Overexpression of Arabidopsis FLOWERING LOCUS T (FT) gene improves floral development in cassava (Manihot esculenta, Crantz)

Abstract

Cassava is a tropical storage-root crop that serves as a worldwide source of staple food for over 800 million people. Flowering is one of the most important breeding challenges in cassava because in most lines flowering is late and non-synchronized, and flower production is sparse. The FLOWERING LOCUS T (FT) gene is pivotal for floral induction in all examined angiosperms. The objective of the current work was to determine the potential roles of the FT signaling system in cassava. The Arabidopsis thaliana FT gene (atFT) was transformed into the cassava cultivar 60444 through Agrobacterium-mediated transformation and was found to be overexpressed constitutively. FT overexpression hastened flower initiation and associated fork-type branching, indicating that cassava has the necessary signaling factors to interact with and respond to the atFT gene product. In addition, overexpression stimulated lateral branching, increased the prolificacy of flower production and extended the longevity of flower development. While FT homologs in some plant species stimulate development of vegetative storage organs, atFT inhibited storage-root development and decreased root harvest index in cassava. These findings collectively contribute to our understanding of flower development in cassava and have the potential for applications in breeding.

Fig 1. Schematic representation of the transformation vector.

Arabidopsis FT cDNA was inserted into the construct through Gateway cloning. pAnos, nopaline synthase polyadenylation signal; pat, phosphinothricin acetyltransferase; Tnos, terminator of nopaline synthase; pAlcA, promoter of alcohol dehydrogenase I (Adh-I) encoded by the alcA gene; FT cDNA, cDNA of Flowering Locus (FT) gene; pA35S, polyadenylation sequence of Cauliflower mosaic virus 35S gene; nos, nopaline synthase terminator; ALCR, transcriptional factor which binds to AlcA promoter; p35S, Cauliflower Mosaic Virus 35S promoter; LB, left border; RB, right border.

Fig 2. Expression of Arabidopsis FT gene in cassava.

The qRT-PCR results were obtained from four biological replicates and two technical replicates for each sample. 60444 represents the non‐transformed wildtype line and FT-02, FT-11, FT-13 and FT-17 represent the four independent transformants. The levels of detected amplification were normalized using 18S and Ubiquitin as reference genes. The expression cassette had an ethanol‐inducible promoter. In each case, potted cassava transgenic plants were either watered normally (H₂O), or the soil was drenched with 1% (v/v) ethanol for two weeks before leaves were harvested and analyzed.

Fig 3. Flowering traits in non‐transformed wildtype line (60444) and in the four independent transformants.

(a) Flowering time in days from establishment in soil to flowering at the 1st, 2nd, and 3rd tier of flowering, as defined by fork-type branching at the apical meristems. (b) Number of shoot nodes to forking events where inflorescences develop. The number of nodes between the soil surface and the first fork, between the first-tier and second-tier forks, and between the second- and third-tier forks. (c) Number of flowers per tier, per plant. (d) Time to start of floral and/or inflorescence senescence. Floral traits were recorded weekly to determine the date of inflorescence appearance, and initial date of floral senescence. The total number of days from flower appearance to start of inflorescence and/or flower senescence was calculated from these weekly records. Shown are the means ± SEM.

Fig 4. Transformed and non-transformed plants at various stages of floral development.

(a): FT-17 transgenic plant at 2 months in vitro. (b and c): FT-17 transgenic plantlet at one month after transfer from in vitro to culture box and soil respectively. (d): Advanced stage transgenic plants flowering at 3 months. (e): Non-transformed (left) vs. transformed (right) plants at 5 months old. (f and g): Close up view of the apical region of 5-month old non-transformed (f) and transformed (g) plants, respectively. Arrows indicate flowers.

Fig 5. Lateral branch development in the axils of leaves on the main stem.

Lateral branches and flowers that formed in fork-type branches at the apex of these lateral branches were counted in the non‐transformed wildtype line (60444) and in the four independent transformants. (a) Number of lateral branches per plant. (b) Total number of flowers on lateral branches. Shown are the means ± SEM.

Fig 6. Root and shoot production in non‐transformed wildtype (60444) and the four independent transformants at harvest.

(a) Storage-root dry weight; (b) total plant dry weight; (c) harvest index (HI), calculated as HI = (storage-root dry mass)/ [(storage-root dry mass) + (above-ground dry mass)]; (d) number of storage-roots. Shown are the means ± SEM.