Distribution of Calcium in Fruit Tissues of Loquat Wenpei Song 1, Huicong Wang 1, Shunquan Lin 1, Xuming Huang 1 and Xizhen Zhu 2 1 College of Horticulture, South China Agricultural University, Guangzhou 510642, China 2 College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China Keywords: Eriobotrya japonica, calcium mapping, fruit development, calcium oxalate, pedicel Abstract Fruits are a good source of various minerals for humans. Calcium in fruits is important for normal development and quality maintenance. The present study examined calcium distribution in different fruit tissues of Zhaozhong No. 6 loquat (Eriobtorya japonica Lindl.). Calcium concentration in young fruit was high but declined throughout fruit development, while in the pedicel calcium concentration was relatively constant and became far higher than in fruit flesh and seed as fruit matured. Calcium mapping in the flesh using X-ray microanalyzer revealed that a larger amount of Ca was distributed in the outer portion or exocarp, where a large number of calcium-rich particles were found in young fruit. These calcium-rich particles disappeared as fruit grew and matured. Within the pedicel, calcium was more abundant in the phloem and pith than in the cortex and xylem. There were a lot of calcium oxalate crystals, either in styloids or druses, in the phloem and pith cells neighboring the xylem and fiber clusters, where Ca level was the lowest. There was a good positive correlation between Ca content in the pedicel and that in the flesh. It was concluded that Ca is likely transported to fruit through the phloem tissues and that the formation of calcium oxalate in the pedicel may not interfere with calcium transport to fruit. INTRODUCTION Calcium is one of the essential macro-minerals in plants, playing three irreplaceable roles: (1) structural role, where calcium participates the construction of cell walls and membranes; (2) signaling role, where calcium serves as a signal involved in plant responses to environmental and developmental cues; (3) ion balancing role, where calcium sequesters and detoxifying oxalate (White and Broadley, 2004; Hepler, 2005). Fruits are terminal organs with succulent tissues and poorly developed vascular system and are thus highly susceptible to calcium deficiency that results in various symptoms associated with quality loss (Shear, 1975). As an important structural element in bones and teeth, Ca plays a crucial role in human well-being. Fresh fruits are a good source of this mineral (Naazir, 2013). There is dispute over the pathway of calcium transport towards fruit. It is generally believed that calcium is fed to fruit via xylem (Saure, 2005). However, there are authors suggested that calcium transport to fruit depends upon phloem pathway (Himelrich and McDuffie, 1983). Distribution of calcium in fruit tissues might reflect the pathway of calcium transport. Electron probe microanalyzer (EPMA) enables in situ observation of element distribution in micro regions of plant tissues. We used this technique to observe the changes in Ca distribution pattern in litchi fruit (Huang et al., 2005; Huang et al., 2006). The results showed that fruit pedicel had a significantly higher Ca concentration than the pericarp and in the pericarp, epidermic cells contain rich calcium. In this study, changes in calcium distribution in loquat fruit tissues were studied using EPMA as well as quantitative measurements of calcium concentrations in different tissues. Proc. IV International Symposium on Loquat Eds.: R. Lo Bianco and J. Janick Acta Hort. 1092, ISHS 2015 235
MATERIALS AND METHODS Materials Thirty fruit were harvest from a 10-year-old Zaozhong No.6 tree on January 10, February 15, and March 17, i.e., 40 (before rapid growth phase), 76 (during rapid growth phase), and 108 (ripening phase) days after anthesis. Fifteen fruit were weighed and dissected into flesh, seed and pedicel. Fresh weights of the tissues from individual fruit were collected before they were dried in an oven at 70 C for 72 h. Dried samples were used to measure calcium content. Five of the fruit were used for calcium mapping analysis with EPMA. Fresh pedicels at a length of 0.5 cm of the remaining 10 fruit were used to analyze calcium in different forms. Methods For determination of total calcium contents in the pericarp, seed and pedicel, the dried tissues were ground into powder. Then 0.1 g of the powder was transferred to a 10 ml melting pot, burnt on an electric stove until smoking stopped, and ashed in an ashing furnace at 550 C for 5 h. The ash was dissolved with 0.1 N HCl solution and set to 25 ml for measuring Ca concentration using a Hitachi Z-5000 atomic absorption spectrometer. For observation of in situ calcium distribution in fruit, pedicels or pericarp from 3 fruit were cut into 0.1 mm thick slices, stuck onto a cupper sample stand with electricity-conductive carbon glue, and coated with platinum in a JFC-1600 vacuum autocoater for 90 s. The structure and calcium distribution were observed with a JXA-8100 electron probe microanalyzer. During the analysis, working distance of all samples was kept at 11 mm, with a probe accelerating voltage of 20 kv and an exciting current of 2 10-8 A. Under a magnification of over 100, secondary electron image of the samples and map image of calcium-characteristic X-ray signal (calcium mapping image) were separately collected. Intensity of calcium-characteristic X-ray signal which reflects calcium abundance was displayed by density of bright dots. Soluble calcium, structural calcium in the cell walls and calcium oxalate in the pedicel were extracted with water 2% (v/v) solution of acetic acid and 5% 5% hydrogen chloride solution, respectively, according to a step-wise procedure modified from Chen and Uemoto (1976). The calcium content different fractions were measured with an atomic absorption spectrometer (Hitachi Z-5000) using 1, 2, 5 and 10 mg L -1 CaCl 2 in distilled water, 2% (v/v) glacial acetic acid solution and 5% hydrogen chloride solution as standards for water soluble Ca, structural Ca and Ca oxalate, respectively. Samples from 10 fruit (n=10) were analyzed. RESULTS AND DISCUSSION Calcium concentration