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1 Article

2 An ABA Synthesis Enzyme Allele OsNCED2 Promotes the

3 Aerobic Adaption in Upland 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

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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

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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 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

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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 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

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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

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123 mature leaves, stems and roots and exhibited relatively lower expression in young

124 panicles and seedlings (Fig. 1, A). Although differences in the OsNCED2 promoter

125 were found between upland and irrigated rice, no significant differences in expression

126 was detected between the upland rice variety IRAT104 and the irrigated rice variety

127 Yueguang (Fig. 1, B). GUS histochemical staining of OsNCED2Pro::GUS reporter

128 transgenic plants also showed consistent expression in related tissues (Fig. 1, C).

129 Interestingly, OsNCED2 was mainly expressed in new or young but not old roots (Fig.

130 1, D). These results suggest that OsNCED2 might play different roles in different

131 tissues.

132

133 OsNCED2 Overexpression can Promote ABA Synthesis and Root Development

134 To verify the function of OsNCED2, two types of OsNCED2 alleles cloned from

T 135 IRAT104 (upland rice with OsNCED2 ) and Nipponbare (irrigated rice with

C 136 OsNCED2 ) were overexpressed in Nipponbare (Fig. S3). An analysis of the root

T C 137 phenotype showed that the overexpression of both OsNCED2 and OsNCED2 can

138 promote crown root development, which results in a denser root system at both the

139 seedling and tillering stages (Fig. 2, A and B; Table 1). Moreover, the overexpression

T C 140 of both OsNCED2 and OsNCED2 can enhance the ABA level compared with that

141 found in non-transgenic plants (Fig. 2, C and D). The lack of a positive correlation

T C 142 between the expression of OsNCED2 or OsNCED2 (Fig. 2, E) and the ABA content

T 143 in the transgenic plants implies that the catalytic activity of OsNCED2 is higher than

C 144 that of OsNCED2 , which is consistent with the result that near-isogenic lines (RILs)

T 145 with OsNCED2 exhibit significantly higher ABA levels and denser lateral roots than

146 C-type RIL families (Lyu et al., 2013). Plant root injury discharge can indicate the

147 vitality and water-absorption ability of roots. Therefore, we detected the plant root

T C 148 injury discharge of the transgenic lines overexpressing OsNCED2 or OsNCED2 . As

149 shown in Fig. S4, almost all the transgenic lines exhibited significantly higher injury

150 discharge than the WT plants. These results further prove that OsNCED2 can mediate

151 rice root development by catalysing ABA synthesis.

152

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153 OsNCED2 Overexpression can Enhance the Drought Tolerance of Rice

154 Stomatal closure is one of the first responses to drought conditions that might

T 155 control plant dehydration. The stomatal status of the OsNCED2 -OE1 and OE3 leaf

156 surfaces at the seedling stage under both normal and drought stress was examined by

157 scanning electron microscopy (SEM), and the results indicated that the leaf surfaces

T 158 of the OsNCED2 -OE1 and OE3 plants exhibited significantly higher stomatal closure

159 rates than those of the WT plants (Fig. 3, A).

T 160 To investigate the physiological responses mediated by OsNCED2 and

C 161 OsNCED2 , several indices of drought-induced effects on OE and WT leaves at the

162 shooting stage under both normal and drought conditions were measured. The water

163 loss rate (WLR) from excised leaves was determined, and the results showed that the

164 OE leaves showed a relatively lower WLR than the WT leaves (Fig. 3, B).

T 165 Accordingly, the relative water contents (RWCs) in the OsNCED2 -OE1 and OE3

166 plants after 1 h of exposure to drought stress (77.9% and 78.6%) were significantly

167 higher than that in the WT plants (67.4%) (Fig. 3, C). The OE seedlings experienced

168 less extensive cell membrane injury (relative electrolyte leakage) and exhibited lower

169 malondialdehyde (MDA) concentrations after drought treatment compared with the

170 WT seedlings (Fig. 3, D and E). Additionally, under drought stress conditions, the

T 171 catalase (CAT), POD and SOD activities in the OsNCED2 -OE1 and OE3 plants were

172 significantly higher than those in the WT plants (Fig. 3, F, G and H), which indicates

T 173 that the OsNCED2 -OE plants exhibit active detoxification by reactive oxygen

174 scavenging regulation in response to drought (Sofo et al., 2015). Taken together, these

T 175 results demonstrate that the DT of OsNCED2 -OE1 and OE3 plants was significantly

176 improved when compared with that of the WT plants, which indicates that the

177 overexpression of OsNCED2 can enhance DT via physiological regulation.

