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OsMADS23 phosphorylated by SAPK9 confers drought and salt tolerance by regulating ABA biosynthesis in rice

['Xingxing Li', 'Key Laboratory Of Biorheological Science', 'Technology Of Ministry Of Education', 'Bioengineering College Of Chongqing University', 'Chongqing', 'Bo Yu', 'Qi Wu', 'Qian Min', 'Rongfeng Zeng', 'Zizhao Xie']

Date: 2021-10

Some of MADS-box transcription factors (TFs) have been shown to play essential roles in the adaptation of plant to abiotic stress. Still, the mechanisms that MADS-box proteins regulate plant stress response are not fully understood. Here, a stress-responsive MADS-box TF OsMADS23 from rice conferring the osmotic stress tolerance in plants is reported. Overexpression of OsMADS23 remarkably enhanced, but knockout of the gene greatly reduced the drought and salt tolerance in rice plants. Further, OsMADS23 was shown to promote the biosynthesis of endogenous ABA and proline by activating the transcription of target genes OsNCED2, OsNCED3, OsNCED4 and OsP5CR that are key components for ABA and proline biosynthesis, respectively. Then, the convincing evidence showed that the OsNCED2-knockout mutants had lower ABA levels and exhibited higher sensitivity to drought and oxidative stress than wild type, which is similar to osmads23 mutant. Interestingly, the SnRK2-type protein kinase SAPK9 was found to physically interact with and phosphorylate OsMADS23, and thus increase its stability and transcriptional activity. Furthermore, the activation of OsMADS23 by SAPK9-mediated phosphorylation is dependent on ABA in plants. Collectively, these findings establish a mechanism that OsMADS23 functions as a positive regulator in response to osmotic stress by regulating ABA biosynthesis, and provide a new strategy for improving drought and salt tolerance in rice.

MADS-box TFs have been well documented to play diverse roles in plant growth and development [ 32 , 33 ]. In recent years, MADS-box proteins have been shown to function as key regulators in various environmental stress responses [ 34 – 37 ]. However, the mechanisms that MADS-box proteins regulate plant response to abiotic stress have just begun to be revealed. In this study, we explored the roles of OsMADS23 as a positive regulator in response to drought and salt stress. Then, we found that OsMADS23 directly targets OsNCED2, OsNCED3, and OsNCED4 to enhance ABA biosynthesis, and the knockout mutants of OsNCED2 exhibit increased sensitivity to oxidative stress. More importantly, our results indicated that SAPK9, an upstream protein kinase, phosphorylates OsMADS23 and increases its stability and transcriptional activity in plants, in an ABA-dependent manner. These results reveal a regulatory mechanism that how OsMADS23 regulates plant response to osmotic stress through ABA signaling pathway. The data also help us to dissect the components on stress-responsive pathways and provide new insights, leading to novel strategies for the improvement of drought and salt tolerance in agricultural and economic crops.

It is well described that 9-cis-epoxycarotenoid dioxygenase (NCED) is the key rate-limiting enzyme in ABA biosynthesis in higher plants, and its activity affects ABA accumulation [ 19 – 23 ]. Currently, increasing evidence has demonstrated that the enhanced expression of NCEDs could promote ABA biosynthesis, and therefore confers the abiotic stress tolerance [ 24 – 26 ]. In contrast, nced mutants exhibit reduced ABA accumulation, repressed seed dormancy as well as abiotic stress-sensitive phenotypes to harmful environmental conditions [ 27 , 28 ], which is similar to that of aba mutants [ 29 , 30 ]. The first identified NCED gene from maize, VP14, is shown to be responsible for promoting seed dormancy and water stress resistance by controlling ABA levels in plants, and vp14 mutant displays early seed germination, reduced ABA biosynthesis and elevated water loss of detached leaves [ 21 ]. In Arabidopsis, nced3, a loss-of-function mutant, exhibits a water deficiency-sensitive phenotype [ 28 ], and nced3nced5 double mutant with much less ABA content displays more serious wilting phenotype than single mutant under drought stress [ 27 ]. In another research, introduction of PvNCED1 into Nicotiana plumbaginifolia increases ABA levels and enhances drought tolerance [ 24 ], and MhNCED3 into Arabidopsis results in enhanced tolerance to osmotic and cadmium stresses [ 26 ]. Recent reports have demonstrated that overexpression of OsNCED3 in rice enhances ABA accumulation, and therefore increases drought tolerance; however, the loss-of-function mutant osnced3 exhibits increased sensitivity to osmotic stress, accompanied by reduced ABA levels and increased stomata aperture under water stress [ 25 , 31 ].

