Professor Dr Apichart Vanavichit, Director of the Rice Science Center, reflects on the decade since the most important gene of fragrance rice was uncovered
Fragrant rice is much sought after worldwide and as such, sells for higher prices than non-fragrant rice. There are two popular fragrant rice classes in the global market, soft- cooked long grain Jasmine rice and fluffy-cooked long grain Basmati rice. In Asia, soft-cooked fragrant rice is more prevalent among middle and wealthy-income populations. Developing new fragrant rice varieties is a strategic goal in rice-consuming countries.
Characteristics of rice fragrance
Cooked rice aroma possesses around 200 volatile compounds, including hydrocarbons, alcohols, aldehydes, ketones, acids, esters, phenols, pyridines, pyrazines and other compounds. In 1983, scientists identified the potent aromatic compound in rice known as 2-acetyl-1-pyrroline (2AP). Fragrant rice accumulated much higher levels of 2AP compared to non-fragrant rice. However, the detection of 2AP requires laborious analytical methods not suitable for rice breeding programmes. Using aroma-specific DNA markers, breeders can select better fragrant plants with more robust throughput. For the first time, the most important gene was cloned from Thai Hom Mali Rice, the world-renown fragrant rice, in 2004. The knockout mutation was identified for precision breeding of fragrant rice.
Function & diversity of the fragrant gene in rice
The gene involved in making rice fragrance is a dysfunction of amino aldehyde dehydrogenase (AMADH), the specific enzyme converting 4-amino butanol to 4-amino butyric acid (GABA) found in non-fragrant rice. Consequently, the building-up of the toxic butanol is converted into 2AP in fragrant rice (1). Is such a biochemical pathway unique for the Thai Hom Mali rice?
A genetic survey concerning four classes of fragrant rice, Basmati (Central Asia), Paw San Hmwe (Myan- mar), Jasmine (Southeast Asia), and Japonica (East Asia) showed a similar dysfunctional enzyme AMADH knock-out by specific mutations.
Precision breeding of fragrant rice
Gene sequences of inactivated mutations were used for designing specific DNA markers for precision breeding. Since 2008, many rice-consuming countries have released newly improved fragrant rice varieties. More than 50 new fragrant rice varieties in Thailand were improved for resistance to biotic and abiotic stresses. In India, 18 new Basmati rice varieties have been released since 2008. Also, improving high-yielding and early maturity fragrant rice has been the major goal In Vietnamese rice.
Discovering fragrant genes in other plants & microbes
The rice fragrance was also detected in other plant species. It is interesting to know if they also carried similar genetic dysfunction of AMADH as rice. The AMADH-like genes from 12 fragrant plant species were analysed for genetic similarity, including rice, bread flour, pandan, vegetable soybean, sorghum, mung bean, taro, spinach, cucumber, ridge gourd, sponge gourd and winter melon. The result revealed different inactivated mutations that did not express the AMADH-like genes yet created 2AP. In addition, a similar genetic mechanism was also responsible for 2AP bio-synthesis in bacteria and fungi such as Bacillus cereus and Bakers’ yeast. These results led us to conclude that 2AP-producing fragrant plant and microbial species shared similar evolutionary paths.
Innovative technology to generate new fragrant plants
Besides precision breeding, molecular inactivation of specific AMADH can generate a new fragrant plant and microbial variants. The initial invention utilised RNA interference technology (RNAi) to convert Nipponbare, non-fragrant japonica rice, to fragrant rice (2,3). Genome editing is the latest technology that precisely creates a specific double DNA strand break at a target genomic site. Gene editing of native AMADH-like genes by utilising TALEN (4) and CRISPR- CAS9 (5, 6) generated innovative 2AP-producing plants.
Recent & future research challenges
Fragrant rice landraces are narrowly adaptable to their specific niche environments; otherwise, low productivity in modern cultural practices. High-yielding fragrant rice varieties do not guarantee strong grain aroma because environmental conditions substantially impact grain aroma. Under salinity, cold, and drought stresses, grain-2AP is enhanced, but grain yield is reduced. Moreover, the overall productivity of RNAi-derived fragrant rice was affected compared to the wild type(3). Therefore, grain yield and strong aroma seem to require the opposite environmental conditions. To overcome such problem, rice farmers need to add innovative steps, including alternating wetting and drying during the vegetative stage (7), exogenous applications of NaCl (8), GABA (9), Silicon (10), and Copper (11) applied during the vegetative and early reproductive stages. These new cultivation practices enhanced grain-2AP at least 1.5-2.0 times compared to the control. Therefore, after a decade of aromatic gene discovery has strong impact on breeding-by-design and precision agriculture to significantly improve fragrance rice productivity in modern agriculture.
