Characterization of germline development and identification of genes associated with germline specification in pineapple

Abstract

Understanding germline specification in plants could be advantageous for agricultural applications. In recent decades, substantial efforts have been made to understand germline specification in several plant species, including Arabidopsis, rice, and maize. However, our knowledge of germline specification in many agronomically important plant species remains obscure. Here, we characterized the female germline specification and subsequent female gametophyte development in pineapple using callose staining, cytological, and whole-mount immunolocalization analyses. We also determined the male germline specification and gametophyte developmental timeline and observed male meiotic behavior using chromosome spreading assays. Furthermore, we identified 229 genes that are preferentially expressed at the megaspore mother cell (MMC) stage during ovule development and 478 genes that are preferentially expressed at the pollen mother cell (PMC) stage of anther development using comparative transcriptomic analysis. The biological functions, associated regulatory pathways and expression patterns of these genes were also analyzed. Our study provides a convenient cytological reference for exploring pineapple germline development and a molecular basis for the future functional analysis of germline specification in related plant species.

References

[1] Ogata, T., Yamanaka, S., Shoda, M., Urasaki, N. & Yamamoto, T. Current status of tropical fruit breeding and genetics for three tropical fruit species cultivated in Japan: pineapple, mango, and papaya. Breed. Sci. 66, 69-81, https://doi.org/10.1270/jsbbs.66.69 (2016).

[2] Lv, L. et al. Isolation and characterization of a FLOWERING LOCUS T homolog from pineapple (Ananas comosus (L.) Merr). Gene 505, 368-73, https://doi.org/10.1016/j.gene.2012.06.011 (2012).

[3] Ma, H. & Sundaresan, V. Development of flowering plant gametophytes. Curr. Top. Developmental Biol. 91, 379-412, https://doi.org/10.1016/S0070-2153(10)91013-2 (2010).

[4] Zhang, D., Luo, X. & Zhu, L. Cytological analysis and genetic control of rice anther development. J. Genet. Genomics = Yi chuan xue bao 38, 379-90, https://doi.org/10.1016/j.jgg.2011.08.001 (2011).

[5] Ito, T. et al. The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS. Nature 430, 356-60, https://doi.org/10.1038/nature02733 (2004).

[6] Sieber, P. et al. Pattern formation during early ovule development in Arabidopsis thaliana. Dev. Biol. 273, 321-34, https://doi.org/10.1016/j.ydbio.2004.05.037 (2004).

[7] Lieber, D., Lora, J., Schrempp, S., Lenhard, M. & Laux, T. Arabidopsis WIH1 and WIH2 genes act in the transition from somatic to reproductive cell fate. Curr. Biol. 21, 1009-17, https://doi.org/10.1016/j.cub.2011.05.015 (2011).

[8] Balasubramanian, S. & Schneitz, K. NOZZLE regulates proximal-distal pattern formation, cell proliferation and early sporogenesis during ovule development in Arabidopsis thaliana. Development 127, 4227-38, https://www.ncbi.nlm.nih.gov/pubmed/10976054 (2000).

[9] Bencivenga, S., Simonini, S., Benkova, E. & Colombo, L. The transcription factors BEL1 and SPL are required for cytokinin and auxin signaling during ovule development in Arabidopsis. Plant Cell 24, 2886-97, https://doi.org/10.1105/tpc.112.100164 (2012).

[10] Nonomura, K. I. Small RNA pathways responsible for non-cell-autonomous regulation of plant reproduction. Plant Reprod. 31, 21-29, https://doi.org/10.1007/s00497-018-0321-x (2018).

[11] Olmedo-Monfil, V. et al. Control of female gamete formation by a small RNA pathway in Arabidopsis. Nature 464, 628-32, https://doi.org/10.1038/nature08828 (2010).

[12] Hernandez-Lagana, E., Rodriguez-Leal, D., Lua, J. & Vielle-Calzada, J. P. A multigenic network of ARGONAUTE4 clade members controls early megaspore formation in arabidopsis. Genetics 204, 1045-1056, https://doi.org/10.1534/genetics.116.188151 (2016).