Plants are often exposed to various environmental stresses, and drought and high salinity are major stress factors that impair plant growth and productivity of crops [ 1 ]. Environmental challenges activate a complex signaling network in plants, which determine plants to achieve optimal adaption to these unfavorable stress conditions ultimately [ 2 , 3 ]. The adaptation response is accomplished via regulating gene expression that alters plant metabolism and growth [ 4 , 5 ]. Particularly, osmotic stress due to drought or salinity triggers the biosynthesis of the phytohormone abscisic acid (ABA) which, in turn, regulates a range of plant physiological processes in response to various abiotic stresses [ 3 ]. It is widely accepted that ABA binding to PYR/PYL/RCAR proteins leads to deactivation of PP2Cs, which releases and activates SnRK2 kinases [ 6 , 7 ]. Activated SnRK2s further pass the signals to AREB/ABF TFs through protein phosphorylation on their conserved motifs like R-X-X-S/T (where X means any amino acids) [ 8 ], thus promoting the activity of downstream TFs to modulate the expression of various ABA-responsive genes [ 9 – 11 ]. In Arabidopsis, SnRK2.2, SnRK2.3 and SnRK2.6 have been shown to play essential roles in regulating ABA signaling [ 12 ]. In rice, there are 10 SnRK2s (designated as SAPK1-10, osmotic stress/ABA-activated protein kinase 1–10) that are found to be activated by osmotic stress, and only SAPK8, SAPK9 and SAPK10 are activated by ABA, suggesting their functions in osmotic stress and ABA signaling [ 13 ]. As homologs of SnRK2.2, SnRK2.3 and SnRK2.6 of Arabidopsis, SAPK8, SAPK9, and SAPK10 are able to phosphorylate and activate the downstream ABRE TFs [ 14 ]. In other studies, SAPK9 is shown to activate OsbZIP46 by phosphorylation under ABA or drought stress treatment in rice [ 15 , 16 ]. It is demonstrated SAPK10 phosphorylates TRAB1 and OsbZIP77 in vitro [ 14 , 17 ]. Actually, SAPK6 is found to be able to phosphorylate OsbZIP46, responding to ABA signaling in vivo [ 15 , 16 ]. More interestingly, SAPK2 is also found to be able to activate OsbZIP23 and OsbZIP46 by phosphorylation and promote the transcription of a large number of genes with functions in stress responses [ 15 , 16 , 18 ], which further expands our understanding on the functions of SAPKs in ABA signaling.

