Correlation between Fruit Characters and Degree of Polysomaty in Fruit Tissues of Capsicum

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1 J. Japan. Soc. Hort. Sci. 79 (2): Available online at JSHS 2010 Correlation between Fruit Characters and Degree of Polysomaty in Fruit Tissues of Capsicum Daisuke Ogawa 1 *, Keiko Ishikawa 1, Osamu Nunomura 1 and Masahiro Mii 2 1 Japan Horticultural Production and Research Institute, Matsudo , Japan 2 Plant Cell Technology Laboratory, Faculty of Horticulture, Chiba University, Matsudo , Japan The correlation between several fruit characters and the degree of polysomaty, i.e., the number of peaks in flow cytometric analysis, was examined using mature fruits of 12 genotypes from three species of Capsicum. Capsicum chacoense PI260429, which had the smallest fruit with the thinnest pericarp, showed the least number of peaks in ploidy levels, i.e., four ploidies ranging from 2C to 16C, whereas Capsicum annuum Édes alma had the thickest pericarp and the highest peak numbers of eight ploidy levels, ranging from 2C to 256C. Among the morphological traits of fruit examined, pericarp thickness showed the highest correlation with the degree of polysomaty (r = 0.88). Key Words: Capsicum, flow cytometory, fruit characters, polysomaty, Solanaceae. Introduction Capsicum fruits are used worldwide as chili peppers, vegetables, folk medicines, and as a source of food additives. Corresponding with their various uses, Capsicum fruits also vary in size and shape, and are classified into several categories, such as elongated, oblated, round, conical, campanulate, and bell (or blocky) types (Andrews, 1995). Fruit length varies from the tiny ovoid Chiltecpin (0.9 cm) to the extremely long Anaheim (33.0 cm) (Andrews, 1995). Fruit morphology was once used by taxonomists as the principal means to classify domesticated Capsicums (Hirose et al., 1956), but is of little taxonomic value at present because of parallel variations that have occurred among different species (Andrews, 1995). Irrespective of these taxonomic problems, fruit size and shape have constantly been important as agricultural traits for breeding. Recently, many reports have been published on the relationship between organ size and DNA contents of cell nuclei determined by flow cytometry analysis. DNA contents of cell nuclei are considered to directly correlate with cell volume and organ size (Mizukami, 2001) and to increase by DNA replication without nuclear and cell division, which is called endoreduplication. The direct correlation between endoreduplication and cell volume Received; July 24, Accepted; October 16, * Corresponding author ( d-ogawa@enken.or.jp). was shown by Hase et al. (2000) using Arabidopsis mutant FRILL1 (FRL1). Although the distal portion of wild-type Arabidopsis petal epidermis consists of diploid cells, cells in the same part of the frl1 mutant exhibit abnormal endoreduplication during development, giving rise to enlarged mutant petals with large polyploid cells. Kudo and Kimura (2002) examined the DNA contents of nuclei from different stages of petal tissues in cabbage and showed that the proximal part of the petal was composed of enlarged cells with highly endoreduplicated nuclei, while cells the distal part of the petal contained normal diploid nuclei. Polysomaty has been observed in fruits of the Solanaceae. In tomato, nuclei in the cells of young green fruits were predominantly 2C and 4C, whereas mature red fruits predominantly contained cells with nuclei of as high as 256C (Bergervoet et al., 1996; Joubes et al., 1999). The diameter of the nuclei of tomato pericarp tissues also increased about six times with the growth of fruits from the young green stage to the red mature stage. Tomato fruits are spherical, and these reports showed that the cells of tomato fruit have increased levels of polysomaty as they increased size. As a member of the Solanaceae, Capsicum is also expected to have high levels of polysomaty. Comparing with tomato and other Solanaceae plants, Capsicum fruits have considerably wide variations in size and shape; therefore, it is interesting to clarify the relationship between polysomaty levels and the variation of fruit characters. In this study, we investigated the 168

