Genome-Wide Identiﬁcation and Analysis of Stress Response of Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase Genes in Quinoa

: Saline-alkali stress seriously affects the yield and quality of crops, threatening food security and ecological security. Improving saline-alkali land and increasing effective cultivated land are conducive to sustainable agricultural development. Trehalose, a nonreducing disaccharide, is closely related to plant growth and development and stress response. Trehalose 6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) are key enzymes catalyzing trehalose biosynthesis. To elucidate the effects of long-term saline-alkali stress on trehalose synthesis and metabolism, we conducted an integrated transcriptome and metabolome analysis. As a result, 13 TPS and 11 TPP genes were identiﬁed in quinoa ( Chenopodium quinoa Willd.) and were named CqTPS1-13 and CqTPP1-11 according to the order of their Gene IDs. Through phylogenetic analysis, the CqTPS family is divided into two classes, and the CqTPP family is divided into three classes. Analyses of physicochemical properties, gene structures, conservative domains and motifs in the proteins, and cis-regulatory elements, as well as evolutionary relationships, indicate that the TPS and TPP family characteristics are highly conserved in quinoa. Transcriptome and metabolome analyses of the sucrose and starch metabolism pathway in leaves undergoing saline-alkali stress indicate that CqTPP and Class II CqTPS genes are involved in the stress response. Moreover, the accumulation of some metabolites and the expression of many regulatory genes in the trehalose biosynthesis pathway changed signiﬁcantly, suggesting the metabolic process is important for the saline-alkali stress response in quinoa.


Introduction
The increasing soil saline-alkalization affects ecological environments and seriously reduces crop yields. On the contrary, the growing population in the world would require greater food production. This will lead to a contradiction between the supply and demand of food in the near future. The improvement of saline-alkali land can increase the area of the effectively cultivated land, which is very important for the sustainable development of agriculture. The total area of saline-alkali land in China is about 9.91 × 10 4 km 2 [1]. The saline-alkali land in northeast China, also known as soda saline-alkali land, contains a high concentration of carbonate, including Na 2 CO 3 and NaHCO 3 , etc. [2,3].
Salt stress leads to ion toxicity and osmotic stress, disrupting metabolism and ion homeostasis, resulting in impaired photosynthesis, disordered metabolic process, and retarded root development, and thus inhibits plant growth and development which leads to a decline in crop yields [4][5][6][7]. In addition to ion toxicity and osmotic stress, alkali stress aggravates the damage to cell structure and activities. Saline-alkali stress causes Int. J. Mol. Sci. 2023, 24, 6950 2 of 16 serious interference with metabolic processes (e.g., carbohydrate degradation and nitrogen metabolism retardation) and significant changes in the contents of inorganic and organic ions, carbohydrates (amino acids and sugars, e.g., proline, sucrose, trehalose, and fructose), ROS, and MDA, etc. [8][9][10].
The carbohydrates are mainly synthesized by starch and sucrose metabolism and related pathways, e.g., glycolysis/gluconeogenesis, and amino sugar and nucleotide sugar metabolism [11]. The starch and sucrose metabolic pathway plays important roles in plant growth and development, yield quality, and stress response. The products and intermediate products not only provide carbon sources for plant growth and development but also participate in many biological processes, acting as signal molecules [12]. Various enzymes, e.g., sucrose phosphate synthase (SPS), sucrose synthase (SUS), and invertase (INV), participate in sucrose biosynthesis, distribution, and accumulation in plants [13]. The content of sucrose is an important factor for controlling the transport tempo of nutrients, directly affecting the growth, yield, and quality of crops [14]. In plants, starch is the most abundant carbohydrate reserve as the source of carbon and energy. Glucose is the precursor of starch biosynthesis in the chloroplasts and amyloplasts [15,16]. Starch metabolism is a complex and tightly regulated process involving several classes of enzymes, including ADP glucose phosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE), starch debranching enzyme (DBE), phosphorylase (PHO), and disproportionation enzyme (DPE).
In addition to catalyzing trehalose biosynthesis, TPSs and TPPs can improve plant stress tolerance. Salt stress induces TPS expression in wheat and improves OsTPP1 expression in rice [22,23]. Overexpression of OsTPS1 in rice increases the aglucon and proline content and promotes the upregulation of some stress-response genes, thereby enhancing tolerance to cold, drought, and salinity in rice [23,24]. Ectopic overexpression of the yeast TPS1 gene in potato also enhances drought tolerance [25]. The Arabidopsis TPS1 gene plays a key role in embryo development and flowering, and AtTPS1 depletion mutants are embryonically lethal [26]. Although the TPS/TPP pathway has been well-characterized in many species, little is known about the TPPs and TPSs in quinoa.
Quinoa (Chenopodium quinoa Willd.) is an annual dicotyledonous herbaceous crop of the Amaranthaceae family. Quinoa grains have high protein contents and excellent amino acid composition, including all essential amino acids required for the human body. Quinoa grains contain abundant natural antioxidant substances, vitamins, dietary fiber, and minerals [27]. Moreover, quinoa is cholesterol-and gluten-free and is an ideal food for certain patients or sensitive people. Based on these characteristics, the research on quinoa has received extensive attention. Moreover, the publication and upgrading of the quinoa genome sequence greatly promoted the research on quinoa [28].
In this study, we identified 13 TPS genes and 11 TPP genes in quinoa by genome sequence search and conserved domain search. The CqTPS and CqTPP genes were classified according to conservative domain and motif analysis. Furthermore, transcriptome and metabolome analyses were performed and clarified the important roles of CqTPS and CqTPP genes as well as starch and sucrose metabolism in the saline-alkali stress response in quinoa.
Our study provides basic information for understanding the characteristics of the CqTPS and CqTPP gene families in quinoa and their functions in response to saline-alkali stress.

