CSIRO PUBLISHING Functional Plant Biology https://doi.org/10.1071/FP18295

Comparative transcriptome analysis reveals unique genetic adaptations conferring salt tolerance in a xerohalophyte

Wei-Wei Chai A, Wen-Ying Wang A, Qing MaA, Hong-Ju Yin A, Shelley R. Hepworth A,B and Suo-Min Wang A,C

AState Key Laboratory of Grassland Agro-ecosystems; Key Laboratory of Grassland Livestock Industry Innovation, Ministry of Agriculture and Rural Affairs; College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou, 730020, PR China. BDepartment of Biology, Institute of Biochemistry, Carleton University, Ottawa, ON, Canada. CCorresponding author. Email: [email protected]

Abstract. Most studies on salt tolerance in plants have been conducted using glycophytes like Arabidopsis thaliana (L.) Heynh., with limited resistance to salinity. The xerohalophyte Zygophyllum xanthoxylum (Bunge) Engl. is a salt- accumulating desert plant that efficiently transports Na+ into vacuoles to manage salt and exhibits increased growth under salinity conditions, suggesting a unique transcriptional response compared with glycophytes. We used transcriptome profiling by RNA-seq to compare gene expression in roots of Z. xanthoxylum and A. thaliana under 50 mM NaCl treatments. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis suggested that 50 mM NaCl was perceived as a stimulus for Z. xanthoxylum whereas a stress for A. thaliana. Exposure to 50 mM NaCl caused metabolic shifts towards gluconeogenesis to stimulate growth of Z. xanthoxylum, but triggered defensive systems in A. thaliana. Compared with A. thaliana, a vast array of ion transporter genes was induced in Z. xanthoxylum, revealing an active strategy to uptake Na+ and nutrients from the environment. An ascorbate-glutathione scavenging system for reactive oxygen species was also crucial in Z. xanthoxylum, based on high expression of key enzyme genes. Finally, key regulatory genes for the biosynthesis pathways of abscisic acid and gibberellin showed distinct expression patterns between the two species and auxin response genes were more active in Z. xanthoxylum compared with A. thaliana. Our results provide an important framework for understanding unique patterns of gene expression conferring salt resistance in Z. xanthoxylum.

Additional keywords: glycophyte, RNA-seq, salt response, stimulus, xerohalophyte, Zygophyllum xanthoxylum.

Received 9 November 2018, accepted 11 March 2019, published online 8 May 2019

Introduction ions, often coupled with specialised morphologies, such as Soil salinity is a major abiotic stress that severely constrains plant succulent leaves for storing excess salt ion. growth and productivity (Zhu 2001; Munns and Tester 2008). Zygophyllum xanthoxylum (Bunge) Engl. is a succulent A high level of Na+ is toxic to plants because it disturbs xerohalophyte, native to desert areas of China and Mongolia. cytoplasmic Na+/K+ homeostasis and leads to secondary This species has the great capacity to absorb Na+ into succulent osmotic and oxidative stresses (Taji et al. 2004). Fortunately, leaves for osmotic adjustment, exhibiting improved growth and plants have evolved various mechanisms to combat salinity, excellent photosynthetic capacity in salty soils (Ma et al. 2012; including the regulation of ion homeostasis (Zhu 2003), Yue et al. 2012). Additionally, Na+ ions promote the uptake of reactive oxygen species (ROS) scavenging systems (Das and important nutrients in Z. xanthoxylum, such as N, P, Fe, Si, Ca2+ Roychoudhury 2014) and hormone responses (Bari and Jones and Mg2+, thereby stimulating growth (Wang et al. 2019). Salt 2009). To date, much effort has aimed at deciphering molecular exposure also increases enzyme activities important for ROS mechanisms of plant salt tolerance and identifying the relevant scavenging in Z. xanthoxylum (Cai et al. 2011). Further, several genes. However, most of these studies have focussed on studies have been performed to elucidate the molecular glycophytes like Arabidopsis thaliana (L.) Heynh. with limited mechanism of salt tolerance in Z. xanthoxylum. Key tolerance to salinity. In recent years, it is experimentally proven functional genes involved in Na+ and K+ homeostasis that halophytes are more appropriate models for studying salt have been explored, such as tonoplast Na+/H+ antiporter tolerance (Shabala 2013; Flowers et al. 2015; Song and Wang ZxNHX (Wu et al. 2011; Yuan et al. 2015), plasma 2015). Halophytes have evolved unique strategies, such as membrane Na+/H+ antiporter ZxSOS1 (Ma et al. 2014), stelar efficient control of uptake and compartmentalisation of salt K+ outward rectifying channel ZxSKOR (Hu et al. 2016),

