Abstract

   Gac is a carotenoid-rich, healthful tropical fruit; however, its
   productivity is limited by soil salinity, a growing environmental
   stress. This study aimed to evaluate the effects of salinity stress on
   key physiological traits and metabolites in 30-day-old gac seedling
   leaves, treated with 0, 25-, 50-, 100-, and 150-mM sodium chloride
   (NaCl) for four weeks to identify potential alarm, acclimatory, and
   exhaustion responses. Electrolyte leakage increased with increasing
   NaCl concentrations (p < 0.05) indicating loss of membrane permeability
   and conditions that lead to reactive oxygen species production. At 25
   and 50 mM NaCl, superoxide dismutase (SOD) activity, starch content,
   and total soluble sugar increased. Chlorophyll a, and total chlorophyll
   increased at 25 mM NaCl but decreased at higher NaCl concentrations
   indicating salinity-induced thylakoid membrane degradation and
   chlorophyllase activity. Catalase (CAT) activity decreased (p < 0.05)
   at all NaCl treatments, while ascorbate peroxidase (APX) and guaiacol
   peroxidase (GPX) activities were highest at 150 mM NaCl.
   GC-MS-metabolite profiling showed that 150 mM NaCl induced the largest
   changes in metabolites and was thus distinct. Thirteen pathways and
   7.73% of metabolites differed between the control and all the
   salt-treated seedlings. Salinity decreased TCA cycle intermediates, and
   there were less sugars for growth but more for osmoprotection, with the
   latter augmented by increased amino acids. Although 150 mM NaCl level
   decreased SOD activity, the APX and GPX enzymes were still active, and
   some carbohydrates and metabolites also accumulated to promote salinity
   resistance via multiple mechanisms.

   Keywords: gac, salinity stress, physiological traits, metabolic profile

1. Introduction

   Gac (Momordica cochinchinensis (Lour.) Spreng.), also known as a sweet
   gourd, belongs to the Cucurbitaceae and is popular in South and
   South-East Asia because of its intense red color and high carotenoid
   content [[32]1], which is 10- and 70-fold higher than in carrots and
   tomatoes, respectively. While β- carotenes, and lycopene are abundant
   in gac, zeaxanthin and β-cryptoxanthin also accumulate to high levels
   [[33]2]. Gac fruit is therefore an effective and new and valuable
   source of carotenoids which may contribute significant benefits to
   human health [[34]3]. Daily consumption of “gac” enhanced plasma levels
   of retinol, α- and β- carotenes, and, lycopene in children [[35]4], and
   gac seed membranes promote healthy vision [[36]1]. Therefore, the
   cultivation of the plant should be encouraged
   [[37]1,[38]3,[39]4,[40]5,[41]6,[42]7,[43]8,[44]9,[45]10,[46]11].

   Soil salinity is a major factor limiting crop productivity worldwide
   [[47]12,[48]13,[49]14,[50]15]. Salt accumulation in soils develops from
   unbalanced irrigation, inferior drainage, incorrect application of
   fertilizer, and overcultivation [[51]16]. Plants growing in saline
   soils develop complex but highly coordinated morphological,
   physiological, and metabolic processes to cope with stress
   [[52]13,[53]14,[54]15,[55]17,[56]18,[57]19,[58]20]. High salt
   concentrations induce loss of intracellular water, because of the lower
   soil water potential, hyperionic stress due to the increased levels of
   ions such as Na^+ and Cl^−, and nutrient imbalances caused by the
   inhibition of essential nutrient uptake, and/or shoot transport.
   Salinity stress also leads to the overproduction and overaccumulation
   of reactive oxygen species (ROS) in plant cells, which results in
   oxidative damage to cellular components such as membranes, proteins,
   and DNA [[59]21,[60]22,[61]23,[62]24,[63]25]. Plants, however, can
   modulate ROS accumulation during salinity stress via non-enzymatic and
   enzymatic antioxidants such as guaiacol peroxidase, (GPX), superoxide
   dismutase, (SOD), catalase, (CAT), ascorbic peroxidase (APX)
   [[64]26,[65]27,[66]28,[67]29,[68]30,[69]31,[70]32,[71]33].

