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Email: [email protected]. Received: 26 February .... Specifically, the first-strand cDNA was used as a template, ... wheat database (http://plants.ensembl.org/common/Tools/Blast? ... gov/gorf/gorf.html) and Conserved Domain Search (http://.
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Altered expression of TaRSL4 gene by genome interplay shapes root hair length in allopolyploid wheat Yao Han*, Mingming Xin*, Ke Huang, Yuyun Xu, Zhenshan Liu, Zhaorong Hu, Yingyin Yao, Huiru Peng, Zhongfu Ni and Qixin Sun State Key Laboratory for Agrobiotechnology and Key Laboratory of Crop Heterosis and Utilization (MOE) and Key Laboratory of Crop Genomics and Genetic Improvement (MOA), Beijing Key Laboratory of Crop Genetic Improvement, China Agricultural University, Yuanmingyuan Xi Road No. 2, Haidian District Beijing 100193, China

Summary Authors for correspondence: Zhongfu Ni Tel: +86 010 62734421 Email: [email protected] Qixin Sun Tel: +86 010 62731452 Email: [email protected] Received: 26 February 2015 Accepted: 22 July 2015

New Phytologist (2016) 209: 721–732 doi: 10.1111/nph.13615

Key words: gene expression, homoeologue, polyploid wheat, root hair length, TaRSL4.

 Polyploidy is a major driving force in plant evolution and speciation. Phenotypic changes often arise with the formation, natural selection and domestication of polyploid plants. However, little is known about the consequence of hybridization and polyploidization on root hair development.  Here, we report that root hair length of synthetic and natural allopolyploid wheats is significantly longer than those of their diploid progenitors, whereas no difference is observed between allohexaploid and allotetraploid wheats.  The expression of wheat gene TaRSL4, an orthologue of AtRSL4 controlling the root hair development in Arabidopsis, was positively correlated with the root hair length in diploid and allotetraploid wheats. Moreover, transcript abundance of TaRSL4 homoeologue from A genome (TaRSL4-A) was much higher than those of other genomes in natural allopolyploid wheat. Notably, increased root hair length by overexpression of the TaRSL4-A in wheat led to enhanced shoot fresh biomass under nutrient-poor conditions.  Our observations indicate that increased root hair length in allohexaploid wheat originated in the allotetraploid progenitors and altered expression of TaRSL4 gene by genome interplay shapes root hair length in allopolyploid wheat.

Introduction Polyploidy or whole genome duplication is a major driving force in plant evolution and speciation (Otto, 2007; Madlung, 2012). Polyploids often show novel phenotypes that are not present in their diploid progenitors or that exceed the range of the contributing progenitor species (Osborn et al., 2003). A well-known effect of polyploidy in many organisms is cell enlargement (Knight & Beaulieu, 2008; Balao et al., 2011). More interestingly, polyploidy is also able to enhance plant tolerance to biotic and abiotic stresses, possibly due to an improvement in physiological processes such as photosynthetic capacity, transpiration and metabolism (Levin, 2002; Balao et al., 2011; Yang et al., 2014). Variation in these and other phenotypic traits, such as organ size and biomass, have been selected for use in agriculture (Osborn et al., 2003). Despite increased knowledge about the effect of hybridization and polyploidization on phenotypic changes, the vast majority of studies have focused on aerial portions, and little is known about the ‘hidden half’ of a plant, especially the root hairs. Consisting of a single cell, root hairs play an important role in nutrient and water uptake (Gilroy & Jones, 2000; Libault et al., *These authors contributed equally to this work. Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

2010). To this end, root hairs undergo a phase of cell initiation after cell specification, where hair-forming cells (H cells) and non-hair cells (N cells) are arranged at regular intervals (Ishida et al., 2008; Schiefelbein et al., 2009). This is followed by a period of elongation, during which the tip growth of hair cells continues and finally results in root hair formation (Schiefelbein & Somerville, 1990). Recent studies have identified a subset of genes that regulate root hair initiation and elongation. For example, basic helix-loop-helix (HLH) transcription factor- ROOT HAIR DEFECTIVE 6 (RHD6) interacts with ROOT HAIR DEFECTIVE6-LIKE 4 (RSL4) to control root hair differentiation, initiation and elongation in Arabidopsis (Menand et al., 2007; Yi et al., 2010). In addition, genes involved in cell-wall organization and modification, such as EXPA7 and EXPA18, are also required for root hair growth (Cho & Cosgrove, 2002). Hexaploid wheat (Triticum aestivum L.) is a typical allopolyploid species that has undergone two separate allopolyploidization events. It arose from the convergence of three diploid ancestors (AA, SS/BB and DD) that have diverged from a common ancestor. The diploid wheats T. urartu (AA genomes) and Aegilops speltoides (SS genomes, most closely related to the B genome) hybridized to form the tetraploid wheat T. turgidum (BBAA). Approximately 8000 years ago, T. turgidum hybridized with the wild diploid goatgrass Aegilops tauschii (DD genomes) New Phytologist (2016) 209: 721–732 721 www.newphytologist.com

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to form the hexaploid wheat (BBAADD; Kihara, 1944; McFadden & Sears, 1946; Dvorak et al., 1998; Huang et al., 2002). To investigate variability in the root hair length and how this phenotypic change was regulated during the evolution of allopolyploid wheat, we compared the root hair length of different wheat species. Our study found that the increased root hair length in allohexaploid wheat originated in the allotetraploid progenitors and was retained during subsequent evolution. One of the direct consequences of polyploidy is an alteration of gene expression (Jackson & Chen, 2010), and a key finding of this study is that transcriptional alteration of TaRSL4 is correlated with the observed variation in the root hair length of different wheat species, and the A-homoeologue may contribute the largest proportion of root hair length variation in the natural allopolyploid wheat. In addition, the longer root hair length of Ubi-TaRSL4 transgenic wheat contributes to increased shoot fresh biomass under nutrient-poor conditions compared with the wild-type. These data provided the evolutionary evidence as well as the molecular basis for the variation in the root hair length during wheat evolutionary history and shed light on the underlying molecular mechanisms and future potential breeding utilization.

