Abstract

   Highland barley (Hordeum vulgare L. var. nudum) is a grain crop that
   grows on the plateau under poor and high salt conditions. Therefore, to
   cultivate high-quality highland barley varieties, it is necessary to
   study the molecular mechanism of strong resistance in highland barley,
   which has not been clearly explained. In this study, a high
   concentration of NaCl (240 mmol/L), simulating the unfavorable
   environment, was used to spray the treated highland barley seeds.
   Transcriptomic analysis revealed that the expression of more than 8,000
   genes in highland barley seed cells was significantly altered,
   suggesting that the metabolic landscape of the cells was deeply changed
   under salt stress. Through the KEGG analysis, the phenylpropane
   metabolic pathway was significantly up-regulated under salt stress,
   resulting in the accumulation of polyphenols, flavonoids, and lignin,
   the metabolites for improving the stress resistance of highland barley
   seed cells, being increased 2.71, 1.22, and 1.17 times, respectively.
   This study discovered that the phenylpropane metabolic pathway was a
   significant step forward in understanding the stress resistance of
   highland barley, and provided new insights into the roles of molecular
   mechanisms in plant defense.

Introduction

   Highland barley (Hordeum vulgare L. var. nudum), barley genus [[38]1],
   is mainly distributed in northwest and southwest China’s highland
   areas. It is not only the main grain for Tibetan residents in China,
   but also the primary raw material for producing of beer, medicine, and
   health products [[39]2]. Compared with conventional barley varieties,
   highland barley has the properties of high protein, high dietary fiber,
   and low fat structure [[40]3], which meet the requirements of modern
   people for food. Therefore, highland barley has a broad application
   prospect in the functional food processing industry. Presently,
   research on highland barley mainly focuses on determining nutritional
   composition, extracting active ingredients, and analysing [[41]4,
   [42]5]. For example, Dang [[43]6] studied the content and antioxidant
   capacity of β-glucan, melatonin, total phenols and flavonoids in
   highland barley seed during the germination stage, and analyzed the
   antioxidant capacity of different tissues of highland barley.
   Therefore, the study of the resistance of highland barley to the unique
   living environment is significant for cultivating high-quality highland
   barley varieties and improving the resistance of different plant
   species.

   Salt stress is an important means of studying the stress resistance of
   plants [[44]7]. Soil salinization and soil osmotic pressure can be
   increased by the changes in the concentration of Na^+ and K^+, thus
   causing physiological drought and inhibiting normal plants growth
   [[45]8]. Excessive salt ions accumulate in the plant tissues, resulting
   in ion antagonism, obstruction and destruction of normal physiological
   metabolism, and plant malformation or death [[46]9]. In addition, the
   redox balance in plant cells is destroyed under salt stress. Therefore,
   to effectively reduce the damage of reactive oxygen species, cells need
   to regulate the expression of a protective enzyme system to form a
   self-defense system [[47]10]. In recent years, omics technology has
   developed rapidly, and it is wildly used to excavate stress tolerance
   genes in plants, especially in the analysis of the molecular mechanism
   of salt tolerance of plant cells [[48]11–[49]13]. However, the results
   of the molecular mechanism of salt stress in highland barley are still
   unsatisfactory [[50]14]. Therefore, the analysis of the molecular
   mechanism of salt stress in highland barley is very important for
   researching and cultivating plant varieties with high-stress
   resistance.

