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Key Message: This study, using multi-omics combined with physiologic assays, found that calcium-ion signaling can regulate phenolic acid accumulation in R. chrysanthum leaves in response to UV-B stress. UV-B stress is a severe abiotic stress capable of destroying cellular structures and affecting plant growth. Rhododendron chrysanthum Pall. (R. chrysanthum) is a plant that has been exposed to high levels of UV-B radiation for an extended period, leading to the development of adaptive responses to mitigate UV-B stress. As such, it serves as a valuable experimental material for studying plant resilience to UV-B stress. We utilized R. chrysanthum as the experimental material and subjected it to UV-B stress. We conducted a comprehensive analysis of the changes in R. chrysanthum under both control and UV-B stress conditions using multi-omic and physiologic assays. Our aim was to investigate the molecular mechanism underlying R. chrysanthum's resistance to UV-B stress, with a focus on calcium-ion signaling. UV-B stress was found to impact the photosynthesis of R. chrysanthum by decreasing the maximum photosynthetic efficiency of photosystem II, reducing Fm, and increasing F0. In addition, the composition of numerous phenolic acid compounds was significantly altered. Genes and proteins related to calcium signaling showed significant differences, with some proteins (CML, CPK1, CRK3, ATP2C, ERG3, CAR7) being modified by acetylation. The correlation between genes and proteins involved in calcium signaling and phenolic compounds suggested that calcium signaling may play a role in regulating the accumulation of phenolic compounds under UV-B stress to help R. chrysanthum adapt. This study examines the impact of calcium-ion signaling on the accumulation of phenolic acid compounds, offering insights for future research on the molecular mechanisms underlying plant resilience to UV-B stress., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rhododendron simsii Planchon is an important ornamental species in the northern hemisphere. Flower color is an important objective of Rhododendron breeding programs. However, information on anthocyanin synthesis in R. simsii is limited. In this research, the regulatory mechanism of anthocyanin biosynthesis in R. simsii was performed through the integrated analysis of metabolome and RNA-seq. A total of 805 and 513 metabolites were screened by positive and negative ionization modes, respectively, In total, 79 flavonoids contained seven anthocyanidins, 42 flavanones, 10 flavans, 13 flavones, and seven flavonols. Methylated and glycosylated derivatives took up the most. Differentially accumulated metabolites were mainly involved in "flavone and flavonol biosynthesis", "cyanoamino acid metabolism", "pyrimidine metabolism", and "phenylalanine metabolism" pathways. For flavonoid biosynthesis, different expression of shikimate O-hydroxycinnamoyltransferase , caffeoyl-CoA O-methyltransferase , flavonoid 3'-monooxygenase , flavonol synthase , dihydroflavonol 4-reductase/flavanone 4-reductase , F3'5'H , chalcone synthase , leucoanthocyanidin reductase , and 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase genes ultimately led to different accumulations of quercetin, myricetin, cyanidin, and eriodictyol. In flavone and flavonol biosynthesis pathway, differential expression of F3'5'H , flavonoid 3'-monooxygenase and flavonol-3-O-glucoside/galactoside glucosyltransferase genes led to the differential accumulation of quercetin, isovitexin, and laricitrin. This research will provide a biochemical basis for further modification of flower color and genetic breeding in R. simsii and related Rhododendron species.

Aluminum (Al) toxicity is an important factor restricting the normal growth of plants in acidic soil. Rhododendron (Ericaceae) can grow relatively well in acidic soil. To uncover the adaptive mechanisms of photosynthesis under Al stress, the influence of Al stress on the photosynthetic activities of Al-sensitive (Baijinpao) and Al-resistant (Kangnaixin) rhododendron cultivars was examined by measuring gas exchange, chlorophyll fluorescence, and the modulated reflection of light at 820 nm. Under Al stress conditions, the net photosynthetic rate and stomatal conductance of the rhododendron leaves decreased, whereas the intercellular CO2 concentration increased. The Al stress treatment damaged the oxygen-evolving complex of the rhododendron seedlings, while also inhibiting electron transport on the photosystem II (PSII) donor side. In addition, the exposure to Al stress restricted the oxidation of plastocyanin (PC) and the photosystem I (PSI) reaction center (P700) and led to the re-reduction of PC+ and P700+. The comparison with Kangnaixin revealed an increase in the PSII connectivity in Baijinpao. Additionally, the donor-side electron transport efficiency was more inhibited and the overall activity of PSII, PSI, and the intersystem electron transport chain decreased more extensively in Baijinpao than in Kangnaixin. On the basis of the study findings, we concluded that Al stress adversely affects photosynthesis in rhododendron seedlings by significantly decreasing the activity of PSII and PSI. Under Al stress, Kangnaixin showed stronger tolerance compared with Baijinpao., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang 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.)

Lysine acetylation of proteins plays a critical regulatory function in plants. A few advances have been made in the study of plant acetylproteome. However, until now, there have been few data on Rhododendron chrysanthum Pall. ( R. chrysanthum ). We analyzed the molecular mechanisms of photosynthesis and stress resistance in R. chrysanthum under UV-B stress. We measured chlorophyll fluorescence parameters of R. chrysanthum under UV-B stress and performed a multi-omics analysis. Based on the determination of chlorophyll fluorescence parameters, R. chrysanthum Y(NO) (Quantum yield of non-photochemical quenching) increased under UV-B stress, indicating that the plant was damaged and photosynthesis decreased. In the analysis of acetylated proteomics data, acetylated proteins were found to be involved in a variety of biological processes. Notably, acetylated proteins were significantly enriched in the pathways of photosynthesis and carbon fixation, suggesting that lysine acetylation modifications have an important role in these activities. Our findings suggest that R. chrysanthum has decreased photosynthesis and impaired photosystems under UV-B stress, but NPQ shows that plants are resistant to UV-B. Acetylation proteomics revealed that up- or down-regulation of acetylation modification levels alters protein expression. Acetylation modification of key enzymes of the Calvin cycle (Rubisco, GAPDH) regulates protein expression, making Rubisco and GAPDH proteins expressed as significantly different proteins, which in turn affects the carbon fixation capacity of R. chrysanthum . Thus, Rubisco and GAPDH are significantly differentially expressed after acetylation modification, which affects the carbon fixation capacity and thus makes the plant resistant to UV-B stress. Lysine acetylation modification affects biological processes by regulating the expression of key enzymes in photosynthesis and carbon fixation, making plants resistant to UV-B stress.

The azalea ( Rhododendron simsii Planch .) is an important ornamental woody plant with various medicinal properties due to its phytochemical compositions and components. However little information on the metabolite variation during flower development in Rhododendron has been provided. In our study, a comparative analysis of the flavonoid profile was performed in Rhododendron pulchrum sweet at three stages of flower development, bud (stage 1), partially open flower (stage 2), and full bloom (stage 3). A total of 199 flavonoids, including flavone, flavonol, flavone C-glycosides, flavanone, anthocyanin, and isoflavone were identified. In hierarchical clustering analysis (HCA) and principal component analysis (PCA), the accumulation of flavonoids displayed a clear development stage variation. During flower development, 78 differential accumulated metabolites (DAMs) were identified, and most were enriched to higher levels at the full bloom stage. A total of 11 DAMs including flavone (chrysin, chrysoeriol O-glucuronic acid, and chrysoeriol O-hexosyl-O-pentoside), isoflavone (biochanin A), and flavonol (3,7-di-O-methyl quercetin and isorhamnetin) were significantly altered at three stages. In particular, 3,7-di-O-methyl quercetin was the top increased metabolite during flower development. Furthermore, integrative analyses of metabolomic and transcriptomic were conducted, revealing that the contents of isoflavone, biochanin A, glycitin, and prunetin were correlated with the expression of 2-hydroxyisoflavanone dehydratase ( HIDH ), which provide insight into the regulatory mechanism that controls isoflavone biosynthesis in R. pulchrum . This study will provide a new reference for increasing desired metabolites effectively by more accurate or appropriate genetic engineering strategies., Competing Interests: The authors declare that they have no competing interests., (© 2024 Yang et al.)

