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Journal of Experimental Botany, Vol. 67, No. 5 pp. 1397–1410, 2016 doi:10.1093/jxb/erv542 Advance Access publication 27 December 2015 RESEARCH PAPER TaCYP78A5 regulates seed size in wheat (Triticum aestivum) Meng Ma1, Huixian Zhao1,2,*, Zhaojie Li1, Shengwu Hu2,3, Weining Song2,3 and Xiangli Liu1,* 1 College of Life Sciences, Northwest A & F University, Yangling, Shaanxi 712100, China State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A & F University, Yangling, Shaanxi 712100, China 3 College of Agronomy, Northwest A & F University, Yangling, Shaanxi 712100, China 2 * Correspondence: [email protected]; [email protected] Received 3 August 2015; Accepted 25 November 2015 Editor: Gerhard Leubner, Royal Holloway, University of London Abstract Seed size is an important agronomic trait and a major component of seed yield in wheat. However, little is known about the genes and mechanisms that determine the final seed size in wheat. Here, we isolated TaCYP78A5, the orthologous gene of Arabidopsis CYP78A5/KLUH in wheat, from wheat cv. Shaan 512 and demonstrated that the expression of TaCYP78A5 affects seed size. TaCYP78A5 encodes the cytochrome P450 (CYP) 78A5 protein in wheat and rescued the phenotype of the Arabidopsis deletion mutant cyp78a5. By affecting the extent of integument cell proliferation in the developing ovule and seed, TaCYP78A5 influenced the growth of the seed coat, which appears to limit seed growth. TaCYP78A5 silencing caused a 10% reduction in cell numbers in the seed coat, resulting in a 10% reduction in seed size in wheat cv. Shaan 512. By contrast, the overexpression of TaCYP78A5 increased the number of cells in the seed coat, resulting in seed enlargement of ~11–35% in Arabidopsis. TaCYP78A5 activity was positively correlated with the final seed size. However, TaCYP78A5 overexpression significantly reduced seed set in Arabidopsis, possibly due to an ovule development defect. TaCYP78A5 also influenced embryo development by promoting embryo integument cell proliferation during seed development. Accordingly, a working model of the influence of TaCYP7A5 on seed size was proposed. This study provides direct evidence that TaCYP78A5 affects seed size and is a potential target for crop improvement. Key words: BSMV-VIGS, cell proliferation, CYP78A, seed size, Triticum aestivum L., TaCYP78A5. Introduction Wheat (Triticum aestivum L.) is a major food crop. The agronomic characteristics of wheat, particularly seed size, have been studied extensively (Smidansky et al., 2002; Khalid et al., 2004; Shomura et al., 2008; Wang et al., 2012). Seed size is a major component of seed yield and an important trait selected during domestication and modern crop breeding (Shomura et al., 2008). Small-seeded species produce more seeds for a given amount of energy than large-seeded species (Aarssen and Jordan, 2001; Henery and Westoby, 2001), and studies suggest that small seeds have a greater tendency to accumulate as persistent seed banks in the soil (Thompson et al., 2001). However, larger seeds, by offering a greater supply of nutrients, have favorable effects on seeding vigor to tolerate many of the stresses encountered during seedling establishment, thus promoting and stabilizing yield (Leishman et al., 2000; Moles et al., 2005). Therefore, increasing seed size is a major goal of crop breeding globally. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected] 1398 | Ma et al. Seed size is determined by the growth and development of the embryo, endosperm and maternal integument (Ohto et al., 2005; Sundaresan, 2005; Berger et al., 2006). For example, ABSCISIC ACID DEFICIENT2 (ABA2) regulates embryo and endosperm size by promoting cell proliferation and early cellularization of the endosperm during early seed development, respectively, ultimately affecting seed size (Cheng et al., 2014). Gain-of-function mutants of HAIKU1 (IKU1), HAIKU2 (IKU2) and MINISEED3 (MINI3) produce larger seeds due to enhanced endosperm proliferation and delayed endosperm cellularization (Garcia et al., 2003; Luo et al., 2005; Zhou et al., 2009; Wang et al., 2010). TRANSPARENT TESTA GLABRA 2 (TTG2) and APETALA 2 (AP2) promote seed growth by increasing cell expansion in the integuments; the integuments limit the size of the seed coat (Johnson, 2002; Garcia et al., 2005; Jofuku et al., 2005); AUXINRESPONSE FACTOR2 (ARF2) and DA1 (DA means ‘large’ in Chinese) act on seed size by restricting cell proliferation in the integuments (Jofuku et al., 2005; Ohto et al., 2005; Schruff et al., 2006; Li et al., 2008). CYP78A is a plant-specific gene family that is highly conserved in land plants from bryophytes to angiosperms (Mizutani and Ohta, 2010; Nelson, 1999, 2006). In Arabidopsis thaliana and rice, several CYP78As have been implicated in the control of seed development. For example, CYP78A6, CYP78A8 and CYP78A9 exhibit redundant functions in controlling floral organ growth and integument development by promoting cell proliferation in Arabidopsis (Fang et al., 2012; Sotelo-Silveira et al., 2013). Overexpression of these genes increases Arabidopsis seed size. In rice, CYP78A13 affects the proper size balance between the embryo and endosperm by controlling cell size in the embryo and cell death in the endosperm (Nagasawa et al., 2013). Loss-offunction mutants of CYP78A13 exhibit a large embryo and small endosperm; by contrast, CYP78A13 overexpression results in a small embryo and enlarged endosperm, resulting in a large seed (Nagasawa et al., 2013; Yang et al., 2013; Xu et al., 2015). CYP78A5/KLUH acts independently of other tested maternal factors that influence integument cell proliferation; the expression of CYP78A5/KLUH is limiting for seed growth in Arabidopsis (Adamski et al., 2009). Similarly, SlKLUH, an orthologous gene of Arabidopsis CYP78A5 in tomato (Solanum lycopersicum), controls fruit size by promoting integument cell proliferation (Chakrabarti et al., 2013; Monforte et al., 2014; van der Knaap et al., 2014). However, the influence of potential wheat homologs of CYP78A5 on seed development has not been reported. Because of the importance of seed size, quantitative trait loci (QTLs) related to seed size characteristics have been identified in wheat (Nakamura et al., 2007; Somyong et al., 2011; Williams and Sorrells, 2014). However, only a few genes associated with seed size have been isolated (Ma et al., 2012a; Yang et al., 2012), and little is