VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant

Abstract

The molecular mechanism underlying dormancy release and the induction of flowering remains poorly understood in woody plants. Mu-legacy is a valuable blueberry mutant, in which a transgene insertion caused increased expression of a RESPONSE REGULATOR 2-like gene (VcRR2). Mu-legacy plants, compared with nontransgenic ‘Legacy’ plants, show dwarfing, promotion of flower bud formation, and can flower under nonchilling conditions. We conducted transcriptomic comparisons in leaves, chilled and nonchilled flowering buds, and late-pink buds, and analyzed a total of 41 metabolites of six groups of hormones in leaf tissues of both Mu-legacy and ‘Legacy’ plants. These analyses uncovered that increased VcRR2 expression promotes the expression of a homolog of Arabidopsis thaliana ENT-COPALYL DIPHOSPHATE SYNTHETASE 1 (VcGA1), which induces new homeostasis of hormones, including increased gibberellin 4 (GA₄) levels in Mu-legacy leaves. Consequently, increased expression of VcRR2 and VcGA1, which function in cytokinin responses and gibberellin synthesis, respectively, initiated the reduction in plant height and the enhancement of flower bud formation of the Mu-legacy plants through interactions of multiple approaches. In nonchilled flower buds, 29 differentially expressed transcripts of 17 genes of five groups of hormones were identified in transcriptome comparisons between Mu-legacy and ‘Legacy’ plants, of which 22 were chilling responsive. Thus, these analyses suggest that increased expression of VcRR2 was collectively responsible for promoting flower bud formation in highbush blueberry under nonchilling conditions. We report here for the first time the importance of VcRR2 to induce a suite of downstream hormones that promote flowering in woody plants.

References

[1] Olsen, J. E. Light and temperature sensing and signaling in induction of bud dormancy in woody plants. Plant Mol. Biol. 73, 37-47 (2010).

[2] Samish, R. M. Dormancy in woody plants. Annu. Rev. Plant Physiol. 5, 183-204 (1954).

[3] Tanino, K. K., Kalcsits, L., Silim, S., Kendall, E. & Gray, G. R. Temperature-driven plasticity in growth cessation and dormancy development in deciduous woody plants: a working hypothesis suggesting how molecular and cellular function is affected by temperature during dormancy induction. Plant Mol. Biol. 73, 49-65 (2010).

[4] Wilkie, J. D., Sedgley, M. & Olesen, T. Regulation of floral initiation in horticultural trees. J. Exp. Bot. 59, 3215-3228 (2008).

[5] Arora, R., Rowland, L. J. & Tanino, K. Induction and release of bud dormancy in woody perennials: a science comes of age. Hortscience 38, 11 (2003).

[6] Lobos, G. A. & Hancock, J. F. Breeding blueberries for a changing global environment: a review. Front. Plant Sci. 6, https://doi.org/10.3389/fpls.2015.00782 (2015).

[7] Atkinson, C. J., Brennan, R. M. & Jones, H. G. Declining chilling and its impact on temperate perennial crops. Environ. Exp. Bot. 91, 48-62 (2013).

[8] Luedeling, E., Girvetz, E. H., Semenov, M. A. & Brown, P. H. Climate change affects winter chill for temperate fruit and nut trees. PLoS ONE 6, e20155 (2011).

[9] Melke, A. The physiology of chilling temperature requirements for dormancy release and bud-break in temperate fruit trees grown at mild winter tropical climate. J. Plant Stud. 4, 47 (2015).

[10] Fornara, F., de Montaigu, A. & Coupland, G. SnapShot: control of flowering in arabidopsis. Cell 141, https://doi.org/10.1016/j.cell.2010.04.024 (2010).

[11] Michaels, S. D. & Amasino, R. M. FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11, 949-956 (1999).

[12] Mateos, J. L. et al. Combinatorial activities of SHORT VEGETATIVE PHASE and FLOWERING LOCUS C define distinct modes of flowering regulation in Arabidopsis. Genome Biol. 16, 31 (2015).

[13] Greenup, A., Peacock, W. J., Dennis, E. S. & Trevaskis, B. The molecular biology of seasonal flowering-responses in Arabidopsis and the cereals. Ann. Bot. 103, 1165-1172 (2009).