Background: This work explored the characteristics of the WRKY transcription factor family in Rhododendron henanense subsp. lingbaoense ( Rhl ) and the expression patterns of these genes under abiotic stress by conducting bioinformatics and expression analyses., Methods: RhlWRKY genes were identified from a gene library of Rhl . Various aspects of these genes were analyzed, including genetic structures, conserved sequences, physicochemical properties, cis -acting elements, and chromosomal location. RNA-seq was employed to analyze gene expression in five different tissues of Rhl : roots, stems, leaves, flowers, and hypocotyls. Additionally, qRT-PCR was used to detect changes in the expression of five RhlWRKY genes under abiotic stress., Result: A total of 65 RhlWRKY genes were identified and categorized into three subfamilies based on their structural characteristics: Groups I, II, and III. Group II was further divided into five subtribes, with shared similar genetic structures and conserved motifs among members of the same subtribe. The physicochemical properties of these proteins varied, but the proteins are generally predicted to be hydrophilic. Most proteins are predicted to be in the cell nucleus, and distributed across 12 chromosomes. A total of 84 cis -acting elements were discovered, with many related to responses to biotic stress. Among the identified RhlWRKY genes, there were eight tandem duplicates and 97 segmental duplicates. The majority of duplicate gene pairs exhibited Ka/Ks values <1, indicating purification under environmental pressure. GO annotation analysis indicated that WRKY genes regulate biological processes and participate in a variety of molecular functions. Transcriptome data revealed varying expression levels of 66.15% of WRKY family genes in all five tissue types (roots, stems, leaves, flowers, and hypocotyls). Five RhlWRKY genes were selected for further characterization and there were changes in expression levels for these genes in response to various stresses., Conclusion: The analysis identified 65 RhlWRKY genes, among which the expression of WRKY&#95;42 and WRKY&#95;17 were mainly modulated by the drought and MeJA, and WRKY&#95;19 was regulated by the low-temperature and high-salinity conditions. This insight into the potential functions of certain genes contributes to understanding the growth regulatory capabilities of Rhl ., Competing Interests: The authors declare that they have no competing interests., (© 2024 Guo et al.)

Our study aims to identify the mechanisms involved in regulating the response of Rhodoendron Chrysanthum Pall. (R. chrysanthum) leaves to UV-B exposure; phosphorylated proteomics and metabolomics for phenolic acids and plant hormones were integrated in this study. The results showed that UV-B stress resulted in the accumulation of salicylic acid and the decrease of auxin, jasmonic acid, abscisic acid, cytokinin and gibberellin in R. chrysanthum. The phosphorylated proteins that changed in plant hormone signal transduction pathway and phenolic acid biosynthesis pathway were screened by comprehensive metabonomics and phosphorylated proteomics. In order to construct the regulatory network of R. chrysanthum leaves under UV-B stress, the relationship between plant hormones and phenolic acid compounds was analyzed. It provides a rationale for elucidating the molecular mechanisms of radiation tolerance in plants., (© 2024. The Author(s).)

With the depletion of the ozone layer, the intensity of ultraviolet B (UV-B) radiation reaching the Earth's surface increases, which in turn causes significant stress to plants and affects all aspects of plant growth and development. The aim of this study was to investigate the mechanism of response to UV-B radiation in the endemic species of Rhododendron chrysanthum Pall. ( R. chrysanthum ) in the Changbai Mountains and to study how exogenous ABA regulates the response of R. chrysanthum to UV-B stress. The results of chlorophyll fluorescence images and OJIP kinetic curves showed that UV-B radiation damaged the PSII photosystem of R. chrysanthum , and exogenous ABA could alleviate this damage to some extent. A total of 2148 metabolites were detected by metabolomics, of which flavonoids accounted for the highest number (487, or 22.67%). KEGG enrichment analysis of flavonoids that showed differential accumulation by UV-B radiation and exogenous ABA revealed that flavonoid biosynthesis and flavone and flavonol biosynthesis were significantly altered. GO analysis showed that most of the DEGs produced after UV-B radiation and exogenous ABA were distributed in the cellular process, cellular anatomical entity, and catalytic activity. Network analysis of key DFs and DEGs associated with flavonoid synthesis identified key flavonoids (isorhamnetin-3-O-gallate and dihydromyricetin) and genes (TRINITY&#95;DN2213&#95;c0&#95;g1&#95;i4-A1) that promote the resistance of R. chrysanthum to UV-B stress. In addition, multiple transcription factor families were found to be involved in the regulation of the flavonoid synthesis pathway under UV-B stress. Overall, R. chrysanthum actively responded to UV-B stress by regulating changes in flavonoids, especially flavones and flavonols, while exogenous ABA further enhanced its resistance to UV-B stress. The experimental results not only provide a new perspective for understanding the molecular mechanism of the response to UV-B stress in the R. chrysanthum , but also provide a valuable theoretical basis for future research and application in improving plant adversity tolerance.

Background: Rhododendron chrysanthum Pall. (R. chrysanthum) is a plant that lives in high mountain with strong UV-B radiation, so R. chrysanthum possess resistance to UV-B radiation. The process of stress resistance in plants is closely related to metabolism. Lysine acetylation is an important post-translational modification, and this modification process is involved in a variety of biological processes, and affected the expression of enzymes in metabolic processes. However, little is known about acetylation proteomics during UV-B stress resistance in R. chrysanthum., Results: In this study, R. chrysanthum OJIP curves indicated that UV-B stress damaged the receptor side of the PSII reaction center, with a decrease in photosynthesis, a decrease in sucrose content and an increase in starch content. A total of 807 differentially expressed proteins, 685 differentially acetylated proteins and 945 acetylation sites were identified by quantitative proteomic and acetylation modification histological analysis. According to COG and subcellular location analyses, DEPs with post-translational modification of proteins and carbohydrate metabolism had important roles in resistance to UV-B stress and DEPs were concentrated in chloroplasts. KEGG analyses showed that DEPs were enriched in starch and sucrose metabolic pathways. Analysis of acetylation modification histology showed that the enzymes in the starch and sucrose metabolic pathways underwent acetylation modification and the modification levels were up-regulated. Further analysis showed that only GBSS and SSGBSS changed to DEPs after undergoing acetylation modification. Metabolomics analyses showed that the metabolite content of starch and sucrose metabolism in R. chrysanthum under UV-B stress., Conclusions: Decreased photosynthesis in R. chrysanthum under UV-B stress, which in turn affects starch and sucrose metabolism. In starch synthesis, GBSS undergoes acetylation modification and the level is upregulated, promotes starch synthesis, making R. chrysanthum resistant to UV-B stress., (© 2024. The Author(s).)

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes ( RdCHS1-3 ) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p -coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi .

Main Conclusion: The post-transcriptional gene regulatory pathway and small RNA pathway play important roles in regulating the rapid and long-term response of Rhododendron moulmainense to high-temperature stress. The Rhododendron plays an important role in maintaining ecological balance. However, it is difficult to domesticate for use in urban ecosystems due to their strict optimum growth temperature condition, and its evolution and adaptation are little known. Here, we combined transcriptome and small RNAome to reveal the rapid response and long-term adaptability regulation strategies in Rhododendron moulmainense under high-temperature stress. The post-transcriptional gene regulatory pathway plays important roles in stress response, in which the protein folding pathway is rapidly induced at 4 h after heat stress, and alternative splicing plays an important role in regulating gene expression at 7 days after heat stress. The chloroplasts oxidative damage is the main factor inhibiting photosynthesis efficiency. Through WGCNA analysis, we identified gene association patterns and potential key regulatory genes responsible for maintaining the ROS steady-state under heat stress. Finally, we found that the sRNA synthesis pathway is induced under heat stress. Combined with small RNAome, we found that more miRNAs are significantly changed under long-term heat stress. Furthermore, MYBs might play a central role in target gene interaction network of differentially expressed miRNAs in R. moulmainense under heat stress. MYBs are closely related to ABA, consistently, ABA synthesis and signaling pathways are significantly inhibited, and the change in stomatal aperture is not obvious under heat stress. Taken together, we gained valuable insights into the transplantation and long-term conservation domestication of Rhododendron, and provide genetic resources for genetic modification and molecular breeding to improve heat resistance in Rhododendron., (© 2024. The Author(s).)

