Cytokinin but not gibberellin application had major impact on the phenylpropanoid pathway in grape

Abstract

Cytokinin and gibberellic acid (GA) are growth regulators used to increase berry size in seedless grapes and it is of interest to understand their effects on the phenylpropanoid pathway and on ripening processes. GA₃ and synthetic cytokinin forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea, CPPU) and their combination were applied to 6 mm diameter fruitlets of ‘Sable Seedless’, and berries were sampled 51 and 70 days (d) following application. All treatments increased berry size and delayed sugar accumulation and acid degradation with a stronger effect of CPPU. CPPU, but not GA, reduced berry color and the levels of anthocyanins. While CPPU reduced the levels of anthocyanins by more than 50%, the combined treatment of GA+CPPU reduced the levels by about 25% at 51 d. CPPU treatment had minor effects on flavonols content but increased the levels of monomeric flavan-3-ols by more than two-fold. Phloroglucinol analysis using HPLC showed that proanthocyanidin content was significantly increased by CPPU, whereas mean degree of polymerization was reduced from 26 to 19. Volatile analysis by GC-MS showed changes in composition with CPPU or GA treatment with potential impact on flavor. RNA-seq analysis showed that GA had a minor overall effect on the transcriptome whereas CPPU had pronounced effects on gene expression at both 51 and 70 d. Comparing the control and CPPU at similar Brix of ca. 19.7°, a reduced expression of stilbene synthases (STSs) including their regulators MYB14 and MYB15, and other phenylpropanoid-related genes was observed in CPPU-treated grapes. Overall, our study shows that CPPU had a major influence on the phenylpropanoid pathway and affected multiple ripening-related processes.

References

[1] McAtee, P., Karim, S., Schaffer, R. J. & David, K. A dynamic interplay between phytohormones is required for fruit development, maturation, and ripening. Front. Plant Sci. 4, 79 (2013).

[2] Conde, C. et al. Biochemical changes throughout grape berry development and fruit and wine quality. Food 1, 1-22 (2007).

[3] Lavee, S. Effect of gibberellic acid on seeded grapes. Nature 185, 395- 395 (1960).

[4] Kuhn, N. et al. Berry ripening: recently heard through the grapevine. J. Exp. Bot. 65, 4543-4559 (2013).

[5] Falchi, R. et al. The genomics of grape berry ripening. In The Grape Genome (eds. Cantu, D. & Walker, A.) 247-274 (Springer Nature, 2019).

[6] Ebeler, S. E. & Thorngate, J. H. Wine chemistry and flavor: looking into the crystal glass. J. Agric. Food Chem. 57, 8098-8108 (2009).

[7] Maoz, I. et al. Insights into the chemosensory basis of flavor in table grapes. J. Sci. Food Agric. 100, 1405-1417 (2020).

[8] Peppi, M. C. & Fidelibus, M. W. Effects of forchlorfenuron and abscisic acid on the quality of ‘Flame Seedless’ grapes. HortScience 43, 173-176 (2008).

[9] Weaver, R. & McCune, S. Effect of gibberellin on seedless Vitis vinifera. Hilgardia 29, 247-275 (1959).

[10] Acheampong, A. K. et al. Abnormal endogenous repression of GA signaling in a seedless table grape cultivar with high berry growth response to GA application. Front. Plant Sci. 8, 850 (2017).

[11] Acheampong, A. K. et al. Functional characterization and developmental expression profiling of gibberellin signalling components in Vitis vinifera. J. Exp. Bot. 66, 1463-1476 (2015).

[12] Giacomelli, L. et al. Gibberellin metabolism in Vitis vinifera L. during bloom and fruit-set: functional characterization and evolution of grapevine gibberellin oxidases. J. Exp. Bot. 64, 4403-4419 (2013).

[13] Blank, R. et al. Effect of a forchlorfenuron dip on kiwifruit fruit size. N. Zeal. J. Crop Hort. Sci. 20, 73-78 (1992).

[14] Stern, R. A., Ben-Arie, R., Neria, O. & Flaishman, M. CPPU and BA increase fruit size of ‘Royal Gala’(Malus domestica) apple in a warm climate. J. Hortic. Sci. Biotech. 78, 297-302 (2003).

[15] Reynolds, A., Wardle, D., Zurowski, C. & Looney, N. Phenylureas CPPU and thidiazuron affect yield components, fruit composition, and storage potential of four seedless grape selections. J. Am. Soc. Hortic. Sci. 117, 85-89 (1992).

[16] Zoffoli, J. P., Latorre, B. A. & Naranjo, P. Preharvest applications of growth regulators and their effect on postharvest quality of table grapes during cold storage. Postharvest Biol. Technol. 51, 183-192 (2009).

[17] Carvajal-Millán, E. et al. Polyphenol oxidase activity, color changes, and dehydration in table grape rachis during development and storage as affected by N-(2-chloro-4-pyridyl)-N-phenylurea. J. Agric. Food Chem. 49, 946-951 (2001).

[18] Zabadal, T. J. & Bukovac, M. J. Effect of CPPU on fruit development of selected seedless and seeded grape cultivars. HortScience 41, 154-157 (2006).

