ipa1 improves rice drought tolerance at seedling stage mainly through activating abscisic acid pathway

ipa1 enhances rice drought tolerance mainly through activating the ABA pathway. It endows rice seedlings with a more developed root system, smaller leaf stomata aperture, and enhanced carbon metabolism. Drought is a major abiotic stress to crop production. IPA1 (IDEAL PLANT ARCHITECTURE 1)/OsSPL14 encodes a transcription factor and has been reported to function in both rice ideal plant architecture and biotic resistance. Here, with a pair of IPA1 and ipa1-NILs (Near Iso-genic Lines), we found that ipa1 could significantly improve rice drought tolerance at seedling stage. The ipa1 plants had a better-developed root system and smaller leaf stomatal aperture. Analysis of carbon–nitrogen metabolism-associated enzyme activity, gene expression, and metabolic profile indicated that ipa1 could tip the carbon–nitrogen metabolism balance towards an increased carbon metabolism pattern. In both the control and PEG-treated conditions, ABA content in the ipa1 seedlings was significantly higher than that in the IPA1 seedlings. Expression of the ABA biosynthesis genes was detected to be up-regulated, whereas the expression of ABA catabolism genes was down-regulated in the ipa1 seedlings. In addition, based on yeast one-hybrid assay and dual-luciferase assay, IPA1 was found to directly activate the promoter activity of OsHOX12, a transcription factor promoting ABA biosynthesis, and OsNAC52, a positive regulator of the ABA pathway. The expression of OsHOX12 and OsNAC52 was significantly up-regulated in the ipa1 plants. Combined with the previous studies, our results suggested that ipa1 could improve rice seedling drought tolerance mainly through activating the ABA pathway and that regulation of the ipa1-mediated ABA pathway will be an important strategy for improving drought resistance of rice.


Introduction
Plants live in fixed locations and face diverse abiotic stresses (such as drought, salinity, and cold) negatively affecting plant growth and seed production. To survive, plants have evolved high plasticity and complex mechanisms to respond to these stimuli over a long period of time (Hu and Xiong 2014). The understanding of plant responses to stresses in physiology, genetics, and molecular biology will be greatly helpful in improving the tolerance of plants to abiotic stresses through genetic engineering (Huang et al. 2009).
Metabolic adaption to abiotic stress is important to plant surviving under unfavorable conditions (Barnaby et al. 2019;Ma et al. 2016). Generally, nitrogen promotes plant shoot growth rather than root, while carbon does oppositely, and high carbon/nitrogen (C/N) ratio enhances plant root development with a high root/shoot ratio (Osuna et al. 2015), which allows plant root access to water profoundly and decreases shoot water losses. Accumulation of carbohydrates resulting from enhanced photosynthesis protects plants from membrane damage and accounts, in part, for the more vigorous growth during stress (Garg et al. 2002). On the contrary, increasing nitrogen levels increased the degree of water stress, resulting in decreased leaf water potential, especially when the total water applied was minimal (Aragon Communicated by Emmanuel Guiderdoni. and De Datta 1982). Moreover, high C/N status may act as a stress condition, which induces a series of stress-related genes, including transcription factors such as OsMYB4, CHS (encoding key enzyme in flavonoid biosynthesis) and genes involved in the jasmonate signaling pathway .
Abscisic acid (ABA) is a multifunctional plant hormone that regulates many physiological processes, including seed dormancy and germination, stomatal movement, and plant responses to abiotic stress. NCED (9-cis-epoxycarotenoid dioxygenase) is the key rate-limiting enzyme in ABA biosynthetic pathway. Overexpression of OsNCED3 in Arabidopsis results in increased accumulation of ABA, reduced relative water loss, delayed seed germination, and greater drought tolerance relative to that of wild-type (Hwang et al. 2010). Rice nced3 mutants had increased sensitivity to water and H 2 O 2 stress, increased stomata aperture, delayed leaf senescence, and decreased ABA content, while overexpression of OsNCED3 could enhance rice water stress tolerance, promote leaf senescence and increase ABA content (Huang et al. 2018). ABA 8'-hydroxylase is considered as the main ABA catabolic enzyme. OsABA8ox3 RNAi lines showed significant improvement in drought stress tolerance with increased ABA content. In contrast, overexpression seedlings were hypersensitive to drought stress with decreased ABA content, indicating OsABA8ox3 gene plays an important role in controlling ABA level and drought stress resistance in rice . Stress response at the molecular level involves induction of stress-responsive and stress-tolerant genes. Many transcription factors have been identified to be involved in plant adaptation to abiotic stresses (Baillo et al. 2019). SQUA-MOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family transcription factors sharing a highly conserved SQUAMOSA PROMOTER BINDING PROTEIN (SBP) domain are plant specific, and their functions are surprisingly diverse, covering virtually every aspect of plant growth and development and response to stresses . In rice genome, about 19 SPL genes have been identified. Among these, as a multifunctional gene in regulating plant development, IPA1/OsSPL14 has attracted extensive attention. It was reported first to function in rice "Ideal Plant Architecture (IPA)" characterized by fewer unproductive tillers, larger panicles and stronger culms (Jiao et al. 2010;Miura et al. 2010). Since then, IPA1 was also identified to play a vital role in rice biotic resistance (Liu et al. 2019;Wang et al. 2018b). Overexpressing of IPA1 could enhance rice resistance to Xanthomonas oryzae pv. oryzae, partially through gibberellin signaling, including interacting with SLR1 and enhancing GA metabolism by activating EUI1 expression (Liu et al. 2019).
Although great progress has been made in understanding the roles of IPA1 in rice plant development and biotic resistance, its function in rice abiotic stress tolerance is still unknown. Here, using a pair of the IPA1-and ipa1-NILs, we found that ipa1 could significantly improve rice drought tolerance at the seedling stage mainly through activating ABA pathway.

