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Article

Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice

by
Se Eun Jung
1,
Seung Woon Bang
1,
Sung Hwan Kim
1,
Jun Sung Seo
1,
Ho-Bin Yoon
1,
Youn Shic Kim
1,2 and
Ju-Kon Kim
1,*
1
Crop Biotechnology Institute, GreenBio Science and Technology, Seoul National University, Pyeongchang 25354, Korea
2
Agriculture and Life Sciences Research Institute, Kangwon National University, Chuncheon 24341, Korea
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(14), 7656; https://doi.org/10.3390/ijms22147656
Submission received: 18 June 2021 / Revised: 12 July 2021 / Accepted: 14 July 2021 / Published: 17 July 2021
(This article belongs to the Special Issue Drought-Stress Induced Physiological and Molecular Changes in Plants)
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Abstract

:
Abiotic stresses severely affect plant growth and productivity. To cope with abiotic stresses, plants have evolved tolerance mechanisms that are tightly regulated by reprogramming transcription factors (TFs). APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors are known to play an important role in various abiotic stresses. However, our understanding of the molecular mechanisms remains incomplete. In this study, we identified the role of OsERF83, a member of the AP2/ERF transcription factor family, in response to drought stress. OsERF83 is a transcription factor localized to the nucleus and induced in response to various abiotic stresses, such as drought and abscisic acid (ABA). Overexpression of OsERF83 in transgenic plants (OsERF83OX) significantly increased drought tolerance, with higher photochemical efficiency in rice. OsERF83OX was also associated with growth retardation, with reduced grain yields under normal growth conditions. OsERF83 is predominantly expressed in the vascular tissue of all organs. Transcriptome analysis revealed that OsERF83 regulates drought response genes, which are related to the transporter (OsNPF8.10, OsNPF8.17, OsLH1), lignin biosynthesis (OsLAC17, OsLAC10, CAD8D), terpenoid synthesis (OsTPS33, OsTPS14, OsTPS3), cytochrome P450 family (Oscyp71Z4, CYP76M10), and abiotic stress-related genes (OsSAP, OsLEA14, PCC13-62). OsERF83 also up-regulates biotic stress-associated genes, including PATHOGENESIS-RELATED PROTEIN (PR), WALL-ASSOCIATED KINASE (WAK), CELLULOSE SYNTHASE-LIKE PROTEIN E1 (CslE1), and LYSM RECEPTOR-LIKE KINASE (RLK) genes. Our results provide new insight into the multiple roles of OsERF83 in the cross-talk between abiotic and biotic stress signaling pathways.

1. Introduction

Abiotic stresses, including drought, cold, salinity, and nutrient stress, adversely affect the cellular homeostasis of plants and ultimately impair their growth and productivity. Among these abiotic stresses, extreme heat and water deficits frequently damage plants [1]. As extreme weather disasters associated with climate change have become steadily more common, crop losses have also increased over the past several decades [2,3]. Rice (Oryza sativa. L.) productivity is severely affected by drought because rice typically requires more water than other crops [4]. It is, therefore, essential to develop rice with enhanced tolerance to drought and heat stress.
To avoid drought stress, plants have evolved a series of sophisticated strategies. Plants respond to drought stresses through stress-specific signaling pathways, leading to morphological, physiological, and biochemical changes. Under drought stresses, plants first perceive external signals through sensors, and then various transcription factors are induced by the transduction of signals [5,6]. These drought-responsive transcription factors, such as members of the AP2/ERF, MYB, bZIP, and NAC families, regulate drought-inducible genes, and consequently, plants show a tolerance to abiotic stresses [5].
The OsERF83 gene belongs to the APETALA2/ethylene-responsive factor (AP2/ERF) family, which is only present in the plant kingdom [7]. The AP2/ERF superfamily commonly possesses a highly conserved AP2 DNA-binding domain, and can be classified into four subfamilies: APETALA2 (AP2), related to abscisic acid insensitive 3/viviparous 1 (RAV), ethylene-responsive factor (ERF), and dehydration-responsive element binding protein (DREB) [8]. In the AP2/ERF superfamily, the ERF family has a single DNA-binding AP2 domain and is classified into ten subgroups (Ⅰ–X) based on putative functional motifs [7]. Many TFs belonging to the ERF family have been reported that specifically bind to the GCC box (AGCCGCC), an ethylene-responsive element (ERE) [7], to play a vital role in plant development, abiotic and biotic stresses, and hormonal signal transduction [9,10,11,12,13,14]. Recently, OsERF48 and OsERF71 were shown to be involved in drought tolerance in rice by enhancing root growth [15] and altering the root structure [16], respectively. Additionally, overexpression of OsEREBP1 confers tolerance to both biotic and abiotic stresses [17]. In Arabidopsis, AtERF019 has been reported to play an important role in plant growth and drought tolerance by delaying plant growth and senescence [18]. However, despite increasing evidence that OsERF genes enhance drought tolerance, the molecular mechanisms have not yet been entirely elucidated.
TFs belonging to group Ⅸ are associated with defense against various pathogens [7]. For example, overexpression of AtERF1 and ORA59 enhances resistance to necrotrophic pathogens in Arabidopsis. AtERF1 is a regulator of ethylene responses after pathogen attack, and ORA59 is an essential integrator of jasmonic acid (JA) [19,20]. In rice, OsERF92 negatively regulates tolerance to Magnaporthe oryzae and salt stress, which is integrated into the cross-talk between biotic and abiotic stress signaling networks [21]. OsERF83, which belongs to Group Ⅸ, is induced by drought, high salinity, low temperature, and ABA treatments [9]. A previous study demonstrated that OsERF83 is a transcription factor that positively regulates resistance to Magnaporthe oryzae and is induced by multiple phytohormone treatments, such as methyl jasmonate, ethephon, and salicylic acid. Furthermore, several PATHOGENESIS-RELATED (PR) protein genes, including PR1, PR2, PR3, PR5, and PR10, are up-regulated in OsERF83 overexpression (OsERF83OX) transgenic rice [22]. These results prompted us to study the function of OsERF83 in biotic and abiotic stresses. In this study, we found that OsERF83 confers drought tolerance and positively regulates abiotic and biotic stress-associated genes.

2. Results

2.1. OsERF83 Is a Drought-Inducible Transcription Factor

In our previous rice 3′-tiling microarray analysis, OsERF83 was induced by drought treatments [9]. Based on the results, we investigated the function of OsERF83 in response to drought. We analyzed the expression patterns using quantitative real-time PCR (qRT-PCR) (Figure 1a). We treated 2-week-old rice seedlings with various stresses, including drought, high salinity, low temperature, and ABA. The transcript level of OsERF83 was significantly induced in both leaves and roots under drought and high salinity conditions, and the degree of induction was higher in the roots than in the leaves. In contrast, the low-temperature (4 °C) and ABA (100 μM) treatments enhanced the transcript levels of OsERF83, specifically in the leaves and roots, respectively. Under drought treatments, the transcript levels of OsERF83 were increased in both the roots and leaves (Figure 1a). The regulation of OsERF83 expression by various stresses shows that it might have multiple roles in response to environmental stresses.
To investigate the spatiotemporal expression patterns of OsERF83, we conducted qRT-PCR with various tissues of rice. The results revealed that OsERF83 was expressed at all developmental stages, and at particularly high levels in the roots (Figure 1b).
One study reported that OsERF83 was localized to the nucleus in onion epidermal cells [22]. To confirm the subcellular localization of OsERF83 in rice, we transformed the full-length OsERF83, fused to green fluorescent protein (GFP), into rice protoplasts (Figure S1a). We also used OsNF-YA7-mCherry as a positive control for nucleus localization [23]. Both constructs were transiently co-expressed in rice protoplasts, and the signals of both green fluorescence and mCherry fluorescence were observed in the nucleus of the rice protoplasts (Figure 1c). This indicated that the OsERF83 was localized at the nucleus. Collectively, OsERF83 is a transcription factor responsive to multiple stresses.

