Genome-wide identification and expression analysis of the WRKY gene family in Rhododendron henanense subsp. lingbaoense.

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_42 and WRKY_17 were mainly modulated by the drought and MeJA, and WRKY_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.

Metabolite analysis reveals flavonoids accumulation during flower development in Rhododendron pulchrum sweet (Ericaceae).

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.

Acetylation proteomics and metabolomics analyses reveal the involvement of starch synthase undergoing acetylation modification during UV-B stress resistance in Rhododendron Chrysanthum Pall.

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.

Molecular Mechanism of Exogenous ABA to Enhance UV-B Resistance in Rhododendron chrysanthum Pall. by Modulating Flavonoid Accumulation.

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_DN2213_c0_g1_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.

Deciphering mycobiota and its functional dynamics in root hairs of Rhododendron campanulatum D. Don through Next-gen sequencing.

The Himalayas provide unique opportunities for the extension of shrubs beyond the upper limit of the tree. However, little is known about the limitation of the biotic factors belowground of shrub growth at these cruising altitudes. To fill this gap, the present study deals with the documentation of root-associated microbiota with their predicted functional profiles and interactions in the host Rhododendron campanulatum, a krummholz species. While processing 12 root samples of R. campanulatum from the sites using Omics we could identify 134 root-associated fungal species belonging to 104 genera, 74 families, 39 orders, 17 classes, and 5 phyla. The root-associated microbiota members of Ascomycota were unambiguously dominant followed by Basidiomycota. Using FUNGuild, we reported that symbiotroph and pathotroph as abundant trophic modes. Furthermore, FUNGuild revealed the dominant prevalence of the saptroptroph guild followed by plant pathogens and wood saprotrophs. Alpha diversity was significantly different at the sites. The heatmap dendrogram showed the correlation between various soil nutrients and some fungal species. The study paves the way for a more in-depth exploration of unidentified root fungal symbionts, their interactions and their probable functional roles, which may serve as an important factor for the growth and conservation of these high-altitude ericaceous plants.

Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

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.

Integrated analysis of transcriptome and small RNAome reveals regulatory network of rapid and long-term response to heat stress in Rhododendron moulmainense.

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.

Integrated metabolomics and transcriptomics reveal molecular mechanisms of corolla coloration in Rhododendron dauricum L.

Rhododendron dauricum L. is a semi-evergreen shrub of high ornamental and medicinal values in Northeast China. To study the molecular mechanisms of corolla coloration in R. dauricum, integrated metabolomics and transcriptomics were performed in R. dauricum featuring purple flowers and R. dauricum var. album featuring white flowers. Comparative metabolomics revealed 25 differential metabolites in the corolla of the two distinct colors, enriched in flavonoids that are closely related to pigmentation in the flower. Differential analysis of the transcriptomics data revealed enrichment of structural genes for flavonoid biosynthesis (99 up- and 58 down-regulated, respectively, in purple corollas compared to white ones). Significantly, CHS and CHI, key genes in the early stage of anthocyanin synthesis, as well as F3H, F3'H, F3'5'H, DFR, ANS, and UFGT that promote the accumulation of pigments in the late stage of anthocyanin synthesis, were up-regulated in R. dauricum (purple color). In R. dauricum var. album, FLS were key genes determining the accumulation of flavonols. In addition, transcriptome-metabolome correlation analysis identified 16 R2R3 MYB transcription factors (out of 83 MYBs) that are important for corolla coloration. Five negative and four positive MYBs were further identified by integrated transcriptional and metabolic network analysis, revealing a key role of MYBA and MYB12 in regulating anthocyanins and flavonols, respectively. Moreover, we validated the function of RdMYBA by creating stable transgenic plants and found that RdMYBA promotes anthocyanin biosynthesis. In summary, we systematically characterized the transcriptome and metabolome of two R. dauricum cultivars with different flower colors and identified MYBs as key factors in modulating corolla coloration.

Study on the characteristics of genetic diversity of different populations of Guizhou endemic plant Rhododendron pudingense based on microsatellite markers.