Results

Performance of osmads23 mutant and OsMADS23-overexpressing plants in growth OsMADS23 has been reported to be preferentially expressed in the root cylinder in rice previously [38]. In our study, two T-DNA insertion mutant alleles of OsMADS23, M1 (osmads23-1) and M2 (osmads23-2), were obtained, and they have different DNA insertion sites in the third intron of OsMADS23 (S1A Fig). We found that both M1 (-/-) and M2 (+/-) showed reduced growth indicated by plant height, compared to their corresponding wild type Zhonghua 11 (Z11) (Figs 1A–1G and S1B–S1D). No homozygous seeds of M2 were obtained, possibly because of the DNA deletion in M2 (S1E Fig). Then constitutive expression of OsMADS23 was performed in rice (Nip) and transgenic rice plants were obtained. Surprisingly, OsMADS23-overexpressing lines (OE1~OE20) also exhibited repressed growth (S2 Fig). Two OsMADS23-overexpressing lines (OE13 and OE14) were used for further evaluation. The shoot length at seedling stage as well as plant height at maturity in overexpression lines were markedly reduced (Fig 1H–1M), but their agronomic traits such as yield per plant and 1000-grain weight were not changed greatly (S1 Table). These results indicate that OsMADS23 plays important roles in plant growth and development, and too high or low expression of OsMADS23 affects plant growth. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Morphological phenotypes of osmads23 mutant and OsMADS23-overexpressing lines. (A) Schematic diagram indicating the T-DNA insertion sites in genomic region in osmads23-1 mutant (M1). Black boxes represent exons; lines between black boxes are introns. The arrow indicates the transcription orientation. (B) Quantitative PCR analysis of different regions of OsMADS23 in osmads23-1. (C) and (D) Phenotypes of wild type (Z11) and osmads23-1 for 10 and 80 days, respectively. (E) Internode morphology in images in (D). (F) and (G) Quantification of shoot length and internode length in wild type and osmads23-1. (H) Quantitative PCR analysis of OsMADS23 in OsMADS23-overexpressing lines (OE13 and OE14). (I) and (J) Phenotypes of wild type (Nip) and OsMADS23-overexpressing lines for 10 and 80 days, respectively. (L) Internode morphology in images in (J). (K) and (M) Quantification of shoot length and internode length of Nip and OsMADS23-overexpressing lines. The significant difference between osmads23-1 or OsMADS23-overexpressing lines and their corresponding wild type was determined by Student’s t test. *p < 0.05, **p < 0.01 or ***p < 0.001. All data displayed as a mean ± SD. M1, osmads23-1 mutant. In (G) and (M), two-way ANOVA was performed, followed by Bonferroni’s post-hoc test. Different letters indicate significant differences (p < 0.05). Three independent experiments were performed (n = 30 plants per genotype in each independent experiment). https://doi.org/10.1371/journal.pgen.1009699.g001

Responses of osmads23 mutant and OsMADS23-overexpressing plants to osmotic stress OsMADS23 was greatly induced by PEG, NaCl and mannitol (S3 Fig), which suggests that it is also likely to be crucial for improving plant tolerance to osmotic stress. We therefore investigated the responses of osmads23 mutant and OsMADS23-overexpressing lines to NaCl or PEG in medium, which mimics the salt or drought stress. In control medium for 7 days, overexpression lines grew more slowly than their corresponding wild-type plants (Nip); however, in the medium supplemented with NaCl or PEG, the performance of overexpression lines was remarkably better than that of wild type (S4A–S4C Fig), suggesting that the overexpression lines were less severely affected by osmotic stress than wild type. Expectedly, osmads23 mutant was substantially more sensitive to NaCl and PEG than its corresponding wild type (Z11) (S4D and S4E Fig), indicating that disruption of OsMADS23 caused hypersensitivity to osmotic stress in plants. After exposed to NaCl or PEG for 14 days, the difference between overexpression lines and wild type is much more apparent. The shoot growth in wild type was significantly inhibited under osmotic stress conditions, compared with that in untreated plants; however, the repression of shoot length in overexpression lines by NaCl or PEG is not severe (S4F and S4G Fig). The total chlorophyll content, which reflects the rate of chlorosis in seedlings under osmotic stress conditions, was reduced slightly in OsMADS23-overexpressing lines, but drastically in wild type, compared with that in their corresponding untreated plants (S4H Fig). These results suggest that OsMADS23 may play an important role in abiotic stress tolerance in plants.