178

T 179 OsNCED2 -NILs Exhibit Higher ABA Levels and More Developed Root Systems

C 180 than the OsNCED2 Background

T 181 Although RILs with OsNCED2 display a significantly higher ABA content and

182 denser lateral roots than C-type RIL families (Lyu et al., 2013), other loci can

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T 183 influence the phenotype. To evaluate whether only the OsNCED2 locus contributes

184 to ABA enhancement and root development, we generated three NILs named

T 185 T--1~3 that contain the OsNCED2 locus in the Koshihikari genetic

186 background (C-Koshihikari), which was selected from the BC4F9 population lines by

187 marker assisted selection (MAS) with a CAPs marker (Supplemental Table S2). A root

T 188 phenotype analysis indicated that NILs with OsNCED2 contained a stronger root

189 system, particularly during crown root development (Fig. 4, A; Table 2). Interestingly,

190 although the leaf ABA content was markedly higher than that in the root, the ABA

191 level showed sharp increases in the root but not the leaves in both genotypes in

192 response to drought stress (Fig. 4, B). Furthermore, the ABA content was significantly

193 higher in the T-Koshihikari-1 leaves than in the C-Koshihikari leaves under both

194 normal and drought conditions (Fig. 4, B). In contrast, the ABA content in the

195 T-Koshihikari1 roots was significantly higher than that in the C-Koshihikari roots

T 196 only under drought conditions (Fig. 4, B). To clarify the DT of the OsNCED2 -NIL

197 plants, we compared the growth status and survival rate of Koshihikari and

T 198 OsNCED2 -NIL seedlings under polyethylene glycol (PEG) treatment. When the

T 199 OsNCED2 -NIL and C-Koshihikari seedlings were grown in hydroponic culture

T 200 under osmotic stress imposed by PEG, the OsNCED2 -NIL plants also exhibited

201 improved DT compared with the C-Koshihikari plants (Fig. 4, C and D), which

T 202 suggests that OsNCED2 contributes to dehydration tolerance in plants. These results

T 203 indicate that OsNCED2 might promote root development by regulating ABA

204 synthesis under drought conditions and that this effect might contribute to drought

205 avoidance and DT, which is as a kind of aerobic adaptation in upland rice.

206

T 207 Under Aerobic Conditions with Drought Stress, OsNCED2 can Enhance

208 Drought Tolerance, which Results in Higher Yield

T 209 To investigate the effect of OsNCED2 and its potential application in rice breeding,

210 we compared the grain yields of C-Koshihikari and T-Koshihikari plants under normal

211 and aerobic conditions with no, moderate (natural upland condition) and severe

212 drought (artificially controlled by rain proof installations). Under normal conditions,

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213 both lines exhibited similar yields, and no significant differences in other agronomic

214 traits except the panicle number per plant were detected (Fig. 5, A, B, C and D;

215 Supplemental Table S3). Although moderate drought significantly reduced the tiller

216 number (even panicle per plant), percentage of filled grains and 1000-grain weight in

T 217 all the tested plants, the OsNCED2 -NIL plants exhibited a higher grain weight per

218 plant than the C-Koshihikari plants because of the above three yield-related factors

219 (Fig. 5, A, B, C and D). Under severe drought, more prominent physiological damage,

220 such as leaf wilting and delayed flowering, was detected in the C-Koshihikari plants

T 221 compared with the OsNCED2 -NIL plants (Fig. 5, G; Supplemental Table S3).

T 222 Furthermore, the yield per plant or unit area of the OsNCED2 -NILs was significantly

223 higher than that of the C-Koshihikari plants (Fig. 5; Supplemental Fig. S5). These

224 results confirm that under drought conditions, both the enhanced rooting and the

T 225 dehydration tolerance promoted by OsNCED2 facilitate improvements in growth

226 vigour and grain filling, which results in higher yield.

227

228 The SNP Mutation C to T Contributes to the Functional Differentiation of

229 OsNCED2 between Irrigated Rice and Upland Rice

230 To define whether the SNP mutation, which is C-to-T from irrigated rice to upland

231 rice is responsible for the functional variation of OsNCED2, a further mutation

232 analysis was conducted with the aim of verifying whether the SNP that produces the

233 amino acid change from valine to isoleucine results in the observed difference in

234 catalytic activity and plays important roles in the ecotypic differentiation of upland

235 rice. A newly derived CRISPR/Cas9 gene editing technology was used to edit the

236 C-SNP of OsNCED2 in Nipponbare (see the Materials and Methods section). Eight

237 positive independent lines identified as heterozygous in this site were identified from

