CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice

Takuya Ogata; Takuma Ishizaki; Miki Fujita; Yasunari Fujita

doi:10.1371/journal.pone.0243376

Abstract

Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants. Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the β-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.

Citation: Ogata T, Ishizaki T, Fujita M, Fujita Y (2020) CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice. PLoS ONE 15(12): e0243376. https://doi.org/10.1371/journal.pone.0243376

Editor: Keqiang Wu, National Taiwan University, TAIWAN

Received: August 19, 2020; Accepted: November 19, 2020; Published: December 3, 2020

Copyright: © 2020 Ogata et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported in part by Japan Society for the Promotion of Science (JSPS) of Japan (Grants-in-Aid for Scientific Research (C) JP16K07412 to Y.F.) and the Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. URL of each funder website: JSPS: https://www.jsps.go.jp/english/e-grants/ MAFF: https://www.maff.go.jp/e/index.html

Competing interests: The authors have declared that no competing interests exist.

Introduction

The increased occurrence of extreme weather events due to climate change severely hinders crop production. Drought is a major abiotic stress limiting rice productivity in rainfed lowland rice agro-ecosystems [1, 2]. A number of physiological and molecular studies have revealed that phytohormone signaling pathways, such as those of abscisic acid (ABA), auxin, and brassinosteroids, play key roles in regulating the drought stress response in plants [3]. Of these, ABA signaling components function as central regulators in the drought stress response [4, 5]. ABA coordinates the plant’s responses to decreased water availability. Cellular dehydration during seed maturation and post-germination growth increases endogenous ABA levels, which triggers multiple developmental and physiological responses, including stomatal closure and the activation of dehydration-responsive gene expression [6]. Numerous genes involved in ABA signaling have been targeted in efforts to engineer improved drought tolerance [7].

The Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1), which encodes the β-subunit of the protein farnesyltransferase, regulates ABA signaling and the dehydration response [8–10]. Protein farnesylation is a post-translational modification by which a farnesyl isoprenoid is attached to a C-terminal CaaX motif of the target protein, where “C” is the farnesylated cysteine, “a” is usually an aliphatic amino acid, and “X” is typically an alanine, cysteine, glutamine, methionine, or serine residue [11]. The farnesyl modification promotes protein association with membranes [11]. Among the approximately 700 Arabidopsis thaliana proteins identified as potential targets of ERA1-mediated farnesylation, CYP85A2, ASG2, and HSP40 proteins have been shown to function as negative regulators of ABA signaling [12–14]. CYP85A2 is a cytochrome P450 enzyme involved in brassinosteroid biosynthesis, whereas ASG2 is a WD40 protein implicated in seed germination and HSP40 functions as a molecular chaperone [12–14]. Another potential farnesylation target protein, AtNAP1;1, modulates cell proliferation and cell expansion during leaf development [15]. As such, ERA1 has been shown to have pleiotropic roles in different biological processes, such as defense against pathogens [16–18], heat-stress responses [14, 19, 20], hormonal responses [13, 21], and growth and development [22–25]. However, initially, the era1 mutant was identified as a negative regulator of ABA signaling in seed germination [8]. In addition, era1 mutant plants showed an enhanced response to ABA in stomatal closure [9, 10]. Therefore, ERA1 is considered to be a candidate gene for increasing drought tolerance in a variety of crops [26–29].

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system is a vital tool for editing the genomes of a wide range of organisms, including plants [30]. CRISPR/Cas9-mediated targeted mutagenesis is a crucial tool for functional analyses of plant genes and for crop improvement [31]. In this study, we used a CRISPR/Cas9-mediated genome editing approach to functionally characterize the role of ERA1 in plants. We showed that rice osera1 mutant lines harboring CRISPR/Cas9-induced frameshift mutations in OsERA1 display similar leaf growth as wild-type (WT) plants but enhanced primary root growth. The osera1 mutant lines also exhibit an enhanced response to ABA and drought stress via stomatal regulation. These findings suggest that OsERA1 acts as a negative regulator of primary root growth under nonstressed conditions and as a negative regulator of responses to ABA and drought stress in rice.

Materials and methods

Plasmid construction and transformation

Target sites and single guide RNAs (gRNAs) for the first, third, and fifth exons of OsERA1 (LOC_Os01g53600) adjacent to a protospacer-adjacent motif (PAM) were designed using the CRISPR-P design tool (http://crispr.hzau.edu.cn/CRISPR/) [32]. Each gRNA was designed to contain a restriction enzyme (RE) recognition site for the downstream PCR/RE-based selection steps [33]. To construct Cas9-gRNA plasmids, the primer pairs (S1 Table) were annealed and inserted into BsaI sites of the pCAMBIA1300-based pRGEB31 vector [34] (Addgene plasmid #51925). The resultant binary constructs, pRGEB31-gRNA1, pRGEB31-gRNA2, and pRGEB31-gRNA3, were introduced into Agrobacterium tumefaciens strain LBA4404, and used for transformation of immature rice (Oryza sativa L. ssp japonica cv. Nipponbare) embryos as described [35], except that a hygromycin concentration of 50 mg L^–1 was used. To confirm that the putative T₀ transformants harbored the transgene, genomic DNA was analyzed by PCR using primers that amplify the Cas9 fragment. PCR/RE assays were performed to detect mutations around the gRNA target sites as described [33] with minor modifications. Samples were PCR-amplified using Blend Taq-plus- (Toyobo, Osaka, Japan) and the products were digested with SacI, TaqI, and Hpy166II for gRNA1, 2, and 3, respectively. The PCR products obtained from PCR/RE-positive T₀ events were subjected to direct sequencing. The genotypes of T₀ events were analyzed using DNA sequencing chromatograms. Then, Cas9-free T₁ plants were selected by PCR analysis using the Cas9-specific primer pair, and the PCR products amplified from the Cas9-free T₁ plants were used to determine the segregation of the targeted mutations by direct sequencing. All primers used in this study are listed in S1 Table.

Plant growth conditions

T₃ and T₄ plants were subjected to physiological tests. After seven days of imbibition at 13°C, rice seeds were germinated in plastic dishes with enough water for five days and then grown in a CO₂-regulated growth chamber (Biotron LH-410-S: Nippon Medical & Chemical Instruments, Osaka, Japan) to ensure that CO₂ concentrations did not fall below 400 ppm [36], under controlled conditions of 16 h light at 28°C/8 h darkness at 25°C and a light intensity of 150 μmol photons m^–2 s^–1. For ABA treatments, the seeds were incubated with 1 μM ABA one day after sowing for 5 days in 9 cm plastic petri dishes in the same temperature-controlled growth chamber.

