Identification of genes preferentially expressed in wild strawberry receptacle fruit and demonstration of their promoter activities

Abstract

Fragaria vesca (F. vesca), the wild strawberry, is a diploid model for the commercial, octoploid strawberry as well as other members of the economically relevant Rosaceae family. Unlike the fruits of tomato and Arabidopsis, the fleshy fruit of strawberry is unique in that it is derived from the floral receptacle and has an external seed configuration. Thus, identification and subsequent characterization of receptacle-expressed genes may shed light on novel developmental processes or provide insight into how developmental regulation differs between receptacle-derived and ovary-derived fruits. Further, since fruit and flower tissues are the last organs to form on a plant, the development of receptacle fruit-specific promoters may provide useful molecular tools for research and application. In this work, we mined previously generated RNA-Seq datasets and identified 589 genes preferentially expressed in the strawberry receptacle versus all other profiled tissues. Promoters of a select subset of the 589 genes were isolated and their activities tested using a GUS transcriptional reporter. These promoters may now be used by the F. vesca research community for a variety of purposes, including driving expression of tissue-specific reporters, RNAi constructs, or specific genes to manipulate fruit development. Further, identified genes with receptacle-specific expression patterns, including MADS-Box and KNOX family transcription factors, are potential key regulators of fleshy fruit development and attractive candidates for functional characterization.

References

[1] Darrow, G. M. The Strawberry: History, Breeding, and Physiology (Holt, Rinehart and Winston, New York, 1966).

[2] Slovin, J. P., Schmitt, K. & Folta, K. M. An inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae. Plant Methods 5, 15 (2009).

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

[4] 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, 1-7 (2018).

[5] 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).

[6] Hollender, C. A., Geretz, A. C., Slovin, J. P. & Liu, Z. Flower and early fruit development in a diploid strawberry, Fragaria vesca. Planta 235, 1123-1139 (2012).

[7] Aharoni, A. & O’Connell, A. P. Gene expression analysis of strawberry achene and receptacle maturation using DNA microarrays. J. Exp. Bot. 53, 2073-2087 (2002).

[8] García-Gago, J. A., Posé, S., Muñoz-Blanco, J., Quesada, M. A. & Mercado, J. A. The polygalacturonase FaPG1 gene plays a key role in strawberry fruit softening. Plant Signal. Behav. 4, 766-768 (2009).

[9] Estrada-Johnson, E. et al. Transcriptomic analysis in strawberry fruits reveals active auxin biosynthesis and signaling in the ripe receptacle. Front. Plant Sci. 8, 889 (2017).

[10] Sánchez-Sevilla, J. F. et al. Gene expression atlas of fruit ripening and transcriptome assembly from RNA-seq data in octoploid strawberry (Fragaria×ananassa). Sci. Rep. 7, 13737 (2017).

[11] Hu, P. et al. Transcriptome profiling by RNA-Seq reveals differentially expressed genes related to fruit development and ripening characteristics in strawberries (Fragaria×ananassa). PeerJ 6, e4976 (2018).

[12] Irish, V. The ABC model of floral development. Curr. Biol. 27, R887-R890 (2017).

[13] Potenza, C., Aleman, L. & Sengupta-Gopalan, C. Targeting transgene expression in research, agricultural, and environmental applications: promoters used in plant transformation. Vitr. Cell Dev. Biol. Plant 40, 1-22 (2004).

[14] Schaart, J., Salentijn, E. & Krens, F. Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants. Plant Cell Rep. 21, 313-319 (2002).

[15] Spolaore, S., Trainotti, L., Pavanello, A. & Casadoro, G. Isolation and promoter analysis of two genes encoding different endo‐β‐1,4‐glucanases in the non‐climacteric strawberry. J. Exp. Bot. 54, 271-277 (2003).

[16] Rosin, F. M. et al. Expression patterns of a putative homolog of AGAMOUS, STAG1, from strawberry. Plant Sci. 165, 959-968 (2003).

[17] Agius, F., Amaya, I., Botella, M. A. & Valpuesta, V. Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression. J. Exp. Bot. 56, 37-46 (2005).

[18] Bustamante, C. A., Civello, P. M. & Martínez, G. A. Cloning of the promoter region of β-xylosidase (FaXyl1) gene and effect of plant growth regulators on the expression of FaXyl1 in strawberry fruit. Plant Sci. 177, 49-56 (2009).

[19] Schaart, J., Salentijn, E., Pelgrom, Aharoni, A. & Krens, F. Isolation and characterization of a strawberry fruit-specific promoter. In Genomics, Transgenics, Molecular Breeding and Biotechnology of Strawberry 5, 108-114 (eds. Husaini AM and Mercado JA) (Global Science Books Ltd. UK, Ikenobe, Japan, 2011).

[20] Kang, C. et al. Genome-scale transcriptomic insights into early-stage fruit development in woodland strawberry Fragaria vesca. Plant Cell 25, 1960-1978 (2013).

[21] Hollender, C. A. et al. Floral transcriptomes in Woodland strawberry uncover developing receptacle and anther gene networks. Plant Physiol. 165, 1062-1075 (2014).

[22] Hawkins, C. et al. An eFP browser for visualizing strawberry fruit and flower transcriptomes. Hortic. Res. 4, 17029 (2017).

[23] Hawkins, C., Caruana, J., Schiksnis, E. & Liu, Z. Genome-scale DNA variant analysis and functional validation of a SNP underlying yellow fruit color in wild strawberry. Sci. Rep. 6, 29017 (2016).

[24] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

[25] Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010).

