In vitro regeneration, photomorphogenesis and light signaling gene expression in Hydrangea quercifolia cv. 'Harmony' under different LED environments.

MAIN CONCLUSION: Plant growth regulators, sucrose concentration, and light quality significantly impact in vitro regeneration of 'Harmony'. Blue light promotes photomorphogenesis by enhancing light energy utilization, adjusting transcription of light signal genes, and altering hormone levels. Hydrangea quercifolia cv. 'Harmony', celebrated for lush green foliage and clusters of white flowers, has been extensively researched for its regenerative properties. Regeneration in stem segments, leaves, and petioles is facilitated by exogenous auxin and cytokinins (CTKs), with the concentration of sucrose (SC) being a key determinant for shoot regeneration from leaves. The study also highlights the significant impact of light conditions on photomorphogenesis. With an increase in the proportion of red (R) light, there is an inhibitory effect, leading to a reduction in leaf area, a decrease in the quantum yield of PSII (ΦPSII), and an increase in non-photochemical quenching (ΦNPQ) and non-regulated energy dissipation in PSII (ΦNO). Conversely, blue (B) light enhances growth, characterized by an increase in leaf area, elevated ΦPSII, and stable ΦNPQ and ΦNO levels. Additionally, B light induces the upregulation of HqCRYs, HqHY5-like, HqXTH27-like, and HqPHYs genes, along with an increase in endogenous CTKs levels, which positively influence photomorphogenesis independent of HqHY5-like regulation. This light condition also suppresses the synthesis of endogenous gibberellins (GA) and brassinosteroids (BR), further facilitating photomorphogenesis. In essence, B light is fundamental in expediting photomorphogenesis in 'Harmony', demonstrating the vital role in plant growth and development.

Regulation of blue infertile flower pigmentation by WD40 transcription factor HmWDR68 in Hydrangea macrophylla 'forever summer'.

BACKGROUND: WD40 transcription factors are crucial in plant growth and developmental, significantly impacting plant growth regulation. This study investigates the WD40 transcription factor HmWDR68's role in developing the distinctive blue infertile flower colors in Hydrangea macrophylla 'Forever Summer'. METHODS AND RESULTS: The HmWDR68 gene was isolated by PCR, revealing an open reading frame of 1026 base pairs, which encodes 341 amino acids. Characterized by four WD40 motifs, HmWDR68 is a member of the WD40 family. Phylogenetic analysis indicates that HmWDR68 shares high homology with PsWD40 in Camellia sinensis and CsWD40 in Paeonia suffruticosa, both of which are integral in anthocyanin synthesis regulation. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that HmWDR68 expression in the blue infertile flowers of 'Forever Summer' hydrangea was significantly higher compared to other tissues and organs. Additionally, in various hydrangea varieties with differently colored infertile flowers, HmWDR68 expression was markedly elevated in comparison to other hydrangea varieties, correlating with the development of blue infertile flowers. Pearson correlation analysis revealed a significant association between HmWDR68 expression and the concentration of delphinidin 3-O-glucoside, as well as key genes involved in anthocyanin biosynthesis (HmF3H, HmC3'5'H, HmDFR, and HmANS) in the blue infertile flowers of 'Forever Summer' hydrangea (P < 0.01). CONCLUSION: These findings suggest HmWDR68 may specifically regulate blue infertile flower formation in hydrangea by enhancing delphinidin-3-O-glucoside synthesis, modulating expression of HmF3H, HmC3'5'H, HmDFR and HmANS. This study provides insights into HmWDR68's role in hydrangea's blue flowers development, offering a foundation for further research in this field.

Metabolomics reveals a key role of salicylic acid in embryo abortion underlying interspecific hybridization between Hydrangea macrophylla and H. arborescens.

