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Current Research Advances in Molecular Characterization of Ornamental Camellias

Jiyuan Li, Yingkun Sun, Hengfu Yin*

Research Institute of Subtropical Forestry, Chinese Academy of Forestry,Fuyang City,Zhejiang Province 311400, China.
Email: jiyuan_li@126.com

1Figure 1. A top-view of wild and cultivated camellia flowers. In the middle is wild Camellia japonica (collected from Zhejiang Province, China). Cultivars from right to left (clock-wise) are: ‘Velvet’, ‘Songzi,’ ‘Hongluzhen’, ‘Jinpanguizhi’ and ‘Shibaxueshi’.

Intensive studies of homeotic mutants of floral development in Arabidopsis and Antirrhinum have set up the ABC model of floral organ identity specification (Bowman et al., 1991; Coen and Meyerowitz, 1991). This model proposes a group of genes, categorized into A, B, and C, a regulating master switch of floral organ identities. The ABC model has been successfully demonstrated in a wide range of angiosperm and gymnosperm species (Ng and Yanofsky, 2001; Becker and Theissen, 2003). Nevertheless, studies in some species have also shown variation in expression and function in many instances, which implies evolutionary alterations have been involved in the formation of unique floral phenotypes (Cseke and Podila, 2004). The classic ABC model provides an excellent genetic model to predict the formation of double flowers by modification of gene functions. Indeed, studies in several ornamental species suggest C class genes play central roles in formation of double flowers. In the rose, contractions of C class gene expression domains have contributed to generate multiple petals in two independent domesticated varieties (Dubois et al. 2010). In the ranunculid Thalictrum thalictroides, a mutation of AG ortholog (ThtAG1) in double flower variety was identified which abolished its interaction with E class gene (Galimba et al. 2012). How ABC genes are re-orchestrated to direct the development of double flower in camellias remains largely unknown.

Gene cloning and expression analysis

To isolate MADS-box like homologs in C. japonica, we designed degenerate primers based on sequence similarity of MADS conserved domain (Malcomber and Kellogg 2004). After sequencing of multiple PCR fragments we identified two partial sequences which displayed very high similarities with AP1 family genes. To obtain the full-length coding sequences, we performed 5’ and 3’ RACE experiments by designing gene specific primers accordingly. To access the phylogenetic placements, we performed phylogenic analysis by grouping 59 MADS genes of Arabidopsis (acquired from PLAZA version 2.0) (Proost et al. 2009; Van Bel et al. 2012) with these two together (Figure 1 A). It showed CjAPL1 and CiAPL2 were nested within the A function clade of MADS family. CjAPL2 and AP1 share75.61 % amino acid identity, while CjAPL1 and FUL share 64.04% amino acid identity. Then we selected several AP1/FUL family members from monocot and closely related eudicot species to construct a phylogenic tree, and showed CjAPL2 and CjAPL1 were classified into AP1 and FUL clades respectively (Figure 1 B).

2Functional analysis of CjAPL1/2 in A. thaliana: Due to gene duplication, AP1-like and FUL-like genes displayed distinct functional divergences across species. To address whether CjAPL1/2 have similar functions in floral patterning to other species and what differences they have, we generated transgenic A. thaliana plants in which CjAPL1 and CjAPL2 were over-expressed respectively. Two constructs for ectopic expression of CjAPL1/2 were driven by the cauliflower mosaic virus (CaMV) 35S promoter. The positive T1 transgenic lines were screened and identified by selectable maker tests and PCR analysis with construct specific primers. Potential single insertion T2 lines were identified by genetic segregation analysis, and three lines of each transgenic were selected for southern blotting analysis which confirmed the single insertions of target constructs. qRT-PCR using gene specific primers were performed to detect the expression levels in transgenic lines, and ectopic expression levels of target genes were evident.

3Figure 2. Ectopic expression of CjAPL1 and CjAPL2 in Arabidopsis. A, wild type flower; B, typical flower of CjAPL1 overexpression with greater number of stamens; C, typical flower of CjAPL2 overexpression with greater number of stamens and carpels. D-F, Overexpression of CjAPL1 produce less rosette leaves (D) and terminal flowers (D, E). A flower of CjAPL1 overexpression with greater number of stamens and carpels is shown (F). G-I, Overexpression of CjAPL2 produces terminal flowers (G), and flowers with more petals, stamens and carpels (H, I). A-C, bar=1mm; D, G, bar=1cm; E-F, bar=1mm; H, I, bar=1mm.