Genome-Wide Identification of TPS and TPP Gene Family in Quinoa
In order to identify TPS and TPP family members in quinoa, the amino acid sequences of AtTPS1 (Accession No.: XM_002889154.1) and AtTPP1 (Accession No.: NM_001344938.1) were used as references to perform local BLAST based on the genome database of quinoa (Chenopodium quinoa Willd.). The family members were further confirmed according to the conservative domains. As a result, thirteen TPS proteins and eleven TPP proteins were identified in quinoa (Tables 1 and 2) and were named CqTPS1-CqTPS13 and CqTPP1-CqTPP11 according to the Gene IDs. The CqTPS protein length is between 501 (CqTPS6) and 1173 (CqTPS12) amino acid residue numbers (aa), but most of the CqTPSs contained between 830 and 1173. This might be associated with differences in the protein domain. And the molecular weight of CqTPSs is between 56.16 kDa (CqTPS12) and 130.87 kDa (CqTPS12). The isoelectric point (pI) of all CqTPS proteins is less than 7; namely, they belong to acidic proteins. The CqTPP protein length is between 324 (CqTPP9) and 387 (CqTPP6) aa, and the molecular weight is between 37.08 kDa (CqTPP9) and 43.28 kDa (CqTPP6). Most CqTPPs belong to basic amino acids with an IP higher than 7, apart from TPP9 and TPP11, the two acidic proteins with pI5.97 and 6.80, respectively.

Phylogenetic Analysis of TPS and TPP Family Genes in Quinoa
To assess the TPS or TPP evolutionary relationship of Chenopodium quinoa Willd, Arabidopsis thaliana (L.), and Gossypium hirsutum Linn. [29][30][31][32], the phylogenetic trees of the TPS and TPP gene families were constructed, respectively. According to the phylogenetic tree, the TPS family members were divided into two classes ( Figure 1A). Class I includes four AtTPSs, six GrTPSs, and six CqTPSs, and Class II includes seven AtTPSs, nine GrTPSs, and seven CqTPSs. The TPP family is divided into three classes ( Figure 1B). Class I includes seven AtTPPs, six GrTPPs, and four CqTPPs, Class II includes three AtTPPs, four GrTPPs, and four CqTPPs, and Class III only includes three CqTPPs and two GrTPPs without an AtTPP homologue.

Phylogenetic Analysis of TPS and TPP Family Genes in Quinoa
To assess the TPS or TPP evolutionary relationship of Chenopodium quinoa Willd, Arabidopsis thaliana (L.), and Gossypium hirsutum Linn. [29][30][31][32], the phylogenetic trees of the TPS and TPP gene families were constructed, respectively. According to the phylogenetic tree, the TPS family members were divided into two classes ( Figure 1A). Class I includes four AtTPSs, six GrTPSs, and six CqTPSs, and Class II includes seven AtTPSs, nine GrTPSs, and seven CqTPSs. The TPP family is divided into three classes ( Figure 1B). Class I includes seven AtTPPs, six GrTPPs, and four CqTPPs, Class II includes three AtTPPs, four GrTPPs, and four CqTPPs, and Class III only includes three CqTPPs and two GrTPPs without an AtTPP homologue.

