Planta (2015) 241:29–42 DOI 10.1007/s00425-014-2217-9

ORIGINAL ARTICLE

Expression divergence of cellulose synthase (CesA) genes after a recent whole genome duplication event in Populus Naoki Takata • Toru Taniguchi

Received: 12 August 2014 / Accepted: 27 November 2014 / Published online: 9 December 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Main conclusion Secondary cell wall-associated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules. Gene duplication is an important mechanism for functional divergence of genes. Secondary cell wall-associated cellulose synthase genes (CesA4, CesA7 and CesA8) are duplicated in Populus plants due to a recent whole genome duplication event. Here, we demonstrate that duplicate CesA genes show tissue-dependent expression divergence in Populus plants. Real-time PCR analysis of Populus CesA genes suggested that Pt 9 tCesA8-B was more highly expressed than Pt 9 tCesA8-A in phloem and secondary xylem tissue of mature stem. Histochemical and histological analyses of transformants expressing a GFP-GUS fusion gene driven by Populus CesA promoters revealed that the duplicate CesA genes showed different expression patterns in phloem fibers, secondary xylem, root cap and leaf trichomes. We predicted putative cis-regulatory motifs that regulate expression of secondary cell wall-associated CesA genes, and identified 19 motifs that are highly conserved in the CesA gene family of eudicotyledonous plants. Furthermore, a transient Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2217-9) contains supplementary material, which is available to authorized users. N. Takata (&)  T. Taniguchi Forest Bio-Research Center, Forestry and Forest Products Research Institute, Hitachi, Ibaraki 319-1301, Japan e-mail: [email protected] T. Taniguchi Forest Tree Breeding Center, Forestry and Forest Products Research Institute, Hitachi, Ibaraki 319-1301, Japan

transactivation assay identified candidate transcription factors that affect levels and patterns of expression of Populus CesA genes. The present study reveals that secondary cell wallassociated CesA genes in Populus have undergone a functional differentiation in expression pattern that may be attributable to evolutionary alteration of regulatory modules. Keywords Cellulose synthase  Functional divergence  Gene duplication  Secondary cell wall  Whole genome duplication Abbreviations CaMV35S Cauliflower mosaic virus 35S CesA Cellulose synthase CSC Cellulose synthase complex GFP Green fluorescent protein EmGFP Emerald green fluorescent protein GUS b-Glucuronidase JTT Jones–Taylor–Thornton LBD LOB domain-containing protein LUC Luciferase M46RE MYB46-responsive cis-element MBSIIG MYB binding site IIG NAC NAM, ATAF1/2 and CUC2 NJ Neighbor-joining RLUC Renilla reniformis luciferase UBQ Ubiquitin WGD Whole genome duplication ZF Zinc-finger protein

Introduction Polyploidy and whole genome duplication (WGD) provide an important source of genetic material for functional

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divergence and innovation (Taylor and Raes 2004). Angiosperm genomes have undergone several ancient WGD events that have been proposed as a driving force for phenotypic and developmental diversity of a wide variety of plant species (Jiao et al. 2011). In Populus species, a polyploidy event called salicoid WGD took place approximately 65 million years ago and affected roughly 92 % of the Populus genome (Tuskan et al. 2006). Syntenic blocks derived from WGD have been widely identified within the extant Populus genome, although genomic rearrangement occurred during evolution of the Salicaceae. The syntenic relationships clearly demonstrate that more than 8,000 pairs of paralogous genes can be identified in the genome of Populus trichocarpa. Paralogous genes accumulate postWGD evolutionary information in genomic sequences such as nucleotide substitutions and insertion-deletion mutations that affect gene expression patterns and protein functions. In P. trichocarpa, whole genome microarray analysis reveals that nearly half of salicoid duplicate genes show tissue-dependent expression divergence (Rodgers-Melnick et al. 2012), indicating that the paralogous genes are under functional differentiation in expression levels and patterns. Duplicate genes diverge in function over time after a gene duplication event. Paralogous gene pairs may undergo divergent fates such as subfunctionalization (subdivision of original functions), neofunctionalization (random acquisition of a new function) and nonfunctionalization (loss of original functions) (Force et al. 1999). Functional divergence is due to genetic mutations in regulatory sequences that affect regulation of gene expression, in coding sequences that affect protein function, or both. Gene expression divergence between duplicate genes, which is attributed to alterations in regulatory sequences such as cisregulatory elements, is widely believed to be an important driver of morphological change in organisms (Ohta 2003; Jime´nez-Delgado et al. 2009). In the model plant Arabidopsis thaliana, an evo-devo approach using a wide range of gene expression data and morphological observations revealed that differential expression patterns among paralogous gene pairs partly contribute to plant developmental diversification (Hanada et al. 2009). Although tissuedependent expression patterns are observed in many salicoid duplicate genes (Rodgers-Melnick et al. 2012), how evolutionary changes in gene regulation mechanisms are introduced is not clear in paralogous gene pairs of Populus. Cellulose synthase (CesA), the catalytic subunit involved in cellulose biosynthesis, constitutes a multigene family in land plants (Yin et al. 2009). There are ten CesA genes in the genome of A. thaliana, with three of them (CesA4, CesA7 and CesA8) required for secondary cell wall biosynthesis (Taylor et al. 2003). Tissue-dependent expression patterns of the secondary cell wall-associated CesA genes are coordinately controlled by transcription