References
- Vanavichit, A., Tragoonrung, S., Toojinda, T., Wanchana, S., Kamolsukyunyong, W. 2005. Transgenic rice plants with reduced expression of Os2AP enhance the expression of the 2-acetyl -1- pyrro- line. (US 7319181, EP 1683869, CN1810977, AU 2005203356).
- Vanavichit, A. and Yoshihashi, T. 2010. Molecular aspects of fragrance and aroma in rice. Adv Bot Rev 56:49-79. (https://www.researchgate.net/publication/229432928_Molecu lar_Aspects_of_Fragrance_and_Aroma_in_Rice)
- Xiangli Niu, Wei Tang, Weizao Huang, Guangjun Ren, Qilin Wang, Di Luo, Uyingyong Xiao, Shimei Yang, Feng Wang, Bao Rong Lu, Fangyuan Gao, Tiegang Lu, and Yongsheng Liu. 2008. RNAi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1-pyrroline) production in rice (Oryza sativa L.). BMC Pl Biol 8:100.
- Qiwei Shan, Yi Zhang, Kunling Chen, Kang Zhang, and Caixia Gao. 2015. Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology. Pl Biotech J 13:791.
- Shao GaoNeng, Xie LiHong, Jiao Guiai, Wei Xiangjin, Sheng ZhongHua, Tang ShaoQing, Hu PeiSong. 2017. CRISPR/CAS9- mediated edting of the fragrant gene BADH2 in rice. Chi J Rice Sci 31(2):216.
- Shanthinie Ashokkumar, Deepa Jaganathan, Valarmathi Ramanathan, Hifzur Rahman, Rakshana Palaniswamy, Rohit Kambale, Raveendran Muthurajan. 2020. Creation of novel alle- les of fragrance gene OsBADH2 in rice through CRISPR/CAS9- mediated gene editing. PLOS ONE https://doi.org/10.1371/ journal.pone.0237018.
- Gegan Baok Umair Ashraf, Chunling Wang, Longxin He, Xiaoshan Wei, Axiang Zheng, Zhaowen, Mo, and Xiangru Tang. 2018. Molecular basis for increased 2-acetyle-1-pyrroline contents under alternate wetting and drying (AWD) conditions in fragrant rice. Pl Physiol Biochem. 133:149.
- Renuka, N., Barvkar, V.T., Ansari, Z., Zhao, C., Wang C., Zhang Y., and Nadaf, A.C. 2022. Co-functioning of 2AP precursor amino acids enhances 2-acetyl-1-pyrroline under salt stress in aromatic rice (Oryza sativa L.) cultivars. Sci Rep 12:3911.
- Wenjun Xie, Leilei Kong, Lin Ma, Umair Ashraf, Shenggang Pan, Meiyang Duan, Hua Tian, Longmei Wu, Xiangru Tang, and Zhowen Mo. 2020. Enhancement of 2-acetyl-1-pyrroline (2AP) concentration, total yield, and quality in fragrant rice through exogenous 4-aminobutyric acid (GABA) application. J Cereal Sci. 91:102900.
- Zhaowen Mo, Shun Lei, Umair Ashraf, Imran Khan, Yuan Li, Shenggang Pan, Meiyang Duan, Hua Tian, and Xiangru Tang. 2017. Silicon fertilization modulates 2-acetyl-1-pyrroline con- tent, yield formation and grain quality of aromatic rice. J Cereal Sci. 75:17.
- Yong Ren, Yulin Zhu, Fang Liang, Qingqing Li, Quanhu Zhao, Ying He, Xueer Lin, Xiao Qin, and Siren Cheng. 2022. Effect of foliar copper application on grain yield, 2-acetyl-1-pyrroline and copper content in fragrant rice. Pl Physio. Biochem. 182:154.
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