Mild drought stress treatment

Rice plants were grown in an isolation greenhouse under 28°C day/24°C night temperatures. The germinating seedlings were grown in soil-filled, open-bottomed small plastic tubes for 16 days. At 16 days after sowing, the plants were transferred to 1/5000a Wagner pots (17 cm diameter; 19 cm height) filled with 2.2 kg of mixed soil (1.56 kg of soil dry weight per pot) made by mixing Bonsol No.2 (Sumitomo Chemical, Osaka, Japan), which is an artificial granular cultivation soil, and red clay at a volume ratio of 1:1 with a soil water content of 29.5% (weight of moisture content/soil dry weight + weight of moisture content + plant weight). Twelve, ten, and twelve pots were prepared for the osera1 mutant lines (M1G and M2T), and WT, respectively. The pots were covered with transparent polyethylene shower caps to prevent water loss. All pots were continually saturated with water for 10 days. At 27 days after sowing, the pots in the water-deficit stress (WS) treatment were drained of excess water overnight. The weight of individual pots was recorded every few days, starting on the day after draining. Experiments were designed based on the definition of total mass-based soil water content (%) that was calculated as [weight of moisture content/total soil weight (soil dry weight + weight of moisture content) + plant weight] × 100. In preliminary tests, we confirmed that plants were subjected to drought stress in WS pots with a soil water content of 40% (S1 Fig). As the water-holding capacity of the soil was 49%, the soil water content was adjusted to approximately 60% (water weight/total soil weight plus plant weight) for the well-watered (WW) treatment and 40% for the WS treatment to compensate for water loss due to transpiration at the time of weight measurement. A soil water content of 30%, 40%, and 49% was confirmed to be equivalent to a soil matric potential of –54 kPa, –11 kPa, 0 kPa, respectively, by measurements using a tensiometer (pressure gauge type DIK-8343, Daiki Rika Kogyo Co., Ltd., Saitama, Japan). Based on the results of our preliminary tests (S1 Fig) and a previous report [37], we determined that the soil water content was adjusted to 60% in WW pots and 40% in WS pots, respectively. The effect of the plant weight on soil water content could be disregarded during the experimental period, since the plant weight was much smaller than the total soil weight. Relative growth rates during the period from p to q days after sowing were calculated as [(HWSq–HWSp)/(HWWq–HWWp)] × 100, where HWSp and HWSq are plant height in WS pots at p and q days after sowing, respectively, and HWWp and HWWq are plant height in WW pots at p and q days after sowing, respectively.

Stomatal conductance measurement

Stomatal conductance (mol H₂O m^–2 s^–1) was measured for the longest leaf of each plant with a LI-6400XT portable photosynthesis system (LI-COR Biosciences, Lincoln, NE, USA). As the ambient CO₂ concentration measured using the LI-6400XT was 480 ppm, the CO₂ concentration of the input flow, chamber block temperature, and intensity of the LED light source were set to 480 μmol mol^–1, 28.5°C, and 1,800 μmol m^–2 s^–1, respectively. Relative stomatal conductance rates during the period from p to q days after sowing were calculated as (CWSq/CWSp) × 100, where CWSp and CWSq are stomatal conductance in WS pots at p and q days after sowing, respectively.

Statistical analysis

A one-way ANOVA with Dunnett’s multiple-comparison test were performed using R software version 3.6.3 [38].

Results

CRISPR/Cas9-taregted mutagenesis of OsERA1

To understand the role of OsERA1 in the plant’s response to ABA and drought stress, we created rice OsERA1 mutants using CRISPR/Cas9. OsERA1 encodes two putative alternative transcripts, OsERA1.1 and OsERA1.2, both of which consist of 14 exons (Fig 1A). To obtain OsERA1 mutants, three gRNAs, gRNA1, gRNA2, and gRNA3, were designed to target the first, third, and fifth exons, respectively (Fig 1A).

Download:

Fig 1. CRISPR/Cas9-mediated mutagenesis of OsERA1 in rice.

(A) Schematic diagram of OsERA1 and the target sites. OsERA1 encodes two putative alternative transcripts, OsERA1.1 and OsERA1.2. The guide RNAs (gRNAs) gRNA1, gRNA2, and gRNA3 were designed to target exon-1, -3, and -5, respectively, to induce mutations in the coding region of OsERA1. (B) The insertion mutations of alleles identified from sequence analysis of PCR amplicons from four osera1 mutant lines, M1T, M1G, M2T, and M3T, with homozygous mutations at the gRNA1 target site. The inserted base (G/T) and protospacer-adjacent motif (PAM; boxed regions) are in red and blue font, respectively. (C) DNA sequence chromatogram of the homozygous T2 progenies of the four mutant lines containing a single nucleotide insertion (T or G) at the same position in the gRNA1 target site. Representative sequence data are shown. The inserted base (G/T) of the alleles are shaded in gray.

https://doi.org/10.1371/journal.pone.0243376.g001

Three OsERA1 CRISPR/Cas9 constructs containing each gRNA were used to transform the rice cultivar Nipponbare, which is widely used as a standard cultivar for studies of lowland rice. We generated 7, 201, and 65 T₀ transformants for the constructs containing gRNA1, gRNA2, and gRNA3, respectively, and identified multiple T₀ transformants harboring target mutations for all three constructs using PCR-based genotyping (S2 Table). However, we failed to obtain homozygous progeny plants with the desired mutations at the gRNA2 and gRNA3 target sites (S2 Table). For example, the growth of the homozygous progenies of the M4 and M5 mutant lines (S2 Table), M4-HM and M5-HM, with deletions at the gRNA2 target site was arrested at the plumule stage, whereas the heterozygous progenies of M4 and M5, M4-HT and M5-HT, grew normally (S2 Fig). Thus, mutations at the gRNA2 and gRNA3 target sites appear to be lethal. By contrast, four independent lines, M1T, M1G, M2T, and M3T, with homozygous mutations at the gRNA1 target site, were obtained (Fig 1B and S2 Table). Since the M1 line had a biallelic mutation in the T₀ generation, two independent alleles, M1T and M1G, segregated at subsequent generations. All four mutant alleles had a single nucleotide insertion (T or G) at the same position in the gRNA1 target site, inducing frameshift mutations that introduce premature stop codons in the OsERA1.1 transcript (Fig 1B and 1C), whereas the mutant mRNAs may still have the ability to produce N-terminal truncated proteins with sequence similarity to OsERA1, except in the first 28 amino acids (S3 Table).

