Genome-wide identification and expression pattern analysis of the kiwifruit GRAS transcription factor family in response to salt stress

Abstract

Background: GRAS is a family of plant-specific transcription factors (TFs) that play a vital role in plant growth and development and response to adversity stress. However, systematic studies of the GRAS TF family in kiwifruit have not been reported.

Results: In this study, we used a bioinformatics approach to identify eighty-six AcGRAS TFs located on twenty-six chromosomes and phylogenetic analysis classified them into ten subfamilies. It was found that the gene structure is relatively conserved for these genes and that fragmental duplication is the prime force for the evolution of AcGRAS genes. However, the promoter region of the AcGRAS genes mainly contains cis-acting elements related to hormones and environmental stresses, similar to the results of GO and KEGG enrichment analysis, suggesting that hormone signaling pathways of the AcGRAS family play a vital role in regulating plant growth and development and adversity stress. Protein interaction network analysis showed that the AcGRAS51 protein is a relational protein linking DELLA, SCR, and SHR subfamily proteins. The results demonstrated that 81 genes were expressed in kiwifruit AcGRAS under salt stress, including 17 differentially expressed genes, 13 upregulated, and four downregulated. This indicates that the upregulated AcGRAS55, AcGRAS69, AcGRAS86 and other GRAS genes can reduce the salt damage caused by kiwifruit plants by positively regulating salt stress, thus improving the salt tolerance of the plants.

Conclusions: These results provide a theoretical basis for future exploration of the characteristics and functions of more AcGRAS genes. This study provides a basis for further research on kiwifruit breeding for resistance to salt stress. RT-qPCR analysis showed that the expression of 3 AcGRAS genes was elevated under salt stress, indicating that AcGRAS exhibited a specific expression pattern under salt stress conditions.

Keywords: Expression patterns; GRAS transcription factors; Genome-wide analysis; Kiwifruit; Salt stress; Transcriptome.

Fig. 1

Phylogenetic tree of the GRAS TF family in Arabidopsis thaliana and kiwifruit. The phylogenetic tree was constructed using the maximum likelihood method and 1000 replicate bootstrap values. Black indicates 1000 replicates in which bootstrap values > 90, gray indicates 1000 replicates in which 90 > bootstrap values > 70, and white‒gray indicates bootstrap values < 70. This plant is labeled separately using different shapes and colors, with triangular purple for kiwifruit and round green for A. thaliana. Different groupings are indicated by different colors

Fig. 2

Phylogenetic relationships, gene structure, structural domains, and conserved motif distribution of AcGRAS TF family genes. A Phylogenetic analysis of 86 GRAS TF family gene constructs by MEGA. B Distribution of conserved motifs of AcGRAS, with motifs indicated by colored boxes and black lines indicating relative lengths of proteins. C Exon‒intron structures and conserved structural domains of AcGRAS genes

Fig. 3

Distribution of cis-acting elements of the 2000 bp sequence upstream of the 86 AcGRAS TF family genes, with different cis-acting elements indicated by different colors

Fig. 4

Covariance analysis of kiwifruit AcGRAS genes. Gray lines indicate all duplicated genes, dark green indicates fragmented duplicated GRAS gene pairs, and red lines connecting genes outside the chromosome indicate tandem duplicated pairs. The heatmap and line graph are gene densities, with line graph densities increasing from blue to white to red, and yellow rectangles are chromosomes, with chromosome names shown between each chromosome and gene density

Fig. 5

Results of the annotation of GO-Level 3 of the AcGRAS gene. MF denotes molecular function, CC denotes cellular component, and BP denotes a biological process, indicated in green, yellow, and blue, respectively. The x-axis presents the number of gene functions, and the y-axis is the annotated function of a gene

Fig. 6

PPI reciprocal relationship diagram. Each circle represents a protein. The blue and green mixed-colored balls indicate genes enriched in the KEGG pathway for the hormone signaling process

Fig. 7

Heatmap of the expression profiles of 81 AcGRAS genes under salt stress. The different colored boxes indicate different log2 (FPKM) values, with expression gradually increasing from blue to yellow to red at a time. CK is the control group, and T is the treatment group

Fig. 8

Expression pattern and functional annotation of 16 AcGRAS significantly differentially expressed genes under salt stress. The GO string diagram is divided into two parts, with genes on the left and arranged according to logFC and GO terms on the right, with different colors indicating different functions

Fig. 9

Diagram of the KEGG phytohormone signaling pathway of the AcGRAS61 gene under salt stress. The figure is drawn with reference to the KEGG phytohormone signaling pathway map [43]. The red part of the diagram shows the regulation of the GA response by the DELLA protein

Fig. 10

Expression analysis of AcGRASs under salt stress. a Response of kiwifruit to NaCl stress, b relative expression of AcGRAS6, c relative expression of AcGRAS21, and (d) relative expression level of AcGRAS61 were determined by RT-qPCR. The standard error is expressed as an error bar. The data is expressed as the mean ± standard deviation of the three biological replicates. *indicates p-value <0.05; N indicate p-value >0.05; STUDENT's T test. CK is the control group, and T is the treatment group

Fig. 11

Flowchart of the experimental methodology of this study, where shapes indicate specific content steps and arrows indicate next steps