Materials and Methods

were used directly for root hair length measurement with Photoshop software. The 20 longest root hairs were measured for each plant and at least five seedlings for each genotype were used for the measurement. Each genotype was performed with three biological replicates. RNA isolation and first-strand cDNA synthesis For expression analysis, 1.5-d-old roots of three sets of synthetic allotetraploid wheat and 50 natural wheat accessions of varying ploidy were collected for RNA extraction (Table S6). The total RNA was extracted with TRIzol (Invitrogen, USA) according to the manufacturer’s instructions. The RNA samples were digested with RNase-free DNase I (Promega, Madison, WI, USA) for 30 min and extracted twice with chloroform to eliminate residual DNA. Nucleic acids were precipitated with 0.3 M NaAc (pH 5.2) and a two-fold volume of ethanol, and then collected by centrifugation (c. 12 000 g, 15 min at 4°C). The total RNA (2 lg) from each sample was used for first-strand cDNA synthesis in 20 ll reactions containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 50 lM dNTPs, 200 U MMLV reverse transcriptase (Promega) and 50 pmol T15 oligonucleotides primer. Reverse transcription was performed at 37°C for 60 min with a final denaturation at 95°C for 5 min.

Plant materials Three sets of synthetic allotetraploid wheat (S1S1A1A1, S2S2A2A2 and S1S1A3A3), three sets of synthetic allohexaploid wheat (DM4/Y199, SCAUP/SQ523 and AS2378/AS82) and their respective parents, 109 natural wheat accessions with different ploidy levels (Supporting Information Tables S1–S5) and the bread wheat (BBAADD), its extracted tetraploid wheat (BBAA) and resynthetic allohexaploid wheat (BBAAD1D1) (detailed in Zhang et al., 2014) were used to examine the difference of root hair length between different ploidy wheats. Besides synthetic allopolyploid wheats and their respective parents, we randomly selected 50 of 109 natural wheat accessions with different ploidy levels for expression analysis (Table S6) and 20 allopolyploid wheats for cleaved amplified polymorphic sequence (CAPS) analysis. In addition, Ji5265, an elite common wheat cultivar in the Northern China winter wheat region, was employed for transformation of the TaRSL4 gene. Root hair length measurement The sterilized seeds were incubated at 4°C for 3 d in the dark and then exposed to white light. Seeds with uniform germination were sown on medium containing 1% agar and grown in a glasshouse at a relative humidity of 75% and a 16 h : 8 h, light : dark cycle, maintaining temperatures of 24 and 16°C for the light and dark cycles, respectively. The seedling root hair length was measured according to the methods described in Yi et al. (2010). Briefly, the seedling root hairs at 2 d after germination (DAG) were visualized and recorded with a fluorescent stereomicroscope (SZX16; Olympus, Tokyo, Japan) attached to a digital imaging system (DP72; Olympus). The digital images New Phytologist (2016) 209: 721–732 www.newphytologist.com

Identification of cDNA Sequences of TaRSL4, TaRHD6 and TaEXPB1 Sequences of Arabidopsis AtRHD6 (AT1G66470), AtRSL4 (AT1G27740) and barley HvEXPB1 (AAQ57591) were used as queries to search against the wheat expressed sequence tag (EST) database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) for those of wheat TaRSL4, TaRHD6 and TaEXPB1, respectively. For TaRHD6, an EST of CA654295 was identified as its partial sequence. Next, to obtain its complete sequence, we performed 50 rapid amplification of the cDNA ends (50 -RACE) assay according to the manufacturer’s instructions for the GeneRacer Kit (Invitrogen). Specifically, the first-strand cDNA was used as a template, and amplification was performed for 35 cycles (5 min at 94°C; cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C; followed by 10 min at 72°C). Specific primer pairs for RACE are listed in Table S7. The gel-purified PCR product was cloned into the pGEM-T Easy Vector (Promega) and was sequenced. Finally, a 1100 bp sequence with a complete open reading frame (ORF) was obtained (Fig. S1a). For TaRSL4, two overlapping ESTs (BJ283368 and CK200622) were identified as its target sequence. Then, the assembly of them was further used to search against the wheat database (http://plants.ensembl.org/common/Tools/Blast? db=core). Finally, TaRSL4 transcripts from diploid wheat and the three homoeologues in allohexaploid wheat were isolated and confirmed by sequencing (Fig. S1b). The TaRSL4 sequence from the S diploid wheat was cloned and sequenced based on their homology. Similarly, three overlapping ESTs (BE405600, CA597386 and CD373673) were found for TaEXPB1 and its sequence with a complete ORF was obtained by sequence assembling, which was also confirmed by sequencing (Fig. S1c). Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

