2020 08 24 264556v1 Full

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1 Short title
2 idr1-1 mutation enhances drought tolerance in rice
3
4 Author for contact details

5 Corresponding author:
6 Honggui La
7 College of Life Sciences,
8 Nanjing Agricultural University,
9 Nanjing 210095, China
10 Tel: (86) 025-84395869; Fax: (86) 025-84395869
11 Email: hongguila@njau.edu.cn
12
bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.264556; this version posted August 24, 2020. The copyright holder for this preprint
13 Title
14 Mutation of IDR1 enhances drought tolerance by reducing ROS production and activating ROS
15 scavenging in rice
16
17 Authors
18 Xiaofeng Zu1†, Yanke Lu2†, Qianqian Wang2†, Yumei La2, Feng Tan2, Jiayu Niu2, Huihui Xia2, Xinyue
19 Hong2, Yufeng Wu3, Shaoxia Zhou4, Kun Li5, Huhui Chen2, Sheng Qiang2, Qi Rui2, Huaqi Wang1,
20 Honggui La2*
21
22
1
College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
23
2
College of Life Sciences, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
24
3
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Bioinformatics Center, Nanjing
25 Agricultural University, Nanjing 210095, China
26
4
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
27
5
State Key Laboratory of Cotton Biology, Henan Joint International Laboratory for Crop Multi-Omics
28 Research, Henan University, Kaifeng 475004, China
29
†
30 Contributed equally to the article
31
*
Corresponding author
32
33 One-sentence summary
34 Mutation of IDR1 significantly enhances drought tolerance in an upland cultivar IAPAR9 by decreasing
35 apoplastic and chloroplastic ROS production and increasing ROS-scavenging ability
36 Footnotes
37 Author contributions
38 H. L, X. Z and Y. L conceived and designed the original research plans; H. L. supervised the entire
39 experiments; X. Z., Y. L. and Q. W. performed most of the experiments; Y. L. analyzed the mRNA-seq
40 data; F. T., J. N., H. X., X. H., K. L. and H. C. helped prepare samples; Y. W., S. Z., S. Q. and Q. R.
41 provided analytical and experimental assistance; H. L., X. Z., Y. L. and Q. W. wrote the article; H. L. and
42 H. W. revised the manuscript.
43
44 Responsibilities of the author for contact

45 Honggui La
46 Email: hongguila@njau.edu.cn
47
48 Funding information
49 This work was supported by grants from the National Key Research and Development Program of China
50 (2016YFD0100604) (to H. L.) and Foundation for Jiangsu Postdoctoral Fellowship (2019K146) (to Q.
51 W.).
52
53 Email address of author for contact

54 Xiaofeng Zu: xfzu917@163.com
55 Yanke Lu: luyanke2009@126.com
56 Qianqian Wang: wangqianqian@njau.edu.cn
57 Yumei La: 908098469@qq.com
58 Feng Tan: tanfeng@njau.edu.cn
59 Jiayu Niu: 447576453@qq.com
60 Huihui Xia: 2551230065@qq.com
61 Xinyue Hong: 853350009@qq.com
62 Yufeng Wu: yfwu@njau.edu.cn
63 Shaoxia Zhou: sxzhou@njau.edu.cn
64 Kun, Li: likun@henu.edu.cn
65 Huhui Chen: chenhuh@njau.edu.cn
66 Sheng Qiang: qiangs@njau.edu.cn
67 Qi Rui: ruiqi@njau.edu.cn
68 Huaqi Wang: wanghuaqi@cau.edu.cn
69
70 Abstract
71 To discover new mutant alleles conferring enhanced tolerance to drought stress, we screened a
72 mutagenized rice population (cv. IAPAR9) and identified a mutant, named idr1-1 (for increased drought
73 resistance 1-1), with obviously increased drought tolerance under upland field conditions. The idr1-1
74 mutant possessed a significantly enhanced ability to tolerate high-drought stress in different trials.
75 Map-based cloning revealed that the gene LOC_Os05g26890 (corresponding to D1 or RGA1 gene),
76 residing in the mapping region of IDR1 locus, carried a single-base deletion in the idr1-1 mutant, which
77 caused a frameshift and premature translation termination. Complementation tests indicated that such a
78 mutation was indeed responsible for the elevated drought tolerance in idr1-1 mutant. IDR1 protein was
79 localized in nucleus and to plasma membrane or cell periphery. Further investigations indicated that the
80 significantly increased drought tolerance in idr1-1 mutant stemmed from a range of physiological and
81 morphological changes occurring in such a mutant, including greater leaf potentials, increased proline
82 contents, heightened leaf thickness, and upregulation of antioxidant-synthesizing and drought-induced
83 genes, etc., under drought-stressed conditions. Especially, ROS production from NADPH oxidases and
84 chloroplasts might be remarkably impaired, while ROS-scavenging ability appeared to be markedly
85 enhanced as a result of significantly elevated expression of a dozen ROS-scavenging enzyme genes in
86 idr1-1 mutant under drought-stressed conditions. Besides, IDR1 physically interacted with TUD1, and
87 idr1-1 mutant showed impaired EBR responsiveness. Altogether, these results suggest that mutation of
88 IDR1 leads to alterations of multiple layers of regulations, which ultimately confers obviously enhanced
89 drought tolerance to the idr1-1 mutant.
90
91 Keywords: idr1-1 (increased drought resistance 1-1), drought tolerance, reactive oxygen species
92 (ROS), brassinosteroid (BR), upland rice
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104 Introduction
105 Rice is one of the most important crops worldwide. Mainly due to the increasing shortage of water
106 resources, drought has become the major limiting factor for rice growth and production. Hence, breeding
107 and cultivation of drought-tolerance rice varieties are important measures to cope with outcomes of
108 drought stress. Thus, understanding the mechanisms of how rice plants respond to drought stress and what
109 factors play critical role in contributing to drought tolerance has become an important step towards
110 breeding drought-tolerant rice by means of conventional cross and genetic engineering.
111 Over the past decades, much progress had been made in understanding the mechanisms of how drought
112 stress damages plants and how plants defend against the drought stress to safeguard themselves from
113 severe damage. Plant stomata are mainly responsible for water transpiration and gas exchange. Stomatal
114 movements regulate water status in response to drought stress (Hong et al., 2008). Phytohormone abscisic
115 acid (ABA) performs important functions in modulating stomata movements when plants are subjected to
116 drought stress (Tuteja and Sopory, 2008). It is generally known that stomatal closing regulated by ABA
117 plays essential roles in preventing plants from excessive water loss at the early stages of drought stress.
118 Besides, other phytohormones, like GA, brassinosteroid (BR), ethylene, auxin, etc., are all involved in
119 plant drought responses (Desikan et al., 2006; Ge et al., 2015; Shi et al., 2015). Growing evidence
120 demonstrates that disruption of GA signaling pathway brings about transcriptional elevation of GA2ox, a
121 gene encoding GA-inactivating enzyme, and RGL3 that encodes a DELLA protein, eventually leading to
122 plant growth retardation and enhanced drought tolerance (Colebrook et al., 2014). DELLA protein was
123 found to restrain the accumulation of ROS which is usually overaccumulated when plants are exposed to
124 abiotic stresses. BRs are a class of polyhydroxylated sterol derivatives, and they play vital roles in
125 promoting cell expansion and division, regulating senescence, and modulating plant responses to various
126 environmental cues (Hu et al., 2013; Feng et al., 2015). A recent report showed that 24-epibrassinolide
127 (EBR) induced stomatal closure in Arabidopsis by triggering a signal transduction pathway including
128 ethylene synthesis, activation of Gα protein, and hydrogen peroxide (H2O2) and nitric oxide (NO)
129 production (Shi et al., 2015). Another study reported that BR was able to induce H2O2 production, and the
130 H2O2 played vital roles in BR signaling (Tian et al., 2018). In Brachypodium distachyon,
131 BRASSINOSTEROID INSENSITIVE 1 (BdBRI1) RNAi lines exhibited markedly enhanced drought
132 tolerance, accompanied by highly elevated expression of drought-responsive genes, such as
133 BdCOR47/BdRD17, BdERD1 and BdRD26 (Feng et al., 2015). Arabidopsis wrky46 wrky54 wrky70 triple
134 mutant had defects in BR-regulated growth and was more tolerant of drought stress, suggesting that
135 WRKY46/54/70 is positively involved in BR-regulated growth and negatively involved in the drought
136 responses (Chen et al., 2017).
137 Accumulating evidence shows that stomatal closure is just an early response when drought stress
138 occurs (Distefano et al., 2015). If the stress becomes severe, plants activate other mechanisms to protect
139 proteins and membrane structures in order to avoid cell death caused by water deficit, such as
140 accumulation of cuticular wax crystals and prolines, control of ROS homeostasis, as well as
141 transcriptional activation of large numbers of transcription factors (Distefano et al., 2015). The rice
142 drought-induced wax accumulation 1 (dwa1) mutant was impaired in cuticular wax accumulation under
143 drought stress, which resulted in significantly increased drought sensitivity (Zhu and Xiong, 2013).
144 Proline is thought to be a scavenger of free radicals and plays an important role in enhancing drought
145 tolerance via osmotic adjustment. In BdBRI1 RNAi lines, expression of BdP5CS was highly induced in
146 response to drought stress, which might be one of the reasons for significantly enhanced drought
147 tolerance in such RNAi lines (Feng et al., 2015).
148 Production of ROS is a frequent event in plants that suffer from diverse abiotic stresses. There are
149 many potential sources of ROS in plant cells, including chloroplasts, mitochondria, peroxisomes, plasma
150 membrane NADPH oxidases, cell wall peroxidases, apoplastic oxalate oxidases, and amine oxidases
151 (Zhang et al., 2010). Mounting evidence showed that ROS generated by NADPH oxidases play crucial
152 roles in plant abiotic stresses, defense and hormonal responses. However, excessive amounts of ROS are
153 harmful to the plants because it causes damage to cells through oxidizing proteins, lipids, and DNA,
154 ultimately leading to retarded growth and eventual cell death (Bargmann and Munnik, 2006; Ning et al.,
155 2010; You et al., 2014). ROS is also able to cause photoinhibition occurring in PSII reaction center, which,
156 in turn, triggers undue production of the ROS (Ferrero-Serrano et al., 2018). Thus, to counteract the
157 ROS’s deleterious effects, plants have evolved the mechanisms for scavenging these excessive ROS,
158 including the involvement of a variety of ROS-scavenging enzymes, superoxide dismutase (SOD),
159 catalase (CAT), glutathione reductase, and peroxidase (POX), etc. (You et al., 2014). Mutation of ROS
160 scavenger’s genes reportedly leads to drought sensitivity in plants. For example, a dsm1 mutant defective
161 in a putative MAPK kinase kinase gene in rice, showed more sensitive to oxidative stresses due to an
162 increase in ROS level caused by reduced expression of POXs (OsPOX22.3 and OsPOX8.1) (Ning et al.,
163 2010). The ospp18 mutants exhibited increased sensitivity to oxidative stress because several genes
164 encoding ROS-scavenging enzymes were downregulated in such mutant plants (You et al., 2014).
165 Overexpression (OE) of OsGH3-2 in rice plants decreased free IAA level and thus impaired SOD activity,
166 therefore conferring drought sensitivity to such plants (Du et al., 2012). OsLG3, a ERF family
167 transcription factor, played a positive role in drought stress tolerance in rice by inducing ROS scavenging;
168 overexpression of the OsLG3 could reduce overaccumulation of ROS as a total of 10 ROS-scavenging
169 genes (APXs, CATB, PODs, SODs, etc.) were upregulated in the OE lines (Bargmann and Munnik, 2006).
170 Heterotrimeric GTP-binding proteins (G proteins), comprising three subunits Gα, Gβ and Gγ, are key
171 signaling molecules in all eukaryotes. Classically, signal perception by a G protein-coupled receptor
172 results in the dissociation of Gα from Gβγ subunits, and then Gα or Gβγ dimer can separately activate
173 downstream signaling (Ferrero-Serrano and Assmann, 2016). Arabidopsis genome has only one Gα
174 (GPA1), one Gβ (AGB1), and three Gγ (AGG1 to AGG3) genes; rice genome contains one Gα (RGA1),
175 one Gβ (RGB1), four Gγ subunit (RGG1, RGG2, GS3, and DEP1), and one Gγ-related gene (or
176 pseudogene) OsGGC2 (Ferrero-Serrano and Assmann, 2016). G proteins play vital roles in the
177 transduction of extracellular signals into intracellular responses; in plants, they have been implicated in
178 multiple signaling pathways, including phytohormone responses such as GA, ABA, jasmonic acid, auxin,
179 and BR (Ritchie and Gilroy, 2000; Wang et al., 2001; Wang et al., 2006; Oki et al., 2009; Hu et al., 2013;
180 Ge et al., 2015; Shi et al., 2015; Ferrero-Serrano and Assmann, 2016). In Arabidopsis, the GPA1 is a
181 modulator of plant cell proliferation, because gpa1 null mutant has reduced cell division in aerial tissues
182 throughout development (Ullah et al., 2001). The GPA1 also regulates stomatal density by controlling
183 epidermal cell sizes and stomata formation (Nilson and Assmann, 2010). Importantly, G proteins not only
184 participate in stomatal movements, but also act as key regulators of H2O2 production in guard cells
185 induced by ABA (Ge et al., 2015). Furthermore, gpa1 mutation resulted in insensitivity of stomatal
186 opening to inhibition triggered by ABA, but did not cause significant impairment of ABA-induced
187 stomatal closing, with the result that the rates of water loss from gpa1 mutants were greater than those
188 from the wild-type control plants (Wang et al., 2001). In gpa1-3 and gpa1-4 guard cells, ABA failed to
189 induce ROS production, suggesting that the GPA1 acts upstream of ROS production in the ABA signaling
190 pathway (Zhang et al., 2011). The gpa1 mutants also showed defects in ethylene-induced H2O2
191 production and stomatal closure, whereas wGα and cGα OE lines showed faster stomatal closure and
192 greater H2O2 production in response to ethylene, indicating that Gα mediates ethylene-induced stomatal
193 closure via H2O2 production (Ge et al., 2015). Moreover, gpa1-4 leaves displayed less damage when
194 treated with O3, and O3-induced H2O2 was found to be greatly impaired in the mutant leaves,
195 demonstrating that the resistance of the gpa1-4 mutants to O3-induced cell damage is attributable to the
196 defect in Gα-mediated activation of cell death-associated late component of the oxidative burst (Joo et al.,
197 2005). Likewise, cell death of gpa1 was mitigated as well in phyA-mediated cell-death pathway (Wei et
198 al., 2008). In addition, gpa1 showed approximately 100-fold less responsiveness to GA (Ullah et al.,
199 2002), and reduced responsiveness to brassinolide (BL), suggesting that GA and BR signal transduction is
200 likely coupled by the Gα protein (Ullah et al., 2001; Ullah et al., 2002).
201 The rice Gα gene (named D1 or RGA1) was first identified from a rice Dwarf 1 (D1) mutant by using a
202 map-based cloning strategy (Ashikari et al., 1999). d1 mutant showed a dwarf phenotype; the elongation
203 of the second leaf sheaths of d1 seedlings was much less responsive to exogenous GA3 than that of the
204 wild-type seedlings, indicating that the d1 mutation partially impairs GA-signaling pathway
205 (Ueguchi-Tanaka et al., 2000). Two independent studies all indicated that D1 participated in BR responses,
206 and the d1 mutant displayed a remarkably reduced responsiveness to EBR, suggesting that the D1 is
207 involved in BR signaling (Wang et al., 2006; Oki et al., 2009). Further, a D1-interacting protein Taihu
208 Dwarf1 (TUD1), a U-box E3 ubiquitin ligase, was reported to act in conjunction with the D1 to affect the
209 BR-signaling pathway, because tud1 mutant was also less responsive to BR treatment as was the d1
210 mutant (Hu et al., 2013; Ferrero-Serrano and Assmann, 2016). Another study provided a line of evidence
211 that epidermal cell death in rice was dependent on signaling through Gα proteins (Steffens and Sauter,
212 2009). A recent study demonstrated that d1 mutants exhibited significantly reduced sensitivity to drought
213 stress; after a long-term progressive drought stress, the d1 plants showed obviously more tolerance to the
214 stress than wild-type plants (Ferrero-Serrano and Assmann, 2016). Moreover, during the course of
215 drought stress, the decrease in photosynthetic rates in the d1 mutants was markedly slower compared to
216 the control plants, implicating that limitation of photosynthesis caused by drought are less severe in the
217 d1 mutants (Ferrero-Serrano and Assmann, 2016). Additionally, under NaCl-treated conditions the d1
218 mutants displayed remarkable alleviation of leaf senescence, chlorophyll degradation, and cytoplasm
219 electrolyte leakage (Urano et al., 2014).
220 In this study, we identified a highly drought-tolerant rice mutant in upland fields, named idr1-1, by
221
60
screening a rice population undergoing Co γ-ray radiation. idr1-1 mutants consistently showed
222 obviously enhanced drought tolerance under different growth conditions; map-based cloning revealed that
223 the idr1-1 was a new mutant allele of Gα. Detailed investigations demonstrated that the enhanced drought
224 tolerance of idr1-1 mutant appeared to stem from altered morphology, impaired ROS production,
225 increased ROS scavenging, weakened leaf senescence, elevated antioxidant levels, etc.; in addition, IDR1
226 physically interacted with TUD1, and the idr1-1 mutant showed reduced BR responsiveness, which is
227 probably associated with the enhanced drought tolerance of idr1-1 mutant. Given the significantly
228 enhanced drought tolerance caused by the idr1-1 mutation, the idr1-1 mutant allele has considerable
229 potential for rice drought-tolerant breeding.
230
231 Results
232 Identification of an idr1-1 Mutant that Shows Significantly Enhanced Drought Tolerance in Upland
233 Field Conditions
234 To study the mechanisms of rice drought tolerance, we performed a genetic screening in upland fields
235 to find candidate lines with enhanced drought tolerance from a γ-ray-mutagenized upland rice (cv.
236 IAPAR9) population. Eventually, a candidate mutant plant was discovered to have obviously increased
237 drought tolerance, and was thus referred to as idr1-1 (for increased drought resistance 1-1). The
238 drought-tolerant phenotype of idr1-1 mutant was further reconfirmed in pots and concrete tanks,
239 respectively (Fig. 1, A and C). For drought treatments in pots, the idr1-1 mutants exhibited enhanced
240 drought tolerance to severe drought stress compared to IAPAR9 (Fig. 1A), and the survival rate of idr1-1
241 mutants following rewatering was approximately 64% higher than that of IAPAR-9 plants (Fig. 1B).
242 Further, for drought treatments in concrete tanks, under moderate drought stress conditions, IAPAR9
243 plants became severely wilted, whereas idr1-1 mutants grew quite normally (Fig. 1C). Under severe
244 drought stress conditions, most of IAPAR9 leaves became dry while only a quite small fraction of idr1-1
245 leaves became wilted or dry. After rewatering, about 50% of IAPAR9 plants undergoing moderate
246 drought stress died but more than 95% of idr1-1 mutants still stay alive (Fig. 1D). Except for
247 drought-tolerant phenotype, idr1-1 mutants also displayed other developmental phenotypes, such as
248 noticeable dwarfism, shorter lengths of grains as well as panicles and leaves, compared to IAPAR9 plants
249 (Supplemental Fig. S1). However, grain width of the idr1-1 mutants exhibited no difference from that of
250 the IAPAR9 plants (Supplemental Fig. S1E, right panel). When germinated in the presence of different
251 concentrations of glucoses, idr1-1 seeds showed significantly reduced germination rates under the
252 conditions of 6% and 8% glucoses (Supplemental Fig. S2, A and B), suggesting that the idr1-1 mutants
253 have increased sensitivity to high concentrations of glucoses, which is in agreement with the previous
254 observations in Arabidopsis (Ritchie and Gilroy, 2000). When grown in 150 mM NaCl solution, idr1-1
255 mutants appeared higher sensitivity than IAPAR9 plants as approximately 10% of mutant plants and 47%
256 of IAPAR9 plants remained alive following the treatments (Supplemental Fig. S2, C and D). Taken
257 together, these results demonstrate that mutation of IDR1 leads to a wide range of morphological and
258 physiological changes, and the idr1-1 mutants show obviously increased drought tolerance under different
259 growth conditions.
260
261 Map-Based Cloning of IDR1 Gene

