Expression Pattern of FT/TFL1 and miR156-Targeted SPL Genes Associated with Developmental Stages in Dendrobium catenatum

Abstract

Time to flower, a process either referring to juvenile-adult phase change or vegetative-reproductive transition, is strictly controlled by an intricate regulatory network involving at least both FT/TFL1 and the micro RNA (miR)156-regulated SPL family members. Despite substantial progresses recently achieved in Arabidopsis and other plant species, information regarding the involvement of these genes during orchid development and flowering competence is still limited. Dendrobium catenatum, a popular orchid species, exhibits a juvenile phase of at least three years. Here, through whole-genome mining and whole-family expression profiling, we analyzed the homologous genes of FT/TFL1, miR156, and SPL with special reference to the developmental stages. The FT/TFL1 family contains nine members; among them, DcHd3b transcribes abundantly in young and juvenile tissues but not in adult, contrasting with the low levels of others. We also found that mature miR156, encoded by a single locus, accumulated in large quantity in protocorms and declined by seedling development, coincident with an increase in transcripts of three of its targeted SPL members, namely DcSPL14, DcSPL7, and DcSPL18. Moreover, among the seven predicted miR156-targeted SPLs, only DcSPL3 was significantly expressed in adult plants and was associated with plant maturation. Our results might suggest that the juvenile phase change or maturation in this orchid plant likely involves both the repressive action of a TFL1-like pathway and the promotive effect from an SPL3-mediated mechanism.

Keywords: Dendrobium catenatum; TFL1-like; flowering; juvenile; miR156; phosphatidylethanolamine-binding protein (PEBP) family; squamosa promoter binding protein-like (SPL) transcription factor.

Figure 1

The nine FT/TFL1 proteins encoded by the phosphatidylethanolamine-binding protein (PEBP) gene family in the Dendrobium cantenatum genome. (a) A phylogenetic tree of FT proteins from D. cantenatum (highlighted in red), Arabidopsis thaliana (highlighted in green), and other species. Only selected FTs from Oryza sativa (Os, in blue), Phalaenopsis equestris (Phe), and Apostasia shenzhenica (Ash) are included; (b) sequence alignment for motif comparisons. The secondary structures are indicated at top of the alignment; the conserved exon boundaries are marked by triangles. The four segments (A, B, C, and D) of the fourth exon important for ligand binding and protein–protein interaction in FT/TFL1, as revealed by Ahn et al., 2006, are line-labeled under the sequences. Known activator FTs and repressor TFL1-like proteins are shaded with light-blue and light-gray, respectively. Red-boxed residues correspond to positions at 85, 134, 138, and 140 for AtFT, and the black-boxed residues denote the highly conserved fragment B in inducer FTs but divergent in the repressor TFLs [7,43]; (c) numbers of PEBP genes identified by BLAST survey in some plant species and the single-cell eukaryotes.

Figure 2

Twelve SPL genes and one microRNA (miR)156 gene are found in Dendrobium catenatum. (a) A phylogenetic tree of SPL proteins from D. catenatum, A. thaliana, and S. lycopersicum. The genome of D. catenatum encodes 12 SPLs (highlighted in red), seven of which are predicted targets of miR156 (marked with red triangles). Arabidopsis contains 16 SPL proteins (highlighted in green), 10 of which are targeted by miR156 (with green triangles). The tomato genome has more SPL-coding genes than listed here, two of which were experimentally shown to be regulated by miR156 (gray triangles); (b) a single locus was identified in the genome which transcribes into three precursor non-coding RNA (ncRNA) variants. Broken lines denote introns, and light-green boxes denote exons; (c) miR156-targeted sites in messenger RNAs (mRNAs) of SPLs. Note that the DcSPL3 is possibly inhibited by mRNA cleavage and/or translational blockage.

Figure 3

D. catenatum plants in the three developmental stages. (a) Protocorm clusts on proliferation medium (½ MS + 0.269 μM NAA + 2.22 μM BA + 5% potato pulp + 2.5% sucrose); (b) proliferating protocorms on proliferation medium; (c) differentiating protocorms at 40 days on germination medium (½ MS +2.69 μM NAA +5% banana pulp + 3% sucrose); (d) one-year-old young plants in juvenile stage which were incapable of flowering; (e) adult three-year-old plants cultured in pots. Red arrows indicate the lateral floral bud sets. Green circles indicate sampling positions for RNA isolation from leaves and stems.

Figure 4

Expression profiles of FT/TFL1 family members in (a) protocorms at proliferation and differentiation stages, (b) stems of young and adult plants, and (c) leaves of young and adult plants. The 18S gene was used a reference. Statistical significance was determined using a paired Student’s t-test with alpha = 0.05; the adjusted p-value is shown above the data.

Figure 5

Expression profiles of miR156 in different tissues and of SPLs in the protocorms. (a) Quantitative stem–loop RT-PCR analysis of miR156 in protocorms, leaves, and stems; (b) RT-qPCR analysis of SPL transcript levels in the protocorms. For comparison, a value of 10⁻² × miR156 was also included. Error bars represent the SD for at least three biological replicates. Statistical significance was determined using a paired Student’s t-test with alpha = 0.05; the adjusted p-value is shown above the bars.

Figure 6

Expression profiles of five SPLs that were not targeted by miR156 in protocorms, leaves, and stems. Error bars represent the SD for at least three biological replicates. For statistical significance, the adjusted p-values are given on top of the bars.

Figure 7

Expression profiles of miR156-targeted SPLs in leaves and stems of one-year-old and three-year-old plants compared with miR156 levels as in Figure 5a. Error bars represent the SD of at least three biological replicates. For statistical significance, the adjusted p-values are given above the bars.