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.

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.

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.

Targeted generation of polyploids in Hydrangea macrophylla through cross-based breeding.

BACKGROUND: Up to now, diploid and triploid cultivars were reported for the ornamental crop Hydrangea macrophylla. Especially, the origin of triploids and their crossing behaviors are unknown, but the underlying mechanisms are highly relevant for breeding polyploids. RESULTS: By screening a cultivar collection, we identified diploid, triploid, tetraploid and even aneuploid H. macrophylla varieties. The pollen viability of triploids and tetraploids was comparable to that of diploids. Systematic crosses with these cultivars resulted in viable diploid, triploid, tetraploid and aneuploid offspring. Interestingly, crosses between diploids produced diploid and 0 or 1-94% triploid offspring, depending on the cultivars used as pollen parent. This finding suggests that specific diploids form unreduced pollen, either at low or high frequencies. In contrast, crosses of triploids with diploids or tetraploids produced many viable aneuploids, whose 2C DNA contents ranged between the parental 2C values. As expected, crosses between diploid and tetraploid individuals generated triploid offspring. Putative tetraploid plants were obtained at low frequencies in crosses between diploids and in interploid crosses of triploids with either diploid or tetraploid plants. The analysis of offspring populations indicated the production of 1n = 2x gametes for tetraploid plants, whereas triploids produced obviously reduced, aneuploid gametes with chromosome numbers ranging between haploid and diploid level. While euploid offspring grew normally, aneuploid plants showed mostly an abnormal development and a huge phenotypic variation within offspring populations, most likely due to the variation in chromosome numbers. Subsequent crosses with putative diploid, triploid and aneuploid offspring plants from interploid crosses resulted in viable offspring and germination rates ranging from 21 to 100%. CONCLUSIONS: The existence of diploids that form unreduced pollen and of tetraploids allows the targeted breeding of polyploid H. macrophylla. Different ploidy levels can be addressed by combining the appropriate crossing partners. In contrast to artificial polyploidization, cross-based polyploidization is easy, cheap and results in genetically variable offspring that allows the direct selection of more robust and stress tolerant polyploid varieties. Furthermore, the generation of polyploid H. macrophylla plants will favor interspecific breeding programs within the genus Hydrangea.

Rapid identification of inflorescence type markers by genotyping-by-sequencing of diploid and triploid F1 plants of Hydrangea macrophylla.

BACKGROUND: The ornamental crop Hydrangea macrophylla develops highly attractive lacecap (wild type) or mophead inflorescences. The mophead trait, which is mostly favored by consumers, is recessively inherited by the INFLORESCENCE TYPE locus (INF). If lacecap cultivars are crossed with mophead cultivars, then either 50% or all progenies develop lacecap inflorescences, depending on the zygosity at the INF locus. For most cultivars, the zygosity at the INF locus is unknown. Furthermore, the determination of the inflorescence type in offspring populations is time-consuming, because seedlings flower the first time in the 2nd year after sowing. Within this study, we aimed to develop DNA-based markers that allow to determine the zygosity at the INF locus of prospective parental plants and to predict the inflorescence phenotype of seedlings already in the non-flowering stage. RESULTS: By crossing a mophead and a lacecap cultivar of H. macrophylla, we produced a pseudo-backcross F1 population consisting of 422 plants. These plants segregated into 279 lacecap, 73 mophead, 3 intermediate and 67 non-flowering plants, differing significantly from the expected 1:1 segregation ratio. Surprisingly, 75% of these plants were triploid, although both parents were diploid. We found that the lacecap parent produced unreduced pollen, which induced the formation of triploids. 380 randomly selected F1 plants were genotyped by genotyping-by-sequencing (GbS). Using a genome assembly of cultivar 'Sir Joseph Banks', we performed subsequently a bulk sequence analysis with pooled GbS data of diploid versus mophead plants. We identified directly 2 markers tightly linked with the INF locus, each of them explaining 99.7% of the inflorescence phenotype. Using a collection consisting of 56 diploid, triploid or tetraploid H. macrophylla varieties, we detected 6 sequence variants for one of these markers. Two variants were associated with the mophead phenotype. Furthermore, we found by marker analysis a co-segregation between the mophead and the non-flowering trait, which indicates a major flowering time locus next to the INF locus. CONCLUSION: Through bulk sequence analysis of pooled GbS data from diploid and polyploid F1 plants, we identify rapidly tightly linked markers for the inflorescence type, a dominant-recessively inherited trait in the non-model plant species H. macrophylla.

