Next Article in Journal
NopAA and NopD Signaling Association-Related Gene GmNAC27 Promotes Nodulation in Soybean (Glycine max)
Next Article in Special Issue
Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review
Previous Article in Journal
The Potential Utility of Salivary and Tear Proteomics to Discriminate Sjögren’s Disease from Non-Sjögren’s Sicca
Previous Article in Special Issue
Immunocytochemical Analysis of Bifid Trichomes in Aldrovanda vesiculosa L. Traps
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 24
10.3390/ijms242417495
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Rice OsANN9 Enhances Drought Tolerance through Modulating ROS Scavenging Systems

by
Yangyang Jia
1,2,†,
Xiangyang Gu
1,2,†,
Jiaxin Chai
1,2,
Xiaohong Yao
1,2,
Shoutao Cheng
1,2,
Lirui Liu
1,2,
Saiya He
1,2,
Yizhuo Peng
1,2,
Qian Zhang
1,2,* and
Zhengge Zhu
1,2,*
1
Ministry of Education Key Laboratory of Molecular and Cellular Biology, Hebei Research Center of the Basic Discipline of Cell Biology, College of Life Sciences, Hebei Normal University, Shijiazhuang 050024, China
2
Hebei Collaboration Innovation Center for Cell Signaling and Environmental Adaptation, Hebei Key Laboratory of Molecular and Cellular Biology, Shijiazhuang 050024, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(24), 17495; https://doi.org/10.3390/ijms242417495
Submission received: 14 November 2023 / Revised: 11 December 2023 / Accepted: 13 December 2023 / Published: 15 December 2023
(This article belongs to the Special Issue Recent Advances in Abiotic Stress Signaling)
Browse Figures
Review Reports Versions Notes

Abstract

:
Drought is a critical abiotic stress which leads to crop yield and a decrease in quality. Annexins belong to a multi-gene family of calcium- and lipid-binding proteins and play diverse roles in plant growth and development. Herein, we report a rice annexin protein, OsANN9, which in addition to regular annexin repeats and type-II Ca2+ binding sites, also consists of a C2H2-type zinc-finger domain. We found that the expression of OsANN9 was upregulated by polyethylene glycol (PEG) or water-deficient treatment. Moreover, plants that overexpressed OsANN9 had increased survival rates under drought stress, while both OsANN9-RNAi and osann9 mutants showed sensitivity to drought. In addition, the overexpression of OsANN9 increased superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activities, which regulate reactive oxygen species homeostasis. Collectively, these findings indicate that OsANN9 may function as a positive regulator in response to drought stress by modulating antioxidant accumulation. Interestingly, the setting rates of osann9 mutant rice plants significantly decreased in comparison to wild-type plants, suggesting that OsANN9 might be involved in other molecular mechanisms in the rice seed development stage.
Keywords:
rice; OsANN9; drought stress; ROS

1. Introduction

Drought is one of the major abiotic stresses that affect plant growth and development and therefore crop yield and quality [1]. Rice, a staple food, is needed around the world, and rice production is of great significance to global food security [2]. However, due to climate change and erratic rainfall patterns, drought stress increased globally and led to crop yields decreased [3]. To adapt to drought stress, plants have evolved a series of multifaceted mechanisms which consist of modifying photosynthetic pathways, regulating leaf transpiration [4], upregulating the expression of enzyme genes involved in scavenging reactive oxygen species (ROS) [5], sensing plant hormone signals and so on [6,7]. Thus, identifying drought resistance genes and revealing their molecular mechanisms are crucial goals for crop breeding scientists.
Annexins belong to a subfamily of Ca2+-dependent phospholipid-binding proteins that are prevalent in plants and animals [8,9], and they are highly conserved in evolution. In general, the C-terminal core of annexin contains four annexin repeats, and each repeat has five short α-helices [9,10]. Previous studies have shown that annexins participate in regulating diverse aspects of plant growth and development as well as responses to stress [11,12]. For example, the overexpression of TdANN6 enhanced salt/osmotic stress tolerance by increasing catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD) activities in durum wheat [13]. AtANN1 and AtANN2 affect the growth of primary roots by regulating silique sucrose transport [14]. The expression of ZmANN33 and ZmANN35 was up-regulated during seed germination but partially suppressed by chilling, and the heterologous expression of ZmANN33 or ZmANN35 in Arabidopsis was also beneficial to plasma membrane (PM) recovery under chilling stress [15]. In other examples, AtANN1, a Ca2+-permeable transporter, was involved in responses to drought, salinity, abscisic acid (ABA), ROS and cold stress by mediating the accumulation of cytosolic-free Ca2+ [16,17,18,19]. AtANN5 accumulated at sites of membrane damage to maintain membrane integrity in pollen grains under osmotic or ionic imbalances [20]. Furthermore, the overexpression of AtANN8 compromised RPW8.1-mediated resistance to powdery mildew and cell death [21]. These results demonstrate that plant annexins are related to diverse physiological pathways.
In recent years, annexins have been reported to be involved in regulating abiotic and biotic stress responses in rice [22]. For example, OsANN5 is reported to be involved in cold stress tolerance at the rice seedling stage [23]. OsANN1 positively regulates heat stress tolerance by modulating ROS homeostasis [24]. Under normal conditions, OsANN1 interacts with OsHAN1 and promotes JA-Ile to exhibit an inactive form. Upon pathogen infection, OsANN1 and HAN1 are separated, leading to HAN1 degradation and JA-Ile accumulation, thus promoting disease resistance [25]. The overexpression of OsANN3 significantly increases drought tolerance by regulating ABA-dependent stress response pathways [26]. RNA silencing of OsANN4 caused hypersensitivity to ABA by modulating ROS production and Ca2+ influx [27]. OsANN10 negatively regulates osmotic stress tolerance by increasing POD and CAT activities to remove excess ROS and by maintaining plasma membrane integrity [28]. However, the physiological roles of most rice annexins are still unclear.
ROS, including singlet oxygen (1O2), superoxide anions (O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (·OH) [29], play an essential role in plant responses to abiotic stress. Under normal conditions, the production and scavenging of ROS remain balanced. However, under drought-stress conditions, this balance is disturbed, resulting in an increase in the production of ROS [30,31], which can damage macromolecules such as proteins, lipids and nucleic acids, ultimately leading to programmed cell death (PCD) [32,33]. Plants have evolved an enzymatic antioxidant defense system to maintain ROS levels. The major ROS scavenging enzymes include SOD, CAT, peroxiredoxin (PrxR), ascorbate peroxidase (APX) and glutathione peroxidase (GPX) [31]. OsRbohB encodes an NADPH oxidase and regulates ROS production to enhance drought tolerance in rice [34]. The overexpression of OsPYL6 can improve drought tolerance by increasing ABA content and reducing H2O2 accumulation [35]. OsAAI1 positively regulates drought tolerance by enhancing the activity of ROS scavenging enzymes such as CAT, APX, GPX and GR (glutathione peroxidase) in rice [36]. Interestingly, some reports also suggest that plant annexins respond to drought stress by regulating ROS homeostasis. For instance, the overexpression of TdANN12 in tobacco improves drought tolerance through the removal of excess ROS [37]. The heterologous expression of VvANN1 enhances drought tolerance in Arabidopsis by increasing the ROS scavenging activities of SOD, POD and CAT [38]. Therefore, plant annexins have critical roles in drought stress and ROS homeostasis, but how annexins are directly involved in this process remains unclear.
In this study, the putative rice annexin OsANN9 was characterized, and its functions in response to drought stress were explored. We constructed a series of transgenic rice plants including OsANN9-OE, OsANN9-Ri and osann9 mutant lines for the experiments used in this study. Our results indicate that OsANN9 is induced by drought stress, and OsANN9 participates in the response to drought stress by modulating the level of malondialdehyde (MDA) and the activities of antioxidant enzymes (SOD, POD and CAT). Overall, our results provide a physiological basis for the role of plant annexins during environmental stress.

