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Bletilla striata has been used in traditional Chinese medicine for thousands of years to treat a variety of health diseases. Currently, metabolic causes of differences in medicinal values are unknown, due to the lack of a large-scale and comprehensive investigation of metabolites in Bletilla species. In order to gain a better understanding of the major chemical constituents responsible for the medicinal value, this study aimed to explore the metabolomic differences among three Bletilla species (Bletilla striata: Bs, Bletilla ochracea: Bo and Bletilla formosana: Bf). There were 258 different metabolites between 'Bo' and 'Bf', the contents of 109 metabolites had higher abundance, while 149 metabolites showed less accumulation. There were 165 different metabolites between the 'Bs' and 'Bf', content of 72 metabolites was increased and content of 93 metabolites was decreased. There were 239 different metabolites between the 'Bs' and 'Bo', content of 145 metabolites was increased and content of 94 metabolites was decreased. In the Bo_vs_Bf, Bs_vs_Bf and Bs_vs_Bo groups, the major differential categories were flavonoids, phenolic acids, organic acids and alkaloids. Moreover, the differential metabolites were clustered into clear and distinct profiles via K-means analysis. In addition, the major differential categories were flavonoids, phenolic acids, organic acids and alkaloids. The 'Flavonoid biosynthesis' (ko00941) and 'Phenylalanine metabolism' (ko00360) pathways were significantly enriched in Bo_vs_Bf, Bs_vs_Bf and Bs_vs_Bo comparisons. These results clarify the metabolomics in different Bletilla species, as well as providing basis for the phamaceutical value of novel species of Bletilla., (© 2024. The Author(s).)

Structural and physiological leaf traits and their plasticity were compared in the hemiepiphyte Vanilla phaeantha . This species grows along a phorophyte reaching different understorey positions and exhibiting diverse responses to environment changes. We analysed three height strata above the ground, establishing a light gradient, and considering seasonal water fluctuations. The upper leaves had higher area and mass and were less pigmented. The dry season induced a reduction of approximately 2h of stomatal opening over the diel 24h crassulacean acid metabolism (CAM) cycle in the leaves at all understorey positions. The leaves more exposed to sunlight were larger with higher titratable acidity during the rainy season, while the leaves near the ground maintained the same rates of stomatal conductance and nocturnal acidification between seasons, with lowest values of carbon isotopes in the rainy season. Our research showed that some structural leaf traits (such as specific leaf mass, biomass, and saturated water content) are sensitive to variation in understorey position. In contrast, other physiological traits (stomatal conductance, transpiration, and fluorescence parameters) are more sensitive to seasonal variations. The results are a novelty in assessing the variation of CAM along the same plant in a height gradient and under field conditions.

The plant-specific INDETERMINATE DOMAIN ( IDD ) gene family is important for plant growth and development. However, a comprehensive analysis of the IDD family in orchids is limited. Based on the genome data of Phalaenopsis equestris , the IDD gene family was identified and analyzed by bioinformatics methods in this study. Ten putative P. equestris IDD genes ( PeIDD s) were characterized and phylogenetically classified into two groups according to their full amino acid sequences. Protein motifs analysis revealed that overall structures of PeIDDs in the same group were relatively conserved. Its promoter regions harbored a large number of responsive elements, including light responsive, abiotic stress responsive elements, and plant hormone c is -acting elements. The transcript level of PeIDD genes under cold and drought conditions, and by exogenous auxin (NAA) and abscisic acid (ABA) treatments further confirmed that most PeIDD s responded to various conditions and might play essential roles under abiotic stresses and hormone responses. In addition, distinct expression profiles in different tissues/organs suggested that PeIDD s might be involved in various development processes. Furthermore, the prediction of protein-protein interactions (PPIs) revealed some PeIDDs (PeIDD3 or PeIDD5) might function via cooperating with chromatin remodeling factors. The results of this study provided a reference for further understanding the function of PeIDD s., Competing Interests: The authors declare there are no competing interests., (©2024 Zhang et al.)

Aluminum-activated malate transporter ( ALMT ) genes play an important role in aluminum ion (Al 3+ ) tolerance, fruit acidity, and stomatal movement. Although decades of research have been carried out in many plants, there is little knowledge about the roles of ALMT in Orchidaceae. In this study, 34 ALMT genes were identified in the genomes of four orchid species. Specifically, ten ALMT genes were found in Dendrobium chrysotoxum and D. catenatum , and seven were found in Apostasia shenzhenica and Phalaenopsis equestris . These ALMT genes were further categorized into four clades (clades 1-4) based on phylogenetic relationships. Sequence alignment and conserved motif analysis revealed that most orchid ALMT proteins contain conserved regions (TM1, GABA binding motif, and WEP motif). We also discovered a unique motif (19) belonging to clade 1, which can serve as a specifically identified characteristic. Comparison with the gene structure of AtALMT genes ( Arabidopsis thaliana ) showed that the gene structure of ALMT was conserved across species, but the introns were longer in orchids. The promoters of orchid ALMT genes contain many light-responsive and hormone-responsive elements, suggesting that their expression may be regulated by light and phytohormones. Chromosomal localization and collinear analysis of D. chrysotoxum indicated that tandem duplication (TD) is the main reason for the difference in the number of ALMT genes in these orchids. D. catenatum was chosen for the RT-qPCR experiment, and the results showed that the DcaALMT gene expression pattern varied in different tissues. The expression of DcaALMT1-9 was significantly changed after ABA treatment. Combining the circadian CO 2 uptake rate, titratable total acid, and RT-qPCR data analysis, most DcaALMT genes were highly expressed at night and around dawn. The result revealed that DcaALMT genes might be involved in photosynthate accumulation. The above study provides more comprehensive information for the ALMT gene family in Orchidaceae and a basis for subsequent functional analysis.