Journal compilation CSIRO 2019 www.publish.csiro.au/journals/fpb B Functional Plant Biology W. W. Chai et al. inward-rectifying K+ channel ZxAKT1 (Ma et al. 2017). For hydroculture, 3-week-old seedlings grown on 1/2 MS In contrast to A. thaliana, Z. xanthoxylum has superior media plates were transferred to containers containing mechanisms for thriving in salty soils. modified Hoagland nutrient solution (same as Z. xanthoxylum). Collective studies suggest that quantitative changes in gene The opaque vinyl chloride containers (175 Â 95 Â 35 mm) were expression are mainly responsible for salt tolerance or sensitivity covered with an opaque vinyl chloride board (1 mm) that had 36 (Bartels and Dinakar 2013; Volkov 2015; Mishra and Tanna holes (3 mm) at regular intervals to diameter 30-mm shield the 2017). Thus, comparative analysis of gene expression in root zone from sunlight (to make sure roots got plenty of air, Z. xanthoxylum and A. thaliana, is a promising strategy for we chose nine holes to fix seedlings, one seedling per hole). The revealing key differences responsible for salt tolerance. RNA nutritional solution was replaced with fresh solution every 3 days. sequencing (RNA-seq) is an effective tool because it provides Plants were grown in a greenhouse at 22C under long detailed information about a broad spectrum of genes (Halimaa days (200 mmol m–2Ás–1 photon flux density). Five-week-old et al. 2014). A. thaliana seedlings were used for the same treatments as Roots are the primary tissue exposed to soil salinity (Ouyang Z. xanthoxylum. The roots of A. thaliana seedlings were et al. 2007). This pivotal organ has key functions in absorbing collected after 6 and 24 h treatment respectively. water and nutrients, secreting enzymes and organic acids, as well as producing hormones (Zhai et al. 2013). Root growth of Data acquisition glycophytes is typically inhibited by salinity (Zolla et al. Eight samples in total were marked as follows: ZxC6R, ZxC24R, 2010,Huet al. 2015). By contrast, the roots of halophytes can ZxS6R, ZxS24R, AtC6R, AtC24R, AtS6R, AtS24R where Zx maintain normal growth or even improved growth under salinity denotes Z. xanthophyllum and At denotes A. thaliana, conditions (Hu et al. 2012). Therefore, it is attractive to examine respectively; ‘C’ and ‘S’ denote control and salt treatments contrasting gene regulation strategies in roots of halophytes with 50 mM NaCl, respectively, 6 and 24 denote 6 and 24 h versus glycophytes. Here, we compared patterns of transcript of treatment, respectively, and R denotes roots. Transcriptome abundance between salt-treated Z. xanothxylum and A. thaliana, data of Z. xanthoxylum were preliminarily published by Ma et al. fi fi and identi ed genes with a unique transcription pro le in (2016). RNA isolation and first-strand cDNA synthesis for Z. xanthoxylum. A. thaliana were performed as described previously for Z. xanthoxylum (Ma et al. 2016). Materials and methods Cultivation of plants and salt exposure Differentially expressed genes (DEGs) analysis Zygophyllum xanthoxylum (Bunge) Engl. plants were cultivated Complementary DNA libraries were constructed in parallel as previously described by Ma et al.(2016). Briefly, seeds of using a tag-based digital gene expression (DGE) system (Xue Z. xanthoxylum were collected from wild plants in the Alxa et al. 2010). Genes were determined to be differentially Desert of the Inner Mongolia Autonomous Region, China. Seeds expressed using a false discovery rate (FDR) adjusted value < > were sterilised for 3 min with 5% (v/v) sodium hypochlorite, of P 0.001 and an absolute value of log2 ratio 1 as the rinsed five times with distilled water, then soaked in distilled threshold. water at 4C for 1 day, and then germinated at 25C in the dark on filter paper wetted with distilled water. Upon sprouting, uniform Re-annotation, functional categorisation of DEGs seedlings weretransplanted intoplugged holes in5 Â5cm square To obtain orthologous gene pairs, unigenes from the plastic containers (two seedlings per container) filled with sand transcriptome library of Z. xanthoxylum (Ma et al. 2016) were and irrigated with modified Hoagland nutrient solution aligned to the Arabidopsis genome assembly TAIR10 (www. containing 2 mM KNO3, 0.5 mM KH2PO4, 0.5 mM arabidopsis.org/, accessed 2 January 2019) using blastn MgSO4*7H2O, 0.5 mM Ca(NO3)2*4H2O, 60 mM Fe-citrate, 50 with stringent parameters ‘blastn.exe -task blastn -query All- mMH3BO3,10mM MnCl2*4H2O, 1.6 mM ZnSO4*7H2O, 0.6 mM Unigene.fa -db TAIR10.fasta -outfmt 7 -max_target_seqs CuSO4*5H2O and 0.05 mMNa2MoO4*2H2O. Seedlings were 1 -evalue 1e-5 -out raw_result. fasta’ to obtain the closest grown under long days (16 h light/8 h dark; photon flux density A. thaliana homologues. The unigene of Z. xanthoxylum with 800 mmol m–2Ás–1) in a greenhouse where the temperature was the lowest E value was selected when multiple Z. xanthoxylum 28 Æ 2C/23 Æ 2C (day/night). Three-week-old seedlings were unigenes matched to the same A. thaliana orthologue. Annotated used for the following treatments: (i) control (C): seedlings were Z. xanthoxylum genes with unique A. thaliana orthologs were irrigated with modified Hoagland nutrient solution (including compared with DEGs in A. thaliana to identify the common 0.1 mMNa+); and (ii) salt treatment (S): seedlings were treated genes in response to salt treatment. These data were further with modified Hoagland nutrient solution plus 50 mM NaCl. annotated by Gene Ontology (GO) functional sorting (http:// Arabidopsis thaliana (L.) Heynh. Col-0 ecotype seeds were www.geneontology.org/, accessed 18 March 2019) and Kyoto sterilised by incubating in 75% (v/v) ethanol and 5% (v/v) Encyclopedia of Genes and Genomes (KEGG) pathway analysis sodium hypochlorite for 3 min each, rinsed 5-7 times (http://www.genome.jp/kegg/, accessed 1 April 2019) to identify with distilled water, and then stratified by soaking in which categories were significantly enriched in GO terms distilled water at 4C for 2 days. The seeds were then sown (P-value < 0.05) and metabolic pathways (Q-value < 0.05). on vertically-oriented 0.8% (w/v) agar plates containing 1/2 Hierarchical cluster analysis was performed using the TIGR strength Murashige and Skoog (MS) supplemented with multi experiment viewer 4.0 (MEV 4.0) software program 0.5% (w/v) sucrose (pH 5.7 adjusted using 1 M Tris-HCl). (Howe et al. 2010). Gene response to salt in Zygophyllum xanthoxylum Functional Plant Biology C

Quantitative real-time PCR validation the transcriptome of roots was analysed and a parallel A total of 30 genes from Z. xanthoxylum and 16 genes from investigation was conducted with A. thaliana. Salinity at 50 A. thaliana were randomly selected for verification of RNA-seq mM NaCl was chosen for this experiment because data using quantitative real-time PCR. Total RNA was extracted Z. xanthoxylum seedlings experience growth stimulation under from seedling samples as described above. DNase-treated total this exposure (Yue et al. 2012). RNA from seedlings was used to synthesise first-strand cDNA using PrimeScript as the reverse transcriptase (TaKaRa Summary of RNA-seq reads collected from Z. xanthoxylum Biotechnology). Quantitative real-time PCR was performed and A. thaliana as well as mapping statistics using SYBR Premix Ex TaqII (TaKaRa Biotechnology) on a Summary statistics of RNA-seq reads collected from StepOne Real-Time PCR Thermocyler (Applied Biosystems) Z. xanthoxylum and A. thaliana and mapping rates were  with the following parameters: 95 C for 30 s followed by 40 calculated (Table S2). A majority of reads passed a quality     cycles of 95 C for 5 s, 60 C for 34 s, then 95 C for 15 s, 60 C filter and were mapped back to the single gene (Table S2).  for 1 min, 95 C for 15 s. Each 20 mL reaction mixture contained Approximately 60% of clean reads in Z. xanthoxylum and 93% 3 mL cDNA, 10 mL SYBR Premix EX TaqII, 0.4 mL ROX of clean reads in A. thaliana mapped uniquely to only one location Reference Dye II, 0.8 mL each of primers (10 mM), and 5 mL and couldbe assigned to a single annotated (Table S2). The results water. Primers for quantitative real-time PCR (see Table S1, allowed us to proceed with downstream analysis of gene available as Supplementary Material to this paper) were designed expression and deep analysis based on gene expression. using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/ primer-blast, accessed 15 February 2019) to have a Tm of 59- Overview of salt responsive gene expression 61C, with a length of 22-26 nts and a product length of 60-150 bps (Udvardi et al. 2008). ZxACTIN (GenBank: EU019550.1) Some Z. xanthoxylum unigenes had no Arabidopsis Information and AtACTIN2 (At3g18780) were used as the internal control Resource Genome match or multiple Z. xanthoxylum unigenes gene in Z. xanthoxylum and A. thaliana respectively. The relative sometimes matched with the same A. thaliana gene, resulting in expression levels of unigene transcripts normalised to ACTIN 40062 Z. xanthoxylum unigenes matched to 15160 transcript were calculated using the 2–DDCt method (Dang et al. 2013). isoforms of homologous genes in A. thaliana, which accounts for Three independent biological samples of each were used in the 38% in all unigenes of Z. xanthoxylum (see Fig. S1, available as analysis. Data reported represent the means Æ s.d. Supplementary Material to this paper). To establish an overview of salt responsive gene expression, a comparison of eight cDNA libraries constructed from the roots of Z. xanthoxylum Results and A. thaliana was performed. In Z. xanthoxylum, 6315 To investigate the unique basis of salt-tolerance in the transcripts were differentially upregulated at 6 h increasing to xerohalophyte Z. xanthoxylum compared with a glycophyte, 6963 transcripts at 24 h (Fig. 1). In A. thaliana, 1033 transcripts