262 F1 heterozygous plants produced from a cross between idr1-1 mutant female and IAPAR9 male
263 showed similar performance of drought tolerance as the IAPAR9 plants, and were noticeably less tolerant
264 of drought stress compared with the idr1-1 mutants (Supplemental Fig. S3A), suggesting that idr1-1
265 mutation is recessive. For cloning IDR1 gene, we crossed the idr1-1 mutant females separately with each
266 of 10 upland rice cultivar males, including HD297, HD385, HD119, etc. (Supplemental Table S1). The
267 resulting F2 populations were planted in the above-mentioned concrete tanks for the moderate drought
268 treatment to distinguish drought-tolerant and drought-sensitive individuals. The results indicated that the
269 F2 progeny produced by a cross between idr1-1 mutant and HD119 plant yielded a segregation of 160
270 highly tolerant and 522 mildly tolerant plus sensitive plants (M+S) (in a ratio of 1:3.2), which is in good
271 agreement with the expected 1:3 ratio (X2 = 2.01, P < 0.05) (Supplemental Fig. S3; Supplemental Table
272 S1), suggesting that the idr1-1 mutation are recessive. Furthermore, we chose 160 F2 individuals with
273 highly drought tolerance from such a population for the primary mapping. Eventually we mapped the
274 candidate mutation to a ~0.78Mb genomic interval between markers RM18402 and RM18421 on
275 chromosome 5 (Fig. 2A). Further, 3800 F2 individuals were used for narrowing down the candidate
276 region. Finally, IDR1 was delimited to an interval of ~30.7 kb between markers IM63 and IM59, which
277 included four predicted genes (Fig. 2A). After sequencing, a single-base deletion (position 886
278 downstream of translation initiation codon ATG) in the fifth exon of LOC_Os05g26890 (corresponding
279 to D1 or RGA1 gene) in idr1-1 mutant was identified, which as a result led to a truncated protein
280 consequent upon a premature stop codon (TGA) newly created on the open reading frame (ORF) of D1
281 (Fig. 2B) (Ashikari et al., 1999; Wang et al., 2006). The D1 had been previously reported to encode α
282 subunit of heterotrimeric G proteins (also known as RGA1) (Wang et al., 2006; Oki et al., 2009);
283 nevertheless, there were very few reports on the involvement of D1 in rice drought tolerance.
284 Complementation of the mutant with a wild-type IDR1 genomic DNA fragment completely corrected
285 the dwarfism defect of idr1-1 mutants (Fig. 2C); by contrast, the transformed plants carrying empty
286 vector (EV) bore striking similarity in plant height to the idr1-1 mutants (Fig. 2C). Similarly,
287 overexpression of IDR1 cDNA in the idr1-1 mutant background also totally rescued the idr1-1’s
288 dwarfism phenotype (Fig. 2C). Moreover, the seeds harvested from IDR1 complementation lines or OE
289 lines reverted to approximately their wild-type sizes (Fig. 2D). As expected, the complementation lines
290 grown in pots exhibited obviously reduced drought tolerance, which was comparable to the performance
291 as was observed in IAPAR9 plants (Fig. 2E); that is, both wild-type IAPAR9 plants and IDR1
292 complementation lines were almost completely dead, while a fraction of idr1-1 mutants remained alive
293 (Fig. 2E). Likewise, when grown in the concrete tanks, such complementation lines displayed remarkable
294 sensitivity to moderate and severe drought treatments (Supplemental Fig. S4). Hence, it can be concluded
295 from the aforementioned results that reintroduction of IDR1 into the idr1 mutants does indeed lower the
296 drought tolerance of the idr1-1 mutants to close to the wild-type level. When all of the genotypes (which
297 underwent severe drought stress) grown in the above-mentioned concrete tanks grew to maturity, the
298 average seed setting rate of idr1-1 mutant plants was approximately 40%, which was around 2 times as
299 high as that of IAPAR9 plants (which achieved about 20% average seed setting rate) (Fig. S5). This
300 suggests that loss of function of IDR1 is able to reduce loss of grain yield under the conditions of severe
301 drought stress.
302
303 Expression Patterns and Subcellular Localization of IDR1

304 Expression analyses revealed that transcript levels of IDR1 gradually declined alongside the increasing
305 drought stresses (from control to severe drought stress), which points to the possibility that the expression
306 of IDR1 is suppressed by drought stress (Fig. 3A). Twenty percent PEG treatments also led to significant
307 drop in IDR1 expression to around half of the level prior to treatment (0 h), at 4 and 8 h following
308 treatments (Fig. 3B). By contrast, 150 mM NaCl treatments caused continuous induction of IDR1
309 expression during the first 4 hours after treatments, followed by a reduction to the level similar to that at 0
310 h (Fig. 3C). Cold treatments (4℃) brought about weak upregulation of IDR1 expression at 1 h upon
311 treatment, but the expression was downregulated from 4 hour onwards (Fig. 3D); similarly, IDR1
312 expression was also induced by heat treatments (40℃), peaking at 4 h, declining at 8 h (Fig. 3E). Fifty
313 micromolar ABA treatments made IDR1 downregulated, which is similar to the case in the foregoing
314 drought treatments (Fig. 3, F and A). Altogether, these data suggest that IDR1 expression is affected by
315 different types of stresses and downregulation of IDR1 may contribute to drought tolerance of rice plants.
316 In order to get a clearer picture of expression patterns of IDR1, tissue-specific expression patterns of
317 IDR1 were examined by qRT-PCR assays. The results demonstrated that IDR1 was ubiquitously
318 expressed in all the three tested tissues, with the highest expression in leaves and the lowest expression in
319 roots (Fig. 3G). Assays of subcellular localization of IDR1 revealed that IDR1 was definitely localized in
320 nucleus, and also at plasma membrane or cell periphery (Fig. 3H). In addition, it seems likely that IDR1
321 was localized in cytoplasm as well (Fig. 3H). These observations suggest that IDR1 presumably performs
322 particular functions on plasma membrane and nucleus.
323
324 idr1-1 Mutation Results in Physiological and Morphological Changes that are Associated with
325 Drought Tolerance
326 To learn about physiological changes caused by idr1-1 mutation, we determined the water potentials of
327 the genotypes as indicated in Fig. 4A. Under control conditions, leaves of idr1-1 mutants had relatively
328 higher water potential values in comparison with those of IAPAR9 plants and the complementation lines
329 at 10 o’clock onwards (Fig. 4A, left panel); a similar trend was observed when under moderate drought
330 conditions: the idr1-1 mutant leaves possessed quite obviously higher water potential values between 10
331 to 18 o’clock (Fig. 4A, right panel), which collectively indicates that the idr1-1 mutant leaves has greater
332 capability to retain water than do leaves of the IAPAR9 plants and the complementation lines. Besides,
333 water loss rates of detached leaves from idr1-1 mutants were markedly lower than those from IAPAR9
334 plants and the three complementation lines, from 2 h onwards after sampling; at time point 12 h, the water
335 loss rate was approximately 72% for IAPAR9 leaves, as against about 60% for idr1-1 leaves (Fig. 4B),
336 suggesting that the increased drought tolerance of the idr1-1 mutant may be, at least in part, a result of
337 attenuated water loss rates of leaves. Although proline contents exhibited no differences among these
338 genotypes under control conditions, the content in idr1-1 leaves was significantly higher than the contents
339 in the leaves of other 4 genotypes under the moderate drought conditions (Fig. 4C), indicating that
340 drought stress might have promoted biosynthesis of proline in the idr1-1 mutant background. Further, we
341 measured malonaldehyde (MDA) contents as well as relative electrolyte leakage (REL) of leaves, and
342 found that idr1-1 mutants possessed a significantly lower MDA level and REL compared to the remaining
343 genotypes under the moderate drought conditions (Fig. 4, D and E). In addition, idr1-1 mutants, which
344 constantly possessed dark-green leaves, were of significantly higher chlorophyll contents compared with
345 the other genotypes when grown in both control and moderate drought conditions (Fig. 4F). All these data
346 together demonstrate that idr1-1 mutants exhibit significantly enhanced capability of retaining water and
347 preserving integrity of plasma membrane, which is presumably one of the important reasons why idr1-1
348 mutants show elevated drought tolerance.
349 Under control conditions, it appeared that stomatal apertures of idr1-1 leaves showed a slight, but not
350 significant, reduction compared to the other genotypes as indicated (Fig. 5, A and B); however, they were
351 significantly greater than those of the other genotypes under drought stress conditions (Fig. 5, A and B,
352 upper panel), which may stems from an impairment of signaling of stomatal closure. There were no
353 evident differences in stomatal densities between idr1-1 mutant and the rest of genotypes under control or
354 drought conditions (Fig. 5B, lower panel). Taken together, these results suggest that mutation of IDR1
355 leads to attenuated stomatal closure, but does not significantly alter the stomatal density.
356 It is widely known that thicker leaves have beneficial effects on plant tolerance to drought stress as
357 they are able to retain more water to provide protection against soil water deficiency. As depicted in Fig. 5,
358 C and D, idr1-1 leaves looked thicker than IAPAR9 leaves at the position in the proximity of upper
359 portion of the main vein; average thickness of idr1-1 leaves reached 65 μm, being 10 μm longer than that
360 of IAPAR9 leaves (Fig. 5D), which may be one of the reasons that idr1-1 mutants exhibits strongly
361 enhanced drought tolerance relative to IAPAR9 plants. Besides, deposition of wax crystals on the front
362 and back of idr1-1 leaves was slightly, but not significantly, less than that of IAPAR9 leaves, implicating
363 that mutation of IDR1 has little effects on the deposition of wax crystals on leaves (Supplemental Fig. S6).
364 To know the differences in roots between idr1-1 mutants and IAPAR9 plants, we determined the several
365 traits of roots that were derived from PVC tube-grown idr1-1 mutants and IAPAR9 plants (Supplemental
366 Fig. S7A). Average root lengths of both idr1-1 mutants and IAPAR9 plants grown in moderate drought
367 conditions were slightly increased compared to that of both genotypes grown in control conditions (Fig. 5,
368 E and F); however, idr1-1 mutants and IAPAR9 plants showed no significant differences in the average
369 root length when both genotypes were grown in either control or drought conditions (Fig. 5, E and F).
370 Although idr1-1 mutants showed a slight decrease in average root volume and a significant decrease in
371 average root number relative to IAPAR9 plants, moderate drought treatments seemingly had only slight or
372 no significant impacts on root volumes or root numbers of both genotypes, respectively (Fig. 5G;
373 Supplemental Fig. S7B); as shown in Fig. 5G, the root volumes of both idr1-1 mutants and IAPAR9
374 plants undergoing the drought stress were a little larger than those of both the genotypes grown in control
375 conditions. Furthermore, the drought treatments also induced slight increases in root dry weights and root
376 diameters of idr1-1 mutants and IAPAR9 plants in comparison with non-drought treatments (control)
377 (Supplemental Fig. S7, C and D); it appeared that mutation of IDR1 led to small increases in root
378 diameters, but subtle decreases in root dry weights under both control and drought conditions
379 (Supplemental Fig. S7, C and D). Considering that idr1-1 mutants have a little changed average root
380 length, average root volume, average root dry weight and average root diameter, together with thicker
381 leaves and remarkably reduced plant heights, the mutant plants must have less transpirational water loss
382 but comparable water absorption from soil when compared to IAPAR9 plants, which therefore makes
383 idr1-1 more tolerant of drought stress.
384
385 idr1-1 Mutation Impairs Apoplastic and Chloroplatic ROS Production Provoked by Drought Stress
386 and Oxidative Stress Inducer
387 To gain insights into the status of ROS accumulation and cell death in idr1-1 mutants when challenged
388 by drought stress, we performed DAB, NBT, and Trypan blue staining of the leaves from different
389 genotypes [treated with or without (control) drought stress] as indicated in Fig. 6A. The results from DAB
390 staining demonstrated that the leaves from control did not show visible H2O2 accumulation as they lacked
391 staining, but moderate drought treatments provoked a stronger accumulation of H2O2 in the leaves of
392 IAPAR9 plants, complementation lines and the OE lines when compared with the idr1-1 leaf (sample 2)
393 that was less stained (Fig. 6A, left panel). NBT staining revealed that O2- production in idr1-1 leaf was
394 less than in leaves of the remaining genotypes after drought treatments (Fig. 6A, middle panel). A similar
395 trend was observed when the leaves from these genotypes were stained with Trypan blue to detect the
396 occurrence of cell death under control and drought stress conditions, that is to say, idr1-1 leaf
397 accumulated less amounts of Trypan blue stain compared with the rest of these genotypes under drought
398 stress conditions (Fig. 6A, right panel). These data collectively suggest that idr1-1 mutation attenuates
399 accumulation of the major species (H2O2 and O2-) of leaf ROS induced by drought stress, and such
400 mutation mitigates cell death elicited by the stress.
401 We further examined subcellular localization of H2O2 accumulation in the leaves undergoing moderate
402 drought treatments by means of a cytochemical technique. It is apparent from the Fig. 6B that, under
403 control (unstressed) conditions, leaf apoplasts of IAPAR9 plants did not accumulate visible CeCl3
404 deposits, while those of idr1-1 mutants did indeed accumulate a certain number of CeCl3 deposits,
405 indicative of higher H2O2 accumulation in the idr1-1 leaf apoplasts (Zhang et al., 2010). This suggest that
406 mutation of IDR1 might have evoked biosynthesis of a particular amount of H2O2 in leaf apoplasts
407 without drought stress, which may provide a reasonable explanation for the above observation that
408 stomatal apertures of idr1-1 mutant leaves slightly smaller than those of IAPAR9 leaves under control
409 conditions (Fig. 6B). By contrast, under drought stress conditions, a relatively larger number of CeCl3
410 deposits were observed in the leaf apoplasts of IAPAR9 plants than of idr1-1 mutants, suggesting that
411 more H2O2 in the leaf apoplasts are accumulated in response to drought stress in the IAPAR9 leaves. It
412 should be noted that the accumulation of CeCl3 deposits showed no significant differences between the
413 idr1-1 samples with or without the treatment of drought stress, suggesting that drought stress does not
414 trigger the H2O2 accumulation efficiently in the idr1-1 leaves, which is in agreement with the results of
415 DAB staining (Fig. 6B).
416 After 2-week-old idr1-1 mutant and IAPAR9 seedlings were treated with or without (control) 30 μM
417 methyl viologen (MV) for 72 h, the seedlings of IAPAR9 became noticeably wilted and/or yellowing
418 following treatments, while those of idr1-1 mutants were affected to a lesser extent (Fig. 6C). DAB
419 staining revealed that detached leaves from IAPAR9 plants experiencing MV treatments showed much
420 greater ROS production than those from idr1-1 mutants, which was marked by a much larger area of
421 intense staining existing on IAPAR9 leaves than on idr1-1 leaves (Fig. 6D). In addition, the IAPAR9
422 leaves subjected to MV treatment possessed a noticeably lower chlorophyll content than those without
423 MV treatment; however, there were almost no differences in chlorophyll contents between MV-treated
424 and untreated idr1-1 leaves, indicating that chlorophyll in IAPAR9 leaves is obviously broken down after
425 treatment with MV (Fig. 6E). Given the fact that MV is able to elicit rapid burst of chloroplastic H2O2
426 production by inhibiting photosynthetic electron transport (PET), the results mentioned above thus
427 suggest that idr1-1 mutation markedly attenuates MV-induced H2O2 generation in chloroplasts, thus
428 diminishing oxidative damage to leaf tissues and chloroplasts themselves.
429 When idr1-1 mutant seeds developed to full maturity (husks turned fully yellow) in the upland fields,
430 idr1-1 mutant leaves remained green and became hardly senescent, while the IAPAR9 control leaves had
431 become almost totally yellow as a result of senescence at the same developmental stage, indicating that
432 idr1-1 mutants have much delayed leaf senescence and breakdown of proteins as well as membranous
433 structures following the seed maturity. Moreover, upon 3-d-darkness induction, IAPAR9 leaves displayed
434 greater leaf senescence than did idr1-1 leaves (Supplemental Fig. S8, top panel). DAB staining revealed
435 that idr1-1 leaves lacked staining compared with strongly stained IAPAR9 leaves (Supplemental Fig. S8,
436 middle panel); NBT staining also showed that idr1-1 leaves were stained less than IAPAR9 leaves
437 (Supplemental Fig. S8, bottom panel). Altogether, these data suggest that the delayed leaf senescence
438 induced by darkness may result from lack or attenuation of ROS accumulation in idr1-1 mutants, which is
439 in accord with the notion that idr1-1 mutation impairs ROS production and/or increases ROS-scavenging
440 ability.
441 A recent genome-wide analysis revealed that there existed ten and nine respiratory burst oxidase
442 homologs (RBOHs) in Arabidopsis and rice, respectively. All the 9 rice OsRBOHs (OsRBOHA-OsRBOHI)
443 were differentially expressed during the entire life cycle of rice (Chang et al., 2016). The expression of
444 five of the rice OsRBOHs, i.e. A-C, E and I, were induced to higher levels by drought stress, with
445 expression levels under drought stress being about 10, 4, and 8.5 times as high as those under normal
446 conditions, respectively (Wang et al., 2013). Our data, however, showed that among the five tested genes,
447 only the OsRBOHB in idr1-1 mutants was induced to a level approximately twice as high as that in
448 IAPAR9 plants under severe drought stress conditions (Supplemental Fig. S9; Supplemental Table S2).
449 Thus, it appears that drought stress fails to induce expression of OsRBOHs efficiently in the idr1-1 mutant
450 background, which may be one of the reasons why idr1-1 mutant produces less amounts of ROS upon
451 exposure to drought stress.
452 Chloroplast is known as one of the major sources of ROS. During photosynthesis, especially when
453 plants are subjected to abiotic stress, ROS are generated in abundance as a result of impaired electron
454 transport (Challabathula et al., 2018). To know if the enhanced drought tolerance of idr1-1 mutant is
455 associated with altered photosynthetic process, we examined expression levels of 11 genes that were
456 involved in chloroplastic PET or redox reactions. Nine of the eleven tested genes, i.e. OsLFNR1, OsFd4,
457 OsNTRC, OsPC, OsGLT2, OsCDSP32, OsTrxX, OsTrxM5 and OsABC1, showed higher expression levels
458 in idr1-1 mutants than in IAPAR9 plants under moderate and severe drought stress conditions
459 (Supplemental Fig. S10; Supplemental Table S2). Among the 9 genes, five genes (OsLFNR1, OsPC,
460 OsGLT2, OsTrxM5 and OsABC1) were expressed at least 2 times as high in idr1-1 mutants as they were
461 in IAPAR9 plants under moderate and/or severe drought stress conditions (Supplemental Fig. S10). These
462 results together suggest that under drought stress conditions, the expression of these 9
463 photosynthesis-related genes in idr1-1 mutants was induced to higher levels when compared to the levels
464 in IAPAR9 plants. OsLFNR1 encodes a chloroplast-localized leaf-type ferredoxin-NADP+-oxidoreductase,
465 and it is able to transfer an electron from ferredoxin (Fd) molecules to NADPH for use in Calvin-Benson
466 cycle. OsNTRC encodes a bifunctional thioredoxin reductase that is required for reduction of oxidized
467 thioredoxins. OsFd encodes a 2Fe-2S iron-sulfur cluster binding domain containing protein, and it
468 receives electrons from PSI and then transfers them to OsFNR (or OsLFNR) or to O2 to form O2-. OsPC
469 encodes a plastocyanin, which transfers electrons from Cytb6f complex to PSI. The remainder are
470 thioredoxin- or thioredoxin-related genes that function in Calvin-Benson cycle or H2O2-detoxifying
471 system (Gong et al., 2020). It should be noted that OsFd can transfer electrons from PSI to OsLFNR in
472 normal circumstances, or to O2 to produce excessive O2- under stress conditions due to inhibition of
473 Calvin-Benson cycle. Thus, it appears that the elevated expression levels of the genes (e.g. OsLFNR1,
474 OsFd4 and OsPC) may contribute to enhanced PET for successful generating NADPH and ATP for
475 Calvin-Benson cycle, and thereby diminish the transfer of electrons to O2 for eventual H2O2
476 overproduction. Moreover, the upregulation of thioredoxin- or thioredoxin-related genes may
477 continuously maintain Calvin-Benson cycle in an active state and limit the overaccumulation of H2O2
478 against undue oxidative stress on chloroplasts.

