Comparative transcriptome analysis revealed the cooperative regulation of sucrose and IAA on adventitious root formation in lotus (Nelumbo nucifera Gaertn)

Abstract

Background: In China, lotus is an important cultivated crop with multiple applications in ornaments, food, and environmental purification. Adventitious roots (ARs), a secondary root is necessary for the uptake of nutrition and water as the lotus principle root is underdeveloped. Therefore, AR formation in seedlings is very important for lotus breeding due to its effect on plant early growth. As lotus ARs formation was significantly affected by sucrose treatment, we analyzed the expression of genes and miRNAs upon treatment with differential concentrations of sucrose, and a crosstalk between sucrose and IAA was also identified.

Results: Notably, 20 mg/L sucrose promoted the ARs development, whereas 60 mg/L sucrose inhibited the formation of ARs. To investigate the regulatory pathway during ARs formation, the expression of genes and miRNAs was evaluated by high-throughput tag-sequencing. We observed that the expression of 5438, 5184, and 5345 genes was enhanced in the GL20/CK0, GL60/CK0, and CK1/CK0 libraries, respectively. Further, the expression of 73, 78, and 71 miRNAs was upregulated in the ZT20/MCK0, ZT60/MCK0, and MCK1/MCK0 libraries, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the differentially expressed genes and miRNAs in the GL20/GL60 and ZT20/ZT60 libraries were involved in signal transduction. A large number of these genes (29) and miRNAs (53) were associated with plant hormone metabolism. We observed an association between five miRNAs (miR160, miR156a-5p, miR397-5p_1, miR396a and miR167d) and nine genes (auxin response factor, protein brassinosteroid insensitive 1, laccase, and peroxidase 27) in the ZT20/ ZT60 libraries during ARs formation. Quantitative polymerase chain reaction (qRT-PCR) was used to validate the high-throughput tag-sequencing data.

Conclusions: We found that the expression of many critical genes involved in IAA synthesis and IAA transport was changed after treatment with various concentration of sucrose. Based on the change of these genes expression, IAA and sucrose content, we concluded that sucrose and IAA cooperatively regulated ARs formation. Sucrose affected ARs formation by improving IAA content at induction stage, and increased sucrose content might be also required for ARs development according to the changes tendency after application of exogenous IAA.

Keywords: Adventitious roots; Gene; Lotus; Sucrose; miRNA.

Fig. 1

The changes of morphology and microstructure of ARs after treatment of sucrose. a The morphology change of ARs in lotus seedlings after 20 mg/L and 60 mg/L sucrose treatment within 5 days (T20 or T60 represented seedlings treated with 20 mg/L or 60 mg/L sucrose for 1-5 d, CK represented the seedling growing in the water for 1-5 d). b The microstructure changes of the ARs in lotus seedlings after 20 mg/L sucrose treatment within 5s days by paraffin section (T20 represented seedlings treated with 20 mg/L sucrose for 1-5 d, CK represented the seedlings growing in the water for 1-5 d)

Fig. 2

The detailed information of miRNAs sequenced by RNA-seq technology. a the number of raw tags or clean tags in four libraries of ARs (MCK0 represented the tags number in the germinated stage, MCK1, ZT20 or ZT20 represented the tags number in the seedlings after water, 20 mg/L or 60 mg/L sucrose treatment for 1 d respectively. b The proportion of all kinds of small RNA after comparison against known sRNA database. c The number of small RNAs in different distribution length of small RNAs. d Statistics of the first base of predicted miRNAs with 18-32 nucleotides

Fig. 3

The expression change of mRNAs or miRNAs after treatment with 20 mg/L and 60 mg/L sucrose. a The changed number of mRNAs or miRNAs after treatment with 20 mg/L and 60 mg/L sucrose. b The expression of genes involved in CK1/CK0, GL60/CK0 and GL20/CK0 libraries. c The most desirable expression profile in CK0, GL20 and GL60 libraries. d the fold change of miRNAs in MCK1/MCK0, ZT60/MCK0 and ZT20/MCK0 libraries

Fig. 4

KEGG classification of differentially expressed mRNAs or miRNAs in GL20/GL60 and ZT20/ZT60 libraries. The X axis showed the number of differentially expressed mRNAs or miRNAs, and the Y axis showed the second KEGG pathway terms. The first pathway terms were indicated using different colors, and second pathway terms were subgroups of the first pathway terms, and were grouped together on the X axis on the right side. a The number of differentially expressed mRNAs in GL20/GL60 libraries. b The number of differentially expressed miRNAs in ZT20/ZT60 libraries

Fig. 5

The display of the top 20 enriched pathway terms in GL20/GL60 and ZT20/ZT60 libraries. The rich factor was the ratio of differentially expressed mRNAs or miRNAs numbers annotated in this pathway term to all gene numbers annotated in this pathway term, and the greater the rich factor, the greater the degree of enrichment. a The display of the top 20 enriched pathway terms in GL20/GL60 libraries. b The display of the top 20 enriched pathway terms in ZT20/ZT60 libraries

Fig. 6

Associated analysis of miRNAs and mRNAs in ZT20/ZT60 libraries. The red and the green ellipse representing up-regulated or down-regulated genes. The red and green five-pointed star representing up-regulated genes or miRNAs or down-regulated miRNAs. LCO 104608184 was protein brassinosteroid insensitive 1; LCO104594385 was auxin response factor 17; LCO104595695 and LOC104597151 were Laccase; LCO104585678 was peroxidase 27; LCO104596390, LCO104604663, LCO1045 93,690 and LCO104609332 were lysine-specific demethylase 3

Fig. 7

The expression of genes and miRNAs in C0, C1 and C2 stages after treatment of 60/L (GL60 or ZT60) and 20 mg/L (GL20 or ZT20) sucrose. a The expression of nine genes by qRT-PCR technology in in C0, C1 and C2 stages; b The expression of nine miRNAs by qRT-PCR technology in C0, C1 and C2 stages

Fig. 8

Genes or miRNAs involved in auxin metabolism. Yellow rectangle repressed genes involved in auxin metabolism, and green rectangle repressed miRNAs involved in auxin metabolism. Blue rectangle repressed the processes of auxin metabolism