The osera1 mutants display increased primary root growth and sensitivity to ABA

Under nonstressed conditions, the seedlings of four osera1 mutant lines, M1T, M1G, M2T, and M3T, showed similar leaf growth but significantly enhanced primary root growth compared with WT plants (Fig 2). T₃ and T₄ homozygous plants of the M1G and M2T mutant lines harboring a single nucleotide insertion (T or G) at the same position at the gRNA1 target site were selected for further study. The seedlings were treated with or without 1 μM ABA for five days to evaluate the effect of mutations at the gRNA1 target site on ABA sensitivity. The leaf and root growth of the M1G and M2T mutant lines were significantly more sensitive to ABA treatment than the WT (Fig 3), supporting the hypothesis that, like Arabidopsis thaliana ERA1 [8–10], OsERA1 is a negative regulator of ABA signaling. A recent study showed that the Arabidopsis era1-2 mutant has a permeable cuticle [18]. In maize (Zea mays L.), cuticle-dependent leaf permeability is regulated by ABA signaling pathways [39]. We thus assessed the leaf cuticle permeability of rice osera1 lines. No significant difference in cuticle permeability was observed between the osera1 line and WT plants (S3 Fig).

Download:

Fig 2. The osera1 homozygous mutant lines show similar leaf growth but significantly enhanced primary root growth.

(A) Growth phenotype of young seedlings of four osera1 mutant lines, M1T, M1G, M2T, and M3T. Seeds were germinated and grown hydroponically for two weeks; representative plants are shown. Scale bar = 1 cm. Leaf length (B) and root length (C) of the seedlings were measured at nine days after germination. Values are presented as means ± SD (n = 6, *p < 0.05 from WT by a one-way ANOVA with a Dunnett’s multiple-comparison test and ns means no significance).

https://doi.org/10.1371/journal.pone.0243376.g002

Download:

Fig 3. The osera1 mutant lines display enhanced sensitivity to ABA in leaf and root growth.

(A) Responses of M1G and M2T mutant lines to ABA solution. One-day-old germinated rice seeds were grown in 1 μM ABA solution in plastic dishes for five days. Photographs were taken at five days after the start of growth in 1 μM ABA solution; representative seedlings are shown. Scale bar = 1 cm. (B) Relative leaf length of the osera1 mutant lines at five days after the start of growth in 1 μM ABA solution. (C) Relative root length of the osera1 mutant lines at five days after the start of growth in 1 μM ABA solution. Values are presented as means ± SD (*p < 0.05 from WT by a one-way ANOVA with a Dunnett’s multiple-comparison test, n = 10).

https://doi.org/10.1371/journal.pone.0243376.g003

The osera1 mutants show an enhanced response to mild drought stress

Since OsERA1 is involved in ABA signaling at the seedling stage, we analyzed the physiological responses of the M1G and M2T mutant lines to mild drought stress during later growth stages in a greenhouse under controlled temperature conditions (Fig 4A). Seedlings were subjected to water-deficit stress (WS) or control (well-watered, WW) treatment and their heights were compared at different time points. Although no substantial differences were observed in the relative growth rates of the WT and mutant seedlings at 27 to 35 days after sowing, the relative growth rates of the M1G and M2T plants at 35 to 44 days after sowing were significantly lower than those of WT plants (Fig 4B). These data suggest that M1G and M2T plants exhibit an enhanced response to drought stress in comparison with WT plants (Fig 4B).

Download:

Fig 4. The osera1 mutant lines exhibit an enhanced response to drought stress.

(A) Soil water content (water weight/total soil weight). For water-deficit stress (WS) treatment, water was drained from the pots at 27 days after sowing and watering was withheld between 27 and 35 days after sowing. The soil water content was roughly maintained between 30% and 40% in WS pots from 35 days after sowing onwards, and between 50% and 60% in well-watered (WW) pots. (B) Relative growth rates of M1G and M2T mutant lines from 27 to 35 days and from 35 to 44 days after sowing. See Materials and Methods for details. (C) Variation of soil water content (% by weight). Soil water content was measured daily from 70 to 73 days after sowing. At 70 and 73 days after sowing, the soil water content was adjusted to 60% in WW pots and 40% in WS pots, respectively. Arrows indicate the time points at which water was supplied. Dashed lines indicate 30, 40, 50, and 60% of the soil water contents. Light blue and pale orange regions between dashed lines indicate the variation range of the soil water contents of WW and WS pots, respectively. (D) Relative stomatal conductance rates of M1G and M2T mutant lines from 70 to 71 days and from 71 to 72 days after sowing. In B and D, values are means ± SD (n = 5 or 6, *p < 0.05 from WT by a one-way ANOVA with a Dunnett’s multiple-comparison test). See Materials and Methods for details.

https://doi.org/10.1371/journal.pone.0243376.g004

Next, to examine the physiological response to drought stress in the osera1 mutants, we measured the stomatal conductance of M1G and M2T mutant lines through the rate of gas exchange in the longest leaf of the individual plants. Soil water content was measured daily from 70 to 73 days after sowing (Fig 4C). At 70 and 73 days after sowing, soil water content was adjusted to 60% in WW pots and 40% in WS pots, respectively (Fig 4C). The relative stomatal conductance rates of M1G and M2T plants between 70 and 71 days after sowing were significantly reduced compared with those of the WT, whereas the stomatal conductance between 71 and 72 days after sowing did not differ between the mutant and WT lines (Fig 4D). Thus, the osera1 mutant plants respond to drought stress more rapidly than WT plants through accelerated stomatal closure. These observations support the view that OsERA1 is a negative regulator of the plant’s response to drought stress.