New Phytologist RT-PCR and cleaved amplified polymorphic sequence (CAPS) analysis The first-strand cDNA was used as a template, and amplification was performed for 30 cycles (5 min at 94°C; cycles of 30 s at 94°C, 30 s at 56–62°C, and 1 min at 72°C; followed by 10 min at 72°C). Specific primer pairs are listed in Table S7. For the CAPS analysis, each amplified CAPS reaction (10 ll) was digested with SphI restriction enzymes according to the manufacturer’s instructions and the digestion products were separated in 2% agarose gels and visualized under ultraviolet light after staining with ethidium bromide. Quantitative RT-PCR analysis Quantitative RT-PCR (qRT-PCR) using a SYBR Green PCR master mix (TaKara, Tokyo, Japan) was performed by CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Specific primer pairs for qRT-PCR analysis are listed in Table S7. The PCR efficiency of all primer pairs was determined from standard curve experiments. The PCR conditions consisted of an initial step at 95°C for 3 min followed by 40 cycles of 95°C for 15 s, 60°C for 15 s and 72°C for 30 s. For the amplification product specificity, a melting curve was generated at the end of each run, and verified by agarose gel electrophoresis of the PCR products. All reactions were performed in triplicate. The Ct values were determined using the CFX96 software with default settings. Differences between the Ct values of the target gene and Actin were calculated as DCt ¼ Cttarget gene  CtActin , and the relative expression levels of the target genes were determined as 2DCt . The average values of 2DCt were used to determine the difference in gene expression. Actin was amplified as an endogenous control. Statistical analysis of the difference in the relative expression levels of target genes was performed using Student’s t-test. Gene cloning and sequence analysis The PCR was performed using the ORF-TaRSL4-F/R primer pairs (Table S7) from two 2-d-old roots of A1A1 and S1S1. The PCR products were confirmed by visualization of the samples using electrophoresis on 1% agarose gel and stained with ethidium bromide. The samples were extracted with MinElute Gel Extraction Kit (Qiagen, Germany), and these purified PCR products were cloned into pGEM-T plasmid vector (Promega). Three clones were subsequently sequenced (Invitrogen). The online tools, Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and Conserved Domain Search (http:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), were used to analyse the nucleotide and amino acid sequences.

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using restriction enzymes SmaI and SacI. pAHC25 was designed as a monocot expression vector for particle bombardment transformation (Christensen & Quail, 1996). The fusion plasmid was transformed into 2-wk-old immature wheat embryos by particle bombardment performed using a PDS1000/He particle bombardment system (Bio-Rad) with a target distance of 90 mm from the stopping plate and helium pressure of 1300 psi (Becker et al., 1994). In total, 119 independent T0 transgenic lines were obtained according to the PCR analysis for the presence of the bar gene. At the T4 generation, partial seeds of 50 independent positive transgenic plants were planted for further characterization, of which all progenies of five lines were verified to be positive based on PCR results, indicating that they are homozygous. Thus, the remaining seeds of these five transgenic wheat lines were used for further investigation. Shoot fresh mass measurement Three-day-old seedlings of uniform size (seeds were removed) were grown in constantly aerated hydroponic medium containing quarter-strength (nutrient-rich) and one-eightieth (nutrientpoor) Hoagland’s solution (Hoagland & Arnon, 1950). On day 15, the shoot fresh mass of the seedlings was measured using an automated electronic scale. The experiments were performed with three biological replicates and five plants of each genotype for each replicate. The mid-parent heterosis (MPH) was calculated using the following formula: MPH = (F1  Mid-parental value)/Mid-parental value in %, where F1 is the average value of the synthetic allotetraploid wheat, and the mid-parental value is the average value of the two parents. Promoter cloning and bisulfite sequencing of TaRSL4 Nucleotide sequence of TaRSL4 was used to search against wheat database (http://plants.ensembl.org/common/Tools/Blast?db=core) and the promoter sequence of TaRSL4 was identified by in silico cloning and confirmed by sequencing using homoeologue conserved primer pairs (promoter-TaRSL4-F, promoter-TaRSL4-R, Table S7; Fig. S2). Homoeologous gene specific primers were designed as follows for the DNA methylation experiment: bioTaRSL4-A-F, bio-TaRSL4-A-R, bio-TaRSL4-S/B-F, bio-TaRSL4S/B-R (Table S7; Fig. S2). Then, the EZ DNA Methylation-Gold kit was used for bisulphite treatment of genomic DNA according to the manufacturer’s instructions (ZYMO Research, Irvine, CA, USA). The bisulphite-treated DNAs were then used for PCR amplification. The PCR products were cloned into the pGEM-T plasmid vector and sequenced. Sequencing data were analysed using Kismeth software (Gruntman et al., 2008).

Results Vector construction and plant transformation First, the coding region of the TaRSL4 gene was amplified by using Ubi-TaRSL4-F/R primer pairs (Table S7) and ligated to pGEM-T plasmid vector for sequencing. Then the confirmed gene sequence was transferred into pAHC25 vector and digested Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

Significant variations in root hair length were observed in diploid and allopolyploid wheats To investigate changes in the seedling root traits after the formation of allopolyploid wheat, we compared the maximum New Phytologist (2016) 209: 721–732 www.newphytologist.com

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root length, lateral root number and the root hair length of synthetic allotetraploid wheat (S1S1A1A1) and its diploid parents (S1S1 and A1A1). The root hair length of S1S1A1A1 was significantly longer than that of the diploid parents at 2 DAG (Figs 1a, S3). This growth vigour was examined in another two sets of synthetic allotetraploid wheat (S2S2A2A2 and S1S1A3A3), which showed consistent results (Fig. 1a). Next, we examined the root hair length of three sets of synthetic allohexaploid wheat (BBAADD) and their parents (BBAA and DD). Although the allohexaploid wheat exhibited superior root hair length relative to the diploid parents, no growth vigour was observed between the allohexaploid and allotetraploid wheats (Fig. 1b), suggesting that the contribution of the D genome to the root hair length in polyploidy wheat was limited. To confirm our observation, we further measured the root hair length of a bread wheat (BBAADD), its extracted tetraploid wheat (BBAA) and resynthetic allohexaploid wheat (BBAAD1D1). As expected, the effect of the

