Lan LUO Le GAO Yanhua MIAO Fangying XU Jianzhong TAN

Abstract ［Objectives］ This study was conducted to explore the color variation mechanism of Anthurium andraeanum spathe at the protein level.

［Methods］ The differential proteins of wild type and its white mutant were separated and identified by using one-dimensional gel electrophoresis and mass spectrometry (1-DE/MS).

［Results］ Compared with leaves and spadices, the 1-DE patterns of two kinds of spathe proteins were significantly different, and two different bands were detected in wild type spathes and mutant spathes respectively. The four significantly differential bands were selected and analyzed by mass spectrometry, and 138, 111, 70 and 427 proteins were identified respectively. The results of GO functional annotation analysis showed that the molecular functions of the proteins were mainly catalytic activity and binding, and the main biological processes involved were cellular process and metabolic process. Many proteins involved in the synthesis of anthocyanins and flavonoids, sugar metabolism and some resistance proteins were screened, indicating that the spathe color difference of A. andraeanum ‘Pink champion is not only related to anthocyanin anabolism, but also regulated by various metabolic pathways.

［Conclusions］ The study provides a new experimental basis for elucidating the molecular mechanism of the regulation of A. andraeanum flower color.

Key words Anthurium andraeanum ; Flower color mutant; Differential protein; Gel electrophoresis; Mass spectrometry

Received: February 27, 2020Accepted: April 16, 2020

Supported by Suzhou Agricultural Applied Basic Research Program(SNG201605).

Lan LUO (1996-), female, P. R. China, master, devoted to research about landscape plant resources and applications.

*Corresponding author. E-mail:sudatanjz@163.com.

Anthurium andraeanum is one of the famous tropical flowers, known for its peculiar shape and colorful spathes. As a key ornamental quality, the spathe color of A. andraeanum has high research value. Its anthocyanidins are mainly anthocyanins, flavonoids and flavonols, while green and brown spathes also contain chlorophyll and carotenoids［1］. Such difference is not only closely related to anthocyanidin content, but also regulated by the expression of genes related to anthocyanin metabolism［2］. Studies have shown that in A. andraeanum varieties and mutants of different color series, with the lightening of spathe color, the expression levels of CHS, F3H, F3餒, DFR, UFGT and LAR genes are reduced［3］. Transcriptome analysis also showed that the expression levels of CHS, CHI, F3H, F3餒, DFR, ANS, LAR and C4H genes are highest in the flower bud stage, and the expression levels of these genes continue to decline with the maturity of spathes［4］. In addition, the anthocyanin synthesis of A. andraeanum is also regulated by the expression of genes encoding transcription factors such as MYB, bHLH and WD40. For example, AaMYB2 is highly expressed in spathes with a high anthocyanin content, but almost not expressed in white and green spathes［5］. These all explain the colouration mechanism of the A. andraeanum 餾 spathe color from the level of gene expression, but the understanding of the regulatory mechanism at the protein level is still lacking. To this end, this study used the wild type (pink) and white mutant of A. andraeanum ‘Pink champion as materials, and explored the differential expression of proteins related to their spathe color variation applying the 1-DE/MS proteomics experimental technique. The study provides a new experimental basis for elucidating the molecular mechanism of the regulation of A. andraeanum flower color.

Materials and Methods

Plant materials

The experimental materials were the wild type A. andraeanum ‘Pink champion (with pink spathes, denoted as CF) and its white mutant (denoted as CW). The leaves, spadices and spathes with similar developmental stages were cut from potted A. andraeanum . The cut materials were washed with double distilled water and dried with filter paper. After quickly frozen in liquid nitrogen, the materials were stored in an ultra-low temperature refrigerator.

