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.

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.

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).

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.

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).

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).

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).

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).

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.

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.

Primers used in this study.

(XLSX)

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

(XLSX)

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Predicted OsERA1.1 amino acid sequences in osera1 mutants.

(XLSX)

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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.

(PDF)

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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.

(PDF)

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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.

(PDF)

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25 Sep 2020

PONE-D-20-25974

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

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Reviewer #1: The manuscript ” 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” by Takuya Ogata et al, describe the making and characterization of rice era1 mutants.

I have a few major and minor comments:

Major comments:

1. The description of ERA1 protein function and era1 mutant phenotypes in Introduction is very short. The only role described role for ERA1 relates to ABA signaling. This is unfortunate as there is considerable information available for Arabidopsis era1, both mutant phenotypes and about protein function. For example, the Arabidopsis era1 mutant is defective in meristem and flower formation (http://www.plantcell.org/content/12/8/1267), pathogen responses (http://www.plantphysiol.org/content/148/1/348.short), heat stress (https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14212). The ERA1 protein has been placed to regulate farnesylation of an enzyme involved in brassinosteroid synthesis [https://www.nature.com/articles/nplants2016114]. Thus the results presented in the current manuscript that some of the obtained rice mutants are lethal (the gRNA2 and gRNA3 target sites) is not surprising as ERA1 regulates protein farnesylation which is involved in many different biological pathways. I suggest that the authors expand the Introduction to more broadly illustrate that ERA1 is doing much more than only regulate ABA signaling. This can also help to explain why some of the mutants were lethal.

2. The Arabidopsis era1 mutant has a permeable cuticle (https://academic.oup.com/jxb/article/70/20/5971/5536716). This would influence loss of water and drought responses. Have the authors considered testing if the rice era1 also has permeable cuticle? If rice era1 has permeable cuticle it might change the conclusions from some of the experiments. For example interpretation of stomatal conductance data might change if rice era1 mutants have permeable cuticles. The toluidine blue stain to test for cuticle permeability is a very easy experiment to do.

Minor comment:

3. The rice era1 mutant was sensitive to drought (Fig. 4). This is opposite to the Arabidopsis era1 phenotype (drought tolerant). This is not surprising as the Arabidopsis era1 mutant is highly pleiotropic and involved in many signaling pathways. However, similar to Introduction I am missing in the Discussion some text related to the many functions that ERA1 and protein farnesylation has in different plant signaling pathways. See also point 2 above – if rice era1 has permeable cuticles this could also be an explanation for why it is drought sensitive.

Reviewer #2: Major Concerns/Suggestions:

1) The assumption of single base insertion at gRNA1 site in “M1T, M1G, M2T, and M3T” led to premature termination of stop codon, and that is the cause of the phenotype appears to wrong due to the following reasons:

Singe base insertion at the site shown in “M1T, M1G, M2T, and M3T” lead to the premature stop codon after 45 amino acid. Whereas mutations in gRNA2 and gRNA3 will produce a longer proteins. The authors need to translate the mutant sequence and show the protein produced in each mutant. I have translated the “M1T, M1G, M2T, and M3T” mutant sequence and found that in reading frame 2, the mutant mRNA can produce a protein with complete homology to ERA1 except for the first 25 amino acid. Since mutants of gRNA2 and gRNA3, which produce longer protein than “M1T, M1G, M2T, and M3T” mutant, are unable to grow normally, the assumption that premature termination is cause of the phenotype in sRNA1 is wrong. A Western blotting may show what is the length of protein produced. I think that the protein produced from reading frame2 without the first 25 amino acid may be responsible for the phenotype. whether the the gain of function rather than the loss of function is responsible for the phenotype?

2) The single base insertion mutants are more sensitive to ABA (Fig 3) and drought stress (Figure 4). Earlier studies have shown that reduction in expression levels of ERA1 leads to enhanced drought tolerance (Plant Journal 43, 413–424; J Exp Bot. 2013 Mar; 64(5): 1381–1392). How this can be explained?

3) Line #98: “ ----- 550 ppm CO2, and a light intensity of 150” Currently ambient CO2 concentration if only 410 ppm? Why a higher CO2 was used?

4) Some problem with soil moisture content and stress imposition: Line #108-109: “red clay with a soil water content of 29.5% (water weight/total soil weight)” – Whether the “total soil weight” is total soil dry weight? Figure 4A. Please check the formula used for soil moisture content. The correct formula for soil moisture content % = (Soil water content/soil dry weight)*100

5) Line #113: It says “Water-holding capacity of the soil was 49%” This value at what soil matric potential? Why 60% for well watered (about 11% higher than the WHC) and Drought 40% (about 9% lower than the WHC) were selected? Whether 40% SMC was stress? What was the soil type and soil matric potential? Since for seed germination only 29.5% was used (Line #108-109), 40% may not be a stress.

