Direct evidence for dynamics of cell heterogeneity in watercored apples: turgor-associated metabolic modifications and within-fruit water potential gradient unveiled by single-cell analyses

Abstract

Watercore is a physiological disorder in apple (Malus × domestica Borkh.) fruits that appears as water-soaked tissues adjacent to the vascular core, although there is little information on what exactly occurs at cell level in the watercored apples, particularly from the viewpoint of cell water relations. By combining picolitre pressure-probe electrospray-ionization mass spectrometry (picoPPESI-MS) with freezing point osmometry and vapor pressure osmometry, changes in cell water status and metabolisms were spatially assayed in the same fruit. In the watercored fruit, total soluble solid was lower in the watercore region than the normal outer parenchyma region, but there was no spatial difference in the osmotic potentials determined with freezing point osmometry. Importantly, a disagreement between the osmotic potentials determined with two methods has been observed in the watercore region, indicating the presence of significant volatile compounds in the cellular fluids collected. In the watercored fruit, cell turgor varied across flesh, and a steeper water potential gradient has been established from the normal outer parenchyma region to the watercore region, retaining the potential to transport water to the watercore region. Site-specific analysis using picoPPESI-MS revealed that together with a reduction in turgor, remarkable metabolic modifications through fermentation have occurred at the border, inducing greater production of watercore-related volatile compounds, such as alcohols and esters, compared with other regions. Because alcohols including ethanol have low reflection coefficients, it is very likely that these molecules would have rapidly penetrated membranes to accumulate in apoplast to fill. In addition to the water potential gradient detected here, this would physically contribute to the appearance with high tissue transparency and changes in colour differences. Therefore, it is concluded that these spatial changes in cell water relations are closely associated with watercore symptoms as well as with metabolic alterations.

References

[1] Tanaka, F., Hayakawa, F. & Tatsuki, M. Flavor and texture characteristics of ‘Fuji’ and related apple (Malus domestica L.) cultivars, focusing on the rich watercore. Molecules 25, 1114 (2020).

[2] Marlow, G. C. & Loescher, W. H. Sorbitol metabolism, the climacteric, and watercore in apples. J. Am. Soc. Hortic. Sci. 110, 676-680 (1985).

[3] Yamada, H., Ohmura, H., Arai, C. & Terui, M. Effect of preharvest fruit temperature on ripening, sugars, and watercore occurrence in apples. J. Am. Soc. Hortic. Sci. 119, 1208-1214 (1994).

[4] Sugiura, T., Ogawa, H., Fukuda, N. & Moriguchi, T. Changes in the taste and textural attributes of apples in response to climate change. Sci. Rep. 3, 2418 (2013).

[5] Wang, S. Y. & Faust, M. Variation in lipid-composition of apples in relation to watercore. J. Am. Soc. Hortic. Sci. 117, 829-833 (1992).

[6] Yamada, H., Takechi, K., Hoshi, A. & Amano, S. Comparison of water relations in watercored and non-watercored apples induced by fruit temperature treatment. Sci. Hortic. 99, 309-318 (2004).

[7] Yamada, H., Mukai, T. & Fukasawa-Akada, T. Comparison of the sub-cellular compartmentation of sugars in mature apples of two cultivars susceptible to different types of watercore and grown in different climates. J. Hortic. Sci. Biotechnol. 87, 17-22 (2012).

[8] Kasai, S. & Arakawa, O. Antioxidant levels in watercore tissue in ‘Fuji’ apples during storage. Postharvest Biol. Technol. 55, 103-107 (2010).

[9] Fukuda, H. Relationship of watercore and calcium to the incidence of internal storage disorders of ‘Fuji’ apple fruit. J. Jpn Soc. Hortic. Sci. 53, 298-302 (1984).

[10] Williams, M. W. Relationship of sugars and sorbitol to watercore in apples. Proc. Am. Soc. Hortic. Sci. 88, 67-75 (1966).

[11] Gao, Z., Jayanty, S., Beaudry, R. & Loescher, W. Sorbitol transporter expression in apple sink tissues: implications for fruit sugar accumulation and watercore development. J. Am. Soc. Hortic. Sci. 130, 261-268 (2005).

[12] Yamada, H. & Kobayashi, S. Relationship between watercore and maturity or sorbitol in apples affected by preharvest fruit temperature. Sci. Hortic. 80, 189-202 (1999).

[13] Zhang, L. Y. et al. Evidence for apoplasmic phloem unloading in developing apple fruit. Plant Physiol. 135, 574-586 (2004).

