Gene 539 (2014) 58–67

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Identification and characterization of the Populus sucrose synthase gene family Xinmin An a,⁎,1, Zhong Chen a,1, Jingcheng Wang a,b, Meixia Ye a, Lexiang Ji a, Jia Wang a, Weihua Liao a, Huandi Ma a a National Engineering Laboratory for Tree Breeding, NDRC, Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, MOE, The Tree and Ornamental Plant Breeding and Biotechnology Laboratory, SFA, College of Biological Sciences and Biotechnology, Beijing Forestry University, Qinghua East Road No.35, Haidian District, Beijing 100083, China b Chinese Academy for Environmental Planning, Ministry of Environmental Protection of the People's Republic of China, Beijing 100012, China

a r t i c l e

i n f o

Article history: Received 29 November 2013 Received in revised form 14 January 2014 Accepted 24 January 2014 Available online 4 February 2014 Keywords: Populus Sucrose synthase RT-qPCR Expression profiles Water-deficit

a b s t r a c t In this study, we indentified 15 sucrose synthase (SS) genes in Populus and the results of RT-qPCR revealed that their expression patterns were constitutive and partially overlapping but diverse. The release of the most recent Populus genomic data in Phytozome v9.1 has revealed the largest SS gene family described to date, comprising 15 distinct members. This information will now enable the analysis of transcript expression profiles for those that have not been previously reported. Here, we performed a comprehensive analysis of SS genes in Populus by describing the gene structure, chromosomal location and phylogenetic relationship of each family member. A total of 15 putative SS gene members were identified in the Populus trichocarpa (Torr. & Gray) genome using the SS domain and amino acid sequences from Arabidopsis thaliana as a probe. A phylogenetic analysis indicated that the 15 members could be classified into four groups that fall into three major categories: dicots, monocots & dicots 1 (M & D 1), and monocots & dicots 2 (M & D 2). In addition, the 15 SS genes were found to be unevenly distributed on seven chromosomes. The two conserved domains (sucrose synthase and glycosyl transferase) were found in this family. Meanwhile, the expression profiles of all 15 gene members in seven different organs were investigated in Populus tomentosa (Carr.) by using RT-qPCR. Additional analysis indicated that the poplar SS gene family is also involved in response to water-deficit. The current study provides basic information that will assist in elucidating the functions of poplar SS family. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Sucrose, an important carbohydrate, is transported in the phloem of plants (Goren et al., 2011). Sucrose synthase (SS, UDP-glucose–D-fructose 2-alpha-glucosyltransferase, EC 2.4.1.13) catalyzes the chemical reaction: Sucrose + UDP (uridine diphosphate) ⇌ UDP-glucose + D-fructose (Winter and Huber, 2000). SS participates in sucrose and starch metabolism and is a member of the glycosyltransferase family of enzymes, or more specifically, the hexosyltransferases. Other names in common use for SS include sucrose-UDP glucosyltransferase, sucrose-uridine diphosphate glucosyltransferase, and uridine diphosphoglucose-fructose glucosyltransferase. Previous studies have demonstrated that SS plays a critical role in energy metabolism, and regulating the assignment of sucrose into various pathways for metabolic, structural and storage Abbreviations: RT-qPCR, real-time quantitative RT-PCR; SS, sucrose synthase; UDP, uridine diphosphate; gDNA, genomic DNA; CDS, coding sequence; Mw, molecular weight; pI, isoelectric point; bp, base pair; nts, nucleotides; kDa, kilodalton (pH = 7.0). ⁎ Corresponding author at: No.35 Qinghua East Road, Haidian District, P.O.Box 118, Beijing Forestry University, Beijing 100083, China. Tel./fax: +86 10 62336248. E-mail address: [email protected] (X. An). 1 These authors contributed equally to this work. 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2014.01.062

functions (Hesse and Willmitzer, 1996; Sturm and Tang, 1999). For an example, SS, in association with the cellulose synthase complex, plays an important role in supplying UDP-glucose for cell wall biosynthesis (Delmer and Amor, 1995; Haigler et al., 2001; Ruan et al., 2003). SS also participates in supplying energy to companion cells for phloem loading by providing a substrate for respiration (Fu and Park, 1995; Hänggi and Fleming, 2001). Additionally, SS provides a substrate for starch synthesis in storage organs of plants (Déjardin et al., 1997; Zrenner et al., 1995). The genome sequence of several species has revealed that the SS gene family in each species contains multiple members. There are six SS genes in Arabidopsis (Barratt et al., 2001; Baud et al., 2004), five in Vitis vinifera (Vvi01015018001, Vvi01035106001, Vvi01028043001, Vvi01029388001, Vvi01035210001), five in Zea mays (Zm2G152908_ T01, Zm2G089713_T04, Zm2G318780_T02, Zm2G045171_T01, Zm2G060659_T02), six in Oryza sativa (LOC_Os02g58480, LOC_ Os04g17650, LOC_Os04g24430, LOC_Os07g42490, LOC_Os03g22120, LOC_Os06g09450, LOC_Os03g28330) (Harada et al., 2005), six in Lotus japonicus (Horst et al., 2007), and eight, eight and fifteen in Gossypium arboreum L., Gossypium raimondii Ulbr., and Gossypium hirsutum L., respectively (Zou et al., 2013). Within each species, the spatio-temporal expression patterns of the different SS genes across tissue types raise

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gene family were also used as a query in order to obtain a comprehensive list of putative SS genes in the poplar genome. Bioinformatics, such as composition, physical and chemical characterization, and conserved functional domains of the SS gene family in P. trichocarpa were performed using the Expert Protein Analysis System (ExPASy) on the website of the Swiss Institute of Bioinformatics (SIB) (http://cn.expasy.org/).