[14] Bielenberg, D. G. et al. Sequencing and annotation of the evergrowing locus in peach [Prunus persica (L.) Batsch] reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation. Tree Genet. Genomes 4, 495-507 (2008).

[15] Wang, Y., Georgi, L. L., Reighard, G. L., Scorza, R. & Abbott, A. G. Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch]. J. Hered. 93, 352-358 (2002).

[16] Jimenez, S., Lawton-Rauh, A. L., Reighard, G. L., Abbott, A. G. & Bielenberg, D. G. Phylogenetic analysis and molecular evolution of the dormancy associated MADS-box genes from peach. BMC Plant Biol. 9, 81 (2009).

[17] Jimenez, S., Reighard, G. L. & Bielenberg, D. G. Gene expression of DAM5 and DAM6 is suppressed by chilling temperatures and inversely correlated with bud break rate. Plant Mol. Biol. 73, 157-167 (2010).

[18] Sasaki, R. et al. Functional and expressional analyses of PmDAM genes associated with endodormancy in Japanese apricot. Plant Physiol. 157, 485-497 (2011).

[19] Vander Kloet, S. P. The genus Vaccinium in North America. Res. Branch Agric. Can. Publ. 1828, 201 (1988).

[20] Song, G. Q. & Hancock, J. F. Vaccinium. In Kole, C. (ed) Wild Crop Relatives: Genomic and Breeding Resources, 197-221 (Springer, Berlin Heidelberg, 2011).

[21] Kovaleski, A. P., Williamson, J. G., Olmstead, J. W. & Darnell, R. L. Inflorescence bud initiation, development, and bloom in two southern highbush blueberry cultivars. J. Am. Soc. Hortic. Sci. 140, 38-44 (2015).

[22] Song, G. Q. & Chen, Q. Comparative transcriptome analysis of nonchilled, chilled, and late-pink bud reveals flowering pathway genes involved in chilling-mediated flowering in blueberry. BMC Plant Biol. 18, 98 (2018).

[23] Gao, X., Walworth, A. E., Mackie, C. & Song, G. Q. Overexpression of blueberry FLOWERING LOCUS T is associated with changes in the expression of phytohormone-related genes in blueberry plants. Hortic. Res. 3, 16053 (2016).

[24] Walworth, A. E., Chai, B. & Song, G. Q. Transcript profile of flowering regulatory genes in VcFT-overexpressing blueberry plants. PLoS ONE 11, e0156993 (2016).

[25] Song, G.-Q. & Walworth, A. An invaluable transgenic blueberry for studying chilling-induced flowering in woody plants. BMC Plant Biol. 18, 16 (2018).

[26] Song, G.-Q. & Chen, Q. Overexpression of the MADS-box gene K-domain increases the yield potential of blueberry. Plant Sci. 276, 10 (2018).

[27] Peng, J. & Harberd, N. P. Gibberellin deficiency and response mutations suppress the stem elongation phenotype of phytochrome-deficient mutants of Arabidopsis. Plant Physiol. 113, 1051-1058 (1997).

[28] Huang, S. et al. Overexpression of 20-oxidase confers a gibberellin-overproduction phenotype in Arabidopsis. Plant Physiol. 118, 773-781 (1998).

[29] Magome, H., Yamaguchi, S., Hanada, A., Kamiya, Y. & Oda, K. The DDF1 transcriptional activator upregulates expression of a gibberellin-deactivating gene, GA2ox7, under high-salinity stress in Arabidopsis. Plant J. 56, 613-626 (2008).

[30] Magome, H., Yamaguchi, S., Hanada, A., Kamiya, Y. & Oda, K. Dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor. Plant J. 37, 720-729 (2004).

[31] Kang, H. G. et al. Overexpression of FTL1/DDF1, an AP2 transcription factor, enhances tolerance to cold, drought, and heat stresses in Arabidopsis thaliana. Plant Sci. 180, 634-641 (2011).

[32] Schomburg, F. M., Bizzell, C. M., Lee, D. J., Zeevaart, J. A. & Amasino, R. M. Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants. Plant Cell 15, 151-163 (2003).

