The APETALA2 homolog CaFFN regulates flowering time in pepper

Abstract

Flowering time is an important agronomic trait that contributes to fitness in plants. However, the genetic basis of flowering time has not been extensively studied in pepper. To understand the genetics underlying flowering time, we constructed an F₂ population by crossing a spontaneous early flowering mutant and a late-flowering pepper line. Using bulked segregant RNA-seq, a major locus controlling flowering time in this population was mapped to the end of chromosome 2. An APETALA2 (AP2) homolog (CaFFN) cosegregated with flowering time in 297 individuals of the F₂ population. A comparison between the parents revealed a naturally occurring rare SNP (SNP2T > C) that resulted in the loss of a start codon in CaFFN in the early flowering mutant. Transgenic Nicotiana benthamiana plants with high CaFFN expression exhibited a delay in flowering time and floral patterning defects. On the other hand, pepper plants with CaFFN silencing flowered early. Therefore, the CaFFN gene acts as a flowering repressor in pepper. CaFFN may function as a transcriptional activator to activate the expression of CaAGL15 and miR156e and as a transcriptional repressor to repress the expression of CaAG, CaAP1, CaSEP3, CaSOC1, and miR172b based on a qRT-PCR assay. Direct activation of CaAGL15 by CaFFN was detected using yeast one-hybrid and dual-luciferase reporter assays, consistent with the hypothesis that CaFFN regulates flowering time. Moreover, the CaFFN gene association analysis revealed a significant association with flowering time in a natural pepper population, indicating that the CaFFN gene has a broad effect on flowering time in pepper. Finally, the phylogeny, evolutionary expansion and expression patterns of CaFFN/AP2 homologs were analyzed to provide valuable insight into CaFFN. This study increases our understanding of the involvement of CaFFN in controlling flowering time in pepper, thus making CaFFN a target gene for breeding early maturing pepper.

References

[1] Henderson, I. R. & Dean, C. Control of Arabidopsis flowering: the chill before the bloom. Development 131, 3829-3838 (2004).

[2] Richter, R. et al. Floral regulators FLC and SOC1 directly regulate expression of the B3-type transcription factor TARGET OF FLC AND SVP 1 at the Arabidopsis shoot apex via antagonistic chromatin modifications. PLoS Genet. 15, e1008065 (2019).

[3] MacAlister, C. A. et al. Synchronization of the flowering transition by the tomato TERMINATING FLOWER gene. Nat. Genet. 44, 1393-1398 (2012).

[4] Fornara, F., de Montaigu, A. & Coupland, G. SnapShot: control of flowering in Arabidopsis. Cell 141, 550 (2010).

[5] Blazquez, M. A. & Weigel, D. Integration of floral inductive signals in Arabidopsis. Nat. 404, 889-892 (2000).

[6] Moon, J. et al. The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis. Plant J. 35, 613-623 (2003).

[7] Liu, C. et al. Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis. Development 135, 1481-1491 (2008).

[8] Lee, J. & Lee, I. Regulation and function of SOC1, a flowering pathway integrator. J. Exp. Bot. 61, 2247-2254 (2010).

[9] Kardailsky, I. et al. Activation tagging of the floral inducer Ft. Sci. 286, 1962-1965 (1999).

[10] Yant, L. et al. Orchestration of the floral transition and floral development in Arabidopsis by the bifunctional transcription factor APETALA2. Plant Cell 22, 2156-2170 (2010).

[11] Abelenda, J. A., Navarro, C. & Prat, S. Flowering and tuberization: a tale of two nightshades. Trends Plant Sci. 19, 115-122 (2014).

[12] Samach, A. & Lotan, H. The transition to flowering in tomato. Plant Biotechnol. 24, 71-82 (2007).

[13] Weng, L., Bai, X. D., Zhao, F. F., Li, R. & Xiao, H. Manipulation of flowering time and branching by overexpression of the tomato transcription factor SlZFP2. Plant Biotechnol. J. 14, 2310-2321 (2016).

