Manipulation of Lipophilic Antioxidants to Enhance Abiotic Stress Tolerance and Nutritional Quality in Sweetpotato = 고구마의 지용성 항산화물질 대사조절을 통한 복합재해내성 및 기능성 향상 연구

다국어 초록 (Multilingual Abstract)

The dramatic increase of global population combined with rapid industrialization has led to great strains on global food, nutrition and energy supplies. According to estimates of the Food and Agriculture Organization of the United Nations (UN-FAO), the world population will be more than 9.7 billion by 2050 and 11 billion by 2100. FAO anticipate that global food and energy requirements in 2050 will exceed current needs by more than 1.7-fold and 3.5-fold, respectively. In order to solve these global issues such as food and nutrition security in the face of climate change and aging society, it is necessary to develop food crops that are resistant to environmental stress with enhanced essential nutrients.
Sweetpotato [Ipomoea batatas (L.) Lam] is the sixth most important starch crop in the world and has rich source of starch, low molecular weight (LMW) antioxidants, dietary fiber and potassium. High levels of LMW antioxidants are an important for both the plant protection to environmental stress and nutritional merits for human being as anti-aging and anti-disease agents. LMW antioxidants in plants can be divided into lipid-soluble antioxidants such as carotenoids and tocopherols (vitamin E) and water-soluble antioxidants such as ascorbic acid (vitamin C), glutathione and polyphenols.
In previous study, sweetpotato IbOr protein isolated form an orange-fleshed sweetpotato, with strong holdase chaperone activity, protects a key enzyme, phytoene synthase (IbPSY), in the carotenoid biosynthetic pathway and stabilizes a photosynthetic component, oxygen-evolving enhancer protein 2-1 (IbPsbP), under heat and oxidative stresses in plants. In addition, previously, five tocopherol biosynthetic genes were isolated and investigated their expression levels in leaves under drought, salt, and oxidative stresses.
In this study, gene manipulation of lipid-soluble antioxidants such as carotenoids and tocopherols was conducted to develop transgenic sweetpotato plants with enhanced abiotic stress tolerance and nutrition quality through plant biotechnology. The major results are as follows.
1) Site-directed mutagenesis of the IbOr gene, leading to Arg to His substitution at position 96 in the IbOr protein (IbOr-R96H), was performed and IbOr-R96H transgenic sweetpotato calli and plants using a white-fleshed sweetpotato cultivar, Xushu 29, were successfully generated. Transgenic sweetpotato calli overexpressing IbOr-R96H with a dark-orange color exhibited 13.3-fold higher total carotenoid contents and 39.3-fold higher levels of β-carotene than those in IbOr-WT calli with a light-yellow color. Interestingly, transgenic calli overexpressing IbOr-R96H showed increased tolerance to salt (150 mM NaCl) and heat (47°C) stresses.
In addition, storage roots of transgenic sweetpotato plants overexpressing IbOr-WT and IbOr-R96H exhibited a light-yellow color and light-orange color, respectively. The total carotenoid contents and β-carotene content of IbOr-R96H storage roots were up to 6.1- and 99.3-fold higher, respectively, than those of IbOr-WT storage roots. Particularly, the β-carotene content of IbOr-R96H storage roots was up to 186.2-fold higher than that of NT storage roots. In addition, leaf discs excised from IbOr-R96H plants showed greater tolerance to heat stress (47°C) than NT and IbOr-WT plants.
2) To further analyze the regulatory mechanisms of IbOr, through yeast two hybrid (Y2H) screening, I found several potential candidates interacting with IbOr protein. I conducted to elucidate the mechanism for regulating environmental stress resistance in relation to the interaction between IbOr and chlorophyll a,b-binding protein (CAB) which is one of the proteins constituting the photosystem. Sweetpotato CAB protein (IbCAB) were isolated and confirmed interaction with IbOr-WT and IbOr-R96H protein using co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assay. In order to resolve the regulatory mechanism through this interaction, IbCAB expression pattern analysis under various environmental stress conditions and light-related stress tolerance assay are under investigation.
3) Previously, isolation of 4-hydroxyphenylpyruvate dioxygenase (IbHPPD) and tocopherol cyclase (IbTC) genes in tocopherol biosynthesis pathway has been conducted. Transgenic sweetpotato plants overexpressing IbHPPD (referred as HP plants) and IbTC (referred as TC plants) were generated to understand the function of tocopherol biosynthetic genes in sweetpotato. Compared with non-transgenic (NT) plants, HP plants exhibited enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses. In addition, HP plants showed increased tolerance to the herbicide sulcotrione, which is involved in the inhibition of the HPPD. Interestingly, under dehydrated condition, HP plants displayed an elevated α-tocopherol content to 19–27% in leaves compared with NT plants. Additionally, the content of α-tocopherol was 1.6–3.3-fold higher in TC leaves than in NT leaves. Compared with NT plants, TC plants showed enhanced tolerance to multiple environmental stresses, including salt, drought, and oxidative stresses.
In conclusion, (1) my results demonstrate that overexpression IbOr-R96H in sweetpotato exhibited enhanced production of carotenoids and environmental stress tolerance and will facilitate the development of new cultivars with enhanced nutritional content and environmental stress tolerance. (2) I anticipate that site-specific mutagenesis of IbOr combined with CRISPR-Cas9-mediated base-editing techniques could be an effective tool for the improvement of carotenoid contents in various plant species. (3) It has been suggested that the interaction of IbOr and IbCAB protein plays an important role in photosynthesis. (4) In addition, IbHPPD and IbTC overexpressing transgenic sweetpotato plants showed an enhanced tolerance to various abiotic stresses. (5) Taken together, results of this study suggest that the manipulation of lipophilic antioxidants represents useful resource for developing crops with increased antioxidant levels and abiotic stress resistance. And it would help mitigate the global food, nutrition and energy security issues in the face of global climate change, thus facilitating the establishment of a sustainable society.

