Domestication-driven Gossypium profilin 1 (GhPRF1) gene transduces early flowering phenotype in tobacco by spatial alteration of apical/floral-meristem related gene expression

Abstract

Background: Plant profilin genes encode core cell-wall structural proteins and are evidenced for their up-regulation under cotton domestication. Notwithstanding striking discoveries in the genetics of cell-wall organization in plants, little is explicit about the manner in which profilin-mediated molecular interplay and corresponding networks are altered, especially during cellular signalling of apical meristem determinacy and flower development.

Results: Here we show that the ectopic expression of GhPRF1 gene in tobacco resulted in the hyperactivation of apical meristem and early flowering phenotype with increased flower number in comparison to the control plants. Spatial expression alteration in CLV1, a key meristem-determinacy gene, is induced by the GhPRF1 overexpression in a WUS-dependent manner and mediates cell signalling to promote flowering. But no such expression alterations are recorded in the GhPRF1-RNAi lines. The GhPRF1 transduces key positive flowering regulator AP1 gene via coordinated expression of FT4, SOC1, FLC1 and FT1 genes involved in the apical-to-floral meristem signalling cascade which is consistent with our in silico profilin interaction data. Remarkably, these positive and negative flowering regulators are spatially controlled by the Actin-Related Protein (ARP) genes, specifically ARP4 and ARP6 in proximate association with profilins. This study provides a novel and systematic link between GhPRF1 gene expression and the flower primordium initiation via up-regulation of the ARP genes, and an insight into the functional characterization of GhPRF1 gene acting upstream to the flowering mechanism. Also, the transgenic plants expressing GhPRF1 gene show an increase in the plant height, internode length, leaf size and plant vigor.

Conclusions: Overexpression of GhPRF1 gene induced early and increased flowering in tobacco with enhanced plant vigor. During apical meristem determinacy and flower development, the GhPRF1 gene directly influences key flowering regulators through ARP-genes, indicating for its role upstream in the apical-to-floral meristem signalling cascade.

Keywords: Apical meristem determinacy; Flower development; Flowering genes; Gene expression; Profilin.

Fig. 1

Schematic representation of different gene constructs used in the present study. a The T-DNA diagramme of nos:nptII:pA::CaMV35S:GhPRF1:pA binary construct for the overexpression of proflin gene in tobacco. b The T-DNA diagramme of nos:nptII:pA::CaMV35S:gus:pA binary construct. c The T-DNA diagramme of RNAi binary construct nos:nptII:pA::CaMV35S:GhPRF1-intron-GhPRF1:pA gene construct for the down-expression of proflin gene in tobacco. The orientations of different gene cassettes are shown as per their respective cloning sites in the binary vector. The horizontal bars are not to scale

Fig. 2

Profilin gene expression analysis in vegetative and reproductive tissues of two transgenic overexpression lines Pf-Ox4 and Pf-Ox17 of tobacco. a Semi-quantitative expression analysis of profilin in vegetative tissues of Pf-Ox4 and Pf-Ox17 in comparison to two independently in vitro regenerated control lines. b Semi-quantitative expression analysis of profilin in reproductive tissues of Pf-Ox4 and Pf-Ox17 in comparison to two independently in vitro regenerated control lines. c & d Average expression values of profilin gene in both vegetative and reproductive tissues of the two overexpression lines in comparison to control lines, respectively, by densitometry imaging analysis avoiding any biases visible in the band intensities on an agarose gel. e Significant increase in the plant height through elongated internodal regions of over-expression lines Pf-Ox4 and Pf-Ox17 (labeled 1 & 2) in comparison to two control lines (labeled 3&4) after 120 dpt. f In vitro regenerated control line after 100 days of vegetative growth post-transplantation. g Transgenic Pf-Ox4 line showing early conversion of apical shoot meristem into floral meristem after 100 days of vegetative growth post-transplantation. h Relative size differences in 20^th leaf from the top of transgenic and control lines. i Relative size differences in 13^th leaf from the top of transgenic and control lines