Analysis of Primary Structures of Genes and Proteins of CqTPSs and CqTPPs
The conservative structure analysis indicates that the TPS domain (Pfam: PF00982) is unique to the CqTPS family proteins, and the TPP domain (Pfam: PF02358) is shared by CqTPS and CqTPP proteins (Figure 2A,B). Class I CqTPS enzymes contain a catalytic triad of residues, Arg (R)/Lys (K)/Glu (E), that is required for T6P synthesis. Moreover, Class I CqTPSs have a SUMOylation site at the C-terminal. Class II CqTPSs have an incomplete triad lacking 'R' residue in TPS domain, and no sumoylation site at the C-terminal ( Figure 2A, Supplemental Figure S1). Then, we used MEME online tools (https://meme-suite.org/meme/doc/meme.html, accessed on 16 December 2022.) to analyze the motif distribution in CqTPS and CqTPP proteins. Ten conservative motifs were identified in CqTPS proteins, with a length of 21-50 aa ( Figure 2C), and their sequence information is shown in Supplemental Figure S2A and Supplemental Table S1. Most CqTPS proteins contain all ten motifs, except for CqTPS7 lacking motif 10, TPS6 lacking motifs 1, 4, 6, and 5, and resulting in shorter proteins. According to the annotation, motifs 4, 6, 1, 5, 8, and 3 constitute the TPS domain, and motifs 10, 2, 9, and 7 constitute the TPP domain. The gene structure analysis based on GFF3 annotation indicates that the number of introns has a significant difference between the two classes. Class II CqTPS genes have only two introns. However, Class I CqTPS genes have a large number of introns, e.g., TPS6 has eight introns, and TPS5, 9, 10, 11, and 12 genes have ≥14 introns ( Figure 2E).
The motif comparison indicates that Class I and Class II CqTPP proteins share some common motifs. All CqTPPs contain motifs 4, 1, 3, 7, 2, and 5. Class I and Class II CqTPPs also have motifs 8 and 6 at the N terminal, while Class II CqTPPs have a motif 9 in the middle of the proteins. In Class III, CqTPP5 has no additional motif, but CqTPP4 and 9 have a motif 10 inserted after motif 1 ( Figure 2F). There is no significant difference in the gene structure of CqTPPs. The above results indicate that CqTPS and CqTPP genes have undergone functional differentiation during evolution. The motif distribution analysis identified ten conservative motifs in CqTPP proteins ( Figure 2D). The length of these conservative motifs ranged from 15 to 50 aa, and their sequence information is shown in Supplemental Figure S2B and Supplemental Table S2. The motif comparison indicates that Class I and Class II CqTPP proteins share some common motifs. All CqTPPs contain motifs 4, 1, 3, 7, 2, and 5. Class I and Class II CqTPPs also have motifs 8 and 6 at the N terminal, while Class II CqTPPs have a motif 9 in the middle of the proteins. In Class III, CqTPP5 has no additional motif, but CqTPP4 and 9 have a motif 10 inserted after motif 1 ( Figure 2F). There is no significant difference in the gene structure of CqTPPs. The above results indicate that CqTPS and CqTPP genes have undergone functional differentiation during evolution.

