Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

pH-Dependent Selective Colorimetric Detection of Proline and Hydroxyproline with Meldrum’s Acid-Furfural Conjugate

Activated 2-furfural gives intense color formation when reacted with amines, due to a ring opening reaction cascade that furnishes a conjugated molecular system. Unique colorimetric characteristic of this reaction makes it an interesting candidate for developing chemosensors operating in visible range. Among many activated 2-furfural derivatives, Meldrum’s acid furfural conjugate (MAFC) recently gained significant interest as colorimetric chemosensor. MAFC has been explored as selective chemosensor for detecting amines in solution, secondary amines on polymer surfaces and even nitrogen rich amino acids (AA) in aqueous solution. In this work, the pH dependency of MAFC-AA reaction is explored. It was found that proline gives an exceptionally fast colored reaction at pH 11, whereas at other pHs, no naked eye color product formation was observed. The reaction sequence including ring opening reaction upon nucleophilic addition of cyclic amine of proline resulting in a conjugated triene was confirmed by NMR titrations. The highly pH dependent reaction can e.g., potentially be used to detect proline presence in biological samples. An even more intense color formation takes place in the reaction of natural proline derivative 4-hydroxyproline. The detection limit of proline and 4-hydroxyproline with MAFC solution was found to be 11 µM and 6 µM respectively.

Determination of color formation of multicomponent fruit and vegetable pastes and dried powder fractions during low temperature treatment

The aim of the work is to determine the color formation of multicomponent fruit and vegetable pastes and dried powder fractions at the stages of low-temperature pre-concentration and drying, as one of the factors, maintaining the quality of the products. In the production of organic plant semi-finished products an important factor is the implementation of high-quality technological operations, including heat and mass transfer, which affects the final organoleptic characteristics of products. It is important to take into account the color of raw materials at the stages of blending puree in the production of paste and powder semi-finished products, which requires the introduction of a unified method for assessing the color of raw materials at each stage of the technological operation. An analysis of traditional methods for determining color formation has been performed, as a result of which it has been found, that the most effective method of evaluation is digital, based on photo processing of the prototype. According to this method, the evaluation of color formation in the manufacture of semi-finished fruits and vegetables in accordance with the proposed recipes has been conducted. Color indices of multicomponent pastes and dried fractions based on them for three prototypes were obtained. The brightness of all samples of pastes is in the range - 36.4… 37.0 % with a purity of tone 64.7… 78.2 %, which corresponds to the reddish-orange color, was obtained. After drying the test samples of pastes to the dried fraction, it has been found, that the brightness, depending on the percentage of raw materials in a sample falls in the range of 30.5… 33.2 %, at that the coloration corresponds to colors from bluish-purple to bluish-red with a purity of tone within 34.7… 34.9 %. As a result of evaluation of organoleptic indicators, it has been found, that according to the presented research samples, the best indicators have a sample with 40 % of raw apples, 20 % of pumpkin, 30 % of cranberries and 10 % of hawthorn. The obtained research data will be useful in the development of methods for the production of semi-finished products from vegetable raw materials. The applied digital method of color determination differs in simplicity and economy in comparison with colorimetric and spectrometric

Regulation of MYB Transcription Factors of Anthocyanin Synthesis in Lily Flowers

Flower color is the decisive factor that affects the commercial value of ornamental flowers. Therefore, it is important to study the regulation of flower color formation in lily to discover the positive and negative factors that regulate this important trait. In this study, MYB transcription factors (TFs) were characterized to understand the regulatory mechanism of anthocyanin biosynthesis in lily. Two R2R3-MYB TFs, LvMYB5, and LvMYB1, were found to regulate anthocyanin biosynthesis in lily flowers. LvMYB5, which has an activation motif, belongs to the SG6 MYB protein subgroup of Arabidopsis thaliana. Transient expression of LvMYB5 indicated that LvMYB5 can promote coloration in Nicotiana benthamiana leaves, and that expression of LvMYB5 increases the expression levels of NbCHS, NbDFR, and NbANS. VIGS experiments in lily petals showed that the accumulation of anthocyanins was reduced when LvMYB5 was silenced. Luciferase assays showed that LvMYB5 can promote anthocyanin synthesis by activating the ANS gene promoter. Therefore, LvMYB5 plays an important role in flower coloration in lily. In addition, the transient expression experiment provided preliminary evidence that LvMYB1 (an R2R3-MYB TF) inhibits anthocyanin synthesis in lily flowers. The discovery of activating and inhibitory factors related to anthocyanin biosynthesis in lily provides a theoretical basis for improving flower color through genetic engineering. The results of our study provide a new direction for the further study of the mechanisms of flower color formation in lilies.

A novel transcription factor CmMYB012 inhibits flavone and anthocyanin biosynthesis in response to high temperatures in chrysanthemum

Flavones are among the major colorless pigments synthesized through branches of the flavonoid pathway in plants. However, due to the absence of a gene encoding flavone synthase (FNS) in the model plant Arabidopsis thaliana species, the regulatory mechanism of FNS-catalyzed flavone biosynthesis has rarely been studied in plants. Here, it was found that flavones play a predominant role in the elimination of excess reactive oxygen species (ROS) at high temperatures in colorless plant organs. A novel atypical subgroup 7 (SG7) R2R3-MYB transcription factor, CmMYB012, was found to be induced in response to prolonged high temperatures and to inhibit flavone biosynthesis by directly regulating CmFNS. Moreover, CmMYB012 was also found to inhibit anthocyanin biosynthesis by suppressing the expression of CmCHS, CmDFR, CmANS, and CmUFGT. CmMYB012 overexpression exerted a negative influence on plant fitness and pink flower color formation, while CmMYB012 suppression had the opposite effect in response to high temperatures. Our findings provide new insights into the mechanisms by which high temperatures regulate the metabolism of flavones and anthocyanins to affect plant fitness and flower color formation.

Color Formation of Musical Compositions of Children’s Choir “Vocalista Angels”

Vocalista Angels Children's Choir is one of the children's choir groups in Klaten Regency, Central Java, Indonesia, established in 1997. Most of the members are children from villages who do not understand singing techniques correctly or conform to classical vocal techniques. So that when practicing, this group has a different method from the classical vocal method that is standard in their coaching. This study seeks to reveal the typical character traits and the principles in practice to produce a unique character distinctive. The method in carrying out this research is mostly in the form of observations involved in data collection. Written records and audiovisual recordings are the most likely ways to obtain valid data. This method produces descriptive data in the form of written, spoken, or musical words for the group members who are the object of observation. As a result, in terms of sound production, it is very clear that phrasing, articulation, resonance, and intonation are important concerns in every practice in choral processing. In the final packaging of production, this group pays serious attention to dynamics, interpretation, and expression. This kind of knowledge is very useful for developing productive and practical knowledge for the presentation of music, especially the production of music in a chorus that has complexities that are not simple.