[13] Su, Z. X. et al. The THO complex non-cell-autonomously represses female germline specification through the TAS3-ARF3 module. Curr. Biol. 27, 1597−+, https://doi.org/10.1016/j.cub.2017.05.021 (2017).

[14] Su, Z. et al. Regulation of female germline specification via small RNA mobility in Arabidopsis. Plant Cell 32, 2842-2854, https://doi.org/10.1105/tpc.20.00126 (2020).

[15] Zhao, L. et al. KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis. Proc. Natl Acad. Sci. USA 115, E526-E535, https://doi.org/10.1073/pnas.1716054115 (2018).

[16] van der Linde, K. et al. Pathogen trojan horse delivers bioactive host protein to alter maize anther cell behavior in situ. Plant cell 30, 528-542, https://doi.org/10.1105/tpc.17.00238 (2018).

[17] Lora, J., Yang, X. & Tucker, M. R. Establishing a framework for female germline initiation in the plant ovule. J. Exp. Bot. 70, 2937-2949, https://doi.org/10.1093/jxb/erz212 (2019).

[18] Cao, L. et al. Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule. Plos Genet 14, e1007230, https://doi.org/10.1371/journal.pgen.1007230 (2018).

[19] Zhao, X. et al. RETINOBLASTOMA RELATED1 mediates germline entry in Arabidopsis. Science 356, https://doi.org/10.1126/science.aaf6532 (2017).

[20] Su, Z. et al. The THO complex non-cell-autonomously represses female germline specification through the TAS3-ARF3 module. Curr. Biol.: CB 27, 1597-1609 e2, https://doi.org/10.1016/j.cub.2017.05.021 (2017).

[21] Yao, X. et al. The canonical E2Fs are required for Germline Development in Arabidopsis. Front Plant Sci. 9, 638, https://doi.org/10.3389/fpls.2018.00638 (2018).

[22] Zhao, D. Z., Wang, G. F., Speal, B. & Ma, H. The excess microsporocytes1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes Dev. 16, 2021-31, https://doi.org/10.1101/gad.997902 (2002).

[23] Yang, S. L. et al. Tapetum determinant1 is required for cell specialization in the Arabidopsis anther. Plant Cell 15, 2792-804, https://doi.org/10.1105/tpc.016618 (2003).

[24] Yang, S. L. et al. Overexpression of TAPETUM DETERMINANT1 alters the cell fates in the Arabidopsis carpel and tapetum via genetic interaction with excess microsporocytes1/extra sporogenous cells. Plant Physiol. 139, 186-91, https://doi.org/10.1104/pp.105.063529 (2005).

[25] Canales, C., Bhatt, A. M., Scott, R. & Dickinson, H. EXS, a putative LRR receptor kinase, regulates male germline cell number and tapetal identity and promotes seed development in Arabidopsis. Curr. Biol. 12, 1718-27, https://doi.org/10.1016/s0960-9822(02)01151-x (2002).

[26] Colcombet, J., Boisson-Dernier, A., Ros-Palau, R., Vera, C. E. & Schroeder, J. I. Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation. Plant Cell 17, 3350-61, https://doi.org/10.1105/tpc.105.036731 (2005).

[27] Jia, G., Liu, X., Owen, H. A. & Zhao, D. Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase. Proc. Natl Acad. Sci. USA 105, 2220-5, https://doi.org/10.1073/pnas.0708795105 (2008).

[28] Kelliher, T. & Walbot, V. Hypoxia triggers meiotic fate acquisition in maize. Science 337, 345-8, https://doi.org/10.1126/science.1220080 (2012).

[29] Chaubal, R. et al. The transformation of anthers in the msca1 mutant of maize. Planta 216, 778-88, https://doi.org/10.1007/s00425-002-0929-8 (2003).

[30] Nonomura, K. et al. The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in rice. Plant Cell 15, 1728-39, https://doi.org/10.1105/tpc.012401 (2003).

[31] Zhao, X. et al. OsTDL1A binds to the LRR domain of rice receptor kinase MSP1, and is required to limit sporocyte numbers. Plant J. 54, 375-87, https://doi.org/10.1111/j.1365-313X.2008.03426.x (2008).