Multilocus coalescent species delimitation to evaluate traditionally defined morphotypes in Hydrangea sect. Asperae (Hydrangeaceae).

The number of species recognized in section Asperae of the flowering plant genus Hydrangea differs widely between subsequent revisions. This variation is largely centered around the H. aspera species complex, with numbers of recognized species varying from one to nearly a dozen. Despite indications of molecular variation in this complex, no sequence-based species delimitation methods have been employed to evaluate the primarily morphology-based species boundaries. In the present study, a multi-locus coalescent-based approach to species delimitation is employed in order to identify separate evolutionary lines within H. sect. Asperae, using four chloroplast and four nuclear molecular markers. Eight lineages were recovered within the focal group, of which five correspond with named morphotypes. The other three lineages illustrate types of conflict between molecular species delimitation and traditional morphology-based taxonomy. One molecular lineage comprises two named morphotypes, which possibly diverged recently enough to not have developed sufficient molecular divergence. A second conflict is found in H. strigosa. This morphotype is recovered as a separate lineage when occurring in geographic isolation, but when occurring in sympatry with two other morphotypes (H. aspera and H. robusta), the coalescent species delimitation lumps these taxa into a single putative species.

A genome-scale mining strategy for recovering novel rapidly-evolving nuclear single-copy genes for addressing shallow-scale phylogenetics in Hydrangea.

BACKGROUND: Identifying orthologous molecular markers that potentially resolve relationships at and below species level has been a major challenge in molecular phylogenetics over the past decade. Non-coding regions of nuclear low- or single-copy markers are a vast and promising source of data providing information for shallow-scale phylogenetics. Taking advantage of public transcriptome data from the One Thousand Plant Project (1KP), we developed a genome-scale mining strategy for recovering potentially orthologous single-copy markers to address low-scale phylogenetics. Our marker design targeted the amplification of intron-rich nuclear single-copy regions from genomic DNA. As a case study we used Hydrangea section Cornidia, one of the most recently diverged lineages within Hydrangeaceae (Cornales), for comparing the performance of three of these nuclear markers to other "fast" evolving plastid markers. RESULTS: Our data mining and filtering process retrieved 73 putative nuclear single-copy genes which are potentially useful for resolving phylogenetic relationships at a range of divergence depths within Cornales. The three assessed nuclear markers showed considerably more phylogenetic signal for shallow evolutionary depths than conventional plastid markers. Phylogenetic signal in plastid markers increased less markedly towards deeper evolutionary divergences. Potential phylogenetic noise introduced by nuclear markers was lower than their respective phylogenetic signal across all evolutionary depths. In contrast, plastid markers showed higher probabilities for introducing phylogenetic noise than signal at the deepest evolutionary divergences within the tribe Hydrangeeae (Hydrangeaceae). CONCLUSIONS: While nuclear single-copy markers are highly informative for shallow evolutionary depths without introducing phylogenetic noise, plastid markers might be more appropriate for resolving deeper-level divergences such as the backbone relationships of the Hydrangeaceae family and deeper, at which non-coding parts of nuclear markers could potentially introduce noise due to elevated rates of evolution. The herein developed and demonstrated transcriptome based mining strategy has a great potential for the design of novel and highly informative nuclear markers for a range of plant groups and evolutionary scales.

Plasma membrane-localized Al-transporter from blue hydrangea sepals is a member of the anion permease family.

In hydrangea sepals, an aluminum complex of delphinidin-3-O-glucoside is responsible for the development of the blue color, and co-existing copigments mediate the solubilization and stabilization of the blue Al-anthocyanin complex which is localized in the sepal vacuole. In addition, hydrangeas are Al-hyperaccumulators and exhibit tolerance to acidic soils, in which the toxicity is due to soluble Al ion. Therefore, an Al-absorbing transport and storage system must exist in hydrangea. Recently, we cloned vacuolar and plasma membrane-localized Al-transporters, HmVALT, and HmPALT1, which are both members of the aquaporin family. However, HmPALT1 was only expressed in the sepals, indicating that a different Al-transporter should exist for absorption and long-distance transportation in the hydrangea plant. Using genetic information and microarray analysis, we identified an additional aluminum transporter gene, HmPALT2, which belongs to a member of the anion permease. The transcript was expressed in all tissues of hydrangea plants, and a transient expression study indicated that the gene product is localized to the plasma membrane. The results of an aluminum tolerance assay using yeast cells showed that the HmPALT2 is also involved in the transport of other metal(loid)s. The over-expression of HmPALT2 in Arabidopsis resulted in aluminum-hypersensitivity, suggesting that HmPALT2 should work as an aluminum transporter into cells in planta.