[33] Porri, A., Torti, S., Romera-Branchat, M. & Coupland, G. Spatially distinct regulatory roles for gibberellins in the promotion of flowering of Arabidopsis under long photoperiods. Development 139, 2198-2209 (2012).

[34] Hwang, I. & Sheen, J. Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413, 383-389 (2001).

[35] Mason, M. G. et al. Multiple type-B response regulators mediate cytokinin signal transduction in Arabidopsis. Plant Cell 17, 3007-3018 (2005).

[36] Fleet, C. M. et al. Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins. Plant Physiol. 132, 830-839 (2003).

[37] Matias-Hernandez, L., Aguilar-Jaramillo, A. E., Marin-Gonzalez, E., Suarez-Lopez, P. & Pelaz, S. RAV genes: regulation of floral induction and beyond. Ann. Bot. 114, 1459-1470 (2014).

[38] Pabon-Mora, N., Ambrose, B. A. & Litt, A. Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development. Plant Physiol. 158, 1685-1704 (2012).

[39] Smith, M. W., Yamaguchi, S., Ait-Ali, T. & Kamiya, Y. The first step of gibberellin biosynthesis in pumpkin is catalyzed by at least two copalyl diphosphate synthases encoded by differentially regulated genes. Plant Physiol. 118, 1411-1419 (1998).

[40] Werner, T. et al. Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15, 2532-2550 (2003).

[41] Frebort, I., Kowalska, M., Hluska, T., Frebortova, J. & Galuszka, P. Evolution of cytokinin biosynthesis and degradation. J. Exp. Bot. 62, 2431-2452 (2011).

[42] Widemann, E. et al. Sequential oxidation of Jasmonoyl-Phenylalanine and Jasmonoyl-Isoleucine by multiple cytochrome P450 of the CYP94 family through newly identified aldehyde intermediates. Phytochemistry 117, 388-399 (2015).

[43] Dempsey, D. A., Vlot, A. C., Wildermuth, M. C. & Klessig, D. F. Salicylic acid biosynthesis and metabolism. Arab. Book 9, e0156 (2011).

[44] Muller, B. & Sheen, J. Arabidopsis cytokinin signaling pathway. Sci. STKE 2007, cm5 (2007).

[45] Kolachevskaya, O. O. et al. Auxin synthesis gene tms1 driven by tuber-specific promoter alters hormonal status of transgenic potato plants and their responses to exogenous phytohormones. Plant Cell Rep. 36, 419-435 (2017).

[46] Liu, X. D. et al. Auxin controls seed dormancy through stimulation of abscisic acid signaling by inducing ARF-mediated ABI3 activation in Arabidopsis. Proc. Natl Acad. Sci. USA 110, 15485-15490 (2013).

[47] Hu, Y. & Yu, D. BRASSINOSTEROID INSENSITIVE2 interacts with ABSCISIC ACID INSENSITIVE5 to mediate the antagonism of brassinosteroids to abscisic acid during seed germination in Arabidopsis. Plant Cell 26, 4394-4408 (2014).

[48] Nakamura, M. et al. Activation of the cytochrome P450 gene, CYP72C1, reduces the levels of active brassinosteroids in vivo. J. Exp. Bot. 56, 833-840 (2005).

[49] Bai, S. L. et al. Transcriptome analysis of Japanese Pear (Pyrus pyrifolia Nakai) flower buds transitioning through endodormancy. Plant Cell Physiol. 54, 1132-1151 (2013).

[50] Li, J., Li, Y., Chen, S. & An, L. Involvement of brassinosteroid signals in the floral-induction network of Arabidopsis. J. Exp. Bot. 61, 4221-4230 (2010).

[51] Jin, S. H. et al. Overexpression of glucosyltransferase UGT85A1 influences trans-zeatin homeostasis and trans-zeatin responses likely through O-glucosylation. Planta 237, 991-999 (2013).

[52] Pineda Rodo, A. et al. Over-expression of a zeatin O-glucosylation gene in maize leads to growth retardation and tasselseed formation. J. Exp. Bot. 59, 2673-2686 (2008).

[53] Veach, Y. K. et al. O-glucosylation of cis-zeatin in maize. Characterization of genes, enzymes, and endogenous cytokinins. Plant Physiol. 131, 1374-1380 (2003).