known about the mechanisms that determine final seed size in wheat. Here, we isolated TaCYP78A5, an ortholog of Arabidopsis CYP78A5/KLUH in wheat that encodes wheat CYP78A5 and can rescue the phenotype of the Arabidopsis deletion mutant cyp78a5. We characterized the features of TaCYP78A5 including gene structure, genome localization and expression pattern. TaCYP78A5 silencing caused a reduction in cell numbers in the wheat seed coat, leading to a 10% decrease in wheat seed size. By contrast, TaCYP78A5 overexpression resulted in the production of more cells in the seed coat, resulting in seed enlargement of ~11–35% in Arabidopsis. Moreover, TaCYP78A5 also influenced embryo development by promoting embryo integument cell proliferation. These results, which demonstrate that TaCYP78A5 affects embryo development, provide new insights on the possible function of CYP78A5 gene in seed development. In summary, our results indicate that TaCYP78A5 affects wheat seed size by promoting integument cell proliferation in the developing ovule and seed and that seed size is positively associated with the expression level of TaCYP78A5. Materials and methods Plant materials and growth Wheat (Triticum aestivum L.) cultivars ‘Shaan 512’ and ‘Shaanmai 159’, which exhibit large-grain and small-grain phenotypes, respectively, were utilized to clone the target gene TaCYP78A5 and to knockdown the target in this study. To investigate the genomic origin of the target gene, different wheat species, including T. urartu (AA), Aegilops tauschii (DD), T. turgidum ssp. durum (AABB) and T. aestivum (AABBDD), were selected for gene-specific PCR using their genomic DNA as templates. To further confirm the chromosome location of the target gene, genomic DNA from a set of nulli-tetrasomic (NT) and double ditelosomic (DT) lines of wheat accession ‘Chinese Spring’ was used as the template for gene-specific PCR. The wheat growth conditions for target gene silencing by the barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) technique were as described previously (Ma et al., 2012b). The wild-type (WT) Arabidopsis thaliana ecotype Columbia-0 (Col) and mutant cyp78a5 (Salk_024697C) in the Columbia-0 background obtained from the Arabidopsis Biological Resource Center (ABRC, https://abrc.osu.edu/) were used to transform Arabidopsis with the target gene TaCYP78A5. Its seeds were germinated and then grown under suitable conditions with a 16-h-light/8-h-dark cycle and 70% relative humidity (light intensity 170–200 mmol m−2 s−1) at 20–22°C for 70 d until the last silique on the inflorescences were dry. Cloning and sequence analysis of TaCYP78A5 To clone wheat TaCYP78A5, the Arabidopsis CYP78A5 (GenBank accession no. EFH69027.1) protein was used as a query probe to search the wheat and barley (Hordeum vulgare L.) expressed sequence tag (EST) databases in GenBank (http://www.ncbi.nlm. nih.gov/). A wheat EST (GenBank accession no. BE424262.1) and a barley full-length cDNA (GenBank accession no. BAK05332.1) were retrieved and assembled as a putative TaCYP78A5 mRNA. The predicted mRNA sequence was used to design the gene-specific PCR primers TaCYP78A5-R and -F (Supplementary Table S1 available at JXB online) to amplify the genomic DNA of TaCYP78A5 in wheat. Details of TaCYP78A5 and its promoter sequence analysis are provided in Supplementary Method S1. Total RNA extraction and cDNA synthesis To clone TaCYP78A5 cDNA from wheat, total RNA was extracted from immature seeds one week after anthesis using RNAiso-mate (TaKaRa, Dalian, China). The details of the total RNA extraction TaCYP78A5 genes influence wheat seed size | 1399 are provided in Supplementary Method S1. cDNA was recovered from total RNA using Oligotex-dT30 (TaKaRa, Dalian, China). TaCYP78A5 cDNA was cloned using the primers TaCYP78A5-R and TaCYP78A5–F (Supplementary Table S1) and the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). To analyze TaCYP78A5 silencing in wheat spikes or seeds, spikelets or seeds were collected from the middle part of BSMV-infected spikes, and total RNA and TaCYP78A5 cDNA were prepared from the spikelets or seeds as described above. BSMV-mediated TaCYP78A5 gene silencing To knockdown the expression of TaCYP78A5 in wheat, the BSMV-VIGS technique was used as previously reported (Ma et al., 2012b). The construction of the barley stripe mosaic virus (BSMV)-derived vector for target gene silencing in wheat seeds is described in Supplementary Method S1. The BSMV construct BSMV:TaCYP78A5 (carrying a fragment of the TaCYP78A5 cDNA sequence) was developed as previously described (Ma et al., 2012b). Ten plants of each wheat cultivar were infected with the BSMV:TaCYP78A5, BSMV:PDS (BSMV vector carrying a 185 bp fragment of a barley phytoene desaturase gene PDS) or BSMV:00 (empty vector) constructs on the spikes at the heading stage; BSMV:00 was used as a negative control, and BSMV:PDS was used as a positive control (Ma et al., 2012b). The spikelets or seeds from the middle part of the BSMV-infected spikes were collected to quantify the transcript abundance of the target gene. This experiment was repeated five times, and the data were averaged. Construction of the TaCYP78A5 expression vector and Arabidopsis transformation To investigate the effect of TaCYP78A5 overexpression on seed development, three expression vectors with TaCYP78A5 under the control of three promoters with different expression patterns in developing Arabidopsis ovules or/and seeds were constructed, respectively. The CYP78A5 promoter (pCYP78A5 or pKLU) has high activity specifically in developing ovules and seeds (Zondlo and Irish, 1999; Adamski et al., 2009). The CaMV 35S promoter (p35S) has relatively low activity in the ovules but high activity in developing seeds (Jenik and Irish, 2000; Hraška et al., 2008), and the INNER NO OUTER promoter (pINO) is highly expressed specifically in the ovule integument (Villanueva et al., 1999). To determine the subcellular localization of TaCYP78A5, the TaCYP78A5 coding sequence without a stop codon was inserted upstream of the GUS coding sequence in the pCAMBIA3301 expression vector to obtain the fusion protein TaCYP78A5-GUS upon target gene expression. The details of the construction of the three TaCYP78A5 expression vectors, p35S::TaCYP78A5:GUS, pKUL::TaCYP78A5:GUS and pINO::TaCYP78A5:GUS are described in Supplementary Method S1. Arabidopsis transformation was performed according to Zhang et al. (Zhang et al., 2006). Transgenic plants