Flowering locus T chimeric protein induces floral precocity in edible citrus

Authors: SINN, JUDITH - Pennsylvania State University; HELD, JEREMY - Pennsylvania State University; VOSBURG, CHAD - Pennsylvania State University; KLEE, SARAH - Pennsylvania State University; ORBOVIC, VLADIMIR - University Of Florida; Taylor, Earl; Gottwald, Timothy; Stover, Eddie; MOORE, GLORIA - University Of Florida; MCNELLIS, TIMOTHY - Pennsylvania State University

Submitted to: Plant Biotechnology Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/2/2020
Publication Date: 8/9/2020
Citation: Sinn, J., Held, J., Vosburg, C., Klee, S., Orbovic, V., Taylor, E.L., Gottwald, T.R., Stover, E.W., Moore, G., McNellis, T. 2020. Flowering locus T chimeric protein induces floral precocity in edible citrus. Plant Biotechnology Journal. https://doi.org/10.1111/pbi.13463.
DOI: https://doi.org/10.1111/pbi.13463

Interpretive Summary: Citrus breeding efforts are hampered by a long juvenility period of 6 or more years. We expressed the FT flowering gene fused to an antibody in ‘Duncan’ grapefruit. The full-length FT fusion protein (FT-scFv) was detected with levels varying among individual lines. Level of juvenile character reduction generally correlated with FT-scFv protein level. Precocious flowering was observed in FT-scFv transgenic lines and fell into three categories, ranging from plants with large blooming flushes nearly continuously to these which did not bloom during the four-year monitoring period. Flowers produced by FT-scFv trees were normal and resulting pollen produced fruit and seed set. Our results document the successful use of an FT fusion protein to reduce flowering time in an edible citrus cultivar. In addition, we developed eight ‘Carrizo’ FT-scFv transformants, of which three had a precocious blooming phenotype, and one transformant of a hybrid of ‘Jackson’ grapefruit, which had a precocious blooming phenotype. Moderate expression of the protein resulted in precocious blooming largely without negative effects. FT fusion proteins may have attenuated flowering promotion activity relative to native FT, possibly accounting for the success of our approach. The reduced juvenility offered by transgenic expression of chimeric FT proteins may provide an additional valuable tool for rapid-cycle citrus breeding.

Technical Abstract: Citrus breeding efforts are hampered by a long juvenility period of 6 or more years. To expedite more rapid citrus breeding, we expressed Poncirus trifoliata FT1 (PtFT1) as a translational fusion with a single-chain variable fragment antibody (scFv). A constitutive expression cassette was used that included the Cauliflower mosaic virus (CaMV) 35S promoter with a double enhancer region, the Tobacco etch virus 5¢ untranslated region, and the CaMV 35S polyadenylation signal. The PtFT1-scFv coding region included the PtFT1 cDNA sequence, a flexible linker sequence ([gly4ser]4), the scFv sequence, and a C-terminal cMyc epitope tag. Agrobacterium tumefaciens was used to transform ‘Duncan’ grapefruit with the PtFT1-scFv expression construct. Fifteen successful transformants were grafted onto ‘Carrizo’ citrange rootstock. A transgenic control was transformed with a construct lacking PtFT1-scFv. Full-length PtFT1-scFv protein was detected with levels varying among the lines. PtFT1-scFv protein was detected in both shoot tips and in mature leaves, and was detectable over the course of the four-year experiment period. Grapefruit trees transformed with PtFT1-scFv displayed varying reductions in juvenile characters that generally correlated with PtFT1-scFv protein level. Severe growth phenotypes were observed in the highest expressing lines, displaying a highly branched, prostrate, dwarfed growth form with greatly reduced thorniness. Precocious flowering was observed in PtFT1-scFv transgenic lines and fell into three blooming phenotype categories: strongly precocious, with large blooming flushes nearly continuously; mildly precocious, blooming 1 – 4 times a year with isolated flowers; and non-precocious, which did not bloom during the four-year monitoring period. Phenotype generally correlated with PtFT1-scFv protein levels. Flowers produced by PtFT1-scFv trees were fragrant and morphologically normal, with viable pollen which resulted in fruit production and seed set. Our results document the successful use of a chimeric FT protein to reduce flowering time in an edible citrus cultivar. In addition, we developed eight ‘Carrizo’ PtFT1-scFv transformants, of which three had a precocious blooming phenotype, and one transformant of a hybrid of ‘Jackson’ grapefruit, which had a precocious blooming phenotype. Moderate expression of the protein resulted in precocious blooming largely without negative effects. FT fusion proteins may have attenuated flowering promotion activity relative to native FT, possibly accounting for the success of our approach. The reduced juvenility offered by transgenic expression of chimeric FT proteins may provide an additional valuable tool for rapid-cycle citrus breeding.