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Identification and Characterization of Two Paralogous Plastid Terminal Oxidase Genes in Soybean.

Byline: Xin Sun, Tao Lei, Jun-Bo Du and Wen-Yu Yang


Plastid terminal oxidase (PTOX) is a plastoquinol oxidase, which plays several important roles in plants. Previous studies suggested that PTOX is encoded by a single gene with only one copy in higher plants. Here we report the identification of two possible paralogous PTOX genes on different chromosomes of soybean, both of which are highly homologous to known PTOX genes in other species. These two paralogs have quite different introns and nearly the same exons and were predicted to encode membrane protein with chloroplast transit peptide. The deduced PTOX protein encoded by both paralogs were proposed to be functional, since the existence of highly conserved amino acid sites necessary for a typical PTOX, including six iron-binding sites and Exon 8 domain.

Moreover, soybean PTOX also exhibit clear sequence similarity to alternative oxidase (AOX). Organ-specific expression analysis showed high transcript levels of soybean PTOX in stems, leaves and flowers, while the levels in pods and roots were relatively low. In addition, a light-inducible character was also suggested for soybean PTOX in the present study.

Keywords: Alternative oxidase (AOX); IMMUTANS; PTOX; Phytozome; Soybean


Plastid terminal oxidase (PTOX), a thylakoid membrane-located quinol oxidase, exists widely in photosynthetic species including higher plants and algae (McDonald et al., 2011). It transfers electrons from plastoquinol to O2 with formation of H2O and acts as the terminal oxidase of chlororespiration, which represents a respiratory electron transport chain in thylakoid membrane. Moreover, PTOX was also regarded as an important co-factor of carotenoid biosynthesis by transferring the electrons derived from precursors to O2 via plastoquinol (McDonald et al., 2011).

Function of plant PTOX was indicated to be important for chloroplast biogenesis and beneficial for stressed-plants (Aluru et al., 2006; Sun and Wen, 2011). But the exact physiological role of PTOX is still unclear, since the limited data are available from only a few model species. More informationfromextensivespecies, especially important crops, is necessary for understanding the properties of plant PTOX. In the present study, we identified the PTOX gene in soybean, an important crop worldwide and analyzed the characters of soybean PTOX gene as well as the deduced protein. In addition, we also detected the expression of soybean PTOX in different organs and compared the transcript levels under light with that in the dark.

Materials and Methods

Seeds of soybean (Glycine max L. Merr. cv. Gongxuan 1, supplied by the Key Laboratory of Crop Eco-physiology and Farming System in Southwest China, Ministry of Agriculture) were germinated and grown in regular growing season. For light and dark treatments, soybean plants were divided into two groups and grown under continuous light or in complete darkness for 12 h, respectively, in growth chambers. Different organs, including roots, stems, leaves, flowers and pods were collected to extract total RNA as described by Lei et al. (2010).

To obtain cDNAs, reverse transcription (RT) was carried out with total RNA using M-MLV reverse transcriptase (TaKaRa Biotech. Co. Ltd., Dalian, P.R. China) and universal Oligo (dT)16 primer. Then, degenerate primers designed by Amirsadeghi et al. (2006) were used for PCR. The product was subsequently ligated into pMD19-T vector (TaKaRa Biotech. Co. Ltd., Dalian, P.R. China) and sequenced. Resulting sequence was applied to pick out candidate soybean PTOX through BLAST using the soybean database in Phytozome (Goodstein et al., 2012).

The identity of soybean PTOX was confirmed by alignment of the candidate sequences with known PTOX genes. Based upon the sequence of soybean PTOX, gene-specific primers were used for RT-PCR with total RNA to detect PTOX transcripts in different organs.

Sequence data of PTOX and alternative oxidase (AOX) used in the present study was obtained from Phytozome and GenBank database. Transit peptide was predicted by Target P 1.1 (Emanuelsson et al., 2007) and transmembrane domains were predicted by TMHMM 2.0 (Krogh et al., 2001). Sequence alignment was performed by Clustal X 2 (Larkin et al., 2007). Cis-acting regulatory elements were searched for soybean PTOX 1.0 kb promoter at PLACE database (Higo et al., 1999).


A product of 432 bp was obtained by PCR amplification with degenerate primers and then sequenced. After BLAST, two candidate loci with names of Glyma09g01130 and Glyma15g11950 were picked out. Transcript sequences of these two candidate loci display high homologies with known PTOX genes, e.g. about 80% sequence identity with Arabidopsis PTOX gene (also known as IMMUTANS, GenBank accession number: AF098072), confirming the identity of soybean PTOX. The existence of two PTOX sequences in soybean genome suggested that there are two copies of PTOX gene appearing as paralogs on different chromosomes (9 and 15, respectively).

Glyma09g01130 and Glyma15g11950 both have eight introns and nine exons (Fig. 1). Their introns display clearly sequence differences, especially for the 4th and 6th introns. Glyma15g1195 has much longer 4th and 6th introns, with insert fragments more than 2400 bp and 300 bp, respectively, compared with Glyma09g01130. However, their nine exons have nearly the same sequences, indicating almost the same amino acid-encoding.

