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Article

OsABT Negatively Regulates the Abscisic Acid Signal Transduction Pathway in Rice Seedling Roots

by
Lingran Bao
,
Yi Shen
,
Yijie Yan
,
Xuanzhu Huang
,
Danni Wen
* and
Bo Shen
*
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(11), 2683; https://doi.org/10.3390/agronomy14112683
Submission received: 10 October 2024 / Revised: 11 November 2024 / Accepted: 12 November 2024 / Published: 14 November 2024
(This article belongs to the Special Issue Hormone Metabolism and Signaling in Rice)
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Abstract

:
Rice (Oryza sativa L.) is a main food crop in China and is crucial for the maintenance of national food security. The growth of rice seedling roots is regulated by a variety of genes and is closely related to abscisic acid (ABA) metabolism and ABA signaling pathways. In this study, we found that OsABT could increase the length of rice root tip meristem cells and upregulate root development-related genes, thereby alleviating ABA’s inhibitory effects on rice root growth and seed germination. The overexpression of OsABT reduced the ABA content by downregulating ABA synthesis genes (OsNCED3 and OsNCED5) and upregulating the ABA catabolic gene (OsABA8ox2). In addition, OsABT interacted with OsPYL4, OsPYL10, and OsABIL2 via the ABA signal transduction pathway. By inhibiting the expression of positive regulatory genes (OsPYL9 and Rab16a) and increasing the expression of a negative regulatory gene (OsABIL1), OsABT negatively regulates the ABA signal transduction pathway. Transcriptome analysis revealed that OsABT inhibited the activity of Gene Ontology entries in response to ABA. Thus, OsABT increased the length of the rice root meristem, reduced the accumulation of ABA in the roots, and negatively regulated the ABA signal transduction pathway by interacting with key proteins in this pathway, ultimately relieving the inhibitory effect of ABA on rice root length and seed germination.

