TY - JOUR
AU - Wang, Chunguo
AB - Abstract In Larix and in some crops, heterosis is prevalent and has been widely used in breeding to produce excellent varieties. However, the molecular basis of heterosis in Larix remains ambiguous. LaAP2L1, a member of the AP2/EREBP transcription factor family, has been suggested to be involved in heterosis in Larix hybrids. Here, the function and regulation of LaAP2L1 were further explored. Overexpression of LaAP2L1 led to markedly enlarged organs and heterosis-like traits in Arabidopsis. Fresh weight of leaves was almost twice as great as in vector controls. Likewise, seed yield of 35S::LaAP2L1 individual plants was >200% greater than that of control plants. The enlarged organs and heterosis-like traits displayed by 35S::LaAP2L1 plants were mainly due to enhanced cell proliferation and prolonged growth duration. At the molecular level, LaAP2L1 upregulated the expression of ANT, EBP1, and CycD3;1 and inhibited the expression of ARGOS in 35S::LaAP2L1 plants, suggesting an important molecular role of LaAP2L1 in regulating plant organ development. These findings provide new insights into the formation of heterosis in woody plants and suggest that LaAP2L1 has potential applications in breeding high-yielding crops and energy plants. In addition, 50 AP2/EREBP transcription factors, including LaAP2L1, in Larix were identified by transcriptome sequencing, and phylogenetic analysis was conducted. This provided information that will be important in further revealing the functions of these transcription factors. Introduction Heterosis or hybrid vigor, prevalent in higher plants, refers to the phenomenon whereby hybrid progeny display improved grain yield, growth potential, resistance to pests, and environmental stresses compared with their parents (Guo 2006, Schnable and Springer 2013). As a direct consequence of hybridization events, heterosis has been demonstrated to play a critical role in promoting speciation and evolution of various plants. Moreover, this phenomenon has been widely used in breeding crops and other economic plants. F1 hybrids can produce yields two to three times as high as their inbred parents in rice, maize, wheat, and other crops (Duvick 1997, Springer et al. 2007, Flint-Garcia et al. 2009, Baranwal et al. 2012). As a result, large-scale cultivation of hybrid varieties, particularly the worldwide promotion and planting of hybrid rice, has made significant contributions to food production and provided great economic and societal benefits (Duvick 1997, Guo et al. 2006, Yao et al. 2013). However, although three main genetic hypotheses—the dominance, overdominance and epistasis hypotheses—have been proposed to explain the origin of heterosis (Li ZK et al. 2001, Birchler et al. 2003, 2006, Hochholdinger and Hoeker 2007, Li LZ et al. 2008), the underlying mechanisms of heterosis in plants, especially woody plants, is still largely unknown. Studies focusing on gene transcription have provided further insights into the molecular basis of heterosis, demonstrating that differences in gene expression, especially nonadditive expression, are tightly associated with the formation of heterotic traits in many plant species (Swanson-Wagner et al. 2006, Li X et al. 2009, Wei et al. 2009, Li A et al. 2012, Zhai et al. 2013). Several genes involved in the formation of heterosis have been studied. For example, the higher expression levels of Gibberellin insensitive dwarf 1 (GID1), Gibberellin insensitive (GAI), Gibberellin-induced protein (GIP) and GAMYB compared with their parents could account for heterosis in plant height by regulating the gibberellin metabolic pathway (Zhang et al. 2007). Circadian rhythm genes, including Circadian clock associated 1 (CCA1), Late elongated hypocotyl (LHY), and Timing of CAB 1 (TOC1), have been shown to participate in the regulation of heterotic traits in Arabidopsis thaliana, mainly by increasing starch synthesis (Ni et al. 2009). Ghd7, encoding a CCT domain protein, significantly improved the yield and adaptability of indicia rice (Xue et al. 2008). However, to date only a few genes have been shown to function in heterosis and thus our understanding of the mechanism of heterosis remains poor. Heterosis is mainly manifested in two ways. One is improved biomass, e.g. improved seed yield, which is frequently exploited in breeding. The other is higher environmental adaptability, such as resilience (Baranwal et al. 2012). Improved biomass results mainly from increased organ size and/or organ number (Schnable and Springer 2013). Therefore, the genes involved in regulating organ development, especially organ size, may potentially contribute to the formation of heterotic traits as shown by improved yield or biomass, although no evidence of direct relationship between organ size-associated genes and heterosis has been reported. Several such key genes/transcription factors have been discovered (Horvath et al. 2006, Schruff et al. 2006, Krizek 2009, Feng et al. 2011, Powell and Lenhard 2012). For instance, Auxin-regulated gene involved in organ size (ARGOS) was first detected in A. thaliana, and functional analysis has revealed that overexpression of ARGOS results in increased plant organ size (Hu et al. 2003). AINTEGUMENTA (ANT), an AP2-domain-containing transcription factor, appears to coordinate cell proliferation and lateral organ development (Elliott et al. 1996, Klucher et al. 1996, Krizek 1999, Mizukami and Fischer 2000, Dash and Malladi 2012). Overexpression of ANT increases leaf size and floral organ growth, whereas loss of ANT function reduces the sizes of leaves and floral organs (Elliott et al. 1996, Klucher et al. 1996, Krizek 1999). Other genes, e.g. ERBB3 binding protein 1 (EBP1), Target of rapamycin (TOR), KLUH (KLU), and GRF-INTERACTING FACTOR1/ANGUSTIFOLIA3 (GIF1/AN3) in A. thaliana (Horváth et al. 2006, Anastasiou et al. 2007, Deprost et al. 2007, Lee et al. 2009, Powell and Lenhard 2012), fw2.2 in tomato (Frary et al. 2000, Cong and Tanksley 2006, Nesbitt and Tanksley 2001) and Cell number regulator 1 (CNR1) in maize (Guo et al. 2010), have also been shown to play roles in regulating organ size, mainly by regulating cell number, not cell size. Therefore, heterosis should be understood from the perspective of organ development regulation in order to provide new information about its molecular basis. Larix, one of the most important afforestation and timber species in the world, has important economic and ecological value. Like some herbaceous plants, such as rice, maize and Brassica campestris, Larix hybrids also exhibit heterosis, and this phenomenon has been widely exploited in Larix breeding. However, owing in part to its long life cycle and complex genetic background, the intrinsic mechanism of heterosis in Larix remains largely unexplored. In a previous study, we have indicated that differential gene expression, especially nonadditive expression, is also closely associated with heterosis in Larix, and a series of nonadditive expression genes have been detected (Li A et al. 2012). In the current study, to further elucidate the function of these nonadditive expression genes in heterosis, a transcription factor, named LaAP2L1, belonging to the plant-specific APETALA2/ethylene-responsive element binding protein (AP2/EREBP) transcription factor family and possessing possible roles in regulating organ development, was selected to conduct gene architecture and transcription expression analysis. Subsequently, the function of LaAP2L1 was explored by overexpression in Arabidopsis, and a series of heterosis-like traits, e.g. enlarged organ size and improved seed yield, were observed. In addition, the cellular and molecular bases of heterosis-like traits in LaAP2L1 overexpression transgenic lines were analyzed. Transcriptomic sequencing to identify other members of the AP2/EREBP transcription factor family was also conducted, and the polygenetic characteristics of these transcription factors were discussed. Results LaAP2L1 is associated with heterosis in Larix hybrids In our previous study, the genome-wide transcript profiles of two Larix genotypes and their reciprocal hybrids were investigated using the microarray method to understand heterosis in Larix kaempferi at the molecular level. A series of genes/transcription factors that may function in heterosis were also identified (Supplementary Table S1). Among these candidate genes, a gene with an AP2 domain named LaAP2L1, a member of the AP2/EREBP