Discussion

Here, we demonstrated that the osera1 mutant lines M1G and M2T display an enhanced response to ABA and drought stress via stomatal regulation (Figs 3 and 4), consistent with previous reports of ERA1 homologs in other plant species [26–29]. Thus, OsERA1 acts as a negative regulator of responses to ABA and drought stress in rice, suggesting that, like Arabidopsis thaliana ERA1 [8, 9], OsERA1 fulfills a key role in the drought stress response in rice. However, since the M1G and M2T osera1 mutant lines have enhanced stomatal closure under drought stress (Fig 4B), these lines may have low yields and might thus not be suitable drought-tolerant lines for agricultural purposes. Considering that transgenic canola (Brassica napus) plants expressing ERA1 antisense mRNAs driven by a drought-inducible promoter showed significantly improved drought tolerance under field conditions without negatively affecting yield [26], it may be worth exploring the effects of CRISPR/Cas9-targeted mutagenesis of the promoter region of OsERA1. Additionally, the M1G and M2T osera1 mutations may be used to engineer improved drought tolerance in combination with other alleles that compensate for reduced yield [40].

The osera1 mutant seedlings exhibited similar leaf growth but increased primary root growth under nonstressed conditions, implying that OsERA1 functions as a negative regulator of primary root growth under normal conditions. Although the relationship between era1 mutation and lateral root growth have been reported in terms of ABA and auxin signaling via ABI3 [41, 42], the role of ERA1 in primary root growth was hitherto unknown. Low ABA concentrations promote primary root growth both by promoting the quiescence of the quiescent center and suppressing stem cell differentiation in root meristems [43]. Furthermore, low ABA concentrations activate the HY5-ERF11 regulon, a two-tiered transcriptional cascade that represses the expression of genes involved in ethylene biosynthesis and thereby promotes primary root growth [44]. Thus, the increased primary root growth of the osera1 mutant under nonstressed conditions may be due to an enhanced sensitivity to ABA. The basal role of ABA signaling under nonstressed conditions has been the focus of recent research [45, 46], and further research is required to clarify the roles of OsERA1 in this context.

This is the first report of CRISPR/Cas9-targeted mutagenesis being used to functionally characterize the role of ERA1 in plants. Although we designed three gRNAs for OsERA1 mutagenesis using the CRISPR-P design tool [32] to select appropriate target sites and avoid off-target effects, we were unable to obtain homozygous progeny plants with the desired mutations at two of the three gRNA target sites located in the first and fifth exons of the putative alternative transcripts, OsERA1.1 and OsERA1.2, whose transcriptional start codons are in the first and second exons, respectively (Fig 1A and S2 Table). Considering that we generated four independent lines (M1T, M1G, M2T, and M3T) with the desired mutations at the gRNA1 target site located in the first exon of OsERA1.1 and also in the 5′ UTR of OsERA1.2 (Fig 1A and S2 Table), these data suggest that OsERA1.2 may be essential for plant survival. This is consistent with previous reports showing that ERA1 has pleiotropic roles in different biological processes, including defense against pathogens [16–18], drought and heat-stress responses [8, 9, 14, 19, 20], hormonal responses [13, 21], and growth and development [22–25]. At the gRNA1 target site, we obtained only mutant lines with single insertion mutations (G or T) at the same nucleotide position (Fig 1A and S2 Table), causing frameshift mutations that create premature stop codons in the OsERA1.1 transcript (Fig 1B and 1C). The mutant mRNAs may still retain the ability to produce N-terminal truncated proteins with sequence similarity to OsERA1 except for the first 28 amino acids (S3 Table). Therefore, the lack of an N-terminal truncated region consisting of at least 28 amino acids may influence the mutant phenotype. Alternatively, the difference at the protein level might underlie the phenotype. However, since ERA1 has not yet been analyzed at the protein level, nor has an antibody been reported for this protein, more research is needed to determine why this mutation results in the observed phenotype. Thus, although ERA1 has been studied extensively using RNA interference, T-DNA insertion mutagenesis, and chemical mutagenesis [8–10, 26–29], our study using CRISPR/Cas9-targeted mutagenesis of OsERA1 suggests the novel potential role of alternative transcripts and of a specific region of the gene in ERA1 function. Collectively, our findings improve our understanding of the role of ABA signaling in the drought stress response in rice and provide information that may be useful in efforts to genetically engineer drought tolerance in rice.

Supporting information

S1 Table. Primers used in this study.

https://doi.org/10.1371/journal.pone.0243376.s001

(XLSX)

S2 Table. Genotypes of T₀ events with CRISPR/Cas9-induced mutations in OsERA1.

https://doi.org/10.1371/journal.pone.0243376.s002

(XLSX)

S3 Table. Predicted OsERA1.1 amino acid sequences in osera1 mutants.

https://doi.org/10.1371/journal.pone.0243376.s003

(XLSX)

S1 Fig. Growth retardation and reppression of rice plants in the mild drought stress test.

Representative photographs of WT plants exposed to mild drought stress for 58 days after sowing. Scale bars = 10 cm.

https://doi.org/10.1371/journal.pone.0243376.s004

(PDF)

S2 Fig. The growth of homozygous progenies of M4 and M5 mutant lines was arrested at the plumule stage.

(A) Deletions at the border of the third exon of alleles identified from a sequence analysis of PCR amplicons from the osera1 mutant lines M4 and M5. The deleted bases of the alleles and the protospacer-adjacent motif (PAM) are in red and blue, respectively. (B) Growth phenotype of seedlings of the homozygous and heterozygous progenies of the M4 mutant lines, M4-HM and M4-HT, with deletions at the gRNA2 target site. Seeds were germinated and grown hydroponically for two weeks; representative plants are shown. Scale bar = 1 cm. (C) Growth phenotype of seedlings of the homozygous and heterozygous progenies of the M5 mutant lines, M5-HM and M5-HT, with deletions at the gRNA2 target site. Seeds were germinated and grown hydroponically for two weeks; representative plants are shown. Scale bar = 1 cm.

https://doi.org/10.1371/journal.pone.0243376.s005

(PDF)

S3 Fig. A cuticle permeability assay for the osera1 lines.