479
480 Increased Expression of the Genes Associated with ROS Scavenging in idr1-1 Mutant Diminishes
481 ROS Accumulation Caused by Drought Stress
482 In order to understand the molecular mechanisms underlying enhanced drought tolerance observed in
483 idr1-1 mutant, we conducted gene expression analysis to examine the levels of those genes associated
484 with ROS scavenging and PET. Three-week-old seedlings were treated with moderate or severe drought
485 stress and subsequently used for qRT-PCR assays. The results demonstrated that two marker genes,
486 RAB16C and RAB16A, were highly induced by severe drought treatment, indicating that drought
487 treatment is quite effective (Supplemental Fig. S11; Supplemental Table S2); another three genes tested,
488 OsLEA3-1, OsLG3, and DSM1, were all induced to higher expression levels in idr1-1 mutants than in
489 IAPAR9 plants under severe drought stress conditions (Fig. 7A; Supplemental Table S2). OsLG3 was
490 discovered to positively regulate drought tolerance in rice by inducing gene expression of a dozen ROS
491 scavengers (Xiong et al., 2018). DSM1 was required for the normal expression of two ROS-scavenging
492 genes, OsPOX22.3 and OsPOX8.1 (Ning et al., 2010). LEA3-1 was considered as a protectant for
493 desiccation and oxidation stress (Jangam et al., 2016). To see whether idr1-1 mutant has increased
494 expression levels of the ROS-scavenging genes due to mutation of IDR1, we further checked the
495 transcript levels of a dozen or so genes implicated in ROS scavenging. It is quite interesting that 11 genes,
496 including 7 APX-family genes (OsAPX1-OsAPX2, OsAPX4-OsAPX8), two CAT-family genes (OsCAT1
497 and OsCAT3), one OsFeSOD as well as one OsPOX22.3 genes, showed higher expression levels in idr1-1
498 mutants than in IAPAR9 plants under moderate and/or severe drought stress conditions (Fig. 7B;
499 Supplemental Table S2). APX genes encode ascorbate peroxidases that are able to use ascorbate to reduce
500 H2O2 to H2O; CAT genes encode catalases that are highly efficient at catalyzing breakdown of H2O2 to
501 H2O and O2; FeSOD is very active in converting O2- into H2O2, and POX actively acts on H2O2 to make it
502 harmless H2O via redox reactions (Ning et al., 2010). Furthermore, measurements of POD enzymatic
503 activity revealed that under either control or moderate drought conditions, POD activities in idr1-1
504 mutants were always significantly higher than in IAPAR9 plants (Fig. 7C). Altogether, these data provide
505 a clear demonstration that mutation of IDR1 might have led to activation of ROS-scavenging system, and
506 thereby induces elevated expression of such important genes for highly efficient ROS scavenging and rise
507 in POD activity, which is presumably one of the important causes of the significantly enhanced tolerance
508 of idr1-1 mutant to drought stress.
509
510 Transcriptome Analyses of Drought-stressed idr1-1 Mutants Reveals Several Mechanisms

