Plastid analysis of pigmented undifferentiated cells of marigold Tagetes erecta L. by transmission electron microscopy

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ “This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: https://doi.org/10.1007/s11627-011-9401-4 ” 1 Running title: Ultrastructural analysis in marigold PLASTID ANALYSIS OF PIGMENTED UNDIFFERENTIATED CELLS OF Tagetes erecta L. BY TRANSMISSION ELECTRON MICROSCOPY Vanegas-Espinoza PE,1 Ramos-Viveros V,1 Jiménez-Aparicio AR,1 López-Villegas EO,2 Heredia FJ, 3 Cruz-Hernández A,1 Quintero-Gutiérrez AG,1 Paredes-López O,4 Del Villar-Martínez AA1* 1Centro de Desarrollo de Productos Bióticos del IPN. Km. 6 Carretera Yautepec-Jojutla, Calle Ceprobi 8, Col. San Isidro, Yautepec, Mor. México, C.P. 62731. 2Escuela Nacional de Ciencias Biológicas del IPN, México, 3Universidad de Sevilla, Facultad de Farmacia, Sevilla, España, 4Centro de Investigación y de Estudios Avanzados del IPN, México. *Correspnding autor: Del Villar Martínez Alma Angélica. Centro de Desarrollo de Productos Bióticos del IPN (CEPROBI-IPN). Km. 6 Carretera Yautepec-Jojutla, Calle Ceprobi 8, Col. San Isidro, Yautepec, Mor. C.P. 62731. México. Phone: +52 (735) 394 2020, Fax +52 (735) 394 1856. e-mail: [email protected] 2 Abstract Marigold (Tagetes erecta) flower has been used in industry because of its high pigment content. Flower color development implies chloroplast-chromoplast transition associated to carotenoids biosynthesis. In this work, undifferentiated pigmented marigold cells were recovered by somaclonal variation and analyzed by transmission electron microscopy in order to observe the pigment accumulating structures. Callus were achieved from leaf explants and developed on MS medium added with 2, 4-D and BA. After several rounds of recurrent selection for pigmented calli, green, yellow and brown (oxidized) calli were obtained. The carotenoids profile changed in the obtained calli, at green material violaxanthin, lutein, zeaxanthin and -carotene were produced, while yellow calli produced mainly lutein (80%) as well as the brown calli (30%). Chloroplast-chromoplast transition was followed by measuring plastids size and shape of undifferentiated marigold cells development by digital image analysis. Cellular alterations were evident in brown callus. Chloroplasts were the main structure in green callus, and in yellow calli were clearly observed the formation of plastoglobules, correlating with a chloroplast-chromoplast transition. The high number of plastoglobulli observed in yellow callus, might be directly related to pigment synthesis and accumulation. Morphometrical analysis showed that chromoplasts of T. erecta cells are a combination of the globular and reticulotubular type. Key words: calli, chromoplast, marigold, TEM 3 Introduction Plant tissue culture has been used as an important technique for the study of different cell processes contributing to simplify the breeding procedures and overcome some agronomic and environmental problems (Rout et al. 2006; Vanegas et al. 2002). Added to these important facts, these tools have not been exploited for studies at subcellular level. In specialized tissues, it has been reported the differentiation of plastids during plant development, also was found a correlationship between pigmentation and chloroplast to chromoplast differentiation (Suzuki et al. 2005; Vázquez-Caicedo et al. 2006). This process is associated with chlorophyll degradation and synthesis of new pigments; carotenoids accumulation is carried out in chloroplastic specialized structures named plastoglobules and the development of new pigment-bearing structures was observed (Bréhélin et al. 2007; Vothknecht and Soll, 2005). In terms of subcellular analysis, development of plastids in specialized tissues of some species has been reported. In these studies, during cell development the growth phase is accompanied with plastid division, as well as structural and biochemical changes yielding several interconvertible plastid types of specialized functions (Bonora et al. 2000; Bréhélin et al. 2007; Vásquez-Caicedo et al. 2006). Chromoplasts mostly occur in mature tissues and are generally classified as globular, tubular, reticulotubular, membranous, and crystalline types (Vásquez-Caicedo et al. 2006). The carotenoids may differ in their physical properties