were selected by spraying 0.1% glufosinate (BASTA) on seedling leaves. At least three independent transgenic lines bearing each construct were obtained. Reverse transcriptase-polymerase chain reaction and quantitative real-time RT-PCR To determine the efficacy of TaCYP78A5 silencing at the RNA level in wheat, seeds on the middle of wheat spikes from BSMV:TaCYP78A5-, BSMV:PDS- or BSMV:00-infected plants were collected at 5, 10, 15, 20 and 25 days post inoculation (dpi) for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The amount of RNA in each reaction was normalized using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is constitutively expressed in wheat. To determine the expression level of TaCYP78A5 in different transgenic T3 lines of Arabidopsis, RT-PCR analysis was performed with total RNA isolated from the top 2 cm of the inflorescence or siliques from each transgenic line, and ubiquitin-conjugating 21 (UBC) was used as a reference. All primers are listed in Supplementary Table S1. Details of the quantitative real-time RT-PCR are provided in Supplementary Method S1. PCR-based genotyping The cyp78a5 mutant allele was identified by PCR analysis using the LBa1 primer on the T-DNA left border. The CYP78A5 wildtype allele was identified using the primers SALK_024697-LP and SALK_024697-RP. The molecular identification of TaCYP78A5transgenic plants was performed by PCR analysis using the primers TaCYP78A5-RT-F and TaCYP78A5-RT-R. Detailed information on the primers is provided in Supplementary Table S1. Morphological and cytological characterization and GUS staining of transgenic plants Morphological differences in the transgenic plants obtained above were characterized as previously described (Disch et al., 2006; Sotelo-Silveira et al., 2013). To measure ovule size, the dissected pistil was stained with a 0.01% solution of Fluorescent Brightener 28 (SIGMA, China) as previously described (Adamski et al., 2009). The cell number and cell size in the integument of the seed coat and cotyledons were measured as previously described (Alonso-Blanco et al., 1999). To measure the biomass and seed yield, transgenic plants and five controls were planted in identical pots and cultured under well-watered conditions. Detailed protocols for the measurements of floral organs, main inflorescence stems, seeds, embryos, cells, histochemical staining for TaCYP78A5 activity and microscopy are described in Supplementary Method S1. Results Isolation and characterization of TaCYP78A5 in wheat To clone TaCYP78A5 in wheat, a putative TaCYP78A5 mRNA sequence was obtained by assembling wheat ESTs encoding proteins with similarity to Arabidopsis CYP78A5. Based on the putative mRNA sequence, primers TaCYP78A5-F and TaCYP78A5-R were designed to amplify the complete coding sequence from genomic DNA of wheat cv. Shaan 512 as the template, and the PCR products were subcloned into a T clone vector. Three TaCYP78A5 cDNA sequences were obtained from 20 monoclones, and each of these three sequences contained a complete open reading frame (ORF) encoding a polypeptide. These three sequences were temporarily named TaCYP78A5-1, TaCYP78A5-2 and TaCYP78A5-3 (Supplementary Fig. S1A). To determine the chromosomal locations of the three TaCYP78A5 genes, the PCR mapping method was applied using primers A5-P1, A5-P2 and A5-P3, and genomic DNA from the nulli-tetrasomic (NT) and double ditelosomic (DT) lines of the wheat accession Chinese Spring and wheat species with AA, DD, AABB or AABBDD genomes as templates (Supplementary Fig. S1B, C). The three TaCYP78A5 sequences were also aligned with the chromosome-based draft sequence of hexaploid bread wheat (T. aestivum L.) (International Wheat Genome Sequencing Consortium, http://www.wheatgenome. 1400 | Ma et al. org/) for further confirmation of their chromosome localization. TaCYP78A5-1, -2 and -3 were located on chromosomes 2BS, 2AS and 2DS, respectively, and consequently designated TaCYP78A5-A, TaCYP78A5-B and TaCYP78A5-D, respectively. Their GenBank accession numbers are KP768392, KP768393 and KP768394, respectively. The coding regions of TaCYP78A5-A, -B and -D are 1651, 1663 and 1650 bp, respectively, and contain two exons and one intron (Fig. 1A), identical to the coding regions of other CYP78A family members. The deduced proteins all contained a hydrophobic region in the N terminus and putative oxygenand heme-binding domains that are typical characteristics of CYP78A family members (Fig. 1A; Supplementary Fig. S2A) (Nebert and Gonzalez, 1987). The deduced proteins also exhibited high similarity with each other (Supplementary Fig. S2B). A phylogenetic tree was constructed with the TaCYP78A5 (putative amino acid sequences of TaCYP78A5-B) and other reported CYP78A family members from various plant species (Fig. 1B). TaCYP78A5 exhibits very high similarity to Arabidopsis CYP78A5 (55% identity) and CYP78A10 (53% identity; GenBank accession no. AEE35551) and is most similar to Oryza sativa CYP78A13 (76% identity; GenBank accession no. AB780362.1). CYP78A13 in O. sativa is the ortholog of Arabidopsis CYP78A5, and CYP78A13 can rescue the cyp78a5 mutant phenotype (Xu et al., 2015). Thus, we concluded that TaCYP78A5 encodes a cytochrome P450 protein and is the ortholog of CYP78A5. Most CYP78A family members are involved in seed development (Ito and Meyerowitz, 2000; Anastasiou et al., 2007; Adamski et al., 2009; Fang et al., 2012; Sotelo-Silveira et al., 2013; Xu et al., 2015). TaCYP78A5 expression was mainly detected in immature seeds (0–20 days post flowering) and young panicles (at 1 and 8 cm) in wheat cv. Shaan512 (Supplementary Fig. S3), consistent with the information for the EST (GenBank accession no. BG416626.2, 94% identity with TaCYP78A5) from a H. vulgare seed coat EST library in the NCBI database. These findings suggest that TaCYP78A5 is involved in seed development. Silencing of TaCYP78A5 reduces seed size in wheat To investigate the effect of TaCYP78A5 on seed development, we knocked down the expression of all three TaCYP78A5 genes in wheat cv. Shaan 512 (large-grain) and Shaanmai 159 (small-grain) by the BSMV-VIGS technique. A 291-bp fragment of the TaCYP78A5-A cDNA (TaCYP78A5-VIGS) sequence (corresponding to 45 bp to 335 bp after the translation start codon) was chosen to develop the recombinant BSMV construct BSMV:TaCYP78A5 (Supplementary Method S1; Supplementary Figs S4, S5) to specifically and efficiently silence TaCYP78A5. The infectivity and efficacy