transcription factor family, was considered. Microarray data revealed that the expression patterns of LaAP2L1 in the two Larix parental lines and their reciprocal hybrids were significantly different. The highest expression level of LaAP2L1 was detected in the heterotic hybrids, while the expression level of LaAP2L1 in the nonheterotic hybrids was close to the mid-parent value. To further identify the transcript expression of LaAP2L1, quantitative real-time RT-PCR (qRT-PCR) analysis was conducted. The results indicated that the expression pattern of LaAP2L1 identified experimentally was fully represented by the microarray data (Supplementary Fig. S1a). Quantitative expression analysis in individual plants of heterotic hybrids, nonheterotic hybrids, and their parents also verified that the highest expression level of LaAP2L1 was detected in the heterotic hybrids (Supplementary Fig. S1b). Expression analysis of the three other Larix cross-combinations also indicated that LaAP2L1 exhibited a higher expression level in the hybrids than in their two parental lines (Supplementary Fig. S1a). These results suggested that LaAP2L1 is involved in heterosis in Larix. Cloning and characterization of LaAP2L1 Transcriptome sequencing was performed in Larix heterotic hybrids by the Solexa high-throughput sequencing method to obtain more information about the nature of LaAP2L1 and to uncover all members of the AP2/EREBP transcription factor family in Larix. According to the sequence annotation information, five contigs originating from the transcript products of LaAP2L1 were discovered, and they consisted of a unigene (Fig. 1a). In total, 50 contigs with the AP2/EREBP domain were detected. Abundance analysis of these contigs further revealed that LaAP2L1 exhibited a high expression level in Larix heterotic hybrids (Supplementary Table S2). Based on the unigene sequence of LaAP2L1, a 1,590 bp cDNA containing initiation and terminator code was obtained by RT-PCR, and further confirmed by resequencing. This cDNA encodes a predicted protein containing 529 amino acids. Alignment analysis indicated that LaAP2L1 was highly homologous with the reported APETALA2-like transcription factors in Larix × marschlinsii, Picea abies, Picea glauca and Pinus thunbergii. Further analysis indicated that the two AP2 domains of LaAP2L1, especially the first domain, were highly conserved in almost all plants, confirming that LaAP2L1 is a member of the AP2 transcription factor subfamily (Fig. 1c). To elucidate the gene architecture of LaAP2L1, a DNA fragment of >4 kb containing the full coding region was cloned (accession number JN851868). This indicated that LaAP2L1 was composed of nine exons and eight introns (Fig. 1b). Fig. 1 View largeDownload slide Gene structure and sequence analysis of LaAP2L1. (a) Sequence assembly of LaAP2L1 cDNA by the ESTs/contigs detected by transcriptome sequencing. (b) Gene structure of LaAP2L1. Thick black boxes and thin connectors indicate the nine exons and eight introns of LaAP2L1, respectively. (c) Alignment of LaAP2L1 and other AP2-like transcription factors in several plant species (accession numbers: Arabidopsis thaliana, NP_195410; Glycine max, XP_003524643; Larix × marschlinsii, ABM26974; Medicago truncatula, XP_003611692; Picea abies, AAG32658; Pinus thunbergii, BAD16603; Populus trichocarpa, XP_002310715; Solanum lycopersicum, NP_001233886; Vitis vinifera, NP_001267881). The red, blue, and purple frames indicate the predicted nuclear localizing signal, the first AP2 domain, and the second AP2 domain, respectively. Fig. 1 View largeDownload slide Gene structure and sequence analysis of LaAP2L1. (a) Sequence assembly of LaAP2L1 cDNA by the ESTs/contigs detected by transcriptome sequencing. (b) Gene structure of LaAP2L1. Thick black boxes and thin connectors indicate the nine exons and eight introns of LaAP2L1, respectively. (c) Alignment of LaAP2L1 and other AP2-like transcription factors in several plant species (accession numbers: Arabidopsis thaliana, NP_195410; Glycine max, XP_003524643; Larix × marschlinsii, ABM26974; Medicago truncatula, XP_003611692; Picea abies, AAG32658; Pinus thunbergii, BAD16603; Populus trichocarpa, XP_002310715; Solanum lycopersicum, NP_001233886; Vitis vinifera, NP_001267881). The red, blue, and purple frames indicate the predicted nuclear localizing signal, the first AP2 domain, and the second AP2 domain, respectively. LaAP2L1 overexpression increases organ size LaAP2L1 overexpression vector driven by an enhanced CaMV 35S promoter was constructed and transformed to Arabidopsis to elucidate the function of LaAP2L1 and its possible function in heterosis in Larix. Twenty-four different 35S::LaAP2L1 transgenic lines exhibiting kanamycin resistance were obtained. RT-PCR analysis indicated that LaAP2L1 exhibited strong expression in these transgenic lines. However, five of these T1 transgenic lines were morphologically dwarfish and/or could not produce normal seeds (data not shown). These five lines were discarded in subsequent analyses. The other 19 transgenic lines showed almost similar phenotypes. Five of these lines were randomly selected and used for genetic analysis. The results indicated that all of these T3 transgenic lines were kanamycin-resistant, suggesting that LaAP2L1 was homozygous in these lines (Supplementary Fig. S2). Subsequent analysis was conducted using these T3 homozygous transgenic plants. Compared with the empty vector control lines, transgenic plants overexpressing LaAP2L1 had markedly enlarged leaves, stems, flowers, and siliques (Fig. 2a–d; Table 1). In brief, the width and length of the fifth rosette leaves was greater by approximately 80 and 150%, respectively, in 35::LaAP2L1 plants, and the fresh weight of the leaves was almost twice as great as that of vector control plants (Fig. 3a–c). The relative length of the 35::LaAP2L1 seedling roots 12 d after germination was about five times longer than that of the vector control plants (Fig. 2e). The siliques of the 35::LaAP2L1 plants were also longer than those of the control plants (Fig. 4d). Seed weight data indicated that the seed yield of each individual 35::LaAP2L1 plant was >200% greater than that of the vector control plants (Fig. 4e). In addition, significantly thicker and higher stems, larger flowers, and more sprays were observed in the 35::LaAP2L1 plants (Fig. 4a–c). These data demonstrate that overexpression of LaAP2L1 in Arabidopsis is sufficient to increase the size of aerial organs by enhancing organ growth and development. Fig. 2 View largeDownload slide Phenotypes of 35S::LaAP2L1 plants and CK (vector control) in different growth phases. (a) Growth characters of 2-week-old 35S::LaAP2L1 plants and vector controls. (b) Growth characters of 3-week-old 35S::LaAP2L1 plants and vector controls. (c) Growth characters of 5-week-old 35S::LaAP2L1 plants and vector controls. (d) Growth characters of 7-week-old 35S::LaAP2L1 plants and vector controls. (e) Roots of 12-day-old 35S::LaAP2L1 plants and vector controls grown in MS medium in culture bottles. Bar = 5.0 mm. Fig. 2 View largeDownload slide Phenotypes of 35S::LaAP2L1 plants and CK (vector control) in different growth phases. (a) Growth characters of 2-week-old 35S::LaAP2L1 plants and vector controls. (b) Growth characters of 3-week-old 35S::LaAP2L1 plants and vector controls. (c) Growth characters of 5-week-old 35S::LaAP2L1 plants and vector controls. (d) Growth characters of 7-week-old 35S::LaAP2L1 plants and vector controls. (e) Roots of 12-day-old 35S::LaAP2L1 plants and vector controls grown in MS medium in culture bottles. Bar = 5.0 mm. Fig. 3 View largeDownload slide Growth characters of leaves in 35S::LaAP2L1 plants and CK (vector controls). (a) Morphology of 5-week-old fifth leaves of 35S::LaAP2L1 plants (right) and vector controls (left). Bar = 5.0 mm. (b) Dimensions (mean ± SD) of 5-week-old fifth leaves (n = 10). (c) Leaf fresh weight (mean ± SD) of 6-week-old plants (n = 10). Fig. 3 View largeDownload slide Growth characters of leaves in 35S::LaAP2L1 plants and CK (vector controls). (a) Morphology of 5-week-old fifth leaves of 35S::LaAP2L1 plants (right) and vector controls (left). Bar = 5.0 mm. (b) Dimensions (mean ± SD) of 5-week-old fifth leaves (n = 10). (c) Leaf fresh weight (mean ± SD) of 6-week-old plants (n = 10). Fig. 4 View largeDownload slide Phenotype characterization of sprays (a), flowers (b), inflorescence stems, (c) and siliques (d) of 35S::LaAP2L1 plants (right or bottom) and