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Effect of pH on the expression of RsMYB1 that regulates anthocyanin production in Petunia plants
J Plant Biotechnol 2018;45:30-35
Published online March 31, 2018
© 2018 The Korean Society for Plant Biotechnology.

Deuk Bum Lee, Trinh Ngoc Ai, Aung Htay Naing, and Chang Kil Kim

Department of Horticulture, Kyungpook National University, Daegu 41566, Korea,
Department of Horticulture, Kyungpook National University, Daegu 41566, Korea
Correspondence to: e-mail: ckkim@knu.ac.kr
Received December 14, 2017; Revised February 28, 2018; Accepted March 5, 2018.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

We established an in vitro system to investigate transcription levels of the RsMYB1 gene expressed in T2 20-day-old transgenic Petunia plants (three independent lines: PhRs1, PhRs2, and PhRs3), and the association between those transcription levels and anthocyanin production at various pH values (3.0 to 8.0) for a period of 10 days. All the lines treated with pH 5.0-7.0 exhibited increased anthocyanin content and delays in growth compared to the wild-type (WT) seedlings. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis confirmed that the enhancement of anthocyanin production in the transgenic lines was due to the upregulation of RsMYB1 transcription at various pH values. The results suggest that pH value can control expression of RsMYB1 which is associated with anthocyanin production.

Keywords : Petunia, Anthocyanin, pH, qRT-PCR, RsMYB1
Introduction

Anthocyanins are well-known water-soluble pigments that impart colour to plants (Byamukama et al. 2011; Li et al. 2013), including their fruit, flowers, leaves, stems, and occasionally roots (Davies 2004; Andersen and Jordheim 2006). Anthocyanins protect plant tissues and senescing autumn leaves against damaging photo-oxidation, promote pollination, and facilitate seed distribution. The accumulation of anthocyanin pigments depends on factors such as temperature, light, osmotic stress, co-pigments, and pH (Rodriguez-Saona et al. 1999). Anthocyanins usually accumulate in the vacuoles of epidermal cells, and their colour depends on the pH of the vacuole in which they localize.

Enhancement of anthocyanin production by the different stress conditions, such as high-intensity light, low temperatures, nutrient deficiency, or pathogen attack, and pH stress, had been observed in many plant species (Dixon and Paiva 1995; Chalker-Scott 1999, Zhang et al. 2014). However, role of pH in anthocyanin accumulation have not been well demonstrated in petunia. Recently, Ai et al (2016) claimed that overexpression of RsMYB1 increased anthocyanin accumulation in vegetative and floral tissue of transgenic petunia Hence, it was of interest to investigate the interaction between pH values and RsMYB1 expression controlling anthocyanin production of the transgenic petunia.

In this study, we used the transgenic plant (‘Mirage Rose’) expressing RsMYB1 to investigate the morphological variation and anthocyanin accumulation under different pH conditions.

Materials and Methods

Plant materials

Seeds of three independent T2 transgenic Petunia lines (PhRs1, PhRs2, and PhRs3) and its wild-type (WT) used in this experiment was provided by Dr. Trinh Ngoc Ai (the Department of Horticultural Science, Kyungpook National University, Daegu, South Korea). The seeds were sterilized in 0.05% sodium hypochlorite solution (Yuhan Co, Ltd., Seoul, South Korea) containing 0.01% Tween 20 (Duchefa Biochemie, Haarlem, The Netherlands) for 10 min and rinsed several times with sterile distilled water. The sterile seeds were sown in Murashige and Skoog (MS) medium containing 3% sucrose and 0.3% Gelrite™ (pH 5.8). The cultures were incubated at 25 ± 2°C and subjected to a 16-h photoperiod at an intensity of 50 µmol·m-2·s-1 for 20 days.