of BSMV-VIGS in both wheat cultivars were investigated as described previously (Ma et al., 2012b). We inoculated ten plants of each wheat cultivar by rubbing each spike with the BSMV:00, BSMV:PDS or BSMV:TaCYP78A5 construct at the heading stage. Seed photobleaching was observed in all spikes infected with the BSMV:PDS construct at 20 dpi, whereas the seeds from BSMV:00- and BSMV:TaCYP78A5-infected plants remained green throughout; the representative phenotype of wheat cultivar Shaan 512 is shown in Fig. 2A. RT-PCR analysis indicated that the transcript levels of PDS and TaCYP78A5 were reduced in BSMV:PDS- and BSMV:TaCYP78A5-infected plants, respectively, compared to BSMV:00-infected plants (Fig. 2A). These results indicate that VIGS manipulation of wheat seeds was effective in this experiment. Furthermore, mature seeds from the BSMV:TaCYP78A5-infected spikes were shorter and narrower than those from BSMV:00- and BSMV:PDS-infected spikes (Fig. 2B). Statistical analysis revealed that seed length, width and seed size of the BSMV:TaCYP78A5-infected plants were significantly reduced by ~8%, ~7% and 10% (Fig. 2C; Supplementary Fig. Fig. 1. TaCYP78A5 structures and phylogenetic analysis of the CYP78A family. (A) Exon and intron structures of the TaCYP78A5-A, -B and -D genes. Two black boxes indicate exons; black lines in the middle indicate introns; the split indicates the regions in TaCYP78A5-B that are absent in TaCYP78A5-A and -D; regions encoding hydrophobic domains, oxygen-binding motifs and heme-binding motifs are indicated by oval, circular and rectangular boxes, respectively. (B) The phylogenetic tree of TaCYP78A5 (putative amino acid sequences of TaCYP78A5-B) and other reported CYP78A family proteins from Triticum urartu, Arabidopsis thaliana, Oryza sativa, Zea mays, Pinusradiata, Phalaenopsis and Glycine max, constructed by the neighbor-joining method. (This figure is available in colour at JXB online.) TaCYP78A5 genes influence wheat seed size | 1401 Fig. 2. TaCYP78A5 silencing leads to a reduction in seed size in wheat cv. Shaan 512. (A) RT-PCR analysis of total RNA isolated from the seeds of BSMV:00-, BSMV:PDS (phytoenedesaturase)- and BSMV:TaCYP78A5-infected plants at 20 dpi. (B) Detection of target gene silencing in mature seeds from BSMV:00-, BSMV:PDS- and BSMV:TaCYP78A5-infected plants. (C) Comparison of the length and width of mature seeds from the middle spikelets of the BSMV:00-, BSMV:PDS- and BSMV:TaCYP78A5-infected plants in which the target gene was silenced. Measurements were obtained from three plants from each of five replicates and two seeds from each plant (n=30). (This figure is available in colour at JXB online.) S6C, E), respectively, compared to those of the control group (BSMV:00- and BSMV:PDS-infected plants). Similar results were obtained for wheat cv. Shaanmai 159 (Supplementary Fig. S6B, D, F) and have been reported for cyp78a5 mutants (Adamski et al., 2009). These results suggest that TaCYP78A5 plays an important role in wheat seed development. Cell number in the wheat seed coat is reduced in TaCYP78A5-silenced plants The effect of CYP78A5 on final seed size is mediated by controlling cell proliferation in ovule integuments. The integument is the future seed coat that ultimately provides an upper limit to the final seed size (Adamski et al., 2009). To determine whether the reduced seed size in TaCYP78A5silenced spikes was due to a decreased cell number in the seed coat, we investigated the cell number in the seed coats (by measuring the number of outer layer cells in the seed coat) of BSMV:00- and BSMV:TaCYP78A5-infected plants. The cell numbers of the seed coat were reduced by ~10% in seeds of BSMV:TaCYP78A5-infected plants compared with BSMV:00-infected plants at 20 dpi (Fig. 3A–E). However, no significant change in the cell length of the seed coat was observed (Fig. 3C, F). Therefore, the reduced seed size (Fig. 3D) is most likely attributable to the reduced size of the seed coat (the seed coat size was represented by the seed coat perimeter, which was calculated by multiplying the seed coat cell number by the cell length, n=10), which was due to a 10% reduction in cell number (Fig. 3E). CYP78A5 controls seed size by affecting cell proliferation in the ovule integument (Adamski et al., 2009). Whether TaCYP78A5 also affects cell proliferation in the ovule integument in wheat is unclear. Moreover, CYP78A5 is expressed not only in ovules but also in developing seeds of Arabidopsis (Zondlo and Irish, 1999), identical to the expression pattern observed for TaCYP78A5 in wheat (Supplementary Fig. S3). To characterize the role of TaCYP78A5 during seed development in wheat, we investigated the integument cell numbers of the developing ovule and seed in plants infected with the BSMV:TaCYP78A5 or BSMV:00 construct on the wheat spikes at ~7 days prior to anthesis as described previously (Ma, et al. 2012b). Because the effective silencing of BSMV-VIGS can last up to 25 days as described previously (Ma et al., 2012b), the silencing covered nearly all stages of ovule (0–7 dpi) and seed (8–25 dpi) development. In contrast to CYP78A5, which only affects ovule integument cell proliferation (Adamski et al., 2009), TaCYP78A5 silencing affected both ovule and seed integument cell development (Fig. 3H), resulting in a reduction of seed size (Fig. 3D, G). These results suggest that the expression of TaCYP78A5 may determine the growth of the seed coat by affecting the extent of integument cell proliferation in the developing ovule and seed. Overexpression of TaCYP78A5 increases seed size in Arabidopsis To confirm the effects of TaCYP78A5 on the developing ovule and seed, we characterized TaCYP78A5-overexpressing transgenic T3 lines bearing different expression vectors p35S::TaCYP78A5:GUS, pKLU::TaCYP78A5:GUS and pINO::TaCYP78A5:GUS that exhibit different expression patterns in the developing Arabidopsis ovule or/and seed. At 1402 | Ma et al. Fig. 3. TaCYP78A5 silencing causes a reduction in integument cell proliferation wheat ovules and seeds. (A) Seeds from BSMV:00- and BSMV:TaCYP78A5-infected plants at 20 dpi. (B) Cross-sections (at position of dashed lines in panel A) of seeds stained by Fluorescent Brightener. (C) Magnified view of the cross-section box in panel B. (D–F) Comparison of seed size (D, represented by the projected area of a seed), cell number (E) and cell length (F) of the outer seed coat between BSMV:00- and BSMV:TaCYP78A5-infected plants at 20 