New Phytologist D genome on the root hair length was limited and there was no difference in the root hair length among these three genotypes (Fig. S4). To determine whether the above observation was prevalent in natural wheat species, we evaluated the root hair length of 109 wheat accessions of varying ploidy, including T. urartu (AuAu), T. boeoticum (AbAb), T. monococcum (AmAm), Ae. speltoides (SS), Ae. longissima (SS), Ae. sharonensis (SS), Ae. tauschii (DD), T. dicoccoides (BBAA), T. durum (BBAA), T. dicoccum (BBAA), T. spelta (BBAADD) and T. aestivum (BBAADD) (Tables S1– S5). The average root hair length of the SS group at 2 DAG was significantly longer than that of the AA and DD groups (Fig. 2; Table 1). Although there was no significant difference between the average root hair length of the BBAA and BBAADD groups, these allopolyploid wheats displayed increased root hair length compared with the three diploid groups (AA, SS and DD). Consistent with this observation, three synthetic allotetraploid wheats (SSAA) also displayed longer root hair compared with the three

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Fig. 1 Root hair phenotypes and length of synthetic allopolyploid wheats and their parents. (a) Root hair phenotypes (left) and statistical analysis of root hair length (right) of three synthetic allotetraploid wheats and their diploid parents. S1S1A1A1, S2S2A2A2 and S1S1A3A3, synthetic allotetraploid wheat; S1S1, A1A1, S2S2, A2A2 and A3A3, diploid parents. (b) Root hair phenotype (left) and statistical analysis of root hair length (right) of three synthetic allohexaploid wheats and their parents. DM4/Y199, SCAUP/SQ523 and AS2378/AS82, synthetic allohexaploid wheats, BBAADD; DM4, SCAUP and AS2378, tetraploid parents, BBAA; Y199, SQ523 and AS82, diploid parents, DD. Bars, 1 mm. The values are means ( SE) of three biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05, least significant difference (LSD) test). Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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Research 725 Table 1 Variation of root hair length in 109 natural wheat accessions Root hair length (mm) Species

No.

Genome

Mean  SD

Min

Max

Triticum urartua Aegilops speltoidesb Ae. tauscchii T. dicoccoidesc T. aestivumd

27 8 21 29 24

AA SS DD BBAA BBAADD

0.72  0.15 c 1.37  0.15 b 0.73  0.15 c 2.00  0.33 a 2.10  0.48 a

0.44 1.17 0.53 1.41 1.35

1.09 1.57 1.08 2.67 2.78

Different letters are used to indicate means that differ significantly (P < 0.05, LSD test). No., the number of wheat accessions; Min, minimum value; Max, maximum value. a Including T. monococcum and T. boeoticum. b Including Ae. longissima and Ae. sharonensis. c Including T. dicoccum and T. durum. d Including T. spelta.

Expression of wheat TaRSL4 gene was correlated with root hair length variation

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Fig. 2 Root hair phenotypes and length of natural wheat accessions. (a). Example of root hair phenotype of natural wheat accessions. Triticum urartu, AA13 (AuAu); T. boeoticum, AA3 (AbAb); T. monococcum, AA11 (AmAm); Aegilops speltoides, Z409 (SS), Ae. tauschii, RM235 (DD); T. dicoccoides, IW57 (BBAA); T. durum, DW25 (BBAA); T. dicoccum, Z42 (BBAA); T. aestivum, CS (BBAADD). See Supporting Information Tables S1–S5 for accession names. Bars, 1 mm. (b) Boxplots showing root hair length for 109 natural wheat accessions. Horizontal line indicates different ploidy levels of 109 natural wheat accessions. AA, SS and DD, diploid wheat; BBAA, allotetraploid wheat; BBAADD, allohexaploid wheat. The root hair length ranges of the AA, SS, DD, BBAA, SSAA and BBAADD are 0.43–1.09, 1.17–1.57, 0.53–1.08, 1.91–2.03, 1.41–2.66 and 1.35– 2.78 mm, respectively.

diploid progenitors, whereas no difference was detected among SSAA, BBAA and BBAADD (Fig. 2; Table 1). To test whether the observed differences at 2 DAG among different genotypes still existed at later stages, dynamic growth patterns of the diploid (RM157, DD) and allohexaploid wheat (J411, BBAADD) were investigated and the root hair of J411 was significantly longer than that of RM157 at 2, 3, 4 and 5 DAG (Fig. S5). Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

