Stress-Activated Protein Kinase OsSAPK7 Regulates Salt- Stress Tolerance by Modulating Diverse Stress-Defensive Responses in Rice

Zeng Dan, Wang Chunchao, Xie Junpin, Zhang Fan, Lu Jialing, Shi Xiaorong, Shi Yingyao, Zhou Yongli

Research Paper

Stress-Activated Protein Kinase OsSAPK7 Regulates Salt- Stress Tolerance by Modulating Diverse Stress-Defensive Responses in Rice

Zeng Dan1, Wang Chunchao1, Xie Junpin2, Zhang Fan1, Lu Jialing1, Shi Xiaorong2, Shi Yingyao2, Zhou Yongli1

(Institute of Crop Sciences / National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Sciences, Beijing 100081, China; College of Agronomy, Anhui Agricultural University, Hefei 230036, China)

Soil salinity is an environmental threat limiting rice productivity. Identification of salinity tolerance genes and exploitation of their mechanisms in plants are vital for crop breeding. In this study, the function of stress-activated protein kinase 7 (OsSAPK7), a SnRK2 family member, was characterized in response to salt stress in rice. Compared with variety 9804,-overexpression plants had a greater survival rate, increased chlorophyll and proline contents, and superoxide dismutase and catalase activities at the seedling stage under salt-stress conditions, as well as decreasedsodium potassium ratio (Na+/K+)and malondialdehyde contents. After salt stress, theknockout plants had lower survival rates, increased Na+/K+ratios and malomdiadehyde contents, and decreased physiological parameters compared with 9804. These changes in transgenic lines suggested thatincreased the salt tolerance of rice by modulating ion homeostasis, redox reactions and photosynthesis. The results of RNA-Seq indicated that genes involved in redox-dependent signaling pathway, photosynthesis and zeatin synthesis pathways were significantly down-regulated in theknockout line compared with 9804 under salt-stress condition, which confirmed thatpositively regulated salt tolerance by modulating diverse stress-defensive responses in rice. These findings provided novel insights for the genetic improvement of rice and for understanding the regulatory mechanisms of salt-stress tolerance.

rice;; salt stress tolerance; survival rate

High salinity is major abiotic stress that affects plant growth, resulting in great yield loss in rice, which is a highly salt-sensitive crop compared with other cereals (Munns and Tester, 2008). Plants have evolved a series of sophisticated mechanisms during growth and development in response to external environmental stress (Bhatt et al, 2020). To combat salinity-dependent damage using breeding or biotechnological strategies, it is essential to identify salt-tolerance genes and understand their response mechanisms to salt stress.

Sucrose non-fermentation 1 (SNF1)-associated protein kinase 2s (SnRK2s), which form a family of plant- specific Ser/Thr kinases, play important roles in regulating plant responses to environmental and biotic stress (Kulik et al, 2011). The first SnRK2 cDNA () was isolated from the ABA-treated wheat endosperm cDNA library (Anderberg and Walker- Simmons, 1992), and SnRK2 functions have been widely elucidated. In, there are 10 known SnRK2 genes that have been studied (Boudsocq et al, 2004). AtSnRK2s not only play roles in many developmental processes, but also in responses to salinity and drought (Zheng et al, 2010).- mediated phosphorylation and salicylic acid (SA) signaling act synergistically to activate nonexpressor of pathogenesis-related 1 (NPR1), which initiates immune responses and increases disease resistance (Lee et al, 2015). In maize, 11 ZmSAPK genes have been cloned and most are induced by one or more biotic stresses. For example, the expression ofis significantly increased after high salt and drought stresses (Ying et al, 2011).

Ten homologous genes have been identified in the rice SnRK2 family and designated asto, respectively (Kobayashi et al, 2004). In recent years, expressions and functions of several OsSAPKs in response to biotic and abiotic stresses in ricehave been characterized. For example,is up-regulated after drought treatment and itsoverexpression can increase rice yields (Dey et al, 2016).can respond to salt stress, and its overexpression results in lower Na+and Cl–accumulations and increased photosynthesis (Diédhiou et al, 2008).,,andare up-regulated in a transgenic rice line carrying the non-host resistance genewhen plants have been inoculated withpv.(Xu M R et al, 2013).andmay function collaboratively as positive regulators of salt-stress tolerance at the germination and seedling stages (Lou et al, 2018).