The standout characteristic of the orchid perianth is the transformation of the upper median petal into a distinctively formed lip, which gives orchid flowers their typically zygomorphic symmetry and makes them the most popular ornamental plants worldwide. To study orchid flower development, two WUSCHEL-related homeobox (WOX) genes, PaWOX3 and PaWOX3B, were identified in Phalaenopsis. PaWOX3 and PaWOX3B mRNAs accumulate abundantly during early reproductive development and perianths of young buds, significantly decreasing in mature flowers and absent in vegetative leaves and roots. PaWOX3 and PaWOX3B virus-induced gene silencing (VIGS) knockdown in Phalaenopsis significantly reduces floral bud numbers, suggesting that PaWOX3/PaWOX3B may be involved in flower initiation. Transgenic Arabidopsis ectopically expressing repressor forms of PaWOX3/PaWOX3B and their Oncidium ortholog, OnPRS, exhibit lateral organ development defects, implicating these genes likely have function in regulating growth and differentiation for lateral organs. Neither PaWOX3, PaWOX3B single nor PaWOX3/PaWOX3B double VIGS Phalaenopsis altered the flower morphology. Interestingly, double silencing of PaWOX3 or PaWOX3B with OAGL6-2, which controlled the identity/formation of lips, altered the symmetry of 'BigLip' produced in OAGL6-2 VIGS. This result indicated that the levels of PaWOX3/PaWOX3B are still sufficient to maintain the symmetry for the OAGL6-2 VIGS 'BigLip'. However, the symmetry of the OAGL6-2 VIGS 'BigLip' cannot be maintained once the expression of PaWOX3 or PaWOX3B is further reduced. Thus, in addition to controlling lip identity, this study further found that OAGL6-2 could cooperate with functionally redundant PaWOX3/PaWOX3B in maintaining the symmetric axis of lip., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Both Dendrobium nobile and D. officinale are widely used medicinal plants in China and their major medicinal components are alkaloids and polysaccharides, respectively. It is still unclear why these two closely related orchids synthesize and accumulate different chemical components. Here, we investigated the molecular mechanisms underlying polysaccharide and alkaloid biosynthesis in D. nobile and D. officinale through transcriptome and metabolomic analysis at different growth stages. A total of 1267 metabolites were identified in the juvenile and mature stages of the two species. D. nobile accumulated a large number of alkaloids, benzenoids/phenylpropanoids, flavonoids, and terpenoids during the transition from juvenile to mature plants. In contrast, D. officinale accumulated a small number of those metabolites and an absence of flavonoids. The correlation analysis of polysaccharide contents with the differentially expressed genes suggested that the differential expression of GH1, GH3, and GH9 might be related to the difference in polysaccharide contents between the two Dendrobium species. Meanwhile, the difference in the biosynthesis of dendrobine, the main component of alkaloids in D. nobile, was involved in the differential expression of HMGCR, DXR, DXS, ISPH and eight CYP450s. These findings provided new insights into understanding the biosynthetic mechanisms of the main medicinal components in Dendrobium species., (© 2024 Scandinavian Plant Physiology Society. Published by John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Polysaccharides and various secondary metabolites are the major bioactive ingredients in Bletilla striata tubers and their biosynthesis and accumulation are influenced by light intensity. However, the mechanisms underlying shading effects remain largely unknown. In the present study, we used a combined analysis of the physiology, metabolome, and transcriptome to investigate the physiological activities and bioactive component accumulation of B. striata under different shading treatments (S0, S50, S70, and S90). The dry weight of shoots and tubers, net photosynthetic rate, and polysaccharide content were highest in S50 and lowest in S90. The content of precursors (sucrose, Glucose-6P, and Mannose-6P) for polysaccharide synthesis significantly increased in S50. However, the expression levels of genes involved in starch biosynthesis decreased in S50. Several structural genes involved in secondary metabolism, including cinnamic acid 4-hydroxylase (C4H), chalcone synthase (CHS), and 1-Deoxy-D-xylulose-5-phosphate synthase (DXS), showed decreased expression in S50. However, the shading effect on the biosynthesis of secondary metabolites (phenylpropanoids, flavonoids, and terpenoids) was inconsistent. Our study provides the molecular mechanisms underlying the effects of shading on the biosynthesis of polysaccharides and secondary metabolites in B. striata and offers a theoretical basis for the artificial cultivation and industrial production of bioactive ingredients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare no conflict interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To explore the regulatory mechanism of endogenous hormones in the synthesis of anthocyanins in Anoectochilus roxburghii (Wall.) Lindl ( A. roxburghii ) under different light intensities, this study used metabolomics and transcriptomics techniques to identify the key genes and transcription factors involved in anthocyanin biosynthesis. We also analyzed the changes in and correlations between plant endogenous hormones and anthocyanin metabolites under different light intensities. The results indicate that light intensity significantly affects the levels of anthocyanin glycosides and endogenous hormones in leaves. A total of 38 anthocyanin-related differential metabolites were identified. Under 75% light transmittance (T3 treatment), the leaves exhibited the highest anthocyanin content and differentially expressed genes such as chalcone synthase ( CHS ), flavonol synthase ( FLS ), and flavonoid 3'-monooxygenase ( F3 ' H ) exhibited the highest expression levels. Additionally, 13 transcription factors were found to have regulatory relationships with 7 enzyme genes, with 11 possessing cis-elements responsive to plant hormones. The expression of six genes and two transcription factors was validated using qRT-PCR, with the results agreeing with those obtained using RNA sequencing. This study revealed that by modulating endogenous hormones and transcription factors, light intensity plays a pivotal role in regulating anthocyanin glycoside synthesis in A. roxburghii leaves. These findings provide insights into the molecular mechanisms underlying light-induced changes in leaf coloration and contribute to our knowledge of plant secondary metabolite regulation caused by environmental factors.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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), H 2 O 2 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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (© 2024. The Author(s).)