2. Results

2.1. OsANN9 Encodes an Annexin Family Member

We previously reported that some annexin genes of rice consisting of Os02g51750 (OsANN1), Os07g46550 (OsANN3), Os05g31750 (OsANN4) and Os09g20330 (OsANN10) were involved in responses to heat, drought, ABA and osmotic stress [24,26,27,28]. In this study, we have focused on another rice annexin gene, Os05g31760. Based on the amino acid sequence alignment with annexins from other species, it is 73.2% similar to the ZmANN9 sequence of maize, and was named OsANN9 (Figure 1A).
Bioinformatics indicates that there are 10 putative annexin genes in the rice genome, all annexin proteins have a conserved annexin repeat domain and some of them possess type-II Ca2+-binding sites; OsANN9 is not an exception. This suggests that OsANN9 might have similar conservative functions as other reported rice annexin proteins [24]. Moreover, OsANN9 also has a distinctive character, such as a zinc-finger-type (C2H2) domain (Figure 1B). This implies that OsANN9 may have some properties that differ from other rice annexin proteins.
To assay the subcellular localization of OsANN9, the cassettes 35S::OsANN9-GFP, OsANN9pro::OsANN9-GFP and 35S::GFP were constructed. The corresponding plasmids were introduced into Agrobacterium tumefaciens individually and then transformed into rice calli to obtain transgenic rice plants. After that, 3-day-old rice roots of T3 plants were used to determine the subcellular localization of OsANN9-GFP. The results showed that OsANN9-GFP was mainly localized in the plasma membrane and cell periphery in the rice root tip cells, whereas GFP fluorescence alone was ubiquitously distributed in the rice root tip cells (Figure 2A). To further test the transient expression of OsANN9, 35S::OsANN9-GFP or 35S::GFP was transformed into rice protoplast or tobacco leaf epidermal cells separately. Interestingly, in rice protoplast and tobacco epidermal cells, the signals of OsANN9-GFP were found in the plasma membrane, cytoplasm and nucleus (Figure 2B,C). This suggested that OsANN9 might be widespread in undifferentiated tissues.
Previous research results indicated that plant annexins could bind Ca2+ in participating physiological processes [24,26,27,28]. To investigate whether OsANN9 binds Ca2+, the plasmid pET30a-OsANN9-His was constructed and introduced into the E. coli BL21 strain. Meanwhile, the protein expression was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18 °C. The OsANN9-His recombinant protein was evaluated by SDS-PAGE, with bands appearing at ~45KD that probably represent OsANN9 (Supplementary Figure S1A). To assay OsANN9 binding to Ca2+, the OsANN9-His recombinant protein was collected from the supernatant after lysing E. coli cells with ultrasonic waves. When different concentrations of Ca2+ (5 mM, 10 mM, 20 mM) were added to the supernatant, which contained an equal amount of OsANN9-His, precipitation occurred suddenly. After adding an equal amount of EDTA-Na2 (5 mM, 10 mM, 20 mM), precipitates redissolved due to the binding of EDTA-Na2 and Ca2+. SDS-PAGE demonstrated that for more OsANN9-His protein, a greater concentration of Ca2+ was added to the supernatant (Figure 2D). This result indicates that OsANN9 can bind Ca2+ in this manner.
To confirm that OsANN9 can bind Ca2+ in other scenarios, we performed the following tests. First, the OsANN9-His recombinant protein was purified with Ni-NTA affinity chromatography, and the eluted fraction was tested with a Western blot to ensure that OsANN9-His was represented by a band at ~45 KD (Supplementary Figure S1B,C). Second, to test the ability of OsANN9 to bind Ca2+, the fluorescence level of the OsANN9-His recombinant protein was assayed with a fluorescence spectrophotometer. The fluorescence intensity of purified OsANN9-His with or without Ca2+ was measured. The results showed that OsANN9-His reached a maximum fluorescence intensity of 5812 (A.U.) at 340 nm under excitation at 315 nm. After adding 2 mM Ca2+ to the OsANN9-His solution, the maximum fluorescence intensity increased to 6394 (A.U.) and decreased to 5526 (A.U.) at 340 nm after adding the same amount of EDTA-Na2 (Figure 2E). These results verified that OsANN9 can bind Ca2+ in this manner.

2.2. OsANN9 Expression Is Induced by Osmotic and Drought Stress

The previous studies indicated that OsANN1, OsANN3, OsANN4 and OsANN10 are involved in response to abiotic stresses [24,26,27,28]. It was hypothesized that OsANN9 may also have these kinds of properties. To explore the function of OsANN9, the 2277-bp promoter sequence upstream of OsANN9 ATG was analyzed with the PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 25 September 2020). The results showed that the OsANN9 promoter sequence consists of a series of cis elements, including ABA-responsive elements (ABRE), v-myb avian myeloblastosis viral oncogene homolog (MYB), Dehydration Responsive Element (DRE) and MYB binding site (MBS), which might be controlled by abiotically induced transcription factors (Supplementary Figure S2). We further assessed the transcription level of OsANN9 with PEG treatment in 14-day-old wild type (WT) plants. When treated with 20% PEG6000, the OsANN9 transcript gradually increased approximately 7-fold compared to plants with no treatment (Figure 3A). These results suggested that OsANN9 might have a key role in the response to PEG treatment. Moreover, we used the same WT seedlings to detect OsANN9 transcription levels under drought stress. As expected, the results still showed that the OsANN9 expression increased strikingly at 8 h; it amounted to a 13-fold increase compared to the no-drought treatment (Figure 3B).
To assess whether OsANN9 was involved in the PEG treatment response, the plasmid OsANN9pro::GUS was introduced into Agrobacterium tumefaciens EHA105 and then transformed into rice calli. GUS staining and GUS activity were monitored in OsANN9pro::GUS plants under PEG-induced osmotic stress. The 3-day-old OsANN9pro::GUS rice plants were subjected to 20% PEG6000 treatment for 6 h, and GUS staining and GUS activity in seedlings were greatly increased following the 20% PEG6000 treatment (Figure 3C,D). Taken together, these results indicate that OsANN9 positively responds to drought and osmotic stress in rice plants.

2.3. Construction of OsANN9-OE, OsANN9-Ri, and osann9 Mutants

To explore the function of OsANN9 in response to osmotic stress, we isolated the 347-bp OsANN9 CDS as the target region to construct the genetic vector for RNA interference (Supplementary Figure S3A). Twenty independent transgenic plants were obtained with a semblable phenotype. Meanwhile, the Ubi::OsANN9-HA expression cassette was constructed and inserted into plasmid pCAMBIA1300 (Supplementary Figure S3B), generating further rice OsANN9 overexpression lines. The osann9 mutant lines were generated by the Agrobacterium-mediated method. Two Cas9-free homozygous mutant lines (osann9-cr1 and osann9-cr3) were analyzed by DNA sequencing (Figure 4). In osann9-cr1, a 1-bp insertion was found at the 80 to 81st position upstream of the start codon, and another 1-bp insertion was found at the 596 to 597th position downstream of the start codon, resulting in a frameshift and a truncated protein with 123 aa. In osann9-cr3, a 35-bp deletion was found at the 578 to 612nd position downstream of the start codon, which led to a truncated protein with 111 aa (Figure 4C). After performing RT-qPCR, two RNAi lines (Ri2 and Ri7) with significantly reduced OsANN9 transcript levels and two independent overexpression lines (OE2 and OE7) with prominently increased OsANN9 transcript levels, as well as osann9-cr1 and osann9-cr3 mutant rice lines, were selected for subsequent studies (Supplementary Figure S4).
Very interestingly, the setting rates of osann9 mutant rice plants significantly decreased in comparison to WT or OsANN9-OE plants (Figure 4E,F); the molecular mechanisms of this phenotype are still conducting.

