Genomic survey and expression analysis of LcARFs reveal multiple functions to somatic embryogenesis in Liriodendron

Abstract

Background: Auxin response factors (ARFs) are critical transcription factors that mediate the auxin signaling pathway and are essential for regulating plant growth. However, there is a lack of understanding regarding the ARF gene family in Liriodendron chinense, a vital species in landscaping and economics. Thus, further research is needed to explore the roles of ARFs in L. chinense and their potential applications in plant development.

Result: In this study, we have identified 20 LcARF genes that belong to three subfamilies in the genome of L. chinense. The analysis of their conserved domains, gene structure, and phylogeny suggests that LcARFs may be evolutionarily conserved and functionally similar to other plant ARFs. The expression of LcARFs varies in different tissues. Additionally, they are also involved in different developmental stages of somatic embryogenesis. Overexpression of LcARF1, LcARF2a, and LcARF5 led to increased activity within callus. Additionally, our promoter-GFP fusion study indicated that LcARF1 may play a role in embryogenesis. Overall, this study provides insights into the functions of LcARFs in plant development and embryogenesis, which could facilitate the improvement of somatic embryogenesis in L. chinense.

Conclusion: The research findings presented in this study shed light on the regulatory roles of LcARFs in somatic embryogenesis in L. chinense and may aid in accelerating the breeding process of this tree species. By identifying the specific LcARFs involved in different stages of somatic embryogenesis, this study provides a basis for developing targeted breeding strategies aimed at optimizing somatic embryogenesis in L. chinense, which holds great potential for improving the growth and productivity of this economically important species.

Keywords: ARF genes; Liriodendron chinense; Somatic embryogenesis.

Fig. 1

Phylogenetic tree of ARF proteins in Arabidopsis and L. chinense. The ARFs can be classified into three major classes based on their phylogenetic relationship. The different-colored areas represent distinct classes within the ARF family

Fig. 2

Unrooted Classification tree representing relationships among ARF genes of 10 species. A total of 162 ARF protein sequences from 10 species were selected to construct a Bayesian phylogenetic tree. Different color blocks represent different evolutionary branches

Fig. 3

Analysis of conserved domains of LcARF gene family. A Schematic organization of conserved domains in LcARF proteins. B Amino acid composition of MR domains in LcARF proteins, bars represent the percentage of different amino acid residues in MR domains of LcARFs

Fig. 4

Analysis of intron-exon organization of LcARF gene family. The intron-exon organization of LcARF genes was plotted using Tbtools (Version 1.09832)

Fig. 5

Analysis of cis-acting elements of LcARF gene family. The 2000-bp regulatory region upstream of ATG was analyzed with the PlantCARE software

Fig. 6

Prediction of protein interaction. Protein-protein interaction network of ARFs in L. chinense, the results were based on an Arabidopsis association model

Fig. 7

Subcellular localization of LcARF in L. chinense protoplasts. The red fluorescence signal was the nuclear localization signal of H2B, and the green fluorescent signals of the six GFP fusion proteins were particularly strong in the nucleus

Fig. 8

Expression patterns of LcARFs in different tissues, analyzed by qRT-PCR. A The expression pattern of LcARFs in different tissues. B qRT-PCR was used to detect the expression pattern of LcARFs in different tissues. The purple line graph represents the results of qRT-PCR experiments, with the scale on the right ordinate of each graph. The blue histogram represents the results of FPKM analysis, with the scale on the left ordinate of each graph

Fig. 9

Expression analysis of LcARFs under SE, analyzed by qRT -PCR. A Callus. B Globular embryo. C Heart-shaped embryo. D Torpedo embryo. E Early cotyledonary embryo. F Cotyledon embryo. G The expression pattern of LcARFs in somatic embryos. H qRT -PCR was used to detect the expression pattern of LcARFs in different stages of SE. The purple line graph represents the results of qRT-PCR experiments, with the scale on the right ordinate of each graph. The blue histogram represents the results of FPKM analysis, with the scale on the left ordinate of each graph

Fig. 10

Phenotypic of calli overexpressing LcARFs in Liriodendron. A Cell morphology of calli from WT and overexpression lines. B Cell length, cell width, and cell aspect ratio statistics of calli from WT and overexpression lines. C Acetomagenta and Evans blue staining of calli from WT and overexpressed lines. p < 0.05

Fig. 11

Expression pattern of LcARF1 during somatic embryogenesis. A, D, G, J: Globular embryo. B, E, H, K: Torpedo embryo. C, F, I, L: Cotyledon embryos. A- F: 35S::GFP. G- L: pLcARF1::GFP