   Researchers mainly use transcriptomic strategies to study the
   differences in gene expression levels of highland barley under salt
   stress to excavate stress-related genes. For example, Lai [[51]15]
   utilized transcriptomics and proteomics strategies to analyze the
   molecular mechanism of salt stress response in highland barley seed
   cells during germination stage. They found that highland barley seeds
   could germinate normally under salt stress conditions and detected many
   genes related to salt response, which initially revealed the molecular
   mechanism of highland barley salt tolerance. However, the mechanism of
   salt stress response in related pathways remains unclear. Therefore, it
   is urgent to investigate further the molecular mechanism of salt stress
   in highland barley. Recently, the phenylpropane metabolic pathway,
   which produces polyphenols, flavonoids, lignin, and other secondary
   metabolites, has been reported as an important branch for the
   regulation of stress resistance in Pontederia cordata [[52]16], Walnut
   [[53]17], and watermelons [[54]18]. These metabolites can effectively
   decrease the accumulation of reactive oxygen in plant cells and prevent
   cell morphology, structure, and physiological metabolic function
   destruction. Therefore, the phenylpropane metabolic pathway is closely
   related to the stress resistance of highland barley [[55]19]. In this
   study, NaCl was used to simulate the salt stress environment to
   stimulate the seeds of highland barley during the germination stage.
   Different periods transcriptomics were used to analyze the molecular
   mechanism of highland barley stress resistance ([56]S1 Fig).

Materials and methods

Materials

   Tibet Qing 2000 was provided by the Academy of Agriculture and Animal
   Husbandry Sciences of Tibet Autonomous Region. Gallic acid standard,
   rutin standard, ferulic acid standard, caffeic acid standard,
   Folin-Ciocalteu’s phenol, and lignin content detection kit were
   purchased from Sangon Biotech. RNA Easy Fast RNA extraction kit for
   plant tissue, FastKing cDNA first strand synthesis kit, and
   RealUniversal color fluorescent quantitative premix reagent were
   purchased from TIANGEN (Beijing, China). qRT-PCR primers were
   synthesized at GenScript (Nanjing, China).

Plant growth conditions

   Highland barley seeds full of grains and no mechanical damage were
   selected and soaked in 1 g/L NaClO for 5 min. Then, highland barley
   seeds were cleaned off and soaked for 10 h at 25°C. About 20 g of
   soaked seeds are placed in each marked seedling box. The treated seeds
   were germinated and grown in the intelligent artificial climate chamber
   (relative humidity of 80%, 12 h light/12 h dark, 25°C). Highland barley
   has its unique tolerance to adversity and is very suitable for growing
   on saline soil as a food crop. Seeds were sprayed with 240 mmol/L of
   NaCl and sterilized water daily to collect highland barley seedlings on
   0, 1, 3, 5, and 7 days.

Preparation of highland barley extract solution

   Referring to Aguilera’s method with slight modifications [[57]20], the
   ultrasonic assisted extraction method can dissolve polyphenols,
   flavonoids and other substances contained in plants in methanol as
   completely as possible. Approximately 0.2 g of highland barley
   freeze-dried powder was weighed and added into 5 mL 50% methanol
   solution for shock mixing. The solution was left in darkness at 4°C for
   16–18 hours, then ultrasonic crushed in the water bath for 30 min.
   Finally, the treated solution was centrifuged at 10,000 r/min for 15
   min at 4°C, and the supernatant was filtered through the 0.45 μm
   microporous filter membrane and stored in the brown flask.

Determination of total polyphenol content

   The gallic acid standard solution (10 mg of gallic acid standard was
   weighed and dissolved in distilled water to 50 mL) of 0, 10, 20, 30,
   40, and 50 μL were added into the well plate, respectively, mixing with
   10 μL Folin-phenol reagent or static reaction for 6 minutes. 100 μL of
   7% (W/V) Na[2]CO[3] was added to the solution, distilled water was
   added and the solution volume was adjusted to 290 μL. Absorbance was
   measured at 760 nm and the standard curve was drawn. 50 μL highland
   barley extract solution was added into the well plate and analyzed with
   the above method.

Determination of total flavonoids content

   The rutin standard solution (10 mg of rutin standard was weighed and
   dissolved in 80% ethanol to 50 mL) of 0, 10, 20, 30, 40, and 50 μL were
   added to the well plate, mixing with 20 μL of 5% (W/V) Na[2]CO[3]
   solution for a static response for 6 min. Then, 200 μL 10% (W/V)
   Al[2](NO[3])[3] was added for a static reaction for 6 min. 200 μL 4%
   (W/V) NaOH was added in the end and the mixture solution was reacted
   for 10 min at room temperature for 15 minutes in the dark. Absorption
   was measured at 510 nm and the standard curve was drawn. 50 μL highland
   barley extract solution was added to the well plate and analyzed with
   the above method.