vector controls (left or top). (e) Seed weight (mean ± SD) of 35S::LaAP2L1 plants and vector controls. Fig. 4 View largeDownload slide Phenotype characterization of sprays (a), flowers (b), inflorescence stems, (c) and siliques (d) of 35S::LaAP2L1 plants (right or bottom) and vector controls (left or top). (e) Seed weight (mean ± SD) of 35S::LaAP2L1 plants and vector controls. Table 1 Morphology of 35S::LaAP2L1 transgenic plants Variable  Vector control  35S::LaAP2L1  Flowering time (days)  24.7 ± 2.1 (n = 10)  33.2 ± 3.6 (n = 10)  Silique length (mm)  13.44 ± 0.96 (n = 40)  17.71 ± 1.21 (n = 40)  Flower length (mm)  2.61 ± 0.07 (n = 40)  3.40 ± 0.14 (n = 40)  Seeds/silique (grains)  54.5 ± 2.35 (n = 40)  64.5 ± 6.82 (n = 40)  Plant height (cm)  34.87 ± 1.46 (n = 10)  42.31 ± 3.68 (n = 10)  Variable  Vector control  35S::LaAP2L1  Flowering time (days)  24.7 ± 2.1 (n = 10)  33.2 ± 3.6 (n = 10)  Silique length (mm)  13.44 ± 0.96 (n = 40)  17.71 ± 1.21 (n = 40)  Flower length (mm)  2.61 ± 0.07 (n = 40)  3.40 ± 0.14 (n = 40)  Seeds/silique (grains)  54.5 ± 2.35 (n = 40)  64.5 ± 6.82 (n = 40)  Plant height (cm)  34.87 ± 1.46 (n = 10)  42.31 ± 3.68 (n = 10)  Values are mean ± SD; n indicates the numbers of individual plants at the time of flowering analysis, siliques at the time of silique length and seed count analysis, and flowers at the time of flower length analysis. View Large Larger organ size in 35S::LaAP2L1 plants results from increased cell number The number and size of cells are two key factors controlling the final size of each organ or tissue in plants as well as in other organisms. Consequently, to understand the increased organ size of transgenic plants with overexpressed LaAP2L1 at the cytological level, epidermal cells of the fully expanded fifth leaves were visualized using a microscope. The data revealed that the average number of cells per fixed leaf unit area (0.54 mm2) was not different between the transgenic plants and vector control plants (Fig. 5a, b). Transverse and longitudinal sections of the fifth leaves of 35S::LaAP2L1 plants and vector control plants were further compared (Fig. 5c, d). The results showed that the leaf blades of these two groups of plants contained the same number of cell layers, and the dimensions of palisade cells in 35S::LaAP2L1 plants were also nearly the same as those in vector control plants. However, obvious differences both in palisade cell and mesophyll cell number were detected in the direction of leaf length and leaf width (Fig. 5d). The fifth leaf of 35S::LaAP2L1 plants appeared to contain approximately 100% more cells than that of the vector control plants. These data therefore indicate that the altered organ size of LaAP2L1 transgenic plants was mainly due to changes in cell number, not cell size. Fig. 5 View largeDownload slide Anatomical analysis of fully expanded fifth leaves of 35S::LaAP2L1plants and vector controls. (a) Epidermal pavement cells of the fifth leaves of 35S::LaAP2L1plants (right) and vector controls (left). Bars = 50 μm. (b) Number of epidermal pavement cells (mean ± SD) in an area of 0.54 mm2 of fifth leaves (n = 10). (c) Transverse sections of the fifth leaf blades of 35S::LaAP2L1 plants (bottom) and vector controls (top). Bars = 100 μm. (d) Total number of mesophyll cells (mean ± SD) in leaf length and leaf width (n = 10). Fig. 5 View largeDownload slide Anatomical analysis of fully expanded fifth leaves of 35S::LaAP2L1plants and vector controls. (a) Epidermal pavement cells of the fifth leaves of 35S::LaAP2L1plants (right) and vector controls (left). Bars = 50 μm. (b) Number of epidermal pavement cells (mean ± SD) in an area of 0.54 mm2 of fifth leaves (n = 10). (c) Transverse sections of the fifth leaf blades of 35S::LaAP2L1 plants (bottom) and vector controls (top). Bars = 100 μm. (d) Total number of mesophyll cells (mean ± SD) in leaf length and leaf width (n = 10). LaAP2L1 influences cell proliferation and meristematic competence of tissues The number of cells in each organ is associated with cell proliferation. The growth characteristics of the roots, leaves, and floral organs of 35S::LaAP2L1 plants were analyzed. Under the same conditions, the seedling root growth of 35S::LaAP2L1 plants differed from that of the vector control plants at 6 d after germination. At 12 d after germination, the relative root length of 35S::LaAP2L1 plants was about five times greater than that of the vector control plants, showing an accelerated growth trend (Fig. 2e). Likewise, detailed data on the growth kinetics of the fifth rosette leaves demonstrated that the leaves of the overexpressed LaAP2L1 transgenic plants grew faster than those of the vector control plants (Fig. 6a). Moreover, the growth duration of the leaves of 35S::LaAP2L1 plants was significantly prolonged. Growth of the leaves of 35S::LaAP2L1 plants could still be detected after 35 d; however, the leaves of the vector control plants ceased to elongate 32 d after germination. Additionally, flowering time was delayed by 8–9 d in 35S::LaAP2L1 plants compared with the vector control plants (Table 1). Fig. 6 View largeDownload slide Effect of LaAP2L1 on plant growth and cell meristematic competence. (a) Growth kinetics of the fifth leaves (mean ± SD) in vector controls and 35S::LaAP2L1 plants (n = 10). (b) Growth characters, especially root growth, of expanded leaves from a 35S::LaAP2L1 plant (bottom) and vector control (top) cultured on hormone-free B5 medium 14 d after excision. Bar = 5.0 mm. (c) Callus growth in leaf explants of vector control (top) and 35S::LaAP2L1 (bottom). Photographs were taken 42 d after excision. Bar = 3.0 mm. (d) Expression levels (mean ± SD) of ARGOS, ANT, EBP1, and CycD3;1 in young and fully expanded rosette leaves of vector controls and 35S::LaAP2L1 transgenic plants. 1, 2-week-old leaves; 2, 4-week-old leaves; 3, 6-week-old leaves. C1, C2,C3, individual plants of CK (vector control); T1, T2, T3, individual plants of 35S::LaAP2L1 transgenic lines. Fig. 6 View largeDownload slide Effect of LaAP2L1 on plant growth and cell meristematic competence. (a) Growth kinetics of the fifth leaves (mean ± SD) in vector controls and 35S::LaAP2L1 plants (n = 10). (b) Growth characters, especially root growth, of expanded leaves from a 35S::LaAP2L1 plant (bottom) and vector control (top) cultured on hormone-free B5 medium 14 d after excision. Bar = 5.0 mm. (c) Callus growth in leaf explants of vector control (top) and 35S::LaAP2L1 (bottom). Photographs were taken 42 d after excision. Bar = 3.0 mm. (d) Expression levels (mean ± SD) of ARGOS, ANT, EBP1, and CycD3;1 in young and fully expanded rosette leaves of vector controls and 35S::LaAP2L1 transgenic plants. 1, 2-week-old leaves; 2, 4-week-old leaves; 3, 6-week-old leaves. C1, C2,C3, individual plants of CK (vector control); T1, T2, T3, individual plants of 35S::LaAP2L1 transgenic lines. To further verify the function of LaAP2L1 in cell proliferation, the meristematic competence of leaf explants in 35S::LaAP2L1 transgenic lines and vector controls was examined. When 2-week-old leaves were cultured in hormone-free B5 medium, stronger roots were produced by leaf explants of 35S::LaAP2L1 plants after 14 d. On average, the roots were almost nine times longer than those of vector control leaf explants (Fig. 6b). In addition, vector control calli ceased to grow and enlarge after 4 weeks when leaf explants were cultured on a callus induction medium containing 4.5 μM 2,4-Dichlorophenoxy acetic acid (2,4-D) and 0.5 μM kinetin, although both groups of the leaf explants produced visible calli at 7 d. By contrast, the calli of the LaAP2L1 transgenic plants continued to grow even after 40 d, thereby producing calli that were twice as large as those of the vector control explants (Fig. 6c). Expression pattern of several organ size-associated genes in 35S::LaAP2L1 plants Several genes associated with the regulation of organ size and cell proliferation in plants have been identified in previous studies. Here, to further understand the molecular mechanism by which LaAP2L1 regulates cell proliferation and its possible relationship with known organ size-associated genes, the transcript expression of four representative genes associated with the positive regulation of organ size in plants, ANT, ARGOS, EBP1, and CycD3;1, were quantitatively analyzed. Compared with the vector controls, expression of ANT, EBP1, and CycD3;1, all of which can positively regulate organ size by increasing cell number, was