In this study, we demonstrated thatwas involved in response to salt stress usingoverexpressing and knockouttransgenic rice lines. Furthermore, to elucidate the response mechanisms to salt stress mediated by, physiological changes and transcriptome profiling in transgenic line and wild type (WT, 9804) were investigated.

Results

Subcellular localization and salt-stress-responsive expression patterns of OsSAPK7 in rice

OsSAPK7 was first identified as a SNF1-associated protein kinase consisting of 359 amino acid residues with a 257-amino acid kinase region at the N-terminal and a 25-amino acid Glu-rich region at the C-terminal (Fig. 1-A). To determine the subcellular localization of OsSAPK7, the OsSAPK7-GFP fusion protein was transiently expressed inleaves.OsSAPK7-GFP was localized in the cytoplasm and nucleus as assessed by merged GFP signals and auto fluorescence (Fig. 1-B).

To analyze the stress-responsive expression patterns ofin rice,expression levels at the seedling stage after being treated with 100 mmol/L NaCl were measured by qRT-PCR.was rapidly expressed in rice seedlings after the salt-stress treatment, and the transcription level was increased by greater than 7-fold from 4 h to 36 h after the stress treatment (Fig. 2). Thus,may be involved in the response to salt stress in rice.

Fig. 1. Domain structure and subcellular localization of OsSAPK7.

A, Schematic representation of the domain structure of OsSAPK7. The highly conserved kinase region at the N-terminal and the Glu-rich motif at the C-terminal are shown.

B, Green fluorescence protein (GFP) signals arising from the OsSAPK7- GFP fusion proteins expressed in tobacco leaves were detected using a confocal microscope. Red signals represent chloroplast auto-fluorescence.

The only GFP signal expressed by pCAMBIA-1300 in tobacco leaves served as a negative control.

OsSAPK7 positively regulates salt-stress tolerance at seedling stage in rice

To completely investigate the function of,overexpression transgenic lines (and)andknockout lines (and) were generated (Fig. S1). Without the NaCl treatment, no obvious differences were observed between the WT and the transgenic plants (Fig. 3-A). After 10 d of 100 mmol/L NaCl treatment, the-over- expression plants were obviously more robust than 9804, and conversely, theknockout plants were more wilted than 9804 (Fig. 3-A). After treatment with 100 mmol/L NaCl for 10 d and recovered in Hoagland’s nutrient solution for 7 d, the survival rates of the-overexpression plants ranged from 45%–50% (Fig. 3-B), which was significantly greater than 9804. However, the survival rates of theknockout plants were only 20%–25%, which were significantly lower than that of the WT (Fig. 3-B). Thus,may increase the tolerance to salt stress at the rice seedling stage.

Fig. 2. Expression ofin rice under salt-stress conditions.After 20-day-old rice seedlings were subjected to 100 mmol/L NaCl treatment,expression levels were assessed by qRT-PCR. Means and standard errors were obtained from three biological replicates. ** and *** indicate statistically significant differences at the 0.01 and 0.001 levels, respectively.

To investigate whetheraffectsroot growth and seed germination under salt-stress conditions,-overexpression and knockout lines, as well as WT 9804, were grown in distilled water or 100 mmol/L NaCl. There were no significant differences in the root lengths of the-overexpression and knockout lines compared with 9804 under both salt-stress and normal conditions (Fig. S2-A to -D). In addition, there were no significant differences among the seed germination rates of-overexpression,knockout and WT lines grown in either distilled water or 100 mmol/L NaCl (Fig. S2-E to -H). Thus,did not affect root growth or seed germination under salt-stress.

