The hydrangea plant Hydrangea macrophylla originates from Japan and East Asia. Hydrangea flowers are composed of sepals whose original color is blue, although various breeding efforts have resulted in a wide range of sepal colors Fig. Et Zucc. In general, flower coloration is thought to be genetically controlled by the expression of structural genes involved in anthocyanin biosynthesis. Hydrangea macrophylla and microscopic observation of the transverse section of sepals.
A Hydrangeas that show the color variety present within the species. Transverse sections of B blue and C red sepals. Chemical studies on hydrangea sepal color development started in the 19th century. This knowledge is effectively used by horticulturalists for cultivating differently colored hydrangea. The major anthocyanin in hydrangea sepals is 3- O -glucosyldelphinidin 1 , 12 and blue and red hydrangeas express the same anthocyanin compounds Fig.
Our group has been interested in studying the blue flower color development in a variety of plant species. Reversible chemical reactions of anthocyanin and color development exemplified for 3- O -glucosyldelphinidin depending on pH in aqueous solution. Studying the mechanism of sepal color development required clarification of intravacuolar conditions such as vacuolar pH, the chemical structure of anthocyanins and copigment composition, and content of metal ion: aluminum ion in hydrangea at first, then reproduction of the sepal color using the obtained analytical data is essential.
The epidermal cell layer of hydrangea sepals is colorless, and the pigmented cells are located the second cell layer Fig. Anthocyanin equilibrium depends on pH Fig. There are many methodologies for measuring vacuolar pH pHv , but the easiest one is to measure the pH of fluid isolates from pressed petal or sepal tissues.
However, these isolates include cellular materials from both colored and colorless cell types. Therefore, we have found that using a pH-sensitive microelectrode method established by Felle 33 is the best method to obtain accurate pHv measurements. The quick color change was due to a significant difference in the pHv values of colored epidermal cells and colorless parenchyma cells.
Therefore, the pH of the pressed juice was a result of the mixture of both cell types. Chause and two red cultivars Kasterin and LK were 4. Micrographs of hydrangea protoplast mixtures. Organic components in colored cells of hydrangea sepals were quantified by collecting similarly colored cells. Ideally, single-cell analysis is the best for this purpose; however, this was technically impossible at the time, so we manually collected the same colored protoplasts from a mixture using a micropipette under a microscope.
The number of cells necessary for quantitative analysis depended on the sensitivity of the analytical method used. Anthocyanin was detected at nm, and copigments were detected at nm. The concentration of the organic and inorganic components in the colored cells was calculated using the average cell diameter and number of cells collected.
In blue cells, 13 eq. This strongly indicated that the distribution of anthocyanin in sepals is limited in colored cells, but copigments may also be present in colorless cells. Reducing the number of cells needed for the Al analysis has several challenges, the most significant is how to prevent environmental Al contamination. Hence, it was essential to wash all the plant materials and experimental equipment with aqueous HNO 3 and to use a clean chamber for cell collection.
After the preparation of the protoplast mixture, we collected approximately colored cells. It is well known that sepal color can change in hydrangea varieties. To characterize the mechanisms underlying this color change, we observed the sepal tissue and analyzed the components according to maturation stage. For color measurement in hydrangea plant tissue, reflection spectra were usually recorded; however, the spectra tended to be affected by the cell surface shape.
In comparison, when a tissue section was treated in vacuo with water, the cell air spaces are filled with water, and the noise from the surface structures can be reduced, which allows the measurement of the visible absorption spectra. Except for the first colorless stage, cells in the second stage of chameleon hydrangea sepal development exhibited the same blue color with the same spectra measured in ordinary blue hydrangea cells.
At the third green stage, the spectra showed a decrease in the peak at nm, which appeared at nm instead. The transverse sections of the sepals at each stage indicated that at the first stage, almost all the cells were colorless, while at the second stage, the blue cells were mainly in the second layer of the adaxial epidermis. However, at the third stage, the blue color of the second layer of cells faded, and chloroplasts appeared in the cytoplasm of the cells in the second and inner cell layers.
At the fourth stage, the second layer became red, while the number of chloroplasts decreased. These results suggested that the green stage was due to the appearance of chloroplast and the color of the second and the fourth stages could depend on the accumulation of anthocyanins. At the first stage, almost no pigment was detected, while at the second stage, 3- O -glucosyldelphinidin 1 and 3- O -sambubiosyldelphinidin 5 were detected as the major and minor pigments, respectively Fig.
During the sepal color change to green near the third stage, the amounts of 3- O -glucosyldelphinidin 1 and 3- O -sambubiosyldelphinidin 5 decreased. This result indicated that the blue and red coloration was due to different anthocyanins developing and accumulating in the tissues in a developmentally dependent manner.