Plant materials
A pair of the ipa1 and IPA1-NILs was developed from a cross of an indica cv. Yuetai B (IPA1/IPA1) and a japonica cv. Shaoniejing (SNJ) (ipa1/ipa1). Yuetai B was crossed with SNJ to develop F 1 plants; then, the F 1 plants were backcrossed to Yuetai B to develop BC 1 F 1 plants. The BC 1 F 1 plants were self-crossed for six generations to develop F 7 plants, from which a plant with a genotype IPA1/ipa1 was identified. Let this plant self-cross, and from its offspring, the plants with genotype IPA1/IPA1 were identified as a IPA1-NIL, while the plants with genotype ipa1/ipa1 as a ipa1-NIL. The degree of genomic similarity for the NILs is 95.8%. The ipa1 plants showed fewer tillers and leaves, but more panicle branches (Supplementary table 1), as reported previously (Jiao et al. 2010).

Hydroponic culture conditions
Seeds were disinfected in 20% sodium hypochlorite solution for 30 min, thoroughly washed with deionized water. Sterilized seeds were germinated in distilled water for 48-72 h at 30 °C in darkness, and then transferred to hydroponic culture solution as described previously (Wang et al. 2018c). Fresh solution was changed every 3 days, and pH was adjusted to 5.5 every day.

Drought stress and osmotic stress experiments
In soil drought experiments, two-leaf stage seedlings cultured in sandy soil were treated with dehydration by removing water with PVC pipes for 7 days and then re-watered for 5 days. For PEG treatment, two-leaf stage seedlings were transferred to culture solution containing 25% (w/v) PEG4000 for 5 days, and then recovered for 4 days.

Imaging of rice leaf stomata
Imaging of rice leaf stomata was conducted as described previously ) with some modifications. Leaves of 15 days seedlings under control and 6 h 25% PEG treatment conditions were immediately fixed by 2.5% glutaraldehyde, and stomatal pictures were obtained by scanning electron microscopy (JSM-6390LV, JEOL, Tokyo, Japan).

Carbon-nitrogen metabolism-associated indexes measurement
After cultured for 20, 28, 36, 44 and 52 days in nutrient solution under outdoor conditions, the leaves of IPA1 and ipa1 seedlings were collected for carbon-nitrogen metabolism-associated indexes measurement. Soluble sugar and sucrose contents were determined using the anthrone method (Shields and Burnett 1960) and the resorcinol method (Han et al. 2015), respectively. FBP (Fructose 1,6-bisphosphatase) activity was determined according to kit instructions (Solarbio, Beijing, China). PEPC (Phosphopyruvate carboxylase) activity was measured by the previously method (Blanke and Ebert 1992). SPS (Sucrose Phosphate Synthase) and SS (Sucrose Synthase) activities were measured as described previously .
Soluble protein and free amino acid contents were determined using the Bradford assay (Bradford 1976) and the ninhydrin method (Sun et al. 2006), respectively. Nitrate content was measured as described previously (Doane and Horwáth 2003). NR (Nitrate reductase), GS (Glutamine synthase), GOGAT (Glutamate synthase), and GDH (Glutamate dehydrogenase) activities were estimated based on the previous methods ).