significantly enhanced in 35S::LaAP2L1 plants. In fully differentiated 6-week-old leaves, only weak expression of these genes was detected in the vector control plants, while strong expression was observed in LaAP2L1-overexpressing transgenic plants (Fig. 6d). However, expression of ARGOS was inhibited in these transgenic lines (Fig. 6d). Identification and polygenetic analysis of AP2/EREBP transcription factors in Larix To further elucidate the role of LaAP2L1 and other members of the AP2/EREBP transcription factor family, 50 contigs, representing possible members of the AP2/EREBP transcription factor family in Larix, were identified based on transcriptomic data (Supplementary Table S2). Sequence alignment and polygenetic analysis indicated that all of these members contained a highly conserved 57-amino acid AP2/ERF DNA-binding domain (Supplementary Fig. S3b) and could be divided mainly into five branches based on evolutionary distance (Supplementary Fig. S3a). LaAP2L1 (contig C803980.5) was located in a small branch composed of only three members, while the largest branch contained 37 members (Supplementary Fig. S3a). In addition, to further uncover the evolutionary position of LaAP2L1 and its homologous genes, a polygenetic tree of AP2/EREBP transcription factors from four representative model plants, including A. thaliana, Oryza sativa, Populus trichocarpa, and Vitis vinifera as well as the members of AP2/EREBP transcription factors detected in Larix, was constructed, and five main evolutionary branches were observed (Fig. 7). Similarly, LaAP2L1 was found in a small branch, and showed a close evolutionary distance to some genes involved in regulating organ development and growth of plants (Fig. 7; Supplementary Table S3). The other members of the AP2/EREBP transcription factor family in Larix were unequally distributed in different branches. Most of these members and other members from the four model plants were found in the largest branch, indicating that these members of the AP2/EREBP transcription factor family were not closely related to LaAP2L1 (Fig. 7). However, the data indicated that most of the AP2/EREBP transcription factors from Larix in each branch or subbranch showed a closer genetic relationship with the corresponding members of P. trichocarpa or V. vinifera when compared with those of A. thaliana and O. sativa. This observation suggests that the AP2/EREBP transcription factors in Larix are more conserved in dicotyledonous woody plants. Fig. 7 View largeDownload slide Phylogenic analysis of AP2/EREBP transcription factors from L. kaempferi and four other representative plants: A. thaliana, O. sativa, P. trichocarpa, and V. vinifera. The sequences of AP2/EREBP transcription factors from A. thaliana, O. sativa, P. trichocarpa, and V. vinifera were downloaded from the GenBank database. The red triangle shows the position of LaAP2L1. The solid red arc indicates the possible members of AP2-like subfamily. Arrows indicate reported members of the AP2/EREBP transcription factor family. Green numbers indicate the possible five major branches. Fig. 7 View largeDownload slide Phylogenic analysis of AP2/EREBP transcription factors from L. kaempferi and four other representative plants: A. thaliana, O. sativa, P. trichocarpa, and V. vinifera. The sequences of AP2/EREBP transcription factors from A. thaliana, O. sativa, P. trichocarpa, and V. vinifera were downloaded from the GenBank database. The red triangle shows the position of LaAP2L1. The solid red arc indicates the possible members of AP2-like subfamily. Arrows indicate reported members of the AP2/EREBP transcription factor family. Green numbers indicate the possible five major branches. Discussion AP2/EREBP transcription factors, making up one of the most important families of transcription factors in plants, are characterized by the presence of a plant-specific AP2/EREBP domain, a conserved region of ∼60 amino acids involved in DNA binding (Weigel 1995, Okamuro et al. 1997, Dietz et al. 2010). The first member of the AP2/EREBP transcription factor family was cloned in A. thaliana and was shown to play roles in the establishment of the floral meristem and in floral organ identity (Jofuku et al. 1994, Wollmann et al. 2010, Yant et al. 2010, Xu et al. 2011). Since then, an increasing number of transcription factors from this family have been identified. Pleiotropic functions involved in the regulation of the developmental processes as well as in response to biotic and abiotic stresses have been detected (Klucher et al. 1996, Boutilier et al. 2002, Dubouzet et al. 2003, Gutterson and Reuber 2007, Fu and Xue 2010, Mizoi et al. 2012, Zhang et al. 2012). According to the number of conserved DNA-binding domains, AP2/EREBP transcription factors can be divided in two subfamilies: the AP2-like subfamily and the ERF (ethylene response factor)-like subfamily (Kim et al. 2006). Studies have identified that AP2-like transcription factors contain two AP2/EREBP domains that mainly function in the regulation of plant development (Klucher et al. 1996, Boutilier et al. 2002, Fu and Xue 2010, Licausi et al. 2010, Zhou et al. 2012), while the ERF-like transcription factors containing only one AP2/EREBP domain are involved in plant response to phytohormones and various environmental stresses (Dubouzet et al. 2003, Gutterson and Reuber 2007, Srivasta et al. 2010, Yaish et al. 2010, Xiong et al. 2013). Two AP2/EREBP domains were observed in LaAP2L1 (Fig. 1c), indicating that LaAP2L1 was a member of the AP2-like transcription factor subfamily and implying its potential roles in regulation of development in Larix. Further sequence analysis indicated that LaAP2L1 was more highly homologous to LmAP2L1, an AP-like transcription factor detected in Larix × marschlinsii, as well as PaAP2L1 and PtAP2L1 from P. abies and P. thunbergii, respectively (Nilsson et al. 2007, Guillaumot et al. 2008), suggesting that LaAP2L1 is highly conserved in Pinaceae. LmAP2L1, with the highest homology to LaAP2L1, was investigated during somatic embryogenesis and germination of Larix × marschlinsii. The results indicated that LmAP2L1 was expressed only during late somatic embryogenesis (Guillaumot et al. 2008). However, the function of LmAP2L1 remains unknown. Similarly, PaAP2L1 has also exhibited distinct expression patterns during plant development but no additional information regarding its function has been reported. Consequently, although several explorations have indicated the potential role of AP2L1 in the developmental processes of Pinaceae plants, our knowledge about the function and regulation of these transcription factors is still very poor. LaAP2L1 was first identified during our gene expression profiling analysis performed to investigate the molecular basis of heterosis in Larix hybrids (Li A et al. 2012). The results from the expression pattern analysis and experimental identification both indicated that LaAP2L1 is involved in the formation of heterosis in Larix hybrids (Supplementary Fig. S1; Supplementary Table S1). However, the roles of LaAP2L1 in the formation of heterosis or organ development remain to be elucidated. Here, the functional analysis indicated that overexpression of LaAP2L1 in A. thaliana can significantly and positively accelerate the growth and development of almost all 35S::LaAP2L1 plants, resulting in enlarged organ size, i.e. larger leaves, floral organs, and siliques and thicker stems (Figs 2–4). In addition, the growth duration of these transgenic plants, especially vegetative growth, was also prolonged. These results demonstrated that overexpression of LaAP2L1 could lead to heterosis-like traits in transgenic plants, further suggesting that LaAP2L1 plays important roles in the formation of heterosis, as observed in Larix heterotic hybrids. It is important to understand the mechanism by which LaAP2L1 can cause transgenic Arabidopsis to exhibit heterosis-like traits. The results indicated that overexpression of LaAP2L1 could significantly enhance cell proliferation, resulting in significantly larger organs in 35S::LaAP2L1 plants than in empty vector control plants in the same growth phase, but cell size was not affected (Fig. 5). In addition, stronger meristematic competence of leaf explants and significantly prolonged vegetative growth duration, as well as delayed flowering, were observed in 35S::LaAP2L1 plants. These observations indicated that the increased cell number and prolonged growth duration were critical