Extraction of proteins

A. andraeanum leaf, spadix and spathe samples (0.2 g each) were weighed, added with liquid nitrogen and ground to a powder form, respectively. A protein extracting liquid (3 ml) containing 10% glycerol, 0.1 mol/L Tris-HCl (pH 6.8), 5% β-mercaptoethanol and 2% SDS was added into each sample, which was then ground to homogenate. The homogenate was transferred to a centrifuge tube and centrifuged at 14 000 r/min and 4 ℃ for 30 min. The precipitate was discarded, and the supernatant was transferred to a new centrifuge tube, and centrifuged at 4 ℃ and 14 000 r/min for 20 min. The supernatant was transferred to a new centrifuge tube and added with 80% acetone pre-cooled with a volume three times of the supernatant, followed by mixing well and standing in a -20 ℃ refrigerator overnight. The mixture was then centrifuged at 14 000 r/min and 4 ℃ for 3 min. The supernatant was discarded, and the precipitate was added with the protein extracting liquid (30-100 μl) to re-suspend and dissolve it. After quantifying the protein sample, the suspension was stored in a refrigerator at -20 ℃.

SDS-PAGE of protein

The polyacrylamide gel concentrations were 12% separation gel and 5% spacer gel. The loading quantity of a sample was adjusted according to the protein concentration of the sample. The sample (35 μg) was mixed with bromophenol blue indicator (5 μl) , followed by sample loading. SDS-PAGE was performed at a fixed current of 35 mA. After the electrophoresis, the gel was removed and stained with CBB-R250 for 1 h, and finally decolorized until the gel background was clear.

Identification of protein differential bands

The differential bands of proteins 1-DE gel were selected and analyzed by mass spectrometry.

Results and Analysis

Phenotypic characteristics in spathe color mutant of A. andraeanum ‘Pink champion

According to the description specification of A. andraeanum germplasm resources, the spathes of the two materials were basically the same, both round, leathery, smooth and shiny, and the tip of the spathes was tail tip-shaped (Fig. 1). However, looking from the color of the spathes, the wild type of ‘Pink champion belonged to the red-purple series (RHSCC code: 62A), and the white mutant belonged to the white series (RHSCC code: NN155B).

SDS-PAGE analysis of proteins in different tissues of A. andraeanum ‘Pink champion

From the SDS-PAGE (1-DE) images of the experimental materials, the leaf protein separation was the clearest, and the number of bands detected was also higher; and the spadix protein separation was poor, showing fuzzy band pattern, and there was no obvious difference between the wild type and mutant. In contrast, the band separation of the spathe餾 protein was clearer, and the number of detected bands was higher. The high-abundance protein components were mainly concentrated in the range of 20-42 kD. Two significantly differential bands were detected at 140 and 145 kD (Fig. 2a and Fig. 2b) in the 1-DE image of the wild type ‘Pink champion, and two significantly differential bands were also detected at 130 and 29 kD in the 1-DE image of the white mutant (Fig. 2c and Fig. 2d).

Identification and analysis of differential proteins by mass spectrometry

From the protein 1-DE gel of the wild type ‘Pink champion and its white mutant, such four differential bands as a, b, c and d in Fig. 2 were cut for mass spectrometry analysis. 138 proteins were detected in the differential band a of the wild type, such as xyloglucan endotransglucosylase /hydrolase, catalase, auxin transporter, chalcone synthase, α-1,3-glucosyl-transferase, etc. ; and 111 proteins were detected in the difference band b, including catalase, malate dehydrogenase, α-mannosidase, laccase, disease-resistant protein, etc. ; 70 proteins were detected in the differential band c of the white mutant, such as peroxidase, α-mannosidase, xylose isomerase, disease-resistant protein, etc. ; and 427 proteins were detected in the differential band d, including glutathione S-transferase , isoflavone reductase, UDP-glycosyltransferase, peroxidase, catalase, sucrose synthase, anthocyanin synthase, etc. (Table 1 only lists part of the protein information related to color variation). The results of GO functional annotation analysis showed that the molecular functions of the proteins in the four bands were mainly catalytic activity and binding, and the main biological processes involved were cellular process and metabolic process.