238 32 transgenic T0 lines via both the CAPs marker and Sanger sequencing. The root

239 system and DT of both T-type and C-type Nipponbare plants belonging to the T1

240 generation were evaluated. The results indicated that the T-type plants had a more

241 enhanced root system than the C-type plants at both the seedling (Fig. 6, A) and

242 mature stages (Fig. 6, B). Furthermore, the yield per plant showed no significant

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243 difference between the T-type and C-type Nipponbare plants under normal conditions

244 (Fig. 6, C). Consequently, under upland conditions, the final grain weight per plant

245 obtained for the T-type plants was significantly higher than that of the C-type plants

246 (Fig. 6, C). These results confirm that the SNP mutation site C-to-T from irrigated

247 rice to upland rice results in the functional variation of OsNCED2, and that both the

248 enhanced rooting and the dehydration tolerance mediated by this site promote a higher

249 yield under drought conditions, which indicates that this site plays vital roles in the

250 adaptation and artificial selection of upland rice.

251 252 Pathways and Downstream Genes Involved in Aerobic Adaptation Mediated by 253 OsNCED2 254 To gain insights into the genes and pathways that contribute to aerobic adaptation

255 and are regulated by OsNCED2, we screened the differentially expressed genes

T 256 (DEGs) in the OsNCED2 -OE3 lines compared with the WT plants by transcriptome

257 sequencing. As shown in Supplemental Table S4, 200 and 546 genes were up- and

T 258 downregulated in the leaves of the OsNCED2 -OE3 plants compared with those of the

259 WT plants under normal growth conditions, respectively, and 414 and 297 genes were

T 260 up- and downregulated in the OsNCED2 -OE3 roots compared with the WT roots

261 under normal growth conditions (Supplemental Table S5). Interestingly, a large

262 number of genes encoding retrotransposon or transposon proteins were highly

T 263 upregulated in both the leaves and roots of the OsNCED2 -OE3 lines (Supplemental

264 Table S4; S5), which might influence various effects on the genome.

265 A GO analysis revealed that the DEGs in both the leaves and roots were

266 functionally related to all types of pathways (Supplemental Table S6; S7). In addition,

267 the DEGs were highly differentially enriched between the leaves and roots, which

268 implies that the ABA molecules that are synthesized by OsNCED2 play diverse roles

269 in different tissues. For example, many DEGs in the leaves were specifically enriched

270 in “transcription factor activity”, “calcium ion binding”, “protein ubiquitination”,

271 “oxidoreductase activity” and “oxidoreductase activity” etc, whereas some DEGs in

272 the roots were enriched in “response to stress”, “nucleoside-triphosphatase activity”

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273 and “actin cytoskeleton” etc (Supplemental Table S6; S7). A further KEGG analysis

T 274 also demonstrated that the DEGs identified in the OsNCED2 -OE3 were involved in

275 “Brassinosteroid biosynthesis” “MAPK signalling pathway” and “Starch and sucrose

276 metabolism” and in “Diterpenoid and Flavonoid biosynthesis”, respectively

277 (Supplemental Table S8), which indicates that ABA inhibits leaf growth and

278 development but enhances the development of roots and their response to

279 environment stress. Particularly, a protein phosphatase 2C (PP2C, LOC_Os09g15670)

280 encoding gene in MAPK signalling pathway is down-regulated in the leaves of

T 281 OsNCED2 -OE3 lines (Supplemental Fig. S6), which relieve the inhibition to the

282 SnRK2s and activate ABF transcript factors to close stomata (Lin et al., 2020; Park et

283 al., 2009).

284 285

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286 DISCUSSION

287 Plants modify their development to adapt to the environment and thus protect

288 themselves from detrimental conditions by triggering a variety of signalling pathways.

289 The hormone ABA is usually associated with major plant responses to stress, and its

290 biosynthesis and signalling pathways have been well characterized (Chen et al., 2019).

291 9‐Cis‐epoxycarotenoid dioxygenase encoded by the NCED gene oxidatively cleaves

292 both 9'‐cis‐neoxanthin and 9′‐cis-violaxanthin to xanthoxin, which plays vital roles in

293 ABA biosynthesis (Anstis et al., 1975; Schwartz et al., 1997). In this study, the

294 phylogenetic tree of both rice NCED paralogous and orthologous genes indicated that

295 rice NCED originated from two ancestral genes that might produce orthologous genes

296 via gene duplication stemming from a proto-NCED gene. OsNCED2 is a 9‐cis‐

297 epoxycarotenoid dioxygenase-encoding gene identified that exhibits high

298 differentiation between irrigated rice and upland rice. In our previous study, we found

T 299 that the SNP-T allele (OsNCED2 ) is fixed in upland rice and associated with a higher

300 ABA content and a denser root system in both natural and segregating populations

301 (Lyu et al., 2013). Consistently, the results from this study further confirmed the

T 302 function of OsNCED2 in ABA biosynthesis and root development as well as its

303 downstream responses to aerobic environment stress.