Metabolomics analysis
The shoot bases of IPA1 and ipa1 seedlings grown for 21 days in nutrient solution under outdoor conditions were collected for metabolomics analysis. The sample preparation, extract analysis, metabolite identification and quantification were performed as described previously (Chen et al. 2014) at Wuhan Metware Biotechnology Co., Ltd., Wuhan, China.

Phytohormone measurement
Leaves of 15-day seedlings under control and 6 h 25% PEG treatment conditions were collected for phytohormone measurement. Plant materials were ground into powder in liquid nitrogen, and extracted with 80% methanol at 4 °C. The extract was centrifuged at 12,000×g under 4 °C for 15 min. The supernatant was collected and evaporated to dryness under nitrogen gas stream, and then reconstituted in 30% methanol. The solution was centrifuged, and the supernatant was collected for LC-MS analysis. The LC-MS analysis was conducted with the API6500 QTRAP LC/MS/MS system, equipped with an ESI Turbo Ion-Spray interface, operating in a positive ion mode and controlled by Analyst 1.6 software (AB Sciex).

RNA extraction and real-time PCR
The plants grew in culture room at 28 ℃ under a 16-h light/8-h dark photoperiod. After treated with 25% PEG for 0, 3, 6 and 9 h, leaves of 15 days seedlings were collected for RNA extraction. Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) reagent. According to the manufacturer, RNA sample (~ 2 μg) was treated with DNaseI and then used for cDNA synthesis with the Super-Script III first-strand cDNA synthesis system (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using 2 × SYBR Green PCR Master Mix (Takara, Dalian, China) in a CFX96TM Real-Time System (BIO-RAD, Hercules, CA, USA). Each experiment included three technical replicates and three biological replicates. OsActin was used as an internal control for normalization. Primers used are listed in Supplementary Table 2.

Dual-luciferase assays
The promoters of OsHOX12, OsNAC52, OsNCED1 and OsNCED3 were amplified by PCR from genomic DNA and cloned into pGreenII 0800-LUC reporter vectors in front of luciferase (LUC) gene. Besides, the Renilla luciferase (REN) reporter gene was driven by the CaMV 35S promoter as a control in each transformation. The coding regions of IPA1, OsHOX12 and OsTB1 were cloned behind the Ubi promoter into the effector vector pRGV (He et al. 2018). The reporter and effector were transformed into rice protoplasts. The luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Beijing, China) and compared with empty vector-transformed plants.

Yeast one-hybrid assay
The coding regions of IPA1 SBP and OsHOX12 were amplified and cloned into the pGADT7 prey vector. The promoters of OsHOX12 and OsNAC52 were amplified and cloned into pAbAi bait vectors. The prey vectors, respectively, cotransformed with bait vector into Y1HGOLD strain. These transformed cells were grown on SD/-Leu/-Ura plates and then grown on SD/-Leu/-Ura/ 500 ng/ml AbA plates at 28 °C for 3-5 days.

