Sugarcane is the main source for sugar industry accounting for 79% of sugar production over the world. Developing techniques for controlling growth of sugarcane accelerate the yields and culture biotechnology for sugarcane. GA and DPC are two pairs of chemicals that regulate plant growth in sugar farming with different effects. GA stimulates sugarcane internode elongation by regulating genes associated to zeatin biosynthesis, nitrogen metabolism and plant hormone signal transduction pathway  while the DPC depressed sugarcane growth. However, compared to the clear mechanism of GA stimulated growth, the molecular mechanisms of DPC is blur. Thus, in the present study, we focus on the transcriptomic regulation by DPC on sugarcane and discuss the key genes mediated the growth depressed effect.
First, to obtain high quality reference for gene annotation, we generate full-length transcriptome from sugarcane. The full-length transcriptome from sugarcane was sequenced by PacBio Sequel platform generated 72,671 isoforms. Compared to illumine platforms, PacBio Sequel platform could gain longer transcripts which is facility to construction of high-quality reference for short sequence analysis. The present study generated the reads with N50 as 3,011 bp. These long reads guarantee longer contigs and isoforms for the following transcriptome analysis . It turns out that the N50 was 3,073 for the isoforms in the present study. Sugarcane is a widely cropped plant. Up to date, large amount of different varieties has been developed. The Guitang varieties developed from Guangxi which have become a series of varieties planted in southern China . GT42 belonging to the Guitang varieties is a new breeding line with higher productivities for sugar . Although the genome of sugarcane is reported until 2018, the genome data may differ from varieties . Our present first reported the full-length transcriptome GT42. These data would accelerate the studies on the new high yield crop and provide high-quality reference when analyzed the illumine short reads. Meanwhile, these global transcriptome data gave a chance to illustrate the function of internodes in GT42. The most abundant biological process GO term of GT42 isoforms contained metabolic process and cellular process. Thus, this functional isoform showed similar function assignment with previous results from sugarcane [46–48]. Based on these data, the GT42 had similar functional constitution of genes with other sugarcane varieties. The present full-length transcriptome first generated the general information of GT42 and provided high-quality reference transcriptome for further investigation on this variety.
DPC is one of the most successful and widely used chemicals to regulated plant growth. Internode length and leaf size could be depressed by DPC treatment in cotton and sugarcane . The present study also suggested that DPC inhibited internode length in GT42 which was similar to previous results. After understanding the effects of DPC on the internode growth, the next question is to find out the molecular mechanism of the function of DPC in sugarcane. We used RNA-seq to show the whole profile of regulation on gene expressions in the present study. Using hi-seq technique, we obtained millions of short reads to reveal the expression in different stages by DPC treatment. Thanks to the high-quality full-length transcriptome data, the mapping ratios for these libraries covered 73.97–83.78%. The comparison between C2 and D2 had the most DEGs which was 6,012 genes. This numbers of DEGs was much higher than that in C1-vs-D1 and C3-vs-D3 suggesting the gene expression changes between control and DPC treatment were mainly in the second stages, namely, on 6 days post spraying. In a study in cotton spraying by DPC, the 96 h post spraying significantly had the most DEGs compared to the 48 and 72 h stages. It seems that the DPC resulted changes gene expression could be in a long-term until 4 to 6 days. The gene expression regulation by DPC is not an acute effect. Meanwhile, after 10 days, the effects of DPC on gene expression were diminished. We supposed the best effect period of DPC regulated gene expression is 6 days.
The KEGG enrichment analysis showed that 55 genes in plant hormone signal transduction pathway were increased by DPC treatment. Internodes growth is controlled by several hormones such as G biosynthesis genes, auxin related genes, and ethylene genes. It had been reported that GA treatment significantly up-regulated these genes. Meanwhile, DPC may suppressed hormone expression. In Agapanthus praecox, auxin-related genes could be inhibited by DPC treatment . Surprisingly, the present study also indicated that DPC increased several hormonal genes. This different may due to the different species. The sugarcane may have different response to DPC in molecular levels. We also found that several key pathways could be down-regulated by DPC, such as phenylpropanoid biosynthesis, flavonoid biosynthesis, favone and flavonol biosynthesis, and glucosinolate biosynthesis were enriched. The phenylpropanoid pathway provides metabolites for plant growth which contributes to the requirement of lignin biosynthesis . Favone, flavonol and glucosinolate are key metabolites for internode growth [51, 52]. Flavonol biosynthesis could be affected by light intensity and led to different growth appearances in Ginkgo (Ginkgo biloba) . The glucosinolate concentration influenced by sulfur and nitrogen supplement was associated with growth of broccoli . The down-regulated of genes in these pathways may lead to the shorten effects of sugarcane internodes.
To find out the key gene modules and hub genes by DPC treatment, WGCNA was performed. The most correlated gene module with D2 group was sienna3 containing only 33 genes. Therefore, the most critical genes play key role in the module. Hub genes are the gene that correlate with other genes in expression levels which could be identified by mathematical method. The top three identified in this study were Stf0 sulphotransferase, cyclin-like F-box and HOX12. Stf0 belongs to sulphotransferase family which affects root development processes, elongation growth, and gravitropism . In several plants, including Medicago truncatula, Lotus japonicus, and Arabidopsis thaliana, cyclin-like F-box genes were expressed in all the tissues contained high actively dividing cells. Meanwhile, knockdown of this gene resulted in accumulation of CYCB1:1 suggesting that cyclin-like F-box gene could regulate cell cycle in the dividing cells . It had been reported that HOX12 regulated panicle exsertion via modulating EUI1 gene expression . These three hub genes were correlated with the other genes in the sienna3 modules. Based on this information, it could be concluded that Stf0 sulphotransferase, cyclin-like F-box and HOX12 mediated a gene group and constituted as a gene network which contributed to the DPC effects on sugarcane growth.
In conclusion, the full-length transcriptome of GT42 was first reported providing an informative resource for sugarcane breeding and transcriptome analysis. The RNA-seq suggested that the main effects of DPC on sugarcane gene expression was 6 days post spraying. The significant enriched gene function categories contained several pathways related to internode growth including multiple pathways that participated in production of Metabolic products. The gene modules included 33 genes were high correlated with the stage of 6 days post spraying in DPC group showing a potential role in response to DPC. Among these genes, Stf0 sulphotransferase, cyclin-like F-box and HOX12 were hub genes that may regulate all the other genes in this module. Further studies should focus on determination the function in detail of these key genes, especially in controlling internode growth affected by DPC.