511 Underlying Enhanced Drought Tolerance
512 To further understand the underlying mechanism for the enhanced drought tolerance caused by idr1-1
513 mutation, we examined transcriptome profiles of 5-week-old idr1-1 mutants and IAPAR9 plants that had
514 undergone 14-d progressive drought stress treatment by using whole-genome mRNA sequencing
515 (mRNA-seq) method (Supplemental Fig. S12). The analyses of the transcriptomic data demonstrated that
516 there were 941 and 120 differential expressed genes (DEGs) identified under control and drought-stressed
517 conditions, respectively, which separately generated a [Control idr1-1 vs Control IAPAR9] (Supplemental
518 Data Set S1) and a [Drought idr1-1 vs Drought IAPAR9] data sets (Supplemental Data Set S2). Further
519 analyses revealed that a total of 397 upregulated and 544 downregulated genes were found in the [Control
520 idr1-1 vs Control IAPAR9] data set (Supplemental Data Set S1); likewise, there were 45 upgreulated and
521 75 downregulated genes present in the [Drought idr1-1 vs Drought IAPAR9] data set (Supplemental Data
522 Set S2). This suggests that idr1-1 mutation largely leads to downregulation of a group of genes in the
523 idr1-1 mutant in comparison with IAPAR9 control. We then conducted Gene Ontology (GO) analyses to
524 classify these DEGs. The results demonstrated that the 941 DEGs from the [Control idr1-1 vs Control
525 IAPAR9] data set were significantly enriched for oxygen binding, secondary metabolic process, and cell
526 growth GO terms (Supplemental Fig. S13); the 120 DEGs from the [Drought idr1-1 vs Drought IAPAR9]
527 data set were significantly enriched for secondary metabolic process (Supplemental Fig. S13). Under
528 drought-stressed conditions, 11 upregulated genes and 38 downregulated genes, the expression of which
529 were affected by both idr1-1 mutation and drought stress, were obtained by overlapping the DEGs from
530 the three different data sets as indicated (Supplemental Fig. S13; Supplemental Table S3). To ascertain
531 how transcriptional changes of a collection of genes in idr1-1 mutant increased its drought tolerance, we
532 carefully examined the genes in both [Control idr1-1 vs Control IAPAR9] and [Drought idr1-1 vs
533 Drought IAPAR9] data sets, and discovered a total of 43 genes that were presumably involved in drought
534 tolerance (Supplemental Table S4). Although some genes in Supplemental Table S4 only showed
535 significantly differential expression in control conditions but not in drought conditions, the altered
536 expression of such genes, however, will help idr1-1 mutants to cope better with drought stress, at least in
537 part, at the onset of occurrence of drought stress due to greater capacity of ROS scavenging and
538 antioxidation in the idr1-1 mutants. These genes were then grouped into 7 classes on the basis of reported
539 or putative functions about them: (1) reduction of oxidative stress, ROS production, cell death, and
540 hypersensitive reaction (Class I); (2) increase of ROS scavenging and detoxification (Class II); (3)
541 increase of antioxidant biosynthesis (flavonoid, anthocyanin, etc.) (Class III); (4) enhancement of osmotic
542 adjustment (Class IV); (5) weakening of leaf senescence (Class V); (6) alteration of hormonal
543 biosynthesis, signaling and response (Class VI); and (7) elevation of water retaining, survival ability, and
544 drought resistance (Class VII) (Supplemental Table S4)..
545 There are 11 genes probably associated with the functions shown in Class I (Supplemental Table S4).
546 Six genes, which encode OsOXO4 (Os03g48780), OsDGK8 (Os12g12260), OsCBSX10 (Os01g44250),
547 UDP-glucosyltransferase (Os02g11680), Yr10-like protein (Os11g37774) and OsRING-1 (Os02g52210),
548 respectively, showed decreased expression in drought-stressed conditions (Supplemental Table S4).
549 OsOXO4 was reported to contribute to apoplastic ROS accumulation under drought conditions
550 (Voothuluru and Sharp, 2013; Zhang et al., 2013). OsDGK8 plays a role in generating phosphatidic acid
551 (PA) in response to biotic and abiotic stresses (Li et al., 2015), suggesting that it may participate in
552 PA-dependent ROS production. OsCBSX10, UDP-glycosyltransferase, Yr10-like protein, OsRING-1 and
553 their Arabidopsis homologs were documented to perform functions in mitochondrial ROS accumulation,
554 pathogen-induced oxidative burst as well as hypersensitive responses (Meng et al., 2006; Coram et al.,
555 2010; Shin et al., 2020). Thus, these data collectively suggest that downregulation of such genes in idr1-1
556 mutant presumably attenuates ROS production and cell death in different cellular compartments or tissues.
557 Four genes, which encode OsPLC4 (Os05g03610), OsAAO3 (Os07g18154), NBS-LRR disease
558 resistance protein (Os06g49390) and NB-ARC domain containing protein (Os11g34970), were all
559 downregulated in idr1-1 mutant only under control conditions (Supplemental Table S4). OsPLC4 is a
560 phospholipase C, and it was reportedly associated with ROS production upon infection by bacterial strain
561 Pst DC3000 (D'Ambrosio et al., 2017). OsAAO3 is an aldehyde oxidase 3, and it can oxidize NADH,
562 ultimately leading to generation of O2- (Kundu et al., 2012; Batth et al., 2017). Os06g49390 and
563 Os11g34970 both encode disease resistance proteins; overexpression of this class of genes brings about
564 rapid HR, cell death and growth retardation (Bai et al., 2012). Hence, reduced expression of the
565 above-mentioned 4 genes in idr1-1 mutant is helpful in weakening ROS generation and cell death.
566 Os01g64120 encodes a 2Fe-2S iron-sulfur cluster binding domain containing protein (Fd-like protein) for
567 facilitating electron transfer from PSI to FNR in normal circumstances, and upregulated expression of
568 such genes in idr1-1 mutant under both drought and control conditions may prevent overproduction of O2-
569 (Hanke and Mulo, 2013). Taken together, these results suggest that altered expression of the foregoing
570 genes in idr1-1 mutant may be favorable to lessening ROS production and cell death under drought stress
571 conditions. There are 10 genes (Class II), which have putative functions in ROS scavenging or
572 detoxification, showing altered expression under drought-stressed or control conditions (Supplemental
573 Table S4). Nine genes, i.e. OsPOX71 (Os05g04500), OsANN3 (Os05g31750), OsPOX62 (Os04g59200),
574 OsPOX8.1 (Os07g48010), OsPOX22.3 (Os07g48020), OsPOX3006 (Os07g48050), OsGSTU6
575 (Os01g37750), OsLG3 (Os03g08470), and OsMT2d (Os01g05585) displayed upregulated transcription in
576 idr1-1 mutant under drought or control conditions. Peroxidases (including OsPOX71, OsPOX62,
577 OsPOX8.1, OsPOX22.3 and OsPOX3006) fulfilled the functions of catalyzing conversion of H2O2 to
578 H2O (Atack and Kelly, 2007; Hasanuzzaman et al., 2017; Majumdar and Kar, 2019) (Supplemental Table
579 S4). Overexpression of OsANN3 in rice led to elevated activities of ROS-scavenging enzymes, including
580 APXs, CATs and SODs (Li et al., 2019). OsGSTU6 was involved in scavenging H2O2 and reducing
581 oxidation stress (Ning et al., 2010). OsLG3 was recently reported to contribute positively to upregulation
582 of 10 of ROS scavenging-related genes (like APXs, CATs, PODs, SODs, etc.) upon overexpression of
583 OsLG3 (Xiong et al., 2018). OsMT2d performed a function in decreasing oxidative damage when rice
584 plants were exposed to abiotic stress conditions (Patankar et al., 2019). OsAAO1 (Os06g37080), which
585 showed decreased expression in control conditions, encodes an ascorbate oxidase 1. This enzyme
586 catalyzes active L-ascorbate to dehydroascorbate, and the accumulation of such dehydroascorbates was
587 correlated with leaf senescence and H2O2 elevation (Chen et al., 2013). Altogether, these data demonstrate
588 that expression changes of the foregoing genes in idr1-1 mutant appear to make positive contributions to
589 idr1-1’s tolerance against drought stress.
590 Antioxidants play important roles in reducing oxidative damage and eliminating free radicals when
591 plants are subjected to abiotic stress. Our analyses led to the discovery of 5 genes (Class III) associated
592 with antioxidant biosynthesis or production, which were all upregulated in drought or control conditions
593 (Supplemental Table S4). Four genes, Os07g02100 and OsF3H2 (Os10g39140) (upregulated in drought
594 conditions), as well as Os01g13610 and Os06g44170 (upregulated in control conditions), are all involved
595 in flavonoid biosynthesis (Supplemental Table S4). Flavonoids played important roles in protecting cell
596 wall and membranes when plants underwent stresses (Song et al., 2016). OsF3H2 in woody plant Lycium
597 chinense had strong effects on scavenging free radicals by synthesis of flavan-3-ols (Song et al., 2016).
598 Os04g37820, which encodes UDP-glucuronosyl/UDP-glucosyltransferase-family protein, was reported to
599 function as an antioxidant for detoxification (Williamson et al., 1998). In conclusion, upregulation of
600 these 5 genes seems likely to favor scavenging of ROS and free radicals by increasing the levels of
601 antioxidants in idr1-1 mutant, thus aiding in idr1-1’s drought tolerance. There are 3 genes (Class IV),
602 which are associated with enhancement of osmotic adjustment (Supplemental Table S4). OsLEA18
603 (Os04g52110) was downregulated quite obviously in idr1-1 mutant; LEA proteins were reported to
604 accumulate to high levels during the late stage of seed maturation and in response to water deficit (Liu et
605 al., 2013). However, a recent study revealed that overexpression of ZmLEA3 in tobacco plants increased
606 the hypersensitive cell death triggered by pst DC3000 and enhanced the expression of PR1a, PR2 and
607 PR4 (Liu et al., 2013). So, it seems that downregulation of OsLEA18 may be an indication that water
608 contents of idr1-1 leaves are possibly higher than those of IAPAR9 leaves, and such downregulation may
609 be beneficial for reducing cell death. OsASR1 (Os01g72900) and OsASR2 (Os01g72910) each encode a
610 ABSCISIC ACID-STRESS-RIPENING-INDUCIBLE PROTEIN, and all exhibited increased expression
611 under control conditions. Overexpression of OsASR1 in rice reportedly resulted in improved water
612 regulation under salt and drought stresses, probably as a result of accumulated osmolytes and reduced
613 transpiration rates (Park et al., 2020) All these data suggest that idr1-1 mutant might accumulate higher
614 levels of osmolytes to make it cope better with osmotic stress, which probably, at least in part, result from
615 the expression changes of the aforementioned 3 genes. There are 4 genes (Class V) connected with
616 weakening of leaf senescence, and they all showed downregulation under drought or control conditions
617 (Supplemental Table S4). Os04g32320 encodes a glycerophosphoryl diester phosphodiesterase-family
618 protein, and it was involved in the onset of leaf senescence (Swidzinski et al., 2002). OsMtN3
619 (Os02g30910), which was downregulated in both drought and control conditions, encodes a nodulin
620 MtN3-family protein, and it has a homologous gene in Arabidopsis, named SAG29;
621 SAG29-overexpressing transgenic plants reportedly exhibited an accelerated senescence and were
622 hypersensitive to salt stress (Seo et al., 2011). OsEIL4 (Os08g39830) encodes an ETHYLENE
623 INSENSITIVE-LIKE GENE 4 protein, and might be a positive regulator for ethylene-mediated leaf
624 senescence (Qiu et al., 2015). OsSAG20 (Os11g01850) is a senescence-associated gene (Mahmood et al.,
625 2016). Collectively, these data suggest that the obviously delayed leaf senescence in idr1-1 mutant might
626 have come from the downregulation of such 4 genes.
627 There are 3 OsGH3 genes [OsGH3.3 (Os01g12160), OsGH3.2 (Os01g55940) and OsGH3.8
628 (Os07g40290)] (in Class VI), showing upregulation under drought or control conditions (Supplemental
629 Table S4). GH3-family proteins catalyze IAA conjugation to amino acids, which thus leads to inactivation
630 of IAA (Du et al., 2012). Exogenous NAA is able to induce ROS production in rice tissues, so
631 downregulation of IAA caused by overexpression of OsGH3.2 and the other two OsGH3 genes (OsGH3.3
632 and OsGH3.8) presumably contributes to reduction of ROS production (Du et al., 2012). Os01g50370,
633 which was downregulated in idr1-1 mutant, encodes a STE_MEKK_ste11_MAP3K.4; downregulation of
634 this gene might help enhance resistance to water deficit through altered ABA responses (Choi et al., 2017).
635 Overall, the above data suggest that the expression changes of these 4 genes may alter IAA levels and
636 responses to ABA in idr1-1 mutant, thereby resulting in attenuation of ROS production and significantly
637 higher tolerance to drought stress. There are 6 genes with altered expression under drought or control
638 conditions, which are connected with molecular functions shown in Class VII (Supplemental Table S4).
639 Three genes (Os04g44060, Os01g54030 and Os02g51370) had downregulated expression under drought
640 or control conditions, and the rest (Os09g16950, Os11g02240 and Os08g33150) exhibited upregulated
641 expression under the same conditions. OsPIP2;3 (Os04g44060) encodes an aquaporin. Water transport in
642 plants has been known to greatly dependent on the expression and activity of aquaporins. However, some
643 opposite results had been observed in OE lines of plasma membrane intrinsic protein (PIP) genes,
644 because rapid water loss, due to increased leaf and root hydraulic conductivity, made some plants even
645 more vulnerable to drought stress (Aharon et al., 2003). So, a general downregulation of most of the PIP
646 genes under drought conditions appears to reduce water loss and to help prevent backflow of water to
647 drying soil. OscytME3 (Os01g54030) encodes cytosolic NADP malic enzyme 3, and restricted expression
648 of ZmnpNADP-ME particularly in the guard cells of tobacco reportedly showed reduced stomatal
649 apertures, and enhanced water use efficiency (Muller et al., 2018). OsGRL4 (Os02g51370), OsCRK25
650 (Os09g16950), OsCIPK15 (Os11g02240) and Myb51-like TF (Os08g33150) were known to be associated
651 with stomatal closure, ROS signaling, improved tolerance to various stresses, and enhanced survival
652 ability under stress conditions (Xiang et al., 2007; Cui et al., 2013; Zhang et al., 2013; Hu et al., 2017;
653 Ruan et al., 2018). These data collectively suggest that alteration in expression of 6 such genes appears to
654 contribute to increasing water retaining and survival ability of idr1-1 mutant, thus improving its drought
655 tolerance.
656
657 IDR1 Physically Interacts with TUD1, and idr1-1 Mutant Possesses Impaired BR Responsiveness
658 A recent study showed that rice RGA1 might genetically interact with TUD1, a U-box E3 ligase that
659 was implicated in BR signaling; loss of function of TUD1 brought about a range of BR
660 signaling-defective phenotypes, such as dwarfism, shortened 2nd internode, etc. (Hu et al., 2013). Our
661 protein-protein interaction assays further proved a physical interaction between IDR1 and TUD1,
662 suggesting a possibility that IDR1 may also take part in BR signaling (Fig. 8, A and B). In order to find
663 out whether idr1-1 mutants also had similar BR signaling-defective phenotypes as tud1 mutants, we
664 tested the responses of the idr1-1 mutants to EBR of different concentrations by lamina joint bending and
665 root elongation assays. The results indicated that leaf lamina joint bending in IAPAR9 seedlings was
666 progressively increased in a BR dose-dependent manner, varying from 33ºto 166ºwhen the seedlings
667 were treated with a range of BR concentrations (from 10-9 to 10-5 as indicated) (Fig. 8, C and D); such
668 bending in idr1-1 mutant seedlings also gradually increased, but the bent angles was somewhat smaller
669 than those of IAPAR9 seedlings under the same treatment conditions, varying from 31ºto 126º(Fig. 8,C
670 and D). Besides, root elongation assay showed that elongation of primary roots of IAPAR9 seedlings
671 were gradually inhibited with the increases in EBR concentrations (Fig. 8, E and F); by contrast, the
672 elongation of primary roots of idr1-1 mutant seedlings appeared less inhibited by the increasing
673 concentrations of EBR as compared to that of IAPAR9 seedlings (Fig. 8, E and F), suggesting that such
674 elongation of idr1-1 mutant seedlings were of decreased responsiveness to EBR treatments with higher
675 concentrations. These data together provide a clear demonstration that idr1-1 mutant exhibits impaired
676 responsiveness to higher BR concentrations than does IAPAR9 plant, which is similar to the
677 BR-responsive phenotypes observed in tud1 mutant (Hu et al., 2013).
678 Both idr1-1 and tud1-5 mutants showed growth retardation at various developmental stages compared
679 with their corresponding wild-type controls (Fig. 8G; Supplemental Fig. S1A). Seeds produced from
680 idr1-1 and tud1-5 mutants were about one-half as long as those produced from the wild-type controls (Fig.
681 8, H-I; Supplemental Fig. S1E). qRT-PCR assays revealed that TUD1 expression was downregulated by
682 moderate and severe drought stresses, which showed a similar trend in expression as observed for IDR1
683 (Fig. 8J; Fig. 3A). Furthermore, RT-PCR and transcriptomic results both indicated that BR-deficient
684 DWARF 1 (OsBRD1), which is involved in BR biosynthesis, was downregulated in idr1-1 mutant (Fig.
685 8K) (Hong et al., 2002). Because TUD1 and IDR1 were both discovered to be involved in BR signaling
686 and both proteins physically interact with each other, it appears that TUD1 and IDR1 may function
687 together in the same protein complex to help transduce BR signal through a non-canonical BR signaling
688 pathway, and the disruption of such signal transduction may be associated with enhanced drought
689 tolerance of idr1-1 mutant. This notion is supported by the previous report that knockdown of BR
690 receptor BdBRI1 causes attenuated BR signaling, therefore rendering the plants much more tolerant of
691 drought stress (Feng et al., 2015).
692
693 Discussion
694 As a highly drought-tolerant mutant, idr1-1 mutant has been preserved in our research group for almost
695 20 years. This mutant was grown every year and its drought tolerance was assessed continually in every
696 rice growing season. It is clear that idr1-1 mutant consistently showed very strong tolerance to drought
697 stress under strictly controlled soil moisture regimes each year, indicating that idr1-1 mutant is a real
698 drought-tolerant mutant. In this study, our analyses pointed to the fact that the strong drought tolerance of
699 idr1-1 mutant presumably stemed from several aspects, including altered plant morphology, reduced ROS
700 production, elevated ROS scavenging, possibly increased antioxidant contents, weakened leaf senescence,
701 etc., all of which collectively led to the obviously increased drought tolerance as observed in idr1-1
702 mutant for many years. Thus, our results provide valuable insights into the mechanisms of why loss of
703 function of rice Gα subunit brings so strong drought tolerance to the idr1-1 mutant.
704
705 Morphological and Physiological Changes of idr1-1 mutant Help Improve its Drought Tolerance by
706 Reducing Water Loss
707 It had been reported that Arabidopsis GPA1 is a positive modulator of plant cell proliferation; gpa1
708 mutant had reduced cell division in aerial tissues, which resulted in a reduced number of elongating cells
709 (Ullah et al., 2001). Rice d1 mutant defective in RGA1 displayed marked dwarfism phenotype,
710 demonstrating that the RGA1 serves a similar function as the GPA1 in the cell proliferation (Ashikari et
711 al., 1999; Ueguchi-Tanaka et al., 2000). Our data also indicated that idr1-1 mutants exhibited obvious
712 reduction of plant height and lengths of leaves, panicles, and grains, compared to IAPAR9 plants during
713 the whole period of growing (Fig. 1; Supplemental Fig. S1). In addition, the average leaf thickness of
714 idr1-1 mutants was significantly greater than that of IAPAR9 plants (Fig. 5, C and D). It is worth noting
715 that the average root length and average root volume of idr1-1 mutants were just slight shorter and
716 smaller than those of IAPAR9 plants, respectively, under drought stress conditions (Fig. 5, F and G)
717 Likewise, Arabidopsis GPA1 did not regulate cell proliferation in root meristems as well (Ullah et al.,
718 2001), which is in agreement with our observations that the average root length of idr1-1 mutants was
719 slightly changed compared with that of IAPAR9 plants (Fig. 5F). Because idr1-1 mutants had similar
720 average root length and average root volume as did IAPAR9 plants, the significantly reduced
721 aboveground biomass of idr1-1 mutants must remarkably slow down water loss in such mutants
722 compared to in IAPAR9 plants
723 Measurements of water potentials and leaf water loss rates revealed that idr1-1 mutants possessed
724 markedly higher water potentials from 10:00 to 18:00 than IAPAR9 plants, and the leaf water loss rates of
725 idr1-1 mutants were also decreased relative to those of IAPAR9 plants from 2 h onwards after leaf
726 detachment (Fig. 4, A and B). idr1-1 mutants accumulated higher levels of proline and had higher
727 chlorophyll contents when compared to IAPAR9 plants under moderate drought conditions (Fig. 4, C and
728 F). Moreover, idr1-1 mutants had significantly lower MDA contents and REL (Fig. 4, D and E). Besides,
729 idr1-1 leaves appeared thicker than IAPAR9 leaves at the examined positions (Fig. 5, C and D). Thus,
730 these morphological and physiological changes of idr1-1 mutant help retain more water in tissues when
731 the mutant plants are challenged by drought stress.
732
733 Mutation of IDR1 greatly Impairs Abiotic Stress- or ABA-induced Cytoplasmic and Chloroplastic
734 ROS Burst, which is Derived from NADPH Oxidases and Chloroplasts, therefore Reducing Cell
735 Death
736 It is generally known that ROS generated from chloroplasts and apoplasts are considered as major
737 sources of ROS when plants experience abiotic stresses (drought, salinity or oxidation) or ABA treatment.
738 There were a few reports of the involvement of GPA1 in ROS production when plants are exposed to
739 ABA treatment or oxidative stress. Under ABA treatment, ROS production was found to be evidently
740 impaired in gpa1 guard cells (Zhang et al., 2011). gpa1 mutation still allowed stomata to close to a level
741 similar to that in Col-0 after ABA treatment, but completely abolished ABA inhibition of stomatal
742 opening, which led to more water loss in the gpa1 mutants (Wang et al., 2001). These data collectively
743 suggest that GPA1 acts upstream of ROS production in the ABA signaling pathway. After MV treatment,
744 idr1-1 leaves showed much less ROS production than the leaves from IAPAR9 plants (Fig. 6, C and D),
745 suggesting that chloroplast-derived ROS burst induced by MV is greatly impaired in idr1-1 leaves. In
746 Arabidopsis, O3-treated gpa1 mutants displayed less leaf damage by excessive ROS than Col-0 control
747 plants (Joo et al., 2005). In fact, following exposure to O3, gpa1-4 mutants showed even less damage than
748 the control plants (Joo et al., 2005). It had been observed that O3 treatment triggered ROS generation
749 from chloroplasts and apoplasts of wild-type leaf cells, while the chloroplastic ROS amounts were
750 substantially decreased and the apoplastic ROS (via NADPH oxidases) were hardly produced in the gpa1
751 leaf cells. These facts together demonstrate that Gα (IDR1 and GPA1) is required for both chloroplastic
752 and apoplastic ROS production (Joo et al., 2005).
753 Ethylene is able to induce H2O2 production and stomatal closure, and GPA1 is needed for the
754 production of H2O2 induced by ethylene; gpa1 mutants showed defects in ethylene-induced H2O2
755 production and stomatal closure, whereas GPA1-overexpressing lines showed faster stomatal closure and
756 H2O2 production in response to ethylene (Ge et al., 2015). BR can stimulate ethylene synthesis, which
757 ultimately leads to stomata closure through H2O2; Gα mediated BR-induced stomatal closure via
758 promoting H2O2 and NO production (Shi et al., 2015). In gpa1 mutant, however, ethylene or BR treatment
759 could not effectively trigger ROS production, suggesting that GPA1 serves functions in mediating
760 initiation and amplification for apoplastic and/or chloroplastic ROS triggered by ethylene and BR. Our
761 results consistently showed that drought stress could also not effectively induce effective H2O2 and O2-
762 production in idr1-1 mutant, suggesting that idr1-1 mutation also impairs ROS production in rice (Fig. 6,
763 A and B), which points to the possibility that drought stress fails to induce expression of OsRBOH genes
764 or activate OsRBOHs’ activities efficiently in the idr1-1 mutant background (Supplemental Fig. S9). In
765 d1 mutant, cell death in response to ethylene and H2O2 was nearly completely abolished (Steffens and
766 Sauter, 2009). Similarly, we found that idr1-1 mutation mitigated cell death elicited by drought stress (Fig.
767 6A). Therefore, ROS production and cell death triggered by ethylene, BR and drought stress are
768 considerably impaired by Gα mutations. d1 mutant was documented to have a similar dwarfism
769 phenotype and impaired GA signaling as did GA-deficient mutant, which overaccumulated DELLA
770 proteins (Ueguchi-Tanaka et al., 2000; Colebrook et al., 2014). So, the significantly increased drought
771 tolerance in idr1-1 mutant may partially come from DELLA overaccumulation that limits excessive ROS
772 production (Du et al., 2012). Furthermore, our transcriptome data revealed that in idr1-1 mutant, three
773 GH3 genes (in Class VI) all showed upregulation under drought or control conditions, which might
774 facilitate decreasing IAA level, and thus contribute to reduction of ROS production (Supplemental Table
775 S4) (Du et al., 2012).
776 Our transcriptome analyses revealed 941 and 120 DEGs identified under control and drought-stressed
777 conditions, respectively (Supplemental Data Sets S1 and S2). Under drought-stressed conditions, there
778 were 11 upregulated genes and 38 downregulated genes discovered as being regulated in expression by
779 both idr1-1 mutation and drought stress (Supplemental Fig. S13; Supplemental Table S3). A total of 11
780 genes in Class I showed altered expression in the drought and/or control conditions (Supplemental Table
781 S4). Downregulation of 10 out of the 11 genes aids in reducing ROS production and cell death derived
782 from hypersensitive responses (Supplemental Table S4). In addition, upregulated expression of
783 Os01g64120 (encoding an Fd-like protein) in idr1-1 mutant may prevent overproduction of O2- under
784 drought stress conditions (Hanke and Mulo, 2013). Hence, these results suggest that altered expression of
785 the above-mentioned genes in idr1-1 mutant favors the attenuation of ROS production and cell death
786 under the drought-stressed conditions.
787 A recent study reported that when exposed to high light, d1 mutants exhibited a greater capacity to
788 dissipate excess irradiance relative to wild-type controls (Ferrero-Serrano et al., 2018). Therefore, D1
789 seems to be a regulator of photoavoidance and photoprotection mechanisms in rice (Ferrero-Serrano et al.,
790 2018). During photosynthesis, splitting of water molecules occurs in PSII complex, and the released
791 electrons are then transferred to the PSI complex via electron transport chain (ETC) from plastocyanin to
792 Fd. Under stress conditions, the electrons can be transferred from Fd to O2 for Mehler peroxidase reaction,
793 in which O2 is reduced to O2- and H2O2 (Gururani et al., 2015); as a consequence, these reactions
794 generate a high level of ROS, thereby causing photodamage or photoinhibition of PSII apparatus
795 (Gururani et al., 2015). Currently, a new proposition suggests that environmental stresses do not directly
796 cause photoinhibition but rather facilitate inhibition of PSII damage repair (Gururani et al., 2015).
797 Moreover, stresses also aggravate ROS production within the ETC of PSII and PSI during light reactions
798 when CO2 is limited and ATP synthesis is impaired (Gururani et al., 2015). ROS inhibits repair of
799 photodamaged PSII by primarily suppressing de novo synthesis of PSII proteins, especially reaction
800 center D1 proteins [this D1 protein is completely different from rice Gα (D1) protein] (Murata et al., 2007;
801 Gururani et al., 2015). The damaged D1 proteins caused by photodamage are selectively degraded by a
802 thylakoid membrane-bound metalloprotease, FtsH (Kato et al., 2009). The expression of FtsH was
803 maintained by THF1 or GPA1 because FtsH transcript level decreased in thf1 or gpa1 mutant (Zhang et
804 al., 2009). THF1 was found to physically interact with GPA1 in chloroplasts (Huang et al., 2006). When
805 photodamaged D1 proteins were rapidly cleaved by FstH or Deg proteases into large fragments, ROS
806 production was greatly activated, thus evoking ROS burst in the chloroplasts (Kato and Sakamoto, 2014).
807 D1 proteins could be phosphorylated in a light-dependent manner, and such phosphorylation was able to
808 protect D1 proteins against cleavage by FstH or Deg, which consequently diminished ROS production
809 (Kato and Sakamoto, 2014). gpa1 mutant showed obviously reduced chloroplastic ROS (especially H2O2)
810 production under the conditions of oxidative stress and ABA treatment (Joo et al., 2005; Zhang et al.,
811 2011; Ge et al., 2015), and idr1-1 mutant also showed decreased ROS production either from apoplasts or
812 from chloroplasts (Fig. 6), which presumably contributes to reducing damage to D1 as well as
813 phosphorylated D1 proteins. Our qRT-PCR results demonstrated that 9 out of the 11 tested genes, such as
814 OsLFNR1, OsFd4, OsPC, etc., showed higher expression levels in idr1-1 mutants than in IAPAR9 plants
815 under moderate and severe drought stress conditions (Supplemental Fig. S10). Besides, it is worth noting
816 that impaired stomatal closure in idr1-1 mutants under drought stress was helpful in reducing
817 photorespiration (which leads to a large ROS burst) caused by CO2 shortage as a result of tightly closed
818 stomata (Fig. 5, A and B). Based on the above facts, we propose a hypothesis to explain why idr1-1
819 mutation results in substantial decrease in ROS production derived from PSI and PSII. For PSI, the
820 elevated expression of the 9 genes implicated in ETC (e.g. OsLFNR1, OsFd4, and OsPC) in idr1-1
821 mutant may contribute to successful electron transport to generate NADPH and ATP for Calvin-Benson
822 cycle, and thereby diminish the transfer of electron to O2, thus leading to a marked decrease of H2O2
823 production from PSI in chloroplasts (Gong et al., 2020). For PSII, in the daytime, high light leads to
824 photodamage of D1 and phosphorylated D1 proteins in wild-type plants, and such damaged D1 proteins
825 are soon recognized and cleaved into large fragments, thus leading to excess ROS burst. However,
826 mutation of Gα (GPA1 or IDR1) decreases FtsH level, consequently reducing cleavage on damaged D1
827 proteins, which therefore results in accumulation of more phosphorylated D1 or damaged D1 proteins
828 during the daytime. At night, the phosphorylated damaged D1 proteins are converted to damaged D1
829 proteins through dephosphorylation, and then they are degraded progressively by FstH or Deg proteases,
830 which does not trigger rapid ROS burst from PSII in chloroplasts (Kato and Sakamoto, 2014). Therefore,
831 large-scale ROS burst does not appear in gpa1 or idr1-1 mutant when either mutant is subjected to
832 oxidative stress and ABA treatment.
833
834 Mutation of IDR1 Enhances ROS-Scavenging Ability by Triggering Gene Expression of