Phenotypes of 35S:CjAPL1 transgenic plants included early flowering, formation of terminal flowers, and flowers with greater number of stamens and carpels. To depict the dramatically early flowering phenotype, we counted the number of rosette leaves. Early flowering occurred while there were 7 rosette leaves on average (Figure 2 D, G), and in some cases only 4 rosette leaves were observed (not shown). In long day conditions, the wild type A. thaliana plants produced 12 rosette leaves on average. The terminal flowers were found in transgenic plants (Figure 2 D, E), and inflorescence branches were replaced by solitary flowers (Figure 2 D, E). Typical flowers of 35S:CjAPL1 had increased number of stamens and pistils (Figure 2 F), while the petal number was not affected. Phenotypes of 35S:CjAPL2 transgenic plants were similar to 35S:CjAPL1 which displayed early flowering, formation of terminal flowers and increased number of stamens and carpels (Figure 2 G, H). However, in 35S CjAPL2 transgenic plants, a high frequency of greater number of petals were observed (Figure 2 I).

Gene expression analysis in double flowers of C. japonica varieties: C. japonica is a famous ornamental plant species which has a long history of domestication. As a result of artificial selection of aesthetic traits, varieties with increased number of petals (double flower) and other showy aspects were retained (Figure 3 A). We selected four varieties containing one semi-double variety ‘Velvet’) and three double-flower variety (‘Hongluzhen’, ‘Songzi’, ’Shibaxueshi’) which displayed different degrees of increased number of petals (Figure 3 B). In semi-double and double flower varieties, petals were roughly divided into Inner and Outer based on distinctive shape and size. In ‘Songzi’ and ‘Hongluzhen’, some stamenoid petals were observed in the inner area which were also counted as Inner petals (Figure 3 B). To address whether CjAPL1/2 were related to the formation of double flower, we compared the expression levels of CjAPL1/2 among three different developmental stages of floral bud as small (less than 3cm), medium (3-8cm) and large (8-11cm). We showed the expression levels of CjAPL1/2 were both remarkably induced in double flower varieties (Figure 3 C, D), although in ‘Songzi’ and ‘Shibaxueshi’ the numbers of stamens were significantly reduced (Figure 3 B). This data suggested the transcriptional alteration of CjAPL1/2 was generally required for double flower formation.

4Figure 3. Increased expression levels of CjAPL1/2 genes in double flower varieties of C. japonica.
A, Overview of wild C. japonica and four different double-flower varieties. B, Floral number counting of single and double-flower varieties. a, inner petals of ‘Velvet’ were similar to outer petals in shape; b, inner petals of ‘Hongluzhen’ were smaller and different from outer petals; c, multiple rows of  petals in ‘Songzi’ were counted as outer petals due to its irregular arrangement; d, in ‘Shibaxueshi’, petals were gradually getting small in size; e, pistils with fused structures or deformed shapes were frequently found.  C-D, relative expression of CjAPL1/2 in floral buds of the single and different varieties. A, bar=1cm.

We isolated the CjAPL1 and CjAPL2 from C. japonica, which belong to euAP1 and euFUL clades respectively. We showed CjAPL1 and CjAPL2 contributed differently to the floral development by gene expression profiling and ectopic expression in Arabidopsis. Overexpression of CjAPL1/2 displayed similar phenotypes in Arabidopsis including early flowering, formation of terminal flowers, and increase in stamen and pistil numbers, but only in overexpression plants of CjAPL2 the petal number was increased. Moreover, we also discovered the expression levels of both CjAPL1 and CjAPL2 were generally induced in double flower varieties. In conclusion, our work for the first time characterized the functions of AP1/FUL family genes in Theaceae, and indicated that the alteration of gene expression was a critical point of double flower domestication (Sun et al 2013). 

Acknowledgements

We thank Prof. Zhongchi Liu from University of Maryland (College Park) for the help of experimental design and analysis of data.
This work was financially supported by National Key Twelfth Five-Year Sci.& Tech. Programme (2012BAD01B0703), MOST international Sci. & Tech. Programme (2011DFA304903), Zhejiang Key Flower Breeding Programme (2012C12909-6) and Zhejiang-CAF Cooperation Project (2012SY02)