1. Introduction

Rice (Oryza sativa L.) is the major staple food for more than half of the world’s population. Therefore, ensuring the stability of rice production is essential for global food security [1]. Rice root growth plays key roles in determining rice yield and is affected by internal factors, such as hormones and gene expression, along with external factors, such as the environment. Among plant hormones, abscisic acid (ABA) regulates rice root growth and development [2]. A core ABA signaling pathway has been established in Arabidopsis Thaliana, consisting of three components: PYR/PYL/RCARs, protein phosphatase 2 (cPP2C), and SNF1-associated protein kinase 2 (SnRK2s). In this pathway, PYR/PYL/RCARs act as ABA receptors, PP2Cs as negative regulators of the pathway, and SnRK2s as positive regulators of downstream signaling. In the absence of ABA, PP2Cs is active and has inhibitory effects on SnRK2s activity and downflow signal production. When ABA is present, PYR/PYL/RCARs interact with PP2Cs, inhibit PP2Cs phosphatase activity, activate SnRK2s and the phosphorylation of target proteins, regulate downstream gene transcription, and stimulate anion efflux, cation efflux, and ROS production in the plasma membrane [3,4,5].
Studies have found that ABA treatments significantly inhibit the growth rate of roots and shoots during seed germination, mainly inhibiting seed germination by regulating the loosening and swelling of the cell wall, which is regulated by three core components, including PYR/PYL receptors on the ABA signaling pathway [6,7]. Meanwhile, the effect of ABA on root elongation is influenced by the conserved PYR/PP2C/SnRK2 ABA signaling pathway. ABA can inhibit root elongation, but the ABA receptor pyr1pyl1pyl1pyl2pyl4 quadruple mutants are insensitive to ABA’s effect on root elongation. Conversely, abi1 and abi2 mutants are hypersensitive to ABA [4,5,8]. Studies have demonstrated that overexpression of SAPK10 can lead to the formation of longer root hairs, while the overexpression of OsABIL2 can lead to shorter root hairs [9,10]. Additionally, bZIP transcription factors are essential regulators of the ABA signaling pathway. The overexpression of OsbZIP23 and OsbZIP46 increases the sensitivity of rice to ABA, and the growth of the roots of rice overexpressing these genes is inhibited by ABA, i.e., these roots exhibit slow growth [11,12].
WD40 proteins are much more abundant and highly conserved in eukaryotes and play key roles in the response of rice to hormones. OsRACK1A, a WD40 protein that responds to exogenous ABA and drought stress, positively regulates rice seed germination by enhancing ABA metabolic processes [13,14,15]. The ABA signaling terminator (ABT) is a gene encoding an Arabidopsis WD40 repeat protein, which negatively regulates the ABA signaling pathway [16]. In our laboratory, previous studies have identified OsABT (Os03g0738700) and its regulation by ABA [17]. However, the mechanism by which the OsABT gene responds to ABA and acts on rice roots remains unknown. In this study, wild-type (Nipponbare) and OsABT-overexpression rice plants (OE-3 and OE-4) were used as materials to investigate root length, germination rate, ABA content, and gene expression through transcriptome analysis and yeast two-hybrid (Y2H) experiments. The rice samples in these experiments were treated with ABA and the ABA inhibitor fluridone (Flu) for 7 d. By exploring the physiological and molecular mechanism of OsABT in response to ABA during rice root development and its role in the ABA signal transduction pathway, we suggested that OsABT negatively regulate the ABA signaling pathway and alleviate the inhibition of root elongation and seed germination during the growth stage of rice seedlings.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Rice (Oryza sativa L.) genotype Nipponbare and two 35S::OsABT-overexpression rice lines (designated as OE-3 and OE-4) were used as experimental materials [18]. The rice seeds were soaked in water for 2 d, then germinated on moist gauze for another 2 d. After germination, the rice seeds were transferred to 96-well plates and cultured in a growth chamber at a constant temperature of 30 °C with a 14 h light/10 h dark photoperiod. During the cultivation process, the rice seedlings were exposed to 2 μmol/L ABA and 0.1 μmol/L Flu solution for 7 d. The root length was directly measured using a ruler. The total rice root surface area and average root diameter were measured using a WinRHIZO plant root analyzer (Regent Co., Ltd., Quebec, Canada), and the other root samples were quickly frozen in liquid nitrogen and stored at −80 °C for later use.

2.2. Germination Experiment

To observe the germination of rice seeds, three treatments were established: (1) 2 μM ABA; (2) 0.1 μM Flu; and (3) distilled water (H2O; blank control). After 24 h of water absorption, the 30 ungerminated seeds were placed in a Petri dish lined with two layers of filter paper. Three replicates were set up for each treatment, and the seeds were then cultured in the dark at 30 °C. Rice seed germination was observed and photographed every 12 h.

2.3. Propidium Iodide Staining

To observe the growth of root tip meristem cells, the root tip was cut upwards by approximately 1.5 cm using a blade. The roots were vacuumed in pure water for 30 min, stained with 5 mg/L propidium iodide staining solution for 10 min in the dark, and then rinsed in deionized water for 3–5 min. Finally, the samples were placed on glass slides containing 50% glycerol, covered with a cover slip, and observed and photographed using a laser scanning confocal microscope (Leica SP8, Wetzlar, Germany).

2.4. RNA Extraction and qRT-PCR Analyses

Utilizing the HiFiScript cDNA Synthesis Kit (YEASEN, Shanghai, China), first-strand cDNA was synthesized after extracting total RNA from rice roots with an RNA extraction kit (CWBIO, Jiangsu, China). The SYBR Premix Ex Taq II kit (TaKaRa, Dalian, China), which facilitated the execution of qRT-PCR on the CFX96TM fluorescence quantitative PCR instrument (Bio-Rad, CA, USA). Each reaction was meticulously carried out in triplicate, accounting for both biological and technical replication. The rice actin gene (Os03g0718100) was utilized as an internal standard. The 2−ΔΔCT method was used to calculate the relative expression levels of genes. The primers used for qRT-PCR are presented in Table S1.