OsSAPK7 modulates diverse physiological traits associated with stress

The capability to eliminate Na+and maintain a lower Na+/K+ratio can reflect the salt tolerance of the plants (Zhu, 2003).-overexpression transgenic plants selectively reduced Na+uptake and maintained lower Na+/K+ratios in the roots and shoots after 6 d of salt stress (Fig. 4-A, -B and Fig. S3). In contrast, theknockout plants accumulated more Na+, leading to greater Na+/K+ratios in the roots and shoots compared with 9804.

To elucidate the physiological changes that occurred in transgenic plants under salt-stress conditions, the chlorophyll contents were determined in the 20-day- old seedlings ofoverexpression,knockout and WT plants grown in Hoagland’s solution supplemented with 100 mmol/L NaCl for 6 d. There were no significant differences in the chlorophyll contents between WT and the transgenic plants under normal conditions (Fig. 4-C). Compared with 9804, the chlorophyll contents were significantly higher inoverexpression plants, but markedly lower inknockout lines under salt-stress conditions (Fig. 4-C).

Fig. 3. Phenotypic reactions of-overexpression,knockout and wild type (WT) 9804 lines under salt-stress conditions.

A, Phenotypic comparison of seedings grown under salt stress at the seedling stage.-overexpression lines (and),knockout lines (and) and 9804 plants under normal conditions for 20-day-old seedlings were transferred to Hoagland’s nutrient solution supplemented with 100 mmol/L NaCl for 10 d and then recovered for 7 d.

B, Survival rates of,,,and 9804 plants after recovered for 7 d. Values are Mean ± SE (= 3). **,< 0.05 according to the Student’s-test.

Additionally, several physiological parameters were measured in transgenic and WT plants under salt- stress conditions. Compared with 9804, superoxide dismutase (SOD) activity was significantly greater in theoverexpression plants but markedly lower inknockout plants under salt-stress conditions (Fig. 4-D). Similarly, catalase (CAT) activity was significantly greater inoverexpression plants but lower inknockout plants than in 9804(Fig. 4-E). Salt stress resulted in a significantly decreased malomdiadehyde (MDA) accumulation in-overexpression plants, while MDA accumulation was significantly increased inknockout plants, compared with WT (Fig. 4-F).

Fig. 4. Responses of 20-day-old transgenic lines and wild type 9804 at 6 d after exposure to 100 mmol/L NaCl treatment.

A, Na+/K+ratio in roots. B, Na+/K+ratio in shoots. C, Chlorophyll content in the leaves under control and salt-stress conditions for 6 d. D, Superoxide dismutase (SOD) activity in leaves. E, Catalase (CAT) activity in leaves. F, Malomdiadehyde (MDA) content of leaves. G, Proline content of leaves.

andare the-overexpression lines.andare theknockout lines. Values are Mean ± SE (= 3). * and ** indicate significant differences at the 0.05 and 0.01 levels according to the Student’s-test compared with 9804, respectively.

Proline accumulation is also a common response to salt stress in many plants (Vives-Peris et al, 2017). Compared with 9804, free proline content in- overexpressionplants was significantly increased under salt stress, while it was decreased significantly inknockout plants (Fig. 4-G). Thus, positive regulation of salt-stress tolerance was associated with a decreasing Na+/K+ratio and an increasing free proline content in plants.

Comparative transcriptome profiling of OsSAPK7 knockout and WT

To elucidate the molecular mechanisms ofin salt-stress responses, a comparative transcriptome profiling of theknockout lineand WT 9804 was globally characterized using RNA-Seq. Compared with WT, 247 differentially expressed genes (DEGs) were identified inunder normal conditions (at 0 h) (Fig. 5-A and Table S1). There were 650 DEGs incompared with WT under salt-stress conditions, including 297 and 416 DEGs at 12 and 36 h after the salt-stress treatment (Fig. 5-A and Table S2), respectively. Among these DEGs, 63 were found at both 12 and 36 h. Cluster analysis of DEGs with R studio showed that they were divided into two clusters. The samples after salt stress (12 and 36 h) were clustered together, and the WT (0 h) clustered in another cluster, indicating thatcan respond to rice salt stress (Fig. 5-B).