and bioavailability according to the underlying chromoplast structure. For instance, the crystalline structure of the chromoplast is enhancing the stability of the all-trans configuration (Welsch et al. 2000). Marigold (Tagetes erecta) is appreciated because of its pigmented flowers, they are traditionally used in folk ceremonies and traditional medicine; moreover, they have been 4 linked to some health benefits, such as prevention of cancer, cardiovascular diseases and they are an important source of carotenoids for the food industry. In marigold, the main pigment that accumulates in flowers is lutein, and the color differences between varieties are the result of differences in lutein content (Delgado-Vargas and Paredes-López 2003; Del Villar-Martínez et al., 2005). In subcellular analysis, changes that occurred during plastid differentiation in flower morphogenesis have been analyzed. Also it was demonstrated pigment deposition in specific structures (lipidic vesicles) during flower development (Del Villar-Martínez et al., 2005). Although tissue culture systems for Tagetes have been established, no studies in undifferentiated pigmented cells or subcellular analysis have been conducted so far. It would be important to generate, trough somaclonal variation or genetic manipulation, pigmented undifferentiated cells in order to propose the use of marigold as a bioreactor. However, to validate this assumption, the chromoplast ultrastructure in Tagetes erecta undifferenciated cells remained to be elucidated. The structure and changes that are generated in chloroplast-chromoplast transition in these cells have not been deeply analyzed yet. In this work, undifferentiated pigmented marigold cells were analyzed by transmission electron microscopy in order to observe the pigment accumulating structures and morphometrical characteristics. Materials and Methods Plant material and pigmented calli selection Undifferentiated marigold (Tagetes erecta) cells were obtained from leaf explants cultivated in MS medium (Murashige and Skoog 1962) added with 2,4-D (2.0 mg/L) and BA (2.0 mg/L), and incubated in a dark growth chamber (25±2° C). In order to identify pigmented cells, the culture was separated in small fractions and subcultured several times 5 in the same medium (Figure 1). During calli culture (from seventh round), differentially pigmented portions were observed (yellow, green, and brown). Stable pigmented cell lines were obtained, and re-growth in a fresh medium. For HPLC analysis, samples of the selected lines were frozen in liquid nitrogen and store at -70ºC until use. For ultrastructure analyses of each cell type, fresh samples were collected for the treatment. Carotenoid determination by HPLC Carotenoids were extracted from calli with different pigmentation, according to DelgadoVargas and Paredes-López (1996). Fresh tissue (2g) samples were extracted with HEAT (hexane:absolute ethanol:acetone:toluene, 10:6:7:7 v/v/v/v) and 40% methanolic KOH solution. The HPLC analysis was carried out on an Agilent 1100 system consisting of a quaternary pump, a photodiode array detector, a column temperature control module and an autosampler, which was set to draw 20 µL from the samples (Agilent, Palo alto, California) The pigments were separated on a YMC 30 column (5 µm, 250 x 4.6 mm) (YMC, Wilmington, NC) kept at 17 ºC. Methanol (MeOH), methyl-ter-butil ether (MTBE), and water were used in the mobile phase. The linear gradient elution was the same as described by Meléndez-Martínez et al (2007): 0 min. 90% MeOH + 5% MTBE + 5% water; 12 min, 95% MeOH + 5% MTBE; 25 min, 89% MeOH + 11% MTBE; 40 min, 75% MeOH + 25% MTBE; 60 min, 50% MeOH + 50% MTBE; 62 min, 90% MeOH + 5% MTBE + 5% water. MeOH and MTBE contained small portions of butylated hydroxytoluene and trietylamine (0.1% and 0.05%, respectively) to protect the carotenoids over the HPLC analysis (Hart and Scott, 1995). The mobile phase was pumped at 1 ml / min, and the chromatograms were monitored at 430 nm. Pigments were identified by analyzing their chromatographic and UV/vis spectroscopic characteristics with standards isolated from appropriated sources as described by Meléndez-Martínez et al (2007) 6 Tissue sampling and microscopy (TEM) Small pieces of marigold callus were cut and fixed for 1 h with 2.5% glutaraldehyde in sodium cacodylate buffer (0.05 M). After