2.4. Overexpression of OsANN9 Activates the ROS Scavenging System, Thereby Enhancing Tolerance to Osmotic and Drought Stress

To better understand the function of OsANN9 in response to abiotic stress in rice, we applied osmotic stress to OsANN9-OE, OsANN9-Ri, osann9 mutants and WT rice plants (Figure 5A). The above rice seeds were germinated on 1/2 Murashige and Skoog (MS) medium for 36 h. Then, the germinated seedlings were transferred to fresh 1/2 MS medium with or without 20% PEG6000. The lengths of shoots or roots were measured on the 7th day after treatment (Figure 5B,C). These measurements showed that the lengths of roots and shoots of OsANN9-OE seedlings on medium with 20% PEG6000 were strikingly longer than those of WT plants, while both OsANN9-Ri and osann9 mutants showed weaker growth than WT plants. Meanwhile, no distinct differences were observed among different rice lines on the medium without 20% PEG6000. Overall, these observations suggest that OsANN9 positively affects drought and osmotic stress responses in rice.
To further investigate the function of OsANN9 in response to drought stress in rice, 14-day-old OsANN9-OE, OsANN9-Ri, osann9 and WT rice seedlings were subjected to drought stress. The rice seedlings exhibited similar growth morphology under no stress conditions. After watering was stopped for 10 days, both OsANN9-Ri lines and osann9 mutants showed hypersensitivity to drought and severe wilting, whereas the OsANN9-OE lines showed better growth comparing to the WT plants (Figure 6A). After 7 days of osann9 recovery, 68% of WT plants survived. Meanwhile, the OsANN9-OE lines had the highest survival rate (78% for OsANN9-OE2 and 73% for OsANN9-OE7), and the OsANN9-Ri lines and osann9 mutants exhibited the lowest survival rate (33% for OsANN9-Ri2, 28.6% for OsANN9-Ri7, 31.6% for osann9-cr1 and 25.8% for osann9-cr3) (Figure 6B).
In addition, to confirm the drought-stress response of OsANN9, the rate of water loss was assayed for the shoots of WT, OsANN9-OE and OsANN9-Ri lines as well as osann9 mutant plants. Compared to WT rice plants, the rates of water loss of OsANN9-OE lines were lower at different time points; however, both OsANN9-Ri lines and osann9 mutants had higher rates of water loss (Figure 6C). Based on the above results, the water loss of shoots matched the survival rate.
Drought stress may lead to the production of ROS [30], and high concentrations of ROS result in cell damage and even programmed cell death. As essential ROS, O2.− and H2O2 have key roles in abiotic stress signaling. To confirm whether OsANN9 manipulates ROS levels in response to drought stress, nitro-blue tetrazolium (NBT) staining and 3,3′-diaminobenzidine (DAB) staining were performed on 7-day-old rice seedlings to assay the in situ localization of O2− and H2O2, respectively. The results indicated that there were no differences in plants under normal conditions. However, when rice plants were drought-stressed for 4 h, blue spots (which reflect O2− production) and brown spots (which reflect H2O2 production) were prominent in the mesophyll cells of OsANN9-Ri or osann9 mutant plants, while fewer blue and brown spots were observed in WT and OsANN9-OE plants (Figure 7A,B). This result suggests that the production of ROS in rice might be related to the function of OsANN9 under drought.
The essential ROS scavengers SOD, POD and CAT eliminate excess cellular ROS, thus protecting plant cells from oxidative damage. To further clarify the role of OsANN9 in regulating ROS under drought stress, SOD, POD and CAT activities were examined. The 7-day-old OsANN9-OE, OsANN9-Ri, osann9 and WT rice lines were subjected to drought stress for 4 h. Interestingly, SOD, POD and CAT activities did not differ in any plants. After exposure to drought stress, SOD, POD and CAT were significantly increased in OsANN9-OE plants but decreased in OsANN9-Ri and osann9 plants (Figure 8A–C). In addition, we also analyzed the transcription levels of ROS-scavenging genes, such as OsSODcc2, OsAPX2 and OsCAT. The transcripts of these three genes were increased in the OsANN9-OE line and decreased in OsANN9-Ri and osann9 plants under drought-stress conditions (Figure 8D–F). To sum up, both ROS scavenger activity and related gene expression levels were consistent with the drought tolerance phenotype of the OsANN9 lines.
Both MDA content and electrolyte leakage are indicators of membrane damage and membrane lipid peroxidation [15,39]. According to the membrane lipid-binding ability of annexin, we examined MDA content and electrolyte leakage in OsANN9-OE, OsANN9-Ri, osann9 mutant and WT plants under normal and drought-stress conditions. The MDA content in the above plants was similar under normal conditions; however, following drought treatment for 8 h, the average MDA content was 3.561 nmol/g/FW in OsANN9-OE plants, 6.108 nmol/g/FW in OsANN9-Ri plants and 6.218 nmol/g/FW in osann9 mutants, while WT plants had an average MDA content of 4.299 nmol/g/FW. Furthermore, electrolyte leakage showed a similar pattern (Figure 7C,D).

3. Discussion

Drought stress is one of the most important environmental variables limiting plant growth and productivity [40], and generating drought-resilient crop varieties is essential for withstanding stress [41]. Annexins belong to a multifunctional protein family that exists in plants, animals and fungi [10]. Recent evidence has shown that plant annexins play critical roles in regulating abiotic and biotic stresses [19,21,25,38]. Our previous study showed that OsANN3 enhances tolerance to drought stress by modulating ABA-dependent stress response pathways in rice [26]. Bioinformatics analysis results indicated that OsANN9 consists of four annexin domains (Figure 1B); this suggests that OsANN9 may have functions in response to abiotic stress. Our results indicate that OsANN9 is involved in the process of ROS homeostasis during drought stress in rice. In addition, OsANN9 has a C2H2 domain, which implies that OsANN9 might have other properties which differ from other rice annexins.
In this study, we provide several lines of evidence that OsANN9 is essential for drought tolerance in rice. Firstly, the expression of OsANN9 was strongly induced by PEG treatment and drought stress, implying that OsANN9 is a drought-response factor in rice (Figure 3A,B). Secondly, the OsANN9-OE rice plants demonstrated enhanced osmotic stress, as revealed by longer shoots and roots under PEG treatment, while the OsANN9-Ri and osann9 mutant plants showed the opposite phenotype (Figure 5). Thirdly, OsANN9 enhanced drought-stress tolerance in rice. The OsANN9-OE plants were much less sensitive to drought, as evidenced by lower levels of water loss, EL values, MDA content and ROS accumulation, and the OsANN9-OE plants had higher survival rates compared with WT plants under drought stress (Figure 6C and Figure 7). These results demonstrate that OsANN9 is a promising candidate gene for developing drought-tolerance rice.
ROS production is usually triggered by and dramatically acclimates to drought stress [42]. Low levels of ROS can serve as a signal in regulating pathways for plant growth and stress responses. Excessive ROS accumulation causes oxidative damage to proteins and damage to lipids in the cell membrane [43,44]. In this study, overexpression of OsANN9 reduced the accumulation of ROS caused by drought stress, as illustrated by lower DAB and NBT staining levels in OsANN9-OE plants. This result suggested that O2− and H2O2 contents in OsANN9-OE plants were lower than those in WT plants under drought stress (Figure 7A,B). Antioxidant protection is an effective way to shield plants from stress-induced oxidative damage. In this study, SOD, POD and CAT activities under drought stress were markedly increased in OsANN9-OE plants compared to OsANN9-Ri and osann9 mutant plants (Figure 8A–C). Therefore, OsANN9 may improve drought-stress tolerance by modulating the intracellular level of H2O2.
In addition, some stress biomarkers have been widely used in detecting and monitoring stress tolerances in plant, such as water content, water-use efficiency, hydraulic conductivity, photosystem II (PSII) and photosystem I (PSI) efficiency, and so on [45]. For example, as an essential photosynthetic parameter, electron transport rate (ETR) affects crucial traits such as stress tolerance, especially drought tolerance [46,47].
Calcium ion (Ca2+) is a ubiquitous intracellular second messenger that mediates plant responses to abiotic stress [48]. Annexins are a family of calcium-dependent membrane-binding proteins that can sense and regulate [Ca2+]cyt under abiotic stress [28,49,50]. It has been reported that AtANN4 can be phosphorylated by AtSOS2, mediating salt-induced increases [Ca2+]cyt in Arabidopsis [51]. OsANN4 overexpression can lead to increasing Ca2+ influx in response to ABA in rice [27]. Based on bioinformatics predictions, OsANN9 has two type-II Ca2+-binding sites (Figure 1B). The results of in vitro assays also showed that OsANN9 has Ca2+-dependent phospholipid-binding activity and a Ca2+-binding capacity (Figure 2D,E). Plant hormones are small-molecule compounds widely present in plants. Many endogenous hormones such as ABA, gibberellins (GA), and cytokinin (CYT) are involved in regulating plant growth and development and play crucial rules in stress resistance [52]. For instance, drought stress could suppress GA biosynthesis, which leads to SLR1 accumulation and thus inhibits the degradation of OsPYL10, finally enhancing the ABA signal response to drought stress [53]. Interestingly, SnRK2s were activated by ABA and then phosphorylated the type-A Arabidopsis response regulator (ARR5) proteins, which strengthen the ABA signal under drought stress; however, the cytokinin signal could be repressed by type-A ARR5, which restricts plant development [54]. Nevertheless, the exact mechanism underlying how OsANN9 modulates Ca2+ and plant hormones under drought stress requires further exploration.
Based on our results, drought stress increased the expression of OsANN9, thus enhancing the drought tolerance of rice. OsANN9 not only heightened ROS scavenger activity but also alleviated cell damage upon drought stress. In conclusion, we postulate that OsANN9 is a candidate gene for breeding rice cultivars with tolerance to drought stress. However, we cannot yet specify the exact mechanism by which the OsANN9 protein contributes to drought-stress responses via Ca2+ signaling. Future studies must identify other signaling pathways to fully understand the role of OsANN9 in plant physiological processes. In a word, we first constructed and used CRISPR lines of rice OsANN9 and further demonstrated that OsANN9 might be involved in a few mechanisms for drought-stress responses.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The rice (Oryza sativa subsp. japonica) cultivar Nipponbare, which was planted in the experimental field at Hebei Normal University (Shijiazhuang, China, 38°02′33″ N 114°30′36″ E), was used in this research. Rice plants were grown on 1/2 MS solid medium in a chamber or greenhouse at temperatures of 28/22 °C (day/night) with 60–70% humidity under 14/10 h light/dark cycles.
To perform the PEG treatment assay, rice seeds were planted on 1/2 MS solid medium with or without 20% (w/v) PEG6000 for 7 days. Then, a photograph was taken, and the length of the shoots or roots was measured.
For drought tolerance analysis, rice seeds were sown on 1/2 MS solid medium and grown for 7 days before being transferred to soil and grown in pots (12 × 12 cm) for 7 days with regular water. Then, all plants were dried for 10 days and given a 7-day recovery.
To assay water loss, 14-day-old rice plants were placed on filter paper at room temperature. The weight of the plants was measured at different time points. The water loss rate was calculated as the ratio of total weight lost compared to the initial weight at different time points [55].

