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/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/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 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 provide a stronger material and energy basis for the plants to tolerate external abiotic stress.
Metabolic profile analysis of the IPA1/ipa1 plants
To further explore the effect of ipa1 on plant metabolism, we analyzed the metabolic profiles with the IPA1/ipa1-NILs by 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 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 upregulated 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 GA3 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 GA3 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 GA3 treatments. Comparatively, the ipa1 seedlings were less sensitive to ABA or GA3 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 (Zhang, et al. 2015). In our study, all these marker genes were up-regulated significantly in the ipa1 plants under the control and PEG-treatment conditions (Fig. 6f).
As for GAs, with an exception of a remarkable increase of the GA4 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 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 AtHB21, 40 and 53. 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 TGGGCC/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 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/AbAi plates, indicating that IPA1 can directly bind to the promoters of OsHOX12 and OsNAC52 (Fig. 7c, d). Then, we carried out a dual-luciferase 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).
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 in the ipa1 plants than in the IPA1 plants under control and PEG-treated conditions (Fig. 7j-l).