Expression Profiling and MicroRNA Regulatory Networks of Homeobox Family Genes in Sugarcane Saccharum spontaneum L

Abstract

Homeobox (HB) genes play important roles in plant growth and development processes, particularly in the formation of lateral organs. Thus, they could influence leaf morphogenesis and biomass formation in plants. However, little is known about HBs in sugarcane, a crucial sugar crop, due to its complex genetic background. Here, 302 allelic sequences for 104 HBs were identified and divided into 13 subfamilies in sugarcane Saccharum spontaneum. Comparative genomics revealed that whole-genome duplication (WGD)/segmental duplication significantly promoted the expansion of the HB family in S. spontaneum, with SsHB26, SsHB63, SsHB64, SsHB65, SsHB67, SsHB95, and SsHB96 being retained from the evolutionary event before the divergence of dicots and monocots. Based on the analysis of transcriptome and degradome data, we speculated that SsHB15 and SsHB97 might play important roles in regulating sugarcane leaf morphogenesis, with miR166 and SsAGO10 being involved in the regulation of SsHB15 expression. Moreover, subcellular localization and transcriptional activity detection assays demonstrated that these two genes, SsHB15 and SsHB97, were functional transcription factors. This study demonstrated the evolutionary relationship and potential functions of SsHB genes and will enable the further investigation of the functional characterization and the regulatory mechanisms of SsHBs.

Keywords: Saccharum spontaneum; expression analysis; homeobox family; microRNA regulation; phylogenetic analysis.

Figure 1

Synteny and origin analysis of HB genes. (A) Synteny analysis of alleles (left) and nonalleles (right) of SsHBs. (B) Synteny analysis of HB genes from S. bicolor and S. spontaneum (one set of homologous chromosomes). Gray lines in the background and brown and red lines between sugarcane and sorghum indicate the collinear blocks and syntenic HB pairs in normal and recombinant regions of chromosomes, respectively. (C) Numbers of HB genes from different origins in A. thaliana, S. bicolor, and S. spontaneum.

Figure 2

The phylogenetic tree of SsHBs. The labels marked in different colors represent different subfamilies.

Figure 3

The HBs in 11 species among 5 lineages. The evolutionary relationships of 11 species were obtained from NCBI (https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi, accessed on 4 May 2021). The numbers in parenthesis indicate the number of alleles of SsHBs.

Figure 4

The Ka/Ks ratios of HB gene orthologs between S. bicolor and S. spontaneum. (A) The Ka/Ks of orthologs in HD-ZIP I, HD-ZIP II, HD-ZIP III and PHD subfamilies. (B) The Ka/Ks of orthologs in WOX, DDT, KNOX and PLINC subfamilies. (C) The Ka/Ks of orthologs in HD-ZIP IV, SAWADEE, BEL, PINTOX and NDX subfamilies. * indicates p-value < 0.05 and ** indicates p-value < 0.01.

Figure 5

The expression pattern of HBs based on TPM in different tissues at different stages (A), gradient developmental leaves (B), and circadian rhythms (C) in 2 Saccharum species. The expression patterns of HB15 and HB97 were presented separately (D). Stem 6/9 indicates maturing internodes (internode number 6 for SES208 (Ss) and internode number 9 for LA-Purple (So)), and stem 9/15 indicates mature internodes (internode number 9 for SES208, and internode number 15 for LA-Purple).

Figure 6

The qRT-PCR verification of SsHBs (A), SsAGO10, and miRNA (B) expression patterns in gradient-developing leaf segments. R², the coefficient of determination, indicates the correlation between RNA-seq and qRT-PCR.

Figure 7

The transcriptional activity and subcellular localization of SsHB15 and SsHB97. (A) 1% agarose gel electrophoresis of PCR clones of SsHB15 and SsHB97 coding sequences (each sample is spread across 2 wells). The original image is shown in Supplementary file 16. (B) Growth of yeast cells transformed with BD-SsHB15 and BD-SsHB97. Positive and negative controls were pGBKT7-53 and pGBKT7-lam, respectively. BD-FL indicates yeast containing the full-length target gene fused to the GAL4 DNA-binding domain. (C) The subcellular localization of SsHB15 and SsHB97 in tobacco epidermal cells and the white scale in each picture is 25μm.