ipa1 enhances rice drought tolerance at seedling stage
To investigate the effect of ipa1 on rice drought tolerance, a soil drought experiment was performed with a pair of the IPA1 and ipa1-NILs. The ipa1-NIL obtained a survival rate of 83.5%, while it was only 20.8% for the IPA1-NIL (Fig. 1a, c). As treated with 25% PEG4000 (osmotic stress-simulating drought stress), the ipa1 seedlings exhibited a survival rate of 62.1%, while the IPA1-NIL showed a significantly lower survival rate of 43.5% (Fig. 1b, d). These results indicated that ipa1 could significantly improve rice drought tolerance at seedling stage.
The characteristics of root and leaf stomata for the IPA1 and ipa1 seedlings As compared to that of the IPA1 seedlings, the ipa1 seedlings had significantly increased root length, root and shoot dry weight as well as the dry weight ratio of root to shoot (Fig. 2a-e).
As for leaf stomata, the ipa1 seedlings showed a decreased stoma size as compared with that of the IPA1 seedlings (Fig. 2f, g), despite no significant difference in Fig. 1 Drought tolerance of the IPA1 and ipa1 seedlings. a, b Performance of the IPA1 and ipa1 seedlings under soil drought and 25% PEG4000 treatments, respectively. c, d Survival rates of the IPA1 and ipa1 seedlings treated with soil drought and 25% PEG4000, respectively. Each experiment has three replicates, and 24 (c) or 36 (d) seedlings were tested in each replicate. Data represent the means ± SE. *P < 0.05, t test. **P < 0.01, t test Fig. 2 Characteristics of root and leaf stomata for the IPA1 and ipa1 seedlings. a Plant architecture of the IPA1 and ipa1 seedlings 25 days after planting. Bar, 10 cm. b-e Root length, root dry weight, shoot dry weight and dry weight ratio of root to shoot, respectively. b-e, n = 18. f Scanning electron microscopy (SEM) images of IPA1 and ipa1 seedlings. Bar, 10 µm. g The stomatal diameter (length of sto-mata, n = 12, derived from three seedlings). h Percentage of three types of stomata. Forty-seven to 52 stomata from 3 seedlings were calculated. i The water loss rates, n = 15. PEG, 25% PEG4000 treatment condition. Data represent the means ± SE. *P < 0.05, t test. **P < 0.01, t test stoma density between the NILs (Supplementary Fig. 1). Moreover, the ipa1 plants displayed more stomata completely close (29.3%) and less stomata completely open (18.5%) than that of the IPA1-NIL (14.0% and 34.7%, respectively). When treated with PEG, more leaf stomata tended to close for both the IPA1 and ipa1 seedlings. Even so, there were still more stomata completely close (56.6%) and less stomata completely open (nearly 0.0%) for the ipa1 seedlings than that for the IPA1 seedlings (45.9% and 8.3%, respectively) (Fig. 2h). Consequently, the ipa1 seedlings were found to lose less water (Fig. 2i).
The ipa1 plants had a better-developed root system conducive to enhancing their ability to absorb water from the soil and their leaves with smaller stomatal aperture were beneficial to enhance their moisturizing function, which could play a vital physiological role in improving their drought tolerance.
The ipa1 seedling could adjust its carbon-nitrogen metabolism balance to a metabolic pattern with a relatively strong carbon metabolism It was found that the ipa1 plants had a higher content for soluble sugar and sucrose than the IPA1 plants in both leaves (Fig. 3a, b) and sheaths ( Supplementary Fig. 2). Then, we measured the activity of several carbon metabolism-related enzymes. Except for FBP, three carbon metabolism-related enzymes (PEPC, SPS, and SS) showed a activity level higher in the ipa1 plants than in the IPA1 plants ( Fig. 3c-f).
As for the nitrogen metabolism, soluble protein and free amino acid contents for the ipa1 seedlings were significantly lower than that for the IPA1 plants (Fig. 4a, b). Meanwhile, the ipa1 plants were found to be significantly higher in inorganic nitrate-nitrogen content than the IPA1 plants (Fig. 4c).
Four key nitrogen assimilation enzymes (NR, GS, GOGAT and GDH) were investigated and each of them showed a significantly lower activity level in the ipa1 plants than in the IPA1 plants ( Fig. 4d-g). Accordingly, the expression of genes involved in nitrogen absorption, transport and assimilation was detected to be down-regulated, especially in the roots of the ipa1 plants ( Supplementary Fig. 3).
Combined with the above findings, it seems that the ipa1 plant could change its carbon-nitrogen metabolism balance to a metabolic pattern with a relatively stronger carbon metabolism by enhancing its carbon metabolic activity and down-regulating its nitrogen metabolism, thus benefiting the accumulation of carbohydrates in the plant, which could Fig. 3 Carbon metabolism-associated indices of the IPA1 and ipa1 seedlings. a, b Contents of soluble sugar and sucrose, respectively, in the IPA1 and ipa1 seedling leaves. c-f Activity of FBP, PEPC, SPS and SS, respectively, in the IPA1 and ipa1 seedlings. Data represent the means ± SE (n = 3). *P < 0.05, t test. **P < 0.01, t test provide a stronger material and energy basis for the plants to tolerate external abiotic stress.