   The ability of plants to deal with salt stress is an essential
   determinant of crop distribution and productivity in many areas; thus,
   understanding the mechanisms that provide tolerance is essential for
   ensuring productivity. For example, reconfiguration of plant metabolic
   pathways is an important response [[72]34,[73]35], and identifying key
   compounds altered as a result may contribute to the understanding
   salinity resistance mechanisms in plants [[74]36]. The accumulation of
   antioxidants, amino acids, and sugars can provide protection against
   salinity stress
   [[75]23,[76]37,[77]38,[78]39,[79]40,[80]41,[81]42,[82]43], as
   demonstrated in a variety of species and tissues
   [[83]26,[84]44,[85]45,[86]46,[87]47,[88]48,[89]49,[90]50]. However,
   changes in metabolites will depend on the severity or the length of the
   stress; the metabolites accumulated may be components of the early
   stress perception response, the acclimation response, or may be
   metabolic byproducts after stress exhaustion. A better understanding of
   the relative abundance and spatiotemporal occurrence of these
   metabolites could help identify tolerance mechanisms in species of
   interest [[91]34].

   There is limited knowledge of the response of gac plant to soil
   salinity. Our preliminary data found that 4-month-old gac plants grow
   normally in soil from 25 to 150 mM NaCl, while 200 mM NaCl induces
   abnormal symptoms [[92]51]. The high level of natural antioxidants in
   gac may undergird its apparent tolerance to salinity stress. However,
   metabolic profiles in this plant have been little studied, especially
   under salinity conditions. Therefore, it is important to investigate
   the effect of salinity stress on cellular metabolism, in terms of
   antioxidant enzymes, plant physiological characteristics together with
   metabolic profiles. The finding of this study will fill this current
   gap in knowledge and aid in the understanding of metabolic responses in
   gac plants under salinity stress, which would benefit breeding program
   aimed at developing salt-tolerant cultivars.

2. Materials and Methods

2.1. Plant Materials

   Gac seeds (Momordica cochinchinensis (Lour.) Spreng.) cv. Kaenpayorm1,
   were supplied by the Faculty of Agriculture, Khon Kaen University, Khon
   Kaen, Thailand. Seeds were soaked in distilled water for 2 days and
   then germinated in saturated peat moss for 7 days. Approximately 5 kg
   of natural sandy loam soils were transferred to each plastic pot (8
   inches in diameter), and tap water was added to saturate the soil
   before planting. The gac seedlings were watered with tap water every
   other day in the greenhouse under natural light, temperature, and
   humidity (June 2017 at Khon Kaen, Thailand). At 30 days post
   germination, the salinity treatment was irrigated every other day with
   either 50 mL of 0, 25, 50, 100, or 150 mM of NaCl. Preliminary studies
   showed that 200 mM NaCl was lethal, therefore the highest concentration
   used was 150 mM. There were six individual plants for each salt
   treatment and leaf tissues were harvested 30 days after the application
   of the stress.

2.2. Carbohydrate Analysis

   Total soluble sugar was extracted from approximately 50 mg gac leaf by
   9 mL of 80% (v/v) ethanol. Total sugar content was analyzed using the
   phenol sulfuric method [[93]52]. One hundred microliters of the sugar
   solution were put into a glass tube and mixed with 500 µL 5% (w/v)
   phenol, 1000 µL concentrated sulfuric acid, and then left for 10 min.
   The mixture was vortexed and left for 30 min, and then the absorbance
   was measured at 490 nm with a spectrophotometer (ThermoScientific™
   GENESYS™ 20 Visible Spectrophotometer). A mixture of phenol and
   sulfuric acid without sugar extract was used as a blank. Total sugar
   content was determined from a standard curve using a known
   concentration of glucose as a standard.

   Starch was assayed using the ethanol insoluble fraction. Approximately
   400 mL of 2M potassium hydroxide was added, and the mixture was
   incubated in a 90 °C water bath for an hour, and then cooled to room
   temperature. To each tube, 600 µL of 10% (v/v) acetic acid was added.
   The leaf was added with 1 mL of 280 U amyloglucosidase (Sigma Chemical)
   which had been dialyzed against 0.1 M Na-acetate buffer (pH 4.5), was
   used to digest the starch to glucose, then incubated in a 45 °C water
   bath overnight [[94]52]. Starch content was determined from glucose
   equivalents released after the enzymatic digestion, using hexokinase,
   and glucose-6-phosphate dehydrogenase (G6PDH) [[95]53]. Twenty
   microliters of the starch extract were pipetted into a glass tube and
   mixed with 1 mL glucose assay mixture: 50 mM Tris-HCl (pH 8.0), 100 mM
   ATP, 500 mM MgCl[2], 100 mM NAD, 5 µL hexokinase, and 5 µL G6PDH, then
   mixed by shaking for 30 min. The absorbance was determined at 360 nm
   with a spectrophotometer. The starch content was determined from a
   standard curve using a known concentration of glucose as a standard.

