Functional Characterization of the MsFKF1 Gene Reveals Its Dual Role in Regulating the Flowering Time and Plant Height in Medicago sativa L

Abstract

Alfalfa (M. sativa), a perennial legume forage, is known for its high yield and good quality. As a long-day plant, it is sensitive to changes in the day length, which affects the flowering time and plant growth, and limits alfalfa yield. Photoperiod-mediated delayed flowering in alfalfa helps to extend the vegetative growth period and increase the yield. We isolated a blue-light phytohormone gene from the alfalfa genome that is an ortholog of soybean FKF1 and named it MsFKF1. Gene expression analyses showed that MsFKF1 responds to blue light and the circadian clock in alfalfa. We found that MsFKF1 regulates the flowering time through the plant circadian clock pathway by inhibiting the transcription of E1 and COL, thus suppressing FLOWERING LOCUS T a1 (FTa1) transcription. In addition, transgenic lines exhibited higher plant height and accumulated more biomass in comparison to wild-type plants. However, the increased fiber (NDF and ADF) and lignin content also led to a reduction in the digestibility of the forage. The key genes related to GA biosynthesis, GA20OX1, increased in the transgenic lines, while GA2OX1 decreased for the inactive GA transformation. These findings offer novel insights on the function of MsFKF1 in the regulation of the flowering time and plant height in cultivated M. sativa. These insights into MsFKF1's roles in alfalfa offer potential strategies for molecular breeding aimed at optimizing flowering time and biomass yield.

Keywords: FKF1; alfalfa; blue light; circadian clock; flowering time.

Figure 1

Alignment of MsFKF1 with its homologs from other species. Amino acid sequence alignment of FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) in Medicago sativa (MsFKF1), M. truncatula (MtFKF1), Glycine max (GmFKF1), Arabidopsis thaliana (AtFKF1), and Zea mays (ZmAGO3). Identical amino acids are indicated by a black background, while the gray shade represents amino acids that share a similarity of 50% or more. PAS/LOV (blue), F-box (black), and the KELCH repeat domain (green and orange) are marked in continuous underline, according to the InterPro website tool.

Figure 2

Analysis of the expression characteristics of MsFKF1. (A) The expression levels of MsFKF1 in different tissues. RNA was extracted from the roots, stems, leaves, buds, and flowers of WT plants grown under LD conditions for 45 days. The results represent the average of three independent biological replicates, and the error bars indicate the standard deviation. (B) Subcellular localization of the MsFKF1-GFP fusion protein in the tobacco epidermal layers analyzed by fluorescence microscopy. The fluorescence of GFP, DAPI, and merged images are shown. DAPI represent 4′,6-diamidino-2-phenylindole (nucleus-specific dye). Scale bar = 50 μm. (C) Schematic diagram of the analysis of the transcriptional activation activity of MsFKF1. (D) The MsFKF1 gene lacks transcriptional activation activity in yeast (Y2H Gold). SD/-T refers to synthetic dropout (SD) yeast growth medium that is deficient in tryptophan (-T). SD/-L-H, SD medium without tryptophan and histidine, and supply with 15 mM 3-AT. Positive means the co-transition of yeast with PGBKT7-53 and PGADT7-T, which could activate the HIS3 reporter gene. BD and BD-MsFKF1 indicate the PGBKT7 vector and PGBKT7 fused with MsFKF1, respectively.

Figure 3

Expression pattern of the MsFKF1 gene in response to light signal. (A) Expression level of MsFKF1 in leaves in darkness, and white and blue light. (B,C) Diurnal changes in MsFKF1 levels under LD and SD conditions. (D) The mRNA levels of MsFKF1 (ZT8) in alfalfa leaves that shift from long-day to short-day. ZT, zeitgeber time (hours after dawn). The fully expanded trifoliate leaves were used in this experiment collected from 30 DAS plants. DAS, days after sowing. Values are presented as the mean value derived from three biological replicates. The bars represent the mean ± SE of three biological replicates per condition.

Figure 4

Overexpression of MsFKF1 delays flowering time in alfalfa plants. (A) The image of two independent late-flowering transgenic lines (OE1, OE3) compared to WT flowering plants. Bar = 10 cm. The photo was taken one week after WT flowering. (B) Comparison of phenotypes of alfalfa shoot apices among WT, OE1, and OE3. Bar = 2 mm. (C) Expression of MsFKF1 in WT, OE1, 3 by RT-qPCR. Leaf samples from 5-week-old plants grown under LD conditions were harvested at zeitgeber time 20 (ZT20) for RNA extraction. MsFKF1 expression was quantified by RT-qPCR in two independent transgenic lines and WT controls. (D) Flowering time of two independent transgenic lines compared to WT regenerated controls. The bars indicate the mean ± SE of three biological replicates and eight clonal plants per line were analysis for flowering time assays. Asterisks represent different levels of significance (** p ≤ 0.01). (E–H) Expression analysis of flowering locus T genes in WT and OE1, 3. Fully expendable leaf samples were collected at ZT20 (peak of the MsFTa1 gene) from the long-day condition.

Figure 5

Overexpression of MsFKF1 in alfalfa affects expression of genes related to the circadian clock. (A–L) Relative gene expression of some alfalfa COL genes and some key genes related to circadian rhythm in a long-day (LD) in 35S: MsFKF1 lines. The data included in this study were obtained from fully expanded trifoliate leaves harvested from T1 plants during day 15 at ZT4. The presented gene expression levels reflect the means of three biological replicates, and the standard error (SE) is given. Asterisks represent level of significance (** p ≤ 0.01). These values were normalized to β-actin and are presented relative to the value of the sample with the highest expression.

Figure 6

Phenotype of the WT plant compared to transgenic lines. (A) Data analysis of elongation stem of internode from shoots apical. (B,C) Relative gene expression of Medicago GA signal-related genes. Measurements were taken when the flower buds appeared during growth under long-day photoperiod at 23 °C. Bars represent the mean ±SE of 6 individual-grown clonal plants per line. Asterisks represent different significance levels (* p ≤ 0.05, ** p ≤ 0.01).