ORIGINAL RESEARCH published: 21 January 2022 doi: 10.3389/fpls.2021.797141 Integrative Approach for Precise Genotyping and Transcriptomics of Salt Tolerant Introgression Rice Lines Mireia Bundó 1 , Héctor Martín-Cardoso 1 , Michele Pesenti 2 , Jorge Gómez-Ariza 1 , Laia Castillo 1 , Julien Frouin 3,4 , Xavier Serrat 5 , Salvador Nogués 5 , Brigitte Courtois 3,4 , Cécile Grenier 3,4 , Gian Attilio Sacchi 2 and Blanca San Segundo 1,6* 1 Centre for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Bellaterra, Spain, 2 Department of Agricultural and Environmental Sciences – Production, Landscape, Agroenergy – DiSAA, University of Milan, Milan, Italy, 3 CIRAD, UMR AGAP, Montpellier, France, 4 AGAP, CIRAD, INRAE, Institut Agro, University of Montpellier, Montpellier, France, 5 Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Secció de Fisiologia Vegetal, Universitat de Barcelona, Barcelona, Spain, 6 Consejo Superior de Investigaciones Científicas, Barcelona, Spain Rice is the most salt sensitive cereal crop and its cultivation is particularly threatened by salt stress, which is currently worsened due to climate change. This study reports the development of salt tolerant introgression lines (ILs) derived from crosses between Edited by: the salt tolerant indica rice variety FL478, which harbors the Saltol quantitative trait loci Giampiero Valè, Università del Piemonte Orientale, Italy (QTL), and the salt-sensitive japonica elite cultivar OLESA. Genotyping-by-sequencing Reviewed by: (GBS) and Kompetitive allele specific PCR (KASPar) genotyping, in combination with Elide Formentin, step-wise phenotypic selection in hydroponic culture, were used for the identification University of Padua, Italy of salt-tolerant ILs. Transcriptome-based genotyping allowed the fine mapping of indica Bijayalaxmi Mohanty, National University of Singapore, genetic introgressions in the best performing IL (IL22). A total of 1,595 genes were Singapore identified in indica regions of IL22, which mainly located in large introgressions at *Correspondence: Chromosomes 1 and 3. In addition to OsHKT1;5, an important number of genes Blanca San Segundo blanca.sansegundo@cragenomica.es; were identified in the introgressed indica segments of IL22 whose expression was blanca.sansegundo@csic.es confirmed [e.g., genes involved in ion transport, callose synthesis, transcriptional regulation of gene expression, hormone signaling and reactive oxygen species (ROS) Specialty section: This article was submitted to accumulation]. These genes might well contribute to salt stress tolerance in IL22 Plant Breeding, plants. Furthermore, comparative transcript profiling revealed that indica introgressions a section of the journal caused important alterations in the background gene expression of IL22 plants (japonica Frontiers in Plant Science cultivar) compared with its salt-sensitive parent, both under non-stress and salt- Received: 18 October 2021 Accepted: 13 December 2021 stress conditions. In response to salt treatment, only 8.6% of the salt-responsive Published: 21 January 2022 genes were found to be commonly up- or down-regulated in IL22 and OLESA Citation: plants, supporting massive transcriptional reprogramming of gene expression caused Bundó M, Martín-Cardoso H, Pesenti M, Gómez-Ariza J, Castillo L, by indica introgressions into the recipient genome. Interactions among indica and Frouin J, Serrat X, Nogués S, japonica genes might provide novel regulatory networks contributing to salt stress Courtois B, Grenier C, Sacchi GA and tolerance in introgression rice lines. Collectively, this study illustrates the usefulness of San Segundo B (2022) Integrative Approach for Precise Genotyping transcriptomics in the characterization of new rice lines obtained in breeding programs and Transcriptomics of Salt Tolerant in rice. Introgression Rice Lines. Front. Plant Sci. 12:797141. Keywords: genotyping by sequencing (GBS), Oryza sativa (L.), Saltol, transcriptomics, indica and japonica, doi: 10.3389/fpls.2021.797141 introgression lines (ILs), salinity stress Frontiers in Plant Science | www.frontiersin.org 1 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines INTRODUCTION act as signaling molecules that trigger signal transduction pathways in response to salt stress. However, when in excess, Rice (Oryza sativa L.) is one of the most important cereal crops ROS cause irreversible cellular damage due to their capacity in the world and serves as a staple food for more than half of to cause oxidative damage to proteins, DNA and lipids. Redox the world’s population. Rice is also considered a salt-sensitive homeostasis in plants is maintained by the activity of antioxidant crop, particularly at the seedling and reproductive stages (Zeng enzymes (e.g., superoxide dismutases, ascorbate peroxidases, and Shannon, 2000). The extent and severity of salt-affected and glutathione-S-transferases), and non-enzymatic systems agricultural land is predicted to worsen as a result of inadequate (e.g., reduced glutathione and flavonoids) (Hossain and Dietz, drainage of irrigated land, entry of sea water in coastal rice fields, 2016). Salt stress responses also involve 14-3-3 proteins, and and global warming. protein phosphorylation processes (Tan et al., 2016; Yang and Tolerance to salt stress in plants is a complex trait governed Guo, 2018; Shah et al., 2021). Salt stress-responsive kinases by a multitude of physiological and molecular mechanisms include calcineurin B-like (CBL)-interacting protein kinases (Van Zelm et al., 2020). High salinity imposes an immediate (CIPKs), mitogen-activated protein kinase (MAPK) cascades, osmotic stress, manifested by reduced water uptake and growth and calcium-dependent protein kinases (CPKs), such as the rice retardation, followed by a long-lasting ionic stress due to gradual OsCPK4, OsCPK12, or OsCPK21 (Asano et al., 2011, 2012; accumulation of Na+ , and subsequent secondary stresses, such as Campo et al., 2014; Shah et al., 2021). Additionally, hormone oxidative stress and nutritional disorders (Negrão et al., 2011; Van signaling pathways creates a sophisticated signaling network that Zelm et al., 2020). In order to maintain fairly constant levels of results in salt stress tolerance, with abscisic acid (ABA) being NaCl over time, roots can regulate NaCl levels by exporting it to considered an important hormone in mediating plant responses the soil or to the shoot. Compared with roots, the transcriptional to salt stress (Yu et al., 2020). Salt stress-inducible genes that are reprogramming of plant leaves during adaptation to salt stress has ABA independent have been described (Yoshida et al., 2014). deserved less attention. Leaves are, however, more sensitive to salt Regulation of gene expression during salinity stress is known stress than roots. to be governed by various transcription factor (TF) pathways, In shoots, high concentrations of Na+ provoke disruption including AP2/ERF, bHLH, bZIP, DREB, GATA, HD-Zip, of the ion homeostasis mechanisms which, in turn, causes a Homeo-box, MADS-box, MYB, NAC, Trihelix, WHIRLY, range of metabolic problems. Among other effects, Na+ competes WOX, WRKY, YABBY, and zinc finger TFs (Hussain et al., with K+ for essential cellular functions (e.g., Na+ replaces K+ 2021; Shah et al., 2021). These TFs might control salinity at binding sites on enzymes resulting on enzyme deactivation) tolerance through ABA-dependent and ABA-independent and the chloride ion may interfere with anionic sites involved signaling pathways, and play essential role in ROS production, in binding of RNA and anionic metabolites. Maintenance of chlorophyll content, lipid-peroxidation (Yang and Guo, 2018; low Na+ /K+ ratio in shoots is then essential to circumvent Yoon et al., 2020). They regulate downstream genes by binding Na+ toxicity under salt stress conditions. For this, plants have to cis-regulatory elements in their promoter region. In particular, multiple Na+ transport systems aiming Na+ extrusion out of members of the AP2/ERF TF family have been comprehensively cells, compartmentalization into vacuoles, or sequestration of studied in plant responses to salt stress, including DREB toxic Na+ into older leaves (Wang et al., 2018; Ketehouli et al., (Dehydration-Responsive Element-binding Protein) and ERF 2019; Van Zelm et al., 2020). Members of the high-affinity (Ethylene response element-binding factors) TFs. DREBs are K+ transporter (HKT) transporter/channel family are known to classified into two subgroups, DREB1 and DREB2 and are play crucial roles in maintenance of Na+ homeostasis during induced by salt and dehydration stress in plants (Nakashima salt stress, and also aquaporins which are crucial for water et al., 2014). DREB TFs associated with salinity tolerance homeostasis and transport of certain solutes during adaptation include OsDREB1A, OsDREBB1C, OsDREBB1F, and OsDREB2A to salt stress (Afzal et al., 2016; Wang et al., 2018; Ketehouli (Dubouzet et al., 2003; Wang et al., 2008; Mallikarjuna et al., et al., 2019). Exposure to salinity causes plasma membrane 2011; Shah et al., 2021). Regarding NAC (NAM, ATAF, and remodeling affecting the activity of membrane proteins (and CUC) TFs, their overexpression in transgenic plants has been signaling molecules) that results in alterations in membrane shown to improve salinity tolerance in many plant species. permeability (Guo et al., 2019). Salt stress also induces changes As an example, transgenic rice plants overexpressing either in the cell wall composition that can benefit the plant by OsNAC6 or OsNAC1 were shown to exhibit strong tolerance preventing water loss and altering ion transport pathways to salt stress (Hu et al., 2006; Nakashima et al., 2007). In other (Zagorchev et al., 2014). studies, OsZIP23 was found to function as a transcriptional Increases in cytosolic Ca2+ concentration and production regulator of a wide spectrum of abiotic stress-related genes of