2.3. Enzyme Activities

   Leaf tissue (0.2 g) was ground on ice using a mortar and pestle in 4 mL
   of grinding buffer: 50 mM potassium phosphate buffer (pH 7.8), 0.4 mM
   EDTA, and 1 mM ascorbic acid. The extracts were filtered through 8
   layers of cheesecloth, then transferred to a microfuge, and was
   centrifuged at 8944 g for 1 min. All steps were performed at 4 °C. A 1
   mL aliquot of the supernatant was used as a crude extract for the
   determination of antioxidant enzymes activity including superoxide
   dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), guaiacol
   peroxidase (GPX; EC 1.11.1.7), and ascorbate peroxidase (APX; EC
   1.11.1.11) [[96]54]. Protein content was investigated using the
   Bradford method [[97]55].

   SOD activity was assayed by measuring its ability to inhibit the
   photochemical reduction of nitro blue tetrazolium chloride (NBT)
   [[98]56]. The photo-reduction of NBT was measured at 560 nm. One unit
   of SOD was defined as the amount of enzyme that produced 50% inhibition
   of NBT. CAT activity was determined by measuring the rate of
   disappearance of H[2]O[2] [[99]57]. The subsequence decomposition of
   H[2]O[2] was observed at 240 nm. GPX activity was calculated from the
   amount of tetraguaiacol formed per minute at 470 nm [[100]58]. For the
   APX assay, the subsequent decrease in ascorbic acid was observed at 290
   nm [[101]58].

2.4. Chlorophyll Concentration

   Chlorophyll concentration was determined according to the method
   outlined by Arnon [[102]59]. Leaf samples (20 mg) were extracted in 10
   mL of 80% (v/v) acetone. The absorbance of the extract was determined
   by spectrophotometer at 645 and 663 nm using 80% (v/v) acetone as a
   blank. Chlorophyll was also assayed using the Soil Plant Analysis
   Development (SPAD)-502 m. Ten independent SPAD measurements were made
   per plant using several different leaves at the position 2/3 of the
   distance from the leaf base to the apex [[103]60,[104]61].

2.5. Electrolyte Leakage

   Electrolyte leakage was measured using an electrical conductivity meter
   [[105]62]. Leaf discs were placed in a test tube containing 10 mL
   distilled water. The samples were incubated at room temperature for 24
   h. The electrical conductivity of the solution (EC[1]) was then read
   after incubation. The same samples were then placed in a boiling water
   bath for 20 min and a second EC reading (EC[2]) was taken after cooling
   the solution to room temperature. Electrolyte leakage was then
   calculated as EC[1]/EC[2] and expressed as a percentage.

2.6. Metabolite Profiling

   Analytical grade solvents and reagents were purchased from Sigma
   Aldrich (Australia). Samples were extracted in a pre-chilled solution
   of methanol (pH 7), chloroform, and deionized water (18.2 MΩ) in
   proportions of 5:2:2 (v/v/v). Approximately 20 ± 5 mg fresh weight of
   gac leaves were put into Eppendorf tubes and ground to a fine powder in
   liquid nitrogen. One mL of pre-chilled extraction solution was added to
   each tube to prevent thawing of the sample. The samples were vortexed
   for 10 s and centrifuged for 2 min at 17,530× g. Five hundred µL of
   supernatant was dried in the speed vacuum concentrator (LaboGene Brand)
   and submitted for derivatization to West Coast Metabolomics Center at
   the University of California Davis, Genome Center, Davis, CA, USA.

   Gas chromatography-mass spectrometry analysis was performed on a total
   of 30 samples and multivariate analysis was used to compare the
   metabolite profiles. The data are acquired using the following
   chromatographic parameters, with more details to be found in Fiehn et
   al. [[106]63].

2.7. Statistical Analysis and Data Analysis

   The experimental design was a completely randomized design (CRD) with
   six replications for the evaluation of all variables. For physiological
   data, statistical analysis using one-way ANOVA and all means were
   separated at the p < 0.05 level using Duncan’s multiple range test
   using the SPSS. A student t-test at p < 0.05 were analyzed to detect
   significant changes between the mean of metabolites in different
   concentrations of NaCl. For multivariate statistical analysis,
   metabolites data were first normalized to an internal standard.
   Principal component analysis (PCA), partial least squares discriminant
   analysis (PLS-DA), and orthogonal partial least squares discriminant
   analysis (OPLS-DA) were performed using Simca^®.