2.5. Y2H Analysis

The pBD-GAL4 Cam vector was used to incorporate the full-length encoding sequence of OsABT, while the pAD-GAL4-2.1 vector was linked with the complete coding sequences of OsABIL2, OsPYL4, and OsPYL10. Following this, the constructs were introduced into the yeast strain YRG2 for transformation. Initially, the transformed yeast clones were cultivated on the SD/-Leu/-Trp/-Ura medium, then transferred to the SD/-Leu/-Trp/-His/-Ura medium and incubated at 30 °C for 4 d. The primer sequences for the yeast two-hybrid (Y2H) analysis are presented in Table S1.

2.6. Bimolecular Fluorescence Complementation Assay

The complete coding sequences of OsABT were subcloned into the pCAMBIA1300-YFPC vector and fused with the N- terminal or C-terminal fragment of yellow fluorescent protein (YFP). Similarly, OsABIL2, OsPYL4, and OsPYL10 were subcloned into the pCAMBIA1300-YFPN vector. The corresponding bimolecular fluorescence complementation (BiFC) plasmid was co-expressed with the negative controls in Nicotianabenthamiana leaves. The cells expressing YFP signals were observed and recorded after 48 h using a confocal laser scanning microscope (Leica SP8, Wetzlar, Germany).

2.7. Transcriptome Analysis

Total RNA was separated three times from the roots of the OE-3, OE-4, and Nipponbare samples after treated with 2 μM ABA and 0.1 μM Flu for 7d. TRIzol reagents (Invitrogen, Carlsbad, CA, USA) were used to isolate and purify total RNA from the root samples and then extract mRNA. The cDNA libraries were constructed using reverse transcriptase and DNA polymerase, and paired-end sequencing was conducted using Illumina Novaseq 6000. After the sequencing was completed, the data were analyzed using DEGseq2 software (https://www.omicstudio.cn/index, accessed on 11 October 2024) to identify significantly differentially expressed genes. Genes with a fold change ≥2 or ≤0.5 and a p-value < 0.05 were defined as differentially expressed genes (DEGs). Functional annotation and pathway analysis of these differentially expressed genes was performed using the GO and KEGG databases.

2.8. Statistical Analysis

The mean ± standard deviation (SD) was calculated for all results, and a Student’s t-test was performed to calculate the p-values. All statistical analyses were conducted using SPSS 20.0 software.

3. Results

3.1. OsABT Relieves the Inhibition of ABA on Rice Root Growth

To investigate the mechanism of the OsABT gene response to ABA, OsABT-overexpression (OE-3 and OE-4) and wild-type (Nipponbare) plants were treated with H2O (control), 0.2 μM ABA, 2 μM ABA, 0.05 μM Flu, and 0.1 μM Flu solution for 7 d. After ABA treatment, the rice roots were significantly shorter, and the inhibitory effects of ABA on rice roots were enhanced with the increase in ABA concentrations (Figure 1A). However, ABA had a greater impact on the roots of Nipponbare plants than on those of OE-3 and OE-4 plants. After treated with 2 μM ABA, the total root length of Nipponbare reached only 37.72% of the control (p < 0.01), while that of OE-3 and OE-4 reached 74.75% and 63.84% of the control (p < 0.05), respectively (Figure 1B). Moreover, ABA treatment reduced the root surface area and thickened the roots; however, Nipponbare rice was more affected than OE-3 and OE-4 rice (Figure 1C,D).
After treated with Flu, no significant differences in root phenotype were observed between OE-3 or OE4 rice and Nipponbare. The length of the rice root apical meristem zone was measured using ImageJ software (https://imagej.net/software/fiji/downloads, accessed on 11 October 2024). The meristem zone is defined as the region where the length-to-width ratio of cells from the apical stationary center to the second cell layer is ≤2. After being treated with ABA, the meristem length of OE-3 was 47.15 μm longer than that of Nipponbare (p < 0.05), and the meristem length of OE-4 was 217.56 μm longer than that of Nipponbare (p < 0.01) (Figure 2). These results indicate that OsABT can suppress the inhibitory effects of ABA on the growth of rice root tip meristem cells, ultimately countering the ABA-mediated inhibition of rice root growth.