Effect of pH on anthocyanin accumulation and gene expression

To determine the effect of the pH on pigment content, 20- day-old transgenic seedlings were cultured in liquid MS media containing the different pH values (3.0, 4.0, 5.0, 6.0, 7.0, and 8.0). The cultures were incubated at 25 ± 2°C and subjected to a 16-h photoperiod at an intensity of 50 μmol·m-2·s-1. After 10 days of culture, the seedlings were transferred to new media for next 10 days. After which, RNA samples were collected to investigate the regulation of anthocyanin gene biosynthesis. The effect of pH on anthocyanin content was also monitored in the transgenic seedlings cultured in the media containing different pH values. We performed anthocyanin content analysis and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis according to the protocols described by Ai et al. (2016). The qRT-PCR primers are listed in Table 1.

List of primers used in the quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of anthocyanin biosynthesis

GenePrimer sequences
  TUBFP: 5′-TGG AA CTC AAC CTC CAT CCA-3′
RP: 5′-TTT CGT CCA TTC CTT CAC CTG-3′

  CHSFP: 5′-GAA CAG CCA CAC CTA CAA AC-3′
RP: 5′-AAC CCT GCT GGT ACA TCA TG-3′

  CHIFP: 5′-TTC CAC CGT CCG TCA AAC CT-3′
RP: 5′-CAT GCC TCC ACT GCA ACC AC-3′

  FLSFP: 5′-CCG ATT TGG CTC TTG GTG TT-3′
RP: 5′-TTT GGG ACA AGA ATG GTG ATA TAT GA-3

  DFRFP: 5′-AGC AGG AAC TGT GAA TGT GG-3′
RP: 5′-GTT GGG ATA ATG GTG ATG AAA T-3′

  ANSFP: 5′-TGG GAG GAT TAT TTC TTC CA-3′
RP: 5′-GTT GTA CTT GCC GTT GCT TA-3′

  DPLFP: 5′-TTT GTC CGG AGG AAG TGG AC-3′
RP: 5′-CGG AAG TCT CCC GGC AAT AA-3′

  PHZFP: 5′-GAT GGT CAC TTA TCG CGG GT-3′
RP: 5′-TCC TCT GCA GGT GTG TGT TC-3′

  JAF13FP: 5′-ACG GAT GAT AAT ATG AGT AAC GGT GTG C-3′
RP: 5′-CTT GAT GGT CTA GTG GGG CAG GC-3′

  RsMYB1FP: 5′-ATG GAG GGT TCG TCC AAA GG-3′
RP: 5′-GAA ACA CTA ATC AAA TTA CAC AGT CTC TCC-3′

Effect of pH on the development of transgenic lines

After treatment of 20 days, 10 plants were randomly selected to determine the effect of pH on transgenic lines and compared with the WT. The breadth of the leaves and the root length were measured to the nearest mm with a rule (Absolute Digimatic, Japan). The numbers of shoots and leaves were also recorded. On Day 20, the average areas and the length/breadth measurements of 10 replicates of the fully developed leaves were measured to represent the size and shape of the leaves, respectively. The breadth was measured from the centre of the midrib region. The experiments were repeated three times, and standard errors were determined for the data obtained from each experiment.

Data analysis

All data were subjected to one-way analysis of variance (ANOVA) and the Duncan’s SSR test (shortest significant ranges) to compare differences between the experimental sites at P < 0.05 (Microsoft Excel 2003 and SAS 9.4 software).

Results and Discussion

Effect of pH on the development of transgenic Petunia seedlings

We determined the effect of media pH (six different pH values) on the development of transgenic lines of Petunia seedlings. After 20 days of culture, there are significant differences in growth between the WT and the transgenic plants. The numbers of shoots and leaves, the sizes of the leaves, and the lengths of the roots are given in Table 2, whereas the numbers of shoots and leaves were significantly higher in the transgenic plants compared to that in the WT plants. These results indicate that the optimum pH value for the growth of transgenic Petunia seedlings is 6.0-8.0, and an acidic environment inhibits the development of seedlings.

