Genome-Wide Identification of Ginkgo biloba SPL Gene Family and Expression Analysis in Flavonoid Biosynthesis and Water Stress

Abstract

SQUAMOSA promoter-binding protein-like (SPL) transcription factors specific to plants are vital for regulating growth, development, secondary metabolite biosynthesis, and responses to both biotic and abiotic stresses. Despite their importance, no systematic investigations or identifications of the SPL gene family in Ginkgo biloba have been conducted. In this study, we identified 13 SPL genes within the Ginkgo biloba reference genome, spanning seven chromosomes, and categorized these genes into six groups based on their phylogenetic relationships with Arabidopsis thaliana SPL gene families. Our analysis of gene structure, conserved domains, motifs, and miR156 target predictions indicates that GbSPLs are highly conserved across evolutionary timelines. Furthermore, synteny analysis highlighted that dispersed duplication events have expanded the SPL gene family in Ginkgo biloba. Examination of the cis-regulatory elements revealed that many GbSPL genes possess motifs associated with light, hormones, and stress, implying their involvement in flavonoid biosynthesis and adaptation to environmental conditions. RNA-Seq and qRT-PCR expression profiles of GbSPL genes across various tissues and low- and high-flavonoid leaves and during both short-term and long-term water stress illustrated their roles in flavonoid biosynthesis and responses to water stress. Subcellular localization experiments showed that GbSPL2 and GbSPL11 proteins are situated within the nucleus. Our research offers the first systematic characterization of the SPL gene family in Ginkgo biloba, establishing a valuable foundation for understanding their evolutionary background and functional roles in flavonoid biosynthesis and water stress response.

Keywords: Ginkgo biloba; SPL; flavonoid biosynthesis; gene family; water stress.

Figure 1

The chromosome distribution of GbSPL genes in the genome of Ginkgo biloba. The length of each chromosome was estimated in megabases (Mb).

Figure 2

Phylogenetic analysis of GbSPL proteins with AtSPL proteins from Arabidopsis and OsSPL proteins from rice. The phylogenetic tree was constructed using IQ-Tree software, employing the Maximum Likelihood (ML) method, and bootstrap replications were set at 1000 times.

Figure 3

Phylogenetic trees, motifs, conserved domains, and gene structure analysis of GbSPL genes. (A) Phylogenetic trees of GbSPL proteins. (B) Motifs in the 13 GbSPL proteins, represented by ten colored boxes indicating different motifs. (C) Conserved domains present in the 13 GbSPL proteins. (D) Exon–intron structures of GbSPL genes.

Figure 4

The sequence alignment of the SBP domain of GbSPL proteins and miR156 target gene binding sites. (A) The multiple sequence alignment of SBP domains. The two conserved zinc finger structures (C3H and C2HC) and NLS are indicated. The purple represents conserved bases. (B) The multiple sequence alignment of miR156 and its GbSPL target gene binding sites. The * represents conserved bases.

Figure 5

Evolutionary analysis of the GbSPL gene family. (A) The number of genes resulted from five duplication events, including singleton, dispersed, proximal, tandem, and WGD or segmental duplication. (B) The heatmap illustrates the duplication events of the GbSPL gene family. (C) The distribution and collinearity of the GbSPL gene family in the genome of Ginkgo biloba. The black line indicates the collinear gene pairs of GbSPL genes, while the gray line represents the collinear gene pairs in the Ginkgo biloba genome. (D) The distribution and collinearity of the GbSPL gene family in Arabidopsis, Ginkgo biloba, and Poplar. The blue line depicts the collinear gene pairs of GbSPL genes, whereas the gray line highlights the collinear gene pairs in the Ginkgo biloba genome with Arabidopsis and Poplar.

Figure 6

The cis-acting elements in the promoters of GbSPL genes. (A) The distribution of cis-acting elements in the promoters (upstream 2000 bp) of GbSPL genes. (B) The number of cis-acting elements among the four different types of response elements in the promoters of the GbSPL genes.

Figure 7

The heatmap illustrates the RNA-Seq-based expression patterns of 13 GbSPL genes. (A) Expression patterns of 13 GbSPL genes across eight tissues, including mature fruit (MF), immature fruit (IF), root (R), stem (S), immature leaf (IL), mature leaf (ML), microstrobilus (M), and ovulate strobilus (OS). (B) Expression patterns of the 13 GbSPL genes grouped by contrasting low and high flavonoid content. The yellow boxes represent different groups. (C,D) Expression patterns of 13 GbSPL genes under short-term (C) and long-term (D) water stress. Red indicates high-expression genes, while blue represents low-expression genes.

Figure 8

qRT-PCR analysis and subcellular localization of GbSPL genes. (A) qRT-PCR analysis of GbSPL genes under short-term water stress. All data are presented as means ± SD (n = 3). (B) qRT-PCR analysis of GbSPL genes under long-term water stress. (C) Subcellular localization of GbSPL2 and GbSPL11. mCherry, a nuclear marker. Arrows highlight fluorescent signals in the micrograph.