Ning Wang, Xiangru Wang,Hengheng Zhang, Xiaohong Liu,Jianbin Shi, Qiang Dong,Qinghua Xu, Huiping Gui,Meizhen Song, Gentu Yan

State Key Laboratory of Cotton Biology, Institute of Cotton Research of Chinese Academy of Agricultural Sciences, Anyang 455000, Henan,China

Keywords:

ABSTRACT Mepiquat chloride(MC)priming alleviates the effects of salt stress during seed germination in cotton (Gossypium hirsutum L.), but the mechanisms underlying its effects are unknown.We found that MC priming increases salt tolerance, as evidenced by marked increases in seed vigor and germination rates,and alleviated salt toxicity by reducing Cl?accumulation in germinating seeds.Consistently, electrophysiological experiments revealed that the seeds with MC priming displayed superior Cl?exclusion ability in the root apex.These beneficial effects of MC priming were abolished by the abscisic acid(ABA)-synthesis blocker fluridone under salt stress.MC priming induced an early response to acclimatization and stress,as indicated by rapidly increasing ABA content during initial exposure to salt stress.Transcriptome analyses revealed that MC priming induced an array of differentially expressed genes(DEGs)in germinating seeds.The most noticeable changes in germinating seeds were MC priming-induced increases in the expression of DEGs encoding components of ABA biosynthesis, ABA catabolism, and ABA signaling pathways under salt stress.MC priming also increased the expression of some DEGs encoding Cl?ion transporters(e.g.CCC,SLAC1/SLAH1/SLAH3,CLC,and ALMT9)in germinating seeds.These results indicate that MC priming-induced ABA contributes to Cl?homeostasis in tissues and acts as a positive regulator of salt tolerance via regulation of Cl?transporters (particularly CCC and SLAC1/SLAH1/SLAH3).Taken together, these findings shed light on the molecular mechanism underlying MC-mediated tolerance to salt stress during seed germination.

1.Introduction

Cotton (Gossypium hirsutum L.) is one of the most important fiber crops in China and is widely cultivated owing to its value as a spinnable fiber,protein source,and edible oil[1,2].Currently, Xinjiang is the largest cotton planting area in China, but soil salt stress in the region limits cotton fiber quality and yield [3].Although cotton is moderately salttolerant,its growth is suppressed under high-salt conditions,especially at the germination stage[1,4].Seed germination is a vital life-cycle transition in plants.Successful plant growth is dependent on germination, particularly in salt affected areas.Thus, seeds must remain viable for long durations in high-salt conditions and germinate under salt stress[5].The successful germination of mature seeds in a salt environment is the beginning of salt tolerance in the plant life cycle[6].

High levels of seed germination and establishment under salt stress are key traits in seed breeding.However, breeding for salt tolerance is challenging,owing to variation in climatic conditions and the complex genetic basis of the adaptive responses of plants to salt stress [7,8].Treatment with exogenous substances is an effective method to improve plant abiotic stress resistance.This approach benefits from the availability of regulators and cost effectiveness, with negligible detrimental effects on the environment [1,4,9].Mepiquat chloride (MC), a plant-growth regulator, is a seed priming agent that improves the response of cotton to various abiotic stresses, including salt stress [10].Our previous research has demonstrated that pretreatment with MC can increase cotton seed vigor by increasing the antioxidant enzyme activity and respiratory rate, thereby increasing seed germination and seedling growth in response to salt stress [4,11].These results demonstrate that MC priming is a promising approach for enhancing cotton seed germination under salt stress.However, the detailed mechanisms underlying MCmediated tolerance to salt stress during seed germination remain unclear.

Excess salts in the soil cause an ionic imbalance,resulting in cellular toxicity, with adverse effects on plant growth and productivity [12].Sodium (Na+) is the predominant ion in the vast majority of areas with saline soil.Salt toxicity is often attributed to the excessive accumulation of Na+in plants,and Na+toxicity is also associated with the ability to maintain the acquisition and distribution of K+in plants [4,5].Intracellular K+and Na+homeostasis is important for enzyme activity,maintenance of membrane potential, and osmotic potential[12,13].Thus, maintaining an optimal K+/Na+ratio is a major strategy by which plants cope with salt stress [4,14].Plants have developed complex mechanisms to maintain the intracellular ionic homeostasis of K+/Na+and adapt to salt stress.Conserved salt tolerance mechanisms control net Na+transport across the plasma membrane (SOS1 antiporter/HKT transporters)and/or tonoplast(NHXs antiporters),minimizing cytosolic and organellar Na+toxicity by ensuring the maintenance of K+/Na+homeostasis in plant tissues [12,15,16].The maintenance of K+/Na+homeostasis can also be achieved by improving K+retention in tissues [2,14].Molecular studies have indicated that the inward-rectifier K+channel AKT1 and the high-affinity K+transporter HAK5 are two major pathways for K+uptake by plant root cells [17,18].AKT1 and HAK5 contribute to the physiological adaptation of plants to salt stress[2,4,17].Accordingly,studies of seed germination under salt conditions have focused on the balance between Na+and K+[4,5,19].