Up-regulated at 6 h Down-regulated at 6 h

Z. xanthoxylum Z. xanthoxylum A. thaliana A. thaliana 6086 229 804 81825 610

Up-regulated at 24 h Down-regulated at 24 h

Z. xanthoxylum A. thaliana Z. xanthoxylum A. thaliana 2198 5659 2480 4483 10220 4809

Fig. 1. Venn diagrams showing the intersection of DEGs between Zygophyllum xanthoxylum and Arabidopsis thaliana under 50 mM NaCl. The blue colour represents number of upregulated or downregulated transcripts in Z. xanthoxylum, and the pink colour represents number of upregulated or downregulated transcripts in A. thaliana respectively. D Functional Plant Biology W. W. Chai et al. were differentially upregulated at 6 h increasing to 14703 Metabolic pathway categorisation of DEGs transcripts at 24 h (Fig. 1). In Z. xanthoxylum, 843 transcripts The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a were differentially downregulated at 6 h, increasing to 7007 resource for the systematic analysis of gene function according to transcripts at 24 h (Fig. 1). In A. thaliana, 635 transcripts were metabolic processes (Kanehisa and Goto 2000). We further differentially downregulated at 6 h, increasing to 7857 at 24 h compared metabolic pathways between Z. xanthoxylum and fi (Fig. 1). These ndings suggest a more rapid and measured A. thaliana under salt treatment (Fig. 2). As shown by KEGG response of Z. xanthoxylum to salt compared with A. thaliana analysis, unigenes involved in ‘amino sugar and nucleotide with transcript induction being the primary response to salt sugar metabolism’ and ‘glycolysis/gluconeogenesis’ were treatment. significantly enriched in Z. xanthoxylum under salt treatment at 6 h whereas genes involved in ‘fructose and mannose ’ Functional classification of DEGs metabolism were enriched at 24 h (Fig. 2a). By contrast, pathways most strongly represented in A. thaliana were fi We classi ed the biological functions of salt-responsive ‘plant-pathogen interaction’ and ‘plant hormone signal DEGs in Z. xanthoxylum and A. thaliana using GO transduction’ at 6 h, and as treatment time increased to 24 h, analysis. This system is used to sort genes into groups ‘mismatch repair’ was strengthened, suggesting a response to according to their predicted or experimentally derived DNA damage (Fig. 2b). In summary, 50 mM NaCl caused cellular component (subcellular location), molecular metabolic shifts towards gluconeogenesis with increased function (activity performed by gene products), and sugars compatible with growth stimulation of Z. xanthoxylum, biological process (larger process performed by multiple but triggered defence pathways compatible with a distress gene products) (Ashburner et al. 2000; Niederhuth et al. response in A. thaliana. 2013). The relative abundance of significantly regulated DEGs involved in different functional categories of Z. xanthoxylum and A. thaliana is summarised (Fig. S2). DEGs enriched in key functional categories In salt-treated Z. xanthoxylem at 6 h, for cellular component, DEGs involved in ion transport ‘ ’ DEGs assigned to mitochondrial were dominant; for Ion transport is particularly crucial in response to salt (Zhu fi molecular function, DEGs were signi cantly enriched in 2003). After salt exposure, abundant transcripts encoding ion ‘ ’ ‘ ’ ‘ transition metal ion binding , metal ion binding , cation transporters were detected in the roots of Z. xanthoxylum, ’ ‘ ’ binding , glutathione transferase activity ; and for biological including important genes related to Na+,K+,Ca2+ transport process, ‘response to stimulus’ was highly represented, while À + 3À 2+ and uptake of NO3 ,NH4 ,PO4 ,andMg macroelements few GO terms were over-represented in Z. xanthoxylem at (Ma et al. 2016). To further understand how salt responses 24 h (Fig. S2, A-1, B-1, C-1). The GO enrichment in A. thaliana differ between the two species, we compared ion transport was different from that in Z. xanthoxylem with more GO terms DEGs from Z. xanthoxylum to orthologs in A. thaliana over-represented at 24 h than that at 6 h. For molecular function, (Table 2). Among these DEGs, transcripts associated with ‘ ’ ‘ ’ receptor activity and transmembrane transporter activity were Na+/K+-transport formed the largest proportion (Table S3). ‘ ’ ‘ dominant at 6 h, but oxidoreductase activity , transferase Most key Na+/K+ transporter genes were significantly ’ activity were highly represented at 24 h; for biological upregulated in Z. xanthoxylum roots, including transcripts for ‘ ’ ‘ ’ process, response to drug and drug transport were mainly SOS1 (salt overly sensitive1), NCX4 (Na+/Ca2+ exchanger), ‘ ’ enriched at 6 h, whereas, anatomical structure morphogenesis HKT1;1 (high affinity K+ transporter1;1) and KUP7 was dominant, suggesting a salt stress harm A. thaliana (Fig. S2, (K+ uptake permeases), while orthologs in salt-treated A-2, B-2, C-2). A. thaliana were slightly upregulated or remained constant in ‘ ’ Differences in biological process terms between the two expression (Table 2). Several DEGs related to macroelement species in response to salt were analysed (Table 1). DEGs transport, such as NRT1.5 (nitrate transporters) and PHT1;1, ‘ ’ involved in response to stimulus were highly represented in PHT1;2 (phosphate transporters) were highly induced in Z. xanthoxylum whereas the overwhelming majority of DEGs in Z. xanthoxylum (Table 2). In A. thaliana,onlyAtHKT1;1, ‘ ’ A. thaliana were related to response to stress both at 6 and 24 h. AtNRT1.8,andAtPHT1;2 were upregulated at 6 h and ‘ ’‘ DEGs involved in transition metal ion binding oxidation- AtNRT1.5 was induced at 24 h (Table S3). Based on ’ reduction process were enriched in salt-treated data from Ma et al.(2016), several important trace element ‘ Z. xanthoxylum at 6 h and DEGs involved in hormone transporters were also induced in salt-treated Z. xanthoxylum.In ’ fi stimulus were signi cantly enriched in salt-treated this work, FRO2 (ferric reduction oxidase 2) and ZIP6 (ZRT, fi A. thaliana. These ndings imply that increased ion transport IRT-like proteins) were detected as very highly induced in fi processes and oxidation reduction processes were ef cient salt Z. xanthoxylum by salt, but expression of their orthologs was adaptation strategies in salt-treated Z. xanthoxylum, whereas relatively unchanged in A. thaliana (Table 2). hormone stimulus was dominant response in A. thaliana in initial stage of salt treatment. After salt treatment for 24 h, DEGs involved in ‘stimulus’ were still upregulated in DEGs involved in ROS removal pathway Z. xanthoxylum but downregulated in A. thaliana.In The concentration of 50 mM NaCl significantly increased the summary, GO analysis suggested that 50 mM NaCl was activities of antioxidant SOD (superoxide dismutase), POD perceived as a stimulus for Z. xanthoxylum whereas a stress (peroxidase), and CAT (catalase) enzymes in Z. xanthoxylum for A. thaliana. (Cai et al. 2011). We further focussed on the expression of these Gene response to salt in Zygophyllum xanthoxylum Functional Plant Biology E

