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No reuse allowed without permission. 1 Article 2 An ABA Synthesis Enzyme Allele OsNCED2 Promotes the 3 Aerobic Adaption in Upland Rice a a a a a a 4 Liyu Huang , Yachong Bao , Shiwen Qin , Min Ning , Jun Lyu , Shilai Zhang , a a b b a,* 5 Guangfu Huang , Jing Zhang , Wensheng Wang , Binying Fu and Fengyi Hu 6 a 7 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, 8 Research Center for Perennial Rice Engineering and Technology of Yunnan, School of 9 Agriculture, Yunnan University, Kunming, Yunnan 650091, China; b 10 Institute of Crop Sciences/National Key Facility for Crop Gene Resources and 11 Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, 12 China. 13 14 15 * 16 Correspondence: [email protected] 17 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.146092; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 18 ABSTRACT 19 There are two ecotypes, upland rice and irrigate rice, during its evolution in rice. 20 Upland rice exhibits aerobic adaptive phenotype via its stronger root system and more 21 rapid drought responses compared with its counterpart, irrigated rice. We assessed the T 22 functional variation and applications of the aerobic adaptive allele OsNCED2 cloned 23 from IRAT104 which is a cultivar of upland rice. OsNCED2-overexpressing T 24 transgenic rice and OsNCED2 -NILs exhibited significantly higher ABA contents at 25 the seedling and reproductive stages, which can improve root development (RD) and 26 drought tolerance (DT) to promote aerobic adaptation in upland rice. RNA-Seq-based 27 expression profiling of transgenic versus wild-type rice identified 28 OsNCED2-mediated pathways that regulate RD and DT. Meanwhile, 29 OsNCED2-overexpressing rice exhibited significantly increased reactive oxygen 30 species (ROS)-scavenging abilities and transcription levels of many stress- and 31 development-related genes, which regulate RD and DT. A SNP mutation (C to T) 32 from irrigated rice to upland rice, caused the functional variation of OsNCED2, and 33 the enhanced RD and DT mediated by this site under aerobic conditions could T 34 promote higher yield of upland rice. These results show that OsNCED2 , through 35 ABA synthesis, positively modulates RD and DT which confers aerobic adaptation in 36 upland rice and might serve as a novel gene for breeding aerobic adaptive or 37 water-saving rice. 38 39 Key words: Upland rice; aerobic adaptation; ABA; Root development; Drought 40 tolerance 41 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.146092; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 42 INTRODUCTION 43 Rice is a major food crop and model plant for the genomic study of monocotyledon 44 species. Long-term natural selection has driven the evolution of various ecotype 45 populations in Oryza sativa, such as upland rice and irrigated rice, which have 46 evolved their respective characteristic traits under both natural and artificial selection, 47 which leads to phenotypic adaptation to the respective environment and simultaneous 48 genetic differentiation between ecotypes. Therefore, rice provides a model system for 49 studying adaptation to aerobic and drought environments (Xia et al., 2019). Although 50 the yield of upland rice is comparatively lower than that of irrigated rice, the former 51 serves as a daily staple food on which nearly 100 million people depend (Pomfret, 52 1986). Accordingly, upland varieties are widely utilized under upland conditions to 53 optimize crop yields due to their more efficient usage of water and their adaptation to 54 aerobic upland (Xia et al., 2019). Previous research has proposed three different 55 mechanisms for plant drought resistance: drought escape, dehydration avoidance, and 56 drought tolerance (DT) (Jérôme et al., 2010). Upland rice has multiple special 57 characteristics, such as aerobic adaption, taller height, lower tillering potential, and 58 longer and denser roots, compared with its irrigated counterpart (Chang, 1982; Chang 59 and Vergara, 1975). 