R gene expression changes related to Cercospora hydrangeae L.

Nursery growing as well as common landscape hydrangeas are all susceptible to leaf spot fungus Cercospora hydrangeae. Warm and rainy weather causes the fungal spores to germinate quickly and spread over the plant leaves forming small purple or brown spots. Although Hydrangea plants are not killed by leaf spot, it detracts from the value of plants through the reduction of flowering and plant vigor. The aim of our study was to isolate, characterize and investigate the expression profile of Hydrangea macrophylla resistance (R) gene transcripts under C. hydrangeae fungus infection and examine their evolutionary relationships by phylogenetic analysis. R-genes are thought to be one of the components of the genetic resistance in plants and most of them encode nucleotide binding site-leucine rich repeat (NBS-LRR) proteins. A cDNA-NBS strategy was carried out using as template cDNAs isolated from control and infected plant leaves. The cDNA-NBS profiling gave an excellent bands reproducibility. Twenty new transcripts corresponding to NBS-LRR proteins were identified only in infected plants. The extent of positivity between the aminoacid sequences at NBS region varied from 45 to 90 %, which indicates the diversity among the RGAs. The results of this paper will provide a genomic framework for the further isolation of candidate disease resistance NBS-encoding genes in Hortensia, and contribute to the understanding of the evolutionary mode of NBS-encoding genes in Hydrangeaceae crops.

Genome sequence of Hydrangea macrophylla and its application in analysis of the double flower phenotype.

Owing to its high ornamental value, the double flower phenotype of hydrangea (Hydrangea macrophylla) is one of its most important traits. In this study, genome sequence information was obtained to explore effective DNA markers and the causative genes for double flower production in hydrangea. Single-molecule real-time sequencing data followed by a Hi-C analysis were employed. Two haplotype-phased sequences were obtained from the heterozygous genome of hydrangea. One assembly consisted of 3,779 scaffolds (2.256 Gb in length and N50 of 1.5 Mb), the other also contained 3,779 scaffolds (2.227 Gb in length, and N50 of 1.4 Mb). A total of 36,930 genes were predicted in the sequences, of which 32,205 and 32,222 were found in each haplotype. A pair of 18 pseudomolecules was constructed along with a high-density single-nucleotide polymorphism (SNP) genetic linkage map. Using the genome sequence data, and two F2 populations, the SNPs linked to double flower loci (djo and dsu) were discovered. DNA markers linked to djo and dsu were developed, and these could distinguish the recessive double flower allele for each locus, respectively. The LEAFY gene is a very likely candidate as the causative gene for dsu, since frameshift was specifically observed in the double flower accession with dsu.

Pollen wall development in Hydrangea bretschneiderii Dippel. (Hydrangeaceae): advanced interpretation through physical input, with in vitro experimental verification.

We aimed to unravel the underlying mechanisms of pollen wall development in Hydrangea bretschneiderii. For this, we tested our hypothesis that distinct physical processes, phase separation and micellar self-assembly, underpinned exine development by taking the substances, determined by the genome, through several phase transitions. We traced each developmental stage with TEM; then, we obtained in vitro simulations corresponding to those stages. The main steps of exine ontogeny observed in the microspore periplasmic space were initiated with phase separation, resulting in the conversion of homogeneous contents to heterogeneous two-layered state of the material. After each step of phase, separation self-assembly picked up the initiative and took the substances through the sequence of micellar mesophases which were the base for all the exine structures. These mesophases are as follows: spherical micelles, transforming first into columns, and then to cylindrical micelles which turn to columellae after initial sporopollenin accumulation. The tectum appeared along the interface of the phase separated material. After the tetrad disintegration and the next phase separation, laminate mesophase appeared being the base for the endexine lamellae. Then, a new step of phase separation at aperture sites brought the appearance of a granular endexine layer; the latter became intermixed finally with lamellae. This gives, together with experimental simulation, strong evidence that the genome "shifts a part of work" on exine formation onto physical processes, and the latter are an inherent mechanism of evolution.

Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins.

Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.