Analysis of Cis-Acting Elements in the Promoters of CqTPS and CqTPP Genes
The sequences 2000 bp upstream of the start codon were selected as the promoters for cis-regulatory elements analysis. A total of 50 elements were identified in the promoters of CqTPSs and CqTPPs ( Figure 3A,B). The cis-acting elements are classified into five categories, including Light Response, Plant Hormone, Plant Growth, Stress Response and Other. The composition of cis-acting elements in the CqTPS and CqTPP families is similar. The Light Response group contains the most elements, accounting for more than 60% of all elements. Among the Light Response elements, the number of Box 4 and G-box are the largest, followed by the GT1-motif element. It has been demonstrated that G-box in salt mustard participates in the salt stress response [33], and GT1-motif participates in nitrogen regulation in soybean [34]. In the Plant Hormone group, the number of ABREs, CGTCA-motifs, and TGACG-motifs are the largest. ABRE is demonstrated to be involved in the abscisic acid response, and the TGACG-motif participates in the methyl jasmonate response, indicating that the trehalose synthesis in quinoa is modulated by abscisic acid and jasmonate, which are stress responsive in plants [35]. Among Plant Growth elements, the number of AREs and Circadians are the largest. ARE is required for anaerobic induction, and Circadian element regulates circadian rhythm. Among the Stress Response elements, MBSs and TC-rich repeats are the most abundant. MBS is the MYB binding site involved in drought induction, and TC-rich repeats are involved in the defense and stress responses [36]. These results indicate that CqTPS and CqTPP genes are widely involved in various physiological and biochemical activities in plants, and responses to light, phytohormones, and abiotic stress.
all elements. Among the Light Response elements, the number of Box 4 and G-box are the largest, followed by the GT1-motif element. It has been demonstrated that G-box in salt mustard participates in the salt stress response [33], and GT1-motif participates in nitrogen regulation in soybean [34]. In the Plant Hormone group, the number of ABREs, CGTCA-motifs, and TGACG-motifs are the largest. ABRE is demonstrated to be involved in the abscisic acid response, and the TGACG-motif participates in the methyl jasmonate response, indicating that the trehalose synthesis in quinoa is modulated by abscisic acid and jasmonate, which are stress responsive in plants [35]. Among Plant Growth elements, the number of AREs and Circadians are the largest. ARE is required for anaerobic induction, and Circadian element regulates circadian rhythm. Among the Stress Response elements, MBSs and TC-rich repeats are the most abundant. MBS is the MYB binding site involved in drought induction, and TC-rich repeats are involved in the defense and stress responses [36]. These results indicate that CqTPS and CqTPP genes are widely involved in various physiological and biochemical activities in plants, and responses to light, phytohormones, and abiotic stress.

Transcriptome and Metabolome Analyses of Trehalose Biosynthesis in Quinoa Leaves under Saline-Alkali Stress
To investigate the saline-alkali stress response of CqTPSs and CqTPPs and related metabolites such as trehalose, glucose, and sucrose, etc., we performed integrated transcriptome and metabolome analysis using quinoa leaves undergoing carbonate treatment. Two weeks after the treatment, the quinoa plant height reduced significantly compared with that of untreated plants (Supplemental Figure S3A,B) and the total chlorophyll content declined significantly (Supplemental Figure S3C), indicating the saline-alkali condition seriously affected quinoa growth and development. Transcriptome analyses of CqTPS and CqTPP genes indicate that CqTPS4 was significantly upregulated [log 2 fold change (FC) = 1.77], while CqTPS3 (log 2 FC = −1.40), CqTPS8 (log 2 FC = −1.25), and CqTPP4 (log 2 FC = −1.82) were significantly downregulated, and expression of the other genes changed but not significantly ( Figure 4A,B). GO enrichment analysis indicates that the trehalose-related processes such as trehalose-phosphatase activity, the trehalose metabolic process, and the trehalose biosynthesis process were well enriched ( Figure 4D  Since the TPS-and TPP-regulated trehalose biosynthesis processes are in the starch and sucrose metabolism pathway, GO analysis of this pathway was conducted ( Figure 5A, Supplemental Tables S3 and S4). Among the metabolites, UDP-Glucose (UDPG) content changed the most (log 2 FC = −12.80), and D-Glucose-1P, D-Glucose-6P, and D-Glucose, etc., also reduced. UDPG is mainly synthesized from Glucose-1P catalyzed by UGP2, or from sucrose catalyzed by sucrose synthases (SUSs) [37], the expression of one SUS increased and two declined. TPSs catalyze the synthesis of T6P from UDPG and D-Glucose-6P, and then TPPs dephosphorylate T6P to generate trehalose [38]. The expression of one CqTPS was upregulated and two CqTPSs and one CqTPP were downregulated. Trehalose and glucose/maltose can be converted into each other, and maltose can be converted into glucose catalyzed by maltodextrin glucosidase (malZ) [39], while the expression of malZ increased. In order to verify the transcriptome data, we performed an RT-qPCR analysis to check the expression of the related DEGs, and the results were basically consistent with the transcriptome data ( Figure 5B). These results prove that CqTPSs and CqTPPs and the starch and sucrose metabolism pathway play important roles in the carbonate-induced saline-alkali stress response in quinoa.

