restorer line R were

shown in S1 Table. RNA-seq data of gene expression during fiber development stages at 5,

10, 20, 25 dpa and in root, stem, leaf, petal, stamen, and pistil in G. hirsutum were down-

loaded from ccNET database (http://structuralbiology.cau.edu.cn/gossypium/) [18].

DYW-deaminase proteins sequence identification and domain analysis

Download hidden Markov model information (PF14432) of the unique DYW deaminase

domain from Pfam29.0 database, then use Pfam file PF14432 to retrieve annotated protein

sequences file of Gossypium species and other organisms with Hmmsearch program (E value as

the default) in the HMMER 3.0 software, obtained sequences above the threshold from totally

protein sequences. Domain analysis manually one by one on candidate protein sequences were

performed by using online software SMART (http://smart.embl-heidelberg.de/), removed these

sequences without DYW deaminase domain.

Genome wide synteny analysis of DYW deaminase genes

A comparative syntenic map of the DYW deaminase genes from tetraploid cotton species and

two diploid cotton species was constructed by using circos-0.69–3 software package, with

parameters set as the default.

Conserved domain alignment and analysis of DYW deaminase proteins

in Gossypium

By using MEME online software analysis, DYW deaminase protein sequences was used to

obtain consensus sequence and Logo of DYW domain and DYW deaminase domain, and

compared consensus sequence with tomato and Arabidopsis thaliana.

Phylogenetic tree construction, gene structure analysis, chromosomal

and subcellular location of DYW-deaminase proteins

The multiple Sequence alignment of DYW deaminase domain sequence in four cotton species

was accomplished by using ClustalX2 software with parameters set as default; the nonroot phy-

logenetic tree was constructed by the neighbour joining tree (NJ), the bootstrap set for 1000

replications in MEGA 6 software.

According to GFF information, the gene structure display system of Peking University

(http://gsds.cbi.pku.edu.cn/) was used to draw the gene structure and DYW deaminase gene

were mapped on chromosomes by using Mapchart software.

Using the signal peptide prediction program Target P to predict the subcellular location of

DYW-deaminase proteins.



http://structuralbiology.cau.edu.cn/gossypium/

http://smart.embl-heidelberg.de/

http://gsds.cbi.pku.edu.cn/


The GO and expression analysis of DYW-deaminase genes

GO analysis of DYW-deaminase genes completed by blast2go software, and the picture was

drawn by online software WEGO (http://wego.genomics.org.cn/cgi-bin/wego/index.pl).

The extraction of RNA from root, stem, leaf and flower was performed using RNAprep

Pure Plant kit (TIANGEN, China). Equal amounts of RNA from three biological replicates

were equally pooled together and constructed the total RNA of each sample. Reverse transcrip-

tion was conducted by using 0.5 μg total RNA with PrimeScriptTMRT reagent kit (Takara Bio

Inc., Dalian, China) to 10 μl reaction volume following the manufacturer’s guides and diluted

to 100 μl before use. For real-time PCR, 1 μl diluted cDNA was mixed with 10 μl 2 × SYBR

Green Mix (Takara) and 1.2 μl primers in a total volume of 20 μl. The PCR was performed

using SYBR Green as fluorescence dye and run on Eppendorf Real plex system with melt curve

program. The PCR amplification reaction was performed as follows: 94˚C for 3 min, 40 cycles

of 94˚C for 5 s, 56˚C for 20 s and 72˚C for 25 s, then ending with the melting curve. Ghhis3was used as the reference gene for normalization. All reactions were done in three biological

replicates, the melting curve was used for each qRT-PCR to determine PCR performance,

Ct value in three biological replicates of each gene in four tissues was calculated by 2-44Ct

method [19]. Primers of 16 selected DYW deaminase genes were designed through Primer

Premier 5.0 (Table 1).

Results

Identification and structural analysis of Gossypium genes encoding

DYW deaminases

A total of 227 G. hirsutum genes were identified as encoding DYW deaminases belonging to the

PPR family. Most of these proteins contained multiple PPR domains, and 190 proteins con-

tained intact DYW deaminase domain structures. An analysis of the 227 predicted G. hirsutumDYW deaminases indicated that the number of amino acids in the protein family ranged from

52 to 2016. Furthermore, we identified 126, 97, and 211 DYW deaminases in G. raimondii, G.

arboreum, and G. barbadense, respectively. Information about the DYW deaminase genes in

Table 1. The primers of 16 selected PPR DYW deaminase genes for quantitative RT-PCR.

Gene ID Forward primer(5’- 3’) Reverse primer(5’- 3’)

Gh_D04G0152 CTCCCCTTTGGATTCTCGTT ACCAAGTAAACCCAACTTCGC

Gh_D09G0761 AAATGCCTGGACGGAATGTT AACCGCCTCATCAACTCTACC

Gh_A02G0419 TCCCATCACCTAACATCGTCTC TGGTTGGGGAGAATAGGGTC

Gh_D08G2381 TTTACATGGTCGAGGAAGGGA TGAAGTGCTCAACTCCAGGCT

Gh_D05G0911 AAGCAGGTCTACTGGAAAACGC CGTATATTTTTTCAGATTCGGGGTG

Gh_A03G1665 TGTCGGAACCTGCCTCATT TGACCCGCTTCAACTAAACCT

Gh_A01G1752 CTTGTTCAAAATTGGGTGCC TGCTTTCTTGCCTTGACCAT

Gh_A07G1662 TGTTATCAGTGCTTGTGCGGG CCATTCTTACTGCCCCTGCT

Gh_A12G0594 AACAAACCCGAAACAGCCC CATCAAGTTCCAAGAAACGACG

Gh_D05G0556 TTGCTCTTATGAATACTCCACCAGG CCTTGAAATGGTGAAACCGACT

Gh_D02G1741 GTTACTTGAAGACGGGCGACA GTTTGCTTGATTACTCCCCGTT

Gh_D01G0153 TGGACCCTGATGATACTGCG TCTTGCACCCATCACGCTTC

Gh_A02G0212 TGGTCGTTGATTTGTTAGGCAG GCTCCCAGGATGTTTAGGTTCA

Gh_A12G0486 GCATACGGATGTAGAGGATTTGG ACGGCAGACCTGGCTGTTAT

Gh_A13G1386 AACCGAATGGAGGGCGTAA CCGATGTCCCACAAAGTTCA

Gh_A01G1103 GGGAAACTCTTCATGCTTACACC TGCTGCTTTACCTTGACCGT

https://doi.org/10.1371/journal.pone.0174201.t001



http://wego.genomics.org.cn/cgi-bin/wego/index.pl



Gossypium is summarized in S2 Table. All of the DYW deaminases in the four sequenced cotton

species had similar protein structures and contained the DYW deaminase domain. Addition-

ally, most of the DYW deaminases also contained PPR domain tandem repeats. Most of the

PPR genes had no introns, which is also an important structural feature of the sequences in

DYW deaminase genes. In G. hirsutum, 81% of the genes lacked an intron, while 12% contained

only one intron and 7% consisted of two or more introns (S1 and S2 Figs). Similar proportions

were observed in G. raimondii (i.e., 75, 16, and 9%, respectively) and G. arboreum (i.e., 81, 12

and 7%, respectively). However, in G. barbadense, only 47% of the DYW deaminase genes

lacked introns, while the proportion of genes with introns was relatively high (i.e., 24% with one

intron and 27% with two or more introns).

Conserved sequences in the DYW and DYW deaminase domains

All of the 227 analyzed Upland cotton protein sequences contained the DYW deaminase domain.

Additionally, 190 proteins contained a complete and conserved DYW domain, while the remain-

ing 37 proteins contained only a degenerated E+ domain or part of the DYW domain. Using

Upland cotton as an example, we obtained the consensus sequences and motif logos for the DYW

domains of 68 randomly selected G. hirsutum protein sequences using the MEME online pro-

gram. We also compared the sequences of these proteins with the DYW domain consensus

sequences from tomato and Arabidopsis thaliana (Fig 1). The obtained consensus sequence of the

G. hirsutum DYW domain was as follows: AGYvPDTSFVLHDveEEeKEXMLXYHSEkLAiAFGlis

TPpGTPIRifKNLRVCGDCHtAiKlISKItGREIiVRDsNRFHHFKdGSCSCGDYW.

A comparison of the domain sequences revealed that the DYW domains of the above three

different species were similar (Fig 1). The sequences contained conserved HxEx. . .CxxCH

motifs, which corresponded to the cytidine deaminase active site (i.e., C/HxEx. . .CxxC). Cyti-

dine deaminase is a Zn-dependent enzyme in which the CxxCH motif functions as a binding

site [20]. The conserved His and Cys residues in the DYW domain combine with Zn ions,

while the conserved Glu is required for deamination [8]. Thus, the DYW domain may be

involved in nucleotide deaminations. Additionally, a previous study revealed that if CxxCH in

the DYW domain mutated into GxxGH, the RNA cleavage activity decreases significantly [5],

suggesting that the CxxCH motif in the DYW domain is necessary for endonuclease activity.

Intriguingly, we detected a CxCx motif near the conserved DYW tripeptide or its variants,

which may be crucial for protein functions. These results indicate that the DYW domain may

have multiple functions.

Fig 1. a: The Logo of DYW domain in G. hirsutum. The higher the letter, the higher the conservation; b: The comparison of DYW domain consensus

sequence in upland cotton, tomato and Arabidopsis. The capital letters represent highly conserved, lowercase letters indicate lower conservative.

https://doi.org/10.1371/journal.pone.0174201.g001





We also obtained the complete consensus sequence of the G. hirsutum DYW deaminase

domain containing part of the E domain, the whole E+ domain, and the DYW domain (Fig 2).

We compared this sequence with the DYW deaminase domain sequences from other three

cotton species. Furthermore, we obtained a conserved PG box sequence of the E and E+

domains as described by Hayes et al. (2013). The PG box may bind to different factors to

generate editing components, although the role of the PG box in editing events has not been

clarified [21]. The intact DYW deaminase domain sequences were highly similar among the

different analyzed cotton species, and contained the PG box, HxEx. . .CxxCH, and CxCxDYW

motifs. These results indicate the DYW deaminase and DYW domains may exhibit similar

molecular characteristics and functions.