KEY MESSAGE: Embryo abortion at the heart-shaped stage is the main reason for the failure of interspecific hybridization of hydrangea, and salicylic acid plays a key role during embryo abortion. Difficulties in obtaining seeds from interspecific hybridization between Hydrangea macrophylla and H. arborescens had severely restricted the process of breeding new hydrangea varieties. To clarify the cause of reproductive barriers, an interspecific hybridization was made between H. macrophylla 'Endless Summer' (female parent) and H. arborescens 'Annabelle' (male parent). The results showed that both parents' floral organs developed normally, 'Annabelle' had high pollen viability (84.83% at 8 h after incubation), and the pollen tube could enter into the ovule of 'Endless Summer' at 72 h after pollination. Therefore, the pre-fertilization barrier was not the main reason for the failure of interspecific hybridization. However, observation of the embryo development by paraffin sections showed that the embryo was aborted at the heart-shaped stage. In addition, salicylic acid (SA) content was significantly higher (fourfold, P < 0.01) at 21 days after pollination (DAP) as compared to that of 17 DAP, which means SA may be closely correlated with embryo development. A total of 957 metabolites were detected, among which 78 were significantly different. During the embryo abortion, phenylpropanoids and polyketides were significantly down-regulated, while organic oxygen compounds were significantly up-regulated. Further analysis indicated that the metabolic pathway was enriched in the shikimic acid biosynthesis pathway, which suggests that more SA was synthesized. Taken together, it can be reasonably speculated that SA plays a key role leading to embryo abortion underlying the interspecific hybridization between Hydrangea macrophylla and H. arborescens. The result is helpful to direct the breeding of hydrangea through distant hybridization.

Phyllodulcin from the hexane fraction of Hydrangea macrophylla inhibits glucose-induced lipid accumulation and reactive oxygen species generation in Caenorhabditis elegans.

We confirmed that the hexane layer of Hydrangea macrophylla leaf extract (HLH) is rich in phyllodulcin (PD), an alternative sweetener, through high performance liquid chromatography (HPLC) analysis. To investigate in vivo activity of HLH and its PD, acute toxicity and growth rate of Caenorhabditis elegans were tested and there are no clinical abnormalities at 125-500 µg/mL of HLH. HLH decreased the total lipid and triglyceride contents dose-dependently in glucose-induced obese worms. Also, HLH increased survival rates under oxidative and thermal stress and decreased body reactive oxygen species (ROS) contents significantly. Such antioxidant properties of HLH were attributed to the enhanced activity of the antioxidant enzyme catalase. To determine whether the effect of HLH was due to PD, worms were treated with PD (concentration contained in HLH), and inhibitory effects on total lipids and ROS were observed. Our results suggest that HLH and its PD as a natural alternative sweetener can be used as materials to improve metabolic diseases.

Hydrangea-like TiO2/Bi2MoO6 porous nanoflowers triggering highly sensitive electrochemical immunosensing to tumor marker.

Rapid and sensitive detection of carcinoembryonic antigen (CEA) is of great significance for cancer patients. Here, molybdenum (Mo) was doped into bismuth oxide (Bi2O3) by one-pot hydrothermal method forming porous tremella Bi2MoO6 nanocomposites with a larger specific surface area than the spherical structure. Then, a new kind of hydrangea-like TiO2/Bi2MoO6 porous nanoflowers (NFs) was prepared by doping titanium into Bi2MoO6, where titanium dioxide (TiO2) grew in situ on the surface of Bi2MoO6 nanoparticles (NPs). The hydrangea-like structure provides larger specific surface area, higher electron transfer ability and biocompatibility as well as more active sites conducive to the attachment of anti-carcinoembryonic antigen (anti-CEA) to TiO2/Bi2MoO6 NFs. A novel label-free electrochemical immunosensor was then constructed for the quantitative detection of CEA using TiO2/Bi2MoO6 NFs as sensing platform, showing a good linear relationship with CEA in the concentration range 1.0 pg/mL ~ 1.0 mg/mL and a detection limit of 0.125 pg/mL (S/N = 3). The results achieved with the designed immunosensor are comparable with many existing immunosensors used for the detection of CEA in real samples.

Integrated metabolome and transcriptome analysis provides insights on the floral scent formation in Hydrangea arborescens.