Determination of lignin content

   The lignin content was determined according to the characteristic
   absorption peak at 280 nm after acetylation of the phenolic hydroxyl
   group in the lignin. The operation procedure followed the instructions
   of the lignin content detection kit.

HPLC analysis of ferulic acid and caffeic acid

   The content of ferulic acid in highland barley extract solution was
   determined by C18 (4.6 mm×250 mm, 5 μm) column Arslan [[58]21]. The
   mobile phase was methanol (A): 0.1% (V/V) phosphoric acid solution (B)
   (20: 80). The detection wavelength was 320 nm; the flow rate was 1.0
   mL/min, the column temperature was 30°C and the sample size was 10 μL.
   Ferulic acid standard solution (5.01 mg of standard ferulic acid was
   dissolved in 10 mL of methanol solution. 1 mL of the above solution was
   added into a 100 mL beaker. Methanol was added to the scale line and
   mixed to obtain 5.01 μg/mL of ferulic acid solution) was diluted to
   various concentrations. Then the standard curve was drawn according to
   peak area and peak time. The content of caffeic acid in highland barley
   extract solution was determined by C18 (4.6 mm×250 mm, 5 μm) column
   Wang [[59]22]. The mobile phase was acetonitrile (A): 0.1% phosphoric
   acid solution (B) (10: 90); detection wavelength was 320 nm; flow rate
   was 1.0 mL/min; column temperature was 35°C; sample size was 20 μL.
   Caffeic acid standard solution (21.72 mg ferulic acid standard was
   dissolved in 10 mL methanol solution. 1 mL of the above solution was
   added into a 100 mL beaker. Methanol was added to the scale line and
   mixed to obtain 21.72 μg/mL) it was diluted to various concentrations
   and then the caffeic acid standard curve was drawn. Highland barley
   extract solution was injected after filtering through a 0.22 μm
   microporous filter membrane.

Transcriptomic sequencing

   The germination and cultivation process of highland barley seeds were
   the same as above. Highland barley seedlings with even growth at 0, 1,
   3, 5, and 7 d were snap-frozen in liquid nitrogen and stored at -80°C.
   Plant RNA was extracted and reverse transcribed to obtain cDNA. Three
   biological repeats were set for each sample. These libraries were
   sequenced by paired end (PE) sequencing based on the Illumina
   sequencing platform of Boser Biotech at Personalbio (Shanghai, China).
   KEGG enrichment analysis of differential genes in transcriptome data
   was performed by clusterprofiler.

qRT-PCR

   Total RNA was extracted from highland barley using the RNA Easy Fast
   RNA Extraction kit purchased from TIANGEN (Beijing) according to the
   manufacturer’s instructions. gapdh was used as the housekeeping gene,
   qRT-PCR was performed using a FastKing cDNA first strand synthesis kit
   and RealUniversal color fluorescent quantitative premix reagent
   purchased from Tiangen (Beijing, China) according to the manufacturer’s
   instructions. For each PCR reaction, 10 μL of 2×RealUniversal PreMix,
   0.6 μL of forward and reverse primers ([60]S5 Table), 1.5 μL of cDNA
   sample, and 7.3 μL of RNase-Free ddH[2]O were mixed, and PCR conditions
   were as follows: 95°C for 15 min, followed by 40 cycles of 95°C for 10
   s and 60°C for 30 s. The result was analyzed with the 2^−ΔΔCt method
   and averaged with triplicates.