835 ROS-scavenging Enzymes, thus Lessening Oxidative Stress and Protecting Membrane Integrity
836 It is widely known that ROS scavenging is very important for drought tolerance. Plants with reduced or
837 loss of ROS scavenging ability usually show increased drought and oxidative sensitivity (You et al.,
838 2014). Conversely, increases of activities of ROS scavenging enzymes make a positive contribution to
839 plant’s drought tolerance (Liu et al., 2017). Under the progressive drought treatments, the d1 mutants
840 reportedly exhibited obviously increased tolerance to drought stress, which is consistent with our
841 observations that idr1-1 mutants showed remarkably elevated drought tolerance (Ferrero-Serrano and
842 Assmann, 2016). Our results showed that mutation of IDR1 might have led to activated expression of 11
843 ROS-scavenging genes (Fig. 7). Considering that IDR1 is not only localized in the nucleus, but also at
844 plasma membrane or cell periphery (Fig. 3H), it seems likely that IDR1 enters nucleus to repress
845 expression of some important genes for highly efficient ROS scavenging, which is presumably one of the
846 important causes of the significantly enhanced tolerance of idr1-1 mutant to drought stress.
847 Our transcriptome data revealed that there are 10 genes (Class II), which have putative functions in
848 ROS scavenging or detoxification, showing altered expression under drought-stressed or control
849 conditions (Supplemental Table S4). Upregulation of the five peroxidase genes (OsPOXs), OsANN3,
850 OsGSTU6 and OsLG3, reportedly induced elevated expression of a range of ROS scavenging genes (such
851 as APXs, CATs, PODs, SODs, etc.), which greatly contributed to eliminating excessive ROS and
852 increasing survival rates of transgenic rice plants under drought stress conditions (Atack and Kelly, 2007;
853 Ning et al., 2010; Xiong et al., 2018; Li et al., 2019). In addition, downregulation of OsAAO1 in idr1-1
854 mutant aided in overaccumulation of L-ascorbate for ROS scavenging (Chen et al., 2013). Hence, these
855 data together indicate that expression changes of these genes in idr1-1 mutant must play a very positive
856 part in scavenging excessive ROS induced by drought stress. Moreover, our transcriptome analyses
857 further demonstrated that there are 5 genes (Class III), which were all upregulated in drought or control
858 conditions, associated with biosynthesis of antioxidants (Supplemental Table S4). F3H2 was reported to
859 have a major impact on scavenging free radicals by synthesizing flavan-3-ols, and had essential roles in
860 the survival of woody plant Lycium chinense when grown under extremely drought-stressed conditions
861 (Song et al., 2016). Thus, upregulation of these 5 genes is likely to make substantial contributions to
862 scavenging ROS and improving idr1-1’s drought tolerance.
863 Plant phospholipase D (PLD) has been shown to be involved in the action or production of several
864 plant hormones (like ABA, ethylene, jasmonic acid, and cytokinin), cell growth, abiotic stress responses,
865 oxidative burst etc. (Mane et al., 2007; Li et al., 2009). When plants undergo stresses, such as drought and
866 salinity, PLDα1 hydrolyzes membrane lipids, and therefore produces a high level of PA; PA reportedly
867 contributes to stomatal closing and reduction of transpirational water loss in the presence of ABA (Hong
868 et al., 2008; Hong et al., 2010). PLDα1-suppressed or knockout plants displayed less sensitivity to
869 ABA-induced stomatal closure and lost more water than wild-type controls (Zhang et al., 2005; Mane et
870 al., 2007; Hong et al., 2008). Nevertheless, PLDα1 has adverse effects on plants when they experience
871 progressively increasing drought stress, because increasing PLDα1 activities will break down membrane
872 bilayers, and as a consequence lead to loss of cell membrane integrity, which ultimately promotes rapid
873 water loss and leaf senescence (Hong et al., 2008). So it appears that in the early stages of drought stress
874 or dehydration, increased expression of PLDα1 promotes signaling of stomatal closure and decreases
875 water loss; nevertheless, in the late stages, prolonged severe drought stress accelerates membrane
876 deterioration, ionic leakage, lipid peroxidation and water loss (Hong et al., 2008; Li et al., 2009).
877 Similarly, a recent study showed that Arabidopsis PLDδ knockout plants were much more tolerant of
878 severe drought and displayed retarded ABA-induced leaf senescence, presumably by suppression of
879 membrane lipid degradation (Distefano et al., 2015). It had been reported that GPA1 could bind to PLDα1,
880 and this binding inhibited the PLDα1 activity; the interaction between PLDα1 and GPA1 could
881 substantially decrease PLDα1 activity (Li et al., 2009). PLDα1 preferred to bind to Gα-GDP, and did not
882 bind to Gα-GTP (Li et al., 2009). When plants were treated with ABA or drought stress, Gα-GDP was
883 converted to Gα-GTP, and Gα was dissociated from PLDα1, which therefore relieved the inhibition of
884 PLDα1 (Li et al., 2009). Increased expression or enzymatic activity of PLDα1 promoted signaling of
885 stomatal closure at the earlier stages of drought stress via PA-promoted H2O2 production through RBOHs
886 (Li et al., 2009). Gα then entered nucleus to activate and repress expression of a host of genes (like
887 ROS-scavenging genes) as evidenced by nuclear localization of IDR1 and transcriptome data (Fig. 3H;
888 Supplemental Data Sets S1 and S2; Supplemental Table S4). On the basis of the above facts, we put
889 forward a hypothesis to delineate how Gα mutation abolishes PLDα1-dependent ROS production.
890 Drought stress induces accumulation of ABA, and the ABA promotes dissociation of PLDα1 and Gα
891 (GPA1 or IDR1). The PLDα1 then promotes generation of PA, and PA subsequently binds to RBOHs to
892 activate their enzymatic activities (Zhang et al., 2009). Meanwhile, dissociated Gα enters nucleus to
893 trigger expression of a group of genes, including the genes to modify ROBHs for their normal functions,
894 and the genes to promote ROS production in chloroplasts. However, Gα seems to inhibit expression of
895 ROS-scavenging genes, because 11 out of 13 tested genes (OsAPX1-OsAPX2, OsAPX4-OsAPX8,
896 OsCAT1, OsCAT3, OsFeSOD and OsPOX22.3) were upregulated to varying extents in idr1-1 mutant (Fig.
897 7B); additionally, 10 ROS-scavenging genes were also identified to be upregulated in the idr1-1 mutant
898 by our transcriptome analyses (Supplemental Table S4). This ultimately leads to H2O2 accumulation in
899 cytoplasm and chloroplasts, and subsequently results in stomatal closure. By contrast, mutation of Gα
900 fails to activate gene expression of those genes for ROBHs’ modifications as well as chloroplastic ROS
901 production, and repress expression of ROS-scavenging genes, which eventually leads to a great decrease
902 in ROS production in cytoplasm and chloroplasts, and a substantial increase in ROS-scavenging activity.
903 As a result, the cytoplasmic and chloroplastic ROS levels are considerably diminished in the Gα mutant,
904 thus abolishing or impairing stomatal closure.
905
906 idr1-1 Mutant Has Impaired BR Signaling, which possibly Makes Valuable Contributions to
907 Enhanced Drought Tolerance in such a Mutant
908 BRs play pivotal roles in promoting cell expansion and division, regulating senescence, modulating
909 plant responses to various environmental signals, etc. (Hu et al., 2013). When treated by BL, gpa1
910 mutants showed impaired sensitivity to BL, probably suggesting that GPA1 mediates BR signaling;
911 however, gpa1 mutant had similar responses to ABA and ethylene as did wild-type Col-0 (Ullah et al.,
912 2002). It had been reported that in Arabidopsis, EBR treatment initiated a marked rise in the levels of
913 ethylene, H2O2 and NO, which were necessary for stomatal closure in wild-type Col-0 (Shi et al., 2015);
914 nevertheless, EBR failed to close stomata of gpa1 mutants because EBR-induced H2O2 production was
915 greatly impaired in such mutants (Shi et al., 2015). A recent investigation showed that downregulation of
916 BdBRI1 resulted in reduced plant heights, shortened internodes, narrow and short leaves, reduced
917 expression of some BR-signaling genes, and enhanced expression of some BR biosynthesis genes (Feng
918 et al., 2015). Importantly, BdBRI1 RNAi lines exhibited greatly enhanced drought tolerance, accompanied
919 by highly elevated expression of some drought-responsive genes, such as BdP5CS (for proline synthesis),
920 BdCOR47/BdRD17, BdERD1 and BdRD26 (Feng et al., 2015). In addition, triple mutations in WRKY46,
921 WRKY54, and WRKY70, which were involved in both BR-regulated growth and drought responses in
922 Arabidopsis, rendered Arabidopsis plants more tolerant of drought stress (Feng et al., 2015). Our results
923 showed that IDR1 was involved in rice BR responses, and mutation of IDR1 led to impaired responses of
924 lamina joint bending and elongation of primary roots to BR (Fig. 8, C-F); additionally, idr1-1 mutation
925 also brought about downregulation of OsBRD1 (Fig. 8K). Considering the above facts, it is reasonable to
926 conclude that the defect in BR signaling increase drought tolerance in Brachypodium distachyon,
927 Arabidopsis, and rice.
928 tud1 mutant showed less sensitivity to EBR treatment (Hu et al., 2013), which is similar to the situation
929 observed in gpa1 and idr1-1 mutants (Shi et al., 2015). TUD1 was reported to genetically interact with
930 D1 (Hu et al., 2013), and our protein-protein interaction assays corroborated such an interaction (Fig. 8, A
931 and B). Both idr1-1 and tud1-5 mutants showed growth retardation and pronouncedly shorter grains
932 compared with their corresponding wild-type controls (Supplemental Fig. S2; Fig. 8, G-I). These data
933 collectively suggest a possibility that IDR1 and TUD1 both have a degree of impacts on BR signaling.
934 However, although IDR1 and TUD1 proteins are both involved in BR signaling, they are apparently not
935 canonical components of the BRI-mediated signaling pathway (Hu et al., 2013). Taken together, these
936 data suggest that loss of function of IDR1 impairs BR signaling, but may have beneficial impacts on
937 drought tolerance, probably via attenuation of ROS production and other as yet unidentified mechanisms.
938
939 idr1-1 Mutation Causes a Considerable Delay of Leaf Senescence, thus Enhancing Drought
940 Tolerance
941 Drought-tolerant plants usually have a feature of obviously delayed drought-induced leaf senescence,
942 and as a consequence they are able to maintain higher water contents and retain higher photosynthetic
943 activities under drought stress conditions. Plants’ stay-green phenotype or delay in leaf senescence is
944 known to stem from suppression of ethylene, ABA, BR, and strigolactone signal transduction or
945 activation of cytokinin signaling, indicating that a complex signaling network regulates leaf senescence
946 (Kusaba et al., 2013). DET2 encodes a steroid 5α-reductase that catalyzes an early step of BR synthesis,
947 and det2 mutant showed a stay-green phenotype (Chory et al., 1994). bzr2/bes1, a transcription factor
948 positively regulating BR signaling, also showed a stay-green phenotype (Yin et al., 2002). These and
949 other lines of evidence strongly suggest that the disruption of either BR biosynthesis or signaling
950 remarkably delays leaf senescence. gpa1 mutant showed attenuated leaf senescence, delayed chlorophyll
951 degradation under stress conditions (Urano et al., 2014). PLDα1-deficient transgenic plants were reported
952 to show slower rates of leaf senescence (Fan et al., 1997). Our results indicated that in upland fields,
953 leaves of idr1-1 plants remained green and became hardly senescent even after full seed maturity,
954 indicating that idr1-1 plants have much delayed leaf senescence and breakdown of leaf proteins as well as
955 membranous structures even following seed maturity. Upon 3-d-darkness induction, idr1-1 leaves
956 displayed delayed leaf senescence and weakened ROS and O2- production (Supplemental Fig. S8). As
957 OsBRD1 transcript level was downregulated (Fig. 8K) and BR signaling was impaired in idr1-1 mutants
958 (Fig. 8, C-F), it is possible that the severely delayed leaf senescence occurring in idr1-1 mutants is a
959 result of impaired BR biosynthesis or signaling. Alternatively, impaired functions of PLDα1 may make
960 positive contributions to such delay in leaf senescence.
961 Transcriptome analyses revealed that there are 4 genes (Os04g32320, OsMtN3, OsEIL4 and OsSAG20)
962 (Class V) associated with weakening of leaf senescence; all of them were downregulated under drought or
963 control conditions (Supplemental Table S4). The proteins encoded by the aforementioned genes take part
964 in initiation of leaf senescence, ethylene-mediated leaf senescence, or hormone-promoted leaf senescence.
965 Therefore, the decreases in expression of such genes might have contributed to delay in leaf senescence
966 under drought stress conditions.
967
968 idr1-1 Mutation Increases Gene Expression of Drought-tolerant Genes, therefore Improving
969 Survival Ability of the Mutant under Drought Stress Conditions
970 Data from other studies revealed that RGA1 regulated expression of an abundant group of genes. For
971 example, in rga1 mutant, there were 626 genes, which separately responded to heat (522), drought (101),
972 salt (37), and cold (15) stresses, found to be changed in gene expression levels (Jangam et al., 2016).
973 Among these genes, a few genes were implicated in biosynthetic pathways of osmoprotectants, such as
974 polyamine, glycine-betaine, proline, and trehalose (Jangam et al., 2016). These data indicate that RGA1
975 does indeed participate in regulation of expression of the genes associated with abiotic stresses. Our
976 transcriptome data demonstrated that there are 4 genes (Class IV), which are associated with enhancement
977 of osmotic adjustment, displaying altered expression levels in drought and/or control conditions
978 (Supplemental Table S4). Downregulatio of OsLEA18 seems to help weaken cell death; upregulation of
979 OsASR1 or OsASR2 are linked to accumulation of osmolytes (Supplemental Table S4) (Desikan et al.,
980 2006; Du et al., 2020; Park et al., 2020). Hence, the alteration of expression of these genes in idr1-1
981 mutant might confer better osmotic adjustment to such a mutant, and as a consequence improve the
982 survival ability of the idr1-1 mutant under drought stress conditions. There are 7 genes (Class VII)
983 showing expression changes under drought or control conditions, which participate in increases of water
984 retaining, survival ability, and drought tolerance (Supplemental Table S4). Decreased expression of
985 OsPIP2;3 under drought conditions was thought to aid in reducing water loss by preventing backflow of
986 water to drying soil. Downregulation of OscytME3 or OsGRL4 appears to result in reduced stomatal
987 apertures, and enhanced water use efficiency (Hu et al., 2017; Muller et al., 2018). In addition,
988 upregulation of OsCRK25, OsCIPK15 or Myb51-like TF contributes to improving the damaging effects of
989 cold, drought, and salt stresses, and increasing water retaining and survival ability of idr1-1 mutant under
990 drought-stressed conditions.
991 Altogether, in this study, we detailed the effects of the idr1-1 mutation on the drought tolerance, and
992 found that loss of function of IDR1 very obviously enhanced tolerance to drought stress in rice. Mutation
993 of IDR1 led to expression changes of a few dozen genes that were related to ROS production, ROS
994 scavenging, antioxidation, osmotic adjustment, leaf senescence, etc., suggesting that improved drought
995 tolerance in idr1-1 mutant cannot be simply attributable to its dwarfism phenotype, but rather results from
996 multiple layers of regulations occurring at morphological, physiological and molecular levels. Gα
997 mutation in Arabidopsis and rice affects multiple signaling pathways, suggesting that GPA1 serves to
998 integrate quite a few internal and external signals, which makes it a good material to study the molecular
999 mechanisms of a little important signaling, such as GA, BR, ABA, ET, phospholipids, etc. idr1-1 mutants
1000 showed significantly lower yield loss under severe drought stress conditions, implying that it can be used
1001 directly in breeding to improve drought tolerance of rice cultivars. In fact, our breeding practices have
1002 demonstrated that introduction of idr1-1 mutation to a few cultivars does indeed improve drought
1003 tolerance of such cultivars, but does not cause significant decreases of crop yields because some progeny
1004 carrying idr1-1 mutation have no noticeable reductions in panicle and seed lengths compared to the
1005 corresponding parental cultivars. Therefore, the idr1-1 allele has great potential for applications in rice
1006 breeding to generate elite rice cultivars with significantly increased drought tolerance in the near future.
1007
1008 Materials and methods

1009 Plant materials
1010 The upland rice cultivar IAPAR9 was used for this experiment as a starting material. The idr1-1 mutant
1011 was derived from a M2 population of IAPAR9 M1 seeds undergoing 60Co γ-ray (250 Gy) radiation. Other
1012 upland rice cultivars, such as HD297, HD385, IRAT109, HD119, etc., were used as male parents for
1013 crossing with the idr1-1 mutants to generate F2 populations for mapping IDR1 locus.The tud1-5 was a
1014 newly discovered mutant allele in the Nipponbare background as described previously (Hu et al., 2013).
1015 All important primers used in this study had been listed in Supplemental Table S5.
1016
1017 Drought stress treatments

1018 For drought-stressed treatments in pots (20 cm in diameter and 15 cm in depth), seedlings at a V6 (i.e.
1019 when the sixth leaf had a visible “collar” at the base of the leaf) developmental stage were subjected to
1020 drought stress by withholding water. When most of the IAPAR9 leaves were dried but the idr1-1 mutant
1021 was affected moderately, we defined such a scale as the severe drought stress conditions; at that time soil
1022 moisture was close to 5% [determined by a precise moisture measurement (PMM) with a
1023 TRIME-PICO64 TDR Technology instrument (IMKO Micromodultechnik GmbH Company, Ettlingen,
1024 DE, Germany)]. After severe drought stress, the plants were rewatered, and survival rates of idr1-1
1025 mutant and IAPAR9 plants were analyzed.
1026 For drought-stressed treatments in concrete tanks (length × width × depth: 3.2 × 2 × 2 m), all tanks
1027 were irrigated every three days until the onset of drought treatment after sowing seeds. Soil moisture
1028 content after full irrigation was approximately 50% (v/v) at a depth of 20 cm as determined by the PMM.
1029 The degrees of drought stresses in this experiment were defined as mild, moderate, and severe drought
1030 stresses when the soil moisture contents decreased to 15%, 10%, and 5%, respectively, as measured by
1031 the PMM. For example, in order to create severe drought stress, water withholding was not stopped until
1032 leaves of the idr1-1 mutant and IAPAR9 plants displayed marked differences in leaf damage, and the
1033 bottom line of soil moisture was about 5% as determined by the PMM. Then the tanks were fully irrigated
1034 and the plants were allowed to recuperate. Survival rates were calculated one week after irrigation.
1035 Non-drought control tanks were kept irrigated every 3 days. Thirty plants were measured in each replicate
1036 and all the experiments had three biological replicates. All of the measurements were completed in one
1037 day.
1038
1039 Map-based cloning of IDR1