Fig 2. a: The alignment of the DYW deaminase domain in G. hirsutum; b: The Logo of DYW deaminase domain in G. hirsutum; c: The

consensus sequence in G. hirsutum and comparison of DYW deaminase domain in Gossypium. The capital letters represent highly

conserved, lowercase letters indicate lower conservative.






Chromosomal localization and evolutionary relationships among

Gossypium DYW deaminase genes

Of the identified 227 G. hirsutum DYW deaminase genes, 203 genes were mapped to chromo-

somes, with 97 genes localized in the A subgenome and 106 genes localized in the D subgenome

(Fig 3). Each chromosome contained an average of 7.8 DYW deaminase genes. In the A subge-

nome, chromosomes 4 and 10 carried the fewest DYW deaminase genes (i.e., only four each),

while chromosome 12 contained the most (i.e., 14). In the D subgenome, chromosome 3 and 10

had the fewest DYW deaminase genes (i.e., only 3 and 4, respectively), and chromosome 12 car-

ried the most DYW deaminase genes (i.e., 12). Furthermore, some genes clustered together on

chromosomes. For example, four DYW deaminase genes localized within a 3.2-Mb region on

chromosome 5 in the D subgenome (i.e., Gh_D05G0556,Gh_D05G0696,Gh_D05G0877, and

Gh_D05G0911), while five DYW deaminase genes were mapped to a 2.9-Mb region on chromo-

some 12 of the same subgenome. Additionally, in G. hirsutum, the location of genes on chromo-

somes 9, 10, and 13 in the A subgenome exhibited a good collinearity with that on homoeologous

chromosomes 9, 10, and 13 in the D subgenome. In contrast, the chromosomal localization of

DYW deaminase genes in G. raimondii and G. arboreum did not exhibit a good collinearity with

that in G. hirsutum because of differences in gene evolution (S2 Fig).

Due to the evolutionary relationships between diploid and tetraploid cotton species, The DYW

deaminases in the A and D subgenomes of the tetraploid species are homologous to those in dip-

loid species G. arboreuum and G. raimondii, respectively. We analyzed the evolutionary relation-

ships between the G. hirsutum and G. barbadense DYW deaminases and their corresponding

proteins in G. raimondii and G. arboreum. An analysis of the DYW deaminases in the two tetra-

ploid cotton species revealed that the G. hirsutum proteins had more tandem duplications. A com-

parative syntenic map of the DYW deaminase genes from tetraploid cotton species and the two

diploid cotton species was constructed for a further analysis of genetic origins and evolution (Fig

4). During the evolution of Gossypium DYW deaminase genes, one G. hirsutum gene and seven

G. barbadense genes did not have homologous genes in the two diploid cotton species (S2 Table).

These genes may be new, with distinct functions in tetraploid cotton species. Additionally, 19 and

36 G. raimondii genes and 11 and 25 G. arboreum genes are not present in the G. hirsutum and G.

Gh_A01G01441.3Gh_A01G02442.4

Gh_A01G087620.6

Gh_A01G110342.4

Gh_A01G142088.6Gh_A01G167495.3Gh_A01G175296.9Gh_A01G176697.1Gh_A01G180797.9

Gh_A02G00740.6Gh_A02G01762.0Gh_A02G02122.4Gh_A02G04195.4

Gh_A02G088930.7

Gh_A02G119968.3

Gh_A02G160682.6

Gh_A03G03777.2

Gh_A03G058715.5

Gh_A03G085047.3

Gh_A03G130490.8

Gh_A03G166597.8

Gh_A04G00460.5

Gh_A04G060040.9

Gh_A04G083454.3

Gh_A04G100460.0

Gh_A05G05676.1Gh_A05G07457.7

Gh_A05G163016.9

Gh_A05G291770.6

Gh_A05G344089.5

Gh_A06G00160.1

Gh_A06G056914.7

Gh_A06G100348.7

Gh_A06G112070.7

Gh_A06G153399.3Gh_A06G1611101.1

Gh_A07G01832.3Gh_A07G02903.5


Gh_A07G166267.2

Gh_A07G188874.3Gh_A07G214378.1

Gh_A08G058210.2

Gh_A08G138288.6Gh_A08G149491.5Gh_A08G181098.7Gh_A08G1988101.0Gh_A08G1996101.1

Gh_A09G00260.6

Gh_A09G075953.7

Gh_A09G115663.0Gh_A09G117263.2Gh_A09G130365.7Gh_A09G163870.0Gh_A09G192172.4Gh_A09G2140 Gh_A09G214374.5

Gh_A10G03293.0

Gh_A10G06179.7

Gh_A10G104742.2

Gh_A10G177092.2

Gh_A11G05755.5Gh_A11G06326.1Gh_A11G07797.7Gh_A11G122415.1Gh_A11G132917.1Gh_A11G138418.0

Gh_A11G203360.8

Gh_A11G227878.2Gh_A11G234280.4

Gh_A11G265788.8Gh_A11G282191.5

Gh_A12G04298.8Gh_A12G04519.7Gh_A12G04589.8Gh_A12G048610.8Gh_A12G052412.8Gh_A12G059414.9Gh_A12G064018.2

Gh_A12G072433.6

Gh_A12G083753.8

Gh_A12G129568.6


Gh_A13G03474.6

Gh_A13G066519.8

Gh_A13G076832.1

Gh_A13G094150.4

Gh_A13G103958.7Gh_A13G115563.8

Gh_A13G138670.5

Gh_A13G172276.2

Gh_D01G01531.1Gh_D01G01891.6Gh_D01G02422.1

Gh_D01G091215.3

Gh_D01G117826.9

Gh_D01G165852.4Gh_D01G199658.8Gh_D01G200958.9Gh_D01G204659.5

Gh_D02G00890.6Gh_D02G01291.1Gh_D02G01351.2Gh_D02G02402.8Gh_D02G04726.2

Gh_D02G116435.1

Gh_D02G174159.7

Gh_D02G208065.0

Gh_D03G01170.9

Gh_D03G087130.2

Gh_D03G116538.2

Gh_D04G01522.2

Gh_D04G070314.1

Gh_D04G106134.7

Gh_D04G133343.1Gh_D04G148346.7Gh_D04G155447.6

Gh_D05G00510.6Gh_D05G00780.8Gh_D05G05564.5Gh_D05G06965.7Gh_D05G08777.4Gh_D05G09117.7Gh_D05G181216.5

Gh_D05G368861.4

Gh_D06G02412.6

Gh_D06G064411.0

Gh_D06G137442.9

Gh_D06G190259.9Gh_D06G198161.4

Gh_D07G02382.6Gh_D07G03453.6

Gh_D07G106715.1Gh_D07G107015.2

Gh_D07G138022.3

Gh_D07G165834.2Gh_D07G166234.4

Gh_D07G187645.9Gh_D07G210351.2Gh_D07G235555.2

Gh_D08G03373.4

Gh_D08G06779.1

Gh_D08G167752.6Gh_D08G179254.5Gh_D08G200758.5Gh_D08G217061.2Gh_D08G238163.7Gh_D08G239063.8

Gh_D09G00240.7

Gh_D09G076131.9Gh_D09G116238.3Gh_D09G117838.6Gh_D09G130940.3Gh_D09G173245.1Gh_D09G212948.5Gh_D09G2346 Gh_D09G234850.5

Gh_D10G03352.9

Gh_D10G07839.3

Gh_D10G147233.6

Gh_D10G204256.2

Gh_D11G06605.7Gh_D11G07426.4Gh_D11G09087.8Gh_D11G137113.3Gh_D11G139813.8Gh_D11G148014.8Gh_D11G152615.3

Gh_D11G231244.0

Gh_D11G258453.6Gh_D11G265655.3Gh_D11G301961.7Gh_D11G317664.4

Gh_D12G02503.3Gh_D12G04257.0Gh_D12G04547.5Gh_D12G04617.6Gh_D12G04948.5Gh_D12G05389.9Gh_D12G068814.9Gh_D12G074818.3

Gh_D12G086028.2Gh_D12G090131.5

Gh_D12G141743.5

Gh_D12G187451.3Gh_D12G191852.1Gh_D12G2423 Gh_D12G242757.3Gh_D12G254658.3

Gh_D13G03904.3

Gh_D13G078013.2Gh_D13G090118.2

Gh_D13G119335.6Gh_D13G129140.4Gh_D13G144145.3

Gh_D13G169750.8

Gh_D13G207056.2

A1(Chr1) A2(Chr2) A3(Chr3) A4(Chr4) A5(Chr5) A6(Chr6) A7(Chr7) A8(Chr8) A9(Chr9) A10(Chr10) A11(Chr11) A12(Chr12) A13(Chr13)

D1(Chr15) D2(Chr14) D3(Chr17) D4(Chr22) D5(Chr19) D6(Chr25) D7(Chr16) D8(Chr24) D9(Chr23) D10(Chr20) D11(Chr21) D12(Chr26) D13(Chr18)

Fig 3. Mapping of the DYW deaminase genes in the chromosomes. Partial DYW deaminase genes localized in

scaffolds.






barbadense genomes, respectively. Furthermore, 32 and 30 G. raimondii genes and 15 G. arboreumgenes had more than one homologous gene in G. hirsutum and G. barbadense, respectively. We

also observed that 75 and 60 G. raimondii genes and 71 and 57 G. arboreum genes each had one

homologous gene in G. hirsutum and G. barbadense, respectively. Further studies are necessary to

clarify the functions of the unique genes in the two tetraploid cotton species.

Conserved DYW tripeptides and disordered amino acids in the C-

terminal of DYW deaminases

In this study, we observed that the DYW and DFW tripeptides were the most frequently

observed tripeptides in the C-terminal regions of DYW deaminases (Table 2). For example,

there were 131 and 27 proteins that contained the DYW and DFW tripeptides in the C-termi-

nal of G. hirsutum DYW deaminases, respectively. There were 113 and 28 G. barbadense DYW

deaminases with DYW and DFW tripeptides, respectively. Of the diploid Gossypium species,

74, 19 and 58, 8 proteins contained the DYW and DFW tripeptide in G. raimondii and G. arbo-retum, respectively. Though the number of DYW tripeptides differed among Gossypium spe-

cies, its proportions in the whole gene family were similar, ranging from 53.55% to 59.79%.