Results

Analysis of antioxidants in highland barley seeds under salt stress

   Salt stress will cause plant cells to produce a large amount of free
   oxygen, resulting in oxidative damage to plant cells [[61]23].
   Polyphenols and flavonoids were demonstrated as strong antioxidants,
   which could effectively remove oxygen free radicals in plant cells,
   thus protecting cells from the damage of cell-free toxicity [[62]24].
   Therefore, in this study, the content of total polyphenols and
   flavonoids in highland barley seeds during seed germination stage under
   salt stress and nonsalt stress environment was first detected. The
   contents of total polyphenols and total flavonoids in highland barley
   reached the maximum under the condition of 240 mmol/L NaCl ([63]S2
   Fig). As shown in [64]Fig 1a and 1b, the maximum accumulation of
   polyphenols and flavonoids was 8.38±0.36 mg/g and 1.982±0.08 mg/g on
   the third day ([65]S1 Table), respectively, reaching 2.71 and 1.22
   times of the accumulation of polyphenols and flavonoids in highland
   barley seed cells under normal conditions. These results illustrated
   that polyphenols and flavonoids content of highland barley seed cells
   could be efficiently increased under salt stress, thus resulting in the
   rapid removal of free oxygen in highland barley seed cells.

Fig 1. Content analysis of highland barley seeds under salt stress.

   Fig 1
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   The contents of total polyphenols and total flavonoids changed on
   different germination days (0 day, 1 day, 3 day, 5 day, and 7 day)
   under salt stress. (a) The content of total polyphenols on different
   germination days. (b) The contents of total flavonoids changes on
   different germination days. (c) The contents of ferulic acid content on
   different germination days. (d) The contents of caffeic acid on
   different germination days. Error bars show the standard deviation from
   three independent experiments.

   It is widely known that ferulic acid and caffeic acid are two primary
   polyphenols in the plant [[67]25, [68]26]. Therefore, ferulic acid and
   caffeic acid content in the highland barley seeds was also detected.
   The results exhibited that the maximum accumulation of ferulic acid and
   caffeic acid was 12.22±1.35 μg/g and 4.69±0.95 μg/g on the third day,
   respectively, reaching 1.36 and 1.62 times the accumulation of ferulic
   acid and caffeic acid in highland barley seed cells under non-salt
   environment. These results implied that ferulic acid and caffeic acid
   would probably be the most important contents in highland barley for
   resisting disadvantage environments.

   Besides the antioxidants, physical factors would also enhance the
   mechanical strength of highland barley seeds, thus resulting in
   protection against the external environment. Plant tissues contain a
   large amount of lignin, which is an important component to maintain the
   extremely high hardness of plants and protect tissues from corrosion
   [[69]27]. Therefore, lignin accumulation during germination of highland
   barley seed was also detected in this study. As shown in [70]Fig 2,
   lignin content increased with the germination time of highland barley
   seed and reached the highest accumulation on the seventh day. In
   addition, lignin synthesis capacity was significantly improved under
   salt stress, reaching 378.64±0.91 mg/g on the seventh day, which was
   1.17 times the seed content without salt stress. These results
   indicated that highland barley seeds might resist the external
   environment by enhancing lignin synthesis in the process of stress
   resistance.

Fig 2. Content analysis of lignin in highland barley seeds on different
germination days.

   Fig 2
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   Error bars show the standard deviation from three independent
   experiments.

Transcriptomic analysis of gene expression levels of highland barley seed
cells under salt stress

   Under salt stress treatment, the synthesis capacity of antioxidants
   polyphenols and flavonoids of highland barley seeds, as well as lignin,
   were significantly improved during the germination stage, meaning that
   the transcription level of genes for the regulation and biosynthesis of
   these metabolites was significantly changed under salt stress. To
   confirm this hypothesis, transcriptomic profiles of highland barley
   seeds germinating on the third and fifth days under salt stress were
   evaluated compared to seeds under non-salt stress. A total of 100 Gb of
   clean, high quality data was obtained using Illumina HiSeq platform.
   The ratio of useful reads varied from 91% to 95.35% ([72]S2 Table). As
   shown in [73]Fig 3a and 3b, and [74]S3 Fig, the transcriptional
   patterns of seeds germinated under salt stress on the third and fifth
   days exhibited considerable differences from that of seeds germinated
   under non-salt stress, with 5,220 and 4,788 upregulated genes and 4,293
   and 4,565 downregulated genes (|log[2]FoldChange|> 1; p < 0.05),
   respectively, among about 55,706 total genes. These data clearly
   demonstrated that the global metabolism of highland barley seed cells
   underwent tremendous changes under salt stress. Apart from that, to
   explore the relationship between the changes in genes expression and
   germination times, the transcriptional patterns of seeds germinated at
   1, 3, 5, and 7 days were compared with seeds germinated at 0 days under
   salt stress. The results showed that more than 20% of the genes in
   highland barley seed cells were significantly altered during the
   germination stage under salt stress ([75]Fig 3a and 3d). Except that,
   the number of up-regulated genes was the largest on the third day
   during the germination stage, when the accumulation of antioxidants was
   the maximum. These results illustrated that the metabolism pathways of
   highland barley seed cells were significantly altered under salt
   stress, especially during the germination stage after 3 days.