Dye exclusion experiments were performed using leaves of 12-day-old seedlings as described by Cui et al. (2019). (A) Before the treatment. (B) Thirty minutes after immersion of the leaves in 0.05% (w/v) toluidine blue solution. No significant difference in cuticle permeability was observed between the leaves of the four osera1 lines, M1T, M1G, M2T, and M3T, and the WT. Scale bars = 1 cm.

https://doi.org/10.1371/journal.pone.0243376.s006

(PDF)

Acknowledgments

We thank K. Shimizu, N. Hisatomi, I. Gejima, S. Shu, Y. Masamura, Y. Saito, and K. Ozawa for their excellent technical support, M. Toyoshima for skillful editorial assistance, and Y. Nagatoshi for critical reading of the manuscript.

References

1. Pandey S, Bhandari H. Drought: economic costs and research implications. In: Serraj J J., Bennet J, Hardy B, editors. Drought frontiers in rice: crop improvement for increased rainfed production. Singapore: World Scientific Publishing; 2009. pp. 3–17.
2. Swain P, Raman A, Singh SP, Kumar A. Breeding drought tolerant rice for shallow rainfed ecosystem of eastern India. Field Crops Res. 2017;209: 168–178. pmid:28775653
- View Article
- PubMed/NCBI
- Google Scholar
3. Gupta A, Rico-Medina A, Caño-Delgado AI. The physiology of plant responses to drought. Science. 2020;368: 266–269. pmid:32299946
- View Article
- PubMed/NCBI
- Google Scholar
4. Fujita Y, Yoshida T, Yamaguchi-Shinozaki K. Pivotal role of the AREB/ABF-SnRK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants. Physiol Plant. 2013;147: 15–27. pmid:22519646
- View Article
- PubMed/NCBI
- Google Scholar
5. Zhu JK, Abiotic stress signaling and responses in plants, Cell. 2016;167: 313–324. pmid:27716505
- View Article
- PubMed/NCBI
- Google Scholar
6. Fujita Y, Fujita M, Shinozaki K, Yamaguchi-Shinozaki K. ABA-mediated transcriptional regulation in response to osmotic stress in plants. J Plant Res. 2011;124: 509–525. pmid:21416314
- View Article
- PubMed/NCBI
- Google Scholar
7. Umezawa T, Fujita M, Fujita Y, Yamaguchi-Shinozaki K, Shinozaki K. Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future. Curr Opin Biotechnol. 2006;17: 113–122. pmid:16495045
- View Article
- PubMed/NCBI
- Google Scholar
8. Cutler S, Ghassemian M, Bonetta D, Cooney S, McCourt P. A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science. 1996;273: 1239–1241. pmid:8703061
- View Article
- PubMed/NCBI
- Google Scholar
9. Pei ZM, Ghassemian M, Kwak CM, McCourt P, Schroeder JI. Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss. Science. 1998;282: 287–290. pmid:9765153
- View Article
- PubMed/NCBI
- Google Scholar
10. Allen GJ, Murata Y, Chu SP, Nafisi M, Schroeder JI. Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era1-2. Plant Cell. 2002;14: 1649–1662. pmid:12119381
- View Article
- PubMed/NCBI
- Google Scholar
11. Galichet A, Gruissem W. Protein farnesylation in plants–conserved mechanisms but different targets. Curr Opin Plant Biol. 2003;6: 530–535. pmid:14611950
- View Article
- PubMed/NCBI
- Google Scholar
12. Dutilleul C, Ribeiro I, Blanc N, Nezames CD, Deng XW, Zglobicki P, et al. ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis. Plant Cell Environ. 2016;39: 185–198. pmid:26147561
- View Article
- PubMed/NCBI
- Google Scholar
13. Northey JG, Liang S, Jamshed M, Deb S, Foo E, Reid JB, et al. Farnesylation mediates brassinosteroid biosynthesis to regulate abscisic acid responses. Nat Plants. 2016;2: 16114. pmid:27455172
- View Article
- PubMed/NCBI
- Google Scholar
14. Barghetti A, Siögren L, Floris M, Paredes EB, Wenkel S, Brodersen P. Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance. Genes Dev. 2017;31: 2282–2295. pmid:29269486
- View Article
- PubMed/NCBI
- Google Scholar
15. Galichet A, Gruissem W. Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development. Plant Physiol. 2006;142: 1412–1426. pmid:17041028
- View Article
- PubMed/NCBI
- Google Scholar
16. Goritschnig S, Weihmann T, Zhang Y, Fobert P, McCourt P, Li X. A novel role for protein farnesylation in plant innate immunity. Plant Physiol. 2018;148: 348–357.
- View Article
- Google Scholar
17. Jalakas P, Huang YC, Yeh YH, Zimmerli L, Merilo E, Kollist H, et al. The role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis stomatal responses is beyond ABA signaling. Plant Physiol. 2017;174: 665–671. pmid:28330935
- View Article
- PubMed/NCBI
- Google Scholar
18. Cui F, Wu W, Wang K, Zhang Y, Hu Z, Brosché M, et al. Cell death regulation but not abscisic acid signaling is required for enhanced immunity to Botrytis in Arabidopsis cuticle-permeable mutants. J Exp Bot. 2019;70: 5971–5984. pmid:31328223
- View Article
- PubMed/NCBI
- Google Scholar
19. Wu JR, Wang LC, Lin YR, Weng CP, Yeh CH, Wu SJ. The Arabidopsis heat-intolerant 5 (hit5)/enhanced response to aba 1 (era1) mutant reveals the crucial role of protein farnesylation in plant responses to heat stress. New Phytol. 2017;213: 1181–1193. pmid:27673599
- View Article
- PubMed/NCBI
- Google Scholar
20. Wu JR, Wang TY, Weng CP, Duong NKT, Wu SJ. AtJ3, a specific HSP40 protein, mediates protein farnesylation-dependent response to heat stress in Arabidopsis. Planta. 2019;250: 1449–1460. pmid:31309322
- View Article
- PubMed/NCBI
- Google Scholar
21. Galichet A, Hoyerová K, Kamínek M, Gruissem W. Farnesylation directs AtIPT3 subcellular localization and modulates cytokinin biosynthesis in Arabidopsis. Plant Physiol. 2008;146: 1155–1164. pmid:18184738
- View Article
- PubMed/NCBI
- Google Scholar