The types and number of tripeptides present in the C-terminal of DYW deaminases vary

depending on organisms, including Cyanidioschyzon merolae, Physcomitrella patens, Selagi-nella moellendorffii, A. thaliana, Oryza sativa, and Sorghum bicolor (Table 2). Interestingly,

some sequences contained other types of C-terminal tripeptides (with disordered amino acid

residues), accounting for different proportions of the tripeptides in diverse organisms. For

example, there are no DYW deaminases in C. merolae, while P. patens contained only DYW

deaminases with C-terminal DYW and DFW tripeptides. However, 15 DYW deaminases in S.

moellendorffii (i.e., 8.67%) contained a disordered amino acid residue in the C-terminal region.

a bFig 4. Genome wide synteny analysis of DYW deaminase genes. a: Synteny analysis between G. hirsutum and two diploid cotton

species; b: Synteny analysis between G. barbadense and two diploid cotton species; Blue lines indicate syntenic regions between G.

arboretum and tetraploid cotton species, red lines between G. raimondii and tetraploid cotton species.






Additionally, 17.7% and 5.38% of the Gossypium species and A. thaliana DYW deaminases

contained a disordered amino acid residue, respectively.

Subcellular localization and gene ontology analysis of DYW deaminases

To predict the subcellular locations of DYW deaminases, the Target P online program was

used to examine protein transport. The DYW deaminases were detected in similar subcellular

locations in all four analyzed cotton species. In G. hirsutum, 120 DYW deaminases (i.e., 53%)

Table 2. Summary of conserved tripeptides in cotton and other organisms.

G.

raimondii

G.

arboretum

G.

hirsutum

G.

barbadense

Cyanidioschyzo

Merolae

Physcomitrella

patens

Selaginella

moellendorffii

Arabidopsis

thaliana

Oryza

sativa

Sorghum

bicolor

Total 126 97 227 211 0 10 173 93 82 65

DYW 74 58 131 113 0 7 110 57 42 30

DFW 19 8 27 28 0 3 16 14 12 6

Disorder a 17 18 37 45 0 0 15 5 17 16

DCW 1 1 2 2 0 0 10 0 0 0

CXCX_ 0 0 0 0 0 0 8 1 0 1

DHW 1 2 3 2 0 0 6 1 0 1

DYC 2 1 4 5 0 0 2 0 0 0

NIW 0 0 0 0 0 0 2 0 0 0

DTW 0 0 0 0 0 0 2 0 0 0

NFW 2 0 3 2 0 0 1 1 0 0

DIW 1 1 1 0 0 0 1 0 0 0

GYW 1 1 2 1 0 0 0 2 3 2

EYW 0 0 0 0 0 0 0 1 2 2

GFW 1 1 2 2 0 0 0 2 1 1

CXCXG_ _ 1 1 2 1 0 0 0 0 1 1

EFW 0 0 0 0 0 0 0 1 1 1

NYW 0 0 1 1 0 0 0 1 1 1

CXCXD_ _ 0 1 0 0 0 0 0 1 0 1

DMW 0 0 0 0 0 0 0 0 1 0

DYG 2 1 2 2 0 0 0 0 0 0

DLW 1 0 1 1 0 0 0 1 0 0

DSW 1 1 2 0 0 0 0 2 0 0

DKW 1 0 2 2 0 0 0 0 0 0

DVE 1 0 2 2 0 0 0 0 0 0

DRW 0 0 0 0 0 0 0 1 0 0

NLW 0 0 0 0 0 0 0 1 0 0

DNW 0 1 0 0 0 0 0 0 0 0

GLW 0 1 1 1 0 0 0 0 0 0

YFW 0 0 1 0 0 0 0 0 0 0

DIC 0 0 1 0 0 0 0 0 0 0

DYR 0 0 0 1 0 0 0 0 0 0

DFC 0 0 0 0 0 0 0 1 0 0

NCW 0 0 0 0 0 0 0 0 0 1

DAW 0 0 0 0 0 0 0 0 0 1

DFG 0 0 0 0 0 0 0 0 1 0

a Disorder means disordered amino acid residues contained in C-terminal.






were localized to the mitochondria and chloroplasts, while 16 proteins (i.e., 7%) containing an

N-terminal signal peptide were transported to other locations. However, we were unable to

predict the subcellular locations of 40% of the predicted DYW deaminases, suggesting the

need for further investigations. In other cotton species, the distribution of DYW deaminases

in specific subcellular locations was similar to that of G. hirsutum (S3 Fig).

Gene ontology (GO) analysis revealed that G. hirsutum DYW deaminases may be important

for developmental processes (Fig 5). The cellular component proteins were mainly associated

with intracellular or membrane-bound organelles, which is consistent with the subcellular local-

ization results for DYW deaminases. The primary molecular functions included binding as well

as hydrolase and transferase activities. The main biological process terms were related to cellular

metabolic processes, macromolecule metabolic processes, nitrogen compound metabolic pro-

cesses, and primary metabolic processes.

Phylogenetic of Gossypium DYW deaminases

To clarify the evolutionary relationships among Gossypium DYW deaminases, we constructed

a phylogenetic tree using the MEGA 6 program (Fig 6). The DYW deaminases were divided

into five groups (i.e., A–E) based on protein sequence similarities, with groups C and E con-

taining the most and fewest proteins, respectively.

Gossypium DYW deaminase gene expression patterns at different

developmental stages and tissues

To understand the temporal and spatial expression patterns of these PPR DYW deaminase

genes in Upland cotton, a publicly deposited RNA-seq data were used to assess the expression

across tissues and developmental stages. Among the 227 PPR DYW deaminase genes, there

Fig 5. The GO analysis of DYW deaminase proteins in G. hirsutum.






were 219 genes with an FPKM>0 in at least one of the 10 investigated tissues and developmen-

tal stages (Fig 7). The expression for the remaining 8 genes was not detected and will not be dis-

cussed in this study. We observed that the expression of DYW deaminase domain-containing

PPR genes were different in specific tissues and developmental stages. For example, most of the

genes were up regulated in root, stem, leaf and pistil, and fiber tissues, while they were down

regulated in petal and stamen tissues except for Gh_A12G0429,Gh_A13G1155,Gh_D08G0677and Gh_D12G2546. Some genes were specifically expressed in different tissues. For example,

Gh_A13G1722 gene was petal-specific, while Gh_D05G3688was root-specific, because they

were highly expressed in petal and root, respectively.

Quantitative real-time PCR (qRT-PCR) analysis

A quantitative real-time PCR was used to investigate the expression levels of 16 randomly selected

PPR DYW deaminase genes in roots, stem, leaves, and flowers of Upland cotton (Fig 8). All of

Cotton A 07524

Gh A10G

0617

GOBAR AA02841

Gorai.011G

088900.1G

h D10G0783

Cotton A 22853

Gorai.005G

053800.1G

h A02G0419

Gh D

02G0472

GO

BAR AA25847

GO

BAR DD26018

Gh Sca246681G

01Cotton A 05775

GO

BAR AA33689G

h D08G

2381G

h A08G1988

Cotton A 07988

Gorai.008G

210900.1

Gorai.008G

210900.2G

h D12G

1918G

OBAR DD

10094G

OBAR AA22545

Cotton A 01069G

orai.009G072800.1

Gh D

05G0696

GO

BAR

DD

20705G

h A05G

0567G

orai.001G029200.1

Gh D

07G0238

GO

BAR

DD

30693G

h A07G

0183G

OB

AR AA

12061C

otton A 15541G

orai.013G158100.1

Gh D

13G1441

Gh A

13G1155

GO

BAR

AA38674

GO

BAR

DD

09400C

otton A 01590G

orai.003G012700.1

GO

BAR

DD

07549G

h D03G

0117G

h A02G

1606G

OB

AR AA

32594C

otton A 34976G

orai.011G165700.1

Gh D

10G1472

GO

BAR

DD

27125G

h A10G

1047G

OB

AR AA

11572C

otton A 30003G

h A07G

1125G

OB

AR AA

04161G

orai.001G138400.1

Gh D

07G2446

GO

BAR

DD

35880C

otton A 37812G

h A03G

0850G

OBAR

AA0 80 48G

orai.005G136900.1

Gh D

02G1164

GO

BAR

DD

24355G

orai.005G119200.1

Gh D

02G2396

GO

BAR

DD

03637G

OB

AR AA

12322

Gh A

02G0889

Cotton A 28557

Gh A

01G0876

GO

BAR

AA16153

Gorai.002G

120300.1

Gh D

01G0912

GO

BAR

DD

11066

Cotton A 30897

Gh A

03G1304

Gorai.005G

192500.1

Gorai.005G

192500.2

Gh D

02G1741

GO

BAR

DD

19796

GO

BAR

AA12387

Cotton A 20263

Gorai.012G

021300.1

Gh D

04G0152

GO

BAR

DD

15801

Gh A

05G3440

GO

BAR

AA09751

GO

BAR

DD

10853

Cot

ton

A 10

175

Gh

A01

G17

52G

OB

AR A

A40

102

Gor

ai.0

02G

2387

00.1

GO

BAR

DD

0050

2G

h D

01G

1996

Gor

ai.0

02G

1206

00.1

GO

BAR

AA16

151

Cotto

n A

1773

5G

h A1

2G22

73G

OBA

R AA

2474

0G

orai

.008

G26

9700

.1

GO

BAR

DD10

882

GO

BAR

AA24

739

Gh

D12

G24

23Co

tton

A 25

990

Cotto

n A

2599

2G

orai

.004

G07

6500

.1

GO

BAR

DD19

527

Gh

D08

G06

77G

h A0

8G05

82G

OBA

R AA

1381

3

Cotto

n A

0050

1G

h A0

1G18

07G

OBA

R AA

2816

7

Gor

ai.0

02G

2447

00.1

Gh

D01G

2046

Cotto

n A

3233

9

Gh

A11G

0779

GOB

AR A

A292

15

Gor

ai.0

07G

0962

00.1

Gh

D11G

0908

GOB

AR D

D130

13

Cotto

n A

0695

6

Gor

ai.0

09G

0970

00.1

Gh

D05G

0911

Cotto

n A

1689

6

Gh A05

G3969

Gh D05

G0051

GOBAR

AA39

257

Gorai.