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factors, some of which are master genes controlling vessel and fiber cell development in xylem tissue (Mitsuda et al. 2005; Yamaguchi et al. 2010). One-to-one orthologous genes of the triple subunits (CesA4, CesA7 and CesA8) are conserved in eudicotyledonous and monocotyledonous plants, indicating that the common ancestral gene was triplicated before the expansion of angiosperm species (Yin et al. 2009). Monocot CesA genes corresponding to Arabidopsis CesA4, CesA7 and CesA8 are involved in secondary cell wall formation (Tanaka et al. 2003; Handakumbura et al. 2013), suggesting that this functional property of the genes has been conserved among angiosperms. In Populus plants, the copy number of secondary cell wall-associated CesA has increased to five genes due to a gene duplication event (Kumar et al. 2009). The five CesA genes may constitute cellulose synthase complex (CSC) that contributes to secondary cell wall biosynthesis in developing xylem tissue (Song et al. 2010). Although the CesA genes are coordinately expressed in cells forming secondary cell walls, the duplicate CesA genes show different expression levels in several tissues (Suzuki et al. 2006; Dharmawardhana et al. 2010; Song et al. 2010). It remains unclear, however, whether these CesA members have undergone functional differentiation during Populus evolution and which transcriptional regulators govern the diverged expression patterns. Understanding the evolutionary changes in coordinate expression patterns of the CesA genes would provide insight into the evolutionary fate of duplicate gene pairs in Populus plants. In this study, we investigated expression divergence of secondary cell wall-associated CesA genes in hybrid aspen. Phylogenetic analysis revealed that the number of CesA genes has increased in Populus plants due to a recent WGD event. The CesA genes showed coordinate expression patterns in most tissues examined, while duplicate genes such as CesA7-A and CesA7-B, and CesA8-A and CesA8B exhibited differential expression levels in several tissues. Promoter regions of the paralogous genes diverged genetically during the evolutionary process. Candidate transcriptional regulators that would affect expression divergence of the duplicate gene pairs were identified using a transient transactivation assay. Our data allowed us to explore evolutionary changes in levels and patterns of expression of the duplicate CesA genes.

Materials and methods Phylogenetic analysis and chromosomal synteny CesA genes were retrieved from genomic databases for Arabidopsis thaliana (The Arabidopsis Information Resource, TAIR), Medicago truncatula (Medicago

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Genome/Hapmap resources version Mt3.5), Oryza sativa (Rice Annotation Project Database) and Brachypodium distachyon, Carica papaya, P. trichocarpa, Sorghum bicolor, Selaginella moellendorffii and Vitis vinifera (Phytozome v7.0). Accession numbers or locus IDs of CesA genes are described in Table S1. Amino acid sequences were aligned using the ClustalW program. The number of amino acids substituted between each pair of CesA proteins was estimated by the Jones–Taylor–Thornton (JTT) model with the complete-deletion option (Jones et al. 1992). From the number of estimated amino acid substitutions, a phylogenetic tree was reconstructed by the neighbor-joining (NJ) method. Bootstrap values were calculated with 1,000 replications using the NJ method. The CesA gene of Mesotaenium caldariorum was utilized as an outgroup in the phylogenetic tree to find the root. These procedures were performed using MEGA5 software (Tamura et al. 2011). Chromosomal synteny was reconstructed in the Plant Genome Duplication Database (Lee et al. 2012). Plant material and growth conditions Sterile rooted cuttings of Populus tremula 9 Populus tremuloides (Pt 9 t; wild type clone T89) were provided by Yasunori Ohmiya (Forestry and Forest Products Research Institute) and were cultured in 0.59 Murashige and Skoog (MS) medium (pH 5.7) containing 0.8 % (w/v) agar at 25 °C under a cycle of 18-h light (50 lmol m-2 s-1)/6-h dark. Cultured plants were transplanted to soil mix (3:1 fertilized peat moss: vermiculite, w/w). Hybrid aspen plants were grown at 18 °C under long-day conditions (18-h light at 300 lmol m-2 s-1/6-h dark). Plants were watered and fertilized once a week with 2,000-fold diluted Hyponex 6-10-5 solution (Hyponex, Osaka, Japan). RNA extraction and real-time PCR Plant tissues were collected from hybrid aspen growing for 2 months on soil. The samples were immediately frozen in liquid nitrogen and stored at -80 °C until use. Total RNA was isolated from samples using an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) with in-column DNase I digestion. First-strand cDNA was synthesized using a High Capacity RNA-to-cDNA Kit (Life Technologies, Carlsbad, CA, USA). Real-time PCR was performed using a StepOnePlus Real-Time PCR System with Power SYBR Green PCR Master Mix (Life Technologies). The genespecific primers for real-time PCR are described in Table S2. To examine relative abundance of mRNA of Pt 9 tCesA genes, the amplified fragments were subcloned into vector pCR2.1 (Life Technologies). These inserts were

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introduced in the pTAC1 vector harboring Ubiquitin (Pt 9 tUBQ) by conventional cloning methods (Takata et al. 2009). The vector containing the fragments of Pt 9 tCesA4, Pt 9 tCesA7A, Pt 9 tCesA7B, Pt 9 tCesA8A, Pt 9 tCesA8B and Pt 9 tUBQ was used to generate a standard curve for real-time PCR amplification. The transcript levels of Pt 9 tCesA genes were normalized to that of Pt 9 tUBQ. Each RNA sample was assayed in triplicate. RNAs were assayed from two biological replicates. Plasmid construction and generation of transgenic plants The amplified EmGFP gene (Life Technologies) was introduced in the BamHI site between the Cauliflower mosaic virus 35S (CaMV35S) promoter and GUS of pBI121 (Jefferson et al. 1987) to construct a binary vector harboring an EmGFP-GUS fusion reporter gene. The promoter regions of Pt 9 tCesA4, Pt 9 tCesA7A, Pt 9 tCesA7B, Pt 9 tCesA8A and Pt 9 tCesA8B were isolated from hybrid aspen using primer pairs described in Table S2. We used sequences longer than 1,500 bp for the plasmid construction, since many studies demonstrate that 1–2 kb upstream region of the translation start site of genes determines the specificity of gene expression pattern (Xiao et al. 2010; Zou et al. 2011; Wang et al. 2012). The promoter region of Pt 9 tCesA4 (2.3 kb), Pt 9 tCesA7A (2.5 kb), Pt 9 tCesA7-B (2.8 kb) and Pt 9 tCesA8A (1.5 kb) extends to 30 -UTR of the neighboring upstream gene but not to the coding region and 50 -UTR. The promoter region of Pt 9 tCesA8-B (3.2 kb) does not include the sequence of the neighboring upstream gene. The PCRamplified promoter fragments were introduced into the SbfI/XhoI site of the binary expression vector, replacing CaMV35S. The binary vectors constructed were introduced into Agrobacterium tumefaciens strain GV3101 (pMP90) (Koncz and Schell 1986). Hybrid aspen was transformed with the vectors and transgenic plants were regenerated essentially as described in Eriksson et al. (2000). Three independent transgenic lines were selected for each genotype and used in subsequent experiments. Histochemical assays Histochemical GUS assays were performed according to a modified protocol of Jefferson et al. (1987). Plant tissues were pretreated with 90 % acetone on ice for 30 min and then incubated in GUS staining solution [0.5 mg ml-1 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid, 50 mM sodium phosphate (pH 7.0), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 0.1 % Triton X-100]