Changes in the expression patterns of developmental genes are often correlated with evolved phenotypes (Stern & Orgogozo, 2008). AtRHD6 and AtRSL4 in Arabidopsis and HvEXPB1 in barley were previously reported to be important for root hair development (Kwasniewski & Szarejko, 2006; Menand et al., 2007; Yi et al., 2010). We analysed the expression patterns of three putative wheat orthologous genes of AtRHD6, AtRSL4 and HvEXPB1 (designated TaRHD6, TaRSL4 and TaEXPB1) in three synthetic allotetraploids and their diploid parents using quantitative real-time PCR (qRTPCR). Consistent with our expectation, these three genes were up-regulated in synthetic allotetraploid wheats compared with their diploid parents (Fig. 3a–c), and Pearson’s correlation index between the root hair length and the gene expression was higher for TaRSL4 (r = 0.874, P < 0.01) than for TaRHD6 (r = 0.501, P = 0.169) and TaEXPB1 (r = 0.542, P = 0.132; Fig. S6). In addition, the expression level of TaRSL4 was also higher in synthetic allohexaploid wheat compared with their diploid parents (Fig. S7). Notably, the TaRSL4 was specifically expressed in roots (Fig. 3d), indicating that TaRSL4 could be a major player in controlling root hair growth. Thus, we examined whether the expression pattern of TaRSL4 can explain the variation in the root hair length in 50 natural wheat accessions with varying ploidy, including 26 diploid, 10 allotetraploid and 14 allohexaploid wheats (Table S6). At each separate ploidy level, significantly positive correlation was only observed in the allotetraploid wheat (r = 0.851, P < 0.01), but not in the diploid and allohexaploid wheat (Table 2). Across the three different ploidy levels, the expression of TaRSL4 was also positively correlated with the root hair length of the natural wheat accessions (SS, AA, DD, BBAA and BBAADD; r = 0.377, P < 0.01; Table 2). Across the diploidy and allotetraploidy levels, significant positive correlation between root hair length and the expression of TaRSL4 was also observed (r = 0.578, P < 0.01; Table 2). New Phytologist (2016) 209: 721–732 www.newphytologist.com

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A-homoeologue of TaRSL4 was more abundantly expressed in allopolyploid wheat To further investigate how the expressions of homoeologous genes were regulated in allopolyploid wheat, we identified bioinformatically three TaRSL4 homoeologous genes from the reference genomic sequences of hexaploid wheat Chinese Spring (CS), which were designated as TaRSL4-2AS, TaRSL4-2BS and TaRSL4-2DS, respectively. Sequence analysis revealed that there was a SphI site (CGCATG) mutation in TaRSL4-2AS (CGCATT), which was used to develop cleaved amplified New Phytologist (2016) 209: 721–732 www.newphytologist.com

Fig. 3 Expression patterns of TaRHD6, TaRSL4 and TaEXPB1 genes relative to root hair development. (a–c) qRT-PCR analysis of TaRHD6 (a), TaRSL4 (b) and TaEXPB1 (c) genes related to root hair development. The expression of Actin was used to normalize mRNA levels. The values are means ( SE) of three biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05, LSD test). (d) Tissuespecific expression of TaRSL4 gene in wheat. Actin was used as a loading control. R, roots; EN, endosperm; ST, stamen; PI, pistil; YE, young ear; L, lemma; PA, palea; G, glume; B, blade; A, awn; EM, embryo; SE, seeds.

polymorphic sequence (CAPS) primers (Fig. S8). As expected, 318 bp PCR products could be completely cleaved by SphI in the nullitetrasomic lines N2AT2B and N2AT2D (Fig. S9). Using the primer pairs for CAPS analysis, we cloned the sequences of the TaRSL4 gene from three synthetic allotetraploid wheats (S1S1A1A1, S2S2A2A2 and S1S1A3A3) and five natural allopolyploid wheats, including T. dicoccoides (BBAA), T. durum (BBAA), T. dicoccum (BBAA), T. spelta (BBAADD) and T. aestivum (BBAADD). Sequence comparison showed that the SphI site mutation in TaRSL4-2AS (CGCATT) was conserved in natural allotetraploid and allohexaploid wheat, whereas the SphI Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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Table 2 Correlation coefficients between the root hair length and the expression levels of TaRSL4 among different ploidy wheat accessions Group of accessions

No. of accessions

r

Diploid (29) Allotetraploid (49) Allohexaploid (69) 29 + 49 29 + 49 + 69

24 10 16 34 50

0.189 0.851* 0.256 0.578* 0.377*

*Significant at 1%.

site mutation was not detected in synthetic allotetraploid wheat (Fig. S10). Next, we examined the relative mRNA abundance of three TaRSL4 homoeologues in 13 synthetic and natural allohexaploid wheats using the polymerase chain reaction with reverse transcription (RT-PCR) and CAPS analysis. The expression levels of TaRSL4-2AS were higher than those of TaRSL4-2BS and TaRSL4-2DS (Fig. 4a,b; Table S8). The mRNA abundance of TaRSL4-2AS was also higher than that of TaRSL4-2BS in natural allotetraploid wheats (Fig. 4c; Table S8). In addition, we used cDNA sequencing to investigate the relative expression level of TaRSL4 homoeologous genes in three synthetic allotetraploid wheats. Of the 20 sequenced cDNA fragments, the majority were derived from the S-genome in S2S2A2A2 (70%) and S1S1A3A3 (75%) (Fig. 4d). Conversely, sequencing of 20 cDNA fragments from S1S1A1A1 showed that 75% were derived from the A-genome, and only 25% were from the S-genome (Fig. 4d). Furthermore, to investigate the underlying mechanisms for