An increase in Na+content is always accompanied by Cl?accumulation in plants exposed to salt stress, and the excessive accumulation of cytosolic Cl?is detrimental to several important metabolic processes [20,21].Chloride poisoning hinders development owing to antagonistic anion?anion competition, suppression of root and tuber output,slow shoot growth and germination,and other effects[20,22].Shoot Cl?content is maintained below toxic levels by restricting xylem-driven root-to-shoot transport[21].The key transporters and channels involved in shoot and root Cl?exclusion have not been fully identified.The nitrate transporter 1/peptide transporter (NRT1/PTR) family 2.5 protein NPF2.5 may regulate Cl?exclusion from root cells under salt stress [23].Some candidate proteins, such as cation chloride co-transporters CCC and s-type anion channels SLAC1/SLAH1/SLAH3, may be involved in Cl?retrieval from xylem[20,21,23].In addition to Cl?exclusion, the chloride channels CLC and aluminum-activated malate transporters ALMT9 are involved in vacuolar Cl?compartmentalization and thus reduce cytosolic Cl?accumulation and toxicity in tissues[24,25].However, most previous studies have focused on the effects of salt stress at the seedling stage,and studies linking the maintenance of Cl?homeostasis with seed germination under salt stress are lacking.

The process by which plants adapt to abiotic stress is regulated primarily by abscisic acid (ABA), which plays a crucial role in the response and tolerance of plants to environmental stresses including salt stress [6,26].Transcriptomic studies [15,22]have revealed that many ABA biosynthesis and signaling genes are upregulated under Cl?salt stress and that the ABA signaling pathway is involved in the regulation of salt stress-reponsive genes.Salt stress can increase ABA accumulation in plants by activating the expression of synthetic pathway genes,including zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED),short-chain dehydrogenase reductase (SDR), and abscisicaldehyde oxidase (AAO) [26-28].As ABA increases, it binds to the pyrabactin resistance protein 1/PYR-like proteins (PYR1/PYLs)-type 2C protein phosphatase(PP2C)complex and blocks PP2C dephosphorylation activity, allowing the activation of sucrose-nonfermenting kinase-1-related protein kinase 2 s(SnRK2s) [28,29].Consequently, the activated SnRK2s further phosphorylates downstream substrates,leading to physiological and cellular responses that counter stress responses[19,28].The ABA signaling pathway exercises a positive regulatory function in salt tolerance by regulating ion homeostasis [30].The key roles of ABA in salt stress reflect its ability to increase the activity and/or expression of ion transporters, achieving K+, Na+, and Cl?homeostasis [26-28].However, the detailed mechanisms by which ABA regulates these processes during seed germination under salt stress remain elusive.Can MC priming enhance salt stress tolerance by modulating the homeostasis of ABA operating upstream of ion transport systems?

The aim of this study was to elucidate the mechanism(s)underlying the effects of MC priming on seed germination under salt stress.We investigated the effects of MC priming on seed germination index,ion(Na+,K+,and Cl?)contents,and ABA content during seed germination under salt stress.We applied a noninvasive micro-test technology (NMT) to investigate the electrophysiological patterns of Na+,K+,and Cl?flux during seed germination with and without ABA synthetic inhibitor treatment.We also applied RNA-Seq to identify the molecular events associated with the MC-induced amelioration of salt stress.

2.Materials and methods

2.1.Plant materials and general conditions

Mature cotton seeds of J1020 were collected from the Cotton Research Institute of the Chinese Academy of Agricultural Sciences and stored at ?4 °C until presoaking and germination.Seeds were surface-sterilized with 10% sodium hypochlorite for 5 min and rinsed with distilled water three times.Seeds were presoaked in distilled water or 200 mg L?1MC solution for 12 h at 28°C in the dark[4].Seeds were placed on wet filter paper in Petri dishes with 12 mL of NaCl at four levels:control(0),weak stress(100 mmol L?1),moderate stress(200 mmol L?1), and severe stress (300 mmol L?1) [31].Petri dishes were placed in a growth chamber (Shanghai Yiheng Technology Co., Ltd., Shanghai, China) in the dark and exposed to a 14 h photoperiod, 28 °C/20 °C day/night temperature and ～80% mean humidity for germination.A randomized design was used with three replicates for each treatment.At indicated time points during salt treatment,samples were harvested, growth parameters were determined, and corresponding materials were frozen at ?80 °C for further analysis.To determine whether ABA is involved in MC-enhanced salt tolerance, 10 μmol L?1fluridone (ABA synthesis inhibitor) was added to each treatment during seed germination following Tsai and Gazzarrini [32].

2.2.Germination and growth measurement

Germination and time to germination were determined at 24 h intervals for 4 days.Four days after germination, three germination parameters: germination percentage, mean germination time (MGT), and seed vigor index (VI), were calculated following Guo et al.[33].

