Phosphorylation of 399S at CsHsp70 of Cymbidium sinense is essential to maintain chlorophyll stability.

The Chinese orchids symbolise nobility and gentility in China, and the variation of leaf color makes Cymbidium sinense more diversified and valuable. However, its color variations especially at the protein level still remain largely unexplored. In this study, the proteomics and phosphoproteomics of Cymbidium sinense leaf color variation mutants were studied. A total of 1059 differentially abundant proteins (DAPs) and 1127 differentially abundant phosphorylation sites belonging to 644 phosphoproteins (DAPPs) were identified in the yellow section of leaf variegation mutant of Cymbidium sinense (MY) compared with the green section (MG). Moreover, 349 co-expressing proteins were found in both omics' datasets, while only 26 proteins showed the same expression patterns in the two omics. The interaction network analysis of kinases and phosphatases showed that DAPs and DAPPs in photosynthesis, response to hormones, pigment metabolic process, phosphorylation, glucose metabolic process, and dephosphorylation might contribute to leaf color variation. The abundance of 28 Hsps and 28 phosphorylation sites belonging to 10 Hsps showed significant differences between MG and MY. CsHsp70 was selected to explore the function in Cymbidium sinense leaf variegation. The results showed CsHsp70 is essential for maintaining photosynthetic pigment content and the 399S phosphorylation site is crucial to the function of CsHsp70. Collectively, our findings construct a comprehensive coverage of protein and protein phosphorylation in leaf variegation of C. sinense, providing valuable insights into its formation mechanisms.

Integrated proteomic, transcriptomic, and metabolomic profiling reveals that the gibberellin-abscisic acid hub runs flower development in the Chinese orchid Cymbidium sinense.

The seasonal flowering Chinese Cymbidium produce an axillary floral meristem and require a dormancy period during cold conditions for flower development. However, the bud activation mechanism remains elusive. This study evaluates the multi-omics across six stages of flower development, along with functional analysis of core genes to decipher the innate mechanism of floral bud initiation and outgrowth in the Chinese orchid Cymbidium sinense. Transcriptome and proteome analyses identified 10 modules with essential roles in floral bud dormancy and activation. Gene clusters in the early stages of flower development were mainly related to flowering time regulation and meristem determination, while the late stages were correlated with hormone signaling pathways. The metabolome identified 69 potential hormones in which gibberellin (GA) and abscisic acid (ABA) were the main regulatory hubs, and GA4 and GA53 exhibited a reciprocal loop. Extraneous GA application caused rapid elongation of flower buds and promoted the expression of flower development genes. Contrarily, exogenous ABA application extended the dormancy process and ABA inhibitors induced dormancy release. Moreover, CsAPETALA1 (CsAP1) was identified as the potential target of ABA for floral bud activation. Transformation of CsAP1 in Arabidopsis and its transient overexpression in C. sinense protoplasts not only affected flowering time and floral organ morphogenesis in Arabidopsis but also orchestrated the expression of flowering and hormone regulatory genes. The presence of ABA response elements in the CsAP1 promoter, rapid downregulation of CsAP1 after exogenous ABA application, and the activation of the floral bud after ABA inhibitor treatment suggest that ABA can control bud outgrowth through CsAP1.

Sb3+/Sm3+ Codoped Cs2NaScCl6 All-Inorganic Double Perovskite: Blue Emission of Self-Trapped Excitons and Red-Emission via Energy Transfer.