2.2. Overexpression of OsERF83 in Rice Confers Drought Tolerance at the Vegetative Stage

To investigate the biological role(s) of OsERF83 in rice development and the drought-stress response, we generated over 30 independent OsERF83 overexpression (OsERF83OX) transgenic rice plants using the GOS2 (rice eukaryotic translation initiation factor 1-like gene) promoter (GOS2::OsERF83), which drives expression in the whole plant body [24] (Figure S1b). Initially, we selected single-copy homozygous lines of OsERF83OX using TaqMan PCR, and then somaclonal variations were eliminated by paddy field selection for three generations. Finally, we chose three homozygous elite lines of OsERF83OX (#1, 2, 11) for further study. The transcript levels of OsERF83 were greatly enhanced in both the leaves and roots of all OsERF83OX (#1, 2, 11) plants, compared to non-transgenic (NT) plants. We checked the expression of each transgenic sister line and confirmed their overexpression (Figure S3).
We next tested the performance of OsERF83OX under drought conditions at the vegetative stage. OsERF83OX and NT plants were grown in a greenhouse for 5 weeks and then exposed to drought conditions for 2 days by withholding water. After 1 day of drought treatment, the NT plants started to display slightly rolled leaves. After 2 days of drought treatment, drought-induced visual symptoms, such as leaf rolling, wilting, and chlorosis, appeared more severely in NT plants than OsERF83OX (#1, 2, 11) plants (Figure 2a). Soil moisture consistently decreased during the drought treatment, indicating the drought stress was uniformly applied to both the NT and OsERF83OX (Figure 2b) plants. After 8 days of rewatering, most of the OsERF83OX plants had recovered and showed an 83 to 95% survival rate, while NT plants showed an approximately 20% survival rate (Figure 2c). We repeated the testing three times through T3–T4 and obtained the same results (Figure S4).
To further confirm the tolerance of OsERF83OX plants to drought stress, we measured the activity of photosynthetic machinery damaged by drought stress using the JIP test. The JIP test showed the Fv/Fm (Fv: variable fluorescence; Fm: maximum fluorescence) value for the photochemical efficiency of photosystem (PS) II and the PItotal (performance index) value for the photochemical efficiency of PS I and PS II [25]. OsERF83OX and NT plants were exposed to drought conditions for 7 days after transplanting into big pots, by withholding water for 7 days (Figure 2f). The Fv/Fm values of the NT plants started to decrease at 5 days after drought treatment, whereas the Fv/Fm values of OsERF83OX plants did not decrease (Figure 2d). Additionally, the PItotal values sharply decreased in NT plants at 5 days after drought treatment, whereas the PItotal values of OsERF83OX were maintained during the drought treatment (Figure 2e). Consistent with the photochemical efficiency, after 5 days of drought treatment, the NT plants started to display drought-induced visual symptoms (Figure 2f). These results indicated that OsERF83 overexpression in rice plants protects PS I and PS II from drought stress, thereby enhancing drought tolerance; in this way, OsERF83 positively regulates drought tolerance. We concluded that overexpression of OsERF83 enhanced drought tolerance at the vegetative stage.

2.3. Overexpression of OsERF83 in Rice Leads to Growth Retardation and Affects Grain Yield under Normal Conditions

The OsERF83OX plants showed smaller plant height, including culm length and panicle length at all stages (Figure 3a,b). At the maturity stage, the average plant height of NT, OsERF83OX #1, OsERF83OX #2, and OsERF83OX #11 was 98.86 cm (100%), 75.50 cm (76.4%), 87.63 cm (87.6%), and 81.50 cm (81.5%), respectively (Figure 3b, Table S1). The yield component values of the OsERF83OX plants, such as the number of spikelets per panicle (NSP), the number of total spikelets (NTS), the number of filled grains (NFG), the grain-filling rate (FR), and total grain weight (TGW), were reduced compared to the NT plants (Figure S5, Table S1). Moreover, the OsERF83OX plants showed shorter grain width than the NT plants (Figure 3c,d). Collectively, these results indicate that overexpression of OsERF83 affects plant growth and grain yield negatively in rice.

2.4. CRISPR/Cas9-Mediated Loss-of-Function Study Analysis of OsERF83

For the loss-of-function study analysis, knock-out (OsERF83KO) mutants were generated using a clustered regularly interspaced short palindromic repeats/recombinant codon-optimized Cas9 (CRISPR/Cas9) genome editing system (Figure S1c) [26], with which various types of induced mutations were found. The single-guide RNA (sgRNA) target sites were designed at the upstream region of the AP2/ERF domain for generating mutants edited in OsERF83 specific regions (Figure 4a). Four types of mutants containing 1 bp insertions were generated at the same upstream position of the PAM sequence (Figure 4a,b). Sequencing analysis revealed mutation patterns in transgenic plants, including frameshifts and premature stop codons (Figure 4c). Among 30 independent transgenic plants, 23.3% were null, and 20% were biallelic heterozygous mutant plants. A further 50% were biallelic homozygous, and we selected four independent OsERF83KO mutants for further study (Figure 4b).
We expected that the phenotype of OsERF83KO mutants would show more sensitivity to drought stress compared with NT plants. However, OsERF83KO mutants showed similar phenotypes under drought conditions (Figure 4d). We speculated that the redundancy of other AP2/ERF transcription factor family genes caused such no significant phenotypes to occur in OsERF83KO compared to NT plants.

2.5. OsERF83 Is Expressed Predominantly in the Vascular Tissue

As shown in Figure 1b, OsERF83 was expressed ubiquitously in all the examined tissues, with high expression levels in roots. To further explore the function of OsERF83 in drought tolerance, we investigated the tissue expression pattern of OsERF83 using histochemical GUS staining with Paraplast section. We generated transgenic rice plants with a β-glucuronidase (GUS) reporter gene, driven by the native OsERF83 promoter (OsERF83::GUS) (Figure S1d). We used a coleoptile, stem, leaves, and roots of plants, as exhibited in Figure 5a–k. Interestingly, the strong GUS signals were consistently detected in vascular tissues of the coleoptile (Figure 5a), stem (Figure 5b,c), leaves (Figure 5d–g), and roots (Figure 5h–k), as shown in the longitudinal section of the roots (Figure 5i), despite the difference in developmental stage and organs. These data indicated that OsERF83 was a constitutively expressed gene and was involved in the vascular tissues.
To investigate possible morphological changes in the vascular tissue caused by OsERF83 expression, we observed the vascular tissue in the roots, stems, and leaves in OsERF83OX, OsERF83KO, and NT plants. However, there were no significant differences in morphology among NT, OsERF83 overexpressing, and knock-out plants (Figure S6).

