Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

The rhizosphere microbiome and its influence on the accumulation of metabolites in Bletilla striata (Thunb.) Reichb. f.

BACKGROUND: Bletilla striata (Thunb.) Reichb. f. (B. striata) is a perennial herbaceous plant in the Orchidaceae family known for its diverse pharmacological activities, such as promoting wound healing, hemostasis, anti-inflammatory effects, antioxidant properties, and immune regulation. Nevertheless, the microbe-plant-metabolite regulation patterns for B. striata remain largely undetermined, especially in the field of rhizosphere microbes. To elucidate the interrelationships between soil physics and chemistry and rhizosphere microbes and metabolites, a comprehensive approach combining metagenome analysis and targeted metabolomics was employed to investigate the rhizosphere soil and tubers from four provinces and eight production areas in China. RESULTS: Our study reveals that the core rhizosphere microbiome of B. striata is predominantly comprised of Paraburkholderia, Methylibium, Bradyrhizobium, Chitinophaga, and Mycobacterium. These microbial species are recognized as potentially beneficial for plants health. Comprehensive analysis revealed a significant association between the accumulation of metabolites, such as militarine and polysaccharides in B. striata and the composition of rhizosphere microbes at the genus level. Furthermore, we found that the soil environment indirectly influenced the metabolite profile of B. striata by affecting the composition of rhizosphere microbes. Notably, our research identifies soil organic carbon as a primary driving factor influencing metabolite accumulation in B. striata. CONCLUSION: Our fndings contribute to an enhanced understanding of the comprehensive regulatory mechanism involving microbe-plant-metabolite interactions. This research provides a theoretical basis for the cultivation of high-quality traditional Chinese medicine B. striata.

Physiological and transcriptomic analyses reveal the regulatory mechanisms of Anoectochilus roxburghii in response to high-temperature stress.

BACKGROUND: High temperatures significantly affect the growth, development, and yield of plants. Anoectochilus roxburghii prefers a cool and humid environment, intolerant of high temperatures. It is necessary to enhance the heat tolerance of A. roxburghii and breed heat-tolerant varieties. Therefore, we studied the physiological indexes and transcriptome of A. roxburghii under different times of high-temperature stress treatments. RESULTS: Under high-temperature stress, proline (Pro), H2O2 content increased, then decreased, then increased again, catalase (CAT) activity increased continuously, peroxidase (POD) activity decreased rapidly, then increased, then decreased again, superoxide dismutase (SOD) activity, malondialdehyde (MDA), and soluble sugars (SS) content all decreased, then increased, and chlorophyll and soluble proteins (SP) content increased, then decreased. Transcriptomic investigation indicated that a total of 2740 DEGs were identified and numerous DEGs were notably enriched for "Plant-pathogen interaction" and "Plant hormone signal transduction". We identified a total of 32 genes in these two pathways that may be the key genes for resistance to high-temperature stress in A. roxburghii. CONCLUSIONS: To sum up, the results of this study provide a reference for the molecular regulation of A. roxburghii's tolerance to high temperatures, which is useful for further cultivation of high-temperature-tolerant A. roxburghii varieties.

Perturbation of periodic spot-generation balance leads to diversified pigmentation patterning of harlequin Phalaenopsis orchids: in silico prediction.

BACKGROUND: A retrotransposon HORT1 in the promoter of the anthocyanin activator gene PeMYB11, microRNA858 (miR858) that targets PeMYB11, and a repressor PeMYBx have been implicated in pigmentation patterning diversity of harlequin Phalaenopsis orchids. However, the interrelationship among them remains to be elucidated. RESULTS: To understand how these factors interact to generate anthocyanin spots in Phalaenopsis, we successfully developed a mathematical model based on the known reaction-diffusion system to simulate their interplay and refined the conceptual biological model. Intriguingly, the expression of both PeMYBx and PeMYB11 were in phase for purple spot formation, even though they showed adverse effects on anthocyanin accumulations. An increase in the self-activation rate of PeMYB11 resulted in the increased size of purple spots, but no effects on spot fusion. Decreased degradation rate of miR858 in the purple regions, led to disruption of the formation of spotted pigmentation patterning and a full-red pigmentation pattern. Significantly, the reduced miR858 level promotes the fusion of large dark purple dots induced by the solo-LTR of HORT1, eventually generating the purple patches. In addition, the spatially heterogeneous insertion of HORT1 caused by the remnant solo-LTR of HORT1 derived from random homologous unequal recombination of HORT1 in individual cells of floral organs could explain the diverse pigmentation patterning of harlequin Phalaenopsis. CONCLUSIONS: This devised model explains how HORT1 and miR858 regulate the formation of the pigmentation patterning and holds great promise for developing efficient and innovative approaches to breeding harlequin Phalaenopsis orchids.