Table 1. GO annotation based on biological process ontology in salt-treated Zygophyllum xanthoxylum and Arabidopsis thaliana Gene hits indicate the number of genes in the clusters of up- or down-regulated genes belonging to that particular Gene Ontology (GO) term. P-values are provided as a score of significance (cut-off <0.05)

GO term Gene hits 6 h 24 h Z. xanthoxylum A. thaliana Z. xanthoxylum A. thaliana Response to stimulus 402 (up) 557 (up) 546 (up) 679 (down) Response to chemical stimulus 225 (up) 292 (up) 299 (up) 332 (up) Response to abiotic stimulus 163 (up) 185 (up) 228 (up) 235 (down) Response to stress 0 332 (up) 0 348 (up) Response to biotic stimulus 0 106 (up) 104 (up) 0 Response to osmotic stress 0 84 (up) 0 100 (down) Response to endogenous stimulus 0 148 (up) 0 0 Transition metal ion binding 163 (up) 0 0 0 Oxidation-reduction process 178 (up) 0 235 (up) 0 Response to inorganic substance 0 0 0 143 (down) Response to hormone stimulus 0 136 (up) 0 0 Response to metal ion 0 0 0 122 (up) Secondary metabolic process 0 59 (up) 0 0 Response to water 0 0 44 (up) 0 Response to water deprivation 0 0 41 (up) 0

(a)(b) A. thaliana Z. xanthoxylum Alanine, aspartate and glutamate metabolism Mismatch repair Biotin metabolism Starch and sucrose metabolism One carbon pool by folate 1 Endocytosis Ascorbate and aldarate metabolism Indole alkaloid biosynthesis Glycine, serine and threonine metabolism 1 Nitrogen metabolism 0.5 Ether lipid metabolism Sulfur metabolism alpha-Linolenic acid metabolism Arginine and proline metabolism 0.5 Fatty acid biosynthesis 0 Flavonoid biosynthesis Fructose and mannose metabolism Flavone and flavonol biosynthesis Glyoxylate and dicarboxylate metabolism Selenocompound metabolism Plant hormone signal transduction 0 Pentose phosphate pathway –0.5 Plant-pathogen interaction Carbon fixation in photosynthetic organisms Glutathione metabolism Glycosphingolipid biosynthesis - ganglio series –0.5 Amino sugar and nucleotide sugar metabolism –1 Limonene and pinene degradation Glycosaminoglycan degradation Biosynthesis of secondary metabolites Cysteine and methionine metabolism –1 Glycerolipid metabolism Phenylpropanoid biosynthesis Circadian rhythm-plant Metabolic pathways Glycolysis/Gluconeogenesis Terpenoid backbone biosynthesis Photosynthesis Sphingolipid metabolism Sesquiterpenoid biosynthesis Glucosinolate biosynthesis lsoflavonoid biosynthesis Glycosphingolipid biosynthesis - globo series Biosynthesis of secondary metabolites Phenylalanine metabolism Other glycan degradation Stilbenoid, diarylheptanoid and gingerol biosynthesis Cyanoamino acid metabolism Pentose and glucuronate interconversions Pentose and glucuronate interconversions Pyruvate metabolism Regulation of autophagy Metabolic pathways Carotenoid biosynthesis Starch and sucrose metabolism Tryptophan metabolism Photosynthesis Carotenoid biosynthesis SNARE interactions in vesicular transport Phenylalanine metabolism Zeatin biosynthesis Benzoxazinoid biosynthesis Ribosome Glucosinolate biosynthesis Linoleic acid metabolism Oxidative phosphorylation Purine metabolism Flavonoid biosynthesis Sulfur metabolism Plant-pathogen interaction Biotin metabolism Plant hormone signal transduction Ribosome ABC transporters Glycosylphosphatidylinositol (GPI)-anchor biosynthesis Amino sugar and nucleotide sugar metabolism Phenylpropanoid biosynthesis Glycolysis/Gluconeogenesis Glutathione metabolism Stilbenoid, diarylheptanoid and gingerol biosynthesis Fructose and mannose metabolism Brassinosteroid biosynthesis Benzoxazinoid biosynthesis alpha-Linolenic acid metabolism

CR6 vs CR24 CR6 vs SR6 SR6 vs SR24 CR24vs SR24 Limonene and pinene degradation Flavone and flavonol biosynthesis CR6 vs CR24 SR6 vs SR24 CR24vs SR24 CR6 vs SR6

Fig. 2. Specific significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for DEGs from CR6 vs CR24, CR6 vs SR6, SR6 vs SR24, CR24 vsSR24 in Zygophyllum xanthoxylum (a) and Arabidopsis thaliana (b). Abbreviations: C, control treatment; S, salt treatment with50 mM NaCl; 6, 6 h treatment; 24, 24 h treatment; R, roots. The –log10 (Q-value) of enrichment was used as the value for clustering by MEV 4.0 program. The colours red (upregulated) and blue (downregulated) indicate significantly enriched pathways. F Functional Plant Biology W. W. Chai et al.

Table 2. Key differentially expressed genes related to ion transport, reactive oxygen species (ROS) scavenging, as well as ABA, GA biosynthesis and auxin response Note: ‘/’ indicates not found; Z. xanthoxylum, Zygophyllum xanthoxylum; A. thaliana, Arabidopsis thaliana

Putative Description Gene ID Fold change (6 h) Fold change (24 h) orthologue Z. xanthoxylum A. thaliana Z. xanthoxylum A. thaliana Z. xanthoxylum A. thaliana Ion transport Na+ SOS1 Salt overly sensitive 1 Unigene9810_All AT2G01980 7.8 2.1 0.3 0.9 NCX4 Cation calcium exchanger 4 CL3586.Contig1_All AT1G54115 7.6 0.2 –0.6 0.3 HKT1;1 Sodium transporter Unigene11474_All AT4G10310 3.2 1.2 –0.8 1.1 K+ KUP7 K+ uptake permease 7 CL11345.Contig1_All AT5G09400 7.5 –0.1 5.6 0.2 À NO3 NRT1.5 Nitrate transporter1.5 CL10526.Contig1_All AT1G32450 8.6 –0.7 0.5 1.7 3À PO4 PHT1;1 Phosphate transporter 1;1 Unigene33655_All AT5G43350 5.6 0.4 –0.5 0.7 PHT1;2 Phosphate transporter 1;2 Unigene7644_All AT5G43370 10.7 1.2 0.7 1.7 Zn2+,Fe2+,Mn2+ ZIP6 Zinc transporter 6 CL11327.Contig1_All AT2G30080 4.1 –0.2 0.1 –0.4 Fe FRO2 Ferric reduction oxidase 2 Unigene16933_All AT1G01580 8.4 / / / ROS scavenging GST GSTU1 The tau class of glutathione CL7539.Contig3_All AT2G29450 4.1 1.0 0.9 –0.1 s-transferase GSTU7 The tau class of glutathione CL11683.Contig1_All AT2G29420 4.0 0.7 0.0 –1.0 s-transferase GSTU10 The tau class of glutathione Unigene60082_All AT1G74590 4.1 2.7 0.0 –0.4 s-transferase GSTU19 The tau class of glutathione CL5645.Contig2_All AT1G78380 4.3 0.0 0.0 –1.0 s-transferase GSTU22 The tau class of glutathione Unigene7560_All AT1G78340 4.0 2.7 0.0 –0.5 s-transferase GSTU25 The tau class of glutathione Unigene1141_All AT1G17180 8.8 2.9 0.0 –1.6 s-transferase