2.6. Identification of Genes Involved in the OsERF83-Mediated Drought Tolerance Pathway

To perform further functional analysis of OsERF83, we generated OsERF83-6myc overexpressing transgenic (OsERF83-MYCOX) plants (Figure S1e). We confirmed protein expression levels of OsERF83 in OsERF83-MYCOX using Western blot analysis (Figure S7a,f) and transcript levels using qRT-PCR (Figure S7b). Both levels were higher in OsERF83-MYCOX than in the NT plants. OsERF83-MYCOX plants also showed drought tolerance in the vegetative stage (Figure S7c–e). To identify downstream genes regulated by OsERF83, we performed an RNA-sequencing (RNA-seq) analysis using a 3-week-old OsERF83-MYCOX. We used whole seedlings of OsERF83-MYCOX and NT, with two independent biological replicates, and calculated the reads per transcript kilobase per million fragments mapped reads (FPKM) values for each sample with count data. The heatmap indicated that the biological replicates showed a similar expression pattern (Figure 6a). A total of 33,221 differentially expressed genes (DEGs) were identified, of which 540 were up-regulated (B) and 1109 were down-regulated (A) in OsERF83 overexpressing plants (Figure 6b).
To identify the putative functions of these DEGs, we conducted gene ontology analysis using gProfiler (https://biit.cs.ut.ee/gprofiler/orth). The DEGs were categorized into three categories: biological process, molecular function, and cellular component. As a result, the significantly enriched GO terms were mainly associated with the oxidation–reduction process and stress response under the biological process category, plasma membrane and the extracellular region under the cellular component category, and catalytic activity and ion binding under the molecular function category (Figure 6c). In addition, KEGG pathway analysis showed that DEGs were related to ‘diterpenoid biosynthesis’, ‘phenylpropanoid biosynthesis’, and ‘plant hormone signal transduction’ (Figure 6d). These results are consistent with our proposed role of OsERF83 in the regulation of drought stress tolerance.
Then, we identified that 289 drought-inducible genes were up-regulated in the OsERF83-MYCOX plants by filtering with a public database containing drought-treated RNA-seq data in shoots and roots (Figure 6e) [27]. These drought-inducible genes were classified into several groups: transporters, transcription factors, abiotic stress-related genes, the cytochrome P450 family, terpenoid-associated genes, disease resistance-related genes, hormone-associated genes, lignin biosynthesis-associated genes, and F-box proteins (Table 1), as well as others (Table S2). Some of the genes in each group were analyzed using qRT-PCR analysis for validation (Figure 7). The results confirmed that OsERF83-MYCOX induces a class of transporter genes (OsNPF8.10, OsNPF8.17, and OsLHT1) (Figure 7a), MYB or Myb/SANT transcription factors (Figure 7b), terpenoid-associated genes (OsTPS3 and OsCPS4) (Figure 7c), and lignin biosynthesis-associated genes (OsLAC17, OsLAC10, and CAD8D) (Figure 7d). We drew the co-expression matrix using up-regulated genes in OsERF83-MYCOX using RiceFREND to identify coregulatory networks with a high co-expression frequency (mutual rank (MR): ~20) (Figure S8) [28]. There were many drought-inducible genes in the co-expression matrix (Figure S8).
A previous study reported that OsERF83 interacts with the GCC box [22]. Therefore, we analyzed the presence of the GCC box within 3 kb of the 5′ upstream region of up-regulated drought-inducible genes in OsERF83-MYCOX (Table 1, Table S2). We found that most of them have the GCC box, which indicates that OsERF83 might directly regulate them.

3. Discussion

A number of studies have shown that APETALA2/ethylene-responsive factor (AP2/ERF) TFs are involved in the integration of signaling pathways in abiotic and biotic stress responses [10,11]. AP2/ERF TFs are classified into several subfamilies, including the ERF family [8]. OsERF83 is a member of group IXc, a subgroup of the ERF family reported to play an important role in abiotic stress [19,20,21]. ERF TFs have been reported that regulate stress-inducible genes by binding cis-acting elements, such as GCC box (AGCCGCC) in the promoter [29]. Previous studies have demonstrated that OsERF83 is a positive transcriptional regulator and interacts with the GCC box [22], and we analyzed the cis-element GCC box in the promoter of DEGs in our search for putative direct targets (Table 1 and Table S2).
The expression of OsERF83 was induced by ABA treatment (Figure 1a). In addition, OsERF83 was also reported to be induced by exogenous SA, JA, and ET [22]. These results indicated that OsERF83 is involved in multiple phytohormone signaling pathways during environmental stresses. It is well known that there are complex interactions between various hormones and stress signaling pathways in plants [6]. A single gene may play roles in various signaling pathways at the same time [21]. Therefore, our study suggested that OsERF83 is a positive regulator in plant responses to abiotic and biotic stresses through various hormone signaling pathways.
OsERF83OX plants showed growth retardation and low grain yields (Figure 3, Figure S5, Table S1). Generally, plants exhibiting stress tolerance have been reported to show growth retardation [30,31] because of hypersensitivity in responding to stresses, directing resources to protection from stresses rather than growing or yielding, which is known as a trade-off. Thus, genes improving stress tolerance induce growth retardation under normal growth conditions when they are constitutively overexpressed. In this study, we used the GOS2 promoter for constitutive overexpression (Figure S1b). However, if we used the stress-inducible promoter instead of the constitutive promoter, OsERF83 could be a good candidate for crop biotechnology.
The essential functions of vascular tissue are transporting water and various nutrients and supporting structures [32]. Consistent with this, there have been reports that vascular tissue development is involved in drought tolerance [33,34]. OsERF83 is predominantly expressed in vascular tissue (Figure 5). However, there were no significant differences in phenotypes of vascular tissue in this study (Figure S6). On the contrary, several transcriptome studies have shown that OsERF83 is induced under iron [35,36] and nitrate deficiency [37]. Moreover, our study revealed that various transporters were up-regulated in OsERF83-MYCOX (Table 1). Therefore, we speculated that OsERF83 might also be involved in iron and nitrate absorption.
Previously, it was reported that OsERF83 positively regulates disease resistance, and the constitutive overexpression of OsERF83 in rice enhanced resistance to Magnaporthe oryzae [22]. However, no further report on the function of OsERF83 in abiotic stress tolerance has been reported so far. In the present study, we analyzed the function of OsERF83 as a regulator of responses to drought stress. OsERF83 was strongly induced not only by drought stress, but also other abiotic stresses (Figure 1a). The OsERF83 promoter also contains several stress-related cis-elements, including ABREs, DREs regulated by ABA and DREB (Figure S9) [38]. These results support OsERF83 potentially playing an important role in abiotic stresses. OsERF83OX plants showed drought tolerance under drought treatment in the vegetative stage (Figure 2), which suggested the positive role of OsERF83 in drought tolerance. While OsERF83KO mutants showed similar phenotypes compared with non-transgenic (NT) plants under drought treatment at the vegetative stage (Figure 4), this could be explained by functional redundancy among a large number of the AP2/ERF TFs [7,12].
RNA-seq analysis identified a large number of transporter genes that were up-regulated in OsERF83 overexpression myc-tagged transgenic (OsERF83-MYCOX) plants, consistent with the vascular-specific expression of OsERF83. In a recent report, NRT1.2/NPF4.6 in Arabidopsis was found to affect ABA import activity [39]. This suggested that a group of transporter genes up-regulated in OsERF83-MYCOX (Table 1, Figure 7a), including NPFs (nitrate and peptide transporters), might affect the drought tolerance of rice through the ABA-dependent pathway. On the other hand, recent reports have shown that lignin plays an important role in protection against water losses [40,41]. A group of lignin biosynthesis-associated genes up-regulated in OsERF83-MYCOX (Table 1, Figure 7d), including OsLAC17 [42,43], OsLAC10 [44], and OsCAD8D [45], might be involved in protection against water losses and affect drought tolerance. A number of abiotic stress-related genes containing SENESCENCE-ASSOCIATED PROTEIN (OsSAP), OsLEA14/Wsi18, PCC13-62, and heat shock protein were up-regulated in OsERF83-MYCOX (Table 1, Figure 7e). OsSAP is involved in drought tolerance by virtue of its antiapoptotic activity [46]. OsLEA14/Wsi18 improves drought tolerance through higher proline and soluble sugar accumulation [47]. Desiccation-related protein (DRP)-encoding gene PCC13-62 is involved in desiccation tolerance in Linderniaceae [48]. Heat shock proteins play a role in membrane stability and regulating antioxidant enzymes and are produced under various abiotic and biotic stresses [49,50]. It has been reported that OsHSP23.7 enhances drought and salt tolerance in rice and overexpression transgenic plants show less membrane damage than NT plants [51]. It has also been shown that cytochrome P450 genes are involved in drought tolerance [52,53]. Many disease resistance-related genes, including PATHOGENESIS-RELATED PROTEIN (PR), WALL-ASSOCIATED KINASE (WAK) gene, CELLULOSE SYNTHASE-LIKE PROTEIN E1 (CslE1), LYSM RECEPTOR-LIKE KINASE (RLK), and terpenoid-associated genes, were also up-regulated (Table 1). It has been reported that many stress-responsive genes are induced by both biotic and abiotic stress treatment. PRs correlate with drought tolerance [54,55]. Cell wall-associated genes containing WAK, CslE1, and lignin biosynthesis genes are involved in both abiotic and biotic stresses [56,57]. Accordingly, we speculated that OsERF83 is involved in the core pathways in abiotic and biotic stress responses (Figure 8).