22. Running MP, Lavy M, Sternberg H, Galichet A, Gruissem W, Hake S, et al. Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases. Proc Natl Acad Sci USA. 2004;101: 7815–7820. pmid:15128936
- View Article
- PubMed/NCBI
- Google Scholar
23. Bonetta D, Bayliss P, Sun S, Sage T, McCourt P. Farnesylation is involved in meristem organization in Arabidopsis. Planta. 2000;211: 182–190. pmid:10945212
- View Article
- PubMed/NCBI
- Google Scholar
24. Ziegelhoffer EC, Medrano LJ, Meyerowitz EM. Cloning of the Arabidopsis WIGGUM gene identifies a role for farnesylation in meristem development. Proc Natl Acad Sci USA. 2000;97: 7633–7638. pmid:10840062
- View Article
- PubMed/NCBI
- Google Scholar
25. Yalovsky S, Kulukian A, Rodriguez-Concepcion M, Young CA, Gruissem W. Functional requirement of plant farnesyltransferase during development in Arabidopsis. Plant Cell. 2000;12: 1267–1278. pmid:10948248
- View Article
- PubMed/NCBI
- Google Scholar
26. Wang Y, Ying J, Kuzma M, Chalifoux M, Sample A, McArthur C, et al. Molecular tailoring of farnesylation for plant drought tolerance and yield protection. Plant J. 2005;43: 413–424. pmid:16045476
- View Article
- PubMed/NCBI
- Google Scholar
27. Manmathan H, Shaner D, Snelling J, Tisserat N, Lapitan N. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance. J Exp Bot. 2013;64: 1381–1392. pmid:23364940
- View Article
- PubMed/NCBI
- Google Scholar
28. Ogata T, Nagatoshi Y, Yamagishi N, Yoshikawa N, Fujita Y. Virus-induced down-regulation of GmERA1A and GmERA1B genes enhances the stomatal response to abscisic acid and drought resistance in soybean, PloS ONE. 2017;12(4): e0175650. pmid:28419130
- View Article
- PubMed/NCBI
- Google Scholar
29. Daszkowska-Golec A, Skubacz A, Sitko K, Słota M, Kurowska M, Szarejko I. Mutation in barley ERA1 (Enhanced Response to ABA1) gene confers better photosynthesis efficiency in response to drought as revealed by transcriptomic and physiological analysis. Environ Exp Bot. 2018;148: 12–26.
- View Article
- Google Scholar
30. Chen K, Wang Y, Zhang R, Zhang H, Gao C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol. 2019;70: 667–697. pmid:30835493
- View Article
- PubMed/NCBI
- Google Scholar
31. Sedeek KEM., Mahas A, Mahfouz M. Plant genome engineering for targeted improvement of crop traits. Front Plant Sci. 2019;10: 114. pmid:30809237
- View Article
- PubMed/NCBI
- Google Scholar
32. Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7: 1494–1496. pmid:24719468
- View Article
- PubMed/NCBI
- Google Scholar
33. Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc. 2014;9: 2395–2410. pmid:25232936
- View Article
- PubMed/NCBI
- Google Scholar
34. Xie K, Yang Y. RNA-guided genome editing in plants using a CRISPR–Cas system. Mol Plant. 2013;6: 1975–1983. pmid:23956122
- View Article
- PubMed/NCBI
- Google Scholar
35. Ishizaki T, Kumashiro T. Genetic transformation of NERICA, interspecific hybrid rice between Oryza glaberrima and O. sativa, mediated by Agrobacterium tumefaciens. Plant Cell Rep. 2008;27: 319–327. pmid:17934736
- View Article
- PubMed/NCBI
- Google Scholar
36. Nagatoshi Y, Fujita Y. Accelerating soybean breeding in a CO2-supplemented growth chamber. Plant Cell Physiol. 2019;60: 77–84. pmid:30219921
- View Article
- PubMed/NCBI
- Google Scholar
37. Singh B, Reddy KR, Redoña ED, Walker T. Screening of rice cultivars for morpho-physiological responses to early-season soil moisture stress. Rice Sci. 2017;24: 322–335.
- View Article
- Google Scholar
38. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, 2020.
39. Castorina G, Domergue F, Chiara M, Zilio M, Persico M, Ricciardi V, et al. Drought-responsive ZmFDL1/MYB94 regulates cuticle biosynthesis and cuticle-dependent leaf permeability. Plant Physiol. 2020;184: 266–282. pmid:32665334
- View Article
- PubMed/NCBI
- Google Scholar
40. Serraj R, Kumar A, McNally KL, Slamet-Loedin I, Bruskiewich R, Mauleon R, et al. Improvement of drought resistance in rice. Adv Agron. 2009;103: 41–99.
- View Article
- Google Scholar
41. Brady SM, Sarkar SF, Bonetta D, McCourt P. (2003). The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant J. 2003;34: 67–75. pmid:12662310
- View Article
- PubMed/NCBI
- Google Scholar
42. Johnson CD, Chary SN, Chernoff EA, Zeng Q, Running MP, Crowell DN. Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis. Plant Physiol. 2005;139: 722–733. pmid:16183844
- View Article
- PubMed/NCBI
- Google Scholar
43. Zhang H, Han W, De Smet I, Talboys P, Loya R, Hassan A, et al. ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem. Plant J. 2010;64: 764–774. pmid:21105924
- View Article
- PubMed/NCBI
- Google Scholar
44. Li Z, Zhang L, Yu Y, Quan R, Zhang Z, Zhang H, et al. The ethylene response factor AtERF11 that is transcriptionally modulated by the bZIP transcription factor HY5 is a crucial repressor for ethylene biosynthesis in Arabidopsis. Plant J. 2011;68: 88–99. pmid:21645149
- View Article
- PubMed/NCBI
- Google Scholar
45. Nagatoshi Y, Fujita Y. Protein kinase CK2α subunits constitutively activate ABA signaling in Arabidopsis. Plant Signal Behav. 2018;13: e1525998. pmid:30335565
- View Article
- PubMed/NCBI
- Google Scholar
46. Yoshida T, Christmann A, Yamaguchi-Shinozaki K, Grill E. Fernie AR. Revisiting the basal role of ABA–Roles outside of stress. Trends Plant Sci. 2019;24: 625–635. pmid:31153771
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Pandey S, Bhandari H. Drought: economic costs and research implications. In: Serraj J J., Bennet J, Hardy B, editors. Drought frontiers in rice: crop improvement for increased rainfed production. Singapore: World Scientific Publishing; 2009. pp. 3–17.