2 J. Japan. Soc. Hort. Sci. 79 (2): correlation between several fruit characters and nuclear DNA contents of the cells in the fruit pericarp in 12 Capsicum genotypes, including 10 genotypes of Capsicum annuum, one genotype of Capsicum chacoense PI and Capsicum frutescens LS1839 (Fig. 1). Materials and Methods Plant materials The seeds of 11 Capsicum cultivars or strains including Capsicum chacoense Plant Introduction (PI) , Capsicum frutescens LS1839 (National Institute of Vegetable and Tea Science, Ano, Japan), Capsicum annuum L (inbred line of Japan Horticultural Production and Research Institute (JHPRI)), Takanotsume (Sakata Seed Co., Yokohama, Japan), Nikkou-tougarashi (Tohoku Co., Utsunomiya, Japan), Fig. 1. The various fruits used in the present study. A) Capsicum chacoense PI , B) C. frutescens LS1839, C) C. annuum 5151, D) C. annuum Takanotsume, E) C. annuum Nikkoutougarashi, F) C. annuum Yatsubusa, G) C. annuum Shishitou No. 562, H) C. annuum Fushimi-tougarashi, I) C. annuum Cherry sweet, J) C. annuum Oh-natsume, K) Paprika, L) C. annuum Édes alma Yatsubusa (Sakata Seed Co.), Shishitou No. 562 (inbred line of JHPRI), Fushimi-tougarashi (Tohoku Co.), Cherry sweet (Stokes Seed Ltd., Ontario, Canada), Oh-natsume (inbred line of JHPRI), and Édes alma (Zöldségtermesztési Kutató Intézet, Hungery) were sown in trays in August. After two weeks, the seedlings were transplanted to pots (ca. 0.6 L). After one month, the plants were planted into ground soil in a greenhouse where the ambient temperatures were 30 C maximum and 18 C minimum. They were grown for nine months, and mature fruits were obtained. In addition, mature fruits of bell-type pepper imported from The Netherlands, which are commonly called Paprika pepper in Japan, were purchased at the local market. C. chacoense PI and C. frutescens LS1839 were selected because of their fruit size, which is ranked as the smallest group among the various Capsicum genotypes. After the analysis of fruit characters and polysomaty status using these fruit materials, three genotypes (C. chacoense PI260429, C. annuum Shishitou No. 562, and Oh-natsume ) were cultivated for three successive years to confirm the stability of the fruit characters and polysomaty status. Measurement of fruit characters Fruit characters, i.e., length, diameter, fresh weight, and thickness of the pericarp of mature fruit were measured for each cultivar. Length and diameter were measured in millimeters as the length from the highest part of the shoulder to the bottom and that at the widest part of the fruit. Pericarp thickness was measured at the middle part of the height. Up to 25 fruits were measured with a minimum of six fruits (PI and 5151, n = 20; LS1839, n = 15; Takanotsume ; n = 22, Nikkoutougarashi ; n = 16, Yatsubusa ; n = 25, Shishitou No. 562 ; n = 14, Fushimi-tougarashi, Oh-natsume ; n = 6, Paprika ; n = 8, Cherry sweet, and Édes alma ; n = 18) (Table 1). Table 1. Fruit characters of 12 genotypes in Capsicum. Genotypes z Length (mm) Diameter (mm) Fresh weight (g) Pericarp thickness (mm) PI ± 0.2 y 6.7 ± ± ± 0.0 LS ± ± ± ± ± ± ± ± 0.0 Takanotsume 55.8 ± ± ± ± 0.1 Nikkou-tougarashi ± ± ± ± 0.2 Yatsubusa 63.1 ± ± ± ± 0.1 Shishitou No ± ± ± ± 0.1 Fushimi-tougarashi ± ± ± ± 0.2 Cherry sweet 34.5 ± ± ± ± 0.2 Oh-natsume 71.8 ± ± ± ± 0.8 Paprika 89.8 ± ± ± ± 0.3 Édes alma 49.1 ± ± ± ± 0.3 z PI and 5151, n = 20; LS1839, n = 15; Takanotsume, n = 22; Nikkou-tougarashi, n = 16; Yatsubusa, n = 25; Shishitou No. 562, n = 14; Fushimitougarashi and Oh-natsume, n = 6; Paprika, n = 8; Cherry sweet and Édes alma, n = 18. y Values are the meants ± SE. (n = 6~25 as described z)

3 170 D. Ogawa, K. Ishikawa, O. Nunomura and M. Mii Flow cytometric analysis The ploidy level of pericarp cells of mature fruit was analyzed by flow cytometry (Partec PA cytometer, Partec, Münster, Germany) according to the method described by Galbraith et al. (1983). The fruits investigated for measurement of objective characters were all used for flow cytometric analysis (Table 1). To release nuclei, pericarp tissue of ca. 0.5 mm 0.5 mm was excised from the midpoint of the fruit height and chopped with a razor blade in 0.2 ml of solution A of Plant High Resolution DNA kit type P (Partec) in a plastic Petri dish. Then, 1.0 ml of staining solution consisting of 10 mm Tris, 50 mm sodium citrate, 2 mm MgCl 2, 1% (w/v) PVP, 0.1% (v/v) Triton X-100, and 2.0 mg L 1 4',6-diamidino-2-phenylindole (DAPI), ph 7.5 (Mishiba and Mii, 2000) was added to the sample solution, which was then filtered with 30 μm nylon mesh to remove cell debris. After incubating for 5 min, the stained nuclear suspension was subjected to flow cytometric analysis to determine the relative nuclear DNA contents on a semilogarithmic scale histogram in which the histogram peaks are evenly distributed along the abscissa (Gilissen et al., 1994) and the height of each peak corresponds to the number of nuclei at each ploidy level. At least 5000 nuclei were counted for each sample to obtain the ratio of the cells at each ploidy level. To determine the