4. Materials and Methods

4.1. Plant Materials

To generate OsERF83 overexpressing plants (OsERF83OX), the coding sequence of OsERF83 (Os03g0860100, LOC_Os03g64260) was amplified from rice (Oryza sativa L. ssp. Japonica cv. Dongjin). The Japonica rice cultivar Dongjin (Oryza sativa. L.) was used as the non-transgenic control. The amplified OsERF83 coding sequence was cloned into rice transformation vector p700 carrying GOS2 promoter for constitutive expression using the Gateway system (Invitrogen) (Figure S2). The final constructs were introduced into Agrobacterium tumefaciens LBA4404 by triparental mating and transformed into rice (Oryza sativa cv. Dongjin). Copy numbers were determined in T0 plants through Taq-Man PCR as described by [58].
For the generation of knock-out (OsERF83KO) mutants, we used the web-based tool CRISPR RGEN Tools (http://www.rgenome.net/, accessed on 18 May 2017). The method was performed as described previously [26].

4.2. Stress Treatments

To confirm the expression levels of the OsERF83 gene under various abiotic stresses and phytohormone treatments, non-transgenic (NT) plants (Oryza sativa L. ssp. Japonica cv. Dongjin) were grown in soil for 2 weeks under standard greenhouse conditions (16 h light/8 h dark cycles at 28–30 °C). For stress treatments, the soil was moved from the roots of the seedlings, and drought stress was induced by air-drying the seedlings, while salinity stress and ABA treatment were imposed by incubating the seedlings in water containing 400 mM NaCl and 100 μM ABA, respectively at 28 °C. Low-temperature stress was induced by incubating the seedlings in water at 4 °C.

4.3. Subcellular Localization of OsERF83

To confirm the subcellular localization of OsERF83, we fused the coding regions of OsERF83 without the stop codon to the GFP. The cassette was driven by a 35S promoter and inserted into the pHBT vector (GenBank accession number EF090408) using the In-fusion system (Clonetech, CA, USA). 35S::NF-YA7-mCherry was used as the control for nuclear localization [23]. The final construct (35S::OsERF83-GFP) and the control vector (35S::NF-YA7-mCherry) were transfected into protoplasts (Oryza sativa cv. Dongjin) using a PEG-mediated protoplast transformation system [59]. The methods of rice protoplast preparation and transient gene expression were performed as described previously [60]. GFP and mCherry signals were observed 12 h after transfection using a confocal laser scanning microscope (Leica TCS SP8 STED, Wetzlar, Germany). Images were processed using Leica LAS AF Lite software. GFP was excited at 488 nm and the emitted light was detected between 512 and 560 nm. mCherry was excited at 587 nm and the emitted light was detected at 610 nm.

4.4. RNA Extraction, and Quantitative Real-Time PCR (qRT-PCR) Analysis

To investigate the spatial and temporal expression patterns of OsERF83, total RNA was isolated from the roots, stem, leaf, and flowers from different developmental stages of rice plants using a Hybrid-R RNA purification kit (GeneAll, Seoul, Korea) according to the manufacturer’s instructions. To measure the transcript levels of OsERF83, total RNA samples were extracted from the shoots and roots using a Hybrid-R RNA purification kit (GeneAll, Seoul, Korea) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized with Oligo-dT and random primers using RevertAid M-MuLV Reverse Transcriptase (Thermo Scientific, Massachusetts, USA). qRT-PCR was carried out using 2x qRT-PCR Pre-mix with 20x EvaGreen (SolGent, Seoul, Korea) and ROX dye (Promega, Madison, WI, USA). The amplification reactions were performed at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s in a 10 μL volume mix containing 0.5 μL EvaGreen Mix. The rice Ubiquitin1 (AK121590, Os06g0681400) transcript was used as a normalization control, and two biological and three technical replicates were analyzed for all qRT-PCRs (Table S3).

4.5. Histochemical GUS Assay

The amplified promoter region of OsERF83 was linked to the GUS reporter gene in a rice transformation vector using the Gateway system (Invitrogen, Carlsbad, CA, USA). The resulting plasmid was introduced into A. tumefaciens strain LBA 4404 by triparental mating and transformed into rice. To detect GUS staining, we used 5-day-old, 2-week-old, and 1-month-old seedlings of the OsERF83::GUS transgenic lines. The GUS staining solution contained 4 mM 5-bromo-4-chloro-3-indolyl-glucuronide cyclohexylamine salt (Gold Biotechnology, MO, USA), 0.5 mM K3Fe(CN)6, 0.5 mM C6FeK4N6, 0.1% Triton X-100, 10 mM EDTA, and 100 mM phosphate buffer. Seedlings were placed into GUS staining solution and then vacuum infiltrated for 30 min. Seedlings were then incubated at 37 °C in a dark chamber overnight. Chlorophyll was removed by washing with 70% (v/v) ethanol. We used Paraplast sections for longitudinals and cross-sections.