We concluded that the red sepal coloration in chameleon hydrangea was not caused by the same mechanism as that of other red cultivars during the maturation period but was instead due to de novo synthesis of pigments with a cyanidin chromophore, which was similar to autumn red leaves and usually observed in hydrangea found in mountain areas.
Change in the organic components in chameleon hydrangea sepals during maturation and senescence The compound numbers are indicated in Figs. As we have described, chemical analysis of similarly colored cells protoplasts significantly advanced hydrangea color development studies. However, it is very often observed that when a potted hydrangea is planted in the garden, the sepal color changes, for example, from blue to purple or from red to purple.
Microscopic observation of the purple tissue showed a surprising phenomenon: the single cells of the purple sepals each ranged in color from blue and purple to red, indicating that the purple color was due to a mosaic effect.
To clarify the chemical mechanism of the color differences between neighboring hydrangea sepal cells, the establishment of a new method for single-cell analysis is required Fig. Schematic methodology for single-cell analysis and mechanistic study of the development of different colors in purple hydrangea sepal cells.
A differently colored protoplast mixture was obtained by enzymatic treatment of purple sepals left. Within this mixture, a cell was selected, and the cell color and pHv were recorded using a microspectrophotometer and pH-sensitive microelectrode upper right. Reproduction experiments with synthetic pigment compounds were then conducted. To measure the chromatic compounds in hydrangea sepals, we needed more than cells when an ODS column with an i.
However, there are problems that need to be resolved for this method to be successful, including the optimization of the detection system, exclusion of impurities in the eluting solvent, manipulation of the narrow column, and reliability of the gradient elution system. The problem of the detection system was solved using a capillary flow cell system i. The elution solvent used was HPLC grade, and the acid was changed from trifluoroacetic acid to phosphoric acid.
To reduce base-line fluctuation, a step-wise gradient elution was adopted to establish an HPLC method for quantifying organic components in single hydrangea cells Fig. Nonetheless, these results indicated a clear contribution of 5- O -acylquinic acids 2 , 3 to the blue sepal cell color in hydrangea. The molar equivalent of 5- O -acylquinic acids 2 , 3 to that of total anthocyanins 1 , 5 was 3.
These measurements were significantly different between samples, although the 3- O -caffeoylquinic acid 4 content did not show any significant difference between the blue and red cell samples. This strongly suggested that 5- O -acylquinic acids play a very important role in blue cell coloration in hydrangea sepals. To quantify the aluminum content in a single cell, GFAAS could not be used because of its detection limitation.
The cell color obtained from purple sepals varied from nm red cell to nm blue cell. Indeed, a similar result was obtained in the analysis of conventionally cultivated blue and red sepal cells. Vacuolar pH pHv measurement in differently colored cells from purple sepals resulted different data from our previous study, 35 which reported that there was no significant correlation between pHv and cell color.
Narumi blue was approximately 3. We next wanted to clarify the mechanism of color variation and structural aspects of the blue pigment in hydrangea. For this purpose, we tried to reproduce the natural coloration by mixing vacuolar components under various pH conditions in vitro. Furthermore, to obtain structural information, artificial copigments were synthesized and used in in vitro reconstruction experiments. Finally, instrumental analysis revealed the composition and proposed structure of the hydrangea blue-complex.
Therefore, the reproduction solutions aimed to obtain these spectra. Vis spectra of the sepal and protoplast suspensions. A The absorption spectrum of blue sepal black line and blue protoplast blue line mixtures. B Absorption spectra of red sepal black line and red protoplast red line mixtures.
In the reconstruction of the pigment complexes, the concentration of anthocyanin was critical and had a significant effect on the results because such a simple anthocyanin found in hydrangea is very unstable in diluted aqueous solutions but can be stabilized in a highly concentrated solution through self-association.
However, measurement of UV—vis absorption spectra and CD is not possible because of the limitation of the path length of cuvette. Therefore, in the reproduction experiments, we had to choose a lower concentration for anthocyanin [0. The CD was recorded just after mixing and showed an exciton-type negative Cotton effect, indicating the anticlockwise self-association of anthocyanidin chromophores.
B Solid line: protoplast mixture. C Solid line: protoplast mixture. A mixture of 10 mM 3- O -glucosyldelphinidin 1 , mM 3- O -caffeoylquinic acid 4 , 25 mM 5- O -caffeoylquinic acid 2 , 10 mM 5- O - p -coumaroylquinic acid 3 , and 0. To verify the mechanisms of various color development blue, purple, and red in hydrangea, further reproduction experiments with varying several factors were conducted.