Analysis of CqTPS Genes' Response to Saline-Alkali Stress
To characterize the regulatory roles of CqTPS genes in response to saline-alkali stress, we determined the growth of yeast (Saccharomyces cerevisiae) harboring CqTPS4/8 genes exposed to saline-alkali stress. The results indicate that CqTPS4-and CqTPS8-expressing yeast cells exhibited an increased survival ratio compared to the control (with empty vector) on an SC-Ura medium containing 25 mM NaHCO3 with pH = 5, 7, 8, and 9 ( Figure 6), suggesting that CqTPS4 and CqTPS8 are involved in the saline-alkali stress response. Rectangles with orange frames, metabolites; circles, DEGs; blue solid lines with arrows, directions of the processes; rectangles with green frames, other metabolism pathways; gray dotted lines, connection of the starch and sucrose metabolism pathway with other metabolism pathways. The colors of rectangles and circles indicate significances, which are presented in color scales. The genes highlighted in red were detected by RT-qPCR. (B) RT-qPCR analysis of the DEGs. UBQ9 (AUR62020068) was used as endogenoue control. Three independent experiments per sample, three replicates per experiment. *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t-test. Abbreviations: AMY, α-amylase; GBE1, glycogen branching enzyme; glgA, glycogen synthase; glgC, ADPglucose pyrophosphorylase; ISA, isoamylase; malZ, maltodextrin glucosidase; SUS, sucrose synthase; T6P, trehalose 6-phosphate; TPP, trehalose 6-phosphate phosphatase; TPS, trehalose-6-phosphate synthase; UDP-glucose, uridine diphosphate glucose.

Analysis of CqTPS Genes' Response to Saline-Alkali Stress
To characterize the regulatory roles of CqTPS genes in response to saline-alkali stress, we determined the growth of yeast (Saccharomyces cerevisiae) harboring CqTPS4/8 genes exposed to saline-alkali stress. The results indicate that CqTPS4and CqTPS8-expressing yeast cells exhibited an increased survival ratio compared to the control (with empty vector) on an SC-Ura medium containing 25 mM NaHCO 3 with pH = 5, 7, 8, and 9 ( Figure 6), suggesting that CqTPS4 and CqTPS8 are involved in the saline-alkali stress response.

Discussion
TPSs and TPPs are key enzymes for trehalose biosynthesis and are important for plant development and stress response. To date, TPS and TPP family genes have been identified in all major plant taxa and microbes such as bacteria, fungi, and yeast, etc. [40][41][42]. In this study, we identified 13 TPS genes and 11 TPP genes in the quinoa genome (Chenopodium quinoa Willd.). Compared with cotton and Arabidopsis, CqTPSs were divided into two classes, and CqTPPs were divided into three classes, according to their protein conservative domains. The Class III TPP group does not include Arabidopsis proteins, and the members lack N-terminal motifs 6 and 8, which exist in Class I and Class II TPPs (Figure 2D). This may be due to the gene variation resulting from exon/intron gain/loss, exonization/pseudoexonization, and/or insertion/deletion, which lead to functional differentiation during evolution [43,44].
The AtTPS1 protein has a catalytic triad of residues, Arg (R)/Lys (K)/Glu (E), which is critical to TPS activity, and contains a putative SUMOylation site in the TPP domain [45]. Lack of the catalytic triad results in loss of TPS activity. Class I AtTPS proteins with the catalytic triad residues in the TPS domain are demonstrated to have catalytic activity, except for AtTPS3, a possible pseudogene [46]. These structure characteristics are also conserved in quinoa. Among the 13 CqTPSs, Class I CqTPSs have the catalytic triad of residues in the TPS domain and a SUMOylation site at the C-terminal (Figure 2A) [45,46]. According to the conservative domains and motifs, it is speculated that Class I CqTPSs may have TPS activity, and, of course, this needs to be verified by experiments. The Class Figure 6. The growth activity of CqTPS4and CqTPS8-expressing yeast under saline-alkali stress. Yeast strain INVSC1 was transformed with the pYES2-CqTPS4, pYES2-CqTPS8, or pYES2 empty vector (control), respectively, and grew for 24 hrs on SC-Ura medium containing 25 mM NaHCO 3 with pH value as indicated. pH 5.8 was the control pH value.