4.6. RNA-Sequencing Analysis

Three-week-old NT and OsERF83 overexpression myc-tagged transgenic (OsERF83-MYCOX) plants were grown under standard greenhouse conditions (16 h light/8 h dark cycles at 28–30 °C). Total RNA was extracted from 3-week-old transgenic (OsERF83-cMYCOX) and non-transgenic (NT) roots and leaves using an RNeasy plant kit (Qiagen, Hilden, German). Two biological replicates were used for RNA-seq. Library construction and next-generation sequencing (NGS) were performed by the Macrogen facility (Seoul, Korea). To make expression profiles for OsERF83-cMYCOX plants, the fragment per transcript kilobase per million fragments mapped reads (FPKM) values for each transcript were normalized with the FPKM values. The expression level of each transcript was expressed as the fragment per transcript kilobase per million fragments mapped reads (FPKM) value, which was calculated based on the number of mapped reads.

4.7. Drought Stress Treatment and Measurement of Chlorophyll Fluorescence

We evaluated the drought tolerance of transgenic, knock-out, and non-transgenic (Oryza sativa L. ssp. Japonica cv. Dongjin) plants grown in a greenhouse at the vegetative stage. Transgenic, knock-out and non-transgenic seeds were germinated on Murashige and Skoog (MS) medium (Duchefa Biochemie, Haarlem, Netherlands) with 3% sucrose in the dark for 3 days at 28 °C and transferred into light conditions for 1 day. Thirty seedlings from each transgenic, knock-out mutant, and non-transgenic plant were transplanted into ten soil pots (4 × 4 × 6 cm, three plants per pot) within a container (59 × 38.5 × 15 cm) and grown for 5 weeks in greenhouse conditions (16 h light/8 h dark cycles at 28–30 °C). Drought stress was simultaneously imposed by withholding water and rewatering. Drought-induced symptoms were monitored by imaging plants at the indicated time points using an a5000 camera (Sony, Tokyo, Japan). Soil moisture was measured at the indicated time points using an SM 150 soil moisture sensor (Delta T Devices, Cambridge, United Kingdom).
To evaluate the phenotypes of transgenic, knock-out, and non-transgenic plants exposed to drought conditions, we measured the chlorophyll fluorescence (Fv/Fm) and the performance index (PItotal). To measure Fv/Fm and PItotal, 4-week-old plants were transplanted into 15 cm diameter × 14 cm tall pots within another larger container (66 × 45.3 × 22.5 cm) and grown for 2 weeks. After each plant was subjected to drought stresses, Fv/Fm and PItotal were measured using the Handy-PEA fluorimeter (Plant Efficiency Analyzer; Hansatech Instruments, King’s Lynn, Norfolk, United Kingdom) in dark conditions to ensure sufficient dark adaptation (at least 1 h). Nine leaves of each line were measured and calculated using the Handy PEA software (version 1.31) and analyzed according to the equations of the JIP test [61].

4.8. Statistical Analysis

All data are represented as mean ± standard deviation. Each data point was compared with the control separately to determine whether they were significantly different from each other, using Student’s t-test (* p < 0.05, ** p < 0.01). Data were analyzed by Microsoft Excel software.

4.9. Accession Numbers

Genes from this article can be found in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/, accessed on 19 November 2020) with the following accession numbers: OsERF83 (Os03g0860100), OsNPF8.10 (Os01g0142800), OsNPF8.17 (Os10g0112500), OsLHT1 (Os08g0127100), OsMSL38 (Os11g0282700), OsMyb (Os01g0298400), OsTPS3 (Os02g0121700), OsCPS4 (Os04g0178300), OsLAC17 (Os10g0346300), OsLAC10 (Os02g0749700), OsCAD8D (Os09g0400400), OsSAP (Os09g0425900), PCC13-62 (Os04g0404400), and OsOPR4 (Os06g0215900).

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/ijms22147656/s1.