Mechanism underlying the rapid growth of Phalaenopsis equestris induced by 60Co-γ-ray irradiation.

Gamma (γ)-ray irradiation is one of the important modern breeding methods. Gamma-ray irradiation can affect the growth rate and other characteristics of plants. Plant growth rate is crucial for plants. In horticultural crops, the growth rate of plants is closely related to the growth of leaves and flowering time, both of which have important ornamental value. In this study, 60Co-γ-ray was used to treat P. equestris plants. After irradiation, the plant's leaf growth rate increased, and sugar content and antioxidant enzyme activity increased. Therefore, we used RNA-seq technology to analyze the differential gene expression and pathways of control leaves and irradiated leaves. Through transcriptome analysis, we investigated the reasons for the rapid growth of P. equestris leaves after irradiation. In the analysis, genes related to cell wall relaxation and glucose metabolism showed differential expression. In addition, the expression level of genes encoding ROS scavenging enzyme synthesis regulatory genes increased after irradiation. We identified two genes related to P. equestris leaf growth using VIGS technology: PeNGA and PeEXPA10. The expression of PeEXPA10, a gene related to cell wall expansion, was down-regulated, cell wall expansion ability decreased, cell size decreased, and leaf growth rate slowed down. The TCP-NGATHA (NGA) molecular regulatory module plays a crucial role in cell proliferation. When the expression of the PeNGA gene decreases, the leaf growth rate increases, and the number of cells increases. After irradiation, PeNGA and PeEXPA10 affect the growth of P. equestris leaves by influencing cell proliferation and cell expansion, respectively. In addition, many genes in the plant hormone signaling pathway show differential expression after irradiation, indicating the crucial role of plant hormones in plant leaf growth. This provides a theoretical basis for future research on leaf development and biological breeding.

A review on endophytic bacteria of orchids: functional roles toward synthesis of bioactive metabolites for plant growth promotion and disease biocontrol.

MAIN CONCLUSION: In this review, we have discussed the untapped potential of orchid endophytic bacteria as a valuable reservoir of bioactive metabolites, offering significant contributions to plant growth promotion and disease protection in the context of sustainable agriculture. Orchidaceae is one of the broadest and most diverse flowering plant families on Earth. Although the relationship between orchids and fungi is well documented, bacterial endophytes have recently gained attention for their roles in host development, vigor, and as sources of novel bioactive compounds. These endophytes establish mutualistic relationships with orchids, influencing plant growth, mineral solubilization, nitrogen fixation, and protection from environmental stress and phytopathogens. Current research on orchid-associated bacterial endophytes is limited, presenting significant opportunities to discover new species or genetic variants that improve host fitness and stress tolerance. The potential for extracting bioactive compounds from these bacteria is considerable, and optimization strategies for their sustainable production could significantly enhance their commercial utility. This review discusses the methods used in isolating and identifying endophytic bacteria from orchids, their diversity and significance in promoting orchid growth, and the production of bioactive compounds, with an emphasis on their potential applications in sustainable agriculture and other sectors.

Identification and Analysis of PEPC Gene Family Reveals Functional Diversification in Orchidaceae and the Regulation of Bacterial-Type PEPC.

Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the PEPC gene family in CAM plants. In this study, a total of 33 PEPC genes were identified across 15 orchids. Specifically, one PEPC gene was found in Cymbidium goeringii and Platanthera guangdongensis; two in Apostasia shenzhenica, Dendrobium chrysotoxum, D. huoshanense, Gastrodia elata, G. menghaiensis, Phalaenopsis aphrodite, Ph. equestris, and Pl. zijinensis; three in C. ensifolium, C. sinense, D. catenatum, D. nobile, and Vanilla planifolia. These PEPC genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid PEPC gene, identified as VpPEPC4. Differences in the number of PEPC homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. Cis-elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, VpPEPC4, exhibited a lower expression level than other VpPEPCs in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid PEPC genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of PEPC genes in Orchidaceae.

PeFtsH5 negatively regulates the biological stress response in Phalaenopsis equestris mitochondria.

Mitochondrial homeostasis plays a crucial role in determining cell fate by direct influence on cell apoptosis and autophagy. The ATP and Zn2+-dependent protease FtsH are of paramount importance in maintaining mitochondrial homeostasis. In Phalaenopsis equestris, three mitochondrial FtsH proteases were identified, one of which was encoded by the PeFtsH5 gene. This gene encoded a distinctive mitochondrial protein featuring a unique domain within the FtsH family. Down-regulating the expression of the PeFtsH5 homolog in Nicotiana benthamiana resulted in elevated expression levels of SA synthesis-related genes, leading to enhanced disease resistance. However, this down-regulation also caused cellular damage. Similarly, in P. equestris, the down-regulation of PeFtsH5 expression promoted the expression of defense response genes, leading to accelerated apoptosis and increased ROS levels. Nonetheless, this down-regulation also positively influenced plant resistance to biotic stress. Notably, the PeFtsH5 (i-AAA) protein, as revealed by dual membrane experiments, could form homopolymers exclusively, as it did not interact with the other two mitochondrial FtsH proteases. Consequently, this mitochondrial FtsH protease functioned as a homopolymer within P. equestris cells. The findings of this study elucidated the role of PeFtsH5 in responding to biological stress and provided new insights into its potential molecular mechanism. The result presented in this study hold promise for future research endeavors examining the regulatory effects of mitochondrial proteases on mitochondrial homeostasis and the development of stress-resistant P. equestris varieties through breeding programs.

Orchids acquire fungal carbon for seed germination: pathways and players.

To germinate in nature, orchid seeds strictly rely on seed germination-promoting orchid mycorrhizal fungi (sgOMFs) for provision of carbon nutrients. The underlying delivery pathway, however, remains elusive. We develop here a plausible model for sugar transport from sgOMFs to orchid embryonic cells to fuel germination. Orchids exploit sgOMFs to induce the formation of pelotons, elaborate intracellular hyphal coils in orchid embryos. The colonized orchid cells then obtain carbon nutrients by uptake from living hyphae and peloton lysis, primarily as glucose derived from fungal trehalose hydrolyzed by orchid-specific trehalases. The uptake of massive fungally derived glucose is likely to be mediated by two classes of membrane proteins, namely, sugars will eventually be exported transporters (SWEETs) and H+-hexose symporters. The proposed model serves as a launch pad for further research to better understand and improve orchid seed germination and conservation.

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 multi-omic analysis reveals the carbon metabolism-mediated regulation of polysaccharide biosynthesis by suitable light intensity in Bletilla striata leaves.