Hydrangea (Hydrangea arborescens var. "Annabelle") flowers are composed of sweet aroma sepals rather than true petals and can change color. Floral volatiles plays important roles in plants, such as attracting pollinators, defending against herbivores, and signaling. However, the biosynthesis and regulatory mechanisms underlying fragrance formation in H. arborescens during flower development remain unknown. In this study, a combination of metabolite profiling and RNA sequencing (RNA-seq) was employed to identify genes associated with floral scent biosynthesis mechanisms in "Annabelle" flowers at three developmental stages (F1, F2, and F3). The floral volatile data revealed that the "Annabelle" volatile profile includes a total of 33 volatile organic compounds (VOCs), and VOCs were abundant during the F2 stage of flower development, followed by the F1 and F3 stages, respectively. Terpenoids and benzenoids/phenylpropanoids were abundant during the F2 and F1 stages, with the latter being the most abundant, whereas fatty acid derivatives and other compounds were found in large amounts during the F3 stage. According to ultra-performance liquid chromatography-tandem mass spectrometer analysis, benzene and substituted derivatives, carboxylic acids and derivatives, and fatty acyls play a significant role in the floral metabolite profile. The transcriptome data revealed a total of 17,461 differentially expressed genes (DEGs), with 7585, 12,795, and 9044 DEGs discovered between the F2 and F1, F3 and F1, and F2 and F3 stages, respectively. Several terpenoids and benzenoids/phenylpropanoids biosynthesis-related DEGs were identified, and GRAS/bHLH/MYB/AP2/WRKY were more abundant among transcription factors. Finally, DEGs interlinked with VOCs compounds were determined using Cytoscape and k-means analysis. Our results pave the way for the discovery of new genes, critical data for future genetic studies, and a platform for the metabolic engineering of genes involved in the production of Hydrangea's signature floral fragrance.

Dihydroisocoumarins of Hydrangea macrophylla var. thunbergia inhibit binding of the SARS-CoV-2 spike protein to ACE2.

Binding of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the cognate angiotensin-converting enzyme 2 (ACE2) receptor is the initial step in the viral infection process. In this study, we screened an in-house extract library to identify food materials with inhibitory activity against this binding using enzyme-linked immunosorbent assays and attempted to ascertain their active constituents. Hydrangea macrophylla var. thunbergia leaves were identified as candidate materials. Its active compounds were purified using conventional chromatographic methods and identified as naringenin, dihydroisocoumarins, hydrangenol, and phyllodulcin, which have affinities for the ACE2 receptor and inhibit ACE2 receptor-spike S1 binding. Given that boiled water extracts of H. macrophylla leaves are commonly consumed as sweet tea in Japan, we speculated that this tea could be used as a potential natural resource to reduce the risk of SARS-CoV-2 infection.

Production of Dihydroisocoumarins by Callus Induction from Hydrangea macrophylla var. thunbergii Leaves.

Dihydroisocoumarins, hydrangenol 8-O-ß-D-glucopyranoside (1), phyllodulcin 8-O-ß-D-glucopyranoside (2), hydrangenol (3), and phyllodulcin (4), are well-known as the major secondary metabolites in the leaves of Hydrangea macrophylla var. thunbergii. Dihydroisocoumarins are pharmaceutical compounds with diverse bioactivity. Although dihydroisocoumarins are commonly isolated from Hydrangea plants or via organic chemical synthesis, their production via callus induction is considered a promising alternative. In the present study, callus induction and proliferation of H. macrophylla var. thunbergii, and constituents 1-4 were quantified in calluses cultured in 17 different media. We found that the combination of the phytohormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA) was useful for callus proliferation in H. macrophylla var. thunbergii. The balance and concentrations of indole-3-acetic acid (IAA) and BA greatly affected the contents of 1-4. Particularly, 1 (2.03-3.46% yield from the dry callus) was successfully produced from the callus induced by IAA (0.5 mg/L) and BA (1.0 mg/L) at yields comparable to isolated yields from plants. To the best of our knowledge, this is the first study to show that the calluses of H. macrophylla var. thunbergii contained 1. These findings may be useful for producing bioactive dihydroisocoumarins.

Comparison of Growth in Hydrangea macrophylla var. thunbergii Grown in Different Soil pH and Quantitative Analysis of Its Sweetness-Related Constituents.