dpi. (G–H) Comparison of ovule or seed size (G, represented by the projected area of an ovule or a seed) and cell number of a cross-section of the ovule or outer seed integument (H) between BSMV:00- and BSMV:TaCYP78A5-infected plants from 5 to 25 dpi. The data represent means ±SE of at least ten independently collected seeds in which the target gene was silenced; measurements were obtained from two plants from each of five replicates and one seed from each plant. Asterisks (**) indicate a significant difference from WT at P<0.01 (t-test). Bars: 2 mm (A); 200 μm (B); 100 μm (C). (This figure is available in colour at JXB online.) least three independent Arabidopsis transgenic lines bearing each construct were evaluated in this experiment. The final seed sizes of the pKLU::TaCYP78A5, 35S::TaCYP78A5 and pINO::TaCYP78A5 transgenic lines were all significantly increased compared to WT (Fig. 4A, B, J). Moreover, TaCYP78A5 overexpression caused obvious overgrowth of the reproductive organs, as evidenced by larger and more abundant flowers (Fig. 4C, D), longer and thicker stems (Fig. 4E) and wider, shorter and more plentiful siliques (Fig. 4F, L, M). To conduct histological localization of TaCYP78A5, all constructs expressed TaCYP78A5-GUS fusion proteins. GUS staining indicated that TaCYP78A5 was highly expressed in the seed (Fig. 4G) and/or ovule (Fig. 4H). RT-PCR analysis further confirmed that TaCYP78A5 was significantly expressed in the inflorescence of transgenic T3 lines bearing each construct (Fig. 4I). A moderate increase in TaCYP78A5 expression could increase the growth-promoting effects of each genotype in the transgenic lines. Thus, together with the effect of TaCYP78A5 silencing on seed size and plant height (Fig. 3A, Supplementary Fig. S7), these results indicate that TaCYP78A5 plays an important role in promoting organ and seed growth and that the altered phenotypes are attributable to changes in TaCYP78A5 activity. Similar observations have been reported for CYP78A5- or CYP78A9-overexpressing plants (Zondlo and Irish, 1999; Anastasiou et al., 2007; Adamski et al., 2009; Eriksson et al., 2010). The overexpression of TaCYP78A5 also caused defects in reproductive development, which manifested as shorter siliques in all transgenic lines containing fewer seeds than WT (Fig. 4K). These similar phenotypes are normally observed in cyp78a9 mutants (Ito and Meyerowitz, 2000; Sotelo-Silveira et al., 2013) or 35S::CYP78A5 transgenic plants (Zondlo and Irish, 1999). Pollinating WT flowers with pollen from the TaCYP78A5-overexpressing plants yielded normal numbers of seeds per silique compared to WT, suggesting that TaCYP78A5 genes influence wheat seed size | 1403 Fig. 4. The overexpression of TaCYP78A5 affects the growth of reproductive organs and seeds in Arabidopsis. (A) Open ethanol-decolorized silique from the Col and pINO::TaCYP78A5-4 transgenic lines. Images of mature seeds (B), flowers at stage 14 (C), inflorescences (D), main inflorescence stems (E) and siliques (F) from different genotypes. For panels B, D–F, from left to right are: WT Col, transgenic lines pKLU::TaCYP78A5-5, 35S::TaCYP78A5-3 and pINO::TaCYP78A5-4 as representative transgenic Arabidopsis bearing each construct and mutant cyp78a5. PKLU/CYP78A5, Arabidopsis CYP78A5 promoter is active in the developing ovule and seed; pINO, INNER NO OUTER promoter is specifically active in ovules. (G) Developing seeds exhibiting TaCYP78A5 expression in the seed coat and embryo at 2 days after anthesis as determined by transgenic line p35S::TaCYP78A5::GUS analysis (arrow heads). (H) A developing flower with sepals and stamens removed at stage 13, displaying TaCYP78A5 expression in the developing ovule inside the gynoecium as determined by transgenic line pINO::TaCYP78A5::GUS analysis (arrow heads). (I) RT-PCR analysis of total RNA isolated from a mixture of top inflorescences from ten lines per genotype. (J–M) Quantification of mature seed and silique (at stage 14) characteristics in response to altered TaCYP78A5 expression levels. Thirty plants of the transgenic lines per genotype were grown to maturity without any assisted pollination and harvested for measurements: (J) Seed size as represented by the projected area of a seed; (K) Seed number per silique; (L) Silique number per main inflorescence stem; (M) Silique length. The values represent means ±SE (n=20). Asterisks (*) and (**) indicate significant differences from WT (Col) at P<0.05 and P<0.01 (t-test), respectively. Bars: 2 mm (A–D); 1 mm (E); 200 μm (F, G); and 500 μm (H). Stages are in accordance with those described by Smyth et al. (1990). (This figure is available in colour at JXB online.) the reduced seed set in TaCYP78A5-overexpressing plants is attributable to a defect on the maternal side. Further detailed analysis of the ovules of pINO::TaCYP78A5-4 transgenic plants revealed that the overexpression of TaCYP78A5 caused an over-proliferation of ovule integument cells and tremendous individual differences during ovule development (Supplementary Fig. S8A–D). Some ovules (31/50) exhibited various degrees of wrinkling, asymmetry and decreased size and failed to further develop, resulting in female sterility; by contrast, other ovules (19/50) exhibited a full shape and further developed into mature seeds that were larger than those of WT (Fig. 4E; Supplementary Fig. S8A, B). In general, the reduction in seed set per silique in the TaCYP78A5overexpressing transgenic line was most likely related to a developmental defect of the ovule, which was likely due to the ovule integument cell over-proliferation by the overexpression of TaCYP78A5. However, the increased seed size was not due to reduced seed set in the TaCYP78A5-overexpressing transgenic line because pINO::TaCYP78A5-4 plants produced larger seeds than WT plants with a similar seed set per plant or per silique (Supplementary Fig. S9A, B, D). Overexpression of TaCYP78A5 promotes integument cell proliferation in Arabidopsis In the above gene silencing experiments, we speculated that the effect of TaCYP78A5 on seed size was mediated by promoting integument cell proliferation in the developing wheat ovule and seed. To further confirm this relationship, we analyzed the cell numbers in the seed coat in different TaCYP78A5 transgenic lines. Indeed, TaCYP78A5 overexpression resulted in an increased cell number in the seed coat, and the increased cell number was positively associated with the TaCYP78A5 activity level (Fig. 