Genome-Wide Transcriptomic and Proteomic Exploration of Molecular Regulations in Quinoa Responses to Ethylene and Salt Stress

Quinoa (Chenopodium quinoa Willd.), originated from the Andean region of South America, shows more significant salt tolerance than other crops. To reveal how the plant hormone ethylene is involved in the quinoa responses to salt stress, 4-week-old quinoa seedlings of ‘NL-6′ treated with water, sodium chloride (NaCl), and NaCl with ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) were collected and analyzed by transcriptional sequencing and tandem mass tag-based (TMT) quantitative proteomics. A total of 9672 proteins and 60,602 genes was identified. Among them, the genes encoding glutathione S-transferase (GST), peroxidase (POD), phosphate transporter (PT), glucan endonuclease (GLU), beta-galactosidase (BGAL), cellulose synthase (CES), trichome birefringence-like protein (TBL), glycine-rich cell wall structural protein (GRP), glucosyltransferase (GT), GDSL esterase/lipase (GELP), cytochrome P450 (CYP), and jasmonate-induced protein (JIP) were significantly differentially expressed. Further analysis suggested that the genes may mediate through osmotic adjustment, cell wall organization, reactive oxygen species (ROS) scavenging, and plant hormone signaling to take a part in the regulation of quinoa responses to ethylene and salt stress. Our results provide a strong foundation for exploration of the molecular mechanisms of quinoa responses to ethylene and salt stress.


Introduction
Quinoa (Chenopodium quinoa Willd.), a dicotyledonous plant in the Chenopodiaceae family, originated from the Andean region of South America and has been cultivated for about 7000 years [1]. Quinoa is an allotetraploid plant (2n = 4X = 36), deriving from the genome fusion of two related parent species in the same genus [2]. Quinoa has five major ecotypes depending on its origin centers, including Highlands originating from Peru and Bolivia; Inter-Andean valleys originating from Bolivia, Colombia, Ecuador, and Peru; Salares originating from Bolivia, Chile and Argentina; Yungas originating from Bolivia; and Lowlands originating from Chile [3].
Quinoa has been considered as a pseudo-cereal because of its grain characteristics [4]. Consumption of seeds is the most common use of quinoa. In recent years, quinoa seeds have been reported to have an exceptional balance between oil, protein, and carbohydrate [4,5]. The absence of gluten in its starch allows the development of specific foods for celiac patients [1]. Quinoa seeds are also good sources of vitamins, oil with high linoleate and linolenate content, natural antioxidants, dietary fiber, and minerals [6]. As a result, consumption of quinoa in human diet leads to lower weight gain, improved lipid profile, decreased blood glucose, and increased antioxidant intake [7,8].
In addition to nutritional value in its seeds, quinoa also has resistance to multiple abiotic stresses, including drought, cold and salinity, which allows quinoa to be cultivated in

Transcriptome Sequencing and Data Analysis
In this research, three independent biological replicates were used, and at least three whole quinoa seedlings were mixed in each replicate. Total RNA was extracted and purified using poly-T oligo-attached magnetic beads. cDNA was synthesized, and adaptors with hairpin loop structures were ligated to prepare for hybridization. The samples were then clustered on a cBot cluster generation system using TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform by Novogene Bioinformatics Technology Co. Ltd. (Beijing), and 150 bp paired-end reads were generated. The raw data of FASTQ format were uploaded to the NCBI Sequence Read Archive (SRA), and the SRA accession number is PRJNA726352.
The reference genome was downloaded from the website https://www.ncbi.nlm.nih. gov/genome/?term=quinoa (accessed on 30 June 2022), and the paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Fragments per kilobase of transcript sequence per million (FPKM) of each gene were calculated to estimate gene expression levels based on the length of the gene and reads count mapped to the gene. The genes with corrected p-value < 0.05 and absolute fold change ≥2 were considered as significant DEGs. The DEGs were then analyzed by GO enrichment analysis and KEGG enrichment analysis to predict their functions.