6) Line #129-130: Why a CO2 concentration of 480 ppm was used? Currently ambient CO2 concentration if only 410 ppm?

Minor comments:

Line #113, 216, 237 and other places: Replace “water stress” with “water-deficit stress”

Line #131-134: “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, and CWWp and CWWq are stomatal conductance in WW pots at p and q days after sowing,” - in the formula CWWp and CWWq are not mentioned.

**********

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Reviewer #2: Yes: Viswanathan Chinnusamy

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5 Nov 2020

RESPONSE 1:

We thank the Editor and Reviewers for their valuable comments and suggestions to improve our manuscript. All the changes and additions made in response to the Editor’s and Reviewers’ comments are in red font in the revised manuscript.

The following minor errors have been changed:

We replaced “enhance” with “improve” (Line 37); “Several” with “A number of” (Line 43); “resistance” with “tolerance” (Lines 52 and 303); and “farnesyltransferase” with “the protein farnesyltransferase” (Line 54), respectively.

COMMENT 2:

Reviewer #1: The manuscript ” 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” by Takuya Ogata et al, describe the making and characterization of rice era1 mutants.

I have a few major and minor comments:

Major comments:

1. The description of ERA1 protein function and era1 mutant phenotypes in Introduction is very short. The only role described role for ERA1 relates to ABA signaling. This is unfortunate as there is considerable information available for Arabidopsis era1, both mutant phenotypes and about protein function. For example, the Arabidopsis era1 mutant is defective in meristem and flower formation (http://www.plantcell.org/content/12/8/1267), pathogen responses (http://www.plantphysiol.org/content/148/1/348.short), heat stress (https://nph.onlinelibrary.wiley.com/doi/full/10.1111/nph.14212). The ERA1 protein has been placed to regulate farnesylation of an enzyme involved in brassinosteroid synthesis [https://www.nature.com/articles/nplants2016114]. Thus the results presented in the current manuscript that some of the obtained rice mutants are lethal (the gRNA2 and gRNA3 target sites) is not surprising as ERA1 regulates protein farnesylation which is involved in many different biological pathways. I suggest that the authors expand the Introduction to more broadly illustrate that ERA1 is doing much more than only regulate ABA signaling. This can also help to explain why some of the mutants were lethal.

RESPONSE 2:

Thank you for your nice suggestion. Based on your suggestion, we have expanded the Introduction to include a description of ERA1 as a pleiotropic regulator (Lines 55–73, 78–79).

COMMENT 3:

2. The Arabidopsis era1 mutant has a permeable cuticle (https://academic.oup.com/jxb/article/70/20/5971/5536716). This would influence loss of water and drought responses. Have the authors considered testing if the rice era1 also has permeable cuticle? If rice era1 has permeable cuticle it might change the conclusions from some of the experiments. For example interpretation of stomatal conductance data might change if rice era1 mutants have permeable cuticles. The toluidine blue stain to test for cuticle permeability is a very easy experiment to do.

RESPONSE 3:

Thank you for your interesting suggestion. Based on your comment, we have checked the cuticle permeability of osera1 lines using toluidine blue staining. There were no significant differences in cuticle permeability between the osera1 line and WT plants. We have added a new supplementary figure (S3 Fig) and corresponding text in the Results section (Lines 222–226).

COMMENT 4:

Minor comment:

3. The rice era1 mutant was sensitive to drought (Fig. 4). This is opposite to the Arabidopsis era1 phenotype (drought tolerant). This is not surprising as the Arabidopsis era1 mutant is highly pleiotropic and involved in many signaling pathways. However, similar to Introduction I am missing in the Discussion some text related to the many functions that ERA1 and protein farnesylation has in different plant signaling pathways. See also point 2 above – if rice era1 has permeable cuticles this could also be an explanation for why it is drought sensitive.

RESPONSE 4:

Thank you for your helpful comment. As we describe in RESPONSE 6, our results show that the osera1 mutant plants respond to drought stress more rapidly than do WT plants through accelerated stomatal closure (Fig. 4), consistent with the hypersensitive ABA response seen in primary root length of osera1 mutant plants (Fig. 3). Therefore, our results are not inconsistent with those described in the previous papers mentioned by the reviewer. Considering your comment, it occurred to us that a more accurate description of our results would be “enhanced response to drought stress”, which we used in the title, rather than “sensitive to drought stress”. We have thus altered the descriptions of our results throughout our manuscript (Lines 34, 82, 246, 256–257, 271, 290–291, and 296). In addition, we have added a sentence discussing our results (Lines 328–332).