standard peak position of 2C nuclei, the 2C peak of young leaves was analyzed. Statistical analysis and comparison of cell size Correlation between four parameters of fruit characters, i.e., fruit length, diameter, fresh weight and pericarp thickness, and the maximum nuclear DNA content were analyzed by multiple regression analysis. To estimate the cell size of the pericarp, small segments were excised from the pericarp of C. annuum Shishitou No. 562 and Oh-natsume and placed in a drop of fixative solution (4% paraformaldehyde-1% glutaraldehyde in phosphate buffer). Samples were dehydrated through an ethanol series, and prepared by the paraffin-embedding method of Sainte-Marie (1962) with some modifications. Paraffin blocks were sectioned using a microtome (RR- 50, Yamato Kohki Co., Asaka, Japan). The sections were stained with 1% toluidine blue and observed by microscope. The cells were assumed to be round, and the diameter of the cells at the midpoint of the pericarp was measured in micrometers for the fruits of C. annuum Shishitou No. 562 and Oh-natsume. In each genotype, 25 cells were measured for each of the five fruits. Results and Discussion Mature fruit characters of 12 genotypes Among the 12 genotypes examined, C. chacoense PI had the smallest fruit, whose length, diameter, fresh weight, and pericarp thickness were 9.3 mm, 6.7 mm, 0.3 g, and 0.3 mm, respectively (Table 1). C. annuum Nikkou-tougarashi had the longest fruit (139 mm), while C. annuum Oh-natsume had the largest diameter (79.3 mm). In contrast, Paprika had the heaviest fruit (147.6 g fresh weight) and C. annumm Édes alma had the thickest fruit (8.4 mm) (Table 1). Three genotypes (C. chacoense PI260429, C. annuum Shishitou No. 562, and Oh-natsume ) cultivated for three successive years in the same season under the same cultural conditions showed the same fruit characters, indicating there stability. Degree of polysomaty in pericarp cells of mature fruits Our preliminary experiments showed that the ploidy levels of the fruit pericarp increased with fruit development and the maximum ploidy levels were observed at the mature stage (date not shown). Pericarp tissues of mature fruit consisted of cells with a high degree of polysomaty with 4 to 8 peaks in all genotypes examined, and they were classified into five groups, A E, according to differences in the degree of polysomaty, i.e., the maximum number of peaks obtained. Group A consisted of only one genotype, C. chacoense PI260429, which showed the least number of peaks, i.e. four ploidy levels, ranging from 2C to 16C (Figs. 2A and 3). Group B consisted of C. frutescens LS1839 and C. annuum 5151, which had five peaks of DNA contents, from 2C to 32C in pericarp tissues (Figs. 2B and 3). C. annuum Takanotsume, Nikkou-tougarashi, Yatsubusa, Shishitou No. 562 and Fushimitougarashi belonged to Group C, in which pericarp tisssues had six peaks, from 2C to 64C of DNA contents (Figs. 2C and 3). Group D consisted of C. annuum Cherry sweet, Oh-natsume and Paprika, and their pericarp tissues had seven peaks, from 2C to 128C of DNA contents (Figs. 2D and 3). Group E contained only C. annuum Édes alma, which had the maximum number of 8 peaks, up to 256C (Figs. 2E and 3). The results of three successive years showed that the degree of polysomaty and the ratio of nuclei with different ploidy levels in the pericarp were the same as those obtained in the first year. Despite the variation in the degree of polysomaty in the fruit, pepper leaves showed a small range of polysomaty from 2C to 8C (data not shown) in all cultivars tested. In Solanaceae, tomato fruits were also highly polysomatic and mature fruits showed a maximum DNA peak of 256C (Bergervoet et al., 1996; Joubes et al., 1999), which coincided with the maximum ploidy level of Group E, C. annuum Édes alma. Our results also indicated that tomato fruits of smaller size had the lower degree of polysomty. Correlation between fruit characters and the degree of polysomaty C. chacoense PI260429, which had the smallest fruit, showed the lowest level of polysomaty up to 16C DNA contents, and the highest level of polysomaty, up to 256C, was observed in C. annuum Édes alma, which

4 J. Japan. Soc. Hort. Sci. 79 (2): Fig. 2. Representative histogram of the ploidy distribution of nuclei in mature fruits of pepper. A) C. chacoense PI260429, B) C. frutescens LS 1839 and C. annuum 5151, C) C. annuum Takanotsume, Nikkou-tougarashi, Yatsubusa, Shishitou No. 562, and Fushimi-tougarashi, D) C. annuum Cherry sweet, Oh-natsume, and Paprika, E) C. annuum Édes alma Fig. 3. Frequencies of nuclei with different ploidy levels in the fruits of 12 pepper genotypes.