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in the dark at 37 °C for 16 h. Samples were fixed in 2.5 % (v/v) glutaraldehyde in 50 mM sodium phosphate (pH 7.0) and subsequently bleached in 70 % ethanol. A Leica CM3050 S cryomicrotome (Leica, Solms, Germany) was used for sectioning of internodes, petioles and shoot tips. Roots and leaves were sectioned by a vibratome (Dosaka EM, Kyoto, Japan). Images were captured by an MZ FLIII stereomicroscope (Leica) and a Leica DMR microscope. GFP observation GFP fluorescence was observed in radial sections of mature internodes. Stem cuttings with a 10 mm length were obtained from the 30th internode and cut into 100 lm radial sections using a sliding microtome (Yamatokohki, Saitama, Japan). Sections were mounted onto glass slides and coverslipped with 0.59 MS medium. GFP fluorescence and autofluorescence of the cell wall were monitored using a Leica TCS SPE system (Leica) with a 209 objective, NA = 0.7 (PL APO CS 20x/0.70 HC, Leica). Autofluorescence excited by a 405 nm laser was detected between 425 and 475 nm. GFP was excited with a 488 nm laser and GFP emission was detected between 500 and 520 nm. Images were combined and processed with Adobe Photoshop CS5 (Adobe systems, San Jose, CA, USA).

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Fusion cloning kit (Takara Bio, Shiga, Japan), replacing CaMV35S. Plasmid CaMV35S::RLUC was used as an internal control for transient transformation. Vector pSAT1 was used to construct effector constructs for transient assays (Tzfira et al. 2005). Effector genes PNAC122, MYB055, MYB152, GATA8b and ZF1 were obtained from the Populus nigra full-length cDNA library collection of the RIKEN BioResource Center (Nanjo et al. 2004). Other effectors were isolated from hybrid aspen using primer pairs (Table S2). Effector genes, their salicoid duplicates and Arabidopsis orthologs are shown in Table S3. Coding sequences of effectors and uidA (GUS) were amplified by PCR and subsequently introduced into the NcoI/BamHI site of vector pSAT1 using an In-Fusion cloning kit. Plasmid CaMV35S::GUS was used as a vector control. Transactivation analysis was performed according to a protocol of Ohtani et al. (2011). T89 leaves were transiently transformed with 1.0 lm diameter gold particles (Bio-Rad, Hercules, CA, USA) coated with a mixture of the reporter, effector and reference plasmid DNA at a ratio of 2.0:0.8:1.0 using a PDS-1000/He system (Bio-Rad). The leaf samples were collected after overnight incubation. Luciferase activity was analyzed by a Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA) using a GloMax 20/20 Luminometer (Promega).

Identification of conserved motifs Accession numbers To predict motifs conserved among promoter regions of secondary cell wall-associated CesA genes, we retrieved regions 2,000 bp upstream from the translation start site from genomic databases for A. thaliana (TAIR), M. truncatula (Medicago Genome/Hapmap resources), Eucalyptus grandis and V. vinifera (Phytozome) (Dataset S1). A set of 1,329 motifs that are highly conserved in Arabidopsis genes and are relevant to plant cell wall synthesis was reported by Wang et al. (2012). We examined whether each motif was retained in the promoter regions of P. tremula 9 P. tremuloides and four other plant species using CLC Main Workbench software (CLCBio, Cambridge, MA, USA). We extracted motifs that were conserved in at least 13 of the 17 CesA genes as putative cis-regulatory motifs. Transient expression assay pUC18-based vectors containing CaMV35S::firefly luciferase (LUC) and CaMV35S::Renilla reniformis luciferase (RLUC) (Shimada et al. 2007) were provided by Prof. Masaaki Sakuta (Department of Biology, Ochanomizu University). To construct reporter plasmids, Pt 9 tCesA promoters amplified by PCR were introduced into the HindIII/SalI site of plasmid CaMV35S::LUC using an In-

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Accession numbers of Pt 9 tCesA gene promoters and effector genes are shown in Table S4.

Results Gene duplication and deletion of Populus CesA genes Five CesA genes, CesA4, CesA7-A, CesA7-B, CesA8-A and CesA8-B, may constitute a CSC to synthesize secondary cell wall in xylem tissue of Populus plants (Song et al. 2010). A reconstructed phylogenetic tree clearly showed that three clusters, CesA-IVa, -IVb and -IVc, are composed of secondary cell wall-associated CesA genes of eudicotyledonous and monocotyledonous plants (Fig. 1a; Table S1). Chromosomal synteny within the P. trichocarpa genome clearly showed that two duplicate gene pairs, CesA7-A and CesA7-B, and CesA8-A and CesA8-B, were produced during the salicoid WGD (Fig. 1c, d) (Tuskan et al. 2006). Ancient CesA4 was duplicated into two genes via the WGD; however, one member of the gene pair may have been deleted during subsequent chromosome rearrangement (Fig. 1b). Since several WGD events have occurred in angiosperm evolutionary lineages (Jiao et al.

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33 b Fig. 1 Phylogenetic tree of CesA genes in angiosperms (a) and

(b)

McCesA1

0

100 SmCesA01-A

SmCesA01-B

Isoetopsida

SmCesA02-B SmCesA03-A 100

SmCesA03-B

500 Chromosome 14 (genes)

100 SmCesA02-A 94

100 SmCesA04-A

SmCesA04-B AtCesA7

75

VvCesA7 PtiCesA7-A

95 50 100

PtiCesA7-B

Monocots

BradiCesA09 OsCesA09

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CesA-IVa

Eudicots

MedtrCesA7

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65

61

SbCesA09

1,500

2,000 0

(c)

AtCesA3

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1,000

2011), other angiosperms examined have lost one member of CesA duplicates after WGD and therefore reverted back to three genes, CesA triplet members CesA4, CesA7 and CesA8, that are involved in secondary cell wall formation (Fig. 1a).