the question of biological significance. Individual SS genes have demonstrated to play specific and non-redundant roles in maize and cotton (Chourey et al., 1998; Ruan et al., 2003). Although a loss of function experiment on SS gene members in Arabidopsis did not have a significant impact on plant viability and morphology (Barratt et al., 2009), a recent study reported that AtSS2 (AT5G49190) and AtSS3 (AT4G02280) are strongly and differentially expressed in seeds (Angeles-Núñez and Tiessen, 2010). The latest version of the poplar (Populus trichocarpa) genome sequence in Phytozome v9.1 revealed that there are fifteen putative members in the SS gene family (Tuskan et al., 2006). The complete P. trichocarpa SS (PtrSS) family, which is the largest SS family described to date, provides a good system to elucidate the function of each isoform. In the present study, we report the updated information on genomic DNA, cDNA, transcript, encoded peptide sequence, theoretical Mw and pI, conserved functional domains, and other characteristics of the entire PtrSS gene family. We also provide a phylogenetic analysis of PtrSS family, a comparison of genomic structure, and the location of each member in the genome. Finally, a comprehensive expression profile is provided for all fifteen SS gene members in various organs of Populus tomentosa, as well as the response of specific members to water-deficit.

Genomic, transcript, and CDS sequences of PtrSS members were downloaded from Phytozome v9.1 (http://www.phytozome.net/ poplar). The schematic structures of PtrSS members, based on exon/ intron data, were produced using DNAMAN 5.2 software. The genomic location of each PtrSS gene was determined using GBrowse based on scaffold information for P. trichocarpa — JGI v2.2 (http://www. phytozome.net/cgi-bin/gbrowse/poplar/). A multiple alignment analysis was performed with Clustalx 1.83. Based on the alignments, genetic distance matrices were obtained and an unrooted phylogenetic tree was constructed using the neighborjoining (NJ) and bootstrap method with 1000 replications (Tamura et al., 2011).

2. Materials and methods

2.4. RNA extraction, RT-qPCR analysis and cloning of PtSS members

2.1. Plant materials

Frozen tissues were ground in liquid nitrogen and total RNA was extracted as previously described (Chang et al., 1993) and genomic DNA contaminants were removed with RQ1 DNase I (Promega, Madison, WI, USA). The concentration of total RNA was measured using a NanoDrop 2000 (Thermo Scientific Inc., Waltham, USA). Two independent extractions per organ were performed for the expression analysis in plant organs, while three independent extractions were performed for each data point in the water deficit study. DNA-free RNA was converted into first-strand cDNA using reverse transcription system (Promega, Madison, WI, USA) with oligo (dT)15. In all cases, three independent RTs were performed. The first-strand cDNA was diluted 1:10 with ddH2O, and 2 μL of the diluted cDNA was used as a template for RT-qPCR. The reactions were performed using a DNA Engine Opticon 2 system (MJ Research Inc., Quebec, Canada) in a total volume of 20 μL, with 0.4 μL of 10 μM forward primers and reverse primers (Table S1), and 10 μL 2× SYBR® Green PCR Master Mix (Invitrogen). The RT-qPCR protocol included a preliminary step of 2 min at 50 and 94 °C followed by 40 cycles of 94 °C for 15 s, 58 °C for 10 s, and 72 °C for 15 s, with a final extension at 72 °C for 7 min. No-template controls for each primer pair were included in

Both vegetative and floral bud samples were collected from female (5082) and male (LM50) trees of P. tomentosa growing in the national nursery of Guanxian, Shandong province, China in 2012. After collection, samples were frozen in liquid nitrogen and stored at − 80 °C until further use. Additionally, root, stem and leaf samples were obtained from one-month-old tissue-cultured plantlets of P. tomentosa, followed by immediate RNA extraction. 2.2. Database search and bioinformatics analysis Referenced the method previously reported (Ji et al., 2012), a search of the P. trichocarpa genome database was performed in order to identify all members of the PtrSS family. The strategy used to obtain the entire family of SS genes in the genome was as follows. Using BLASTP, the amino acid sequence of the SS domain in P. tomentosa was used as a query to search the most recent poplar genome data hosted in Phytozome v9.1 (http://www.phytozome.net/search.php). Amino acid sequences of the SS domain of some members of the Arabidopsis SS

2.3. Gene structure and phylogenetic relationship analysis

Table 1 Characteristics of PtrSS family members in P. trichocarpa (updated data according to Phytozome v9.1). Isoforms

PtrSS1.1 PtrSS1.2 PtrSS2.1 PtrSS2.2 PtrSS3.1 PtrSS4.1 PtrSS4.2 PtrSS4.3 PtrSS5.1 PtrSS5.2 PtrSS6.1 PtrSS6.2 PtrSS7.1 PtrSS7.2 PtrSS7.3

Locus name

POPTR_0018s07380 POPTR_0006s13900 POPTR_0002s20340 POPTR_0015s05540

POPTR_0012s03420 POPTR_0004s07940 POPTR_0017s02060

Transcript name

Potri.018G063500.1 Potri.018G063500.2 Potri.006G136700.1 Potri.006G136700.2 Potri.002G202300.1 Potri.015G029100.1 Potri.015G029100.2 Potri.015G029100.3 Potri.012G037200.1 Potri.012G037200.2 Potri.004G081300.1 Potri.004G081300.2 Potri.017G139100.1 Potri.017G139100.2 Potri.017G139100.3

gDNA size (bp)

6045 4135 7048 5335 6440 4401 4488 4401 4524 4110 4326 4326 4272 4540 4404

Transcript size (nts)