009G

0068

00.1

Cotto

n A

2327

2

Gh A13

G0941

GOBAR A

A0658

8

Gorai.

013G

1314

00.1

Gh D13

G1193

GOBAR D

D0183

4

Cotton

A 34

884

GOBAR A

A2984

3

Gh A06

G1873

Gorai.0

10G03

2300

.2

Gorai.0

10G03

2300

.1

Gh D06

G0241

GOBAR DD13

595

Cotton

A 17

776

Gh A12

G2290

GOBAR AA11

285

Gorai.0

08G27

3500

.1

Gh D12

G2427

Cotton A

0957

5

Gh A07

G0989

Gorai.0

01G12

1000

.1

Gh D07

G1067

GOBAR AA1587

0

Cotton A

3772

6

Gorai.0

02G15

1700

.2

Gorai.0

02G15

1700

.1

Gh D01G117

8

Gh A01G1103

GOBAR AA11811

GOBAR DD33682

Gorai.009G198200.3

GOBAR DD04717

GOBAR AA23986

Gorai.009G198200.2

Gorai.009G198200.1

Gh D05G1812

GOBAR DD21204

Gh A05G1630

Cotton A 21752

Gh A01G1420

Gorai.002G200700.1

Gh D01G1658

GOBAR DD13440

Cotton A 08048

Gh A12G1713

GOBAR AA16579

Gorai.008G205900.2

Gorai.008G205900.1

Gh D12G1874

GOBAR DD06747

GOBAR DD37886

Cotton A 32575

GOBAR AA02650

Gorai.008G086300.1

Gh D12G0748

Gh Sca123232G01

Gh A12G0724

Cotton A 27301

Gh A09G1638

Gorai.006G199900.1

Gh D09G1732

GOBAR DD32180

Cotton A 32227

GOBAR AA15945

Gh A06G1533

Gorai.010G214600.1

Gh D06G1902

GOBAR DD19584

Cotton A 16255

Gh A05G3966

Gorai.009G009600.1

Gh D05G0078

GOBAR DD32649

Cotton A 13417

Gh A09G1303

GOBAR AA24276

Gorai.006G154800.1

Gh D09G1309

GOBAR DD06946

Cotton A 30825

Gh A05G2917

Gorai.012G082800.1

Gh D04G0703

Cotton A 36721

Gh A02G1199

GOBAR AA03201

Gorai.002G057400.1

Gh D03G1703

GOBAR DD09520

Cotton A 18227

Gh A06G0016

Gorai.010G001600.1

Gorai.008G028200.1

Gh D12G0250

GOBAR DD00255

GOBAR AA02374

Cotton A 27764

Gh D03G1165

Gh A03G0377GOBAR DD33973GOBAR AA12923Gorai.003G128500.1Cotton A 17523GOBAR AA30383Gh A10G0329Gorai.011G038100.1GOBAR DD29525Gh D10G0335Cotton A 22374Gh A13G1386Gorai.013G198600.1Gh D13G1697GOBAR AA11791GOBAR DD06602Gh Sca181024G01GOBAR DD17455Cotton A 04493Gh A12G1295GOBAR AA32865Gorai.008G156300.1Gh D12G1417GOBAR DD08185Cotton A 21338Gh A12G0486Gorai.008G055100.1Gh D12G0494GOBAR DD34053GOBAR AA08153Cotton A 20564GOBAR AA00732Gh A09G0026Gorai.006G003200.1

Gh D09G0024GOBAR DD24456Cotton A 04367GOBAR DD36164

Gh A02G0074Gorai.005G009200.1Gh D02G0089GOBAR AA24508

Cotton A 39671Gorai.010G158200.1

GOBAR DD03254Gorai.010G153300.1

Gh D06G1374GOBAR DD13959

Gh A06G1120GOBAR AA13100

Cotton A 37555Gorai.007G344400.1



Gorai.005G026900.1


GOBAR AA28361


Cotton A 07464

Gh A04G0046Gorai.009G451200.1

Gh D05G3688

GOBAR DD06176

GOBAR AA30883

Cotton A 10449

GOBAR AA03862

Gorai.011G229900.2

Gorai.011G229900.1

Gh D10G2042

Gh A10G1770

Gorai.011G229900.3

GOBAR DD07612

Cotton A 29248

Gh A06G1611

GOBAR AA32302

Gorai.010G224300.1

Gh D06G1981

GOBAR DD11714

GOBAR DD38242

Cotton A 28020

Gh Sca141989G01

Gh A07G0290

GOBAR AA17450

Gorai.001G040600.1

Gh D07G0345

GOBAR DD15895

Cotton A 06599

Gh A11G2821

GOBAR AA16106

Gorai.007G363000.1

Gh D11G3176

GOBAR DD12502

Cotton A 28368

Gh A11G2278

GOBAR AA24217

Gh D11G2584

GOBAR DD30304

Gorai.007G280600.1

Cotton A 39243

GOBAR AA29285

Gh A07G2181

Gh D07G1662

GOBAR DD36560

GOBAR DD09943

Gorai.001G191900.1

Gh D07G1658

Gh A07G2185

Cotton A 02968

Gh A11G3080

GOBAR AA26382

Gorai.007G152300.1

Gh D11G1398

GOBAR DD10574

Cotton A 01226

GOBAR AA16455

Gorai.009G

057300.1

Gh D05G

0556

GOBAR DD37403

GOBAR DD23854

Cotton A 25415

Gh A04G

0600

GOBAR AA34679

Gorai.009G

414200.1

Gh D04G

1061

Cotton A 16485

Gorai.006G

243300.1

Gh A09G

1921

Gh D

09G2129

GO

BAR DD20453

Cotton A 09744

GO

BAR AA02422

Gh A08G

1382

Gorai.004G

181600.1

Gh D

08G1677

GO

BAR DD19314

Cotton A 07815

Gh A09G

1156

Gorai.006G

137700.1

Gh D

09G1162

GO

BAR DD34428

Cotton A 03411

Gh A04G

0834

GO

BAR AA38340

Gorai.012G

121600.1

Gh D

04G1333

GO

BAR

DD

08834

Cotton A 05942

Gh A

07G1888

GO

BAR

AA24195

Gorai.001G

240800.1

Gh D

07G2103

GO

BAR

DD

20764

Gorai.008G

067300.1

GO

BAR

DD

00789

Gh A

12G0594

GO

BAR

AA12245

Gh S

ca084155G01

Cotton A 17392

GO

BAR

AA22083

Gorai.008G

264200.1

Gh A

12G2221

Gh D

12G2728

Cotton A 12931

GO

BAR

AA23966

Gh A

05G0745

Gorai.009G

092600.1

Gh D

05G0877

GO

BAR

DD

15599

GO

BAR

DD

37880

Gorai.006G

267000.1

Gh D

09G2348

Gh A

09G2143

GO

BAR

AA31790

Gorai.013G

086000.2G

h D13G

0780G

orai.013G086000.1

Gh A

13G0665

GO

BAR

DD

06715C

otton A 09458G

h A11G

0575G

h Sca010496G

01G

h D11G

0660G

OB

AR AA

20521G

orai.007G071200.1

GO

BAR

AA28628

GO

BAR

DD

31628C

otton A 17704G

h A12G

0429G

orai.008G048000.1

GO

BAR

DD

10316G

OB

AR AA

33487G

h D12G

0425G

h Sca157535G

01C

otton A 00058G

h A01G

0144G

OB

AR AA

28654G

orai.002G020900.1

Gh D

01G0189

GO

BAR

DD

24064G

orai.003G097100.1

Gh D

03G0871

GO

BAR

DD

01206G

h A03G

0587G

OB

AR AA

14902C

otton A 09637G

h A09G

2140G

OB

AR AA

31786G

orai.006G266600.1

Gh D

09G2346

GO

BAR

DD

24232

Cot

ton

A 09

578

Gh

A07

G09

92

GO

BAR

AA

2444

3

Gh

D07

G10

70

GO

BAR

DD09

425

Gor

ai.0

01G

1213

00.1

Cotto

n A

3691

0

Gh

A12G

2587

GO

BAR

AA18

315

Gor

ai.0

08G

0795

00.1

Gh

D12

G06

88

GO

BAR

DD04

482

Cotto

n A

0375

9

Gh

A11G

1329

GO

BAR

AA38

069

Gor

ai.0

07G

1608

00.1

Gh

D11

G14

80

GO

BAR

DD15

406

Cotto

n A

0440

7

Cotto

n A

0441

0

Gh

Sca0

0480

2G02

GOB

AR A

A365

68

Gor

ai.0

05G

0144

00.1

Gh

D02G

0135

GOB

AR D

D378

32

Gh

D02G

0129

GOB

AR D

D299

40

GOB

AR D

D299

44

Gor

ai.0

12G

1466

00.1

Gh

D04G

1554

Gh

A04G

1004

GOB

AR D

D315

20

Gor

ai.0

08G

0505

00.1

Gorai.

008G

0505

00.2

Gorai.

008G

0505

00.3

Gh A12

G0451

Gh D12

G0454

GOBAR

DD06

036

Gorai.