2.3.Determination of tissue ion concentrations

To measure Na+,K+,and Cl?contents,intact fresh seed samples at 3 days after germination (DAG3) were rinsed in deionized water, oven-dried at 105 °C for 10 min, and dried to constant weight at 70 °C.Approximately 0.5 g of finely ground plant samples were digested in H2SO4?H2O2as described previously[34].K+and Na+concentrations were determined using an atomic absorption spectrophotometer (SpectAA-50/55; Varian, Sydney,Australia).The Cl?content was determined by silver ion titration with a chloridometer (model 442-5150; Buchler Instruments,Lenexa, KS, USA) according to the manufacturer’s instructions.The elemental compositions of hypocotyl regions were examined with a transmission electron microscope(TEM,JEOL 2100,Tokyo,Japan) equipped with an energy-dispersive X-ray (EDX) attachment at China Agricultural University.The samples of DAG3 were post-fixed, dehydrated, infiltrated, and embedded according to previously published methods [35]and examined using TEM.Samples were collected for X-ray microanalysis and the relative weights of mineral ions in hypocotyl cells of the embryo were quantified automatically according to net K-shell X-ray peak counts after subtraction of background X-ray counts.

2.4.Measurement of net Na+, K+,and Cl?fluxes

Net fluxes of Na+, K+, and Cl?in primary roots of samples at DAG3 were measured non-invasively using the NMT (NMTYG-100; Younger, Amherst, MA, USA) as described by Sun et al.[13]and Wang et al.[11]at the Xuyue(Beijing)BioFunction Institute.Construction of Na+-, K+-, and Cl?-selective microelectrodes followed standard procedures [14,36].Briefly,cleaned intact DAG3 samples were transferred into test solutions (0.5 mmol L?1NaCl, 0.1 mmol L?1KCl, 0.1 mmol L?1CaCl2, 0.1 mmol L?1MgCl2, 0.3 mmol L?1MES, 0.2 mmol L?1Na2SO4, pH 6.0 adjusted with Tris or HCl) to equilibrate for 30 min, with the primary root completely immersed.After equilibration,the samples were immobilized on the bottom of a measuring chamber containing 5 mL of fresh test solution.Transient ion fluxes were measured at the root apex region(about 300 μm from the root tip).Steady ion fluxes were continuously recorded for 5-10 min, and ion flux estimates were calculated with JCal V3.0 (a free MS Excel spreadsheet,youngerusa.com or xbi.org).All values represented net extracellular flux, where positive values indicate efflux and negative values indicate influx.Five or six individual samples from each treatment were measured.

2.5.Determination of plant gibberellic acid (GA) and ABA contents

GA and ABA were determined according to standard protocols described by Pan et al.[37].Germinated seeds (50 mg) were harvested at 0, 12, and 24 h after salt stress treatment, and high-performance liquid chromatography?tandem mass spectrometry determinations of GA and ABA were performed using a liquid chromatography instrument equipped with a mass spectrometer(Shimadzu, Kyoto,Japan).

2.6.RNA isolation and RNA-Seq analysis

After salt stress treatment for 12 and 24 h, samples were collected.Ten germinated seeds were mixed and used as a single biological replicate for each treatment.Tissue samples were immediately frozen in liquid N2and stored at ?80 °C,with three biological replicates for each treatment.Total RNA was extracted from germinated seeds using TRIzol reagent(Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.Libraries were constructed using High Throughput Illumina Strand-Specific RNA Sequencing Library protocols[22].Library construction and sequencing were performed by BGI-Shenzhen Company(Shenzhen,China)on the Illumina HiSeq 2000 platform [6].To obtain clean reads, low-quality reads, adaptors, and reads with ambiguous bases (N) were removed using SOAPnuke software, developed by BGIShenzhen.After quality trimming, clean reads were mapped to the reference genome using HISAT [38]with standard mapping parameters.A total of 69,794 expressed genes were detected, including 11,175 predicted as new.Novel coding transcripts were mapped to reference sequences to generate a complete reference set for detection and mapping using Bowtie 2 [39].The fragments per kilobase of exon per million mapped fragments(FPKM)method was used to quantify gene expression.False discovery rate (FDR) was calculated to determine a P-value threshold in multiple tests.To identify differentially expressed genes (DEGs), FDR ＜0.001 and |log2fold change| ＞ 1 were used as thresholds.The Blast2GO program(https://www.blast2go.com/)was employed to obtain Gene Ontology (GO) annotations.Functional interpretation was further completed by assigning DEGs to metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database (www.genome.jp/dbget/).Pathways with a q-value ＜0.01 were considered significantly enriched.The sequence data have been uploaded to the SRA database of the National Center for Biotechnology Information under accession number PRJNA587164.

