Abstract：15-cis-ζ-carotene isomerase (Z-ISO) can isomerize 9,15,9'-tri-cis-ζ-carotene into 9,9'-di-cis-ζ-carotene, and Z-ISO gene is the last identified gene in plant carotenoid biosynthetic pathway. In order to clone the ORF of Z-ISO genes in Osmanthus fragrans and study their expression profile, ORF sequences of OfZ-ISO1 (GenBank accession: KX120175) and OfZ-ISO2 (GenBank accession: KX120176) from O. fragrans were amplified using PCR based on the sequence information of Unigene from the petal transcriptome database of O. fragrans Yanhong Gui from the Aurantiacus Group. Bioinformatics analysis indicated that both OfZ-ISO1 and OfZ-ISO2 contained 1 107 bp ORF, encoding 368 amino acid residues. Six predicted transmembrane domains were in both OfZ-ISO1 and OfZ-ISO2 proteins, as well as 3 conserved residues (H152, H268 and D296) which were essential for isomerase activity. OfZ-ISO1 and OfZ-ISO2 from O. fragrans shared high sequence similarity with ZmZ-ISO1 from Zea mays and AtZ-ISO1 from Arabidopsis thaliana. Phylogenetic analysis showed that OfZ-ISO1 and OfZ-ISO2 were gathered into one branch with Sesamum indicum Z-ISO, which revealed that OfZ-ISO1 and OfZ-ISO2 showed the closest genetic relationship with Z-ISO from S. indicum. Gene expression showed that during the flower development of O. fragrans Yanhong Gui, the expression level of OfZ-ISO1 was low in the inflorescences at bud stages, dramatically increased at linggeng stage, kept the same at initial flowering stage and greatly decreased at full flowering stage, while that of OfZ-ISO2 was low in the inflorescences at bud stages and linggeng stage, then gradually increased, and reached the maximum at full flowering stage. In the O. fragrans cultivars with different petal color, Yanhong Gui from the Aurantiacus Group showed the darkest petal color with the lowest L* value and the highest a* value and the highest total carotenoid content. In Jin Qiugui from the Luteus Group, the petal color was lighter than that of Yanhong Gui from the Aurantiacus Group and the total carotenoid content was lower than that of Yanhong Gui, and Yu Linglong from the Albus Group showed the lightest petal color with the highest L* value and the lowest a* value and the lowest total carotenoid content. Surprisingly, the expression levels of OfZ-ISO1 and OfZ-ISO2 were not the highest in Yanhong Gui with the highest total carotenoid content. Generally, the expression levels of OfZ-ISO and OfZ-ISO were the highest in Yu Linglong at linggeng stage and initial flowering stage, however, at full flowering stage, the expression levels were the same in the 3 cultivars with different petal color. The results of this study revealed that the expression levels of OfZ-ISO1 and OfZ-ISO2 during flower opening were essential to the carotenoid accumulation of O. fragrans, and total carotenoid content of cultivars with different petal color was not positively related with the expression level of OfZ-ISO1 and OfZ-ISO2. This study has important practical significance to fully reveal the mechanism of petal coloration in O. fragrans.
[1] 侯丹. 2014. 桂花主要品种花香和花色及其对温度变化的响应[D]. 硕士学位论文, 浙江农林大学, 导师: 赵宏波. pp. 58-60. (Hou D. 2014. Analysis of floral scent and pigment constituents and the reflectance to temperature fluctuation in Osmanthus fragrans (Thunb.) Lour. [D]. Thesis for M.S., Zhejiang A & F University, Supervisor: Zhao H B. pp. 58-60.) [2] 向其柏, 刘玉莲. 2008. 中国桂花品种图志[M]. 浙江科学技术出版社, 杭州. pp. 86-88. (Xiang Q B, Liu Y L. 2008. An illustrated monograph of the sweet osmanthus variety in China[M]. Zhejiang Science & Technology Press, Hangzhou. pp. 86-88.) [3] Ampomah-Dwamena C, Dejnoprat S, Lewis D, et al. 2012. Metabolic and gene expression analysis of apple (Malus × domestica) carotenogenesis[J]. Journal of Experimental Botany, 63(12): 4497-4511. [4] Bartley G E, Scolnik P A, Beyer P. 1999. Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and ζ-carotene desaturase, expressed in Escherichia coli, catalyze a poly- cis pathway to yield pro-lycopene[J]. European Journal of Biochemistry, 259(1-2): 396-403. [5] Bartnikas T B, Tosques I E, Laratta W P, et al. 1997. Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3[J]. Journal of Bacteriology, 179(11): 3534-3540. [6] Beltrán Zambrano J A. 2015. Functional characterization of the plant 15-cis-zeta-carotene isomerase z-iso[D]. Thesis for Ph.D., City University of New York, Supervisor: Eleanore T W. pp. 63-64. [7] Beyer P, Mayer M, Kleinig H. 1989. Molecular oxygen and the state of geometric isomerism of intermediates are essential in the carotene desaturation and cyclization reactions in daffodil chromoplasts[J]. European Journal of Biochemistry, 184(1): 141-150. [8] Chen Y, Li F, Wurtzel E T. 2010. Isolation and characterization of the Z-ISO gene encoding a missing component of carotenoid biosynthesis in plants[J]. Plant Physiology, 153(1): 66-79. [9] Fantini E, Falcone G, Frusciante S, et al. 2013. Dissection of tomato lycopene biosynthesis through virus-induced gene silencing[J]. Plant Physiology, 163(2): 986-998. [10] Han Y, Li L, Dong M, et al. 2013. cDNA cloning of the phytoene synthase (PSY) and expression analysis of PSY and carotenoid cleavage dioxygenase genes in Osmanthus fragrans[J]. Biologia, 68(2): 258-263. [11] Han Y, Wang X, Chen W, et al. 2014. Differential expression of carotenoid-related genes determines diversified carotenoid coloration in flower petal of Osmanthus fragrans[J]. Tree Genetics & Genomes, 10(2): 329-338. [12] Li F, Murillo C, Wurtzel E T. 2007. Maize Y9 encodes a product essential for 15-cis-ζ-carotene isomerization[J]. Plant Physiology, 144(2): 1181-1189. [13] Matthews P D, Luo R B, Wurtzel E T. 2003. Maize phytoene desaturase and ζ-carotene desaturase catalyse a poly-Z desaturation pathway: implications for genetic engineering of carotenoid content among cereal crops[J]. Journal of Experimental Botany, 54(391): 2215-2230. [14] Ohmiya A. 2013. Qualitative and quantitative control of carotenoid accumulation in flower petals[J]. Scientia Horticulturae, 163: 10-19. [15] Young P R, Lashbrooke J G, Alexandersson E, et al. 2012. The genes and enzymes of the carotenoid metabolic pathway in Vitis vinifera L.[J]. BMC Genomics, 13: 243. [16] Zhang C, Fu J, Wang Y, et al. 2015. Identification of suitable reference genes for gene expression normalization in the quantitative real-time PCR analysis of sweet osmanthus (Osmanthus fragrans Lour.)[J]. PLoS ONE, 10(8): e0136355. [17] Zhang C, Wang Y, Fu J, et al. 2016. Transcriptomic analysis and carotenogenic gene expression related to petal coloration in Osmanthus fragrans ‘Yanhong Gui’[J]. Trees - Structure and Function, DOI 10.1007/s00468-016-1359-8. (Published online) [18] Zhu C, Bai C, Sanahuja G, et al. 2010. The regulation of carotenoid pigmentation in flowers[J]. Archives of Biochemistry and Biophysics, 504: 132-141.