[14] Cohen, O., Borovsky, Y., David-Schwartz, R. & Paran, I. CaJOINTLESS is a MADS-box gene involved in suppression of vegetative growth in all shoot meristems in pepper. J. Exp. Bot. 63, 4947-4957 (2012).

[15] Cohen, O., Borovsky, Y., David-Schwartz, R. & Paran, I. Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum). N. Phytol. 202, 1014-1023 (2014).

[16] Mimura, Y., Minamiyama, Y., Sano, H. & Hirai, M. Mapping for axillary shooting, flowering date, primary axis length, and number of leaves in pepper (Capsicum annuum). J. Jpn. Soc. Hort. Sci. 79, 56-63 (2010).

[17] Tan, S. et al. Construction of an interspecific genetic map based on InDel and SSR for mapping the QTLs affecting the initiation of flower primordia in pepper (Capsicum spp.). PLoS ONE 10, e0119389 (2015).

[18] Zhang, X. F. et al. Candidate genes for first flower node identified in pepper using combined SLAF-seq and BSA. PLoS ONE 13, e0194071 (2018).

[19] Jeifetz, D., David-Schwartz, R., Borovsky, Y. & Paran, I. CaBLIND regulates axillary meristem initiation and transition to flowering in pepper. Planta 234, 1227-1236 (2011).

[20] Borovsky, Y., Sharma, V. K., Verbakel, H. & Paran, I. CaAP2 transcription factor is a candidate gene for a flowering repressor and a candidate for controlling natural variation of flowering time in Capsicum annuum. Theor. Appl. Genet. 128, 1073-1082 (2015).

[21] Elitzur, T. et al. Co-ordinated regulation of flowering time, plant architecture and growth by FASCICULATE: the pepper orthologue of SELF PRUNING. J. Exp. Bot. 60, 869-880 (2009).

[22] Wu, L. et al. Genome-wide correlation of 36 agronomic traits in the 287 pepper (Capsicum) accessions obtained from the SLAF-seq-based GWAS. Int. J. Mol. Sci. 20, 5675 (2019).

[23] Barchi, L., Lefebvre, V., Sage-Palloix, A. M., Lanteri, S. & Palloix, A. QTL analysis of plant development and fruit traits in pepper and performance of selective phenotyping. Theor. Appl. Genet. 118, 1157-1171 (2009).

[24] Alimi, N. A. et al. Multi-trait and multi-environment QTL analyses of yield and a set of physiological traits in pepper. Theor. Appl. Genet. 126, 2597-2625 (2013).

[25] Zhang, X. F. et al. High-density genetic map construction and QTL mapping of first flower node in pepper (Capsicum annuum L.). BMC Plant Biol. 19, 167 (2019).

[26] Zhu, Z. et al. Construction of a high density genetic map of an interspecific cross of Capsicum chinense and Capsicum annuum and QTL analysis of floral traits. Sci. Rep. 9, 1054 (2019).

[27] Anbessa, Y., Warkentin, T., Vandenberg, A. & Ball, R. Inheritance of time to flowering in chickpea in a short-season temperate environment. J. Hered. 97, 55-61 (2006).

[28] Boni, T. A., Prioli, A. J., Prioli, S. M. A. P., Lucio, L. C. & de Mello, R. Inheritance of aluminum tolerance in maize. Crop Breed. Appl. Biot. 9, 147-153 (2009).

[29] Ou, L. J. et al. Pan-genome of cultivated pepper (Capsicum) and its use in gene presence-absence variation analyses. N. Phytol. 220, 360-363 (2018).

[30] Chung, M. Y. et al. A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening. Plant J. 64, 936-947 (2010).

[31] Zhao, Y. et al. Genome-wide identification and analysis of the AP2 transcription factor gene family in wheat (Triticum aestivum L.). Front. Plant Sci. 10, 1286 (2019).