Bletilla striata, valued for its medicinal and ornamental properties, remains largely unexplored in terms of how light intensity affects its physiology, biochemistry, and polysaccharide formation. In this 5-month study, B. striata plants were exposed to three different light intensities: low light (LL) (5-20 µmol m-2·s-1), middle light (ML) (200 µmol m-2·s-1), and high light (HL) (400 µmol m-2·s-1). The comprehensive assessment included growth, photosynthetic apparatus, chlorophyll fluorescence electron transport, and analysis of differential metabolites based on the transcriptome and metabolome data. The results indicated that ML resulted in the highest plant height and total polysaccharide content, enhanced photosynthetic apparatus performance and light energy utilization, and stimulated carbon metabolism and carbohydrate accumulation. HL reduced Chl content and photosynthetic apparatus functionality, disrupted OEC activity and electron transfer, stimulated carbon metabolism and starch and glucose accumulation, and hindered energy metabolism related to carbohydrate degradation and oxidation. In contrast, LL facilitated leaf growth and increased chlorophyll content but decreased plant height and total polysaccharide content, compromised the photosynthetic apparatus, hampered light energy utilization, stimulated energy metabolism related to carbohydrate degradation and oxidation, and inhibited carbon metabolism and carbohydrate synthesis. Numerous genes in carbon metabolism were strongly related to polysaccharide metabolites. The katE and cysK genes in carbon metabolism were strongly related not only to polysaccharide metabolites, but also to genes involved in polysaccharide biosynthesis. Our results highlight that light intensity plays a crucial role in affecting polysaccharide biosynthesis in B. striata, with carbon metabolism acting as a mediator under suitable light intensity conditions.

Combined metabolomics and transcriptomics reveal the secondary metabolite networks in different growth stages of Bletilla striata (Thunb.) Reichb.f.

BACKGROUND: Bletilla striata (Thunb.) Reichb.f. (B. striata) is a traditional Chinese medicinal herb. B. striata polysaccharides (BSP), stilbenes and 2-isobutyl malic acid glucosoxy-benzyl ester compounds are the main active ingredients in B. striata. However, there is limited report on the changes of medicinal components and their biosynthesis regulation mechanisms in the tubers of B. striata at different stages. METHOD: The tubers of B. striata were collected during the flowering period, fruiting period, and harvest period to determine the total polysaccharide content using the phenol sulfuric acid method. The changes in secondary metabolites in the tubers at these stages were analyzed by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS), and transcriptomics was conducted for further exploration of their biosynthetic pathways. RESULT: The BSP content gradually increases from the flowering period to the fruiting period as the tubers develop, reaching its peak, but subsequently decreases at harvest time, which may be associated with the germination of B. striata buds in later stage. A total of 294 compounds were identified in this study. Among them, a majority of the compounds, such as 2-isobutyl malate gluconoxy-benzyl ester, exhibited high content during the fruit stage, while stilbenes like coelonin, 3'-O-methylbatatasin III, and blestriarene A accumulated during the harvesting period. The transcriptome data also revealed a substantial number of differentially expressed genes at various stages, providing a partial explanation for the complex changes in metabolites. We observed a correspondence between the expression pattern of GDP-Man biosynthesis-related enzyme genes and cumulative changes in BSP. And identified a positive correlation between 9 transcription factors and genes associated with polysaccharide biosynthesis, while 5 transcription factors were positively correlated with accumulation of 2-isobutyl malate gluconoxy-benzyl ester compounds and 5 transcription factors exhibited negative correlated with stilbene accumulation. CONCLUSION: It is imperative to determine the appropriate harvesting period based on the specific requirements of different active ingredients and the accumulation patterns of their metabolites. Considering the involvement of multiple transcription factors in the biosynthesis and accumulation of its active ingredients, a comprehensive investigation into the specific regulatory mechanisms that facilitate high-quality cultivation of B. striata is imperative.

Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of Cattleya tigrina A. Richard / Determinação de AIA e carboidratos endógenos durante a indução e desenvolvimento deestruturas semelhantes a protocormos de Cattleya tigrina A. Richard

Tissue culture techniques have been employed for orchid mass propagation by means of themorphogenetic route of protocorm like-bodies (PLBs). This study aimed to analyze and compare Indoleaceticacid (IAA) and sugar endogenous levels in protocorm-like bodies (PLBs) induction and developmentin Cattleya tigrina, in order to better understand this process and to optimize micropropagation procedureprotocols. Leaves grown on MS (Murashige and Skoog) culture medium, suplemented with 9 μM TDZfor PLBs induction and development were collected after 0, 2, 7, 14, 30, 60 and 100 days of cultivation, forfurther analysis. Increase of IAA and reduction of sugar levels are strongly related to morphogeneticresponse, that is, PLBs formation over the preexisting ones and leaf primordia formation. Sucrose, fructoseand glucose presence in this study is related to cell signaling. Thus, hormonal signals and carbohydratesalter metabolism, triggering PLBs initiation and development in C. tigrina.