Protein Extraction, TMT Labeling, and Proteomics Analysis
In this research, three independent biological replicates were used for protein analysis, and at least three whole quinoa seedlings were mixed in each replicate. The proteomics analyses were performed by Novogene Bioinformatics Technology Co. Ltd. (Beijing, China). In detail, total proteins were extracted by the cold acetone method, labeled by TMT tags, and fractionated using a C18 column on a Rigol L3000 HPLC system. Shotgun proteomics analyses were then performed using an EASY-nLC TM 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with a Q Exactive TM HF-X mass spectrometer (Thermo Fisher) operating in the data-dependent acquisition (DDA) mode.
The resulting spectra from each run were searched separately against the 733788-X101SC20124467-Z02-Chenopodium quinoa Willd.-NCBI.fasta database by Proteome Discoverer 2.4 (PD 2.4, Thermo). Peptide spectrum matches (PSMs), with credibility of more than 99%, were identified, and the identified PSMs and proteins were retained and performed with FDR no more than 1.0%. Proteins with fold changes in a comparison >1.2 or <0.83 and unadjusted significance level p < 0.05 were considered as DEPs. The DEPs were then analyzed by GO and KEGG enrichment analyses. The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org (accessed on 24 May 2021)) via the iProX partner repository [34] with the dataset identifier PXD026210.

Correlation Analysis between Proteomic and Transcriptomic Results
The DEGs and the DEPs were separately counted, and the Venn diagrams were plotted according to the counted results. Correlation analysis was performed for the differential multiples of DEGs or DEPs identified in both transcriptomic analysis and proteomic analysis by R (version 3.5.1).

Quantitative Real Time PCR (qRT-PCR) Analysis
In this research, 12 DEGs were selected randomly for qRT-PCR verification, and the coding sequence (CDS) of these selected 12 DEGs are listed in the Supplementary Material Table S1. CqACTIN was used as the endogenous control. The primers were designed using Primer Premier 5.0 (Premier) and are listed in Supplementary Material Table S2. Total RNA was extracted by the CTAB method and subjected to DNase treatment (Takara, Japan). The first-strand cDNA was synthesized using M-MLV reverse transcriptase (Takara, Japan) with oligo d(T)18 primer. The qRT-PCR program contains a preliminary step of 2 min at 50 • C, 10 min at 95 • C, followed by 40 cycles of 95 • C for 60 s, 56 • C for 20 s, and 72 • C for 15 s. Three independent biological replicates and three technical replicates were used. The primer efficiency was tested by generating standard curves, and the data were analyzed by the comparative ∆∆CT method.

Physiological Indexes Detection
In this research, 4-week-old quinoa seedlings of 'NL-6 treated with water, 300 mM NaCl, or 300 mM NaCl with 100 µM ACC for 2-3 d were used for examination of physiological indexes. The nitrogen content and relative level of total chlorophyll were measured by PJ-4N plant nutrition analyzer, and the relative permeability of cell membrane, damage rate of leaves, malondialdehyde (MDA) content, soluble sugar level, and SOD activity were analyzed as previously described [35]. Three independent biological replicates and three technical replicates were used in the experiments.

Statistical Analyses
Statistical analyses were performed by SAS, and the statistical significance of the difference was evaluated by ANOVA. Means followed by the same letter were not significantly different at the α = 0.05 level.

Gene Identification and DEGs Analysis in Transcriptome
To investigate ethylene-regulated salt responses in quinoa, the 4-week-old H 2 Or, SALTr, and ACCr samples were used for transcriptomic analysis. The principal component analysis (PCA) showed the differences among different treatments and confirmed the reliability of the sequencing results (Supplementary Material Figure S1A). A total of 60,602 genes were identified, and the genes with corrected p-value < 0.05 and absolute fold change ≥2 were considered as significant DEGs. The DEGs between SALTr and H 2 Or were recognized as the components in salt responses of quinoa. The DEGs between SALTr and ACCr were recognized as functioning in ethylene-regulated salt responses of quinoa, and the DEGs between ACCr and H 2 Or were thought to be involved in ethylene responses or salt responses of quinoa. The DEGs in these three comparisons are presented in the Supplementary Material Excel S1. The heat maps with hierarchical clustering, which show relative expression of the DEGs in these comparisons, are presented in Supplementary Material Figures S2-S4.