013G

2284

00.1

Gh A13

G1722

Gh D13

G2070

GOBAR D

D1701

1

GOBAR A

A1409

5

Gorai.0

13G04

3300

.1

Gh D13

G0390

Gh A13

G0347

Cotton

A 27

094

Gorai.0

05G03

1300

.1

Gh A02

G0212

GOBAR AA33

130

Cotton

A 38

340

Gh A06

G1003

GOBAR AA14

724

GOBAR AA14

723

GOBAR DD01

758

Cotton A

0779

3

Gh A09

G1172

GOBAR DD36

365

Gorai.0

06G13

9500

.1

Gh D09

G1178

GOBAR AA0862

9

GOBAR DD25

932

Gorai.0

06G13

9600

.1

Cotton A

3431

2

Gh A07G1269

Gh D07G1380

GOBAR DD10479

Gorai.001G155500.1

GOBAR AA36335

Cotton A 01429

Gh A12G2727

GOBAR AA20943

Gorai.008G289000.1

GOBAR DD18779

GOBAR DD37372

Gh D12G2546

Cotton A 24301

Gh A08G1810

GOBAR AA25515

Gorai.004G235300.1

Gh D08G2170

GOBAR DD03089

Cotton A 03702

GOBAR AA09700

Gh A11G1384

Gorai.007G166400.1

Gh D11G1526

GOBAR AA27240

GOBAR DD30172

Cotton A 18858

Gh A06G0569

GOBAR AA25278

Gorai.010G075400.2

Gorai.010G075400.1Gh D06G0644

GOBAR DD18750Cotton A 00793Gh A03G1665

Gorai.005G237500.1Gh D02G2080

GOBAR DD09792GOBAR DD37733GOBAR AA20269

Gh D08G2007Cotton A 36351

GOBAR AA00296Gh A11G2033

Gorai.007G249300.1Gh D11G2312Cotton A 33828Gh A13G0768GOBAR DD30932

Gorai.013G098500.1Gh D13G0901GOBAR AA12611Gorai.008G051300.1Gh D12G0461Gh A12G0458Cotton A 26302Gh A08G1494GOBAR AA23445GOBAR DD30508Gorai.004G194300.1Gh D08G1792GOBAR DD29858Gorai.008G073100.1Gh D12G0860Gh Sca082327G01Gh A12G0640GOBAR AA30065GOBAR DD02429

Cotton A 23711GOBAR AA21892Gh A09G0759Gorai.006G094600.1

GOBAR DD30523Gh D09G0761

Cotton A 23710GOBAR DD30522GOBAR AA21895

Cotton A 22715Gh A07G2143Gh D07G2355

GOBAR DD11850Gorai.001G275900.1

GOBAR AA26881Cotton A 30439Gh A13G1039

GOBAR AA04344

Gorai.013G142700.1

Gh D13G1291

GOBAR DD11547

Cotton A 05786

Gh A08G1996

GOBAR AA19031

Gorai.004G262700.1

Gh D08G2390

GOBAR DD31556

Gorai.002G032400.1

Gh D01G0242

Gh A01G0244

GOBAR AA25897

Cotton A 17617

GOBAR AA05746

Gorai.004G038400.1

Gh D08G0337

GOBAR DD28118

Gh A01G1674

GOBAR AA09921

Gorai.001G214700.1

Gh D07G1876

GOBAR DD08488

GOBAR AA21405

Gh A07G1662

Cotton A 15955

Gh A12G0837

Gorai.008G102200.1

Gh D12G0901

GOBAR DD04982

GOBAR AA17793

Cotton A 35950

Gh A01G1766

GOBAR AA28237

Gorai.002G240300.1

Gh D01G2009

GOBAR DD16403

Cotton A 15473

Gorai.007G079800.1

Gorai.007G079800.2

Gh D11G0742

GOBAR DD15244

Gh A11G0632

GOBAR AA38953

Gh Sca072123G01

Cotton A 02993

Gh A11G1224

Gorai.007G149300.1

Gh D11G1371

GOBAR DD03135

Cotton A 28264

Gh A04G1432

Gorai.012G138600.1

Gh D04G1483

GOBAR DD26562

Cotton A 15028

Gh A11G2342

Gorai.007G288400.1

Gh D11G2656

GOBAR DD33683

Cotton A 00096

Gh A01G2040

GOBAR AA34559

Gorai.002G016600.1

Gh D01G0153

GOBAR DD04999

Cotton A 11411

Gorai.008G059500.1

Gh D12G0538

GOBAR DD34817

GOBAR AA22792

Gh A12G0524

A

B

C

D

E

Fig 6. Phylogenetic tree of DYW deaminase genes in Gossypium.






the 16 selected genes were expressed in the four analyzed tissues, with different tissue-specific

expression profiles. Among these genes, Gh_D09G0761,Gh_D05G0556, and Gh_A03G1665were

the most highly expressed genes in flowers. Gh_D09G0761 and Gh_A03G1665produced similar

expression profiles (i.e., gradual increase in the four examined tissues). The results imply that

these genes may have a special role during flower development. Gh_A01G1752,Gh_D08G2381,

Gh_D04G0152,Gh_A12G0594,Gh_A02G0419,Gh_A07G1662, and Gh_A13G1386were the most

highly expressed genes in leaves. Additionally, highest expression levels for Gh_D02G1741,

Gh_D01G0153,Gh_A12G0486,Gh_A01G1103, and Gh_A02G0212were observed in stems.

Finally, Gh_D05G0911was highly expressed in the roots. These results indicate that DYW deami-

nases may be important for cotton development.

Differentially expressed PPR genes between CMS-D2 and restorer

In a genomewide transcriptome analysis of floral buds between near-isogenic CMS-D2 (i.e., A

line) and its restorer (R line) differing in the Rf1 locus, 6 differentially expressed (DE) PPR

genes were identified (S1 Table). However, no DYW deaminase domain in C-terminal was

found in these 6 PPR genes. Among these 6 PPR genes, Gh_A02G1102 (PLS subgroup), Gh_A11G0734 and Gh_D11G0852 (E subgroup) belong to the PLS subfamily, while Gh_A06G0542,

Gh_D05G3392 and Gh_D06G0610belong to the P subfamily. It is interesting to note that, as com-

pared with the R line, 3 PLS subfamily genes (Gh_A02G1102,Gh_A11G0734 and Gh_D11G0852)

were up regulated and 3 P subfamily genes (Gh_A06G0542,Gh_D05G3392 and Gh_D06G0610)

were down regulated in the A line. Furthermore, the PPR gene Gh_D05G3392 is located on the

chromosome where the restorer gene Rf1 resides. The Gh_D05G3392 protein belongs to P sub-

family and contain 14 repeats of PPR motifs. According to the previous studies [22–26], we

root

stem

leaf

pistil

stamen

petal

5dpa_fibre

10dpa_fibre

20dpa_fibre

25dpa_fibre

Gh_A09G1303Gh_A05G0567Gh_A05G3969Gh_A02G0889Gh_A07G0183Gh_D02G1164Gh_D12G0860Gh_D13G0901Gh_A04G1004Gh_A09G1172Gh_A02G0212Gh_A11G0632Gh_A01G2040Gh_D03G0871Gh_D09G2346Gh_A02G0419Gh_A06G1533Gh_A04G1432Gh_A03G1304Gh_D01G0912Gh_A04G0834Gh_D12G1874Gh_D11G1480Gh_A13G0768Gh_D09G2129Gh_D07G2355Gh_D07G2446Gh_A11G0575Gh_D02G0089Gh_D07G0238Gh_A13G1386Gh_D06G1902Gh_A12G0429Gh_D03G1703Gh_A12G2727Gh_A02G0176Gh_D04G1333Gh_A08G0582Gh_D07G1658Gh_A02G0074Gh_A13G1155Gh_A12G2221Gh_A07G2181Gh_D13G1697Gh_A05G3966Gh_A12G0724Gh_A12G1295Gh_A01G1103Gh_A09G1156Gh_D05G0877Gh_A10G1770Gh_D01G0189Gh_A09G0026Gh_D08G0337Gh_D05G3688Gh_A10G1047Gh_D05G0556Gh_D08G2170Gh_D07G1876Gh_D01G0242Gh_A13G0347Gh_D13G0390Gh_A07G1662Gh_D04G0152Gh_D05G0078Gh_D08G2390Gh_D12G2546Gh_A11G0779Gh_A12G0640Gh_A13G0941Gh_A12G0458Gh_A02G1199Gh_D10G0783Gh_D11G3176Gh_A03G0377Gh_A07G2143Gh_D10G0335Gh_D12G0454Gh_D03G1165Gh_A10G0329Gh_A09G1921Gh_A12G0451Gh_A07G0290Gh_D10G2042Gh_A11G3080Gh_D12G0901Gh_D07G2103Gh_A08G1996Gh_D09G0024Gh_A06G1120Gh_D05G0051Gh_A12G0524Gh_A01G0244Gh_D05G0696Gh_A08G1494Gh_D10G1472Gh_D07G0345Gh_A04G0046Gh_D12G0461Gh_D01G2046Gh_D12G2423Gh_D04G1483Gh_D11G0742Gh_A06G1003Gh_D12G0688Gh_D01G1996Gh_A01G1752Gh_A08G1810Gh_D08G2381Gh_A12G2273Gh_A09G2140Gh_D11G0908Gh_A05G0745Gh_A11G1384Gh_D09G2348Gh_A01G0144Gh_D08G1792Gh_D13G2070Gh_A13G1722Gh_D09G1732Gh_A11G2821Gh_A12G0486Gh_D13G1291Gh_A11G1329Gh_A13G1039Gh_D06G1374Gh_A11G2278Gh_A01G1766Gh_D01G0153Gh_D11G2656Gh_A03G0587Gh_D12G0494Gh_A13G0665Gh_D11G2312Gh_D09G0761Gh_A11G2342Gh_D01G1658Gh_D02G0135Gh_Sca004802G02Gh_A11G2657Gh_D11G3019Gh_D04G1554Gh_D02G2396Gh_D08G0677Gh_D12G2728Gh_D12G1918Gh_D07G1380Gh_D13G1441Gh_D11G1371Gh_D01G1178Gh_D03G0117Gh_A11G1224Gh_A02G1606Gh_A04G0600Gh_A01G1674Gh_D12G0748Gh_D12G1417Gh_D02G0129Gh_A06G0569Gh_D04G1061Gh_A07G0992Gh_D09G1309Gh_A05G2917Gh_A01G1420Gh_D02G1741Gh_D08G2007Gh_D11G1526Gh_A05G1630Gh_D05G1812Gh_A12G2290Gh_A03G1665Gh_D02G2080Gh_A12G1713Gh_A07G1888Gh_D02G0472Gh_D11G1398Gh_A06G1611Gh_A08G1988Gh_Sca084155G01Gh_A12G2587Gh_A12G0594Gh_A09G1638Gh_D12G0425Gh_A07G2185Gh_A03G0850Gh_A08G1382Gh_D08G1677Gh_A01G0876Gh_A07G1125Gh_A06G0016Gh_A11G2033Gh_D05G0911Gh_D06G1981Gh_D06G0241Gh_D12G2427Gh_A06G1873Gh_D09G1178Gh_D04G0703Gh_D09G1162Gh_A10G0617Gh_D01G2009Gh_D13G1193Gh_A07G1269Gh_D07G1067Gh_A07G0989Gh_D13G0780Gh_A05G3440Gh_D11G2584Gh_A09G0759Gh_A09G2143Gh_A01G1807Gh_D12G0250Gh_D02G0240Gh_D06G0644Gh_D07G1662Gh_D12G0538Gh_D07G1070Gh_A12G0837Gh_Sca010496G01

