Floral organ-specific proteome profiling of the floral ornamental orchid (Cymbidium goeringii) reveals candidate proteins related to floral organ development

Background Cymbidium goeringii, belonging to the Orchidaceae family, is an important ornamental plant with striking petals and lips. Extremely diversified floral patterns and morphologies make C. goeringii good research material to examine floral development of orchids. However, no floral organ-specific protein has been identified yet. To screen floral development associated proteins, four proteomes from petal (PE), lip (LI), gynostemium (GY), and sepal (SE) were analyzed using Tandem Mass Tag-based proteomic analysis. Results A total of 6626 unique peptides encoding 2331 proteins were identified in our study. Proteins in several primary metabolic pathways, including amino acid metabolism, energy metabolism, and lipid metabolism, were identified as differentially expressed proteins. Interestingly, most of the energy metabolism-related proteins highly expressed in SE, indicating that SE is an important photosynthetic organ of C. goeringii flower. Furthermore, a number of phytohormone-related proteins and transcription factors (TFs) were identified in C. goeringii flowers. Expression analysis showed that 1-aminocyclopropane-1-carboxylate oxidase highly expressed in GY, IAA-amino acid hydrolase ILR1-like 4 and gibberellin receptor 1 C greatly expressed in LI, and auxin-binding protein ABP20 significantly expressed in SE, suggesting a significant role of hormones in the regulation of flower morphogenesis and development. For TFs, GY-highly expressed bHLH13, PE-highly expressed WRKY33, and GY-highly expressed VIP1, were identified. Conclusions Mining of floral organ differential expressed enzymes and TFs helps us to excavate candidate proteins related to floral organ development and to accelerate the breeding of Cymbidium plants.

Paenibacillus cymbidii sp. nov., isolated from Cymbidium goeringii roots

A Gram-stain-variable, aerobic, rod-shaped, endospore-forming strain R196T) was isolated from internal tissues of roots of Cymbidium goeringii. Cells were motile with peritrichous flagella. The colonies were light pink on tryptone soya agar medium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain R196T fell into a phylogenetic cluster belonging to the genus Paenibacillus . Strain R196T was closely related to Paenibacillus cavernae C4-5T and Paenibacillus contaminans CKOBP-6T with 93.6 and 93.3% sequence similarities, respectively. The major cellular polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids, two unidentified aminophospholipids and an unidentified aminolipid. The dominant respiratory quinone was MK-7. The main cellular fatty acids were anteiso-C15 : 0 (53.01%), C16 : 0 (13.04%) and iso-C16 : 0 (10.80%). The genome size of R196T was 9.45 Mb, containing 7617 predicted protein-coding genes, with a DNA G+C content of 57.7 mol%. Based on the results of phenotypic, chemotaxonomic and whole-genome analyses, strain R196T represents a novel species of the genus Paenibacillus , for which the name Paenibacillus cymbidii sp. nov. is proposed. The type strain is R196T (=ACCC 61713T=KCTC 33718T).

Functional analysis of CgWRKY57 from Cymbidium goeringii in ABA response

Background The orchid is one of the top ten Chinese flowers and has high ornamental value and elegant color. However, orchids are vulnerable to abiotic stresses during their growth and development, and the molecular mechanism of the abiotic stress response in orchids is unclear. WRKY proteins belong to a transcription factor family that plays important roles in biotic stress, abiotic stress, growth and development in plants, but little is known about the WRKY family in Cymbidium goeringii. Methods The specific fragment of the CgWRKY57 gene of C. goeringii was analyzed by bioinformatics. The expression of the CgWRKY57 gene of C. goeringii under 4 °C, 42 °C water and ABA stress as well as different tissues was detected by real-time fluorescence quantitative PCR. CgWRKY57 gene was overexpressed in wild type Arabidopsis thaliana by inflorescence infection method, and the function of transgenic lines under ABA stress was analyzed. Results CgWRKY57 was cloned from C. goeringii and found to encode 303 amino acids. The CgWRKY57 protein is an acidic, nonsecreted hydrophilic protein without a signal peptide or transmembrane domain. The CgWRKY57 protein is located to the nucleus and may function intracellularly according to its predicted subcellular localization. A domain analysis and homology comparison showed that the CgWRKY57 protein has a “WRKYGQK” domain and belongs to Group III of the WRKY family, and a phylogenetic analysis demonstrated that CgWRKY57 is closely related to OsWRKY47. CgWRKY57 was expressed in the roots, stems, leaves and floral organs of C. goeringii, and its expression level was highest in the roots according to real-time qPCR analysis. There were significant differences in CgWRKY57 expression under 4 °C, 42 °C ABA and water stress treatments, and its expression changed greatly under ABA stress. The expression of CgWRKY57 in transgenic plants was significantly higher than that in wild type plants under ABA stress, and the root length and germination rate were reduced in transgenic plants compared to wild type plants. Conclusions These results indicate that CgWRKY57 overexpression is responsive to ABA stress, and they provide a foundation for future analyses of the biological functions of the WRKY family in C. goeringii.

Different Amino Acids Inhibit or Promote Rhizome Proliferation and Differentiation in Cymbidium goeringii

The proliferation and differentiation of rhizomes are crucial for the propagation of Cymbidium species. We systematically assessed the effects of different concentrations of 20 amino acids on the proliferation and differentiation of C. goeringii rhizomes. Rhizome proliferation rates were significantly higher in media with 2.0 mmol·L−1 cysteine, 0.5 mmol·L−1 arginine, 0.2 mmol·L−1 asparagine, 1.0 mmol·L−1 proline, and 0.5 mmol·L−1 lysine compared with those in the control. Additionally, 1.0 mmol·L−1 tyrosine, 0.5 mmol·L−1 asparagine, and 0.2 mmol·L−1 aspartate were beneficial for rhizome differentiation. Furthermore, two combinations of amino acids, 0.5 mmol·L−1 arginine + 1.0 mmol·L−1 proline and 0.5 mmol·L−1 arginine + 2.0 mmol·L−1 cysteine, resulted in proliferation rates of 3.05 and 3.01, respectively, after 60 days. The highest differentiation rate (5.39 after 60 days) was observed in media with 0.5 mmol·L−1 asparagine + 0.2 mmol·L−1 aspartate. This study demonstrated that certain combinations of amino acids can effectively promote the proliferation and differentiation of rhizomes during the rapid propagation of C. goeringii.

Genetic Diversity and Population Structure of the Spring Orchid Cymbidium goeringii in Korean Distant Islands

The spring orchid (Cymbidium goeringii), found in northeast Asia, is one of the most popular and horticulturally important species of the orchid family. This study analyzed the genetic diversity and population structure of the spring orchid populations in the small islands and mainland South Korea using 11 microsatellite markers. The genetic diversities of spring orchid populations in the distant islands (Heuksan Island and Ulleung Island) were slightly lower than that of the mainland population (Yeonggwang-gun). The population structure in the mainland was genetically separated from the populations in the islands. The population of Ulleung Island, located in the eastern part of the Korean peninsula, was genetically closer to the populations from China and Japan than to the populations from Yeonggwang-gun and Heuksan Island, which are geographically close to China. These results imply that the populations of spring orchids distributed in Yeonggwang-gun and Heuksan Island appear not to be influenced by the yellow dust winds. As the first population genetic study of spring orchids distributed in small distant islands, our study will be useful for understanding the genetic diversity and population structure of isolated C. goeringii populations.