Protein Identification and DEPs Analysis
The H 2 Op, SALTp, and ACCp samples were analyzed by proteomics. The PCA analysis showed the differences among different treatments and confirmed the reliability of the proteomic analysis results (Supplementary Material Figure S1B). A total of 9672 proteins were identified, and the proteins with fold change >1.2 or <0.83 and unadjusted significance level p < 0.05 were considered as DEPs. Similar to the analysis of DEGs, the DEPs between SALTp and H 2 Op were recognized as the components of quinoa salt responses, and the DEPs between SALTp and ACCp were recognized as functioning in ethylene-regulated salt responses of quinoa, and the DEPs between ACCp and H 2 Op were thought to be involved in ethylene responses or salt responses of quinoa. The DEPs in these three comparisons are presented in Supplementary Material Excel S3. The heat maps with hierarchical clustering, which show relative expression of the DEPs in these comparisons, are presented in Supplementary Materials Figures S6-S8.

DEPs Analysis in Ethylene Regulated Salt Responses
In order to study the DEPs in ethylene-regulated salt responses in quinoa, multiple comparisons of DEPs among SALTp-vs-H 2 Op, SALTp-vs-ACCp, and ACCp-vs-H 2 Op were conducted, and nine DEPs were overlapped in the comparisons and may play roles in ethylene-regulated salt responses of quinoa (

Correlation between the Proteomic and Transcriptomic Results
Correlation analysis between the transcriptomic data and the proteomic result identified 184 genes/proteins, which were differentially expressed when treated with salt in

Correlation between the Proteomic and Transcriptomic Results
Correlation analysis between the transcriptomic data and the proteomic result identified 184 genes/proteins, which were differentially expressed when treated with salt in quinoa ( The correlation analysis between the transcriptome and proteome also detected 117, 113, and 69 proteins differentially expressed in the comparisons between SALT and H 2 O samples, between ACC and H 2 O samples, and between ACC and SALT samples, respectively, but no expression difference was detected in their transcript levels, suggesting a possible presence of post-transcriptional modification in the proteins (Figure 3, Supplementary Material Excel S5).
In contrast, it was found that 3934, 2791, and 804 genes were detected differentially expressed at the transcript level but not at the protein level in the comparisons between SALT and H 2 O samples, between ACC and H 2 O samples, and between ACC and SALT samples, respectively ( Figure 3, Supplementary Material Excel S5), suggesting that stressregulated molecules are more likely altered at the transcript level when challenged.

Verification of RNA-seq Results by qRT-PCR
In order to verify the results obtained from the quinoa transcriptomic and proteomic analysis in ethylene-regulated salt responses, 12 DEGs were randomly selected, and their relative expression levels were examined in the quinoa seedlings treated with water, NaCl, and NaCl with ACC for 0, 3, 6, 9, 12, 24, and 36 h. The expressions of the reference genes under the different treatments are shown in Supplementary Material Excel S6. The results showed that the expressions of CqNRT2.1 and CqACO1 were increased to a peak after 6 h of salt treatment and then began to decrease, while the expressions of CqCSI, CqPER12, CqFK, and CqPDP were increased to a peak after 12 h of salt treatment and then began to decrease (Figure 4). The expression of CqABCB kept decreasing in SALT and ACC samples ( Figure 4). Together, the expressions of these 12 DEGs were obviously affected by salt and ethylene, suggesting that they may play important roles i.

Physiological Alterations by Ethylene and Salt Stress
In order to examine the physiological changes in the H 2 O-, SALT-, and ACC-treated samples ( Figure 5A), the nitrogen content, SPAD value, relative permeability of cell membrane, damage rate of leaves, MDA content, soluble sugar level, and SOD activity were detected in these samples. The results indicated that salt treatment rendered higher relative permeability of cell membranes, damage rate of leaves, MDA, and soluble sugar levels, while ethylene treatment reduced them ( Figure 5B-E). The SOD activity was activated by salt treatment, which was enhanced by ethylene ( Figure 5F). However, the relative content of total chlorophyll denoted by the SPAD value, and the N content were reduced due to salt treatment ( Figure 5G). The effects of salt on the SPAD value and N content were alleviated by ethylene treatment, although their contents in the ACC sample were still lower than in the untreated sample ( Figure 5G). Taken together, it was concluded that ethylene may regulate salt responses in different ways in quinoa.  , between ACC and H2O samples (C), and between ACC and SALT samples (E), respectively; all_tran represents all the genes obtained from the transcriptome, diff_tran represents the DEGs identified by transcriptome, all_prot represents all the proteins identified by proteome, and diff_prot represents the DEPs identified by proteome. (B,D,F) Scatter plots of expression correlation between SALT and H2O samples (B) between ACC and H2O samples (D) and between ACC and SALT samples (F), respectively. The abscissa is the differential multiple of proteins, the ordinate is the differential multiple of corresponding genes, and the correlation coefficient and p value of the transcriptome and proteome are also shown in the figures. The points represent proteins/genes: the blue points represent non-differential proteins/genes, and the green points represent DEPs/DEGs. and between ACC and SALT samples (F), respectively. The abscissa is the differential multiple of proteins, the ordinate is the differential multiple of corresponding genes, and the correlation coefficient and p value of the transcriptome and proteome are also shown in the figures. The points represent proteins/genes: the blue points represent non-differential proteins/genes, and the green points represent DEPs/DEGs.