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 Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Anoectochilus roxburghii (Wall.) Lindl is a perennial herb of the Orchidaceae family; a yellow-green mutant and a yellow mutant were obtained from the wild type, thereby providing good material for the study of leaf color variation. Pigment content analysis revealed that chlorophyll, carotenoids, and anthocyanin were lower in the yellow-green and yellow mutants than in the wild type. Transcriptome analysis of the yellow mutant and wild type revealed that 78,712 unigenes were obtained, and 599 differentially expressed genes (120 upregulated and 479 downregulated) were identified. Using the Kyoto Encyclopedia of Genes and Genomes pathway analysis, candidate genes involved in the anthocyanin biosynthetic pathway (five unigenes) and the chlorophyll metabolic pathway (two unigenes) were identified. Meanwhile, the low expression of the chlorophyll and anthocyanin biosynthetic genes resulted in the absence of chlorophylls and anthocyanins in the yellow mutant. This study provides a basis for similar research in other closely related species., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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, 60 Co-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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 VpPEPC s 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.

Mitochondrial homeostasis plays a crucial role in determining cell fate by direct influence on cell apoptosis and autophagy. The ATP and Zn 2+ -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Orchidaceae is one of the largest monocotyledon families and contributes significantly to worldwide biodiversity, with value in the fields of landscaping, medicine, and ecology. The diverse phenotypes and vibrant colors of orchid floral organs make them excellent research objects for investigating flower development and pigmentation. In recent years, a number of orchid genomes have been published, laying the molecular foundation for revealing flower development and color presentation. In this article, we review transcription factors, the structural genes responsible for the floral pigment synthesis pathways, the molecular mechanisms of flower morphogenesis, and the potential relationship between flower type and flower color. This study provides a theoretical reference for the research on molecular mechanisms related to flower morphogenesis and color presentation, genetic improvement, and new variety creation in orchids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO 2 assimilation rates up to 1.6 and 1.5-folds samples at 4 pm and 10 pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD + -linked malic enzyme (NAD + -ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Background: The orchid industry has seen a recent surge in export values due to the floral morphology and versatile applications of orchids in various markets for medicinal, food additive, and cosmetic usages. However, plant-related diseases, including the yellow leaf disease caused by Fusarium solani, have caused significant losses in the production value of Phalaenopsis (up to 30%)., Results: In this study, 203 Phalaenopsis cultivars were collected from 10 local orchid nurseries, and their disease severity index and correlation with flower size were evaluated. Larger flowers had weaker resistance to yellow leaf disease, and smaller flowers had stronger resistance. For the genetic relationship of disease resistance to flower size, the genetic background of all cultivars was assessed using OrchidWiz Orchid Database Software and principal component analysis. In addition, we identified the orthologous genes of BraTCP4, namely PeIN6, PeCIN7, and PeCIN8, which are involved in resistance to pathogens, and analyzed their gene expression. The expression of PeCIN8 was significantly higher in the most resistant cultivars (A7403, A11294, and A2945) relative to the most susceptible cultivars (A10670, A6390, and A10746)., Conclusions: We identified a correlation between flower size and resistance to yellow leaf disease in Phalaenopsis orchids. The expression of PeCIN8 may regulate the two traits in the disease-resistant cultivars. These findings can be applied to Phalaenopsis breeding programs to develop resistant cultivars against yellow leaf disease., (© 2023. The Author(s).)

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Epiphytes with crassulacean acid metabolism (CAM) photosynthesis are widespread among vascular plants, and repeated evolution of CAM photosynthesis is a key innovation for micro-ecosystem adaptation. However, we lack a complete understanding of the molecular regulation of CAM photosynthesis in epiphytes. Here, we report a high-quality chromosome-level genome assembly of a CAM epiphyte, Cymbidium mannii (Orchidaceae). The 2.88-Gb orchid genome with a contig N50 of 22.7 Mb and 27 192 annotated genes was organized into 20 pseudochromosomes, 82.8% of which consisted of repetitive elements. Recent expansions of long terminal repeat retrotransposon families have made a major contribution to the evolution of genome size in Cymbidium orchids. We reveal a holistic scenario of molecular regulation of metabolic physiology using high-resolution transcriptomics, proteomics, and metabolomics data collected across a CAM diel cycle. Patterns of rhythmically oscillating metabolites, especially CAM-related products, reveal circadian rhythmicity in metabolite accumulation in epiphytes. Genome-wide analysis of transcript and protein level regulation revealed phase shifts during the multifaceted regulation of circadian metabolism. Notably, we observed diurnal expression of several core CAM genes (especially βCA and PPC) that may be involved in temporal fixation of carbon sources. Our study provides a valuable resource for investigating post-transcription and translation scenarios in C. mannii, an Orchidaceae model for understanding the evolution of innovative traits in epiphytes., (Copyright © 2023. Published by Elsevier Inc.)

Aerides Lour. (Orchidaceae, Aeridinae) is a group of epiphytic orchids with high ornamental value, mainly distributed in tropical and subtropical forests, that comprises approximately 20 species. The species are of great value in floriculture and garden designing because of their beautiful flower shapes and colors. Although the morphological boundaries of Aerides are clearly defined, the relationship between Aerides and other closely related genera is still ambiguous in terms of phylogeny. To better understand their phylogenetic relationships, this study used next-generation sequencing technology to investigate the phylogeny and DNA barcoding of this taxonomic unit using genetic information from six Aerides plastid genomes. The quadripartite-structure plastomes ranged from 147,244 bp to 148,391 bp and included 120 genes. Among them, 74 were protein coding genes, 38 were tRNA genes and 8 were rRNA genes, while the ndh genes were pseudogenized or lost. Four non-coding mutational hotspots ( rpl20-rpl33, psbM, petB, rpoB-trnC GCA , Pi > 0.06) were identified. A total of 71-77 SSRs and 19-46 long repeats (>30 bp) were recognized in Aerides plastomes, which were mostly located in the large single-copy region. Phylogenetic analysis indicated that Aerides was monophylic and sister to Renanthera . Moreover, our results confirmed that six Aerides species can be divided into three major clades. These findings provide assistance for species identification and DNA barcoding investigation in Aerides, as well as contributes to future research on the phylogenomics of Orchidaceae.