factors and important cytological factors resulting in heterosis-like traits by overexpression of LaAP2L1, although the relationship between cell proliferation and growth duration requires further elucidation. Regardless of the complex genetic foundation and diverse molecular mechanism of heterosis, other studies have indicated that the phenomenon of growth vigor, one of the most important phenotypic characteristics of heterosis, as indicated by enlarged organ size, increased biomass, and improved seed production, could be mimicked by ectopically expressing some organ size-associated genes involved in cell proliferation (Gonzalez et al. 2009, Krizek 2009, Guo et al. 2010, Cheng et al. 2013). Consequently, it is highly possible that LaAP2L1 plays a role in the development of growth vigor traits by enhancing the competence of cell proliferation in Larix heterotic hybrids. The transcript expression of several genes involved in organ size, e.g. ARGOS, ANT, EBP1, and CycD3;1, by regulating cell proliferation was quantitatively analyzed to understand the molecular basis of the increased cell number in transgenic Arabidopsis with overexpressed LaAP2L1. ANT and EBP1, both regulated by auxin, have been shown to play critical roles in enhancing cell proliferation and to upregulate CycD3;1 (Krizek 1999, Mizukami and Fischer 2000, Horváth et al. 2006, Powell and Lenhard 2012). However, the pathways involved in regulating cell proliferation may be different. In the ANT pathway, auxin stimulates the expression of ARGOS, which acts upstream of ANT and positively regulates the expression of ANT, which in turn maintains CycD3;1 expression (Krizek 1999, Mizukami and Fischer 2000, Hu et al. 2003, Powell and Lenhard 2012). In the EBP1 pathway, no relationship between ARGOS and EBP1 or other novel transcription factors regulated by auxin has been reported, while EBP1 has also been shown to upregulate CycD3;1 (Horváth et al. 2006). Interestingly, the present study clearly showed that the expression of both ANT and EBP1 was dramatically upregulated in transgenic lines overexpressing LaAP2L1, suggesting that LaAP2L1 acts as an upstream positive regulator of both ANT and EBP1 (Fig. 6d). However, the expression of ARGOS was significantly inhibited compared with the empty vector controls, indicating that ARGOS may underlie a negative feedback pathway regulated by LaAP2L1. Accordingly, a proposed model of the role of LaAP2L1 in cell proliferation was sketched based on genetic factors and pathways previously reported to be involved in regulating plant organ size (Fig. 8). In this model, LaAP2L1 positively upregulates the ANT and EBP1 pathways, both of which take part in the regulation of plant organ size by enhancing cell proliferation. Thus, overexpression of LaAP2L1 leads to heterosis-like traits such as enlarged organ size and improved biomass. Fig. 8 View large Download slide Proposed model of the role of LaAP2L1 in cell proliferation of 35S::LaAP2L1 transgenic Arabidopsis. Black arrows indicate the reported pathways positively regulating cell proliferation. Blue arrows indicate that LaAP2L1 positively regulates ANT and EBP1, which has been identified in the present study. indicates that the expression of AROGS is inhibited by LaAP2L1. The virtual blue arrow indicates that the relationship of auxin and LaAP2L1 need further confirm. Fig. 8 View large Download slide Proposed model of the role of LaAP2L1 in cell proliferation of 35S::LaAP2L1 transgenic Arabidopsis. Black arrows indicate the reported pathways positively regulating cell proliferation. Blue arrows indicate that LaAP2L1 positively regulates ANT and EBP1, which has been identified in the present study. indicates that the expression of AROGS is inhibited by LaAP2L1. The virtual blue arrow indicates that the relationship of auxin and LaAP2L1 need further confirm. In Arabidopsis, over 140 members of the AP2/EREBP transcription factor family have been reported, and a similar number of AP2/EREBP transcription factors in rice were also detected (Sakuma et al. 2002, Nakano et al. 2006). Consequently, to further understand the characteristics of LaAP2L1 and other AP2/EREBP transcription factors in Larix, transcriptomic analysis was carried out, and 50 AP2/EREBP transcription factors were detected in Larix heterotic hybrids (Supplementary Table S2). This was far less than the number of transcription factors detected in Arabidopsis and rice, implying the evolutionary conservation of the AP2/EREBP transcription factor family in Larix. Certainly, the sequencing depth or differences in temporal and spatial expression of some AP2/EREBP transcription factors may affect the precise numbers. Nevertheless, the present study is the first large-scale analysis of AP2/EREBP transcription factors in Larix as well as in gymnosperms. Consistent with the pleiotropic function and abundant polymorphism of AP2/EREBP transcription factors detected in other plant species, polygenetic analysis indicated that the 50 AP2/EREBP transcription factors identified in Larix are also highly evolved, and five primary branches, each representing one possible functional class group, were observed (Supplementary Fig. S3a). LaAP2L1 was located in a relatively small branch that comprised only three members, and the largest branch contained about 80% of the members (Supplementary Fig. S3a). It suggested that the evolutionary rate of each AP2/EREBP transcription factors in different branches has varied significantly. Similar results were detected in the further polygenetic analysis of AP2/EREBP transcription factors from four representative sequencing model plants (Fig. 7). These results further verified that LaAP2L1 is situated in a relatively conserved branch and shows a closer genetic relationship with ANT, AINTEGUMENTA-like (AIL), BABY BOOM (BBM), and WRINKLED1(WRI1) in Arabidopsis, all of which have been shown to play important roles in the regulation of plant development (Elliott et al. 1996, Klucher et al. 1996, Krizek 1999, Boutilier et al. 2002, Cernac et al. 2004, 2006, Passarinho et al. 2008, Krizek and Eaddy 2012, Mudunkothge and Krizek 2012, Rigal et al. 2012), implying that LaAP2L1 is evolutionarily conserved and may mainly function in the regulation of plant development. The present data also showed that overexpression of a single gene, LaAP2L1, is sufficient to significantly increase organ size, biomass, and especially the seed yield of 35S::LaAP2L1 transgenic Arabidopsis. For example, the fresh weight of all the leaves was almost twice that of the vector control plants, and the seed yield of 35::LaAP2L1 individual plants was over 200% greater than that of vector control plants (Figs 3c, 4e). Overexpression of LaAP2L1 in cauliflower (Brassica oleracea L. var. botrytis) also exhibited remarkably enlarged leaves and a more developed root system, especially the lateral roots (data not shown). These results demonstrated that LaAP2L1 plays a critical role in the regulation of plant organ development and highlight the potential significance of LaAP2L1 in breeding crops, forage plants, and energy plants with much higher yield or biomass using genetic engineering methods. Materials and methods Plant materials and growth conditions Larix kaempferi parental lines and their corresponding hybrids were obtained from the Chinese Academy of Forestry, Beijing, China. The seeds of all materials were sown in Liaoning province, China, in 1990, and the traits, especially tree height and diameter at breast height, were observed continuously. Arabidopsis thaliana ecotype Landsberg erecta (Ler) was used in this study. The seeds (wild-type and transgenic plants) were sterilized in 2% NaClO with 0.02% Triton X-200 for 15 min and then washed five times with sterilized water. Then, the seeds were planted on Murashige & Skoog (MS) medium and vernalized in the darkness at 4°C for 3 d. The plates were transferred to a culture room at 22°C with a 16 h/8 h light/dark photoperiod. At 10 d after germination, the seedlings were transferred to soil and placed in a growth chamber at 22°C with 40–65% relative humidity and a 16 h/8 h light/dark photoperiod. LaAP2L1 sequence cloning and analysis Total RNA was isolated from L. kaempferi leaves using the hexadecyl trimethyl ammonium bromide (CTAB) method. Part of the full-length cDNA sequence of LaAP2L1 was initially identified in the gene expression profile analyzed with an Arabidopsis 70-mer oligo microarray (CapitalBio Corp., Beijing, China). The