4.2. Construction of OsANN9 Expression Vectors

To overexpress OsANN9, the CDS of OsANN9 was inserted into the pCAMBIA1301-Ubi::HA expression vector, using the BamHI site. To construct an RNA interference construct, a 347-bp cDNA fragment of OsANN9 was cloned into the pTCK303 vector using the KpnI and BamHI sites. To construct the CRISPR/Cas9 vector, the specific sgRNA sequences of OsANN9 were synthesized, and the CRISPR/Cas9 vector was generated using the genome editing approach, as reported previously [56].
To construct the 35S::OsANN9-GFP vector, the CDS of OsANN9 was cloned into the pMDC83 vector using the XbaI and BamHI sites. For the OsANN9pro::GUS vectors, the 1879-bp genomic sequence upstream of the OsANN9 ATG was cloned into the pCAMBIA1300-GUS vector using the XbaI and PstI sites.
All constructs were introduced into Agrobacterium strain EHA105 separately and subsequently transformed into Nipponbare by the Agrobacterium-mediated method [57]. All primers for vector construction are listed in Supplementary Table S1.

4.3. Subcellular Localization of OsANN9

Root tips of 3-day-old OsANN9-GFP rice seedlings were used to observe subcellular localization with a fluorescence confocal microscope. Agrobacterium GV3101 carrying the 35S::OsANN9-GFP or 35S::GFP plasmid was injected into tobacco leaves. After 40 h of infiltration, fluorescence signals were observed using a fluorescence confocal microscope. The 35S::OsANN9-GFP or 35S::GFP construct was transformed into rice protoplasts via the PEG-mediated method [58], and the fluorescence signals were observed with a fluorescence confocal microscope (FV3000, Olympus, Tokyo, Japan).

4.4. Recombinant OsANN9-His Protein Purification and Ca2+-Binding Activity

The CDS of OsANN9 was inserted into a pET30a vector to generate OsANN9-His constructs. The construct was transformed into the E. coli strain BL21, and recombinant protein OsANN9-His was induced by adding 0.5 mM IPTG at 18 °C for 6 h. The E. coli cells containing OsANN9-His were lysed via ultrasonication, and samples were ultracentrifuged at 12,000× g for 10 min at 4 °C. The supernatant was used for the Ca2+-binding activity assay, as described previously [24]. Different concentrations of CaCl2 (5 mM, 10 mM, 20 mM) and an equivalent amount of EDTA-Na2 were added successively to the supernatant. SDS-PAGE was used to assay the protein samples.
For fluorescence measurements, the recombinant protein OsANN9-His was purified using Ni-NTA affinity chromatography. The assay medium contained 0.2 mg mL−1 OsANN9-His protein and 0 mM or 2 mM Ca2+ in the buffer (20 mMTris-HCl, pH 8.0). Fluorescence spectroscopy was performed using a fluorescence spectrophotometer (F-4600, Hitachi, Tokyo, Japan).

4.5. GUS Staining and GUS Activity

OsANN9pro::GUS seedlings were incubated with 5-bromo-4-chloro-3-indolyl-β-glucuronic acid buffer at 37 °C under dark conditions for GUS staining. Then, the stained seedlings were immersed in 70% ethanol to remove surface dyes and chlorophyll. For GUS activity detection, the protein was extracted from OsANN9pro::GUS seedlings and incubated with 4-methylumbelliferyl β-D-glucuronide (MUG). The cleavage of MUG was monitored quantitatively.

4.6. Antioxidant Enzyme Activity

The 7-day-old rice seedlings were homogenized in phosphate buffer (50 mM phosphate, 1 mM EDTA-Na2, 1% (w/v) polyvinyl pyrrolidone, pH 7.4) and centrifuged for 30 min at 4 °C at 10,000× g. The supernatant was used for the antioxidant enzyme activity assay. The CAT, POD and SOD activities were tested as reported previously [59].

4.7. Detection of O2− and H2O2 In Situ

To detect O2− in situ, the leaves of 7-day-old rice seedlings were detached and immersed in 6 mM NBT for 24 h at 25 °C. Then, leaves were boiled in ethanol for 30 min to remove chlorophyll. The samples were imaged with a 3D microscope (DVM6, Leica, Wetzlar, Germany).
For the detection of H2O2 in situ, DAB staining was performed on the leaves of 7-day-old rice seedlings. The leaves were immersed in DAB solution for 12 h at 25 °C. To remove chlorophyll, the leaves were incubated with ethanol. Samples were imaged with a 3D microscope (DVM6, Leica, Wetzlar, Germany).

4.8. Measurement of MDA and Electrolyte Leakage

The 14-day-old rice seedlings were homogenized in 0.1% (w/v) trichloroacetic acid (TCA) and then centrifuged at 1500 g for 10 min at 4 °C. The MDA content was determined with thiobarbituric, as reported previously [60].
For the electrolyte leakage assay, 7-day-old rice seedlings were soaked in deionized water for 24 h, and this conductivity was measured as E1. Then, the samples were boiled for 30 min, and conductivity after cooling was measured as E2. The conductivity of deionized water was measured as E0. This conductivity measurement method was described previously [28].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms242417495/s1.