Agricultural Biotechnology2020

Conclusions and Discussion

In this study, the A. andraeanum pink variety ‘Pink Champion and its white mutant were used as experimental materials, and the 1-DE/MS proteomics method was used to explore the expression differences of proteins related to their flower color variation. According to the comparison results of proteins 1-DE images, it was found that the differences in the 1-DE images of the proteins from the two kinds of spathes were more significant than those of leaves and spadices. Four significantly differential bands were selected and analyzed by mass spectrometry. 138, 111, 70 and 427 proteins were identified, respectively. The results of GO functional annotation analysis showed that the molecular functions of the proteins were mainly catalytic activity and binding, and the main biological processes were involved in cellular process and metabolic process.

Two differential bands, specifically up-regulated expressed and large-molecular-weight bands a and b were detected in the protein 1-DE from the wild type ‘Pink champion spathes. In the differential band a, chalcone synthase (CHS) and α-1,3-glucosyltransferase related to flavonoid pigment metabolism were identified. It has been made clear that CHS catalyzes the initial reaction of flavonoid synthesis in phenylpropane metabolic reaction and is a key enzyme in the anthocyanin biosynthesis pathway［6］. It can regulate the expression and accumulation level of anthocyanins. In different varieties of the same plant, the expression of CHS is higher in the red series with a higher anthocyanin content, but less in the yellow and white series［7］. In this study, CHS was detected in the differential band a of the wild type ‘Pink champion, and the corresponding band was not detected in the 1-DE image of the white mutant, indicating that the spathe color change of the wild type ‘Pink champion and its white mutant was also related to the change in the expression of CHS .

Anthocyanin synthase (ANS) is a key enzyme at the end of anthocyanin biosynthesis pathway in plant, catalyzing the conversion of colorless anthocyanins to colored anthocyanins［8］. It is generally believed that the expression of ANS is higher in the part of darker or brighter plant tissue. For example, in variegated peach flowers, the expression of ANS in pink petals is significantly higher than that in white petals［9］. However, in A. andraeanum spathes, even when the anthocyanin level is low at the early stage of development, the ANS transcript always maintains high expression［10］. In this study, the expression of ANS was detected in the differential band d of the white mutant of ‘Pink champion, indicating that the anthocyanin synthesis reaction also occurred in its spathes, but it failed to show red phenotype in appearance. It is speculated that in the white mutant, the amount of anthocyanin synthesis is very few, or it will be degraded or transformed soon after synthesis. Therefore, it can be considered that the shade of the color (pink) of A. andraeanum spathes not only is directly related to the expression level of ANS , but also regulated and influenced by various other factors.

Secondly, a variety of enzyme proteins related to glycometabolism were detected in the differential band d of the white mutant of ‘Pink champion. Studies have shown that carbohydrates can provide energy and metabolic substrates for the synthesis of plant anthocyanins, and also regulate the synthesis of anthocyanins through signal transduction pathways in the form of signal molecules［11］. For example, sucrose synthase is a key enzyme in sucrose anabolic metabolism, and uridine diphosphate glucose, the substrate required for sucrose synthesis, is also the main precursor for anthocyanin synthesis［12］. Since there is a certain substrate competition relationship between plant anthocyanin and sucrose synthesis［13］, it is speculated that the high expression of sucrose synthase in the white mutant may lead to a decrease in anthocyanin synthesis, which makes the color of spathes faded.

Furthermore, in addition to anthocyanin anabolic and sugar metabolism pathways, there are also many various life processes involving the proteins identified from the differential bands, such asstress response, disease resistance, antioxidant response, gene transcription and transcription factors, protein translation and transport, and protein degradation regulation, indicating that in the differential expression profiles (1-DE bands) of the wild type and white mutant of ‘Pink champion, these high-abundance expressed proteins are also intrinsically related to the spathe color variation. They may be related to the regulation of anthocyanin biosynthesis and degradation metabolism, but the mechanism remains to be further studied.

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