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. 2025 Dec;20(1):2527380.
doi: 10.1080/15592324.2025.2527380. Epub 2025 Jul 1.

Identification and characterization of nine PAT1 genes subfamily in Medicago edgeworthii

Gaoping Tang  1   2 , Tingting Ni  1 , García-Caparrós Pedro  3 , Li-Hua Meng  1 , Xudong Sun  2
Affiliations

Identification and characterization of nine PAT1 genes subfamily in Medicago edgeworthii

Gaoping Tang et al. Plant Signal Behav. 2025 Dec.

Abstract

GRAS proteins represent a unique class of transcription factors that are exclusive to plants. Among the various subfamilies within the GRAS family, the phytochrome A signal transduction 1 (PAT1) subfamily is particularly prominent, given its multifaceted regulatory functions in phytochrome signaling pathways and stress response mechanisms, as well as its involvement in plant developmental processes. Despite the recognized importance of GRAS proteins, there are no studies to date that have characterized the GRAS gene family in Medicago edgeworthii. In this study, we performed a comprehensive genome-wide analysis of GRAS genes and identified nine genes belonging to the PAT1 subfamily in M. edgeworthii. Multiple sequence alignment of these proteins revealed the presence of a conserved C-terminal GRAS domain, alongside a highly variable N-terminal region. Additionally, we observed that members of the PAT1 subfamily were expressed in roots, stems, and leaves, indicating their broad involvement in the development of various tissues in M. edgeworthii. Furthermore, functional analysis indicated that PAT1 subfamily proteins in M. edgeworthii activated the expression of MeDOF3.4 gene, indicating that PAT1 subfamily proteins may be associated with the promotion of cell proliferation and graft fusion. In conclusion, this study provided the first comprehensive characterization of PAT1 subfamily genes in M. edgeworthii, establishing a foundation for future research on the functional roles of MeGRAS genes and providing a theoretical basis for the development of high-quality Medicago varieties.

Keywords: GRAS gene family; PAT1 subfamily gene; phylogenetic analysis; phytochrome.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Phylogenetic tree of GRAS proteins from M. edgeworthii, M. truncatula and Arabidopsis thaliana. The unrooted phylogenetic tree was constructed by the Neighbor-Joining (NJ) method using MEGA 7.0 software incorporating 1000 bootstraps iterations. The analysis included 53 GRAS proteins, 32 from Arabidopsis thaliana, 68 from M. truncatula, and 58 from M. edgeworthii. These proteins were clustered into nine distinct subfamilies: LISCL, SHR, PAT1, SCL3, DELLA, SCR, LS, SCL28, and HAM, respectively.
Figure 2.
Figure 2.
Phylogenetic and structural analysis of PAT1 gene subfamily. (a) Phylogenetic tree and MEME protein structure of members of PAT1 gene subfamily in M. edgeworthii. (b) Five most conserved motifs of PAT1 gene subfamily in M. edgeworthii, with the bits representing the score of the site.
Figure 3.
Figure 3.
Distribution and localization of MeGrass gene family members on different chromosomes. Chromosome number is indicated at the left of each chromosome. Blue represents the PAT1 subfamily genes.
Figure 4.
Figure 4.
Cis-acting element analyses of PAT1 subfamily genes. (a) Cis-acting elements of members of PAT1 gene subfamily in M. edgeworthii. Promoter sequences (−2 kb) of 9 MeGRAS genes are analyzed by PlantCARE. Rectangles with different colors indicate different cis-acting elements participating in various abiotic stress regulations. (b) Three-dimensional conformation of members of PAT1 gene subfamily in M. edgeworthii.
Figure 5.
Figure 5.
The relative expression levels of 9 PAT1 genes in M. edgeworthii among different tissues. The error bars indicate standard deviation (SD), and the sample size was 3. The data are analyzed by one-way ANOVA (Duncan’s test). Asterisks above columns indicate the significance of a group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 6.
Figure 6.
Fluorescence map of the subcellular localization of MeGRAS3, MeGRAS14, MeGRAS24, MeGRAS38 and MeGRAS42. 35S: GFP: expression of 35S-GFP/pRI101-GFP empty vector in tobacco leaves. Scale bar = 20 μm.
Figure 7.
Figure 7.
The PAT1 subfamily genes activate the expression of the MeDOF3.4 gene in M. edgeworthii. Transient expression assays (a) and the mean of fluorescence intensity (b) confirmed that MeGRAS14, MeGRAS24, MeGRAS38, MeGRAS41, and MeGRAS42 can activate the expression of the MeDOF3.4 gene. The error bars indicate standard deviation (SD), and the sample size was 3. The data are analyzed by one-way ANOVA (Duncan’s test). Asterisks above columns indicate the significance of a group: **p < 0.01. “1” indicates “pMedof3.4:luc”; “2” indicates “pMedof3.4:luc + MeGRAS14”; “3” indicates “pMedof3.4:luc + MeGRAS24”; “4” indicates “pMedof3.4:luc + MeGRAS38”; “5” indicates “pMedof3.4:luc + MeGRAS41”; “6” indicates “pMedof3.4:luc + MeGRAS42”.
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