국문 초록 (Abstract)

산업혁명 이후 급속한 산업화와 세계 인구 증가는 심각한 글로벌 기후변화뿐만 아니라 식량, 에너지, 보건문제를 초래하고 있다. UN 식량농업기구 (FAO)는 2050년 세계인구는 97억 명, 2100년에는 110억 명에 도달하게 될 것이며 현재 추세대로 식량과 에너지를 사용한다면 2050년에는 식량과 에너지가 각각 지금의 1.7배와 3.5배 이상 필요할 것이라 전망했다. 기후위기시대, 고령화 시대에 대응하기 위해서는 환경스트레스에 잘 견디고 영양성분이 강화된 식량작물 개발이 필요하다.
고구마[Ipomoea batatas (L.) Lam.]는 세계 6대 식량작물로 전분 뿐만아니라 비타민C, 베타카로틴 등 각종 항산화물질, 식이섬유, 칼륨 등이 풍부하여 건강식품으로 재평가되고 있다. 저분자 항산화물질은 환경스트레스로부터 식물체를 보호하고 인간의 노화방지, 각종 질병 예방 등에 중요한 역할을 한다. 저분자 항산화물질은 카로티노이드, 토코페롤 (비타민E)과 같은 지용성 항산화물질과 아스코르브산 (비타민C), 글루타티온, 폴리페놀과 같은 수용성 항산화물질로 크게 분류할 수 있다. 선행연구에서 분리한 고구마 Orange (IbOr) 단백질은 강한 샤페론활성을 가지면서 카로티노이드 축적뿐만 아니라 고온, 건조, 염 등 다양한 환경스트레스에 저항성을 유도하였다. IbOr 단백질은 고온 등 스트레스 조건에서 카로티노이드 생합성에 중요한 phytoene synthase (IbPSY) 단백질과 광합성 광계II 단백질의 하나인 oxygen-evolving enhancer protein 2–1 (IbPsbP) 단백질과 결합하여 식물을 보호하는 것이 확인되었다. 또한 고구마에서 토코페롤 생합성에 중요한 유전자들이 분리되어 환경스트레스 관점에서 유전자 발현 등이 조사된 바 있다.
본 연구에서는 고구마의 지용성 항산화물질인 카로티노이드와 토코페롤을 형질전환기술을 이용한 대사조절로 지용성 항산화물질을 다량 함유하면서 복합 환경스트레스에 잘 견디는 복합기능성 고구마 개발을 위한 기반기술을 확보하고자 연구를 수행하여 다음과 같은 주요 결과들을 도출하였다.
1) 고구마 IbOr 단백질 염기서열에서 96번째 아미노산을 아르기닌 (R)에서 히스티딘 (H)로 치환시킨 IbOr-R96H 변이체를 제작하여 저장뿌리 속이 흰색인 고구마 품종 (Xushu 29)에 과발현 한 형질전환 캘러스와 식물체를 개발하였고 변이체의 기능을 IbOr (IbOr-WT)와 비교하였다. IbOr-R96H 배양세포는 IbOr-WT 배양세포보다 전체 카로티노이드와 베타카로틴 함량이 각 약 13.3배와 약 39.3배 증가하였다. 또한 IbOr-R96H 배양세포는 고염 (150 mM NaCl), 고온 (47°C) 스트레스에서 내성을 나타내었다.IbOr-WT 과발현 고구마와 IbOr-R96H 과발현 고구마의 저장뿌리에서 IbOr-WT은 노란색, IbOr-R96H는 옅은 주황색의 표현형을 띄었다. IbOr-R96H 과발현 저장뿌리의 전체 카로티노이드와 베타카로틴 함량은 IbOr-WT 식물체에 비해 각각 최대 약 6.1배와 99.3배 증가하였다. 특히 IbOr-R96H 식물체 저장뿌리의 베타카로틴 함량은 NT식물체에 비해 최대 약 186.2배 증가되었다. 또한 잎 절편 (leaf disc)를 이용한 고온 (47°C) 스트레스 내성 실험에서 IbOr-R96H 과발현 형질전환 고구마 식물체가 IbOr-WT식물체보다 47°C 고온에 더 강한 저항성을 가지는 것을 확인하였다.