2.7.Statistical analysis

Means were compared by one-way analysis of variance(ANOVA), followed by t-tests or Duncan’s multiple range tests (P＜0.05).SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

3.Results

3.1.MC priming promoted salt tolerance during seed germination stages

Fig.1 –Effect of increasing NaCl concentration on seed germination of W priming(A)and MC priming(B), mean germination time(C)and seed vigor index(D)in the initial 4 days.W,seeds pretreated with water;MC,seeds pretreated with MC.Different lowercase letters denote significant differences between salt treatments within the same priming treatment at P＜0.05, as determined by Duncan’s multiple range test.* indicates significant differences between priming treatments under the same salt treatment at P＜0.05,as determined by t-test.

Under normal conditions, seed germination was more rapid for MC priming than for water(W)priming.The rate of germination was 86.1%for seeds with MC priming after 4 days of germination,10.2% higher than that of W priming (Fig.1A, B).In comparison with normal conditions, the germination of cotton seeds was significantly delayed by NaCl, with more severe inhibition at higher concentrations(Fig.1A,B).MC priming effectively reduced the adverse effects of salt stress on germination, with respectively 41.9%,35.0%,and 8.1%increases in germination rates under mild (100 mmol L?1), moderate (200 mmol L?1), and severe(300 mmol L?1) salt stress, compared to those for W-primed seeds.Salt stress significantly extended mean germination time(MGT) and reduced seed vigor index (VI), while MC priming increased the NaCl-induced aggravation of MGT and VI inhibition(Fig.1C,D).Based on these phenotypes,MC priming significantly alleviated the negative effects of salt stress during seed germination.Moderate salt stress was used in subsequent experiments.

Table 1–Effect of MC priming on the Na+, K +, and Cl- contents in seeds during in vitro germination for 3 days.

3.2.MC priming regulated ion homeostasis during response to salt stress

In seeds with both MC and W priming, salt stress increased Na+concentrations compared to levels in controls, and the increase was weaker for W priming than for MC priming(Table 1).Salt also significantly increased the Cl?concentration in seeds with both MC and W priming compared to levels in controls.As expected, MC priming significantly decreased the Cl?content under salt stress,with a 47.9%decrease during salt stress compared to the content in seeds with W priming.The K+content of seeds with W priming decreased significantly by 19.6% under salt stress, but the decrease was reduced by MC priming (Table 1).Salt is toxic to seeds when it accumulates in embryos[5].Interestingly,no K+or Na+was detected in hypocotyl cells,suggesting that those ions do not play crucial roles in embryos in the response to germination under salt stress (Table 1).However, salt stress markedly increased Cl?concentrations, showing ～8.7-fold and 42.9%increases in hypocotyl cells of seeds with W priming and MC priming,respectively,compared to concentrations in controls.These findings suggest that the beneficial effects of MC priming on salt stress are mediated partially by regulation of Cl?homeostasis.

3.3.Ion fluxes in response to MC priming during salt stress

To elucidate the physiological mechanisms involved in plant response to the combination of MC priming and salt stress,we applied NMT to investigate the electrophysiological patterns of Na+,K+,and Cl?flux.Under control conditions,we detected stable and constant influxes of Na+and K+in germinating seeds; however, rates of influx were significantly higher for the MC priming treatment (Fig.2A, B).In contrast to Na+and K+, the germinating seeds showed slight effluxes of Cl?, with mean values of respectively 199.9 and 509.5 pmol cm?2s?1in W-and MC-priming treatments under control conditions(Fig.2C).Salt stress induced a marked net Na+influx and Cl?efflux in germinating seeds (Fig.2A, C), and a more pronounced effect was observed in the MC priming treatment with steady state values of ?6819 and 1605 pmol cm?2s?1, respectively,compared with those for the W-priming treatment(?1148 and 1145 pmol cm?2s?1,respectively).However,net K+influx rates were markedly decreased by salt stress (Fig.2B).More pronounced effects were observed in the W-priming treatment,and salt stress led to a marked shift in K+influx toward a slight K+efflux(68.9 pmol cm?2s?1).

Fig.2–Effect of MC priming on Na+flux(A),K+flux(B),and Cl?flux(C)with or without addition of fluridone during germination.All values represent net extracellular flux,where positive values indicate efflux and negative values indicate influx.W,seeds pretreated with water;MC,seeds pretreated with MC;CK,germination in water;T,germination in salt;F,with the addition of fluridone.Different lowercase letters denote significant differences between salt treatments within the same priming group at P＜0.05,as determined by Duncan’s multiple range test.*indicates significant differences between priming treatments under the same conditions at P＜0.05,as determined by t-test.

3.4.MC priming rapidly induced ABA production but repressed GA content during salt stress

Under normal conditions, ABA concentrations decreased markedly in seeds during germination and more pronounced effects were observed after MC priming(Fig.3A).ABA content in seeds was increased significantly by W priming after 24 h of salt stress,but the increase was not observed in the first 12 h of salt stress.MC priming rapidly induced ABA production in response to salt stress.For example, after 12 h of salt stress,the ABA content in seeds with MC priming increased 2.91 ng g-?1when compared to that in the control.Simultaneously, the ABA contents were increased 6.30 ng g?1and 3.74 ng g?1by MC- and W priming after 24 h of salt stress.These results indicated that ABA was produced earlier and at higher levels after MC priming.Previous studies have demonstrated that GA also plays a key role in seed germination [9,40].Under normal conditions, MC priming reduced the levels of GA(GA1+ GA7) in seeds in the first 12 h of germination, but significantly increased the GA levels within 24 h after germination (Fig.3B).Salt stress drastically reduced GA biosynthesis after both MC and W priming during germination for 24 h,and more pronounced effects were observed for MC priming.These results suggested that the production of ABA, not GA, plays crucial roles in the response to germination under salt stress.

