Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees

Abstract

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants, particularly for wind-pollinated trees such as poplar. We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE (SVL), a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE (SVP), delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars. Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance, and did not cause morphologically obvious or statistically significant effects on leaf characteristics, tree form, or stem volume. Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific. Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.

References

[1] Daniell, H. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol. 20, 581-586 (2002).

[2] Brunner, A. M. et al. Genetic containment of forest plantations. Tree Genet. Genomes 3, 75-100 (2007).

[3] Häggman, H. et al. Genetically engineered trees for plantation forests: key considerations for environmental risk assessment. Plant Biotechnol. J. 11, 785-798 (2013).

[4] Dodet, M. & Collet, C. When should exotic forest plantation tree species be considered as an invasive threat and how should we treat them? Biol. Invasions 14, 1765-1778 (2012).

[5] Fritsche, S., Klocko, A. L., Boron, A., Brunner, A. M. & Thorlby, G. Strategies for engineering reproductive sterility in plantation forests. Front. Plant Sci. 9, 1671 (2018).

[6] Whitesell, C. D., DeBell, D. S., Schubert, T. H., Strand, R. F., & Crabb, T. B. Short-Rotation Management of Eucalyptus: Guidelines for Plantations in Hawaii (Southwest Research Station, 1992).

[7] Strauss, S. H., Rottmann, W. H., Brunner, A. M. & Sheppard, L. A. Genetic engineering of reproductive sterility in forest trees. Mol. Breed. 1, 5-26 (1995).

[8] Klocko, A. L. et al. Phenotypic expression and stability in a large-scale field study of genetically engineered poplars containing sexual containment transgenes. Front. Bioeng. Biotechnol. 6, 100 (2018).

[9] Vining, K. J., Contreras, R. N., Ranik, M. & Strauss, S. H. Genetic methods for mitigating invasiveness of Woody Ornamental Plants: research needs and opportunities. Hort. Sci. 47, 1210-1216 (2012).

[10] Elorriaga, E. et al. A tapetal ablation transgene induces stable male sterility and slows field growth in Populus. Tree Genet. Genomes 10, 1583-1593 (2014).

[11] Moon, H. S. et al. Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system. Plant Mol. Biol. 75, 621-631 (2011).

[12] Zhang, C. et al. Control of pollen-mediated gene flow in transgenic trees. Plant Physiol. 159, 1319-1334 (2012).

[13] Klocko, A. L. et al. Containment of transgenic trees by suppression of LEAFY. Nat. Biotechnol. 34, 918-922 (2016a).

[14] Azeez, A. & Busov, V. CRISPR/Cas9-mediated single and biallelic knockout of poplar STERILE APETALA (PopSAP) leads to complete reproductive sterility. Plant Biotechnol. J 19, 23 (2020).

[15] Cronk, Q. C. B., Needham, I. & Rudall, P. J. Evolution of catkins: inflorescence morphology of selected salicaceae in an evolutionary and developmental context. Front. Plant Sci. 6, 1030- 1030 (2015).

[16] Brunner, A. M. & Nilsson, O. Revisiting tree maturation and floral initiation in the poplar functional genomics era. N. Phytol. 164, 43-51 (2004).

[17] Pin, P. A. & Nilsson, O. The multifaceted roles of flowering locus T in plant development. Plant Cell Environ. 35, 1742-1755 (2012).

[18] Yarur, A., Soto, E., León, G. & Almeida, A. M. The sweet cherry (Prunus avium) Flowering locus T gene is expressed during floral bud determination and can promote flowering in a winter-annual Arabidopsis accession. Plant Reprod. 29, 311-322 (2016).

[19] Wu, R. et al. SVP-like MADS box genes control dormancy and budbreak in apple. Front. Plant Sci. 8, 477 (2017).

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

[21] Klocko, A. L. et al. FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus. Plant Biotechnol. J. 14, 808-819 (2016b).

[22] Endo, T. et al. Ectopic expression of an FT homolog from citrus confers an early flowering phenotype on trifoliate orange (Poncirus trifoliata L. Raf.). Transgenic Res. 14, 703-712 (2005).