Metabolic profile analysis of the IPA1 and ipa1 plants
To further explore the effect of ipa1 on plant metabolism, we analyzed the metabolic profiles with the IPA1/ipa1-NILs using a liquid chromatography-electrospray ionization-tandem mass spectrometry. There were 357 compounds to be identified. These compounds covered the key components involved in metabolic pathways of sugars, amino acids, nucleotide, organic acids, fatty acids and others (Supplementary Table 3).
The majority of carbohydrates such as sucrose, trehalose 6-phosphate (T6P), glucosamine and glucarate o-phosphoric acid were detected to accumulate more in the ipa1 plants than in the IPA1 plants, except glucose (Fig. 5a). For the key metabolites involved in nitrogen assimilation, the ipa1 plants showed a significantly decrease in the contents of amino acids Tyr and Trp (Fig. 5b) while their precursor shikimic acid mainly accumulated in the ipa1 plants (Fig. 5c). Similarly, the levels of organic acids (2-OG, succinic acid, and malic acid) and amino acids (Gln, Glu, Asn and Asp, as the major forms of nitrogen in xylem sap of rice plant) were significantly decreased in the ipa1 plants (Fig. 5b, c) while their precursor aconitic acid (a major element in TCA cycle) also showed to accumulate significantly in the ipa1 plants (Fig. 5c). These results indicated that the carbon flux to nitrogenous compounds was depressed in the ipa1 plants as compared to the IPA1 plants. Besides that, most of the other identified amino acids, amino acid derivates and nucleotides were decreased to different degrees in the ipa1 plants (Supplementary Fig. 4a, b). Therefore, all these results suggested that the gene ipa1 could significantly influence the balance of carbon-nitrogen metabolism, tipping the carbon/nitrogen metabolism balance towards increased carbon metabolism.
Cysteine is the first carbon/nitrogen-reduced sulfur product resulting from the sulfate assimilation pathway. As a sulfur donor, it plays a major role in the growth and development of plant. Glutathione derived from cysteine protects plants from reactive oxygen species (ROS) damage caused by abiotic stress (Droux 2004). In this study, a significantly increased cysteine content was found in the ipa1 plants, coupled with an increase of reduced glutathione content and a decrease of oxidized glutathione content (Fig. 5d). Moreover, contents of the other antioxidants such as coumarin and curcumin raised dramatically in the ipa1 plants ( Supplementary Fig. 4c). The same situation also happened to glycerophospholipids ( Supplementary Fig. 4d), the cell membrane major components. Therefore, ipa1 may activate sulfate assimilation and the related defense mechanism, which plays an essential role in protecting the ipa1 plants from ROS damage under abiotic stresses. In addition, ferulic Activity of NR, GS, GOGAT and GDH, respectively, in the IPA1 and ipa1 seedlings. Data represent the means ± SE (n = 3). *P < 0.05, t test. **P < 0.01, t test acid is reported to be a marker metabolite for plant drought resistance and high photosynthesis (Ma et al. 2016). The contents of ferulic acid-related metabolites were significantly up-regulated in the ipa1 seedlings ( Supplementary  Fig. 4e).

The enhanced drought tolerance in the ipa1 plants could be mediated by ABA accumulation
ABA and GAs are known to be primary phytohormones that antagonistically regulate plant abiotic stress resistance (Vishal and Kumar 2018). In this study, exogenous ABA application led to an obvious inhibition on plant height, whereas GA 3 treatment significantly promoted the trait for both the NILs under non-drought stress conditions (Supplementary Fig. 5). When PEG was applied to simulate drought conditions (osmotic stress), the ABA application significantly improved survival rates of seedlings, whereas GA 3 decreased survival rates for both the NILs (Fig. 6a,  b). Although so, the two NILs showed significant differences in degree of response to ABA and GA 3 treatments. Comparatively, the ipa1 seedlings were less sensitive to ABA or GA 3 treatment. Exogenous ABA treatment improved PEG resistance of the IPA1 plants to a greater extent, resulting in a fact that survival rate of the IPA1 plants was no longer different from that for the ipa1 plants (Fig. 6a, b).
Then, we measured ABA and GAs contents of the NILs. In both control and PEG-treated conditions, ABA content in the ipa1 seedlings was significantly higher than that in the IPA1 seedlings (Fig. 6c). Meanwhile, the ABA biosynthesis genes such as OsNCED1, OsNCED3, and OsNCED4 were detected to be up-regulated, whereas ABA catabolism genes were down-regulated in ipa1-NIL (Fig. 6e). OsABI5, OsLEA3, OsLIP9, and OsRAB16A are marker genes of the ABA pathway involved in abiotic stress response . In our study, the expression of OsLEA3 (under PEG-treated condition) and the other marker genes (under both the control and PEG-treated conditions) was up-regulated significantly in the ipa1 plants (Fig. 6f).
As for GAs, with an exception of a remarkable increase of the GA 4 content in ipa1-NIL under the control condition, no significant difference has been observed in the contents of GAs investigated between the two NILs under the control or PEG-treated conditions (Fig. 6d), although several genes for GA biosynthesis and catabolism showed some differences Fig. 5 Contents of key metabolites in primary metabolism of the IPA1 and ipa1 seedlings. a-d Contents of carbohydrates, amino acids, organic acids and metabolites in sulfate primary assimilation processes, respectively, in the IPA1 and ipa1 seedlings. Data represent the means ± SE (n = 3). *VIP ≥ 0.5, P < 0.05, t test. **VIP ≥ 0.5, P < 0.01, t test 1 3 in expression levels between the two NILs ( Supplementary  Fig. 6).
The above results suggested that the enhanced drought tolerance of the ipa1-NIL could mainly result from a high level of ABA accumulation in the ipa1 seedlings.