Some Gene Ontology (GO) categories associated with basic biological processes, such as metabolic process (GO:0008152), transporter activity (GO:0005215), enzyme regulator activity (GO:0030234) and localization (GO:0051179), were common betweenand WT at 0, 12 and 36 h after salt stress (Fig. 5-C, Fig. S4 and Table S3). Strikingly, several significant GO terms, including electron carrier activity (GO:0009055), structural molecule activity (GO:0005198) and protein binding transcription factor activity (GO:0000988), were characterized inbut not in WT at 12 h after salt stress (Fig. 5-C and Table S3). Notably, the DEGs betweenand WT at 36 h after salt stress were more significantly enriched in electron carrier activity, structural molecule activity, protein binding transcription factor activity, membrane-enclosed lumen (GO:0031974) and organelle part (GO:0044422) (Fig. 5-C and Table S3).

Fig. 5. Transcriptome profiling analysis ofknockout lineand wild type (WT, 9804) under salt stress conditions.

A, Venn diagram of differentially expressed genes (DEGs) in WT vsat 0, 12 and 36 h under salt-stress conditions.

B, Hierarchical clustering of DEGs between WT andat 12 and 36 h under salt stress. The color scale represents log2 of the fragment per kilo base of exon model per million mapped fragments (FPKM).

C, Gene Ontology analysis of DEGs in WT vsat 12 and 36 h under salt-stress conditions.

D, Kyoto Encyclopedia of Genes and Genomes pathway analysis of DEGs in WT vs.

In addition, significant Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including phenylalanine metabolic pathway and phenylpropanoid biosynthesis pathway were common incompared with WT at 0, 12 and 36 h after salt stress (Fig. 5-D and Table S4). Compared with the sample at 0 h, photosynthesis was significantly enriched in KEGG pathway inat 12 h after salt stress, and zeatin biosynthesis and ribosome pathways were enriched at 36 h after salt stress (Fig. 5-D and Table S4).

DEGs involved in redox signaling and photosynthetic pathways in response to salt stress

The electron carrier activity is important in the redox process, with electrons being transported through a series of reactions from the reductant (electron donor) to the oxidant (electron acceptor), with concomitant energy conversion (Garg et al, 2010). In this study, four genes,,,and,associated with electron carrier activity,exhibited the same expression patterns after salt stress (Fig. 6-A and Table S5). Among them,was down-regulated incompared with 9804 at 12 h, and,andwere down-regulated incompared with 9804 at 36 h (Fig. 6-A and Table S5).

Fig. 6. Expression patterns of differentially expressed genes (DEGs) inknockout linecompared with wild type 9804.

A, Hierarchical clustering of DEGs associated with electron carrier activity, photosynthesis and zeatin synthesis pathway regulated byafter salt-stress treatment. Color scale represents the log2 of the fragment per kilo base of exon model per million mapped fragments (FPKM).

B, Expression patterns of key genes in 9804 andassessed by qRT-PCR and FPKM. Values are Mean ± SE (= 3).

Moreover,,and, which wereinvolved in photosynthesis, were down-regulated incompared with 9804 at 12 h after salt stress (Fig. 6-A and Table S5). Compared with 9804 at 36 h after salt stress,,and, which have roles in the zeatin synthesis pathway, were also down-regulated in(Fig. 6-A and Table S5). The relative expression levels of the five tested DEGs as assessed by qRT-PCR were consistent with their fragment per kilo base of exon model per million mapped fragments (FPKM) values (Fig. 6-B).

Discussion

Ten members of the OsSAPKs family in rice might be activated by hyperosmotic stress in the cellular protoplasts (Anderberg and Walker-Simmons, 1992).,andcan function as positive regulators of salt-stress tolerance in rice (Diédhiou et al, 2008; Lou et al, 2018). However, the mechanism of salt tolerance mediated by this kinase family remains to be elucidated to date. In this study,positively regulated salt tolerance at the rice seedling stage and revealed physiological and molecular mechanisms underlying salt tolerance.

