Abstract
Orchids are some of the most popular ornamental plants worldwide. Orchid floral morphology has increasingly attracted horticultural and commercial attention. Although multiple genes have been shown to be involved in the formation of the orchid flower, the underlying multi-level regulatory networks are largely unknown. In this study, we analyzed the ontogeny of flower development in Cymbidium ensifolium, a traditional orchid in the tropical and subtropical regions of Asia, by performing deep sequencing of the transcriptome of individual flower organs to discover organ-specific genes potentially involved in their growth. We identified 3,017 differentially-expressed genes (DEGs) during the development of various flower organs, and observed over-representation of GROWTH-REGULATING FACTORS (GRFs) specific to flower column (gynostemium). Eleven C. ensifolium GRFs (CeGRFs) from our transcriptome data clustered into five phylogenetic subgroups. Ten of these GRFs shared a region complementary to C. ensifolium microRNA396 (Ce-miR396), and degradome sequencing confirmed the cleavage of transcripts derived from seven CeGRFs. We cloned Ce-miR396 and used a protoplast-based transient expression system to overexpress it in Cymbidium protoplasts. We observed a significant decrease in the transcripts of several CeGRFs in flowers and leaves, indicating a potential role for miR396–GRF module in organ development through the cleavage of distinct CeGRFs. Temporal and spatial expression analysis indicated that most CeGRF transcripts accumulated in flower buds and column tissues, where Ce-miR396 expression was the lowest. Expression dynamics in wild type and floral-defective mutants further confirmed a strong correlation between Ce-miR396, CeGRFs, and flower organ development and column specification. Moreover, overexpression of Ce-miR396 in Nicotiana tabacum resulted in curved pistils and reduced fertility, implying that the conserved role of Ce-miR396 in floral development. These results provide tools to better understand the biological roles of GRFs in orchid development, and open new avenues for the diversification of orchid floral patterns.
Highlights
Orchids are a highly valuable floricultural crop
A crescent bract primordium initiates around the inflorescence meristem (Figure 1A), the flower meristem emerges with a flattened and oval flower meristem (Figure 1B, stage 0), which continues to enlarge and form a floret primordium (FP) (Figure 1C, stage 1)
A central transversal depression can be observed in FP, from which a labellum primordium (LP) initiates in the adaxial region, followed by development of two lateral sepal primordia (SP)
Summary
Comprising more than 25,000 species distributed in ∼800 genera, orchids represent one of the largest families of flowering plants (Ramirez et al, 2007; Givnish et al, 2015). Cymbidium ensifolium, which belongs to subgenus Jensoa, blossoms many times a year and is highly valuable in flower markets of China (Zhang et al, 2015; Yang et al, 2017; Su et al, 2018). The molecular underpinnings of flower development in terms of floral organ number, arrangement, and initiation timing has been widely studied in the orchid family. A unique mechanism underlying perianth patterning of orchid plants is mediated by MADS-box type transcription factors (TFs) with some modifications of the arabidopsis ABCE model of flower development (Hsu et al, 2015). The miRNAs have been detected by deep-sequencing in orchids, such as Abbreviations: GRF, growth regulating factor; GIF, GRF-interacting factor; DEGs, differentially expressed genes; GO, gene ontology; KEGG, Kyoto encyclopedia of genes and genomes; TF, transcription factor; PTES, protoplast-based transient expression system; NF-Y, nuclear factor Y; TALE, three-amino-acid-loopextension; Bzip, basic leucine zipper domain; bHLH, basic helix-loop-helix
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