Peroxidase superfamily POD Peroxidase superfamily CL7046.Contig3_All AT2G39040 8.9 À0.8 / 9.9 protein POD Peroxidase superfamily CL9262.Contig2_All AT5G42180 8.1 À0.5 0.3 1.3 protein POD Peroxidase superfamily CL1944.Contig2_All AT5G66390 8.1 0.7 0.1 0.6 protein POD Peroxidase superfamily CL7488.Contig2_All AT4G11290 7.6 0.4 0.4 0.4 protein SOD SOD1 Manganese superoxide Unigene67157_All AT3G10920 7.7 0.0 / 0.4 dismutase CAT CAT1 Catalyses the reduction of CL8063.Contig1_All AT1G20630 4.2 0.3 0.0 –0.1 hydrogen peroxide ABA, GA biosynthesis and auxin response ABA NCED3 Biosynthesis of abscisic acid CL86.Contig2_All AT3G14440 –6.1 4.6 7.1 0.7 ABA2 The conversion of xanthoxin CL6088.Contig2_All AT1G52340 4.3 –0.2 –2.1 –0.4 to ABA-aldehyde GA GA2 gibberellin biosynthetic Unigene15467_All AT1G79460 5.3 À0.3 / 0.6 GA3 gibberellin biosynthetic Unigene13918_All AT5G25900 4.6 0.3 6.5 1.2 Gene response to salt in Zygophyllum xanthoxylum Functional Plant Biology G

Table 2. (continued )

Putative Description Gene ID Fold change (6 h) Fold change (24 h) orthologue Z. xanthoxylum A. thaliana Z. xanthoxylum A. thaliana Z. xanthoxylum A. thaliana GA2ox2 Negative regulated CL10145.Contig3_All AT1G30040 –6.1 1.0 –0.1 0.1 gibberellin biosynthetic AUXIN/IAA ILR1 IAA-leucine resistant CL2976.Contig1_All AT3G02875 4.6 0.6 / 0.0 ARF7 Auxin response factor CL163.Contig6_All AT5G20730 4.6 0.2 4.6 –0.3 TIR1 mediates auxin-regulated CL1890.Contig1_All AT3G62980 4.6 0.1 –0.1 –0.3 transcription SAUR16 Small auxin upregulated Unigene21036_All AT4G38860 6.5 0.1 7.6 / RNA SAUR32 Small auxin upregulated Unigene21000_All AT2G46690 6.8 –0.7 1.5 1.6 RNA SAUR35 Small auxin upregulated Unigene22027_All AT4G12410 6.0 / –5.3 0.9 RNA Auxin-responsive Auxin-responsive Unigene888_All AT2G04850 7.1 –0.4 1.4 0.6 GH3.11 indole-3-acetic acid- CL3620.Contig2_All AT2G46370 6.8 0.7 –0.7 0.7 amidosynthetase GH3.5 indole-3-acetic acid- Unigene62182_All AT4G27260 6.8 –0.7 –0.1 –0.6 amidosynthetase genes in Z. xanthoxylum and A. thaliana to identity differences. salt-treated Z. xanthoxylum and A. thaliana, ZxNCED3 was Nine POD superfamily proteins were transcribed at a high level identified as downregulated (–6.1-fold) whereas AtNCED3 in Z. xanthoxylum (Table S4), and four of which were was upregulated (4.6-fold) after salt-treatment for 6 h upregulated at least 7-fold (Table 2). In addition, ZxSOD1 and (Table 2). ABA (ABA deficient) genes that functions in first ZxCAT1 were upregulated 7.7- and 4.2-fold respectively step of the biosynthesis of ABA were induced in the two species. (Table 2). By comparison, their orthologs remained nearly ZxABA1 and AtABA1 were both slightly induced at 6 h, and constant in expression in salt-treated A. thaliana (Table 2). downregulated at 24 h (Table S5); ABA2 was highly upregulated Ascorbate-glutathioine (AsA-GSH) cycle plays an important (4.3-fold) in Z. xanthoxylum but expression of its orthologue in role in the ROS scavenging system of Z. xanthoxylum based on A. thaliana was unchanged (Table 2). transcript analysis (Ma et al. 2016). In the AsA-GHS pathway of GA biosynthesis-related genes: GA (GA requiring) required Z. xanthoxylum, six APX (ascorbate peroxidase) genes (ZxSAPX, for GA synthesis showed different expression pattern. ZxGA2 ZxLAC5, ZxLAC10, ZxLAC17, ZxSKS6, ZxSKS15), two GRX and ZxGA3 were strongly upregulated, whereas ZxGA2ox (glutaredoxin) genes (ZxGRXC2, ZxGRXC5), one MDAR (GA-oxidase 2) required for GA catabolism was significantly (monodehydroascorbate reductase) genes (NADP+-linked downregulated; but the corresponding orthologs in A. thaliana oxidoreductase), one DHAR (dehydroascorbate reductase) remained nearly constant in expression under salt treatment gene (ZxDHAR2), two ALDH (aldehyde dehydrogenase) (Table 2). genes (ZxALDH3, ZxALDH7B4), one GSH (glutathione Auxin-related response genes: several DEGs related to reductase) gene (ZxGSH2), and eight GSTs (glutathione auxin response were identified in Z. xanthoxylum. Sixteen s-transferase) genes (ZxGSTs) showed relatively abundant auxin-related DEGs were upregulated during 50 mM NaCl transcript levels (Table S4). Especially six ZxGSTUs (tau treatment for 6 h (Table S5). Among DEGs at 24 h, five auxin- class of glutathione s-transferase) were highly induced above related genes were upregulated and nine were downregulated; 4-fold (Table 2). By contrast, only four AtGSTUs (AtGSTU1, and five were co-upregulated both at 6 and 24 h (Table S5). AtGSTU10, AtGSTU22, AtGSTU25) were changed in expression By contrast, only five orthologue genes were differentially in salt-treated A. thaliana (Table 2). upregulated during salt treatment for 24 h in A. thaliana (Table S5). In particular, the ARF (auxin responsive transcription factor) encoding ZxARF7 gene was abundantly DEGs involved in ABA and GA biosynthesis and response expressed in Z. xanthoxylum under salt treatment both at 6 and to auxin 24 h, whereas AtARF7 did not change in expression Phytohormones also play essential roles in plant responses to (Table 2). Several SAUR (small auxin-up RNA) family salinity (Ryu and Cho 2015). In this work, we focussed on genes, such as ZxSAUR16, ZxSAUR32,andZxSAUR35, abscisic acid (ABA) and gibberellin (GA) biosynthesis and were highly induced at 6 h, whereas ZxSAUR16 was response to auxin (Table S5). maintained at high expression both at 6 and 24 h ABA biosynthesis-related genes: a NCED (nine-cis- (Table 2). In contrast, orthologue genes in A. thaliana epoxycarotenoid dioxygenase) gene encoding a key enzyme remained nearly constant in expression under salt treatment during ABA biosynthesis showed different response in (Table 2). Two DEGs from the GH3 (indole-3-acetic H Functional Plant Biology W. W. Chai et al. acid-amido synthetase) gene family (GH3.11, GH3.5) were protein, was upregulated 3.4-fold; and CL13064.Contig2_All, significantly upregulated in Z. xanthoxylum but downregulated predicted to encode a thioredoxin, was upregulated 8.8-fold. In in A. thaliana (Table 2). addition, 1486 DEGs with no homology to known genome sequences and potentially unique to Z. xanthoxylum were Unigenes of Z. xanthoxylum with no clear counterpart in uncovered. A. thaliana Several unigenes from Z. xanthoxylum (22019) had no clear Validation of sequencing data by quantitative real-time counterpartinA.thaliana(Fig.S1).Asaresult,2883DEGsinsalt- PCR treated Z. xanthoxylum (7%) had no match to sequences in the Random quantitative real-time PCR was used to test the Arabidopsisdatabase(Fig.S1).Thisgroupcomprised1558DEGs reproducibility of RNA-seq datasets used in this study. The upregulated and 1107 DEGs downregulated at 6 h, and 657 DEGs quantitative real-time PCR results for 30 randomly selected upregulated and 518 DEGs downregulated at 24 h. Within this unigenes from Z. xanthoxylum and 16 randomly selected group were several interesting unigenes. Two unigenes (CL1521. genes from A. thaliana showed general agreement with Contig1_All and Unigene18565_All) that encode NAC (NAM/ transcript abundance changes measured by RNA-seq ATAF/CUC) transcription factors were upregulated 7.8- and suggesting reliability of the RNA-seq profiling data. A linear 9.9-fold under salt treatment at 6 h respectively. We also regression analysis of the fold-changes between DGE profiling noted that CL2133.Contig1_All, which encodes a MYB and quantitative real-time PCR exhibited a positive correlation (Myeloblastosis) transcription factor, was upregulated 1.6-fold; (Fig. 3). Detailed information about the primers is provided in CL5341.Contig1_All, predicted toencode anoxidation resistance Table S1.