[14] Clijsters, H. Malic acid metabolism and initiation of the internal breakdown in “Jonathan” apples. Physiol. Plant. 18, 85-94 (1965).

[15] Smagula, J. M., Bramlage, W., Southwick, R. & Marsh, H. Effects of watercore on respiration and mitochondrial activity in Richared Delicious apples. Proc. Am. Soc. Hortic. Sci. 93, 753-761 (1968).

[16] Argenta, L., Fan, X. & Mattheis, J. Impact of watercore on gas permeance and incidence of internal disorders in ‘Fuji’ apples. Postharvest Biol. Technol. 24, 113-122 (2002).

[17] Tanaka, F. et al. Methyl ester generation associated with flesh browning in ‘Fuji’ apples after long storage under repressed ethylene function. Postharvest Biol. Technol. 145, 53-60 (2018).

[18] Beruter, J . Carbohydrate partitioning and changes in water relations of growing apple fruit. J. Plant Physiol. 135, 583-587 (1990).

[19] Sweeney, T. E. & Beuchat, C. A. Limitations of methods of osmometry: measuring the osmolality of biological-fluids. Am. J. Physiol. 264, R469-R480 (1993).

[20] Wolf, A. V., Brown, M. G. & Prentiss, P. G. In CRC Handbook of Chemistry and Physics (eds R. C. Weast & M. J. Astle) D223-D274 (CRC Press, Inc., 1983).

[21] Hüsken, D., Steudle, E. & Zimmermann, U. Pressure probe technique for measuring water relations of cells in higher plants. Plant Physiol. 61, 158-163 (1978).

[22] Boyer, J. S. Measuring the Water Status of Plants and Soils (Academic Press, 1995).

[23] Nakashima, T. et al. Single-cell metabolite profiling of stalk and glandular cells of intact trichomes with internal electrode capillary pressure probe electrospray ionization mass spectrometry. Anal. Chem. 88, 3049-3057 (2016).

[24] Wada, H. et al. Multiple strategies for heat adaptation to prevent chalkiness in the rice endosperm. J. Exp. Bot. 70, 1299-1311 (2018).

[25] Blokhina, O. et al. Ray parenchymal cells contribute to lignification of tracheids in developing xylem of norway spruce. Plant Physiol. 181, 1552-1572 (2019).

[26] Wada, H. et al. On-site single pollen metabolomics reveals varietal differences in phosphatidylinositol synthesis under heat stress conditions in rice. Sci. Rep. 10, 2013 (2020).

[27] Wada, H. et al. Endosperm cell size reduction caused by osmotic adjustment during nighttime warming in rice. Sci. Rep. 11, 4447 (2021).

[28] Rocco, R. M. Volatiles and osmometry. Clin. Chem. 22, 399- 399 (1976).

[29] Tyerman, S. D. & Steudle, E. Comparison between osmotic and hydrostatic water flows in a higher plant cell: determination of hydraulic conductivities and reflection coefficients in isolated epidermis of Tradescantia virginiana. Aust. J. Plant Physiol. 9, 461-479 (1982).

[30] Shackel, K. A., Greve, C., Labavitch, J. M. & Ahmadi, H. Cell turgor changes associated with ripening in tomato pericarp tissue. Plant Physiol. 97, 814-816 (1991).

[31] Tong, C. et al. Comparison of softening-related changes during storage of ‘Honeycrisp’ apple, its parents, and ‘Delicious’. J. Am. Soc. Hortic. Sci. 124, 407-415 (1999).

[32] Thomas, T. R., Matthews, M. A. & Shackel, K. A. Direct in situ measurement of cell turgor in grape (Vitis vinifera L.) berries during development and in response to plant water deficits. Plant Cell Environ. 29, 993-1001 (2006).

[33] Wada, H., Shackel, K. A. & Matthews, M. A. Fruit ripening in Vitis vinifera: apoplastic solute accumulation accounts for pre-veraison turgor loss in berries. Planta 227, 1351-1361 (2008).

[34] Wada, H., Matthews, M. A. & Shackel, K. A. Seasonal pattern of apoplastic solute accumulation and loss of cell turgor during ripening of Vitis vinifera fruit under field conditions. J. Exp. Bot. 60, 1773-1781 (2009).

[35] Wada, H., Matthews, M. A., Choat, B. & Shackel, K. A. In situ turgor stability in grape mesocarp cells and its relation to cell dimensions and microcapillary tip size and geometry. Environ. Control Biol. 49, 61-73 (2011).