Author Contributions

S.E.J. and S.W.B. designed the experiments; S.E.J., S.H.K. and H.-B.Y. performed the experiments; Y.S.K. and S.H.K. helped with the collection of plant materials; S.E.J. prepared the figures; S.E.J. wrote the manuscript; S.E.J. and J.S.S. edited the manuscript; S.E.J., J.S.S. and J.-K.K. review and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01566801 to J.S.S)” Rural Development Administration, Republic of Korea.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We thank the Rural Development Administration and Kyungpook National University for providing the rice paddy fields.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Expression pattern of OsERF83 and subcellular localization of OsERF83 in rice protoplasts. (a) The relative expression level of OsERF83 by quantitative RT-PCR in the roots and shoots of 2-week-old seedlings treated with air-drying (drought), 400 mM NaCl (high salinity), 4 °C (cold), and 100 μM abscisic acid (ABA). OsUbi (Ubiquitin1; Os06g0681400) expression was used as an internal control for normalization. Error bars indicate the standard deviation based on three technical replicates. (h: hour) (b) The expression level of OsERF83 in various tissues and at different growth stages. (d, day; w, week; m, month; M, meiosis; BH, before heading; AH, after heading). OsUbi1 expression was used as an internal control for normalization. Error bars indicate the standard deviation based on three technical replicates. (c) Subcellular localization of the OsERF83 protein in rice protoplasts. 35S::OsERF83-GFP and 35S::OsNF-YA7-mCherry as a control vector were transiently expressed in rice leaf protoplasts. Scale bar, 10 µm.
Figure 1. Expression pattern of OsERF83 and subcellular localization of OsERF83 in rice protoplasts. (a) The relative expression level of OsERF83 by quantitative RT-PCR in the roots and shoots of 2-week-old seedlings treated with air-drying (drought), 400 mM NaCl (high salinity), 4 °C (cold), and 100 μM abscisic acid (ABA). OsUbi (Ubiquitin1; Os06g0681400) expression was used as an internal control for normalization. Error bars indicate the standard deviation based on three technical replicates. (h: hour) (b) The expression level of OsERF83 in various tissues and at different growth stages. (d, day; w, week; m, month; M, meiosis; BH, before heading; AH, after heading). OsUbi1 expression was used as an internal control for normalization. Error bars indicate the standard deviation based on three technical replicates. (c) Subcellular localization of the OsERF83 protein in rice protoplasts. 35S::OsERF83-GFP and 35S::OsNF-YA7-mCherry as a control vector were transiently expressed in rice leaf protoplasts. Scale bar, 10 µm.
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Figure 2. Overexpression of OsERF83 in rice confers drought tolerance. (a) Drought tolerance phenotypes of OsERF83 overexpression (OsERF83OX) transgenic plants (lines 1, 2, 11). The 5-week-old plants (0 d) were exposed to drought by withholding water for 2 days and rewatering for 8 days. (b) Measurement of soil moisture content (%). Error bars represent the means ± SD (n = 10). (c) The survival rate of the transgenic plants, scored 8 days after rewatering. (d,e) Determination of the photosynthetic viability of transgenic and NT plants under drought conditions. At the indicated time point after exposure to drought stresses, the chlorophyll fluorescence (Fv/Fm) (d) and performance index (PI) (e) of overexpression plants and NT plants were measured. The data represent the mean ± SD (n = 10 points per independent line of each genotype). (f) Drought tolerance phenotypes of OsERF83OX transgenic plants (lines 1, 2, 11) in big pots. The 6-week-old plants (0 d) were exposed to drought by withholding water for 7 days and rewatering for 8 days. Asterisks (**) indicate statistically significant differences compared with non-transgenic (NT) plants according to a Student’s t-test ** p < 0.001).
Figure 2. Overexpression of OsERF83 in rice confers drought tolerance. (a) Drought tolerance phenotypes of OsERF83 overexpression (OsERF83OX) transgenic plants (lines 1, 2, 11). The 5-week-old plants (0 d) were exposed to drought by withholding water for 2 days and rewatering for 8 days. (b) Measurement of soil moisture content (%). Error bars represent the means ± SD (n = 10). (c) The survival rate of the transgenic plants, scored 8 days after rewatering. (d,e) Determination of the photosynthetic viability of transgenic and NT plants under drought conditions. At the indicated time point after exposure to drought stresses, the chlorophyll fluorescence (Fv/Fm) (d) and performance index (PI) (e) of overexpression plants and NT plants were measured. The data represent the mean ± SD (n = 10 points per independent line of each genotype). (f) Drought tolerance phenotypes of OsERF83OX transgenic plants (lines 1, 2, 11) in big pots. The 6-week-old plants (0 d) were exposed to drought by withholding water for 7 days and rewatering for 8 days. Asterisks (**) indicate statistically significant differences compared with non-transgenic (NT) plants according to a Student’s t-test ** p < 0.001).
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Figure 3. Morphological analysis of OsERF83OX transgenic plants. (a) The whole plant height of 2-month-old OsERF83OX and NT plants grown in paddy fields. (b) The plant height was measured after the heading stage of the rice. A total of 18 plants for each line were measured. Data represent the mean value ± SD (n = 10). (c,d) The grain size of OsERF83OX line and NT plants. A total of 25 grains from each line were measured. Bar = 1 cm. Two asterisks (**) indicate statistically significant differences at p < 0.01, and one asterisk (*) indicates significant differences at p < 0.05 compared with NT based on the Student’s t-test. Data are shown as the mean ± SD (n = 25).
Figure 3. Morphological analysis of OsERF83OX transgenic plants. (a) The whole plant height of 2-month-old OsERF83OX and NT plants grown in paddy fields. (b) The plant height was measured after the heading stage of the rice. A total of 18 plants for each line were measured. Data represent the mean value ± SD (n = 10). (c,d) The grain size of OsERF83OX line and NT plants. A total of 25 grains from each line were measured. Bar = 1 cm. Two asterisks (**) indicate statistically significant differences at p < 0.01, and one asterisk (*) indicates significant differences at p < 0.05 compared with NT based on the Student’s t-test. Data are shown as the mean ± SD (n = 25).
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Figure 4. Construction of knock-out (OsERF83KO) mutants and their phenotypes. (a) Schematic illustration of the target sites in the OsERF83 genomic sequence. The protospacer adjacent motif (PAM) sequence is marked in red. Mutation sequences are marked in blue. (b) Mutation pattern on OsERF83KO #10, #15, #27, and #29 plants. (c) Amino acid sequence alignment of OsERF83KO plants revealed a frameshift and premature stop codon. (d) Phenotypes of OsERF83KO (#10, #15, #27, #29) and WT plants under drought stress. All plants were grown in soil for 5 weeks under well-watered conditions, exposed to drought stress for 2 days, and then rewatered for 4 days, in the greenhouse. Measurement of the soil moisture contents (%). The data represent the mean value ± SD of five measurements performed at different locations in the soil. The survival rate of transgenic plants was scored 4 days after rewatering.