Fig.3–Effects of MC pretreatment on ABA content(A)and GA content(B)during in vitro germination for 0,12,and 24 h in cotton seeds.W,seeds pretreated with water;MC,seeds pretreated with MC;CK,germination in water; T,germination in 200 mmol L?1 NaCl.Different lowercase letters denote significant differences between salt treatments within the same priming treatment at P＜0.05,as determined by Duncan’s multiple range test.

3.5.Role of rapid ABA accumulation in salt stress response of seeds with MC priming

Fig.4–Effect of MC priming on seed germination rate(A)and seed vigor index(B)with or without addition of fluridone in the initial 4 days.W,seeds pretreated with water;MC,seeds pretreated with MC;CK,germination in water;T,germination in salt;F,with the addition of fluridone.Different lowercase letters denote significant differences between salt treatments within the same priming group at P＜0.05, as determined by Duncan’s multiple range test.* indicates significant differences between priming treatments under the same conditions at P＜0.05,as determined by t-test.

The rapid and strong accumulation of ABA in response to salt stress prompted us to investigate its potential function in combating this environmental stress.To characterize the function of ABA in the response to salt stress, fluridone, an inhibitor of ABA synthesis, was used.Under normal conditions, fluridone treatment sharply increased the germination rate in W and MC priming groups, by 8.4% and 6.7%,respectively (Fig.4A).Seed VI showed similar trends in response to fluridone (Fig.4B).As expected, the addition of fluridone to germinating solutions further reduced seed vigor,expressed as germination rate and VI, with no significant differences between W- and MC-priming treatments.These results supported the hypothesis that tolerance to salt stress conferred by MC priming is mediated, at least in part, by an ABA-dependent pathway.

To further evaluate the contribution of ABA to the response to MC priming,we next performed pharmacological experiments using an ABA-synthesis inhibitor to test transient ion fluxes.The application of fluridone could potentially activate the influx of Na+and K+in germinating seeds under control or salt conditions;however,the influx rates in the MCpriming treatment were markedly higher than those for the W-priming treatment (Fig.2A, B).These results indicate that increases in Na+and K+contents following MC priming under salt conditions are not determined by an ABA-dependent pathway.In the presence of fluridone, Cl?efflux rates under control conditions were increased in W- and MC-priming treatments with steady state values of 772 and 1108 pmol cm-?2s?1, respectively, compared with those in the absence of fluridone (Fig.2C).In stress conditions, fluridone treatment inhibited salt stress-induced Cl?efflux in both W- and MCpriming treatments.No significant differences were observed in Cl?efflux for the W- and MC-priming treatments, suggesting that ABA is required for the induction of Cl?efflux elicited by MC priming under salt stress,at least in part.

3.6.Effects of MC priming on transcriptomes during seed germination stages

To investigate the mechanisms by which MC promotes cotton seed germination under salt stress, we performed an RNA-Seq analysis of W priming and MC priming seeds under control conditions(CK)and salt treatment(T)for 12 and 24 h.RNA-Seq was performed in three biological replicates for each treatment.Twenty-four cDNA libraries with over 1.28 billion raw reads and 168.6 Gb of data were obtained (Table S1).Following the removal of low-quality regions, adapters, and all possible contamination, 1.12 billion clean reads were successfully mapped to the cotton genome, the majority of which were uniquely mapped reads (66.9%).The correlations observed between the three biological replicates for gene expression levels in the W and MC priming were higher than 0.99, indicating reproducibility (Fig.S1).We also performed quantitative real-time PCR analyses of fifteen differentially expressed genes (DEGs) to validate the RNA-Seq results (Fig.S2).The qRT-PCR data were consistent with the RNA-seq data,and a significant positive correlation(R2= 0.895)supports the reliability of the RNA-seq data.

To identify the effects of MC priming on the transcriptome under salt stress,we examined the DEGs in two comparisons:salt treatment versus control conditions (T vs.CK) and MC priming versus W priming(MC vs.W) during subsequent salt stress(at 12 and 24 h).Fig.5A shows a four-way Venn diagram of all 41,937 DEGs detected in all samples of MC primed and W-primed seeds under control and salt stress for 12 h.We identified 37,773 DEGs in seeds with MC priming under salt stress for 12 h (Fig.5A).The number of DEGs in seeds with W priming (31,924 genes) at this stage of stress was lower than that in seeds with MC priming.These findings were suggestive of large,coordinated shifts in gene expression at the early stages of salt stress in MC priming seeds.There were 578 common DEGs for the T vs.CK and MC vs.W comparisons.A total of 342 specific DEGs were influenced only by MC priming under salt stress for 12 h.

