Anatomical and Biochemical Studies of Bicolored Flower Development in Muscari Latifolium
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Protoplasma (2013) 250:1273–1281 DOI 10.1007/s00709-013-0509-8 ORIGINAL ARTICLE Anatomical and biochemical studies of bicolored flower development in Muscari latifolium Yinyan Qi & Qian Lou & Huibo Li & Juan Yue & Yali Liu & Yuejin Wang Received: 1 March 2013 /Accepted: 6 May 2013 /Published online: 16 May 2013 # Springer-Verlag Wien 2013 Abstract The inflorescence of the broad-leafed grape hya- respectively, with the difference being nonsignificant. cinth, Muscari latifolium, shows an interesting, two-tone HPLC results indicate that the dihydroflavonol and flavonol appearance with the upper flowers being pale blue and the contents are also very similar in the two sorts of flower. lower ones purple. To elucidate the mechanism of the dif- However, the upper flowers contained only delphinidin, ferential color development, anatomical research was car- whereas the lower flowers also contained cyanidin. The total ried out and a cytological study of the colored protoplasts in anthocyanin content in the lower flowers was 4.36 mg g−1, which the shapes of the cells accumulating anthocyanin which is approximately seven times higher than in the upper were observed by scanning electron microscopy. Next, vac- flowers, while the delphinidin content is four times higher. uolar pH was recorded using a pH meter with a micro Quantitative real-time PCR analysis established that the combination pH electrode, and the sap’s metal-ion content two-tone flower was a result of different expressions of the was measured by inductively coupled plasma mass spec- F3′5′H, F3′H and DFR genes, and these lead to different trometry. The anthocyanin and co-pigment composition was amounts of anthocyanin. determined by high-performance liquid chromatography (HPLC). Chemical analyses reveal that the difference in Keywords Anthocyanin . Color development . Metal ions . metal-ion content of the two parts was not great. The vacu- Grape hyacinth . Protoplast olar pHs of the upper and lower flowers were 5.91 and 5.84, Abbreviations RHSCC Royal Horticultural Society color chart Handling Editor: Hanns H. Kassemeyer CIE International Commission on Illumination Yinyan Qi and Qian Lou contributed equally to this work. Y. Qi : Q. Lou : Y. Wang College of Horticulture, Northwest A&F University, Yangling, Introduction Xianyang 712100 Shaanxi, People’s Republic of China : : H. Li J. Yue Y. Liu The Muscari, commonly known as grape hyacinths, is a College of Forestry, Northwest A&F University, Yangling, genus of ornamental bulbous plants named after their spikes ’ Xianyang 712100 Shaanxi, People s Republic of China of dense, urn-shaped flowers resembling bunches of grapes. Y. Qi : Q. Lou : H. Li : J. Yue : Y. Liu : Y. Wang They are excellent as garden, bedding, and ground-cover Key Laboratory of Biology and Genetic Improvement of plants because of their profuse flowering, adaptability, at- Horticultural Crops(Northwest Region), Ministry of Agriculture, tractive fragrance, and distinctive blue colors in spring. In Yangling, addition, they have important cultural significances as they Xianyang 712100, People’s Republic of China are referred to in many of the world’s literatures. There are : : : : : Y. Qi Q. Lou H. Li J. Yue Y. Liu (*) Y. Wang (*) about 40 different species of Muscari, and their flower color State Key Laboratory of Crop Stress Biology in Arid Areas, is most commonly a shade of blue (Doussi et al. 2002). Northwest A&F University, Yangling, Muscari latifolium is distinguished from the other grape 712100, Xianyang, Shaanxi, People’s Republic of China e-mail: [email protected] hyacinth species by the production from each bulb of a e-mail: [email protected] single, bicolored, racemose inflorescence. The inflorescence 1274 Y. Qi et al. also shows an unusual two-toned appearance being purple to delphinidin, while the reddish or pale purple colors are over most of its slender length, but with a soft pale blue attributable to a mixture of cyanidin and delphinidin. “cap” (Fig. 1). To determine more precisely the causes of bicolor devel- In nature, a number of species can exhibit more than one opment in M. latifolium inflorescences, we analyzed tepal color in a flower, but these are rare. Bicolored inflorescences structures, protoplast features, and the anthocyanin and fla- attract considerable interest among researchers and breeders, vonol compositions of the flower perianths in relation to and much research has been carried into this, but the mech- their different colors. We also investigated certain physico- anisms involved have not been fully elucidated because of chemical properties of the flowers which could have been their complexity and also the scarcity of multicolored associated with color development, such as pH and metal- flowers. In some species, different anthocyanin chromo- ion content. Through this analysis, we hoped to determine phores are the