Figure 4. Construction of knock-out (OsERF83KO) mutants and their phenotypes. (a) Schematic illustration of the target sites in the OsERF83 genomic sequence. The protospacer adjacent motif (PAM) sequence is marked in red. Mutation sequences are marked in blue. (b) Mutation pattern on OsERF83KO #10, #15, #27, and #29 plants. (c) Amino acid sequence alignment of OsERF83KO plants revealed a frameshift and premature stop codon. (d) Phenotypes of OsERF83KO (#10, #15, #27, #29) and WT plants under drought stress. All plants were grown in soil for 5 weeks under well-watered conditions, exposed to drought stress for 2 days, and then rewatered for 4 days, in the greenhouse. Measurement of the soil moisture contents (%). The data represent the mean value ± SD of five measurements performed at different locations in the soil. The survival rate of transgenic plants was scored 4 days after rewatering.
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Figure 5. Histochemical analysis of OsERF83. (a) The OsERF83 expression in different tissues of the OsERF83::GUS transgenic rice plants by GUS staining analysis, in the coleoptile of 5-day-old seedlings (a), stem cross-sections of 2-month-old plants (b,c), leaves of 2-week-old plants (d), leaf cross-sections of 2-week-old plants (e,g), the root of a 5-day-old seedling (h), longitudinal root section of 2-week-old plants (I), and root cross-sections of 2-week-old plants (j,k). Scale bars represent 1 mm in (a,b), 100 µm in (c,d) and (h), and 50 µm in (e–k). (i) Transverse section images represent structures of the roots and leaves.
Figure 5. Histochemical analysis of OsERF83. (a) The OsERF83 expression in different tissues of the OsERF83::GUS transgenic rice plants by GUS staining analysis, in the coleoptile of 5-day-old seedlings (a), stem cross-sections of 2-month-old plants (b,c), leaves of 2-week-old plants (d), leaf cross-sections of 2-week-old plants (e,g), the root of a 5-day-old seedling (h), longitudinal root section of 2-week-old plants (I), and root cross-sections of 2-week-old plants (j,k). Scale bars represent 1 mm in (a,b), 100 µm in (c,d) and (h), and 50 µm in (e–k). (i) Transverse section images represent structures of the roots and leaves.
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Figure 6. RNA-seq analysis using leaves of OsERF83-MYCOX plants. (a) Heat map analysis of differentially expressed genes (DEGs) between OsERF83-MYCOX and NT plants. (b) Venn diagram of the numbers of up- and down-regulated DEGs in OsERF83-MYCOX plants compared to NT plants. (c) Gene ontology (GO) enrichment analysis of DEGs between OsERF83-MYCOX and NT plants. The most enriched GO term is shown in ‘biological process’, ‘molecular function’, and ‘cellular component’. Asterisks indicate significant enriched GO terms (** p < 0.01, * p < 0.05). (d) KEGG pathway enrichment analysis of DEGs. (e) Venn diagram of up-regulated genes amongst the DEGs identified in OsERF83-MYCOX and DEGs identified in drought-treated NT shoots and roots from public data (TENOR: http://tenor.dna.affrc.go.jp/, accessed on 30 November 2020).
Figure 6. RNA-seq analysis using leaves of OsERF83-MYCOX plants. (a) Heat map analysis of differentially expressed genes (DEGs) between OsERF83-MYCOX and NT plants. (b) Venn diagram of the numbers of up- and down-regulated DEGs in OsERF83-MYCOX plants compared to NT plants. (c) Gene ontology (GO) enrichment analysis of DEGs between OsERF83-MYCOX and NT plants. The most enriched GO term is shown in ‘biological process’, ‘molecular function’, and ‘cellular component’. Asterisks indicate significant enriched GO terms (** p < 0.01, * p < 0.05). (d) KEGG pathway enrichment analysis of DEGs. (e) Venn diagram of up-regulated genes amongst the DEGs identified in OsERF83-MYCOX and DEGs identified in drought-treated NT shoots and roots from public data (TENOR: http://tenor.dna.affrc.go.jp/, accessed on 30 November 2020).
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Figure 7. Expression analysis of up-regulated genes in both OsERF83 overexpression myc-tagged transgenic (OsERF83-MYCOX) plants and drought-treated samples. (a) Relative expression levels of transporter genes (b), transcription factors (c), terpenoid-associated genes (d), lignin biosynthesis-associated genes, (e), abiotic stress-related genes. (f) and hormone associated genes, (g) qRT-PCR analysis of OsERF83 expression in 3-week-old OsERF83OX (line 1). The white bar is the expression value in NT plants, and the black bar is the expression value in OsERF83OX plants. OsUbi (Ubiquitin1; Os06g0681400) expression was used as an internal control for normalization. Error bars indicated the standard deviation based on three technical replicates. Asterisks indicate significant enriched GO terms (* p < 0.01, p < 0.05).
Figure 7. Expression analysis of up-regulated genes in both OsERF83 overexpression myc-tagged transgenic (OsERF83-MYCOX) plants and drought-treated samples. (a) Relative expression levels of transporter genes (b), transcription factors (c), terpenoid-associated genes (d), lignin biosynthesis-associated genes, (e), abiotic stress-related genes. (f) and hormone associated genes, (g) qRT-PCR analysis of OsERF83 expression in 3-week-old OsERF83OX (line 1). The white bar is the expression value in NT plants, and the black bar is the expression value in OsERF83OX plants. OsUbi (Ubiquitin1; Os06g0681400) expression was used as an internal control for normalization. Error bars indicated the standard deviation based on three technical replicates. Asterisks indicate significant enriched GO terms (* p < 0.01, p < 0.05).
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Figure 8. Schematic representation of OsERF83-mediated stress tolerance. The rice recognizes the stress signal through a plant hormone signaling pathway, and OsERF83 is up-regulated in response to stresses. Transporter genes (OsNPF.10, OsLH1, OsNPF8.17), terpenoid synthesis genes (OsTPS14, OsTPS33, OsTPS3), cytochrome P450 genes (Oscyp71Z4, CYP76M10), lignin biosynthesis genes (OsLAC17, OsLAC10, CAD8D), and abiotic stress-related genes (OsSAP, OsLEA14, PCC13-62) were induced by OsERF83.
Figure 8. Schematic representation of OsERF83-mediated stress tolerance. The rice recognizes the stress signal through a plant hormone signaling pathway, and OsERF83 is up-regulated in response to stresses. Transporter genes (OsNPF.10, OsLH1, OsNPF8.17), terpenoid synthesis genes (OsTPS14, OsTPS33, OsTPS3), cytochrome P450 genes (Oscyp71Z4, CYP76M10), lignin biosynthesis genes (OsLAC17, OsLAC10, CAD8D), and abiotic stress-related genes (OsSAP, OsLEA14, PCC13-62) were induced by OsERF83.
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Table
Table 1. List of genes up-regulated (>2-fold) in OsERF83-MYCOX and drought treatment in shoots and roots.
Table 1. List of genes up-regulated (>2-fold) in OsERF83-MYCOX and drought treatment in shoots and roots.
DescriptionsGene IDOX/NT Fc.OX/NT Pval.GCC Box
Transporters
Similar to sugar transport protein 14Os09g032200012.800.000−1858, −1702, −902
OsLTP2.7Os10g05046504.780.002
OsNPF8.10Os01g01428004.270.006
OsATL11Os02g01010003.950.003
ABC transporter-like domain-containing proteinOs08g05643003.140.021
NPF8.17Os10g01125002.530.040
OsACA1Os03g02037002.340.044−1439, −1052, −1049
NPF5.1Os05g03362002.330.040
