Abstract
Cremastra appendiculata has become endangered due to reproductive difficulties. Specifically, vegetative reproduction is almost its only way to reproduce, and, under natural conditions, it cannot grow branches, resulting in an extremely low reproductive coefficient (reproductive percentage). Here, we performed RNA-Seq and a differentially expressed gene (DEG) analysis of the three stages of lateral bud development in C. appendiculata after decapitation—dormancy (D2), transition (TD2), and emergence (TG2)—and the annual axillary bud natural break (G1) to gain insight into the molecular regulatory network of shoot branching in this plant. Additionally, we applied the auxin transport inhibitors N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodibenzoic acid (TIBA) to a treated pseudobulb string of C. appendiculata to verify the conclusions obtained by the transcriptome. RNA-Seq provided a wealth of valuable information. Successive pairwise comparative transcriptome analyses revealed 5988 genes as DEGs. GO (Gene Ontology) and KEGG (Kyoto encyclopedia of genes and genomes) analyses of DEGs showed significant enrichments in phytohormone biosynthesis and metabolism, regulation of hormone levels, and a hormone-mediated signaling pathway. qRT-PCR validation showed a highly significant correlation (p < 0.01) with the RNA-Seq generated data. High-performance liquid chromatography (HPLC) and qRT-PCR results showed that, after decapitation, the NPA- and TIBA-induced lateral buds germinated due to rapidly decreasing auxin levels, caused by upregulation of the dioxygenase for auxin oxidation gene (DAO). Decreased auxin levels promoted the expression of isopentenyl transferase (IPT) and cytochrome P450 monooxygenase, family 735, subfamily A (CYP735A) genes and inhibited two carotenoid cleavage dioxygenases (CCD7 and CCD8). Zeatin levels significantly increased after the treatments. The increased cytokinin levels promoted the expression of WUSCHEL (WUS) and inhibited expression of BRANCHED1 (BRC1) in the cytokinin signal transduction pathway and initiated lateral bud outgrowth. Our data suggest that our theories concerning the regulation of shoot branching and apical dominance is really similar to those observed in annual plants. Auxin inhibits bud outgrowth and tends to inhibit cytokinin levels. The pseudobulb in the plant behaves in a similar manner to that of a shoot above the ground.
Highlights
Cremastra appendiculata is a rare, medicinal perennial plant
We have constructed effective techniques to relieve lateral relieve lateral inhibition coefficient and increase coefficient in this plant,that and budtechniques inhibition to and increase thebud reproductive in the thisreproductive plant, and we previously reported we previously reported that shoot branching of appendiculata is related to phytohormones
80 days post-treatment, the lateral buds grew into seedlings (Figure 2D,F,G)
Summary
Cremastra appendiculata is a rare, medicinal perennial plant. A variety of pharmacologically active compounds, with properties such as anti-angiogenic activity [1], selective blockade activity. In this plant, a small proportion of the(from budsthe (from thepseudobulb) annual pseudobulb) yield only only a small proportion of the buds annual yield branches branches under natural conditions. Strigolactone can regulate shoot branching via the repression of auxin canalization [25,26,27] The interactions among these phytohormones regulate axillary bud outgrowth, but their mechanism of interaction in C. appendiculata and the related interconnected molecular process is unclear. To explore the molecular regulatory network of shoot branching, we harvested biennial pseudobulb lateral buds from the three stages (D2, TD2, and TG2) during the lateral bud breaking process and collected the annual axillary buds of the natural breaking stage (G1, where the number 1 indicates that the sample is from annual pseudobulbs) to perform an RNA-Seq analysis. This study lays the foundation for the molecular breeding of C. appendiculata and for studying the apical dominance of the underground stem
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