2) IbOr 단백질의 기능을 자세히 규명하기 위해 yeast two hybrid (Y2H) screening을 통해 IbOr 단백질과 상호작용하는 새로운 여러 후보 단백질들을 탐색하여, 그 중 하나인 chlorophyll a,b-binding (CAB) 단백질의 기능을 규명하고자 하였다. 고구마에서 IbCAB 유전자를 분리하여 co-immunoprecipitation (Co-IP), bimolecular fluorescence complementation (BiFC) assay 실험으로 IbCAB 단백질이 IbOr-WT 단백질, IbOr-R96H 단백질과 상호작용한다는 것을 확인하였다. 현재 IbOr 단백질과 IbCAB 단백질의 상호작용 조절 메커니즘을 규명하기 위해 빛 등 다양한 환경스트레스 조건에서 IbCAB의 발현 패턴분석 등을 분석 중이다.
3) 고구마에 존재하는 토코페롤 생합성에 관여하는 유전자 가운데 4-hydroxyphenylpyruvate dioxygenase (IbHPPD)와 tocopherol cyclase (IbTC) 유전자의 기능을 이해하고자 이들 유전자를 과발현 하는 형질전환 고구마 (Xushu 29 품종 이용)를 제작하여 분석하였다. IbHPPD 식물체는 NT 식물체에 비해 HPPD 저해 제초제인 sulcotrione (1 mM), 염 (200 mM NaCl), 건조, 산화스트레스 (5 μM MV) 내성을 보였으며 건조스트레스 후에 잎에서 α-tocopherol 함량이 NT식물체에 비해 최대 27%까지 증가하였다. IbTC 식물체는 스트레스가 없는 정상 조건일 때 잎에서 α-tocopherol 함량이 NT 식물체에 비해 최대 3.3배 증가했고, 고염 (200 mM NaCl), 건조, 산화 (3 μM MV) 스트레스에서 내성이 증가한 것을 확인 할 수 있었다.
본 연구에서 얻어진 결과를 토대로 종합적인 고찰과 전망은 다음과 같다. (1) 카로티노이드 축적 관련 IbOr-R96H 유전자가 IbOr-WT와 함께 카로티노이드 함량을 대폭 증가시키면서 고온 등 환경스트레스에 강한 품종을 개발할 수 있음이 강하게 제시되었다. (2) IbOr-R96H 연구결과는 고구마 유전자 편집기술(CRISPR-Cas9-mediated base-editing techniques)에 직접 활용될 수 있을 뿐만 아니라 다양한 식물에도 적용할 수 있을 것으로 기대된다. (3) IbOr 단백질과 상호작용하는 IbCAB 단백질을 처음으로 분리하여 IbOr이 광합성에 매우 중요하게 관여함이 시사되어 후속연구가 기대된다. (4) 토코페롤 생합성 유전자 (IbHPPD와 IbTC) 과발현 형질전환연구에서 토코페롤 대사조절로 복합 환경스트레스 내성식물체 개발에 활용될 수 있음이 시사되었다. 결론적으로 본 연구에서 얻어진 결과를 발전시킨다면 기후위기와 고령화 시대에 대응할 수 있는 환경스트레스에도 강하고 지용성 항산화물질을 고생산하는 복합기능 고구마 개발에 기여할 것으로 기대된다.

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