microarray was used to examine genes expressed differentially between Larix hybrids and their parents. Subsequently, according to the transcriptomic sequencing data of the Larix heterotic hybrids, all expressed sequence tags (ESTs) and contigs of LaAP2L1 were detected, and a unigene containing the full coding sequences of LaAP2L1 was obtained. Primers were designed based on the unigene sequence to amplify the cDNA and DNA sequences of LaAP2L1 (forward, 5′-ATGTGGGATCTGAATCATATGC-3′; reverse, 5′-TCAACCTAGAGACAAATTCAATG-3′). The corresponding PCR products were cloned using the T-A cloning method. The gene architecture of LaAP2L1 was explored by comparative analysis of the DNA and cDNA sequences. Further sequence alignment analysis was carried out using the Blast tool and ClustalW software (Thompson et al., 1994), respectively. To further evaluate the expression pattern of LaAP2L1, total RNA from other three Larix cross combinations and several individual plants were also isolated and reverse-transcribed. Quantitative RT-PCR (qRT-PCR) analysis was conducted in triplicate with iQ SYBR Green Supermix (Roche, Basel, Switzerland) according to the manufacturer’s instructions. All the assays were run on the iCycler iQ5 system (Bio-Rad, California, USA), and the transcript level of LaAP2L1 in each sample was measured by the (Ct) 2−Δ(ΔCt) method. Transfer DNA constructs and plant transformation The full-length cDNA of LaAP2L1 with restriction sites was amplified using the following primers: LaAP2L1 forward, 5′-GTCTCTAGAATGTGGGATCTGAATCATATGC-3′; LaAP2L1 reverse, 5′-GATGAGCTCTCAACCTAGAGACAAATTCAATG-3′. The underlined sequences, TCTAGA and GAGCTC, represent the restriction sites of XbaI and SacI, respectively. The full-length open reading frame of LaAP2L1 was inserted into the pBI121 vector, and the construct 35S::LaAP2L1 was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation. Arabidopsis plants were then transformed using this Agrobacterium strain by vacuum infiltration. T1 seeds of the transgenic plants were germinated on MS medium containing 50 mg/L of kanamycin to select the transformants. After regeneration under kanamycin selection, the transgenic lines were further identified by genomic PCR using unique primers. T3 homozygous plants with the LaAP2L1 insertion were then used for detailed analysis. Phenotypic data collection and analysis Phenotypic data on transgenic Arabidopsis plants and vector controls were collected from individual plants grown in the culture room. The length and width of the fifth leaf were measured every 3 d after germination. After the siliques ceased growing, silique length and the number and weight of seeds were determined from the same plants as those measured previously. Plant height was measured from the ground to the top of the plant. The flowering time of vector controls and 35S::LaAP2L1 plants was observed in at least 10 plants. Cell size and number Fully expanded fifth leaves (35 d after germination) were collected for further analysis to determine the size and number of cells of 35S::LaAP2L1 plants and vector control plants. To examine epidermal cells, a 1-cm2 section was cut from the midpoint of the fifth leaf and fixed in 70% ethanol. The epidermal cells were removed, mounted on a glass slide, and covered with a glass coverslip. Images were captured using a Zeiss LSM510 microscope (Zeiss, Jena, Germany). To compare the size and number of cells in thin sections, specimens of fully expanded fifth leaves were fixed in formalin–-acetic-acid–-alcohol (FAA) fluid, containing 5% of formalin, 5% glacial acetic acid, and 63% ethanol. The specimens were dehydrated with an ethanol series, infiltrated, and embedded using a Leica historesin embedding kit (Leica, Wetzlar, Germany) according to the manufacturer’s instructions. The thin section were stained with 0.05% toluidine blue O. Cell size and cell number were measured in the middle region of the fifth leaves in transverse sections or ∼1 mm from mid-vein in longitudinal sections. Leaf explant culture To examine cell competence, rosette leaves of 2-week-old 35S::LaAP2L1 transgenic plants and vector controls were excised and cultured on hormone-free B5 medium under a 16 h light/8 h dark photoperiod at 22°C. At the same time, other explants were cultured on B5 medium containing 4.5 μM 2,4-D and 0.5 μM kinetin at 22°C in a 16 h light/8 h dark photoperiod. qRT-PCR expression analysis Leaf tissue samples of different stages were ground and total RNA was isolated using Trizol reagent (Invitrogen, USA) following the manufacturer’s protocol. Each RNA sample was treated with DNase using the DNA-free Kit (TaKaRa, Japan). First-strand cDNA was made from DNase-treated RNA from each sample using the M-MLV Kit (Promega, USA). One hundred nanograms of first-strand cDNA was used for real-time PCR analysis. Real-time qPCR was performed with iQ SYBR Green Supermix (Roche, Basel, Switzerland) using the iCycler iQ5 system (Bio-Rad, California, USA) and gene-specific primers (Supplementary Table S4). Three independent biological replicates were analyzed per sample. Expression values were normalized using the housekeeping gene actin and relative expression levels of each sample were determined using the (Ct) 2−Δ(ΔCt) method. Transcriptomic sequencing and polygenetic analysis For transcriptomic sequencing, total RNA was isolated from fresh leaves of Larix heterotic hybrids using the CTAB method and then treated with RNAase-free DNase I (TaKaRa, Japan) for 45 min according to the manufacturer’s protocols. Double-stranded cDNA was synthesized using the cDNA Synthesis System (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Sequencing libraries for the Illumina GA platform were constructed by shearing the enriched cDNA, and then the cDNA library products were sequenced using the Illumina Genome Analyzer (Beijing Genomics Institute, China). For transcriptome reconstruction, the de novo assembly of RNA-Seq reads was generated by Oases/Velvet (Zerbino and Velvet 2008). Functional assignment and classification of transcripts was performed with a sequence similarity search using BLAST (E-value cut-off of 1e−5) against the eggNOG database, the Kyoto Encyclopedia of Genes and Genomes (KEGG) reference database, and the nonredundant GenBank CDS database. To classify the transcripts by protein domain and obtain the Gene Ontology (GO) annotations, we encoded the transcript sequences to a local InterProScan (v4.8) (Mulder and Apweiler 2007) pipeline against the Pfam database (Finn et al. 2008). The protein sequences of AP2/EREBP transcription factors from A. thaliana, O. sativa, P. trichocarpa, and V. vinifera were downloaded from the GenBank database, and the protein sequences of AP2/EREBP transcription factors from L. kaempfer i were obtained from our previous Larix transcriptomic sequencing. Some of the sequences for the AP2/EREBP transcription factors were full-length, while others were not. However, all sequences contained the conserved AP2 domain. Then, all the AP2/EREBP transcription factors detected in Larix and four other representative plants were aligned using ClustalW (Thompson et al. 1994). Phylogenetic trees were constructed using the neighbor-joining method and diagrams of phylogenetic trees were drawn with MEGA5 (http://www.megasoftware.net/). The parameters were set as follows: multiple alignment gap opening penalty, 10; gap extension penalty, 0.2. Funding This work was performed with financial support from the National Key Basic Research Program in China (No. 2009CB119105) and the National Natural Science Foundation of China and Tianjin (No. 31100234 and No. 10JCZDJC17900). Disclosures The authors have no conflict of interest to declare. Acknowledgments We thank Dr. Liwang Qi of the Chinese Academy of Forestry, Beijing, China, for kindly providing the Larix kaempferi parental lines (Larix13 and Larix82) and their reciprocal hybrids (Larix13 × Larix82-21 and Larix82 × Larix13-6). We greatly thank Dr. Sha Jiang at the College of Life Sciences, Nankai University, Tianjin, China, for helping to conduct the cytological analysis. Abbreviations Abbreviations AP2/EREBP APETALA2/ ethylene-responsive element binding protein ERF ethylene response factor CTAB hexadecyl trimethyl ammonium bromide EST expressed sequence tag qRT-PCR quantitative RT-PCR 2,4-D 2,4-Dichlorophenoxy acetic acid MS medium Murashige & Skoog medium KT kinetin FAA formalin-acetic-acid-alcohol KEGG Kyoto Encyclopedia of Genes and Genomes References Anastasiou E,  Kenz S,  Gerstung M,  MacLean D,  Timmer J,  Fleck C, et al.  