3.2. OsABT Relieves the Inhibition of ABA on Rice Seed Germination

We investigated whether OsABT was involved in rice seed germination following exogenous ABA treatment. The seeds of Nipponbare, OE-3, and OE-4 rice were treated with H2O (control), 2 μM ABA, and 0.1 μM Flu solution for 72 h, and germination was recorded every 12 h (Figure 3). ABA treatment significantly inhibited the germination rate of the rice seeds. At 24 h, the germination rate of Nipponbare was 6.67%, whereas the germination rates of OE-3 and OE-4 were 32.22% and 50%, respectively, which were significantly higher than those of Nipponbare. At 72 h, the germination rates of OE-3 and OE-4 were close to 100%, while the germination rate of Nipponbare was only 65.56%. These results show that OsABT can relieve the inhibition of ABA during rice seed germination.

3.3. OsABT Reduces the Accumulation of ABA in Rice Roots

To clarify whether OsABT relieves ABA inhibition by reducing ABA content in rice roots, we analyzed the ABA content in the roots of rice seedling. Exogenous ABA treatment led to an increase in ABA content, with increased ABA accumulation being observed in the roots of Nipponbare. The ABA contents in OE-3 and OE-4 were 84.13% and 91.75% of that in Nipponbare, respectively (Figure 4).
Additionally, expression analysis of ABA metabolism-related genes showed that ABA treatment decreased the expression of OsNCED3 and OsNCED5. Compared with Nipponbare, the expression in OE-3 and OE-4 was lower. OsNCED3 expression in OE-3 and OE-4 was 32.74% and 48.53% of that in Nipponbare, whereas OsNCED5 expression in OE-3 and OE-4 was 88.79% and 71.83% of that in Nipponbare, respectively (Figure 5A,B). Moreover, OsABA8ox2 expression in OE-3 was significantly higher than that in Nipponbare after ABA treatment (Figure 5C). This indicates that OsABT can reduce the accumulation of ABA in rice seedlings by regulating the expression level of ABA metabolism-associated genes.

3.4. OsABT Positively Regulates the Expression of Development-Related Genes

To further investigate whether OsABT regulates root length by regulating root development-related genes, the expression of the root development-related genes OsCRL4 and OsEXPA8 was analyzed. After ABA treatment, the expression of root development-related genes decreased; however, compared with Nipponbare, the expression was higher in OE-3 and OE-4 rice roots. The expression levels of OsCRL4 in OE-3 and OE-4 were 1.02 and 1.17 times higher than those in Nipponbare, respectively, and the expression levels of OsEXPA8 were 1.27 and 1.42 times higher than those in Nipponbare, respectively (Figure 6). These results indicate that OsABT positively regulates rice root length by upregulating root development-related genes.