Author Contributions

Z.Z. designed this study; Y.J., X.G., J.C. and Q.Z. performed most of the experiments; X.Y., S.C., L.L., S.H. and Y.P. assisted in some experiments; Z.Z., Y.J., X.G. and J.C. wrote the manuscript. All authors contributed to the article. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Hebei Natural Science Foundation (C2020205019), the National Natural Science Foundation of China (31571638, 31340046), Hebei Research Center of the Basic Discipline of Cell Biology (C2023205049). The Graduate Innovation Funding Program of Hebei Province (CXZZBS2023087).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We thank Yaoguang Liu, College of Life Sciences, South China Agricultural University for providing pYLCRISPR/Cas9Pubi-H expression vectors, and Kang Chong, Chinese Academy of Science, for providing the pTCK303 vector.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zhu, J.K. Abiotic stress signaling and responses in plants. Cell 2016, 167, 313–324. [Google Scholar] [CrossRef]
  2. He, X.Q.; Batáry, P.; Zou, Y.; Zhou, W.W.; Wang, G.H.; Liu, Z.Y.; Bai, Y.Y.; Gong, S.X.; Zhu, Z.R.; Settele, J.; et al. Agricultural diversification promotes sustainable and resilient global rice production. Nat. Food 2023, 4, 788–796. [Google Scholar] [CrossRef]
  3. Rezvi, H.U.A.; Tahjib-Ul-Arif, M.; Azim, M.A.; Tumpa, T.A.; Tipu, M.M.H.; Najnine, F.; Dawood, M.F.A.; Skalicky, M.; Brestič, M. Rice and food security: Climate change implications and the future prospects for nutritional security. Food Energy Secur. 2022, 12, e430. [Google Scholar] [CrossRef]
  4. He, F.; Wang, H.L.; Li, H.G.; Su, Y.Y.; Li, S.; Yang, Y.L.; Feng, C.H.; Yin, W.L.; Xia, X.L. PeCHYR1, a ubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus. Plant Biotechnol. J. 2018, 16, 1514–1528. [Google Scholar] [CrossRef] [PubMed]
  5. Wei, T.L.; Wang, Y.; Xie, Z.Z.; Guo, D.Y.; Chen, C.W.; Fan, Q.J.; Deng, X.D.; Liu, J.H. Enhanced ROS scavenging and sugar accumulation contribute to drought tolerance of naturally occurring autotetraploids in Poncirus trifoliata. Plant Biotechnol. J. 2019, 17, 1394–1407. [Google Scholar] [CrossRef] [PubMed]
  6. Hassan, M.U.; Mahmood, A.; Awan, M.I.; Maqbool, R.; Aamer, M.; Alhaithloul, H.A.S.; Huang, G.Q.; Skalicky, M.; Brestic, M.; Pandey, S.; et al. Melatonin-induced protection against plant abiotic stress: Mechanisms and prospects. Front. Plant Sci. 2022, 13, 902694. [Google Scholar] [CrossRef] [PubMed]
  7. Gupta, A.; Rico-Medina, A.; Caño-Delgado, A.I. The physiology of plant responses to drought. Science 2020, 368, 266–269. [Google Scholar] [CrossRef]
  8. Clark, G.B.; Roux, S.J. Annexins of plant cells. Plant Physiol. 1995, 109, 1133–1139. [Google Scholar] [CrossRef] [PubMed]
  9. Mortimer, J.C.; Laohavisit, A.; Macpherson, N.; Webb, A.; Brownlee, C.; Battey, N.H.; Davies, J.M. Annexins: Multifunctional components of growth and adaptation. J. Exp. Bot. 2008, 59, 533–544. [Google Scholar] [CrossRef] [PubMed]
  10. Laohavisit, A.; Davies, J.M. Annexins. New Phytol. 2011, 189, 40–53. [Google Scholar] [CrossRef]
  11. Clark, G.B.; Morgan, R.O.; Fernandez, M.P.; Roux, S.J. Evolutionary adaptation of plant annexins has diversified their molecular structures, interactions and functional roles. New Phytol. 2012, 196, 695–712. [Google Scholar] [CrossRef] [PubMed]
  12. Xu, J.W.; Han, L.J.; Xia, S.Y.; Zhu, R.J.; Kang, E.; Shang, Z.L. ATANN3 is involved in extracellular ATP-regulated auxin distribution in Arabidopsis thaliana seedlings. Plants 2023, 12, 330. [Google Scholar] [CrossRef] [PubMed]
  13. Harbaoui, M.; Ben Romdhane, W.; Ben Hsouna, A.; Brini, F.; Ben Saad, R. The durum wheat annexin, TdAnn6, improves salt and osmotic stress tolerance in Arabidopsis via modulation of antioxidant machinery. Protoplasma 2021, 258, 1047–1059. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, J.; Song, J.; Clark, G.; Roux, S.J. ANN1 and ANN2 function in post-phloem sugar transport in root tips to affect primary root growth. Plant Physiol. 2018, 178, 390–401. [Google Scholar] [CrossRef] [PubMed]
  15. He, F.; Gao, C.H.; Guo, G.Y.; Liu, J.; Gao, Y.; Pan, R.H.; Guan, Y.J.; Hu, J. Maize annexin genes ZmANN33 and ZmANN35 encode proteins that function in cell membrane recovery during seed germination. J. Exp. Bot. 2019, 70, 1183–1195. [Google Scholar] [CrossRef] [PubMed]
  16. Huh, S.M.; Noh, E.K.; Kim, H.G.; Jeon, B.W.; Bae, K.; Hu, H.C.; Kwak, J.M.; Park, O.K. Arabidopsis annexins AnnAt1 and AnnAt4 interact with each other and regulate drought and salt stress responses. Plant Cell Physiol. 2010, 51, 1499–1514. [Google Scholar] [CrossRef]
  17. Lee, S.; Lee, E.J.; Yang, E.J.; Lee, J.E.; Park, A.R.; Song, W.H.; Park, O.K. Proteomic identification of annexins, calcium-dependent membrane binding proteins that mediate osmotic stress and abscisic acid signal transduction in Arabidopsis. Plant Cell 2004, 16, 1378–1391. [Google Scholar] [CrossRef]
  18. Richards, S.L.; Laohavisit, A.; Mortimer, J.C.; Shabala, L.; Swarbreck, S.M.; Shabala, S.; Davies, J.M. Annexin 1 regulates the H2O2-induced calcium signature in Arabidopsis thaliana roots. Plant J. 2013, 77, 136–145. [Google Scholar] [CrossRef]
  19. Liu, Q.B.; Ding, Y.L.; Shi, Y.T.; Ma, L.; Wang, Y.; Song, C.P.; Wilkins, K.A.; Davies, J.M.; Knight, H.; Knight, M.R.; et al. The calcium transporter ANNEXIN1 mediates cold-induced calcium signaling and freezing tolerance in plants. EMBO J. 2021, 40, e104559. [Google Scholar] [CrossRef]
  20. Lichocka, M.; Krzymowska, M.; Górecka, M.; Hennig, J. Arabidopsis annexin 5 is involved in maintenance of pollen membrane integrity and permeability. J. Exp. Bot. 2022, 73, 94–109. [Google Scholar] [CrossRef]
  21. Zhao, Z.X.; Xu, Y.J.; Lei, Y.; Li, Q.; Zhao, J.Q.; Li, Y.; Fan, J.; Xiao, S.; Wang, W.M. ANNEXIN 8 negatively regulates RPW8.1-mediated cell death and disease resistance in Arabidopsis. J. Integr. Plant Biol. 2021, 63, 378–392. [Google Scholar] [CrossRef] [PubMed]
  22. Jami, S.K.; Clark, G.B.; Ayele, B.T.; Roux, S.J.; Kirti, P.B. Identification and characterization of annexin gene family in rice. Plant Cell Rep. 2011, 31, 813–825. [Google Scholar] [CrossRef] [PubMed]
  23. Que, Z.Q.; Lu, Q.N.; Li, Q.X.; Shen, C.X. The rice annexin gene OsAnn5 is involved in cold stress tolerance at the seedling stage. Plant Direct 2023, 7, e539. [Google Scholar] [CrossRef]
  24. Qiao, B.; Zhang, Q.; Liu, D.L.; Wang, H.Q.; Yin, J.Y.; Wang, R.; He, M.L.; Cui, M.; Shang, Z.L.; Wang, D.K.; et al. A calcium-binding protein, rice annexin OsANN1, enhances heat stress tolerance by modulating the production of H2O2. J. Exp. Bot. 2015, 66, 5853–5866. [Google Scholar] [CrossRef] [PubMed]
  25. Zhao, Q.Q.; Liu, R.; Zhou, Q.Z.; Ye, J.; Meng, F.W.; Liu, J.; Yang, C. Calcium-binding protein OsANN1 regulates rice blast disease resistance by inactivating jasmonic acid signaling. Plant Physiol. 2023, 192, 1621–1637. [Google Scholar] [CrossRef]
  26. Li, X.F.; Zhang, Q.; Yang, X.; Han, J.B.; Zhu, Z.G. OsANN3, a calcium-dependent lipid binding annexin is a positive regulator of ABA-dependent stress tolerance in rice. Plant Sci. 2019, 284, 212–220. [Google Scholar] [CrossRef]
  27. Zhang, Q.; Song, T.; Guan, C.; Gao, Y.J.; Ma, J.C.; Gu, X.Y.; Qi, Z.G.; Wang, X.J.; Zhu, Z.G. OsANN4 modulates ROS production and mediates Ca2+ influx in response to ABA. BMC Plant Biol. 2021, 21, 474. [Google Scholar] [CrossRef]
  28. Gao, S.X.; Song, T.; Han, J.B.; He, M.L.; Zhang, Q.; Zhu, Y.; Zhu, Z.G. A calcium-dependent lipid binding protein, OsANN10, is a negative regulator of osmotic stress tolerance in rice. Plant Sci. 2020, 293, 110420. [Google Scholar] [CrossRef]
  29. Foyer, C.H.; Hanke, G. ROS production and signalling in chloroplasts: Cornerstones and evolving concepts. Plant J. 2022, 111, 642–661. [Google Scholar] [CrossRef]
  30. Cruz de Carvalho, M.H. Drought stress and reactive oxygen species: Production, scavenging and signaling. Plant Signal. Behav. 2008, 3, 156–165. [Google Scholar] [CrossRef]
  31. Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9, 490–498. [Google Scholar] [CrossRef]
  32. Sies, H.; Jones, D.P. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat. Rev. Mol. Cell Biol. 2020, 21, 363–383. [Google Scholar] [CrossRef]
  33. Ali, S.; Tyagi, A.; Bae, H. ROS interplay between plant growth and stress biology: Challenges and future perspectives. Plant Physiol. Biochem. 2023, 203, 108032. [Google Scholar] [CrossRef]
  34. Shi, Y.; Chang, Y.L.; Wu, H.T.; Shalmani, A.; Liu, W.T.; Li, W.Q.; Xu, J.W.; Chen, K.M. OsRbohB-mediated ROS production plays a crucial role in drought stress tolerance of rice. Plant Cell Rep. 2020, 39, 1767–1784. [Google Scholar] [CrossRef]
  35. Santosh Kumar, V.V.; Yadav, S.K.; Verma, R.K.; Shrivastava, S.; Ghimire, O.; Pushkar, S.; Rao, M.V.; Senthil Kumar, T.; Chinnusamy, V. The abscisic acid receptor OsPYL6 confers drought tolerance to indica rice through dehydration avoidance and tolerance mechanisms. J. Exp. Bot. 2021, 72, 1411–1431. [Google Scholar] [CrossRef]
  36. Long, Q.; Qiu, S.C.; Man, J.M.; Ren, D.H.; Xu, N.; Luo, R. OsAAI1 increases rice yield and drought tolerance dependent on ABA-mediated regulatory and ROS scavenging pathway. Rice 2023, 16, 35. [Google Scholar] [CrossRef]
  37. Ben, S.R.; Harbaoui, M.; Ben, R.W.; Zouari, N.; Giang, K.N.; Ben, H.A.; Brini, F. Overexpression of Triticum durum TdAnn12 gene confers stress tolerance through scavenging reactive oxygen species in transgenic tobacco. Funct. Plant Biol. 2019, 46, 885–895. [Google Scholar]
  38. Niu, S.K.; Gu, X.Y.; Zhang, Q.; Tian, X.M.; Chen, Z.; Liu, J.R.; Wei, X.J.; Yan, C.X.; Liu, Z.W.; Wang, X.J.; et al. Grapevine bZIP transcription factor bZIP45 regulates VvANN1 and confers drought tolerance in Arabidopsis. Front. Plant Sci. 2023, 14, 1128002. [Google Scholar] [CrossRef]
  39. Sharma, P.; Jha, A.B.; Dubey, R.S.; Pessarakli, M. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J. Bot. 2012, 2012, 217037. [Google Scholar] [CrossRef]
  40. Hu, C.; Elias, E.; Nawrocki, W.J.; Croce, R. Drought affects both photosystems in Arabidopsis thaliana. New Phytol. 2023, 240, 663–675. [Google Scholar] [CrossRef] [PubMed]
  41. Liu, S.X.; Qin, F. Genetic dissection of maize drought tolerance for trait improvement. Mol. Breed. 2021, 41, 8. [Google Scholar] [CrossRef]
  42. Mahmood, T.; Khalid, S.; Abdullah, M.; Ahmed, Z.; Shah, M.K.N.; Ghafoor, A.; Du, X.M. Insights into drought stress signaling in plants and the molecular genetic basis of cotton drought tolerance. Cells 2020, 9, 105. [Google Scholar] [CrossRef]
  43. Schmidt, R.; Mieulet, D.; Hubberten, H.M.; Obata, T.; Hoefgen, R.; Fernie, A.R.; Fisahn, J.; San Segundo, B.; Guiderdoni, E.; Schippers, J.H.M.; et al. Salt-responsive ERF1 regulates reactive oxygen species-dependent signaling during the initial response to salt stress in rice. Plant Cell 2013, 25, 2115–2131. [Google Scholar] [CrossRef]
  44. Khan, M.N.; Zhang, J.; Luo, T.; Liu, J.H.; Rizwan, M.; Fahad, S.; Xu, Z.H.; Hu, L.Y. Seed priming with melatonin coping drought stress in rapeseed by regulating reactive oxygen species detoxification: Antioxidant defense system, osmotic adjustment, stomatal traits and chloroplast ultrastructure perseveration. Ind. Crops Prod. 2019, 140, 111597. [Google Scholar] [CrossRef]
  45. Munne-Bosch, S.; Villadangos, S. Cheap, cost-effective, and quick stress biomarkers for drought stress detection and monitoring in plants. Trends Plant Sci. 2023, 28, 527–536. [Google Scholar] [CrossRef]
  46. Terletskaya, N.V.; Shcherban, A.B.; Nesterov, M.A.; Perfil‘ev, R.N.; Salina, E.A.; Altayeva, N.A.; Blavachinskaya, I.V. Drought stress tolerance and photosynthetic activity of alloplasmic lines T. dicoccum x T. aestivum. Int. J. Mol. Sci. 2020, 21, 3356. [Google Scholar] [CrossRef]
  47. Smith, E.N.; van Aalst, M.; Tosens, T.; Niinemets, Ü.; Stich, B.; Morosinotto, T.; Alboresi, A.; Erb, T.J.; Gómez-Coronado, P.A.; Tolleter, D.; et al. Improving photosynthetic efficiency toward food security: Strategies, advances, and perspectives. Mol. Plant 2023, 16, 1547–1563. [Google Scholar] [CrossRef] [PubMed]
  48. Dodd, A.N.; Kudla, J.; Sanders, D. The language of calcium signaling. Annu. Rev. Plant Biol. 2010, 61, 593–620. [Google Scholar] [CrossRef] [PubMed]
  49. Laohavisit, A.; Richards, S.L.; Shabala, L.; Chen, C.; Colaço, R.D.D.R.; Swarbreck, S.M.; Shaw, E.; Dark, A.; Shabala, S.; Shang, Z.L.; et al. Salinity-induced calcium signaling and root adaptation in Arabidopsis require the calcium regulatory protein Annexin1. Plant Physiol. 2013, 163, 253–262. [Google Scholar] [CrossRef] [PubMed]
  50. Boyidi, P.; Trishla, V.S.; Botta, H.K.; Yadav, D.; Kirti, P.B. Heterologous expression of rice annexin OsANN5 potentiates abiotic stress tolerance in transgenic tobacco through ROS amelioration. Plant Stress 2021, 2, 100022. [Google Scholar] [CrossRef]
  51. Ma, L.; Ye, J.M.; Yang, Y.Q.; Lin, H.X.; Yue, L.L.; Luo, J.; Long, Y.; Fu, H.Q.; Liu, X.N.; Zhang, Y.L.; et al. The SOS2-SCaBP8 complex generates and fine-tunes an AtANN4-dependent calcium signature under salt stress. Dev. Cell 2019, 48, 697–709.e5. [Google Scholar] [CrossRef]
  52. Li, C.Y.; Li, J.Y.; Chong, K.; Harter, K.; Lee, Y.; Leung, J.; Martinoia, E.; Matsuoka, M.; Offringa, R.; Qu, L.J.; et al. Toward a molecular understanding of plant hormone actions. Mol. Plant 2016, 9, 1–3. [Google Scholar] [CrossRef]