Physiological Alterations by Ethylene and Salt Stress
In order to examine the physiological changes in the H2O-, SALT-, and ACC-treated samples ( Figure 5A), the nitrogen content, SPAD value, relative permeability of cell levels, while ethylene treatment reduced them ( Figure 5B-E). The SOD activity was activated by salt treatment, which was enhanced by ethylene ( Figure 5F). However, the relative content of total chlorophyll denoted by the SPAD value, and the N content were reduced due to salt treatment ( Figure 5G). The effects of salt on the SPAD value and N content were alleviated by ethylene treatment, although their contents in the ACC sample were still lower than in the untreated sample ( Figure 5G). Taken together, it was concluded that ethylene may regulate salt responses in different ways in quinoa.

Discussion
Quinoa, an ancient crop native to South America, has high nutritional value and health-promoting phytochemicals in seeds and has received increasing world-wide attention in the past decade [8,9,36]. Quinoa is resistant to multiple abiotic stresses including drought, cold, and salinity [9,10]. Salt stress is a major abiotic stress and affects ~6.5% of the total land of the world [9]. The effects of salt stress on plants are mainly di-

Discussion
Quinoa, an ancient crop native to South America, has high nutritional value and health-promoting phytochemicals in seeds and has received increasing world-wide attention in the past decade [8,9,36]. Quinoa is resistant to multiple abiotic stresses including drought, cold, and salinity [9,10]. Salt stress is a major abiotic stress and affects~6.5% of the total land of the world [9]. The effects of salt stress on plants are mainly divided into two components, the nonspecific osmotic stress that causes water deficit, and the specific ion effects that provoke the accumulation of toxic ions. Quinoa plants shows significant salt tolerance, but the research on quinoa responses to salt stress is still limited. High-throughput transcriptomic analysis provides a way to excavate molecular mechanism of the quinoa salt responses at the genomic scale [10,21,30,31]. Considering the importance of differentially expressed proteins in most biological processes, examination of the protein level change is more valuable and practical. Unfortunately, little is known about the molecular regulation of quinoa at the proteomic scale. In this research, 4-week-old quinoa seedlings treated with water, NaCl, and NaCl with ACC were analyzed by transcriptional sequencing and proteomics. The identified DEGs and DEPs were analyzed by GO and KEGG, and their correlation analyses was conducted. The study provides a strong foundation for further research on the molecular regulation of quinoa to ethylene and salt stress.

