3.1 Identification and physicochemical property analysis of AmGRF gene
All protein sequences in A. mongholics genome were extracted and submitted to the PlantTFDB website to obtain candidate transcription factor sequences. After further retrieval and elimination of redundant sequences in A. mongholics genome, nine AmGRF genes were identified and named AmGRF1-AmGRF9 (Supplementary Table 2). Physicochemical analysis showed that the sequence length of all AmGRF proteins ranged 320 (AmGRF4) to 642 (AmGRF2) amino acids, and the molecular mass ranged 36.1–69.9 kDa. The isoelectric points ranged 6.74 (AmGRF8) to 9.16 (AmGRF4). The mean value of AmGRF hydrophilicity was < 0, indicating that AmGRF is a hydrophobic protein (Table1). Subcellular localization revealed that AmGRF proteins were located in the nucleus.
Table 1. Physicochemical properties of the AmGRF gene
Gene ID
|
Rename
|
pI
|
MW
|
AA
|
GRAVY
|
Subcellular Localization
|
Chromosome position
|
Am01G001010.1
|
AmGRF1
|
8.66
|
37790.22
|
330
|
-1.092
|
Nucleus
|
3920824,3922818
|
Am02G003770.1
|
AmGRF2
|
7.77
|
69908.11
|
642
|
-0.690
|
Nucleus
|
14766959,14771445
|
Am03G012480.1
|
AmGRF3
|
7.00
|
55486.16
|
512
|
-0.751
|
Nucleus
|
33675371,33677913
|
Am04G009960.1
|
AmGRF4
|
9.16
|
36089.21
|
320
|
-0.839
|
Nucleus
|
28771317,28774332
|
Am05G010550.1
|
AmGRF5
|
7.72
|
36018.75
|
325
|
-0.759
|
Nucleus
|
83747571,83751451
|
Am05G010590.1
|
AmGRF6
|
7.72
|
36824.78
|
332
|
-0.720
|
Nucleus
|
84151650,84155421
|
Am06G015050.1
|
AmGRF7
|
8.67
|
40208.41
|
353
|
-0.906
|
Nucleus
|
54343425,54346356
|
Am06G023050.1
|
AmGRF8
|
6.74
|
41999.23
|
368
|
-0.941
|
Nucleus
|
137513488,137515282
|
Am09G017960.1
|
AmGRF9
|
8.34
|
39125.40
|
357
|
-0.727
|
Nucleus
|
64370726,64372998
|
3.2 Phylogenetic analysis of the AmGRF gene family
To further understand the evolutionary relationships among members of the AmGRF gene family, MEGA-X11 software was used to analyze A. mongholics, A. thaliana, and O. sativa GRF were compared and analyzed for a total of 29 protein sequences, and a neighbor-joining (NJ) phylogenetic tree was constructed (Fig.1). The results showed that the nine identified AmGRF genes were divided into five of six subfamilies (I–VI). Group I contained AmGRF1, AmGRF7, and AmGRF8; Group II contained AmGRF4, AmGRF5, and AmGRF6; Group III contained AmGRF2; Group IV contained AmGRF3; and Group V contained AmGRF6. However, no AmGRF gene was classified in Group VI. Among these, AtGRF and AmGRF, such as AtGRF7, AmGRF3, AtGRF5 and AmGRF1.
Fig.1 NJ phylogenetic tree constructed with A. thaliana, O. sativa, and AmGRF proteins. The bootstrap value was set to 1000 replicates. The red star represents the AmGRF protein, the blue triangle represents the OsGRF protein, and the green circle represents the AtGRF protein. Different colors represent the various subfamilies.