Rhododendron species provide excellent ornamental use worldwide, yet heat stress (HS) is one of the major threats to their cultivation. However, the intricate mechanisms underlying the photochemical and transcriptional regulations associated with the heat stress response in Rhododendron remain relatively unexplored. In this study, the analyses of morphological characteristics and chlorophyll fluorescence (ChlF) kinetics showed that HS (40 °C/35 °C) had a notable impact on both the donor's and acceptor's sides of photosystem II (PSII), resulting in reduced PSII activity and electron transfer capacity. The gradual recovery of plants observed following a 5-day period of culture under normal conditions indicates the reversible nature of the HS impact on Rhododendron × pulchrum. Analysis of transcriptome data unveiled noteworthy trends: four genes associated with photosynthesis-antenna protein synthesis (LHCb1, LHCb2 and LHCb3) and the antioxidant system (glutamate-cysteine ligase) experienced significant down-regulation in the leaves of R. × pulchrum during HS. Conversely, aseorbate peroxidase and glutathione S-transferase TAU 8 demonstrated an up-regulated pattern. Furthermore, six down-regulated genes (phos-phoenolpyruvate carboxylase 4, sedoheptulose-bisphosphatase, ribose-5-phosphate isomerase 2, high cyclic electron flow 1, beta glucosidase 32 and starch synthase 2) and two up-regulated genes (beta glucosidase 2 and UDP-glucose pyrophosphorylase 2) implicated in photosynthetic carbon fixation and starch/sucrose metabolism were identified during the recovery process. To augment these insights, a weighted gene co-expression network analysis yielded a co-expression network, pinpointing the hub genes correlated with ChlF dynamics' variation trends. The cumulative results showed that HS inhibited the synthesis of photosynthesis-antenna proteins in R. × pulchrum leaves. This disruption subsequently led to diminished photochemical activities in both PSII and PSI, albeit with PSI exhibiting heightened thermostability. Depending on the regulation of the reactive oxygen species scavenging system and heat dissipation, photoprotection sustained the recoverability of R. × pulchrum to HS., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rhododendron pulchrum sweet, a member of the Ericaceae family possessing valuable horticultural properties, is widely distributed in the temperate regions. Though serving as bioindicator of metal pollution, the molecular mechanism regulating flowering in R. pulchrum is very limited. Illumina sequencing was performed to identify critical miRNAs in the synthesis of flavonoids at different developmental stages. Totally, 722 miRNAs belonging to 104 families were screened, and 84 novel mature miRNA sequences were predicted. The miR166, miR156, and miR167-1 families were dominant. In particular, 126 miRNAs were significantly differentially expressed among four different flowering stages. Totally, 593 genes were differentially regulated by miRNAs during the flower development process, which were mostly involved in "metabolic pathways", "plant hormone signal transduction", and "mitosis and regulation of biosynthetic processes". In pigment biosynthesis and signal transduction processes, gra-miR750 significantly regulated the expression of flavonoid 3',5'-hydroxylase; aof-miR171a, aof-miR171b, aof-miR171c, cas-miR171a-3p, and cas-miR171c-3p could regulate the expression of DELLA protein; aof-miR390, aof-miR396b, ath-miR3932b-5p, cas-miR171a-3p, aof-miR171a, and aof-miR171b regulated BAK1 expression. This research showed great potentials for genetic improvement of flower color traits for R. pulchrum and other Rhododendron species., (© 2023. Springer Nature Limited.)

The influence of UV-B stress on the growth, development, and metabolism of alpine plants, such as the damage to DNA macromolecules, the decline in photosynthetic rate, and changes in growth, development, and morphology cannot be ignored. As an endogenous signal molecule, ABA demonstrates a wide range of responses to UV-B radiation, low temperature, drought, and other stresses. The typical effect of ABA on leaves is to reduce the loss of transpiration by closing the stomata, which helps plants resist abiotic and biological stress. The Changbai Mountains have a harsh environment, with low temperatures and thin air, so Rhododendron chrysanthum ( R. chrysanthum ) seedlings growing in the Changbai Mountains can be an important research object. In this study, a combination of physiological, phosphorylated proteomic, and transcriptomic approaches was used to investigate the molecular mechanisms by which abiotic stress leads to the phosphorylation of proteins in the ABA signaling pathway, and thereby mitigates UV-B radiation to R. chrysanthum . The experimental results show that a total of 12,289 differentially expressed genes and 109 differentially phosphorylated proteins were detected after UV-B stress in R. chrysanthum , mainly concentrated in plant hormone signaling pathways. Plants were treated with ABA prior to exposure to UV-B stress, and the results showed that ABA mitigated stomatal changes in plants, thus confirming the key role of endogenous ABA in plant adaptation to UV-B. We present a model that suggests a multifaceted R. chrysanthum response to UV-B stress, providing a theoretical basis for further elaboration of the mechanism of ABA signal transduction regulating stomata to resist UV-B radiation.

Chalcone synthase (CHS) plays a vital role in anthocyanin biosynthesis pathway, which is associated with petal color of flower. To date, lots of CHS genes have been obtained from plants, while few were from Rhododendron genus. In this study we got a new CHS gene named RhCHS (MW358095) from Rhododendron × hybridum Hort. It had a 2040 bp coding region consisting of two exons and one intron. By using the deduced RhCHS protein as a query sequence, 15 CHS homologous family genes with sequence similarity from 60% to 98%, designated as RgCHS-D(x), were retrieved from the genome assembly of Rhododendron griersonianum (RGv1.1) by TBlastN. 12 CHS family genes were found locating in No.9 chromosome arranged in clusters, while only 3 of them exhibited in No.1, 2, and 8 chromosomes, respectively. The results revealed gene duplication of CHS in evolutionary process. Multiple alignment of the deduced amino acid sequence of RhCHS showed high similarity of the active site, the catalytic residue, and the signature motif, the conserved characteristics of which were also exhibited in the tertiary structure prediction of the RhCHS, as well as the phylogenetic tree, all these demonstrated the RhCHS belonging to the type III PKS superfamily. HPLC-MS/MS of flower petals detected the total concentration of CC, DC, and PelC. These anthocyanidins showed an overall increasing trend during the flowering period and reached the peak in the full-blooming stage, which was consistence with the changeable rule of RhCHS expression level. The promoter, which was 1507 bp exhibiting high β-glucuronidase (GUS) staining activity, was predicted containing many cis-acting elements, especially light and transcription factor such as bHLH, MYB, WRKY, Dof, and ERF. In short, this study may provide the help to Rhododendron × hybridum Hort. not only in the mechanism research of petals color exhibition, but also in molecular breeding of CHS practice value., 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 B.V. All rights reserved.)

Phytochemicals with potential to competitively bind to the host receptors or inhibit SARS-CoV-2 replication, may prove to be useful as adjunct therapeutics for COVID-19. We profiled and investigated the phytochemicals of Rhododendron arboreum petals sourced from Himalayan flora, undertook in vitro studies and found it as a promising candidate against SARS-CoV-2. The phytochemicals were reported in various scientific investigations to act against a range of virus in vitro and in vivo, which prompted us to test against SARS-CoV-2. In vitro assays of R. arboreum petals hot aqueous extract confirmed dose dependent reduction in SARS-CoV-2 viral load in infected Vero E6 cells (80% inhibition at 1 mg/ml; IC50 = 173 µg/ml) and phytochemicals profiled were subjected to molecular docking studies against SARS CoV-2 target proteins. The molecules 5-O-Feruloyl-quinic acid, 3-Caffeoyl-quinic acid, 5-O-Coumaroyl-D-quinic acid, Epicatechin and Catechin showed promising binding affinity with SARS-CoV-2 Main protease (M Pro ; PDB ID: 6LU7; responsible for viral replication) and Human Angiotensin Converting Enzyme-2 (ACE2; PDB ID: 1R4L; mediate viral entry in the host). Molecular dynamics (MD) simulation of 5-O-Feruloyl-quinic acid, an abundant molecule in the extract complexed with the target proteins showed stable interactions. Taken together, the phytochemical profiling, in silico analysis and in vitro anti-viral assay revealed that the petals extract act upon M Pro and may be inhibiting SARS-CoV-2 replication. This is the first report highlighting R. arboreum petals as a reservoir of antiviral phytochemicals with potential anti-SARS-CoV-2 activity using an in vitro system.