3.5. OsABT Negatively Regulates the ABA Signal Transduction Pathway

To explore the relationship between OsABT and the ABA signaling pathway, the OsABT protein and key proteins in the ABA signaling pathway were verified using Y2H and BiFC experiments. The Y2H experiments showed that the yeast in the negative control could not cultivated on the four-deficient medium SD/-Trp/-Leu/-His/-Ura, whereas the positive control and experimental groups grew normally (Figure 7A). In the BiFC experiment, the negative control group showed no fluorescence, whereas the positive control and experimental groups showed fluorescence (Figure 7B). These results suggest that OsABT interacts with OsABIL2, OsPYL4, and OsPYL10, and that the interaction between OsABT and OsABIL2 is the strongest.
The expression level of the positive regulatory genes OsPYL9 and Rab16a and the negative regulatory gene OsABIL1 in the ABA signaling pathway were analyzed. The expression of positive regulatory genes in the ABA signaling pathway was induced, and the expression of negative regulatory genes was inhibited by ABA. Compared with Nipponbare, the expression of positive regulatory genes in OE-3 and OE-4 was lower, and the expression of negative regulatory genes was higher (Figure 8).
To further understand the mechanism of the OsABT gene’s response to ABA, transcriptome sequencing was performed on the rice roots. Transcriptome analysis showed that 988 and 554 differential expressed genes (DEGs) were downregulated in OE-3 and OE-4, respectively, compared to Nipponbare after ABA treatment. In GO terms, analysis of the response to ABA showed that most of the DEGs were downregulated in OE-3 and OE-4 rice under ABA treatment (Figure 9). Gene set enrichment analysis (GSEA) also showed that most genes were clustered at the bottom, and a peak appeared at the back of the sorted gene set, indicating that the GO term responding to ABA was inhibited (Figure 10). These results indicate that OsABT negatively regulates the ABA signaling pathway.

4. Discussion

ABA is one of the main hormones in rice, and it plays an essential role in regulating its growth and development. ABT negatively regulates the ABA signaling pathway in Arabidopsis; however, its response to ABA in rice roots is unclear. Previous studies have shown that the expression of OsABT is induced by ABA and salt and that OsABT can significantly enhance tolerance to ABA and NaCl [17,18]. In this paper, OsABT negatively regulated the ABA signaling pathway and alleviated the inhibition of root elongation and seed germination in rice seedlings.

4.1. OsABT Alleviates the Inhibition of ABA on Rice Root Growth and Seed Germination

ABA is a sesquiterpene hormone that inhibits plant growth and development [19], particularly root elongation. Studies in Arabidopsis have found that ABA can inhibit the total root length and elongation of primary roots, and that these inhibitory effects are enhanced with an increase in the ABA concentration [20]. Moreover, the root length of ZH11 seedlings decreased from 10.4 cm to 8.5 and 6.9 cm after treatment with 0.5 and 1 μM ABA, respectively [21]. The cell division and differentiation in root meristems are also regulated by ABA. The root of the arf2 mutant was inhibited by exogenous ABA, while the number of meristem cells decreased and the length of the meristem became shorter [22,23]. In this study, following ABA treatment, the OsABT-overexpressing rice showed greater root growth (length), a larger total root surface area, and a thicker average root diameter compared to Nipponbare. Propidium iodide staining also showed that the meristem length of OsABT-overexpressing rice was longer than that of Nipponbare after ABA treatment. Meanwhile, the expression of the root development-related genes OsCRL4 and OsEXPA8 in OsABT-overexpressing rice was also higher than that in Nipponbare after ABA treatment. This indicates that OsABT could relieve the inhibition of root growth by exogenous ABA in rice seedlings.
Germination plays an important role in seedling growth, and studies have shown that ABA content during seed germination is crucial [24]. As a positive regulator of rice seed germination, OsSEA1 enhances the germination rate of OsSEA1-overexpressing rice by directly binding to the promoter of OsABI5 in the ABA signal transduction pathway, whereas FON1 regulates seed germination by activating ABA-responsive genes [25,26]. In the germination rate experiment of this study, ABA reduced the germination rate of rice, and the germination rate of Nipponbare seeds decreased significantly. Compared with Nipponbare, the germination of OsABT-overexpressing rice seeds was less affected. This indicates that OsABT could relieve the inhibitory effect of ABA on seed germination.

