THE ANTHOCYANIN BIOSYNTHETIC REGULATOR MDMYB1 POSITIVELY REGULATES ASCORBIC ACID BIOSYNTHESIS IN APPLE

Highlights

• The contents of anthocyanin and AsA in red-flesh apples are higher than that in non-red-flesh apples.

• The anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA.

• MdMYB1-MdDHAR module may play a key role in AsA-DHA homeostasis.

Abstract

Ascorbic acid (AsA, vitamin C) is involved in the regulation of many aspects of plant growth and development. It is an essential micronutrient for humans and can prevent scurvy, maintain the health of gums and blood vessels, reduce the level of plasma cholesterol and enhance the immune systen. Apple cultivars Orin and Guanghui were crossed to obtain a group of hybrid offspring with and without red flesh in the course of assessing apple germplasm resources. Unexpectedly, the red-flesh apples had higher AsA contents than other apples. Further studies showed that the anthocyanin biosynthetic regulator MdMYB1 directly activates the expression of dehydroascorbate reductase gene MdDHAR, thus promoting the activity of the DHAR enzyme and the accumulation of AsA. This finding reveals the mechanism leading to high AsA levels in red-flesh apples and suggests a new idea to cultivate red-flesh apples with high AsA contents and produce AsA efficiently and without pollution.

Graphical abstract

Keywords

anthocyanin / apples / ascorbic acid / MdMYB1 / vitamin C

1 Introduction

China is one of the largest countries for poultry production in the world. However, more than half of the poultry are bred in small-scale farms or in backyards. This type of breeding style enables domestic fowls to share the same niches with wild birds, allowing domestic fowls to be an important intermediate host in the transfer of influenza from wild birds to domestic animals [ 1], which poses great difficulties for the control of avian influenza in China. In 2004, a combined culling plus vaccination strategy was used to control outbreaks of highly pathogenic avian influenza (HPAI) in China [ 2]. However, up to 2014, 127 outbreaks in domestic poultry had occurred in over 20 provinces, causing severe economic damage to the poultry industry in China (http://www.oie.int).

2 Outbreaks of H5 avian influenza and vaccines used in China

H5N1 avian influenza viruses were first detected in sick geese in Guangdong province in 1996 [ 3]. This is the first documented H5N1 HPAI virus isolation in China. Since 1999, H5N1 viruses have been repeatedly isolated from apparently healthy ducks in southern China [ 4]. In 2001 and 2003, two H5N1 viruses were isolated from pigs in Fujian Province [ 5]. On January 27, 2004, a highly pathogenic H5N1 virus caused an outbreak in domestic ducks in Guangxi Zhuang Autonomous Region, and then more than 40 outbreaks occurred in 16 provinces within the next month [ 2]. To control the outbreaks of highly pathogenic avian influenza, an inactivated oil-emulsified vaccine was developed using an H5N2 low pathogenic virus, A/Turky/England/N-28/73, as a seed virus [ 2]. The H5N2 vaccines played an important role in the rapid control of the H5N1 outbreaks in China in 2004. However, the H5N2 vaccine seed virus exhibited antigenic diversity with the prevalent H5N1 strains in China. To solve the problem, Re-1, a reassortant virus, was created through plasmid based reverse genetics, with the haemagglutinin (HA) and neuraminidase (NA) genes from the A/goose/Guangdong/1/1996 (GS/GD/1/96) virus and the internal genes from the high growth A/Puerto Rico/8/34 (PR8) virus [ 6]. The vaccine was approved to be used in the field by the end of 2004.

In 2006, there were 10 outbreaks on small-scale, backyard chicken or duck farms, with birds that had not been vaccinated. It is important to note that H5N1 outbreaks occurred in some layer farms in Shanxi Province, although the chickens in these farms were vaccinated with the H5 inactivated vaccines. Samples were sent to the National Avian Influenza Reference Laboratory for disease diagnosis. An H5N1 influenza virus, A/chicken/Shanxi/2/06 (CK/SX/06) was identified, but this strain reacted poorly with the antisera against Re-1 virus-based antigen. Sequence analysis indicated that this H5N1 virus was also genetically quite different from the H5N1 viruses isolated from other locations [ 7]. To control the epidemics caused by the CK/SX/06 virus, a new inactivated vaccine Re-4 was developed by using a recombinant H5N1/PR8 reassortant virus bearing the modified HA and NA genes from the CK/SX/06 virus. The vaccine was applied in the selected provinces in northern China in 2006. In 2006, Smith et al. reported the emergence and predominance in poultry in China since late 2005 of a previously uncharacterized H5N1 virus sublineage (Fujian-like) [ 8]. Viruses from this sublineage gradually replaced those multiple regional distinct sublineages and caused human infection in China. The virus was transmitted to Hong Kong, Laos, Malaysia, and Thailand, resulting in a new wave of transmission and outbreaks in South-east Asia. In May 2008, another new H5N1/PR8 reassortant virus, Re-5, which derives its HA and NA genes from A/duck/Anhui/1/06, was applied in China.