Fig 3. Transcriptomic profiling of highland barley seeds cells under salt
stress.

   [76]Fig 3
   [77]Open in a new tab

   (a) Number of genes significantly (|log[2]FoldChange| > 1; p < 0.05)
   differentially expressed in highland barley seed cells during different
   times of germination stage under salt stress and non-salt stress. (b)
   Heatmap of transcript diversity across highland barley seeds cells
   during different germination stages (1 day, 3 day, 5 day, and 7 day)
   under salt stress. (c) Venn diagram analysis of highland barley seeds
   cells during the germination stage (3 day and 5 day) under salt stress
   and non-salt stress. (d) Heatmap of transcript diversity across
   highland barley seeds cells during the germination stage (3 day and 5
   day) under salt stress and non-salt stress. Three biological replicates
   were performed per sample.

   The Venn diagram was used to confirm the relationship between the
   changed genes. As exhibited in [78]Fig 3c, the transcription levels of
   4453 genes and 4295 genes were just changed on the third and fifth day,
   respectively, illustrating that specific genes’ transcription levels
   differed during different germination stages. Interestingly, the
   transcription level of about 5058 genes was significantly altered both
   on the third day and fifth day, indicating that the transcription level
   of a large number of genes was continuously affected under salt stress.

Changes analysis of metabolic pathways of highland barley seeds cells under
salt stress

   After salt stress treatment, the transcription level of more than 20%
   of the genes in highland barley seed cells was changed. In addition,
   the accumulation of antioxidants, including polyphenols and flavonoids,
   and anticorrosive substance, lignin, of highland barley seeds were
   efficiently increased. Therefore, metabolic pathways in highland barley
   seed cells should be significantly changed. To investigate this
   hypothesis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway
   enrichment analysis was used to identify metabolic differences between
   highland barley seed cells during the germination stage under salt and
   non-salt stress ([79]Fig 4 and [80]S4 Fig). Changes in ribosomal
   pathways stood out among metabolic variations. Among 123 genes involved
   in ribosomal pathways, more than 90 genes exhibited upregulated
   transcriptional levels on the third day during germination stage
   ([81]S3 Table), suggesting that profound changes occurred in the global
   gene translation networks of the highland barley seed cells under salt
   stress. Interestingly, significant changes took place in the
   phenylpropane biosynthesis pathways. Under salt stress, 122 genes and
   48 genes in the phenylpropane biosynthesis pathway (total 351 genes)
   were efficiently up-regulated and down-regulated, respectively, during
   the germination stage (3 day), thus resulting in the improvement of the
   accumulation of its metabolites, such as polyphenols, flavonoids, and
   other substances, in highland barley seeds. It is worth noting that
   significant changes also took place in the glycolysis and TCA cycle
   pathways. Interestingly, some key genes in the TCA cycle were
   significantly downregulated (3 genes up-regulated and 3 genes
   down-regulated), while some key genes of the glycolysis pathway were
   upregulated (19 genes up-regulated and 22 genes down-regulated),
   indicating that additional precursors could be available for the
   biosynthesis above metabolites in highland barley seeds under salt
   stress.

Fig 4. Functional enrichment analysis of transcripts based on KEGG pathways.

   Fig 4
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   Three biological replicates were performed per condition.