Plant Hormones Play Regulatory Roles in Quinoa Responses to Ethylene and Salt Stress
Plant hormones play important roles in various stress responses. For instance, ABA is thought to be essential for plant responses to abiotic stresses in many plant species including wheat, rice, and Magnolia wufengensis [19,25,37]. For example, it was reported that the rice Osnced5 mutant reduced the ABA level and decreased salt tolerance, while OsNCED5 overexpression increased the ABA level and enhanced salt tolerance, indicating the importance of ABA to the salt tolerance of rice [37]. In quinoa, the gene encoding for 9-cis-epoxycarotenoid dioxygenase (NCED) in ABA biosynthesis was strongly induced after salt treatment [30,38]. Several PP2Cs in ABA signaling were detected highly upregulated in quinoa under salt stress by transcriptional sequencing [21]. In the present study, only one abscisic acid receptor, PYL4 (LOC110710755), was detected functioning in ethylene-regulated salt responses, one abscisic acid receptor PYL4 (LOC110714604) detected playing roles in non-ethylene-regulated salt responses, and one abscisic acid receptor PYL4 (LOC110715607) detected playing roles in ethylene responses but not in salt tolerance of quinoa ( Table 2, Supplementary Materials Figure S5, Excels S2-S4, S7 and S8), suggesting that crosstalk between ABA and ethylene may exist in the quinoa stress responses.
Other hormones were also reported to be involved in quinoa stress responses. TIFY 10A, a JA response repressor, was strongly induced in responses to salt, while the GA 3-betadioxygenase (GA3OX4) in gibberellin biosynthesis was strongly inhibited under salt treatment [21]. In this study, three jasmonate-induced proteins (JIPs; LOC110715081, LOC110711071, and LOC110733576) were detected. In addition, it was reported that one ethylene receptor, ETR1, and one ethylene responsive factor (ERF) were strongly induced by salt treatment in quinoa [21,30]. In the present study, three ERFs (LOC110729845, LOC110730331, and LOC110719638) were detected in ethylene-regulated salt responses of quinoa (Table 2, Supplementary Materials Figure S5, Excels S2-S4). These findings broaden our understanding of the phytohormone-mediated regulations in the quinoa stress responses.
In the present study, we also found the other novel genes/proteins responding to salt and ethylene in quinoa. For instance, one auxin response factor (ARF; LOC110719716), one auxin binding protein (ABP; LOC110715799), and one cytokinin riboside 5'-monophosphate phosphoribohydrolase (LOG; LOC110738584) were detected ( Table 2, Supplementary Materials Figure S5, Excels S2-S4). ARF and ABP function in the auxin signaling pathway, and LOG functions in the release of a ribose 5'-monophosphate from a cytokinin nucleotide to form a biologically active cytokinin [39]. Our results indicated that auxin and cytokinin may play roles in ethylene-regulated salt responses of quinoa, and these genes/proteins may be important for the crosstalk of plant hormones.
The auxin efflux carrier (LOC110691454 and LOC110736434), auxin transporter (LOC 110706251), ARF (LOC110736906, LOC110715765, and LOC110714183), and ABP (LOC110691560) were detected from this study, and they may be involved in non-ethylene-regulated salt responses (Supplementary Material Excel S7). In contrast, the detected auxin response factor (LOC110714183), auxin efflux carrier (LOC110691454 and LOC110736434), auxin transporter (LOC110706251), and ARF (LOC110736906 and LOC110715765) may be involved in ethylene responses but not in salt tolerance of quinoa (Supplementary Material Excel S8). Taken together, these results suggest that the plant hormone auxin may play diverse roles in quinoa.

ROS Scavenging Enzymes Function in Quinoa Responses to Ethylene and Salt Stress
Salt stress causes ROS accumulation and oxidative stress aggravation [11]. ROS damage nucleic acids, oxidize proteins, and cause lipid peroxidation, while the antioxidant enzymes including GST, SOD, POD, and CAT neutralize the salt-induced ROS accumulation to protect plants from destructive oxidative reactions [12,17,18]. In detail, SOD dismutates O 2 − into H 2 O 2 , which is decomposed into water and oxygen by CAT in the peroxisomes. POD mainly catalyzes substrate oxidation with H 2 O 2 as an electron acceptor in vacuoles and cell walls in plants [40][41][42]. In plants, GSTs are multifunctional enzymes existed in different classes (Phi, Tau, Zeta, Theta, and others) and play important roles in cellular detoxification of xenobiotic protection against oxidative stress as well as diverse ligand-binding activities [43]. In this research, 9 GSTs (LOC110724460, LOC110696392, LOC110724461, LOC110728060, LOC110711174, LOC110711174, LOC110739278, LOC110713696, and LOC110727188) and 16 PODs (LOC110682117, LOC110682546, LOC110685850, LOC110692926, LOC110699378, LOC110724764, LOC110735668, LOC110694635, LOC110735670, LOC110681844, LOC11068 7369, LOC110690635, LOC110727528, LOC110699380, LOC110684661, and LOC110704239) were detected in the quinoa responses to ethylene and salt stress ( Table 2, Supplementary Materials Figure S5, Excels S2-S4). The ROS scavenging enzymes, POD5, had been reported to be functioning in salt responses of quinoa by RNA-seq [21]. In this study, two POD5 including LOC110692926 and LOC110727528 were detected in ethylene-regulated salt responses of quinoa. On the other hand, 16 PODs including LOC110683143, LOC110729735, and LOC110699379 may play roles in non-ethylene-regulated salt responses (Supplementary Material Excel S7), and 23 PODs including LOC110685846, LOC110704240, LOC110707569, LOC110711884, and LOC110704238 are probably involved in ethylene responses but not in salt responses in quinoa (Supplementary Material Excel S8). The PODs were also detected as core salt-responsive genes in both salt-tolerant quinoa and salt-sensitive quinoa by a previous RNA-seq research [21]. In this study, the SOD activity was also detected and activated by salt treatment in quinoa ( Figure 5F). In addition, it was shown that ethylene enhances the SOD activity in salt responses of quinoa ( Figure 5F), providing evidence that ethylene may mediate ROS to regulate salt tolerance of quinoa.