Salt stress generally impairs the metabolic processes of plants through both osmotic and ionic effects (Munns and Tester, 2008). Osmotic effects result in a reduction in the water absorption capability. Ionic stress causes Na+toxicity, which disrupts photosynthesis, protein synthesis and enzyme activities (Horie et al, 2015). Na+absorption increases in cell and the change in the Na+/K+ratio may cause the degradation or failure of essential enzymes, leading to cell death (Passricha et al, 2019). Reactive oxygen species (ROS) accumulate during abiotic-stress exposure. ROS accumulation acts as a signal in the mediation of stress, but excessive ROS at later stages injures plant cells by damaging lipids, DNA and proteins (Zhang et al, 2016; Li et al, 2020). Plants have evolved efficient enzymatic and nonenzymatic detoxification mechanisms to scavenge ROS (Xu J et al, 2013). SOD and CAT play important roles in reducing ROS to resist stress. In this study,overexpression plants displayedalower Na+/K+ratio compared with 9804under salt-stress conditions, whileknockout plants had a greater Na+/K+ratio (Fig. 4-A and -B). Furthermore,overexpression plants displayed greater SOD and CAT activities compared with 9804under salt-stress conditions, whileknockout plants had decreased SOD and CAT activities (Fig. 4-D and -E). Notably, MDA content, which is an important intermediate in ion leakage and reflects membrane injuryand ROS scavenging (Zhou et al, 2018), was also significantly reduced in- overexpression plants compared with 9804 (Fig. 4-F). Conversely, it was dramatically increased inknockout plants. These physiological changes in thetransgenic plants suggested thatcan improve salt tolerance by regulating ion homeostasis and scavenging ROS through redox reactions.

Plants can synthesize some small molecules, such as sugars, amino acids and other substances, to regulate the osmotic pressure and resist external stress through photosynthesis. In particular, proline has beneficial roles in ROS scavenging and osmotic pressure regulation to protect plants under abiotic stress conditions (Vives-Peris et al, 2017).overexpression plants had increased chlorophyll and proline contents compared with 9804after salt treatments, whileknockout plants had decreased chlorophylland prolinecontents, suggesting that the salt tolerance mediated bymight be related tophotosynthesis in rice.

Comparative transcriptome profiling ofknockoutand WT lines corroborated thatregulated salt-stress tolerance by modulating diverse stress-defensive responses. DEGs associated with the electron carrier activity, photosynthetic pathway and zeatin biosynthesis pathway, which prevent chlorophyll degradation (Rodo et al, 2008), were significantly enriched in WT compared with transgenic plants under salt-stress conditions, which was in accordance with the change trends of investigated physiological parameters. Thus, the salt tolerance mediated byin rice was involved in diverse stress-defensive responses, including modulating ion homeostasis, scavenging ROS and improving photosynthesis. The results of this study provided novel insights that can be used in the genetic improvement in rice.The further characterization of downstream components and signal transduction cascades triggered byand determination of the DEG functions identified in this study will help elucidate the complex molecular networks involved in salt-stress responses in rice.

methods

Plant materials, strain and vectors

Seeds ofL. variety 9804 and transgenic lines were maintained at the experimental fields of Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China. The plasmid pYLCRISPR/Cas9Pubi-H was obtained from Prof. Liu Yaoguang from South China Agricultural University, Guangzhou, China. pMDC43 vector (Curtis and Grossniklaus, 2003) andEHA105 were maintained in our laboratory.

Subcellular localization and quantitative real-time PCR (qRT-PCR) analysis

To localize the SAPK7 protein, the coding sequence ofwas cloned into the pMDC43 vector, in which it was fused to GFP coding sequence. The plasmid construct was transformed intoleaves. The leaves ofwere observed 48–72 h after infiltration using an LSM 700 laser confocal scanning microscope (ZEISS Microsystems, Jena, Germany). Because the pMDC43 empty vector contains CcdB (Controller of cell division or death B) toxic protein, and it is lethal when transformed into tobacco alone, we supplemented pCAMBIA-1300 as a negative control for only GFP. The excitation and detection wavelengths used for GFP were 488 and 509 nm, respectively.