4.2. OsABT Reduces the Accumulation of ABA in Rice Roots and Negatively Regulates the ABA Signal Transduction Pathway

ABA accumulation is mainly regulated by the ABA metabolic process, and NCED3 is a key enzyme involved in ABA synthesis. The overexpression of OsNCED3 can regulate rice growth and tolerance to abiotic stress. The knockout of OsNCED3 can reduce the ABA content in rice, promote embryo growth, and advance seed germination time [27,28]. OsNCED5 is induced by ABA and salt and regulates the expression of ABA-dependent abiotic stress- and senescence-related genes. The ectopic expression of OsNCED5 in Arabidopsis delays seed germination [29,30]. In this study, following ABA treatment, the expression of the ABA synthesis genes OsNCED2 and OsNCED5 in OsABT-overexpressing rice was lower than that in Nipponbare. In addition, the ABA content in the roots of rice seedlings increased after ABA treatment; however, the ABA content in OsABT-overexpressing rice was significantly lower than that in Nipponbare. This indicates that OsABT could reduce ABA accumulation in rice roots.
The effect of ABA on root elongation is influenced by the conserved PYR/PP2C/SnRK2 ABA signaling pathway. The ABA signaling pathway in rice mainly includes OsPYL/RCAR receptors, OsPP2C phosphatase, SAPK protein kinase, downstream transcription factors, and ABA response genes. OsPYL9 positively regulates seed germination, and the overexpression of OsPYL9 can improve drought and cold tolerance in rice. OsPYL10 can also enhance drought tolerance by closing the stomata [31,32]. OsABIL1 and OsABIL2 are homologous genes of ABI1/2/3 in Arabidopsis. The overexpression of OsABIL2 reduces the sensitivity of rice to ABA and salt stress [10], while the overexpression of Rab16 can improve salt tolerance [33]. In this study, the expression of the positive regulatory genes OsPYL9 and Rab16a in OE-3 and OE-4 rice was lower than that in Nipponbare, and the expression of the negative regulatory gene OsABIL1 was higher than that in Nipponbare. In Arabidopsis, ABT has been shown to interact with PYR1/PYL and PP2C to inhibit ABA signal transduction. The Y2H and BiFC experiments also confirmed that the OsABT protein interacted with OsPYL4, OsPYL10, and OsABIL2 in the ABA signal transduction pathway. Enrichment analysis of ABA-responsive GO entries revealed that most of the DEGs were downregulated in OE-3 and OE-4 rice, and GSEA revealed that the overexpression of OsABT inhibited the activity of this pathway. These results indicate that OsABT negatively regulates the ABA signaling pathway by interacting with key proteins in the pathway and reducing ABA accumulation.

5. Conclusions

OsABT was found to negatively regulate the ABA signaling pathway by affecting the expression of ABA metabolism-related genes and interacting with key proteins in this pathway, leading to the expression of ABA signaling pathway-related genes. As a result, OsABT increased the length of the root apical meristem and upregulated the expression of genes related to root development. This alleviated the effects of ABA on rice root growth and seed germination. In summary, OsABT played a crucial role in rice root growth by regulating ABA signaling pathway, suggesting its potential as a target for improving rice yield under challenging environmental conditions.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14112683/s1. Table S1: List of primers used in quantitative real-time PCR analysis.

Author Contributions

D.W. and B.S. designed and planned the research. L.B., Y.S., Y.Y. and X.H. performed the experiments and data analyses. L.B. and D.W. wrote the original draft. D.W. and B.S. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Zhejiang Science and Technology Major Program on Agri cultural New Variety Breeding, grant number 2021C02063-6-4.