primary contributors to the distinctively color the mechanism of color development in their distinctive phenotypes. These occur in Anagallis monelli (Quintana et upper and lower flowers. al. 2007), Delphinium (Hashimoto et al. 2002), and Hydrangea macrophylla cv. Hovariatrade mark “Homigo” (Yoshida et al. 2008). In the case of some tulips, Tulipa Materials and methods gesneriana (Momonoi et al. 2009, 2012; Shoji et al. 2007, 2010) and H. macrophylla (Ito et al. 2009; Schreiber et al. Plant material 2010, 2011), metal ions are required for specific color development. In Ipomoea tricolor, the color variation in Grape hyacinth (M. latifolium) bulbs were purchased from different regions of the same tepal is linked to vacuolar pH Zhejiang Hongyue Seeds Co., Ltd. (Zhejiang, People’s differences (Yoshida et al. 2005, 2009a, b). Thus, it is Republic of China) and planted in an experimental plot at reasonable to infer that the two-tone effect observed in the the Northwest A&F University, Xi’an, Shaanxi, People’s differently colored flowers of the M. latifolium raceme is the Republic of China. The flowers were harvested from the result of certain biochemical or physiological differences spikes of each cultivar just after anthesis and were used but, at this stage, the precise mechanisms involved are immediately for the morphological and anatomy observa- uncertain. Comprehensive studies of color formation in the tions and the preparation of protoplasts. To measure antho- Muscari, or other flowers of the same family, have not yet cyanin, flavonol, and pH, the flowers were cut in half been reported to our knowledge. At this stage, there are a lengthways using a razor blade. The tepals were then care- few reports of anthocyanin composition in 13 genotypes of fully collected from straight-cut flowers and treated with a Muscari ssp. Mori et al. (2002) summarize these, stating that small puffer to blow off any dust or pollen adhering to the the varying shades found in the blue flowers are attributable epidermis. Next, the tepals were quickly frozen in liquid Fig. 1 Phenotypes of M. latifolium. Upper flowers (a) with pale blue tepals and lower flowers (b) with purple tepals. Bar=2mm Studies of bicolored flower development in M. latifolium 1275 nitrogen and stored at −80°C pending flavonoid and pH 80 mM KCl, and 20 mM MES-Tris (pH 6.0). The solution measurement. Additional tepals were collected and dried was warmed to 55 °C for 10 min to enhance enzyme for metal-ion analysis. These were rinsed in sterile water, solubility, then cooled to room temperature, and 10 mM before being placed in a drying oven at 105 °C for 30 min, CaCl2 and 0.1 % BSA were added. The final enzyme solu- followed by one at 80 °C for 5 h. tion was filtered through a 0.45-μm syringe filter device into a Petri dish. Fresh tepals (0.5 g) were taken from a well- Measurement of tepal color opened flower at 9:00 am. Tepal strips (0.5–1 mm wide) were cut from the middle part of a tepal using a sharp razor Tepal color was determined in the mid portion of the urn- blade. They were transferred quickly and gently and im- shaped flowers at noon, indoors and in a north light (i.e., no mersed in enzyme solution. After vacuum infiltration for direct sunlight) using the Royal Horticultural Society Color 30 min without shaking, digestion was continued in the dark Chart (RHSCC). About 60–100 fully pigmented flowers for 3 h at room temperature. The reaction mixture were randomly sampled and placed in a single layer on a containing free protoplasts was filtered through a 75-μm six-well culture plate (Corning Costar, Sigma-Aldrich, nylonmeshandwashedwithbuffer[20mMMES-Tris USA). Their color parameters were measured with a chroma (pH 6.0), 0.8 M mannitol], then centrifuged at 100×g for meter (CR-400, Konica Minolta Investment Ltd., China) 3 min to form a pellet of protoplasts in a 30-ml round- based on the CIE (International Commission on bottomed tube. As much supernatant liquid as possible Illumination) L∗a∗b∗ scale (Hanbury and Serra 2002; was removed, and the protoplast pellet was re-suspended Zhang et al. 2008). Five replicates were recorded. in 2 ml of fresh buffer by gentle swirling. The centrifugal separation/re-suspension procedure was repeated three Microscopic observation of epidermal cells and transverse times, and the released protoplasts were checked under the sections microscope. All the procedures after the filtration were conducted below 4 °C. Fresh urn-shaped flowers were cross-sectioned, and an epi- dermal layer was peeled off by hand using a razor blade and Measurement of metal elements in colored tepals placed on a glass slide with a drop of water. The fresh sections were immediately observed using a light micro- Dried tepals were finely ground to a powder. Next, a 1-g scope (Eclipse 50i, Nikon, Japan), equipped with a DS sample of dried material was digested in 5 ml of concen- cooled camera head with FNIS-Elements image processing trated HNO3 and 1 ml of H2O2, followed by treatment in a software.