Plant materials were collected from wild type (WT) 9804 and transgenic plants that were cultured in Hoagland’s nutrient solution (Yoshida et al, 1976). Tissue samples were frozen in liquid nitrogen at -80 oC. The total RNA was extracted using a TRIzol reagent kit (Invitrogen, CA, USA). First-strand cDNA was extracted using TransScript?RT/RI Enzyme Mix reverse transcriptase (Trans, Beijing, China). The real-time quantification of transcript levels was performed in 20 μL reactions using the SYBR?Green PCR Master Mix kit (Applied Biosystems, CA, USA) following the manufacturer’s protocol on an ABI Prism 7500 Sequence Detection System (Applied Biosystems, CA, USA). Primers used in qRT-PCR are shown in Table S6.gene was used as a reference. Independent biological repetitions of each time point for each sample were performed in triplicate.

Vector construction and transgenic line generation

To generate the-overexpression plants, a 1080-bpcDNA sequence was inserted downstream of the35S promoter of pMDC43 using the Gateway system (Curtis and Grossuiklaus, 2003). The full-lengthsequence was downloaded from the NCBI database (https://www.ncbi.nlm. nih.gov/). Using cDNA of 9804 as the template, the target fragment was amplified by PCR using the primer pair SAPK7-CDS-F and SAPK7-CDS-R. The purified product was cloned into the entry vector pGWC using TA cloning to obtain pGWC-. The correctly sequenced pGWC-plasmid was exchanged with the destination vector pMDC43 to obtain pMDC43-.

To generate theknockout lines, the first exon of thegenomic sequence from 38 to 57 bp was used as a single guide RNA (Table S6). pYLCRISPR/Cas9Pubi-H plasmid and-Cas9 vector were constructed using the method described by Ma et al (2005).

The above constructs were introduced intostrain EHA105 and incorporated into the genome of 9804 using an-mediated transformation method. The positiveoverexpression plants were identified using PCR and qRT-PCR. Two positive T1overexpression lines,and, which exhibited greaterexpression levels, were further confirmed using a western blot assay as described by Kim et al (2017). The positiveknockout T0and T1plants were screened by sequencing with primers Cas9-SAPK7-Test-F and Cas9-SAPK7-Test-F (Table S6). The two homozygousknockout linesandwere obtained (Fig. S1). All the primers are listed in Table S6.

Assaying transgenic line phenotypes under salt-stress

overexpression linesand,knockout linesand, and WT 9804 plants were cultured in Hoagland’s nutrient solution (Yoshida et al, 1976) for 20 d and then treated with 100 mmol/L NaCl. For each line, 40 seedlings were planted in three biological replicates. At 10 d after the salt-stress treatment, the seedlings were transferred to normal Hoagland’s nutrient solution to recover for 7 d, and then the survival rates were measured.

To observe the effect ofon seed germination, the seeds of transgenic lines and WT 9804 were treated with 75% alcohol for 5 min, washed with water for three times, and then-overexpression,knockout and WT seeds were sown in distilled water (control) or distilled water supplemented with 100 mmol/L NaCl solution. Each plate contained 30 seeds with three replications. The plates were incubated at 25 oC under 16 h/8 h (light/dark) cycles.

To evaluate the effect ofon root growth, 3-day-old seedlings that were fully germinated were transferred to germination bags (Phytotc, Beijing, China) containing distilled water or 100 mmol/L NaCl solution. To measure root length, the germination bags were incubated vertically, and the root lengths were measured after 7 d.

Assaying physiological parameters under salt-stress

SOD and CATactivity levels, and the chlorophyll, proline, MDA, Na+and K+contents in transgenic lines,,andas well as WT 9804 were detected under salt-stress conditions. The leaves of 20-day-old seedlings cultured in Hoagland’s nutrient solution supplemented with 100 mmol/L NaCl for 6 d were collected, with leaves of the same lines cultured in Hoagland’s nutrient solution serving as controls. The collected samples were immediately frozen in liquid nitrogen, and stored at -80 oC. The total chlorophyll content, and SOD and CAT activity levels, were measured according to Civello et al (1995). The proline content of the leaves was measured according to Tezvergil-Mutluay et al (2010). MDA content was measured according to Ashraf et al (2010). The relative Na+and K+ion accumulations in the roots and shoots were measured according to Schmidt et al (2013). Three replicates were used for each measurement.