   The phenylpropanoid metabolic pathway is the core pathway for producing
   of polyphenols, flavonoids, and other substances, which have been
   demonstrated as antioxidants in plants to defend against stress. This
   metabolic pathway starts from phenylalanine, catalyzed by phenylalanine
   ammonia lyase (PAL) to produce trans-cinnamic acid. In addition,
   cinnamic acid 4-hydroxylase (C4H) catalyzes trans-cinnamic acid to
   produce coumaric acid, ferulic acid, erucic acid, caffeic acid, and
   others [[83]28]. Finally, 4-coumarate-CoA ligase (4CL),
   cinnamyl-alcohol dehydrogenase (CAD), caffeate 3-O-methyltransferase
   (COMT), caffeoyl CoA 3-O-methyltransferase (COMT), caffeoyl CoA
   3-O-methyltransferase (CCoAOMT), and cinnamoyl-CoA reductase (CCR) work
   together to synthesize phenylpropanoid metabolites, such as lignin,
   flavones, and polyphenols. In the analysis of KEGG data ([84]Fig 5,
   [85]S5 Fig, and [86]S4 Table), we found that more than half of the
   genes related to the phenylpropane metabolic pathways were
   significantly up-regulated, while less than 10% were downregulated.
   These results indicated that highland barley seed cells enhanced the
   synthesis ability of polyphenolic metabolites via enhancing
   phenylpropane metabolic pathways under salt stress, thus reducing free
   oxygen content in cells and defending stress resistance. Interestingly,
   the phenylpropane metabolic pathway was the most critical pathway
   associated with the stress resistance of highland barley, which
   provided a theoretical basis for studying the resistance
   characteristics of highland barley and other plant species and the
   cultivation of resistant strains.

Fig 5. Functional enrichment analysis of phenylpropanoid metabolic pathway.

   Fig 5
   [87]Open in a new tab

   Red and green lines represent the upregulated and downregulated
   pathways, respectively.

Verification of the key genes in phenylpropane pathway

   In order to further verify the key points of the phenylpropane
   metabolic pathway in highland barley seed cells in response to salt
   stress, quantitative reverse transcription polymerase chain reaction
   (qRT-PCR) was performed to analyze the transcriptional level of each
   key metabolic gene, including two PAL genes (D1007_08519 and
   D1007_11280), CH4 gene (D1007_58538), 4CL gene (D1007_39853), COMT gene
   (D1007_58836), CCoAOMT gene (D1007_35630), CCR gene (D1007_03378 and
   D1007_34900), and CAD gene (D1007_24864). Comparison of the relative
   expression levels of these genes evaluated by FPKM and qPCR methods is
   shown in [88]Fig 6. The results showed that the transcriptional levels
   of these 9 genes analyzed by qRT-PCR were consistent with that of RNA
   seq, which verified that the key points of the phenylpropane metabolic
   pathway were indeed significantly changed under salt stress.

Fig 6. qRT-PCR verification of the key genes in the phenylpropane metabolic
pathway.

   Fig 6
   [89]Open in a new tab

   The mRNA expression levels were normalized to the expression level of
   gapdh. Error bars show the standard deviation from three independent
   experiments.

Discussion

   Highland barley is an important grain crop in the plateau area of
   China. It is rich in nutrients and meets people’s demand for a healthy
   diet [[90]29]. In addition, highland barley has a strong stress
   resistance, so as to ensure the normal growth of highland barley crops
   in the adverse environment [[91]30]. Therefore, in recent years, more
   and more researchers have begun to study the stress resistance
   mechanism of highland barley, which not only helps to cultivate
   highland barley crops suitable for different adverse environments, but
   also provides a reference for the improvement of stress resistance of
   other crops. However, the current research on stress resistance in
   highland barley has mainly focused on the content analysis of
   metabolites [[92]31–[93]33], and gene mining and verification through
   omic, the work for the identification of metabolic pathways related to
   stress resistance is unsatisfactory. This study used the germination of
   highland barley seeds under salt stress to simulate the unfavorable
   environment of highland barley crops, and the effective improvement of
   related genes in the phenylpropane metabolic pathway was located
   through the transcriptional level of highland barley seeds during
   different stages under salt stress.