The reproduction conditions referred to the analytical results of the single-cell analysis of purple sepals. Under other conditions, a purple solution was obtained, with the hue depending on the combination of these factors. Synthetic studies are a powerful tool for characterizing natural product chemistry. In the structural study of the hydrangea blue-complex, no analyzable NMR spectra were obtained, and MS analysis did not produce molecular ions in our previous studies.
Therefore, we designed and synthesized various copigments to investigate the essential functional structure in copigments for a stable blue complex: 1 is affected by the position of the acyl moiety when it is 3- O -equatorial or 5- O -axial, 2 is sensitive to the number of phenolic hydroxyl groups in the cinnamate moiety, 3 is affected by the size of the 5- O -aromatic moiety, 4 requires the 1-OH of quinate, 5 requires a 1-COOH, and 6 requires a 5- O -ester structure.
In the synthetic study of 5- O -acylquinic acid, there were two problems that affected the synthetic route and yield. In the first-generation synthesis, 46 , 47 we chose the protocol described by Montchamp et al. We continued the synthetic studies and established a second-generation synthesis protocol Fig. The second-generation synthesis of 5- O -caffeoylquinic acid 2. The 1-dehydroxy and 1-OMe derivatives 1-COOMe compound, 5-cinnamyl ether, and 3- O -caffeoylquinic acid did not produce a blue solution but did make a blue precipitate.
The size of the aromatic component of the 5- O -acyl moiety increased the stability of the complex. Therefore, 5- O -naphthoylquinic acid gave the most stable blue solution, suggesting that the aromatic component of the 5- O -acyl residue may stack with the anthocyanidin chromophore through hydrophobic interactions and has a copigmentation effect on the blue complex. The essential structural parts of copigments required for blue color development in hydrangea.
The circle highlights the importance of structure stabilization from hydrophobic interactions within the anthocyanidin chromophore. However, the ratio of each component is not stoichiometric; it fluctuates within a certain range. The complex could exist only in aqueous solution, and attempts to crystallize the complex were unsuccessful. The NMR measurement condition that included 6 M acetate buffer was found coincidentally, and the spectra gave helpful information for the analysis of the hydrangea blue-complex Fig.
A mixture of 1 and 5- O -caffeoylquinic acid 2 produced a simple combination of the signals attributed to both compounds with a small high-field shift of signals that were attributed to the delphinidin chromophore, which indicated copigmentation between anthocyanin and quinic acid Fig. Many trials to determine the structure of the hydrangea blue-complex failed, and the ratio of the components seemed to fluctuate rather than have stoichiometric characteristics.
To determine the composition of the complex, we tried direct observation of the molecular ion of the complex using electrospray-ionization mass spectrometry ESI-TOF-MS. Combining these results, we proposed a quasi-stable chemical structure for the hydrangea blue-complex Fig.
However, the coordination might not have high stability, and thus, the hydrangea blue-complex may exist in equilibrium with the coordinated and dissociated states of the copigments. The equilibrium point may vary depending on the concentration of the components and pH of the solution.
Hence, the NMR spectrum of the blue solution may give both broad and insufficient signals for components in complexes with free copigments, which are visualized by sharp signals in the spectra. Recent innovations in MS spectrometry have promoted improved MS imaging to map intracellular organic and inorganic components. However, compared with animal tissues, mapping plant tissues has its unique difficulties as high water content and high osmotic pressure can compromise the analyte when cells break.
Among imaging MS techniques, the most promising method for in planta visualization of water-soluble chemicals localized in vacuoles is cryo-time-of-flight secondary ion mass spectrometry cryo-TOF-SIMS of freeze-fixed samples. After Al soil treatment, the Al in stems was more widely distributed, and mainly located in the inner part of the cortex and in the xylem.
We have already discussed the molecular weight of the hydrangea blue-complex. The total positive and negative ion images were consistent with the SEM imaging, indicating that the resolution of the MS imaging was high Fig. A Total ion distribution obtained by negative detection. Most plants are sensitive to acidic soils and this is because aluminum ion in soils becomes water-soluble in acidic environment below pH 5.
However, the molecular mechanisms underlying Al transport and storage are poorly understood. Therefore, we attempted to identify Al transporters in hydrangea. Hydrangea is not a model plant and its complete genome has not yet been determined. Furthermore, no Al transporter gene has been found in yeast or other microorganisms. Thus, to identify candidate Al transporter genes in hydrangea, we used the following approach: 1 preparation and sequencing of a full-length cDNA library from blue hydrangea sepal tissue, 2 generation of a custom microarray based on these cDNAs, 3 selection of candidate genes by hybridization on the microarray, and 4 implementation of a complementation assay using an Al-sensitive yeast strain Fig.
According to the abovementioned strategy, followed by analysis of the subcellular localization to the membrane by SOSUI application, 62 we obtained six candidate genes. We found one gene that conferred Al-tolerance to the transformed yeast.
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