5A, B). However, no obvious difference in cell area was observed between WT and TaCYP78A5overexpressing plants, except pINO::TaCYP78A5 transgenic lines, which exhibited reduced cell area (Fig. 5C). Thus, the formation of large seeds by TaCYP78A5 transgenic plants 1404 | Ma et al. appears to be the result of increased cell numbers in the seed coat. The cell number of the final seed coat is determined by integument cell proliferation during ovule and seed development (Haughn and Chaudhury, 2005). Therefore, we further investigated the integument cell number in the developing ovule and seed of TaCYP78A5-overexpressing plants. TaCYP78A5 overexpression led to increased cell numbers in the ovule integument of the pKLU::TaCYP78A5-5 transgenic plants compared with WT plants (Supplementary Fig. S8A, B). Similar effects have been observed in CYP78A5overexpressing plants (Adamski et al., 2009). Moreover, we investigated the seed integument cells of cyp78a5 mutants and pKLU::TaCYP78A5-5 transgenic plants during seed development at 1, 2, 4, 6 and 8 dap. The stages were in accordance with those described by Cheng et al. (2014). At the initial seed development stage (from 1–4 dap), the rate of cell proliferation was faster in pKLU::TaCYP78A5-5 transgenic plants than in WT plants but was decreased to the same level as in WT plants after 6 dap (Fig. 5D–F). By contrast, relatively slow cell proliferation was observed in cyp78a5 mutants (Fig. 5F). These results suggest that expression of TaCYP78A5 affects seed coat development by promoting integument cell proliferation in the developing ovule and seed. Similar effects of Da1, TTG2, ARF2 and AP2 on seed size via restriction of cell proliferation or cell expansion in the seed integuments Fig. 5. TaCYP78A5 overexpression results in increased cell numbers in the Arabidopsis seed coat. (A) Fluorescence micrographs of the mature seed coat stained by Fluorescent Brightener. Bar, 100 μm. Quantification of the cell number (B) and cell area (C) in the outer integument of mature seeds from each genotype. Light micrographs of alcohol-discolored seeds from (D) WT Col and (E) pKLU::TaCYP78A5-5 transgenic lines at 1–8 dap. (F) The cell numbers in the outer integument of developing seeds in response to TaCYP78A5 activity from 1 dap to 8 dap. The values represent means ±SE (n=20). Asterisks (*) and (**) indicate significant differences from the wild type (Col) at P<0.05 and P<0.01 (t-test), respectively. (This figure is available in colour at JXB online.) TaCYP78A5 genes influence wheat seed size | 1405 have been reported (Garcia et al., 2005; Schruff et al., 2006; Li et al., 2008; Ohto et al., 2009). However, TaCYP78A5 may act independently of these genes because the expression levels of these genes in the transgenic lines were similar to those in WT plants (Supplementary Fig. S10). Abnormally developed embryos were observed in the double-knockout cyp78a5 cyp78a7 mutant (Wang et al., 2008), and enlarged embryos were also observed in seeds from TaCYP78A5-overexpressing plants compared to seeds from WT plants (Fig. 6A–C, G). To examine the potential role of TaCYP78A5 in embryo growth, we measured the cell numbers in the cotyledon integument of pKLU::TaCYP78A5, pINO::TaCYP78A5 and cyp78a5 mutant and WT plants. The enlarged cotyledons in pKLU::TaCYP78A5 transgenic plants exhibited significantly increased cell numbers (~11%) compared to WT (Fig. 6A, B, G, I) but similar cell size (Fig. 6D, E, H). These findings suggest that the enlarged embryo of pKLU::TaCYP78A5 transgenic plants resulted from enhanced cell proliferation rather than cell elongation. By contrast, the reduced cotyledons of the cyp78a5 mutant exhibited a smaller number of integument cells compared to WT (Fig. 6C, F, I). Compared to WT, pINO::TaCYP78A5 transgenic plants with TaCYP78A5 expression only in the ovule exhibited similar cell numbers in the embryo integument (Fig. 6I) but significantly enlarged cell size (~35%) (Fig. 6H), suggesting that the significantly enlarged cotyledon of pINO::TaCYP78A5 transgenic plants was due to the effect of TaCYP78A5 not on embryo growth but on seed size. The effects of TaCYP78A5 on seed size were attributed to the promotion of integument cell proliferation by TaCYP78A5 in the ovule only. These results suggest that TaCYP78A5 affects embryo development by promoting embryo integument cell proliferation. Together with the effects of the silencing and overexpression of TaCYP78A5 in the ovule or/and seed on seed size, these results indicate that the alteration in seed size represents the combined effects of TaCYP78A5 on seed coat size and embryo size. These effects are mediated by the modulation of the extent of integument cell proliferation by the TaCYP78A5 expression level. By contrast, previous studies have demonstrated that CYP78A5 controls seed size by affecting cell proliferation in the ovule integument only (Adamski et al., 2009). TaCYP78A5 can rescue the phenotype of the cyp78a5 deletion mutant of Arabidopsis To further verify the involvement of TaCYP78A5 in promoting organ and seed growth, we transformed three constructs, Fig. 6. TaCYP78A5 promotes cell proliferation in the embryo integument. (A–C) Light micrographs of alcohol-discolored mature embryos from the WT Col, transgenic pKLU::TaCYP78A5-5 and cyp78a5 mutant plants. (D–F) Magnified views of the cross-section boxed areas in panels A, B and C, respectively. (G–I) Quantification of the characteristics of mature cotyledon integument cells in response to altered TaCYP78A5 activity. Asterisks (*) and (**) indicate significant differences from wild type (Col) at P<0.05 and P<0.01 (t-test), respectively. The values represent means ±SE (n=20). Bars: 100 μm (A–C); 10 μm (D–F). (This figure is available in colour at JXB online.) 