Discussion
TPSs and TPPs are key enzymes for trehalose biosynthesis and are important for plant development and stress response. To date, TPS and TPP family genes have been identified in all major plant taxa and microbes such as bacteria, fungi, and yeast, etc. [40][41][42]. In this study, we identified 13 TPS genes and 11 TPP genes in the quinoa genome (Chenopodium quinoa Willd.). Compared with cotton and Arabidopsis, CqTPSs were divided into two classes, and CqTPPs were divided into three classes, according to their protein conservative domains. The Class III TPP group does not include Arabidopsis proteins, and the members lack N-terminal motifs 6 and 8, which exist in Class I and Class II TPPs ( Figure 2D). This may be due to the gene variation resulting from exon/intron gain/loss, exonization/pseudoexonization, and/or insertion/deletion, which lead to functional differentiation during evolution [43,44].
The AtTPS1 protein has a catalytic triad of residues, Arg (R)/Lys (K)/Glu (E), which is critical to TPS activity, and contains a putative SUMOylation site in the TPP domain [45]. Lack of the catalytic triad results in loss of TPS activity. Class I AtTPS proteins with the catalytic triad residues in the TPS domain are demonstrated to have catalytic activity, except for AtTPS3, a possible pseudogene [46]. These structure characteristics are also conserved in quinoa. Among the 13 CqTPSs, Class I CqTPSs have the catalytic triad of residues in the TPS domain and a SUMOylation site at the C-terminal (Figure 2A) [45,46]. According to the conservative domains and motifs, it is speculated that Class I CqTPSs may have TPS activity, and, of course, this needs to be verified by experiments. The Class II CqTPSs have an incomplete triad lacking the 'R' residue in the TPS domain, and no sumoylation site is found at the C-terminal, suggesting they may not have TPS activity. These structural features are consistent with the Arabidopsis Class II members, which have been elucidated to have no TPS catalytic activity [47,48]. The TPP domain in AtTPS1 is likely crucial for plant development, although it lacks some of the residues in the active site of the TPP enzyme [42]. Interestingly, despite the high conservation of TPP activity sites, none of the class II AtTPSs have TPP activity [49][50][51]. Meanwhile, the TPP domain in Class I and Class II CqTPSs is highly similar to that of AtTPSs, implying that they are likely to have similar functions (Supplemental Figure S1). Although the Class II TPS proteins may have no TPS and TPP enzyme activities, they are proposed to regulate Class I TPS enzyme activity based on the association of rice class I and II proteins [52], and act as signaling proteins based on their conservative ligand-binding sites [42]. There is much evidence implicating that Class II TPS proteins are involved in stress response. For instance, TPS5 is involved in ABA signaling [53], TPS5 in thermotolerance [54], TPS5 in basal pathogen defense [43], and TPS11 in cold tolerance [44] and aphid resistance [55]. In quinoa leaves, the expression levels of Class II genes, CqTPS3, CqTPS4, and CqTPS8, were altered significantly under saline-alkali conditions, suggesting that Class II genes in quinoa also respond to stress. On the other hand, all CqTPS proteins do not have a nuclear localization signal (NLS), suggesting they cannot enter the nucleus autonomously.
In addition to TPP catalytic activity, some AtTPP genes are associated with an abiotic stress response. For example, AtTPPD is responsible for salt and oxidative stress resistance [56], and AtTPPF and AtTPPI are involved in drought response [56,57]. Moreover, the rice OsTPP1 and OsTPP2 are involved in the cold stress response [58,59], and OsTPP7 regulates anaerobiosis resistance during germination [60]. In quinoa leaves, expression of CqTPP4 decreased significantly under saline-alkali stress ( Figure 6B), suggesting it is involved in the stress response. The transcription levels of other CqTPP genes also changed under the same conditions but not significantly. Further study is needed to clarify the association of CqTPP genes with different stresses.
When plants are under stress, the related transcription factors (TFs) are activated to bind specific cis-acting elements to regulate the expression of downstream stress-response genes. In this study, many cis-acting elements related to stress (A-box, MBS, LTR, TC-rich repeats, and WUN-motif), phytohormones (ABRE, GCTGA-motif, etc.), and light (G-box, box4, etc.) were identified in promoters of CqTPS and CqTPP family genes. In quinoa leaves under saline-alkali stress, the expression levels of CqTPS4, 3, 9, and CqTPP4 were altered significantly ( Figure 4A,B). Among them, CqTPS4, the only upregulated gene, contains six GATA-motifs, three Box4s, two Gap-boxes, and two G-boxes in its promoter ( Figure 3A), suggesting these elements may be closely related to the response to saline-alkali stress. The other three CqTPS and CqTPP genes were significantly downregulated under saline-alkali stress. This is also observed in previous studies, that show that some TPS or TPP genes are up-and some other genes are downregulated. This may be due to the complexity of regulatory mechanisms of trehalose biosynthesis as well as the functional division of these genes, e.g., some Class I TPS genes are responsible for plant growth and development, and some Class II TPS genes are related to the stress response. Another possible reason is the plant development stage and stress treatment duration. Two-week treatment is a long-term stress, the quinoa plants may have adapted to the condition and have adjusted the metabolic system and regulatory machineries. For example, the stabilized expression levels of CqTPS genes lead to a proper level of T6P, which subsequently stabilizes the abundance of sucrose and trehalose as well as related physiological activities.