Técnicas de cultura de tecidos têm sido empregadas para a propagação em massa de orquídeaspor meio da via morfogenética de estruturas semelhantes a protocormos (ESPs). O estudo teve comoobjetivo analisar e comparar os níveis endógenos de ácido indol-3-acético (AIA) e de açúcares na indução eno desenvolvimento de estruturas semelhantes a protocormos (ESPs) em Cattleya tigrina, visando à melhorcompreensão deste processo e à otimização de protocolos de micropropagação. Folhas cultivadas em meiode cultura MS (Murashige & Skoog), suplementadas com 9 μM de TDZ para indução e desenvolvimentode ESPs foram coletadas após 0, 2, 7, 14, 30, 60 e 100 dias de cultivo, para posterior análise. O aumento dosníveis de AIA e a redução dos níveis de açúcar estão fortemente relacionados à resposta morfogenética, ouseja, à formação de ESPs sobre os pré-existentes e à formação de primórdios foliares. A presença desacarose, frutose e glicose neste estudo está relacionada à sinalização celular. Assim, sinais hormonais ecarboidratos alteram o metabolismo, desencadeando a iniciação e o desenvolvimento de ESPs em C. tigrina.

Evaluación de diferentes medios de cultivo in vitro en el desarrollo de híbridos de Phalaenopsis (Orchidaceae) / Evaluation of different in vitro culture media in the development of Phalaenopsis hybrid (Orchidaceae)

Los híbridos de Phalaenopsis tienen una gran importancia económica a nivel mundial, como flor cortada y planta ornamental, debido a sus flores vistosas y a la capacidad de adaptación a diferentes condiciones ambientales. Las técnicas de cultivo in vitro resultan indispensables para mejorar la eficacia germinativa, el crecimiento y desarrollo de orquídeas con fines comerciales e investigativos. En esta investigación se determinó el medio de cultivo más apropiado para la germinación in vitro de un híbrido de Phalaenopsis. Inicialmente se evaluó la viabilidad de las semillas utilizando la prueba de tetrazolio (TZ). Las semillas se desinfectaron y se cultivaron aplicando el método de la jeringuilla. El porcentaje de viabilidad en promedio fue de 92,2 % (P≤ 0,05: Tukey HSD), con un porcentaje de germinación entre todos los medios de 95,1 % (P≤ 0,05: Tukey HSD). El medio de cultivo más eficiente para la germinación de híbridos de Phalaenopsis a las 18 semanas de cultivo fue el Murashige & Skoog (MS) suplementado con agua de coco, y jugo de piña con diferencias estadísticamente significativas (P≤ 0,05: Tukey HSD), con respecto a los demás medios de cultivo, contribuyendo de esta manera al uso de componentes orgánicos con el fin de mejorar la germinación y desarrollo de Phalaenopsis.

The Phalaenopsis hybrids have a significant economic importance throughout the world, as ornamental flower or plant. It is because of its attractive flowers and its adaptation capacity into different environments. The different culture media in vitro are vital to improve the efficacy of germination, growing and development of the Orchids for commercial and research purposes. In this research, the most appropriated medium for in vitro propagation of Phalaenopsis hybrid was determined. At first, the seeds viability was evaluated by using tetrazolium test (TZ). The seeds were disinfected and cultivated by means of the syringe method. The viability percentage average was 92.2 % (P≤ 0.05: Tukey HSD), with a percentage of germination of 95.1 % (P≤ 0.05: Tukey HSD) in all the environments. The most efficient culture Medium for Phalaenopsis hybrid phenological development, at 16 weeks, was Murashige & Skoog (MS). Coconut water and pineapple juice were used as supplement showing statistically significant differences (P≤ 0,05: Tukey HSD), in comparison with the other culture media, contributing this way to the usage of organic components, which will be employed to improve the germination and development of the Phalaenopsis.