IPA1 directly activated the expression of OsHOX12 and OsNAC52
OsHOX12 is a transcription factor homologous with Arabidopsis HOMEOBOX PROTEIN 21 (HB21), HOME-OBOX PROTEIN 40 (HB40) and HOMEOBOX PRO-TEIN 53 (HB53). It was reported to activate expression of OsNCED1, and promote ABA biosynthesis in rice (Liu et al. 2020). OsNAC52, a transcription factor belonging to NAC family, potentially responds to ABA and confers drought tolerance in transgenic plants (Gao et al. 2010). In addition, as a transcription activator, IPA1 can regulate its target gene by directly binding to the core motif GTAC or indirectly to the core motif TGG GCC /T of the target gene promoter (Lu et al. 2013). Bioinformatics analysis identified twelve and three GTAC motifs in the promoters of OsHOX12 and OsNAC52, respectively (Fig. 7a, b). We searched previously published ChIP-seq data of IPA1 (Lu et al. 2013), and found that OsHOX12 and OsNAC52 were potential targets of IPA1, suggesting that IPA1 Fig. 6 PEG tolerance response to exogenous ABA and GA 3 treatments, endogenous ABA and GAs contents of the IPA1 and ipa1 seedlings and the expression of ABA-associated genes in the IPA1 and ipa1 seedlings. a Phenotypes of the IPA1 and ipa1 seedlings treated by ABA and GA 3 with PEG. The IPA1 and ipa1 seedlings cultured in normal condition were treated with ABA (5 μM) and GA 3 (10 μM), respectively, then transmitted to culture solution with 25% PEG. b Survival rates of the IPA1 and ipa1 seedlings in correspond-ing conditions. Each experiment has 3 replicates, and 18 seedlings were tested in each replicate. c, d ABA and GAs contents in the IPA1 and ipa1 seedlings, respectively. #, undetectable GAs. e The expression levels of genes involved in ABA biosynthesis and catabolism in the IPA1 and ipa1 seedlings at different time points after 25% PEG treatment. f The expression levels of marker genes of the ABA pathway at different time points after 25% PEG treatment. Data represent the means ± SE. *P < 0.05, t test. **P < 0.01, t test may directly activate the expression of OsHOX12 and OsNAC52.
To test the hypothesis, we conducted a yeast one-hybrid assay. Cells co-transformed with bait vectors and prey vectors grew well on SD/-Leu/-Ura/AbA plates, indicating that IPA1 can directly bind to the promoters of OsHOX12 and OsNAC52 (Fig. 7c, d). Then, we carried out a dualluciferase assay using the full length of the OsHOX12 and OsNAC52 promoters in rice protoplasts. Co-transformed reporter vectors and effector vectors activated the expression of LUC gene, suggesting that IPA1 can significantly enhance the activity of the OsHOX12 and OsNAC52 promoters (Fig. 7e, f). Accordingly, the expression of OsHOX12 was increased in the ipa1 seedlings under PEG-treated conditions (Fig. 7h). Moreover, the expression of OsNAC52 was also significantly up-regulated in the ipa1 plants under both the control and PEG-treated conditions (Fig. 7i). In addition, we also found that OsHOX12 could directly bind to the promoter of OsNCED3 and activate its activity (Supplementary Fig. 7a-c).We also tested expression of the other genes involved in abiotic stresses in the NIL plants. OsNAC5, OsNAC6, and OsNAC19 are three other NAC family transcription factors, and overexpression of each of those was reported to enhance rice resistance to abiotic stresses (Hu et al. 2006;Takasaki et al. 2010). The results depicted that these genes' expression showed a significantly higher level Fig. 7 IPA1 directly activates the promoter activity of OsHOX12 and OsNAC52, and up-regulates the expression of the other NAC family genes. a Schematic of OsHOX12 promoter. S6 sequence is GTA CGT ACG TAC , and the other sites sequence is GTAC. b Yeast one-hybrid analysis with the fragment containing S1-S10 as bait. c Schematic of OsNAC52 promoter. The S1-S3 sequence is GTAC. d Yeast onehybrid analysis with the fragment containing S1-S3 as bait. e, f Rela-tive LUC activity in transient transactivation assays using the promoters of OsHOX12 (2000 bp) and OsNAC52 (1964 bp). g-l Relative expression levels of IPA1, OsHOX12, OsNAC52, OsNAC5, OsNAC6 and OsNAC19 at different time points after 25% PEG treatment, respectively. Data represent the means ± SE (n = 3). *P < 0.05, t test. **P < 0.01, t test in the ipa1 plants than in the IPA1 plants under control and PEG-treated conditions (Fig. 7j-l).