reactive oxygen species (ROS) are some of the earliest through an ABA-dependent regulation pathway, and its described salt stress responses (Van Zelm et al., 2020). ROS overexpression in rice confers salt-stress tolerance (Xiang et al., 2008). For additional information on TFs involved in Abbreviations: ABA, abscisic acid; DEG, differentially expressed gene; salinity tolerance in plants we refer to recent reviews by Hussain GBS, genotyping-by-sequencing; GO, Gene Ontology; HKT, high-affinity et al. (2021) and Shah et al. (2021). Although there have been K+ transporter; IL, introgression line; KASPar, Kompetitive allele specific many reports describing mechanisms of salinity tolerance PCR; MABC, marker-assisted backcross; QTL, quantitative trait loci; ROS, reactive oxygen species; SES, Standard Evaluation System; SNP, single in plants, most of these studies have been carried out in the nucleotide polymorphism. dicotyledonous model plant Arabidopsis. Much remains to Frontiers in Plant Science | www.frontiersin.org 2 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines learn about how multiple signaling pathways from different were used to monitor the introgression of indica segments into cellular compartments are coordinated, and how salt stress OLESA and to further characterize advanced ILs. Evaluation responsive genes function during adaptation to salt stress of salt tolerance in the ILs was conducted at the seedling in crop species. stage in hydroponically grown plants. A fine-mapping of indica Many efforts are being made by rice breeders to develop introgressions was performed in the best performing IL (IL22) high-yielding rice cultivars with improved tolerance to salt by transcriptome-based genotyping. Furthermore, we provide stress. A major quantitative trait loci (QTL), named Saltol, a global view of the leaf transcriptome of IL22 plants, both was identified in a recombinant inbred line (RIL) population under control and salt-stress conditions. Evidence is presented derived from a cross between the salt tolerant indica landrace that genes in the introgressed indica regions at Chromosome 1 Pokkali and the salt sensitive indica cultivar IR29 (Gregorio (including Saltol) and Chromosome 3, in the IL22 line, might et al., 1997; Bonilla et al., 2002). Since then, the Saltol QTL well contribute to a phenotype of salt tolerance, either by their has been widely used to introduce seedling stage salt tolerance intrinsic function (e.g., ion transporters and cell wall biosynthesis into modern high yielding rice varieties, through marker-assisted genes), or by functional interactions with other indica or backcrossing (Waziri et al., 2016; Babu et al., 2017; Yadav et al., japonica genes. The information gained in this study extend our 2020). The Saltol QTL has been mapped on Chromosome 1, understanding on the molecular mechanisms involved in salinity and accounts for a low Na+ /K+ ratio in rice shoots under tolerance in introgression rice lines harboring the Saltol QTL salt stress (Bonilla et al., 2002; Gregorio et al., 2002; Thomson which might also be useful for the identification of marker genes et al., 2010). The OsHKT1;5 gene located in the Saltol region for salinity tolerance in rice. has been proposed to be the responsible gene for the salinity tolerance provided by Saltol (Ren et al., 2005; Platten et al., 2013). OsHKT1;5 is expressed in rice roots and encodes for a MATERIALS AND METHODS Na+ -selective transporter that mediates Na+ exclusion out of the xylem vessel into xylem parenchyma, thus, preventing Na+ Plant Material and Marker-Assisted transfer to young leaf blades (Kobayashi et al., 2017). In this way, Backcross Breeding OsHKT1;5 helps in maintaining the Na+ /K+ ratio and minimizes Introgression lines were generated through backcross breeding the harmful effects of Na+ accumulation in shoots. Although the between FL478 (male donor, indica ssp.) harboring the Saltol Saltol QTL has been extensively employed in breeding programs QTL (Thomson et al., 2010) and OLESA, a salt sensitive, to enhance salt tolerance in rice at the seedling stage, to date, long grain japonica cultivar (female recipient) (Supplementary the underlying mechanisms responsible of salinity tolerance in Figure 1). FL478 was obtained from the International Rice Saltol-introgressed lines remain largely unknown. Research Institute (IRRI, accession number IRGC 117406). Most QTLs so far identified for salinity tolerance in rice derive OLESA was provided by Càmara Arrossera del Montsià SCCL from indica accessions (e.g., Pokkali and Nona Bokra). More (Amposta, Spain, accession number NRVC20120346). It involved recently, genome-wide association mapping identified candidate the initial cross and three backcrosses coupled to embryo rescue QTLs for tolerance to mild salinity stress in temperate japonica (Ohnishi et al., 2011), and three selfing steps. Complementary rice (Batayeva et al., 2018; Frouin et al., 2018). Saltol introgression approaches were used to monitor the introgression of indica into japonica varieties was reported to improve salt tolerance in segments into the japonica background of OLESA. The first introgression lines (ILs) (Usatov et al., 2015). In other studies, step consisted in the screening of progenies for the presence however, the Saltol QTL from the indica cultivar IR64-Saltol was of the Saltol QTL using the Saltol linked SSR marker SKC10 introgressed into a japonica rice variety, and no differences could (Thomson et al., 2010) which was found to be polymorphic be observed between Saltol and non-Saltol ILs (Han et al., 2020). between the two parents (FL478 and OLESA) (Supplementary When FL478, a RIL derived from the cross between IR29 and Figure 2). The second genotyping step was carried out on Pokkali, was used as a Saltol donor, some lines that introgressed positive samples for the SKC10 SSR marker and involved only the Saltol QTL were found to exhibit less salinity tolerance the analysis of Saltol single nucleotide polymorphism (SNP) than their donor parent, thus, suggesting the presence of regions flanking markers located at 9.06 and 13.34 Mb in Chromosome in the FL478 genome other than the Saltol region contributing to 1 using KASPar (Kompetitive allele specific PCR) coupled to salinity tolerance (Kim et al., 2009; Thomson et al., 2010; Alam Light Cycler 480 system technology (Roche) (Supplementary et al., 2011). Clearly, many questions still remain unanswered Table 1). Finally, KASPar coupled to the Fluidigm technology regarding the use of the Saltol QTL in salinity tolerance in rice. was used for foreground and background selection. A total of Furthermore, considering that most QTLs so far identified for 68 SNPs distributed along the 12 chromosomes were assayed salinity tolerance are from indica subspecies, it is important (Supplementary Table 1). to investigate the genome-wide impact on gene transcription in a recipient japonica background caused by introgressions of Genotyping-by-Sequencing indica genomic regions. In this study we report the production Genomic DNA was extracted from young leaves of ILs and and characterization of salt tolerant ILs derived from the cross their respective parental lines by high-throughput automated of FL478 (indica cv, donor for the Saltol QTL), and the salt- methods using NucleoMag Plant kit (Macherey-Nagel) and its sensitive elite japonica variety OLESA. Kompetitive allele specific quality verified on 1.5% agarose gel. After Hoechst quantification, PCR (KASPar) genotyping and genotyping by sequencing (GBS) DNA’s concentrations were normalized. DNAs were digested Frontiers in Plant Science | www.frontiersin.org 3 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines individually with the ApeKI restriction enzyme. GBS sequencing RNA-Seq Library Preparation, library was prepared by ligating the digested DNA to unique Sequencing, and Data Analysis nucleotide adapters (barcodes) followed by PCR. Sequencing Plants (IL22, OLESA) were grown in hydroponic cultures as was performed using Illumina HiSeq3000. To detect informative described above (phenotyping for salinity tolerance). Leaves from SNPs, fastq files were analyzed using Tassel V5 pipeline and salt-treated (80 mM NaCl, 24 h of treatment) and untreated an alignment on the O. sativa Nipponbare reference genome control plants were harvested (three replicates per genotype and (MSU7) with Bowtie2. Only polymorphic parental loci were kept condition, four plants per replicate). Samples were immediately and filtered on heterozygous rate (<40%) and then imputed frozen in liquid nitrogen and grinded by TissueLyser (Qiagen). with Beagle v5.0. Total RNA was extracted using the Maxwell RSC Plant RNA Kit (Promega). Details of library preparation and RNA-Seq analysis Phenotyping for Salinity Tolerance are presented in Supplementary Method. Statistical analysis of Introgression lines derived from FL478 × OLESA (30 lines, read counts was performed with R, with the HTSFilter package BC3 F4 ) were screened in hydroponic experiments using the to remove low-expressed genes and the edge R package. Criteria FL478 and OLESA cultivars as tolerant and sensitive checks, used for calling differentially expressed genes (DEGs) were fold- respectively. Rice seeds were pregerminated in water for change (log2 fold change >0.5 or <−0.5, for up-regulated and 7 days and then transferred to hydroponic tanks with modified down-regulated genes), significance of differences in expression Yoshida solution (1.43 mM NH4 NO3 , 0.51 mM K2 SO4 , (P ≤ 0.05), and expression level (fragments per kilobase per 0.85 mM KH2 PO4 , 0.12 mM K2 HPO4 , 0.75 mM CaCl2 ·2H2 O, million mapped reads-FPKMs ≥ 25). Gene Ontology (GO) 1.64 mM MgSO4 ·7H2 O, 9.5 µM MnCl2 ·4H2 O, 0.075 µM enrichment of DEGs was carried out by Singular Enrichment (NH4 )6 ·Mo7 O24 ·4H2 O, 18.9 µM H3 BO3 , 0.15 µM ZnSO4 ·7H2 O, Analysis (SEA) using AgriGO (Tian et al., 2017). Data sets for 0.16 µM CuSO4 ·5H2 O, 35.75 µM FeSO4 -EDTA, pH 5) for RNA-Seq analyses have been deposited at the National Center for 7 days. Salt treatment was applied by supplementing the nutrient Biotechnology Information (NCBI) Gene Expression Omnibus solution with 80 mM NaCl for 14 days. Control plants were not (GEO) with accession number GSE167342). supplemented with NaCl. No aeration was applied. Plants were randomly distributed, in a ratio of 40 plants per 10 L tank (5 and Transcriptome-Based Genotyping 10 plants per genotype, control and salt conditions, respectively). Data sets obtained by RNA-Seq analysis were used for the fine Plants were grown at 28/26◦ C day/night with a 14/10 h light/dark mapping of indica regions introgressed into the genome of cycle. The nutrient solution was replaced weekly. IL22 plants (japonica background). Details on the various steps Fresh weight (FW), dry weight (DW), shoot length (SL), followed for the identification of introgressed indica regions in root length (RL), and SES score (standard evaluation system, IL22 plants are presented in Supplementary Method. IRRI) were examined at 14 days after salt treatment. FW, DW, SL, and RL were calculated as the percentage of FW, Reverse Transcription-Quantitative PCR DW, SL, or RL in salt conditions compared to control Total RNA was obtained as described above (RNA-Seq conditions and then normalized to OLESA sensitive check. In library preparation and sequencing). Total RNA (1 µg) this way, the improvement or worsening of salinity tolerance was retrotranscribed using the High Capacity cDNA reverse in ILs in comparison to its recurrent parent OLESA could be transcription Kit (Applied Biosystems, Thermo Fisher Scientific). determined. Electrolyte leakage (EL) analyses of young leaves Reverse transcription-quantitative PCR (RT-qPCR) analyses were were carried out at 2 days after salt treatment as previously carried out in 96-well optical plates in a LightCycler 480 System R described (Campo et al., 2014). Briefly, leaves were cut in 1 cm (Roche) according to the following program: 10 min at 95◦ C, segments and washed in Milli-Q water. Then, leaf segments 45 cycles of 95◦ C for 10 s and 60◦ C for 30 s, and an additional were incubated for 2 h in Milli-Q water in a shaker and cycle of dissociation curves to ensure a unique amplification. electro conductivity 1 (Ec1) was measured. After autoclaving The reaction mixture contained 5 µl of SYBR Green Master mix the leaf segments for 20 min, Ec2 was measured and the reagent (Roche), 2 µl of 1:4 diluted cDNA samples and 300 nM EL was calculated as (Ec1/Ec2) × 100. Three independent of each gene-specific primer in a final volume of 10 µl. The experiments were performed (three replicates from four plants results for the gene expression were normalized to OsUbi1. Three each). For Na+ /K+ molar ratio evaluation, plants were harvested biological replicates each one from a pool of four different plants, at 7 days of salt treatment. In order to remove apoplastic and three technical replicates for each biological replicate were Na+ and K+ , roots where washed twice for 10 min at 4◦ C analyzed. Primers were designed using Primer-BLAST1 . Primers in a 25 mM Rb2 SO4 solution before dissecting the plant used for RT-qPCR are listed in Supplementary Table 2. tissues (young leaves, old leaves, sheath and stem, and roots). Roots and aerial parts were air-dried and then mineralized Statistical Analysis by a microwave digester system (MULTIWAVE-ECO, Anton Significant differences in FW, DW, SL, and RL, EL, Na+ /K+ Paar GmbH) in 65% (v/v) HNO3 . Aliquots of the mineralized ratio, and RT-qPCR experiments were determined by Student’s samples were adequately diluted in Milli-Q water and the t-test, evaluating pairwise comparisons of mean differences. For concentrations of Na+ and K+ were measured by ICP-MS RT-qPCR analysis, statistical significance was determined by technique (Bruker Aurora M90 ICP-MS, Bruker Daltonik Student’s t-test (salt-treated vs non-treated, each genotype). GmbH). Three independent experiments were performed (three replicates from three plants each). 1 https://www.ncbi.nlm.nih.gov/tools/primer-blast/ Frontiers in Plant Science | www.frontiersin.org 4 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines RESULTS introgression of Saltol. For instance, a large flanking region surrounding the target QTL and some areas on Chromosome 3 Development of Introgression Lines by maintained a significant portion of the donor parents in more than half the progenies, despite the three cycles of backcrossing. Marker-Assisted Backcross Breeding Therefore, GBS allowed us to discover and call SNPs on 30 In this study, marker-assisted backcross (MABC) breeding was advanced inbred lines of rice for salinity tolerance, while defining used to transfer the Saltol QTL from the highly salt tolerant common and specific polymorphic SNPs between the various ILs. indica RIL FL478 (male donor parent) into the salt-sensitive japonica elite cultivar OLESA (female recurrent parent). Three successive cycles of marker-assisted backcrossing coupled to Phenotypic Characterization of embryo rescue (Ohnishi et al., 2011), followed by three cycles of selfing were carried out (Supplementary Figure 1). Several Introgression Lines Introgression lines harboring the Saltol QTL in homozygosis (30 marker-assisted approaches were used for selection of progenies lines) were evaluated for salinity tolerance at the seedling stage through the backcrossing process, which involved the use of in hydroponic cultures. Before assaying salt tolerance of ILs, Saltol SSR marker SKC10 by PCR (Thomson et al., 2010) and the two parental lines were evaluated for salinity tolerance at KASPar markers coupled to the Light Cycler 480 system and three different NaCl concentrations, namely 60, 80, and 100 mM, the Fluidigm technology (Roche) (Supplementary Figure 2 and using the Standard Evaluation System (SES; IRRI). The SES score Supplementary Table 1). A total of 30 BC3 F3 inbred lines rate the symptoms of salt stress on a scale of 1 to 9 based on showing a recurrent parent genome (RPG) recovery ranging morphological symptoms, where a lower score of SES indicates from 92.7 to 100% were identified (average of 96.9% RPG) tolerance and a higher score indicates sensitivity to salt stress. which were advanced to BC3 F4 by selfing. The ILs were Differences between the two parental lines were better seen using all homozygous for the 68 SNPs used for foreground and a NaCl concentration of 80 mM and a period of treatment of background selection at the BC3 F3 generation (Supplementary 14 days (Supplementary Figure 4). Figure 3). In addition to the Saltol region (Chromosome 1), Next, the ILs (30 lines) were evaluated for salinity tolerance these lines had additional introgressions of different lengths in successive rounds of hydroponic assays (80 mM NaCl for distributed in different chromosomes (Supplementary Figure 3). 14 days; six independent assays) (Figure 2A). At the onset of The 30 BC3 F4 lines derived from four different BC3 F1 plants, salt treatment, all the plants were at the same developmental which resulted in four different introgression patterns in the stage (two-leaf stage). In each assay, the most salt-sensitive lines ILs at the BC3 F3 generation. Thus, depending on the pattern of were discarded. SES evaluation revealed different degrees of salt- indica introgressions, the ILs classified into four different groups induced damage among lines from the four groups (I to IV, (groups I, II, III and IV) (Supplementary Figure 3). according to the BC3 F1 parent from which each line derived), as well as among lines in a given group (Supplementary Figure 5A). Genotyping-by-Sequencing Genotyping Thus, even though all the ILs were homozygous for the markers of Introgression Lines linked to Saltol (BC3 F4 plants), there was an extensive variation Genotyping by sequencing (GBS) represents a powerful in phenotypic responses to salt stress among the 30 ILs. For approach to detect genotypic variation, such as SNPs and subsequent analyses, we focused on lines IL22 and IL13, these insertion/deletions in crop species. In the present study, GBS lines performing well in salt tolerance assays compared with was used to identify allelic variations between ILs generated by the salt-sensitive parent OLESA (Supplementary Figures 5A,B). MABC breeding. A total of 15,580 SNPs were identified in the The ILs developed new leaves under salt stress (Supplementary 30 ILs here investigated, its number being variable depending on Figure 6), and were able to flower and set seeds when the stress the rice chromosome (Figure 1 and Supplementary Table 3). was removed (e.g., by transferring the plants from hydroponic Polymorphic SNPs were not randomly distributed among all cultures to soil). the 12 chromosomes, with the highest SNP number found on Based on SES, 73.1% of the OLESA plants showed a SES Chromosome 1 (Saltol QTL). A relatively low number of SNP score of 9 (highly sensitive), but only 40.9 and 47.8% of IL22 polymorphisms were observed in Chromosomes 8, 10, 11, and and IL13, respectively, scored in this category (Figure 2B). Also, 12. ILs genotyping obtained in the GBS data was in accordance 22.5 and 25.4% of the IL22 and IL13 plants, respectively, had a with the genotypes observed using KASPar markers, showing score of 5 (moderately tolerant), while only 6.4% of plants from the Saltol QTL completely introgressed in homozygosis in the the recurrent parent OLESA scored in this category. Analysis 30 inbred lines with some other small introgressions in the of morphological traits also indicated that lines IL22 and IL13 other chromosomes. As previously observed by KASPar analysis, were the best performing ILs in salt tolerance assays (Figure 2B). GBS confirmed four groups of indica introgression patterns (I Compared with the sensitive and tolerant parents (OLESA and to IV) derived from four different