Growth parameters of seedlings after 20 days of treatment at various pH values in wild-type (WT) and transgenic plants. Numbers of shoots and leaves, leaf sizes, and root lengths were determined. The data are the means of 10 replications

Morphological line characteristicspH 3.0pH 4.0pH 5.0pH 6.0pH 7.0pH 8.0
Number of shootsWT 1.0 ± 0.0e 1.2 ± 0.2e 1.0 ± 0.0e 1.2 ± 0.2e 1.8 ± 0.4e 2.8 ± 0.8de
PhRs1 2.8 ± 0.9de 7.2 ± 0.6bcd 6.8 ± 0.9bcd 5.2 ± 0.4cde 3.6 ± 0.5de 3.4 ± 0.8de
PhRs2 2.0 ± 0.6e 3.4 ± 0.9de 3.4 ± 0.9de 3.8 ± 0.5de 3.8 ± 0.8de 5.0 ± 1.1de
PhRs3 3.6 ± 1.0de 9.6 ± 4.3ab 7.4 ± 1.3bcd 9.4 ± 1.9abc 12.2 ± 2.6a 13.6 ± 2.5a
Number of leavesWT8.0 ± 0.8f 14.4 ± 1.7dfce 13.4 ± 2.8dfce 14.0 ± 0.7dfce 13.4 ± 1.2dfce 17.0 ± 1.8dce
PhRs110.4 ± 1.2fe 11.4 ± 1.8dfe 17.8 ± 3.67dc 17.0 ± 1.1dce 16.4 ± 4.2ce 19.2 ± 3.3bc
PhRs2 11.0 ± 0.5dfe 13 ± 1.1dfce 13.8 ± 2.5dfce 15.8 ± 0.7dce 15.4 ± 2.4dce 13.2 ± 1.1dfce
PhRs3 11.8 ± 1.3dfe 14.6 ± 1.3dfce 10.8 ± 0.6dfe 15.2 ± 1.2dce 24.2 ± 3.1ab 25.5 ± 1.3a
Leaf size (mm)WT174.9 ± 51.3i373.4 ± 39.6hi631.2 ± 50.4de759.5 ± 81.5ab 689.3 ± 47.6cd697.9 ± 77.2c
PhRs1 332.5 ± 68.1hi  465.5 ± 78.5fg  450.8 ± 135.9fg  543.5 ± 78.7ef  575.2 ± 41.4ef673.1 ± 96.5de
PhRs2198.4 ± 21.1hi484.4 ± 67.1fg433.4 ± 108.2gh571.7 ± 62.3ef363.38 ± 57.1hi442.1 ± 53.6gh
PhRs3264.5 ± 43.1hi627.8 ± 51.3de380.6 ± 68.0hi804.3 ± 132.3a796.33 ± 86.1a327.5 ± 53.6hi
Root length (mm)WT8.94 ± 2.1f 93.9 ± 13.3cd138.4 ± 6.18a 99.9 ± 22.1bcd  126.8 ± 17.8ab   97.5 ± 12.5cd 
PhRs1 0.0 ± 0.0f 82.5 ± 15.2de 76.5 ± 12.62de123.9 ± 7.6abc 107.2 ± 10.1cd101.0 ± 7.5cd
PhRs26.68 ± 6.8f 94.5 ± 7.4cd102.2 ± 10.8cd105.2 ± 8.2cd 87.4 ± 10.1dc 83.1 ± 15.9de
PhRs313.6 ± 9.1f 89.2 ± 5.2dc 70.1 ± 1.31de 86.6 ± 11.4dc 79.7 ± 8.1de 48.4 ± 16.1e

The leaves of the transgenic seedlings were smaller than those of the WT seedlings, but there were more leaves on the transgenic seedlings. Similar patterns have been detected in Arabidopsis thaliana and in maize (Zea mays) (Wang et al. 2011). Anugoolprasert et al. (2012) reported that the optimum pH for the development of crab apples was 6.0 owing to anthocyanin accumulation. However, in the present experiment pH values over 7.0 were associated with the strongest development. We assume this difference was due to the use of different cultivars of crab apple and Petunia in the present study.