Discussion
IPA1/OsSPL14 is one of the most concerned genes in current studies of rice functional genomics due to its multifunctions in regulating plant development (Wang et al. 2018a). In this study, we found that ipa1 could significantly improve rice drought tolerance at seedling stage. The ipa1 seedlings demonstrated a better-developed root system in terms of phenotypes, which helped to enhance their ability to absorb water from the soil. Their leaves with smaller stomatal aperture improved their moisturizing ability, which could play a crucial physiological role in enhancing their resistance to drought tolerance.
ABA is induced in response to adverse environmental conditions, and it plays a critical role in regulating abiotic stress response in plants (Cutler et al. 2010). Deficit of ABA in nced3 mutants increased rice sensitivity to water stress, while accumulation of ABA in OsNCED3-overexpressing seedlings enhanced rice water stress tolerance (Huang et al. 2018). In this study, the ipa1 seedlings had a significantly higher ABA content than the IPA1 plants (Fig. 6c). The ABA pathway marker genes were up-regulated by ipa1 (Fig. 6f). Exogenous ABA treatment largely promoted PEG resistance for the IPA1 plants (Fig. 6a, b). These results seem to suggest that the improved drought tolerance might result from activation of the ABA pathway in the ipa1 seedlings.
OsHOX12 and OsNAC52 are two of the transcription factors involved in ABA pathway in rice (Gao et al. 2010;Liu et al. 2020). Our yeast one-hybrid assay and dualluciferase test indicated that IPA1 can directly bind to the promoters of OsHOX12 and OsNAC52 and significantly enhance the activity of the OsHOX12 and OsNAC52 promoters ( Fig. 7c-f). Therefore, IPA1 may directly activate OsHOX12, thus promoting ABA biosynthesis. Meanwhile, our study indicated that ipa1 could enhance the ABA pathway by directly regulating OsNAC52, which was reported to be a positive regulator of the ABA pathway (Gao et al. 2010). In addition, Gonzalez-Grandio et al. (2017) reported that the TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) transcription factor BRANCHED1 (BRC1) in Arabidopsis binds to and positively regulates the transcription of three related Homeodomain leucine zipper protein (HD-ZIP) encoding genes HB21, HB40 and HB53, which together with BRC1, enhances NCED3 expression, leading to ABA accumulation and triggering hormone response. In rice, Lu et al. (2013) revealed that IPA1 directly targets OsTB1 (an ortholog of BRC1). Here, we showed that OsTB1 was up-regulated significantly in the ipa1 seedlings, and that OsTB1 could activate the promoter activity of OsHOX12, OsNCED1 and OsNCED3 ( Supplementary Fig. 7d-g). Therefore, we speculate that, as in Arabidopsis, IPA1 could also target OsTB1 to enhance the expression of the ABA biosynthesis genes, thus leading to ABA accumulation in ipa1 seedlings.
ABA is also a key regulator of plant stomatal aperture and root development. In response to drought stress, plants can synthesize ABA, which triggers closing of stomatal pores, thus reducing water loss (Schroeder et al. 2001). The foliagederived ABA promoted root growth relative to shoot growth but inhibited the development of lateral roots (McAdam et al. 2016). Moreover, a very recent paper showed that moderate enhancement of ABA signaling helps maintain the RM (root meristem) size, sustaining root growth by antagonizing the GA-promoted degradation of OsSHR1 through the SnRK2-APC/CTE regulatory module, while mutants of OsABA1 (a ABA biosynthesis gene) displayed a short root phenotype (Lin et al. 2020). Therefore, the smaller stomatal and better-developed root system in ipa1 seedlings could result from the accumulation of ABA.