BC3 F1 plants. Additionally, FL478, respectively), the IL22 and IL13 lines showed intermediate GBS analysis allowed us to identify allelic variations in the responses in the various phenotypical parameters assayed (i.e., introgressions in lines grouped into the same group. The FW, DW, SL, and RL) (Figure 2B). value added by seeing the allelic profile of markers more Then, we examined EL in leaves of IL plants, which was densely distributed on the whole genome, was in a posteriori compared with that of their sensitive and tolerant parents. EL consideration of the choice of the KASPar makers used for the is an indicator of cell membrane injury and it is commonly Frontiers in Plant Science | www.frontiersin.org 5 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 1 | Genotyping-by-sequencing analysis of introgression lines. Scheme representing SNPs variation and its distribution among the 12 chromosomes in BC3 F4 introgression lines. SNPs are indicated in columns according to their chromosomal location. Introgression lines (IL1 to IL31) are clustered in four groups (I to IV) depending on the BC3 F1 parent from which they derive. The Saltol QTL location (and length) is indicated in the upper part. The total number of SNPs found in each chromosome is indicated in the lower part. Donor (FL478) and recurrent (OLESA) homozygous alleles in the 12 chromosomes are depicted in blue and white, respectively, and heterozygous in red. A detailed description of SNPs is provided in Supplementary Table 3. used for the identification of salt tolerant plants (Bajji et al., these plants are less stressed at the cellular level than OLESA 2002; Demidchik et al., 2014). As expected, upon exposure to plants. Thus, values of Na+ /K+ ratios in shoots of IL22 and IL13 salt stress, the parental varieties FL478 and OLESA (salt tolerant plants were consistent with the observed phenotypic responses to and salt sensitive, respectively) showed low and high levels salt treatment in these lines. of EL, respectively (Figure 2B). Compared with the sensitive Collectively, results obtained in salt tolerance assays revealed parent, IL22 and IL13 exhibited reduced EL values, indicating less lower SES scores, reduced EL values and lower Na+ /K+ ratios in membrane damage during salt treatment (Figure 2B). leaves of IL22 and IL13 plants, thus, supporting enhanced salinity Maintenance of adequate Na+ /K+ ratios in plant tissues under tolerance in these introgression rice lines. salt stress is essential for salinity tolerance, and Saltol has been found to be associated with low Na+ /K+ ratio in rice seedlings (Gregorio, 1997; Bonilla et al., 2002; Gregorio et al., 2002; Waziri Fine Mapping of Indica Introgressions in et al., 2016). Accordingly, we estimated the Na+ /K+ molar ratio IL22 by Transcriptome Genotyping in different tissues of IL22 and IL13 plants (e.g., young leaves, old The observation that ILs exhibited extensive phenotypic variation leaves, sheath and stem, and roots), as well as in the whole shoots in response to salt stress, even though all these lines harbor (Table 1 and Supplementary Table 4). The Na+ /K+ ratio in IL Saltol in homozygosis, raised interesting questions. We reasoned plants was compared with that in the parental varieties FL478 that indica introgressions might have an effect on the leaf and OLESA. In roots, the Na+ /K+ ratio of ILs and parent plants transcriptome of the IL, not only because of the introgression was similar (Table 1). Further, the Na+ /K+ ratio in tissues, and in of favorable indica genes from FL478, but also because epistatic particular in the young leaves, of IL plants was significantly lower interactions between indica introgressions and genes from the compared with the salt-sensitive parent (OLESA), indicating that japonica background (OLESA) might occur. In this respect, Frontiers in Plant Science | www.frontiersin.org 6 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 2 | Characterization of salt tolerant introgression lines. Introgression (IL13, IL22) and parental (FL478, OLESA) lines were grown hydroponically in modified Yoshida solution and exposed to salt stress (80 mM NaCl). Student’s t-test (*P < 0.05, **P < 0.001, ***P < 0.0001). (A) Experimental design used to assess salinity tolerance. (B) Analysis of salinity tolerance at 14 days [SES scores, fresh weight (FW), dry weight (DW), shoot length (SL), and root length (RL)] or 2 days (electrolyte leakage, EL) after treatment. SES scores are shown as the percentage of plants at each score value. 1, highly tolerant; 3, tolerant; 5, moderately tolerant; 7, sensitive; 9, highly sensitive. Six independent experiments were carried out (10 plants/genotype each experiment). For FW, DW, SL, RL, and EL, values in each genotype were normalized to those in OLESA. Gray bars represent the fold change of each line in comparison to OLESA values, calculated by the means of six independent hydroponic cultures (blue dots) with 10 plants per genotype in each hydroponic assay (5 plants in control conditions). EL was measured in three independent experiments (12 plants/genotype each experiment). salinity tolerance, and maintenance of low Na+ /K+ ratios at the genetic background of japonica, however, requires knowing the seedling stage, is known to be governed by both additive the precise loci introgressed that are transcriptionally active in and dominant effects in rice (Gregorio and Senadhira, 1993). a particular IL. The IL22 line performing best in salt tolerance Understanding the overall impact of indica introgressions into assays was selected for these studies. Frontiers in Plant Science | www.frontiersin.org 7 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines TABLE 1 | Na+ /K+ molar ratios in different tissues of introgression rice in the introgressed indica regions were arranged in clusters (some lines and parents. examples are shown in Figure 4). Young leaves Old leaves Sheath and stem Shoot Root Interestingly, a large number of the genes identified in FL478 0.22 ± 0.00** 1.19 ± 0.17** 1.21 ± 0.18* 0.90 ± 0.12** 1.89 ± 0.06 introgressed indica regions have been shown to play a role in OLESA 1.94 ± 0.18 2.92 ± 0.17 5.33 ± 0.57 3.29 ± 0.17 1.72 ± 0.17 IL13 1.01 ± 0.09** 2.07 ± 0.16* 2.53 ± 0.09* 2.11 ± 0.11* 1.85 ± 0.13 salt and/or drought stress responses, such as those involved in IL22 0.91 ± 0.18* 2.53 ± 0.06 3.22 ± 0.01* 2.41 ± 0.09* 1.90 ± 0.03 ion homeostasis (e.g., Na+ /K+ transporters) and water channel Data shows mean ± SEM in comparison to OLESA values (Student’s t-test, proteins (aquaporins) (Figure 4 and Supplementary Tables 5, 6). *P < 0.05, **P < 0.01). In addition to the high affinity HKT transporter OsHKT1;5 (SKC1 in the Saltol QTL), the indica region in Block 2 of Chromosome 1 also contained OsHKT2.3/HKT3 and OsHAK3 In this work, a transcriptome-based approach was used (putative K+ uptake transporters) (Figure 4). Other genes with a twofold objective: (i) to get a fine mapping of in indica regions of IL22 were those encoding salt-associated indica genetic introgressions in IL22, and (ii) to assess the TFs (e.g., DREB2A), LEA proteins, antioxidant enzymes (e.g., expression of indica and japonica genes in IL22 plants. For Thioredoxins, Glutathione S-tranferases, and Peroxidases), and this, IL22 and OLESA plants were grown in hydroponic components of the salt stress-induced signal transduction culture under control and moderate salt stress conditions pathways (e.g., calcium sensors, CIPKs, and CDPKs protein (80 mM NaCl, 24 h of salt treatment). Comparisons of RNA- kinases and protein phosphatases) (Figure 4 and Supplementary Seq data from each genotype (IL22, OLESA) and condition Table 5). Finally, genes that function in cell wall biosynthesis (control, salt-treated) were made as shown in Supplementary and modification (xylogalactosyl transferases, expansins, callose Figure 7 (fine mapping of indica introgressions in IL22; synthases, etc.) were highly represented in the indica regions of comparative transcriptomes of IL22 and OLESA, control and salt IL22 (Supplementary Table 6). stress conditions). One important finding of this study is that genes with To get a fine mapping of the indica regions introgressed related functions were present in the various indica regions in into the genome of IL22 plants, RNA-Seq reads from control IL22. For instance, genes involved in brassinosteroid signaling and salt-treated plants were aligned to japonica (cv Nipponbare, were identified in the two indica segments in Chromosome IRGSP-1.0/Ensembl release 42) reference genome. As a control 1. Brassinosteroid Insensitive 1 (BRI1)-associated receptor kinase of natural variation, OLESA transcripts were also mapped 1 (OsBAK1), and two Brassinosteroid receptor kinase (BRI1)- against japonica reference genome. By comparing the number interacting protein genes (OsBIP115, OsBIP128) were present of transcript variants in IL22 and OLESA, three large genomic in Block 1 of Chromosome 1, while BR-signaling kinase 1 regions containing high frequency of transcript variants in IL22 (BSK1) was found in Block 2 of this chromosome (Figure 4 (indicating the presence of indica loci) were identified: two and Supplementary Table 5). Genes involved in modulation large introgressions located in Chromosome 1, while the third of ABA signaling pathway and ABA biosynthesis controlling one located at Chromosome 3 (Figure 3). These results were tolerance to salt stress were also present in indica regions essentially in accordance with those obtained by GBS and KASPar introgressed in IL22 (e.g., the NAC22 TF in Chromosome 3; genotyping of IL22. Most importantly, this analysis allowed us OsDET1 in Chromosome 1) (Supplementary Table 5). Ethylene- to identify the introgression boundaries for each introgressed related genes were also identified in indica regions in IL22 region, hence, the precise loci contained in the various (Supplementary Table 5). Another example of genes