Dried and fermented (processed) leaves of Hydrangea macrophylla Seringe var. thunbergii Makino (Hydrangeae Dulcis Folium) are currently used as a crude drug with a sweet taste for diabetic patients and as an oral refrigerant. The sweet taste of this crude drug is primarily attributed to phyllodulcin. However, there are currently no standards for the cultivation of H. macrophylla var. thunbergii and the isolation and production of the primary constituents of this crude drug. In the present study, we prepared five types of soils with different pH values (pH 7.5-5.0) and investigated the effects of these soils on the growth of this plant. The contents of phyllodulcin and its glycoside, phyllodulcin 8-O-ß-D-glucopyranoside, in the leaves of plants grown in these soils were quantified. Furthermore, the correlation between the sweetness of Hydrangeae Dulcis Folium and phyllodulcin was investigated. The results showed that soils with pH ranging from 7.0 to 5.5 was not only suitable for plant growth but also increased the content of phyllodulcin and phyllodulcin 8-O-ß-D-glucopyranoside in the leaves. Altogether, these findings could be useful for the development of high-quality Hydrangeae Dulcis Folium.

Evaluation of Hydrangea Cultivars for Tolerance Against Root Rot Caused by Fusarium oxysporum.

Root rot caused by Fusarium oxysporum Schltdl. is a newly identified disease in oakleaf hydrangea. Some cultivars such as Pee Wee and Queen of Hearts grown in pot-in-pot container systems showed root rot symptoms after late spring frost in May 2018 with 40 and 60% incidence in the infected nursery, respectively. This experiment was carried out to evaluate the tolerance among different hydrangea cultivars against root rot caused by F. oxysporum. Fifteen hydrangea cultivars from four different species were selected, and rooted cuttings were prepared from new spring flushes. Twelve plants from each cultivar were transplanted in a 1-gallon pot. Half of transplanted plants (six single plants) were inoculated by drenching 150 ml of F. oxysporum conidial suspension to maintain the concentration of 1 × 106 conidia/ml. Half of the plants remain noninoculated (control) and were drenched with sterile water. After 4 months, root rot was assessed using a scale of 0 to 100% root area affected, and recovery of F. oxysporum was recorded by plating 1-cm root sections in Fusarium selective medium. Fusaric acid (FA) and mannitol were extracted from the roots of inoculated and noninoculated plants to see the effect and role on pathogenesis. Further, mannitol concentration was analyzed using absorption wavelength in a spectrophotometer, and FA was analyzed using high-performance liquid chromatography (HPLC). Results indicated that no cultivars were resistant to F. oxysporum. Cultivars from Hydrangea arborescens, H. macrophylla, and H. paniculata were more tolerant to F. oxysporum compared to cultivars from H. quercifolia. Among H. quercifolia, cultivars Snowflake, John Wayne, and Alice were more tolerant to F. oxysporum.

Identification and Chemical and Biological Management of Fusarium Root and Crown Rot Disease of Oakleaf Hydrangea.

Oakleaf hydrangea (Hydrangea quercifolia) is an important ornamental plant grown in Tennessee. In May 2018, after late spring frost, cultivars Pee Wee and Queen of Hearts showed root and crown rot symptoms and identification and management of the disease was a major concern. The objective of this research was to identify the causal organism of this disease and develop management recommendations for nursery growers. Isolates from the infected root and crown parts were subjected to microscopy, and the morphology of fungi resembled Fusarium. Molecular analysis was conducted by amplifying the internal transcribed spacer of ribosomal DNA, ß-tubulin, and translation elongation factor 1-α regions. Fusarium oxysporum was identified as a causal organism based on molecular analysis. A pathogenicity test was done to complete the Koch's postulates by drenching containerized oakleaf hydrangea with a conidial suspension. Experiments were conducted to evaluate different chemical fungicides and biological products with different rates for Fusarium root and crown rot management in container-grown Queen of Hearts. Plants were inoculated by drenching containerized oakleaf hydrangea with 150-ml conidial suspensions of F. oxysporum, maintaining the concentration of 1 × 106 conidia/ml. Root and crown rot were assessed using a scale of 0 to 100%. Recovery of F. oxysporum was recorded by plating root and crown sections. Chemical fungicides such as mefentrifluconazole (BAS75002F), the low rate (1.09 ml/liter) of difenoconazole + pydiflumetofen (Postiva), and the high rate (1.32 ml/liter) of isofetamid (Astun) and biopesticide were applied; the high rate (1.64 g/liter) of ningnanmycin (SP2700 WP) effectively reduced Fusarium root rot severity and pyraclostrobin effectively reduced Fusarium crown rot severity in both trials.

