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Past genetic studies have shown three independent loci designated O, R and M control spathe color in Anthurium andraeanum (Hort.). To evaluate the genetic model and to understand the control of anthocyanin biosynthesis at the molecular level, the expression of the anthocyanin biosynthetic genes, CHS, F3H, DFR, ANS and F3'H, was examined at the mRNA and protein levels and correlated to anthocyanin content and spathe color in eight genetically characterized anthurium cultivars representing different states of the O, R and M loci. The results showed that the expression of F3H and ANS was co-regulated by a putative transcription factor encoded by the R locus, and the expression of DFR was regulated by a putative transcription factor encoded by the O locus. White cultivars, which were in the homozygous recessive state for either O or R or both, exhibited reduced expression of the anthocyanin biosynthetic genes and hence had negligible levels of anthocyanin. Cultivars that were mm displayed reduced expression of F3'H suggesting that it may either encode a defective form of the F3'H gene or a regulator that controls its expression. Additionally, a correlation between anthocyanin abundance and the expression of F3'H in the red cultivars suggested that F3'H expression may be a key control point in the regulation of anthocyanin biosynthesis in anthurium and hence plays a major role in influencing the shade intensity in red cultivars. These findings provide evidence in support of the genetic model for color inheritance in the spathe.

Anthurium andraeanum (Hort.) is an important ornamental in the tropical cut-flower industry. However, there is currently insufficient information to establish a clear connection between the genetic model(s) proposed and the putative genes involved in the differentiation between colors. In this study, 18 cDNA libraries related to the spathe color and developmental stages of A. andraeanum were characterized by transcriptome sequencing (RNA-seq). For the de novo transcriptome, a total of 114,334,082 primary sequence reads were obtained from the Illumina sequencer and were assembled into 151,652 unigenes. Approximately 58,476 transcripts were generated and used for comparative transcriptome analysis between three cultivars that differ in spathe color ('Sasha' (white), 'Honduras' (red), and 'Rapido' (purple)). A large number of differentially expressed genes (8,324), potentially involved in multiple biological and metabolic pathways, were identified, including genes in the flavonoid and anthocyanin biosynthetic pathways. Our results showed that the chalcone isomerase (CHI) gene presented the strongest evidence for an association with differences in color and the highest correlation with other key genes (flavanone 3-hydroxylase (F3H), flavonoid 3'5' hydroxylase (F3'5'H)/ flavonoid 3'-hydroxylase (F3'H), and leucoanthocyanidin dioxygenase (LDOX)) in the anthocyanin pathway. We also identified a differentially expressed cytochrome P450 gene in the late developmental stage of the purple spathe that appeared to determine the difference between the red- and purple-colored spathes. Furthermore, transcription factors related to putative MYB-domain protein that may control anthocyanin pathway were identified through a weighted gene co-expression network analysis (WGCNA). The results provided basic sequence information for future research on spathe color, which have important implications for this ornamental breeding strategies. [ABSTRACT FROM AUTHOR]

Abstract: Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique capable of accurately characterizing gene expression. However, the lack of suitable reference genes for sample normalization is an impediment to reliable expression studies. The aim of this study was to identify stable reference genes for RT-qPCR studies in Anthurium andraeanum (Hort.). Eight putative reference genes were partially cloned from anthurium using degenerate primers and RT-qPCR assays were developed. Total RNA was isolated from spathe tissues from different cultivars, color groups, developmental stages and harvesting times, and the reference genes were assayed using a RT-qPCR. Datasets were analyzed using ANOVA and reference genes were ranked based on stability using the coefficient of variation, geNorm and NormFinder approaches. CYP, UBQ5 and EF1α ranked amongst the most stable genes, except for studies involving development stages, where EF1α ranked poorly. Conversely, GAPDH displayed highly significant differences (P <0.001) in expression across cultivars and color groups and ranked as the least stable. Gene expression levels varied with 18S > UBQ5 >(CYP, EF1α)>(ACT, IF4A)>(TUB, GAPDH). 18S expression levels were found to be 1000 fold greater than expression levels of UBQ5, the second highest expressed gene. geNorm pairwise variation analysis demonstrated that only two of the recommended reference genes were necessary for accurate normalization of RT-qPCR datasets. [Copyright &y& Elsevier]

Flavonoid and anthocyanin levels, and the expression profiles of four flavonoid biosynthetic genes were investigated in 16 anthurium cultivars with varying spathe colours with the objective of understanding the regulation of anthocyanin biosynthesis. The results suggested that F3H and ANS were controlled by a putative regulatory element designated 'R', DFR by another putative regulatory element designated 'O' and CHS by yet another mechanism. The differences between red and pink cultivars and orange and red cultivars were evident in both the anthocyanin and flavonoid levels as well as in the levels of expression of F3H and ANS controlled by the regulatory element 'R'. The higher level of expression of F3H in the red cultivars than in the pinks may be responsible for the different anthocyanins detected in the reds compared to the pinks. A plausible mechanism based on impediment of carbon flow down the flavonoid pathway is discussed. [ABSTRACT FROM AUTHOR]

The relationship between vacuolar pH in Anthurium andraeanum (Hort.) and spathe color, cultivar, developmental stage, spathe location, spathe surface differences, and time after harvest was investigated with the overall long-term objective of developing a methodology for engineering blue-colored spathes. Chromospectral analysis of the pigmentation was also studied. Six experiments were conducted involving 23 cultivars of anthurium with each experiment arranged in a randomized complete block design with five replications. Spathe color was associated with vacuolar pH with the whites and greens having the highest pH (average 5.65) followed by corals (5.38), pinks (5.20), reds (5.10), and oranges (4.5). In general, there was correspondence between the lightness of the pigmentation (L*) and the pH values with the lighter colors having higher pH values. There were, however, significant differences among cultivars within the color groups. Whereas spathe pH decreased with aging, there was no difference in the spectral data, suggesting that factors other than anthocyanin content may be contributing to the pH difference. There were no differences in pH between locations sampled on the spathe nor between the spathe surfaces provided there were no differences in color intensity (L*). The pH increased with vase life in two of three cultivars tested with pH values showing an association with increases in a* and b* (chromospectral data) reflecting a bluing effect. The importance of the results to engineering blue-colored spathes in anthurium is discussed. [ABSTRACT FROM AUTHOR]