[54] Xu, Q. et al. A role for jasmonates in the release of dormancy by cold stratification in wheat. J. Exp. Bot. 67, 3497-3508 (2016).

[55] Bruckhoff, V. et al. Functional characterization of CYP94-genes and identification of a novel jasmonate catabolite in flowers. PLoS One 11, e0159875 (2016).

[56] Widemann, E., Smirnova, E., Aubert, Y., Miesch, L. & Heitz, T. Dynamics of Jasmonate metabolism upon flowering and across leaf stress responses in Arabidopsis thaliana. Plants (Basel) 5, 15 (2016).

[57] Chailakhyan, M. K. New facts in support of the hormonal theory of plant development. Dokl. Akad. Nauk SSSR 4, 79-83 (1936).

[58] Song, G. Q., Walworth, A., Zhao, D. Y., Jiang, N. & Hancock, J. F. The Vaccinium corymbosum FLOWERING LOCUS T-like gene (VcFT): a flowering activator reverses photoperiodic and chilling requirements in blueberry. Plant Cell Rep. 32, 1759-1769 (2013).

[59] Corbesier, L. et al. FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis. Science 316, 1030-1033 (2007).

[60] Notaguchi, M. et al. Long-distance, graft-transmissible action of Arabidopsis FLOWERING LOCUS T protein to promote flowering. Plant Cell Physiol. 49, 1645-1658 (2008).

[61] Zhu, Y., Liu, L., Shen, L. & Yu, H. NaKR1 regulates long-distance movement of FLOWERING LOCUS T in Arabidopsis. Nat. Plants 2, 16075 (2016).

[62] Yoo, S. J., Hong, S. M., Jung, H. S. & Ahn, J. H. The cotyledons produce sufficient FT protein to induce flowering: evidence from cotyledon micrografting in Arabidopsis. Plant Cell Physiol. 54, 119-128 (2013).

[63] Putterill, J. & Varkonyi-Gasic, E. FT and florigen long-distance flowering control in plants. Curr. Opin. Plant Biol. 33, 77-82 (2016).

[64] Jaeger, K. E. & Wigge, P. A. FT protein acts as a long-range signal in Arabidopsis. Curr. Biol. 17, 5 (2007).

[65] Mathieu, J., Warthmann, N., Küttner, F. & Schmid, M. Export of FT protein from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr. Biol. 17, 6 (2007).

[66] Turck, F., Fornara, F. & Coupland, G. Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu. Rev. Plant Biol. 59, 573-594 (2008).

[67] Tamaki, S. et al. FT-like proteins induce transposon silencing in the shoot apex during floral induction in rice. Proc. Natl Acad. Sci. USA 112, E901-E910 (2015).

[68] Beauvieux, R., Wenden, B. & Dirlewanger, E. Bud dormancy in perennial fruit tree species: a pivotal role for oxidative cues. Front. Plant Sci. 9, https://doi.org/10.3389/fpls.2018.00657 (2018).

[69] Yamane, H. Regulation of bud dormancy and bud break in Japanese apricot (Prunus mume Siebold & Zucc.) and peach [Prunus persica (L.) Batsch]: a summary of recent studies. J. Jpn. Soc. Hortic. Sci. 83, 187-202 (2014).

[70] Song, G. Q. Blueberry (Vaccinium corymbosum L.). Methods Mol. Biol. 1224, 121-131 (2015).

[71] Zeng, W. Q. et al. A genetic screen reveals arabidopsis stomatal and/or apoplastic defenses against pseudomonas syringae pv. tomato DC3000. Plos Pathog. 7. https://doi.org/10.1371/journal.ppat.1002291 (2011).

[72] Zamboni, A., Pierantoni, L., De Franceschi, P. & Total, R. N. A. extraction from strawberry tree (Arbutus unedo) and several other woodyplants. Iforest-Biogeosciences For. 1, 122-125 (2008).

[73] Haas, B. J. et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat. Protoc. 8, 1494-1512 (2013).

[74] Song, G. Q. & Gao, X. Transcriptomic changes reveal gene networks responding to the overexpression of a blueberry DWARF AND DELAYED FLOWERING 1 gene in transgenic blueberry plants. BMC Plant Biol. 17, 106 (2017).