BACKGROUND: Rhododendron pudingense, firstly discovered in Puding county of Guizhou province in 2020, have adapted to living in rocky fissure habitat, which has important ornamental and economic values. However, the genetic diversity and population structure of this species have been rarely described, which seriously affects the collection and protection of wild germplasm resources. RESULTS: In the present study, 13 pairs of primers for polymorphic microsatellite were used to investigate the genetic diversity of 65 R. pudingense accessions from six different geographic populations. A total of 254 alleles (Na) were obtained with an average of 19.5 alleles per locus. The average values of polymorphic information content (PIC), observed heterozygosity (Ho), and expected heterozygosity (He) were 0.8826, 0.4501, and 0.8993, respectively, These results indicate that the microsatellite primers adopted demonstrate good polymorphism, and the R. pudingense exhibits a high level of genetic diversity at the species level. The average genetic differentiation coefficient (Fst) was 0.1325, suggested that moderate divergence occurred in R. pudingense populations. The average values of genetic differentiation coefficient and gene flow among populations were 0.1165 and 3.1281, respectively. The analysis of molecular variance (AMOVA) indicated that most of the population differences (88%) were attributed to within-population variation. The PCoA results are consistent with the findings of the UPGMA clustering analysis, supporting the conclusion that the six populations of R. pudingense can be clearly grouped into two separate clusters. Based on Mantel analysis, we speculate that the PD population may have migrated from WM-1 and WM-2. Therefore, it is advised to protect the natural habitat of R. pudingense in situ as much as possible, in order to maximize the preservation of its genetic diversity. CONCLUSIONS: This is the first comprehensive analysis of genetic diversity and population structure of R. pudingense in Guizhou province. The research results revealed the high genetic diversity and moderate population diferentiation in this horticulture plant. This study provide a theoretical basis for the conservation of wild resources of the R. pudingense and lay the foundation for the breeding or cultivation of this new species.

Abscisic Acid Affects Phenolic Acid Content to Increase Tolerance to UV-B Stress in Rhododendron chrysanthum Pall.

The presence of the ozone hole increases the amount of UV radiation reaching a plant's surface, and UV-B radiation is an abiotic stress capable of affecting plant growth. Rhododendron chrysanthum Pall. (R. chrysanthum) grows in alpine regions, where strong UV-B radiation is present, and has been able to adapt to strong UV-B radiation over a long period of evolution. We investigated the response of R. chrysanthum leaves to UV-B radiation using widely targeted metabolomics and transcriptomics. Although phytohormones have been studied for many years in plant growth and development and adaptation to environmental stresses, this paper is innovative in terms of the species studied and the methods used. Using unique species and the latest research methods, this paper was able to add information to this topic for the species R. chrysanthum. We treated R. chrysanthum grown in a simulated alpine environment, with group M receiving no UV-B radiation and groups N and Q (externally applied abscisic acid treatment) receiving UV-B radiation for 2 days (8 h per day). The results of the MN group showed significant changes in phenolic acid accumulation and differential expression of genes related to phenolic acid synthesis in leaves of R. chrysanthum after UV-B radiation. We combined transcriptomics and metabolomics data to map the metabolic regulatory network of phenolic acids under UV-B stress in order to investigate the response of such secondary metabolites to stress. L-phenylalanine, L-tyrosine and phenylpyruvic acid contents in R. chrysanthum were significantly increased after UV-B radiation. Simultaneously, the levels of 3-hydroxyphenylacetic acid, 2-phenylethanol, anthranilate, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, α-hydroxycinnamic acid and 2-hydroxy-3-phenylpropanoic acid in this pathway were elevated in response to UV-B stress. In contrast, the study in the NQ group found that externally applied abscisic acid (ABA) in R. chrysanthum had greater tolerance to UV-B radiation, and phenolic acid accumulation under the influence of ABA also showed greater differences. The contents of 2-phenylethanol, 1-o-p-coumaroyl-ß-d-glucose, 2-hydroxy-3-phenylpropanoic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-o-feruloylquinic ac-id-o-glucoside were significantly elevated in R. chrysanthum after external application of ABA to protect against UV-B stress. Taken together, these studies of the three groups indicated that ABA can influence phenolic acid production to promote the response of R. chrysanthum to UV-B stress, which provided a theoretical reference for the study of its complex molecular regulatory mechanism.

Transcriptome and photosynthetic analyses provide new insight into the molecular mechanisms underlying heat stress tolerance in Rhododendron × pulchrum Sweet.

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.

Multi-Omic Analysis Reveals the Molecular Mechanism of UV-B Stress Resistance in Acetylated RcMYB44 in Rhododendron chrysanthum.

Ultraviolet-B (UV-B) radiation is a significant environmental factor influencing the growth and development of plants. MYBs play an essential role in the processes of plant responses to abiotic stresses. In the last few years, the development of transcriptome and acetylated proteome technologies have resulted in further and more reliable data for understanding the UV-B response mechanism in plants. In this research, the transcriptome and acetylated proteome were used to analyze Rhododendron chrysanthum Pall. (R. chrysanthum) leaves under UV-B stress. In total, 2348 differentially expressed genes (DEGs) and 685 differentially expressed acetylated proteins (DAPs) were found. The transcriptome analysis revealed 232 MYB TFs; we analyzed the transcriptome together with the acetylated proteome, and screened 4 MYB TFs. Among them, only RcMYB44 had a complete MYB structural domain. To investigate the role of RcMYB44 under UV-B stress, a homology tree was constructed between RcMYB44 and Arabidopsis MYBs, and it was determined that RcMYB44 shares the same function with ATMYB44. We further constructed the hormone signaling pathway involved in RcMYB44, revealing the molecular mechanism of resistance to UV-B stress in R. chrysanthum. Finally, by comparing the transcriptome and the proteome, it was found that the expression levels of proteins and genes were inconsistent, which is related to post-translational modifications of proteins. In conclusion, RcMYB44 of R. chrysanthum is involved in mediating the growth hormone, salicylic acid, jasmonic acid, and abscisic acid signaling pathways to resist UV-B stress.