[36] Mills, T. M., Behboudian, M. H. & Clothier, B. E. Water relations, growth, and the composition of ‘Braeburn’ apple fruit under deficit irrigation. J. Am. Soc. Hortic. Sci. 121, 286-291 (1996).

[37] Torres, C. A., Sepulveda, A., Gonzalez-Talice, J., Yuri, J. A. & Razmilic, I. Fruit water relations and osmoregulation on apples (Malus domestica Borkh.) with different sun exposures and sun-injury levels on the tree. Sci. Hortic. 161, 143-152 (2013).

[38] Herremans, E. et al. Comparison of X-ray CT and MRI of watercore disorder of different apple cultivars. Postharvest Biol. Technol. 87, 42-50 (2014).

[39] Nonami, H. & Boyer, J. S. Origin of growth-induced water potential: Solute concentration is low in apoplast of enlarging tissues. Plant Physiol. 83, 596-601 (1987).

[40] Iwanami, H., Moriya, S., Kotoda, N. & Abe, K. Turgor closely relates to postharvest fruit softening and can be a useful index to select a parent for producing cultivars with good storage potential in apple. HortScience 43, 1377 (2008).

[41] Johnston, J. W., Hewett, E. W. & Hertog, M. L. A. T. M. Postharvest softening of apple (Malus domestica) fruit: a review. N.Z. J. Crop Hortic. Sci. 30, 145-160 (2002).

[42] Ting, V. J., Silcock, P., Bremer, P. J. & Biasioli, F. X-ray micro-computer tomographic method to visualize the microstructure of different apple cultivars. J. Food Sci. 78, E1735-E1742 (2013).

[43] Wada, H. et al. Turgor-responsive starch phosphorylation in Oryza sativa stems: a primary event of starch degradation associated with grain-filling ability. PLoS ONE 12, e0181272 (2017).

[44] Castellarin, S. D. et al. Characterization of major ripening events during softening in grape: turgor, sugar accumulation, abscisic acid metabolism, colour development, and their relationship with growth. J. Exp. Bot. 67, 709-722 (2016).

[45] Guerrero, F. D., Jones, J. T. & Mullet, J. E. Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Mol. Biol. 15, 11-26 (1990).

[46] Jones, J. T. & Mullet, J. E. Developmental expression of a turgor-responsive gene that encodes an intrinsic membrane protein. Plant Mol. Biol. 28, 983-996 (1995).

[47] Diamond, J. M. & Wright, E. M. Biological membranes: the physical basis of ion and nonelectrolyte selectivity. Annu Rev. Physiol. 31, 581-646 (1969).

[48] White, J. W. Composition of a volatile fraction of apples. Food Res. 15, 68-78 (1950).

[49] Tanaka, F., Miyazawa, T., Okazaki, K., Tatsuki, M. & Ito, T. Sensory and metabolic profiles of “Fuji” apples (Malus domestica Borkh.) grown without synthetic agrochemicals: the role of ethylene production. Biosci. Biotechnol. Biochem. 79, 2034-2043 (2015).

[50] Janssen, S. et al. 3D pore structure analysis of intact ‘Braeburn’ apples using X-ray micro-CT. Postharvest Biol. Technol. https://doi.org/10.1016/j.postharvbio.2019.111014 (2020).

[51] Lewak, S. Metabolic control of embryonic dormancy in apple seed: seven decades of research. Acta Physiol. Plant. 33, 1-24 (2011).

[52] Nishitani, C. et al. Efficient genome editing in apple using a CRISPR/Cas9 system. Sci. Rep. 6, 31481 (2016).

[53] Wada, H. et al. Polarity of water transport across epidermal cell membranes in Tradescantia virginiana. Plant Physiol. 164, 1800-1809 (2014).

[54] Nonami, H. & Schulze, E. D. Cell water potential, osmotic potential, and turgor in the epidermis and mesophyll of transpiring leaves: Combined measurements with the cell pressure probe and nanoliter osmometer. Planta 177, 35-46 (1989).

[55] Wada, H., Iwaya-Inoue, M., Akita, M. & Nonami, H. Hydraulic conductance in tepal growth and extension of vase life with trehalose in cut tulip flowers. J. Am. Soc. Hortic. Sci. 130, 275-286 (2005).

[56] Tatsuki, M., Hayama, H., Yoshioka, H. & Nakamura, Y. Cold pre-treatment is effective for 1-MCP efficacy in ‘Tsugaru’ apple fruit. Postharvest Biol. Technol. 62, 282-287 (2011).

[57] Wan, W. Y. & Wilkins, T. A. A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal. Biochem. 223, 7-12 (1994).