Fig. 4 Transcript levels of TaRSL4 homoeologous genes in allopolyploid wheat. (a–c) Expression levels of TaRSL4 homoeologous genes in 10 natural allohexaploid wheats (BBAADD) (a), three synthetic allohexaploid wheats (BBAADD) (b) and 10 natural allotetraploid wheats (BBAA) (c). PCR products with genomic DNA (gDNA) as template (left) and RT-PCR products with cDNA as template (right) were digested with SphI restriction enzyme. -SphI, before digestion; +SphI, after digestion. (d). Transcript profiles of TaRSL4 homoeologues in three synthetic allotetraploid wheats. The ratio of expression level derived from Sgenome and A-genome were determined by sequencing of 20 clones for each genotype. Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

unequal expression of TaRSL4 homoeologues, we examined the DNA methylation status in promoter regions of TaRSL4 homoeologues. In both natural and synthetic allotetraploid wheats, the A-homoeologue is hypermethylated, whereas the S/B-homoeologue is hypomethylated (Fig. S11). Overexpression of TaRSL4 gene increased root hair length in wheat To determine the biological function of the TaRSL4 gene, we cloned the complete open reading frame (ORF) of the TaRSL4 gene from A1A1 and S1S1 and obtained the Ae. tauschii (DD) sequence from the reference genome. These homoeologues of the TaRSL4 gene had a striking similarity in nucleotide sequence, ranging between 95% and 100% similarity. Comparison of the deduced amino acid sequences revealed that TaRSL4 was orthologous to a group of proteins possessing a conserved HLH domain, including AtRSL4, OsRSL4 and ZmRSL4 (Fig. S12). Moreover, the TaRSL4 proteins from different wheat species exhibited high similarity (94–100%), especially in the HLH domain (100%) (Fig. S13), indicating that the function of these TaRSL4 homoeologous genes may be redundant. Considering the higher expression of TaRSL4-2AS in natural allopolyploid wheat, we generated transgenic wheat plants overexpressing TaRSL4-2AS of CS under the control of ubiquitin (Ubi) promoter. We confirmed five homozygous lines (#1–5) in T4 transgenic plants by genomic PCR (Fig. 5a). The results of the qRT-PCR showed that the expression of the TaRSL4 gene in transgenic lines #1, #3 and #5 was up-regulated by more than two-fold compared with the wild-type (Fig. 5b). The root hair

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lengths of lines #1, #3 and #5 (1.85  0.06, 1.91  0.03 and 1.73  0.09 mm) were significantly longer than that of the wildtype (1.35  0.03 mm; Fig. 5c,d). Longer root hairs contribute to the increased shoot fresh biomass in Ubi-TaRSL4 transgenic lines under nutrientpoor conditions To investigate whether increased root hair length affects seedling growth, we compared the shoot fresh biomass of the UbiTaRSL4 transgenic lines with the wild-type under variable nutrient conditions. Although no difference was observed under the nutrient-rich condition, the shoot fresh biomass of Ubi-TaRSL4 transgenic lines was significantly higher than that of the wild-type under nutrient-poor conditions (Fig. 6a,c). In addition, we also measured and compared the shoot fresh biomass of the synthetic allotetraploid S1S1A1A1 and its diploid parents. Synthetic allotetraploid S1S1A1A1 displayed growth vigour in terms of shoot fresh biomass under both nutrient-rich and nutrient-poor conditions. Remarkably, mid-parent heterosis (134.12%) under nutrient-poor conditions was considerably higher than that under nutrient-rich conditions (77.83%) (Fig. 6b,d).

Discussion Allopolyploid wheat displays growth vigour in root hair length compared to their diploid progenitors Polyploidization has taken place throughout c. 70% of angiosperms, which has long been recognized as a driving force shaping the evolution of flowering plants (Osborn et al., 2003). Heterozygosity and intergenomic interactions in allopolyploids can induce phenotypic variation and growth vigour (Ha et al., 2009; Chen, 2010; Li et al., 2014). For example, the fibre of New Phytologist (2016) 209: 721–732 www.newphytologist.com

Fig. 5 Overexpression of TaRSL4 increased root hair length in wheat. (a) The genomic PCR results identified five homozygous lines (Ubi-TaRSL4 #1–5) using specific primer designed in recombinant plasmid vector (Fig. S8). (b) Relative expression level of TaRSL4 gene in Ubi-TaRSL4 transgenic lines and wild-type. The expression of Actin was used to normalize mRNA levels. The values are means ( SE) of three biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05, least significant difference (LSD) test). (c) Root hair phenotypes of transgenic lines (#1, #3 and #5) and wild-type. Bars, 1 mm. (d) Statistical analysis of root hair length of transgenic lines (#1, #3 and #5) and wild-type. The values are means ( SE) of three biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05, LSD test).