Aging varies greatly within a single genus: A demographic study of Rhododendron spp. in botanic gardens.

PREMISE: There is mounting evidence that age matters in plant demography, but also indications that relationships between age and demographic rates may vary significantly among species. Age-based plant demographic data, however, are time-consuming to collect and still lacking for most species, and little is known about general patterns across species or what may drive differences. METHODS: We used individual birth and death records for 12 Rhododendron species from botanic gardens and conducted Bayesian survival trajectory analyses to assess how mortality changed with age. We calculated the demographic measures of aging rate, life-span equality, and life expectancy for each species, and assessed their relationships with the climatic conditions at species' sites of ancestral origin and with taxonomic group (subgenus). RESULTS: We found substantial among-species variation in survival trajectories, with mortality increasing, decreasing, or remaining constant with advancing age. Moreover, we found no relationships between demographic measures and ancestral climatic conditions but there were statistically significant differences among taxonomic groups in the rate of change in mortality with age (aging rate). CONCLUSIONS: We conclude that demographic consequences of aging can differ qualitatively, even among species in the same genus. In addition, taxonomic trends in aging rates indicate they may be genetically determined, though evolutionary drivers are still unclear. Furthermore, we suggest there is untapped potential in using botanic garden records in future studies on plant life history.

Small RNA sequencing provides insights into molecular mechanism of flower development in Rhododendron pulchrum Sweet.

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.

Early and Late Transcriptomic and Metabolomic Responses of Rhododendron 'Xiaotaohong' Petals to Infection with Alternaria sp.

In recent years, petal blight disease caused by pathogens has become increasingly epidemic in Rhododendron. Breeding disease-resistant rhododendron is considered to be a more environmentally friendly strategy than is the use of chemical reagents. In this study, we aimed to investigate the response mechanisms of rhododendron varieties to petal blight, using transcriptomics and metabolomics analyses. Specifically, we monitored changes in gene expression and metabolite accumulation in Rhododendron 'Xiaotaohong' petals infected with the Alternaria sp. strain (MR-9). The infection of MR-9 led to the development of petal blight and induced significant changes in gene transcription. Differentially expressed genes (DEGs) were predominantly enriched in the plant-pathogen interaction pathway. These DEGs were involved in carrying out stress responses, with genes associated with H2O2 production being up-regulated during the early and late stages of infection. Correspondingly, H2O2 accumulation was detected in the vicinity of the blight lesions. In addition, defense-related genes, including PR and FRK, exhibited significant up-regulated expression during the infection by MR-9. In the late stage of the infection, we also observed significant changes in differentially abundant metabolites (DAMs), including flavonoids, alkaloids, phenols, and terpenes. Notably, the levels of euscaphic acid, ganoderol A, (-)-cinchonidine, and theophylline in infected petals were 21.8, 8.5, 4.5, and 4.3 times higher, respectively, compared to the control. Our results suggest that H2O2, defense-related genes, and DAM accumulation are involved in the complex response mechanisms of Rhododendron 'Xiaotaohong' petals to MR-9 infection. These insights provide a deeper understanding of the pathogenesis of petal blight disease and may have practical implications for developing disease-resistant rhododendron varieties.

Genome-wide identification and bioinformatics analysis of the WD40 transcription factor family and candidate gene screening for anthocyanin biosynthesis in Rhododendron simsii.

WD40 transcription factors (TFs) constitute a large gene family in eukaryotes, playing diverse roles in cellular processes. However, their functions in the major ornamental plant, Rhododendron simsii, remain poorly understood. In this study, we identified 258 WD40 proteins in the R. simsii genome, which exhibited an uneven distribution across chromosomes. Based on domain compositions and phylogenetic analysis, we classified these 258 RsWD40 proteins into 42 subfamilies and 47 clusters. Comparative genomic analysis suggested that the expansion of the WD40 gene family predates the divergence of green algae and higher plants, indicating an ancient origin. Furthermore, by analyzing the duplication patterns of RsWD40 genes, we found that transposed duplication played a major role in their expansion. Notably, the majority of RsWD40 gene duplication pairs underwent purifying selection during evolution. Synteny analysis identified significant orthologous gene pairs between R. simsii and Arabidopsis thaliana, Oryza sativa, Vitis vinifera, and Malus domestica. We also investigated potential candidate genes involved in anthocyanin biosynthesis during different flower development stages in R. simsii using RNA-seq data. Specifically, we identified 10 candidate genes during the bud stage and 7 candidate genes during the full bloom stage. GO enrichment analysis of these candidate genes revealed the potential involvement of the ubiquitination process in anthocyanin biosynthesis. Overall, our findings provide a valuable foundation for further investigation and functional analysis of WD40 genes, as well as research on the molecular mechanisms underlying anthocyanin biosynthesis in Rhododendron species.