Osmotic Adjustment Is Important for Quinoa Responses to Ethylene and Salt Stress
High concentrations of NaCl in salt stress generate K + and H + fluxes in quinoa roots to the apoplast, so leaf osmoregulation, K + retention, Na + exclusion, and ion homeostasis confer quinoa salt tolerance [44]. In addition to these inorganic ions, the accumulation of organic substances including protein, sugars, proline, and total phenolics is also attributed to improve the quinoa salt tolerance [15,16]. The high affinity K + transporters (HKT1.2) play a key role in Na + load into bladder cells in quinoa, and the Na + in bladder cells is then collected in the bladder hairs and washed off by rain [45]. Quinoa plants were reported to accumulate more Na + than K + under salinity stress, because the K + /Na + ratio was detected to be decreased with increasing of salt concentration [44]. The cell anion channel (SLAH), nitrate transporter (NRT), and chloride channel protein (C1C) were also activated by salt stress, indicating their possible functions in salt responses of quinoa [10].

Cell Wall Structural Proteins Respond to Ethylene and Salt Stress in Quinoa
The levels of principal structural component of the plant cell wall such as lignins, pectins, celluloses, and hemicelluloses are affected by salt stress, which induce the alteration of cell wall elasticity [48,49]. Previously, it was reported that transcriptional changes of the genes involved in cell wall organization could been detected by RNA-seq after salt treatment of quinoa seedlings [30]. The genes involved in suberin and cutin biosynthesis, photosynthesis, and chloroplast were also reported to be significantly changed due to salt treatment in the bladder cells of quinoa [10]. TBLs encode the cell wall polysaccharide specific O-acetyltransferases and are probably involved in maintaining esterification of pectins [50]. In Arabidopsis, the functional study of the cellulose synthesis in salt tolerance had been previously reported [51]. In this research, 2 TBLs (LOC110715157 and LOC110685228) were detected differentially expressed ( Table 2, Supplementary Materials Figure S5, Excels S2-S4), and 5 CESs (LOC110715976, LOC110717430, LOC110689768, LOC110689717, and LOC11072 1870) in cellulose synthesis were detected. In addition, two BGLUs (LOC110739769 and LOC110724275), two BGALs (LOC110682558 and LOC110685863), and four glycine-rich cell wall structural proteins (GRPs) (LOC110732550, LOC110730178, LOC110730179, and LOC110732549), which may be involved in cell wall structure and elasticity in quinoa, were detected (Table 2, Supplementary Material Figure S5, Excels S2-S4). All these findings strongly support the importance of cell wall structure and elasticity in the quinoa stress responses.

Secondary Metabolism-Associated Proteins Respond to Ethylene and Salt Stress in Quinoa
Betalain is a tyrosine-derived, red-violet, and yellow pigment in quinoa with antioxidant activity, which plays important roles in salt responses [52]. For example, CqCYP76AD1-1 was reported in the betalain biosynthesis process in quinoa [53,54]. In this research, one CqCYP76AD1 (LOC110731693) was detected in the ethylene-regulated salt responses, although its molecular mechanism in the responses is unclear. The methyltransferases (MTs), GTs, and GPATs are transferases that transfer methyl, glucosyl, and acyl groups from one compound to another, respectively. The CHSs condense a phenylpropanoid CoA ester with three acetate units from malonyl-CoA molecules and cyclize the resulting intermediate to produce a chalcone, which is the precursor of diverse flavonoids [55]. The GELPs have high potential to be used in the hydrolysis and synthesis of important ester compounds [56]. It was reported that ectopic expression of Arabidopsis glycosyltransferase UGT85A5 enhances salt tolerance in tobacco, but knock down of the corresponding genes decreases salt tolerance at seedling and reproductive stages of rice [57,58].