More DEGs were detected with extended NaCl exposure times (Fig.5B), and more DEGs were detected at the later stage of stress (24 h; 50,045 DEGs) than at the early stages(12 h; 41,937 DEGs).During this stage, 4510 common DEGs were observed in the T vs.CK and MC vs.W comparisons.According to the KEGG pathway enrichment analysis, 203 genes were involved in carotenoid biosynthesis (including ABA metabolism) and plant hormone signal transduction,and 148 of these genes were significantly up-regulated after treatment with MC(Fig.S3;Table S2).In other modules,1354 DEGs were influenced only by MC priming under salt stress for 24 h.Additionally, 251 genes were enriched for the GO term “defense response, response to stimulus, signal transduction and cellular response to stimulus” in this module(Fig.S4).These enrichment results suggest that ABA metabolism and plant hormone signal transduction were substantially modified by MC priming during salt stress.For most DEGs, there was a general correlation (R2= 0.70, P＜0.0001)betweenandlevels (Fig.5C).Thus,the patterns of gene expression were generally similar,reflecting the similar post-germinative metabolic rates in these tissues.We concluded that MC pretreatment induced faster and more intense adjustments to trigger or enhance salt responses.

Fig.5– Venn diagram showing numbers of overlapping genes that were differentially expressed between MC-treated seeds and seeds under salt stress for 12 h (A) and 24 h(B).Correlations between differentiallye xpressed genes inand(C).W,seeds pretreated with water;MC,seeds pretreated with MC;CK,germination in water;T,germination in 200 mmol L?1 NaCl.

3.7.MC priming induces the metabolism and signaling of ABA in response to salt stress

DEGs were enriched for ABA metabolism and signal transduction.A schematic overview of MC priming and its effects on ABA metabolism and signal transduction is provided (Fig.S5; Fig.6).Compared to W priming, ABA biosynthesis-related genes, i.e., ZEP and SDR, in addition to the ABA catabolism gene CYP707A, were increased by MC-priming seeds during the early phase under normal conditions (Fig.6A).However,the biosynthesis and catabolism of ABA(but not of AAO)were repressed at later time points (24 h) after MC priming.MC priming also significantly up-regulated ABA signaling-related genes, particularly during the later phases (Fig.6B).In response to salt stress, the expression of ABA biosynthetic genes (excluding AAO) and catabolism-related genes decreased significantly at 12 and 24 h of seed germination with W priming (Fig.6A).However, genes encoding essential proteins in the ABA signaling pathway, including PP2C and SnRK2s, were induced only in the late phase of salt stress in seeds with W priming(Fig.6B).MC priming up-regulated ABA biosynthesis but down-regulated catabolism in the first 12 h of salt stress compared to W priming(Fig.6A), and this trend matched the more rapid ABA accumulation observed in MC priming (Fig.3A).MC priming significantly induced ABA signaling by increasing the expression of PYR1/PRLs and SnRK2s and repressing the expression of PP2C during later phases of salt stress(Fig.6B).These findings strongly support the theory that earlier and more active ABA homeostasis and ABA signaling occur in response to MC priming under salt stress.

Fig.6 –Heat map of key genes involved in ABA metabolism(A)and ABA signaling(B)in response to MC priming and salt stress in a transcriptome analysis.W,seeds pretreated with water;MC, seeds pretreated with MC;CK,germination in water;T,germination in 200 mmol L?1 NaCl;ZEP,zeaxanthin epoxidase; NCED,9-cis-epoxycarotenoid dioxygenase;SDR,short-chain dehydrogenase reductase; AAO,abscisic-aldehyde oxidase;CYP707A,abscisic acid 8’-hydroxylase; PYR1/PYLs,pyrabactin resistance protein1/PYR-like proteins;PP2C,protein phosphatase 2C; SnRK2s,sucrose nonfermenting kinase-1-related protein kinase 2 s.

3.8.MC priming mediates the expression of genes related to key Cl?transporters under salt stress

Several genes altered by MC priming are involved in Cl?homeostasis; accordingly, we further characterized these during MC priming.We identified 32 genes involved in Cl?transport in cotton seeds during germination(Fig.7).Five CCC homologs, four SLAC1/SLAH1/SLAH3 homologs, fifteen CLC homologs, and eight ALMT9 homologs were found.Under normal conditions, the transcript abundances of CCC and SLAC1/SLAH1/SLAH3 were higher during the later phase for MC priming than for W priming,and this trend corresponded to the higher Cl?efflux rate observed in MC priming (Fig.2C).The opposite trend was observed in seeds with MC priming,in which CLC was significantly repressed compared to levels in seeds with W priming.Salt stress drastically suppressed the levels of these ions transporters (except transcript abundances of CCC after 24 h of salt stress) in seeds with W priming, particularly the expression of SLAC1/SLAH1/SLAH3.For instance, the meanvalues for SLAC1/SLAH1/SLAH3 homologs were ?4.96 and ?2.90 after respectively 12 h and 24 h of salt stress.Compared to W priming, MC priming significantly boosted the expression of genes encoding those ion transporters after 12 h and 24 h of salt stress,particularly during later phases.The mean lvalues for CCC,SLAC1/SLAH1/SLAH3, CLC, and ALMT9 homologs were respectively 0.25, 2.16,?0.15,and 0.17 after 12 h of salt stress,but 1.55, 2.71, 1.08, and 0.72 after 24 h of salt stress.These results suggested that these genes (particularly CCC and SLAC1/SLAH1/SLAH3) involved in Cl?transport are involved in MC-triggered tolerance of salt stress.