Data Availability Statement

The data are available upon request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phenotypic differences between Nipponbare (WT) and OsABT-overexpressing (OE-3 and OE-4) rice roots after different treatments. (A) Phenotypic images of three rice varieties under different treatments for 7 d. Note: H2O, 0.2 μMABA, 2 μMABA, 0.05 μMFlu, and 0.1 μMFlu solutions were applied to rice plants (n = 5 per group) from left to right for 7 d. (B–D) The changes in the total root length, root surface area, and average root diameter of the three groups of rice were measured after treatment with H2O, 2 μMABA, and 0.1 μM Flu solutions for 7 d. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to H2O.
Figure 1. Phenotypic differences between Nipponbare (WT) and OsABT-overexpressing (OE-3 and OE-4) rice roots after different treatments. (A) Phenotypic images of three rice varieties under different treatments for 7 d. Note: H2O, 0.2 μMABA, 2 μMABA, 0.05 μMFlu, and 0.1 μMFlu solutions were applied to rice plants (n = 5 per group) from left to right for 7 d. (B–D) The changes in the total root length, root surface area, and average root diameter of the three groups of rice were measured after treatment with H2O, 2 μMABA, and 0.1 μM Flu solutions for 7 d. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to H2O.
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Figure 2. The images of root apical meristem zone of Nipponbare (WT) and OsABT-overexpressing rice (OE-3 and OE-4). The treatments were with H2O (A–C), 2 μMABA (D–F), and 0.1 μM Flu (G–I). The white arrows in the figure indicate the location of the meristem. Each image is arranged from left to right as the fluorescence channel, bright field, and superposition field. Scale: 100 μm. (J) The changes in meristem length of three groups of rice roots treated with H2O, 2 μMABA, and 0.1 μM Flu for 7 d. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
Figure 2. The images of root apical meristem zone of Nipponbare (WT) and OsABT-overexpressing rice (OE-3 and OE-4). The treatments were with H2O (A–C), 2 μMABA (D–F), and 0.1 μM Flu (G–I). The white arrows in the figure indicate the location of the meristem. Each image is arranged from left to right as the fluorescence channel, bright field, and superposition field. Scale: 100 μm. (J) The changes in meristem length of three groups of rice roots treated with H2O, 2 μMABA, and 0.1 μM Flu for 7 d. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
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Figure 3. Germination rate of Nipponbare (WT) and OsABT-overexpressing rice (OE-3 and OE-4). The germination photos were taken after treatment with H2O (A), 2 μMABA (B), and 0.1 μM Flu (C) solutions for 72 h. The germination rate was measured every 12 h after treatment with H2O, 2 μM ABA, and 0.1 μM Flu solutions (D–F).
Figure 3. Germination rate of Nipponbare (WT) and OsABT-overexpressing rice (OE-3 and OE-4). The germination photos were taken after treatment with H2O (A), 2 μMABA (B), and 0.1 μM Flu (C) solutions for 72 h. The germination rate was measured every 12 h after treatment with H2O, 2 μM ABA, and 0.1 μM Flu solutions (D–F).
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Figure 4. The changes in ABA content of Nipponbare (WT) and OsABT-overexpressing rice (OE-3 and OE-4). The asterisks indicate differences at * p < 0.05 compared to Nipponbare. The data are performed as the means ± SDs of three independent experiments.
Figure 4. The changes in ABA content of Nipponbare (WT) and OsABT-overexpressing rice (OE-3 and OE-4). The asterisks indicate differences at * p < 0.05 compared to Nipponbare. The data are performed as the means ± SDs of three independent experiments.
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Figure 5. Relative expression levels of ABA metabolism-related genes under different treatments. (A) OsNCED3, (B) OsNCED5, and (C) OsABA8ox2. The amplification of Actin was utilized as the internal control. The relative expression levels of the genes were calculated using the 2−ΔΔCt method. The data are presented as the means ± SDs of three independent experiments. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
Figure 5. Relative expression levels of ABA metabolism-related genes under different treatments. (A) OsNCED3, (B) OsNCED5, and (C) OsABA8ox2. The amplification of Actin was utilized as the internal control. The relative expression levels of the genes were calculated using the 2−ΔΔCt method. The data are presented as the means ± SDs of three independent experiments. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
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Figure 6. Relative expression levels of root development-related genes under different treatments. (A) OsCRL4 and (B) OsEXPA8. The amplification of Actin was utilized as the internal control. The relative expression levels of the genes were calculated using the 2−ΔΔCt method. The data are presented as the means ± SDs of three independent experiments. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
Figure 6. Relative expression levels of root development-related genes under different treatments. (A) OsCRL4 and (B) OsEXPA8. The amplification of Actin was utilized as the internal control. The relative expression levels of the genes were calculated using the 2−ΔΔCt method. The data are presented as the means ± SDs of three independent experiments. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
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Figure 7. Interaction between OsABT and key proteins in the ABA signaling pathway. (A) Y2H assay. (B) BiFC assay. Yellow fluorescence indicates the interaction of two fusion proteins.
Figure 7. Interaction between OsABT and key proteins in the ABA signaling pathway. (A) Y2H assay. (B) BiFC assay. Yellow fluorescence indicates the interaction of two fusion proteins.
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Figure 8. Relative expression levels of ABA signaling pathway-related genes under different treatments. (A) OsPYL9, (B) Rab16a, and (C) OsABIL1. The amplification of Actin was utilized as the internal control. The relative expression levels of the genes were calculated using the 2−ΔΔCt method. The data are presented as the means ± SDs of three independent experiments. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
Figure 8. Relative expression levels of ABA signaling pathway-related genes under different treatments. (A) OsPYL9, (B) Rab16a, and (C) OsABIL1. The amplification of Actin was utilized as the internal control. The relative expression levels of the genes were calculated using the 2−ΔΔCt method. The data are presented as the means ± SDs of three independent experiments. The asterisks indicate differences at * p < 0.05 and ** p < 0.01 compared to Nipponbare.
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Figure 9. Cluster analysis of DEGs involved in response to ABA GO terms in each comparison group. N: Nipponbare, S: overexpression rice OE-3, (A): ABA treatment, (B): overexpression rice OE-4.
Figure 9. Cluster analysis of DEGs involved in response to ABA GO terms in each comparison group. N: Nipponbare, S: overexpression rice OE-3, (A): ABA treatment, (B): overexpression rice OE-4.
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Figure 10. GSEA of the response to ABA in Nipponbare (N), OE-3 (S), and OE-4 (B). The x-axis represents the ranked list of the genes; the y-axis represents the running enrichment score (ES); the red curve shows the position of the selected gene set; the green curve shows the position of the gene set with random phenotype labels. (A) NES = −1.621; FDR q-value = 0.108; (B) NES = −1.498; FDR q-value = 0.258.
Figure 10. GSEA of the response to ABA in Nipponbare (N), OE-3 (S), and OE-4 (B). The x-axis represents the ranked list of the genes; the y-axis represents the running enrichment score (ES); the red curve shows the position of the selected gene set; the green curve shows the position of the gene set with random phenotype labels. (A) NES = −1.621; FDR q-value = 0.108; (B) NES = −1.498; FDR q-value = 0.258.
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MDPI and ACS Style