The lack of uniformity in nomenclature often leads to confusion in the interpretation of the evolutionary relationships among HPAI virus (H5N1) isolates. To address the issue, the World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization H5N1 Evolution Working Group developed a unified nomenclature system for the classification of H5N1 HPAI viruses and 10 distinct clades of viruses (0–9) were initially designated to characterize the phylodynamics of the HA gene of the Gs/GD-like H5 viruses [ 9]. After 2008, molecular epidemiological surveys indicated that commercial farms have been well protected by the vaccines, but the H5N1 viruses are still circulating in China, especially in live bird markets, and backyard flocks are at greater risk of infections [ 7, 10]. Phylogenetic analysis indicated that three clades (2.3.4, 2.3.2 and 7) co-circulated in poultry in China in 2007–2009 [ 10]. The variant of clade 2.3.2 was first isolated in Hong Kong SAR and mainland China in 2007 [ 11, 12]. It has since circulated widely in poultry in China and caused a new wave of cross-continental infection, spreading from Asia to Europe [ 13– 15]. Antigenic analysis revealed that the clade 2.3.2 viruses reacted weakly with antisera prepared from the Re-4 and Re-5 vaccines widely used in China [ 10]. The new H5N1/PR8 reassortant virus Re-6 was developed and applied in China from 2011.

After 2007, clade 7.2 viruses were predominantly found in chickens in Northern China. Although the viruses from this clade were genetically similar to the Re-4 virus, they reacted poorly with the antisera raised against Re-4 virus-based antigen [ 10]. In December 2013, there was an outbreak of H5N2 HPAI in a chicken farm in Hebei Province. Phylogenetic analysis revealed that the virus was a reassortant of the H5N1 and H9N2 viruses [ 16]. To control the epidemics, Re-7 vaccine was approved for use in China. In addition to H5N1 HPAI viruses, the emergence of novel H5N2, H5N5, H5N6, and H5N8 subtype HPAI viruses adds more evidence for the active evolution of H5 viruses in China [ 17– 20]. These novel H5 viruses reacted poorly with antisera prepared from the Re-5 vaccine, although they belong to the same clade. To control these novel H5 viruses, a new vaccine Re-8 was developed. This new vaccine is expected to be approved for use in the future. The present facts indicate that although vaccination has been implemented in China for the past ten years, H5 avian influenza still continues to be found in poultry populations.

Table 1 shows H5 avian influenza outbreaks and vaccines used in China from 2004 to 2014.

Tab.1 H5 avian influenza outbreaks and vaccines used in China from 2004 to 2014

Year	Outbreaks	Vaccine
Subtype	Seed name
2004	50	H5N2	A/turky/England/N-28/73
2005	32	H5N1	H5N1(A/goose/Guangdong/1/1996)/PR8 (Re-1)
2006	12
2007	4	H5N1	H5N1(A/chicken/Shanxi/2/2006)/PR8 (Re-4)
2008	6	H5N1	H5N1(A/duck/Anhui/1/2006)/PR8 (Re-5)
2009	2
2010	0
2011	1	H5N1	H5N1(A/duck/Guangdong/S1322/2010)/PR8 (Re-6)
2012	8
2013	2
2014	9	H5N1	H5N1(A/Chicken/Hebei/2012)/PR8 (Re-7)
total	126

3 Problems in implementing the vaccination strategy

3.1 Farm management

Clinical data have shown that vaccination was not ideal and had some drawbacks. Most of the influenza vaccines could prevent clinical signs but not infection [ 21– 23]. The culling plus vaccination policy for preventing HPAI H5N1 viruses was implemented in China in 2004 [ 2], requiring all poultry to be vaccinated. However, there is still a large proportion of poultry raised in small-scale, free-range contexts in China. Shoddy rearing equipment, poor prevention conditions and extensive management are the main problems for such poultry production. Furthermore, it is not easy to carry out an effective vaccination program for these farms. All these deficiencies resulted in ineffective immunization in these poultry. As a consequence of small-scale, free-range poultry, it is difficult for the government to supervise and provide epidemic prevention services. Therefore, free-range poultry are often missed in programs for preventing and controlling avian influenza epidemics, and therefore represent a disease risk.

3.2 Quality of the vaccine

The government has required 100% vaccine coverage in domestic poultry, and financial support from the government ensures the implementation of this strategy. However, many farmers lack the enthusiasm to use the vaccine. They complain of low quality vaccines containing suboptimal concentration of antigens that are passed the quality guarantee dates. In addition, inactivated vaccines induce an antibody peak at least 14 days after immunization [ 24]. The short growing period of meat poultry and vaccine residues in the tissue of immunized poultry restrict its comprehensive usage in poultry meat production.