3.3 AmGRF gene structure, domain, and motifs
To further understand the diversity and similarity of AmGRF, the CD Search tool and Pfam data were used to screen the conserved domains of the candidate sequences. Based on the QLQ (PF08880) and WRC (PF08879) conserved domains of the GRF gene, we analyzed the AmGRF protein using the MEME tool and identified ten motifs (Fig.3B). Among these, nine AmGRF proteins containing motif1, motif2, motif1, and motif2 were QLQ and WRC domains, respectively (Fig.3C), with lengths of 45 and 41 amino acids present in the GRF protein, and were highly conserved in the AmGRF protein sequence (Fig.2A&B). The GRF protein contains 2–9 different conserved motifs, and members grouped in the same branch have similar conserved motifs, such as AmGRF7/AmGRF8 and AmGRF4/AmGRF5. In subfamilies I, II, and III containing motif4 and motif6, only AmGRF6 in subfamily II exhibited the phenomenon of motif6 preceding motif4. Simultaneously, motif3 was missing during the evolutionary process in AmGRF3 and AmGRF6. AmGRF4 and AmGRF5 have the highest number of conserved motifs. AmGRF3 contains the least number of conserved motifs, with only two conserved motifs, motif1 and motif2. Among the three subfamilies, motif1, motif2, motif3, motif4, and motif6 are common. We further analyzed the distribution of introns and exons in the AmGRF gene family and found that the number of introns and exons in all AmGRF is approximately 2–3 (Fig.3D). The AmGRF protein contains UTR and CDS.
Fig.2 Conservative sequence alignment of AmGRF gene family proteins. A: QLQ conservative domain. B: WRC conserved domain.
Fig.3 f the AmGRF protein. (A) Representation of the AmGRF protein phylogenetic tree. Green, pink, yellow, light blue, and dark blue backgrounds represent groups I, II, III, IV, and V, respectively. (B) The MEME website was used to identify the composition of AmGRF protein motifs, with different colors representing the various motifs. (C) The AmGRF gene has a conserved domain, with green representing the WRC domain and yellow representing the QLQ domain. (D) AmGRF gene structure. CDS: encoding sequence, UTR: In the untranslated region; lines represent introns. (E) The conservative structural domains motif1 and motif2 of AmGRFs.
3.4 Chromosome location, duplication, and synteny analysis of AmGRF gene family
The distribution of AmGRF genes on the chromosomes was visualized using gene annotation (Fig.4). Nine AmGRF genes were randomly distributed across seven chromosomes. AmGRF was primarily distributed at both ends of the chromosome, with less distribution in the middle. The AmGRF gene has up to two genes distributed on the fifth chromosome. There was no chromosomal distribution on the seventh and eighth chromosomes, whereas there was gene distribution on all the other chromosomes (Fig.5A). Three pairs of gene replicates were identified on chromosomes one and six, namely AmGRF1/AmGRF8, AmGRF1/AmGRF7, and AmGRF7/AmGRF8. The AmGRF gene replication events may have played a crucial role in the evolution of AmGRF.
Fig.4 Chromosomal distribution and localization of AmGRFs. All nine AmGRF genes are distributed on the chromosomes, and the scale on the left represents the length of the chromosome.
To further understand the homologous relationships between different species of AmGRFs, we compared five dicotyledonous plants and one monocotyledonous plant O. Sativa, as well as five dicotyledonous plants, including two medicinal plants, C. lanceodata and C. sativa, and three types of fruit, V. vinifera, S. lycopersicum, and M. domestica (Fig.5B). The results showed the closest collinearity between the AmGRF and MdGRF genes, with 17 pairs of collinearity genes, 12 pairs of AmGRF and SlbGRF, ten pairs of AmGRF and VvGRF, six pairs of AmGRF and ClGRF, five pairs of AmGRF and OsGRF, and four pairs of collinearity between AmGRF and CsGRF. The number of collinear genes was lower in monocotyledonous plants is less than in dicotyledonous plants.
Fig.5 A Chromosomal distribution and gene replication events of the AmGRF gene. The red line represents the tandem replication relationship between the AmGRF genomes. The two outer circles show gene density information, with red indicating high gene density and blue indicating low gene density.B Collinear relationship between A. mongholicus and other species. The light blue lines represent collinear gene pairs. The green section represents the chromosome information of A. mongholicus, and the blue sections represent the chromosome information of M. domestica, S. lycopersicum, V. vinifera, C. lanceolata, O. sativa, and C.sativa.