[32] van der Linde, K. & Walbot, V. Pre-meiotic anther development. Curr. Top. Developmental Biol. 131, 239-256, https://doi.org/10.1016/bs.ctdb.2018.11.001 (2019).

[33] Wang, C. J. et al. Maize multiple archesporial cells 1 (mac1), an ortholog of rice TDL1A, modulates cell proliferation and identity in early anther. Dev. Dev. 139, 2594-603, https://doi.org/10.1242/dev.077891 (2012).

[34] Nonomura, K. et al. A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice. Plant Cell 19, 2583-94, https://doi.org/10.1105/tpc.107.053199 (2007).

[35] Nonomura, K. et al. A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L.). Plos Genet. 7, e1001265, https://doi.org/10.1371/journal.pgen.1001265 (2011).

[36] Cai, W. & Zhang, D. The role of receptor-like kinases in regulating plant male reproduction. Plant Reprod. 31, 77-87, https://doi.org/10.1007/s00497-018-0332-7 (2018).

[37] Makinen, Y., Upadhya, M. D. & Brewbaker, J. L. Cytotoxic effects of extracts from gamma-irradiated pineapples. Nature 214, 413, https://doi.org/10.1038/214413a0 (1967).

[38] B. Van de Poel *, J.C., M.P. De Proft . Determination of pineapple (Ananas comosus, MD-2 hybrid cultivar) plant maturity, the efficiency of flowering induction agents and the use of activated carbon. Scientia Horticulturae 120, 6 https://doi.org/10.1016/j.scienta.2008.09.014 (2009).

[39] Ming, R. et al. The pineapple genome and the evolution of CAM photosynthesis. Nat. Genet. 47, 1435-42, https://doi.org/10.1038/ng.3435 (2015).

[40] Chen, L. Y. et al. The bracteatus pineapple genome and domestication of clonally propagated crops. Nat. Genet. 51, 1549-1558, https://doi.org/10.1038/s41588-019-0506-8 (2019).

[41] Escobar-Guzman, R., Rodriguez-Leal, D., Vielle-Calzada, J. P. & Ronceret, A. Whole-mount immunolocalization to study female meiosis in Arabidopsis. Nat. Protoc. 10, 1535-42, https://doi.org/10.1038/nprot.2015.098 (2015).

[42] Qin, Y. et al. ACTIN-RELATED PROTEIN6 regulates female meiosis by modulating meiotic gene expression in Arabidopsis. Plant Cell 26, 1612-1628, https://doi.org/10.1105/tpc.113.120576 (2014).

[43] Zhao, L. et al. Comparative expression profiling reveals gene functions in female meiosis and gametophyte development in Arabidopsis. Plant J. 80, 615-28, https://doi.org/10.1111/tpj.12657 (2014).

[44] Kurzbauer, M. T., Uanschou, C., Chen, D. & Schlogelhofer, P. The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis. Plant Cell 24, 2058-70, https://doi.org/10.1105/tpc.112.098459 (2012).

[45] Cao, L. et al. Arabidopsis ICK/KRP cyclin-dependent kinase inhibitors function to ensure the formation of one megaspore mother cell and one functional megaspore per ovule. Plos Genet. 14, http://dx.doi.org/ARTN e100723010.1371/journal.pgen.1007230 (2018).

[46] Wang, L. et al. Floral transcriptomes reveal gene networks in pineapple floral growth and fruit development. Commun. Biol. 3, 500, https://doi.org/10.1038/s42003-020-01235-2 (2020).

[47] Ming, R., Wai, C. M. & Guyot, R. Pineapple genome: a reference for monocots and CAM Photosynthesis. Trends Genet. 32, 690-696, https://doi.org/10.1016/j.tig.2016.08.008 (2016).

[48] Wai, C. M. et al. Temporal and spatial transcriptomic and microRNA dynamics of CAM photosynthesis in pineapple. Plant J. 92, 19-30, https://doi.org/10.1111/tpj.13630 (2017).

