Identification of ARF transcription factor gene family and its defense responses to bacterial infection and salicylic acid treatment in sugarcane

Abstract

Auxin response factor (ARF) is a critical regulator in the auxin signaling pathway, involved in a variety of plant biological processes. Here, gene members of 24 SpapARFs and 39 SpnpARFs were identified in two genomes of Saccharum spontaneum clones AP85-441 and Np-X, respectively. Phylogenetic analysis showed that all ARF genes were clustered into four clades, which is identical to those ARF genes in maize (Zea mays) and sorghum (Sorghum bicolor). The gene structure and domain composition of this ARF family are conserved to a large degree across plant species. The SpapARF and SpnpARF genes were unevenly distributed on chromosomes 1-8 and 1-10 in the two genomes of AP85-441 and Np-X, respectively. Segmental duplication events may also contribute to this gene family expansion in S. spontaneum. The post-transcriptional regulation of ARF genes likely involves sugarcane against various stressors through a miRNA-medicated pathway. Expression levels of six representative ShARF genes were analyzed by qRT-PCR assays on two sugarcane cultivars [LCP85-384 (resistant to leaf scald) and ROC20 (susceptible to leaf scald)] triggered by Acidovorax avenae subsp. avenae (Aaa) and Xanthomonas albilineans (Xa) infections and salicylic acid (SA) treatment. ShARF04 functioned as a positive regulator under Xa and Aaa stress, whereas it was a negative regulator under SA treatment. ShARF07/17 genes played positive roles against both pathogenic bacteria and SA stresses. Additionally, ShARF22 was negatively modulated by Xa and Aaa stimuli in both cultivars, particularly LCP85-384. These findings imply that sugarcane ARFs exhibit functional redundancy and divergence against stressful conditions. This work lays the foundation for further research on ARF gene functions in sugarcane against diverse environmental stressors.

Keywords: auxin response factor; biotic and abiotic stress; expression pattern; stress response; sugarcane.

Figure 1

Orthologous ARF family gene clusters among S. spontaneum AP85-441 (Spap), S. spontaneum Np-X (Spnp), S. bicolor (Sb), and Z. mays B73 (ZmB73). The number in each sector of the diagram indicates the number of homologous clusters and the numbers in parentheses indicate the total number of genes contained within the associated clusters. The numbers in parentheses below the species names indicate the number of species-specific singletons (genes with no homologs).

Figure 2

Phylogenetic tree of ARF family genes in S. spontaneum AP85-441 (Spap) and Np-X (Spnp), Sorghum bicolor (Sb), Fagopyrum tataricum (Ft), and Zea mays (Zm). This tree was constructed with the neighbor-joining method and 1,000 bootstraps implemented in the MEGA 6.0 software. Four phylogenetic Groups (I, II, III, and IV) are marked with different colored backgrounds and lines. SpapARF and SpnpARF genes are marked red and blue colors, respectively.

Figure 3

Conserved domains (A), exon-intron structure (B), and conserved motifs (C) of ARF genes in S. spontaneum AP85-441 (Spap) and Np-X (Spnp). (A) The Clades (I/II/III/IV) of ARF genes are identical to those phylogenetic groups (A, B, C, and D) in Supplementary Figure S2, respectively. The functional domain was obtained from model gene annotation and results of the NCBI CDD-batch search. B3: B3 DNA-binding domain; Auxin-resp: ARF domain; AUX_IAA: C-terminal dimerization domain. (B) The exons and introns in each gene are represented by lines and boxes, respectively. The sizes of exons, introns, and untranslated regions are drawn on the bottom. (C) Conserved motifs of SpapARF and SpnpARF proteins were identified using MEME (suite 4.11.4). Each motif is indicated with a colored box numbered 1 to 12 at the bottom. Protein length is estimated using the scale (amino acid) at the bottom in (A) and (C) panels, while gene length is estimated using the scale (bp) at the bottom in (B) panel.

Figure 4

Circos illustration of duplicated ARF genes on chromosomes of S. spontaneum AP85-441 (Spap) (A) and Np-X (Spnp) (B), and multicollinearity analysis of ARF genes in four genomes including two S. spontaneum clones, Z. mays B73 (ZmB73), and S. bicolor (Sb) (C). The grey ribbon indicates a collinear relationship among blocks in the whole genome and the red ribbon shows ARF paralogs in (A) and (B) panels. The chromosomes are arranged in a colored arc. Genes with red color represent segmentally duplicated events. Blue lines represent syntenic ARFs in the (C) panel and gray lines in the background represent all orthologous genes among four genomes.

Figure 5

Network map of predicted miRNAs targeting SpapARFs and SpnpARFs (A) and schematic diagram of miRNAs targeting sites in some ARFs (B). (A) Green, blue, and orange boxes stand for miRNAs and their targeted genes of SpapARFs and SpnpARFs, respectively. (B) The thick blue bar indicates the location of the ARF gene harbored at the specific chromosome. The thick red bar indicates the location of the miRNA targeted at the gene sequence. The RNA sequence of each complementary site from 5′ to 3′ and the predicted miRNA sequence from 3′ to 5′ are shown with red lines below the gene sequences. All miRNAs predicted to target SpapARF and SpnpARF genes are shown in Supplementary Table S5. Sof: S. officinarum.

Figure 6

Transcript expression profiles of SpapARF and SpnpARF genes in sugarcane cultivars infected by Acidovorax avenae subsp. avenae (Aaa). (A) Transcriptome dataset of two sugarcane cultivars MT11-610 (S: susceptible to red stripe) and ROC22 (R: resistant to red stripe) triggered by Aaa infection. RNA-seq data were recorded for each cultivar at 0, 24, 48, and 72 h post-inoculation (hpi). Colored boxes in each column represent relative expression levels of individual genes with log₂(Fold Change). (B) qRT-PCR-based expression profiling of six candidate ARF genes in sugarcane cultivars (ROC20 and LCP85-384) under Aaa infection. Leaf samples were collected at 0, 24, 48, and 72 hpi. All data are shown as the mean ± standard error (SE).

Figure 7

Transcript expression profiles of SpapARF and SpnpARF genes in sugarcane cultivars infected by Xanthomonas albilineans (Xa). (A) Transcriptome dataset of two sugarcane cultivars ROC20 (S: susceptible to leaf scald) and LCP85-384 (R: resistant to leaf scald) triggered by Xa infection. RNA-seq data were recorded for each cultivar at 0, 24, 48, and 72 h post-inoculation (hpi). Colored boxes in each column represent relative expression levels of individual genes with log₂(Fold Change). (B) qRT-PCR-based expression profiling of six candidate ARF genes in sugarcane cultivars (ROC20 and LCP85-384) under Xa infection. Leaf samples were collected at 0, 24, 48, and 72 h post treatment (hpt). All data are shown as the mean ± standard error (SE).

Figure 8

The qRT-PCR-based expression profiling of six candidate ARF genes in two sugarcane cultivars ROC20 (susceptible to leaf scald) and LCP85-384 (resistant to leaf scald) under exogenous salicylic acid (SA) stress. Leaf samples were collected at 0, 24, 48, and 72 h post treatment (hpt). All data are shown as the mean ± standard error (SE).