allopolyploid cotton is vastly superior to that of its diploid relatives (Paterson, 2005). Triticum is an allopolyploid complex of agricultural importance and allohexaploid wheat has superior grain quality and broader adaptability compared with their progenitors (Dubcovsky & Dvorak, 2007). Here, we found that the root hair length of synthetic allotetraploid wheat (SSAA) was considerably longer than that of their diploid parents (Fig. 1). Increasing evidence has indicated that phenotypic traits have undergone human selection under domestication during the evolution of allopolyploid wheat, for example grain shape (Chantret et al., 2005; Gegas et al., 2010; Meyer et al., 2012). In this study, both allotetraploid and allohexaploid wheats displayed an increased average root hair length compared with three diploid groups (AA, SS and DD), but no significant difference was observed between the allotetraploid and allohexaploid wheats (Fig. 2; Table 1). Furthermore, no differences in the root hair length were found among the allohexaploid bread wheat (BBAADD), its extracted tetraploid wheat (BBAA) and resynthetic allohexaploid wheat (BBAAD1D1) (Fig. S4). Collectively, we proposed that the increased root hair length in allohexaploid wheat originated in the allotetraploid progenitors and was retained during subsequent evolution. Remarkably, root hair length is dependent on both intrinsic genetic programmes and environmental factors (M€uller & Schmidt, 2004). The controllable conditions on a medium containing 1% agar are clearly different from those in nature. Therefore, it will be interesting to compare the root hair length of different wheat genotypes when the seeds germinate in soil. TaRSL4 played an important role in the evolution of the root hair length of wheat species Polyploidization is accompanied by rapid and dynamic changes of genome and chromatin structures, as well as alterations of gene Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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Fig. 6 Increased root hair length affects fresh biomass in shoots. (a) Seedlings of UbiTaRSL4 transgenic lines and wild-type under nutrient-rich and -poor conditions. Bars, 1 cm. #1, #3 and #5, Ubi-TaRSL4 transgenic lines. (b) Seedlings of synthetic allotetraploid wheat and its diploid parents under nutrientrich and nutrient-poor conditions. Bars, 1 cm. S1S1A1A1, synthetic allotetraploid wheat; S1S1 and A1A1, diploid parents. (c) Statistical analysis of shoot fresh biomass for UbiTaRSL4 transgenic lines and wild-type under nutrient-rich conditions. The values are means ( SE) of three biological replicates. Different letters are used to indicate means that differ significantly (P < 0.05, LSD test). (d) Shoot fresh biomass and mid-parent heterosis of synthetic allotetraploid wheat and its diploid parents. The values are means ( SE) of three biological replicates. H, midparent heterosis (MPH); MP, mid-parental value; **, significant at P < 0.01.

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expression and epigenetic variation (Osborn et al., 2003; Chen, 2007; Gaeta et al., 2007; Akhunova et al., 2010). Studies have documented that changes in gene expression play an important role in the growth vigour associated with polyploidy (Osborn et al., 2003; Chen, 2007). For instance, growth vigour in Arabidopsis allotetraploid is partially controlled by the altered expression of circadian regulator genes (Ni et al., 2009). In synthetic allohexaploid wheat, an immediate transcriptional reprogramming of the D-homoeologue of HKT1;5 contributed to its higher fitness under salt stress conditions (Yang et al., 2014). In this study, altered expression of the TaRSL4 gene is one of the principal molecular variables contributing to the quantitative variation of root hair length in wheat species. First, the relative expression level of the TaRSL4 in synthetic allotetraploid wheat was significantly higher than those of their diploid parents (Fig. 3b), which was consistent with the observed growth vigour in terms of root hair length (Fig. 1a); second constitutive Ó 2015 The Authors New Phytologist Ó 2015 New Phytologist Trust

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expression of the TaRSL4 in wheat resulted in longer root hair compared with the wild-type (Fig. 5); Finally, the expression level of TaRSL4 was positively correlated to the root hair length of the natural wheat accessions with different ploidy levels (Table 2). Remarkably, the correlation coefficients were distinctly improved for the allotetraploid, the combined diploid and allotetraploid data, but no significant correlation was observed at each ploidy level of diploid and allohexaploid (Table 2), indicating that the altered expression of the TaRSL4 might represent a ploidy specific effect. Most notably, although growth vigour in terms of the root hair length was not detected in synthetic allohexaploid wheats, the TaRSL4 gene displayed the high-parent or over-dominance expression patterns (Fig. S7), which coincided with the lack of correlation between the TaRSL4 gene expression and the root hair length of diploid and allohexaploid wheat, suggesting that complexity of the underlying mechanism for growth vigour of root hair in allohexaploid wheat. Thus, rather than studying New Phytologist (2016) 209: 721–732 www.newphytologist.com

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differential gene expression on a gene-by-gene basis, genomewide analysis will be a valuable method for identifying genes important for variation of root hair length in allopolyploid wheat (Renny-Byfield & Wendel, 2014). Nutrients are critical elements for plant growth and productivity (L opez-Bucio et al., 2003). Roots hairs are known to be important for the uptake of sparse soluble nutrients (Keyes et al., 2013). For example, the rsl4 and rhd6 mutants of Arabidopsis showed impaired root hair growth (Menand et al., 2007; Yi et al., 2010), which inhibited the capacity for phosphate uptake under phosphate-limited conditions (Bates & Lynch, 1996; Narang & Altmann, 2001). In this study, constitutive expression of the TaRSL4 in allohexaploid wheat increased fresh biomass of the shoot under nutrient-poor conditions compared with the wildtype, which provides further evidence that increased root hair length benefits plant growth (Fig. 6a,c). Theoretically, increased root hair length in allopolyploid wheat provides advantages for aerial growth under nutrient-poor conditions compared with its parents. Consistent with this hypothesis, the synthetic allotetraploid (S1S1A1A1) displayed much higher mid-parent heterosis of the shoot fresh biomass under nutrient-poor conditions compared with nutrient-rich conditions (Fig. 6b,d). However, the root hair is just one of the factors that affect the biomass vigour of allotetraploid wheat. Many other factors including photosynthesis and carbon metabolic activities may also play important roles in the observed biomass heterosis under nutrient-poor conditions (Warner & Edwards, 1993; Ni et al., 2009). Unequal expression of TaRSL4 homoeologues is prevalent in allopolyploid wheat Although most homoeologues are assumed to be expressed, homoeoloci frequently make unequal contributions to the total gene expression levels in allopolyploid wheat (Pumphrey et al., 2009; Akhunova et al., 2010; Chague et al., 2010; Leach et al., 2014). For example, Bx genes involved in benzoxazinone biosynthesis had a biased transcription towards the homoeologues of the B genome (Nomura et al., 2005). Transcription of the Gli-B2 gene was underrepresented compared with their homoeologues from A and D genomes (Salentijn et al., 2009). Here, we noted a bias in the transcript level of the TaRSL4 gene from the S genome in synthetic allotetraploid wheat S2S2A2A2 and S1S1A3A3, whereas the transcript of TaRSL4 gene from the A genome was much higher than that of the S genome in S1S1A1A1 (Fig. 4d). This indicates that different combinations of hybridizing genomes could lead to activation of the genes from different parental origins. An additional aspect of polyploid evolution that has garnered considerable attention is genomic asymmetry in the control of a variety morphological, physiological and molecular traits, that is the complete or predominant control of certain traits by one of the constituent genomes (Feldman et al., 2012). In allopolyploid wheat, the A genome preferentially controlled the morphological traits, whereas the B and/or D genome preferentially controlled the reaction to biotic and abiotic factors (Feldman et al., 2012). Interestingly, transcripts of TaRSL4 homoeologue from the A New Phytologist (2016) 209: 721–732 www.newphytologist.com