[ref2] 2. Swain P, Raman A, Singh SP, Kumar A. Breeding drought tolerant rice for shallow rainfed ecosystem of eastern India. Field Crops Res. 2017;209: 168–178. pmid:28775653
View Article
PubMed/NCBI
Google Scholar

[3] View Article

[4] PubMed/NCBI

[5] Google Scholar

[ref3] 3. Gupta A, Rico-Medina A, Caño-Delgado AI. The physiology of plant responses to drought. Science. 2020;368: 266–269. pmid:32299946
View Article
PubMed/NCBI
Google Scholar

[7] View Article

[8] PubMed/NCBI

[9] Google Scholar

[ref4] 4. Fujita Y, Yoshida T, Yamaguchi-Shinozaki K. Pivotal role of the AREB/ABF-SnRK2 pathway in ABRE-mediated transcription in response to osmotic stress in plants. Physiol Plant. 2013;147: 15–27. pmid:22519646
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Zhu JK, Abiotic stress signaling and responses in plants, Cell. 2016;167: 313–324. pmid:27716505
View Article
PubMed/NCBI
Google Scholar

[15] View Article

[16] PubMed/NCBI

[17] Google Scholar

[ref6] 6. Fujita Y, Fujita M, Shinozaki K, Yamaguchi-Shinozaki K. ABA-mediated transcriptional regulation in response to osmotic stress in plants. J Plant Res. 2011;124: 509–525. pmid:21416314
View Article
PubMed/NCBI
Google Scholar

[19] View Article

[20] PubMed/NCBI

[21] Google Scholar

[ref7] 7. Umezawa T, Fujita M, Fujita Y, Yamaguchi-Shinozaki K, Shinozaki K. Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future. Curr Opin Biotechnol. 2006;17: 113–122. pmid:16495045
View Article
PubMed/NCBI
Google Scholar

[23] View Article

[24] PubMed/NCBI

[25] Google Scholar

[ref8] 8. Cutler S, Ghassemian M, Bonetta D, Cooney S, McCourt P. A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science. 1996;273: 1239–1241. pmid:8703061
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 9. Pei ZM, Ghassemian M, Kwak CM, McCourt P, Schroeder JI. Role of farnesyltransferase in ABA regulation of guard cell anion channels and plant water loss. Science. 1998;282: 287–290. pmid:9765153
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 10. Allen GJ, Murata Y, Chu SP, Nafisi M, Schroeder JI. Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era1-2. Plant Cell. 2002;14: 1649–1662. pmid:12119381
View Article
PubMed/NCBI
Google Scholar

[35] View Article

[36] PubMed/NCBI

[37] Google Scholar

[ref11] 11. Galichet A, Gruissem W. Protein farnesylation in plants–conserved mechanisms but different targets. Curr Opin Plant Biol. 2003;6: 530–535. pmid:14611950
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 12. Dutilleul C, Ribeiro I, Blanc N, Nezames CD, Deng XW, Zglobicki P, et al. ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis. Plant Cell Environ. 2016;39: 185–198. pmid:26147561
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Northey JG, Liang S, Jamshed M, Deb S, Foo E, Reid JB, et al. Farnesylation mediates brassinosteroid biosynthesis to regulate abscisic acid responses. Nat Plants. 2016;2: 16114. pmid:27455172
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 14. Barghetti A, Siögren L, Floris M, Paredes EB, Wenkel S, Brodersen P. Heat-shock protein 40 is the key farnesylation target in meristem size control, abscisic acid signaling, and drought resistance. Genes Dev. 2017;31: 2282–2295. pmid:29269486
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Galichet A, Gruissem W. Developmentally controlled farnesylation modulates AtNAP1;1 function in cell proliferation and cell expansion during Arabidopsis leaf development. Plant Physiol. 2006;142: 1412–1426. pmid:17041028
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 16. Goritschnig S, Weihmann T, Zhang Y, Fobert P, McCourt P, Li X. A novel role for protein farnesylation in plant innate immunity. Plant Physiol. 2018;148: 348–357.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref17] 17. Jalakas P, Huang YC, Yeh YH, Zimmerli L, Merilo E, Kollist H, et al. The role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis stomatal responses is beyond ABA signaling. Plant Physiol. 2017;174: 665–671. pmid:28330935
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref18] 18. Cui F, Wu W, Wang K, Zhang Y, Hu Z, Brosché M, et al. Cell death regulation but not abscisic acid signaling is required for enhanced immunity to Botrytis in Arabidopsis cuticle-permeable mutants. J Exp Bot. 2019;70: 5971–5984. pmid:31328223
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref19] 19. Wu JR, Wang LC, Lin YR, Weng CP, Yeh CH, Wu SJ. The Arabidopsis heat-intolerant 5 (hit5)/enhanced response to aba 1 (era1) mutant reveals the crucial role of protein farnesylation in plant responses to heat stress. New Phytol. 2017;213: 1181–1193. pmid:27673599
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref20] 20. Wu JR, Wang TY, Weng CP, Duong NKT, Wu SJ. AtJ3, a specific HSP40 protein, mediates protein farnesylation-dependent response to heat stress in Arabidopsis. Planta. 2019;250: 1449–1460. pmid:31309322
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref21] 21. Galichet A, Hoyerová K, Kamínek M, Gruissem W. Farnesylation directs AtIPT3 subcellular localization and modulates cytokinin biosynthesis in Arabidopsis. Plant Physiol. 2008;146: 1155–1164. pmid:18184738
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref22] 22. Running MP, Lavy M, Sternberg H, Galichet A, Gruissem W, Hake S, et al. Enlarged meristems and delayed growth in plp mutants result from lack of CaaX prenyltransferases. Proc Natl Acad Sci USA. 2004;101: 7815–7820. pmid:15128936
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref23] 23. Bonetta D, Bayliss P, Sun S, Sage T, McCourt P. Farnesylation is involved in meristem organization in Arabidopsis. Planta. 2000;211: 182–190. pmid:10945212
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref24] 24. Ziegelhoffer EC, Medrano LJ, Meyerowitz EM. Cloning of the Arabidopsis WIGGUM gene identifies a role for farnesylation in meristem development. Proc Natl Acad Sci USA. 2000;97: 7633–7638. pmid:10840062
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref25] 25. Yalovsky S, Kulukian A, Rodriguez-Concepcion M, Young CA, Gruissem W. Functional requirement of plant farnesyltransferase during development in Arabidopsis. Plant Cell. 2000;12: 1267–1278. pmid:10948248
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref26] 26. Wang Y, Ying J, Kuzma M, Chalifoux M, Sample A, McArthur C, et al. Molecular tailoring of farnesylation for plant drought tolerance and yield protection. Plant J. 2005;43: 413–424. pmid:16045476
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref27] 27. Manmathan H, Shaner D, Snelling J, Tisserat N, Lapitan N. Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance. J Exp Bot. 2013;64: 1381–1392. pmid:23364940
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref28] 28. Ogata T, Nagatoshi Y, Yamagishi N, Yoshikawa N, Fujita Y. Virus-induced down-regulation of GmERA1A and GmERA1B genes enhances the stomatal response to abscisic acid and drought resistance in soybean, PloS ONE. 2017;12(4): e0175650. pmid:28419130
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref29] 29. Daszkowska-Golec A, Skubacz A, Sitko K, Słota M, Kurowska M, Szarejko I. Mutation in barley ERA1 (Enhanced Response to ABA1) gene confers better photosynthesis efficiency in response to drought as revealed by transcriptomic and physiological analysis. Environ Exp Bot. 2018;148: 12–26.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref30] 30. Chen K, Wang Y, Zhang R, Zhang H, Gao C. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu Rev Plant Biol. 2019;70: 667–697. pmid:30835493
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref31] 31. Sedeek KEM., Mahas A, Mahfouz M. Plant genome engineering for targeted improvement of crop traits. Front Plant Sci. 2019;10: 114. pmid:30809237
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref32] 32. Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL. CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant. 2014;7: 1494–1496. pmid:24719468
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref33] 33. Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc. 2014;9: 2395–2410. pmid:25232936
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref34] 34. Xie K, Yang Y. RNA-guided genome editing in plants using a CRISPR–Cas system. Mol Plant. 2013;6: 1975–1983. pmid:23956122
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref35] 35. Ishizaki T, Kumashiro T. Genetic transformation of NERICA, interspecific hybrid rice between Oryza glaberrima and O. sativa, mediated by Agrobacterium tumefaciens. Plant Cell Rep. 2008;27: 319–327. pmid:17934736
View Article
PubMed/NCBI
Google Scholar