1040 To generate mapping populations, idr1-1 mutant females were separately crossed with 10 upland rice
1041 cultivar males, including IAPAR9, HD297, HD385, IRAT109, HD119, etc. The resulting F2 progeny
1042 derived from such crosses were used to test segregation ratios of drought-tolerant to drought-sensitive
1043 individuals. To select drought-tolerant individuals, water was withheld from 4-week-old F2 seedlings for
1044 drought treatment. Finally, a total of 160 F2 individuals with the tolerance to moderate drought stress were
1045 selected for primary mapping using simple sequence repeat (SSR) markers
1046 (http://www.gramene.org/microsat/ssr.html) distributed across all the 12 chromosomes. Fine mapping was
1047 performed using 3800 segregants selected from 20310 F2 plants. The new Indel markers were developed
1048 using RiceVarMap database (http://ricevarmap.ncpgr.cn/). CAPs and dCAPs markers were designed by
1049 comparing sequences between IAPAR9 and HD119 (Yang et al., 2016). To find genomic lesions of the
1050 candidate genes, genomic DNA of the four candidate genes located between markers IM63 and IM59
1051 from idr1-1 mutant and IAPAR9 plant was PCR-amplified, and then the PCR products were sequenced
1052 and analyzed by DNAMAN software.
1053
1054 Generation of transgenic lines

1055 To generate IDR1 complementation and OE lines, a 4020-bp genomic DNA sequence plus 2500-bp
1056 5’-terminal region upstream of ATG codon and one 1173-bp ORF sequence of IDR1 were cloned into the
1057 binary vector pCAMBIA1305 and pCAMBIA1300, respectively; the IDR1 ORF sequence was driven by
1058 a maize ubiquitin promoter. Both the resulting constructs were separately transformed into calli derived
1059 from mature idr1-1 seeds through the Agrobacterium (EHA105)-mediated transformation. Rice
1060 transformation was performed according to the method described previously (Hiei et al., 1994).
1061
1062 Subcellular localization of IDR1

1063 To investigate the subcellular localization of IDR1, the 1170-bp ORF sequence of IDR1 without the
1064 stop codon was cloned into the pCaMV35S:GFP binary vector to generate pCaMV35S:IDR1-GFP
1065 construct. This construct was subsequently co-transformed with a construct carrying marker gene plasma
1066 membrane intrinsic protein 2A (PIP2A-RFP) (for plasma membrane localization) or MADS-box
1067 transcription factor 3-like protein (MADS3L-RFP) (for nucleus localization) into Nicotiana benthamiana
1068 leaf cells by using Agrobacterium-mediated transformation methods. The transformed cells were then
1069 examined under a confocal fluorescence microscope (Zeiss LSM 710, Germany) after 48-72 h of
1070 incubation. The excitation/emission wavelengths were 488 nm/500–550 nm for GFP, and 543 nm/620–
1071 630 nm for mCherry.
1072
1073 Measurements of morphological traits

1074 For observation of stomata, 5-week-old leaves were harvested after severe drought stress treatment in
1075 concrete tanks, and stored in 2.5% glutaraldehyde stationary liquid (pH 7.3). The leaves were then
1076 transferred to a solution containing 1% osmic acid for 1 h before photography. Scanning electron
1077 microscopy (SEM) was used to examine stomata as previously described (Luo et al., 2013). Stomata were
1078 imaged by an Olympus BX53 microscope with IpExp60C software. Stomatal aperture was expressed as
1079 average width of a number of stomata and the apertures were measured by using the free software ImageJ
1080 (National Institutes of Health) (Ling et al., 2017). To measure thickness of IAPAR9 and idr1-1 leaves,
1081 flag leaves of both genotypes were cut into about 1 × 1 mm by hand. The thickness of transverse sections
1082 of IAPAR9 and idr1-1 leaves at the position in the proximity of upper portions of the main veins were
1083 observed and measured with a light microscope with CCD camera. For observation of deposition of wax
1084 crystals, the flag leaves of 30-d-old seedlings were cut into pieces of 1 × 1 cm. After being quickly frozen
1085 with liquid nitrogen, cuticular wax crystals of idr1-1 mutant and IAPAR9 leaves were viewed by scanning
1086 electron microscope (S-3000N, Hitachi). To investigate root traits, we planted one seedling in each PVC
1087 tube, which contained mixed soil [soil/vermiculite/peat, 1:1:1 (v/v/v)]. The soil moisture was about 50%
1088 before drought treatment. When the seedlings reached a V8 developmental stage, water was withheld for
1089 drought stress. When the soil moisture decreased to about 10% (moderate drought stress), where the
1090 IAPAR9 plants began to flower, whole roots of both the genotypes were washed and the clean roots were
1091 used for measuring root traits
1092
1093 Measurements of physiological traits

1094 After moderate drought treatment in the concrete tanks, leaf water potentials of idr1-1 mutant and
1095 IAPAR9 leaves were determined with a Pressure Chamber Instrument (Model 600) (PMS Instrument
1096 Company, Albany, OR, USA), and manipulations of the instrument were performed as described by the
1097 manufacture. Thirty leaves from each genotype were selected randomly, and water potentials of the leaves
1098 were measured at 6, 10, 14, and 18 o’clock, respectively. For measurement of water loss rate, detached
1099 leaves from idr1-1 mutant and IAPAR9 plants as well as the three complementation lines were weighed
1100 immediately and recorded as the initial weights, and then they were incubated at room temperature to lose
1101 water gradually. Water loss was monitored by weighing these leaves at the indicated time points. Proline
1102 contents were measured as previously reported (Verslues, 2010). Briefly, leaf tissues were cut into pieces
1103 and extracted with 3% sulfosalicylic acid, and then the extract was analyzed using a spectrophotometer
1104 (UV762/762PC) at 520 nm. For each genotype, 30 plants were used for measuring the leaf proline
1105 contents in each of the three replicates. MDA was extracted as previously described (Meir et al., 1992).
1106 Briefly, 0.5 grams of leaf tissues were ground into homogenate in 5 mL solution that contained 20%
1107 trichloroacetic acid (TCA) and 1.5 mM EDTA. MDA was determined using the thiobarbituric acid (TBA)
1108 test as described in the reference. Chlorophyll contents were measured by the SPAD method (Wu et al.,
1109 2015). Briefly, total chlorophyll in the leaves was extracted with 80% acetone. The extract was analyzed
1110 using a spectrophotometer (UV762/762PC) at 470 nm. Besides, leaf electrolyte leakage was measured as
1111 described previously (Zhu and Xiong, 2013). For measurement of POD activities, briefly, 0.2 grams of
1112 leaves were ground to homogenate by adding 2 mL of PBS solution, and then the homogenate was
1113 centrifuged at 12,000 g for 20 min at 4℃. The supernatant was used for extracting POD enzyme.
1114 Peroxidase activity of POD was determined according to the method described by Kwak et al. with minor
1115 modifications (Kwak et al., 1995). The reaction mixture (in a total volume of about 200 mL) contained
1116 0.2 M PBS (200 mL, pH 6.0), 30% (v/v) H2O2 (0.112 mL), and 0.076 mL guaiacol as substrates. Thirty
1117 microliter of enzyme extract was mixed with 3 mL of reaction mixture for each reaction. The oxidation of
1118 guaiacol was monitored and the absorbance was measured at 470 nm every 30 s.
1119
1120 Measurements of responsiveness of idr1-1 mutants to various experimental treatments

1121 For glucose treatment, idr1-1 mutant and IAPAR9 seeds were placed in petri dishes with different
1122 concentrations of glucose and cultured in greenhouse for 5 days. Seeds were considered to be germinated
1123 when the radicles completely emerged from husks and then photographs were taken (Chen et al., 2006).
1124 For salt treatment, 2-week-old idr1-1 mutant and the IAPAR9 seedlings were treated with 150 mM NaCl
1125 until the salt-sensitive phenotype appeared. For MV treatment, roots of 2-week-old idr1-1 mutant and
1126 IAPAR9 seedlings were submerged into 30 μM MV solution for 3 days. Detached leaves from both the
1127 genotypes were stained with DAB and photographed. For examination of responsiveness to EBR, 5-d-old
1128 idr1-1 mutant and IAPAR9 seedlings grown in Hoagland solution were treated with different
1129 concentrations of EBR for 3 days in the dark. Photographs of the second lamina joint were taken and the
1130 angles of leaf lamina joint bending were measured and plotted against different EBR concentrations. For
1131 root elongation inhibition assay, 5-d-old idr1-1 mutant and IAPAR9 seedlings were treated with EBR of
1132 different concentrations for 3 days. Lengths of the primary roots were measured and plotted against
1133 different EBR concentrations (Wang et al., 2006). For test of darkness-induced leaf senescence, detached
1134 leaves of idr1-1 mutant and IAPAR9 plants were incubated in the dark for 3 days. DAB and NBT staining
1135 of the leaves were performed and such leaves were then photographed.
1136
1137 RNA extraction and gene expression analyses

1138 Generally, 150 mg of tissues from plant materials in each experiment were used for total RNA isolation
1139 by TRIpure Reagent (Aidlab, Beijing, China). Then the first-strand cDNA synthesis was primed with an
1140 oligo (dT) primer by using a PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time)
1141 (Takara, Dalian, China) according to the manufacturer’s instructions. The resulting cDNA was then used
1142 for quantitative real-time PCR (qRT-PCR) by using a Hieff qPCR SYBR Green Master Mix (Low Rox
1143 Plus) (YEASEN, Shanghai, China) in an Applied Biosystems Q5 Real-time PCR System with primers
1144 corresponding to each detected gene. The results were normalized using Ubiquitin5 as an internal control.
1145 RT-PCR (qRT-PCR) amplification was carried out as follows: 95℃ for 5 min, followed by 18-40
1146 amplification cycles [95℃ for 30 (5) s, 55 (60)℃ for 30 s, and 72℃ for 30 (0) s]. The primers were listed
1147 in Supplemental Table S5. To analyze expression levels of IDR1 under different abiotic stresses,
1148 2-week-old IAPAR9 seedlings grown in 1/2 MS solution were subjected to different treatments with 20%
1149 (w/v) PEG 6000, NaCl (150 mM), cold (4℃), heat (40℃) or ABA (50 μM), respectively. Leaves were
1150 harvested at 0, 1, 4, 8 hours after treatment (Xiong et al., 2018). To examine expression levels of IDR1 in
1151 different tissues and of OsBRD1 and OsBRD2 as well as TUD1, total RNA was isolated from 4-week-old
1152 idr1-1 mutant and IAPAR9 seedlings, and then subjected to qRT-PCR assays. To check expression levels
1153 of ROS-scavenging and OsRBOH genes, total RNA was isolated from leaves of both idr1-1 mutant and
1154 IAPAR9 plants undergoing moderate, severe or no stress, and used for qRT-PCR assays (Ning et al., 2010;
1155 Lu et al., 2020).
1156
1157 Detection of ROS accumulation and cell death

1158 For measurement of H2O2 accumulation in leaves, rice leaves were treated with DAB staining solution
1159 (1 mg/mL, pH 3.8) for 12 h in the dark. After 2-3 rinses, the leaves were decolorized with 100%, 90%,
1160 80%, and 70% (v/v) alcohol each for 10 min at 100℃ to gradually eliminate chlorophyll thoroughly. At
1161 least 5 leaves from different plants were analyzed for each experiment, and the representative images
1162 were shown (Xiong et al., 2018). O2- was detected by NBT staining. Briefly, rice leaves were treated with
1163 NBT staining solution (1 mg/mL) for 12 hours in the dark and then they were decolorized. Trypan blue
1164 staining was performed following a protocol reported previously (Xiong et al., 2018). Leaves were treated
1165 with 0.1% Trypan blue solution for 12 h at room temperature in the dark followed by decolorization.
1166 CeCl3 was used for localizing and visualizing H2O2 at the subcellular level by transmission electron
1167 microscopy (TEM) as described previously (Zhang et al., 2010). Briefly, leaf tissues undergoing drought
1168 stress were excised to pieces (1-2 mm2) and incubated in 5 mM CeCl3 for 1 h at 4℃. Then the leaf
1169 sections were subjected to a series of manipulations, i.e. fixation, wash, postfixation, dehydration and
1170 embedment. The resulting blocks were then sectioned (70-90 nm) on a Reichert-Ultracut E microtome
1171 (Reichert-Jung, Leica, Bensheim, Germany) and mounted on uncoated copper grids (300 mesh). Sections
1172 were examined by using a TEM at an accelerating voltage of 75 kV (Zhang et al., 2010).
1173
1174 mRNA-Seq analyses

1175 Leaves of idr1-1 mutant and IAPAR9 plants were harvested and used for mRNA-seq after moderate
1176 drought treatment. To prepare samples for mRNA-seq, 5-week-old idr1-1 mutant and IAPAR9 plants
1177 were grown in concrete tanks for nonstressed or drought-stressed treatment. Samples were collected when
1178 soil moisture content was decreased to 10%. Total RNA was extracted from seedling using RNAiso Plus
1179 (Takara, D9108B) for mRNA-seq. mRNA-seq was carried out according to procedures provided by the
1180 sequencing company (BGI, Beijing, China). The trimmed high-quality clean data were aligned to the
1181 entire Col-0 reference genome (TAIR10) by using Hisat2 software (Kim et al., 2019), whereby
1182 parameters were set as default parameters. Subsequently, the reads were counted by using softwares
1183 FeatureCounts and Subread (Liao et al., 2013). DESeq2 was then used to analyze the gene expression
1184 levels of differentially expressed genes; meanwhile, the critical thresholds for screening for differentially
1185 expressed genes were P-value < 0.05 and the log2 fold change > 2 (Love et al., 2014).
1186
1187 Yeast two-hybrid and LCI assays

1188 Protein-protein interaction assays were performed as described previously (Wang et al., 2018). Briefly,
1189 for yeast two-hybrid assay, the entire coding sequences of IDR1 and TUD1 were in-frame cloned into
1190 pGADT7 (AD) or pGBKT7 (BD) vectors to generate fusion constructs, respectively. Then the
1191 pGADT7:IDR1 and pGBKT7:TUD1 constructs were co-transformed into yeast strain AH109. Later on,
1192 the co-transformed yeast cells were first spotted onto a synthetic dropout medium without Leu and Trp
1193 (SD/-Leu/-Trp) to grow for 3 days and then transferred to a stringent selection medium [synthetic dropout
1194 medium lacking Trp, Leu, His and adenine (SD/-Trp/-Leu/-His/-Ade) to grow for an additional 2 days at
1195 28℃ prior to observation. For LCI assays, the coding sequences of IDR1 and TUD1 were cloned into
1196 pCAMBIA1300-nLUC/cLUC vectors. LCI assays were performed by transiently co-expressing both
1197 constructs in tobacco leaves through Agrobacterium-mediated infiltration. Three days after
1198 agroinfiltration, luciferase activity was detected with a CCD camera (Tanon, Shanghai, China).
1199
1200 Accession number

1201 All the mRNA-seq data used in this article have been deposited in GenBank as accession number
1202 SRP273943.
1203
1204 Acknowledgments
1205 Data analyses were supported by the high-performance computing platform of Bioinformatics Center,
1206 Nanjing Agricultural University.
1207
1208 Supplemental materials

1209 Supplemental Figure S1. idr1-1 mutants display noticeably reduced elongation in major agronomic
1210 traits.
1211 Supplemental Figure S2. idr1-1 mutants show enhanced sensitivity to glucose and NaCl treatments.
1212 Supplemental Figure S3. Tests of drought tolerance of idr1-1 mutant and other relevant lines.
1213 Supplemental Figure S4. Tests of drought tolerance of IDR1 complementation lines.
1214 Supplemental Figure S5. Performance of panicles and seed setting rates of idr1-1 mutant under severe
1215 drought stress conditions.
1216 Supplemental Figure S6. Accumulation of cuticular wax crystals on flag leaves.
1217 Supplemental Figure S7. Performance of root traits under drought-stressed conditions.
1218 Supplemental Figure S8. Darkness-induced senescent performance in idr1-1 mutant leaves.
1219 Supplemental Figure S9. Transcript levels of a few OsRBOH genes in idr1-1 mutant.
1220 Supplemental Figure S10. Expression changes of a dozen genes involved in PET chain.
1221 Supplemental Figure S11. Induction of expression of two marker genes, OsRAB16C and OsRAB16A.
1222 Supplemental Figure S12. Materials of idr1-1 mutant and IAPAR9 plants for transcriptome profiling.
1223 Supplemental Figure S13. Analyses of the genes regulated by IDR1.
1224 Supplemental Table S1. Phenotype segregation analyses of the F2 progeny.
1225 Supplemental Table S2. Information of the genes detected by (q)RT-PCR assays.
1226 Supplemental Table S3. A list of genes affected by both idr1-1 mutation and drought stress.
1227 Supplemental Table S4. Seven classes of genes exhibited significantly differential expression.
1228 Supplemental Table S5. Primer sequences used in this study.
1229 Supplemental Data Set S1: Differentially expressed genes between nonstressed idr1-1 mutant and
1230 IAPAR9 plants.
1231 Supplemental Data Set S2: Differentially expressed genes between drought-stressed idr1-1 mutant and
1232 IAPAR9 plants.
1233 Figure legends

1234 Figure 1. idr1-1 mutation confers obviously enhanced drought tolerance to upland rice plants of cv.
1235 IAPAR9 under different growth conditions.
1236 (A and B) Drought tolerance tests of idr1-1 mutant and IAPAR9 seedlings in pots. Both the idr1-1
1237 mutant and IAPAR9 seedlings at a V6 (when the sixth leaf has a visible “collar” at the base of the leaf)
1238 developmental stage were subjected to drought stress for approximately 2-3 weeks (severe drought),
1239 followed by rewatering for 7 days to examine their drought tolerance and calculate survival rates (A).
1240 Survival rates of idr1-1 mutant and IAPAR9 seedlings as described in A were calculated from rewatered
1241 plants (B). Scale bar = 5 cm. (C and D) Drought tolerance tests of idr1-1 mutant and IAPAR9 seedlings
1242 in concrete tanks. Both idr1-1 mutant and IAPAR9 seedlings at the V6 developmental stage grown in
1243 concrete tanks with a rain-off shelter were subjected to moderate (withholding water for approximately 2
1244 weeks) or severe drought stress (withhold water for approximately 3 weeks) to examine their drought
1245 tolerance (C). Survival rates of idr1-1 mutant and IAPAR9 plants were calculated from rewatered plants
1246 from the experiment depicted in C (D). The pictures were taken after drought treatment. Data are means
1247 (±SD) for three biological replicates. Significant differences from IAPAR9 were determined by Student’s
1248 t-test: **P < 0.01, t-test.
1249
1250 Figure 2. Map-based cloning of IDR1 and complementation of idr1-1 mutant by IDR1 transgenes.
1251 (A) Mapping of IDR1 locus on the chromosome 5 by using molecular markers. IDR1 locus was primarily
1252 mapped to the interval between molecular markers RM18402 and RM18421 on the chromosome 5, and it
1253 was further delimited to a ~30.7 kb genomic region between markers IM63 and IM59. Marker names and
1254 the numbers of recombinants were shown above and below each line, respectively. Four candidate genes
1255 were indicated by arrows, among which the red arrow represented the IDR1 gene (LOC_05g26890). The
1256 two genes without locus names above them were predicted by RAP-DB database but not by MSU RGAP
1257 database. A single-base deletion occurring on the fifth exon of IDR1 was shown, whereby a premature
1258 stop codon (TGA) was created on the ORF sequence of such a gene. (B) A close-up view of the
1259 single-base deletion at the position 886 downstream of the ATG start codon on the IDR1 genomic DNA
1260 (left panel). The newly created premature stop codon TGA on the IDR1 ORF sequence was underlined.
1261 The full-length and truncated proteins corresponding to the entire ORF sequence and the mutated ORF
1262 sequence of IDR1 were shown in right panel. Black bar represents a portion of protein with a changed
1263 sequence due to the frameshift resulting from the single-base deletion. The numbers above the diagrams
1264 indicate the coordinates relative to the start codon ATG (left panel) or start amino acid 1 (right panel). (C
1265 and D) Reversion of plant heights (C) and grain lengths (D) of idr1-1 mutants to wild-type sizes by
1266 introduction of IDR1 genomic DNA or OE constructs to the mutants. Com1 to Com3, IDR1
1267 complementation lines; OE2, OE3, and OE5, IDR1 cDNA-overexpressing lines; EV, empty vector. Scale
1268 bars = 50 cm (C) and 1 cm (D). (E) Comparisons of drought tolerance to severe drought stress among
1269 seedlings of the idr1-1 mutants, IAPAR9 plants, and IDR1 complementation lines grown in pots. Control,
1270 no drought stress; drought, severe drought stress. Scale bar = 10 cm.
1271
1272 Figure 3. Expression patterns and subcellular localization of IDR1.