A haplotype-resolved genome assembly of Rhododendron vialii based on PacBio HiFi reads and Hi-C data.

Rhododendron vialii (subgen. Azaleastrum) is an evergreen shrub with high ornamental value. This species has been listed as a plant species with extremely small populations (PSESP) for urgent protection by China's Yunnan provincial government in 2021, due to anthropogenic habitat fragmentation. However, limited genomic resources hinder scientifically understanding of genetic threats that the species is currently facing. In this study, we assembled a high-quality haplotype-resolved genome of R. vialii based on PacBio HiFi long reads and Hi-C reads. The assembly contains two haploid genomes with sizes 532.73 Mb and 521.98 Mb, with contig N50 length of 35.67 Mb and 34.70 Mb, respectively. About 99.92% of the assembled sequences could be anchored to 26 pseudochromosomes, and 14 gapless assembled chromosomes were included in this assembly. Additionally, 60,926 protein-coding genes were identified, of which 93.82% were functionally annotated. This is the first reported genome of R. vialii, and hopefully it will lay the foundations for further research into the conservation genomics and horticultural domestication of this ornamentally important species.

Integration of Phosphoproteomics and Transcriptome Studies Reveals ABA Signaling Pathways Regulate UV-B Tolerance in Rhododendron chrysanthum Leaves.

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.

Identification and characterization of genome-wide long terminal repeat retrotransposons provide an insight into elucidating the trait evolution of five Rhododendron species.

Rhododendron is well-known for its beauty and colourful corolla. Although some high-quality whole-genome sequencing of it has been completed, there are few studies on long terminal repeat (LTR) retrotransposons in Rhododendron, which limits our ability to elucidate the causes of genetic variations in Rhododendron species. Properties of the intact Rhododendron LTR retrotransposons were investigated at a genome-wide level. Based on available data, the high-quality genomes from five species, i.e. R. griersonianum, R. simsii, R. henanense subsp. lingbaoense, R. mucronatum var. ripense and R. ovatum, were selected as targets with good assembly continuity. A total of 17,936 intact LTR retrotransposons were identified; these belong to superfamilies Copia and Gypsy, with 17 clades. The insertion time of these transposons was later than 120 million years ago (Mya), and the outbreak period was concentrated more recently than 30 Mya. Phylogenetic analysis revealed that many LTR retrotransposons might originate from intraspecific duplication. Current evidence also suggests that most LTR retrotransposons were inserted in the interstitial part of genes in R. griersonianum, R. simsii, R. henanense, and R. ovatum, and the functions of the inserted genes mainly involve starch metabolism, proteolysis, etc. The effect of the LTR retrotransposon on gene expression depends on its insertion site and activation. Highly expressed LTR retrotransposons tend to be younger. The results herein improve our knowledge of LTR retrotransposons in Rhododendron genomes and facilitate further study of genetic variation and trait evolution in Rhododendron.

IRAPs in Combination with Highly Informative ISSRs Confer Effective Potentials for Genetic Diversity and Fidelity Assessment in Rhododendron.

The species belonging to the Rhododendron genus are well-known for their colorful corolla. Molecular marker systems have the potential to elucidate genetic diversity as well as to assess genetic fidelity in rhododendrons. In the present study, the reverse transcription domains of long terminal repeat retrotransposons were cloned from rhododendrons and used to develop an inter-retrotransposon amplified polymorphism (IRAP) marker system. Subsequently, 198 polymorphic loci were generated from the IRAP and inter-simple sequence repeat (ISSR) markers, of which 119 were derived from the IRAP markers. It was shown that in rhododendrons, IRAP markers were superior to the ISSRs in some polymorphic parameters, such as the average number of polymorphic loci (14.88 versus 13.17). The combination of the IRAP and ISSR systems was more discriminative for detecting 46 rhododendron accessions than each of the systems on their own. Furthermore, IRAP markers demonstrated more efficiency in genetic fidelity detection of in-vitro-grown R. bailiense Y.P.Ma, C.Q.Zhang and D.F.Chamb, an endangered species recently recorded in Guizhzhou Province, China. The available evidence revealed the distinct properties of IRAP and ISSR markers in the rhododendron-associated applications, and highlighted the availability of highly informative ISSR and IRAP markers in the evaluation of genetic diversity and genetic fidelity of rhododendrons, which may facilitate preservation and genetic breeding of rhododendron plants.