The ABA pathway also plays a vital role in regulating plant metabolism. The presence of ABA releases and activates the SNF1-RELATED KINASES2 (SnRK2s), which phosphorylate the downstream targets to induce ABA responses (Fujii et al. 2009;Nakashima et al. 2009). Overexpression of SnRK2.6 promotes plant carbon assimilation with drastically boosted sucrose and total soluble sugar levels in the leaves through increasing SPS activity (Zheng et al. 2010). SnRK2s-mediated phosphorylation of NRT1.1 was reported to be involved in the inhibitory effect of ABA on nitrate uptake in Arabidopsis (Su et al. 2021). In rice, the effect of ABA on plant metabolism was fond to be concentration-dependent. Lower concentrations of ABA significantly stimulated the accumulation of sucrose and total soluble sugars, and increased SPS and SS activity, while higher concentrations of ABA exerted inhibitory effects on SPS and SS activities (Liang et al. 1996;Tang et al. 2009). In addition, exogenous ABA incubation decreased the GS and GDH activities, and soluble protein contents in rice leaves in a dose-dependent manner (Zakari et al. 2020). Our results indicated that ipa1 plants could adjust the balance of carbon-nitrogen metabolism by enhancing their carbon metabolic activity, but relatively down-regulating their nitrogen metabolism (Fig. 3, 4). Metabolic profile analysis further supported such a change of carbon-nitrogen metabolism in ipa1 plants (Fig. 5a, b). Thus, it seems that, for the ipa1 seedlings, tipping the carbon/nitrogen balance towards increased carbon metabolism might also, at least partially, be associated with the enhancement of ABA signaling, which contributes to their enhanced drought tolerance. Further detailed analyses are required to understand the interaction of ABA pathway and C/N metabolism balance in the ipa1 plants.
It should be noted that the results of the current study revealed the effect of the gene ipa1 on drought tolerance of rice plants only at seedling stage. At this stage, OsTB1 is the most important target of IPA1, and this target gene is mainly expressed at seedling stage (Lu et al. 2013). As rice plants develop into panicle differentiation stage, the main targets of IPA1 turn into genes such as OsDEP1 (Lu et al. 2013). Accordingly, rice plants' hormone regulation and metabolic pattern could be changed remarkably, which is worthy of further study in the future. In addition, the gene ipa1 is one of the most essential yield-increasing genes reported in rice so far (Wang et al. 2018a;Yu et al. 2020). It increases rice yield mainly by shaping ideal plant type with fewer tillers and larger panicles (Jiao et al. 2010). In this study, the ipa1 seedlings were observed to have a larger dry weight per plant (Fig. 2c, d), although their nitrogen metabolism was down-regulated relative to its WT seedlings. Obviously, the ipa1 rice plants with larger biomass at the seedling stage would be more likely to develop larger panicles later, thus contributing to increased yields.
In conclusion, this study elucidated that ipa1 could significantly improve rice drought tolerance at seedling stage. The ipa1 plants had a better-developed root system and smaller leaf stomatal aperture. They could tip the carbon-nitrogen metabolism balance towards an increased carbon metabolism pattern. Meanwhile, the ABA biosynthesis genes were up-regulated, whereas the ABA catabolism genes were down-regulated in the ipa1 seedlings, resulting in accumulation of endogenous ABA. Based on yeast one-hybrid assay and dual-luciferase assay, IPA1 was found to directly activate the expression of OsHOX12 and OsNAC52, a transcription factor promoting ABA biosynthesis and a positive regulator of the ABA pathway, respectively. These results suggested that ipa1 could improve rice seedling drought tolerance mainly through activating the ABA pathway, and that it may has potential applications in improving drought resistance of rice.