Background: We previously found that total flavones of Rhododendron (TFR) protected against the cerebral ischemia/reperfusion (I/R) injury. But the detailed mechanism is not clear. Recent research revealed that reactive astrocytes were divided into A1 and A2 phenotypes for their morphological and functional remodeling and neurotoxic- vs-neuroprotective effect on the injury of the central nervous system (CNS)., Purpose: The present study was undertaken to explore the role and mechanism of TFR on the phenotypic change of astrocytes following cerebral I/R in vivo and oxygen glucose deprivation/re-oxygenation (OGD/R) in vitro., Study Design and Methods: We tested the expression of astrocytes marker glial fibrillary acidic protein (GFAP), A1 astrocytes marker C3 protein and A2 astrocytes marker S100a10, as well as the BrdU/GFAP-positive cells, GFAP/S100a10-positive cells and GFAP/C3-positive cells in mice hippocampal tissues to evaluate the phenotypic change of astrocytes. Besides, we assessed the change of astrocyte phenotypes following OGD/R in vitro., Results: We found that mice cerebral I/R promoted the astrocytes proliferation of both A1 and A2 phenotypes in hippocampal tissues. While treatment with TFR could promote the proliferation of A2 astrocytes but inhibit the A1 astrocytes proliferation in mice hippocampal tissues, suggesting that TFR could accelerate the astrocytes transformation into A2 subtype following cerebral I/R. Whereas, in OGD/R model of astrocytes, we found that TFR inhibited the proliferation of both A1 and A2 astrocytes. Besides, we found that TFR could up-regulate the release of cystathionine β-synthase (CBS)-produced hydrogen sulfide (H 2 S) and inhibit RhoA/Rho kinase pathway, and revealed that the inhibitory effect of TFR on astrocytes proliferation could be blocked by aminooxyacetic acid (AOAA), an CBS inhibitor. Furthermore, TFR could ameliorate the mice cerebral I/R injury and the OGD/R-induced astrocytic damage., Conclusion: These findings suggested that TFR could affect the transformation of astrocytes subtypes following cerebral I/R, which may be related to up-regulation of CBS-produced H 2 S and subsequent inhibition of RhoA/ROCK pathway., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier GmbH. All rights reserved.)

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R . hybridum flavanone 3-hydroxylase ( RhF3H ) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a- RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302- RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium -mediated method. The results showed that the R . hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe 2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R . hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R . hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a- RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A . thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A . thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R . simsiib Planch.

Rhododendron chrysanthum ( Rhododendron chrysanthum Pall.), an alpine plant, has developed UV-B resistance mechanisms and has grown to be an important plant resource with the responsive capacity of UV-B stress. Our study uses acetylated proteomics and proteome analysis, together with physiological measurement, to show the Rhododendron chrysanthum seedling's reaction to UV-B stress. Following a 2-day, 8-h radiation therapy, 807 significantly altered proteins and 685 significantly altered acetylated proteins were discovered. Significantly altered proteins and acetylated proteins, according to COG analysis, were mostly engaged in post-translational modification, protein turnover, and chaperone under UV-B stress. It indicates that protein acetylation modification plays an important role in plant resistance to UV-B. The experimental results show that photosynthesis was inhibited under UV-B stress, but some photosynthetic proteins will undergo acetylation modification, which can alleviate the UV-B damage of plants to a certain extent. These results will serve as the basis for more research into the intricate molecular mechanisms underlying plant UV-B adaptation.

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1 , TPS10 , TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1 , TPS4, TPS9 , TPS10 , TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R . fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R . fortunei .

Background: Rhododendron nivale Hook. f (R.n), one of the four Manna Stash used in Tibetan medicine to delay aging, possesses anti-aging pharmacological activity. However, which R.n ingredients contain anti-aging properties and the underlying mechanisms involved are unclear., Hypothesis/purpose: Based on interactions between gut microbiota and natural medicines and the important role of gut microbiota in anti-aging, the study investigated the hypothesis that R.n possesses anti-aging properties and the interaction of gut microbiota with R.n is responsible for its anti-aging effects., Study Design: The primary active ingredients of R.n and their target function and pathway enrichment were explored. An aging mouse model was used to clarify the underlying anti-aging mechanisms of R.n., Methods: Chromatography, spectroscopy, nuclear magnetic technology, and pharmacology were used to reveal the major active ingredients of ethanol extract residues of R.n (RNEA). The target function and pathway enrichment of these active ingredients were explored. Plasma metabolomics coupled with intestinal flora evaluation and bioinformatics analysis was used to clarify the underlying anti-aging mechanisms of RNEA., Results: Myricetin-3-β-D-xylopyranoside, hyperin, goospetin-8-methyl ether 3-β-D-galactoside, and diplomorphanin B were separated and identified from RNEA. The network pharmacology study revealed that the active ingredients' target function and pathway enrichment focused mainly on the glutathione antioxidant system. In a D-galactose-induced mouse model of aging, RNEA was shown to possess suitable anti-aging pharmacological activity, as indicated by the amelioration of memory loss and weakened superoxide dismutase and glutathione peroxidase activities. Plasma metabolomics coupled with intestinal flora examination and bioinformatics analysis revealed that RNEA could regulate the expression of glutathione-related enzymes and ameliorate D-galactose-induced imbalances in methionine, glycine, and serine, and betaine and galactose metabolism. The results showed that RNEA reshaped the disordered intestinal flora and mitigated the D-galactose-mediated decline in glutathione oxidase expression, further confirming that the anti-aging effect of RNEA was closely related to regulation of the glutathione antioxidant system., Conclusion: RNEA, consisting of myricetin-3-β-D-xylopyranoside, hyperin, goospetin-8-methyl ether 3-β-D-galactoside, and diplomorphanin B, possesses anti-aging activity. The anti-aging effect of RNEA might be due to reshaping intestinal flora homeostasis, increasing the expression of glutathione peroxidase 4 in the intestines and liver, enhancing glutathione peroxidase activity, and reinforcing the glutathione antioxidant system., (Copyright © 2022. Published by Elsevier GmbH.)

Background: Color is the major ornamental feature of the Rhododendron genus, and it is related to the contents of flavonoid in petals. However, the regulatory mechanism of flavonoid biosynthesis in Rhododendron pulchrum remains unknown. The transcriptome and metabolome analysis of Rhododendron pulchrum with white, pink and purple color in this study aimed to reveal the mechanism of flavonoid biosynthesis and to provide insight for improving the petal color., Results: Flavonoids and flavonols are the major components of flavonoid metabolites in R.pulchrum, such as laricitrin, apigenin, tricin, luteolin, isoorientin, isoscutellarein, diosmetin and their glycosides derivatives. With transcriptome and metabolome analysis, we found CHS, FLS, F3'H, F3'5'H, DFR, ANS, GT, FNS, IFR and FAOMT genes showed significantly differential expression in cultivar 'Zihe'. FNS and IFR were discovered to be associated with coloration in R.pulchrum for the first time. The FNS gene existed in the form of FNSI. The IFR gene and its related metabolites of medicarpin derivatives were highly expressed in purple petal. In cultivar 'Fenhe', up-regulation of F3'H and F3'5'H and down-regulation of 4CL, DFR, ANS, and GT were associated with pink coloration. With the transcription factor analysis, a subfamily of DREBs was found to be specifically enriched in pink petals. This suggested that the DREB family play an important role in pink coloration. In cultivars 'Baihe', flavonoid biosynthesis was inhibited by low expression of CHS, while pigment accumulation was inhibited by low expression of F3'5'H, DFR, and GT, which led to a white coloration., Conclusions: By analyzing the transcriptome and metabolome of R.pulchrum, principal differential expression genes and metabolites of flavonoid biosynthesis pathway were identified. Many novel metabolites, genes, and transcription factors associated with coloration have been discovered. To reveal the mechanism of the coloration of different petals, a model of the flavonoid biosynthesis pathway of R.pulchrum was constructed. These results provide in depth information regarding the coloration of the petals and the flavonoid metabolism of R.pulcherum. The study of transcriptome and metabolome profiling gains insight for further genetic improvement in Rhododendron., (© 2022. The Author(s).)