References

Bowman JL, Smyth DR, Meyerowitz EM,1989. Genes directing flower development in Arabidopsis. The Plant cell 1: 37-52
Chen SY,1985. Camellias of Zhejiang Province. Zhejiang Sci.&Tech. Press, Hangzhou, China
Clough SJ, Bent AF,1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant journal : for cell and molecular biology 16: 735-743
Coen ES, Meyerowitz EM, 1991. The war of the whorls: genetic interactions controlling flower development. Nature 353: 31-37
Dubois A, Raymond O, Maene M, Baudino S, Langlade NB, Boltz V, Vergne P, Bendahmane M, 2010. Tinkering with the C-function: a molecular frame for the selection of double flowers in cultivated roses. PloS one 5: e9288
Galimba KD, Tolkin TR, Sullivan AM, Melzer R, Theissen G, Di Stilio VS, 2012. Loss of deeply conserved C-class floral homeotic gene function and C- and E-class protein interaction in a double-flowered ranunculid mutant. Proceedings of the National Academy of Sciences of the United States of America 109: E2267-2275
Gao JY, 2005. Collected Species of The Genus Camellia- An Illustrated Outline. Zhejiang Science and Technology Publishing House, Hangzhou
Jaakola L, Poole M, Jones MO, Kamarainen-Karppinen T, Koskimaki JJ, Hohtola A, Haggman H, Fraser PD, Manning K, King GJ, Thomson H, Seymour GB, 2010. A SQUAMOSA MADS box gene involved in the regulation of anthocyanin accumulation in bilberry fruits. Plant physiology 153: 1619-1629
Kaufmann K, Wellmer F, Muino JM, Ferrier T, Wuest SE, Kumar V, Serrano-Mislata A, Madueno F, Krajewski P, Meyerowitz EM, Angenent GC, Riechmann JL, 2010. Orchestration of floral initiation by APETALA1. Science 328: 85-89
Lamb RS, Hill TA, Tan QK, Irish VF, 2002. Regulation of APETALA3 floral homeotic gene expression by meristem identity genes. Development 129: 2079-2086
Li JY, Sui N., Li XL, Fan ZQ , 2013. UPOV/TG/275/1:Guidelines for the Conduct of Tests for Distinctness, Uniformity and Stability Camellia excluding C.sinensis. www.upov.int.
Litt A, 2007. AN EVALUATION OF A-FUNCTION: EVIDENCE FROM THE APETALA1 AND APETALA2 GENE LINEAGES. Int. J. Plant Sci. 168: 73–91
Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25: 402-408
Malcomber ST, Kellogg EA, 2004. Heterogeneous expression patterns and separate roles of the SEPALLATA gene LEAFY HULL STERILE1 in grasses. The Plant cell 16: 1692-1706
Mandel MA, Gustafson-Brown C, Savidge B, Yanofsky MF, 1992. Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature 360: 273-277
Ng M, Yanofsky MF, 2001. Activation of the Arabidopsis B class homeotic genes by APETALA1. The Plant cell 13: 739-753
Pabon-Mora N, Ambrose BA, Litt A, 2012. Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development. Plant physiology 158: 1685-1704
Preston JC, Kellogg EA, 2007. Conservation and divergence of APETALA1/FRUITFULL-like gene function in grasses: evidence from gene expression analyses. The Plant journal : for cell and molecular biology 52: 69-81
Proost S, Van Bel M, Sterck L, Billiau K, Van Parys T, Van de Peer Y, Vandepoele K, 2009. PLAZA: a comparative genomics resource to study gene and genome evolution in plants. The Plant cell 21: 3718-3731
Savige TJ, 1993. The International Register Volume One. International Camellia Society. Fine Arts Press Pty Limited Sydney, Australia
Sun YK, FAN ZQ, LI JY, YIN HF, 2013. The APETALA1 and FRUITFUL homologs in Camellia japonica and their roles in double flower domestication. Molecular Breeding,DOI 10.1007/s11032-013-9995-9
Sealy JR, 1958. A revision of the genus Camellia. Royal Horticultural Society
Van Bel M, Proost S, Wischnitzki E, Movahedi S, Scheerlinck C, Van de Peer Y, Vandepoele K, 2012. Dissecting plant genomes with the PLAZA comparative genomics platform. Plant physiology 158: 590-600
Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann KA, Meyerowitz EM, 1990. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346: 35-39
Zhang Y. Flower Encyclopedia (Ancient Chinese literature). 221
Zhu GP, Li JY, Ni S, Fan ZQ, Yin HF, Li XL, Zhou XW, 2011. The potential role of B-function gene involved in floral development for double flowers formation in Camellia changii Ye. Afr J Biotechnol 10: 16757-16762

 
 

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