with introgressed indica intervals. In Chromosome 1, a total of 1,213 related functions identified in indica introgression refers to genes genes were identified: 583 and 630 genes in Block 1 and Block 2, involved in callose deposition, such as Phospholipase D (PLD) respectively, Block 2 containing the Saltol QTL (Supplementary in Block 1 (PLDa1) and Block 2 (PLDz2) of Chromosome Table 5). The indica region introgressed in Chromosome 3 1, respectively, and callose synthase in Chromosome 1 (Block contained 354 genes (Supplementary Table 5). Additionally, 28 2) and Chromosome 3 (Figure 4). Phospholipase D has indica genes distributed among other rice chromosomes, except been shown to regulate callose deposition in response to salt Chromosome 9, were identified (Supplementary Table 5). stress (Hunter et al., 2019). There is then the possibility Singular enrichment analysis (SEA) of molecular functions that indica genes in different introgressed genomic regions using AgriGO (Tian et al., 2017) revealed enriched GO terms might function in concerted action for salinity tolerance at the in indica regions introgressed into the IL22 genome. Genes in seedling stage in IL22 plants. They include genes related to the category of “Protein kinase activity” and “Iron ion binding” hormone signaling, such as ABA, ethylene, or brassinosteroid were overrepresented in Chromosome 1 (Block 1 and Block 2, signaling. In this respect, sophisticated crosstalk is known respectively). Genes in the “Iron ion binding” category included to occur among the different hormones during adaptation peroxidase, Respiratory Burst Oxidase Homolog B (RBOHB), to salt stress in plants, including rice, with cooperative or cytochrome P450, ferredoxin oxygenase, as well as protein antagonistic relationships between them (Yu et al., 2020). In phosphatase genes (Supplementary Figure 8 and Supplementary particular, a number of studies revealed cross-talk between Table 5). Regarding the indica region in Chromosome 3, brassinosteroids and ABA in Arabidopsis and rice (Hu and Yu, this region was enriched in genes in the category of “Anion 2014; Dong et al., 2020). transmembrane transporter activity” (Supplementary Figure 8 As for TF genes, previous studies investigated expression and Supplementary Table 5). We also noticed that many genes dynamics of genes encoding TFs that localize within the Saltol Frontiers in Plant Science | www.frontiersin.org 8 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 3 | Transcriptome-based genotyping of the introgression line IL22. Scatter plots showing the number of transcript variants for each IL22 transcript with respect to the natural variation of OLESA transcripts (both compared to japonica reference genome). Transcripts identified as indica (red dots) are those showing 10 or more variants in IL22 than in OLESA. Transcripts showing less than 10 transcript variants in IL22 compared to OLESA were considered japonica. Each chromosome (1–12) show their scatter plot and transcripts are sorted by chromosomal position, indicated in the x-axis. QTL (named as SalTFs) in contrasting genotypes of rice under regulated in contrasting genotypes, these TFs also showing control and salt stress conditions (Nutan et al., 2017). In that unique features in relation to their response to salt stress and study, the authors demonstrated that SalTFs are differentially during plant growth and development. Frontiers in Plant Science | www.frontiersin.org 9 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 4 | Scheme of indica introgressions identified in Chromosome 1 (A, Block 1 and Block 2) and Chromosome 3 (B) of IL22 plants. Black regions denote clusters of genes (the number of genes in each cluster is shown in parenthesis). 2OG-Fe(II) (2-oxoglutarate Iron 2), ABC (ATP Binding Cassette transporters), AP2/EREBP (APETALA2/ethylene-responsive element binding protein TF), BAK [Brassinosteroid Insensitive 1 (BRI1)-associated receptor kinase], BBI (Bowman–Birk proteinase Inhibitor), bHLH (Basic helix–loop–helix TF), BIP [Brassinosteroid receptor kinase (BRI1)-interacting protein], BSK1 (BR-signaling kinase 1), Cam (Calmodulin), CATC (Catalase C), CCC2 (Cation-Chloride cotransporter 2), CDPK7 (Calcium-dependent protein kinase 7), CIPK (Calcineurin B-like interacting protein kinase), COIN (Cold inducible), DET1 (De-Etiolated 1), DREB2A (Dehydration responsive element binding 2A), DSM2 (drought-sensitive mutant 2), ERF (Ethylene Responsive Factor TF), GST (Glutathione-S-transferase), HAK (High-affinity Potassium Transporter), HKT (High-affinity Potassium Transporter), HSP (Heat Shock Protein), JRL (jacalin-related mannose-binding lectin), LEA (Late Embryogenesis Abundant protein), Lsi2 (Low Silicon 2), LTI6A (Low temperature-induced 6A), MCO (Multicopper Oxidase), MKK6 [mitogen activated protein kinase (MAPK) kinase 6], MRS2-5 (Mitochondrial RNA Splicing, Mg2+ transporter 2-5), MTP9 (Metal Tolerance Protein 9), NAC (NAM/ATAF/CUC TF), PDR6 (Pleiotropic Drug Resistance 6), PEX11-1 (Peroxin 11-1), PLD (Phospholipase D), PP2C (Protein Phosphatase 2C), Rab (Ras-related in brain), RBOHB (Respiratory burst oxidase homolog B), RLK (Receptor-Like Kinase), SIET4 (Silicon efflux transporter 4), SIP1 (Small and basic intrinsic protein 1), SKCD1 (Suppressor of K+ transport growth defect-like protein 1), TIP1;1 (Tonoplast intrinsic protein 1;1), TRX (Thioredoxin), UGPases (UDP-glucose pyrophosphorylases), VDAC (voltage-dependent anion cannel), and WAK (Wall-Associated kinase). Frontiers in Plant Science | www.frontiersin.org 10 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines Genes identified in indica regions of IL22 were arbitrarily was compared with that of the corresponding genes in the selected and their expression analyzed by RT-qPCR analysis. salt-sensitive japonica parent OLESA. For calling DEGs, a They included genes encoding proteins involved in salt stress log2 fold change (FC) >0.5 or <−0.5, and a P-value ≤ 0.05 tolerance (i.e., TF DREB2A, K+ efflux 5 transporter, tonoplast were applied. In this way, 770 and 830 genes were found to intrinsic protein TIP1;1, and Calcineurin B-Like Protein- be up-regulated and down-regulated, respectively, in IL22 Interacting Protein Kinase 9-CIPK9) (Liu et al., 1994; Dubouzet compared with the salt-sensitive parent OLESA (Figure 6A et al., 2003; Li et al., 2008; Mallikarjuna et al., 2011; Zhu et al., and Supplementary Table 7). GO enrichment in biological 2018; Kurowska, 2021; Shabala et al., 2021). RT-qPCR analysis processes was performed using AgriGO in up-regulated and confirmed that indica genes are expressed in leaves of IL22 plants, down-regulated genes in IL22 plants, and visualized through the and also revealed differences in the expression level of these genes REVIGO tool (Supek et al., 2011). The GO category “Response between IL22 and its salt-tolerant indica parent FL478 when to Abiotic Stimulus” was specifically enriched in up-regulated grown under control conditions (Figure 5, gray bars). Thus, genes of IL22, though no salt treatment was applied to these under control conditions, some of these genes were found to plants (Figure 6B, upper panel and Supplementary Table 7). be expressed at either a higher level, or a lower level, in IL22 Other GO categories highly represented in up-regulated genes in plants compared with the salt tolerant parent FL478 (Figure 5, IL22 plants were those related to “Transport,” “Transcription,” gray bars). Knowing this, we considered the possibility that “Small GTPase-mediated signal transduction,” and “Regulation the expression of salt-associated genes might have a differential of Ras protein signal transduction” (Figure 6B, upper panel regulation in IL22 plants (e.g., stronger induction) under salt and Supplementary Table 7). Genes down-regulated in IL22 stress conditions, as opposed to being constitutively up-regulated plants grouped into diverse categories with “Translation” being as in FL478. Supporting this possibility, salt-associated genes the dominant term in this set of genes (Figure 6B, lower panel exhibited stronger induction in response to salt treatment in and Supplementary Table 7). Other genes down-regulated IL22 plants than in FL478 plants (e.g., salt-treated vs non- in IL22 plants categorized in “Gene expression,” “Protein treated conditions) (Figure 5, black bars). Some examples deubiquitination,” “Transport,” and “Metabolic Processes” are: OsDREB2A (an important regulatory TF controlling salt (Figure 6B, lower panel and Supplementary Table 7). Together, stress responses), Cation efflux protein OsMTP9, aquaporin these results demonstrated that, in the absence of salt stress, the OsTIP1;1, glutathione-S-transferase OsGSTF1, Catalase CAT- indica introgressions had a high impact on the global japonica C, Brassinosteroid receptor kinase (BRI1)-interacting protein leaf transcriptome of IL22 plants. OsBIP128, Calcineurin B-like interacting protein kinase OsCIPK9, Using the same criteria described above (log2 FC >0.5 or Ras-related in brain 18B2 RAB18B2, Phospholipase PDLalpha1 <−0.5, P-value ≤ 0.05), we investigated differences in the OsPLDalpha1, and raffinose synthase 5 RS5. There were also leaf transcriptome of IL22 and OLESA plants under moderate examples of genes that were induced by salt treatment in IL22, but salt stress conditions (80 mM NaCl, 24 h; see Supplementary not in FL478 (K+ efflux transporter 5, Bowman–Birk proteinase Figure 7). Pair-wise comparisons of the leaf transcriptional inhibitor 2-2 RBBI2-2), as well as genes showing an opposite response to salt treatment with their respective controls revealed regulation by salt stress in IL22 and FL478 plants [Bowman– 1,749 and 2,021 genes as differentially expressed in IL22 and Birk proteinase inhibitor 3-1 RBBI3-1, Brassinosteroid Insensitive OLESA plants, respectively (Figure 7A and Supplementary 1 (BRI1)-associated receptor kinase 1 OsBAK1, Glucan Synthase- Table 8). Notably, a