SPL transcription factors regulate important processes such as plant growth and development, metabolic regulation, and abiotic stress. They play crucial roles in the development of flower organs. However, little is known about the characteristics and functions of the SPL s in the Orchidaceae. In this study, Cymbidium goeringii Rchb. f., Dendrobium chrysotoxum Lindl., and Gastrodia elata BI. were used as research objects. The SPL gene family of these orchids was analyzed on a genome-wide scale, and their physicochemical properties, phylogenetic relationships, gene structures, and expression patterns were studied. Transcriptome and qRT-PCR methods were combined to investigate the regulatory effect of SPL s on the development of flower organs during the flowering process (bud, initial bloom, and full bloom). This study identifies a total of 43 SPL s from C. goeringii (16), D. chrysotoxum (17), and G. elata (10) and divides them into eight subfamilies according to the phylogenetic tree. Most SPL proteins contained conserved SBP domains and complex gene structures; half of the genes had introns longer than 10 kb. The largest number and variety of cis-acting elements associated with light reactions were enriched, accounting for about 45% of the total (444/985); 13/43 SPL s contain response elements of miRNA156 . GO enrichment analysis showed that the functions of most SPL s were mainly enriched in the development of plant flower organs and stems. In addition, expression patterns and qRT-PCR analysis suggested the involvement of SPL genes in the regulation of flower organ development in orchids. There was little change in the expression of the CgoSPL in C. goeringii , but DchSPL9 and GelSPL2 showed significant expression during the flowering process of D. chrysotoxum and G. elata , respectively. In summary, this paper provides a reference for exploring the regulation of the SPL gene family in orchids.

The pollen grains of Phalaenopsis orchids are clumped tightly together, packed in pollen dispersal units called pollinia. In this study, the morphology, cytology, biochemistry, and sucrose transporters in pollinia of Phalaenopsis orchids were investigated. Histochemical detection was used to characterize the distribution of sugars and callose at the different development stages of pollinia. Ultra-performance liquid chromatography-high resolution-tandem mass spectrometry data indicated that P. aphrodite accumulated abundant saccharides such as sucrose, galactinol, myo-inositol, and glucose, and trace amounts of raffinose and trehalose in mature pollinia. We found that galactinol synthase (PAXXG304680) and trehalose-6-phosphate phosphatase (PAXXG016120) genes were preferentially expressed in mature pollinia. The P. aphrodite genome was identified as having 11 sucrose transporters (SUTs). Our qRT-PCR confirmed that two SUTs (PAXXG030250 and PAXXG195390) were preferentially expressed in the pollinia. Pollinia germinated in pollen germination media (PGM) supplemented with 10% sucrose showed increased callose production and enhanced pollinia germination, but there was no callose or germination in PGM without sucrose. We show that P. aphrodite accumulates high levels of sugars in mature pollinia, providing nutrients and enhanced SUT gene expression for pollinia germination and tube growth., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The pseudobulb is a storage organ for water and nutrients that plays a crucial role in the growth and survival of epiphytic orchids. However, the role of water and metabolites in pseudobulb during adaptation to environmental stress are rarely detected through control experiments. In the present study, water-related physiological traits and metabolite changes in the pseudobulbs at the flowering stage and full leaf expansion stage for Pleione aurita were investigated after drought stress and recovery treatments. We found that the composition of non-structural carbohydrates (starch vs. soluble sugar) varied over the lifetime of pseudobulbs, and older pseudobulbs stored more water, whereas younger pseudobulbs stored more dry matter. When plants were subjected to drought stress and subsequent recovery, multiple metabolites in the pseudobulbs including non-structural carbohydrates, flavonoids, phenolic acids, as well as amino acids and their derivatives responded positively to these water level fluctuations. For those metabolites that differently accumulated in both stress and recovery processes, old pseudobulbs contained a higher number of these key metabolites than did the connected younger pseudobulbs. In addition, young and old pseudobulbs use different metabolic pathways to both respond and recover to drought. These results indicate that orchid pseudobulbs cope with water level fluctuations by mobilizing metabolite reserves and that pseudobulbs of different ages exhibit different physiological and metabolic responses to drought stress. These findings broadens our understanding of the role pseudobulbs play in the survival of orchids growing in epiphytic habitats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Phalaenopsis equestris is an ornamental plant with very large leaves. In this study, we identified genes related to the regulation of leaf development in Phalaenopsis and explored their mechanism of action. Sequence alignment and phylogenetic analyses revealed that PeGRF6 in the PeGRF family of P. equestris has similarities with the Arabidopsis genes AtGRF1 and AtGRF2, which are known to be involved in the regulation of leaf development. Among the PeGRFs, PeGRF6 was continuously and stably expressed at various stages of leaf development. The functions of PeGRF6 and of its complex formed with PeGIF1 in leaf development were verified by virus-induced gene silencing (VIGS) technology. The results show that the PeGRF6-PeGIF1 complex forms in the nucleus and positively regulates leaf cell proliferation via influencing cell size. Interestingly, VIGS suppression of PeGRF6 resulted in anthocyanin accumulation in Phalaenopsis leaves. Analyses of the regulatory mechanism of the miR396-PeGRF6 model based on the P. equestris small RNA library constructed here suggested that PeGRF6 transcripts are cleaved by Peq-miR396. These results show that, compared with PeGRF6 or PeGIF1 alone, the PeGRF6-PeGIF1 complex plays a more important role in the leaf development of Phalaenopsis, possibly by regulating the expression of cell cycle-related genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Squalene epoxidase catalyzes the oxidation of squalene to 2,3-oxo-squalene (BsSE1), and is the key rate limiting enzyme in the synthesis of triterpenoids and sterols in plants. This study focused on the basic aspects of BsSE1 including the sequence information, sub-cellular localization expression patterns of BsSE1. Using to the sequence information of Bletilla striata transcriptome, the full-length CDS of BsSE1 gene was amplified. The physicochemical properties and structural characteristics of BsSE1 protein were analyzed by bioinformatics analysis software, and vector was constructed to analyze the protein locations and expression patterns. The results showed that the CDS of BsSE1 gene was 1542 bp, encoding 513 amino acids. BsSE1 protein is a hydrophobic protein with two transmembrane domains but no signal peptides. It is localied in the endoplasmic reticulum membrane and belongs to the typical squalene epoxidase gene. BsSE1 has the closest genetic relationship with SE protein of Dendrobium officinale and Phalaenopsis equestris. The expression level of BsSE1 was higher in pseudobulblet of Bletilla striata seedlings, followed by roots, and lower in seedling stems. After SA induction, the expression of BsSE1 in Bletilla striata showed significant changes, increased first, then decreased, finally increase again. The results provide a basis for further study of this gene family in plants., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The purpose of our study was to determine the role of flower structure and nectar composition in shaping the reproductive success (RS) of the generalist orchid Epipactis helleborine in natural and anthropogenic populations. We supposed that the distinct character of two groups of habitats creates different conditions for plant-pollinator relationships, thus influencing reproductive success in E. helleborine populations. Both pollinaria removal (PR) and fruiting (FRS) were differentiated between the populations. On average, FRS was almost two times higher in the anthropogenic than in the natural populations. The difference between the two population groups in PR was smaller but still statistically significant. RS parameters were correlated with some floral display and flower traits. Floral display influenced RS only in three anthropogenic populations. Flower traits had a weak influence on RS (10 of the 192 cases analyzed). The more important trait in shaping RS was nectar chemistry. The nectar of E. helleborine is relatively diluted with a lower sugar concentration in the anthropogenic than in the natural populations. In the natural populations, domination of sucrose over hexoses was found, while in the anthropogenic populations, hexoses were more abundant and the participation of sugars was balanced. In some populations, sugars influenced RS. In E. helleborine nectar, 20 proteogenic and 7 non-proteogenic amino acids (AAs) were found with a clear domination of glutamic acid. We noted relationships between some AAs and RS, but distinct AAs shaped RS in different populations, and their impact was independent of their participation. Our results indicate that the flower structure and nectar composition of E. helleborine reflect its generalistic character and meet the requirements of a wide range of pollinators. Simultaneously, the differentiation of flower traits suggests a variation in pollinator assemblages in particular populations. Knowledge about the factors influencing RS in distinct habitats helps to understand the evolutionary potential of species and to understand mechanisms and processes crucial for shaping interactions between plants and pollinators.