High-throughput transcriptome analyses and DEG identification

To determine the expression profiles of genes modulated by, high-throughput transcriptome analyses were performed for the salt stress-challengedknockoutand WT lines. Briefly, 20-day-old seedlings ofand WT were grown in Hoagland’s nutrient solution supplemented with 100 mmol/L NaCl, and the plant leaves were harvested at 0, 12 and 36 h after salt stress. The total RNA of each sample was extracted using a TRIzol extraction kit (Invitrogen, CA, USA). Two replicated RNA samples for each plant were mixed equally and used to construct the library. The produced libraries underwent cluster generation on cBot were sequenced on a HiSeq 2000 platform (Illumina) with paired-end 100-bp reads by CapitalBio Corporation, Beijing, China. The raw sequence data reported in this study have been deposited in the Genome Sequence Archive (Wang et al, 2017) in the BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number PRJCA001237 and are publicly accessible at http://bigd.big.ac.cn/gsa.

The raw reads were first filtered by removing adaptor sequences and low-quality sequences using an in-house perl script. The retained high-quality reads in each sample were mapped to the Rice Genome Note Project (RGAP) of Michigan State University (MSURGAP7, ftp://ftp.plantbiology.msu.edu/ pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomo lecules/version_7.0) using Bowtie (Trapnell et al, 2010). The DEGs were usually identified using a Chi-square test when there was a statistically significant difference in the relative abundance between the two samples (using IDEG6 software, based on a-value threshold ≤ 0.001). Cuffdiff was used to identify DEGs. GO (www.geneontology.org/) and KEGG enrichment (http://bioinfo.sibs.ac.cn/carmo/Gene_Annotation. php) analyses of the DEGs were performed using an online website. The Venn diagram was built using software available online (http://bioinfogp.cnb.csic.es/tools/venny/index.html), and the heat map was created by R studio. According to the GO and KEGG enrichment analyses, candidate genes were selected and verified by qRT-PCR.

Acknowledgements

This study was supported by the National Key Research and Development Program of China (Grant No. 2016YFD0100101), the National High-Tech Program of China (Grant No. 2014AA10A603), the Bill & Melinda Gates Foundation (Grant No. OPP1130530). We thank Professor Liu Yaoguang from South China Agricultural University for providing plasmid pYLCRISPR/Cas9Pubi-H. We thank Lesley Benyon from Liwen Bianji Edanz Group China (www.liwenbianji.cn/ac) for editing the English text.

Supplemental data

The following materials are available in the online version of this article at http://www.sciencedirect.com/journal/rice-science; http://www.ricescience.org.

Fig. S1. Graphic representation of vector constructs and expression analysis ofin transgenic plants.

Fig. S2. Root growth and germination rates oftransgenic lines and wild type 9804 under salt-stress conditions.

Fig. S3. Potassium and sodium ion concentrations in plant tissues of transgenic and wild type 9804 plants.

Fig. S4. Gene Ontology analysis of differentially expressed genes in theknockout linecompared with wild type 9804 at 0 h.

Table S1. Differentially expressed genes incompared with wild type at 0 h after exposure to salt stress.

Table S2. Differentially expressed genes incompared with wild type at 12 and 36 h after exposure to salt stress.

Table S3. Gene ontology analysis of differentially expressed genes inat 0, 12 and 36 h after exposure to salt stress.

Table S4. KEGG analysis of differentially expressed genes inat 0, 12 and 36 h after exposure to salt stress.

Table S5. Differentially expressed genes in electron carrier activity, photosynthesis and zeatin biosynthesis processes under salt-stress condition.

Table S6. Primers used in this study.

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Zhou Yongli (zhouyongli@caas.cn)

1 July 2020;

12 October 2020

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http://dx.doi.org/10.1016/j.rsci.2021.09.003

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