COMMENT 5:

Reviewer #2: Major Concerns/Suggestions:

1) The assumption of single base insertion at gRNA1 site in “M1T, M1G, M2T, and M3T” led to premature termination of stop codon, and that is the cause of the phenotype appears to wrong due to the following reasons:

Singe base insertion at the site shown in “M1T, M1G, M2T, and M3T” lead to the premature stop codon after 45 amino acid. Whereas mutations in gRNA2 and gRNA3 will produce a longer proteins. The authors need to translate the mutant sequence and show the protein produced in each mutant. I have translated the “M1T, M1G, M2T, and M3T” mutant sequence and found that in reading frame 2, the mutant mRNA can produce a protein with complete homology to ERA1 except for the first 25 amino acid. Since mutants of gRNA2 and gRNA3, which produce longer protein than “M1T, M1G, M2T, and M3T” mutant, are unable to grow normally, the assumption that premature termination is cause of the phenotype in sRNA1 is wrong. A Western blotting may show what is the length of protein produced. I think that the protein produced from reading frame2 without the first 25 amino acid may be responsible for the phenotype. whether the the gain of function rather than the loss of function is responsible for the phenotype?

RESPONSE 5:

Thank you for pointing this out. Based on your suggestion, we have translated the “M1T, M1G, M2T, and M3T” mutant sequence in silico and confirmed that the mutant mRNA can indeed produce a protein with homology to ERA1 except for the first 28 amino acids (S3 Table). This suggests that the N-terminal truncated region may influence the phenotype. In addition, as pointed out, the difference at the protein level may also affect the phenotype. However, ERA1 has not been analyzed at the protein level, nor have its antibodies been reported. Therefore, more research is needed to determine why this mutation results in the observed phenotype. In the revised version of our manuscript, we have thus added a new supplementary table (S3 Table), phrases (Lines 194–196), and sentences (Lines 332–342) to explain the current situation and have altered a sentence for clarity (Lines 328–329, 342–346). In addition, we have removed “that cause premature stop codons” in the Introduction (Line 80).

COMMENT 6:

2) The single base insertion mutants are more sensitive to ABA (Fig 3) and drought stress (Figure 4). Earlier studies have shown that reduction in expression levels of ERA1 leads to enhanced drought tolerance (Plant Journal 43, 413–424; J Exp Bot. 2013 Mar; 64(5): 1381–1392). How this can be explained?

RESPONSE 6:

Thank you for raising this good point. As we described in RESPONSE 4, our results show that the osera1 mutant plants respond to drought stress more rapidly than do WT plants through accelerated stomatal closure (Fig. 4), consistent with the hypersensitive ABA response seen in primary root length of osera1 mutant plants (Fig. 3). Therefore, our results are not inconsistent with those described in the previous papers mentioned by the reviewer. Upon further consideration, it occurred to us that a more accurate description of our results would be “enhanced response to drought stress”, which we used in the title, rather than “sensitive to drought stress”. Accordingly, we have altered the descriptions of our results throughout our manuscript (Lines 34, 82, 246, 256–257, 271, 290–291, and 296).

COMMENT 7:

3) Line #98: “ ----- 550 ppm CO2, and a light intensity of 150” Currently ambient CO2 concentration if only 410 ppm? Why a higher CO2 was used?

RESPONSE 7:

Thank you for pointing this out. Our group (Nagatoshi et al., 2019) and other independent groups (Ohnishi et al. 2011 PCP 52:1249–1257; Tanaka et al. 2016 Breed Sci. 66:542–551) have shown that CO2 concentrations inside growth chambers that comprise a closed and limited space are much lower than those of the ambient atmosphere during the growth phase of rice and soybean, and that CO2 supplementation enhances rice and soybean growth in plant growth chambers. In our case, by setting the CO2 concentration to 550 ppm, the actual CO2 concentration inside the chamber used to grow rice and soybean plants was maintained above 400 ppm. We have altered this sentence to convey this information and have added a reference in the Materials and Methods section (Lines 115–118).

COMMENT 8:

4) Some problem with soil moisture content and stress imposition: Line #108-109: “red clay with a soil water content of 29.5% (water weight/total soil weight)” – Whether the “total soil weight” is total soil dry weight? Figure 4A. Please check the formula used for soil moisture content. The correct formula for soil moisture content % = (Soil water content/soil dry weight)*100

RESPONSE 8:

Thank you for your comment. In this paper, “total soil weight” does not mean total soil dry weight but soil dry weight plus the weight of the moisture content. Therefore, the formula for soil water content (%) was calculated as [weight of moisture content/total soil weight (soil dry weight plus weight of moisture content) + plant weight] × 100. To use round numbers in this study, the values calculated on a total mass basis are presented as soil water contents; however, we have also added a dry weight value that readers can use to calculate the dry weight-based soil water content. We have provided clear definitions of these terms to avoid confusion (Lines 125–130; 136–140).