Cymbidium sinense represents a distinctive Orchidaceae plant that is more tolerant than other terrestrial orchids. Studies have shown that many members of the MYB transcription factor (TF) family, especially the R2R3-MYB subfamily, are responsive to drought stress. This study identified 103 CsMYBs ; phylogenetic analysis classified these genes into 22 subgroups with Arabidopsis thaliana . Structural analysis showed that most CsMYB genes contained the same motifs, three exons and two introns, and showed a helix-turn-helix 3D structure in each R repeat. However, the members of subgroup 22 contained only one exon and no intron. Collinear analysis revealed that C. sinense had more orthologous R2R3-MYB genes with wheat than A. thaliana and rice. Ka/Ks ratios indicated that most CsMYB genes were under purifying negative selection pressure. Cis -acting elements analysis revealed that drought-related elements were mainly focused on subgroups 4, 8, 18, 20, 21, and 22, and Mol015419 (S20) contained the most. The transcriptome analysis results showed that expression patterns of most CsMYB genes were upregulated in leaves in response to slight drought stress and downregulated in roots. Among them, members in S8 and S20 significantly responded to drought stress in C. sinense . In addition, S14 and S17 also participated in these responses, and nine genes were selected for the real-time reverse transcription quantitative PCR (RT-qPCR) experiment. The results were roughly consistent with the transcriptome. Our results, thus, provide an important contribution to understanding the role of CsMYBs in stress-related metabolic processes.