304 The SNP site evolved from a C residue in irrigated rice to a T residue in upland rice

305 under upland selection and contributes to plant aerobic adaptation. Although sequence

306 differences, such as SNPs and indels, were detected between the upland rice and

307 irrigated rice varieties, the expression of OsNCED2 was not significantly different

308 between the two ecotypes under both normal and drought conditions, which implies

309 that the amino acid change in the SNP site might have resulted in functional variation.

T C 310 Both OsNCED2 and OsNCED2 have functions in catalysing ABA synthesis, and

T 311 OsNCED2 might exhibit higher enzyme activity and thus induce elevated ABA

312 levels in both leaves and roots of upland rice. Additional studies are needed to

T 313 determine whether the expression of OsNCED2 exhibits tissue specificity and plays

314 cell type-dependent roles.

315 Under aerobic conditions with drought stress, defects in osmotic homeostasis and

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316 the ion balance of plant cells trigger a signal transduction pathway for the recovery of

317 cellular homeostasis and the repair of damaged proteins and membrane systems

318 (Viswanathan et al., 2004). In plants, MAPKs play important roles in signal

319 transduction in response to drought, reactive oxygen species (ROS), pathogen defence,

320 wounding, and low temperature (Jonak et al., 1996; Mizoguchi et al., 1997).

321 Particularly a down-regulated PP2C gene related to the MAPK signalling pathway,

T 322 which indicates that ABA-dependent downstream pathways in OsNCED2 -OE3

323 plants such as AREB/ABF transcription factors might be regulated by active SnRK2s,

324 result in rapid stomatal closure (Lin et al., 2020; Park et al., 2009).

325 As part of the adaptive response to stress, ABA can inhibit vegetative growth and

326 thus oppose the growth-promoting properties of BRs (Clouse, 2016; Wang et al.,

T 327 2018). BR-related genes were markedly downregulated in OsNCED2 -OE3 plants,

328 which suggests that OsNCED2 might also be involved in the inhibition of plant

329 growth and development through antagonization of the BR pathway.

330 To improve aerobic adaption with drought resistance, plants develop deep and

331 denser roots and undergo rapid stomatal closure to absorb water from soil, which have

332 positive effects on drought avoidance (Fukai and Cooper, 1995; Wang et al., 2020b;

333 Yoshida and Hasegawa, 1982; Zhou et al., 2020). Our results indicate that the aerobic

334 adaptation of upland rice involves an enhanced root system due to

T 335 OsNCED2 -mediated improvements in root development and rapid stomatal closure,

T 336 which partially endow the OsNCED2 containing rice with increased drought

337 avoidance.

338 The aerobic with DT system, including osmotic adjustment, membrane-protection

339 proteins and redox homeostasis maintenance, also plays vital roles in plant survival

340 and reproduction. DEGs involved in osmotic adjustment-related functions, such as

341 carbohydrate metabolism and ion transport, were also enriched in the

T 342 OsNCED2 -OE3 plants. In plants, abiotic stress can induce the production of cellular

343 ROS, which have dual functions dependent on the amount: at a low level, ROS trigger

344 defences and developmental responses at early stages, and at a high level, ROS attack

345 the cell membrane to break down the defence barrier and thus destroy the cell (Guo et

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T 346 al., 2018; Jaspers and Jaakko, 2010; Miao et al., 2006). The OsNCED2 -OE3 plants

347 strengthened their antioxidant systems, including enzymatic and nonenzymatic

348 antioxidants, such as superoxide dismutase, catalase, ascorbate peroxidase,

349 glutathione reductase, ascorbic acid, tocopherol, glutathione, and phenolic compounds,

350 which can scavenge or reduce excessive deleterious ROS, in response to an imbalance

351 between ROS production and antioxidant defence (Gupta et al., 1993; Sharma and

T 352 Dietz, 2009), which indicates that OsNCED2 contributes to DT through the

353 maintenance of redox homeostasis in plants.