The lead-free halide perovskites possess nontoxicity and excellent chemical stability, whereas relatively weak luminescence intensity limits their potential in practical applications. Therefore, strengthening the luminescence intensity and expanding application fields are urgent tasks for the development of lead-free halide perovskites. In this paper, antimony-doped Cs2NaScCl6 crystals synthesized by a solvothermal method emit bright, deep blue photoluminescence at 447 nm. The photoluminescence (PL), photoluminescence excitation (PLE), and absorption spectra demonstrate that Sb3+ doping effectively activate the intrinsic "dark self-trapped exciton (STE)," leading to an impressive photoluminescence quantum yield (PLQY) value of 78.31% for 1% Sb3+ doping. Furthermore, the luminescence intensity remains above 92% compared with the fresh sample without secondary phases detected even after 90 days under environmental conditions. To expand the emission spectra, rare-earth Sm3+ is further incorporated into Cs2NaScCl6:1% Sb3+ crystals. The results show that Sb ions not only enhance intrinsic STE luminescence but also serve as sensitizers to boost the red-light emission of Sm3+, leading to a significant 500-fold increase in red emission intensity. Finally, the PLQY reaches a stunning 86.78%. These findings provide valuable insights in the design of Sb ion-doped lead-free double perovskites, broadening the application fields in various optoelectronic devices.

First Report of Agroathelia rolfsii Causing Southern Blight on Lippia (Phyla canescens) in Guangzhou, China.

Lippia (Phyla canescens) is a fast-growing, mat-forming, and prostrate perennial plant well adapted to infertile, high-saline, and drought environments (Leigh, et al. 2004). It arrived in China from Japan as a flowering ground cover in 2001 (Cai, et al. 2004). In June 2022, southern blight appeared in our nursery of the Floriculture Research Institute of Guangdong Academy of Agricultural Sciences. High temperature and damp environment are major factors for this disease. The symptoms of top-layer plants were not easily detected, but they were slightly yellowed. A yellowish-brown water-soak lesion appeared on the stems and lowest leaves exposed to soil. White mycelium appeared in the middle stage. Finally, the surface plants showed water-soak decay, and a mass of beige to black-brown rapeseed-shaped sclerotia appeared on the residue and surrounding soil; these plants died. Sclerotia and mycelia were collected from disease tissue, and after surface sterilization, sclerotia was cultured on potato dextrose agar (PDA) at 28±2°C in an incubator without light. Eight fungal isolates with similar colony morphologies were consistently isolated by purifying from different sampling areas. The isolates exhibited obvious septa and a clamp connection structure within the white mycelium. The average growth rate was 26.86±0.06 mm/day. Numerous white granular sclerotia were produced on the mycelium 6 days later. The sclerotia with a diameter of 1.24±0.07mm (n=189) gradually changed from diage to yellow to brown. A typical strain B1 was selected for further identification, targeting its 18S rRNA and LSU rRNA sequences (Yang, et al. 2011; Xue, et al. 2019). Its 18S rRNA sequence (GenBank Accession No. OR517233, 1626 bp) is 99.63% and 99.57% identical to Athelia rolfsii (AY665774, 1179bp; KC670714, 1775bp; JF819726, 1781bp). Its LSU rRNA sequence (OR539570, 757 bp) is 99.87% identical to Agroathelia rolfsii (OR526537, 904 bp). For Athelia rolfsii, a synonym of Agroathelia rolfsii, by combining the morphological characteristics and molecular identification, the isolate pathogen B1 was confirmed to be Agroathelia rolfsii (the teleomorph of Sclerotium rolfsii). To fullfill Koch's postulates, we inoculated the mycelial plugs to healthy lippia stems and leaves which has grown for one year, with PDA plugs free of mycelium as the control. All the plants were kept in a greenhouse at 28±2°C with a 14-h photoperiod and 80% relative humidity. Each treatment was repeated thrice and vaccinated with 6 points. At 7 d following inoculation, all plants inoculated with B1 showed typical symptoms, but the control group was asymptomatic, and sclerotia appeared 17d after inoculation. Using the same protocol mentioned above, pathogenic fungal was reisolated only from treated groups, but not from the control group. Chose three of the pathogens for 18S rRNA and LSU rRNA sequencing, the results showed 100% identity to B1, the same as its microstructure. There are few reports about the disease on P. canescens. Sosa (2007) investigated the pathogens on P. canescens in Argentina, 16 fungi were found but no A. rolfsii. Sclerotium rolfsii were identified on P. nodiflora or P. lanceolata (Michaux) Greene in America (Farr, et al. 1989). To our knowledge, this is the first report in China. Because this pathogen has wide-ranging hosts and causes serious damage, the results from this study will offer guidance for the prevention and treatment of this disease.