   Scree plot, one-way ANOVA analysis, box plots, heat map, volcano plots,
   and pathway impact were performed using MetaboAnalyst Version 5.0
   (statistical, functional, and integrative analysis of metabolomics
   data) is freely available at [107]http://www.metaboanalyst.ca/
   (accessed on 15 January 2022) [[108]64,[109]65,[110]66]. The identified
   metabolites were mapped on the KEGG pathway of the model plant
   Arabidopsis thaliana, which demonstrated the role of different
   metabolites in various biochemical pathways.

3. Results

3.1. Electrolyte Leakage

   There was a significant positive relationship between electrolyte
   leakage in gac seedling leaves and soil salinity (p < 0.01; R^2 =
   0.3679) ([111]Figure S2). Membrane permeability differed from the
   control only at 100 mM (p < 0.05) and plants treated with 150 mM NaCl
   were distinct from all others ([112]Figure S1) as well as soil
   electroconductivity ([113]Figure 1A).

Figure 1.

   [114]Figure 1
   [115]Open in a new tab

   Gac seedlings leaf response to increasing soil salinity. (A) Rate of
   electrolyte leakage, (B) starch content, (C) total soluble sugar.
   Different lowercase letters indicate significant differences between
   treatments (p < 0.05). (D) Chlorophyll a, b and total chlorophyll in
   gac leaf at the seedling stage. Values are means ± SE (n = 6). Lower
   case letters, numbers, and capital letters denote significant
   differences in chlorophyll a, chlorophyll b, and total chlorophyll
   content, respectively, between treatments (p < 0.05).

3.2. Total Sugar and Starch Content

   Starch content in gac seedling leaves increased when grown under 25 mM
   NaCl compared to the control plant, while treating with 50 mM NaCl
   showed similar starch content to the control plant. At 100 and 150 mM
   NaCl, however, starch content decreased compared to the control plant
   and 50 mM NaCl but it was not significant (p > 0.05) ([116]Figure 1B).
   Sugar content increased only at 50 mM compared to the control plant.
   Although 100 and 150 mM NaCl led to reductions in sugar content, they
   were not significant (p > 0.05) ([117]Figure 1C).

3.3. Chlorophyll Concentration in Gac Seedling Leaf

   Although not significant, the total chlorophyll, chlorophyll a content,
   and SPAD readings of gac seedling leaves showed an increase at 25 mM
   but decreased (p > 0.05) at 50 and 100 mM NaCl compared to the control
   ([118]Figure 1D). Unlike the chlorophyll readings obtained from the
   spectrophotometer, SPAD showed an unambiguous decrease at 150 mM NaCl
   compared to the control ([119]Figure S3), and in contrast to the
   chlorophyll a and total chlorophyll measurements, chlorophyll b, the
   content was similar at all NaCl concentrations.

3.4. Antioxidant Enzyme Activities

   Antioxidant enzyme activities including APX, CAT, GPX, and SOD were
   investigated, and they showed contrapuntal responses to salinity
   stress. APX and GPX activity increased, but only at the highest salt
   concentration of 150 mM—increasing three- and four-fold, respectively
   ([120]Figure 2A,B). CAT activity in contrast decreased under all saline
   treatment conditions ([121]Figure 2C). SOD activity was higher at 25
   and 50 mM NaCl compared to 100 mM of NaCl, while at 150 mM, enzyme
   activity was the same as the control treatment ([122]Figure 2D).

Figure 2.

   [123]Figure 2
   [124]Open in a new tab

   Antioxidant enzyme activity in gac leaves in response to salinity
   stress. Activity assayed included (A) APX, (B) GPX, (C) CAT, and (D)
   SOD. Values are means ± SE (n = 6). Different lowercase letters
   indicate significant differences between treatments (p < 0.05).