genome were much higher than those of other genomes in the roots of seedlings from natural allotetraploid and allohexaploid wheats (Fig. 4a,c). This phenomenon has also been observed in synthetic allohexaploid wheat (Fig. 4b). Collectively, our data demonstrated that the A-homoeologue of TaRSL4 may play a major role in the root hair length of natural allopolyploid wheat than of other homoeologues. However, there were some variations of the amino acids among the three TaRSL4 homoeologues, which possibly affects their functions in root hair development and deserves further investigation. DNA methylation has been demonstrated to mediate the epigenetic regulation of gene expression, growth and development in plants and animals (Berger, 2007; Li et al., 2007; Zhang, 2008). Recently, it was reported that DNA methylation changes occur among wheat homoeologues after polyploid formation, which is associated with gene expression and phenotype (Liu et al., 1998; Wolffe & Matzke, 1999; Jenuwein & Allis, 2001; Shaked et al., 2001; Kashkush et al., 2002). For example, a higher level of cytosine methylation was detected in the promoter region of TaEXPA1-B, which may contribute to its silencing in leaves (Hu et al., 2013). Gene-specific silencing of the WLHS1-B homoeologue is caused by cytosine methylation (Shitsukawa et al., 2007). On the contrary, we found that the promoter of the highly expressed A-homeologue of TaRSL4 was hypermethylated. Consistent with this observation, promoter methylation (CHH islands in 50 upstream regions and promoters) could be related to the activation of genes (Ha et al., 2009; Gent et al., 2013). Collectively, these data indicate the complexity underlying the regulation mechanisms for biased expression of homoeologues in allopolyploid wheat, which will require further investigation.

Acknowledgements We thank Bao Liu (Northeast Normal University), Dengcai Liu (Sichuan Agricultural University), Dongfa Sun (Huazhong Agricultural University), Fangpu Han (Chinese Academy of Sciences), Chaojie Xie (China Agricultural University) and Yuming Wei (Sichuan Agricultural University) for providing wheat seeds of varying ploidy levels. This work was supported by the Major Program of the National Natural Science Foundation of China (31290212), the 863 Project Grant (2012AA10A309), the National Natural Science Foundation of China (31271709) and Chinese Universities Scientific Fund (15054038).

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Nucleotide sequences of the TaRHD6, TaRSL4 and TaEXPB1. Fig. S2 TaRSL4 promoter sequences of S1S1A1A1, S2S2A2A2 and IW53 (BBAA) used for DNA methylation experiment and corresponding primer position. Fig. S3 Statistical analysis of root traits of synthetic allohexaploid wheat and its diploid parents. Fig. S4 Root hair phenotypes and length of a bread wheat (AABBDD), its extracted tetraploid wheat (AABB) and resynthetic allohexaploid wheat (AABBD1D1). Fig. S5 Dynamic growth patterns of root hair of allohexaploid (J411) and diploid (RM157) wheat.

Fig. S7 Expression patterns of TaRSL4 in three synthetic allohexaploids and their diploid parents using qRT-PCR. Fig. S8 Nucleotide sequences comparison of TaRSL4 homoeologs genes in CS. Fig. S9 The examination of SphI site by CAPS analysis in CS nullitetrasomics lines. Fig. S10 Nucleotide sequences comparison of TaRSL4 homoeologs in diploid and allopolyploid wheat. Fig. S11 Methylation status of CG/CHG/CHH sites of TaRSL4 homoeologous genes in synthetic (S1S1A1A1 and S2S2A2A2) and natural allotetraploid wheats (IW53, BBAA). Fig. S12 Comparison of TaRSL4 amino acid sequence to homologs in multiple species. Fig. S13 Comparison of TaRSL4 protein sequences in diploid and allohexaploid wheat. Table S1 Origin and identity of diploid (genome AA) wheat accessions used in this study Table S2 Origin and identity of diploid (genome SS) wheat accessions used in this study Table S3 Origin and identity of diploid (Ae. tauschii, DD) wheat accessions used in this study Table S4 Origin and identity of allotetraploid (genome BBAA) wheat accessions used in this study Table S5 Origin and identity of allohexaploid (genome BBAADD) wheat accessions used in this study Table S6 Natural wheat accessions of varying ploidy used for correlation analysis Table S7 Primers used in this study Table S8 Quantification of the electrophoretic bands of Fig. 4 Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

Fig. S6 Correlation analysis between root hair length and TaRHD6, TaRSL4 and TaEXPB1 gene expression levels.

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