2807 2786 3016 2996 3560 3094 3055 3175 2978 2838 3056 3265 2732 3080 2956

CDS size (nts)

Peptide residues

Theoretical Mw (kDa)

Theoretical pI

Functional domains (start–end) Sucrose synthase

Glycosyl transferase

2418

805

92.50

6.23

7–554

565–728

2412

803

92.16

6.17

5–552

563–736

2475 2601 2433

824 866 810

94.09 97.63 91.78

5.86 6.51 6.48

8–555 9–557

567–739 566–734

2766 2403 2508 2454 2520 1965 2448

921 800 835 817 839 654 815

104.08 90.43 95.15 92.80 95.36 74.39 92.40

6.34 5.98 6.76 7.17 6.49 7.53 6.13

9–548 9–558 6–545 6–555 6–555 6–370 6–545

559–724 569–734 554–719 564–729 564–729 377–544 554–719

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cultured served as controls. All plantlets were immediately frozen in liquid nitrogen, and followed by immediate total RNA extraction as previously described. RT-qPCR was also performed as previously described. 3. Results 3.1. The SS gene family in P. trichocarpa

Fig. 1. A distance tree of SS isoform proteins in P. trichocarpa and P. tomentosa. A bootstrap analysis with 1000 replicates was performed to assess the statistical reliability of the tree topology. The blue squares indicate SS isoforms derived from P. tomentosa. The red, blue, purple red and blue green branches represent Class I, Class II, Class III and Class IV, respectively.

each run. According to a previous study, the poplar ACTIN gene (GenBank accession: AY261523.1) can serve as a stably expressed internal control (Zhang et al., 2008; Zheng et al., 2011), therefore it was employed as an internal reference gene to normalize small differences in template amounts using the primers listed in Table S1. At least three different RNA isolations and cDNA syntheses were used as replicates for the RT-qPCR. A set of primers (Table S1) was designed, based on PtrSS1.1, PtrSS2.1 and PtrSS3.1, and used for amplification of PtSS homologues. PtSS1.1, PtSS2.1 and PtSS3.1 were obtained by RT-PCR. Thermal cycling was performed at 94 °C for 3 min, 94 °C for 30 s, 60 °C/58 °C for 30 s, 72 °C for 120 s, for 35 cycles, then at 72 °C for 5 min. The resulting PCR products were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA) and were subsequently transformed into Escherichia coli cells (Top10) for sequencing. 2.5. Water-deficit treatment and RT-qPCR analysis According to an earlier report (Pelah et al., 1995, 1997), whole rooted plantlets (3–4 weeks after subculturing) of tissue cultured P. tomentosa was removed from the culture medium, wilted from their original fresh status at room temperature by continuous monitoring of weight loss on a balance, and collected plantlets after 1 h, 2 h, and 4 h of dewatering treatment respectively. The fresh plantlets of tissue

Based on data obtained from the most recent poplar database in Phytozome v9.1 and the program ExPASy, a bioinformatics analysis of the entire PtrSS gene family is presented in Table 1. Bioinformatics information for each gene includes the length of genomic DNA sequence, length of the transcript, length of the CDS, number of amino acids, theoretical Mw, theoretical pI, and the location of the functional domains (Table 1). The information on poplar SS gene in Phytozome v 9.1 (http://www.phytozome.net/getSequenceQueryResults.php?id= 1175361) varies considerably from the previous genome database and assembly (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html). For example, the transcripts previously listed as PtrSS1, PtrSS2, PtrSS5 and PtrSS6 are now named PtrSS1.1 and PtrSS1.2, PtrSS2.1 and PtrSS2.2, PtrSS5.1 and PtrSS5.2, PtrSS6.1 and PtrSS6.2, respectively, and PtrSS4 and PtrSS7 are each listed with three transcripts. In contrast, the PtrSS3 gene is now listed as a single transcript (PtrSS3.1) rather than two (PtrSS3.1 and PtrSS3.2). The total number of PtrSS genes has increased from 8 to 15, and consequently, the corresponding total number of coding peptide sequences has increased to 12. It is worthy to note that the sizes of the genomic DNA, transcript, CDS, as well as the number of peptide residues have also been updated, along with the theoretical Mw and pI, of PtrSS gene family members. To determine the evolutionary relationships of the PtrSS family in poplar, a phylogenetic distance tree of 12 PtrSS isoform proteins from P. trichocarpa and 3 PtSS isoform proteins deduced by CDS of PtSS1.1, PtSS2.1 and PtSS3.1 (unpublished data) from P. tomentosa was constructed using Clustalx 1.83 and MEGA 5.0. Four distinct branches were evident in the resulting dendrogram (Fig. 1). One (Class I) was composed of PtrSS1.1/1.2, PtrSS2.1/2.2, PtSS1.1 and PtSS2.1. PtrSS3.1 and PtSS3.1 belonged to an independent branch (Class II), while the other two branches (Classes III and IV) each consist of two subgroups, one subgroup (Class III) contains PtrSS4.1 and PtrSS4.2/4.3, PtrSS5.1 and PtrSS5.2, while the other subgroup (Class IV) contains PtrSS6.1and PtrSS6.2, PtrSS7.1, PtrSS7.2 and PtrSS7.3 (Fig. 1). A matrix of amino acid sequence similarity for the PtrSS gene family is presented in Table 2. A high percentage of amino acid identity/ positive exists between PtrSS1.1/1.2 and PtrSS2.1/2.2 (93.1% and 97.3%, respectively), PtrSS4.1 and PtrSS4.2/4.3 (99.3% and 99.6%, respectively), PtrSS4.1and PtrSS5.1 (89.0% and 94.5%, respectively), PtrSS4.1 and PtrSS5.2 (89.0% and 95.9%, respectively), PtrSS4.2/4.3 and PtrSS5.1 (90.8% and 95.9%, respectively), PtrSS4.2/4.3 and PtrSS5.2 (89.5% and