OsLHT1Os08g01271002.210.008−1281
Transcription factors
OsMSL38Os11g028270025.080.000−2906
Zinc finger, RING-type domain-containing proteinOs06g053480011.730.002−2989, −2975
OsC3H32Os04g067180010.060.000−2976, −2918, −2879, −2876, −2835, −1283, −1144
Myb/SANT-like domain-containing proteinOs08g04967003.510.005−1455, −1191, −900
OsC3H17, OsWD40–49Os02g06777003.440.015
OsbZIP89Os12g06345003.270.028−483
OsABI5, OsbZIP10Os01g08593003.040.010−1554
Zinc finger, RING/FYVE/PHD-type domain-containing proteinOs09g05047002.670.030−1835, −1803, −1740
ONAC79Os04g04370002.630.038−2800, −506
OsERF86Os07g04107002.620.029−2589, −2495, −657
OsSPL1, OsDLN8Os01g02929002.470.000−2338, −2204, −1991
OsMybOs01g02984002.450.011−1348, −1325, −1089, −889
OsbHLH025Os01g01963002.410.046−1581, −1578
Zinc finger, RING/FYVE/PHD-type domain-containing proteinOs07g04843002.160.045−2923, −2920, −888
Abiotic stress-related genes
OsDjA1Os02g065650026.990.000−2460, −2354, −2351
OsSAP, OsTET13Os09g042590012.490.000−908
OsTHICOs03g067970010.840.000−1881, −1878, −1875
OsLEA14, wsi18Os01g07052009.950.000−2526
Similar to sorting nexin 1Os01g08623006.860.000−2778, −1217, −982, −979, −690, −544, −487, −481, −470, −359, −329, −298, −278, −188, −29
OsMPK14Os05g01435006.850.013
PCC13-62, OsEnS-64Os04g04044005.600.000−2980, −2977, −2974, −2223
OsHSP23.7Os12g05697003.680.015−1065, −1018, −988, −957, −444, −441, −379, −174
OsFAD2-3Os07g04169003.430.005−1476
Similar to class III peroxidase 136Os12g01118003.290.001
OsBBX28Os09g05097002.650.009−2975
OsNTP3Os01g08465002.630.040−1783, −1780, −1685, −1653
OsPEX11−5Os06g01270002.490.019−2371, −2368, −2054, −2051, −2048
Glycine-rich RNA-binding proteinOs04g06537002.370.032−2957, −2901, −2895, −2892, −2307, −2684, −2680, −1677, −1610, −1607, −1604, −1577, −1478, −1475, −1442, −1415, −1322, −1319, −1316, −1313, −710, −678
OsSKIPOs02g07598002.230.001−2986, −2831, −1142, −1139, −1136, −990, −906, −840, −837, −800
OsRZFP34, OsRFP1Os01g07191002.210.027−1128, −1114, −346, −120
OsVPE3Os02g06440002.200.000−2797, −2751
OsASMT1Os09g03445002.100.007
Cytochrome P450 family
Oscyp71Z4Os10g04398005.790.001−2642, −2625
Cytochrome P450 family proteinOs12g05826664.860.001
CYP76M10Os08g05071004.090.007−1515, −1512, −1354, −1297, −1008, −572
CYP450-like GeneOs08g01057004.090.000−2870, −2337, −2065. −1006, −998, −995, −974
Similar to cytochrome P450 family protein, expressedOs12g06402003.530.010−1064, −1061, −1050, −989, −940, −240, −190, −187
OsCYP71AA2Os01g09578003.500.022−2440, −2437, −2434, −2410, −2224, −2029
Cytochrome P450 family proteinOs02g01847003.010.047−1217
Cytochrome P450 family proteinOs03g05941002.540.022−2178
CYP76M8Os02g05694002.280.039
CYP99A2Os04g01804002.260.046−2113, −2010, −1980, −1959
CYP76M2Os08g05080002.150.012−2928, −937
Terpenoid-associated genes
OsTPS33Os12g04918004.050.001−608
OsTPS14Os03g04282002.830.024−1604
OsTPS3Os02g01217002.800.027
OsCPS4Os04g01783002.680.000−1983, −1933, −1926, −1897, −1825, −1797, −1794, −1786
OsTPS16Os04g01797002.610.036
Disease resistance-related genes
OsCSN5AOs04g065470025.760.000
Similar to blight-associated protein p12Os09g047270023.770.000−2580, −1500, −1353, −1308, −1305, −1302, −1069, −1066, −1063, −995, −992, −927, −924, −130, −98, −37
OsLysM-RLK8Os11g054930013.440.000−2808, −2279, −2093, −2021, −1995, −1992, −1972
OsCslE1Os09g047810010.690.000−2802
eIF4Gtsv1Os07g05552009.140.000−1520
OsAGP29Os01g06071007.420.000−2606
OsWAK89bOs09g05614506.550.005
OsTHI7Os06g05141005.480.004−1693, −995
OsGLP4-1Os04g06179005.220.000−738
Similar to blight-associated protein p12 precursorOs09g04729004.910.000−2469, −2376, −178
Similar to receptor-like kinase Xa21-binding protein 3.Os05g01120004.860.016−2973
Similar to herbicide safener binding proteinOs11g03064004.670.042
Leucine-rich repeat domain-containing proteinOs04g01220004.570.010−1725
OsPR8Os10g04165004.500.000−2994, −2939, −871, −726
OsGSL3Os01g07542004.450.018−2773, −2770, −584, 581, −578, −575, 572, −569
OsEXO70B3Os05g04735004.310.003−2788, −2764, −2748, −508, −468, −465, −438, −413, −264, −159
Leucine-rich repeat, typical subtype domain-containing proteinOs11g05688003.830.009−2429, −2426, −1239, −1210, −1185, −1130
Leucine-rich repeat, N-terminal domain-containing proteinOs12g02115003.800.017
Similar to pathogen-related proteinOs01g02483003.800.000−2698, −2680, −2644, −2637, −1700, −1620, −1560, −1557, −1534, −1317
OsDXS3Os07g01900003.280.000
Multi-antimicrobial extrusion protein MatE family proteinOs09g05243003.270.026
OsLOX9Os08g05088003.200.000−1626
OsWAKL21.2Os12g05958002.930.016−959, −842, −839, −836, −833, −308, −305, −302, −299, −296, −183, −167, −164, −161, −92
Similar to resistance protein candidate (fragment)Os03g02580002.840.031−577, −478, −391
IAI2Os01g01320002.780.000−1174, −1167
OsTHI9Os06g05148002.740.025−1718, −1715, −1697
Leucine-rich repeat, N-terminal domain-containing proteinOs01g08917002.700.031
OsPR4dOs11g05918002.260.001−2134, −993
Allergen V5/Tpx-1 related family proteinOs07g01264012.240.014
Allergen V5/Tpx-1 related family proteinOs07g01256002.230.015−553, −386, −336, −333, −215
OsPR1bOs07g01250002.220.015
OsPR1-73Os07g01276002.150.018
Similar to thaumatin-like proteinOs12g06305002.150.000−2453
PR5Os03g06635002.120.006
OsGLN1Os01g09467002.060.000−2549, −1263, −760, −669, −616, −457, −374, −323
OsAOS2Os03g02259002.010.007−2843, −2202, −349
Hormone-associated genes
OsLUGL, OsWD40-17Os01g060740011.970.001−2980, −2906, −2903, −2900, −2897, −2836, −2833
OsARF12Os04g067190010.330.000
OsJAZ8Os09g04013006.010.000−2541, −2538
OsGA20ox4Os05g04219004.850.015
OsARF13, OsARF12Os04g06906002.740.030
OsOPR4Os06g02159002.460.000−2863, −1540, −1481, −1478, −1459, −1431, −1428, −1279, −1276, −1238, −1206, −1133, −1129, −1126, −1070, −1040, −1016, −1013
Lignin biosynthesis-associated genes
OsUGT707A5Os07g05033004.530.000−1544, −1507, −1470
OsUGT93BOs04g05564003.990.003−2364
UDP-glucuronosyl/UDP-glucosyltransferase family proteinOs03g08049003.230.027−2977, −805, −777, −774, −740, −737, −690, −473, −470, −407, −404, −376
OsLAC17Os10g03463003.020.010−1525, −1481, −1468, −151
OsLAC10Os02g07497002.950.026−1963, −1907
OsCAD8DOs09g04004002.940.000−2137
F-box protein
OsFbox331Os07g01189008.110.013
OsFbox228Os04g05713004.070.003−1080
OsFbox552Os10g03964003.340.036
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Jung, S.E.; Bang, S.W.; Kim, S.H.; Seo, J.S.; Yoon, H.-B.; Kim, Y.S.; Kim, J.-K. Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice. Int. J. Mol. Sci. 2021, 22, 7656. https://doi.org/10.3390/ijms22147656

AMA Style

Jung SE, Bang SW, Kim SH, Seo JS, Yoon H-B, Kim YS, Kim J-K. Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice. International Journal of Molecular Sciences. 2021; 22(14):7656. https://doi.org/10.3390/ijms22147656

Chicago/Turabian Style

Jung, Se Eun, Seung Woon Bang, Sung Hwan Kim, Jun Sung Seo, Ho-Bin Yoon, Youn Shic Kim, and Ju-Kon Kim. 2021. "Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice" International Journal of Molecular Sciences 22, no. 14: 7656. https://doi.org/10.3390/ijms22147656

APA Style

Jung, S. E., Bang, S. W., Kim, S. H., Seo, J. S., Yoon, H. -B., Kim, Y. S., & Kim, J. -K. (2021). Overexpression of OsERF83, a Vascular Tissue-Specific Transcription Factor Gene, Confers Drought Tolerance in Rice. International Journal of Molecular Sciences, 22(14), 7656. https://doi.org/10.3390/ijms22147656

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