[133] View Article

[134] PubMed/NCBI

[135] Google Scholar

[ref36] 36. Nagatoshi Y, Fujita Y. Accelerating soybean breeding in a CO2-supplemented growth chamber. Plant Cell Physiol. 2019;60: 77–84. pmid:30219921
View Article
PubMed/NCBI
Google Scholar

[137] View Article

[138] PubMed/NCBI

[139] Google Scholar

[ref37] 37. Singh B, Reddy KR, Redoña ED, Walker T. Screening of rice cultivars for morpho-physiological responses to early-season soil moisture stress. Rice Sci. 2017;24: 322–335.
View Article
Google Scholar

[141] View Article

[142] Google Scholar

[ref38] 38. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, 2020.

[ref39] 39. Castorina G, Domergue F, Chiara M, Zilio M, Persico M, Ricciardi V, et al. Drought-responsive ZmFDL1/MYB94 regulates cuticle biosynthesis and cuticle-dependent leaf permeability. Plant Physiol. 2020;184: 266–282. pmid:32665334
View Article
PubMed/NCBI
Google Scholar

[145] View Article

[146] PubMed/NCBI

[147] Google Scholar

[ref40] 40. Serraj R, Kumar A, McNally KL, Slamet-Loedin I, Bruskiewich R, Mauleon R, et al. Improvement of drought resistance in rice. Adv Agron. 2009;103: 41–99.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref41] 41. Brady SM, Sarkar SF, Bonetta D, McCourt P. (2003). The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant J. 2003;34: 67–75. pmid:12662310
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref42] 42. Johnson CD, Chary SN, Chernoff EA, Zeng Q, Running MP, Crowell DN. Protein geranylgeranyltransferase I is involved in specific aspects of abscisic acid and auxin signaling in Arabidopsis. Plant Physiol. 2005;139: 722–733. pmid:16183844
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref43] 43. Zhang H, Han W, De Smet I, Talboys P, Loya R, Hassan A, et al. ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem. Plant J. 2010;64: 764–774. pmid:21105924
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref44] 44. Li Z, Zhang L, Yu Y, Quan R, Zhang Z, Zhang H, et al. The ethylene response factor AtERF11 that is transcriptionally modulated by the bZIP transcription factor HY5 is a crucial repressor for ethylene biosynthesis in Arabidopsis. Plant J. 2011;68: 88–99. pmid:21645149
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref45] 45. Nagatoshi Y, Fujita Y. Protein kinase CK2α subunits constitutively activate ABA signaling in Arabidopsis. Plant Signal Behav. 2018;13: e1525998. pmid:30335565
View Article
PubMed/NCBI
Google Scholar

[168] View Article

[169] PubMed/NCBI

[170] Google Scholar

[ref46] 46. Yoshida T, Christmann A, Yamaguchi-Shinozaki K, Grill E. Fernie AR. Revisiting the basal role of ABA–Roles outside of stress. Trends Plant Sci. 2019;24: 625–635. pmid:31153771
View Article
PubMed/NCBI
Google Scholar

[172] View Article

[173] PubMed/NCBI

[174] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Plasmid construction and transformation

Plant growth conditions

Mild drought stress treatment

Stomatal conductance measurement

Statistical analysis

Results

CRISPR/Cas9-taregted mutagenesis of OsERA1

The osera1 mutants display increased primary root growth and sensitivity to ABA

The osera1 mutants show an enhanced response to mild drought stress

Discussion

Supporting information

S1 Table. Primers used in this study.

S2 Table. Genotypes of T0 events with CRISPR/Cas9-induced mutations in OsERA1.

S3 Table. Predicted OsERA1.1 amino acid sequences in osera1 mutants.

S1 Fig. Growth retardation and reppression of rice plants in the mild drought stress test.

S2 Fig. The growth of homozygous progenies of M4 and M5 mutant lines was arrested at the plumule stage.

S3 Fig. A cuticle permeability assay for the osera1 lines.

Acknowledgments

References

S2 Table. Genotypes of T₀ events with CRISPR/Cas9-induced mutations in OsERA1.