[26] Vrebalov, J. et al. A MADS-box gene necessary for fruit ripening at the tomato ripening-inhibitor (rin) locus. Science 296, 343-346 (2002).

[27] Pelaz, S., Ditta, G. S., Baumann, E., Wisman, E. & Yanofsky, M. F. B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405, 200-203 (2000).

[28] Bemer, M. et al. The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening. Plant Cell 24, 4437-4451 (2012).

[29] Dutt, M., Dhekney, S. A., Soriano, L., Kandel, R. & Grosser, J. W. Temporal and spatial control of gene expression in horticultural crops. Hortic. Res. 1, 14047 (2014).

[30] Chow, C.-N. et al. PlantPAN3.0: a new and updated resource for reconstructing transcriptional regulatory networks from ChIP-seq experiments in plants. Nucleic Acids Res. 47, D1155-D1163 (2019).

[31] Kaplan-Levy, R. N., Brewer, P. B., Quon, T. & Smyth, D. R. The trihelix family of transcription factors--light, stress and development. Trends Plant. Sci. 17, 163-171 (2012).

[32] Shahan, R. et al. Consensus co-expression networks provide insights into wild strawberry flower and fruit development. Plant Physiol. 178, 202-216 (2018).

[33] Butelli, E. et al. Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat. Biotechnol. 26, 1301-1308 (2008).

[34] Klee, H. J. Improving the flavor of fresh fruits: genomics, biochemistry, and biotechnology. New Phytol. 187, 44-56 (2010).

[35] Zhang, Y. et al. Anthocyanins double the shelf life of tomatoes by delaying overripening and reducing susceptibility to gray mold. Curr. Biol. 23, 1094-1100 (2013).

[36] Conley, T. R., Park, S. C., Kwon, H. B., Peng, H. P. & Shih, M. C. Characterization of cis-acting elements in light regulation of the nuclear gene encoding the A subunit of chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from Arabidopsis thaliana. Mol. Cell. Biol. 14, 2525-2533 (1994).

[37] Tjaden, G., Edwards, J. W. & Coruzzi, G. M. Cis elements and trans-acting factors affecting regulation of a nonphotosynthetic light-regulated gene for chloroplast glutamine synthetase. Plant Physiol. 108, 1109-1117 (1995).

[38] Brown, R. L., Kazan, K., McGrath, K. C., Maclean, D. J. & Manners, J. M. A role for the GCC-box in Jasmonate-mediated activation of the PDF1.2 gene of Arabidopsis. Plant Physiol. 132, 1020-1032 (2003).

[39] Arabidopsis Genome Initiative . Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796-815 (2000).

[40] Yamamoto, Y. Y. et al. Prediction of transcriptional regulatory elements for plant hormone responses based on microarray data. BMC Plant Biol. 11, 39 (2011).

[41] Jefferson, R. A., Kavanagh, T. A. & Bevan, M. W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901-3907 (1987).

[42] Garceau, D. C., Batson, M. K. & Pan, I. L. Variations on a theme in fruit development: the PLE lineage of MADS-box genes in tomato (TAGL1) and other species. Planta 246, 313-321 (2017).

[43] Yin, W. et al. Suppression of the MADS-box gene SlMBP8 accelerates fruit ripening of tomato (Solanum lycopersicum). Plant. Physiol. Biochem. 118, 235-244 (2017).

[44] Ireland, H. S. et al. Apple SEPALLATA1/2-like genes control fruit flesh development and ripening. Plant J. 73, 1044-1056 (2013).

[45] Feng, X., An, Y., Zheng, J., Sun, M. & Wang, L. Proteomics and SSH analyses of ALA-promoted fruit coloration and evidence for the involvement of a MADS-Box gene, MdMADS1. Front Plant Sci 7, 1615 (2016).

[46] Li, J. et al. Characteristics and regulatory pathway of the PrupeSEP1 SEPALLATA gene during ripening and softening in peach fruits. Plant Sci. 257, 63-73 (2017).

[47] Schaffer, R. J., Ireland, H. S., Ross, J. J., Ling, T. J. & David, K. M. SEPALLATA1/2-suppressed mature apples have low ethylene, high auxin and reduced transcription of ripening-related genes. AoB Plants 5, pls047 (2012).

[48] Karlova, R. et al. Transcriptional control of fleshy fruit development and ripening. J. Exp. Bot. 65, 4527-4541 (2014).

[49] Proost, S. et al. PLAZA 3.0: an access point for plant comparative genomics. Nucleic Acids Res. 43, D974-D981 (2015).

[50] Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462-469 (2003).

[51] Chatterjee, M., Bermudez-Lozano, C. L., Clancy, M. A., Davis, T. M. & Folta, K. M. A strawberry KNOX gene regulates leaf, flower and meristem architecture. PLoS ONE 6, e24752 (2011).

[52] Caruana, J. C., Sittmann, J. W., Wang, W. & Liu, Z. Suppressor of runnerless encodes a DELLA protein that controls runner formation for asexual reproduction in strawberry. Mol. Plant 11, 230-233 (2018).

[53] Takahashi, N. et al. Cytokinins control endocycle onset by promoting the expression of an APC/C activator in Arabidopsis roots. Curr. Biol. 23, 1812-1817 (2013).

[54] Sessions, A., Weigel, D. & Yanofsky, M. F. The Arabidopsis thaliana MERISTEM LAYER 1 promoter specifies epidermal expression in meristems and young primordia. Plant J. 20, 259-263 (1999).

[55] Du, Z., Zhou, X., Ling, Y., Zhang, Z. & Su, Z. agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res. 38, W64-W70 (2010).