Sucrose is the final product of the photosynthesis of green plants and functions as an important carbon source for cell activities. Carbohydrates from sucrose account for 90% of plant biomass. At the same time, sucrose and starch metabolism, which produces signal molecules such as glucose, fructose, and T6P, is one of the key pathways to regulate plant growth and development and abiotic stress response [61][62][63]. Trehalose and T6P are important intermediates in sucrose and starch metabolism, and trehalose biosynthesis is essential for plant growth and development [64]. In most flowering plants, the trehalose content is extremely low and is barely detectable [65,66]. Under stress conditions, trehalose functions as an osmoprotectant to protect the cellular structure and cell membrane from stress-induced damage [38] and improve plant tolerance to various abiotic stresses, especially salinity and drought stresses [36]. T6P links plant growth and development with the status of sucrose metabolism [67]. In Arabidopsis seedlings, the T6P content is highly correlated with the level of sucrose, and dynamically responds to the fluctuation of sucrose and gives feedback. Therefore, T6P is considered a key signaling metabolite, acting as a plant 'insulin' to regulate sucrose utilization. [68].
Surprisingly, despite being under saline-alkali treatment and despite the alteration of expression of CqTPS and CqTPP genes and significant decrease of contents of UDPG and D-Glucose-6P, the contents of sucrose, trehalose, and T6P in quinoa leaves did not change significantly, whereas the contents of UDPG and D-Glucose-6P, the substrates for T6P generation, decreased significantly ( Figures 4C and 5A). Similar phenomena were also observed in maize ra3 mutants, the TPP loss-of-function alleles, and there were no significant differences in T6P or trehalose levels compared to wild-type [66]. Moreover, there were no significant differences in T6P levels between wild-type, ra3, and ra3 tpp4 mutants [69]. Since T6P plays a central role in controlling growth and development, a modicum change in T6P level may cause serious effects on plants. Significant changes have also occurred in the transcription levels of many genes in starch and sucrose metabolic pathways, such as amylase genes, SUS (sucrose synthase), glgA (glycogen synthase), and glgC (ADPglucose pyrophosphorylase) genes, etc. (Figure 5), which consequently led to changes in metabolites. GO Enrichment analysis indicates that the trehalose-related processes such as trehalose-phosphatase activity, the trehalose metabolic process, and the trehalose biosynthesis process were well enriched in DEGs ( Figure 4D, arrows). On the other hand, the contents of some metabolites in starch and sucrose metabolism pathway altered significantly. Therefore, there may be tight regulatory machineries to rescue the change of T6P, such as the complementation from other functional TPS and/or TPP genes, or other unknown pathway(s). In the case of this study, the two-week treatment belongs to a long-term stress, and the saline-alkali stress was moderate, thus the quinoa plants may have adapted to the stress and have adjusted the T6P level, and subsequently stabilized the contents of trehalose and sucrose. Quinoa is a species with strong resistance to stress; whether the adjustment of T6P is due to this characteristic of quinoa or the nature of many plants is unknown. In source leaves, much of the UDP-Glc is consumed by sucrose synthesis [70]; whereas in growing tissues, it is the direct substrate for the synthesis of cellulose and hemicelluloses, and a precursor for other nucleotide sugars that are needed for the synthesis of noncellulosic cell wall polysaccharides [71,72]. Therefore, the remarkable decrease of UDPG in leaves of developing quinoa plants may be due to consumption in sucrose synthesis and cell wall deposition, as well as in other metabolic pathways, such as amino sugar and nucleotide sugar metabolism ( Figure 5A).
Moreover, the expression of many regulatory genes in the sucrose and starch metabolism pathway changed, including sucrose synthases (SUSs) and amylases, etc. ( Figure 5A). SUSs catalyze the reversible conversion of sucrose and fructose and produce UDPG [73]. The abundance of SUSs affects the thickness of the secondary cell wall, sharevegetative growth, and mechanical strength [74]. On the other hand, the expression of SUSs is affected by salt stress [75]. The amylases include alpha-amylase (AMY) and beta-amylase (β-amylase). AMY is a crucial enzyme that functions throughout the whole life cycle of angiosperm [76]. Alteration of the expression of these genes suggests that the sucrose and starch metabolism pathway is crucial for the saline-alkali stress response. The phylogenetic trees of TPS and TPP families, containing quinoa, Arabidopsis, and cotton proteins, were constructed based on multiple sequence alignments using MAGA 11 software [78] and the maximum likelihood model (bootstrap is 1000). iTOL (http://itol.embl.de/, accessed on 12 December 2022.) website was used to optimize the evolutionary tree.