Anoectochilus roxburghii is a rare and precious plant with medicinal and healthcare functions. Embryo abortion caused the lack of resources. Polyamine promoted its flowering and stress resistance in our previous study. But the mechanism remains unclear. The WRKY transcription factor family has been linked to a variety of biological processes in plants. In this study, two WRKY TFs (ArWRKY5 and ArWRKY20) of A. roxburghii, which showed significant response to Spd treatment, were identified and functionally analyzed. Tissue specific expression analyzation showed both of them mostly present in the flower. And ArWRKY5 expressed highest in the flower bud stage (-1 Flowering), while ArWRKY20 showed the highest expression in earlier flower bud stage (-2 Flowering) and the expression gradually decreased with flowering. The transcriptional activation activity assay and subcellular localization revealed that ArWRKY5 and ArWRKY20 were located in the nucleus and ArWRKY20 showed transcriptional activity. The heterologous expression of ArWRKY5 in Arabidopsis thaliana showed earlier flowering, while overexpression of ArWRKY20 delayed flowering. But the OE-ArWRKY20 lines had a robust body shape and a very significant increase in the number of rosette leaves. Furthermore, stamens and seed development were positively regulated by these two ArWRKYs. These results indicated that ArWRKY5 and ArWRKY20 not only play opposite roles in the floral development, but also regulate the plant growth and seed development in A. thaliana. But their specific biological functions and mechanism in A. roxburghii need to be investigated further., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Bletilla striata (Thunb.) Reichb.f., is a traditional Chinese medicine, and the Bletilla striata polysaccharide (BSP) is one of the principal components extracted from Bletilla striata with various biological activities. Previous studies have shown that many natural polysaccharides have significant immunomodulatory activities. However, as a plant polysaccharide, the research of BSP on immunomodulatory activities is limited. In this study, we aim to investigate the immunomodulatory effect of BSP in vivo and further explore its underlying mechanism in vitro . In vivo , a cyclophosphamide (CTX)-induced immunosuppression mice mode was established by intraperitoneal injection of CTX, and the immune-enhancing effect of BSP (25, 50 and 100 mg/kg) on immunosuppressed mice were evaluated. The result indicated that BSP could significantly improve the immune organ index and the content of immunoglobulin, TNF-α and IL-4 in serum. It was also found that BSP could clearly ameliorate the spleen damage induced by CTX. Meanwhile, the result showed that BSP could not only improve the proliferation of splenocytes, but also activate the lactate dehydrogenase (LDH) and acid phosphatase (ACP) in mouse spleen tissue. In vitro , potential mechanism was further revealed in macrophages. The result supported that BSP could activate macrophages with high phagocytic ability, and induce macrophages to secrete cytokines. Finally, it revealed that activation of NF-κB and MAPK signalling pathway should be the underlying mechanism of the immunoenhancment of BSP.

Phalaenopsis spp., one genus of Orchidaceae, have become very popular worldwide for their fascinating flowers with various colors and pigmentation patterns. Several R2R3-MYB transcription factors have been reported to function in anthocyanin accumulation in Phalaenopsis spp. However, its molecular mechanism underlying the detailed regulatory pathway remains poorly understood. In this study, we identified a novel subgroup 2 R2R3-MYB transcription factor PeMYB4L, the expression profile of which was concomitant with red color formation in Phalaenopsis spp. flowers. Virus-induced gene silencing (VIGS) and transient overexpression assay verified that PeMYB4L promotes anthocyanin accumulation in flower tissues. In addition, PeMYB4L could directly regulates the expression of Phalaenopsis spp. chalcone synthase gene (PeCHS) through MYBST1 (GGATA) binding site. It's interesting that the basic-helix-loop-helix (bHLH) protein PeMYC4 shows opposite expression pattern from PeMYB4L in anthocyanin accumulation. Furthermore, PeMYC4 was verified to form MYB-bHLH complex with PeMYB4L, and attenuated the expression of PeCHS and weakened anthocyanin production, indicating a novel regulatory model of MYB-bHLH complex. Our findings uncover the detailed regulatory pathway of MYB-bHLH, and might provide a new insight into the complicated anthocyanin pigmentation in Phalaenopsis spp., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: To adapt the periodic fluctuation of environmental factors, plants are subtle to monitor the natural variation for the growth and development. The daily activities and physiological functions in coordination with the natural variation are regulated by circadian clock genes. The circadian emission of floral scents is one of the rhythmic physiological activities controlled by circadian clock genes. Here, we study the molecular mechanism of circadian emission pattern of ocimene and linalool compounds in Oncidium Sharry Baby (Onc. SB) orchid., Results: GC-Mass analysis revealed that Onc. SB periodically emitted ocimene and linalool during 6 to 14 o'clock daily. Terpene synthase, one of the key gene in the terpenoid biosynthetic pathway is expressed in coordination with scent emission. The promoter structure of terpene synthase revealed a circadian binding sequence (CBS), 5'-AGATTTTT-3' for CIRCADIAN CLOCK ASSOCIATED1 (CCA1) transcription factor. EMSA data confirms the binding affinity of CCA1. Transactivation assay further verified that TPS expression is regulated by CCA1. It suggests that the emission of floral scents is controlled by CCA1., Conclusions: The work validates that the mechanism of circadian emission of floral scents in Onc. Sharry Baby is controlled by the oscillator gene, CCA1(CIRCADIAN CLOCK ASSOCIATED 1) under light condition. CCA1 transcription factor up-regulates terpene synthase (TPS) by binding on CBS motif, 5'-AGATTTTT-3' of promoter region to affect the circadian emission of floral scents in Onc. SB., (© 2022. The Author(s).)

For all organs at all Cymbidium faberi stages, ACT , UBQ3 and GAPDH can be selected as reference genes. For organs of the vegetative stage, UBQ2 and UBQ3 can be chosen for analysis of normalized gene expression. For the bud stage, ACT and UBQ3 can be used for analysis of gene expression. For the full blossom stage, ACT , UBQ3 and UBQ2 can be introduced into relative gene expression analysis. For vegetative organs, UBQ2 and ACT can be used as reference genes. For reproductive organs, ACT , UBQ3 and UBQ2 can be used as a reference for data processing. CfAG1 gene expression is more consistent when UBQ3 , GAPDH and ACT are used as reference genes.