fixation, samples were washed 5 min in washing solution (sucrose 0.25 M in the same buffer). Post-fixation was carried out in OsO4:bidestiled water solution (1:50 w/v) for 1 h. Tissue samples were washed and dehydrated through graded (10%, 10 min steps) ethanol series from 50% to absolute; propylene oxide was the intermediate solvent for infiltration for 20 min. The first infiltration step was carried out with propylene oxide and EPONTM resin (2:1 v/v) for 1 h. A second infiltration step was carried out with propylene oxide and resin (1:1 v/v) for 1 h. Finally, propylene oxide and resin (1:3 v/v) for 1 h. After infiltration steps, embedded samples were transferred to embedding moulds filled with fresh undiluted resin and polymerized in a convection oven at 60º C for 24 h. To select samples for TEM observation, 200-500 nm thick sections were obtained with ultramicrotome. Thin sections were attached to a 200-mesh copper grid, contrasted with lead citrate and uranyl acetate, and observed at 60 kV in a JEOL 1010 transmission electron microscope (JEOL, Inc., Boston, USA). Each image was recorded in Tiff format with size of 640 X 480 pixels. Digital image analysis and morphometrical parameters evaluation The captured images were processed according the method proposed by Chanona et al. (2003). Corel Photo Paint software (Corel Draw V11.0, Corel Corporation, USA) was used for this purpose. First, images were transformed to a gray scale of 8 bits in a bit map format (*.bpm extension). Intensity, brightness and contrast of transformed images were adjusted in order to achieve a precise definition of the border of plastids (perimeter). Afterwards, 7 plastids images were collected as individual objects, copied and transfer to a new template of 640 x 480 pixels. Images were binarized (2 bits) as shows in Figure 2. Individual plastids were analyzed with the Sigma Scan Pro software (V 5.0, SPSS Inc., USA), the morphometrical parameters evaluated were: a) perimeter (P), which is the longest distance between the pixels that delimitate the border of the shape; b) area (A), it corresponds to pixels contained by the object; c) shape factor (Sf), given by A)/(P2) in which Sf = 1 corresponds to a circle or round shape; d) compacity factor (Cf), defined as P2/A, which correspond to a circ ; this factor gives useful information on the rugosity of the perimeter, and therefore on the irregularity of an object; and, e) elliptic factor, determined by the maximal and minimal length relationship of the object (SSPS 1999). Results and Discussion Somaclonal variation and pigment production Somaclonal variation is an interesting toll used for the recovery of material with desired characteristics, such as biotic and abiotic stress resistance or metabolite production. In the technique is used the natural cell response and adaptation to different factors. We screened the cultures for pigment production using a repetitive growth of the calli trying to expose the natural variation of the cultures. After seven rounds of cultivation early differentiated cells were observed. Then small sections of calli were isolated and established as differentiated cultures. The stability of the selected cells was maintained after several rounds of growth. At this time the analysis for pigment content and subcellular structures were done. 8 Pigment production In order to explore the changes in carotenoids content at the undifferentiated pigmented lines (Table 1,), the first step was to analyze the carotenoid profile of each tipe of calli (Figure 2). The analysis was established with standards, violaxantin, lutein, zeaxantin and -carotene were eluted at 19. 52, 23.49, 25.77 and 48.01 minutes. The HPLC profile of an extract from the green calli is shown in the Figure 2A; two mayor peaks eluted early; a peak was seen to elute at 19 minutes similarly to violaxantin, and a second peak eluted to 23.4 minutes, similar to the lutein. Two other peaks with a smaller area elute later at 26.5 and 47.9, corresponding to zeaxantin and -carotene. The HPLC profile for the yellow calli (Figure 2B) shown the presence of a main peak at 23.6 min and the small one at 26.7 minutes of elution, corresponding to lutein and zeaxantin. For the brown material (oxidized) only a small peak near to 23 min was observed, which could correspond to lutein (data not shown). The advanced