Identification of key genes responsible for green and white colored spathes in Anthurium andraeanum (Hort.)

Abstract

Modern anthuriums, Anthurium andraeanum (Hort.) are among the most popular flowering plants and widely used for interior decoration. Their popularity is largely attributed to the exotic spathes with different colors. Previous studies have reported color development in red spathe cultivars, but limited information is available on key genes regulating white and green colored spathes. This study analyzed anthocyanin, chlorophyll, and carotenoid contents as well as transcript differences in spathes of eight cultivars that differed in spathe colors ranging from red to white and green. Results showed that increased expression of a transcription factor AaMYB2 was associated with elevated levels of anthocyanin in spathes, but decreased expression of AaMYB2 and increased expression of AaLAR (leucoanthocyanidin reductase) and AaANR (anthocyanidin reductase) were accompanied with the accumulation of colorless proanthocyanidin, thus the white spathe. As to the green colored spathe, chlorophyll content in the green spathe cultivar was substantially higher than the other cultivars. Correspondingly, transcripts of chlorophyll biosynthesis-related genes AaHemB (porphobilinogen synthase) and AaPor (protochlorophyllide oxidoreductase) were highly upregulated but almost undetectable in white and red spathes. The increased expression of AaHemB and AaPor was correlated with the expression of transcription factor AaMYB124. Subsequently, qRT-PCR analysis confirmed their expression levels in nine additional cultivars with red, white, and green spathes. A working model for the formation of white and green spathes was proposed. White colored spathes are likely due to the decreased expression of AaMYB2 which results in increased expression of AaLAR and AaANR, and the green spathes are attributed to AaMYB124 enhanced expression of AaHemB and AaPor. Further research is warranted to test this working model.

Keywords: Anthurium andraeanum; MYB transcription factor; anthocyanin; chlorophyll; flavonoid; spathe color.

Figure 1

Spathe phenotype and pigment analysis of eight anthurium cultivars. Phenotypes of anthurium spathes (A), the total content of anthocyanin (B), chlorophyll (C), and carotenoid (D) in spathes of eight cultivars are shown as means with corresponding standard error (n = 5). Different letters above bars indicate significant differences analyzed by Fisher’s LSD test at P< 0.05 level.

Figure 2

DEGs involved in the flavonoid metabolism and their expression levels. Heatmaps were constructed based on Fragments per kilobase per million mapped reads (FPKM) of eight anthurium cultivars with different spathe colors. Color bar: Log2 (fold changes).

Figure 3

Molecular analysis of key genes in the flavonoid biosynthesis pathway. (A) Expression analysis of AaMYB2, AaLAR, and AaANR in the spathe tissue of six cultivars. Expression levels were normalized based on the expression of the GADPH gene. Data are presented as means of three biological replicates and error bar shows standard error (n = 5). Different letters above bars indicate significant differences determined by Fisher’s LSD test at P< 0.05 level. (B) Prediction of MYB2 transcription factor binding sites in the promoters of AaLAR and AaANR. The binding sites were shown as red ovals (positive strand).

Figure 4

Molecular analysis of key genes in the chlorophyll metabolism pathway. (A) Heatmap of MYBs related to chlorophyll metabolism. (B) Expression analysis of AaMYB124, AaHemB, and AaPor in the spathe tissue of six cultivars. Expression levels were normalized to the expression of the GADPH gene. Data are presented as means of three biological replicates and error bar shows standard error (n = 5). Different letters above bars indicate significant differences analyzed by Fisher’s LSD at P< 0.05 level. (C) DEGs involved in chlorophyll metabolism and their expression levels. Each colored cell represents the average FPKM value standardized by the Z-Score of each gene. (D) Prediction of MYB124 transcription factors binding sites in the promoters of AaHemB and AaPor. The binding sites were shown in red (MYB124) and purple ovals (positive strand).

Figure 5

Expression analysis of candidate genes in nine additional anthurium cultivars with different spathe colors. (A) Spathe phenotypes of red cultivars TLS, 2016, A302, green cultivars KXG, A166, A086, and white cultivars BM, A231BAI, and A168 at early bud (S7 stage). (B) The transcript level of AaMYB2, AaMYB124, AaLAR, AaANR, AaHemB, and AaPor in the spathe tissue of nine cultivars. Expression levels were normalized based on the expression of the GADPH gene. Data are presented as means of three biological replicates and the error bar shows standard error (n = 5). Different letters above bars indicate significant differences analyzed by Fisher’s LSD test at P< 0.05 level.

Figure 6

A working model for the formation of red, white, and green spathes in A. andraeanum (Hort.). AaMYB3 interacts with AabHLH1 to regulate proanthocyanidin accumulation. AaMYB3 was coexpressed with AaCHS, AaF3H, AaDFR, AaANS, AaLAR, and AaANR in the developing red spathe. The different expression of AaUFGT might explain the formation of anthocyanin-loss mutant. Increased expression of AaMYB2 enhances the expression of AaCHS, AaF3’H, and AaANS as well as anthocyanin accumulation in reddish spathes. Meanwhile, decreased expression of AaMYB2 activates AaLAR and AaANR expression and promotes biosynthesis of proanthocyanidins, leading to spathes with white color. Under the suppressed expression of AaMYB2, two other TFs AaMYB124 upregulated HemB and Por expression, thus chlorophyll accumulation and spathe with green coloration.