OsMADS23 confers rice with the salinity tolerance In many cases, plants with improved drought tolerance can also resist salt stress [40,41]. In our salt tolerance tests, wild type (Nip) exhibited earlier and more severe wilting symptoms than OsMADS23-overexpressing lines, and the latter showed an obvious salt resistance phenotype (Fig 3A). Compared with an about 11% survival rate in wild type, about 50% of overexpression plants survived after a 8-day NaCl treatment followed by a 5-day recovery period (Fig 3B). In parallel to the salt-sensitive phenotype of seedlings, the detached leaves of wild type were observed to bleach more quickly than that of overexpression lines under salt stress (Fig 3C). Expectedly, much less ROS accumulated in overexpression lines after NaCl treatment (Fig 3D and 3E). Moreover, under salt stress, the activities of antioxidant enzymes and transcription of ROS-scavenging genes were significantly enhanced in overexpression plants, compared to that in wild type (Fig 3F–3H). In accordance with the enhanced salt tolerance, proline content was drastically higher but MDA levels were markedly lower in the overexpression lines than that in wild type (Fig 3I and 3J). Together, these data indicate that OsMADS23 is also an essential positive regulator in salt tolerance, and OsMADS23 can enhance the ability of adaption to osmotic stress in plants. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Phenotypes of OsMADS23-overexpressing lines under salt stress. (A) Images showing the phenotypes of wild type (Nip) and OsMADS23-overexpressing lines (OE13 and OE14) under salt stress. Twenty-eight-day-old plants were subjected to 300 mM salt stress and then resumed growth. Scale bars, 5 cm. (B) The survival rates of wild type and overexpression lines after 8 days of salt stress and then 5 days of resuming growth. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 48 plants). (C) The leaves detached from 60-day-old plants were exposed to 200 mM NaCl for 3 days to indicate the salt stress tolerance. Scale bars, 2 cm. (D) DAB staining for the leaves of plants exposed to salt stress for 5 days to indicate H 2 O 2 levels. Scale bars, 1.5 cm. (E) Quantification of H 2 O 2 content in the leaves from plants exposed to salt stress for 5 days. (F) and (G) Activities of SOD and CAT in plants exposed to salt stress for 5 days, respectively. (H) Expression of ROS-scavenging genes in plants exposed to salt stress for 3 days. (I) and (J) Content of proline and MDA in plants exposed to salt stress for 5 days, respectively. In (E) to (J), error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). All data are means ± SD. Three independent experiments were performed. https://doi.org/10.1371/journal.pgen.1009699.g003

Overexpression of OsMADS23 reduces the sensitivity to oxidative stress in plants The increased drought and salt resistance of OsMADS23-overexpressing plants (Figs 2 and 3) suggests that they might have the enhanced tolerance to oxidative stress. To further confirm this, the response of OsMADS23-overexpressing lines to oxidative stress was investigated by using MV, an oxidative stress inducer in plants. Two-day-old seedlings were grown on half-strength MS medium supplemented with 2 mM MV. In the medium without MV, overexpression plants grow more slowly than wild type; however, after 7 days of growth in MV, the growth impairment in wild type (Nip) was much more severe than that of overexpression lines, indicated by the shoot length (Fig 4A and 4B). On the contrary, osmads23 mutant was much more hypersensitive to oxidation stress than its corresponding wild type (Z11) (Fig 4A and 4C). Oxidation can cause degradation of chlorophyll and etiolating phenotypes [42]. Here, the detached leaves of OsMADS23-overexpressing plants exhibited much less sensitivity to oxidative stress, whereas osmads23 mutant had a quicker bleaching rate than its corresponding wild type (Fig 4D). After MV treatment, wild type (Nip) showed a severe reduction of chlorophyll (only 30% of chlorophyll of untreated plants retained), whereas the chlorophyll content of overexpression plants just decreased slightly (about 70% of untreated plants retained) (Fig 4E). By contrast, osmads23 mutant had more significant chlorophyll reduction than its wild type (Z11) (Fig 4E). These results further confirmed that OsMADS23 positively regulated the oxidation tolerance in plants, and its overexpression can attenuate oxidative damage under oxidative stress, suggesting that OsMADS23 is a promising candidate gene for improving the oxidation tolerance in plants. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Overexpression of OsMADS23 promoted plant adaption to oxidative stress. (A) Performance of OsMADS23-overexpressing plants (OE13 and OE14) or osmads23-1 mutant (M1) in half-strength medium supplemented with 2 μM MV for 7 days. Two-day-old seedlings were grown in medium with or without MV. Scale bars, 2 cm. (B) and (C) Shoot length of OsMADS23-overexpressing plants and osmads23-1 mutant in the medium with or without MV for 7 days, respectively. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 20 plants). (D) The detached leaves from 60-day-old plants were exposed to 5 μM MV for 3 days to indicate the oxidative tolerance. Scale bars, 1.5 cm. (E) The chlorophyll content of 3-day-old plants growing in the medium with or without MV for 7 days, respectively. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). MV, methyl viologen. *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). All data are means ± SD. Three independent experiments were performed. https://doi.org/10.1371/journal.pgen.1009699.g004