5 172 D. Ogawa, K. Ishikawa, O. Nunomura and M. Mii had the thickest pericarp among the 12 genotypes examined. Statistical analysis revealed that the number of peaks in the pericarp was more strongly correlated with pericarp thickness (r = 0.88) than the length (r = 0.35), diameter (r = 0.75), and fresh weight (r = 0.64) of the fruit (Table 2). These results suggest that fruits with higher levels of polysomaty might have a thicker pericarp and tend to become larger than those with lower level of polysomaty. Since pericarp thickness is one of the most important characters for the breeding of vegetable pepper fruits, the selection for fruits with a thick pericarp might have been conducted during the process of domestication. It might consequently have resulted in the selection of genotypes with high polysomaty levels. In order to increase the size of some organs and tissues, there are two possible methods, to increase cell numbers or cell size. Several reports have shown that the final fruit size is related to the number of cells, not to cell size (Cowan et al., 2001; Higashi et al., 1999; Zhang et al., 2006). In the present study, however, microscopic observation revealed that the cell size of the pericarp tissues of C. annuum Oh-natsume was larger than that of C. annuum Shishitou No. 562 (Fig. 4). Cell size expressed as cell diameter, and the cells from middle of the pericarp tissues of C. annuum Oh-natsume and Shishitou No. 562 were about 190 μm and 110 μm, respectively, provided that the cells were spherical (Fig. 4). C. annuum Oh-natsume has a thicker pericarp and a higher degree of polysomaty (128C) than C. annuum Shishitou No. 562 (64C) (Table 1 and Fig. 3). Moreover, C. annuum Oh-natsume had the higher proportion of cells with 32C or higher ploidy levels than C. annuum Shishitou No. 562 (Fig. 3). These results suggest that pericarp thickness was increased by larger cell size with higher polysomaty levels in some groups of Capsicum. Cells with a higher ploidy level usually become larger than diploid cells and consequently give rise to organs of larger size (Kondorosi et al., 2000; Kudo and Kimura, 2002). In Arabidopsis, the hyp7 mutant, which is deficient in endoreduplication with a maximum ploidy level of 8C (Sugimoto-Shirasu et al., 2005), showed a reduced cell size and dwarf morphology compared with wild-type plants with a maximum ploidy level of 32C. In cabbage leaves, the cells of proximal parts showed a higher degree of polysomaty than those of distal part, and cells from the proximal part were larger than from the distal part (Kudo and Kimura, 2002). It is reported that cells with high polysomaty are preferable for containing metabolic substrates and are considered to be suitable sink organs (Dhawan and Lavania, 1996); therefore, the high polysomaty level of the pericarp is also considered to be preferable for nutritional and functional metabolites, and consequently, for highquality fruits. In the present study, the degree of polysomaty of pericarp tissues had a stronger correlation Table 2. Correlation between fruit characters and the number of peaks by multiple regression analysis. Length Diameter Fresh weight Pericarp thickness No. of peaks Length 1.00 Diameter Fresh weight ** Z 1.00 Pericarp thickness ** 0.83** 1.00 No. of peaks ** 0.64* 0.88** 1.00 Z,*,** denote significance at 5% and 1% levels, respectively. Fig. 4. Comparison of cell size of pericarp in pepper fruits. A) C. annuum Shishitou No. 562, B) C. annuum Oh-natsume. The right sides of both sections are the surface of the fruit pericarp, and the left sides of both sections are inside (placentate) the fruit. Bar is 200 µm.