Phalaenopsis orchids are popular worldwide due to their high ornamental and economic value; the spike and inflorescence formation of their flowers could be efficiently controlled under proper conditions. In this study, transcriptomic profiles and endogenous hormone changes were investigated to better understand the spike formation of Phalaenopsis . Morphological observations revealed four spike initiation statuses (i.e., S0: the status refers to axillary buds remaining dormant in the leaf axils; S1: the status refers to the 0.5 cm-long initial spike; S2: the status refers to the 1 cm-long spike; S3: the status refers to the 3 cm-long spike) during the process of spike development, while anatomical observations revealed four related statuses of inflorescence primordium differentiation. A total of 4080 differentially expressed genes were identified based on pairwise comparisons of the transcriptomic data obtained from the S0 to S3 samples; high levels of differential gene expression were mostly observed in S1 vs. S2, followed by S0 vs. S1. Then, the contents of 12 endogenous hormones (e.g., irindole-3-acetic acid (IAA), salicylic acid (SA), abscisic acid (ABA), gibberellins, and cytokinins) were measured. The results showed that the ABA content was decreased from S0 to S1, while the gibberellic acid 1 (GA1) content exhibited an opposite trend, indicating the reduction in ABA levels combined with the increase in GA1 levels in S0 promoted the axillary bud dormancy breaking, preparing for the following spike initiation. The GA20 oxidase and ABA 8'-hydroxylase genes, which are involved in endogenous hormone metabolism and signaling pathways, displayed similar expression patterns, suggesting they were probably the key genes participating in the GA and ABA regulation. Taken together, the findings of this study indicate that GA and ABA may be the key endogenous hormones breaking the dormancy and promoting the germination of axillary buds in Phalaenopsis ., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; nor in the decision to publish the results.

SWEETs (sugars will eventually be exported transporters), a well-known class of sugar transporters, are involved in plant growth and development, sugar transport, biotic and abiotic stresses, etc. However, to date, there have been few investigations of SWEETs in Orchidaceae. In this study, 23 SWEET genes were identified in Bletilla striata for the first time, with an MtN3/saliva conserved domain, and were divided into four subgroups by phylogenetic tree. The same subfamily members had similar gene structures and motifs. Multiple cis -elements related to sugar and environmental stresses were found in the promoter region. Further, 21 genes were localized on 11 chromosomes and 2 paralogous pairs were found via intraspecific collinearity analysis. Expression profiling results showed that BsSWEETs were tissue-specific. It also revealed that BsSWEET10 and BsSWEET18 were responsive to low temperature and oxidative stresses. In addition, subcellular localization study indicated that BsSWEET15 and BsSWEET16 were localized in the cell membrane. This study provided important clues for the in-depth elucidation of the sugar transport mechanism of BsSWEET genes and their functional roles in response to abiotic stresses.

The protocorm is a structure that is formed upon germination of an orchid seed. It lacks cotyledons and is ovoid in shape. The protocorm-like body (PLB), on the other hand, is a protocorm-like organ induced from somatic tissues. PLBs have been widely used for orchid micropropagation. Because of its unique structure and its application in the orchid industry, PLB development has drawn considerable interest from orchid and developmental biologists. Our previous genome-wide comparative transcriptome study demonstrated that protocorms and PLBs share similar molecular signatures and suggested that SHOOT MERISTEMLESS (STM)-dependent organogenesis is important for PLB development. Here, we show that overexpression of Phalaenopsis aphrodite STM (PaSTM) greatly enhances PLB regeneration from vegetative tissue-based explants of Phalaenopsis orchids, confirming its regulatory role in PLB development. Expression of PaSTM restored shoot meristem function of the Arabidopsis (Arabidopsis thaliana) stm-2 mutant. Moreover, we identified class S11 MYB transcription factors (TFs) as targets downstream of PaSTM. A cis-acting element, TTGACT, identified in the promoters of S11 MYB TFs was found to be important for PaSTM binding and activation. Overexpression of PaSTM or its downstream targets, PaMYB13, PaMYB14, and PaMYB17, enhanced de novo shoot regeneration in Arabidopsis, indicating the active role of the PaSTM-S11 PaMYB module in organogenesis. In summary, our data demonstrate that PaSTM is important for PLB development. The STM-S11 MYB regulatory module is evolutionarily conserved and may regulate shoot or shoot-related organ development in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

As part of our continuous studies on natural cholinesterase inhibitors from plant kingdom, the 95% ethanol extract from tubers of Bletilla striata showed promising butyrylcholinesterase (BChE) inhibition (IC 50  = 8.6 μg/mL). The extracts with different polarities (petroleum ether, ethyl acetate, n-butanol, and water) were prepared and evaluated for their inhibition of cholinesterases. The most active ethyl acetate extract was subjected to a bioassay-guided isolation and afforded twenty-two bibenzyls and phenanthrenes (1-22). All isolates were further evaluated for their BChE inhibition activity, and five phenanthrenes presented promising capacity (IC 50  < 10 μM). Further kinetic studies indicated their modes of inhibition. Compounds 6, 8, and 14 were found to be mixed-type inhibitors, while compounds 10 and 12 could be classified as non-competitive inhibitors. The potential interaction mechanism of them with BChE was demonstrated by molecular docking and molecular dynamics simulation, showing that they could interact with catalytic active site and peripheral anionic site of BChE. These natural phenanthrenes provide new scaffold for the further design and optimization, with the aim to discover new selective BChE inhibitors for the treatment of AD., (© 2022. The Author(s).)

Background: Anoectochilus roxburghii and Anoectochilus formosanus , belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites., Methods: The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis . The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR)., Results: AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii . The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis ., Discussion: The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus . Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii , which can improve their medicinal active ingredients and be used for drug discovery in plants., Competing Interests: The authors declare there are no competing interests., (©2022 Yang et al.)