CesA-V

PtiCesA3-D

100

CpCesA3 72

Chromosome 18 (genes)

PtiCesA3-C

97

200

Eudicots

PtiCesA3-B

VvCesA3-B SbCesA02

80 50

Monocots

OsCesA02 BradiCesA02

98

BradiCesA08 OsCesA08

99 81

400 600 800 1,000

PtiCesA7-B 1,200 1,400

SbCesA08

0

AtCesA1

99

AtCesA10

CpCesA1

90

PtiCesA1-A

83

PtiCesA1-B

100

OsCesA01

Monocots

97

BradiCesA01

80

(d)

CesA-VI

Eudicots

MedtrCesA1 VvCesA1

66

SbCesA01

500 1,000 1,500 2,000 2,500 Chromosome 6 (genes) PtiCesA7-A

3,000

0

500 PtiCesA8-A

CesA-VII (including MedtrCesA11, VvCesA11,SmCesA11-A, SmCesA11-B, BradiCesA11, OsCesA11 and SbCesA11)

Chromosome 11 (genes)

77

100

2,500 PtiCesA4

PtiCesA3-A

100

VvCesA3-A

52

1,000 1,500 2,000 Chromosome 2 (genes)

0

MedtrCesA3 95

500

1,000

1,500

VvCesA6-A

0

AtCesA2

99

AtCesA9

98

AtCesA5

100 92

100

AtCesA6

MedtrCesA6-A

54

PtiCesA6-A

53 97

CesA-II

Eudicots

CpCesA6-A

66

PtiCesA6-B MedtrCesA6-B VvCesA6-B

96

98

CpCesA6-B PtiCesA6-C 100

PtiCesA6-D MedtrCesA6-C

55

PtiCesA6-E

94

PtiCesA6-F

52 96

Eudicots

CpCesA6-C 100

VvCesA6-C

69

VvCesA6-D

CesA-I

BradiCesA06

82

OsCesA06 97

SbCesA06

Monocots

100

BradiCesA03-A BradiCesA03-B

65

OsCesA03

50

SbCesA03

57

OsCesA05

70 56

chromosomal synteny of P. trichocarpa chromosomes harboring secondary cell wall-associated CesA genes (b–d). The phylogenetic tree was reconstructed by the NJ method based on the number of amino acid substitutions estimated by the JTT model (a). The numerals at the branch indicate bootstrap values calculated by the NJ method with 1,000 replications. Bootstrap values [50 % are shown. The clade names refer to Yin et al. (2009). Syntenic relationships between chromosome (chr.) 2 and chr. 14 (b), between chr. 6 and chr. 18 (c) and between chr. 4 and chr. 11 (d) were described by a synteny plot. Red arrows indicate the chromosomal location of the CesA genes

SbCesA05

500 1,000 1,500 2,000 PtiCesA8-B Chromosome 4 (genes)

2,500

Tissue-dependent expression levels of CesA genes in hybrid aspen To detail coordinate expression patterns of the five CesA genes and the expression divergence of duplicate genes such as CesA7-A and CesA7-B, and CesA8-A and CesA8-B, gene expression levels were estimated in several tissues using quantitative real-time PCR. In P. tremula 9 P. tremuloides, the CesA genes showed higher expression in mature stem tissue, such as the 10th and 20th internodes, than in leaves, petioles, shoot tips and younger stems (Fig. 2). In the 10th and 20th internodes, higher expression was observed in xylem tissue than phloem tissue. For the duplicate genes, Pt 9 tCesA8-B was more highly expressed than Pt 9 tCesA8-A in all tissues examined and was highly abundant in xylem tissue of the mature stem. The expression of Pt 9 tCesA7-B was slightly higher than that of Pt 9 tCesA7-A in leaves, petioles, shoot tips and younger stems. In xylem tissues of the mature stem, there was no difference in expression between Pt 9 tCesA7A and Pt 9 tCesA7-B. The expression analysis suggests that the duplicate gene pairs, Pt 9 tCesA7-A and Pt 9 tCesA7-B, and Pt 9 tCesA8-A and Pt 9 tCesA8-B, are differentially regulated in several tissues of hybrid aspen.

AtCesA4 MedtrCesA4

57

CpCesA4

61

VvCesA4

92

SbCesA07

Monocots

BradiCesA07 OsCesA07

100

MedtrCesA8

90 98

PtiCesA8-A

100

CpCesA8

100 96

VvCesA8

58

OsCesA04 SbCesA04

Monocots

BradiCesA04 100

CesA-IVc

PtiCesA8-B

64

Eudicots

AtCesA8

76

0.05

Developmental and tissue expression patterns of Pt 9 tCesA genes

CesA-IVb

PtiCesA4

84 100

Eudicots

88

To investigate tissue-specific expression patterns of CesA genes, we generated transgenic hybrid aspen plants that express a GFP-GUS fusion gene driven by Pt 9 tCesA promoters. The upstream region of Pt 9 tCesA4, Pt 9 tCesA7-A, Pt 9 tCesA7-B and Pt 9 tCesA8-B (2.3–3.1 kb

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Planta (2015) 241:29–42 1.2

(a)

PtxtCesA4 PtxtCesA7-A PtxtCesA7-B PtxtCesA8-A PtxtCesA8-B

0.04

(b) 1.0

0.8

0.03 0.6 0.02 0.4 0.01

0.2

0

Relative expression (PtxtCesAs/UBQ)

Relative expression (PtxtCesAs/UBQ)

0.05

0 Young leaf

Petioles Petioles Mature leaf Shoot tip (Young leaves) (Mature leaves)

2nd and 3rd 4th and 5th internodes internodes

Phloem Xylem 10th internode

Phloem Xylem 20th internode

Fig. 2 Relative expression levels of Pt 9 tCesA genes in hybrid aspen. Real-time PCR was performed to estimate transcript accumulation of each gene in young leaves, mature leaves, petioles, shoot tips, 2nd and 3rd internodes and 4th and 5th internodes (a), and in phloem and xylem of 10th internodes and 20th internodes (b).

Expression levels of the Pt 9 tCesA genes were normalized to that of Pt 9 tUBQ. Each RNA sample was assayed in triplicate. Results are representative, and experiments were repeated with similar results. Values are mean ± SD

from the start codon) was isolated to generate a binary expression vector. For construction of Pt 9 tCesA8-A, the upstream region from the start codon to the 30 UTR of the neighboring upstream gene (1.5 kb region) was used. In younger shoots, the five CesA genes were expressed in primary xylem of the third internode but not in other tissues such as the shoot apical meristem, pith or primary phloem (Fig. 3e, f and Suppl. Fig. S1). The expression of the duplicate gene pair Pt 9 tCesA7-A and Pt 9 tCesA7B was weaker than the others in the primary xylem. In the mature stem, the Pt 9 tCesA genes were expressed in phloem fibers and secondary xylem tissue (Fig. 3g, h and Suppl. Fig. S1). The promoter activity of Pt 9 tCesA8A was lower than that of the others in the secondary phloem and xylem tissues. In leaves, Pt 9 tCesA4, Pt 9 tCesA8A and Pt 9 tCesA8-B were expressed in leaf veins, xylem of the main vein and petiole xylem vessels (Fig. 3a–d and Suppl. Fig. S1). Lower expression was detected for the gene pair Pt 9 tCesA7-A and Pt 9 tCesA7-B in leaf veins and petioles. GUS staining of trichome cells was detected in Pt 9 tCesA4, Pt 9 tCesA7-B, Pt 9 tCesA8-A and Pt 9 tCesA8-B plants; however, weaker staining was observed in Pt 9 tCesA7-A plants. In root tissue, promoter activities of the genes were observed in the root vascular bundle (Fig. 3i, j and Suppl. Fig. S2). Intriguingly, gene expression diverged among transgenic constructs and transgenic lines in the root cap. Positive GUS staining was observed in the root cap of 50 % of Pt 9 tCesA4 lines, 31.6 % of Pt 9 tCesA7-A lines, 11.8 % of Pt 9 tCesA7-B lines and 75 % of Pt 9 tCesA8-A lines; however, no lines of Pt 9 tCesA8-B showed staining. These results indicate

that the secondary cell wall-associated CesA genes showed differential tissue-dependent expression patterns in hybrid aspen.

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Expression patterns of the Pt 9 tCesA genes in secondary xylem differentiation To examine expression patterns of the five Pt 9 tCesA genes in developing xylem, GFP fluorescence was imaged in radial sections of mature stems at the 30th internode. The Pt 9 tCesA genes were expressed in phloem fiber cells and secondary xylem, including xylem fibers and ray parenchyma cells (Fig. 4 and Suppl. Fig. S3). GFP fluorescence was extensively detected in fiber cells of secondary xylem tissue. The fiber cells that exhibited GFP fluorescence had autofluorescence excited by UV light, which represents lignification of the cell wall. The GFP fluorescence of Pt 9 tCesA8-A plants was weaker than that of the other transgenic plants in phloem fibers, xylem fibers and xylem ray parenchyma cells, which was consistent with the GUS staining patterns in mature stems. These results suggest that the duplicate gene pair Pt 9 tCesA8-A and Pt 9 tCesA8-B shows expression divergence throughout secondary xylem differentiation. Divergence of promoter regions among Populus CesA genes Our histochemical and histological analyses suggest that the expression divergence of CesA genes is due to evolutionary alteration of their promoter sequences. To

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Fig. 3 Histochemical GUS analysis in transgenic hybrid aspen. The images were captured in leaf blade (a), trichome (b), cross section of main vein (c), cross section of petiole (d), radial section of shoot tip (e), cross section of 3rd internode (f), 10th internode (g) and 20th internode (h), root (i) and cross section of root (j). Primary xylem in 3rd internodes is shown in high-magnification images. CZ cambial zone, PF phloem fiber, Ph phloem, Pi pith, PX primary xylem, SX secondary xylem, Xy xylem. Bars indicate 1 cm (i), 200 lm (a, c–h and j) and 100 lm (b and high-magnification images in f)

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(a)

(b)

Ep PF CZ+EZ Co Ph

Ep

SX

Pi

Co

PF

Ph

CZ+EZ

SX

PX Pi

SX

Pi

Bright field

GFP

Autofluorescence

(c) Ep

Co

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SX

Ep PF CZ+EZ Pi Co Ph

Bright field

GFP

Autofluorescence

(e) Ep PF CZ+EZ Co Ph

SX

PX Pi

Bright field

GFP

Autofluorescence

Fig. 4 Expression of GFP in mature stem of transgenic hybrid aspen. Bright field image, GFP fluorescence and autofluorescence excited by 405 nm laser were captured in radial section of transgenic hybrid aspen samples; Pt 9 tCesA7-Apro::GFP-GUS (a), Pt 9 tCesA7Bpro::GFP-GUS (b), Pt 9 tCesA8-Apro::GFP-GUS (c), Pt 9 tCesA8-Bpro::GFP-GUS (d) and Pt 9 tCesA4pro::GFP-GUS

(e). GFP fluorescence in xylem ray parenchyma cells are shown in high-magnification images (insets pits are seen on the radial wall of the ray parenchyma cell). Co cortex, CZ cambial zone, Ep epidermis, EZ expansion zone, PF phloem fiber, Ph phloem, Pi pith, PX primary xylem, SX secondary xylem. Bars indicate 200 and 20 lm (highmagnification images)

investigate genetic variation in the promoter sequences, we first compared sequences by nucleic acid dot plots. Genetic mutations such as nucleotide substitution, insertion and

deletion had accumulated in the promoter regions of the duplicate genes, although the coding sequences were highly conserved (Suppl. Fig. S4).

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Planta (2015) 241:29–42

We next predicted putative cis-regulatory motifs that regulate expression patterns of the CesA genes. In A. thaliana, 1,329 motifs have been reported as putative cisregulatory motifs that would be relevant to cell wall formation (Wang et al. 2012). CesA4, CesA7 and CesA8 of A. thaliana, for example, respectively, have 42, 13 and 41 unique motifs in their 1,000 bp upstream regions. To clarify phylogenetic conservation of regulatory motifs among secondary cell wall-associated CesA genes, we examined which motifs are conserved among CesA genes of eudicots such as A. thaliana, E. grandis, M. truncatula, P. tremula 9 P. tremuloides and V. vinifera. The computational analysis showed that 19 motifs were highly retained in the secondary cell wall-associated CesA gene family (Table 1; Suppl. Fig. S5). The motifs corresponded to characterized cis-elements such as four MYB binding sites, a GAGA element, an ABI4 binding site, a TCP binding site, CAACTC regulatory elements, JASE2, an L1 element, IDE1, an AGAMOUS binding site, an ALS-1 binding site, a STY1 binding site and a BOX I consensus sequence (Table S5). Nine putative motifs were conserved in the promoter sequences of all secondary cell wallassociated CesA genes of Populus, some of which were relevant to MYB binding sites (Table 1). For the duplicate gene pairs, there were, respectively, four and three motifs retained in one member of the CesA7 gene pair and the CesA8 gene pair. Transcription factors regulating expression of Populus CesA genes Many transcription factors such as the MYB and NAC (NAM, ATAF1/2 and CUC2) genes control biosynthesis of cell wall components (cellulose, hemi-cellulose and lignin) in xylem tissue (Zhong et al. 2011; Hussey et al. 2013). To identify transcription factors affecting differential expression levels of the Populus CesA genes, we conducted transactivation assays using 19 transcription factors including the MYB, NAC, GATA, LBD (LOB domaincontaining protein) and ZF (zinc-finger protein) genes, which are downstream targets of secondary wall-associated NAC master genes (Fig. 5a) (Ohashi-Ito et al. 2010; Yamaguchi et al. 2011; Zhong et al. 2011). The assay showed that 15 of the transcription factors positively regulated at least one of the secondary cell wall-associated CesA genes in Populus (Fig. 5b). The MYB transcription factors MYB003, MYB010, MYB125, MYB152 and MYB192 induced luciferase activities of all reporter constructs of CesA genes, indicating that these factors act as transactivators. The Pt 9 tCesA4 and Pt 9 tCesA8 gene pairs were, respectively, activated by MYB199, PNAC124, PNAC161 and LBD15, and by MYB055, MYB148, MYB199 and PNAC124. For the duplicate genes,

37

MYB026, MYB175 and PNAC128 were positive regulators for Pt 9 tCesA8-B but not for Pt 9 tCesA8-A. Likewise, MYB026 and MYB158, respectively, activated the expression of Pt 9 tCesA7-B and Pt 9 tCesA7-A.

Discussion Half of the salicoid duplicates exhibit functional divergence at the expression level (Rodgers-Melnick et al. 2012). However, evolutionary alterations of the regulatory system responsible for expression divergence remain to be determined for many gene pairs. In the present study, we targeted secondary cell wall-associated CesA genes CesA4, CesA7 and CesA8, which coordinately function in xylem differentiation in angiosperms (Gardiner et al. 2003; Tanaka et al. 2003; Taylor et al. 2003; Creux et al. 2008, 2013; Song et al. 2010). In Populus plants, the duplicate CesA genes exhibited different expression patterns in several tissues such as phloem fibers, secondary xylem, root cap and leaf trichomes. The expression divergence may be due to genetic mutation of the promoter sequences. The transcription factors activating Populus CesA genes differed for the duplicated genes. Our results provide insight into the evolutionary alteration of gene expression regulation within duplicate gene pairs after a WGD event. In A. thaliana, CesA4, CesA7 and CesA8 are required for protein assembly of a cellulose synthesis complex in secondary cell wall biosynthesis (Gardiner et al. 2003; Taylor et al. 2003). One-to-one orthologous genes of the triple subunit are widely identified not only in eudicots such as E. grandis, M. truncatula and V. vinifera but also in monocots such as B. distachyon, Hordeum vulgare, O. sativa and S. bicolor (Fig. 1) (Burton et al. 2003; Creux et al. 2008, 2013; Handakumbura et al. 2013). In these plants, the components of the CesA triplet are coexpressed and presumed to have a role in secondary wall synthesis similar to Arabidopsis CesA4, CesA7 and CesA8 (Tanaka et al. 2003; Creux et al. 2008; Handakumbura et al. 2013). Most angiosperms retain three CesA genes when they have undergone WGD events (Jiao et al. 2011). Arabidopsis and Oryza, for example, show evidence of three and two rounds of a recent WGD event, respectively. These observations indicate that the numbers of WGD-derived duplicate CesA genes have been reduced to a single gene during postWGD evolution and that the CesA triplet members CesA4, CesA7 and CesA8 are an essential gene set for secondary cell wall biosynthesis. The gene loss events affecting CesA genes might be supported by a gene dosage balance hypothesis; upstream regulators of the genetic network would be retained after large-scale chromosomal duplication compared to end products of a genetic network (Freeling and Thomas 2006). In Populus plants, duplicate

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123

ACATAC

TGGAGC

TTCACC

AAGCAT

CATCAC

TATAAG

CAAGCA

GGKNGAG

GAGGG

CACCAA

Cluster_237

Cluster_522

Cluster_586

Cluster_707

Cluster_778

Cluster_797

Cluster_811

Cluster_812

Cluster_1010

2

5

–

2

–

1

2

–

–

2

3

7

2

4

2

2

3

8

1

1

2

4

1

1

1

3

–

1

3

2

–

–

3

–

2

2

3

1

2

4

2

3

1

1

3

2

1

3

3

2

2

1

1

3

4

3

1

3

3

4

1

1

–

1

2

1

1

1

2

1

4

4

2

3

–

3

7

6

3

1

2

2

2

1

2

–

1

2

2

1

2

3

7

1

2

CesA8B

–

3

1

2

3

1

1

–

–

1

3

5

1

3

1

1

2

3

1

5

2

2

3

–

1

2

2

1

3

1

1

1

2

3

1

3

–

1

CesA7

–

2

1

–

1

–

3

2

1

3

–

2

–

2

–

1

–

2

–

CesA8

2

1

5

2

2

1

2

2

–

1

1

4

3

1

4

–

3

1

4

CesA4

1

–

2

–

2

1

–

2

1

1

–

4

2

2

2

1

4

3

2

CesA7

CesA4

CesA8A

CesA4

CesA7B

A. thaliana

M. truncatula

P. tremula 9 P. tremuloides CesA7A

Malvids

Fabids

The numerals indicate the motif number identified within each promoter sequence * CesA1, CesA2 and CesA3 in E. grandis are orthologous to Arabidopsis CesA8, CesA4 and CesA7, respectively

GAGTTG

GNTTGGT

Cluster_56

Cluster_208

CYCCANNNNC

Cluster_32

Cluster_149

CCAACC

Cluster_14

AGCAAC

CCACC

Cluster_9

CCTCNA

CCTCC

Cluster_8

Cluster_124

GAGAGAG

Cluster_0

Cluster_57

Motif sequence

Motif IDs

Table 1 Putative motifs that are highly conserved in the secondary cell wall-associated CesA gene family

3

1

2

1

3

2

–

2

1

–

2

2

1

3

2

–

3

2

1

CesA8

2

12

2

–

3

1

2

1

1

–

2

3

–

2

1

2

4

7

17

CesA1*

3

6

5

3

3

2

2

–

1

1

1

1

1

1

1

–

6

9

6

CesA2*

E. grandis

2

4

1

2

4

–

2

2

1

–

3

7

1

2

3

3

7

9

9

CesA3*

–

3

3

1

–

–

1

1

–

2

1

1

1

2

4

–

6

4

3

CesA4

1

8

–

1

3

4

1

2

1

1

–

6

1

2

4

1

8

8

9

CesA7

V. vinifera

Vitales in rosids

2

4

1

3

–

1

3

1

1

1

3

6

3

1

4

3

7

8

3

CesA8

38 Planta (2015) 241:29–42

Planta (2015) 241:29–42

39

(a) Reporters

Effectors PtxtCesA4pro PtxtCesA7-Apro PtxtCesA7-Bpro PtxtCesA8-Apro PtxtCesA8-Bpro

PtxtCesApro::LUC

Luciferase

MYB003 MYB010 MYB026 MYB055 MYB090 MYB125 MYB148

NOSter

Internal control CaMV35S::RLUC

(b)

CaMV35S

Renilla luciferase

PtxtCesA4pro

NOSter

PtxtCesA7-Apro

MYB152 MYB158 MYB175 MYB192 MYB199 PNAC122 PNAC124

PNAC128 PNAC161 GATA8 LBD15 ZF1

Transcriptional factors

2xCaMV35S

TF

CaMV35Ster

Control

2xCaMV35S

uidA

CaMV35Ster

PtxtCesA7-Bpro

PtxtCesA8-Apro

PtxtCesA8-Bpro

GUS MYB003

**

MYB010

* *

**

**

**

MYB026

**

**

*

**

*

*

MYB055

**

*

MYB090 MYB125

*

**

*

**

*

MYB148

*

**

MYB152

*

MYB158

*

**

**

*

*

MYB175

*

MYB192

*

**

MYB199

*

*

*

**

**

*

PNAC122 PNAC124

**

*

*

PNAC128 PNAC161

** *

GATA8 LBD15

*

ZF1 0.1 1 10 100 0.1 1 10 100 1000 1 10 100 0.1 1 10 0.1 1 10 100 0.1 Relative luciferase activity Relative luciferase activity Relative luciferase activity Relative luciferase activity Relative luciferase activity

(c) PNAC161 LBD15

MYB199 PNAC124

CesA4

MYB158

CesA7-A

MYB003, MYB010 MYB125, MYB152 MYB192

CesA7-B

MYB026

CesA8-A

MYB055 MYB148

MYB175 PNAC128

CesA8-B

Fig. 5 Transcriptional activation of Pt 9 tCesA genes by selected transcription factors. a Schematic diagrams of the effector, reporter and internal control plasmids used in the transactivation assay. The reporter plasmids contain LUC reporter genes driven by promoters of the Pt 9 tCesA genes. The effector plasmids consist of the selected transcription factors (Table S3) and GUS driven by the CaMV35S promoter. The effector construct containing the GUS gene was used as a vector control. The plasmid containing CaMV35S::RLUC was used as an internal control for transient transformation. b Relative

luciferase activity after transient co-transformation of the reporter and effector plasmid with internal control plasmid. The luciferase activity, representing Pt 9 tCesA gene promoter activity, was normalized to the RLUC activity. The luciferase activity when co-transformed with the vector control (CaMV35S::GUS) was set to 1. A Student’s t test was used with significance indicated as **P \ 0.01 or *P \ 0.05. Values are mean ± SD (n = 3). c Summary of transcriptional activation of secondary cell wall-associated CesA genes in Populus. Black arrows indicate positive regulation

CesA genes were retained after the salicoid WGD event (Fig. 1a). Ancient CesA4, CesA7 and CesA8 had increased to six copies; however, one member of the CesA4 gene pair was eliminated by subsequent chromosome rearrangement (Fig. 1b–d). Consequently, five CesA genes, CesA4, CesA7-A, CesA7-B, CesA8-A and CesA8-B, are involved in

secondary cell wall formation in extant Populus plants (Fig. 3, Suppl. Figs. S1 and S2) (Song et al. 2010). The Populus CesA genes are mainly coexpressed in leaf veins, trichomes, petiole xylem vessels, primary xylem, phloem fibers, xylem fibers, xylem ray parenchyma cells, the root vascular bundle and the root cap. Likewise, secondary cell

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40

wall-associated CesA genes of other angiosperms are expressed in vascular bundles in the whole plant system (Holland et al. 2000; Creux et al. 2008; Betancur et al. 2010; Kim et al. 2011). In trichomes on the leaf surface, expression of secondary cell wall-associated CesA genes is likely to vary in a species-dependent manner; the cotton CesA4 ortholog of Arabidopsis CesA8 is expressed in trichomes, whereas Arabidopsis CesA4, CesA7 and CesA8 are not (Wu et al. 2009; Betancur et al. 2010; Kim et al. 2011). Taken together, the increased number of CesA genes of Populus plants shows coordinate and tissue-specific expression patterns. Duplicated CesA genes such as CesA7-A and CesA7-B, and CesA8-A and CesA8-B showed distinct levels and patterns of expression in hybrid aspen. Several reports demonstrated that salicoid duplicate gene pairs undergo functional differentiation at the levels of expression and protein function (Kalluri et al. 2007; Oakley et al. 2007; Bocock et al. 2008; Wilkins et al. 2009; Hsu et al. 2011; Rodgers-Melnick et al. 2012). For the duplicate gene pair of Populus CesA8, CesA8-B was more highly abundant in mature xylem and phloem tissues than CesA8-A (Figs. 2, 3, 4, Suppl. Figs. S1 and S3). Histological GUS assay and GFP observation clearly showed the predominant expression of CesA8-B in phloem fibers, xylem fibers and xylem ray parenchyma cells (Figs. 3, 4, Suppl. Figs. S1 and S3), implying that CesA8-B plays a key role in cell wall biosynthesis during secondary xylem and phloem formation. In contrast to the higher expression of CesA8-B in mature stem, CesA8-A may have a major function in root cap development (Fig. 3 and Suppl. Fig. S2). For the duplicate gene pair of CesA7, a difference in expression between CesA7-A and CesA7-B was found in trichomes on the leaf surface (Fig. 3 and Suppl. Fig. S1). Since the other CesA genes CesA4, CesA8-A and CesA8-B are present in trichomes, the expression of CesA7-A must have declined after the gene duplication event. Our expression data indicate that the paralogous gene pairs CesA7-A and CesA7-B, and CesA8-A and CesA8-B underwent subfunctionalization in both the level and pattern of tissue expression after the salicoid WGD event and became distinct from the original expression pattern of the ancestral genes. The promoter region is constituted by a number of cisregulatory elements and controls spatiotemporal expression of every gene. Our promoter analysis identified 19 putative motifs that are highly conserved among the secondary cell wall-associated CesA gene family of eudicots (Table 1; Suppl. Fig. S5). Three motifs (cluster_14, 56 and 1010) corresponding to MYB46-responsive cis-element (M46RE) are retained in the promoter regions of all Populus CesA genes, CesA4, CesA7-A, CesA7-B, CesA8-A and CesA8B. M46RE is the key element for transcriptional activation by MYB46, a direct transcriptional activator of secondary

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Planta (2015) 241:29–42

cell wall-associated CesA genes in A. thaliana and Populus (Kim et al. 2012a, b; Zhong et al. 2013). Consistent with our motif prediction, all CesA genes are positively regulated by Populus MYB003, an ortholog of Arabidopsis MYB46 (Fig. 5b) (Wilkins et al. 2009). Similar to this result, the Populus CesA genes possess a CCACC motif (cluster_9) included in the MYB binding site IIG (MBSIIG) that is controlled by MYB15 in A. thaliana (Romero et al. 1998) and were predictably activated by Populus MYB192, which is orthologous to Arabidopsis MYB15 (Fig. 5b) (Wilkins et al. 2009). For the Populus CesA8 gene pair, MYB026 and MYB175, which are members of an R2R3-MYB subgroup including Arabidopsis MYB52 and MYB54, positively controlled CesA8B but not CesA8-A in hybrid aspen. Given that MYB52 and MYB54 control secondary cell wall synthesis and activate gene expression of CesA8 in A. thaliana (Zhong et al. 2008), the transcriptional activation by MYB026 and MYB175 appears to have declined in the regulation of CesA8-A after the gene duplication event but provides higher expression of CesA8-B in mature xylem and phloem tissues of hybrid aspen (Figs. 3, 4, Suppl. Figs. S1 and S3). For the Populus CesA7 gene pair, a putative motif (cluster_32) that is retained in the promoter region of CesA7B but not of CesA7-A is similar to a class I TCP transcription factor binding site (Tables 1 and S5). In cotton, GbTCP, a member of the class I TCP family, is expressed in trichomes and regulates cotton fiber development (Hao et al. 2012). The CesA7-A gene, which has lost a motif corresponding to the class I TCP binding site, is not expressed in trichome cells of hybrid aspen (Fig. 3 and Suppl. Fig. S1). Collectively, our data imply that the secondary cell wall-associated CesA genes have shared several key regulatory modules during post-WGD evolution in Populus plants and that the evolutionary alteration of regulatory modules may cause the differential expression between members of the duplicate gene pairs. In the extant genome of Populus species, many salicoid duplicates have been retained and undergone functional differentiation (Tuskan et al. 2006; Rodgers-Melnick et al. 2012). These gene pairs could be an important resource for studying functional divergence of duplicate genes and evolutionary alteration of regulation of gene expression after large-scale chromosomal duplication. Our gene expression data revealed that CesA genes underwent subfunctionalization in Populus plants. We identified several candidate transcriptional factors that may be responsible for the expression divergence of the CesA7 and CesA8 pairs. The present study implies that the evolutionary change of cis-regulatory modules has been achieved after the WGD event and led to subfunctionalization of the duplicate gene pairs at the expression level in several tissues. Further analyses are clearly needed to elucidate

Planta (2015) 241:29–42

whether the duplicate CesA proteins play a distinct role in a catalytic unit. Author contribution NT and TT conceived and designed research. NT conducted experiments and drafted the manuscript. Both authors read and approved the final manuscript. Acknowledgments We thank Prof. Masaaki Sakuta for a gift of pUC18-CaMV35S::LUC and pUC18-CaMV35S::RLUC constructs. We thank Dr. Misato Ohtani (Nara Institute of Science and Technology) for helpful advice on luciferase transient expression assays. We are grateful to Ms. Maki Konnai and Tomoko Okuyama in our laboratory for their technical assistance. This work was supported in part by a Grant-in-Aid for Young Scientists (Start-up) from the Japan Society for the Promotion of Science (23880029 to NT). Conflict of interest of interest.

The authors declare that they have no conflict

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