3.5 Cis-acting element analysis of the AmGRF gene
To understand the functions of members of the AmGRF gene family, we used the upstream 2000 bp region of nine AmGRF motifs as the promoter region to perform a predictive analysis of cis-acting elements. We obtained the cis-elements of the AmGRF gene using the PlantCRAE website, summarized their possible functions, and plotted them (Fig.6). The 211 cis-acting elements identified could be divided into three categories: stress-, hormone-, and growth-related. Among these, the number of light-responsive elements is the highest, with a total of 121 active elements. Endosperm expression is a cis-acting element of AmGRF7, whereas flavonoid biosynthetic gene regulation is a cis-acting element of AmGRF3, which may be involved in flavonoid synthesis. These results indicate that the AmGRF gene may be involved in various biological processes in plants and plays an indispensable role.
Fig.6 Prediction of the cis-acting elements in the upstream 2000-bp promoter of the nine AmGRF genes.
3.5 GO analysis of the AmGRF gene family
GO classification was used to identify the possible functions of the nine AmGRFs. Analysis of the annotation results showed that AmGRF gene functions could be divided into three categories: 25 functional groups of cellular components (CC), molecular functions (MF), and biological processes (BP) (Fig.7). The primary biological processes involving these genes are affected by hormones, light, and low temperatures. AmGRF genes were primarily found in the Golgi cytoplasm, plasma membrane, nuclear envelope, chloroplast matrix, and nucleus. In the molecular function category, these genes promoted the transcription and binding of protein sites at the molecular level and promoted their expression.
Fig.7 GO annotation of members of the AmGRF gene family.
3.6 qRT-PCR verification of AmGRF gene expression patterns in different tissues of A. mongolicus
To explore the expression patterns of AmGRF genes in different tissues, the expression levels of nine AmGRF genes were analyzed based on the transcriptome data of the leaves, roots, and stems of A. mongholics. Five genes were expressed in the stems and leaves (RPKM>0.5), and all genes showed low expression in the roots. AmGRF4 and AmGRF8 were highly expressed in the stems. AmGRF2 , and AmGRF4 are expressed in various tissue sites, and these genes may be involved in the growth and development cycle of A.mongholics (Figure 8). The expression of the AmGRF gene was higher in the leaves than in the roots. Three genes (AmGRF5, AmGRF6 and AmGRF8) were not listed as expressed in all tissue sites (RPKM<0.5), which may be pseudogenes or require specific conditions to activate their expression(Fig.8A).
Based on the transcriptome data, these nine AmGRF genes were selected as candidate genes for qRT-PCR analysis. The results showed that the expression patterns of the five candidate genes were consistent with the expression trends obtained from RNA sequencing (RNA-Seq) data. Notably, compared to the expression of AmGRF5 and AmGRF6 in the transcriptome data, the RT-qPCR results indicated that the AmGRF5 and AmGRF6 gene was expressed in all root leaves. AmGRF1 and AmGRF8 exhibited higher expression in roots, and AmGRF1 and AmGRF9 exhibited higher expressed in stems. These results indicate that these nine AmGRF genes may be closely related to the growth and development of A. mongolicus (Fig.8B).
Fig.8 A.Expression patterns of AmGRF genes in roots, stems, and leaves. Expression patterns were generated based on Roes scale 1 and analyzed using heatmap hierarchical clustering. Different color codes represent the relative expression levels of genes, and a color gradient from purple to yellow indicates a decrease in gene expression levels. B.The relative expression levels of differentially expressed genes (DEGs). AmGRFs in the roots, stems, and leaves of 50-d hydroponic seedlings of A.mongolicus under normal growth conditions. The X-axis represents the location of plant tissue, whereas the Y-axis represents the relative expression level of genes. The error bars represent standard deviations (SD; n = 3). The significance analysis was performed using a t-test. The *, **, and *** show significance at p < 0.05, p < 0.01, and p < 0.001, respectively.