Feeding potential of adult Systena frontalis (Coleoptera: Chrysomelidae) on leaves of Hydrangea paniculata (Cornales: Hydrangeaceae).

Systena frontalis (F.) (Coleoptera: Chrysomelidae), also known as the red-headed flea beetle, is a defoliating pest of a variety of crop systems, such as ornamentals and food crops. Leaf consumption by this beetle renders ornamental nursery plants, such as hydrangeas (Hydrangea paniculata Siebold, Hydrangeaceae), unsaleable. In Virginia, this insect has become a major pest at commercial nurseries, and their feeding potential on affected crops has not been quantified. In this study, the extent of their damage to individual leaves and host preference between leaf ages were determined. The rate of defoliation on mature and young hydrangea leaves was measured over 24 and 48 h and between different numbers of adults. A single adult caused up to 10% damage to a young leaf or 5% to a whole mature leaf in 24 h. Without choice, there was a higher percent damage to young leaves. When the size of leaves was controlled by cut-out mature leaves, the area damaged was still higher in young leaves when compared with mature leaves. Adult feeding between mature or young leaves was further investigated by choice assays on a caged plant and within a containerized system. In these choice assays, adults inflicted higher percent damage on mature leaves in both caged plant assays and containerized direct choice assays. The choice assays were more similar to field conditions than the nonchoice assays. This demonstrates that S. frontalis showed a preference for mature leaves over young leaves within hydrangeas.

Study on the Flower Induction Mechanism of Hydrangea macrophylla.

The flower induction of Hydrangea macrophylla "Endless Summer" is regulated by a complex gene network that involves multiple signaling pathways to ensure continuous flowering throughout the growing season, but the molecular determinants of flower induction are not yet clear. In this study, genes potentially involved in signaling pathway mediating the regulatory mechanism of flower induction were identified through the transcriptomic profiles, and a hypothetical model for this regulatory mechanism was obtained by an analysis of the available transcriptomic data, suggesting that sugar-, hormone-, and flowering-related genes participated in the flower induction process of H. macrophylla "Endless Summer". The expression profiles of the genes involved in the biosynthesis and metabolism of sugar showed that the beta-amylase gene BAM1 displayed a high expression level at the BS2 stage and implied the hydrolysis of starch. It may be a signaling molecule that promotes the transition from vegetative growth to reproductive growth in H. macrophylla "Endless Summer". Complex hormone regulatory networks involved in abscisic acid (ABA), auxin (IAA), zeatin nucleoside (ZR), and gibberellin (GA) also induced flower formation in H. macrophylla. ABA participated in flower induction by regulating flowering genes. The high content of IAA and the high expression level of the auxin influx carrier gene LAX5 at the BS2 stage suggested that the flow of auxin between sources and sinks in H. macrophylla is involved in the regulation of floral induction as a signal. In addition, flowering-related genes were mainly involved in the photoperiodic pathway, the aging pathway, and the gibberellin pathway. As a result, multiple pathways, including the photoperiodic pathway, the aging pathway, and the gibberellin pathway, which were mainly mediated by crosstalk between sugar and hormone signals, regulated the molecular network involved in flower induction in H. macrophylla "Endless Summer".

Comparative physiology and transcriptome analysis reveals that chloroplast development influences silver-white leaf color formation in Hydrangea macrophylla var. maculata.