Light stress is one of the important stresses for winter survival in evergreens, especially for plants with broad leaves, like evergreen rhododendrons. Photoprotection has been shown to upregulate dramatically in rhododendrons during winter, but whether it directly contributes to enhancing the freezing tolerance is still unknown. In this study, we found that the expression and circadian rhythm of an early light-induced protein (ELIP)-RhELIP3-which exerts photoprotection in Rhododendron 'Elsie Lee', could be impacted by both photoperiod and low temperature, with low temperature being the predominant inducer. Arabidopsis overexpressing RhELIP3 displayed significantly stronger freezing tolerance and better photosystem II function after a 3-day recovery from freezing treatment. Moreover, RhHY5 binds with the RhELIP3 promoter to activate its expression. Arabidopsis overexpressing RhHY5 exhibited stronger freezing tolerance and better photosystem II function. AtELIP1 and AtELIP2 were significantly induced in RhHY5-overexpressed Arabidopsis at low temperatures. We also discovered that RhBBX24 binds directly to RhELIP3 promoter and suppresses its expression. RhBBX24 can also interact with RhHY5 and inhibit the interaction of RhHY5-RhELIP3. RhELIP3, RhHY5, and RhBBX24 exhibited similar circadian rhythms under low temperature with short period. Overall, our investigation highlights that photoprotection is involved in improving the freezing tolerance of evergreen rhododendrons., (© 2022 John Wiley & Sons Ltd.)

Understanding the molecular mechanisms and evolutionary process of plant adaptation to the heterogeneous environment caused by altitude gradients in plateau mountain ecosystems can provide novel insight into species' responses to global changes. Flower color is the most conspicuous and highly diverse trait in nature. Herein, the gene expression patterns, evolutionary adaptation and metabolites changes of different-colored flowers of alpine Rhododendron L. species along altitude gradients were investigated based on a combined analysis of transcriptomics and metabolomics. Differentially expressed genes were found to be related to the biosynthesis of carbohydrates, fatty acids, amino acids and flavonoids, suggesting their important roles in the altitude adaptability of Rhododendron species. The evolution rate of high-altitude species was faster than that of low-altitude species. Genes related to DNA repair, mitogen-activated protein kinase and ABA signal transduction, and lipoic acid and propanoate metabolism were positively selected in the flowers of high-altitude Rhododendron species and those associated with carotenoid biosynthesis pathway, ABA signal transduction and ethylene signal transduction were positively selected in low-altitude species. These results indicated that the genes with differentiated expressions or functions exhibit varying evolution during the adaptive divergence of heterogeneous environment caused by altitude gradients. Flower-color variation might be attributed to the significant differences in gene expression or metabolites related to sucrose, flavonoids and carotenoids at the transcription or metabolism levels of Rhododendron species. This work suggests that Rhododendron species have multiple molecular mechanisms in their adaptation to changing environments caused by altitude gradients., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The aim of the present study was to investigate the role of Rhododendron luteum extract (RLE) in the induction of Nrf2‑related oxidative stress and endoplasmic reticulum (ER) stress in human cervical cancer (HeLa) cells. The antiproliferative effect of RLE on HeLa and fibroblast cells was determined using the MTT assay. The effects of RLE on the cell cycle, apoptosis, and production of reactive oxygen species (ROS) in HeLa cells were evaluated using fluorescent probes. The mRNA expression levels of Nrf2 [and its targets glutamate-cysteine ligase catalytic subunit (GCLC), and glucose-6-phosphate dehydrogenase (G6PD)], and C/EBP homologous protein (CHOP, an ER stress marker were determined using reverse transcription‑quantitative polymerase chain reaction (RT-PCR). The results demonstrated that RLE exhibited a selective cytotoxic effect (2.9-fold) on HeLa cells compared to fibroblast cells. RLE arrested the cell cycle at the S phase, and induced apoptosis, ER stress, and ROS formation. In addition, RLE significantly suppressed the expression levels of Nrf2, GCLC and G6PD (0.65, 0.69, and 0.54-fold, respectively) and increased the expression of CHOP (4.48-fold) in HeLa cells at 72 h of treatment ( p  < 0.05). These results show that the antiproliferative effect of RLE occurs through the Nrf2 and ER stress pathways, and the results should now be supported by further in vivo studies.

Background: As an alpine plant, Rhododendron chrysanthum (R. chrysanthum) has evolved cold resistance mechanisms and become a valuable plant resource with the responsive mechanism of cold stress., Methods and Results: We adopt the phosphoproteomic and proteomic analysis combining with physiological measurement to illustrate the responsive mechanism of R. chrysanthum seedling under cold (4 °C) stress. After chilling for 12 h, 350 significantly changed proteins and 274 significantly changed phosphoproteins were detected. Clusters of Orthologous Groups (COG) analysis showed that significantly changed phosphoproteins and proteins indicated cold changed energy production and conversion and signal transduction., Conclusions: The results indicated photosynthesis was inhibited under cold stress, but cold induced calcium-mediated signaling, reactive oxygen species (ROS) homeostasis and other transcription regulation factors could protect plants from the destruction caused by cold stress. These data provide the insight to the cold stress response and defense mechanisms of R. chrysanthum leaves at the phosphoproteome level., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Rhododendron delavayi is a popular ornamental plant with globular flowers noted for their bright red color, but very limited studies have been reported on its flower color formation. In this study, we successfully isolated a novel DFR gene (RdDFR1) from red flowers of Rhododendron delavayi. Multiple sequence alignments revealed that RdDFR1 had the conserved NADP and substrate binding domain, and was classified into Asn-type DFR. Meanwhile, quantitative real-time PCR analysis showed that transcript levels of RdDFR1 matched the accumulation patterns of anthocyanins during flower development, hinting its potential role involved in anthocyanin biosynthesis. Then in vitro enzymatic analysis indicated that recombinant RdDFR1 protein could catalyze the production of leucoanthocyanidins from dihydroquercetin and dihydromyricetin. Furthermore, the in planta assay, using Arabidopsis thaliana dfr mutant (tt3-1) and tobacco, displayed that RdDFR1 transgenes recovered the defective proanthocyanidin and anthocyanin biosynthesis at seed coats, hypocotyl as well as cotyledon, and altered the flowers color of tobacco from pale pink to dark pink which demonstrated its function as dihydroflavonol 4-reductase in vivo. In summary, our findings suggest that RdDFR1 plays a crucial role in the biosynthesis of anthocyanin and will also make a contribution to understand the mechanisms of flower color formation in Rhododendron delavayi., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Rhododendron (Ericaceae) extracts contain flavonoids, chromones, terpenoids, steroids, and essential oils and are used in traditional ethnobotanical medicine. However, little is known about the immunomodulatory activity of essential oils isolated from these plants. Thus, we isolated essential oils from the flowers and leaves of R. albiflorum (cascade azalea) and analyzed their chemical composition and innate immunomodulatory activity. Compositional analysis of flower (REO Fl ) versus leaf (REO Lv ) essential oils revealed significant differences. REO Fl was comprised mainly of monoterpenes (92%), whereas sesquiterpenes were found in relatively low amounts. In contrast, REO Lv was primarily composed of sesquiterpenes (90.9%), with a small number of monoterpenes. REO Lv and its primary sesquiterpenes (viridiflorol, spathulenol, curzerene, and germacrone) induced intracellular Ca 2+ mobilization in human neutrophils, C20 microglial cells, and HL60 cells transfected with N -formyl peptide receptor 1 (FPR1) or FPR2. On the other hand, pretreatment with these essential oils or component compounds inhibited agonist-induced Ca 2+ mobilization and chemotaxis in human neutrophils and agonist-induced Ca 2+ mobilization in microglial cells and FPR-transfected HL60 cells, indicating that the direct effect of these compounds on [Ca 2+ ] i desensitized the cells to subsequent agonist activation. Reverse pharmacophore mapping suggested several potential kinase targets for these compounds; however, these targets were not supported by kinase binding assays. Our results provide a cellular and molecular basis to explain at least part of the beneficial immunotherapeutic properties of the R. albiflorum essential oils and suggest that essential oils from leaves of this plant may be effective in modulating some innate immune responses, possibly by inhibition of neutrophil migration.