6 J. Japan. Soc. Hort. Sci. 79 (2): with pericarp thickness than other traits such as the length, diameter, and fresh weight of fruits (Table 2). Pericarp was also strongly correlated with the diameter and fresh weight of fruits (r = 0.91 and 0.83, respectively) (Table 2). Multiple regression analysis in the present study showed that the four fruit characters (length, diameter, fresh weight, and thickness of the fruit) and the maximum ploidy levels of the fruit pericarp could be divided into two groups. One group consisted of the length of the fruit. Fruit length had a low correlation with other fruit characters and the peak numbers (Table 2). High correlations were found between the fresh weight and diameter (r = 0.96), thickness and diameter (r = 0.91), and thickness and fresh weight (r = 0.83) (Table 2). Considering the typical fruit characters of the 12 Capsicum genotypes, these two groups are expected to be determined genetically. Genetic analysis between the genotype with a thin pericarp (low ploidy group) and thickness pericarp (high ploidy group), or the investigation of physiological treatments, such as the application of plant growth substances to change the degree of ploidy, will give more profound information on fruit characters. In breeding, it is important to assess fruit characters, which are controlled by genotypes and environmental conditions. Clarifying the factors which determine fruit characters will be useful in critical breeding. Acknowledgements We thank Prof. Takato Koba of the Faculty of Horticulture, Chiba University, for critical suggestions for statistical analysis. Literature Cited Andrews, J PEPPERS. University of Texas Press, Austin, USA. Bergervoet, J. H. W., H. A. Verhoeven, L. J. W. Gilissen and R. J. Bino High amounts of nuclear DNA in tomato (Lycopersicon esculentum Mill.) pericarp. Plant Sci. 116: Cowan, A. K., R. F. Cripps, E. W. Richings and N. J. Tayjor Fruit size: Towards an understanding of the metabolic control of fruit growth using avocado as a model system. Physiol. Plant. 111: Dhawan, O. P. and U. C. Lavania Enhancing the productivity of secondary metabolites via induced polyploidy: a review. Euphytica 87: Galbraith, D. W., K. R. Harkins, J. M. Maddox, N. M. Ayres, D. P. Sharma and E. Firoozabady Rapid flow cytometric analysis of the cell cycle in intact plant tissue. Science 220: Gilissen, L. J. W., M. J. Staveren, J. C. Hakkert, M. J. M. Smulders, H. A. Verhoeven and J. Creemers-molenaar The competence of cells for cell division and regeneration in tobacco explants depends on cellular location, cell cycle phase and ploidy level. Plant Sci. 103: Hase, Y., A. Tanaka, T. Baba and H. Watanabe FRL1 is required for petal and sepal development in Arabidopsis. Plant J. 24: Higashi, K., K. Hosoya and H. Ezura Histological analysis of fruit development between two melon (Cucumis melo L. reticulates) genotypes setting a different size of fruit. J. Exp. Bot. 50: Hirose, T., S. Ukita and S. Takashima Studies on the characteristics of pepper varieties. Sci. Rep. of Saikyo Univ. Agr., Japan. No. 8: (In Japanese). Joubes, J., T. H. Phan, D. Just, C. Rothan, C. Bergounioux, P. Raymond and C. Chevalier Molecular and biochemical characterization of the involvement of cyclin-dependent kinase A during the early development of tomato fruit. Plant Physiol. 121: Kondorosi, E., F. Roudier and E. Gendreau Plant cell-size control: growing by ploidy? Curr. Opin. Plant Biol. 3: Kudo, N. and Y. Kimura Nuclear DNA endoreduplication during petal development in cabbage: relationship between ploidy levels and cell size. J. Exp. Bot. 53: Mishiba, K. and. M. Mii Polysomaty analysis in diploid and tetraploid Portulaca grandiflora. Plant Sci. 156: Mizukami, Y A matter of size: developmental control of organ size in plants. Curr. Opin. Plant Biol. 4: Sainte-marie, G A paraffin embedding technique for studies employing immunofluorescence. J Histochem. Cytochem. 10: Sugimoto-shirasu, K., G. R. Roberts, N. J. Stacy, M. C. Mccann, A. Maxwell and K. Roberts RHL1 is an essential component of the plant DNA topoisomerase VI complex and is required for ploidy-dependent cell growth. Proc. Natl. Acad. Sci. USA 102: Zhang, C., K. Tanabe, S. Wang, F. Tamura, A. Yoshida and K. Matsumoto The impact of cell division and cell enlargement on the evolution of fruit size in Pyrus pyrifolia. Ann. Bot. 98: