Abstract
Key message
Overexpressing miR529a can enhance oxidative stress resistance by targeting OsSPL2 and OsSPL14 genes that can regulate the expression of their downstream SOD and POD related genes.
Abstract
MicroRNAs are involved in the regulation of plant developmental and physiological processes, and their expression can be altered when plants suffered environment stresses, including salt, oxidative, drought and Cadmium. The expression of microRNA529 (miR529) can be induced under oxidative stress. However, its biological function under abiotic stress responses is still unclear. In this study, miR529a was overexpressed to investigate the function of miR529a under oxidative stress in rice. Our results demonstrated that the expression of miR529a can be induced by exogenous H2O2, and overexpressing miR529a can increase plant tolerance to high level of H2O2, resulting in increased seed germination rate, root tip cell viability, reduced leaf rolling rate and chlorophyll retention. The expression of oxidative stress responsive genes and the activities of superoxide dismutase (SOD) and peroxidase (POD) were increased in miR529a overexpression plant, which could help to reduce redundant reactive oxygen species (ROS). Furthermore, only OsSPL2 and OsSPL14 were targeted by miR529a in rice seedlings, repressing their expression in miR529aOE plants could lead to strengthen plant tolerance to oxidation stress. Our study provided the evidence that overexpression of miR529a could strengthen oxidation resistance, and its target genes OsSPL2 and OsSPL14 were responsible for oxidative tolerance, implied the manipulation of miR529a and its target genes regulation on H2O2 related response genes could improve oxidative stress tolerance in rice.
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Introduction
Rice is usually subjected to various abiotic stresses in the process of growth and development, such as high and low temperature, drought, heavy metals and salt (Cui et al. 2015; Dionisio-Sese and Tobita 1998; Nigam et al. 2015; Tang et al. 2014). The plants exhibit growth retardation under stresses, which can lead to reduce crop yield and quality, even cause death. Reactive oxygen species (ROS) including hydrogen peroxide (H2O2), superoxide radical (O2 ·−), and hydroxyl radicals (.OH) are by-products of aerobic metabolism, which are controlled by antioxidant defense systems (Gill and Tuteja 2010; Mittler 2002; You and Chan 2015). When plants constantly suffer from environmental stresses, the generated ROS will be beyond the ability of the active oxygen scavenging system, which will cause oxidative damage, eventually resulting in cell apoptotic death. H2O2, acting as a signaling molecule or a second messenger (Quan et al. 2008; Vranova et al. 2002), can adjust plant development, adaptation of environmental stresses, respiration and programmed cell death (PCD) (Neill et al. 2002; Orozco-Cardenas et al. 2001).
Recently, many H2O2 stress related response genes have been discovered. Constitutive expression of pathogenesis-related genes 5 (CPR5), a new type of regulatory factor (Orjuela et al. 2013), is involved in the regulation of seed germination, seedling development and plant ROS signaling pathway of oxidative stress (Bowling et al. 1997; Gao et al. 2011). The vacuole was known to affect oxidative stress response, and the vacuolar processing enzymes (VPEs) are participated in H2O2-induced PCD pathway (Deng et al. 2011). In addition, Os04g0376100 and Os10g0519700, encoding for translocase of the inner/out mitochondrial membrane (TIM/TOM) complexes, were also found to be related to H2O2 signaling pathway (Pegoraro et al. 2012).
MicroRNA (miRNA) is one of the most important regulators to control gene expression, which is involved in many fundamental biological processes, and plays the key role in the process of plant development (Jones-Rhoades et al. 2006). A lot of miRNAs have emerged as important players in plant responses to biotic and abiotic stresses (Sunkar et al. 2007). Among them, miR398 mediated responses to oxidative stress through regulating the expression of its target genes (Dugas and Bartel 2008; Lu et al. 2011). MiR529 was down-regulated in response to cold and drought stresses (Gupta et al. 2014; Zhou et al. 2010), and it was identified to be responsive to exogenous H2O2 stress in rice (Li et al. 2011). However, the mechanism of miR529 response to oxidative stress is still not clear. MiR529 was present in the common ancestor of embryophytes but lost in eudicots (Cuperus et al. 2011), which contains miR529a and miR529b two members (Zhang et al. 2015). MiR529 targets SQUAMOSA promoter-binding protein like (SPL) genes, a plant specific transcription factor in regulating plant growth and development (Chen et al. 2010). Previous studies showed that SPLs, especially SPL2 and SPL11 promote the expression of heat stress (HS)-responsive genes, which is critical only after exposure to HS, and SPL3, SPL14 and SPL17 are involved in regulating the expression of cold stress responsive genes (Cui et al. 2014, 2015; Stief et al. 2014). Whereas, the network of miR529a-SPLs in plant ROS signaling pathway of oxidative stress is still not clear.
Here, we reported a systematic investigation of H2O2-regulated miR529a and its target genes in rice. Overexpressing miR529a showed that only OsSPL2 and OsSPL14 two genes were targeted by miR529a in rice seedlings. The expression of H2O2-responsive genes were increased by repressing the expression of miR529a targeted genes OsSPL2 and OsSPL14, which could lead to increase the expression of ROS-responsive genes and finally elevate the activities of SOD and POD to strengthen plant tolerance to oxidation stress. Our results suggested that manipulation of miR529a and its target genes could improve oxidative stress tolerance in rice.
Materials and methods
Plants materials and growth conditions
Two japonica cultivars Nipponbare and Aikawa1 were used in this study. Seeds were shelled and sterilized in 1% NaClO for 20–30 min, washed with distilled water for at least four times, placed on sterile MS solid culture medium, and kept at the conditions of 28 °C/21 °C, 14 h light/10 h dark, and 75% relative humidity in growth cabinet for germination and growth. The seedlings were then placed in MS liquid culture solution after 7 days growth.
Vector construction and rice transformation
To overexpress miR529a in rice, the 456 bp pri-miR529a sequence including stem-loop structure was amplified by PCR with specific primers (Table S1), PCR product was then cloned into the SacI–SmaI sites of the binary vector pCAMBIA1300, which was driven by a maize Ubiquitin promoter (Fig. S1). And the constructed vector was then transferred to Agrobacterium tumefaciens strain EHA105 for transformation. A modified high-efficiency transformation system by co-cultivating rice calli with Agrobacterium was used in our experiment (Ozawa 2009). The transgenic rice plants carrying MIR529a was confirmed by PCR using the specific primers (Table S1). The homozygotes of T3 generation of transgenic plants (miR529aOE3, OE5, and OE6) were chosen for further study.
Seed germination test and phenotype analysis of rice seedling
Rice seeds were soaked at 37 °C in the dark for 24 h, were then moved on filter paper wetted by ddH2O supplemented with different concentrations of H2O2 in petri dishes, and incubated at 27 °C with 14/12 h light/dark cycles for three days. The germination rate was calculated using 30 seeds in each treatment (Deng et al. 2011).
The four-leaf seedlings were immersed in the solutions of different concentrations of H2O2 (0, 0.6, 3, and 15 mM) for 6 h to detect phenotype response. The number of rolling leaf was calculated and equal amounts of leaves (0.15 g) were harvested and frozen in liquid nitrogen, then stored at −80 °C.
Detection of ROS formation in root tip with FDA staining
The rice root tips were treated with different concentrations of H2O2 for 3 days, and the primary roots were then incubated in 20 µM H2DCF-DA [initially dissolved in dimethyl sulfoxide (DMSO)] at 4 °C for 1 h (Foreman et al. 2003). The roots were washed and immersed with 0.1 mM KCl and 0.1 mM CaCl2 (pH 6.0) at 22 °C for 1 h, and were immediately mounted and photographed under a fluorescence microscope (Nikon AZ100, Japan) equipped with a filter set (Fluorescence was detected using wavelength of 488 nm excitation and emission wavelength of 505–545 nm band pass filter).
The activities of antioxidant enzymes analysis
Leaf was grounded to fine powder by grinding machine (Shanghai Jingxin, China), then transferred to 10 mL tubes and added 5 mL of 50 mM phosphate buffer (PBS, pH 7.8). The samples were then violently oscillated for 1 min, and homogenates were centrifuged at 8000g for 25 min at 4 °C. Supernatants were then collected into fresh tubes and stored at 4 °C for enzyme analysis.
SOD (EC 1.15.1.1) activity was measured according to nitro blue tetrazolium (NBT) method by measuring the photo-reduction of NBT at OD560 (Beers and Sizer 1952; Qiao et al. 2010); activity of POD (EC1.11.1.7) was assayed by monitoring guaiacol at 470 nm by spectrophotometry (Egley et al. 1983).
Histochemical detection of H2O2 and O2 ·−
The content of H2O2 in the leaf was measured spectrophotometrically as described previously (Willekens et al. 1997). And 3, 3′-diaminobenzidine (DAB) and NBT staining were used to determine the accumulation of H2O2 and O2 ·−, respectively (Wohlgemuth et al. 2002).
For H2O2 detection, the treated rice leaf was immediately immersed into 1 mg/mL DAB solution (pH 3.8) and incubated for 6 h in light at 25 °C. And leaf was then bleached in 95% boiling ethanol for at least 10 min. This step decolorized the leaf with the exception of the brown polymerization spots produced by the reaction of DAB with H2O2. As for observation of the visualized O2 ·− accumulation, NBT staining solution was prepared by dissolving NBT with 25 mM HEPES free acid (pH 7.8) to a concentration of 0.5 mg/mL. Leaf was then collected and completely submerged into NBT solution. The following steps were the same as the procedures of DAB staining except for incubation in dark for 6 h instead of light.
Chlorophyll content assay
The flag leaf was treated with varied concentrations of H2O2 (0, 0.6, 3, and 15 mM) for 5 days. 0.1 g leaf was grind into powder, and then infiltrated in 10 mL 95% ethanol solution at room temperature in dark for 3–5 days. A 665 and A 649 of the extracts were monitored using a Multifunction Microplate Reader (BioTek Synery H1, Vermont, USA). The chlorophyll content was calculated using the formula:
The total content of chlorophyll (mg/g) = [chlorophyll concentration × extraction volume/sample (fresh weight). All experiments were repeated three times.
RNA extraction, qRT-PCR and stem-loop qRT-PCR
Total RNA was extracted from rice seedlings using TRIzol reagent (Invitrogen, USA). PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Japan) was used to remove residues genomic DNA, then the first strand of cDNA was synthesized. The qRT-PCR was performed on a Roche LightCycler® 96 Real-time PCR System (Roche, Switzerland) by using SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Japan). The conditions were as follows: denature at 95 °C for 30 s, 40 cycles at 94 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s. OsEf1-α (LOC_Os03g08050) was used as an internal reference. To enable statistical analysis, three independent biological replicates were obtained and subjected to qRT-PCR in triplicate.
Stem-loop qRT-PCR was performed to measure the expression of miR529a (Chen et al. 2005; Kramer 2011; Varkonyi-Gasic and Hellens 2011). U6 snRNA was used as the internal control for normalizing the raw data. Gene-specific primers are shown in supporting information Table S1.
Results
Expression profile of miR529a and miR529a overexpression
At vegetative stage, miR529a was highly expressed in seedling, and lowly expressed in shoot, root and leaf (Fig. 1). Previous study showed that miR529a was up regulated under H2O2 treatment (Li et al. 2011), four-leaf stage rice seedling was then treated with different concentrations of H2O2 (0, 0.6, 3, and 15 mM) for 6 h, the expression of miR529a was increased gradually with the rising of concentration of H2O2 (Fig. 2a), which was consistent with the previous study (Li et al. 2011).
To clarify the function of miR529a responding to H2O2, miR529a overexpression (miR529aOE) vector was constructed to generate transgenic plants, six independent transgenic lines with similar phenotype were obtained (Fig. S1b). The results displayed that the expression of miR529a was around 15 times higher in miR529aOE plants than in WT plants (Fig. 2b), while there was no significant difference in the phenotype of WT and miR529aOE plants at seedling stage.
MiR529a overexpression promotes plant tolerance to H2O2 stress
No significant difference of germination capacity was exhibited in WT, miR529aOE and Aikawa1 under normal conditions. While the germination rate was dramatically reduced in WT (58 ± 1.4%) and Aikawa1 (38.9 ± 9.4%), compared to miR529aOE (83.3 ± 6.9%) after treated with 15 mM H2O2 for 3 days (Fig. 3).
The fluorescein diacetate (FDA) method was performed to detect the ROS formation in root tip, and the miR529aOE maintained higher cell viability after exposure to 15 mM H2O2 for 5 days compared to WT, while Aikawa1 showed the lowest cell viability (Fig. 3c).
The WT showed obvious severely wilted symptoms, such as water loss and leaf roll, whereas miR529aOE remained relatively healthy when the seedlings (four-leaf stage) were subjected to oxidation stress (15 mM H2O2) for 6 h (Fig. 4a). Statistical analysis showed that miR529aOE plants exhibited significantly less rolling leaf than WT under oxidative stress (Fig. 4b).
The ROS levels can be controlled by synthesize antioxidant enzymes to prevent their toxicity. Therefore, the activities of two enzymes SOD (the first enzyme scavenges oxygen radical) and POD (the inducible enzyme) were detected, which can eliminate the metabolic process of hydrogen peroxide (Jacob and Burri 1996). The SOD and POD activities were increased under oxidation stress, while the activities of SOD and POD in miR529aOE were much higher than that in WT after H2O2 treatment, especially under 15 mM (Fig. 5a, b), suggesting that overexpressing miR529a could be endowed with the ability to keep balance of ROS. Furthermore, the accumulation of H2O2 and O2 ·− in young leaf from WT and miR529aOE was assessed by DAB and NBT staining, respectively. The WT leaf displayed more staining spots than the leaf from miR529aOE (Fig. 5c, d), demonstrating that the former had higher ROS than the latter. Subsequently, quantitative measurement of H2O2 showed that the endogenous H2O2 was significantly higher in WT than in miR529aOE (Fig. 5e).
The flag leaf was treatment with 15 mM H2O2 for 5 days that led to severe chlorosis in WT, while maintained green in miR529aOE (Fig. 5f), and the chlorophyll level in miR529aOE was significantly higher than that in WT (Fig. 5g).
Expression patterns of H2O2 responsive genes
Above results showed a potential role of miR529a in ROS scavenging. To gain more insight into the role of miR529a in response to H2O2 stress, we further examined the expression patterns of ROS-responsive genes in both WT and miR529aOE, including one organic osmotic regulation related gene LOC_Os05g38150 (OsP5CS) and two scavenging ROS genes LOC_Os01g22249 and LOC_Os04g59150 (Chen et al. 2009; Cui et al. 2015; Maruyama et al. 2014), which can be response to adverse stimulus. All three genes were up-regulated under H2O2 stress and displayed similar expression profile in miR529aOE, with the highest expression level at concentration of 3 mM after 6 h H2O2 treatment (Fig. 6). However, the expression of genes at 15 mM after 6 h treatment were decreased, even significantly lower than untreated (Fig. 6a–c), indicating that scavenging enzymes had been accumulated enough at concentration of 15 mM.
Additionally, H2O2-induced closely related genes were further selected to discover the possible path of H2O2 responses. CPR5, Os10g0519700 and OsVPE3 three genes displayed up-regulation after exposure to H2O2 treatment for 6 h in WT, while in miR529aOE, the expression were much higher than in WT at 15 mM (Fig. 6d–f), but not in others (Fig. S2), these results demonstrated that CPR5, Os10g0519700 and OsVPE3 might directly respond to H2O2 stress.
Expression patterns of miR529a target genes
MiR529a was predicted to target five SPL genes-OsSPL2, OsSPL14, OsSPL16, OsSPL17 and OsSPL18 using psRNATarget software with the cut-off threshold as 2.5 (Dai and Zhao 2011). However, only OsSPL2 and OsSPL14 genes were down-regulated in the seedling of miR529aOE compared to WT (Fig. 7a), indicating that only OsSPL2 and OsSPL14 genes were targeted by miR529a in rice seedlings. And the expression of OsSPL2 and OsSPL14 was decreased with the increasing of the concentration of exogenous H2O2 (Fig. 7b, c), resulting from the increased expression of miR529a (Fig. 1b). The expression of OsSPL2 and OsSPL14 were then investigated under oxidative stress with 15 mM H2O2 in WT and miR529aOE, their expression were significantly lower in miR529aOE than in WT after H2O2 treatment (Fig. 7d, e), which implied that low expression of SPLs significantly contribute to resist oxidation, and they were negatively responded to H2O2 stress.
Up-regulation of miR529a target gene OsSPL14 can weaken oxidative tolerance
The seedlings of Aikawa1 and Nipponbare were treated with 15 mM H2O2 to test oxidative tolerance, the result showed that Aikawa1 has a severe syndrome (Fig. 8a), and the leaf rolling rate of Aikawa1 was significantly higher than Nipponbare (Fig. 8b). Moreover, the expression level of OsSPL14 in Aikawa1 seedling of both control and H2O2 treatment was much higher than in Nipponbare (Fig. 8c). These results implied that OsSPL14 was a negative regulator to control ROS balance, and down-regulated OsSPL14 by overexpression of miR529a could enhance oxidative tolerance.
Discussion
Many studies showed that miRNAs not only were involved in plant growth and development, but also affected plant stress responses (Liu et al. 2008; Sunkar and Zhu 2004; Wang et al. 2013; Zhou et al. 2008). MiR529 is an ancient microRNA in plants, which is present from moss to vascular plant, especially enriched in monocots (Chavez Montes et al. 2014). It has miR529a and miR529b two members in rice (Liu et al. 2005; Zhu et al. 2008). The expression of miR529a was much higher than miR529b, and it is the miR529a-5p (miR529a*), not miR529a-3p, acts as an executive in rice development (Jeong et al. 2011). Recent studies have shown that the expression of miR529a can be induced by the environmental stimuli of H2O2 and Cd stresses, which could help to reduce redundant ROS (Li et al. 2011; Tang et al. 2014). The expression of miR529a was up-regulated under H2O2 treatment, and the higher the concentration of H2O2, the higher the expression of miR529a (Fig. 2a), demonstrating that miR529a was positive response to H2O2 stress and the induced ROS by H2O2 was significantly dependent on exogenous H2O2 (Deng et al. 2011), while miR529 was negatively response to drought, arsenate and arsenite stresses (Sharma et al. 2015; Zhou et al. 2010).
ROSs are by-products of the aerobic metabolic pathways including respiration and photosynthesis in plant chloroplast, mitochondria and peroxisome (D’Autreaux and Toledano 2007). Furthermore, exogenous H2O2 can also induce a typical ROS in the suspension cultured cells (Desikan et al. 1998; Houot et al. 2001). The plant has its own system to modulate the internal ROS level to maintain its homeostasis (Del Rio and Lopez-Huertas 2016). Once the balance is broken, the high level of ROS will cause plant oxidative stress. The flag leaf of WT accumulated much more ROS than transgenic line (Fig. 5c, d); which had less chlorosis degree treated with exogenous H2O2 (Fig. 5f), suggested that overexpressing miR529a had the ability to keep ROS at suitable levels.
The high concentration of ROS is reactive and toxic in plants, although the accumulation of stress-induced ROS can be counteracted by intrinsic antioxidant systems. The antioxidant systems include various enzymatic scavengers, which can convert toxic superoxide radicals to harmless ion, and at last eliminate hydrogen peroxide in plants (Mittler et al. 2004). SOD and POD are two important ROS-scavengers in antioxidant defense system (Bansal and Kanwar 2013; Wang et al. 2016), their activities in transgenic line were significantly higher than that in WT (Fig. 5a, b), which were confirmed by highly expressed ROS scavenging genes (Fig. 6b, c). In other respect, the free proline accumulation can directly protect plants from various stresses (Szabados and Savoure 2010). And then, the expression of proline synthase gene (LOC_Os05g38150) was significantly higher in transgenic line than WT, which may partly explain the increased tolerance to oxidative stress (Fig. 6a). These results revealed that miR529a could enhance the tolerance ability to exogenous H2O2 stress.
Not surprisingly, the expression of H2O2-responsive genes (Liu et al. 2013) in rice seedling were up-regulated in transgenic lines at the concentration of 15 mM H2O2 (Fig. 6d–h), and these genes might positive regulate H2O2 stress in ROS signaling pathway.
The SPL genes family is a class of highly conserved transcription factors involved in multiple physiological and biochemical processes, including plant morphosis, flowering time, juvenile-to-adult transition and stresses response (Chen et al. 2010; Gou et al. 2011; Wang et al. 2009; Yang et al. 2008), which can be targeted by miR529 (Ling and Zhang 2012; Morea et al. 2016; Zhang et al. 2015). Five SPL genes were predicted to be targeted by miR529a, while overexpressing miR529a only led to decrease the expression of OsSPL2 and OsSPL14 in rice seedlings (Fig. 8a). It indicated that OsSPL2 and OsSPL14 might be targets of miR529a in rice seedlings, while OsSPL14 and OsSPL17 were regulated by miR529a to affect tillering and panicle branching (Wang et al. 2015), implying that SPL genes were tissue-specifically regulated by miR529a. In addition, the expression of OsSPL2 and OsSPL14 were decreased with the increasing of H2O2 concentration (Fig. 7b, c), and their expression were all significantly low in WT and transgenic line under H2O2 treatment (Fig. 7d, e), suggested that miR529a might play an important role in response to oxidative stress in rice, and its targets are negatively response to exogenous H2O2 in rice seedlings.
The japonica cultivar Aikawa1 has a single nucleotide change from C to A at the miR156-targeted site in OsSPL14 gene (Miura et al. 2010), displayed higher transcript of OsSPL14, low seed germination rate and low ROS level of root tip, suffered more severe rolling syndrome than Nipponbare under H2O2 stress because of increased transcript level of OsSPL14 (Fig. 8a). Furthermore, OsSPL14 was reported to be regulated directly by miR529a and miR156 (Jeong et al. 2011). These findings confirmed that OsSPL14 gene was likely the target of miR156/miR529 and negatively regulated exogenous H2O2 stress.
Overall, the expression of H2O2 responsive genes CPR5, Os10g0519700 and OsVPE3 were increased by repressing the expression of miR529a target genes OsSPL2 and OsSPL14, which could lead to increase the expression of LOC_Os05g38150, LOC_Os01g22249 and LOC_Os04g59150, and finally elevated SOD and POD activities to strengthen the plant tolerance to oxidation stress (Fig. 9). These results indicated a potential complex network of miR529a-SPLs-downstream genes in ROS signaling pathway of oxidative stress response (Fig. 9).
Author contribution statement
JHX and EY designed research; EY, ZL, CL, YL, and QL performed research; EY, ZL, CL, and JHX analyzed data; EY and JHX wrote and edited the manuscript.
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We are grateful to Dr. Motoyuki Ashikari from Nagoya University for his offering the Aikawa1 seeds.
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Communicated by Da-Bing Zhang.
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Yue, E., Liu, Z., Li, C. et al. Overexpression of miR529a confers enhanced resistance to oxidative stress in rice (Oryza sativa L.). Plant Cell Rep 36, 1171–1182 (2017). https://doi.org/10.1007/s00299-017-2146-8
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DOI: https://doi.org/10.1007/s00299-017-2146-8