Table 2 Identity/positive matrix for twelve PtrSS amino acid sequences in P. trichocarpa. Positive (%)

Identity (%)

PtrSS1.1/1.2 PtrSS2.1/2.2 PtrSS3.1 PtrSS4.1 PtrSS4.2/4.3 PtrSS5.1 PtrSS5.2 PtrSS6.1 PtrSS6.2 PtrSS7.1 PtrSS7.2 PtrSS7.3

PtrSS1.1/1.2

PtrSS2.1/2.2

PtrSS3.1

PtrSS4.1

PtrSS4.2/4.3

PtrSS5.1

PtrSS5.2

PtrSS6.1

PtrSS6.2

PtrSS7.1

PtrSS7.2

PtrSS7.3

– 93.1 68.1 55.2 55.3 55.8 55.1 54.3 55.1 55.1 61.5 54.3

97.3 – 69.9 55.8 56.0 56.5 55.8 55.2 55.9 55.4 61.7 54.7

83.5 83.8 – 57.9 58.1 57.5 56.9 55.3 55.9 55.4 61.6 54.8

72.9 72.5 75.4 – 99.3 89.0 89.0 72.7 73.8 70.9 73.1 71.0

73.1 72.7 75.8 99.6 – 90.8 89.5 72.9 73.9 73.3 76.4 72.4

73.0 72.1 75.0 94.5 95.9 – 98.8 72.3 73.6 73.3 76.4 72.5

71.6 70.9 74.0 94.3 94.4 98.8 – 72.8 72.5 72.6 75.5 73.6

72.1 71.5 74.0 84.4 84.7 83.9 84.7 – 98.5 89.1 87.2 72.9

73.2 72.5 74.9 85.6 85.5 85.4 84.2 98.6 – 89.6 87.8 88.5

73.2 72.5 74.6 83.2 85.6 85.4 84.2 94.2 95.0 – 99.2 98.8

79.0 78.2 78.5 83.3 86.2 86.2 84.6 93.1 94.1 99.4 – 97.7

72.1 71.4 73.6 83.2 84.4 84.2 85.3 84.7 93.7 98.8 97.8 –

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Fig. 2. Schematic of the gene structure of members of the PtrSS gene family in poplar. Blue rectangle blocks and blue boxes indicate coding exons and non-coding exons, respectively. Blue lines connecting the boxes and blocks denote introns. Sizes of exons are given above the blue boxes, and sizes of first intron in PtSS1.1, PtSS2.1 and PtSS2.2 are given above the corresponding lines, respectively.

94.4%, respectively), PtrSS5.1 and PtrSS5.2 (98.8% and 98.8%, respectively), PtrSS6.1 and PtrSS6.2 (98.5% and 98.6%, respectively), PtrSS6.1 and PtrSS7.1 (89.1% and 94.2%, respectively), PtrSS6.1 and PtrSS7.2 (87.2% and 93.1%, respectively), PtrSS6.1 and PtrSS7.3 (72.9% and 82.7%, respectively), PtrSS6.2 and PtrSS7.1 (89.6% and 95.0%, respectively), PtrSS6.2 and PtrSS7.2 (87.8% and 94.1%, respectively), PtrSS6.2 and PtrSS7.3 (88.5% and 93.7%, respectively), PtrSS7.1 and PtrSS7.2 (99.2% and 99.4%, respectively), PtrSS7.1 and PtrSS7.3 (98.8% and 98.8%, respectively), and PtrSS7.2 and PtrSS7.3 (97.7% and 98.8%, respectively). The isoform, PtrSS3, is very different from the other eleven PtrSS isoforms (Table 2). The PtrSS isoforms could be classified into 4 groups based on similarity indices. Group-I would be composed of PtrSS1.1/ 1.2 and PtrSS2.1/2.2, group-II would contain only PtrSS3.1, group-III would consist of PtrSS4.1, PtrSS4.2/4.3, PtrSS5.1 and PtrSS5.2, and

group-IV would be composed of PtrSS6.1, PtrSS6.2, PtrSS7.1, PtrSS7.2 and PtrSS7.3 (Table 2). 3.2. Genomic structure and location of PtrSS gene family members The genomic organization of PtrSS genes was determined using the genomic and transcript sequences deposited in the Phytozome v9.1 database. As indicated in Fig. 2, all of the predicted exon/intron junctions conform to the GT–AG rule. A comparison of PtrSS orthologs revealed a high level of conservation in genomic structure, although differences in the length and number of exons/introns were evident among the genes. The first introns in PtrSS1.1, PtrSS2.1 and PtrSS2.2 were 1889 bp, 2667 bp and 974 bp, respectively, while the size of the first intron in the other family members is relatively smaller.

Fig. 3. Location of sucrose synthase gene family members on the P. trichocarpa chromosomes. Scale represents a 2.0 Mb chromosomal distance. Scaffold numbers and sizes (Mb) are indicated at the bottom end of each scaffold.

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Fig. 4. Deduced amino acid sequence alignments of fifteen sucrose synthase gene family members from P. trichocarpa. The multiple sequences alignment was showed as normal view mode in color. The lines beneath indicate conserved domains. The red and blue lines indicate a sucrose synthase domain and a glycosyl transferase domain, respectively. Asterisks and dots indicate conserved and conservatively replaced amino acids, respectively.