Genome-wide association analysis identified molecular markers and candidate genes for flower traits in Chinese orchid (Cymbidium sinense).

The orchid, the champagne of flowers, brings luxury, elegance, and novelty to nature. Cymbidium sinense is a symbol of gigantic floral variability on account of wavering shapes and sizes of floral organs, although marker-trait association (MTA) has not been studied for its floral traits. We evaluated markers associated with 14 floral traits of C. sinense through a genome-wide association study (GWAS) of 195 accessions. A total of 65 318 522 single-nucleotide polymorphisms (SNPs) and 3 906 176 insertion/deletion (InDel) events were identified through genotyping-by-sequencing. Among these, 4694 potential SNPs and 477 InDels were identified as MTAs at -log10 P > 5. The genes related to these SNPs and InDels were largely associated with floral regulators, hormonal pathways, cell division, and metabolism, playing essential roles in tailoring floral morphology. Moreover, 20 candidate SNPs/InDels linked to 11 genes were verified, 8 of which were situated on exons, one was located in the 5'-UTR and two were positioned in introns. Here, the multitepal trait-related gene RABBIT EARS (RBE) was found to be the most crucial gene. We analyzed the role of CsRBE in the regulation of flower-related genes via efficient transient overexpression in C. sinense protoplasts, and found that the floral homeotic genes CsAP3 and CsPI, as well as organ boundary regulators, including CsCUC and CsTCP genes, were regulated by CsRBE. Thus, we obtained key gene loci for important ornamental traits of orchids using genome-wide association analysis of populations with natural variation. The findings of this study can do a great deal to expedite orchid breeding programs for shape variability.

Genome-Wide Identification, Expression, and Molecular Characterization of the CONSTANS-like Gene Family in Seven Orchid Species.

The orchid is one of the most distinctive and highly valued flowering plants. Nevertheless, the CONSTANS-like (COL) gene family plays significant roles in the control of flowering, and its functions in Orchidaceae have been minimally explored. This research identified 68 potential COL genes within seven orchids' complete genome, divided into three groups (groups I, II, and III) via a phylogenetic tree. The modeled three-dimensional structure and the conserved domains exhibited a high degree of similarity among the orchid COL proteins. The selection pressure analysis showed that all orchid COLs suffered a strong purifying selection. Furthermore, the orchid COL genes exhibited functional and structural heterogeneity in terms of collinearity, gene structure, cis-acting elements within their promoters, and expression patterns. Moreover, we identified 50 genes in orchids with a homology to those involved in the COL transcriptional regulatory network in Arabidopsis. Additionally, the first overexpression of CsiCOL05 and CsiCOL09 in Cymbidium sinense protoplasts suggests that they may antagonize the regulation of flowering time and gynostemium development. Our study will undoubtedly provide new resources, ideas, and values for the modern breeding of orchids and other plants.

Functional conservation and divergence of SEPALLATA-like genes in floral development in Cymbidium sinense.

Cymbidium sinense is one of the most important traditional Chinese Orchids due to its unique and highly ornamental floral organs. Although the ABCDE model for flower development is well-established in model plant species, the precise roles of these genes in C. sinense are not yet fully understood. In this study, four SEPALLATA-like genes were isolated and identified from C. sinense. CsSEP1 and CsSEP3 were grouped into the AGL9 clade, while CsSEP2 and CsSEP4 were included in the AGL2/3/4 clade. The expression pattern of CsSEP genes showed that they were significantly accumulated in reproductive tissues and expressed during flower bud development but only mildly detected or even undetected in vegetative organs. Subcellular localization revealed that CsSEP1 and CsSEP4 were localized to the nucleus, while CsSEP2 and CsSEP3 were located at the nuclear membrane. Promoter sequence analysis predicted that CsSEP genes contained a number of hormone response elements (HREs) and MADS-box binding sites. The early flowering phenotype observed in transgenic Arabidopsis plants expressing four CsSEP genes, along with the expression profiles of endogenous genes, such as SOC1, LFY, AG, FT, SEP3 and TCPs, in both transgenic Arabidopsis and C. sinense protoplasts, suggested that the CsSEP genes played a regulatory role in the flowering transition by influencing downstream genes related to flowering. However, only transgenic plants overexpressing CsSEP3 and CsSEP4 caused abnormal phenotypes of floral organs, while CsSEP1 and CsSEP2 had no effect on floral organs. Protein-protein interaction assays indicated that CsSEPs formed a protein complex with B-class CsAP3-2 and CsSOC1 proteins, affecting downstream genes to regulate floral organs and flowering time. Our findings highlighted both the functional conservation and divergence of SEPALLATA-like genes in C. sinense floral development. These results provided a valuable foundation for future studies of the molecular network underlying floral development in C. sinense.