Fig. 3

CLV1 gene expression analysis in vegetative and reproductive tissues of two transgenic overexpression lines. a Semi-quantitative expression analysis of CLV1 gene in vegetative tissues of Pf-Ox4 and Pf-Ox17 in comparison to two control lines (C1 and C2). b Semi-quantitative expression analysis of CLV1 gene in reproductive tissues of Pf-Ox4 and Pf-Ox17 in comparison to the control lines. c & d Average expression values of CLV1 gene in both vegetative and reproductive tissues of the two overexpression lines in comparison to control lines, respectively, by densitometry imaging analysis avoiding any biases visible in the band intensities on agarose gel

Fig. 4

a In vitro organogenesis of tobacco on MS medium supplemented with NAA (0.01 mg/l) and BAP (1.0 mg/l) phytohormones. Three different concentrations i.e., minimal (Mi; <0.1 mM), optimal (O; 0.1 mM) and maximal (Mx; >0.1 mM) of micro-nutrient Boron was supplied with MS medium and leaf explants were cultured. b Induction of shoot primordia on the edges of leaf explants after15 days of culture on MS shoot induction medium. c An enlarged view of a microscopic shoot meristem (shown by an arrow) which was harvested for meristem-determinacy gene expression analysis. d Temporal expression of CLV1 gene analyses in 7 days and 15 days old shoot primordia harvested from explants cultured on minimal, optimal and maximal boron- supplemented medium. Similarly, semi-quantitative expression of WUS gene was analysed in 7 days and 15 days shoot primordia harvested from explants cultured on minimal, optimal and maximal boron- supplemented medium. e Average expression values of CLV1 and WUS genes in shoot primordia after 7 days and 15 days of culture, measured by densitometry imaging analysis avoiding any biases visible in the band intensities on an agarose gel

Fig. 5

Influence of GhPRF1 overexpression on the shoot induction (organogenesis). a Untransformed Xanthi explants cultured on MS medium supplemented with phytohormones required for shoot induction. b Explants transformed with nos:nptII:pA::35S:gus:pA gene cassettes and cultured on MS medium + Phytohormones + kanamycin (100 mg/l). c Explants transformed with nos:nptII:pA::35:GhPRF1 gene cassettes and cultured on MS medium + Phytohormones + kanamycin (100 mg/l). The lower panel shows significant changes in the rate of organogenesis and % plantlet formation in GhPRF1 transformed explants in comparison to 35S:gus transformed explants and untransformed explants

Fig. 6

Genetic manipulation of profilin gene in tobacco. a Transgenic plants are developed with ectopic constitutive overexpression line, constitutive down-expression line, and control line, respectively. All three lines are photographed at the same age. The inset picture shows the relative expression of proflin gene in control, overexpression, and RNAi silencing line. b Flower number per plant is shown for Pf-Ox4 line. c Flower number per plant is shown for the Pf-Si23 line. d Flower number per plant is shown for control line. e A number of flowers produced by Pf-Ox4 and Pf-Si23 transgenic lines along with control plant. One way ANOVA analysis was performed for statistical analysis of differences using Graphpad Prism resulted in R² value 0.9467. f The onset of flowering in Pf-Ox4 and Pf-Si23 transgenic lines along with control plant after transplantation. Unpaired t-test was performed (p < 0.05) to analyze significant differences in flowering time