−2

−1

0

1

2

Fig 7. Expression profiles of DYW deaminase genes during fiber development and in different

tissues. The color bar represents the expression values.






adopted the three number PPR code as proposed by Yin et al [24] and predicted the amino acids

in 3 particular positions (1, 4 and ii; residues 2, 5 and 35, respectively, as used in this study) within

Gh_D05G3392 protein motifs. In Fig 9, we aligned the PPR motif and highlighted the residues

at 2, 5 and 35 positions. Additionally, we also predicted the RNA sequence (UUUUUUUUUU

CUU) recognized by the Gh_D05G3392 according to the rule proposed by Yagi et al [23]. In-

terestingly, one of the blast results in NCBI showed that this sequence (UUUUUUUUUUCUU)

is a partial sequence of a pseudogene in G. hirsutum and SSR marker GNCOT-C1-F (GenBank:

KX090570.1). However, it is outside of the Rf1 locus [27], while other five genes were even not

located on the Rf1-carrying chromosome (Gh_D05). Thus, with the introduction of the dominant

restorer gene Rf1 to the A line containing the CMS-inducing cytoplasm, the expression of the

Root stem leaf flower0

2

4

6

8

10

12noiss

erpx

e evit

ale

R

Gh_D09G0761a

Root stem leaf flower0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0Gh_D05G0556


2

4

6

8

10

12

14

16Gh_A03G1665


1

2

3

4

5

6noisser

pxe

evital

eR

Gh_A01G1752b


1

2

3

4

5

6 Gh_D08G2381


200

400

600

800

1000

1200 Gh_D04G0152


50

100

150

200

250

Gh_A12G0594


5

10

15

20

25

30

35noiss

erpx

e evit

ale

R

Gh_A02G0419


2

4

6

8

10

12

14

16

18

20Gh_A07G1662


10

20

30

40

50

60

70 Gh_A13G1386


1

2

3

4

5

6

7noiss

erpx

e evit

ale

R

Gh_D02G1741c


2

4

6

8

10

Gh_D01G0153


10

20

30

40

50

60Gh_A12G0486


1

2

3

4

5

6noiss

erpx

e evit

ale

R

Gh_A02G0212


2

4

6

8

10

12 Gh_A01G1103

Root stem leaf flower0.0

0.5

1.0

1.5

2.0

noiss

erpx

e evit

a le

R

Gh_D05G0911d

Fig 8. Expression analysis of 16 selected DYW deaminase genes in different tissues through

qRT-PCR. a: High expression in the flower; b: Expression peaks in the leaf; c: High expression in the stem; d:

High expression in the root.






three PLS subfamily genes (PLS and E subgroup) was suppressed, while other three genes in the

P subfamily were activated for male fertility restoration in the R line. These results indicated that

DYW deaminase domain-containing PPR genes may not directly function in the CMS occur-

rence or fertility restoration. This is consistent with previous results in cloned restorer genes from

other plant species.

Discussion

Relationship between DYW deaminase and DYW domains

In this study, we identified 227 DYW deaminase genes in the G. hirsutum genome. We also

obtained the consensus sequence of the Gossypium DYW deaminase domain. Compared with

the DYW domain, the complete DYW deaminase domain contained the E and E+ domains,

which was consistent with the findings of a previous study [7]. Additionally, a PG-box was

detected in the DYW deaminase domain, spanning the E and E+ domains. The functions of

this PG box have not been characterized.

Molecular evolution of DYW deaminases

Allotetraploid Gossypium species evolved from two diploid cotton species through hybridiza-

tions and genome doubling that occurred 1–2 million years ago [11]. Therefore, diploid and

allotetraploid cotton species are useful models for investigating the evolution of plants. The

whole genome sequences of Gossypium species have enabled researchers to study the molecu-

lar evolutionary characteristics of Gossypium DYW deaminases. In this study, we analyzed the

types and number of DYW tripeptides or their variants in four Gossypium species (Table 2).

We observed that DYW tripeptides are the most conserved and abundant, followed by the

DFW tripeptide. Additionally, some tripeptide types are specific to different Gossypium spe-

cies. Furthermore, in contrast to the diploid species, the number of tripeptide types increased

and the proportion of conserved tripeptides in the whole DYW deaminase family decreased in

allotetraploid Gossypium species.

By comparing the types and number of tripeptides present in the C-terminal of DYW

deaminases from red algae (C. merolae), bryophytes (P. patens), lycophyta (S. moellendorffii),monocots (Sorghum bicolor and Oryza sativa), and eudicots (A. thaliana and Gossypium spe-

cies), we determined that diverse plant species evolved differently in terms of the types and

number of tripeptides. For example, C. merolae lacks DYW deaminases, while P. patens pro-

duces 10 DYW deaminases with a conserved C-terminal tripeptide (i.e., either DYW or

DFW). Selaginella moellendorffii rapidly evolved 10 types of tripeptides, and the proportion of

Fig 9. Sequence alignment of the 14 repeats of Gh_D05G3392. The residues at 2, 5 and 35 positions

were predicted to be the molecular determinants for RNA binding specificity are red. The RNA sequences

recognized are listed on the right, 5’ to 3’ from top to bottom.






conserved DYW tripeptides decreased. Additionally, a disordered amino acid residue is pres-

ent in the C-terminal of some S. moellendorffii DYW deaminases, similar to corresponding

DYW deaminases in monocots and eudicots. This indicates that the types of conserved DYW

tripeptide increased during the evolution from algae to angiosperms. Selection pressures may

have induced the adaptation of DYW tripeptide types to evolutionary processes. Previous stud-

ies indicated that the phylogenetic distribution of DYW domains is highly correlated with

RNA editing, with the number of RNA editing events increasing during evolution [6, 28].

Thus, the types and number of conserved tripeptides in DYW domains may be highly corre-

lated with RNA editing events.

The frequency of residue changes differs among conserved tripeptides. In the DYW tripep-

tide, Trp is the most conserved residue, followed by Asp. We observed that most DYW deami-

nases contain a CxCx motif near the conserved tripeptide. However, some protein sequences

lacking the CxCx motif may also be missing the HxExx. . .CxxCH motif. This may be because

during early plant evolutionary processes, the conserved DYW tripeptide sequence became

flexible and distinct tripeptides due to residue changes increased the number of tripeptide

types. Some flexible tripeptides were gradually lost during evolution, which resulted in the loss

of the CxCx motif and other sequences in the DYW domain following the tripeptide. Based on

the relationship between the conserved DYW tripeptide and CxCx motif, we hypothesized that

the conserved DYW tripeptide functions like a protective cap to prevent the loss of the CxCx

motif. As distinct parts of DYW deaminases, the roles of the CxCx motif and conserved DYW

tripeptide should be more thoroughly characterized in future studies.

PPR gene family and male fertility restoration

The PPR gene family constitutes a large family of RNA binding in plants, it involves in many cel-

lular functions and biological processes in organelles including gene expression, RNA stabiliza-

tion, RNA cleavage and RNA editing [25, 29]. Previous studies indicated the PPR proteins

involved in RNA editing events mostly present a characteristic motif, which contain a PPR tract

followed by C-terminal motifs (E, E+ and DYW), although the E+ and DYW domain are often

missing [30]. Additionally, a conserved motif HxE(x)nCxxCH exist in DYW domain is required

for C to U conversion, rather than for recognition the editing site [31]. In this study, we found

the complete DYW deaminase domain contained the part of E, E+ and DYW domains, and

most DYW deaminases contain a CxCx motif near the conserved tripeptide. However, some pro-

tein sequences lacking the CxCx motif may also be missing the whole or part of HxExx. . .CxxCH

motif. The DYW deaminase domain as the catalytic domain maybe promote C to U conversion.

As the tandem arrays of PPR motifs, Yagi et al. (2013) found amino acid residues at 3 particular

position (1, 4, and ii) within the PPR motif recognizing the upstream nucleotide sequence for the

editing sites [23]. Furthermore, Takenaka et al. (2013) found that combining the P, L and S

motifs will improve the prediction of RNA editing target sites [26]. In this study, we predicted

the PPR code in 3 particular positions (1, 4 and ii; residues 2, 5 and 35, respectively, as used in

this study) within Gh_D05G3392 motifs and predicted the RNA sequence (UUUUUUUUUU

CUU) recognized by Gh_D05G3392, which are part of a pseudogene in Gossypium hirsutum and

SSR marker GNCOT-C1-F (GenBank: KX090570.1).

