Comprehensive identification, stress responses and protein-protein interactions of the ferredoxin gene family in the fast-growing tree species Paulownia fortunei

Abstract

Paulownia fortunei is a significant fast-growing economic tree species with wide industrial value, and studies have revealed that its rapid growth mechanism is associated with its C3-CAM photosynthetic characteristics. Ferredoxin, a key protein in the plant photosynthetic electron transport chain, plays a crucial role in both C3 and CAM pathways. In addition to participating in plant photosynthesis and carbon assimilation, ferredoxin also plays important roles in plant nitrogen assimilation, sulfur metabolism, and responses to biotic and abiotic stresses. In this study, bioinformatics analysis was utilized to identify eight members of the ferredoxin gene family in Paulownia fortunei, categorized into four subfamilies distributed across eight different chromosomes. All members contain a 2Fe-2 S domain and light-responsive elements, displaying high conservation among subfamily members. Transcriptome analysis and real-time quantitative PCR (qRT-PCR) assays indicated that photosynthetic ferredoxins PfFd1 and PfFd2 play important roles in the response of Paulownia fortunei to droughts, salt and pathogen stresses. Yeast two-hybrid(Y2H)results showed that PfFd1 and PfFd2 can form heterodimers. Interaction predictions, Y2H, and bimolecular fluorescence complementation (BiFC) experiments verified direct interactions between the major photosynthetic ferredoxin PfFd2 and ferredoxin-NADP + reductase (FNR) as well as sulfite reductase (SIR) in Paulownia fortunei, suggesting that PfFd2 may play a significant role in photosynthetic electron transport and sulfur metabolism in Paulownia fortunei, implying its potential association with the fast-growing characteristics of Paulownia fortunei and its influence on normal growth and metabolism. Additionally, the interaction between the Paulownia witches' broom phytoplasma effector pawb3 and PfFd2 was verified through Y2H and GST pull-down experiments, suggesting that PfFd2 may be related to disease resistance in Paulownia fortunei. This study systematically analyzed the family characteristics of ferredoxins in Paulownia fortunei and preliminary explored their biological functions, providing a theoretical basis for future in-depth exploration of molecular mechanisms related to plant fast growth and the cultivation of high-resistance, fast-growing germplasm resources.

Keywords: Paulownia fortunei; Fast-growing; Ferredoxin; Stress response.

Fig. 1

Phylogenetic analysis of PfFd genes in Paulownia fortunei, Arabidopsis thalian, Oryza sativa and Zea mays. Different color blocks correspond to different subfamilies respectively

Fig. 2

Motifs, conserved domain, gene structures and cis acting elements between the PfFds members in Paulownia fortunei. A Architecture of motifs in 8 PfFds. Each motif is represented by the number on the colored box; B Architecture of conserved domain in 8 PfFds. Each conserved domain is represented by the number on the colored box; C Exon/intron structures of 8 PfFds; D Cis acting elements of 2000 bp promoter region upstream of 8 PfFds. Each cis-regulatory element type was marked by different colored boxes

Fig. 3

Chromosome distribution and collinearity analysis of the PfFds genes in Paulownia fortunei. A Distribution of PfFds genes in chromosomes; B Syntent analysis of Paulownia foutunei. The green line represents the collinearity gene pair in the Paulownia foutunei genome; C Collinearity analysis of PfFds genes between Paulownia fortunei and Arabidopsis thalian, Paulownia fortunei and Sesamum indicum. The gray line represents the collinear blockin the genome. The collinear gene pairs of PfFds gene are highlighted by blue lines

Fig. 4

Expression analysis of PfFds under biotic and abiotic stress and subcellular localization of PfFd2. A Heatmap of PfFds genes expression in the occurrence of drought, salt and Paulownia witches’ broom disease. Scaled log2 expression values based on transcriptomics data were shown from green to red, indicating low to high expression; B qRT-PCR detection of expression of PfFds genes under pathogen stress. Significant and highly significant difference compared with the gene expression in PF are shown as * (P < 0.05) and ** (P < 0.01), respectively; C Subcellular localization of PfFd2. Leaf tissues of N. benthamiana transiently expressing the PfFd2-GFP and GFP alone were examined by epifluorescence microscopy. GFP: GFP fluorescence detection, Cy5: Chloroplast autofluorescence detection, BF: Bright fields, Merged: superposition fields. Bar = 50 μm

Fig. 5

Prediction and Verification of Interaction Proteins of photosynthetic ferredoxin PfFd1 and PfFd2. A Prediction of protein interactions in PfFd1 and PfFd2 through String database; B, C Prediction of interactions between PfFds and interacting proteins through AlphaFold3 database; D Transcriptional activity analysis of PfFd1 and PfFd2 proteins; E Verification of the interaction between PfFd1, PfFd2 and PfFNR, PfSIR using Y2H; F Verification of the interaction between PfFd2 and interacting proteins using BiFC. Detection of the PfFd2-PfFNR and PfFd2-PfSIR interaction in N. benthamiana leaves transiently expressing the marked constructs. Bar = 20 μm

Fig. 6

Heterodimer and PaWB Disease Regulation of PfFd2. A Verification of the heterodimer between PfFd1 and PfFd2 using Y2H; B Verification of the interaction between PfFd2 and pawb3 using Y2H; C Purification of PfFd2 and pawb3 proteins. Left lane (His-PfFd2): 1: Pellet after sonication and centrifugation of induced bacterial culture; 2: Supernatant after sonication and centrifugation of induced bacterial culture; 3: Flow-through from Ni-IDA column purification; 4: Wash with Buffer D2 (removal of contaminating proteins); 5: Elution of target protein with Buffer C; 6: Proteins retained on the column; M: Mid-range molecular weight protein marker; Right lane (GST-pawb3): 1: Supernatant; 2: Flow-through; 3: Column-bound; The molecular mass of GST-pawb3 and His-PfFd2 bands was 48 and 33kD, respectively; D GST pull-down verification the interaction of pawb3 and PfFd2 proteins