Fig. 7

Dynamicity of coordinated profilin interaction network with key flowering regulators predicted in silico and analyzed at the transcript level in GhPRF1 over- expression and RNAi lines. The genes encoding for positive and negative flowering regulators were analysed in vegetative tissue (VT) and reproductive tissue (RT) of control plant, Pf-Ox4 overexpression line and Pf-Si23 silencing line. a In silico prediction of profilin interaction with key flowering genes based on STRING 10. This panel also shows the positive flowering regulator AP1 gene expression data in vegetative tissue (VT) and reproductive tissue (RT) of the control plant, Pf-Ox4 line and Pf-Si23 line. In the in silico predicted interaction map, line thickness represents the confidence level of protein-protein interaction. b This panel represents the positive flowering regulators FT4 and SOC1 gene expression data in VT and RT of different lines. c This panel represents the negative flowering regulators FLC1 and FT1 gene expression data in VT and RT of different lines

Fig. 8

Gene expression analyses of ARP genes and prediction of their interaction with other genetic factors. a ARP4 gene expression analyses in Pf-Ox4 and Pf-Si23 transgenic lines along with control plant in VT and RT tissues. b ARP6 gene expression analyses in Pf-Ox4 and Pf-Si23 transgenic lines along with control plant in VT and RT tissues. c In silico prediction of profilin-ARP interaction based on STRING 10. This analysis shows the interaction of profilin with actin proteins and one of the key flowering regulator FLC via ARP6 protein. The line thickness represents the confidence level of protein-protein interaction

Fig. 9

Comparative morphological characters are shown in control flowers and Pf-Ox4 overexpression line. a Complete flower of control and Pf-Ox4 line. b Longitudinal section of both control and Pf-Ox4 line. c & d Calyx of control and Pf-Ox4 line, respectively. e Androecia of control and Pf-Ox4 line showing filament and anthers. f Gynoecia of control and Pf-Ox4 line. g Pollen of control line stained with aniline blue. h Pollen of Pf-Ox4 line stained with aniline blue. i & j Maturing fruit and their number in control and Pf-Ox4 line, respectively

Fig. 10

Comparative anatomical features shown in control flowers and Pf-Ox4 overexpression line. a & b Microscopic view of stamen of control and Pf-Ox4 line. c & b Anther of both control and Pf-Ox4 line. e & f Stigma of control and Pf-Ox4 line, respectively. g & h Ovary of control and Pf-Ox4 line. i & j T.S. of a mature ovary of control and Pf-Ox4 line, respectively

Fig. 11

A molecular framework for profilin-mediated activation of apical and reproductive meristem. Different roles of profilin are shown: (i) including its classical role in cellular architecture mainly through actin polymerization and depolymerization; cellular signalling mainly through actin-related proteins (ARPs). In association with ARPs, profilin polymerizes actin, and certain ARPs have also been reported for their role in flowering phenomenon. Also, ARP6 induces FLC gene expression leading to the repression of flowering [88, 89]. Such coordinated regulation of flowering time mainly through ARP genes with FLC1 regulator directly influence flower genes expression cascade. (ii) its novel roles in apical meristem determinacy via transcriptional activation of CLV1 gene in the homeodomain trans-factor WUS- dependent manner; and (iii) activation of key flowering regulators for floral development. The latter are known to largely initiate reproductive meristem activation through flowering time controlling genes such as flowering locus T4 (FT4) gene which travels from vegetative leaf cells to the initiating floral meristem and in turn up-regulates other flower controlling regulators mainly SOC1, LFY and ultimately AP1 which is a class ‘A’ gene and is responsible for the activation of class ‘B’ genes during floral development. Here, we identify important genes whose expression is directly induced by profilin overexpression that furthermore jointly regulate flower primordium initiation. These genes encode known regulators of flower development: FT4 gene, which specifies the flowering time, SOC1 transcription factor, which in collaboration with AGL24 and LEAFY (LFY) gene up-regulates AP1 gene, which is a class ‘A’ gene and works as a key regulator of floral development. In parallel, overexpression of profilin down-regulates negative flowering regulators: FLC1 gene, which suppresses the expression of SOC1 trans-factor; and FT1 gene, which acts as transcriptional inhibitor exclusively in tobacco [67]. Our study reveals a link between profilin and flower primordium initiation mainly via up-regulation of ARP genes