Fig.7–Heat map of key genes involved in Cl?transport in response to MC priming and salt stress in a transcriptome analysis.W,seeds pretreated with water;MC,seeds pretreated with MC;CK,germination in water;T,germination in 200 mmol L?1 NaCl.CCC,cation chloride co-transporter;SLAC1/SLAH1/SLAH3,s-type anion channel-associated;ALMT9,aluminum-activated malate transporters;CLC,chloride channels.

4.Discussion

In addition to being a critical stage in the initiation of the plant life cycle, seed germination is the stage most sensitive to environmental factors during the plant growth process[6,19].As a major abiotic stress, salinity strongly influences seed germination, causing low rates of germination and seedling establishment[1,4].Similar phenomena were observed in our study, including reductions in seed vigor and germination rates under salt stress(Fig.1).The exogenous use of chemicals to reduce the adverse effects of abiotic stresses has implications from both a theoretical and practical perspective[7,9,27,30].In this study, pretreatment of seeds with MC effectively alleviated the impact of salt stress on seed germination (Fig.1).MC priming improved salt tolerance in cotton by enabling Cl?homeostasis in tissues, and the regulation of Cl?homeostasis was conferred by an ABAdependent signaling pathway required to activate stressresponsive genes and increasing the expression of Cl?transporters (particularly CCC and SLAC1/SLAH1/SLAH3).

Increasing K+accumulation, reducing Na+uptake, and reducing the Na+/K+ratio might be a crucial strategy for plants to overcome salt stress [12-14].We predicted that the mechanism underlying MC-induced salinity tolerance would differ between germinating seeds and seedlings.We found that MC priming efficiently increased Na+accumulation under salt stress owing to higher influx rates of Na+,thus resulting in a significant reduction in K+/Na+ratios in germinating seeds(Table 1; Fig.2A, B).Our results are in accord with previous observations in G.hirsutum and rice seeds under salt-stress conditions [4,19].No K+or Na+was detected in both MC- and W-priming hypocotyl cells after 3 days of salt stress.These findings suggest that Na+/K+homeostasis plays an important but not a crucial role in MC-mediated tolerance to salt stress during seed germination stages.This phenomenon can probably be explained by varying physiochemical responses to salt stress in plants at different developmental stages[8].A newly published study [6]has also shown that many these ionic transporters in seeds are not differentially expressed under salt stress, implying that a completely different mechanism may protect plant against salt stress during seed germination.

In general,crops(such as cotton,rice,and barley)are more sensitive to Na+than to Cl?[10].However,Cl?is toxic to plants when it accumulates to high levels,and even for reported Cl?-tolerant species, a tissue content of 15-50 mg g?1DW can severely inhibit plant growth [24].In the present study, Cl?accumulation reached nearly 95 mg g?1DW in seeds under salt treatment (Table 1).We inferred that a high Cl?concentration in tissues was the principal cause of salt-induced growth reduction.This dilemma may be resolved by restricting xylem-driven root-to-shoot transport.We showed that MC priming plays a positive regulatory role in salt tolerance by regulating this process.Electrophysiological experiments revealed that seeds with MC priming displayed superior Cl?exclusion ability in the root apex (Fig.2C).This finding is consistent with the observation (Fig.7) that MC priming increased the expression levels of most DEGs encoding Cl?ion transport genes (CCC and SLAC1/SLAH1/SLAH3), a development expected [21-24]to increase Cl?exclusion from shoot and salinity resistance.The transporter NPF2.5 is involved in the excretion of Cl?from root cells under salt stress [22,23].The finding that NPF2.5 is not expressed in germinating seeds indicates that the gene does not play a vital role in salt tolerance during germination.These results suggested that MC priming decreases salt-induced Cl?accumulation, mainly by increasing shoot Cl?exclusion transporters, rather than by increasing NPF2.5 expression.However, the involvement of other unknown transporters in Cl?exclusion from root cells cannot be ruled out.MC priming increased the transcript abundances of CLC and ALMT9 under salt stress (Fig.7), helping to compartmentalize Cl?into the vacuole and reducing Cl?toxicity in the cytosol [23-25].The regulation of Cl?uptake and translocation in plants is a significant issue,particularly under salt conditions,where Cl?toxicity may be present [22].Thus, the maintenance of Cl?homeostasis by MC priming may represent an efficient line of defense against salt stress.