60 In our previous study, the genomes of upland and irrigated rice accessions were 61 analysed via resequencing and population genetics (Lyu et al., 2014). Multiple 62 pathways or ecotype differentiated genes (EDGs) that potentially contribute to the 63 aerobic adaptation of upland rice to dryland were identified, but only a few of these 64 have been functionally verified. Interestingly, a 9-cis-epoxycarotenoid dioxygenase 65 gene (Os12g0435200) named OsNCED2, which plays key roles in the abscisic acid 66 (ABA) synthesis pathway, was identified as a domestication gene which contributes 67 to the aerobic adaptation of upland rice (Lyu et al., 2013). The ABA-mediated root 68 tropic response and stomatal closure under water stress are key responses of plants 69 that ensure their survival under water-limited conditions. ABA signalling also plays a 70 critical role in the regulation of root growth and the architecture of the root system 71 and likely interacts with other hormones, such as auxins, gibberellins, or 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.146092; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 brassinosteroids (BRs), to regulate in these processes (Deak and Malamy, 2005; 73 Rodrigues et al., 2009; Swarup et al., 2005). ABA is a stress-responsive 74 phytohormone that inhibits seed germination and seedling growth to adapt to 75 unfavourable environmental conditions, whereas gibberellic acid (GA) and BRs are 76 major growth-promoting phytohormones that promote seed germination, seedling 77 growth, flowering and leaf expansion (Clouse, 2016; Cutler et al., 2010; Golldack et 78 al., 2013; Wang et al., 2018; Weiner et al., 2010). 79 In this study, OsNCED2 was functionally identified as a 9-cis-epoxycarotenoid 80 dioxygenase that mediates ABA synthesis. Furthermore, OsNCED2 can promote root 81 development while reducing plant height in OsNCED2-overexpressing transgenic 82 lines. Although both types of OsNCED2 (C/T) have a 9-cis-epoxycarotenoid 83 dioxygenase function, they might exhibit significant differences in catalytic activity, 84 which result in the different ABA concentrations between upland rice and irrigated T 85 rice. Thus, rice with T-type OsNCED2, OsNCED2 , exhibits increased adaptation to 86 aerobic conditions due to its higher ability to regulate the synthesis and/or distribution 87 of ABA in response to osmotic stress. Consequently, both the overexpression of T 88 OsNCED2 and the import of OsNCED2 into irrigated rice enhance plant yield and 89 therefore can potentially be used in the breeding of aerobic adaptive or water-saving 90 rice. 91 92 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.146092; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 93 RESULTS 94 Identification and Characterization of OsNCED2 from Upland Rice and 95 Irrigated Rice 96 In our previous comparative genomic study, we found that OsNCED2 on 97 chromosome 12, was highly differentiated between the two ecotypes (Lyu et al., 98 2013). OsNCED2 differs in abundance between upland and irrigated rice and 99 produces two haplotypes of the SNP (C/T), namely, T-type in upland rice and C-type 100 in irrigated rice (Lyu et al., 2013). The full-length OsNCED2 gene sequence consists 101 of 1731 bp without any intron and encodes a 576-amino-acid polypeptide annotated as 102 a 9-cis-epoxycarotenoid dioxygenase. A phylogenetic analysis indicated that all 103 NCED paralogous genes might have originated from two ancestral genes in rice and 104 that OsNCED2 clustered with other paralogous genes, which implies that OsNCED2 105 might have arisen from gene duplication in the rice genome (Supplemental Fig. S1). 106 OsNCED2 contains several signal-responsive cis-elements in its promoter region (1.5 107 kb upstream of the start codon), and these include light, anaerobic, GA, MeJA and 108 ABA response elements (Supplemental Table S1), which implies that the expression 109 of OsNCED2 could be regulated by environmental and hormone signalling. A Target P 110 analysis confirmed that OsNCED2 might be located in chloroplasts and our 111 cytological analyses indicate that OsNCED2 is localized in the chloroplast 112 (Supplemental Fig. S2), which is consistent with its function as a 113 9-cis-epoxycarotenoid dioxygenase that catalyses the synthesis of ABA in 114 chloroplasts (Qin and Zeevaart, 1999). 115 116 Spatial-temporal Expression Pattern of OsNCED2 in Rice 117 Because OsNCED2 has seven paralogous genes in the whole genome and might 118 play different roles in rice development and response to the environment, we 119 examined the OsNCED2 expression patterns in different tissues at different 120 developmental stages by both qRT-PCR and histochemical staining, which indicated 121 the occurrence of OsNCED2 promoter activity in OsNCED2Pro::GUS reporter 122 transgenic plants. The results showed that OsNCED2 was mainly expressed in the 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.11.146092; this version posted June 12, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 123 mature leaves, stems and roots and exhibited relatively lower expression in young 124 panicles and seedlings (Fig.