Genetic insights into the regulatory pathways for continuous flowering in a unique orchid Arundina graminifolia.

BACKGROUND: Manipulation of flowering time and frequency of blooming is key to enhancing the ornamental value of orchids. Arundina graminifolia is a unique orchid that flowers year round, although the molecular basis of this flowering pattern remains poorly understood. RESULTS: We compared the A. graminifolia transcriptome across tissue types and floral developmental stages to elucidate important genetic regulators of flowering and hormones. Clustering analyses identified modules specific to floral transition and floral morphogenesis, providing a set of candidate regulators for the floral initiation and timing. Among candidate floral homeotic genes, the expression of two FT genes was positively correlated with flower development. Assessment of the endogenous hormone levels and qRT-PCR analysis of 32 pathway-responsive genes supported a role for the regulatory networks in floral bud control in A. graminifolia. Moreover, WGCNA showed that flowering control can be delineated by modules of coexpressed genes; especially, MEgreen presented group of genes specific to flowering. CONCLUSIONS: Candidate gene selection coupled with hormonal regulators brings a robust source to understand the intricate molecular regulation of flowering in precious orchids.

Transcriptional Cascade in the Regulation of Flowering in the Bamboo Orchid Arundina graminifolia.

Flowering in orchids is the most important horticultural trait regulated by multiple mechanisms. Arundina graminifolia flowers throughout the year unlike other orchids with a narrow flowering span. However, little is known of the genetic regulation of this peculiar flowering pattern. This study identifies a number of transcription factor (TF) families in five stages of flower development and four tissue types through RNA-seq transcriptome. About 700 DEGs were annotated to the transcription factor category and classified into 35 TF families, which were involved in multiple signaling pathways. The most abundant TF family was bHLH, followed by MYB and WRKY. Some important members of the bHLH, WRKY, MYB, TCP, and MADS-box families were found to regulate the flowering genes at transcriptional levels. Particularly, the TFs WRKY34 and ERF12 possibly respond to vernalization and photoperiod signaling, MYB108, RR9, VP1, and bHLH49 regulate hormonal balance, and CCA1 may control the circadian pathway. MADS-box TFs including MADS6, 14, 16, AGL5, and SEP may be important regulators of flowering in A. graminifolia. Therefore, this study provides a theoretical basis for understanding the molecular mechanism of flowering in A. graminifolia.

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.

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.

Comparative Metabolomic Analysis Reveals Distinct Flavonoid Biosynthesis Regulation for Leaf Color Development of Cymbidium sinense 'Red Sun'.

The colorful leaf is an important ornamental character of Cymbidium sinense (C. sinense), especially the red leaf, which has always been attracted by breeders and consumers. However, little is documented on the formation mechanism of the red leaf of C. sinense. In this study, the changing patterns of flavonoid-related metabolites, corresponding enzyme activities and genes expression in the leaves of C. sinense 'Red Sun' from red to yellow and finally to green was investigated. A total of 196 flavonoid-related metabolites including 11 anthocyanins metabolites were identified using UPLC-MS/MS-based approach. In the process of leaf color change, 42 metabolites were identified as having significantly different contents and the content of 28 differential metabolites turned to zero. In anthocyanin biosynthetic pathway, content of all 15 identified metabolites showed downregulation trend in the process of leaf color change. Among the 15 metabolites, the contents of Naringenin chalcone, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-d-glucoside decreased to zero in the green leaf stage. The changing pattern of enzyme activity of 10 enzymes involved in the anthocyanin biosynthetic pathway showed different trends from red leaves that have turned yellow and finally green, while the expression of genes encoding these enzymes was all down-regulated in the process of leaf color change. The results of this study revealed the types of flavonoid-related metabolites and the comprehensive analysis of metabolites content, enzyme activities and genes expression providing a new reference for breeders to improve the leaf color of C. sinense 'Red Sun'.