CMS in flowering plants is characterized by a maternally inherited trait that lacks functional

pollen grains [32], and this trait can be restored by nuclear genes known as restorer-of-fertility

(Rf) genes [33]. Previous studies indicated that RNA editing events may have certain relation-

ship with CMS occurrence in many crops. For example, Wei et al. (2008) found that the editing

of atp9 change an arginine codon into a termination codon, shorting the protein to the stan-

dard in Ying xiang B while in Ying xiang A, which has no termination codon, cannot be a




normal protein [34]. Wang et al. (2009) found that RNA editing occurrence rates in atp6 and

cox2 generally increase in sterile lines [35]; Chakraborty et al. (2015) indicated that an over-

expression of the unedited mitochondrial orfB gene generated male sterility in fertile rice lines

in a dose-dependent manner, in which the unedited orfB gene expression increased, resulting

in a low level of ATP synthase activity of F1F0-ATP synthase (complex V) [36]. In cotton,

Suzuki et al. (2013) identified RNA editing sites in eight mitochondrial genes among CMS-D8

three line systems, and found that the restorer gene may change RNA editing efficiency [37].

However, little is known about the RNA editing events of mitochondrial genes in CMS-D2.

Wu et al. (2011) found a C-U and a C-G editing site at position 1178 and 1214 bp downstream

of the ATG codon in atpA and a A-U and a C-U editing site at position 18 and 35 bp upstream

of ATG codon in nad6 between maintainer and CMS-D2 lines, respectively [16]. Thus, we

speculate that RNA editing events in D2 mitochondrial genes may regulate ATP synthase

activity, and the impaired ATP synthesis results in CMS occurrence.

Fertility restoration generally requires the reduction of CMS-associated RNAs or proteins

by the action of the Rf gene (s). Most of the restorer genes cloned so far are reported to encode

a PPR protein. For instance, the radish Rfo [38], and two PPR genes in sorghum [14, 15], and

Rf4 and Rf6 gene in rice [12, 13] each encodes a different PPR protein. Additionally, previous

studies indicated that most of them belong to P subfamily, except for that fact that the Rf1 gene

in sorghum encoded a PPR protein which is classified as member of E subgroup of PLS sub-

family [14, 39][14, 33]. In this study, 6 differentially expressed (DE) PPR genes were identified

through a genomewide transcriptome analysis between A and R lines. Interestingly, 3 PLS sub-

family genes (Gh_A02G1102,Gh_A11G0734 and Gh_D11G0852) were up regulated and 3 P

subfamily genes (Gh_A06G0542,Gh_D05G3392 and Gh_D06G0610) were down regulated in

the A line. These results indicated that DYW deaminase domain-containing PPR genes may

not directly function in the CMS occurrence or fertility restoration, while P subfamily genes

might have critical role in the process of fertility restoration. And this is consistent with the

previous studies that most of Rf genes belong to P subfamily [39]. Previous studies indicated a

small subgroup of PPR genes existed in plant genomes that share high similarity with the iden-

tified Rf-PPRs, called as Rf-like PPRs (RFL) genes [38, 40]. For example, in radish Rfo loci,

three similar PPRs were identified, which may arise through gene duplication events [38]. In

this study, five genes were not located on the Rf1-carrying chromosome (Gh_D05), while the

PPR gene Gh_D05G3392 is located on the chromosome where the restorer gene Rf1 resides

[27], though it is outside of the Rf1 locus and its protein was predicted to target in the chloro-

plasts, and the physical distance is short. Therefore, we speculate that the Gh_D05G3392 gene

may share a high similarity with Rf1 in cotton but it is not the candidate gene for Rf1. As for

the mechanism of CMS restoration in cotton, due to the limited study, we speculate that the

Rf1 may be a PPR gene belonging to the P subfamily. Takenaka et al. (2013) proposed that the

P subfamily PPR proteins often bind tightly to their RNA targets and cannot be removed [26].

And Suzuki et al. (2013) indicated that the restorer gene may alter RNA editing efficiency

among the CMS-D8 three line systems [37]. Thus, Rf1-PPR protein maybe binds tightly to

their CMS gene or product, resulting in that the DYW deaminase gene cannot edit the CMS

gene product, so the male fertility is restored. However, the RNA editing events in CMS-D2

cotton and the role of Rf1 gene in RNA editing still need further studies.

Conclusion

We identified DYW deaminases based on four sequenced Gossypium species genomes. We

conducted chromosomal and subcellular localization experiments as well as gene structure,

expression, and GO analyses for the identified genes and proteins. Most of the Gossypium




DYW deaminases contained the conserved Asp-Tyr-Trp tripeptide (or variants) in the C ter-

minal. The DYW deaminases lacking the conserved tripeptide may have been gradually lost

during evolution. The PPR proteins containing the DYW domain are ancient RNA-editing

proteins. With increasing numbers of editing sites, some amino acids in the DYW domain

were gradually lost during evolution, resulting in an increase in the abundance of proteins

from other subgroups (e.g., when the DYW domain was converted to an E domain). There-

fore, the DYW deaminase domain might influence nucleotide deamination, which is consis-

tent with the predicted molecular function based on GO analysis. Furthermore, regarding

subcellular localizations, DYW deaminases were predicted to mainly localize in mitochondria

or chloroplasts, which was consistent with GO analysis results related to cell components. In

addition, the transcriptome analysis indicated no differentially expressed DYW deaminase-

coding PPRs were directly associated with male sterility and restoration in the CMS-D2 sys-

tem. In summary, our study provides basic information regarding the structural and evolu-

tionary characteristics of DYW deaminases in cotton. Our findings may be useful for

improving cotton production in future studies.

Supporting information

S1 Fig. The number of introns in DYW deaminase genes in Gossypium.

(TIF)

S2 Fig. The gene structure of DYW deaminase in Gossypium.

(EPS)

S3 Fig. Mapping of the DYW deaminase genes in the chromosomes of G. arboretum, G. rai-mondii and G. barbadense.

(EPS)

S4 Fig. Subcellular localization of DYW deaminases in Gossypium.

(TIF)

S1 Table. Differentially expressed genes between CMS-D2 and restorer.

(XLSX)

S2 Table. Information about the DYW deaminase genes in Gossypium.

(XLSX)

Acknowledgments

We thank Xihua Li and Nuohan Wang, Qi Gong for analyzing the genome data, figure and

helpful comments on the manuscript.

Author Contributions

Conceptualization: CX JW.

Data curation: CX JW JZ.

Formal analysis: BZ GL XL.

Funding acquisition: CX JW.

Investigation: BZ GL XL LG XZ.

Methodology: CX JW TQ HW.



http://www.plosone.org/article/fetchSingleRepresentation.action?uri=info:doi/10.1371/journal.pone.0174201.s001







Project administration: CX JW LG.

Resources: XZ HW HT XQ.

Supervision: LG TQ XZ.

Visualization: BZ GL XL JZ.

Writing – original draft: BZ JW JZ.

Writing – review & editing: BZ JW JZ.

References

1. Wedekind JE, Dance GSC, Sowden MP, Smith HC. Messenger RNA editing in mammals: new mem-

bers of the APOBEC family seeking roles in the family business. Trends in Genetics. 2003; 19(4):207–

16. https://doi.org/10.1016/S0168-9525(03)00054-4 PMID: 12683974

2. Shikanai T. RNA editing in plants: Machinery and flexibility of site recognition. Biochimica et Biophysica

Acta (BBA)—Bioenergetics. 2015; 1847(9):779–85.

3. Small ID, Peeters N. The PPR motif–a TPR-related motif prevalent in plant organellar proteins. Trends

in Biochemical Sciences. 2000; 25(2):45–7.

4. Lurin C, Andres C, Aubourg S, Bellaoui M, Bitton F, Bruyere C, et al. Genome-wide analysis of Arabi-

dopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell.

2004; 16(8):2089–103. Epub 2004/07/23. PubMed Central PMCID: PMCPmc519200. https://doi.org/

10.1105/tpc.104.022236 PMID: 15269332

5. Nakamura T, Sugita M. A conserved DYW domain of the pentatricopeptide repeat protein possesses a

novel endoribonuclease activity. FEBS Lett. 2008; 582(30):4163–8. Epub 2008/12/02. https://doi.org/

10.1016/j.febslet.2008.11.017 PMID: 19041647

6. Salone V, Rudinger M, Polsakiewicz M, Hoffmann B, Groth-Malonek M, Szurek B, et al. A hypothesis

on the identification of the editing enzyme in plant organelles. FEBS Lett. 2007; 581(22):4132–8. Epub

2007/08/21. https://doi.org/10.1016/j.febslet.2007.07.075 PMID: 17707818

7. Iyer LM, Zhang D, Rogozin IB, Aravind L. Evolution of the deaminase fold and multiple origins of eukary-

otic editing and mutagenic nucleic acid deaminases from bacterial toxin systems. Nucleic Acids Res.

2011; 39(22):9473–97. Epub 2011/09/06. PubMed Central PMCID: PMCPmc3239186. https://doi.org/

10.1093/nar/gkr691 PMID: 21890906

8. Hayes ML, Dang KN, Diaz MF, Mulligan RM. A conserved glutamate residue in the C-terminal deami-

nase domain of pentatricopeptide repeat proteins is required for RNA editing activity. J Biol Chem.

2015; 290(16):10136–42. Epub 2015/03/06. PubMed Central PMCID: PMCPmc4400329. https://doi.

org/10.1074/jbc.M114.631630 PMID: 25739442

9. Wagoner JA, Sun T, Lin L, Hanson MR. Cytidine deaminase motifs within the DYW domain of two pen-

tatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing. J Biol

Chem. 2015; 290(5):2957–68. Epub 2014/12/17. PubMed Central PMCID: PMCPmc4317000. https://

doi.org/10.1074/jbc.M114.622084 PMID: 25512379

10. Boussardon C, Salone V, Avon A, Berthome R, Hammani K, Okuda K, et al. Two interacting proteins

are necessary for the editing of the NdhD-1 site in Arabidopsis plastids. Plant Cell. 2012; 24(9):3684–

94. https://doi.org/10.1105/tpc.112.099507 PMID: 23001034

11. Hu G, Hawkins JS, Grover CE, Wendel JF. The history and disposition of transposable elements in

polyploid Gossypium. 2010; 53(8):599–607. https://doi.org/10.1139/g10-038 PMID: 20725147

12. Kazama T, Toriyama K. A fertility restorer gene, Rf4, widely used for hybrid rice breeding encodes a

pentatricopeptide repeat protein. Rice. 2014; 7(1):1–5.