3.3 Antigenic diversity

The H5N1 HPAI viruses in the natural environment are not a homogeneous population, and they may have different antigenicity [ 7, 10]. Thus, after exposure to the field viruses, vaccinated birds become infected without signs of disease but excrete small amount of the viruses from the respiratory tract and/or in the feces [ 10, 21– 23]. This phenomenon may also cause some new problems for the control of the disease. First, infections with the HPAI viruses in vaccinated birds may cause silent spread of the virus between flocks when biosecurity is weak [ 25]. Secondly, antibody pressure may drive the viral evolution [ 26, 27]. Influenza A virus is an RNA virus which has an polymerase enzyme lacks proofreading activity, resulting in a replication mistake in every 10^-4 nucleotides per se [ 28]. Populations of viruses in a host then appear as quasi-species. The quasi-species which are the most fit in a particular environment would become the predominant population. Thus, new viral strains would have a selective advantage for spreading in vaccinated flocks, if they have become antigenically different from the vaccine strains [ 26].

In addition, many low level biosecurity farms ignore effective management strategies and readily use vaccine to prevent diseases. Controlling the infection source, cutting off the route of transmission, and protecting susceptible populations are three prevention strategies to control the infectious disease. When biosecurity is compromised, poultry are often exposed to viruses in the field. In this situation, vaccination alone will not prevent the spread of the viruses because the transmission rate of HPAI viruses is very high [ 25, 29].

4 Suggestions for controlling avian influenza

4.1 Improving traditional poultry breeding practices

Good farm management and strict biosecurity measures help prevent virus entry into farms and transmission between sheds within farms [ 30]. A retrospective analysis showed that most of the H5N1 outbreaks in China occurred on small-scale or backyard farms [ 2]. Some small-scale farms showed a low immunity level. Also, free-range domestic fowls have potential contact with wild waterfowls and other domestic animals such as pigs. Therefore, these farms are important in transmission of virus from wild birds to other domestic poultry and other animals, including humans [ 1, 31]. Thus, the traditional poultry raising practices in China must be improved to control H5N1 HPAI. A poultry farm should only raise one kind of poultry, and avoid mixed production of chickens with either ducks or geese. We strongly suggest that the government set up an “admission threshold ” for approval to breed poultry.

4.2 Stopping live poultry markets

Epidemiology surveillance has shown that the live poultry markets have an important role in the amplification and dissemination of influenza viruses [ 32, 33], and the exposure history in live poultry markets is the key risk factor for human H5N1 and H7N9 cases [ 34– 36]. In the 1997 Hong Kong influenza incident and the 2013 H7N9 human infection in Shanghai, stopping the live poultry markets proved to be a very useful measure to control human infection. Therefore, the following measures are suggested: slaughtering the poultry in specialized factories with refrigeration, and eventually sending the fresh-kept poultry product to supermarkets for sale. The strategy may not only break the transmission route of the virus, but also prevent human contact with live poultry, thereby stopping infection of people.

4.3 Improving the prevention system for animal epidemics

An effective animal epidemic prevention and control system is the best option to control avian influenza [ 30]. However, China’s animal epidemic prevention system does not satisfy the requirements yet. Although mechanism for emergency reactions are ready for the prevention and control of major animal diseases such as the avian influenza, and planning has been undertaken, the relatively poor system in rural areas makes it impossible to meet the demand of higher standard of prevention and control of avian influenza. Problems have appeared in some districts where the suspected avian influenza could not be diagnosed in time, which eliminated the best opportunity for prevention. It is essential to find and eradicate outbreaks promptly to control HPAI [ 30]. In addition, the government should establish regional avian influenza laboratories in each province in addition to the National Avian Influenza Reference Laboratory. The China Animal Health and Epidemiology Center could then manage the prevalence and distribution of avian influenza based on annual epidemiological surveys, and consequently perform risk assessment and guide prevention work.

Furthermore, the government should also put in place comprehensive mechanisms of culling compensation. Once culling measures are performed, full compensation should be immediately provided to the farmers so they do not suffer a financial loss. Only in this way they will report an outbreak quickly instead of concealing the truth, and this will help the government to take prompt measures to control outbreaks of HPAI.

5 Conclusions

China has employed a culling plus vaccination strategy to control H5N1 avian influenza for a decade. The use of influenza vaccine increased the resistance of poultry to the disease and played an important role in controling the transmission of avian influenza. However, clinical data have shown some problems during implementation of the vaccination strategy. Avian influenza has been the biggest barrier in the development of the poultry industry. Therefore, great efforts should be made to change avian production practices, develop standardized and large-scale poultry farms, change poultry consumption habits, and improve the animal epidemic prevention and control systems. With these problems resolved, vaccination programs can be stopped, and monitoring and culling policies adopted, as carried out in developed countries, and avian influenza ultimately controlled.

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Acknowledgements

This work was funded by grants from the Agricultural Variety Improvement Project of Shandong Province (2019LZGC007), Fruit quality improvement of Yunnan Province (2019ZG002), and the Natural Science Foundation of Shandong Province (ZR2019PC004).

Compliance with ethics guidelines

Jianping An, Xiaofei Wang, Chunxiang You, and Yujin Hao declare that they have no conflicts of interest or financial conflicts to disclose. This article does not contain any studies with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2020. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)