[23] Kotoda, N. et al. Molecular characterization of Flowering Locus T-like genes of apple (Malus × domestica Borkh.). Plant Cell Physiol. 51, 561-575 (2010).

[24] Ratcliffe, O. J., Kumimoto, R. W., Wong, B. J. & Riechmann, J. L. Analysis of the Arabidopsis Mads Affecting Flowering gene family: MAF2 prevents vernalization by short periods of cold. Plant Cell 15, 1159 (2003).

[25] Castelán-Muñoz, N. et al. MADS-Box genes are key components of genetic regulatory networks involved in abiotic stress and plastic developmental responses in plants. Front. Plant Sci. 10, 853 (2019).

[26] Moser, M. et al. The MADS-box gene MdDAM1 controls growth cessation and bud dormancy in apple. Front. Plant Sci. 11, 1003 (2020).

[27] Wu, R. et al. RNAi-mediated repression of dormancy-related genes results in evergrowing apple trees. Tree Physiol. https://doi.org/10.1093/treephys/tpab007. (2021).

[28] Singh, R. K. et al. A genetic network mediating the control of bud break in hybrid aspen. Nat. Commun. 9, 4173 (2018).

[29] Mohamed, R. et al. Populus CEN/TFL1 regulates first onset of flowering, axillary meristem identity and dormancy release in Populus. Plant J. 62, 674-688 (2010).

[30] Melzer, R., Wang, Y.-Q. & Theißen, G. The naked and the dead: the ABCs of gymnosperm reproduction and the origin of the angiosperm flower. Semin. Cell Dev. Biol. 21, 118-128 (2010).

[31] Filichkin, S. et al. Enhancer trapping in woody plants: Isolation of the ET304 gene encoding a putative AT-hook motif transcription factor and characterization of the expression patterns conferred by its promoter in transgenic Populus and Arabidopsis. Plant Sci. 17, 206-216 (2006).

[32] Sundell, D. et al. The plant genome integrative explorer resource: PlantGenIE.org. N. Phytol. 208, 1149-1156 (2015).

[33] Hartmann, U. et al. Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J. 21, 351-360 (2000).

[34] Tuskan, G. A. et al. The Genome of Black Cottonwood, Populus trichocarpa (Torr. & Gray). Science 313, 1596 (2006).

[35] Brunner, A. M., Varkonyi-Gasic, E., & Jones, R. C. in Comparative and Evolutionary Genomics of Angiosperm Trees (eds. Groover, A. & Cronk, Q.) 227-274 (Springer, 2017).

[36] Rajeevkumar, S., Anunanthini, P. & Ramalingam, S. Epigenetic silencing in transgenic plants. Front. Plant Sci. 6, 693 (2015).

[37] de Felippes, F. et al. The key role of terminators on the expression and post-transcriptional gene silencing of transgenes. Plant J. 104, 96-112 (2020).

[38] Strauss, S. H., Ma, C., Ault, K., & Klocko, A. L. in Biosafety of Forest Transgenic Trees: Improving the Scientific Basis for Safe Tree Development and Implementation of EU Policy Directives (eds. Vettori, C. et al. ) 101-124 (Springer, 2016).

[39] Kim, Y. et al. The immediate upstream region of the 5′-UTR from the AUG start codon has a pronounced effect on the translational efficiency in Arabidopsis thaliana. Nucleic Acids Res. 42, 485-498 (2014).

[40] Liu, Y. et al. Targeted deletion of floral development genes in Arabidopsis with CRISPR/Cas9 using the RNA endoribonuclease Csy4 processing system. Hortic. Res. 6, 99 (2019).

[41] Meilan, R., Sabatti, M., Ma, C. & Kuzminsky, E. An early-flowering genotype of Populus. J. Plant Biol. 47, 52-56 (2004).

[42] Han, K., Meilan, R., Ma, C. & Strauss, S. H. An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Rep. 19, 315-320 (2000).

[43] Schneider, C. A., Rasband, W. S. & Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671-675 (2012).

[44] 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).

[45] Edgar, R. C. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinform. 5, 1-19 (2004).

[46] Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evolution 35, 1547-1549 (2018).