relatively low number of genes were found Like 10 GSL10 also known as callose synthase 10]. Differences to be commonly regulated in the two genotypes (171 and 129 observed in the expression of indica genes among IL22 and FL478 genes, up- and down-regulated, respectively) suggesting that suggest that regulation of salt stress responses might be distinctly there is a genotype-specific response to salt treatment in an different in IL22 relative to FL478 plants. important number of genes (Figure 7A). GO terms in Biological Collectively, these results suggested that indica regions Processes specifically enriched in the set of up-regulated genes introgressed in IL22 were enriched in salt-associated genes with in IL22 plants were: “Cell redox homeostasis,” “Transmembrane different functions and that these genes, very likely, contribute to transport,” “Transport” (Lipid and Ion transport), Metabolic salinity tolerance in IL22 plants at the seedling stage. These genes Processes (“Cellular catabolic processes,” “Nucleotide metabolic might function either alone or in combination with other indica process,” “ATP biosynthetic process,” “Hexose metabolic and japonica genes. process,” and “Cofactor biosynthetic process”), and “Translation initiation” (Figure 7B and Supplementary Figure 9). Biological Process terms specifically enriched in up-regulated genes in Comparative Transcript Profiling of the OLESA plants were “Response to water,” “Vesicle-mediated Salt Tolerant Line IL22 and Its transport,” “Protein folding,” “Cellular aromatic compound Salt-Sensitive Parent metabolic process,” and “Transcription initiation” (Figure 7B To obtain further insights into the molecular mechanisms and Supplementary Figure 10). Finally, genes that were underlying salinity tolerance in IL22, we compared the leaf down-regulated by salt stress classified into a broad spectrum transcriptome of IL22 and OLESA plants grown under control of Biological Processes in both genotypes (IL22 and OLESA) conditions in both cases. We focused on transcriptional (Supplementary Figures 11, 12). Gene expression correlation alterations in loci not included in the introgression intervals between RNA-Seq and RT-qPCR analyses in IL22 and OLESA in IL22 plants (hereafter japonica genes), whose expression plants could be observed for selected genes (Figure 7C). Frontiers in Plant Science | www.frontiersin.org 11 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 5 | Salt-responsiveness of genes identified in indica regions introgressed in IL22. Plants were grown in hydroponic cultures under control and salt-conditions (24 h of salt treatment, 80 mM NaCl). Transcript levels of the indicated genes were determined by RT-qPCR. OsUbi1 was used as the reference gene. Bars represent means of three biological replicates, each one from a pool of four different plants, ±SEM (Student’s t-test, *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005; salt-treated vs un-treated plants). DREB2A (Dehydration responsive element binding 2A), OsMTP9 (Metal Tolerance Protein 9, manganese transporter), K+ efflux 5 (K+ antiporter efflux 5), OsTIP1;1 (Tonoplast intrinsic protein 1-1), RBBI2-2 (Bowman–Birk proteinase Inhibitor 2-2), RBBI3-1 (Bowman–Birk proteinase Inhibitor 3-1), OsGSTF1 (Glutathione-S-transferase F1), CAT-C (Catalase C), OsBAK1 [Brassinosteroid Insensitive 1 (BRI1)-associated receptor kinase 1], OsBIP128 [Brassinosteroid receptor kinase (BRI1)-interacting protein 128], OsCIPK9 (Calcineurin B-like interacting protein kinase 9), OsRab18B2 (Ras-related in brain 18 B2), OsGSL10 (Glucan Synthase-Like 10, callose synthase 10), OsPLDalpha1 (Phospholipase D alpha 1), RS5 (Raffinose synthase 5), OsABCC13 (ATP Binding Cassette transporter B 13). Primers and locus ID are listed in Supplementary Table 2. For better understanding of salt-responsive mechanisms in software. This analysis showed that the salt stress response IL22 plants, all salt-responsive genes of IL22 and OLESA mechanisms were complex and involved multiple mechanism plants were visualized using the MapMan (Version 3.6.0RC1) and signal transduction pathways while confirming as a Frontiers in Plant Science | www.frontiersin.org 12 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines differential regulation of salt responses in genes in multiple processes. For example, multiple genes related to hormone signaling, transcriptional regulation, stress responses or ion transport, among others, were differentially regulated in IL22 and OLESA plants (Supplementary Figure 13 and Supplementary Table 8). This differential regulation of salt stress responses might well be the consequence of interactions between introgressed indica genes and genes in the recipient genome of IL22. Additional studies are, however, needed to understand gene regulatory networks operating in IL22 plants. Collectively, comparative transcriptome analysis of IL22 and OLESA plants revealed that introgression of indica genes in IL22 is accompanied by a wide-range of alterations in gene expression under non-stress and salt stress conditions. The expression of genes involved in ion/water transport, redox homeostasis, protein phosphorylation/dephosphorylation processes, lipid transport, transcriptional regulation, hormone signaling, as well as genes involved in diverse metabolic processes showed differential regulation in IL22 plants relative to its salt- sensitive parent OLESA. DISCUSSION In this work, MABC breeding and step-wise phenotypic selection in hydroponic culture was used for the generation of salt tolerant ILs derived from crosses between the salt tolerant indica FL478 and the salt sensitive japonica OLESA. Based on phenotypic analyses (fresh weight, dry weight, shoot length, and root length) and salt injury symptoms (SES score), two ILs (IL22 and IL13) were identified as the best performing lines under salt stress conditions. Compared with the recurrent parent OLESA, tolerance to salt stress in IL22 and IL13 plants correlated with reduced leaf EL, and indicator of reduced membrane injury, and lower Na+ /K+ ratios in shoot tissues, which is also a hallmark of salinity tolerance in rice plants (Gregorio and Senadhira, 1993). Presumably, the introgression of favorable genes from the donor FL478 would cause a reduction of Na+ /K+ ratios in leaves, which would alleviate the cytotoxic effects of Na+ during salt stress. An important phenotypic variation was observed among inbred lines that are homozygous for Saltol, which can be explained by the different patterns of indica introgressions (as revealed by KASPar and GBS genotyping). Variation in salinity tolerance among ILs is consistent with the complexity of the FIGURE 6 | Differentially expressed genes in leaves of IL22 plants relative to genetic control of salinity tolerance in plants, a process in which OLESA plants. Plants grown in hydroponic culture under control conditions as a plethora of genes distributed through the rice genome are indicated in Figure 2. Leaves of 2-week-old plants were used for RNA-Seq expected to be implicated. It is worth mentioning that, despite analysis. (A) Number of DEGs (black, up-regulated genes; gray, the Saltol QTL has been extensively employed for improvement down-regulated genes). (B) Biological processes altered in IL22 plants relative to OLESA plants. GO terms enriched in up- and down-regulated genes in IL22 of salinity tolerance in rice breeding, the specific loci in Saltol plants were visualized by REVIGO (Supek et al., 2011). The most enriched GO responsible for salinity tolerance is still a matter of debate terms in the set of up-regulated and down-regulated genes are presented (Walia et al., 2005; Cotsaftis et al., 2011; Li et al., 2018; López- (upper and lower panels, respectively). Circles represent GO terms and those Cristoffanini et al., 2020). So far, only OsHKT1;5 located in the clustered closer to each other indicate similar GO terms. Color scale (yellow to Saltol QTL has been shown to play a role in conferring salinity red) represents the degree of GO enrichment (log10 P-value). Larger and smaller disc size represent more general and more specific terms, respectively. tolerance at the seedling stage (Kobayashi et al., 2017). In other studies, however, no significant differences in salinity tolerance Frontiers in Plant Science | www.frontiersin.org 13 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 7 | Comparison of OLESA and IL22 transcriptomes in response to salt stress (80 mM NaCl, 24 h of treatment). (A) Venn diagrams indicate the number of genes that are specifically and commonly regulated by salt stress in each genotype, up-regulated and down-regulated genes (log2 fold change >0.5 or <–0.5; P-value ≤ 0.05). (B) Enriched GO terms in biological processes in the up-regulated genes in each genotype performed using AgriGO (Tian et al., 2017). Light and dark blue bars indicate common and genotype-specific GO terms, respectively. The scale shows the statistical significance (–log10 P-value). (C) Salt-responsiveness of genes identified in japonica regions of IL22 and OLESA. Plants were grown in hydroponic cultures under control and salt-conditions (24 h of salt treatment, 80 mM NaCl). Transcript levels of the indicated genes were determined by RT-qPCR. OsUbi1 was used as the reference gene. Bars represent means of three biological replicates, each one from a pool of four different plants, ±SEM (Student’s t-test, *P ≤ 0.05, **P ≤ 0.005). OsLEA23 (Late Embryogenesis Abundant protein 23), OsAPX2 (Ascorbate peroxidase 2), OsNAC6 (NAM/ATAF/CUC 6 TF), OsRab7b3 (Ras-related in brain 7 B3); OsLTP1.2 (Lipid transfer protein 1.2), and OsOCPI2 (Oryza sativa chymotrypsin inhibitor-like 2). Primers and locus ID are listed in Supplementary Table 2. Frontiers in Plant Science | www.frontiersin.org 14 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines could be observed between Saltol-containing rice lines and non- causes alterations in