[32] Lee, D. Y. & An, G. Two AP2 family genes, supernumerary bract (SNB) and Osindeterminate spikelet 1 (OsIDS1), synergistically control inflorescence architecture and floral meristem establishment in rice. Plant J. 69, 445-461 (2012).

[33] Maher, C., Stein, L. & Ware, D. Evolution of Arabidopsis microRNA families through duplication events. Genome Res. 16, 510-519 (2006).

[34] De Grassi, A., Lanave, C. & Saccone, C. Genome duplication and gene-family evolution: the case of three OXPHOS gene families. Gene 421, 1-6 (2008).

[35] Qiao, X. et al. Different modes of gene duplication show divergent evolutionary patterns and contribute differently to the expansion of gene families involved in important fruit traits in pear (Pyrus bretschneideri). Front. Plant Sci. 9, 161 (2018).

[36] Qin, C. et al. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization. Proc. Natl Acad. Sci. U. S. A. 111, 5135-5140 (2014).

[37] Aukerman, M. J. & Sakai, H. Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell 15, 2730-2741 (2003).

[38] Mlotshwa, S., Yang, Z., Kim, Y. & Chen, X. Floral patterning defects induced by Arabidopsis APETALA2 and microRNA172 expression in Nicotiana benthamiana. Plant Mol. Biol. 61, 781-793 (2006).

[39] Axtell, M. J. & Bartel, D. P. Antiquity of microRNAs and their targets in land plants. Plant Cell 17, 1658-1673 (2005).

[40] Wurschum, T., Gross-Hardt, R. & Laux, T. APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem. Plant Cell 18, 295-307 (2006).

[41] Ohto, M. A., Fischer, R. L., Goldberg, R. B., Nakamura, K. & Harada, J. J. Control of seed mass by APETALA2. Proc. Natl Acad. Sci. U. S. A. 102, 3123-3128 (2005).

[42] Kim, S. et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat. Genet. 46, 270-278 (2014).

[43] Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009).

[44] Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).

[45] Mlotshwa, S. et al. Transgenic plants expressing HC-Pro show enhanced virus sensitivity while silencing of the transgene results in resistance. Virus Genes 25, 45-57 (2002).

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

[47] 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).

[48] Jian, W. et al. SlMYB75, an MYB-type transcription factor, promotes anthocyanin accumulation and enhances volatile aroma production in tomato fruits. Hortic. Res. 6, 22 (2019).

[49] Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-1797 (2004).

[50] Bradbury, P. J. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633-2635 (2007).

[51] Yuan, X. J. et al. Association analysis of resistance to Phytophthora capsici Leonian in pepper (Capsicum annuum L.) and exploration of elite alleles. J. Plant Genet. Resour. 20, 1026-1040 (2019).

[52] El-Gebali, S. et al. The Pfam protein families database in 2019. Nucleic Acids Res. 47, 427-432 (2019).

[53] Nakano, T., Suzuki, K., Fujimura, T. & Shinshi, H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 140, 411-432 (2006).

[54] Wang, Y. P. et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 40, e49 (2012).

[55] Xia, X. DAMBE6: new tools for microbial genomics, phylogenetics, and molecular evolution. J. Hered. 108, 431-437 (2017).

[56] Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729 (2013).

[57] Yang, Z. PAML 4: phylogenetic analysis by maximum likelihood. Mol. Biol. Evol. 24, 1586-1591 (2007).

[58] Koch, M. A., Haubold, B. & Mitchell-Olds, T. Comparative evolutionary analysis of chalcone synthase and alcohol dehydrogenase loci in Arabidopsis, Arabis, and related genera (Brassicaceae). Mol. Biol. Evol. 17, 1483-1498 (2000).

[59] Park, S. J., Jiang, K., Schatz, M. C. & Lippman, Z. B. Rate of meristem maturation determines inflorescence architecture in tomato. Proc. Natl Acad. Sci. U.S.A. 109, 639-644 (2012).

[60] Winter, D. et al. An “Electronic Fluorescent Pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS ONE 2, e718 (2007).