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Although a large variation exists in intron size among PtrSS genes, the lengths and numbers of exons are relatively similar or even identical. The fifteen members of the PtrSS gene family could be classified into 4 groups based on the similarity of their genomic structure. Group-I includes PtrSS1.1, PtrSS1.2, PtrSS2.1 and PtrSS2.2. Apart from differences in the length of the first intron, the exon/intron numbers and length are almost identical among them. Group-II contains only PtrSS3.1 which is composed of 15 exons. Group-III contains five PtrSS orthologs, PtrSS4.1, PtrSS4.2, PtrSS4.3, PtrSS5.1, and PtrSS5.2. PtrSS4.1 is composed of 14 exons/13 introns, while PtrSS4.2, PtrSS4.3, and PtrSS5.2 all contain 13 exons/12 introns. PtrSS5.2 contains 16 exons/15 introns. The length of most of the exons in the five orthologues in this group is highly similar or even identical. Group-IV consists of two subgroups; one subgroup contains PtrSS6.1 and PtrSS6.2, which are composed of 12 exons/11 introns, while the other subgroup contains three orthologs. PtrSS7.1 and PtrSS7.2, share a high level of similarity in genomic structure at their 3′ end while PtrSS7.1 and PtrSS7.3 are similar at their 5′ end. Overall, the high level of conservation in the genomic structure within the four PtrSS groups is striking (Fig. 2). Based on the current poplar genome database in Phytozome v9.1 (http://www.phytozome.net/poplar), we mapped the position of members of the PtrSS gene family. PtrSS1.1/1.2, PtrSS2.1/2.2, PtrSS3.1, PtrSS4.1/ 4.2/4.3, PtrSS5.1/5.2, PtrSS6.1/6.2, and PtrSS7.1/7.2/7.3 are located on chromosome nos.18, 6, 2, 15, 12, 4, and 17, respectively (Fig. 3). The fifteen members share the two characteristic functional domains of SS genes, a sucrose synthase domain and a glucosyl-transferase domain (Table 1, Fig. 4). 3.3. Phylogenetic relationships of PtrSS gene family An unrooted tree was constructed by analysis of the multiple alignment of SS amino acid sequences using Clustalx 1.83 and MEGA 5.0. Results indicated a grouping into four sub-families (Fig. 5), and provided information about the evolution of the PtrSS gene family. The analysis indicated that PtrSS1.1/1.2 and PtrSS2.1/2.2 are paralogs of AtSS1 and AtSS4 within the dicot SS group. PtrSS3.1 is most likely a member of the monocots and dicots, M & D 1 group, which includes SS genes from diverse monocot and dicot species, such as AtSS2, AtSS3, and a Z. mays (ZmSS) homolog (Zm2G318780_T02) and an O. sativa (OsSS) ortholog (LOC_Os03g22120). The remaining PtrSS family members are contained in two subgroups within the larger M & D 2 group also consisting of a mixture of monocots and dicots. Subgroup 1 contains PtrSS4.1, PtrSS4.2/4.3, and PtrSS5.1/5.2. Subgroup 2 contains PtrSS6.1/ 6.2, PtrSS7.1/7.2, and PtrSS7.3. The apparent diversification within the Populus family could imply discrete biological roles for the paralogs despite their high sequence similarity. 3.4. Expression profiles of PtSS gene members in P. tomentosa A set of specific primers was designed to examine the expression profile of each member of the PtSS gene family in various tissues and organs of P. tomentosa. The specificity of each primer was checked by a blast search on the Phytozome v9.1 website (http://www.phytozome. net/search.php?show=blast&targetType=genome&method=Org_ Ptrichocarpa), and sequencing of the RT-PCR products. PtACTIN gene was employed as an internal reference gene to standardize the level of gene expression. Fig. 6 presents the expression profiles of all 15 PtSS gene members in various tissues and organs of P. tomentosa. Although transcript levels varied among the family members, the graphs illustrate that all 15 genes were expressed in roots, stems, leaves, vegetative buds, both male and female floral buds, and floral catkins. The majority of PtSS genes presented exhibited a similar expression pattern characterized by a high level of transcript in roots, vegetative buds and floral catkins, and relatively lower level of transcript in stems, leaves, male and female floral buds. The members with similar patterns and levels of expression include PtSS1.1, PtSS1.2, PtSS2.1,

Fig. 5. Unrooted cladogram showing the phylogenetic structure of the SS gene family. The neighbor-joining (NJ) tree of SS sequences was constructed from complete amino acid sequences using Clustalx 1.83 and MEGA 5.0. Bootstrap values were generated based on 1000 bootstrap trials. Peptide sequences were obtained from the following SS isozymes: Populus trichocarpa PtrSS isozymes 1–7 (POPTR_0018s07380, POPTR_0006s13900, POPTR_0002s20340, POPTR_0015s05540, POPTR_0012s03420, POPTR_0004s07940, POPTR_0017s02060), Arabidopsis thaliana AtSS isozymes 1–6 (AT5G20830, AT5G49190, AT4G02280, AT3G43190, AT5G37180, AT1G73370); Solanum lycopersicum SlSS isozymes 1, 3 & 4 (L19762, HM180942, HM180943); Solanum tuberosum StSS isozymes 1, 3 & 4 (AY205302, AY205084, M18745); Zea mays ZmSS isozymes 1–5 (Zm2G152908_T01, Zm2G089713_T04, Zm2G318780_T02, Zm2G045171_T01, Zm2G060659_T02); Vitis vinifera VviSS isozymes 1–5 (Vvi01015018001, Vvi01035106001, Vvi01028043001, Vvi01029388001, Vvi01035210001); Oryza sativa OsSS isozymes (LOC_Os02g58480, LOC_Os04g17650, LOC_Os04g24430, LOC_Os07g42490, LOC_Os03g22120, LOC_ Os06g09450, LOC_Os03g28330), Pisum sativum PsSS isozymes 1, 3 & 4 (AF07985, AJ311496, AJ001071); Citrus unshiu CtiSS (AB029401, AB022092). Genes enclosed in solid lines denote 4 major SS groups: monocots, dicots, monocots & dicots 1 (M & D1) and monocots & dicots 2 (M & D 2); genes enclosed by dashed lines represent poplar family subgroups within the four main groups of all SS genes. The bar indicates evolutionary distance.