3.5. Relative Levels of Metabolites in Gac Seedling Leaf Concerning NaCl
Concentration

   We determined the extent to which salt concentrations affected gac leaf
   polar GC-MS-TOF-generated metabolic profiles using multivariate
   analyses. On these plots, samples with similar metabolic profiles will
   group together, while those with distinct compositions will be more
   distant. There was no differentiation of samples by PCA analysis
   suggesting that the metabolite composition of the control and the
   salt-treated samples were similar ([125]Figure S4). We, therefore, used
   the partial least squares discriminant analysis (PLS−DA), a supervised
   method, in an attempt to achieve better separation of the treatments
   [[126]67,[127]68]. There was no distinction between the control, 25 mM,
   50 mM, and 100 mM samples, whereas the 150 mM group was significantly
   split from the others as shown in [128]Figure 3A. OPLS-DA was used to
   find the difference between the two groups (control and each stress
   group) shown in [129]Figure 3B–E. It shows a very clear separation
   between each paired sample; therefore, only the control and the 150 mM
   OPLS−DA model were valid [[130]69].

Figure 3.

   [131]Figure 3
   [132]Open in a new tab

   Multivariate analysis of metabolites assayed by GC−MS in gac leaf grown
   under salinity. (A) PLS−DA, (B) OPLS−DA of control and 25 mM groups,
   (C) OPLS−DA of control and 50 mM groups, (D) OPLS-DA of control and 100
   mM groups and (E) OPLS−DA of control and 150 mM groups. Each of six
   biological replicates was individually plotted and the samples were
   projected onto a bi-plot showing the first two PCs. Each symbol on the
   plot represents data from 637 metabolites reduced to a single data
   point defined by the first (PLSC[1]) and second (PLSC[2]) PLSC. Samples
   that have similar metabolite compositions will cluster together while
   samples that are different will be further apart. The percentage
   variation explained by each PC is shown in parentheses on each axis.
   Please note the x-axes were automatically drawn to differing scales and
   do not allow for a true comparison of the distinction between control
   and salt treatment across experiments.

   To identify the metabolites in gac leaf that differed due to NaCl
   treatments, the mean of each metabolite was compared using one-way
   ANOVA analysis. Approximately 8% of the metabolites, i.e., 52 of the
   637 metabolites differed (p value < 0.05 and FDR < 0.05; [133]Figure
   S9). Using a more stringent p value, i.e., p < 10^−5 as a cut-off, we
   found ten metabolites that differed, four of which were unknowns
   (‘95440’, ‘34149’, ‘89221’, and ‘4219’), while the others were all
   amino acids, i.e., valine, isoleucine, phenylalanine, leucine,
   tryptophan, and glycine ([134]Figure 4). Interestingly, all amino acids
   except glycine increased due to the 150 mM salt treatment. A heat map
   depicting the 52 metabolites clearly showed that the 150 mM treatment
   was distinct from the others because of the drastic change in
   metabolite level. The heat map also showed that the ten highly
   differentiated metabolites by ANOVA also showed a similar pattern of
   change in response to salinity ([135]Figure 5).

Figure 4.

   [136]Figure 4
   [137]Open in a new tab

   Metabolites that differed in gac samples due to salinity. One−way ANOVA
   analysis of significant metabolites measured by GC−MS−TOF in gac leaf
   grown under 0, 25, 50, 100, and 150 mM NaCl. Each metabolite
   (represented by a black dot) was individually plotted at the adjusted
   p−value = 0.05, post hoc analysis: Fisher’s LSD.

Figure 5.

   [138]Figure 5
   [139]Open in a new tab

   A heat map provides an intuitive visualization of metabolites found in
   this study. Each colored cell on the map corresponds to a concentration
   value in the data table, with samples in rows and metabolites in
   columns. Displayed are the top 52 metabolites ranked by t−tests with
   p−values by ANOVA of < 0.05 and FDR < 0.05.

   We next sought to determine the specific metabolites that differed from
   the control in the plants grown under each saline condition. The
   fold−change analysis revealed that 27, 43, 47, and 205 metabolites
   differed significantly at 25, 50, 100, and 150 mM NaCl, (p < 0.05),
   respectively, when compared to the control ([140]Figure S8 and
   [141]Table S2). To obtain an overview of the similarities and/or
   differences of the identified metabolites, the behavior of the
   identified metabolites was analyzed using pathway impact. Pathway
   impact scores provide a measure of the importance of a given pathway
   relative to others assessed (See Liu et al., 2019 for additional
   details). Pathway analyses were conducted using the MetaboAnalyst 5.0
   computational platform to better understand the impact of salinity
   levels on gac metabolic profiles [[142]65,[143]66,[144]67]. A single
   p-value for each metabolic pathway was computed by pathway enrichment
   analysis, as opposed to a t-test [[145]70]. The results showed that
   there was no pathway impact in the 50 mM treatment, i.e., there were no
   metabolic pathways enriched or distinguishable from others due to this
   treatment. In the 25 mM−treated plants, two pathways (p-values < 0.05)
   were enriched, however, the FDR values were too high (0.66353), so they
   were not considered ([146]Table S3). There were nine pathways of
   identified metabolites with higher impact (p < 0.05) when the 100 mM
   and 150 mM treatments were examined ([147]Figures S6 and S7). The
   metabolites were shown in the schematic metabolic pathway ([148]Figure
   6). At mild stress, many carbohydrates including starch, trehalose,
   maltose, sucrose-6-P, glucose-6-P, fructose-6-P, and TCA substances
   increased or remained the same as the control, then decreased in higher
   NaCl concentration, while for amino acids, the opposite trend was
   found.