Transcript sequences of Glyma09g01130 and Glyma15g11950 both have an open reading frame of 999 bp, which encodes a protein of 332 amino acids with a 36-amino acid chloroplast transit peptide (Fig. 2). The mature protein of 296 amino acids has a calculated molecular mass of about 34.3 kDa. Two transmembrane domains were predicted in the deduced polypeptide (Fig. 2), indicating the membrane protein property. Six iron-binding sites, including four glutamate and two histidine residues (E116, E155, H158, E207, E276, H279), were also proposed (Fig. 2).

These iron-binding sits are conserved in all PTOX proteins examined to date and showed no change (Fu et al., 2005; McDonald et al., 2011). Moreover, E155 and H158, E276 and H279 also display as the EXXH motifs, which have strong iron-binding property. Exon 8 domain is crucial for activity and stability of PTOX proteins (McDonald et al., 2011). This domain has not been found in other proteins (Fu et al., 2005), further confirming the identity of soybean PTOX. Besides, another five vital sites (L115, H131, Y192, Y214, D274), which have functional importance for PTOX activity such as substrate binding (Fig. 2).

Sequence alignment revealed that the deduced amino acid sequences of Glyma09g01130 and Glyma15g11950 share 19-22% identity with the three members of soybean AOX (Fig. 2), which act as the terminal oxidase of alternative pathway in mitochondria. Many residues are conserved between PTOX and AOX, including six iron-binding sites and five conserved activity sites (Fig. 2). However, PTOX and AOX also have their own unique domains. For example, the Exon 8 domain in PTOX is missing in all the members of soybean AOX (Fig. 2). On the other hand, a conserved cysteine in all of the AOX members, which participates in disulde bond formation between adjacent monomers and gives rise to a dimer, is not found in PTOX (Fig. 2), implying that PTOX cannot dimerize and exists only as a monomer.

Data further detected PTOX transcripts in different soybean organs (i.e. roots, stems, leaves, flowers and pods). Primers used for RT-PCR were designed based on the same coding sequences from both paralogs, in order to detect total transcripts. Results showed high transcript levels in stems, leaves and flowers, while the levels in pods and roots were relatively low (Fig. 3a). Cis-acting regulatory elements analysis indicated a widely distribution of light-responsive motifs (e.g. GATA-box, GT1-motif, REalpha) in the promoters of both Glyma09g01130 and Glyma15g11950 (data not shown) and suggested a light-inducible character for soybean PTOX. Subsequently, we detected the transcript levels of PTOX under light and dark conditions, respectively.

Results showed a higher level in leaves under light compared with that in the dark (Fig. 3b), further confirmed the light-inducible character of soybean PTOX. But transcripts remained at the same level in roots under both light and dark conditions (Fig. 3b), suggesting a different expression pattern other than light-induction in roots.


Soybean is a diploidized ancient tetraploid, whose genes are often present as multiple copies since the chromosome duplication events in evolution process (Schmutz et al., 2010). So, existence of two paralogous PTOX genes in soybean can be considered as a result of this duplication events. However, Glyma09g01130 and Glyma15g11950 are both described as AOX, which encodes mitochondria-located protein, in Phytozome database. Many studies indicated that PTOX and AOX are homologs, which have the same origin (McDonald and Vanlerberghe, 2006). But they have distinct subcellular localizations and unique conserved domains. Since the existence of chloroplast transit peptide and Exon 8 domain in deduced amino acid sequences, we hold the opinion that these two loci should be described as PTOX.

Previous studies suggested that PTOX always appears as a single gene with only one copy in the genomes of higher plants (Wu et al., 1999; Kong et al., 2003), while a recent study showed a second gene with homology to PTOX in rice genome (Tamiru et al., 2014). But this gene was predicted to encode a polypeptide which lacks many of the conserved residues necessary for PTOX, such as iron-binding sites and the Exon 8 domain and is therefore unlikely to execute function (Tamiru et al., 2014). In the present study, deduced amino acid sequences of the two soybean PTOX paralogs have all of the conserved sites, involving the six iron-binding sites and Exon 8 domain. Therefore, both of these paralogs can be proposed to encode functional PTOX protein.

Our present study showed that soybean PTOX has an organ-dependent expression pattern. The expression of PTOX gene was also indicated to be organ-dependent in Arabidopsis and rice (Aluru et al., 2001; Tamiru et al., 2014), implying that PTOX has distinct roles in different organs. In addition, transcript detection and cis-acting regulatory elements analysis suggested a light-inducible character for soybean PTOX. But this character is not proposed for underground organ, the roots.


This study identified two possible paralogous PTOX genes in a higher plant for the first time. Both of these paralogs in soybean were suggested to encode functional PTOX protein, which possess all of the conserved amino acid sites necessary for PTOX. The expression of soybean PTOX was suggested to be organ-dependent and light-inducible.


This work was supported by the National Natural Science Foundation of China (31000682, 31371555 and 31401308) and the Applied Basic Research Project of Sichuan Province (2014JY0103).


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Author:Sun, Xin; Lei, Tao; Du, Jun-Bo; Yang, Wen-Yu
Publication:International Journal of Agriculture and Biology
Article Type:Report
Date:Dec 31, 2015
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