At-SR6 vs CR6 (a) Zx-SR6 vs CR6 (b) 12 12

10 10

2 8 8 R = 0.7633 R2 = 0.8851 6 6 4 4 2 2 0 0 –4 –2 0 2 4 6 8 10 –4 –2 0 2 4 6 8 10 –2 –2 –4

(c)(12 Zx-SR24 vs CR24 d) At-SR24 vs CR24

Fold change (RNA-Seq) Fold 12

10 10

8 2 R = 0.9729 8 R2 = 0.8416 6 6 4 4 2 2 0 –4 –2 0 2 4 6810 0 –2 –4 –2 0 2 4 6 8 10 –2 Fold change (qPCR)

Fig. 3. Correlation of gene expression between quantitative real-time PCR and RNA-seq results. Overall, there is good concordance between log2 values derived from RNA-seq and the experimental values derived from quantitative real-time PCR measures. Total RNA was extracted from three replicated samples individually for each treatment of roots (n = 3). (a) Zx-SR6 vs CR6, (b) At-SR6 vs CR6, (c) Zx-SR24 vs CR24, (d) At-SR24 vs CR24. Abbreviations: Zx, Zygophyllum xanthophyllum; At, Arabidopsis thaliana; C, control treatment; S, salt treatment with 50 mM NaCl; 6, 6 h treatment; 24, 24 h treatment; R, roots. Gene response to salt in Zygophyllum xanthoxylum Functional Plant Biology I