Figure 6.

   [149]Figure 6
   [150]Open in a new tab

   A Schematic showing the coordinate biochemical and physiological
   changes in gac seedling leaves in response to salinity. Shown are the
   relative metabolite levels, antioxidant enzyme activities, chlorophyll
   content and electrolyte leakage in response to increasing soil
   electrical conductivity. Arrows indicate the direction of the reaction.
   Heat maps showing the difference in each parameter under 0, 25, 50,
   100, and 150 mM. Parameters in white boxes are present at relative low
   levels, while those in darker red boxes are higher.

4. Discussion

   The aim of this study was to investigate key physiological and
   metabolic responses of gac to salinity, including potential
   acclimation, resistance, and exhaustion responses. Understanding the
   dynamics of plant response to salinity would help to develop solutions
   for more resilient plants, either through cultural practices or plant
   breeding. Saline soils are classified as those having an EC value of
   4000 µScm^−1 or more [[151]71,[152]72,[153]73], but many crops are
   sensitive to EC values lower than 4000 µScm^−1 [[154]74]. In this
   study, the NaCl corresponded to 800 to 5000 µScm^−1 which spans low
   (25–50 mM) to moderate (100–150 mM) salinity stress levels
   [[155]75,[156]76] as shown in [157]Figure S1. Electrolyte leakage is
   used as a proxy for stress-induced injury and tolerance, as it often
   co-occurs with other stress physiological traits
   [[158]62,[159]77,[160]78,[161]79]. Electrolyte leakage was only
   impaired at higher NaCl levels, i.e., at 100 and 150 mM ([162]Figure
   1A), indicating that there were adaptative mechanisms at 25 and 50 mM.
   It was therefore of interest to identify biochemical changes that
   differed at these time points. At low salinity stress, we found that
   there was carbohydrate accumulation that may function in salt tolerance
   by playing an osmoprotective, and ROS scavenging role [[163]80]. At a
   moderate level, many amino acids were increased ([164]Figure 6).

   Antioxidant activity. Salinity stress induces electrolyte leakage which
   in turn, is associated with ROS accumulation [[165]81,[166]82], and
   cellular oxidative damage [[167]83,[168]84]. APX, CAT, GPX, and SOD,
   are often deployed to detoxify ROS and engender stress tolerance
   [[169]33,[170]36,[171]85,[172]86,[173]87,[174]88]. The relative
   proportion of these enzymes determines the intracellular levels of ROS,
   and changes in their relative balance may induce stress tolerance
   mechanisms [[175]89]. SOD is the first line of defense against ROS by
   catalyzing the dismutation of O[2] to H[2]O[2] [[176]90,[177]91]. SOD
   activity increased when the seedlings were exposed to moderate salinity
   ([178]Figure 2D), but decreased at high salinity. Similar results were
   seen in Brassica napus L., where 100 to 150 mM NaCl decreased SOD
   activity [[179]92]. ROS can be generated by CAT, which is responsible
   for the removal of H[2]O[2] by reducing H[2]O[2] to 2H[2]O. Our
   observations also strongly imply, that SOD, APX, and GPX enzymes are
   the salinity-induced ROS scavenging mechanisms in gac seedling leaves.
   Moreover, increased plant antioxidants is positively associated with
   decreased oxidative damage and improved salinity tolerance
   [[180]93,[181]94,[182]95,[183]96]. From the heat map ([184]Figure 5),
   putrescine increased at 100 and 150 mM NaCl. Zhang et al. [[185]97]
   reported that putrescine plays a positive role in salt-tolerance
   mechanisms by reducing oxidative damage in soybean. Jiménez-Bremont et
   al. also reported that as free putrescine increased in maize leaves
   oxidative damage during NaCl stress decreased [[186]98]. Tang and
   Newton [[187]99] proposed that exogenous putrescine could improve plant
   growth under NaCl stress, by inhibiting Na^+ and Cl^− uptake, and
   accelerating the accumulation of K^+, Ca^2+, and Mg^2+ in the leaves,
   thereby protecting plants from salt stress damage.