PtSS4.2, PtSS4.3, PtSS5.1, PtSS5.2, PtSS6.1, PtSS6.2, PtSS7.1, PtSS7.2 and PtSS7.3. In contrast to the above pattern, transcript levels of PtSS2.2 and PtSS4.1 were relatively lower in floral catkins while the transcript level of f PtSS3.1 was relatively high in all tissues examined.

3.5. Response of PtSS gene members to water-deficit To investigate the response of PtSS genes to water deficit, each primary transcript of PtSS1.1, PtSS2.1, PtSS3.1, PtSS4.1, PtSS5.1, PtSS6.1 and PtSS7.1 was measured by RT-qPCR (Fig. 7). Results indicated that PtSS2.1, PtSS3.1, PtSS4.1 and PtSS7.1 exhibited a similar response pattern where the transcript was sharply upregulated at 1 h after the establishment of water stress. Transcript levels were slightly reduced 2 h after water stress was imposed and sharply downregulated at 4 h. PtSS5.1 was downregulated after 1 h, upregulated at 2 h and then sharply downregulated at 4 h. No variation in transcript level was observed for PtSS6.1 after the first hour of water stress after which the gene became upregulated, reaching a peak at 2 h and was sharply down

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regulated at 4 h. Interestingly, PtSS1.1 exhibited stable expression levels during the entire 4 h period after the initiation of water stress. 4. Discussion 4.1. Structure and phylogenetic relationships of SS family in poplar Multiple SS isoforms have been characterized in both dicot and monocot species. These studies, as well as the publication of the complete genome sequences of Arabidopsis thaliana, Z. mays and O. sativa,

have provided abundant data for the identification of additional SS genes (Baud et al., 2004; Bieniawska et al., 2007; Hirose et al., 2008). Poplar (P. trichocarpa) was the first perennial, woody plant species to be sequenced (Tuskan et al., 2006), and released as version 1.1 by the U.S. Department of Energy Joint Genome Institute (DOE-JGI) (http:// genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html). Recently, the poplar genome data and information have been updated and deposited in Phytozome v9.1 (http://www.phytozome.net/poplar). As a result of the updated data, information on the poplar SS family has changed considerably. Compared to a previous report (Zhang et al., 2011), the total

Fig. 6. The expression profiles of members of the PtSS gene family obtained by RT-qPCR. R, S and L (root, stem and leaf from one-month-old tissue-cultured plantlets, respectively), VB (vegetative bud), EmFB (early stage-male floral bud, on July 5th), EfFB (early stage female floral bud, on July 5th), LmFB (late stage male floral bud, on December 10th), LfFB (late stage-female floral bud, on December 10th).

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number of gene family members has now increased from 7 to 15. Genomic DNA, cDNA and transcripts lengths, as well as deduced amino acid sequences, have been modified (Table 2). Concomitant with these modifications are the changes in the theoretical MW and pI of PtrSS members (Table 2). The added gene members have a high degree of similarity and are located next to previously identified SS genes and have therefore received appropriate sequential names; for example, PtrSS1.1 and PtrSS1.2 (Fig. 3). In comparison to SS gene families in other dicots (e.g., Arabidopsis, V. vinifera) and monocots (e.g., Zea may, O. sativa), the PtrSS gene family is the largest reported to date. Nevertheless, we found a high level of conservation in coding exon structures among PtrSS members. For examples, the numbers and length of coding exons are almost identical in PtrSS1.1, PtrSS1.2, PtrSS2.1 and PtrSS2.2, even though they differ greatly in the distance to the site of the first intron. Similarly, most of the coding exons of PtrSS4.1, PtrSS4.2 PtrSS4.3, PtrSS4.1, and PtrSS4.2 shared a high level of similarity and only differed near the 3′ end. In Arabidopsis, SS genes can be classified into four distinct groups, based on a comparison of the predicted amino acid sequences, as well as intron/exon structure (Baud et al., 2004). The amino acid sequences of PtrSS family members were highly similar to those of the SS gene families in Arabidopsis, Z. mays, O. sativa, and Hordeum vulgare (Barrero-Sicilia et al., 2011; Baud et al., 2004;

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Bieniawska et al., 2007; Hirose et al., 2008). Like the SS gene family, the PtrSS gene family was also classified into four sub-groups (Fig. 1), categorized as dicots, M & D1, and M & D 2 (Fig. 5). However, in contrast to Arabidopsis, genome-wide duplication events have occurred in the evolution of poplar. These duplication events and the conservation in the structure and composition of PtrSS genes during its phylogenetic history may have allowed poplar to adapt its growth and development to a diversity of environments. The improved genomic data provides essential information to enable a better understanding regarding the functional role of the individual members of this important gene family. 4.2. Expression patterns of SS family members in poplar The function of sucrose synthase, a glycosyltransferase belonging to the GT4 family (Henrissat et al., 2001), as a sucrose lyase has been well established in plants (Winter and Huber, 2000). Sucrose synthase is a ubiquitous enzyme found in all plant tissues, with high levels being particularly prevalent in sink tissues, signifying the importance of sucrose synthase in sink tissue metabolism (Coleman et al., 2009, 2010; Martin et al., 1993). In several sequenced plant genomes, sucrose synthase presents as many isoforms. For example, in Arabidopsis there are six isoforms, designated AtSS1 through AtSS6 (Bieniawska et al.,

Fig. 7. The expression profile of seven PtSS gene members in response to dehydrative stress as obtained by RT-qPCR.

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2007). The proteins coded by them are all capable of performing the same biochemical functions in sucrose metabolism. The role of the functional redundancy of sucrose synthase family members is still an important question. The diversity in gene structure among the poplar sucrose synthase family members, however, suggests that members may differ in their expression level or and/or function. The expression profiles of the 15 poplar PtSS members obtained by RT-qPCR in our study will be helpful in understanding their individual functions. The 15 PtSS genes exhibited different but partially overlapping expression patterns (Fig. 6). Translational regulation is primarily comprised of a multipronged approach for controlling protein output by manipulating the translational characteristics of an mRNA, which resides in its UTRs (untranslated regions) located at its 5′- and 3′-ends (Chatterjee and Pal, 2009). Differences in the 5′-UTRs and 3′-UTRs of poplar PtrSS gene family members (Fig. 2) suggest that these duplicated genes may differ in transcript level, spatio-temporal expression patterns, and mRNA subcellular localization. The importance of SS, especially in sink tissues, has been demonstrated in studies with transgenic plants with altered expression. For example, the use of transgenic and mutant (knock-out) Arabidopsis plants has demonstrated that Arabidopsis SS isoforms have different specific functions in Arabidopsis (Bieniawska et al., 2007).

4.3. Response of SS family members to water-deficit The accumulation of soluble sugars in higher plants experiencing a water deficit is a well-known response. Sugars help to maintain osmotic balance under dehydrative conditions (Bray, 1997). SS and sucrose phosphate synthase (SPS) in plants have shown to be vital for acclimation to dehydration (Ramanjulu and Bartels, 2002). For example, sucrose accumulation during water deficit occurs in the drought-tolerant plant Craterostigma plantagineum where dehydration induces the conversion of 2-octulose, an eight-carbon sugar, to sucrose (Bianchi et al., 1991). This conversion is correlated with the upregulation of both SS and SPS (Ingram et al., 1997; Kleines et al., 1999). SS is considered to be a key enzyme in sucrose synthesis and metabolism (Zou et al., 2013). The expression of SS genes has been generally observed to be up-regulated in response to dehydration/osmotic stress (Déjardin et al., 1997; Pelah et al., 1997). In our study, members of the PtSS gene family exhibited different response patterns during the dehydration of poplar plantlets (Fig. 7). While the transcript level of PtSS1.1 remained relatively stable, transcript levels of PtSS2.1, PtSS3.1, PtSS4.1, and PtSS7.1 were significantly upregulated after 1 h of the dehydration treatment and were then gradually downregulated at 2 and 4 h later. The response patterns of PtSS5.1 and PtSS6.1 were different from the abovementioned members. Collectively the data indicate that the PtSS gene family is involved in response to water stress in poplar plantlets. In summary, a relatively complete bioinformatics analysis of the Populus sucrose synthase family was performed. Fifteen genes were identified as putative PtrSS genes and their expressions in seven different tissue types of P. tomentosa were assessed by RT-qPCR. The ubiquitous, partially overlapping but distinctly different expression profiles of members of the PtSS gene family suggest that these genes may play vital and complex roles in development and stress response. Our studies provide baseline information for elucidating the functions of the Populus SS family; however, further studies of each member, using gene silencing and overexpression, will be necessary to decipher their molecular function. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.01.062.

Conflict of interest The authors declare no potential conflict of interest with respect to the authorship and/or publication of this paper.