Control of plant organ size by KLUH/CYP78A5-dependent intercellular signaling,  Dev. Cell ,  2007, vol.  13 (pg.  843- 856) Google Scholar CrossRef Search ADS PubMed  Baranwal VK,  Mikkilineni V,  Zehr UB,  Tyagi AK,  Kapoor S.  Heterosis: emerging ideas about hybrid vigour,  J. Exp. Bot. ,  2012, vol.  63 (pg.  6309- 6314) Google Scholar CrossRef Search ADS PubMed  Birchler JA,  Auger DL,  Riddle NC.  In search of the molecular basis of heterosis,  Plant Cell ,  2003, vol.  15 (pg.  2236- 2239) Google Scholar CrossRef Search ADS PubMed  Birchler JA,  Yao H,  Chudalayandi S.  Unraveling the genetic basis of hybrid vigor,  Proc. Natl Acad. Sci. U. S. A. ,  2006, vol.  103 (pg.  12957- 12958) Google Scholar CrossRef Search ADS PubMed  Boutilier K,  Offringa R,  Sharma VK,  Kieft H,  Ouellet T,  Zhang L, et al.  Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth,  Plant Cell ,  2002, vol.  14 (pg.  1737- 1749) Google Scholar CrossRef Search ADS PubMed  Cernac A,  Benning C.  WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis,  Plant J. ,  2004, vol.  40 (pg.  575- 785) Google Scholar CrossRef Search ADS PubMed  Cernac A,  Andre C,  Hoffmann-Benning S,  Benning C.  WRI1 is required for seed germination and seedling establishment,  Plant Physiol. ,  2006, vol.  141 (pg.  745- 757) Google Scholar CrossRef Search ADS PubMed  Cheng Y,  Cao L,  Wang S,  Li Y,  Shi X,  Liu H, et al.  Down-regulation of multiple CDK inhibitor ICK/KRP genes up-regulates E2F pathway and increases cell proliferation, organ and seed sizes in Arabidopsis,  Plant J. ,  2013  doi: 10.1111/tpj.12228 Cong B,  Tanksley SD.  FW2.2 and cell cycle control in developing tomato fruit: a possible example of gene co-option in the evolution of a novel organ,  Plant Mol. Biol. ,  2006, vol.  62 (pg.  867- 880) Google Scholar CrossRef Search ADS PubMed  Dash M,  Malladi A.  The AINTEGUMENTA genes, MdANT1 and MdANT2, are associated with the regulation of cell production during fruit growth in apple (Malus × domestica Borkh.),  BMC Plant Biol. ,  2012, vol.  12 pg.  98  Google Scholar CrossRef Search ADS PubMed  Deprost D,  Yao L,  Sormani R,  Moreau M,  Leterreux G,  Nicolaï M, et al.  The Arabidopsis TOR kinase links plant growth, yield, stress resistance and mRNA translation,  EMBO Rep. ,  2007, vol.  8 (pg.  864- 870) Google Scholar CrossRef Search ADS PubMed  Dietz KJ,  Vogel MO,  Viehhauser A.  AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling,  Protoplasma ,  2010, vol.  245 (pg.  3- 14) Google Scholar CrossRef Search ADS PubMed  Dubouzet JG,  Sakuma Y,  Ito Y,  Kasuga M,  Dubouzet EG,  Miura S, et al.  OsDREB genes in rice, Oryza sativa L, encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression,  Plant J. ,  2003, vol.  33 (pg.  751- 763) Google Scholar CrossRef Search ADS PubMed  Duvick DN.  Coors JG,  Pandey S.  Heterosis: feeding people and protecting natural resources,  The Genetics and Exploitation of Heterosis in Crops ,  1997 WI ASA, Madison(pg.  19- 29) Elliott RC,  Betzner AS,  Huttner E,  Oakes MP,  Tucker WQ,  Gerentes D, et al.  AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth,  Plant Cell ,  1996, vol.  8 (pg.  155- 168) Google Scholar CrossRef Search ADS PubMed  Feng G,  Qin Z,  Yan J,  Zhang X,  Hu Y.  Arabidopsis ORGAN SIZE RELATED1 regulates organ growth and final organ size in orchestration with ARGOS and ARL,  New Phytol ,  2011, vol.  191 (pg.  635- 646) Google Scholar CrossRef Search ADS PubMed  Finn RD,  Tate J,  Mistry J,  Coggill PC,  Sammut SJ,  Hotz HR, et al.  The pfam protein families database,  Nucleic Acids Res. ,  2008, vol.  36 (pg.  281- 288) Google Scholar CrossRef Search ADS   Flint-Garcia SA,  Buckler ES,  Tiffin P,  Ersoz E,  Springer NM.  Heterosis is prevalent for multiple traits in diverse maize germplasm,  PloS One ,  2009, vol.  4 pg.  e7433  Google Scholar CrossRef Search ADS PubMed  Frary A,  Nesbitt TC,  Grandillo S,  Knaap E,  Cong B,  Liu J, et al.  fw2.2: a quantitative trait locus key to the evolution of tomato fruit size,  Science ,  2000, vol.  289 (pg.  85- 88) Google Scholar CrossRef Search ADS PubMed  Fu FF,  Xue HW.  Coexpression analysis identifies rice starch regulator1, a rice AP2/EREBP family transcription factor, as a novel rice starch biosynthesis regulator,  Plant Physiol. ,  2010, vol.  154 (pg.  927- 938) Google Scholar CrossRef Search ADS PubMed  Gonzalez N,  Beemster GT,  Inzé D.  David and Goliath: what can the tiny weed Arabidopsis teach us to improve biomass production in crops?,  Curr. Opin. Plant Biol. ,  2009, vol.  12 (pg.  157- 164) Google Scholar CrossRef Search ADS PubMed  Guillaumot D,  Lelu-Walter MA,  Germot A,  Meytraud F,  Gastinel L,  Riou-Khamlichi C.  Expression patterns of LmAP2L1 and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix × marschlinsii),  J. Plant Physiol. ,  2008, vol.  165 (pg.  1003- 1010) Google Scholar CrossRef Search ADS PubMed  Guo M,  Rupe MA,  Dieter JA,  Zou J,  Spielbauer D,  Duncan KE, et al.  Cell Number Regulator1 affects plant and organ size in maize: implications for crop yield enhancement and heterosis,  Plant Cell ,  2010, vol.  22 (pg.  1057- 1073) Google Scholar CrossRef Search ADS PubMed  Guo M,  Rupe MA,  Yang X,  Crasta O,  Zinselmeier C,  Smith OS, et al.  Genome-wide transcript analysis of maize hybrids: allelic additive gene expression and yield heterosis,  Theor. Appl. Genet. ,  2006, vol.  113 (pg.  831- 845) Google Scholar CrossRef Search ADS PubMed  Gutterson N,  Reuber TL.  Regulation of disease resistance pathways by AP2/ERF transcription factors,  Curr. Opin. Plant Biol. ,  2007, vol.  17 (pg.  465- 471) Hochholdinger F,  Hoecker N.  Towards the molecular basis of heterosis,  Trends Plant Sci. ,  2007, vol.  12 (pg.  427- 432) Google Scholar CrossRef Search ADS PubMed  Horváth BM,  Magyar Z,  Zhang Y,  Hamburger AW,  Bakó L,  Visser RG, et al.  EBP1 regulates organ size through cell growth and proliferation in plants,  EMBO J. ,  2006, vol.  25 (pg.  4909- 4920) Google Scholar CrossRef Search ADS PubMed  Hu Y,  Xie Q,  Chua NH.  The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size,  Plant Cell ,  2003, vol.  15 (pg.  1951- 1961) Google Scholar CrossRef Search ADS PubMed  Jofuku KD,  den Boer BG,  Van Montagu M,  Okamuro JK.  Control of Arabidopsis flower and seed development by the homeotic gene APETALA2,  Plant Cell ,  1994, vol.  6 (pg.  1211- 1225) Google Scholar CrossRef Search ADS PubMed  Kim S,  Soltis PS,  Wall K,  Soltis DE.  Phylogeny and domain evolution in the APETALA2-like gene family,  Mol. Biol. Evol ,  2006, vol.  23 (pg.  107- 120) Google Scholar CrossRef Search ADS PubMed  Klucher KM,  Chow H,  Reiser L,  Fischer RL.  The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2,  Plant Cell ,  1996, vol.  8 (pg.  137- 153) Google Scholar CrossRef Search ADS PubMed  Krizek BA.  Ectopic expression of AINTEGUMENTA in Arabidopsis plants results in increased growth of floral organs,  Dev. Genet. ,  1999, vol.  25 (pg.  224- 236) Google Scholar CrossRef Search ADS PubMed  Krizek BA.  Making bigger plants: key regulators of final organ size,  Curr. Opin. Plant Biol. ,  2009, vol.  12 (pg.  17- 22) Google Scholar CrossRef Search ADS PubMed  Krizek BA,  Eaddy M.  AINTEGUMENTA-LIKE6 regulates cellular differentiation in flowers,  Plant Mol. Biol. ,  2012, vol.  78 (pg.  199- 209) Google Scholar CrossRef Search ADS PubMed  Lee BH,  Ko JH,  Lee S,  Lee Y,  Pak JH,  Kim JH.  The Arabidopsis GRF-INTERACTING FACTOR gene family performs an overlapping function in determining organ size as well as multiple developmental properties,  Plant Physiol. ,  2009, vol.  151 (pg.  655- 668) Google Scholar CrossRef Search ADS PubMed  Li A,  Fang MD,  Song WQ,  Chen CB,  Qi LW,  Wang CG.  Gene expression profiles of two intraspecific Larix lines and their reciprocal hybrids,  Mol. Biol. Rep. ,  2012, vol.  39 (pg.  3773- 3784) Google Scholar CrossRef Search ADS PubMed  Li LZ,  Lu KY,  Chen ZM,  Mu TM,  Hu ZL,  Li XQ.  Dominance, overdominance and epistasis condition the heterosis in two heterotic rice hybrids,  Genetics ,  2008, vol.  180 (pg.  1725- 1742) Google Scholar CrossRef Search ADS PubMed  Li X,  Wei Y,  Nettleton D,  Brummer EC.  Comparative gene expression profiles between heterotic and non-heterotic hybrids of tetraploid Medicago sativa,  BMC Plant Biol. ,  2009, vol.  9 pg.  107  Google Scholar CrossRef Search ADS PubMed  Li ZK,  Luo LJ,  Mei HW,  Wang DL,  Shu QY,  Tabien R, et al.  