OsMADS23 mediates ABA sensitivity and is involved in ABA-induced stomatal closure in plants ABA has been widely considered as a stress hormone, and plant drought and salt responses are closely related to ABA sensitivity [7,30,43]. To further investigate whether OsMADS23 is involved in ABA responses, we investigated the seed germination as well as shoot and primary root (PR) elongation in different genotypes of OsMADS23 in response to exogenous ABA. Compared to control, both the seed germination and plant growth in OsMADS23-overexpressing lines were repressed more severely, whereas was much less severe in osmads23 mutant than their corresponding wild type (Figs 5A–5D and S6A–S6F). The results indicate that overexpression of OsMADS23 increases the sensitivity of plants to exogenous ABA, and suggest its potential role in ABA signaling. Meanwhile, the endogenous ABA accumulation in plants in response to drought stress was evaluated. As shown in Fig 5E, after drought for 3 days, the ABA levels were much higher in overexpression plants, but lower in osmads23 mutant than their corresponding wild type. PPT PowerPoint slide

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TIFF original image Download: Fig 5. OsMADS23 mediates ABA sensitivity and ABA-induced stomatal movement in rice. (A) Seed germination of OsMADS23-overexpressing plants (OE13 and OE14) or osmads23-1 mutant (M1) compared to their corresponding wild type (Nip or Z11) on half-strength MS medium without or with ABA for 4 days, respectively. Scale bars, 2 cm. (B) Seed germination rates scored from day 1 to day 4 after stratification on medium supplemented without or with 1 μM ABA, respectively. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 50 seeds). (C) Plant growth in half-strength MS medium without or with ABA for 4 days, respectively. Two-day-old seedlings were transferred on medium with or without ABA. Scale bars, 2 cm. (D) Decrease rate of shoot and root length in 1 μM ABA compared to mock on day 4. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 20 plants). (E) ABA content in OsMADS23-overexpressing lines and osmads23 mutant, and their corresponding wild type after exposed to drought stress. Two-week-old plants were subjected to drought stress for 3 days, and leaves were collected for measurement of ABA content. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 5 plants). (F) Percentages of completely open, partially open, and completely closed stomata in wild type (Nip) and OsMADS23-overexpressing plants under normal and ABA treatment conditions. Results represent means ± SD (n = 300) from 10 plants per genotype. Two-way ANOVA was performed, followed by Bonferroni’s post-hoc test. Scale bars, 10 μm. (G) Relative transcription levels of key genes involved in ABA-dependent stress response pathway in 2-week-old plants under drought conditions for 3 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). *p < 0.05, **p < 0.01 or ***p < 0.001 (Student’s t test). Three independent experiments were performed. https://doi.org/10.1371/journal.pgen.1009699.g005 It is well recognized that ABA promotes stomatal closure to avoid water loss under drought or salt stress. To explore possible cellular processes affected by OsMADS23 in improving plant osmotic stress tolerance, we compared the ABA-induced stomatal movement in OsMADS23-overexpressing lines with that in wild type (Nip). Under daylight conditions, stomata in rice leaves can be classified into three typical status categories: completely open, partially open, and completely closed [44] (Fig 5F). In the absence of ABA, there was little difference in the proportions of the three categories of stomata between overexpression lines and wild type (Fig 5F). However, after ABA treatment, the proportions of the completely and partially open stomata in overexpression lines were significantly lower than that in wild type, while the proportion of the completely closed stomata was markedly higher (Fig 5F). Hence, we concluded that OsMADS23 was involved in ABA-induced stomatal closure. Elevated endogenous ABA accumulation as well as ABA-induced stomatal closure suggests that the expression of ABA-responsive genes might be altered in overexpression plants. To verify this supposition, expression of stress-inducible marker genes that function in the ABA-dependent pathway was analyzed. As shown in Figs 5G and S6G, compared to mock, the expression of ABA-biosynthetic genes such as OsNCED2, OsNCED3, OsNCED4 and ABA-inducible genes OsP5CR and OsP5CS1 in overexpression lines was much higher than that in wild type under drought conditions. Together, these results clearly show that OsMADS23 regulates the drought and salt tolerance in plants, at least partially, through the ABA-dependent pathway.