Acknowledgments This work was supported by the Major State Basic Research Development Program (2012CB114505), the National Natural Science Foundation (31170631, J1103516), and the National Hightech Research and Development Program (2013AA102703, 2011AA100201) of China. References Angeles-Núñez, J.G., Tiessen, A., 2010. Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds. Planta 232, 701–718. Barratt, D.P., Barber, L., Kruger, N.J., Smith, A.M., Wang, T.L., Martin, C., 2001. Multiple, distinct isoforms of sucrose synthase in pea. Plant Physiol. 127, 655–664. Barratt, D.P., et al., 2009. Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase. Proc. Natl. Acad. Sci. 106, 13124–13129. Barrero-Sicilia, C., Hernando-Amado, S., González-Melendi, P., Carbonero, P., 2011. Structure, expression profile and subcellular localisation of four different sucrose synthase genes from barley. Planta 234, 391–403. Baud, S., Vaultier, M.N., Rochat, C., 2004. Structure and expression profile of the sucrose synthase multigene family in Arabidopsis. J. Exp. Bot. 55, 397–409. Bianchi, G., Gamba, A., Murelli, C., Salamini, F., Bartels, D., 1991. Novel carbohydrate metabolism in the resurrection plant Craterostigma plantagineum. Plant J. 1, 355–359. Bieniawska, Z., et al., 2007. Analysis of the sucrose synthase gene family in Arabidopsis. Plant J. 49, 810–828. Bray, E.A., 1997. Plant responses to water deficit. Trends Plant Sci. 2, 48–54. Chang, S., Puryear, J., Cairney, J., 1993. A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep. 11, 113–116. Chatterjee, S., Pal, J.K., 2009. Role of 5′- and 3′-untranslated regions of mRNAs in human diseases. Biol. Cell 101, 251–262. Chourey, P., Taliercio, E., Carlson, S., Ruan, Y.-L., 1998. Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis. Mol. Gen. Genet. MGG 259, 88–96. Coleman, H.D., Yan, J., Mansfield, S.D., 2009. Sucrose synthase affects carbon partitioning to increase cellulose production and altered cell wall ultrastructure. Proc. Natl. Acad. Sci. 106, 13118–13123. Coleman, H.D., Beamish, L., Reid, A., Park, J.-Y., Mansfield, S.D., 2010. Altered sucrose metabolism impacts plant biomass production and flower development. Transgenic Res. 19, 269–283. Déjardin, A., Rochat, C., Wuilleme, S., Boutin, J.P., 1997. Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds. Plant Cell Environ. 20, 1421–1430. Delmer, D.P., Amor, Y., 1995. Cellulose biosynthesis. Plant Cell 7, 987. Fu, H., Park, W.D., 1995. Sink-and vascular-associated sucrose synthase functions are encoded by different gene classes in potato. Plant Cell Online 7, 1369–1385. Goren, S., Huber, S.C., Granot, D., 2011. Comparison of a novel tomato sucrose synthase, SlSUS4, with previously described SlSUS isoforms reveals distinct sequence features and differential expression patterns in association with stem maturation. Planta 233, 1011–1023. Haigler, C.H., et al., 2001. Carbon partitioning to cellulose synthesis. Plant Mol. Biol. 47, 29–51. Hänggi, E., Fleming, A.J., 2001. Sucrose synthase expression pattern in young maize leaves: implications for phloem transport. Planta 214, 326–329. Harada, T., Satoh, S., Yoshioka, T., Ishizawa, K., 2005. Expression of sucrose synthase genes involved in enhanced elongation of pondweed (Potamogeton distinctus) turions under anoxia. Ann. Bot. 96, 683–692. Henrissat, B., Coutinho, P.M., Davies, G.J., 2001. A census of carbohydrate-active enzymes in the genome of Arabidopsis thaliana. Plant Mol. Biol. 47, 55–72. Hesse, H., Willmitzer, L., 1996. Expression analysis of a sucrose synthase gene from sugar beet (Beta vulgaris L.). Plant Mol. Biol. 30, 863–872. Hirose, T., Scofield, G.N., Terao, T., 2008. An expression analysis profile for the entire sucrose synthase gene family in rice. Plant Sci. 174, 534–543. Horst, I., et al., 2007. TILLING mutants of Lotus japonicus reveal that nitrogen assimilation and fixation can occur in the absence of nodule-enhanced sucrose synthase. Plant Physiol. 144, 806–820. Ingram, J., Chandler, J.W., Gallagher, L., Salamini, F., Bartels, D., 1997. Analysis of cDNA clones encoding sucrose-phosphate synthase in relation to sugar interconversions associated with dehydration in the resurrection plant Craterostigma plantagineum Hochst. Plant Physiol. 115, 113–121. Ji, L., et al., 2013. Identification and characterization of the Populus AREB/ABF subfamily. J. Integr. Plant Biol. 55, 177–186. Kleines, M., Elster, R.-C., Rodrigo, M.-J., Blervacq, A.-S., Salamini, F., Bartels, D., 1999. Isolation and expression analysis of two stress-responsive sucrose-synthase genes from the resurrection plant Craterostigma plantagineum (Hochst.). Planta 209, 13–24. Martin, T., Frommer, W.B., Salanoubat, M., Willmitzer, L., 1993. Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs. Plant J. 4, 367–377. Pelah, D., Shoseyov, O., Altman, A., 1995. Characterization of BspA, a major boiling-stable, water-stress-responsive protein in aspen (Populus tremula). Tree Physiol. 15, 673–678. Pelah, D., Wang, W., Altman, A., Shoseyov, O., Bartels, D., 1997. Differential accumulation of water stress-related proteins, sucrose synthase and soluble sugars in Populus species that differ in their water stress response. Physiol. Plant. 99, 153–159. Ramanjulu, S., Bartels, D., 2002. Drought- and desiccation-induced modulation of gene expression in plants. Plant Cell Environ. 25, 141–151.

X. An et al. / Gene 539 (2014) 58–67 Ruan, Y.-L., Llewellyn, D.J., Furbank, R.T., 2003. Suppression of sucrose synthase gene expression represses cotton fiber cell initiation, elongation, and seed development. Plant Cell Online 15, 952–964. Sturm, A., Tang, G.-Q., 1999. The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon partitioning. Trends Plant Sci. 4, 401–407. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Tuskan, G.A., et al., 2006. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596–1604. Winter, H., Huber, S.C., 2000. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit. Rev. Plant Sci. 19, 31–67.

67

Zhang, Q., et al., 2008. Characterization of resistance gene analogs with a nucleotide binding site isolated from a triploid white poplar. Plant Biol. 10, 310–322. Zhang, D., Xu, B., Yang, X., Zhang, Z., Li, B., 2011. The sucrose synthase gene family in Populus: structure, expression, and evolution. Tree Genet. Genomes 7, 443–456. Zheng, H.Q., Zhang, Q., Li, H.X., Lin, S.Z., An, X.M., Zhang, Z.Y., 2011. Over-expression of the triploid white poplar PtDrl01 gene in tobacco enhances resistance to tobacco mosaic virus. Plant Biol. 13, 145–153. Zou, C., et al., 2013. Genome-wide analysis of the Sus gene family in cotton. J. Integr. Plant Biol. 55, 643–653. Zrenner, R., Salanoubat, M., Willmitzer, L., Sonnewald, U., 1995. Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant J. 7, 97–107.