   Leaf chlorophyll content. High salt interferes with leaf photosynthetic
   machinery which can be manifested as reduced chlorophyll. Our results
   showed a small reduction in chlorophyll a, and SPAD values when gac
   seedlings were subjected to 50, 100, and 150 mM NaCl, which agrees with
   previous reports [[188]100,[189]101]. Salinity−induced decreases in
   chlorophyll levels is associated with thylakoid membrane degradation,
   increased chlorophyllase activity, and inhibition of
   magnesium-protoporphyrin IX monomethyl ester (oxidative) cyclase due to
   oxidative stress [[190]102,[191]103,[192]104,[193]105,[194]106]. In our
   study, salinity affected chlorophyll a but not chlorophyll b content,
   an observation made in other studies [[195]107,[196]108]. Chlorophyll b
   can be synthesized from chlorophyll a [[197]109]. In Cymbopogon nardus,
   chlorophyll a appears to be more sensitive than chlorophyll b to
   salinity [[198]110] and salinity could trigger chlorophyll a conversion
   to chlorophyll b. Interestingly, chlorophyll a, total chlorophyll, and
   SPAD value of gac seedling leaf exposed to 25 mM NaCl were increased
   which were supported by Elhaak et al. [[199]111], Gomes et al.
   [[200]112], and Gong et al. [[201]108].

   Although photosynthetic pigments decreased when gac seedlings were
   exposed to 150 mM NaCl, the heat map results ([202]Figure 5) showed an
   increase in 1-deoxyerythriol which is involved in the biosynthesis of
   chlorophyll side chains [[203]113]. Gamma, delta, and alpha-tocopherol,
   which are in chloroplasts and which act as antioxidants, also increased
   [[204]114]. Salt stress also increases plants’ respiration rate due to
   a need for higher energy for adaptive mechanisms such as ion exclusion,
   compatible solute synthesis, and ROS detoxification [[205]115]. In
   wheat, higher NaCl concentration increased the respiration rate
   [[206]116], moreover, more and ATP was higher in a salt-tolerant wheat
   cultivar compared to a salt-sensitive one [[207]117,[208]118].
   Therefore, plants may show tolerance to salinity by increasing
   photosynthetic pigments for higher photosynthetic capacity and ATP
   production. The resistance of photosynthetic systems to salinity is
   associated with the ability of the plant to effectively sequester the
   ions in the chloroplast and the other organelles [[209]119].

   Carbohydrate metabolism plays an important role as an adaptive response
   to salinity [[210]120,[211]121,[212]122,[213]123,[214]124] and it also
   has been associated with salinity-tolerant mechanisms in many plants,
   via osmotic adjustments [[215]125]. Accumulation of sugars as
   compatible solutes is accompanied by the influx of water into a plant
   cell [[216]126]. Exposure to 25 mM NaCl triggered higher starch content
   and was also associated with the accumulation of some photosynthetic
   pigments ([217]Figure 1D and [218]Figure S3). It is possible to
   speculate that higher photosynthetic activity was linked to an increase
   in starch content at low salinity. Moreover, starch increased at 25 mM
   and total soluble sugar content at 50 mM NaCl compared to the control
   and the other groups. Pattanagul and Thitisaksakul [[219]46] also
   reported that sugar content was induced by salinity stress in
   salt-sensitive rice as an osmotic adjustment. Therefore, these results
   agree with the previous studies on the responses of plants under
   salinity stress [[220]127,[221]128]. Leaf starch may hyperaccumulate to
   prevent feedback inhibition of photosynthesis and then under prolonged
   stress act as a store of sugars for osmoprotection [[222]127]. In
   addition to carbohydrate accumulation, there were increases in other
   compounds that serve as osmoprotectants: glycine, 5-hydroxynorvaline,
   glucose-6-phosphate, fructose-6-phosphate, and proline
   [[223]129,[224]130,[225]131,[226]132,[227]133].