13. Huang W, Yu C, Hu J, Wang L, Dan Z, Zhou W, et al. Pentatricopeptide-repeat family protein RF6 func-

tions with hexokinase 6 to rescue rice cytoplasmic male sterility. Proc Natl Acad Sci U S A. 2015; 112

(48):14984–9. https://doi.org/10.1073/pnas.1511748112 PMID: 26578814

14. Klein RR, Klein PE, Mullet JE, Minx P, Rooney WL, Schertz KF. Fertility restorer locus Rf1of sorghum

(Sorghum bicolor L.) encodes a pentatricopeptide repeat protein not present in the colinear region of

rice chromosome 12. Theoretical and Applied Genetics. 2005; 111(6):994–1012. https://doi.org/10.

1007/s00122-005-2011-y PMID: 16078015

15. Jordan DR, Mace ES, Henzell RG, Klein PE, Klein RR. Molecular mapping and candidate gene identifi-

cation of the Rf2 gene for pollen fertility restoration in sorghum [Sorghum bicolor (L.) Moench].



https://doi.org/10.1016/S0168-9525(03)00054-4

http://www.ncbi.nlm.nih.gov/pubmed/12683974

https://doi.org/10.1105/tpc.104.022236

https://doi.org/10.1105/tpc.104.022236


https://doi.org/10.1016/j.febslet.2008.11.017





https://doi.org/10.1093/nar/gkr691

https://doi.org/10.1093/nar/gkr691


https://doi.org/10.1074/jbc.M114.631630






https://doi.org/10.1105/tpc.112.099507


https://doi.org/10.1139/g10-038


https://doi.org/10.1073/pnas.1511748112


https://doi.org/10.1007/s00122-005-2011-y

https://doi.org/10.1007/s00122-005-2011-y



Theoretical and Applied Genetics. 2010; 120(7):1279–87. https://doi.org/10.1007/s00122-009-1255-3

PMID: 20091293

16. Wu J, Gong Y, Cui M, Qi T, Guo L, Zhang J, et al. Molecular characterization of cytoplasmic male steril-

ity conditioned by Gossypium harknessii cytoplasm (CMS-D2) in upland cotton. Euphytica. 2011; 181

(1):17–29.

17. Suzuki H, Rodriguez-Uribe L, Xu J, Zhang J. Transcriptome analysis of cytoplasmic male sterility and

restoration in CMS-D8 cotton. Plant Cell Reports. 2013; 32(10):1531–42. https://doi.org/10.1007/

s00299-013-1465-7 PMID: 23743655

18. You Q, Xu W, Zhang K, Zhang L, Yi X, Yao D, et al. ccNET: Database of co-expression networks with

functional modules for diploid and polyploidGossypium. Nucleic Acids Research. 2017;45(Database

issue):D1090–D9.

19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR

and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4):402–8. https://doi.org/10.1006/meth.2001.

1262 PMID: 11846609

20. Sugita M, Ichinose M, Ide M, Sugita C. Architecture of the PPR gene family in the moss Physcomitrella

patens. RNA Biol. 2013; 10(9):1439–45. Epub 2013/05/07. PubMed Central PMCID:

PMCPmc3858427. https://doi.org/10.4161/rna.24772 PMID: 23645116

21. Hayes ML, Giang K, Berhane B, Mulligan RM. Identification of two pentatricopeptide repeat genes

required for RNA editing and zinc binding by C-terminal cytidine deaminase-like domains. Journal of Bio-

logical Chemistry. 2013; 288(51):36519–29. https://doi.org/10.1074/jbc.M113.485755 PMID: 24194514

22. Shen C, Zhang D, Guan Z, Liu Y, Yang Z, Yang Y, et al. Structural basis for specific single-stranded

RNA recognition by designer pentatricopeptide repeat proteins. Nature Communications. 2016;

7:11285. https://doi.org/10.1038/ncomms11285 PMID: 27088764

23. Yagi Y, Hayashi S, Kobayashi K, Hirayama T, Nakamura T. Elucidation of the RNA recognition code for

pentatricopeptide repeat proteins involved in organelle RNA editing in plants. PLoS One. 2013; 8(3):

e57286. Epub 2013/03/09. PubMed Central PMCID: PMCPmc3589468. https://doi.org/10.1371/

journal.pone.0057286 PMID: 23472078

24. Yin P. Structural basis for the modular recognition of single-stranded RNA by PPR proteins. Nature.

2013; 504(7478):168. https://doi.org/10.1038/nature12651 PMID: 24162847

25. Prikryl J, Rojas M, Schuster G, Barkan A. Mechanism of RNA stabilization and translational activation

by a pentatricopeptide repeat protein. Proc Natl Acad Sci U S A. 2011; 108(1):415–20. https://doi.org/

10.1073/pnas.1012076108 PMID: 21173259

26. Takenaka M, Zehrmann A, Brennicke A, Graichen K. Improved computational target site prediction for

pentatricopeptide repeat RNA editing factors. Plos One. 2013; 8(6):e65343. https://doi.org/10.1371/

journal.pone.0065343 PMID: 23762347

27. Wu J, Cao X, Guo L, Qi T, Wang H, Tang H, et al. Development of a candidate gene marker for Rf 1

based on a PPR gene in cytoplasmic male sterile CMS-D2 Upland cotton. Molecular Breeding. 2014; 34

(1):231–40.

28. Knoop V, Rudinger M. DYW-type PPR proteins in a heterolobosean protist: plant RNA editing factors

involved in an ancient horizontal gene transfer? FEBS Lett. 2010; 584(20):4287–91. Epub 2010/10/05.

https://doi.org/10.1016/j.febslet.2010.09.041 PMID: 20888816

29. Schmitzlinneweber C, Small I. Pentatricopeptide repeat proteins: a socket set for organelle gene

expression. Trends in Plant Science. 2008; 13(12):663–70. https://doi.org/10.1016/j.tplants.2008.10.

001 PMID: 19004664

30. Okuda K, Myouga F, Motohashi R, Shinozaki K, Shikanai T. Conserved domain structure of pentatrico-

peptide repeat proteins involved in chloroplast RNA editing. Proc Natl Acad Sci U S A. 2007; 104

(19):8178–83. Epub 2007/05/08. PubMed Central PMCID: PMCPmc1876591. https://doi.org/10.1073/

pnas.0700865104 PMID: 17483454

31. Boussardon C, Avon A, Kindgren P, Bond CS, Challenor M, Lurin C, et al. The cytidine deaminase sig-

nature HxE(x)n CxxC of DYW1 binds zinc and is necessary for RNA editing of ndhD-1. New Phytologist.

2014; 203(4):1090–5. https://doi.org/10.1111/nph.12928 PMID: 25041347

32. Hanson MR, Bentolila S. Interactions of Mitochondrial and Nuclear Genes That Affect Male Gameto-

phyte Development. The Plant Cell. 2004; 16(suppl 1):S154–S69.

33. Wang F, Stewart JM, Zhang J. Molecular markers linked to the Rf2 fertility restorer gene in cotton.

Genome / National Research Council Canada = Genome / Conseil national de recherches Canada.

2007; 50(9):818–24.

34. Wei L, Ding YY. Mitochondrial RNA editing of F0-ATPase subunit 9 gene (atp9) transcripts of Yunnan

purple rice cytoplasmic male sterile line and its maintainer line. Acta Physiologiae Plantarum. 2008; 30

(5):657–62.



https://doi.org/10.1007/s00122-009-1255-3


https://doi.org/10.1007/s00299-013-1465-7

https://doi.org/10.1007/s00299-013-1465-7


https://doi.org/10.1006/meth.2001.1262

https://doi.org/10.1006/meth.2001.1262


https://doi.org/10.4161/rna.24772




https://doi.org/10.1038/ncomms11285





https://doi.org/10.1038/nature12651










https://doi.org/10.1016/j.tplants.2008.10.001

https://doi.org/10.1016/j.tplants.2008.10.001





https://doi.org/10.1111/nph.12928



35. Wang J, Cao MJ, Pan GT, Lu YL, Rong TZ. RNA editing of mitochondrial functional genes atp6 and

cox2 in maize (Zea mays L.). Mitochondrion. 2009; 9(5):364–9. https://doi.org/10.1016/j.mito.2009.07.

005 PMID: 19666144

36. Chakraborty A, Mitra J, Bhattacharyya J, Pradhan S, Sikdar N, Das S, et al. Transgenic expression of

an unedited mitochondrial orfB gene product from wild abortive (WA) cytoplasm of rice (Oryza sativa L.)

generates male sterility in fertile rice lines. Planta. 2015; 241(6):1463–79. https://doi.org/10.1007/

s00425-015-2269-5 PMID: 25754232

37. Suzuki H, Yu J, Ness SA, O’Connell MA, Zhang J. RNA editing events in mitochondrial genes by ultra-

deep sequencing methods: a comparison of cytoplasmic male sterile, fertile and restored genotypes in

cotton. Molecular Genetics and Genomics. 2013; 288(9):445–57. https://doi.org/10.1007/s00438-013-

0764-6 PMID: 23812672

38. Brown GG, Formanova N, Jin H, Wargachuk R, Dendy C, Patil P, et al. The radish Rfo restorer gene of

Ogura cytoplasmic male sterility encodes a protein with multiple pentatricopeptide repeats. Plant Jour-

nal. 2003; 35(2):262–72. PMID: 12848830

39. Dahan J, Mireau H. The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes.

Rna Biology. 2012; 10(9):1469–76.

40. Fujii S, Bond CS, Small ID. Selection patterns on restorer-like genes reveal a conflict between nuclear

and mitochondrial genomes throughout angiosperm evolution. Proc Natl Acad Sci U S A. 2011; 108

(4):1723–8. https://doi.org/10.1073/pnas.1007667108 PMID: 21220331



https://doi.org/10.1016/j.mito.2009.07.005

https://doi.org/10.1016/j.mito.2009.07.005


https://doi.org/10.1007/s00425-015-2269-5

https://doi.org/10.1007/s00425-015-2269-5


https://doi.org/10.1007/s00438-013-0764-6

https://doi.org/10.1007/s00438-013-0764-6






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