Woodland strawberry WRKY71 acts as a promoter of flowering via a transcriptional regulatory cascade

Abstract

The WRKY proteins are a large family of transcription factors that play important roles in stress responses and plant development. However, the roles of most WRKYs in strawberry are not well known. In this study, FvWRKY71 was isolated from the woodland strawberry ‘Ruegen’. FvWRKY71 was highly expressed in the shoot apex and red fruit. Subcellular localization analysis showed that FvWRKY71 was located in the nucleus. Transactivation analysis showed that FvWRKY71 presented transcriptional activation activity in yeast. Overexpression of FvWRKY71 in Arabidopsis and woodland strawberry revealed early flowering in the transgenic plants compared with the wild-type control. Gene expression analysis indicated that the transcript levels of the flowering time and development integrator genes AP1, LFY, FT, AGL42, FUL, FPF1, SEP1, SEP2, and SEP3 were increased in FvWRKY71-overexpressing Arabidopsis and strawberry plants compared with the wild-type controls, which may result in accelerated flowering in transgenic plants. Furthermore, FvWRKY71 was proven to directly bind to the W-boxes (TTGACT/C) of the FvFUL, FvSEP1, FvAGL42, FvLFY, and FvFPF1 promoters in vitro and in vivo. Taken together, our results reveal a transcriptional regulatory cascade of FvWRKY71 involved in promoting flowering in woodland strawberry.

References

[1] Ishiguro, R. & Nakamura, K. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5′ upstream regions of genes coding for sporamin and β-amylase from sweet potato. Mol. Gen. Genet. 244, 563-571 (1994).

[2] Rushton, P. J., Somssich, I. E., Patricia, R. & Shen, Q. J. WRKY transcription factors. Plant Signal. Behav. 15, 247-258 (2010).

[3] Bakshi, M. & Oelmüller, R. WRKY transcription factors: Jack of many trades in plants. Plant Signal. Behav. 9, e27700 (2014).

[4] Banerjee, A. & Roychoudhury, A. WRKY proteins: signaling and regulation of expression during abiotic stress responses Scientific World J. 2015, 807560 (2015).

[5] Zheng, Z. et al. Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J. 48, 592-605 (2010).

[6] Birkenbihl, R. P., Diezel, C. & Somssich, I. E. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection. Plant Physiol. 159, 266-285 (2012).

[7] Hu, Y., Dong, Q. & Yu, D. Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae. Plant Sci. 185-186, 288-297 (2012).

[8] Jiang, Y. & Deyholos, M. Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol. Biol. 69, 91-105 (2009).

[9] Ren, X. et al. ABO3, a WRKY transcription factor, mediates plant responses to abscisic acid and drought tolerance in Arabidopsis. Plant J. 63, 417-429 (2010).

[10] Li, S. et al. Arabidopsis thaliana WRKY25, WRKY26, and WRKY33 coordinate induction of plant thermotolerance. Planta 233, 1237-1252 (2011).

[11] Hu, Y. et al. Arabidopsis transcription factor WRKY8 functions antagonistically with its interacting partner VQ9 to modulate salinity stress tolerance. Plant J. 74, 730-745 (2013).

[12] Silke, R. & Imre, E. S. Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev. 16, 1139-1149 (2002).

[13] Wang, H. et al. Mutation of WRKY transcription factors initiates pith secondary wall formation and increases stem biomass in dicotyledonous plants. Proc. Natl Acad. Sci. USA 107, 22338-22343 (2010).

[14] Guo, D. et al. The WRKY transcription factor WRKY71/EXB1 controls shoot branching by transcriptionally regulating RAX genes in Arabidopsis. Plant Cell. 27, 3112-3127 (2015).

[15] Liu, J. et al. Alternative splicing of rice WRKY62 and WRKY76 transcription factor genes in pathogen defense. Plant Physiol. 171, 1427-14429 (2016).

[16] Qiu, D. et al. OsWRKY13 mediates rice disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. Mol. Plant Microbe Interact. 20, 492-499 (2007).

[17] Qiu, Y. & Yu, D. Over-expression of the stress-induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis. Environ. Exp. Bot. 65, 35-47 (2009).