   The phenylpropane metabolic pathway is ubiquitous in plants, and its
   secondary metabolites play an important role in plant growth,
   development, and defending stress [[94]34]. The biosynthesis of
   phenylpropane metabolites starts from the common metabolic pathway of
   phenylpropane. Several branches of phenylpropane metabolism are
   downstream, including the lignin synthesis pathway, polyphenol
   synthesis pathway, and flavonoid synthesis pathway. Lignin is the main
   component of the cell wall [[95]35] that supports the plant upright and
   allows the xylem to withstand the negative pressure generated during
   water transport. Under salt stress, the lignification degree of plant
   root cell wall will increase, which can not only effectively prevent
   the internal ion absorption of cells, but also enhance the structural
   rigidity and firmness of conduction tissue and improve the plant salt
   tolerance [[96]36]. Polyphenols and flavonoids have been demonstrated
   as strong antioxidants, effectively removing oxygen free radicals in
   plant cells, thus participating in plant stress resistance. Therefore,
   the phenylpropane metabolic pathway is probably the most important
   method for stress resistance in highland barley.

   The phenylpropane metabolic pathway has not been reported to be related
   to stress resistance in highland barley, but it has been studied in
   other crops [[97]37]. For example, Kiani [[98]38] found that flavonoids
   in Tartary buckwheat flowers increased significantly after 12 h under
   salt stress, which may indicate that anthocyanins were synthesized from
   the corresponding products of salt stress to resist the adverse effects
   of salt stress [[99]39]. Benincasa [[100]40] found that the content of
   polyphenols in rapeseed was increased under salt stress, improving
   plants’ resistance ability to salt stress. Therefore, in this study,
   the barley seeds were treated with 240 mmol/L NaCl, and the total
   polyphenols, flavonoids, and lignin in highland barley seed cells were
   measured. It was found that under salt stress, the yield of highland
   barley seeds polyphenols, flavonoids, and lignin increased by about
   50%, 25%, and 20%, respectively (Figs [101]1 and [102]2). The results
   indicated that highland barley seed cells could effectively increase
   the biosynthesis capacity of polyphenols, flavonoids, and lignin by
   enhancing the phenylpropane metabolism pathway under salt stress, thus
   improving the ability of cells to degrade free oxygen generated under
   adverse conditions and resistance to mechanical forces.

   Considering that the biosynthesis capacity of these metabolites was
   directly according to the transcription level of metabolism pathways,
   therefore, the transcriptome sequencing method was used to analyze the
   differences of genes transcription. The results showed that about the
   transcriptional level of 20% total genes were significantly changed
   under salt stress ([103]Fig 3), illustrating the global metabolism of
   highland barley seed cells could be influenced by salt stress. Through
   the KEGG analysis, we found that about half of the genes in the
   phenylpropane biosynthesis pathway were significantly changed ([104]Fig
   4 and [105]S4 Fig), thus resulting in the improvement of the
   accumulation of its metabolites, such as polyphenols, flavonoids, and
   other substances, in highland barley seeds. Therefore, in order to
   clarify the work mechanism of the response of salt stress resistance in
   highland barley, the detailed analysis of the phenylpropane
   biosynthesis pathway was indispensable.