1273 (A-F) Expression patterns of IDR1 after treatment with drought, PEG, NaCl, 4℃, 40℃ or ABA. Leaves
1274 were sampled from 2-week-old IAPAR9 seedlings treated with moderate or severe drought stress (A), 20%
1275 PEG (B), 150 mM NaCl (C), 4℃ (D), 40℃ (E) or 50 μM ABA (F) at the indicated time points (0, 1, 4, 8
1276 h) following the treatments, and then total RNA was isolated and subjected to qRT-PCR assays. Control,
1277 no drought stress. (G) Tissue-specific expression patterns of IDR1 at seedling stage. Total
1278 RNA extracted from roots, stems and leaves of 4-week-old seedlings was used for qRT-PCR assays. (H)
1279 Subcellular localization of IDR1. IDR1 cDNA was fused to GFP, and the resulting IDR1-GFP construct
1280 was co-transfected with PIP2A-RFP (plasma membrane localization marker) (upper panels) or
1281 MADS3L-RFP (nuclear localization marker) (lower panels) into 30-d-old N. benthamiana leaves for
1282 fluorescence observation. PIP2A, plasma membrane intrinsic protein 2A; MADS3L, MADS-box
1283 transcription factor 3-like protein. Scale bar = 20 μm. Data are means (±SD) for three biological
1284 replicates. Significant differences from control (A), 0 h (B-F) or root (G) were determined by Student’s
1285 t-test: *P < 0.05, **P < 0.01, t-test.
1286
1287 Figure 4. Physiological changes of idr1-1 mutant help retain more water in its leaves.
1288 (A) Measurements of leaf water potentials of idr1-1 mutants, IAPAR9 plants, and IDR1 complementation
1289 lines grown under control or moderate drought stress conditions. Leaf samples for measuring water
1290 potentials were collected at the 4 time points as indicated. Control, no drought stress. (B) Measurements
1291 of water loss rates of detached leaves from idr1-1 mutants, IAPAR9 plants, and IDR1 complementation
1292 lines at the indicated time points. Water loss was expressed as the percentage of decreases in fresh
1293 weights (FW) of the detached leaves at the different time points compared to their own at 0 h. Data are
1294 means (±SD) for three biological replicates; for each genotype, 6 leaves were used for measuring leaf
1295 water potential (A) or leaf FW (B) in each of the three replicates. (C-F) Measurements of proline contents
1296 (C), MDA contents (D), REL (E) and relative chlorophyll contents (F) in leaves of idr1-1 mutants,
1297 IAPAR9 plants and IDR1 complementation lines under control or moderate drought stress conditions.
1298 Data are means (±SD) for three biological replicates. Significant differences from IAPAR9 in each group
1299 (control or drought) of every bar chart were determined by Student’s t-test: *P < 0.05, **P < 0.01, t-test.
1300 Control, no drought stress.
1301
1302 Figure 5. Morphological changes contribute to enhanced drought tolerance in idr1-1 mutant.
1303 (A) Representative images for stomatal apertures of leaves of idr1-1 mutants, IAPAR9 plants and IDR1
1304 complementation lines under control or drought conditions. The photographs were taken with a scanning
1305 electron microscope. Scale bar = 10 μm. Control, no drought stress. (B) Statistics of stomatal apertures
1306 (upper panel) and stomatal numbers (lower panel) of idr1-1 mutants, IAPAR9 plants and IDR1
1307 complementation lines, which were derived from more stomatal images taken simultaneously with those
1308 in A. Data are means (±SD) for three biological replicates; for each genotype, 6 stomata were used for
1309 measuring stomatal apertures in each of the three replicates. (C and D) Average thickness of transverse
1310 sections of idr1-1 mutant and IAPAR9 leaves. The thickness of idr1-1 mutant and IAPAR9 flag leaves at
1311 the positions in the proximity of upper portions of the main vein were measured (C) and shown (D).
1312 Scale Bar = 50 μm. (E-G) Effects of idr1-1 mutation on root traits. Plants were grown in PVC tubes for 1
1313 month, and then they were treated with or without (control) moderate drought stress for another 2-3
1314 weeks by withholding water. The whole roots were isolated from soils by washing them with running
1315 water, and then imaged (E). Root lengths and root volumes of idr1-1 mutant and IAPAR9 plants were
1316 measured and the means of them were shown (F and G). Data are means (±SD) for three biological
1317 replicates; for each genotype, 5 leaves (D) or 3 plants (F and G) were used for measuring leaf thickness or
1318 root lengths as well as root volumes, respectively, in each of the three replicates. Significant differences
1319 from IAPAR9 were determined by Student’s t-test: **P < 0.01, t-test. Scale Bar = 20 cm.
1320
1321 Figure 6. idr1-1 mutation attenuates apoplastic and chloroplastic ROS production triggered by
1322 drought stress and MV.
1323 (A) DAB, NBT and Trypan blue staining of detached leaves from idr1-1 mutant and IAPAR9 plants.
1324 Five-week-old idr1-1 mutant and IAPAR9 seedlings underwent drought stress for about 2 weeks
1325 (moderate stress) and the detached leaves of IAPAR9, idr1-1, Com1, Com2, Com3, OE2, OE3 and OE5
1326 (1-8, respectively) were used for such staining. Scale Bar = 10 cm. (B) Cytochemical localization of
1327 drought-induced H2O2 in intercellular space between mesophyll cells of idr1-1 mutant or IAPAR9 leaves.
1328 Leaf samples for sectioning were from the experiment depicted in A. The H2O2 was stained with CeCl3
1329 and the photographs were taken with transmission electron microscopy (TEM). Red arrows indicate
1330 CeCl3 precipitates. Scale Bar = 1 μm. (C-E) Performance of idr1-1 mutant and IAPAR9 seedlings after
1331 MV treatments. Two-week-old idr1-1 mutant and IAPAR9 seedlings cultured in liquid Hoagland medium
1332 were treated with 30 μM MV for 3 days (C). DAB staining of leaves from idr1-1 mutant and IAPAR9
1333 seedlings following MV treatments were performed to detect amounts of H2O2 (D). In parallel, total
1334 chlorophyll contents of the idr1-1 mutant and IAPAR9 leaves from the experiment shown in C were
1335 measured and shown (E). Data are means (±SD) for three biological replicates; for each genotype, 30
1336 plants were used for measuring leaf chlorophyll contents in each of the three replicates. Significant
1337 differences from IAPAR9 were determined by Student’s t-test: **P < 0.01, t-test. Scale Bars = 10 cm (C
1338 and D).
1339
1340 Figure 7. idr1-1 mutation increases expression of a group of ROS-scavenging genes under
1341 drought-stressed conditions.
1342 (A) Relative expression levels of three genes that were reportedly associated with activation of
1343 ROS-scavenging system. (B) Relative expression levels of a dozen genes involved in ROS scavenging
1344 under control or drought stress (moderate or severe) conditions. For qRT-PCR analyses in A and B, leaves
1345 from 4-week-old seedlings of idr1-1 and IAPAR9 experiencing nonstressed (control) and
1346 drought-stressed treatments, were used for RNA isolation and qRT-PCR assays to examine expression of
1347 the genes as indicated. Rice ubiquitin 5 gene was amplified and used as an internal control. (C) POD
1348 activity assays in idr1-1 mutant and IAPAR9 leaves under control or moderate drought stress conditions.
1349 Leaf samples for POD activity assays were from the experiment described in B. Data are means (±SD)
1350 for three biological replicates. Significant differences from IAPAR9 were determined by Student’s t-test:
1351 *P < 0.05, **P < 0.01, t-test.
1352
1353 Figure 8. IDR1 physically interacts with TUD1, and idr1-1 mutant exhibits impaired BR
1354 responsiveness.
1355 (A and B) Physical interactions between IDR1 and TUD1 in yeast cells (A) and in N. benthamiana leaves
1356 (B). Yeast cells (strain AH109), which was co-transfected with BD-TUD1 plus AD-IDR1 and other
1357 combinations of constructs as indicated, were grown on YPDA medium lacking Trp and Leu for 3 d (left
1358 panel in A) and then transferred to a stringent selection medium lacking Trp, Leu, His and adenine
1359 (SD/-Trp/-Leu/-His/-Ade) (right panel in A), and allowed them to grow for 2 days at 28℃ prior to
1360 observation. In parallel, luciferase complementation image (LCI) system was used to test interactions
1361 between IDR1 and TUD1 (B). Upper panels, bright field; lower panel, dark field. 1, AtSGT1a-nLUC +
1362 cLUC; 2, AtRAR1-nLUC + AtSTG1a-cLUC; 3, nLUC + AtRAR1-cLUC; 4 and 8, nLUC + cLUC; 5,
1363 IDR1-nLUC + cLUC; 6, IDR1-nLUC + TUD1-cLUC; 7, nLUC + TUD1-cLUC. The number “2” in B
1364 indicates a positive control, and the number “4” and “8” indicate negative controls. (C and D) Effects of
1365 different concentrations of EBR on the degrees of inclination of the leaf lamina. Five-d-old idr1-1 mutant
1366 and IAPAR9 seedlings grown in Hoagland solution were treated by different concentrations of EBR (0,
-9 -8 -7 -6 -5
1367 10 , 10 , 10 , 10 and 10 M) for 3 days. Photographs of the second lamina joints were taken (C) and
1368 angles of the leaf lamina joint bending were measured and plotted against varying EBR concentrations
1369 (D). Data are means (±SD) for three biological replicates; for each genotype, 3 seedlings for control (0 M
1370 EBR) or each EBR concentration were used for measuring the angles of leaf lamina joint bending in each
1371 of the three replicates. Scale Bar = 5 cm. (E and F) Responses of primary roots of idr1-1 mutant and
1372 IAPAR9 seedlings to different concentrations of EBR. Seeds of both the genotypes were germinated in
1373 Hoagland solution for 5 days, and the selected seedlings with uniform lengths of primary roots were
-8 -7 -6
1374 treated with different concentrations of EBR (0, 10 , 10 and 10 M) for 3 days. Photographs of the
1375 primary roots following treatment were taken (E), and lengths of primary roots were plotted against
1376 varying EBR concentrations (F). Data are means (±SD) for three biological replicates; for each genotype,
1377 5 seedlings for control (0 M EBR) or each EBR concentration were used for measuring root lengths in
1378 each of the three replicates. Scale Bar = 5 cm. (G) Comparisons of plant heights between Nipponbare
1379 (NIP) and tud1-5. Seedlings of the NIP and tud1-5 were grown in pots for 40 days prior to photography.
1380 Scale Bar = 5 cm. (H and I) Comparisons of grain lengths between idr1-1 mutant and IAPAR9 plants and
1381 between tud1-5 mutant and NIP plants. Mature seeds of IAPAR9, idr1-1, NIP and tud1-5 were harvested
1382 simultaneously and photographed (H). Grain lengths were measured and compared (I). Scale Bar = 1 cm.
1383 (J) Relative expression levels of TUD1 in IAPAR9 leaves that underwent moderate, severe or no (control)
1384 drought stress as revealed by qRT-PCR assays. (K and L) Expression levels of OSBRD1, OSBRD2 and
1385 TUD1 in idr1-1 mutant as shown by RT-PCR assays (K). Meanwhile, OSBRD1 transcript abundance from
1386 the transcriptome data was shown as mean log2 fold change ± SE (n = 3) of three independent
1387 experiments (L); values were plotted relative to the log2 of normalized IAPAR9 transcript abundance
1388 which was set at 1.0. RNA samples prepared from idr1-1 mutant and IAPAR9 leaves without stress
1389 treatment (K) or leaves of both the genotypes undergoing moderate drought stress (L) were used for such
1390 assays. Data are means (±SD) for three biological replicates. Significant differences from IAPAR9 (I, L)
1391 or control (J) were determined by Student’s t-test: **P < 0.01, t-test.
1392
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1394
A No drought After drought Rewatering
B
IAPAR9 idr1-1 IAPAR9 idr1-1 IAPAR9 idr1-1
120
IAPAR9 idr1-1
100
**
Survival rate (%)

80
60
40
20
0
Before After
C No drought Moderate drought Severe drought
drought drought
IAPAR9 idr1-1 IAPAR9 idr1-1 IAPAR9 idr1-1 D
120
IAPAR9 idr1-1
**
100
Survival rate (%)

80
60
40
20
0
Before After
drought drought
Figure 1. idr1-1 mutation confers obviously enhanced drought tolerance to upland rice plants of cv. IAPAR9
under different growth conditions. (A and B) Drought tolerance tests of idr1-1 mutant and IAPAR9 seedlings in
pots. Both the idr1-1 mutant and IAPAR9 seedlings at a V6 (when the sixth leaf has a visible “collar” at the base
of the leaf) developmental stage were subjected to drought stress for approximately 2-3 weeks (severe drought),
followed by rewatering for 7 days to examine their drought tolerance and calculate survival rates (A). Survival
rates of idr1-1 mutant and IAPAR9 seedlings as described in A were calculated from rewatered plants (B). Scale
bar = 5 cm. (C and D) Drought tolerance tests of idr1-1 mutant and IAPAR9 seedlings in concrete tanks. Both
idr1-1 mutant and IAPAR9 seedlings at the V6 developmental stage grown in concrete tanks with a rain-off
shelter were subjected to moderate (withholding water for approximately 2 weeks) or severe drought stress
(withhold water for approximately 3 weeks) to examine their drought tolerance (C). Survival rates of idr1-1
mutant and IAPAR9 plants were calculated from rewatered plants from the experiment depicted in C (D). The
pictures were taken after drought treatment. Data are means (±SD) for three biological replicates. Significant
differences from IAPAR9 were determined by Student’s t-test: **P < 0.01, t-test.
A RM18383 RM18402 RM18421 RM3437
Marker 150kb
Chr. 5 short Chr. 5 long
IM9 C32 IM15 IM42 IM23 IM39 IM43 C21 IM6
Marker 70kb
R= 34 29 25 4 11 19
52 45 20 21 27
C52 C73 IM81 IM63 IM59 C77 C59

Marker 15kb
R= 10 7 4 2 2 2 3
LOC_Os05g26890 LOC_Os05g06902 4kb
1 bp deletion TGA
5’ 3’
B 886 1 391
IAPAR9: GATTCCTCA……CTGA…
1 113 142
idr1-1: GAT CCTCA……CTGA…
C D
IAPAR9
idr1-1
Com1
Com2
Com3
OE2
OE3
OE5
E
Control
Drought
IAPAR9 IAPAR9 IAPAR9
idr1-1 Com1 idr1-1 Com2 idr1-1 Com3

Figure 2. Map-based cloning of IDR1 and complementation of idr1-1 mutant by IDR1 transgenes.
(A) Mapping of IDR1 locus on the chromosome 5 by using molecular markers. IDR1 locus was primarily mapped to
the interval between molecular markers RM18402 and RM18421 on the chromosome 5, and it was further delimited
to a ~30.7 kb genomic region between markers IM63 and IM59. Marker names and the numbers of recombinants
were shown above and below each line, respectively. Four candidate genes were indicated by arrows, among which
the red arrow represented the IDR1 gene (LOC_05g26890). The two genes without locus names above them were
predicted by RAP-DB database but not by MSU RGAP database. A single-base deletion occurring on the fifth exon
of IDR1 was shown, whereby a premature stop codon (TGA) was created on the ORF sequence of such a gene. (B)
A close-up view of the single-base deletion at the position 886 downstream of the ATG start codon on the IDR1
genomic DNA (left panel). The newly created premature stop codon TGA on the IDR1 ORF sequence was
underlined. The full-length and truncated proteins corresponding to the entire ORF sequence and the mutated ORF
sequence of IDR1 were shown in right panel. Black bar represents a portion of protein with a changed sequence due
to the frameshift resulting from the single-base deletion. The numbers above the diagrams indicate the coordinates
relative to the start codon ATG (left panel) or start amino acid 1 (right panel). (C and D) Reversion of plant heights
(C) and grain lengths (D) of idr1-1 mutants to wild-type sizes by introduction of IDR1 genomic DNA or OE
constructs to the mutants. Com1 to Com3, IDR1 complementation lines; OE2, OE3, and OE5, IDR1 cDNA-
overexpressing lines; EV, empty vector. Scale bars = 50 cm (C) and 1 cm (D). (E) Comparisons of drought
tolerance to severe drought stress among seedlings of the idr1-1 mutants, IAPAR9 plants, and IDR1
complementation lines grown in pots. Control, no drought stress; drought, severe drought stress. Scale bar = 10 cm.
A Drought B PEG
C
1.2 NaCl
1.2 1.8 **
Relative expression level