[18] Wu, X. et al. Enhanced heat and drought tolerance in transgenic rice seedlings overexpressing OsWRKY11 under the control of HSP101 promoter. Plant Cell Rep. 28, 21-30 (2009).

[19] Zhang, L. et al. Transcription factor WRKY75 interacts with DELLA proteins to affect flowering. Plant Physiol. 176, 790-803 (2018).

[20] Li, W. et al. Arabidopsis WRKY transcription factors WRKY12 and WRKY13 oppositely regulate flowering under short-day conditions. Mol. Plant 9, 1492-1503 (2016).

[21] Yu, Y. et al. MlWRKY12, a novel Miscanthus transcription factor, participates in pith secondary cell wall formation and promotes flowering. Plant Sci. 212, 1-9 (2013).

[22] Yu, Y. et al. WRKY71 accelerates flowering via the direct activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana. Plant J. 85, 96-106 (2016).

[23] Huang, R. W. et al. CpWRKY71, a WRKY transcription factor gene of Wintersweet (Chimonanthus praecox), promotes flowering and leaf senescence in Arabidopsis. Int. J. Mol. Sci. 20, 5325 (2019).

[24] Darrow, G. The Strawberry: History, Breeding and Physiology. (Holt, Rinehart and Winston, NY, 1966).

[25] Rousseau-Gueutin, M. et al. Comparative genetic mapping between octoploid and diploid Fragaria species reveals a high level of colinearity between their genomes and the essentially disomic behavior of the cultivated octoploid strawberry. Genetics 179, 2045-2060 (2008).

[26] Nakajima, R. et al. Molecular cloning and expression analyses of FaFT, FaTFL, and FaAP1 genes in cultivated strawberry: their correlation to flower bud formation. Biol. Plant. 58, 641-648 (2014).

[27] Whitaker, V. M. et al. A roadmap for research in octoploid strawberry. Hortic. Res. 7, 33 (2020).

[28] Brown, T. et al. Genetical control of everbearing habit and 3 other characters in varieties of Fragaria vesca. Euphytica 14, 97-112 (1965).

[29] Koskela, E. A. et al. Mutation in TERMINAL FLOWER1 reverses the photoperiodic requirement for flowering in the wild strawberry Fragaria vesca. Plant Physiol. 159, 1043-1054 (2012).

[30] Mouhu, K. et al. The Fragaria vesca homolog of SUPPRESSOR OF OVER-EXPRESSION OF CONSTANS1 represses flowering and promotes vegetative growth. Plant Cell. 25, 3296-3310 (2013).

[31] Gaston, A. et al. PFRU, a single dominant locus regulates the balance between sexual and asexual plant reproduction in cultivated strawberry. J. Exp. Bot. 64, 1837-1848 (2013).

[32] Zhou, H. et al. Genome-wide analysis of the expression of wrky family genes in different developmental stages of wild strawberry (Fragaria vesca) fruit. PLoS ONE 11, e0154312 (2016).

[33] Wei, W. et al. The WRKY transcription factors in the diploid woodland strawberry Fragaria vesca: Identification and expression analysis under biotic and abiotic stresses. Plant Physiol. Biochem. 105, 129-144 (2016).

[34] Eulgem, T., Rushton, P. J., Robatzek, S. & Somssich, I. E. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 5, 199-206 (2000).

[35] Hellens, R. P. et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1, 13 (2005).

[36] Richard, A. Seasonal and developmental timing of flowering. Plant J. 61, 1001-1013 (2010).

[37] Gu, X. et al. Arabidopsis FLC clade members form flowering-repressor complexes coordinating responses to endogenous and environmental cues. Nat. Commun. 4, 1947 (2013).

[38] Amasino, R. M. & Michaels, S. D. The timing of flowering. Plant Physiol. 154, 516-520 (2010).

[39] Andrés, F. & Coupland, G. The genetic basis of flowering responses to seasonal cues. Nat. Rev. Genet. 13, 627-639 (2012).

[40] Wang, J. W. Regulation of flowering time by the miR156-mediated age pathway. J. Exp. Bot. 65, 4723-4730 (2014).