Plants usually experience fluctuating light conditions at chilling temperatures during the autumn season. We hypothesized that photosystem I (PSI) and PSII are more susceptible to photoinhibition under fluctuating light at chilling temperatures in deciduous species relative to evergreen species. We measured the photosynthetic performances under fluctuating light at 6 °C in two evergreen and two deciduous broadleaf tree species. Within the first 10 s after light increased at 6 °C, none of these species could generate an enough trans-thylakoid proton gradient. Meanwhile, PSI was highly oxidised in evergreen species but was highly reduced in deciduous species. This transient over-reduction of PSI in deciduous species was mainly caused by the higher electron flow from PSII. Furthermore, the deciduous species showed a significantly smaller violaxanthin pool and lower non-photochemical quenching under high light conditions at 6 °C, leading to more excess light energy could not be dissipated in PSII. Hence, we propose that fluctuating light combined with chilling temperature cause the over-reduction of photosynthetic electron chain in deciduous species., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: The Rhododendron sanguineum complex is endemic to alpine mountains of northwest Yunnan and southeast Tibet of China. Varieties in this complex exhibit distinct flower colors even at the bud stage. However, the underlying molecular regulations for the flower color variation have not been well characterized. Here, we investigated this via measuring flower reflectance profiles and comparative transcriptome analyses on three coexisting varieties of the R. sanguineum complex, with yellow flush pink, bright crimson, and deep blackish crimson flowers respectively. We compared the expression levels of differentially-expressed-genes (DEGs) of the anthocyanin / flavonoid biosynthesis pathway using RNA-seq and qRT-PCR data. We performed clustering analysis based on transcriptome-derived Single Nucleotide Polymorphisms (SNPs) data, and finally analyzed the promoter architecture of DEGs., Results: Reflectance spectra of the three color morphs varied distinctively in the range between 400 and 700 nm, with distinct differences in saturation, brightness, hue, and saturation/hue ratio, an indirect measurement of anthocyanin content. We identified 15,164 orthogroups that were shared among the three varieties. The SNP clustering analysis indicated that the varieties were not monophyletic. A total of 40 paralogous genes encoding 12 enzymes contributed to the flower color polymorphism. These anthocyanin biosynthesis-related genes were associated with synthesis, modification and transportation properties (RsCHS, RsCHI, RsF3H, RsF3'H, RsFLS, RsANS, RsAT, RsOMT, RsGST), as well as genes involved in catabolism and degradation (RsBGLU, RsPER, RsCAD). Variations in sequence and cis-acting elements of these genes might correlate with the anthocyanin accumulation, thus may contribute to the divergence of flower color in the R. sanguineum complex., Conclusions: Our results suggested that the varieties are very closely related and flower color variations in the R. sanguineum complex correlate tightly with the differential expression levels of genes involved in the anabolic and catabolic synthesis network of anthocyanin. Our study provides a scenario involving intricate relationships between genetic mechanisms for floral coloration accompanied by gene flow among the varieties that may represent an early case of pollinator-mediated incipient sympatric speciation.

Azaleas (Ericaceae) comprise one of the most diverse ornamental plants, renowned for their cultural and economic importance. We present a chromosome-scale genome assembly for Rhododendron simsii, the primary ancestor of azalea cultivars. Genome analyses unveil the remnants of an ancient whole-genome duplication preceding the radiation of most Ericaceae, likely contributing to the genomic architecture of flowering time. Small-scale gene duplications contribute to the expansion of gene families involved in azalea pigment biosynthesis. We reconstruct entire metabolic pathways for anthocyanins and carotenoids and their potential regulatory networks by detailed analysis of time-ordered gene co-expression networks. MYB, bHLH, and WD40 transcription factors may collectively regulate anthocyanin accumulation in R. simsii, particularly at the initial stages of flower coloration, and with WRKY transcription factors controlling progressive flower coloring at later stages. This work provides a cornerstone for understanding the underlying genetics governing flower timing and coloration and could accelerate selective breeding in azalea.

Plants invest floral resources, including nectar and pigment, with likely consequent reproductive costs. We hypothesized that plants, whose flowers abscise with age, reabsorb nectar and pigment before abscission. This was tested with flowers of Rhododendron decorum, which has large, conspicuous white flowers that increasingly abscise corollas as flowers age. As this species is pollinated by bees, we also hypothesized that nectar concentration would be relatively high (i.e., > 30% wt/vol) and petals would contain UV-absorbing pigment. Floral nectar volume and concentration were sampled on successive days until abscission (up to ten days old, peak at five days) and for sub-sample of four-day-old flowers. Flowers just abscised were similarly sampled. Flower colours were measured using a modified camera, with recordings of spectral reflectance for abscised and open non-abscised flowers. Pigment content was summed values of red, green, blue channels of false color photos. As expected, flowers reabsorbed almost all nectar before abscission, separately reabsorbing nectar-sugar and nectar-water, and petals contained UV-absorbing pigment. However, flowers did not reabsorb pigment and nectar-concentration was < 30% wt/vol. That flowers reabsorb nectar, not pigment, remains unexplained, though possibly pigment reabsorption is uneconomical. Understanding floral resource reabsorption therefore requires determination of biochemical mechanisms, plus costs/benefits for individual plants.

Background: Rhododendron molle (Ericaceae) is a traditional Chinese medicinal plant, its flower and root have been widely used to treat rheumatism and relieve pain for thousands of years in China. Chemical studies have revealed that R. molle contains abundant secondary metabolites such as terpenoinds, flavonoids and lignans, some of which have exhibited various bioactivities including antioxidant, hypotension and analgesic activity. In spite of immense pharmaceutical importance, the mechanism underlying the biosynthesis of secondary metabolites remains unknown and the genomic information is unavailable., Results: To gain molecular insight into this plant, especially on the information of pharmaceutically important secondary metabolites including grayanane diterpenoids, we conducted deep transcriptome sequencing for R. molle flower and root using the Illumina Hiseq platform. In total, 100,603 unigenes were generated through de novo assembly with mean length of 778 bp, 57.1% of these unigenes were annotated in public databases and 17,906 of those unigenes showed significant match in the KEGG database. Unigenes involved in the biosynthesis of secondary metabolites were annotated, including the TPSs and CYPs that were potentially responsible for the biosynthesis of grayanoids. Moreover, 3376 transcription factors and 10,828 simple sequence repeats (SSRs) were also identified. Additionally, we further performed differential gene expression (DEG) analysis of the flower and root transcriptome libraries and identified numerous genes that were specifically expressed or up-regulated in flower., Conclusions: To the best of our knowledge, this is the first time to generate and thoroughly analyze the transcriptome data of both R. molle flower and root. This study provided an important genetic resource which will shed light on elucidating various secondary metabolite biosynthetic pathways in R. molle, especially for those with medicinal value and allow for drug development in this plant.

Cold acclimation (CA) is a well-known strategy employed by plants to enhance freezing tolerance (FT) in winter. Global warming could disturb CA and increase the potential for winter freeze-injury. Thus, developing robust FT through complete CA is essential. To explore the molecular mechanisms of CA in woody perennials, we compared field and artificial CAs. Transcriptomic data showed that photosynthesis/photoprotection and fatty acid metabolism pathways were specifically enriched in field CA; carbohydrate metabolism, secondary metabolism and circadian rhythm pathways were commonly enriched in both field and artificial CAs. When compared with plants in vegetative growth in the chamber, we found that the light signals with warm air temperatures in the fall might induce the accumulation of leaf abscisic acid (ABA) and jasmonic acid (JA) concentrations, and activate Ca 2+ , ABA and JA signaling transductions in plants. With the gradual cooling occurrence in winter, more accumulation of anthocyanin, chlorophyll degradation, closure/degradation of photosystem II reaction centers, and substantial accumulation of glucose and fructose contributed to obtaining robust FT during field CA. Moreover, we observed that in Rhododendron 'Elsie Lee', ABA and JA decreased in winter, which may be due to the strong requirement of zeaxanthin for rapid thermal dissipation and unsaturated fatty acids for membrane fluidity. Taken together, our results indicate that artificial CA has limitations to understand the field CA and field light signals (like short photoperiod, light intensity and/or light quality) before the low temperature in fall might be essential for complete CA., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Rhododendron lapponicum L. is a familiar ornamental plant worldwide with important ornamental and economic value. However, a full-length R. lapponicum transcriptome is still lacking. In the present study, we used the Pacific Biosciences single-molecule real-time sequencing technology to generate the R. lapponicum transcriptome. A total of 346,270 full-length non-chimeric reads were generated, from which we obtained 75,002 high-quality full-length transcripts. We identified 55,255 complete open reading frames, 7,140 alternative splicing events and 2,011 long non-coding RNAs. In gene annotation analyses, 71,155, 33,653, 30,359 and 31,749 transcripts were assigned to the Nr, GO, COG and KEGG databases, respectively. Additionally, 3,150 transcription factors were detected. KEGG pathway analysis showed that 96 transcripts were identified coding for the enzymes associated with anthocyanin synthesis. Furthermore, we identified 64,327 simple sequence repeats from 45,319 sequences, and 150 pairs of primers were randomly selected to develop SSR markers. This study provides a large number of full-length transcripts, which will facilitate the further study of the genetics of R. lapponicum.