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

Orchids are among the most precious flowers in the world. Regulation of flowering time is one of the most important targets to enhance their ornamental value. The beauty of Arundina graminifolia is its year-round flowering, although the molecular mechanism of this flowering ability remains masked. Therefore, we performed a comprehensive assessment to integrate transcriptome and miRNA sequencing to disentangle the genetic regulation of flowering in this valuable species. Clustering analyses provided a set of molecular regulators of floral transition and floral morphogenesis. We mined candidate floral homeotic genes, including FCA, FPA, GI, FT, FLC, AP2, SOC1, SVP, GI, TCP, and CO, which were targeted by a variety of miRNAs. MiR11091 targeted the highest number of genes, including candidate regulators of phase transition and hormonal control. The conserved miR156-miR172 pathway of floral time regulation was evident in our data, and we found important targets of these miRNAs in the transcriptome. Moreover, endogenous hormone levels were determined to decipher the hormonal control of floral buds in A. graminifolia. The qRT-PCR analysis of floral and hormonal integrators validated the transcriptome expression. Therefore, miRNA-mediated mining of candidate genes with hormonal regulation forms the basis for comprehending the complex regulatory network of perpetual flowering in precious orchids. The findings of this study can do a great deal to broaden the breeding programs for flowering time manipulation of orchids.

Genome-wide identification of Cymbidium sinense WRKY gene family and the importance of its Group III members in response to abiotic stress.

Transcription factors (TFs) of the WRKY family play pivotal roles in defense responses and secondary metabolism of plants. Although WRKY TFs are well documented in numerous plant species, no study has performed a genome-wide investigation of the WRKY gene family in Cymbidium sinense. In the present work, we found 64 C. sinense WRKY (CsWRKY) TFs, and they were further divided into eight subgroups. Chromosomal distribution of CsWRKYs revealed that the majority of these genes were localized on 16 chromosomes, especially on Chromosome 2. Syntenic analysis implied that 13 (20.31%) genes were derived from segmental duplication events, and 17 orthologous gene pairs were identified between Arabidopsis thaliana WRKY (AtWRKY) and CsWRKY genes. Moreover, 55 of the 64 CsWRKYs were detectable in different plant tissues in response to exposure to plant hormones. Among them, Group III members were strongly induced in response to various hormone treatments, indicating their potential essential roles in hormone signaling. We subsequently analyzed the function of CsWRKY18 in Group III. The CsWRKY18 was localized in the nucleus. The constitutive expression of CsWRKY18 in Arabidopsis led to enhanced sensitivity to ABA-mediated seed germination and root growth and elevated plant tolerance to abiotic stress within the ABA-dependent pathway. Overall, our study represented the first genome-wide characterization and functional analysis of WRKY TFs in C. sinense, which could provide useful clues about the evolution and functional description of CsWRKY genes.

The Transcriptome Profiling of Flavonoids and Bibenzyls Reveals Medicinal Importance of Rare Orchid Arundina graminifolia.