osnced2 mutants exhibit reduced tolerance to drought and oxidation stress Having elucidated OsMADS23 regulates the osmotic stress tolerance by regulating ABA and proline biosynthesis via binding to the promoter regions of OsNCED2, OsNCED3, OsNCED4, and OsP5CR, we are interested in their potential contribution to osmotic stress resistance. OsNCED3 and OsNCED4 have been shown to promote ABA biosynthesis and abiotic stress tolerance [25,45]. OsP5CR is found to play crucial roles in salt tolerance [37,46]. The roles of OsNCED2 in drought stress tolerance in plants still remain elusive. Here we explored the contribution of OsNCED2 to water stress tolerance by using two independent homozygous mutants (osnced2-1 and osnced2-2, Z11 background) produced by CRISPR/Cas9 system. osnced2-1 has a two-nucleotide deletion, and osnced2-2 has a one-nucleotide insertion in the position of 87 bp after ATG, respectively, and these lead to frameshift mutations that promote early termination of protein translation (Figs 8A and S7A). OsNCED2, which has only one exon of 1710 bp in length, was constitutively expressed in various tissues in rice plants (S7B Fig). Knockout of OsNCED2 significantly impaired plant growth, indicated by the reduced shoot and root length (Fig 8B and 8C). Given NCED as the key rate-limiting enzyme in ABA biosynthesis, therefore, we first investigated the ABA accumulation in osnced2 mutants. As shown in Fig 8D, the ABA levels were significantly reduced in osnced2 mutants compared to that in wild type (Z11). Then, the contribution to the drought stress tolerance made by OsNCED2 was evaluated. After withdrawing water and then rewatering, approximately 35.2% of the wild type plants survived, but only 3.2% of osnced2-1 and 8.6% of osnced2-2 plants recovered, respectively (Fig 8E and 8F). The water stress sensitivity of the osnced2 mutants was confirmed by the oxidation stress assay. When exposed to MV, the detached leaves of osnced2 mutants were observed to bleach more quickly than that of wild type (Fig 8G). Additionally, the osnced2 mutants had more severe decrease of chlorophyll than wild type in the presence of MV (Fig 8H). These results indicate that osnced2 mutants have a reduced capacity for osmotic and oxidant stress tolerance. PPT PowerPoint slide

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TIFF original image Download: Fig 8. osnced2 mutants were more sensitive to drought and oxidation stress than wild type. (A) Schematic diagram of CRISPR-Cas9-mediated target mutagenesis of OsNCED2. (B) Phenotypes of osnced2 mutants and wild type (Z11) in half-strength medium for 7 days. Scale bars, 4 cm. (C) Shoot and root length of wild type and osnced2 mutants in half-strength medium for 7 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 20 plants). (D) ABA content in the leaves of wild type (Z11) and osnced2 mutants growing for 20 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). (E) Images showing the phenotypes of wild type and osnced2 mutants under drought stress. Twenty-day-old plants were subjected to drought stress and then rewatering. Scale bars, 5 cm. (F) The survival rates of wild type and osnced2 mutants after drought stress and rewatering. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 48 plants). (G) The detached leaves from 70-day-old plants were exposed to 5 μM MV for 3 days to indicate the oxidative tolerance. Scale bars, 2 cm. (H) The chlorophyll content of wild type and osnced2 mutants in MV for 7 days. Error bars indicate SD with biological triplicates (n = 3, each replicate containing 3 plants). MV, methyl viologen. *p < 0.05, **p < 0.01 (Student’s t test). Three independent experiments were performed. https://doi.org/10.1371/journal.pgen.1009699.g008

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