developmental stage and cell disorganization of the cells may contribute to the degradation of the carotenoids. Table 1 resumes the results for carotenoids analysis, as seen, some important differences are notorious in the carotenoids profiles in the different selected calli, at green four different carotenoids, at yellow just two carotenoids. A notorious change in lutein content is evident between different lines, in green calli a 0.17 ug/g FW concentration was determined, while in the yellow line 0.64 ug/g FW was found. It represents a 350% in the lutein concentration (increase of 3.5 times). In brown calli (oxidized) the lutein content diminished to 0.05 ug/g FW, that correspond to 20% (a decrese of 5 times) of the lutein content at green calli. In green calli, violaxantin and lutein are the predominant carotenoids while in the yellow selected materials, the main carotenoid was lutein. In marigold flowers 15 - Meléndez-Martínez AJ, Vicario IM, Heredia FJ (2007) Carotenoids, color, and ascorbic acid content of a novel frozen-marketed orange juice. J Agric Food Chem 55:13471355 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plantarum 15:472-497 Rout GR, Mohapatra A, Mohan-Jain S (2006) Tissue culture of ornamental pot plant: A critical review of present scenario and future prospects. Biotechnol Adv 24:531-560 Seemann P, Barriga P (1993) Utilización de técnicas de micropropagación. Avances en producción y sanidad vegetal. Cultivos no tradicionales. Ed Universitaria. 230 Sitte P, Falk H, Liedvogel B (1980) Chromoplasts. In: Pigments in Plants. (Ed) Czygan F. 2nd ed. Gustav Fischer Verlag, Stuttgart Inc USA Suzuki Y, Kimura T, Takahashi D, Terai H (2005) Ultrastructural evidence for the inhibition of chloroplast to chromoplast conversion in broccoli floret sepals by ethanol vapor. Postharv Biol Tech 32:237-243 Vanegas PE, Cruz-Hernández A, Valverde ME, Paredes-López O (2002) Plant regeneration via organogenesis en marigold. Plant Cell Tiss Org 69:279-283 Vásquez-Caicedo AL, Heller A, Neidhart S, Carle R (2006) Chromoplast morphology and J Agr Food Chem 54:5769-5766 Vishnevetsky M, Ovadis M, Vainstein A (1999) Carotenoid sequestration in plants: The role of carotenoidassociated proteins. Trends Plant Sci 4:232-235 Vothknecht UC, Soll J (2005) Chloroplast membrane transport: interplay of prokaryotic and eukaryotic traits. Gene 354:99-109 16 Vothknecht UC, Westhoff P (2001) Biogenesis and origin of thylakoid membranes. Biochim Biophys Acta 1541:91-101 Welsch R, Beyer P, Hugueney P, Kleining H, von Lintig J (2000) Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis. Planta 211:846-854 19 Figure 1. Undifferentiation process on leaf explant of Tagetes erecta a) leaf cutting, b) Leaf explant on culture media. Inset: undifferentiating explant, c) Undifferentiated tissue. Figure 2: Reversed-phase C30 of undiferentiated marigold cells. A) Profile from an extract of green calli; 1: violaxanthin, 2: lutein, 3: zeaxanthin, 4: b-carotene, 5: esterified carotenoids. B) Profile from an extract of yellow calli; 1:lutein, 2: esterified carotenoids. Detection was carried at 450 nm Figure 3. Plastids found in different pigmented marigold calli . a) Green callus, (cloroplasts) b) Yellow callus (chromoplasts) c) Brown callus (No plastid found). ic: intercellular space, chl: cloroplast, chr: chromoplast, pg: plastoglobuli, mi: mitocondrium. bar represents 500 nm. Figure 4. TEM pictures of marigold undifferentiated cells. a) panoramic view of whole cell, b) panoramic view of three neigborg cells, c) proplastid appereance, d) chloroplast presence, e) chromoplast and motochondirium view. v: vacuole, n: nucleous, ic: intercellular space, pp: proplastid, cl: cloroplast, cr: chromoplast, pg: plastoglobuli, mi: mitochondrium (bar a:10 µm; b: 2 µm, c, d, e: 500 nm). Figure 5. Morphological changes occurring along plastid development in marigold (Tagetes erecta) calli. a) proplastid; b) chloroplast; c) intermediate stage; Proplastid-chromoplast; d) intermediate stage, chloroplast.-chromoplast; e) chromoplasts. (bar = 500 nm). ic: intercellular space, pp: proplastid, chl: cloroplast, chr: chromoplast, pg: plastoglobuli, mi: mitocondrium. 20 Figure1 21 1 2 Figure2 A) mA 17.5 A B S O R 10 B A N C E 0 B) mA 20 A B S O R 10 B A N C E 0 1 2 3 4 5 6 min 1 2 3 4 5 6 RETENTION min 1 2 5 3 4 5 22 c) a) b) Figure 3 23 Figure 4