miRNA is a non-coding, yet crucial entity in remodeling the genetic architecture. Rhododendron arboreum of Himalayas grows and even flower under fluctuating climate. sRNA from leaves of vegetative and reproductive periods was sequenced to elucidate its seasonal associations. Conserved (256) and novel (210) miRNAs and their precursors were located based on homology with plant databases and transcriptome of the species. 27,139 predicted targets were involved with metabolism, reproduction, and response to abiotic stimuli. A comparative analysis showed differential expression of 198 miRNAs with season-specific abundance of 103 miRNAs. Specific isoforms of 11 miRNA families exhibited a temporal expression and targeted different genes implying a complex regulation. The variable miRNA expression among the tissues of different conditions can be associated with the adaptability of the species, which will prove essential for further study on miRNAs mediating seasonal response. Moreover, exogenous cues also mediate phase transition via networking of flowering pathways and their components. In this context, 18 known families and 77 novel miRNAs modulating 117 genes crucial in circadian entrainment were filtered. A negative correlation was obtained between the expression of 18 of these miRNAs and their targets when tested through quantitative-PCR. It highlighted the role of miRNA-target pairs in perceiving environmental variabilities and monitoring flowering growth. Furthermore, a phylogenetic clustering was performed, which supported the lineage-specific evolution and function of putative miR156 sequence in the species. This documentation of genome-wide profiling of miRNA, their targets, and expression will enhance the understanding of developmental and climate-tolerance strategies in high-altitude trees., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rhododendron ponticum L. is a damaging invasive alien species in Britain, favouring the moist, temperate climate, and the acidic soils of upland areas. It outshades other species and is thought to create a soil environment of low pH that may be higher in phytotoxic phenolic compounds. We investigated native vegetation restoration and R. ponticum regeneration post-clearance using heathland sites within Snowdonia National Park, Wales; one site had existing R. ponticum stands and three were restoring post-clearance. Each site also had an adjacent, uninvaded control for comparison. We assessed whether native vegetation restoration was influenced post-invasion by soil chemical properties, including pH and phytotoxic compounds, using Lactuca sativa L. (lettuce) bioassays supported by liquid chromatography-mass spectroscopy (LC-MS n ). Cleared sites had higher shrub and bare ground cover, and lower grass and herbaceous species cover relative to adjacent uninvaded control sites; regenerating R. ponticum was also observed on all cleared sites. No phenolic compounds associated with R. ponticum were identified in any soil water leachates, and soil leachates from cleared sites had no inhibitory effect in L. sativa germination assays. We therefore conclude that reportedly phytotoxic compounds do not influence restoration post R. ponticum clearance. Soil pH however was lower beneath R. ponticum and on cleared sites, relative to adjacent uninvaded sites. The lower soil pH post-clearance may have favoured shrub species, which are typically tolerant of acidic soils. The higher shrub cover on cleared sites may have greater ecological value than unaffected grass dominated sites, particularly given the recent decline in such valuable heathland habitats. The presence of regenerating R. ponticum on all cleared sites however highlights the critical importance of monitoring and re-treating sites post initial clearance.

Molecular regulatory mechanism of heat stress response (HSR) in Ericaceae remains unknown. Here, we sought to identify HSR mechanisms in Rhododendron hainanense, a Ericaceae species, through a combination of physiological and transcriptomic studies. The levels of MDA, H 2 O 2 , Pro, SOD, CAT and APX in leaves of R. hainanense were analyzed to characterize a dramatic difference in varied temperature treatment. Also, three sequencing libraries, including one control and two heat stress (HS)-treated samples, were constructed for comparative transcriptomic analysis. By Illumina sequencing and Trinity strategy, 350 million clean reads (average length = 149 bp) was assembled into 183,486 unigenes. According to analysis of differential expression genes (DEGs), a total of 2658 DEGs were obtained. Moreover, a complex interaction network of 982 DEGs was established, of which master portions were comprised of 109 transcription factors (TFs). Importantly, integrated differential expression profiling, qRT-PCR and functional analysis, several TFs of R. hainanense (ABR1, IAA26, OBF1, LUX, SCL3, DIV, NAC29, NAC72 and TCP3) and their potential regulations for the crosstalk between hormonal signal and HSR were identified. These findings will contribute to our understanding of the regulatory mechanisms of HSR in R. hainanense, breeding cultivars with improved thermotolerance., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rhododendron schlippenbachii Maxim. is a garden plant that is also used for natural medicines as a consequence of the biological activities of its diverse metabolites. We accordingly profiled two anthocyanins and 40 primary and secondary metabolites in the three different colored flowers. The major anthocyanins found in the flowers were cyanidins. The red flowers exhibited the highest accumulation of anthocyanins (1.02 ± 0.02 mg/g dry weight). Principal component analysis was applied to the GC‒TOFMS data. The levels of key tricarboxylic acid cycle intermediates in red flowers, such as succinic acid, fumaric acid, and malic acid, were found to be highly significantly different ( p < 0.0001) from those in the flowers of other colors. In this study, we aimed to determine metabolite interactions and phenotypic variation among white, violet, and red flowers of R. schlippenbachii by using gas chromatography time-of-flight mass spectrometry (GC‒TOFMS) and high-performance liquid chromatography (HPLC).

Hydroxycinnamates including free hydroxycinnamic acids and their chlorogenic acid derivatives and glycosides have been profiled in leaf extracts of 98 Rhododendron species using LC-MS techniques. In total, 69 hydroxycinnamic acid derivatives were identified in the leaves of 98 Rhododendron species. Some derivatives serve as unique phytochemical marker for a single species, whereas other compounds are limited to certain subgenera. The distribution of compounds among six different subgenera of Rhododendron was studied using PCA and PLS-DA. This contribution presents data that provide unique metabolomic insight in the distribution of a class of secondary metabolites within a large selection of species from the botanically diverse plant genus Rhododendron., (Copyright © 2017. Published by Elsevier Ltd.)

Daurichromenic acid (DCA) synthase catalyzes the oxidative cyclization of grifolic acid to produce DCA, an anti-HIV meroterpenoid isolated from Rhododendron dauricum We identified a novel cDNA encoding DCA synthase by transcriptome-based screening from young leaves of R. dauricum The gene coded for a 533-amino acid polypeptide with moderate homologies to flavin adenine dinucleotide oxidases from other plants. The primary structure contained an amino-terminal signal peptide and conserved amino acid residues to form bicovalent linkage to the flavin adenine dinucleotide isoalloxazine ring at histidine-112 and cysteine-175. In addition, the recombinant DCA synthase, purified from the culture supernatant of transgenic Pichia pastoris , exhibited structural and functional properties as a flavoprotein. The reaction mechanism of DCA synthase characterized herein partly shares a similarity with those of cannabinoid synthases from Cannabis sativa , whereas DCA synthase catalyzes a novel cyclization reaction of the farnesyl moiety of a meroterpenoid natural product of plant origin. Moreover, in this study, we present evidence that DCA is biosynthesized and accumulated specifically in the glandular scales, on the surface of R. dauricum plants, based on various analytical studies at the chemical, biochemical, and molecular levels. The extracellular localization of DCA also was confirmed by a confocal microscopic analysis of its autofluorescence. These data highlight the unique feature of DCA: the final step of biosynthesis is completed in apoplastic space, and it is highly accumulated outside the scale cells., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Premise: We explored trait-trait and trait-climate relationships for 27 Rhododendron species while accounting for phylogenetic relationships and within-species variation to investigate whether leaf and root traits are coordinated across environments and over evolutionary time, as part of a whole-plant economics spectrum., Methods: We examined specific leaf area (SLA) and four root traits: specific root length (SRL), specific root tip abundance (SRTA), first order diameter, and link average length, for plants growing in a cold, seasonal climate (Kirtland, Ohio) and a warmer, less seasonal climate (Federal Way, Washington) in the United States. We estimated a phylogeny and species' climate of origin, determined phylogenetic signal on mean traits and within-species variation, and used phylogenetically informed analysis to compare trait-trait and trait-climate relationships for deciduous and evergreen lineages., Results: Mean SLA and within-species variation in SRL were more similar between close relatives than expected by chance. SLA and root traits differed according to climate of origin and across growth environments, though SLA differed within- and among-species less than roots. A negative SRL-SRTA correlation indicates investment in foraging scale vs. precision as a fundamental trade-off defining the root economic spectrum. Also, the deciduous clade exhibited a strong negative relationship between SLA and SRL, while evergreen clades showed a weaker positive or no relationship., Conclusions: Our work suggests that natural selection has shaped relationships between above- and belowground traits in genus Rhododendron and that leaf and root traits may evolve independently. Morphological decoupling may help explain habitat diversity among Rhododendron species, as well as the changes accompanying the divergence of deciduous and evergreen lineages., (© 2017 Botanical Society of America.)