1.6

1
1
1.4
0.8 * 0.8 1.2
1
0.6 0.6
* * 0.8
*
0.4 0.4 0.6
0.4
0.2 0.2
0.2
0 0 0
0h 1h 4h 8h 0h 1h 4h 8h
D 4℃ E 40℃ F ABA
1.4 1.8 1.2
*
1.2 1.6

1
1.4
1 *
1.2 0.8
0.8 1
0.6
0.6 0.8
0.6 0.4
0.4
0.4
0.2 0.2
0.2
0 0 0
0h 1h 4h 8h 0h 1h 4h 8h 0h 1h 4h 8h
G
3.5
** H
GFP field Bright field
3
2.5 ** IDR1-GFP +
2 PIP2A-RFP
1.5
1
IDR1-GFP +
0.5 MADS3L-RFP
0
Root Stem Leaf
Figure 3. Expression patterns and subcellular localization of IDR1. (A-F) Expression patterns of IDR1
after treatment with drought, PEG, NaCl, 4℃, 40℃ or ABA. Leaves were sampled from 2-week-old IAPAR9
seedlings treated with moderate or severe drought stress (A), 20% PEG (B), 150 mM NaCl (C), 4℃ (D), 40℃ (E)
or 50 μM ABA (F) at the indicated time points (0, 1, 4, 8 h) following the treatments, and then total RNA
was isolated and subjected to qRT-PCR assays. Control, no drought stress. (G) Tissue-specific expression patterns
of IDR1 at seedling stage. Total RNA extracted from roots, stems and leaves of 4-week-old seedlings was used for
qRT-PCR assays. (H) Subcellular localization of IDR1. IDR1 cDNA was fused to GFP, and the resulting IDR1-
GFP construct was co-transfected with PIP2A-RFP (plasma membrane localization marker) (upper panels) or
MADS3L-RFP (nuclear localization marker) (lower panels) into 30-d-old N. benthamiana leaves for fluorescence
observation. PIP2A, plasma membrane intrinsic protein 2A; MADS3L, MADS-box transcription factor 3-like
protein. Scale bar = 20 μm. Data are means (±SD) for three biological replicates. Significant differences from
control (A), 0 h (B-F) or root (G) were determined by Student’s t-test: *P < 0.05, **P < 0.01, t-test.
A Control Drought B
6:00 10:00 14:00 18:00 6:00 10:00 14:00 18:00
0 0
Water potential (MPa)
100 IAPAR9
-2 idr1-1
Water loss rate (%)

-5 80 Com1
-4
Com2
-6 -10 60 Com3
-8
-10 -15 40
-12 20
-20
-14
-16 -25 0
0h 1h 2h 3h 4h 5h 6h 7h 8 h 10 h 12 h
IAPAR9 idr1-1 Com1
Com2 Com3
C D E F IAPAR9
Chlorophyll content (SPAD value)

Relative electrolyte leakage (%)
50 IAPAR9 ** 1.4 IAPAR9 * * 100 IAPAR9 60 idr1-1
MDA content (μmol/g FW)
idr1-1 idr1-1 * Com1

Proline content (μg/g FW)
1.2 * idr1-1 Com2

40 Com1 Com1 50
80 Com1 Com3
Com2 1 Com2
Com3
Com2 40 *
30 Com3 Com3 *
0.8 60
* * 30
20 0.6
40
20
0.4
10 20 10
0.2
0 0 0 0
Control Drought Control Drought Control Drought Control Drought
Figure 4. Physiological changes of idr1-1 mutant help retain more water in its leaves. (A)
Measurements of leaf water potentials of idr1-1 mutants, IAPAR9 plants, and IDR1 complementation lines grown
under control or moderate drought stress conditions. Leaf samples for measuring water potentials were collected at
the 4 time points as indicated. Control, no drought stress. (B) Measurements of water loss rates of detached leaves
from idr1-1 mutants, IAPAR9 plants, and IDR1 complementation lines at the indicated time points. Water loss was
expressed as the percentage of decreases in fresh weights (FW) of the detached leaves at the different time points
compared to their own at 0 h. Data are means (±SD) for three biological replicates; for each genotype, 6
leaves were used for measuring leaf water potential (A) or leaf FW (B) in each of the three replicates. (C-F)
Measurements of proline contents (C), MDA contents (D), REL (E) and relative chlorophyll contents (F) in leaves
of idr1-1 mutants, IAPAR9 plants and IDR1 complementation lines under control or moderate drought stress
conditions. Data are means (±SD) for three biological replicates. Significant differences from IAPAR9 in each
group (control or drought) of every bar chart were determined by Student’s t-test: *P < 0.05, **P < 0.01, t-test.
Control, no drought stress.
A B
IAPAR9 idr1-1 Com1 Com2 Com3 EV 0.12 IAPAR9
idr1-1
Stomatal aperture
0.1 Com1
Control
Com2
0.08 Com3
*
0.06
0.04
Moderate drought
0.02
0
600
Stomatal number (/mm2)

500
400
Severe drought
300
200
100
0
Control Moderate
drought
C IAPAR9 idr1-1 D 70 ** E
Average leaf thickness (μM)

60
50
40
30
20
10
0
F G IAPAR9 idr1-1
160 IAPAR9 450 IAPAR9
Average root length (cm)
idr1-1
Average root volume (cm3)
140 400 idr1-1

350 Control Drought
120
300
100
250
80
200
60
150
40 100
20 50
0 0
Control Drought Control Drought
Figure 5. Morphological changes contribute to enhanced drought tolerance in idr1-1 mutant. (A)
Representative images for stomatal apertures of leaves of idr1-1 mutants, IAPAR9 plants and IDR1
complementation lines under control or drought conditions. The photographs were taken with a scanning electron
microscope. Scale bar = 10 μm. Control, no drought stress. (B) Statistics of stomatal apertures (upper panel) and
stomatal numbers (lower panel) of idr1-1 mutants, IAPAR9 plants and IDR1 complementation lines, which were
derived from more stomatal images taken simultaneously with those in A. Data are means (±SD) for three
biological replicates; for each genotype, 6 stomata were used for measuring stomatal apertures in each of the three
replicates. (C and D) Average thickness of transverse sections of idr1-1 mutant and IAPAR9 leaves. The thickness
of idr1-1 mutant and IAPAR9 flag leaves at the positions in the proximity of upper portions of the main vein were
measured (C) and shown (D). Scale Bar = 50 μm. (E-G) Effects of idr1-1 mutation on root traits. Plants were grown
in PVC tubes for 1 month, and then they were treated with or without (control) moderate drought stress for another
2-3 weeks by withholding water. The whole roots were isolated from soils by washing them with running water, and
then imaged (E). Root lengths and root volumes of idr1-1 mutant and IAPAR9 plants were measured and the means
of them were shown (F and G). Data are means (±SD) for three biological replicates; for each genotype, 5 leaves
(D) or 3 plants (F and G) were used for measuring leaf thickness or root lengths as well as root volumes,
respectively, in each of the three replicates. Significant differences from IAPAR9 were determined by Student’s t-
test: **P < 0.01, t-test. Scale Bar = 20 cm.
DAB NBT Trypan Blue
A B
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 IAPAR9 idr1-1
Control
Control
Moderate drought
Moderate drought
C D Control 30 μM MV
Control 30 μM MV
IAPAR9 idr1-1 IAPAR9 idr1-1 E
Total chlorophyll content (mg/g)

IAPAR9
No staining
2 idr1-1
**
*
1.5 *
DAB staining
0.5
0
Control 30 MV
Control μM MV
Figure 6. idr1-1 mutation attenuates apoplastic and chloroplastic ROS production triggered by drought
stress and MV. (A) DAB, NBT and Trypan blue staining of detached leaves from idr1-1 mutant and IAPAR9
plants. Five-week-old idr1-1 mutant and IAPAR9 seedlings underwent drought stress for about 2 weeks (moderate
stress) and the detached leaves of IAPAR9, idr1-1, Com1, Com2, Com3, OE2, OE3 and OE5 (1-8, respectively)
were used for such staining. Scale Bar = 10 cm. (B) Cytochemical localization of drought-induced H2O2 in
intercellular space between mesophyll cells of idr1-1 mutant or IAPAR9 leaves. Leaf samples for sectioning were
from the experiment depicted in A. The H2O2 was stained with CeCl3 and the photographs were taken with
transmission electron microscopy (TEM). Red arrows indicate CeCl3 precipitates. Scale Bar = 1 μm. (C-E)
Performance of idr1-1 mutant and IAPAR9 seedlings after MV treatments. Two-week-old idr1-1 mutant and
IAPAR9 seedlings cultured in liquid Hoagland medium were treated with 30 μM MV for 3 days (C). DAB staining
of leaves from idr1-1 mutant and IAPAR9 seedlings following MV treatments were performed to detect amounts of
H2O2 (D). In parallel, total chlorophyll contents of the idr1-1 mutant and IAPAR9 leaves from the experiment
shown in C were measured and shown (E). Data are means (±SD) for three biological replicates; for each genotype,
30 plants were used for measuring leaf chlorophyll contents in each of the three replicates. Significant differences
from IAPAR9 were determined by Student’s t-test: **P < 0.01, t-test. Scale Bars = 10 cm (C and D).
A OsLEA3-1 OsLG3 DSM1
6 20 1.4
IAPAR9 **
Relative mRNA levels

5 * 1.2
idr1-1 15 1
4
0.8 **
3 10
0.6
2 0.4
5
1 0.2
0 0 0
Control Severe Control Severe Control Severe
drought drought drought
B
OsAPX1 OsAPX2 OsAPX3 OsAPX4 OsAPX5
2.5 2 3 1.2
** ** 2.5
IAPAR-9 * 1.8 **
Relative mRNA level
2 1.6 2.5 1 2 *
idr1-1 1.4
1.2 2 0.8 **
1.5 1.5
1 1.5 0.6
1 0.8 1
0.6 1 0.4
0.5 0.4 0.5 0.5
0.2
0.2
0 0 0 0 0
OsAPX6 OsAPX7 OsAPX8 OsCAT1 OsCAT2

2.5 1.2 1.8 1.4 12
** 1.6 ** **
Relative mRNA level
1 * 1.2 10
2 * 1.4
* 0.8 1
1.2 8
1.5 0.8
0.6 1
6
1 0.8 0.6
0.4 0.6 4
0.4
0.5 0.4 ** 2
0.2 0.2
0.2 ** ** **
0 0 0 0 0
OsCAT3 OsFeSOD OsPOX22.3 C

POD activity (U g-1FW min-1)
1.6 1.4 7
** * 60000
1.4 1.2 6 * IAPAR9
Relative mRNA level
idr1-1 *
1.2 ** 1 50000
5
1 0.8 4 40000
0.8 ** 30000
0.6 3 **
0.6 *
0.4 2 20000
0.4
0.2 0.2 1 10000
**
0 0 0 0
Control Moderate
drought
Figure 7. idr1-1 mutation increases expression of a group of ROS-scavenging genes under drought-stressed
conditions. (A) Relative expression levels of three genes that were reportedly associated with activation of ROS-
scavenging system. (B) Relative expression levels of a dozen genes involved in ROS scavenging under control or
drought stress (moderate or severe) conditions. For qRT-PCR analyses in A and B, leaves from 4-week-old
seedlings of idr1-1 and IAPAR9 experiencing nonstressed (control) and drought-stressed treatments, were used for
RNA isolation and qRT-PCR assays to examine expression of the genes as indicated. Rice ubiquitin 5 gene was
amplified and used as an internal control. (C) POD activity assays in idr1-1 mutant and IAPAR9 leaves under
control or moderate drought stress conditions. Leaf samples for POD activity assays were from the experiment
described in B. Data are means (±SD) for three biological replicates. Significant differences from IAPAR9 were
determined by Student’s t-test: *P < 0.05, **P < 0.01, t-test.
A B C
BD/AD 0 10-9 10-8 10-7 10-6 10-5
IAPAR9
P53 /T
TUD1/IDR1
Lam/T 1 2 5 6
idr1-1
Vec/IDR1
TUD1/Vec 3 4 7 8
SD/-Trp-Leu SD/-Trp-Leu-His-
Ade
D E IAPAR9 idr1-1 F
0 10-8 10-7 10-6 0 10-8 10-7 10-6
Length of primary roots (cm)

200 IAPAR9 20 IAPAR9
idr1-1 idr1-1
Leaf angle (°)
150
15
100
10
50
0 5
0 10-9 10-8 10-7 10-6 10-5 0 10-8 10-7 10-6
EBR EBR
G H I J
NIP tud1-5
1 1.2 TUD1

0.8 1
Grain length (cm)
0.8
0.6 **
**
** 0.6
0.4
0.4
0.2 **
0.2
0 0
K L
IAPAR9 idr1-1 1.4 OsBRD1 IAPAR9
Fold change (log2)
1.2 idr1-1
OsBRD1
1
OsBRD2 0.8
0.6
TUD1
0.4
UBQ 0.2
0
Control Drought
Figure 8. IDR1 physically interacts with TUD1, and idr1-1 mutant exhibits impaired BR
responsiveness. (A and B) Physical interactions between IDR1 and TUD1 in yeast cells (A) and in N.
benthamiana leaves (B). Yeast cells (strain AH109), which was co-transfected with BD-TUD1 plus AD-IDR1 and
other combinations of constructs as indicated, were grown on YPDA medium lacking Trp and Leu for 3 d (left
panel in A) and then transferred to a stringent selection medium lacking Trp, Leu, His and adenine (SD/-Trp/-Leu/-
His/-Ade) (right panel in A), and allowed them to grow for 2 days at 28℃ prior to observation. In parallel,
luciferase complementation image (LCI) system was used to test interactions between IDR1 and TUD1 (B). Upper
panels, bright field; lower panel, dark field. 1, AtSGT1a-nLUC + cLUC; 2, AtRAR1-nLUC + AtSTG1a-cLUC; 3,
nLUC + AtRAR1-cLUC; 4 and 8, nLUC + cLUC; 5, IDR1-nLUC + cLUC; 6, IDR1-nLUC + TUD1-cLUC; 7, nLUC
+ TUD1-cLUC. The number “2” in B indicates a positive control, and the number “4” and “8” indicate negative
controls. (C and D) Effects of different concentrations of EBR on the degrees of inclination of the leaf lamina.
Five-d-old idr1-1 mutant and IAPAR9 seedlings grown in Hoagland solution were treated by different
concentrations of EBR (0, 10-9, 10-8, 10-7, 10-6 and 10-5 M) for 3 days. Photographs of the second lamina joints were
taken (C) and angles of the leaf lamina joint bending were measured and plotted against varying EBR
concentrations (D). Data are means (±SD) for three biological replicates; for each genotype, 3 seedlings for control
(0 M EBR) or each EBR concentration were used for measuring the angles of leaf lamina joint bending in each of
the three replicates. Scale Bar = 5 cm. (E and F) Responses of primary roots of idr1-1 mutant and IAPAR9
seedlings to different concentrations of EBR. Seeds of both the genotypes were germinated in Hoagland solution for
5 days, and the selected seedlings with uniform lengths of primary roots were treated with different concentrations
of EBR (0, 10-8, 10-7 and 10-6 M) for 3 days. Photographs of the primary roots following treatment were taken (E),
and lengths of primary roots were plotted against varying EBR concentrations (F). Data are means (±SD) for three
biological replicates; for each genotype, 5 seedlings for control (0 M EBR) or each EBR concentration were used
for measuring root lengths in each of the three replicates. Scale Bar = 5 cm. (G) Comparisons of plant heights
between Nipponbare (NIP) and tud1-5. Seedlings of the NIP and tud1-5 were grown in pots for 40 days prior to
photography. Scale Bar = 5 cm. (H and I) Comparisons of grain lengths between idr1-1 mutant and IAPAR9 plants
and between tud1-5 mutant and NIP plants. Mature seeds of IAPAR9, idr1-1, NIP and tud1-5 were harvested
simultaneously and photographed (H). Grain lengths were measured and compared (I). Scale Bar = 1 cm. (J)
Relative expression levels of TUD1 in IAPAR9 leaves that underwent moderate, severe or no (control) drought
stress as revealed by qRT-PCR assays. (K and L) Expression levels of OsBRD1, OsBRD2 and TUD1 in idr1-1
mutant as shown by RT-PCR assays (K). Meanwhile, OsBRD1 transcript abundance from the transcriptome data
was shown as mean log2 fold change ± SE (n = 3) of three independent experiments (L); values were plotted
relative to the log2 of normalized IAPAR9 transcript abundance which was set at 1.0. RNA samples prepared from
idr1-1 mutant and IAPAR9 leaves without stress treatment (K) or leaves of both the genotypes undergoing moderate
drought stress (L) were used for such assays. Data are means (±SD) for three biological replicates. Significant
differences from IAPAR9 (I, L) or control (J) were determined by Student’s t-test: **P < 0.01, t-test.

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bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.264556; this version posted August 24, 2020.

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4 Author for contact details

44 Responsibilities of the author for contact

53 Email address of author for contact

261 Map-Based Cloning of IDR1 Gene

303 Expression Patterns and Subcellular Localization of IDR1

478 against undue oxidative stress on chloroplasts.

510 Transcriptome Analyses of Drought-stressed idr1-1 Mutants Reveals Several Mechanisms

834 Mutation of IDR1 Enhances ROS-Scavenging Ability by Triggering Gene Expression of

1008 Materials and methods

1017 Drought stress treatments

1039 Map-based cloning of IDR1

1054 Generation of transgenic lines

1062 Subcellular localization of IDR1

1073 Measurements of morphological traits

1093 Measurements of physiological traits

1120 Measurements of responsiveness of idr1-1 mutants to various experimental treatments

1137 RNA extraction and gene expression analyses

1157 Detection of ROS accumulation and cell death

1174 mRNA-Seq analyses

1187 Yeast two-hybrid and LCI assays

1200 Accession number

1208 Supplemental materials

1233 Figure legends

1272 Figure 3. Expression patterns and subcellular localization of IDR1.

Survival rate (%)

Survival rate (%)

C52 C73 IM81 IM63 IM59 C77 C59

LOC_Os05g26890 LOC_Os05g06902 4kb

IAPAR9 IAPAR9 IAPAR9

idr1-1 Com1 idr1-1 Com2 idr1-1 Com3

Relative expression level

Relative expression level

Relative expression level

Water loss rate (%)

Chlorophyll content (SPAD value)

idr1-1 idr1-1 * Com1

1.2 * idr1-1 Com2

Stomatal number (/mm2)

Average leaf thickness (μM)

140 400 idr1-1

Total chlorophyll content (mg/g)

Relative mRNA levels

OsAPX6 OsAPX7 OsAPX8 OsCAT1 OsCAT2

OsCAT3 OsFeSOD OsPOX22.3 C

Length of primary roots (cm)

Relative expression level

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