Transcriptome Profiles Reveal the Crucial Roles of Auxin and Cytokinin in the "Shoot Branching" of Cremastra appendiculata

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.

Keywords: Cremastra appendiculata; phytohormone signaling; shoot branching; transcription factors; transcriptome.

Figure 1

Pseudobulb string formation process: the pictures in (A–F) illustrate the development and growth processes of a newborn bulb. Annually, C. appendiculata forms into a biennial plant (F) through this development and growth process. Once per year, the biennial plant grows into a triennial plant through recycling (G). After repeated growth cycles, this plant forms a pseudobulb string.

Figure 2

The decapitation, NPA, and TIBA treatments promoted lateral bud outgrowth: (A–C) morphological pictures of the lateral buds at three representative time points (0, 6, and 18 days post-decapitation); (D–G) morphological pictures of decapitated and intact plants, and those treated with NPA and TIBA after for 80 days; and (H) the statistical chart of bud length. Values are means ± SDs, n = 3. Error bars indicate standard deviations obtained from three biological replicates.

Figure 3

The phytohormone content of C. appendiculata lateral buds was tested during the bud elongation process by HPLC: (A) IAA; (B) zeatin; and (C) zeatin/IAA ratio. Values are means ± SD, n = 3. Error bars indicate the standard deviations obtained from three biological replicates. * and ** indicate significant differences based on one-way ANOVA tests at p < 0.05 and p < 0.01, respectively, compared with the intact group.

Figure 4

Characteristics of the similarity search of unigenes against the NR database: (A) similarity distribution of the top BLAST hit for each gene; and (B) E-value distribution of BLAST hits for each unigene with a cutoff E-value.

Figure 5

KEGG enrichments of the annotated differentially expressed genes (DEGs). The left Y-axis indicates the KEGG pathway. The X-axis indicates the rich factor (the number of genes enriched in this pathway compared to the number of genes on this pathway.). High q values are shown in blue, and low q values are shown in red.

Figure 6

Hierarchical cluster analysis of auxin- (A), cytokinin- (B), and transcription factor-related (C) DEGs during lateral bud elongation in C. appendiculata. Red indicates high relative gene expression and green indicates low relative gene expression.

Figure 7

Comparison of expression levels measured by RNA-Seq and qRT-PCR for 12 selected differentially expressed genes for TG2 vs. D2 (nine DEGs) and TD2 vs. D2 (three DEGs).

Figure 8

The relative expression levels of 10 candidate DEGs were measured during the lateral bud elongation process in C. appendiculata by qRT-PCR. Auxin synthesis and metabolic genes: CaYUCCA and CaDAO; cytokinin synthesis and metabolic genes: CaCYP735A, CaIPT, and CaCKX; strigolactone synthesis genes: CaCCD7 and CaCCD8; and transcription factors: CaBRC1, CaWUS, and CaWRKY71. Values are means ± SD, n = 3. Error bars indicate the standard deviations obtained from three biological replicates.

Figure 8

The relative expression levels of 10 candidate DEGs were measured during the lateral bud elongation process in C. appendiculata by qRT-PCR. Auxin synthesis and metabolic genes: CaYUCCA and CaDAO; cytokinin synthesis and metabolic genes: CaCYP735A, CaIPT, and CaCKX; strigolactone synthesis genes: CaCCD7 and CaCCD8; and transcription factors: CaBRC1, CaWUS, and CaWRKY71. Values are means ± SD, n = 3. Error bars indicate the standard deviations obtained from three biological replicates.

Figure 9

A hypothetical model to investigate the molecular mechanism of shoot branching in C. appendiculata. Auxin, cytokinin, and strigolactone might play crucial roles in regulating lateral bud outgrowth. BRC1 acts as the integrator of these three hormones. Promotion and inhibition regulatory actions are indicated by arrows and lines with bars, respectively.