membrane properties (Hunter et al., 2019; Saltol-containing backcross lines (Alam et al., 2011; De Leon Figure 8). In line with this, Phospholipase D (PLD) genes were et al., 2017; Han et al., 2020). identified in the introgressed indica regions in IL22, these genes Results presented on the fine mapping and transcriptome potentially contributing to salinity tolerance in these plants. analysis of IL22 demonstrated that introgression of large indica The plant response to salt stress also involves cell wall segments from FL478 in Chromosomes 1 and 3 of the japonica modification which can benefit the plant by preventing water cultivar OLESA has important implications in salinity tolerance. loss and modulating ion transport pathways. Along with this, A variety of genes with a function in salinity tolerance were the introgressed indica regions in IL22 contained many cell wall- identified in the introgressed indica regions (e.g., genes involved related genes (see Supplementary Table 6). The set of indica in signaling, ROS detoxification, ion transport, etc.). This genes in IL22 also included the two key genes responsible of information will help in the identification of novel genotype- callose biosynthesis, namely UDP-glucose pyrophosphorylase transcriptome-phenotype correlations in tolerance to salt stress and callose synthase. Callose deposition at plasmodesmata is in rice. Since the plant response to salt stress is physiologically known to occur in salt-stressed Arabidopsis plants for the and genetically complex, genes in the Saltol genomic region, and regulation of plasmodesmal permeability (Hunter et al., 2019). As genes located outside the Saltol region but interacting with genes it was observed for genes related to BRs signaling, genes involved in Saltol, are expected to be implicated in salinity tolerance in rice in callose biosynthesis are located in independent introgression (Thomson et al., 2010; Nutan et al., 2017). regions. This fact points to the possibility of interactions between indica genes in conferring salinity tolerance in IL22 plants. An overview of genes identified in the indica segments in IL22, Fine Mapping of the Genomic and the regulatory networks in which these genes participate Introgressions in the Salt-Tolerant Line in the plant response to salt stress, is presented in Figure 8. IL22 Based on the information available on mechanisms involved in Transcriptome genotyping allowed us to know the exact number salinity tolerance in plants, it can be hypothesized that salinity and identity of indica genes introgressed in IL22 plants. This perception would trigger an increase in intracellular Ca2+ in IL22 study also allowed us to characterize the behavior of introgressed plants that would then be decoded by Ca2+ sensors and Ca2+ – loci in their new japonica genomic background, thus, supporting dependent protein kinases for the activation of downstream the usefulness of RNA-based genotyping for the analysis of protein phosphorylation/dephosphorylation cascades (e.g., introgression rice lines. Loci contained in introgressed segments genes encoding CaM1-3, CIPKs, CDPK7, MKK6, and PP2C) at Chromosomes 1 and 3 included genes related to: (i) ion (Na+ , (Figure 8). Ca2+ is also a key regulator for PLD activity, K+ ) and water transport (aquaporins), and plasma membrane- and the PLDalpha1 is known to be involved in membrane or tonoplast-localized H+ -ATPses; (ii) production of H2 O2 lipid modification for the regulation of plasma membrane and ROS detoxification; (iii) hormone signaling (e.g., ABA and function. Perception of salt stress would also trigger ROS and brassinosteroid pathways); (iv) signal transduction, such production through the activity of the plasma membrane- as protein kinases (CDPK7, CIPKs) and protein phosphatase localized RBOHB in IL22 plants (Figure 8). In turn, IL22 plants PP2C; (v) transcriptional regulation (e.g., DREB2A); and (vi) salt- would activate the expression of enzymatic antioxidant systems responsive genes [e.g., LEAs (OsLEA9) and Heat Shock proteins to protect themselves from excessive levels of ROS (e.g., CAT, (HSPs)]. These genes are expected to contribute to the phenotype PRX, GST, and TRX genes) (Figure 8). Furthermore, CDPKs of salinity tolerance in IL22 plants. For instance, indica regions have been shown to regulate the production of ROS in plants in IL22 contained an important number of ion transporter genes (Kobayashi et al., 2007). Presumably, ion transporters (Na+ and with a function in salt stress tolerance, such as Na+ , K+ , and K+ transporters) and water transporters identified in indica Cl− transporters (Van Zelm et al., 2020). The introgressed indica regions of IL22 plants that are known to localize at the plasma regions in IL22 also contained phosphate transporters and metal membrane, tonoplast, mitochondria or chloroplast, would help transporters, but the functional relevance of these transporters in in maintaining appropriate cellular Na+ concentrations in IL22 salinity tolerance remains to be investigated. plants (e.g., KHT1;5, HKT3, HAK3, Aquaporins, H+ -ATPase Moreover, indica regions in IL22 included genes involved in and VDACs, among others) (Figure 8). Moreover, UGPase and the apoplastic production of H2 O2 (NADPH oxidase) and ROS callose synthase which are key genes in callose deposition at homeostasis, and regulatory genes in brassinosteroids (BRs) and plasmodesmata were identified in indica regions, and expressed, ABA signaling (Figure 8). In Arabidopsis, it has been shown that in IL22 plants (Figure 8). Then, a regulation of the symplastic H2 O2 mediates the crosstalk of BRs and ABA pathways (Zhou transport of ions, and other molecules, can be expected which et al., 2014; Tian et al., 2018). Equally, a role of BRs in salinity might confer good adaptability to salt stress in IL22. tolerance and connections between BRs signaling, ABA signaling, Importantly, a large number of TFs were identified in and ROS homeostasis have been described in rice (Sharma et al., indica introgressed regions in IL22, of which OsDREB2A might 2013, 2017; Gui et al., 2016; Kaur et al., 2016). have important implications for salinity tolerance. Constitutive Membrane remodeling has been documented in plants expression of OsDREB2A in rice was reported to confer salt exposed to salinity (Guo et al., 2019). In Arabidopsis, it is known stress tolerance (Mallikarjuna et al., 2011). ONAC022 (a NAC that the salt-induced increase in cytoplasmic Ca2+ concentration family member) and OsCOIN (a member of the zinc finger activates PLDα1 for phosphatidic acid production which, in turn, TF family) were also identified among indica genes in IL22 Frontiers in Plant Science | www.frontiersin.org 15 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines FIGURE 8 | Summary of the salt-related genes located in the indica introgressed regions of IL22. Thick black arrows indicate entrance or exit of molecules. Thin black arrows indicate steps in a process or chemical reaction. Gray arrows indicate interactions or regulations between components. ABC (ATP Binding Cassette transporters), AP2/EREBP (APETALA2/ethylene-responsive element binding protein TF), BAK1 (Brassinosteroid Insensitive 1 (BRI1)-associated receptor kinase1), BBI (Bowman–Birk proteinase Inhibitor), bHLH (Basic helix–loop–helix TF), BIP [Brassinosteroid receptor kinase (BRI1)-interacting protein], BSK1 (BR-signaling kinase 1), CaM1-3 (Calmodulin 1-3), CATC (Catalase C), CCC2 (Cation-Chloride cotransporter 2), CDPK7 (Calcium-dependent protein kinase 7), CIPK (Calcineurin B-like interacting protein kinase), DET1 (De-Etiolated 1), DREB2A (Dehydration responsive element binding 2A), GST (Glutathione-S-transferase), HAK3 [High-affinity Potassium(K+ ) Transporter 3], HKT (High-affinity Potassium Transporter), HSP (Heat Shock Protein), LEA (Late Embryogenesis Abundant protein), Lsi2 (Low Silicon 2), MKK6 [mitogen activated protein kinase (MAPK) kinase 6], MTP9 (Metal Tolerance Protein 9), NAC (NAM/ATAF/CUC TF), PLDalpla1 (Phospholipase D alpha 1), PP2C (Protein Phosphatase 2C), PRX (Peroxidase), Rab GTPase (Ras-related in brain GTPase), RBOHB (Respiratory burst oxidase homolog B), SIP1 (Small and basic intrinsic protein 1), TIP1;1 (Tonoplast intrinsic protein 1;1), TRX (Thioredoxin), UGPAse (UDP-glucose pyrophosphorylase), and VDAC (voltage-dependent anion cannel). Scheme created with BioRender.com. (Supplementary Table 5). The overexpression of these TFs has and salt stress conditions. These observations point to an effect been shown to improve salt tolerance in rice (Liu et al., 2007; of japonica background genes on the expression of introgressed Hong et al., 2016). Moreover, ONAC022-overexpressing rice indica loci. The basal expression of salt-associated indica genes plants exhibited up-regulation of OsDREB2 and OsbZIP23 (Hong in IL22 plants in the absence of salt stress may help IL22 et al., 2016). As for OsbHLH035 (also identified among indica plants to cope more effectively with subsequent salt stress. genes in IL22), it was reported that this TF mediates seedling Furthermore, the observed superinduction of indica genes in recovery after salt stress (Chen et al., 2018). Moreover, the Saltol IL22 plants suggest that these plants might be in a “priming QTL-localized TF OsGATA8 was reported to be essential for status,” a mechanism that plants use to mount effective defense salinity tolerance in rice seedling (Nutan et al., 2020). responses to pathogen attack (Martinez-Medina et al., 2016). Transcriptome analyses confirmed that indica genes in IL22 Priming of salt-associated genes would help the plants to cope are expressed in the new genomic background. Different with a situation of salt stress. Likely, the expression of indica expression patterns of indica genes were, however, found between genes identified in introgression segments, either individually IL22 and its salt tolerant parent FL478, both under non-stress