354 Unlike mobile animals, sessile plants cannot evade but rather have to tolerate

355 unfavourable environmental conditions, such as drought, salinity and cold. Our results

356 reveal a key gene that functions in the ABA biosynthesis pathway and subsequent

357 ABA signalling pathway and contributes to ABA-triggered DA and DT. Consistent

358 with the viewpoint that ABA increases resistance to abiotic stresses in land plant

359 evolution and terrestrial adaptation, which originated or expanded in the common

360 ancestor of Zygnematophyceae and embryophytes (Cheng et al., 2019; Wang et al.,

361 2020a; Zhang et al., 2020), it is conceivable that the ABA responses meditated by

T 362 OsNCED2 might offer an advantageous strategy used by upland rice to survive and

T 363 breed under aerobic conditions. Furthermore, OsNCED2 -containing rice exhibit a

364 higher yield under both rainfed upland and severe drought conditions, and these

365 findings provide new genetic basis for the breeding of aerobic adaptation rice. 366 367 MATERIALS AND METHODS

368 Plant Materials, Growth Conditions and Stress Treatments

369 L. cv. Nipponbare and Koshihikari were used in this study as the WT

T 370 and background rice, respectively. Three NILs containing the OsNCED2 locus

371 (named T-Koshihikari-1~3) were obtained by crossing the upland variety IRAT104

372 with the irrigated variety Koshihikari and selected from the BC4F9 population lines

373 with Koshihikari as the recurrent parent by MAS with a CAPs marker (Supplemental

374 Table S2).

375 For phenotype analysis at the seedling stage, were grown in Hoagland’s

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376 nutrient solution under controlled conditions in a growth chamber with 14-h of

377 daylight at 28 °C and a 10-h dark period at 25 °C. PEG6000 was added to the nutrient

378 solution to a final concentration of 20% to simulate drought stress. The transgenic rice

T 379 and OsNCED2 -NIL plants and their background parents, Nipponbare and

380 Koshihikari, were evaluated in term of their root system and DT performances.

381 Phenotyping was also performed in the field under both irrigated and upland

382 conditions in Xishuangbanna, Yunnan Province. For irrigated condition, seeds were

383 germinated in a seedbed, and the resulting seedlings were transplanted to a paddy

384 field, where water was ponded on the soil surface throughout the growth and

385 developmental period. For upland condition, we conducted direct seeding by dibbling

386 the seeds in dry soil, during growth stages, no any irrigation was applied but depend

387 on rainfall. Another aerobic with drought stress treatment experiment was performed

388 using waterproof installations at the breeding centre of Yunnan University. For each

389 line, we planted three replicates, and each replicate consisted of 12 individuals in two

390 rows (six individuals in each row), with a row spacing of 20 centimetres and a plant

391 spacing of 20 centimetres. From each line, approximately eight individuals were

392 randomly selected and phenotyped. Leaf tissues were harvested at the indicated times

393 and stored at -80 °C for further analysis.

394

395 Measurement of ABA

396 For the ABA content assays, the shoots (containing the coleoptile and first leaves)

397 and roots of seedlings were harvested. For each sample, approximately 0.2 g of fresh

398 tissue was homogenized under liquid nitrogen, weighed, and extracted for 24 h with

2 399 cold methanol containing antioxidant and 6 ng of H6-ABA (internal standard;

400 OlChemIm). The endogenous ABA was purified and measured as previously

401 described (Fu et al., 2012) with some changes in the detection conditions (Yin et al.,

402 2015).

403

404 RNA Extraction and Quantitative Real-time PCR (qRT-PCR)

405 Total RNA from rice tissues of the three genotypes was extracted using the TRIzol

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406 reagent (Invitrogen, USA). cDNA synthesis was performed using EasyScript

407 First-strand cDNA Synthesis SuperMix (TransGene, Beijing, China) according to the

408 manufacturer’s instructions. qRT-PCR analysis was carried out using TaKaRa SYBR

TM 409 Premix Ex Taq according to the manufacturer’s instructions. The relative

-∆Ct -∆∆Ct 410 expression of each gene was calculated using the 2 and 2 method (Livak and

411 Schmittgen, 2001). The primers (Supplemental Table S2) used for qRT-PCR were

412 subsequently tested using a dissociation curve analysis, and the results confirmed the

413 absence of nonspecific amplification. The ubiquitin gene was used as the reference.