   The first three catalytic reactions in the phenylpropane metabolic
   pathway were demonstrated to be the core reactions of the whole
   metabolic pathway [[106]41]. PAL is the first reactive enzyme in the
   phenylpropane metabolism pathway, and also a rate-limiting enzyme in
   catalyzing phenylpropane to cinnamic acid, which is the precursor for
   the synthesis of downstream secondary metabolites [[107]42]. Sang
   [[108]43] found that the PAL activity was increased in wheat roots with
   the increase of Na^+, especially in the salt-sensitive wheat genotypes.
   This study found that the transcription of three PAL genes (a total of
   9 PAL genes) was significantly up-regulated under salt stress
   conditions, particularly D1007_11280, with a maximum increase in gene
   transcription level (Log[2]FoldChange = 4.945). With the coordinated
   expression of C4H and PAL, p-hydroxycinnamic acid can be produced by
   hydroxylation of the C4 position of cinnamic acid, which is the second
   step of phenylpropane metabolism. Baek [[109]44] cloned C4H gene from
   black raspberry and found that it had a differential expression pattern
   during fruit development. The gene regulation of C4H could change the
   content of black raspberry flavonoids. In this study, we found that the
   transcriptional level of one of the C4H genes, D1007_58538, in highland
   barley seed cells was significantly up-regulated, reaching about 8
   (23.003) times on the third day during germination stage, respectively
   ([110]Fig 5 and [111]S4 Table). 4CL is the last catalytic enzyme in the
   three standard steps of the phenylpropane metabolic pathway, and it is
   also the main fulcrum enzyme in the secondary metabolism of the
   life-generating thioester [[112]45]. Our study found that transcription
   levels of two 4CL genes (a total of three) in highland barley seed
   cells were significantly up-regulated ([113]S4 Table).

   Several key enzymes, including CCR, CAD, COMT, and CCoAOMT, work to
   produce phenylpropane secondary metabolites, such as polyphenols,
   flavonoids, lignin and methyl cinnamate. In this study, three CAD
   genes, four CCR genes, five COMT genes, and one CCoAOMT gene were
   demonstrated to respond to salt stress ([114]S4 Table). The results
   showed that transcription levels of these key enzymes reached the
   maximum on the third day during germination stage. Except that, the
   transcriptional level of each key metabolic gene, including two PAL
   genes, CH4 gene, 4CL gene, COMT gene, CCoAOMT gene, CCR gene, and CAD
   gene, was accurately analyzed through qRT-PCR method ([115]Fig 6),
   which further verify that the key points of the phenylpropane metabolic
   pathway in highland barley seed cells could sensitively response to
   salt stress. It has been shown that the expression of these genes was
   considered as the regulatory factors controlling the phenylpropane
   pathway, and overexpression of these genes in highland barley would
   improve the resistance of highland barley to stress [[116]46].

Conclusion

   Indeed, the resistance mechanism of highland barley cannot focus on
   just one metabolic pathway. For example, it was found in this study
   that the glutathione synthesis pathway and energy synthesis pathway of
   highland barley seed cells were significantly improved after salt
   stress. What’s more, it’s necessary to analyze the salt tolerance of
   different varieties of highland barley in the next step, and then
   screen out susceptible plants for further understanding of salt
   tolerance mechanism as well as new pathway. These issues need to be
   further addressed in our future studies.

Supporting information

   S1 Fig. Phenotypic characterization of highland barley.

   (TIF)
   [117]Click here for additional data file.^ (1MB, tif)
   S2 Fig. Content analysis of highland barley seeds under different salt
   concentration.

   (TIF)
   [118]Click here for additional data file.^ (126.8KB, tif)
   S3 Fig. Volcano plot analysis of differentially expressed genes.

   (TIF)
   [119]Click here for additional data file.^ (87.1KB, tif)
   S4 Fig. GO enrichment analysis of differentially expressed genes.

   (TIF)
   [120]Click here for additional data file.^ (173.1KB, tif)
   S5 Fig. KEGG analysis of phenylpropane metabolic pathway.

   (TIF)
   [121]Click here for additional data file.^ (123.5KB, tif)
   S1 Table. The content of antioxidants.

   (DOCX)
   [122]Click here for additional data file.^ (12KB, docx)
   S2 Table. Characteristics of libraries.

   (DOCX)
   [123]Click here for additional data file.^ (13.7KB, docx)
   S3 Table. Genes altered significantly in transcription level.

   (DOCX)
   [124]Click here for additional data file.^ (12.3KB, docx)
   S4 Table. Transcriptional level analysis of key genes in the
   phenylpropane metabolic pathway.

   (DOCX)
   [125]Click here for additional data file.^ (12.4KB, docx)
   S5 Table. Primers used for qRT-PCR.

   (DOCX)
   [126]Click here for additional data file.^ (12.7KB, docx)

Acknowledgments