1406 | Ma et al. Fig. 7. TaCYP78A5 can rescue the phenotype of mutant cyp78a5. For panels A–F, from left to right: WT Col, transgenic pKLU(CYP78A5)::TaCYP78A52;cyp78a5, p35S::TaCYP78A5-5;cyp78a5 and pINO::TaCYP78A5-3;cyp78a5 as representative Arabidopsis transgenic lines bearing each construct and mutant cyp78a5 plants. Phenotypes of WT, cyp78a5 mutant and TaCYP78A5 transgenic plants at: (A) the seedling stage (Arabidopsis plants at 12 days post germination, with leaf numbers indicated by the numbers in white); (B) stage 6.00; and (C) stage 6.50. In panels B, C the multi-branched phenotype of the cyp78a5 mutant was rescued by TaCYP78A5 overexpression. Elongated main inflorescence stems in response to the overexpression of TaCYP78A5 are also visible. (D) Silique phenotypes. (E) The expression level of TaCYP78A5 transgenic lines against a cyp78a5 mutant background was detected by semi-quantitative RT-PCR analysis of total RNA isolated from a mixture of several top inflorescences per genotype. (F) Phenotypes of mature seeds. (G, H) Quantification characteristics of mature seeds and siliques (at stage 14) in response to altered TaCYP78A5 expression levels. Thirty plants from each transgenic line per genotype were grown to maturity without any assisted pollination and harvested for measurements. (G) Silique length. (H) Seed size. The values represent means ±SE (n=20). Asterisks (*) and (**) indicate significant differences from WT (Col) at P<0.05 and P<0.01 (t-test), respectively. Bars: 1 cm (A); 2 cm (B, C); 2 mm (D, F). The stages are in accordance with those described by Boyes et al. (2001). (This figure is available in colour at JXB online.) pKLU/CYP78A5::TaCYP78A5, 35S::TaCYP78A5 and pINO::TaCYP78A5, into homozygous cyp78a5 mutants exhibiting a phenotype of multi-branched, fast leaf initiation rate and small seed size (Supplementary Fig. S11A) (Anastasiou et al., 2007). A homozygous mutant of cyp78a5 (Salk_024697C) in the Columbia-0 background obtained from the ABRC was identified (Supplementary Fig. S11B, C). The fast leaf initiation rate and multi-branching phenotype of the cyp78a5 mutants was rescued to normal WT levels by transferring pKLU::TaCYP78A5 and 35S::TaCYP78A5 constructs (Fig. 7A–D, G). However, the phenotype of cyp78a5 mutants at the seedling stage was not rescued by pINO::TaCYP78A5 because TaCYP78A5 was expressed only in the ovules of pINO::TaCYP78A5transgenic plants. Furthermore, the overexpression of TaCYP78A5 in the cyp78a5 mutant rescued the slightly reduced apical dominance phenotype of the cyp78a5 mutant (Adamski et al., 2009) (Fig. 7C). RT-PCR analysis further confirmed that TaCYP78A5 was significantly expressed in the inflorescence of transgenic T3 lines bearing each construct (Fig. 7E). The overexpression of TaCYP78A5 in the cyp78a5 mutant resulted in the production of larger seeds compared to WT plants (Fig. 7F, H). These results suggest that TaCYP78A5 can rescue the phenotype of cyp78a5 mutants and has a conserved function in promoting organ and seed growth in both monocots and dicots. TaCYP78A5 genes influence wheat seed size | 1407 Overexpression of TaCYP78A5 increases growth duration but not seed yield First flower formation, bolting and growth stoppage occur a few days (2–5 d) earlier in the cyp78a5 mutant compared to WT plants (Anastasiou et al., 2007). These results suggest that the functional deletion of CYP78A5 might induce a shortened growth duration. To determine if TaCYP78A5 also affects growth duration, we investigated the effects of TaCYP78A5 activity on reproductive organ growth duration by measuring the main inflorescence length and the silique number of WT, cyp78a5 mutant and transgenic pINO::TaCYP78A5-4 plants. The cyp78a5 mutant and WT plants stopped growing at 5 and 6 weeks of growth, respectively. However, the main inflorescences of transgenic line pINO::TaCYP78A5-4 continued growing at 8 weeks of growth to produce more flowers and siliques (Supplementary Fig. S12A–D). Moreover, these overgrowth effects were positively associated with the level of TaCYP78A5 expression in transgenic plants. Thus, the main reason pINO::TaCYP78A5-4 transgenic plants produced a longer main inflorescence and more siliques may be not only the overgrowth of the floral organ but also extension of growth duration (~2 more weeks) in the transgenic plants induced by TaCYP78A5 overexpression. Similar effects were observed in CYP78A9-overexpressing plants (Sotelo-Silveira et al., 2013). Overexpression of TaCYP78A5 increases longevity in Arabidopsis, but the effects on seed yield remain unclear. We therefore investigated the effect of TaCYP78A5 activity on overall seed yield. TaCYP78A5 silencing caused a 10% reduction in seed size (Fig. 2) and did not significantly affect seed set. Both the 100-seed weight and total seed weight per panicle were 10% lower in BSMV::TaCYP78A5-silenced plants compared to the control groups (BSMV:00 and BSMV:PDS infected plants) (Supplementary Fig. S13A, C). By contrast, the overexpression of TaCYP78A5 in the WT background significantly increased seed size and the 100-seed weight of transgenic plants (Fig. 4J; Supplementary Fig. S13B) but did not lead to an increased total seed yield per plant (Supplementary Fig. S13D). The increased seed size at the whole-plant level may have been offset by a reduced seed number per silique and per plant; the reduced effects of TaCYP78A5 on seed set were even stronger than the increased effects on seed size (Fig. 4K; Supplementary Fig. S13B). Moreover, we further investigated the harvest index (the proportion of total seed yield to total aerial biomass; n=20) of TaCYP78A5-silencing or -overexpressing plants. The alteration of TaCYP78A5 activity in both wheat and Arabidopsis led to a reduced harvest index (Supplementary Fig. S13E, F). These results indicate that either silencing or overexpression of TaCYP78A5 leads to a reduced seed yield due to reduced seed size or reduced seed set. Discussion CYP78A family members have similar functions in influencing reproductive development in plants Cytochrome P450 is one of the largest families of plant proteins (http://drnelson.uthsc.edu/cytochromeP450.html). The CYP78A class of the CYP family appears to be involved in plant-specific reactions (Nelson 1999). Several CYP78A family members, such as CYP78A5, CYP78A6 and CYP78A9 in Arabidopsis (Ito and Meyerowitz, 2000; Adamski et al., 2009; Fang et al., 2012; Sotelo-Silveira et al., 2013), CYP78A13 in rice (Nagasawa et al., 2013; Yang et al., 2013) TaCYP78A3 (Ma et al., 2015) and TaCYP78A5 in wheat, which was characterized in the present study, exhibit growth-promoting effects during reproductive organ and seed development. The overexpression of these genes results in overgrowth of seeds and reproductive organs; by contrast, the homozygous cyp78a5, cyp78a6 cyp78a9, cyp78a8 and cyp78a9 mutants exhibit smaller reproductive organs and seeds compared to WT plants (Ito and Meyerowitz, 2000; Anastasiou et al., 2007; Fang et al., 2012; Sotelo-Silveira et al., 2013). In the present experiment, TaCYP78A5-silenced wheat plants produced smaller seeds than the control plants (Fig. 2). However, some