Genome-wide characterization and expression analysis of the growth-regulating factor family in Mikania micrantha

Abstract

Background: Growth-regulating factors (GRFs) are plant-specific transcription factors involved in growth, development, and stress responses. Mikania micrantha, a highly invasive weed, displays rapid growth and adaptability, yet the GRF gene family in this species remains largely unexplored.

Results: We identified and characterized 16 GRF genes in M. micrantha, analyzing their phylogeny, gene structure, collinearity, and expression patterns. These genes were classified into six subfamilies and showed conserved synteny with GRFs from Arabidopsis thaliana and Asteraceae species, with all gene pairs under purifying selection. Expression profiling revealed high transcript levels of most MmiGRFs in stem tips and young tissues. Hormone treatments demonstrated that MmiGRFs responded differentially to GA₃, ABA, IAA, BR, 6-BA, and MeJA, with MmiGRF8 notably upregulated by all treatments. Subcellular localization confirmed nuclear localization of MmiGRF proteins. Additionally, five miR396 members were predicted to target 15 MmiGRFs, with MmiGRF10 containing two distinct sites.

Conclusion: This study provides the first comprehensive analysis of the M. micrantha GRF gene family, revealing their potential roles in rapid growth and environmental adaptation. These findings offer molecular insights into the species' invasive capacity and support future strategies for its control.

Keywords: Mikania micrantha; Expression analysis; Growth and development; Growth-regulating factors.

Fig. 1

Phylogenetic tree of GRF proteins from A. thaliana, M. cordata, L. sativa, M. micrantha, H. annuus and A. annua. Based on bootstrap values and evolutionary distances, the tree was classified into six subfamilies. GRF proteins from different species are indicated by distinct shapes and colors

Fig. 2

Phylogenetic relationship, gene structure, and conserved motif pattern of MmiGRF genes in M. micrantha. (A) Phylogenetic tree of MmiGRF genes constructed using the NJ method. (B) Gene structures of 16 MmiGRF genes. Introns and coding sequences are depicted as black lines and yellow blocks, respectively. (C) Distribution of conserved motifs in GRF proteins. Motifs 1–10 are represented by rectangles in different colors

Fig. 3

Chromosomal localization and synteny analysis of MmiGRF genes. Colored columns represent chromosomes, gene IDs are shown on the outer sides, and red lines indicate syntenic gene pairs among MmiGRF family members

Fig. 4

Collinearity analysis of MmiGRF genes in M. micrantha and four other plant species: A. thaliana, M. cordata, L. sativa, and H. annuus. Gray lines in the background represent all collinear blocks between the M. micrantha genome and the genomes of the other species, while orange lines indicate collinear gene pairs involving MmiGRF genes

Fig. 5

Expression profiles of MmiGRF genes across various tissues. Relative expression levels were determined by qRT-PCR using roots as the reference tissue. Data represent the mean of three biological replicates. Values were log₂-transformed and normalized by row, and heat maps were generated using TBtools. The color scale is shown in the top right corner

Fig. 6

Relative expression levels of MmiGRF genes under various plant hormone treatments. (A) Relative expression levels at different time points following GA₃ treatment. (B) Relative expression levels 3 h after treatment with ABA, 6-BA, IAA, BR, and MeJA. Data represent the mean ± standard deviation (SD) of three biological replicates. Statistical significance was assessed using ANOVA. ns indicates no significant difference; p < 0.01 (*), p < 0.05 (**), and p < 0.001 (***)

Fig. 7

Subcellular localization of MmiGRF8, MmiGRF9, MmiGRF11, and MmiGRF12 proteins. Images from left to right show green fluorescent protein (GFP), nuclear marker, bright field, and a merged overlay of all three channels from the same sample. The scale bar represents 100 μm

Fig. 8

Predicted miRNA targets in MmiGRF genes. (A) Schematic representation of Mmi-miR396 target sites within MmiGRF genes. (B) Alignment of Mmi-miR396 binding sites across different MmiGRF genes. Mismatched or non-complementary bases are indicated in white