[41] Mouhu, K. et al. Identification of flowering genes in strawberry, a perennial SD plant. BMC Plant. Biol. 9, 122 (2009).

[42] Hu, J. Y. & Meaux, J. D. miR824-regulated AGAMOUS-LIKE16 contributes to flowering time repression in Arabidopsis. Plant Cell 26, 2024-2037 (2014).

[43] Hartmann, U. et al. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J. 21, 351-360 (2010).

[44] Markus, S. et al. Dissection of floral induction pathways using global expression analysis. Development 130, 6001-6012 (2003).

[45] Cao, S. et al. A distal CCAAT/NUCLEAR FACTOR Y complex promotes chromatin looping at the FLOWERING LOCUS T promoter and regulates the timing of flowering in Arabidopsis. Plant Cell 26, 1009-1017 (2014).

[46] Wang, C. Q. et al. BBX19 interacts with CONSTANS to repress FLOWERING LOCUS T transcription, defining a flowering time checkpoint in Arabidopsis. Plant Cell 26, 3589-3602 (2014).

[47] Sawa, M. & Kay, S. A. GIGANTEA directly activates FLOWERING LOCUS T in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 108, 11698-11703 (2011).

[48] Wickland, D. P. & Hanzawa, Y. The FLOWERING LOCUS T/TERMINAL FLOWER 1 gene family: functional evolution and molecular mechanisms. Mol. Plant 8, 983-997 (2015).

[49] Blázquez, M. A. et al. LEAFY expression and flower initiation in Arabidopsis. Development 124, 3835-3844 (1997).

[50] Ferrándiz, C. et al. Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development 127, 725-734 (2000).

[51] Mandel, M. A. & Yanofsky, M. F. A gene triggering flower formation in Arabidopsis. Nature 377, 522-524 (1995).

[52] Iwata, H. et al. The TFL1 homologue KSN is a regulator of continuous flowering in rose and strawberry. Plant J. 69, 116-125 (2012).

[53] Randoux, M. et al. Gibberellins regulate the transcription of the continuous flowering regulator, RoKSN, a rose TFL1 homologue. J. Exp. Bot. 63, 6543-6554 (2012).

[54] Rantanen, M. et al. Strawberry homolog of TERMINAL FLOWER1 integrates photoperiod and temperature signals to inhibit flowering. Plant J. 82, 163-173 (2015).

[55] Melzer, S. et al. FPF1 modulates the competence to flowering in Arabidopsis. Plant J. 18, 395-405 (1999).

[56] Yamaguchi, A. & Wu, M. L. The microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL and APETALA1. Dev. Cell 17, 268-278 (2009).

[57] Becker, A. & Theiben, G. The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol. Phylogenet. Evol. 29, 464-489 (2003).

[58] Dornelas, M. C. et al. MADS: the missing link between identity and growth? Trends Plant Sci. 16, 89-97 (2011).

[59] Pelaz, S. et al. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405, 200-203 (2000).

[60] Dorca-Fornell, C., Gregis, V. & Grand., V. The Arabidopsis SOC1-like genes AGL42, AGL71 and AGL72 promote flowering in the shoot apical and axillary meristems. Plant J. 67, 1006-1017 (2011).

[61] Tamura, K. et al. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729 (2013).

[62] Shulaev, V. et al. The genome of woodland strawberry (Fragaria vesca). Nat. Genet. 43, 109-116 (2011).

[63] Edger, P. P. et al. Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity. Gigascience 7, gix124 (2017).

[64] Gietz, R. D. & Schiest, R. H. High-efficiency yeast transformation using the LiAC/SS carrier DNA/PEG method. Nat. Protoc. 2, 31-34 (2007).

[65] Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735-743 (2010).

[66] Li, W. J. et al. FveRGA1, encoding a DELLA protein, negatively regulates runner production in Fragaria vesca. Planta 247, 941-951 (2018).

[67] Chang, L. et al. Detection of strawberry RNA and DNA viruses by RT-PCR using total nucleic acid as a template. J. Phytopathol. 155, 431-436 (2010).

[68] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT Method . Methods 25, 402-408 (2001).