414 All the analyses were performed with three biological replicates.

415

416 Vector Construction and Genetic Transformation

417 The coding region of OsNCED2 was amplified from the genomic DNA of two

418 types of rice (IRAT104 and Nipponbare) by PCR using Pst I and Spe I linker primers

419 (Supplemental Table S2). The resulting OsNCED2 fragments were inserted into the

420 Pst I and Spe I sites of pCUbi1390 (Peng et al., 2009) to generate

T C 421 UbiPro::OsNCED2 and UbiPro::OsNCED2 , respectively. Fragments of 3.4 kb in

422 the OsNCED2 promoter of IRAT104 were amplified from rice genomic DNA

423 (Nipponbare) by PCR and inserted into the pMDC162 vector (Curtis and

424 Grossniklaus, 2003) using Gateway technology to generate OsNCED2Pro::GUS. All

425 the vectors were introduced into Agrobacterium tumefaciens strain EHA105 and then

426 transferred into Nipponbare plants via Agrobacterium-mediated transformation in

427 accordance to the standard protocol (Duan et al., 2012).

428

429 Physiological Traits of the Three Genotypes under Drought Stress

430 Fresh leaves (0.5 g) of the seedlings under normal and stress conditions were

431 harvested and used to measure the MDA content and the SOD, POD and CAT activity

432 levels according to described methods (Shin et al., 2012; Shukla et al., 2012). The

433 RWC was calculated using the following formula: RWC (%) = [(FM − DM)/(TM −

434 DM)] × 100, where FM, DM, and TM are the fresh, dry, and turgid masses of the

435 weighed leaves, respectively. The relative electrolyte leakage (REL) or solute leakage

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436 from the sampled rice leaves was evaluated using the method described by Arora et al.

437 (Arora et al., 1998). The percent injury induced by each treatment was calculated

438 from conductivity data using the following equation: % injury = [(% L(t)-%

439 L(c))/(100-% L(c))] × 100), where % L (t) and % L(c) are the percent conductivity of

440 the treated and control samples, respectively. The second leaves from 1-hour

441 drought-stressed plants were used for SEM analyses, as described by (You et al., 2012)

442 with minor modifications. Fresh leaf samples were prefixed for 3 h in 3%

443 glutaraldehyde-sodium phosphate buffer (0.1 M) at room temperature and rinsed three

444 times with 0.1 M sodium phosphate buffer. Postfixation was performed with 2% OsO4

445 at 4°C. The samples were dehydrated through an ethanol series and infiltrated with an

446 isoamyl acetate series. The samples were then coated with metal particles for SEM

447 analysis to observe the guard cells. A Hitachi S750 scanning electron microscope

448 (http://www.hitachi-hitec.com/global/em/) was used to obtain photographs, and the

449 numbers of guard cells in randomly selected fields were counted and statistically

450 analysed.

451

452 Subcellular Localization of GFP-OsNCED2 Fusion Proteins

453 The open reading frames (ORFs) of OsNCED2 were inserted into pMDC43 as

454 C-terminal fusions to the green fluorescent protein (GFP) reporter gene driven by the

455 CaMV 35S promoter (Curtis and Grossniklaus, 2003). For transient expression, the

456 GFP- OsNCED2 fusion vector constructs were transformed into the leaves of

457 3-week-old tobacco (Nicotiana benthamiana) by A. tumefaciens infiltration (Sparkes

458 et al., 2006). The green fluorescence resulting from GFP-OsNCED2 expression was

459 observed using a confocal laser scanning microscope (LSM700, Zeiss, Jena,

460 Germany). The 35S::GFP construct was used as a control.

461

462 Histochemical GUS Assay

463 For GUS staining analysis, sample tissues were submerged in GUS staining buffer

464 (containing 2 mM 5-bromo-4-chloro-3-indolyl glucuronide, 0.1 M sodium phosphate

465 buffer [pH 7.0], 0.1% [v/v] Triton X-100, 1 mM potassium ferricyanide, 1 mM

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466 potassium ferrocyanide, and 10 mM EDTA), vacuum infiltrated for 10 min, and then

467 incubated overnight at 37 °C. The staining buffer was removed, and the samples were

468 then cleared with 95% (v/v) ethanol and observed using a stereoscope (LEICA,

469 10447157, Germany).

470

471 Gene Editing for the SNP Mutation Site in OsNCED2

472 The 23-bp targeting sequences (including PAM) (Supplemental Table S2) were

473 selected within the target gene OsNCED2, and their targeting specificity was

474 confirmed through a BLAST search against the rice genome

475 (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The designed targeting sequences were

476 synthesized and annealed to form oligo adaptors. The pCSGAPO1 vector (Cong et al.,

477 2013) was digested with Bsa I and purified using a DNA purification kit (Tiangen,

478 China). A ligation reaction (10 µl) containing 10 ng of the digested pCSGAPO1

479 vector and 0.05 mM oligo adaptor was conducted and directly transformed into E. coli

480 competent cells to produce CRISPR/Cas9 plasmids. The CRISPR/Cas9 plasmids were

481 introduced into A. tumefaciens strain EHA105. The transformation of rice and the

482 genotyping of the SNP site of OsNCED2 were performed as described above.