As one of the important species belonging to the Bletilla genus of Orchidaceae, Bletilla striata (Thunb.) Rchb. f., possesses both ornamental and medicinal values. Its dried tubers are used as a traditional Chinese medicine, and several secondary metabolites have been indicated to be the active ingredients. However, the molecular mechanisms related to the regulation of secondary metabolism have not been characterized in B. striata. In this study, integrated analysis of RNA sequencing (RNA-seq), small RNA sequencing (sRNA-seq), and degradome sequencing (degradome-seq) data from three organs (leaf, root, and tuber) of B. striata provided us with a comprehensive view of the microRNA (miRNA)-mediated regulatory network. Firstly, based on the RNA-seq data, the organ-specific expression patterns of the protein-coding genes, especially for those related to secondary metabolism, were investigated. Secondly, 342 conserved miRNA candidates were identified from B. striata. These miRNAs were assigned to 88 families, some of which were selected for expression pattern analysis. Additionally, 31 hairpin-structured precursors encoding 23 novel miRNAs were uncovered from the transcriptome assembly. Thirdly, based on the degradome signatures, 1,142 validated miRNA-target pairs (involving 167 conserved miRNAs and six novel miRNAs and 51 target genes) were included in the regulatory network. Organ-specific expression level comparison between the miRNAs and their targets revealed some interesting miRNA-target pairs. Fourthly, some valuable subnetworks were extracted for further functional studies. Additionally, some regulatory pathways were indicated to be monocot specific. Summarily, our results lay a solid basis for in-depth studies on the regulatory mechanisms underlying the production of the medicinal ingredients in B. striata., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy., (© 2022. The Author(s).)

Shoot multiplication induced by exogenous cytokinins (CKs) has been commonly used in Phalaenopsis micropropagation for commercial production. Despite this, mechanisms of CKs action on shoot multiplication remain unclear in Phalaenopsis . In this study, we first identified key CKs metabolic genes, including six isopentenyltransferase ( PaIPTs ), six cytokinin riboside 5' monophosphate phosphoribohydrolase ( PaLOGs ), and six cytokinin dehydrogenase ( PaCKXs ), from the Phalaenopsis genome. Then, we investigated expression profiles of these CKs metabolic genes and endogenous CKs dynamics in shoot proliferation by thidiazuron (TDZ) treatments (an artificial plant growth regulator with strong cytokinin-like activity). Our data showed that these CKs metabolic genes have organ-specific expression patterns. The shoot proliferation in vitro was effectively promoted with increased TDZ concentrations. Following TDZ treatments, the highly expressed CKs metabolic genes in micropropagated shoots were PaIPT1 , PaLOG2, and PaCKX4 . By 30 days of culture, TDZ treatments significantly induced CK-ribosides levels in micropropagated shoots, such as t ZR and iPR (2000-fold and 200-fold, respectively) as compared to the controls, whereas c ZR showed only a 10-fold increase. Overexpression of PaIPT1 and PaLOG2 by agroinfiltration assays resulted in increased CK-ribosides levels in tobacco leaves, while overexpression of PaCKX4 resulted in decreased CK-ribosides levels. These findings suggest de novo biosynthesis of CKs induced by TDZ, primarily in elevation of t ZR and iPR levels. Our results provide a better understanding of CKs metabolism in Phalaenopsis micropropagation.

Petals of the monocot Phalaenopsis aphrodite (Orchidaceae) possess conical epidermal cells on their adaxial surfaces, and a large amount of cuticular wax is deposited on them to serve as a primary barrier against biotic and abiotic stresses. It has been widely reported that subgroup 9A members of the R2R3-MYB gene family, MIXTA and MIXTA-like in eudicots, act to regulate the differentiation of conical epidermal cells. However, the molecular pathways underlying conical epidermal cell development and cuticular wax biosynthesis in monocot petals remain unclear. Here, we characterized two subgroup 9A R2R3-MYB genes, PaMYB9A1 and PaMYB9A2 (PaMYB9A1/2), from P. aphrodite through the transient overexpression of their coding sequences and corresponding chimeric repressors in developing petals. We showed that PaMYB9A1/2 function to coordinate conical epidermal cell development and cuticular wax biosynthesis. In addition, we identified putative targets of PaMYB9A1/2 through comparative transcriptome analyses, revealing that PaMYB9A1/2 acts to regulate the expression of cell wall-associated and wax biosynthetic genes. Furthermore, a chemical composition analysis of cuticular wax showed that even-chain n-alkanes and odd-chain primary alcohols are the main chemical constituents of cuticular wax deposited on petals, which is inconsistent with the well-known biosynthetic pathways of cuticular wax, implying a distinct biosynthetic pathway occurring in P. aphrodite flowers. These results reveal that the function of subgroup 9A R2R3-MYB family genes in regulating the differentiation of epidermal cells is largely conserved in monocots and dicots. Furthermore, both PaMYB9A1/2 have evolved additional functions controlling the biosynthesis of cuticular wax., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The genus Paphiopedilum , belonging to the Orchidaceae, has high ornamental value. Leaf variations can considerably improve the economic and horticultural value of the orchids. In the study, a yellow leaf mutant of a Paphiopedilum hybrid named P. SCBG COP15 was identified during the in vitro plant culture process; however, little is known about their molecular mechanisms. For this, RNA-seq libraries were created and used for the transcriptomic profiling of P. SCBG COP15 and the yellow mutant. The Chl a , Chl b , and carotenoid contents in the yellow leaves decreased by approximately 75.99%, 76.92%, and 56.83%, respectively, relative to the green leaves. Decreased chloroplasts per cell and abnormal chloroplast ultrastructure were observed by electron microscopic investigation in yellowing leaves; photosynthetic characteristics and Chl fluorescence parameters were also decreased in the mutant. Altogether, 34,492 unigenes were annotated by BLASTX; 1,835 DEGs were identified, consisting of 697 upregulated and 1138 downregulated DEGs. HEMA , CRD , CAO , and CHLE , involved in Chl biosynthesis, were predicted to be key genes responsible for leaf yellow coloration. Our findings provide an essential genetic resource for understanding the molecular mechanism of leaf color variation and breeding new varieties of Paphiopedilum with increased horticultural value.