members of the CYP78A family perform overlapping functions in controlling reproductive organ and seed development. For example, the seed size and weight phenotype of cyp78a6 or cyp78a8 was synergistically enhanced by cyp78a9, suggesting that CYP78A9 functions redundantly with CYP78A6 and CYP78A8 to control seed growth (Fang et al., 2012; Sotelo-Silveira et al., 2013). Embryos of the double-knockout mutant cyp78a5 cyp78a7 do not develop correctly, yet no loss-of-function phenotype has been described for cyp78a7 mutants. CYP78A5 and CYP78A7 appear to play redundant roles in regulating embryo growth in Arabidopsis (Wang et al., 2008). In addition, two CYP78A members, CYP78A27 and CYP78A28 in Physcomitrella patens, act redundantly during reproductive organ development (Katsumata et al., 2011). Thus, CYP78A family members have similar functions in the regulation of reproductive development. Expression of CYP78A family members affects female fertility in plants Many studies have indicated that CYP78As are involved in female fertility. Either loss-of-function or gain-of-function of CYP78A members lead to reduced seed set in Arabidopsis. These alterations of fertility are all related to the female reproductive organ ovule (Ito and Meyerowitz, 2000; Adamski et al., 2009; Fang et al., 2012; Sotelo-Silveira et al., 2013). For example, in a reciprocal cross experiment, Adamski and co-workers observed that the reduced seed set per silique in pINO::CYP78A5 transgenic plants appears to be attributable to a defect on the maternal side (Adamski et al., 2009). In the present study, overexpression of TaCYP78A5 also caused a decrease in fertility due to a defect in ovule development that was probably due to the promotion of integument cell over-proliferation during ovule development (Supplementary Fig. S8). Similarly, the overexpression or functional deletion of CYP78A9 also induced female sterility in Arabidopsis. Scanning electron microscopy analysis of the ovules revealed that most of the ovules are shriveled in CYP78A9-overexpressing mutants (Ito and Meyerowitz, 2000). By contrast, the double homozygous mutant cyp78a8cyp78a9 exhibits a short integument of the ovule that cannot 1408 | Ma et al. Fig. 8. Working model for the influence of TaCYP7A5 on seed size. During ovule and seed development, TaCYP78A5 affects ovule, seed coat and embryo development by promoting integument cell proliferation, ultimately increasing the mature seed size. (This figure is available in colour at JXB online.) accommodate the developing embryo sac (Sotelo-Silveira et al., 2013). Thus, the expression of CYP78As not only significantly affects seed size but also impacts female plant fertility, which requires a normal expression pattern of CYP78A members during ovule development. The effect of TaCYP78A5 on seed size in wheat and the underlying mechanisms In wheat, a large number of QTLs have been reported to control seed size and yield components (Nakamura et al., 2007; Somyong et al., 2011; Williams and Sorrells, 2014). Thirty QTLs for seed yield have been detected on ten chromosomes (Su et al., 2006). Most (24/30) of these QTLs were mapped to the chromosomes of homologous groups 2, 5 and 7. Interestingly, the three TaCYP78A5 genes were also located on the short arms of the homologous group 2 chromosomes. TaGW2, like rice grain width-associated gene 2 (OsGW2) (Song et al., 2007), is involved in grain development in wheat. Its haplotype, Hap-6A-A, has a significantly positive effect on grain size and is considered a potentially superior allele for the improvement of grain yield in wheat (Su et al., 2011). TaCwi-A1, which is highly homologous to the rice cell wall invertase (CWI) gene (Hirose et al., 2002), has been characterized by allelic variations and QTL analysis. QTL analysis indicated that TaCwi-A1 is associated with kernel weight in Chinese commercial wheat varieties and landraces (Ma et al., 2012a). However, little is known about the mechanisms underlying final seed size in wheat. Here, we isolated a seed size-related gene, TaCYP78A5, and provided direct evidence for a role of TaCYP78A5 in determining seed size. Seeds from TaCYP78A5-silenced wheat plants were 10% smaller than those from control plants (Supplementary Fig. S6C), whereas TaCYP78A5-overexpressing Arabidopsis plants produced seeds that were larger than the seeds of WT plants by ~11–35% (Fig. 4J). The effects of TaCYP78A5 on final seed size are mediated by reducing or increasing integument cell numbers during ovule and seed development. In summary, TaCYP78A5, the ortholog of Arabidopsis CYP78A5 in wheat, affects seed size by influencing integument cell proliferation during ovule and seed development. TaCYP78A5 also influences embryo development by affecting embryo integument cell proliferation during seed development. The expression level of TaCYP78A5 is positively correlated with the final seed size but also affects female fertility. Accordingly, we have proposed a working model for the effects of TaCYP7A5 on seed size (Fig. 8). This study provides direct evidence for control of seed size by TaCYP78A5. These results suggest that TaCYP78A5 may be a target gene for crop improvement. Supplementary Data Supplementary data are available at JXB online.Figure S1. Chromosome location of TaCYP78A5. Figure S2. Alignment of three TaCYP78A5 proteins. Figure S3. TaCYP78A5 expression pattern in wheat organs or tissues as detected by quantitative real time-PCR (qRT-PCR). Figure S4. Multiple sequence alignment of TaCYP78A5VIGS and three TaCYP78A5 cDNA sequences. Figure S5. Schematic organization of the Barley stripe mosaic virus (BSMV) genomes and the inserts used for BSMV-induced gene silencing. Figure S6. Comparison of seed size, seed length and seed width of BSMV:00-, BSMV:PDS- and BSMV:TaCYP5infected plants of the wheat cultivars Shaan 512 (Large-seed) and Shaanmai 159 (Small-seed). Figure S7. The effect of TaCYP78A5 silencing by barley stripe mosaic virus (BSMV)-induced gene silencing at the seedling stage. Figure S8. TaCYP78A5 promotes cell proliferation in the ovule integument of Arabidopsis. Figure S9. Additional seed measurements of Arabidopsis in response to increased TaCYP78A5 activity. TaCYP78A5 genes influence wheat seed size | 1409 Figure S10. Expression levels of Da1, TTG2, ARF2 and AP2 in wild type Col and pINO::TaCYP78A5-4, -8 transgenic lines as detected by quantitative real-time PCR. Figure S11. 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