[19] Weaver, R., Van Overbeek, J. & Pool, R. Effect of kinins on fruit set and development in Vitis vinifera. Hilgardia 37, 181-201 (1966).

[20] Vogt, T. Phenylpropanoid biosynthesis. Mol. Plant. 3, 2-20 (2010).

[21] Constabel, C. Molecular controls of proanthocyanidin synthesis and structure: prospects for genetic engineering in crop plants. J. Agric. Food Chem. 66, 9882 (2018).

[22] Maoz, I. et al. Effect of the cytokinin forchlorfenuron on tannin content of Thompson Seedless table grapes. Am. J. Enol. Viticult. 65, 230-237 (2014).

[23] Tyagi, K. et al. Girdling of table grapes at fruit set can divert the phenylpropanoid pathway towards accumulation of proanthocyanidins and change the volatile composition. Plant Sci. 296, 110495 (2020).

[24] Coombe, B. G. Growth stages of the grapevine: adoption of a system for identifying grapevine growth stages. Aust. J. Grape Wine Res. 1, 104-110 (1995).

[25] Ruzin, S. E. Plant Microtechnique and Microscopy. Vol. 198, 93-95 (Oxford University Press, 1999).

[26] Maoz, I., Beno‐Moualem, D., Kaplunov, T., Lewinsohn, E. & Lichter, A. Uneven distribution of flavour components in tablegrape berries. Aust. J. Grape Wine Res. 22, 343-349 (2016).

[27] Tyagi, K. et al. Enhancement of table grape flavor by postharvest application of monoterpenes in modified atmosphere. Postharvest Biol. Technol. 159, 111018 (2020).

[28] Chong, J., Wishart, D. S. & Xia, J. Using MetaboAnalyst 4.0 for comprehensive and integrative metabolomics data analysis. Curr. Protoc. Bioinformatics 68, e86 (2019).

[29] Mitsuda, N. et al. NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell 19, 270-280 (2007).

[30] Reynolds, A., Robbins, N., Lee, H.-S. & Kotsaki, E. Impacts and interactions of abscisic acid and gibberellic acid on sovereign Coronation and Skookum seedless table grapes. Am. J. Enol. Viticult. 67, 327-338 (2016).

[31] Wang, W., Khalil-Ur-Rehman, M., Feng, J. & Tao, J. RNA-seq based transcriptomic analysis of CPPU treated grape berries and emission of volatile compounds. J. Plant Physiol. 218, 155-166 (2017).

[32] Czemmel, S. et al. The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol. 151, 1513-1530 (2009).

[33] Downey, M. O., Harvey, J. S. & Robinson, S. P. Analysis of tannins in seeds and skins of Shiraz grapes throughout berry development. Aust. J. Grape Wine Res. 9, 15-27 (2003).

[34] Bogs, J., Jaffé, F. W., Takos, A. M., Walker, A. R. & Robinson, S. P. The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development. Plant Physiol. 143, 1347-1361 (2007).

[35] Terrier, N. et al. Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway. Plant Physiol. 149, 1028-1041 (2009).

[36] Deikman, J. & Hammer, P. E. Induction of anthocyanin accumulation by cytokinins in Arabidopsis thaliana. Plant Physiol. 108, 47-57 (1995).

[37] Schnablová, R. et al. Transgenic ipt tobacco overproducing cytokinins overaccumulates phenolic compounds during in vitro growth. Plant Physiol. Biochem. 44, 526-534 (2006).

[38] Vannozzi, A., Dry, I. B., Fasoli, M., Zenoni, S. & Lucchin, M. Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses. BMC Plant Biol. 12, 130 (2012).

[39] Vannozzi, A. et al. Combinatorial regulation of stilbene synthase genes by WRKY and MYB transcription factors in grapevine (Vitis vinifera L.). Plant Cell Physiol. 59, 1043-1059 (2018).

[40] Höll, J. et al. The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera. Plant Cell 25, 4135-4149 (2013).

[41] Wong, D. C. J. et al. A systems-oriented analysis of the grapevine R2R3-MYB transcription factor family uncovers new insights into the regulation of stilbene accumulation. DNA Res. 23, 451-466 (2016).

[42] Walker, A. R. et al. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J. 49, 772-785 (2007).

[43] Xiao, Z., Rogiers, S. Y., Sadras, V. O. & Tyerman, S. D. Hypoxia in grape berries: the role of seed respiration and lenticels on the berry pedicel and the possible link to cell death. J. Exp. Bot. 69, 2071-2083 (2018).

[44] Brenner, W. G., Ramireddy, E., Heyl, A. & Schmülling, T. Gene regulation by cytokinin in Arabidopsis. Front. Plant Sci. 3, 8 (2012).

[45] Böttcher, C., Harvey, K., Forde, C., Boss, P. & Davies, C. Auxin treatment of pre-veraison grape (Vitis vinifera L.) berries both delays ripening and increases the synchronicity of sugar accumulation. Aust. J. Grape Wine Res. 17, 1-8 (2011).

[46] Nelson, S. K. & Steber, C. M. Gibberellin hormone signal perception: down-regulating DELLA repressors of plant growth and development. Annu. Plant Rev. 1, 153-187 (2018).