[49] Bolanos-Villegas, P. & Arguello-Miranda, O. Meiosis research in orphan and non-orphan tropical crops. Front. Plant Sci. 10, 74, https://doi.org/10.3389/fpls.2019.00074 (2019).

[50] Kim, J. & Choi, K. Signaling-mediated meiotic recombination in plants. Curr. Opin. Plant Biol. 51, 44-50, https://doi.org/10.1016/j.pbi.2019.04.001 (2019).

[51] Mercier, R. & Grelon, M. Meiosis in plants: ten years of gene discovery. Cytogenetic Genome Res. 120, 281-90, https://doi.org/10.1159/000121077 (2008).

[52] Klucher, K. M., Chow, H., Reiser, L. & Fischer, R. L. The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2. Plant Cell 8, 137-53, https://doi.org/10.1105/tpc.8.2.137 (1996).

[53] Habib, S., Waseem, M., Li, N., Yang, L. & Li, Z. Overexpression of SlGRAS7 affects multiple behaviors leading to confer abiotic stresses tolerance and impacts gibberellin and auxin signaling in tomato. Int J. Genomics 2019, 4051981, https://doi.org/10.1155/2019/4051981 (2019).

[54] Liu, Y. et al. Overexpression of SlGRAS40 in tomato enhances tolerance to abiotic stresses and influences auxin and gibberellin signaling. Front. Plant Sci. 8, 1659, https://doi.org/10.3389/fpls.2017.01659 (2017).

[55] Jin, Y. et al. OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues. Plant Mol. Biol. 98, 51-65, https://doi.org/10.1007/s11103-018-0762-5 (2018).

[56] Ranjan, R. et al. bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice. Sci. Rep. 7, 43397, https://doi.org/10.1038/srep43397 (2017).

[57] Farquharson, K. L. A domain in the bHLH transcription factor DYT1 is critical for anther development. Plant Cell 28, 997-8, https://doi.org/10.1105/tpc.16.00331 (2016).

[58] Phan, H. A., Iacuone, S., Li, S. F. & Parish, R. W. The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana. Plant Cell 23, 2209-24, https://doi.org/10.1105/tpc.110.082651 (2011).

[59] Mitsuda, N., Seki, M., Shinozaki, K. & Ohme-Takagi, M. The NAC transcription factors NST1 and NST2 of Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant Cell 17, 2993-3006, https://doi.org/10.1105/tpc.105.036004 (2005).

[60] Chen, L. G. et al. BZR1 family transcription factors function redundantly and indispensably in BR signaling but exhibit BRI1-independent function in regulating anther development in Arabidopsis. Mol. Plant 12, 1408-1415, https://doi.org/10.1016/j.molp.2019.06.006 (2019).

[61] Chen, W. et al. BES1 is activated by EMS1-TPD1-SERK1/2-mediated signaling to control tapetum development in Arabidopsis thaliana. Nat. Commun. 10, 4164, https://doi.org/10.1038/s41467-019-12118-4 (2019).

[62] Schmidt, A. et al. Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development. PLoS Biol. 9, e1001155, https://doi.org/10.1371/journal.pbio.1001155 (2011).

[63] Zhao, H. et al. Comparative expression profiling reveals genes involved in megasporogenesis. Plant Physiol. 182, 2006-2024, https://doi.org/10.1104/pp.19.01254 (2020).

[64] Singh, S. K. et al. The TRAF mediated gametogenesis progression (TRAMGaP) gene is required for megaspore mother cell specification and gametophyte development. Plant Physiol. 175, 1220-1237, https://doi.org/10.1104/pp.17.00275 (2017).

[65] Aw, S. J., Hamamura, Y., Chen, Z., Schnittger, A. & Berger, F. Sperm entry is sufficient to trigger division of the central cell but the paternal genome is required for endosperm. Dev. Arabidopsis. Dev. 137, 2683-90, https://doi.org/10.1242/dev.052928 (2010).

[66] Ingouff, M., Jullien, P. E. & Berger, F. The female gametophyte and the endosperm control cell proliferation and differentiation of the seed coat in Arabidopsis. Plant Cell 18, 3491-501, https://doi.org/10.1105/tpc.106.047266 (2006).