Discussion salt stress (Fig. 2). Another transcriptome analysis in root of Construction of an informative comparison of RNA-seq A. thaliana also indicated 50 mM NaCl triggered genes involved analysis and the unique unigenes of Z. xanthoxylum in defence systems and induced ROS in root observed by histochemical analyses (Zhao et al. 2009). Quantitative differences in gene expression play a key role in salt These data above were consistent with phenotypic data tolerance (Mishra and Tanna 2017). Therefore, gene expression showing that Na+ stimulates Z. xanthoxylum root growth analysis is an important tool in deciphering how different species (Wang et al. 2014) whereas salt inhibits the elongation of adapt to extreme environments. In this study, the expression of A. thaliana roots (Zolla et al. 2010; Tsou et al. 2012; Irani 15 160 gene pairs were compared with explore the genetic basis and Todd, 2016). The positive effect of moderate salt had also of salt response in a xerohalophyte compared with a model been reported in xerohalophyte Atriplex halimus (Bendaly et al. glycophyte plant species. In so doing, we uncovered a set of 2016) and Nitraria roborowskii (Lu et al. 2016). DEGs in salt-treated Z. xanthoxylum with no homologous sequence in the genome of A. thaliana plants. We detected Ion transport systems are more active in Z. xanthoxylum some DEGs predicted to encode transcription factors, showed based on transcriptome analysis fi signi cantly increased expression, such as two NAC Generally, the plant’s first response to salinity is either ion- transcription factors (at least upregulated 8-fold) and a MYB exclusion (glycophytes) or sequestration of ions in cell vacuoles (upregulated 1.6-fold) transcription factors. Transcription (halophytes) leading to tissue tolerance (Deinlein et al. 2014). factors belonging to NAC and MYB families are prevalent in Our data highlight key differences for DEGs related to ion regulatory networks responding to biotic and abioticstresses, and transport in Z. xanthoxylum and A. thaliana, compatible with were found to confer resistance to salt stress in plants (Wang et al. distinct coping strategies for salt (Table S3). 2017; Jiang et al. 2018). Therefore, NAC and MYB transcription For Z. xanthoxylum, the large absorption of Na+ ions while factors might function as positive regulators of salt response in maintaining a stable K+ status are central mechanisms of salt Z. xanthoxylum. A similar pattern of results was reported for response (Yuan et al. 2015). This regulation suggests that another xerophyte, Caragana korshinskii, with NAC Z. xanthoxylum has ion channels/transporters with specific transcriptions were dramatically differentially expressed under features that lead to superior K+/Na+ homeostasis. Cation salt treatment (Li et al. 2016). In addition, oxidation resistance transporters and channels such as CNGCs are involved in the protein genes, likeCL5341.Contig1, may be a critical component transport of Na+ across membranes (Kakar et al. 2017). Notably, of the defence system of Z. xanthoxylum. Thioredoxins, such as CNGC10 is proposed to function as a channel providing Na+ the one encoded by CL13064.Contig2_All, may play a role in influx (Jin et al. 2015). ZxCNGC10 was induced by 1.5-fold protecting against oxidative damage and lipid peroxidation (Dos (Table S3), suggesting that ZxCNGC10 may be involved in Santos and Rey 2006). mediating Na+ transport. The overall transcript levels for CNGCs Several Z. xanthoxylum salt-induced DEGs could not be in salt-treated A. thaliana were lower than Z. xanthoxylum matched to any database sequence and were of unknown (Table S3). One possible reason is that Na+ influx is improved function. These unannotated unigenes could be novel genes in Z. xanthoxylum during salt-stress. The SOS pathway is an found only in Z. xanthoxylum. Nevertheless, this unmatched established pathway for control of Na+ homeostasis (Qiu et al. set of unigenes represents a group of potentially unique genes in 2004). SOS1 is the first identified core gene in this pathway. This Z. xanthoxylum contributing to salt response, worthy of future gene is directly involved in the transport of Na+ across the plasma study. membrane and plays a key role in the efflux of Na+ and xylem loading (Shi et al. 2002). ZxSOS1 is essential for long-distance fi Differential gene expression pro les in roots of transport and spatial distribution of Na+ in Z. xanthoxylum (Ma Z. xanthoxylum and A. thaliana et al. 2014). In this work, ZxSOS1 transcripts were increased 7.8- The initial responses of salt stress are generally focussed on fold, compared with 2.1-fold induction of AtSOS1 transcripts seedling roots (Julkowska et al. 2017). An overview of salt under salt treatment for 6 h(Table 2).This difference may explain responsive DEGs in Z. xanthoxylum and A. thaliana was the superior ability for transport of Na+ from root to shoot in obtained using Venn diagrams (Fig. 1). These data suggest a Z. xanthoxylum compared with A. thaliana. The regulation of more rapid response of Z. xanthoxylum to salt compared with Na+ uptake and transport in plants under salt conditions is widely A. thaliana. The GO analysis of these DEGs suggests that interpreted in the context of maintaining high tissue K+/Na+, Z. xanthoxylum perceives moderate salt as a stimulus for which is a key salt tolerance trait (Almeida et al. 2017). adaptive processes whereas A. thaliana activates defence AtHKT1;1 and OsHKT1;5, the homologues of ZxHKT1;1, pathways (Table 1; Fig. S2). KEGG provides a platform for not only mediate Na+ unloading from xylem vessels but also the systematic analysis of gene function according to metabolic promote K+ accumulation in leaves (Lin et al. 2004; Ren et al. networks of gene products. KEGG enrichment analysis suggests 2005; Sunarpi et al. 2005; Horie et al. 2009). The expression that enhanced sugar metabolism generates energy to promote induction of ZxHKT1;1 (3.2-fold increase) was higher than that growth of Z. xanthoxylum under 50 mM NaCl treatment (Fig. 2). of AtHKT1;1 (1.2-fold increase) (Table 2), which may be at least Similarly, KEGG pathway of the halophyte Salvadora persica partially related to an outstanding capacity for maintaining the also showed the salinity-induced metabolites are associated with stability of K+ concentrations in Z. xanthoxylum under salt the major biological pathways related to sugar metabolism treatment (Yuan et al. 2015;Huet al. 2016). Furthermore, as (Kumari and Parida 2018). In contrast, KEGG enrichment a possible mechanism to maintain adequate K+ nutrition, high- revealed that A. thaliana activates defence systems to combat affinity K+ transporter genes KT/KUP/HAKs were highly J Functional Plant Biology W. W. Chai et al. induced, such as KUP7, which is involved in high-affinity K+ upregulated in the roots of salt-treated Z. xanthoxylum (Ma et al. uptake (Han et al. 2016). ZxKUP7 was maintaining a high 2016). In the present work, nine genes encoding peroxidase expression both at salt treatment for 6 h (7.5-fold) and 24 h superfamily proteins were highly induced in salt-treated (5.6-fold) (Table 2), which indicate an important role in K+ Z. xanthoxylum in initial response to salt (Table S4). uptake. In contrast, genes relating to K+ transport remained Peroxidases are involved in lignin formation, the cross-linking nearly constant in expression in salt-treated A. thaliana of cell wall components, the removal of H2O2, the oxidation of (Table S3), implying that K+ uptake was not enhanced. This toxic reductants, and defence against biotic stresses (Welinder expression pattern fits with a decline in K+ concentration in roots 1992). These findings suggest that peroxidases may be the of A. thaliana under 50 mM NaCl treatment (Ghars et al. 2008). main scavenger of ROS under short-term salt treatment in Salt reduces nutrient uptake in glycophytes (e.g. nitrogen, Z. xanthoxylum. phosphate, calcium, magnesium and iron) (Tuna et al. 2008;Wu Salt tolerance in salt-tolerant wild tomato has been attributed 2+ – et al. 2013). Several important genes for transport of Ca ,NO3 , to an outstanding AsA-GSH cycle (Shalata et al. 2001). Ma et al. + 3– 2+ NH4 ,PO4 ,andMg ,suchasNRTs, AMTs, PHTs and MGTs (2016) also inferred an important role for GSH in the ROS- were significantly induced in salt-treated Z. xanthoxylum but not scavenging system of Z. xanthoxylum. Our results showed that in A. thaliana (Table S3). NRTs mediate nitrate transport (Lin AsA-GSH cycle related genes were more active in et al. 2008) and PHT1 belongs to a family of known phosphate Z. xanthoxylum than in A. thaliana (Table S4). In this cycle, transporters (Nussaume et al. 2011). ZxNRT1.5, ZxPHT1;1, GSH must be catalysed by glutathione transferases (GSTs) to act ZxPHT1;2 and ZxPHT1;3 were highly induced by 50 mM as an antioxidant (Dixon et al. 2002). GSTs are known to play NaCl, which may contribute to nitrogen and phosphate uptake direct roles in minimising oxidative damage (Roxas et al. 2000), in salt-treated Z. xanthoxylum.Liet al.(2017) found that NRT1.5/ enhancing tolerance to stresses (Nianiou-Obeidat et al. 2017), as NPF7.3 functions as a proton-coupled H+/K+ antiporter for K+ well as reducing secondary toxic substances generated during loading into the xylem. PHT1 proteins are known for transporting oxidative stress (Sappl et al. 2009). GSTs may be grouped into 3– ions other than PO4 , including phosphite, sulfate, nitrate, four classes and termed the phi, zeta, theta and tau classes (Dixon chloridion and selenite (Preuss et al. 2010). MGT family et al. 2002). Tau class glutathione transferases (GSTU) are plant proteins, such as MGT1 and MGT10, have been characterised specific, and important for protecting plants against oxidative as Mg2+ transporters(Li etal. 2001).ZxMGT2 was induced by salt damage (Jha et al. 2011). It has been confirmed that GSTU (Table S3), indicating that Mg2+ transport is likely enhanced in contribute to detoxifying in plant cells (Mano et al. 2017). Z. xanthoxylum under 50 mM NaCl. A study in rice showed that ZxGSTUs were maintained at a high expression level under OsMGT1 confers salt tolerance by enhancing the transport salt treatment, whereas most AtGSTUs showed lower activity of OsHKT1;5 (Chen et al. 2017). In the future, expression (Table 2). Therefore, we infer that ZxGSTUs play synergistic functions of ZxMGT and ZxHKT should be key roles in detoxifying cells under salinity and improve salt considered. FRO2, encoding a root ferric-chelate reductase, is resistance of Z. xanthoxylum. Because of the important roles of involved in high-affinity Fe2+ transport in roots (Fourcroy et al. GSTs, overexpression of GST genes may increase plant tolerance 2016). Notably, ZxFRO2 was highly induced by 50 mM NaCl to salinity (Sharma et al. 2014; Jia et al. 2016). Therefore, (Table 2), which might explain an increase of Fe concentration in ZxGSTUs may be used as candidate genes for enhancing roots of salt-treated Z. xanthoxylum (Wang et al. 2019). It was abiotic stress tolerance of plants. reported that ZIP family metal transporters are linked to micronutrient uptake, including zinc, iron and manganese (Lin et al. 2009; Milner et al. 2013). Highly-expressed ZxZIPs may ABA and GA biosynthesis genes, as well as auxin response play important roles in micronutrient uptake in salt-treated genes show distinct expression patterns between Z. xanthoxylum roots. Z. xanthoxylum and A. thaliana Our analysis showed that many DEGs related to classic plant hormones like ABA, GA and auxin have contrasting patterns of Peroxidases and glutathione transferases have a predicted expression in Z. xanthoxylum and A. thaliana under salt treatment major ROS-scavenging role in Z. xanthoxylum (Table S5). Salt induces secondary ionic and osmotic stresses in plant cells, ABA is a well characterised stress hormone because of its leading to the further accumulation of ROS (Pang and Wang, rapid accumulation in response to stresses and its mediation of 2008; Miller et al. 2010). ROS such as hydrogen peroxide many responses that help plant survival during stresses (Zhang À (H2O2), superoxide radical (O2* ), and hydroxyl radical (OH*) et al. 2006). Cellular ABA biosynthesis occurs mainly in can alter membrane function by changing lipid composition, and chloroplasts and the cytoplasm (Liu and Hou 2018). First, the by affecting enzyme activities (Bose et al. 2014). Plants have carotenoid zeaxanthin is catalysed to the all-trans violaxanthin in evolved complex antioxidant defence mechanisms that consist of the chloroplast by zeaxanthin epoxidase, known as ABA1 (Liu enzymatic and non-enzymatic pathwaysto eliminateexcess ROS and Hou 2018). ABA1 transcripts were slightly elevated both in accumulation (Bose et al. 2014). In a previous study, superoxide Z. xanthoxylum and A. thaliana at 6 h post-treatment with salt to dismutase, peroxidase, and catalase activities were induced in play roles in ABA biosynthesis (Table S5). Second, the salt -treated Z. xanthoxylum (Cai et al. 2011) and the expression intermediate violaxanthin is catalysed to xanthoxin by NCED of more than 70 DEGs involved in four major ROS-scavenging (Zhang et al. 2014). NCED3 is the key regulator in NaCl-induced pathways, such as AsA-GSH cycle, glutathione peroxidase(GRX), ABA biosynthesis (Barrero et al. 2006). In the present study, catalase (CAT) and peroxiredoxin/thioredoxin (PrxR/Trx), were NCED3 was induced in A. thaliana but repressed in Gene response to salt in Zygophyllum xanthoxylum Functional Plant Biology K