483

484 RNA-Seq Analysis

T 485 Total RNA from rice seedlings belonging to the OsNCED2 -OE3 and WT lines was

486 extracted using the TRIzol reagent according to the manufacturer’s instructions

487 (Invitrogen, Waltham, MA, USA). For each sequencing library, 100 mg of RNA from

488 each replicate was mixed together. The libraries were sequenced using an Illumina

489 HiSeq 2000 Sequencing System. Low-quality nucleotides (< Q20) were trimmed from

490 the raw sequences obtained for each sample, and pair-end reads with at least one end

491 with a length < 30 bp were removed using an in-house Perl script. The retained

492 high-quality reads were mapped to the Michigan State University Rice Genome

493 Annotation Project database

494 (ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs

495 /pseudomolecules/version_7.0) using Bowtie (Ouyang et al., 2007). The Cuffdiff

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496 module was used for identification of the DEGs. Chi-square tests were used to

497 identify genes with significant differences in their relative abundance levels (as

498 reflected by the total counts of individual sequence reads) between two samples using

499 IDEG6 software based on the criteria p ≤ 0.001 and fold change > 2 (Romualdi et al.,

500 2003; Z.N. et al., 2003). The pathway and GO enrichment analyses of rice DEGs were

501 conducted using EXPath 2.0 (Chien et al.; Jia et al., 2018).

502

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503 doi: https://doi.org/10.1101/2020.06.11.146092 ; this versionpostedJune12,2020. The copyrightholderforthispreprint

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504 Figure Legends: 505

506 507 Figure 1. Expression pattern of OsNCED2 in different tissues of rice. A Relative -∆∆Ct 508 expression level (2 ) of OsNCED2 in different rice tissues at the seedling, tillering 509 and booting stages. Young panicles were used as reference controls. B Koshihikari 510 and IRAT104 exhibit similar OsNCED2 expression levels and models. C OsNCED2 511 expression in different tissues of OsNCED2Pro::GUS transgenic rice plants 512 determined by GUS staining analysis. D Novel young roots exhibit high OsNCED2 513 expression (left), whereas old roots show low OsNCED2 expression (right). Scale 514 bars, 0.5 cm. 515 516 517 518 519

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520 T C 521 Figure 2. Both OsNCED2 and OsNCED2 can promote ABA synthesis and enhance 522 root development. Root phenotype (A) and plant phenotype (B) of transgenic rice T C 523 overexpressing both OsNCED2 and OsNCED2 . C ABA content in leaves of T C -∆Ct 524 OsNCED2 -OE and OsNCED2 -OE transgenic plants. D Expression level (2 ) of 525 OsNCED2 in transgenic rice. The ABA content exhibited no correlation with the T C 526 OsNCED2 expression levels in lines overexpressing OsNCED2 and OsNCED2 . 527 Scale bars, 2 cm (A), 20 cm (B). 528

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529 T 530 Figure 3. Physiological traits of OsNCED2 -OE lines under normal (CK) conditions T 531 and 1-h drought treatment. A Stomatal closure rate of leaves of OsNCED2 -OE and 532 WT lines (both the upper and lower epidermis were counted in 10 randomly selected 533 high-power fields). B Water loss rate of the transgenic and WT lines. From each 534 replicate, 30 fully expanded leaves at the seedling stage were used in a triplicate 535 experiment. Relative water content (C) and relative electrolyte leakage (D) of T 536 OsNCED2 -OE lines under normal conditions and drought treatment. (E) MDA T 537 contents of OsNCED2 -OE and WT plants under normal conditions and after 1 h of 538 drought treatment. Activity of ROS-scavenging enzymes, including CAT (F), SOD (G) T 539 and POD (H), in OsNCED2 -OE and WT plants under normal conditions and after 1 540 h of drought treatment. The values are presented as the means and standard errors 541 from three replicates. *p < 0.05 versus the WT plants, as determined by Student’s 542 t-test. 543 544 545

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546 T 547 Figure 4. OsNCED2 -NIL plants have an enhanced root system and a stronger ABA 548 synthesis ability compared with the background C-Koshihikari plants with C T 549 OsNCED2 . A Plant and root phenotypes of OsNCED2 -NIL and C-Koshihikari T 550 plants. B Leaf and root ABA contents in OsNCED2 -NIL and C-Koshihikari plants T 551 under both normal conditions and drought treatment. C Phenotype of OsNCED2 -NIL 552 and C-Koshihikari plants under both normal and drought (1 week of treatment with 20% 553 PEG treatment) conditions. D Survival rate under re-watered conditions after 1 week 554 of PEG stress treatment. Scale bars, 5 cm. 555 556