BACKGROUND: Hydrangea macrophylla var. Maculata 'Yinbianxiuqiu' (YB) is an excellent plant species with beautiful flowers and leaves with silvery white edges. However, there are few reports on its leaf color characteristics and color formation mechanism. RESULTS: The present study compared the phenotypic, physiological and transcriptomic differences between YB and a full-green leaf mutant (YM) obtained from YB. The results showed that YB and YM had similar genetic backgrounds, but photosynthesis was reduced in YB. The contents of pigments were significantly decreased at the edges of YB leaves compared to YM leaves. The ultrastructure of chloroplasts in the YB leaves was irregular. Transcriptome profiling identified 7,023 differentially expressed genes between YB and YM. The expression levels of genes involved in photosynthesis, chloroplast development and division were different between YB and YM. Quantitative real-time PCR showed that the expression trends were generally consistent with the transcriptome data. CONCLUSIONS: Taken together, the formation of the silvery white leaf color of H. macrophylla var. maculata was primarily due to the abnormal development of chloroplasts. This study facilitates the molecular function analysis of key genes involved in chloroplast development and provides new insights into the molecular mechanisms involved in leaf coloration in H. macrophylla.

Physiological and metabolomics responses of Hydrangea macrophylla (Thunb.) Ser. and Hydrangea strigosa Rehd. to lead exposure.

Hydrangea is a potential remediation plant for lead (Pb) pollution. Plant roots communicate with soil through the release of root exudates. It is crucial to study rhizoremediation mechanisms to understand the response of root exudates to contamination stress. Here, we investigated the physiological responses and metabolomic profiling of two Hydrangea species, a horticultural cultivar (Hydrangea macrophylla (Thunb.) Ser.) and a wild type (Hydrangea strigosa Rehd.), under Pb-free and Pb-stressed conditions for 50 days. The results showed that Pb treatment adversely affected the biomass and root growth of the two species. H. strigosa was a Pb-tolerant species with higher superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities and more ascorbic acid (AsA) content in roots. Metabolomic profiling showed that 181 and 169 compounds were identified in H. macrophylla and H. strigosa root exudates, respectively, among which 18 showed significant differences between H. macrophylla and H. strigosa under Pb exposure. H. strigosa showed significantly (P < 0.05) higher secretion of sucrose, glycolic acid, and nonanoic acid than H. macrophylla after Pb treatment. Pb stress promoted fatty acid metabolism in H. strigosa, suppressed amino acid metabolism in H. macrophylla, and promoted a higher carbohydrate metabolism in H. strigosa compared with H. macrophylla. This study provides a possible mechanism for the high Pb absorption potential of Hydrangea.

Comparative Transcriptome Analysis Unveils the Molecular Mechanism Underlying Sepal Colour Changes under Acidic pH Substratum in Hydrangea macrophylla

The hydrangea (Hydrangea macrophylla (Thunb). Ser.), an ornamental plant, has good marketing potential and is known for its capacity to change the colour of its inflorescence depending on the pH of the cultivation media. The molecular mechanisms causing these changes are still uncertain. In the present study, transcriptome and targeted metabolic profiling were used to identify molecular changes in the RNAome of hydrangea plants cultured at two different pH levels. De novo assembly yielded 186,477 unigenes. Transcriptomic datasets provided a comprehensive and systemic overview of the dynamic networks of the gene expression underlying flower colour formation in hydrangeas. Weighted analyses of gene co-expression network identified candidate genes and hub genes from the modules linked closely to the hyper accumulation of Al3+ during different stages of flower development. F3'5'H, ANS, FLS, CHS, UA3GT, CHI, DFR, and F3H were enhanced significantly in the modules. In addition, MYB, bHLH, PAL6, PAL9, and WD40 were identified as hub genes. Thus, a hypothesis elucidating the colour change in the flowers of Al3+-treated plants was established. This study identified many potential key regulators of flower pigmentation, providing novel insights into the molecular networks in hydrangea flowers.

Exploring the Molecular Mechanism of Sepal Formation in the Decorative Flowers of Hydrangea macrophylla 'Endless Summer' Based on the ABCDE Model.