   Our study found that six metabolites were significantly decreased when
   gac seedlings were grown under salinity conditions. These metabolites
   included isocitric acid, citric acid [[228]134], and maleic acid
   [[229]135], which are found in the TCA cycle ([230]Figure 6). In a
   study of salt-treated wheat [[231]116], higher respiration rates and
   extensive metabolite changes were shown. The activity of key enzymes of
   the TCA cycle was shown to be directly salt-sensitive by increased GABA
   shunt activity, which provides an alternative carbon source for
   mitochondria that bypasses salt-sensitive enzymes, to facilitate
   increased respiration [[232]116]. In accordance with our carbohydrate
   metabolism results, high salinity increased starch degradation and
   sucrose and glucose, and fructose content, which could enhance flux
   through the respiratory pathways and increase ATP. However, if key
   enzymes of the TCA cycle are inhibited, then it is unlikely that ATP
   would be generated.

   Lysine and citrulline are important indicators of salt tolerance, and
   both increased in gac seedling leaves, [[233]135,[234]136]. Under high
   salinity (150 mM), there are 33 metabolic changes, which could be a
   response to the cellular damage induced by higher NaCl concentrations
   [[235]137,[236]138], precursors of IAA [[237]139,[238]140], compatible
   solutes protecting cells from osmotic stress
   [[239]141,[240]142,[241]143,[242]144], precursors of biosynthesis of
   photosynthetic pigments [[243]114], antioxidant molecules in
   chloroplasts [[244]114].

   Pathway impact was used to identify metabolic pathway associated with
   changes in salinity. Many metabolic pathways were altered, especially
   amino acid metabolism pathways, which have roles in stress response
   [[245]141,[246]145,[247]146,[248]147,[249]148,[250]149,[251]150,[252]15
   1,[253]152,[254]153,[255]154]. Our study found that many organic acids
   including TCA cycle intermediates such as pyruvic acid, oxalic acid,
   malic acid, citric acid, and succinic acid declined under salinity. The
   reduction in organic acids, and TCA cycle intermediates may be due to
   the diversion of carbon flux towards the synthesis of osmoprotectants
   to cope with salt stress [[256]155,[257]156]. Salinity-induced
   reductions in organic acids are a metabolic phenotype reported in
   halophytes such as Thellungiella halophila, Suaeda salsa, Salvadora
   persica, and Chenopodium quinoa [[258]155,[259]156,[260]157]. Under
   salt stress, the regulation of organic acid metabolism is intimately
   related to growth reduction, and it might be an energy-saving strategy
   by inducing changes in starch metabolism and sugar export in the source
   [[261]158]. Salinity stress at 100 mM affected glucosinolate, lysine
   biosynthesis [[262]159], cyanoamino acid metabolism [[263]160], and
   valine, leucine, and isoleucine degradation ([264]Figure S6). However,
   higher salinity stress had no impact on these metabolic pathways. Our
   study also found that starch and sucrose, galactose, and β-alanine
   metabolism were changed when gac seedlings were grown under 150 mM NaCl
   ([265]Figure S7). Sugar and sugar derivatives found in these pathways
   are likely an adaptive response that helps plants cope with water
   stress via an osmotic mechanism, energy metabolism, and ROS scavenging.

5. Conclusions

   This study observed changes in physiological characteristics and
   metabolic profiles in response to mild and moderate salinity stress in
   gac leaf. At mild stress, which may be the alarm response, SOD and POX
   enzyme activity increased and were probably associated with ROS
   scavenging in stress tolerance ([266]Figure 7). Photosynthetic pigments
   slightly increased presumably for maintaining energy stores
   ([267]Figure 7). Increases in starch may prevent feedback inhibition of
   photosynthesis and then, serve as a store of sugars for osmoprotection
   ([268]Figure 7). Higher salt concentrations revealed the acclimatory
   and exhaustion responses, POX enzymes were still active while SOD
   enzymes decreased. Moreover, metabolites in the TCA cycle decreased,
   presumably because of a greater flux of carbon towards amino acids
   production, to act as compatible solutes. Further research should focus
   on the mechanisms regulating the level of metabolites involved in
   salinity stress tolerance, especially amino acids for the determination
   of key pathways controlling high salinity tolerance in plants.

Figure 7.

   [269]Figure 7
   [270]Open in a new tab

   A Schematic comparing mild salinity stress and moderate salinity stress
   responses of gac plant. Figure is based on data generated in this
   study.

Acknowledgments