Orchids are very important flowering plants that spend long juvenile phases before flowering. Along with aesthetic importance, they are rich sources of medicinal components. However, their long reproductive cycle is the major hurdle to study the medicinal efficacy. Arundina graminifolia is a rare orchid that grows fast, unlike other orchids, and this characteristic makes it an ideal plant to study the medicinal enrichment of orchids. Therefore, this study presents the identification of important medicinal components in various parts of A. graminifolia. Transcriptome analysis was performed for five stages (FD1-FD5) of flower development and four tissue types (mature flower, silique, root, and leaf) to ascertain genetic regulators of flavonoids and bibenzyls. Most of the genes showed the highest expression in roots as compared with other tissues. Weighted gene coexpression network analysis (WGCNA) was performed to identify the coexpression modules and the candidate genes involving biosynthesis pathways of these chemicals. MEyellow module contained the highly coexpressed genes. Moreover, the concentrations of phenylpropanoid, bibenzyls, and flavone were ascertained through high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Phenylpropanoid and bibenzyl were comparatively high in the leaf, while flavone showed a high concentration in the stem. The selected candidate genes [bibenzyl biosynthesis (BIBSY212), CYP84A1, CYP73A4, 4CLL7, UGT88B1, UGT73C3, anthocyanin synthase (ANS), phenylalanine ammonia-lyase (PAL), flavanone synthase FLS, and CHS8] were validated through quantitative real-time PCR (qRT-PCR). Most of these genes showed high expression in leaf and root as compared with other tissue. Therefore, the presence of bibenzyls and flavonoids in different parts of A. graminifolia and their molecular regulators can provide a quick source to decipher the medicinal efficacy of orchids.

Exogenous GA3 promotes flowering in Paphiopedilum callosum (Orchidaceae) through bolting and lateral flower development regulation.

Paphiopedilum orchids have a high ornamental value, and their flower abundance and timing are both key horticultural traits regulated by phytohormones. All one-flowered Paphiopedilum have additional lateral buds in the apical bract that fail to develop. In this study, an exogenous gibberellin (GA3) application promoted flowering of Pathiopedilum callosum by inducing its early bolting instead of the floral transition of dominant flowers. Applying GA3 effectively promoted lateral flower differentiation, resulting in a two-flowered inflorescence. GA-promoted lateral flower formation involved GA interacting with indole-3-acetic acid (IAA) and cytokinins (CTKs), given the decreased CTK content and downregulated expression of CTK synthesis genes, the increased IAA content and downregulated expression of IAA degradation, and the upregulated expression of transport genes. Further, GA acted via PcDELLA, PcTCP15, and PcXTH9 expressed in stage 5 to promote bolting, and via expression of PcAP3, PcPI, and PcSEP to promote flowering. This study provides insight into mechanisms regulating flower development of P. callosum.

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.

The Cymbidium genome reveals the evolution of unique morphological traits.

The marvelously diverse Orchidaceae constitutes the largest family of angiosperms. The genus Cymbidium in Orchidaceae is well known for its unique vegetation, floral morphology, and flower scent traits. Here, a chromosome-scale assembly of the genome of Cymbidium ensifolium (Jianlan) is presented. Comparative genomic analysis showed that C. ensifolium has experienced two whole-genome duplication (WGD) events, the most recent of which was shared by all orchids, while the older event was the τ event shared by most monocots. The results of MADS-box genes analysis provided support for establishing a unique gene model of orchid flower development regulation, and flower shape mutations in C. ensifolium were shown to be associated with the abnormal expression of MADS-box genes. The most abundant floral scent components identified included methyl jasmonate, acacia alcohol and linalool, and the genes involved in the floral scent component network of C. ensifolium were determined. Furthermore, the decreased expression of photosynthesis-antennae and photosynthesis metabolic pathway genes in leaves was shown to result in colorful striped leaves, while the increased expression of MADS-box genes in leaves led to perianth-like leaves. Our results provide fundamental insights into orchid evolution and diversification.

Stage Specificity, the Dynamic Regulators and the Unique Orchid Arundina graminifolia.