Overdominant epistatic loci are the primary genetic basis of inbreeding depression and heterosis in rice. I. Biomass and grain yield,  Genetics ,  2001, vol.  158 (pg.  1737- 1753) Google Scholar PubMed  Licausi F,  Giorgi FM,  Zenoni S,  Osti F,  Pezzotti M,  Perata P.  Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera,  BMC Genomics ,  2010, vol.  11 pg.  719  Google Scholar CrossRef Search ADS PubMed  Mizoi J,  Shinozaki K,  Yamaguchi-Shinozaki K.  AP2/ERF family transcription factors in plant abiotic stress responses,  Biochim. Biophys. Acta ,  2012, vol.  1819 (pg.  86- 96) Google Scholar CrossRef Search ADS PubMed  Mizukami Y,  Fischer RL.  Plant organ size control: AINTEGUMENTA regulates growth and cell numbers during organogenesis,  Proc. Natl Acad. Sci. U. S. A. ,  2000, vol.  97 (pg.  942- 947) Google Scholar CrossRef Search ADS PubMed  Mudunkothge JS,  Krizek BA.  Three Arabidopsis AIL/PLT genes act in combination to regulate shoot apical meristem function,  Plant J. ,  2012, vol.  71 (pg.  108- 121) Google Scholar CrossRef Search ADS PubMed  Mulder N,  Apweiler R.  InterPro and InterProScan: tools for protein sequence classification and comparison,  Methods Mol. Biol. ,  2007, vol.  396 pg.  59  Google Scholar PubMed  Nakano T,  Suzuki K,  Fujimura T,  Shinshi H.  Genome-wide analysis of the ERF gene family in Arabidopsis and rice,  Plant Physiol. ,  2006, vol.  140 (pg.  411- 432) Google Scholar CrossRef Search ADS PubMed  Nesbitt TC,  Tanksley SD.  fw2.2 directly affects the size of developing tomato fruit, with secondary effects on fruit number and photosynthate distribution,  Plant Physiol. ,  2001, vol.  127 (pg.  575- 583) Google Scholar CrossRef Search ADS PubMed  Ni Z,  Kim ED,  Ha M,  Lackey E,  Liu J,  Zhang Y, et al.  Altered circadian rhythms regulate growth vigour in hybrids and allopolyploids,  Nature ,  2009, vol.  457 (pg.  327- 331) Google Scholar CrossRef Search ADS PubMed  Nilsson L,  Carlsbecker A,  Sundås-Larsson A,  Vahala T.  APETALA2 like genes from Picea abies show functional similarities to their Arabidopsis homologues,  Planta ,  2007, vol.  225 (pg.  589- 602) Google Scholar CrossRef Search ADS PubMed  Okamuro JK,  Caster B,  Villarroel R,  Van Montagu M,  Jofuku KD.  The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis,  Proc. Natl Acad. Sci. U. S. A. ,  1997, vol.  94 (pg.  7076- 7081) Google Scholar CrossRef Search ADS PubMed  Passarinho P,  Ketelaar T,  Xing M,  van Arkel J,  Maliepaard C,  Hendriks MW, et al.  BABY BOOM target genes provide diverse entry points into cell proliferation and cell growth pathways,  Plant Mol. Biol. ,  2008, vol.  68 (pg.  225- 237) Google Scholar CrossRef Search ADS PubMed  Powell AE,  Lenhard M.  Control of organ size in plants,  Curr. Biol. ,  2012, vol.  22 (pg.  360- 367) Google Scholar CrossRef Search ADS   Rigal A,  Yordanov YS,  Perrone I,  Karlberg A,  Tisserant E,  Bellini C, et al.  The AINTEGUMENTA LIKE1 homeotic transcription factor PtAIL1 controls the formation of adventitious root primordia in poplar,  Plant Physiol. ,  2012, vol.  160 (pg.  1996- 2006) Google Scholar CrossRef Search ADS PubMed  Sakuma Y,  Liu Q,  Dubouzet JG,  Abe H,  Shinozaki K,  Yamaguchi-Shinozaki K.  DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression,  Biochem. Biophys. Res. Commun. ,  2002, vol.  290 (pg.  998- 1009) Google Scholar CrossRef Search ADS PubMed  Schnable PS,  Springer NM.  Progress toward understanding heterosis in crop plants,  Annu. Rev. Plant Biol. ,  2013, vol.  64 (pg.  71- 88) Google Scholar CrossRef Search ADS PubMed  Schruff MC,  Spielman M,  Tiwari S,  Adams S,  Fenby N,  Scott RJ.  The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs,  Development ,  2006, vol.  133 (pg.  251- 261) Google Scholar CrossRef Search ADS PubMed  Springer NM,  Stupar RM.  Allelic variation and heterosis in maize: how do two halves make more than a whole?,  Genome Res ,  2007, vol.  17 (pg.  264- 275) Google Scholar CrossRef Search ADS PubMed  Srivasta A,  Mehta S,  Lindlof A,  Bhargava S.  Over-represented promoter motifs in abiotic stress-induced DREB genes of rice and sorghum and their probable role in regulation of gene expression,  Plant Signal Behav. ,  2010, vol.  5 (pg.  775- 784) Google Scholar CrossRef Search ADS PubMed  Swanson-Wagner RA,  Jia Y,  Cook R,  Borsuk LA,  Nettleton D,  Schnable PS.  All possible modes of gene action are observed in a global comparison of gene expression in a maize F1-hybrid and its inbred parents,  Proc. Natl Acad. Sci. U. S. A. ,  2006, vol.  103 (pg.  6805- 6810) Google Scholar CrossRef Search ADS PubMed  Thompson JD,  Higgins DG,  Gibson TJ.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,  Nucleic Acids Res. ,  1994, vol.  22 (pg.  4673- 4680) Google Scholar CrossRef Search ADS PubMed  Wei G,  Tao Y,  Liu G,  Chen C,  Luo R,  Xia H, et al.  A transcriptomic analysis of superhybrid rice LYP9 and its parents,  Proc. Natl Acad. Sci. U. S. A. ,  2009, vol.  106 (pg.  7695- 7701) Google Scholar CrossRef Search ADS PubMed  Weigel D.  The APETALA2 domain is related to a novel type of DNA binding domain,  Plant Cell ,  1995, vol.  7 (pg.  388- 389) Google Scholar CrossRef Search ADS PubMed  Wollmann H,  Mica E,  Todesco M,  Long JA,  Weigel D.  On reconciling the interactions between APETALA2, miR172 and AGAMOUS with the ABC model of flower development,  Development ,  2010, vol.  137 (pg.  3633- 3642) Google Scholar CrossRef Search ADS PubMed  Xiong AS,  Jiang HH,  Zhuang J,  Peng RH,  Jin XF,  Zhu B, et al.  Expression and function of a modified AP2/ERF transcription factor from Brassica napus enhances cold tolerance in transgenic Arabidopsis,  Mol. Biotechnol. ,  2013, vol.  53 (pg.  198- 206) Google Scholar CrossRef Search ADS PubMed  Xu ZS,  Chen M,  Li LC,  Ma YZ.  Functions and application of the AP2/ERF transcription factor family in crop improvement,  J. Integr. Plant Biol. ,  2011, vol.  53 (pg.  570- 585) Google Scholar CrossRef Search ADS PubMed  Xue W,  Xing Y,  Weng X,  Zhao Y,  Tang W,  Wang L, et al.  Natural variation in Ghd7 is an important regulator of heading date and yield potential in rice,  Nature Genet ,  2008, vol.  40 (pg.  761- 767) Google Scholar CrossRef Search ADS PubMed  Yaish MW,  El-Kereamy A,  Zhu T,  Beatty PH,  Good AG,  Bi YM, et al.  The APETALA-2-like transcription factor OsAP2-39 controls key interactions between abscisic acid and gibberellin in rice,  PLoS Genet. ,  2010, vol.  6 pg.  e1001098  Google Scholar CrossRef Search ADS PubMed  Yant L,  Mathieu J,  Dinh TT,  Ott F,  Lanz C,  Wollmann H, et al.  Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2,  Plant Cell ,  2010, vol.  22 (pg.  2156- 2170) Google Scholar CrossRef Search ADS PubMed  Yao H,  Dogra Gray A,  Auger DL,  Birchler JA.  Genomic dosage effects on heterosis in triploid maize,  Proc. Natl Acad. Sci. U. S. A. ,  2013, vol.  110 (pg.  2665- 2669) Google Scholar CrossRef Search ADS PubMed  Zerbino DR,  Velvet EB.  Algorithms for de novo short read assembly using de bruijn graphs,  Genome Res. ,  2008, vol.  18 (pg.  821- 829) Google Scholar CrossRef Search ADS PubMed  Zhai R,  Feng Y,  Wang H,  Zhan X,  Shen X,  Wu W, et al.  Transcriptome analysis of rice root heterosis by RNA-Seq,  BMC Genomics ,  2013, vol.  14 pg.  19  Google Scholar CrossRef Search ADS PubMed  Zhang CH,  Shangguan LF,  Ma RJ,  Sun X,  Tao R,  Guo L, et al.  Genome-wide analysis of the AP2/ERF superfamily in peach (Prunus persica),  Genet. Mol. Res. ,  2012, vol.  11 (pg.  4789- 4809) Google Scholar PubMed  Zhang Y,  Ni Z,  Yao Y,  Nie X,  Sun Q.  Gibberellins and heterosis of plant height in wheat (Triticum aestivum L.),  BMC Genet. ,  2007, vol.  8 pg.  40  Google Scholar CrossRef Search ADS PubMed  Zhou Y,  Lu D,  Li C,  Luo J,  Zhu BF,  Zhu J, et al.  Genetic control of seed shattering in rice by the APETALA2 transcription factor shattering abortion1,  Plant Cell ,  2012, vol.  24 (pg.  1034- 1048) Google Scholar CrossRef Search ADS PubMed  © The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com
TI - LaAP2L1, a Heterosis-Associated AP2/EREBP Transcription Factor of Larix, Increases Organ Size and Final Biomass by Affecting Cell Proliferation in Arabidopsis
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pct124
DA - 2013-10-06
UR - https://www.deepdyve.com/lp/oxford-university-press/laap2l1-a-heterosis-associated-ap2-erebp-transcription-factor-of-larix-yGNyTlgR2L
SP - 1822
EP - 1836
VL - 54
IS - 11
DP - DeepDyve
ER -