Gene Structure and Cis-Acting Element Analysis
The gene structure of the CqTPS and CqTPP family genes was analyzed using TBtools combined with the GFF3 gene annotation data, and to plot the exon-intron diagram.
The 2kb sequences upstream of the start codons of CqTPS and CqTPP genes were screened using TBtools and used as promoter sequences for analysis. plantCARE [81] (https: //bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 December 2022.) was used to investigate the cis-acting elements in promoters to predict the roles of genes in stress responses.

Plant Materials, Growth Conditions, and Stress Treatments
Quinoa (Jiaqi #3) plants grew under a 16 h light/8 h dark cycle at 22 • C. Two-week-old quinoa seedlings were treated with a solution containing 100 mM Na 2 CO 3 :NaHCO 3 = 1:9 once every 5 days, three times in total. The control group was treated with water. The leaves were harvested randomly five days after the third treatment, then sent for RNA sequencing and an ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) analysis.

RT-qPCR Validation
The total RNA used for RT-qPCR (reverse transcription quantitative PCR) was consistent with that for RNA sequencing. RT-qPCR was performed following the manufacturer's instructions for Ultra SYBR Mixture (Low ROX) on the ABI7300 real-time PCR system (Applied Biosystems, Waltham, MA, USA). UBQ9 (AUR62020068) was used as the reference gene for normalizing mRNA transcription [82]. The relative expression level was calculated by the 2 − CT method [83]. All RT-qPCR analyses were set with 3 technical repeats. The primers are listed in Supplemental Table S5.

Stress Tolerance Assays of CqTPS4 and CqTPS8 in Yeast
The cds of CqTPS4 and CqTPS8 were amplified from the quinoa cDNA library using specific primers (listed in Supplemental Table S4). The PCR products were inserted into the pYES2 vector, and the recombined pYES2-TPS4 and pYES2-TPS8 plasmids were transformed into yeast (Saccharomyces cerevisiae) strain INVSC1, respectively. The pYES2 empty vector was used as a control.
The transformed yeast was cultured overnight in SC-Ura liquid medium containing 2% glucose at 30 • C. After adjustment of OD600 to 0.4, the culture was diluted according to the indicated gradient on an SC-Ura medium containing 25 mM NaHCO 3 with pH 5, 6, 7, 8, and 9, respectively, at 30 • C for 24 h.

Conclusions
In this study, we identified 13 CqTPS genes and 11 CqTPP genes in the quinoa genome and analyzed their physicochemical properties, gene structures, conservative domains and motifs in the proteins, and cis-regulatory elements, as well as evolutionary relationships.
The results indicate that the TPS and TPP family characteristics are highly conserved in quinoa. Transcriptome and metabolome analyses of the sucrose and starch metabolism pathway in leaves indicate that CqTPP4 and Class II CqTPS4, 3, 9 genes, and trehalose biosynthesis are important for the saline-alkali stress response in quinoa. This study may help to explain the biological activities of CqTPS and CqTPP proteins in developmental processes and stress responses in quinoa. However, our knowledge of their precise biological role is still lacking. Thus, in order to provide important insights to help others for developing crop cultivars resistant to unfavorable stress conditions, an extensive functional validation study of CqTPSs andCqTPPs is necessary.