Early Response Genes and Late Response Genes in Quinoa
It was reported that many genes are divided into two categories, namely early response genes and late response genes, depending on their different activation patterns in response to stimuli. The early response genes, which are also called primary response genes, are induced without de novo protein synthesis, while the late response genes, which are also called secondary response genes, require de novo protein synthesis and are induced more slowly because that synthesis needs signaling molecules or cytokines [59,60].
In this research, the correlation between proteome and transcriptome was analyzed, and the results are shown in Figure 3, Table 2, and Supplementary Materials Figure S5, Excels S2, S4 and S5. For example, the genes/proteins (GST (LOC110739278) and GLU1 (LOC110717177)) differentially expressed in both transcript and protein levels, belong to early response genes, and their proteins had already been synthesized and could be detected at early times.
The genes/proteins that were differentially expressed in transcript levels but not protein levels belonged to late response genes. Their protein levels did not accumulate within 24 h of treatment. It was suggested that their protein levels could be changed in later hours. In this research, the genes/proteins including JIPs (LOC110715081, LOC110711071, and LOC110733576), GSTs (LOC110724460, LOC110696392, LOC110724461, LOC110728060, and LOC110711174), and PODs (LOC110682546, LOC110685850, LOC110692926, LOC110699378, LOC110724764, LOC110735668, LOC110694635, LOC110735670, LOC110681844, LOC11068 7369, LOC110690635, LOC110727528, LOC110699380, and LOC110684661) were differentially expressed in transcript levels but not in protein levels, suggesting that these genes may belong to the late response genes.

Conclusions
The proposed molecular mechanism of ethylene-regulated salt responses in quinoa is complex. Under salt stress, ROS scavenging enzymes including GSTs and PODs; transporters and solutes in osmotic adjustment including HKT, PT, Na + /metabolite cotransporter, high-affinity Na + transporters, cation/H + antiporter, Na + /Ca 2+ exchanger, aquaporin, bidirectional sugar transporters, polyol transporter, and sucrose synthases; cell wall structural proteins including GLCs, β-GALs, CESs, TBLs, and GRPs; and secondary metabolism-associated proteins including GTs, GPATs, CHSs, GELPs, CYPs, and MTs are activated in responses to ethylene and salt stress in quinoa. Plant hormones, including AUX, ABA, JA, and CK, also play important roles in the responses. Considering the large number of transporters in osmotic adjustment identified in the ethylene-regulated salt responses in quinoa, it is concluded that osmotic adjustment is probably one of the main regulations for quinoa when challenged by salt stress.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants10112281/s1. Figure S1: The PCA analysis in transcriptomic (A) and proteomic analysis (B), Figure S2: The heat map with hierarchical clustering of DEGs in comparisons between SALTr and H 2 Or, Figure S3: Supplementary Material S6: The heat map with hierarchical clustering of DEGs in comparisons between SALTr and ACCr, Figure S4: The heat map with hierarchical clustering of DEGs in comparisons between ACCr and H 2 Or, Figure S5: The heat map of candidate proteins/genes in ethylene and salt responses of quinoa, Figure S6: Supplementary Material S11: The heat map with hierarchical clustering of DEPs in comparisons between SALTp and H 2 Op., Figure S7: The heat map with hierarchical clustering of DEPs in comparisons between SALTp and ACCp, Figure S8: The heat map with hierarchical clustering of DEPs in comparisons between ACCp and H 2 Op, Table S1: The sequence templates of randomly selected DEGs in qRT-PCR confirmation, Table S2: Oligonucleotide primers used in qRT-PCR confirmation, Excel S1: The summary of DEGs in single comparisons, Excel S2: The DEGs annotation in the ethylene and salt responses of quinoa, Excel S3: The summary of DEPs in single comparisons, Excel S4: The DEPs annotation in ethylene and salt responses of quinoa, Excel S5: The genes/proteins annotation in correlation analysis, Excel S6: The expression of the reference gene CqACTIN under the different treatments in this research, Excel S7: The genes/proteins playing roles in non-ethylene-regulated salt responses, Excel S8: The genes/proteins playing roles in ethylene responses but not related with salt tolerance in quinoa.