or in combination with other indica or japonica genes, would Frontiers in Plant Science | www.frontiersin.org 16 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines contribute to improve salinity tolerance in IL22 plants. On this Phytohormones, besides controlling plant growth and point, an interesting finding of our study was the observation development under normal conditions, also mediate plant that two NAC6 genes, OsNAC6 and SNAC1 (similar to OsNAC6) responses to salt stress (Yu et al., 2020). In this way, plants were induced by salt treatment in both IL22 and OLESA, but develop adaptive responses to salt stress by orchestrating the their expression reached a higher level in salt-treated IL22 than synthesis, signaling and metabolism of various hormones with in salt-treated OLESA plants (OsNAC6, FC = 2.20 and 1.52 in multiple levels of crosstalk. ABA is one of the most important IL22 and OLESA, respectively; SNAC1, FC 2.56 and 2.19 in hormones in salt stress response that integrates numerous IL22 and OLESA, respectively) (Supplementary Tables 8A,B). processes to cope with Na-induced osmotic stress and adaptive Furthermore, OsZIP23 was among the set of salt-responsive genes signaling cascades. Salinity and osmotic stress cause an increase in IL22 (FC = 2.20), but not in the set of salt-responsive OLESA in endogenous ABA levels for the activation of phosphorylation genes. For OsNAC6 and OsZIP23, a positive role in regulating the signaling cascades and ABA-responsive TFs belonging to rice response to salt stress was previously reported (Nakashima different TF families. Accumulating evidence also uncovers et al., 2007; Xiang et al., 2008). the role of brassinosteroids in plant adaptation to salt stress, On the other hand, information available in the literature including genes associated to brassinosteroid synthesis and indicates the presence of retro-transposons and transposons signaling (Yu et al., 2020). Brassinosteroids are perceived by within the Saltol QTL (Soda et al., 2013; Waziri et al., 2016). cell surface-localized receptor kinases, BRI1 and BAK1 and The presence of transposons in this region might well affect trigger a series of phosphorylation/dephosphorylation events the expression of indica genes in the salt tolerant ILs. In for the activation of brassinosteroid-responsive genes through particular, a MITE has been found to localize upstream of the regulation of distinct TFs (BZR1, BES1) (Mao and Li, 2020; OsHKT1;5, and deletion of this MITE using the CRISPR/Cas9 Yu et al., 2020). Besides, brassinosteroid signaling on plasma system results in reduced OsHKT1;5 expression (Wang et al., membrane has been shown to be important in salinity tolerance 2020). Finally, in this study, the RNA-Seq method was not in rice (Dong et al., 2020). Thus, the presence of genes involved used for the fine mapping of other salt-tolerant ILs. Further in brassinosteroid signaling in indica regions introgressed in research is, however, needed to identify common and specific IL22 (e.g., BAK1, BIP, and BSK) might well contribute to salinity gene expression patterns among the various ILs and to correlate tolerance in these plants (Figure 8). Brassinosteroid signaling genomic and phenotypic data of ILs. also regulate salt tolerance by interacting with other hormone signaling pathways, such as ABA or ethylene pathways (Zhang et al., 2009; Divi et al., 2010; Li et al., 2012). Effect of Indica Introgressions on the Salt-Responsive Transcriptome of IL22 Plants CONCLUSION Currently, limited efforts have been performed to identify Saltol QTL x background interactions contributing to salinity tolerance. To conclude, the results of this study indicated important For instance, it is still unclear which are the effects of the transcriptional reprogramming caused by indica introgressions Saltol introgression into the transcriptome of a recipient genome, in IL22 plants which might well underlie the phenotype of or which genes within each genetic background (donor and salinity tolerance at the seedling stage in IL22 plants. It will be recipient genomes) should be combined to effectively increase now of interest to use the transcriptome approach for the fine salinity tolerance. Results here presented revealed differences mapping and expression analysis of additional salt tolerant ILs in the transcriptome of IL22 plants beyond the expression of generated in this study. This will help in deciphering precise loci in the introgressed indica segments, not only under control functional interactions between indica and japonica genes in ILs conditions, but also under moderate salt stress conditions in contributing to salinity tolerance in rice. A better understanding comparison to the salt-sensitive parent OLESA. We showed of these mechanisms will be the basis for the development of new that a small number of genes are commonly regulated (up- salt-tolerant rice varieties. regulated or down-regulated) by salt stress in the IL22 and OLESA plants, supporting a genotype-specific salt-responsive expression pattern in IL22 plants caused by indica introgressions. DATA AVAILABILITY STATEMENT For instance, the set of salt-responsive genes in IL22, but not in OLESA, was enriched in genes involved in cell redox homeostasis The datasets presented in this study can be found in online and transport (i.e., transmembrane, ion, and lipid transport). repositories. The names of the repository/repositories and Differences in the salt-responsive transcriptome of IL22 relative accession number(s) can be found below: Gene Expression to OLESA also suggest that the expression of genes in the Omnibus, GSE167342. indica introgressed regions of IL22 has an important impact on the landscape of japonica genes from the recipient parent. Clearly, interactions between indica and japonica genes might AUTHOR CONTRIBUTIONS well be responsible for the creation of novel expression profiles in IL22 plants and/or the modification of signaling pathways and BS and MB conceived the research plans and wrote the regulatory networks involved in salinity tolerance. manuscript. MB performed the most experiments, genotyping Frontiers in Plant Science | www.frontiersin.org 17 January 2022 | Volume 12 | Article 797141 Bundó et al. Transcriptomics of Rice Introgressed Lines (PCR, KASPar), phenotyping for salinity tolerance, RNA- Programme/Generalitat de Catalunya”. We acknowledge support Seq analysis, and analyzed the data. XS and SN performed of the publication fee by the CSIC Open Access Publication FL478 × OLESA crosses. JG-A, HM-C, and LC contributed with Support Initiative through its Unit of Information Resources for hydroponic experiments. HM-C contributed to the validation of Research (URICI). RNA-Seq data by RT-qPCR. JF, BC, and CG performed the GBS and analyzed the data. MP and GS performed Na+ /K+ analyses and analyzed the data. All authors supervised and complemented ACKNOWLEDGMENTS the writing. BS agreed to serve as the author responsible for contact and ensures communication. We thank M. Bernardo-Faura and V. M. González (CRAG, Bioinformatics Unit) for support in RNA-Seq data analysis. FUNDING We also thank Càmara Arrossera del Montsià SCCL (Amposta, Spain) for providing the salt-sensitive elite This project has received funding from the European Union’s japonica variety OLESA. Horizon 2020 Research and Innovation Programme under grant agreement No. 678168; NEURICE. MB, HM-C, JG-A, LC, and XS were funded by H2020-SFS-2015-2 – NEURICE – 678168. SUPPLEMENTARY MATERIAL We acknowledge financial support from the Spanish Ministry of Science and Innovation-State Research Agency (AEI), through The Supplementary Material for this article can be found the “Severo Ochoa Programme for Centres of Excellence in online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. R&D” SEV-2015-0533 and CEX2019-000902-S, and the “CERCA 797141/full#supplementary-material REFERENCES De Leon, T. B., Linscombe, S., and Subudhi, P. K. (2017). Identification and validation of QTLs for seedling salinity tolerance in introgression lines of a salt Afzal, Z., Howton, T. C., Sun, Y., and Mukhtar, M. S. (2016). The roles of tolerant rice landrace “Pokkali”. PLoS One 12:e0175361. doi: 10.1371/journal. aquaporins in plant stress responses. J. Dev. 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Bioeng. 3, 1067–1072. and do not necessarily represent those of their affiliated organizations, or those of Xiang, Y., Tang, N., Du, H., Ye, H., and Xiong, L. (2008). Characterization the publisher, the editors and the reviewers. Any product that may be evaluated in of OsbZIP23 as a key player of the basic leucine zipper transcription this article, or claim that may be made by its manufacturer, is not guaranteed or factor family for conferring abscisic acid sensitivity and salinity and drought endorsed by the publisher. tolerance in rice. Plant Physiol. 148, 1938–1952. doi: 10.1104/pp.108.12 8199 Copyright © 2022 Bundó, Martín-Cardoso, Pesenti, Gómez-Ariza, Castillo, Frouin, Yadav, A. K., Kumar, A., Grover, N., Ellur, R. K., Krishnan, S. G., Bollinedi, H., et al. Serrat, Nogués, Courtois, Grenier, Sacchi and San Segundo. This is an open-access (2020). Marker aided introgression of ‘Saltol’, a major QTL for seedling stage article distributed under the terms of the Creative Commons Attribution License salinity tolerance into an elite Basmati rice variety ‘Pusa Basmati 1509’. Sci. Rep. (CC BY). The use, distribution or reproduction in other forums is permitted, provided 10:13877. doi: 10.1038/s41598-020-70664-0 the original author(s) and the copyright owner(s) are credited and that the original Yang, Y., and Guo, Y. (2018). Unraveling salt stress signaling in plants. J. Integr. publication in this journal is cited, in accordance with accepted academic practice. No Plant Biol. 60, 796–804. doi: 10.1111/jipb.12689 use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 20 January 2022 | Volume 12 | Article 797141