Background: Rhododendron chrysanthum Pall., an endangered species with significant ornamental and medicinal value, is endemic to the Changbai Mountain of China and can also serve as a significant plant resource for investigating the stress tolerance in plants. Proteomics is an effective analytical tool that provides significant information about plant metabolism and gene expression. However, no proteomics data have been reported for R. chrysanthum previously. In alpine tundra, the abiotic stress will lead to a severe over-accumulation of reactive oxygen species (ROS). Many alpine plants overcome the severe stresses and protect themselves from the oxidative damage by increasing the ratio and activity of antioxidant enzymes., Results: In our study, wild type and domesticated Rhododendron chrysanthum Pall. were used as experimental and control groups, respectively. Proteomics method combined with biochemical approach were applied for the stress tolerance investigation of R. chrysanthum at both protein and molecular level. A total of 1,395 proteins were identified, among which 137 proteins were up-regulate in the experimental group. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidases (APXs), and glutathione peroxidase (GPX) were significantly higher and the expression of APXs and GPX were also increased in the experimental group. Moreover, the interaction network analysis of these enzymes also reveals that the antioxidant enzymes play important roles in the stress resistance in plants., Conclusions: This is the first report of the proteome of Rhododendron chrysanthum Pall., and the data reinforce the notion that the antioxidant system plays a significant role in plant stress survival. Our results also verified that R. chrysanthum is highly resistant to abiotic stress and can serve as a significant resource for investigating stress tolerance in plants., Reviewers: This article was reviewed by George V. (Yura) Shpakovski and Ramanathan Sowdhamini.

This study investigated altitudinal changes in leaf-lamina hydraulic conductance (K L ) and leaf morphological traits related to K L using five Rhododendron species growing at different altitudes (2500-4500 m above sea level) in Jaljale Himal region in eastern Nepal. Sun leaves were collected from the highest and the lowest altitude populations of each species, and K L was measured with a high pressure flow meter method. Leaf-lamina hydraulic conductance ranged from 7.7 to 19.3 mmol m -2 s -1 MPa -1 and was significantly positively correlated with altitude. The systematic increase with altitude was also found in K L , leaf nitrogen content and stomatal pore index. These relationships suggest that plants from higher-altitude habitats had a large CO 2 supply to the intercellular space in a leaf and high CO 2 assimilation capacity, which enables efficient photosynthesis at high altitude. The variation in K L was associated with the variation in several leaf morphological traits. High K L was found in leaves with small leaf area and round shape, both of which result in shorter major veins. These results suggest that the short major veins were important for efficient water transport in unlobed leaves of Rhododendron species. The extent of lignification in bundle sheaths and bundle sheath extension was associated with K L Lignified compound primary walls inhibit water conduction along apoplastic routes. All species analyzed had heterobaric leaves, in which bundle sheath extensions developed from minor veins, but strongly lignified compound primary walls were found in Rhododendron species with low K L It is still unclear why cell walls in bundle sheath at minor veins were markedly lignified in Rhododendron species growing at lower altitude. The lignified cell wall provides a high pathogenic resistance to infection and increases the mechanical strength of cell wall. The data imply that lignified bundle sheath may provide a trade-off between leaf hydraulic efficiency and leaf mechanical toughness or longevity., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Flower color is one of the major ornamental characteristics of the genus Rhododendron, but few studies on flower color in alpine Rhododendron have been reported. In our study, the flower colors and the pigment constituents of petals from 10 Rhododendron species sampled in the Sygera Mountains of Southeast Tibet were analyzed using high-performance liquid chromatography-diode array detection and mass spectrometry (HPLC-DAD-ESI-MS(2)). The color analysis showed that the 10 Rhododendron species could be divided into five color groupings: yellow, red, red-purple, purple-violet, and purple. A total of 5 anthocyanin compounds and 23 flavonol compounds were tentatively identified and quantified. There were obvious differences in the composition of anthocyanin and flavonol among the petals of the 10 Rhododendron species. The color parameter L* decreased as the TA (total anthocyanin) content increased in the red-purple group. However, there was no obvious correlation between the L* value and the TA content in the other sampled Rhododendron species. In this study, the TA values of most of the Rhododendron species were quite low, but the TF (total flavonol) content was high. These results indicate the existence of copigmentation effects in these 10 Rhododendron species., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Germins and germin-like proteins (GLPs) comprise large families of extracellular plant glycoproteins that are structurally similar, yet they have been reported to have distinct biochemical activities: oxalate oxidase and superoxide dismutase activities, respectively. We expressed an azalea GLP (RmGLP2) in cultured cells of tobacco, and determined that the extracellular protein fraction and the recombinant RmGLP2 protein purified from these cells catalyzed the oxidation of oxalate. Notably, this activity is purportedly restricted to germin and has not been demonstrated for a GLP. Although the specific activity of the purified RmGLP2 protein was low compared with that of a previously characterized barley germin/oxalate oxidase, tobacco cells expressing RmGLP2 exhibited significantly reduced oxalate levels. Thus, RmGLP2 represents the first reported GLP with oxalate oxidase activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rhododendron dauricum L., a flowering tree popular in Hokkaido, produces daurichromenic acid (DCA), a terpenophenol with a potent anti-HIV activity. The DCA-producing enzyme, named DCA synthase, could be detected in the soluble protein fraction prepared from the young leaves of R. dauricum. DCA synthase catalyzed oxidocyclization of the farnesyl group of grifolic acid to form (+)-DCA as the major reaction product. The DCA synthase reaction proceeds without the need for any cofactors and coenzymes except for molecular oxygen. Interestingly, these catalytic properties of DCA synthase are quite similar to those reported for cannabinoid synthases in the marijuana plant Cannabis sativa L.

Farrerol, isolated from Rhododendron dauricum L., has been proven to be an important multifunctional physiologically active component, but its vasoactive mechanism is not clear. The present study was performed to observe the vasoactive effects of farrerol on rat aorta and to investigate the possible underlying mechanisms. Isolated aortic rings of rat were mounted in an organ bath system and the myogenic effects stimulated by farrerol were studied. Intracellular Ca2+ ([Ca2+]in) was measured by molecular probe fluo-4-AM and the activities of L-type voltage-gated Ca2+ channels (LVGC) were studied with whole-cell patch clamp in cultured vascular smooth muscle cells (VSMCs). The results showed that farrerol significantly induced dose-dependent relaxation on aortic rings, while this vasorelaxation was not affected by NG-nitro-l-arginine methylester ester or endothelium denudation. In endothelium-denuded aortas, farrerol also reduced Ca2+-induced contraction on the basis of the stable contraction induced by KCl or phenylephrine (PE) in Ca2+-free solution. Moreover, after incubation with verapamil, farrerol can induce relaxation in endothelium-denuded aortas precontracted by PE, and this effect can be enhanced by ruthenium red, but not by heparin. With laser scanning confocal microscopy method, the farrerol-induced decline of [Ca2+]in in cultured VSMCs was observed. Furthermore, we found that farrerol could suppress Ca2+ influx via LVGC by patch clamp technology. These findings suggested that farrerol can regulate the vascular tension and could be developed as a practicable vasorelaxation drug.

Flower bud dormancy in azalea (Rhododendron simsii) is broken by artificial cold treatment and this will have its consequences on carbon reserves and photosynthesis. The effect of cold storage at 7 °C on carbohydrate and starch content in leaves and flower buds of an early ('Nordlicht') and semi-early ('M. Marie) flowering cultivar was quantified. Carbon loss due to respiration was lowest for 'M. Marie'. Photosynthetic measurements on 'Nordlicht' showed that photosynthesis 3 days after cold treatment (plants ready to flower) was improved compared to before cold treatment (plants with dormant flower buds).

Background: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population., Results: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H., Conclusions: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.