Inflorescence Development and the Role of LsFT in Regulating Bolting in Lettuce (Lactuca sativa L.)

Abstract

Lettuce (Lactuca sativa L.) is one of the most important leafy vegetable that is consumed during its vegetative growth. The transition from vegetative to reproductive growth is induced by high temperature, which has significant economic effect on lettuce production. However, the progression of floral transition and the molecular regulation of bolting are largely unknown. Here we morphologically characterized the inflorescence development and functionally analyzed the FLOWERING LOCUS T (LsFT) gene during bolting regulation in lettuce. We described the eight developmental stages during floral transition process. The expression of LsFT was negatively correlated with bolting in different lettuce varieties, and was promoted by heat treatment. Overexpression of LsFT could recover the late-flowering phenotype of ft-2 mutant. Knockdown of LsFT by RNA interference dramatically delayed bolting in lettuce, and failed to respond to high temperature. Therefore, this study dissects the process of inflorescence development and characterizes the role of LsFT in bolting regulation in lettuce.

Keywords: LsFT; bolting; floral transition; lettuce; morphology.

FIGURE 1

Capitulum structure and floral transition in lettuce. (A–D) The four developmental stages of lettuce. From left to right, vegetative stage, bolting stage, inflorescence stage, and flowering stage. (E) The morphology of the capitulum in lettuce. (F) The structure of a ray floret. An, anther, Ov, ovary; Pe, petal; Sti, stigma; Pa, pappus. (G) The transverse section of a capitulum in lettuce. Sta, stamen; Sty, style. (H–O) Histological sections showing the stages of floral transition progression in lettuce. (H) Stage 1 vegetative shoot apical meristem (SAM) (20 DAP [days after planting]). (I) Stage 2 dome-shaped SAM (28 DAP). (J) Stage 3 elongated meristem showing the transition from vegetative to reproductive growth (35 DAP). (K) Stages 4 indicating the development of involucre primordia from the inflorescence meristem (IM) (45 DAP). (L) Stage 5 showing the development of capitulum primordia from the IM (55 DAP). (M) Stage 6 highlighting the development of floret primordia (75 DAP). (N) Stage 7 indicating the formation of ray floret (85 DAP). (O) Stage 8 showing the ovary development (95 DAP). Scale bars represent 6 cm in (A–D), 2 mm in (E,F), 200 μm in (G), and 50 μm in (H–O).

FIGURE 2

Expression analysis and subcellular localization of LsFT in lettuce. (A) Quantitative real time RT-PCR (qRT-PCR) analysis of LsFT in different tissues of lettuce. YL, young leaves; ML, mature leaves; CB, capitulum buds; OC, opening capitulum; CC, closing capitulum; R, root; S, stem. Lettuce 18S ribosomal RNA (HM047292.1) was used as an internal reference to normalize the expression data. (B) The top row is the diagram of the fusion protein construct used for subcellular localization. The open reading frame (ORF) of LsFT cDNA was introduced into PUC-19 vector using the XbaI and SmaI sites, and fused with GFP in frame. The 35S promoter directs the expression of fusion genes. The bottom row is the subcellular localization of LsFT fusion protein in onion epidermal cells. Plasmid with green fluorescent protein (GFP) alone served as the control (top). Scale bar represents 50 μm. (C) The morphology of lettuce varieties with different bolting times. The days to bolting in the growth chamber were as follows: S24 (75 days), S43 (58 days), S7 (55 days), S1 (50 days), S3 (48 days), S8 (46 days), S28 (43 days), S26 (42 days), S39 (38 days). (D) qRT-PCR analysis of LsFT in different lettuce varieties before and after heat treatment (35°C/25°C) for 48 h. (E) The number of days to bolting under heat treatment (35°C/25°C) and mock treatment. Error bars represent standard errors. Significant difference were determined by student’s t-test (^∗represents P < 0.05 and ^∗∗indicates P < 0.01).

FIGURE 3

Ectopic expression of LsFT in ft-2 mutant Arabidopsis plants. (A) Ectopic expression of LsFT can fully restore the late-flowering phenotype in ft-2 mutant plants. (B) Quantification of flowering phenotypes in LsFT transgenic lines. (C) Expression analyses of FT and LsFT by qRT-PCR in ft-2, WT, and overexpression lines of 35S-LsFT/ft-2 (OV-1, OV-6, OV-14). Error bars represent standard errors. Significant difference were determined by student’s t-test (^∗represents P < 0.05 and ^∗∗indicates P < 0.01). Scale bar represents 6 cm.

FIGURE 4

Functional characterization of LsFT in lettuce. (A) qRT-PCR analysis of LsFT expression in different LsFT-RNAi lines in lettuce. (B) LsFT-RNAi resulted in significant delay in bolting in lettuce. Scale bar represents 6 cm. (C) Quantification of the delayed bolting phenotypes in LsFT-RNAi lines. (D) Expression analyses of LsAP1, LsAP3 and LsLFY in LsFT-RNAi lines. Lettuce 18S ribosomal RNA (HM047292.1) was used as an internal reference to normalize the expression data. (E) qRT-PCR analysis of LsFT in WT and LsFT-RNAi lines at 1, 2, 3, 4 days after heat treatment (DAH). (F) The number of days to bolting in WT and LsFT-RNAi lines under heat treatment and mock treatment. Error bars represent standard errors. Significant difference were determined by student’s t-test (^∗represents P < 0.05 and ^∗∗indicates P < 0.01).