Accumulation of anthocyanin responds to pH in transgenic Petunia seedlings

We used a spectrometer to determine anthocyanin accumulation in transgenic Petunia seedlings at various environmental pH values. We used 0.5 g of leaves after 20 days of cultivation. Three transgenic lines (PhRs1, PhRs2, and PhRs3) exhibited different levels of anthocyanin expression. As shown in Figure 1, anthocyanin accumulation was visually observable in the three transgenic lines, but not in the WT seedlings. The pigment was clearly observed when seedlings were cultured in environments with higher pH values (pH 6.0-8.0). The leaves collected from the WT seedlings were green, whereas those from the transgenic seedlings were red (Fig. 1). The anthocyanin levels were significantly higher in the transgenic plants than in the WT plants (Fig. 2). Of the three transgenic lines, anthocyanin content was highest in PhRs3 over all pH values, however, the anthocyanin levels in the WT seedlings remained unchanged at all pH values.

Fig. 1.

Anthocyanin accumulation after 20 days of treatment at various medium pH values in wild-type (WT) and transgenic plants


Fig. 2.

Accumulation of anthocyanin at various pH values in wild-type (WT) and transgenic seedlings after 20 days of treatment. The data are the means of three replications, and the bars indicate standard error


Bakowska et al. (2003) reported that a pH of 3.5 was suitable for anthocyanin co-pigmentation in Japanese morning glory (Ipomea nil), and a pH of 4 can affect anthocyanin accumulation in Petunia plants (Tanaka et al. 2000; Quattrocchio et al. 2006; Koes et al. 2005). Our results indicated that enhancement of anthocyanin in three transgenic lines seemed to be associated with pH, but such association was not observed for WT plants. Higher anthocyanin accumulation was clearly observed when the seedlings from all the transgenic lines were cultured in media with higher pH values.

Effect of pH on the expression levels of anthocyanin biosynthesis genes

According to results of qRT-PCR, expression of anthocyanin biosynthesis genes in the three transgenic lines and the WT line had the same expression trends (Fig. 3), and that higher anthocyanin accumulation was associated with higher levels of gene expression. The PhRs3 line exhibited the highest levels of expression. We found low levels of expression in the WT line. Transcription levels of the anthocyanin biosynthetic structural genes CHS and CHI, which are involved in the first reaction of the anthocyanin biosynthetic pathway, were significantly increased in the PhRs2 and PhRs3 lines at pH 6.0-8.0, compared to those in the WT seedlings. The expression levels of DFR and ANS (which control the last step in the biosynthetic anthocyanin pathway and encode anthocyanin or flavonol biosynthesis) were significantly higher in PhRs2 and PhRs3 than in PhRs1 or the WT plants. Expression levels of anthocyanin biosynthetic structural genes ANS and JAF13 (responsible for the last reaction in the anthocyanin biosynthetic pathway) were significantly higher in PhRs2 and PhRs3 at higher pH values, compared to those in the WT seedlings. The expression levels of DFR and FLS, which control the biosynthetic direction of the anthocyanin and flavonol pathways, encoding anthocyanin or flavonol biosynthesis, were significantly higher in PhRs2 and PhRs3 than in PhRs1 or the WT plants. Zhang et al. (2014) indicated that, under high pH condition, the high expression levels of flavonol biosynthetic genes are associated with flavonol accumulation in crab apple cultivars, grapevine leaves (Gutha et al. 2010), and Kiwifruit (Actinidia) (Fraser et al. 2013).

Fig. 3.

Effect of pH on the expression of anthocyanin-related genes in wild-type (WT) and transgenic seedlings. Gene expression levels were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR), as described in the text. The data are the means of three replications, and the bars indicate standard error


The present study showed that in transgenic plants expressing the RsMYB1 transcription factor, the content of anthocyanin pigments increased significantly at low pH values in vegetative plants owing to the expression of anthocyanin biosynthesis genes, and high pH values increased the growth parameters. However, enhancement of anthocyanin significantly increased the number of shoots and leaves. In contrast, the leaf size decreased. Furthermore, anthocyanin accumulation dramatically (and visibly) increased in the RsMYB1 transgenic plants.

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