was highest in fruit flesh in the early stage of its development. It declined constantly in the flesh and seed during fruit growth, while in the pedicel it was relatively constant (Fig. 1A). As a result, at the end of the experiment the pedicel had a significantly higher calcium concentration than the flesh and the seed. Similar result was reported in litchi (Huang et al., 2006). However, calcium content on per fruit basis increased in all tissues with time (Fig. 1B), suggesting constant accumulation of calcium during fruit development. The decrease in calcium concentration in the flesh and seed was therefore, a result of dilution effect due to fruit expansion. Calcium was less distributed in the inner portion of the flesh than the outer portion (Fig. 2), where a lot of calcium-rich particles were observed. As fruit developed, calciumrich particles became sparse and most of them disappeared as fruit matured. In the pedicel, calcium abundance was always greater in the phloem than in the xylem (Fig. 3), indicating that phloem might be an important pathway for calcium transport. A large number of calcium-rich bodies could be observed in the phloem, while they were much less seen in the xylem. Crystal structures could be found in the calciumrich sites, suggesting these crystals were calcium oxalate, which is the most common 236
form of crystals found in plant cells (Webb, 1999). Concentration and composition of calcium in different forms in the pedicel were analyzed. The results (Fig. 4) showed that water soluble calcium was the lowest form. Its concentration and percentage declined during fruit development. There was an equal amount of structural calcium and oxalate calcium in the young fruit sampled on January 10. In premature fruit sampled on February 15, structural calcium became the predominant form. However, in mature fruit sampled on March 17, calcium in oxalate turned to be the major form. The results suggest that considerable amount of calcium in the pedicel was sequestered by forming crystals of calcium oxalate, which contributed the major part of calcium accumulated in the pedicel. This suggestion generates one question. Does calcium sequestration in the pedicel interfere the calcium transport towards fruit? To answer this question, correlation of the amount of calcium accumulated in the pedicel and the content of calcium in fruit was analyzed. The results showed that there was a significant positive correlation (R 2 =0.527, P<0.001) between calcium contents in the pedicel and in the fruit, indicating that calcium sequestration in the pedicel might not interfere with calcium transport to fruit, instead it might have a positive role in calcium uptake by fruit. In litchi, we drew a similar conclusion that the presence of oxalate in the pericarp and fruit pedicel is not linked to a shortage of fruit calcium. Formation of calcium oxalate in fruit calcium uptake awaits further study. ACKNOWLEDGEMENTS This study was fund supported by the National Natural Science Foundation of China (31372009). Literatures Cited Chen, W.S. and Uemoto, S. 1976. Studies on calcium absorption in vegetable crops. I. The absorption and physiological significance of calcium in vegetative and reproductive phases of plant growth. J. Japan. Soc. Hort. Sci. 45:33-42. Hepler, P.K. 2005. Calcium: a central regulator of plant growth and development. Plant Cell 17:2142-2155. Himelrich, D.G. and McDuffie, R.F. 1983. The calcium cycle: uptake and distribution in apple trees. HortScience 18:147-151. Huang, X.M., Wang, H.C., Li, J.G., Yuan, W.Q., Lu, J.M., Huang, H.B., Luo, S. and Yin, J.H. 2006. The presence of oxalate in the pericarp and fruit pedicel is not linked to a shortage of fruit calcium and increase in cracking incidence in litchi. J. Hort. Sci. Biotechnol. 81(2):231-237. Huang, X.M., Yuan, W.Q., Wang, H.C., Li, J.G., Luo, S., Yin, J.H. and Huang, H.B. 2005. A study of calcium accumulation and distribution in the pericarp of litchi cultivars differing in cracking resistance. Acta Hort. Sinica 32(4):578-583. Naazir, S. 2013. http://www.livestrong.com/article/248315-fruit-sources-of -calcium -ina-vegetarian-diet. Saure, M.C. 2005. Calcium translocation to fleshy fruit: its mechanism and endogenous control. Scientia Hort. 105:65-89. Shear, C.B. 1975. Calcium-related disorders of fruits and vegetables. HortScience 10:361-365. Webb, M.A. 1999. Cell-mediated crystallization of calcium oxalate in plants. Plant Cell 11:751-761. White, P.J. and Broadley, M.R. 2003. Calcium in plants. Ann. Bot. 92:487-511. Wilsdorf, R.E., Lotze, E., Mesjasz-Przybylowicz, J. and Przbylowicz, W.J. 2012. Mapping the distribution of calcium on apple tissue with proton-induced X-ray emission-after application of additional pre-harvest foliar or soil calcium. Acta Hort. 984:347-356. 237
Figures Fig. 1. Changes in calcium concentrations (A) and content (B) in different tissues of loquat fruit. Error bars indicate standard errors of means (n=15). 238
A B C D E F Fig. 2. Calcium distribution in flesh tissue of loquat fruit. A, C and E are the secondary electron images of flesh tissue of fruit sampled on January 10, February 15 and March 17, respectively; B, D and F are the calcium mapping images corresponding to tissues displayed in A, C and E respectively. 239
D A G Pith Pith Cortex Cortex B Pith E Pith H Fiber C Fiber F I fiber Ploem fiber Ploem Fig. 3. Distribution of calcium in the pedicel of loquat fruit. A, B and C are the secondary electron images of pedicel tissue from fruit sampled on January 10, February 15 and March 17, respectively; D, E and F are the calcium mapping image in pedicel tissues displayed in A, B and C respectively. G, H and I are pedicel tissues with detailed structures in samples taken on January 10, February 15 and March 17, respectively. Arrows show crystal structures that are likely calcium oxalate. 240
A B Fig. 4. Concentrations (A) and percentages (B) of different forms of calcium in the pedicel. Fig. 5. Linear association between calcium content in the pedicel and that in the fruit harvested at different stages. Each data point represents the value from a single fruit. 241
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