Orchids take years to reach flowering, but the unique bamboo orchid (Arundina graminifolia) achieves reproductive maturity in six months and then keeps on year round flowering. Therefore, studying different aspects of its growth, development and flowering is key to boost breeding programs for orchids. This study uses transcriptome tools to discuss genetic regulation in five stages of flower development and four tissue types. Stage specificity was focused to distinguish genes specifically expressed in different stages of flower development and tissue types. The top 10 highly expressed genes suggested unique regulatory patterns for each stage or tissue. The A. graminifolia sequences were blasted in Arabidopsis genome to validate stage specific genes and to predict important hormonal and cell regulators. Moreover, weighted gene co-expression network analysis (WGCNA) modules were ascertained to suggest highly influential hubs for early and late stages of flower development, leaf and root. Hormonal regulators were abundant in all data sets, such as auxin (LAX2, GH3.1 and SAUR41), cytokinin (LOG1), gibberellin (GASA3 and YAB4), abscisic acid (DPBF3) and sucrose (SWEET4 and SWEET13). Findings of this study, thus, give a fine sketch of genetic variability in Orchidaceae and broaden our understanding of orchid flower development and the involvement of multiple pathways.

The genome of Cymbidium sinense revealed the evolution of orchid traits.

The Orchidaceae is of economic and ecological importance and constitutes ˜10% of all seed plant species. Here, we report a genome physical map for Cymbidium sinense, a well-known species belonging to genus Cymbidium that has thousands of natural variation varieties of flower organs, flower and leaf colours and also referred as the King of Fragrance, which make it arose into a unique cultural symbol in China. The high-quality chromosome-scale genome assembly was 3.52 Gb in size, 29 638 protein-coding genes were predicted, and evidence for whole-genome duplication shared with other orchids was provided. Marked amplification of cytochrome- and photosystem-related genes was observed, which was consistent with the shade tolerance and dark green leaves of C. sinense. Extensive duplication of MADS-box genes, and the resulting subfunctional and expressional differentiation, was associated with regulation of species-specific flower traits, including wild-type and mutant-type floral patterning, seasonal flowering and ecological adaption. CsSEP4 was originally found to positively regulate gynostemium development. The CsSVP genes and their interaction proteins CsAP1 and CsSOC1 were significantly expanded and involved in the regulation of low-temperature-dependent flowering. Important genetic clues to the colourful leaf traits, purple-black flowers and volatile trait in C. sinense were also found. The results provide new insights into the molecular mechanisms of important phenotypic traits of Cymbidium and its evolution and serve as a powerful platform for future evolutionary studies and molecular breeding of 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.

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops.

Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4-0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 µM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 106/g fresh weight (FW)], flower pedicels (∼5.26 × 106/g FW), and young root tips (∼7.66 × 105/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 107/g FW), Phalaenopsis (1.83 × 107/g FW), Paphiopedilum (1.10 × 107/g FW), Dendrobium (8.21 × 106/g FW), Arundina (3.78 × 106/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 107/g FW) and rice (Oryza sativa) (4.31 × 107/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.

Orchid Bsister gene PeMADS28 displays conserved function in ovule integument development.

The ovules and egg cells are well developed to be fertilized at anthesis in many flowering plants. However, ovule development is triggered by pollination in most orchids. In this study, we characterized the function of a Bsister gene, named PeMADS28, isolated from Phalaenopsis equestris, the genome-sequenced orchid. Spatial and temporal expression analysis showed PeMADS28 predominantly expressed in ovules between 32 and 48 days after pollination, which synchronizes with integument development. Subcellular localization and protein-protein interaction analyses revealed that PeMADS28 could form a homodimer as well as heterodimers with D-class and E-class MADS-box proteins. In addition, ectopic expression of PeMADS28 in Arabidopsis thaliana induced small curled rosette leaves, short silique length and few seeds, similar to that with overexpression of other species' Bsister genes in Arabidopsis. Furthermore, complementation test revealed that PeMADS28 could rescue the phenotype of the ABS/TT16 mutant. Together, these results indicate the conserved function of Bsister PeMADS28 associated with ovule integument development in orchid.