With its large inflorescences and colorful flowers, Hydrangea macrophylla has been one of the most popular ornamental plants in recent years. However, the formation mechanism of its major ornamental part, the decorative floret sepals, is still not clear. In this study, we compared the transcriptome data of H. macrophylla 'Endless Summer' from the nutritional stage (BS1) to the blooming stage (BS5) and annotated them into the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases. The 347 identified differentially expressed genes (DEGs) associated with flower development were subjected to a trend analysis and a protein-protein interaction analysis. The combined analysis of the two yielded 60 DEGs, including four MADS-box transcription factors (HmSVP-1, HmSOC1, HmAP1-2, and HmAGL24-3) and genes with strong connectivity (HmLFY and HmUFO). In addition, 17 transcription factors related to the ABCDE model were screened, and key candidate genes related to the development of decorative floret sepals in H. macrophylla were identified by phylogenetic and expression pattern analysis, including HmAP1-1, HmAP1-2, HmAP1-3, HmAP2-3, HmAP2-4, and HmAP2-5. On this basis, a gene regulatory network model of decorative sepal development was also postulated. Our results provide a theoretical basis for the study of the formation mechanism of decorative floret sepals and suggest a new direction for the molecular breeding of H. macrophylla.

Separation of Dihydro-Isocoumarins and Dihydro-Stilbenoids from Hydrangea macrophylla ssp. serrata by Use of Counter-Current Chromatography.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar 'Yae-no-amacha' using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.

Single-cell analysis clarifies mosaic color development in purple hydrangea sepal.

Hydrangea sepals exhibit a wide range of colors, from red, through purple, to blue; the purple color is a color mosaic. However, all of these colors are derived from the same components: simple anthocyanins, 3-O-glycosyldelphinidins, three co-pigment components, acylquinic acids and aluminum ions (Al3+ ). We show the color mosaic is a result of graded differences in intravacuolar factors. In order to clarify the mechanisms of mosaic color, we performed single-cell analyses of vacuolar pH, and anthocyanin, co-pigment and Al3+ content. From the sepals, a protoplast mixture of various colors was obtained. The cell color was evaluated by microspectrophotometry and vacuolar pH then was recorded by using a pH microelectrode. The organic and Al3+ contents were quantified by micro-HPLC. We found that the bluer the cell, the greater the ratio of 5-O-acylquinic acids and Al3+ to anthocyanins. Furthermore, reproducing experiments were conducted by mixing the components under various pH condition; all the colors could be reproduced in the various mixing conditions. Based on the above, we provide experimental evidence for cell color variation in hydrangea. Our study demonstrates the expression of phenotypic differences without any direct genomic control.

Inferring historical survivals of climate relicts: the effects of climate changes, geography, and population-specific factors on herbaceous hydrangeas.

Climate relicts hold considerable importance because they have resulted from numerous historical changes. However, there are major interspecific variations among the ways by which they survived climate changes. Therefore, investigating the factors and timing that affected population demographics can expand our understanding of how climate relicts responded to historical environmental changes. Here, we examined herbaceous hydrangeas of genus Deinanthe in East Asia, which show limited distributions and a remarkable disjunction between Japan and central China. Chloroplast genome and restriction site-associated DNA sequencing revealed that speciation event occurred in the late Miocene (ca. 7-9 Mya) in response to global climate change. Two lineages apparently remained not branched until the middle Quaternary, and afterwards started to diverge to regional population groups. The narrow endemic species in central China showed lower genetic diversity (He = 0.082), as its population size rapidly decreased during the Holocene due to isolation in montane refugia. Insular populations in the three Japanese islands (He = 0.137-0.160) showed a genetic structure that was inconsistent with sea barriers, indicating that it was shaped in the glacial period when its range retreated to coastal refugia on the exposed sea floor. Demographic modelling by stairway-plot analysis reconstructed variable responses of Japanese populations: some experienced glacial bottlenecks in refugial isolation, while post-glacial range expansion seemingly exerted founder effects on other populations. Overall, this study demonstrated the involvement of not just one, but multiple factors, such as the interplay between climate changes, geography, and other population-specific factors, that determine the demographics of climate relicts.