Organ-Specific Gene Expression Reveals the Role of the Cymbidium ensifolium-miR396/Growth-Regulating Factors Module in Flower Development of the Orchid Plant Cymbidium ensifolium

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.

Transcriptome mining of hormonal and floral integrators in the leafless flowers of three cymbidium orchids

“…Our recent studies have found a number of TFs related to flowering regulation in orchids, especially the Arundina graminifolia (Ahmad et al, 2020;Ahmad et al, 2021a;Ahmad et al, 2022a). Our study also showed a number of MADS-box and zinc finger TFs with significant difference in the leafless flowers and healthy leaves of three Cymbidium species (Figures 5, 6), which are known floral regulators in orchids (Teo et al, 2019;Valoroso et al, 2019;Ahmad et al, 2021b;Chen et al, 2021;Yang et al, 2021b;Ahmad et al, 2022a;Ahmad et al, 2022b). However, more flowering related genes and TFs were upregulated in C. sinense as compared to other two species (Figure 5), suggesting that C. sinense may have different body plans than other Cymbidium orchids.…”

The Integrated mRNA and miRNA Approach Reveals Potential Regulators of Flowering Time in Arundina graminifolia

“…The miR319 positively regulates auxin signaling [ 77 , 78 ], and its overexpression in Arabidopsis and rice affects cell differentiation by inhibiting GA biosynthesis [ 79 , 80 ]. Auxin upregulated miR319 production in Brassica while cytokinin repressed its expression in rice, suggesting a critical role of miR319 in coordinating antagonistic auxin-cytokinin pathways [ 81 , 82 ]. Similarly, it could also control antagonistic ABA-GA pathways, as previous studies showed that ABA treatment in rice seedlings inhibited the expression of miR319 [ 81 ].…”

Identification and analysis of miRNAs differentially expressed in male and female Trichosanthes kirilowii maxim

“…Unfortunately, KEGG pathway enrichment has not been annotated with this gene, and the specific metabolic pathway involved in this gene is unknown. Yang et al [ 41 ] found that Nicotiana tabacum overexpressing CemiR396 showed pistil bending and reduced fertility, implying the conserved role of CemiR396 in floral development. Liang et al [ 42 ] showed that miR396 can control the number of carpels and pistil development of Arabidopsis by regulating the GRF/GIF complex.…”