Z. xanthoxylum (Table 2), suggesting NCED3 induction (Table 2). GH3 proteins regulate the levels of phytohormones, represented an early step in controlling osmotic stress-induced including auxin and jasmonates (Okrent and Wildermuth 2011). ABA biosynthesis in A. thaliana. Then, xanthoxin is converted to These two hormones are responsible for regulating the balance abscisic aldehyde and oxidised to bioactive ABA by short-chain between growth and defence (Withers et al. 2012; Ren and Gray dehydrogenase reductase and abscisic aldehyde oxidase in the 2015). cytoplasm (Seo and Koshiba 2002). Transcripts for ABA2, encoding a short-chain dehydrogenase reductase, were Conclusion induced in Z. xanthoxylum but had constant expression in A. thaliana (Table 2), which suggests a more active process of This work presents a comparative RNA-seq analysis between xanthoxin conversion to bioactive ABA in Z. xanthoxylum. Z. xanthoxylum and A. thaliana under 50 mM NaCl treatment. ABA and GA antagonistically regulate various These data suggest that 50 mM NaCl is perceived as a stimulus for Z. xanthoxylum whereas a stress for A. thaliana. developmental processes, such as root growth, leaf fi development, and responses to abiotic stresses (Golldack In support of this nding, 50 mM NaCl caused metabolic shifts et al. 2013). Several transcripts related to GA biosynthesis in towards gluconeogenesis in Z. xanthoxylum, whereas induced defensive systems in A. thaliana. A vast array of ion transporters Z. xanthoxylum and A. thaliana showed contrasting expression + fi patterns in response to salt (Table S4). GA3 (ent-kaurene related to Na and nutrient uptake was signi cantly induced in oxidase) catalyses a key step in gibberellin biosynthesis Z. xanthoxylum but not in A. thaliana. ROS scavenging system (Helliwell et al. 1999). ZxGA3 was strongly upregulated by components also featured strongly in Z. xanthoxylum with the salt but remained at nearly constant expression in salt-treated high expression of key metabolic genes in this pathway A. thaliana (Table 2). Bioactive GAs are rendered inactive by compared with A. thaliana. Moreover, important genes related GA2 oxidases (GA2ox) to control overall GA accumulation (Sun to ABA and GA biosynthesis, as well as genes response to auxin 2011). Our data showed that GA2ox transcripts were induced in showed distinct patterns of expression between Z. xanthoxylum A. thaliana but repressed in Z. xanthoxylum (Table 2). Thus, and A. thaliana. Finally, numerous salt-responsive DEGs unique to Z. xanthoxylum were identified, representing a new resource higher levels of GA3 and lower levels of GA2ox2 in salt-treated fi Z. xanthoxylum might contribute to biosynthesis and accumulate for further studies. Candidate genes identi ed in Z. xanthoxylum GA, which could stimulate growth. will facilitate the genetic engineering of salt-adapted crop plants. Environmental signals control auxin homeostasis, redistribution, and signalling (Ren and Gray 2015). Auxin is a Conflicts of interest key hormone responsible for altering growth and development The authors declare no conflicts of interest. and inducing genes for stress tolerance (Su et al. 2015). Three fi major classes of auxin-responsive genes have been identi ed, Acknowledgements including ARF, SAUR and GH3 families (Hagen and Guilfoyle 2002). ARF7 and ARF19 are key components in a developmental This research was supported by the National Natural Science Foundation of pathway regulating lateral root formation (Okushima et al. China (31730093 and 31470503) and the Fundamental Research Funds for 2007). In the present study, ZxARF7 was significantly induced the Central Universities (lzujbky-2018-k01). at both 6 and 24 h under salt treatment (Table 2), suggesting an important function to regulate root growth of Z. xanthoxylum. References Most SAURs are reported to promote cell expansion and Almeida DM, Oliveira MM, Saibo NJM (2017) Regulation of Na+ and K+ hypocotyl elongation (Spartz et al. 2012; Kong et al. 2013). homeostasis in plants: towards improved salt stress tolerance in crop However, few have studied SAURs under salt treatment. Our plants. Genetics and Molecular Biology 40, 326–345. doi:10.1590/ data show that ZxSAUR16, ZxSAUR32 and ZxSAUR35 were 1678-4685-gmb-2016-0106 significantly upregulated in the initial stage of salt treatment Ashburner M,Ball CA, BlakeJA, Botstein D, ButlerH, Cherry JM,Davis AP, (Table 2), indicating potential involvement of these SAURs in Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, salt responses. 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