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557 T 558 Figure 5. Effect of OsNCED2 on yield during exposure to aerobic and drought stress. 559 Panicle number per plant (A), grain number per plant (B), thousand-grain weight (C), 560 percentage of filled grains (D) and grain weight per plant (F) obtained for T 561 C-Koshihikari and OsNCED2 -NIL plants grown during exposure to no, moderate 562 and severe drought. The values in the histograms represent the means ± SDs (n = 24). 563 The panicle number refers to the number of effective tillers that were fertile. The 564 panicle phenotypes (E) include the primary and secondary branch numbers and the 565 grain number under moderate-drought conditions and the plant performance (G) under 566 severe-drought conditions. The yellow arrows indicate abortive grains. * and ** 567 indicate significant differences from the C-Koshihikari plants at p < 0.05 and p < 0.01, 568 respectively, as determined by Student's t-test. Scale bars, 5 cm (E), 10 cm (G) 569 570

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571

572 573 Figure 6. The SNP mutation (C to T) results in the functional differentiation of 574 OsNCED2. A The root phenotype data, including total length, total superficial area 575 (surface area), total root volume and root tip number, indicate that T-type plants have 576 denser roots than C-type plants. B Comparison of the root phenotypes of C-type and 577 T-type plants. C Grain weight per plant obtained for C-type and T-type Nipponbare 578 plants grown under normal and upland conditions. The values in the histograms 579 represent the means ± SDs (n = 24). * indicates significant differences between 580 C-type and T-type plants at p < 0.05, as determined by Student's t-test. Scale bars, 0.5 581 cm. 582 583 584

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585 Supplemental Data:

586

587 Supplemental Figure S1. Phylogenetic analysis of the NCED proteins in plants. The 588 maximum parsimony tree of the NCED proteins from various plants was based on the 589 sequence database in the UniProt Knowledgebase (UniProtKB). 590

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591

592 Supplemental Figure S2. Subcellular localization analysis of OsNCED2 in plants. A 593 GFP was extensively localized in both the nucleus and cytoplasm in N. benthamiana 594 protoplasts, while OsNCED2-GFP was positively localized in the chloroplast. The 595 chlorophyll fluorescence is shown in red. The scale bars represent 10 μm. B 596 Prediction of subcellular location via Target P analysis. 597

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598

599 Supplemental Figure S3. Analysis of OsNCED2 expression in transgenic plants T C 600 overexpressing OsNCED2 and OsNCED2 . 601

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602

603 Supplemental Figure S4. Bleeding intensity of transgenic plants overexpressing T C 604 OsNCED2 and OsNCED2 . 605

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606

T 607 Supplemental Figure S5. Phenotyping of OsNCED2 -NIL and C-Koshihikari plants 608 in both paddy fields and aerobic conditions (severe-drought conditions under a rainout 609 shelter). Comparisons of rice performance in paddy fields (A) and aerobic land (B). C T 610 Yield per unit area of OsNCED2 -NIL and C-Koshihikari plants. * indicates a 611 significant difference from the C-Koshihikari plants at p < 0.05, as determined by T 612 Student’s t-test. D Performance of OsNCED2 -NIL and C-Koshihikari plants in 613 drylands at the maturation stage. 614

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615

616 Supplemental Figure S6. MAPK signaling pathway and hormone signal transduction 617 pathway rendered by Pathview. 618

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619 Supplemental Tables:

620 Supplemental Table S1. Cis-acting elements in the OsNCED2 promoter.

621 Supplemental Table S2. Information on the primers used in this study.

T 622 Supplemental Table S3. Agronomic traits of OsNCED2 -NIL plants.

T 623 Supplemental Table S4. DEGs in the leaves of OsNCED2 -OE3, when compared to 624 WT under the normal growth conditions.

T 625 Supplemental Table S5. DEGs in the roots of OsNCED2 -OE3, when compared to 626 WT under the normal growth conditions.

T 627 Supplemental Table S6. Function categories list of leaves DEGs in OsNCED2 -OE3 628 compared with WT via GO analysis.

T 629 Supplemental Table S7. Function categories list of roots DEGs in OsNCED2 -OE3 630 compared with WT via GO analysis.

631 Supplemental Table S8. List of pathways and related DEGs enriched in T 632 OsNCED2 -OE3 vs WT via KEGG analysis. 633

634

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