COMMENT 9:

5) Line #113: It says “Water-holding capacity of the soil was 49%” This value at what soil matric potential? Why 60% for well watered (about 11% higher than the WHC) and Drought 40% (about 9% lower than the WHC) were selected? Whether 40% SMC was stress? What was the soil type and soil matric potential? Since for seed germination only 29.5% was used (Line #108-109), 40% may not be a stress.

RESPONSE 9:

Thank you for raising this question. We measured the soil matric potential at the water-holding capacity of the soil using a tensiometer. Our measurements confirmed that a soil water content of 30%, 40%, and 49% was equivalent to a soil matric potential of –54 kPa, –11 kPa, and 0 kPa, respectively. We used mixed soil, made by combining Bonsol No.2 (Sumitomo Chemical, Osaka, Japan), which is an artificial granular cultivation soil, and red clay at a volume ratio of 1:1. We have added sentences to provide this new information (Lines 125–130; 143–148).

In preliminary tests, we confirmed that plants were subjected to drought stress in WS pots with a soil water content of 40% (S1 Fig). Based on the results of our preliminary tests (S1 Fig) and a previous report (Singh et al., 2007), we determined that the soil water content was adjusted to 60% in WW pots and 40% in WS pots, respectively. In pots with a soil water content of 60%, about 1–2 cm of water accumulates on the soil surface. The weight of individual pots was recorded every few days, and the soil water content was adjusted to approximately 60% for the WW treatment and 40% for the WS treatment to compensate for water loss due to transpiration at the time of weight measurement. 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 WW pots. Therefore, the water content in WW pots was usually maintained above the water-holding capacity during the test. As shown in Fig. 4A, since the plants are grown in sunlight, the temperature rose depending on the weather and, consequently, the soil water content occasionally dipped below the water-holding capacity. We have added these data (S1 Fig) and the corresponding descriptions (Lines 146–148).

Seeds were germinated in plastic dishes with enough water for 5 days and then the germinated seedlings were transferred to soil-filled plastic tubes as described in Lines 125–126. For clarity, we have added descriptions of this procedure in the Materials and Methods section (Line 114–115).

COMMENT10:

6) Line #129-130: Why a CO2 concentration of 480 ppm was used? Currently ambient CO2 concentration if only 410 ppm?

RESPONSE 10:

Thank you for raising these questions. For the experiments, we set a CO2 concentration of LI-6400XT at 480 ppm, since 480 ppm was the ambient CO2 concentration monitored by LI-6400XT in the greenhouse where we grew our plants. We have added text to explain this point (Lines 159–160).

COMMENT 11:

Minor comments:

Line #113, 216, 237 and other places: Replace “water stress” with “water-deficit stress”

Line #131-134: “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, and CWWp and CWWq are stomatal conductance in WW pots at p and q days after sowing,” - in the formula CWWp and CWWq are not mentioned.

RESPONSE 11:

Thank you for your suggestions. In response to your comment, we have replaced “water stress” with “water-deficit stress” (Lines 134, 251, and 272). In addition, we have removed the unneeded phrase (Lines 162–164).

20 Nov 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

PONE-D-20-25974R1

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #2: Partly

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Reviewer #1: In the revised manuscript, the authors have answered all of my previous comment. I have only one additional comment:

1. In the revised version, lines 192 - 196 , there is a description of the effect of the Crispr mutations on the OsERA1.1 transcript and corresponding protein. This was very difficult to understand, and would benefit from careful editing. As far as I can tell from S3 Table, the crispr mutations leads to a truncated protein - if translation is initiated from the first ATG. However, if translation is started at the second ATG, the only effect is a somewhat shorter protein. If this is a correct interpretation, then I suggest to edit the text on lines 192-196 to make this easier to understand. For example, instead of trying to have all this information in one long sentence, it could be edited to several shorter sentences.

Reviewer #2: Most concerns were addressed.

1) The statistical significant in Fig 3 and and in few bars of Figure 4 may be checked as the error bars indicate the different may not be significant in few cases.

2) Relative stomatal conductance is also not a good measure of drought stress response as even in the well watered plants, stomatal conductance will change as the VPD changes.

**********

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25 Nov 2020

PONE-D-20-25974R1

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

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