Bao, L.; Shen, Y.; Yan, Y.; Huang, X.; Wen, D.; Shen, B. OsABT Negatively Regulates the Abscisic Acid Signal Transduction Pathway in Rice Seedling Roots. Agronomy 2024, 14, 2683. https://doi.org/10.3390/agronomy14112683

AMA Style

Bao L, Shen Y, Yan Y, Huang X, Wen D, Shen B. OsABT Negatively Regulates the Abscisic Acid Signal Transduction Pathway in Rice Seedling Roots. Agronomy. 2024; 14(11):2683. https://doi.org/10.3390/agronomy14112683

Chicago/Turabian Style

Bao, Lingran, Yi Shen, Yijie Yan, Xuanzhu Huang, Danni Wen, and Bo Shen. 2024. "OsABT Negatively Regulates the Abscisic Acid Signal Transduction Pathway in Rice Seedling Roots" Agronomy 14, no. 11: 2683. https://doi.org/10.3390/agronomy14112683

APA Style

Bao, L., Shen, Y., Yan, Y., Huang, X., Wen, D., & Shen, B. (2024). OsABT Negatively Regulates the Abscisic Acid Signal Transduction Pathway in Rice Seedling Roots. Agronomy, 14(11), 2683. https://doi.org/10.3390/agronomy14112683

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