As in our previous studies [10,11,41], MC-induced changes in GA and ABA metabolism modulate plant physiological responses; for example, they promote cell division, increase lateral root formation, and increase cotton seed vigor.ABA catabolism and GA biosynthesis play well-established roles in seed germination [9,29,40].These findings prompted us to investigate whether MC-induced tolerance against salt stress is associated with modifications in GA and ABA metabolism.Our results supported the notion that MC priming overcomes dormancy in cotton seeds by increasing GA and decreasing ABA under normal conditions (Fig.3), as evidenced by the increased VI (Fig.1D) and ion homeostasis (Fig.2).These findings were in accord with our previous observation [4,11]that seed vigor and K+uptake by roots in cotton seedlings were significantly improved by MC priming.These results collectively suggested that the role of MC priming in improving seed vigor was partly explained by its effect on GA and ABA contents.However, under salt conditions, the levels of GA (Fig.3B) and the ABA/GA ratio (data not shown) appeared less vital in seed germination than previously [29,40]suggested.Studies [6,19,42]have also shown that ABA plays a dominant role in ABA/GA balance, particularly under unfavorable conditions.

Salt stress-induced ABA production is essential in early stress signaling and subsequent adaptation to hypersaline conditions [27-29,43].It has been demonstrated [6,19]that ABA can alleviate the effects of salt stress on seed germination in several plant species.The seeds of ABA-deficient mutants are hypersensitive to salt stress [44].Some recent studies [15,24,28]have indicated that ABA plays a positive regulatory role in salt tolerance by regulating Cl?homeostasis.In the present study, the application of the ABA synthetic inhibitor fluridone abolished the beneficial effects of MC priming on Cl?exclusion (Fig.2C), suggesting that ABA is required for the induction of Cl?efflux elicited by MC priming under salt stress.Consistently, MC priming rapidly elevated ABA biosynthesis in germinating seeds under salt stress (Fig.3A),a process that might be mediated by the promotion of Na+accumulation[19].An increase in ABA abundance in stressed tissues has been linked to the expression of one or more ABA metabolic genes, particularly genes encoding NCED and CYP707A enzymes [15,27-29].In agreement with these previous reports, the rapid induction of ABA in seeds with MC priming was caused by the activation of ABA biosynthesisrelated genes as well as the inhibition of ABA catabolic genes under salt stress (Fig.6A).Genes encoding essential proteins for the ABA signaling pathway were strongly induced by MC priming under salt stress(Fig.6B),suggesting that MC priming regulation was also involved in the ABA signaling pathway.Thus, our results strongly support the theory that earlier and more active ABA biosynthesis and signaling occur in seeds with MC priming, increasing Cl?exclusion and avoiding excessive Cl?accumulation in seeds.However, the exact mechanisms underlying the up-regulation of Cl?transporter activity in MC-induced ABA production remain to be determined.

Finally, ABA clearly mediates the inhibition of seed germination.The question remains as to how ABA accumulation increases seed germination under salt stress.The transcription factor abscisic acid-insensitive 5 (ABI5) is a key regulator of ABA-mediated seed germination [29,40].When seeds are in unfavorable environments,elevated endogenous ABA levels result in ABI5 accumulation, preventing seeds from germinating [28,45].Accordingly, we observed that the expression of ABI5 decreased significantly in germinating seeds by MC priming under salt stress (Table S3).Thus, we speculate that maintaining a low level of ABI5 in seeds by MC priming would also contribute to higher seed germination rates under salt stress, and this prospect should be investigated further.

5.Conclusions

MC priming regulated various responses to salt stress at the molecular and physiological levels and plays an important role in salt tolerance.These results suggest that MC priming efficiently prevents Cl?accumulation in tissues under salt stress by increasing Cl?exclusion, thus building a line of defense against salt stress.These effects may be explained by the rapid activation of metabolism and signal transduction of ABA,thereby regulating physiological responses involving Cl?ion homeostasis.This study sheds light on the mechanism underlying MC-mediated tolerance to salt stress during seed germination and provides evidence linking ABA biosynthesis,ABA signaling,and Cl?homeostasis under abiotic stresses.

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2020.08.004.

CRediT authorship contribution statement

Gentu Yan, Meizhen Song, and Ning Wang conceived and designed the experiments.Ning Wang, Xiangru Wang, and Hengheng Zhang performed the experiments and analyzed the data.Jianbin Shi, Qiang Dong, Qinghua Xu, and Huiping Gui contributed reagents and materials.Ning Wang and Xiangru Wang wrote the manuscript.Meizhen Song and Gentu Yan reviewed and edited the manuscript.All authors read and approved the final manuscript.

Declaration of competing interest

Authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31801312), the National Key Research and Development Program of China (2017YFD0101600), Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences,the China Agriculture Research System (CARS-18-05), and Xinjiang Production and Construction Corps Science&Technology NOVA Program(2020CB029).