[67] Wang, C. J. & Tseng, C. C. Recent advances in understanding of meiosis initiation and the apomictic pathway in plants. Front Plant Sci. 5, 497, https://doi.org/10.3389/fpls.2014.00497 (2014).

[68] Ma, J., Skibbe, D. S., Fernandes, J. & Walbot, V. Male reproductive development: gene expression profiling of maize anther and pollen ontogeny. Genome Biol. 9, R181, https://doi.org/10.1186/gb-2008-9-12-r181 (2008).

[69] Draeger, T. et al. Dmc1 is a candidate for temperature tolerance during wheat meiosis. Theor. Appl. Genet. 133, 809-828, https://doi.org/10.1007/s00122-019-03508-9 (2020).

[70] Deng, Z. Y. & Wang, T. OsDMC1 is required for homologous pairing in Oryza sativa. Plant Mol. Biol. 65, 31-42, https://doi.org/10.1007/s11103-007-9195-2 (2007).

[71] Wang, J., Qiu, X., Li, Y., Deng, Y. & Shi, T. A transcriptional dynamic network during Arabidopsis thaliana pollen development. BMC Syst. Biol. 5(Suppl 3), S8, https://doi.org/10.1186/1752-0509-5-S3-S8 (2011).

[72] Gibalova, A. et al. Characterization of pollen-expressed bZIP protein interactions and the role of ATbZIP18 in the male gametophyte. Plant Reprod. 30, 1-17, https://doi.org/10.1007/s00497-016-0295-5 (2017).

[73] Mou, W. et al. Ethylene-independent signaling by the ethylene precursor ACC in Arabidopsis ovular pollen tube attraction. Nat. Commun. 11, 4082, https://doi.org/10.1038/s41467-020-17819-9 (2020).

[74] Maruyama, D. et al. Rapid elimination of the persistent synergid through a cell fusion mechanism. Cell 161, 907-18, https://doi.org/10.1016/j.cell.2015.03.018 (2015).

[75] Zhang, X. S. & O’Neill, S. D. Ovary and gametophyte development are coordinately regulated by auxin and ethylene following pollination. Plant Cell 5, 403-418, https://doi.org/10.1105/tpc.5.4.403 (1993).

[76] Zhu, Y. et al. Genome-wide identification and expression analysis reveal the potential function of ethylene responsive factor gene family in response to Botrytis cinerea infection and ovule development in grapes (Vitis vinifera L.). Plant Biol. 21, 571-584, https://doi.org/10.1111/plb.12943 (2019).

[77] Khaskheli, A. J. et al. RhERF113 functions in ethylene-induced petal senescence by modulating cytokinin content in rose. Plant Cell Physiol. 59, 2442-2451, https://doi.org/10.1093/pcp/pcy162 (2018).

[78] Song, X., Li, Y. & Hou, X. Genome-wide analysis of the AP2/ERF transcription factor superfamily in Chinese cabbage (Brassica rapa ssp. pekinensis). BMC Genomics 14, 573, https://doi.org/10.1186/1471-2164-14-573 (2013).

[79] Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-20, https://doi.org/10.1093/bioinformatics/btu170 (2014).

[80] Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562-78, https://doi.org/10.1038/nprot.2012.016 (2012).

[81] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550, https://doi.org/10.1186/s13059-014-0550-8 (2014).

[82] Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923- 30, https://doi.org/10.1093/bioinformatics/btt656 (2014).

[83] Tian, T. et al. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res. 45, W122-W129, https://doi.org/10.1093/nar/gkx382 (2017).

[84] Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792-7, https://doi.org/10.1093/nar/gkh340 (2004).

[85] Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-9, https://doi.org/10.1093/molbev/mst197 (2013).

[86] Chen, C. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 13, 1194-1202, https://doi.org/10.1016/j.molp.2020.06.009 (2020).

[87] Wu, M. F. & Wagner, D. RNA in situ hybridization in Arabidopsis. Methods Mol. Biol. 883, 75-86, https://doi.org/10.1007/978-1-61779-839-9_5 (2012).