  53. Liao, Z.G.; Zhang, Y.C.; Yu, Q.; Fang, W.C.; Chen, M.Y.; Li, T.F.; Liu, Y.; Liu, Z.C.; Chen, L.; Yu, S.W.; et al. Coordination of growth and drought responses by GA-ABA signaling in rice. New Phytol. 2023, 240, 1149–1161. [Google Scholar] [CrossRef] [PubMed]
  54. Huang, X.Z.; Hou, L.Y.; Meng, J.J.; You, H.W.; Li, Z.; Gong, Z.Z.; Yang, S.H.; Shi, Y.T. The antagonistic action of abscisic acid and cytokinin signaling mediates drought stress response in Arabidopsis. Mol. Plant 2018, 11, 970–982. [Google Scholar] [CrossRef] [PubMed]
  55. Zhang, L.; Xiao, S.S.; Li, W.Q.; Feng, W.Q.; Li, J.; Wu, Z.D.; Gao, X.W.; Liu, F.Q.; Shao, M. Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance. J. Exp. Bot. 2011, 62, 4229–4238. [Google Scholar] [CrossRef] [PubMed]
  56. Li, X.T.; Xie, Y.Y.; Zhu, Q.L.; Liu, Y.G. Targeted genome editing in genes and cis-regulatory regions improves qualitative and quantitative traits in crops. Mol. Plant 2017, 10, 1368–1370. [Google Scholar] [CrossRef] [PubMed]
  57. Toki, S.; Hara, N.; Ono, K.; Onodera, H.; Tagiri, A.; Oka, S.; Tanaka, H. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J. 2006, 47, 969–976. [Google Scholar] [CrossRef]
  58. Tang, N.; Ma, S.Q.; Zong, W.; Yang, N.; Lv, Y.; Yan, C.; Guo, Z.L.; Li, J.; Li, X.; Xiang, Y.; et al. MODD mediates deactivation and degradation of OsbZIP46 to negatively regulate ABA signaling and drought resistance in rice. Plant Cell 2016, 28, 2161–2177. [Google Scholar] [CrossRef]
  59. Jiang, Y.J.; Qiu, Y.P.; Hu, Y.R.; Yu, D.Q. Heterologous expression of AtWRKY57 confers drought tolerance in Oryza sativa. Front. Plant Sci. 2016, 7, 145. [Google Scholar] [CrossRef]
  60. Zhou, Y.B.; Liu, C.; Tang, D.Y.; Yan, L.; Wang, D.; Yang, Y.Z.; Gui, J.S.; Zhao, X.Y.; Li, L.G.; Tang, X.D.; et al. The receptor-like cytoplasmic kinase STRK1 phosphorylates and activates CatC, thereby regulating H2O2 homeostasis and improving salt tolerance in rice. Plant Cell 2018, 30, 1100–1118. [Google Scholar] [CrossRef]
Ijms 24 17495 g001
Figure 1. Characterization of OsANN9. (A) Phylogenic tree of annexins of rice, maize and wheat. The phylogenetic tree was constructed with MEGA7. All polypeptide sequences of annexins were derived from NCBI. (B) Sequence alignment of OsANN9 and other reported annexins in rice. The yellow box indicates the C2H2 domain. The red boxes indicate type-II Ca2+-binding sites.
Figure 1. Characterization of OsANN9. (A) Phylogenic tree of annexins of rice, maize and wheat. The phylogenetic tree was constructed with MEGA7. All polypeptide sequences of annexins were derived from NCBI. (B) Sequence alignment of OsANN9 and other reported annexins in rice. The yellow box indicates the C2H2 domain. The red boxes indicate type-II Ca2+-binding sites.
Ijms 24 17495 g001
Ijms 24 17495 g002
Figure 2. Subcellular localization and Ca2+ binding activity of OsANN9. (A–C). Subcellular localization of OsANN9-GFP in root tips of 3-day-old rice seedlings (A), tobacco leaf epidermal cells (B), and rice protoplasts (C). GFP alone was used as a control. Bar = 20 μm. (D) Total protein of the E. coli cultures transformed with pET30a-OsANN9 (lane 1). When different concentrations of CaCl2 (5 mM, 10 mM, 20 mM) were added to the supernatants separately, the OsANN9-His protein bound to Ca2+ and formed pellets (lanes 3, 5, 7). After adding equal amounts of EDTA-Na2 to the pellets, OsANN9-His was released (lanes 4, 6, 8). Lane 2 shows the molecular weight marker. (E) Fluorescence intensity of OsANN9 without (blue curve) or with (green curve) 2 mM CaCl2. The brown curve shows the fluorescence intensity of OsANN9 with CaCl2 and an equal amount of EDTA-Na2.
Figure 2. Subcellular localization and Ca2+ binding activity of OsANN9. (A–C). Subcellular localization of OsANN9-GFP in root tips of 3-day-old rice seedlings (A), tobacco leaf epidermal cells (B), and rice protoplasts (C). GFP alone was used as a control. Bar = 20 μm. (D) Total protein of the E. coli cultures transformed with pET30a-OsANN9 (lane 1). When different concentrations of CaCl2 (5 mM, 10 mM, 20 mM) were added to the supernatants separately, the OsANN9-His protein bound to Ca2+ and formed pellets (lanes 3, 5, 7). After adding equal amounts of EDTA-Na2 to the pellets, OsANN9-His was released (lanes 4, 6, 8). Lane 2 shows the molecular weight marker. (E) Fluorescence intensity of OsANN9 without (blue curve) or with (green curve) 2 mM CaCl2. The brown curve shows the fluorescence intensity of OsANN9 with CaCl2 and an equal amount of EDTA-Na2.
Ijms 24 17495 g002
Ijms 24 17495 g003
Figure 3. Expression pattern of OsANN9 under PEG or drought treatment. (A,B) RT-qPCR results of OsANN9 expression in WT seedlings under 20% PEG treatment and drought stress. OsACTIN1 was used as the internal reference gene for normalization. (C) GUS staining of tissues from OsANN9pro::GUS transgenic rice lines. Bar = 1 mm. (D) Relative GUS activity with or without 20% PEG6000 for 6 h in OsANN9pro::GUS lines. T1, T3 and T7 were represented by three independent lines. Values are presented as the means ± SE of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.
Figure 3. Expression pattern of OsANN9 under PEG or drought treatment. (A,B) RT-qPCR results of OsANN9 expression in WT seedlings under 20% PEG treatment and drought stress. OsACTIN1 was used as the internal reference gene for normalization. (C) GUS staining of tissues from OsANN9pro::GUS transgenic rice lines. Bar = 1 mm. (D) Relative GUS activity with or without 20% PEG6000 for 6 h in OsANN9pro::GUS lines. T1, T3 and T7 were represented by three independent lines. Values are presented as the means ± SE of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.
Ijms 24 17495 g003
Ijms 24 17495 g004
Figure 4. CRISPR-targeted mutagenesis of OsANN9. (A) Diagram of gene structure. Black boxes and lines indicate exons and introns, respectively, and gray and red boxes indicate UTRs and gRNA targeting regions, respectively. (B) The osann9 mutants were generated by CRISPR/Cas9 gene editing. The sequence marked in red exhibits the gRNA target sequence. The yellow-labeled T shows the insertion site in the osann9-cr1 mutant. Short red lines show the deletion of 35-bp in the osann9-cr3 mutant. (C) Amino acid sequence alignment of OsANN9 in WT and osann9 mutants. The amino acids marked in red are encoded by the gRNA target sequence. Black asterisks represent premature protein termination. (D) DNA sequencing results showed an insertion of 1-bp T (the black arrow points) and a deletion of 35-bp (the black arrow points between two G) in the genomic DNA of osann9-cr1 and osann9-cr3. (E,F) Seed setting rate of osann9, WT and OsANN9-OE plant. Values are presented as the means ± SE of three biological replicates. n > 30. *** p < 0.001, Student’s t-test.
Figure 4. CRISPR-targeted mutagenesis of OsANN9. (A) Diagram of gene structure. Black boxes and lines indicate exons and introns, respectively, and gray and red boxes indicate UTRs and gRNA targeting regions, respectively. (B) The osann9 mutants were generated by CRISPR/Cas9 gene editing. The sequence marked in red exhibits the gRNA target sequence. The yellow-labeled T shows the insertion site in the osann9-cr1 mutant. Short red lines show the deletion of 35-bp in the osann9-cr3 mutant. (C) Amino acid sequence alignment of OsANN9 in WT and osann9 mutants. The amino acids marked in red are encoded by the gRNA target sequence. Black asterisks represent premature protein termination. (D) DNA sequencing results showed an insertion of 1-bp T (the black arrow points) and a deletion of 35-bp (the black arrow points between two G) in the genomic DNA of osann9-cr1 and osann9-cr3. (E,F) Seed setting rate of osann9, WT and OsANN9-OE plant. Values are presented as the means ± SE of three biological replicates. n > 30. *** p < 0.001, Student’s t-test.
Ijms 24 17495 g004
Ijms 24 17495 g005
Figure 5. Osmotic stress test with OsANN9-OE, OsANN9-Ri, osann9 and WT. (A) Performance of OsANN9-OE, OsANN9-Ri, osann9 and WT seedings grown in 1/2 MS medium with or without 20% PEG6000 for 7 days. Bars = 1 cm. (B,C) Shoot and root length of plants in (A). Values are presented as the means ± SE of three biological replicates. n > 30. Different letters indicate significant differences among genotypes (on two-way ANOVA, p < 0.05).
Figure 5. Osmotic stress test with OsANN9-OE, OsANN9-Ri, osann9 and WT. (A) Performance of OsANN9-OE, OsANN9-Ri, osann9 and WT seedings grown in 1/2 MS medium with or without 20% PEG6000 for 7 days. Bars = 1 cm. (B,C) Shoot and root length of plants in (A). Values are presented as the means ± SE of three biological replicates. n > 30. Different letters indicate significant differences among genotypes (on two-way ANOVA, p < 0.05).
Ijms 24 17495 g005
Ijms 24 17495 g006
Figure 6. Drought-stress test with OsANN9-OE, OsANN9-Ri, osann9 and WT. (A) Seedlings grown for 14 days under normal conditions (top panel) and were subjected to drought-stress for 10 days (middle panel), followed by re-watering for 7 days (bottom panel). Bars = 10 cm. (B) Survival rates of OsANN9-OE, OsANN9-Ri, osann9 and WT plants after re-watering for 7 days. (C) Water loss by 14-day-old OsANN9-OE, OsANN9-Ri, osann9 and WT whole plants. Fresh weight was monitored at the indicated times. Values are presented as the means ± SE of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.
Figure 6. Drought-stress test with OsANN9-OE, OsANN9-Ri, osann9 and WT. (A) Seedlings grown for 14 days under normal conditions (top panel) and were subjected to drought-stress for 10 days (middle panel), followed by re-watering for 7 days (bottom panel). Bars = 10 cm. (B) Survival rates of OsANN9-OE, OsANN9-Ri, osann9 and WT plants after re-watering for 7 days. (C) Water loss by 14-day-old OsANN9-OE, OsANN9-Ri, osann9 and WT whole plants. Fresh weight was monitored at the indicated times. Values are presented as the means ± SE of three biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.
Ijms 24 17495 g006
Ijms 24 17495 g007
Figure 7. OsANN9 overexpression reduced ROS accumulation and membrane lipid peroxidation. (A,B) In situ detection of H2O2 and O2− in the leaves of transgenic plants under normal or drought-stress conditions with DAB and NBT staining. Bar = 1 mm. (C,D) MDA content and relative electrolyte leakage from the detached leaves of 14-day-old plants under normal or drought-stress conditions. Values are presented as the means ± SE of three biological replicates. n = 9; different letters indicate significant differences among genotypes (on two-way ANOVA, p < 0.05).
Figure 7. OsANN9 overexpression reduced ROS accumulation and membrane lipid peroxidation. (A,B) In situ detection of H2O2 and O2− in the leaves of transgenic plants under normal or drought-stress conditions with DAB and NBT staining. Bar = 1 mm. (C,D) MDA content and relative electrolyte leakage from the detached leaves of 14-day-old plants under normal or drought-stress conditions. Values are presented as the means ± SE of three biological replicates. n = 9; different letters indicate significant differences among genotypes (on two-way ANOVA, p < 0.05).
Ijms 24 17495 g007
Ijms 24 17495 g008
Figure 8. OsANN9 modulates ROS scavenging by regulating SOD, POD and CAT activity. (A–C) SOD, POD and CAT enzyme activity in 7-day-old plants under normal or drought-stress conditions. (D–F) The expression of OsSODcc2, OsAPX2 and OsCAT in OsANN9-OE, OsANN9-Ri, osann9 and WT plants under normal or drought-stress conditions. Values are presented as the means ± SE of three biological replicates. Different letters indicate significant differences among genotypes (on two-way ANOVA, p < 0.05).
Figure 8. OsANN9 modulates ROS scavenging by regulating SOD, POD and CAT activity. (A–C) SOD, POD and CAT enzyme activity in 7-day-old plants under normal or drought-stress conditions. (D–F) The expression of OsSODcc2, OsAPX2 and OsCAT in OsANN9-OE, OsANN9-Ri, osann9 and WT plants under normal or drought-stress conditions. Values are presented as the means ± SE of three biological replicates. Different letters indicate significant differences among genotypes (on two-way ANOVA, p < 0.05).
Ijms 24 17495 g008
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Jia, Y.; Gu, X.; Chai, J.; Yao, X.; Cheng, S.; Liu, L.; He, S.; Peng, Y.; Zhang, Q.; Zhu, Z. Rice OsANN9 Enhances Drought Tolerance through Modulating ROS Scavenging Systems. Int. J. Mol. Sci. 2023, 24, 17495. https://doi.org/10.3390/ijms242417495

AMA Style

Jia Y, Gu X, Chai J, Yao X, Cheng S, Liu L, He S, Peng Y, Zhang Q, Zhu Z. Rice OsANN9 Enhances Drought Tolerance through Modulating ROS Scavenging Systems. International Journal of Molecular Sciences. 2023; 24(24):17495. https://doi.org/10.3390/ijms242417495

Chicago/Turabian Style

Jia, Yangyang, Xiangyang Gu, Jiaxin Chai, Xiaohong Yao, Shoutao Cheng, Lirui Liu, Saiya He, Yizhuo Peng, Qian Zhang, and Zhengge Zhu. 2023. "Rice OsANN9 Enhances Drought Tolerance through Modulating ROS Scavenging Systems" International Journal of Molecular Sciences 24, no. 24: 17495. https://doi.org/10.3390/ijms242417495

APA Style

Jia, Y., Gu, X., Chai, J., Yao, X., Cheng, S., Liu, L., He, S., Peng, Y., Zhang, Q., & Zhu, Z. (2023). Rice OsANN9 Enhances Drought Tolerance through Modulating ROS Scavenging Systems. International Journal of Molecular Sciences, 24(24), 17495. https://doi.org/10.3390/ijms242417495

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop