Table 1 reports the essential oil compositions produced at various levels of salinity stress and melatonin treatment. Accordingly, twenty-two compounds were identified in all the plant cultures of H. longiflorus; however, the percentages of the essential oil components significantly differed by the treatments used. The results indicated that the largest percentages were exhibited by d-limonene (2.55-15.65%), α-pinene (0.57-15.06%), α-thujene (0.96-14.37%), copaene (3.24-6.46%), eremophila ketone (2.01-4.78%), terpinen-4-ol (1.69-4.68%), and γ-terpinene (0.56-4.67%).
Essential oil components
Our results showed that salinity stress significantly affected all the essential oil components identified in H. longiflorus volatile compounds (Table 1). As the levels of NaCl were raised to 50 and 100 mM, significant increases were observed in the concentrations of α-thujene, α-calacorene, abietatriene, α-cadinene, cadalene, cadine-1,4-diene, d-limonene، γ-terpinene, ylangene, and 5,15-rosadiene. In contrast, decreases were exhibited by amorpha-4,11-diene, terpinen-4-ol, ar-curcumene, copaene, β-eudesmol, epicubenol, and α-pinene contents with the increase in NaCl levels. Moreover, the use of NaCl (up to 50 mM) showed significant positive effects on the contents of pimaradiene, α-cedrene, epi-γ-eudesmol, 1,7-di-epi-β-cedrene, and eremophila ketone, which then subsided with further increase in NaCl concentration, however (100 μM). Enriching the culture medium with different melatonin levels also changed the percentages of H. longiflorus essential oil compounds. Applying 15 μM melatonin with 50 mM NaCl increased the contents of abietatriene, cadalene, cadine-1,4-diene, and 5,15-rosadiene by 1.94, 2.80, 2.56, and 1.65 times, respectively, as compared to those with the application of NaCl alone. In addition, the amounts of α-cadinene and α-calacorene in the culture medium containing a mixture of 50 mM NaCl and 5 and 25 μM melatonin showed a significant increase (by 51.02% and 51.18%, respectively) compared to those in the salt-stressed plants without melatonin. The highest contents of amorpha-4,11-diene, terpinen-4-ol, β-eudesmol, and copaene were recorded in the culture medium containing 5 and 25 μM melatonin without NaCl treatment. The content of ar-curcumene showed the highest increase in the culture medium comprising 50-mM NaCl and 25 μM melatonin. 15 μM melatonin treatment alone increased the content of epicubenol by 24.19% compared to that in the control plants. At the same time, it did not show any significant difference from those in melatonin treatments (15 and 25 μM) in the presence of 50 mM NaCl. Similarly, under 100 mM salinity stress, the contents of ylangene and α-pinene with the addition of 15 and 25 μM of melatonin to the medium were 36.32% and 26.33 times higher than those in 200 mM NaCl treatment alone. The culture medium containing 50 mM NaCl enriched with 15 μM melatonin strongly increased the percentage of pimaradiene (by 25.48%). The application of 50 mM NaCl without melatonin and in combination with different levels thereof (5, 15, and 25 μM) resulted in higher α-cedrene contents (by 96.95%, 99.13%, 106.74%, and 100.21%, respectively) compared to that in the control treatment. With 15 μM melatonin treatment and no stress, the eremophila ketone, epi-γ-eudesmol, and 1,7-di-epi-β-cedrene contents increased by 46.18%, 63.51%, and 61.64%, respectively, as compared to those in the normal conditions. The contents of d-limonene, α-thujene, and γ-terpinene noted similar patterns and reached their highest levels in a combination of 15 μM melatonin and 100 mM NaCl (Table 1).
Table 1 Effect of application of melatonin and NaCl on essential oil components (%) of H. longiflorus plants grown in in vitro conditions
|
Treatments
|
|
Compound
|
S1M1
|
S1M2
|
S1M3
|
S1M4
|
S2M1
|
S2M2
|
S2M3
|
S2M4
|
S3M1
|
S3M2
|
S3M3
|
S3M4
|
RI
|
α-Pinene
|
13.29±0.50b
|
8.47±0.54d
|
3.80±0.09g
|
2.92±0.18g
|
7.28±0.70e
|
5.48±0.15f
|
8.74±0.15d
|
11.79±0.49c
|
0.57±0.04h
|
9.28±0.48d
|
14.17±0.46ab
|
15.06±0.10a
|
952
|
D-Limonene
|
4.47±0.21d
|
3.53±0.51ef
|
2.55±0.20g
|
6.65±0.54c
|
5.75±0.33c
|
2.83±0.12fg
|
3.82±0.37de
|
3.31±0.21e-g
|
6.65±0.28c
|
10.24±0.33b
|
15.65±0.19a
|
5.86±0.27c
|
1027
|
Copaene
|
5.27±0.06cd
|
5.44±0.23bc
|
3.24±0.15f
|
6.46±0.29a
|
3.26±0.13f
|
4.26±0.22e
|
4.25±0.16e
|
4.65±0.43de
|
3.44±0.25f
|
5.37±0.38c
|
6.06±0.12ab
|
5.14±0.12cd
|
1376
|
α-Thujene
|
2.58±0.11e
|
0.96±0.08i
|
1.99±0.20fg
|
2.25±0.09ef
|
3.61±0.15d
|
1.74±0.14gh
|
3.81±0.07cd
|
1.37±0.10h
|
4.19±0.12c
|
6.13±0.11b
|
14.37±0.16a
|
1.73±0.18g
|
946
|
Eremophila ketone
|
3.27±0.01de
|
3.34±0.06de
|
4.78±0.10a
|
3.81±0.19c
|
4.01±0.08bc
|
3.70±0.13cd
|
3.16±0.10ef
|
4.30±0.17b
|
2.81±0.12f
|
2.01±0.24g
|
2.08±0.15g
|
3.20±0.27ef
|
1576
|
β-Eudesmol
|
3.08±0.03c
|
3.22±0.11bc
|
1.99±0.08ef
|
3.86±0.12a
|
1.46±0.08g
|
3.45±0.09b
|
1.20±0.11gh
|
1.93±0.09f
|
1.15±0.08h
|
2.57±0.07d
|
0.80±0.02i
|
2.20±0.13e
|
1657
|
Terpinen-4-ol
|
2.52±0.26cd
|
4.68±0.11a
|
2.11±0.15d-f
|
2.96±0.17b
|
2.35±0.06c-e
|
1.72±0.07fg
|
1.72±0.15fg
|
2.38±0.03cd
|
1.93±0.19fg
|
2.56±0.06bc
|
1.96±0.08e-g
|
1.69±0.18g
|
1180
|
Amorpha-4,11-diene
|
2.78±0.13b
|
3.17±0.08a
|
1.59±0.09e
|
0.97±0.02fg
|
1.02±0.08f
|
2.06±0.07d
|
2.61±0.11bc
|
2.34±0.06c
|
1.34±0.08e
|
0.85±0.07fg
|
0.74±0.03g
|
0.92±0.05fg
|
1490
|
Pimaradiene
|
1.28±0.05fg
|
1.69±0.09e
|
1.68±0.07e
|
1.06±0.12g
|
2.59±0.09c
|
2.97±0.08b
|
3.25±0.15a
|
2.00±0.12d
|
2.16±0.10d
|
2.02±0.05d
|
1.44±0.04ef
|
1.20±0.05fg
|
2014
|
α-Cadinene
|
1.40±0.10d
|
2.07±0.10bc
|
2.14±0.13b
|
1.79±0.15c
|
1.96±0.06bc
|
2.96±0.06a
|
2.79±0.13a
|
2.70±0.15a
|
1.97±0.12bc
|
1.05±0.09e
|
1.17±0.11de
|
1.32±0.06de
|
1539
|
α-Cedrene
|
0.92±0.11e
|
1.04±0.07de
|
1.49±0.09b
|
1.34±0.11bc
|
1.81±0.04a
|
1.83±0.06a
|
1.91±0.08a
|
1.84±0.09a
|
1.47±0.04b
|
1.23±0.08cd
|
1.21±0.05cd
|
1.76±0.09a
|
1386
|
γ-Terpinene
|
0.61±0.01f
|
0.72±0.07ef
|
0.56±0.05f
|
1.14±0.03d
|
1.25±0.09d
|
0.92±0.06e
|
1.95±0.08c
|
0.74±0.04ef
|
1.18±0.12d
|
2.38±0.10b
|
4.67±0.09a
|
1.23±0.08d
|
1045
|
Cadine-1,4-diene
|
0.87±0.06fg
|
1.26±0.08e
|
1.59±0.06d
|
0.97±0.07f
|
1.02±0.10f
|
2.06±0.06c
|
2.61±0.09a
|
2.34±0.05b
|
1.34±0.07e
|
0.85±0.05fg
|
0.74±0.07g
|
0.92±0.08fg
|
1652
|
1,7-Di-epi-β-cedrene
|
0.83±0.05de
|
0.74±0.03de
|
1.34±0.12a
|
1.17±0.05ab
|
1.04±0.10bc
|
0.93±0.04cd
|
0.80±0.06de
|
0.90±0.06cd
|
0.69±0.04e
|
0.86±0.03c-e
|
0.91±0.01cd
|
1.17±0.11ab
|
1414
|
Abietatriene
|
0.76±0.01f-h
|
0.84±0.01d-f
|
0.68±0.04gh
|
0.44±0.02i
|
0.96±0.03d
|
1.42±0.07b
|
1.87±0.04a
|
0.91±0.02de
|
1.21±0.12c
|
0.77±0.02e-g
|
0.67±0.08gh
|
0.62±0.02h
|
2064
|
α-Calacorene
|
0.72±0.02g-i
|
1.14±0.06a-c
|
1.00±0.08c-e
|
0.93±0.05d-f
|
0.81±0.01f-h
|
1.04±0.04b-d
|
1.18±0.11ab
|
1.22±0.10a
|
0.84±0.02e-g
|
0.65±0.01h-j
|
0.55±0.01j
|
0.59±0.01ij
|
1545
|
Cadalene
|
0.45±0.01ij
|
0.91±0.04cd
|
0.88±0.01de
|
0.72±0.01fg
|
0.60±0.01gh
|
0.76±0.02ef
|
1.69±0.09a
|
1.15±0.10b
|
0.65±0.00f-h
|
0.57±0.02hi
|
1.04±0.06bc
|
0.38±0.02j
|
1681
|
Ar-curcumene
|
0.76±0.05cd
|
0.81±0.03b-d
|
0.76±0.02cd
|
0.86±0.02b
|
0.75±0.03d
|
0.52±0.01f
|
0.83±0.02b-d
|
1.00±0.07a
|
0.41±0.01g
|
0.62±0.02e
|
0.62±0.01e
|
0.85±0.03bc
|
1485
|
epi-γ-Eudesmol
|
0.74±0.01de
|
0.65±0.02ef
|
1.21±0.15a
|
0.68±0.02d-f
|
0.80±0.03cd
|
0.56±0.01f
|
0.72±0.02de
|
0.89±0.02bc
|
0.57±0.01f
|
0.96±0.02b
|
0.33±0.02g
|
0.56±0.00f
|
1642
|
Epicubenol
|
0.58±0.01d
|
0.63±0.01c
|
0.72±0.02a
|
0.51±0.02e
|
0.52±0.01e
|
0.68±0.01b
|
0.71±0.02ab
|
0.75±0.02a
|
0.25±0.01h
|
0.33±0.02g
|
0.63±0.01c
|
0.46±0.01f
|
1622
|
Ylangene
|
0.33±0.01f
|
0.48±0.01bc
|
0.34±0.01f
|
0.47±0.01b-d
|
0.41±0.01e
|
0.35±0.01f
|
0.44±0.01de
|
0.35±0.02f
|
0.45±0.02c-e
|
0.50±0.02b
|
0.61±0.02a
|
0.59±0.01a
|
1352
|
5,15-Rosadiene
|
0.20±0.00g
|
0.32±0.00e
|
0.28±0.00f
|
0.20±0.01g
|
0.34±0.01d
|
0.38±0.01bc
|
0.57±0.00a
|
0.40±0.01b
|
0.35±0.01cd
|
0.28±0.00f
|
0.18±0.02h
|
0.18±0.01h
|
1904
|
The values indicated by different letters (means ± SE) within each column show a significant difference (p < 0.05)
S1M1: control, S1M2: melatonin 5 μM, S1M3: melatonin 15 μM, S1M4: melatonin 25 μM, S2M1: NaCl 50 mM, S3M1: NaCl 100 mM, S2M2: NaCl 50 mM and melatonin 5 μM, S2M3: NaCl 50 mM and melatonin 15 μM, S2M4: NaCl 50 mM and melatonin 25 μM, S3M2: NaCl 100 mM and melatonin 5 μM, S3M3: NaCl 100 mM and melatonin 15 μM, S3M4: NaCl 100 mM and melatonin 25 μM. RI: Retention Index
Our results also demonstrated that 2,4-(D2)menth-2-ene, sativene, and caryophyllene were detected only in the plants grown under treatment with 5 μM of melatonin. On the other hand, 4-epi-α-acoradiene, dehydroaromadendrene, dl-neoisolongifolene, eremophilene, and epizonarene were observed only in the plants treated with 15-μM melatonin. However, 100 mM NaCl in the growth medium led to the biosynthesis of α-amylcinnamaldehyde, α-phellandrene, and thujopsene-(I2). In addition, diisooctyl phthalate was identified only in the medium containing 50 mM of NaCl (Table 2). The chromatogram of these detected compounds is shown in supplementary Fig. 1.
Table 2 Effect of application of melatonin and NaCl on new component identified in essential oil of H. longiflorus plants grown in in vitro conditions
Number
|
Compound
|
Formula
|
Mwt
|
|
|
|
|
|
|
Treatment
|
|
|
|
|
|
S1M1
|
S1M2
|
S1M3
|
S1M4
|
S2M1
|
S2M2
|
S2M3
|
S2M4
|
S3M1
|
S3M2
|
S3M3
|
S3M4
|
1
|
2,4-(D2)menth-2-ene
|
C10H16D2
|
140.00
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
2
|
Sativene
|
C15H24
|
204.35
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
3
|
Caryophyllene
|
C15H24
|
204.35
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
4
|
4-epi-α-Acoradiene
|
C15H24
|
204.35
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
5
|
Dehydroaromadendrene
|
C15H24
|
204.35
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
6
|
dl-Neoisolongifolene
|
C15H24
|
204.35
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
7
|
Eremophilene
|
C15H24
|
204.35
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
8
|
Epizonarene
|
C15H24
|
204.35
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
9
|
Diisooctyl phthalate
|
C24H38O4
|
390.6
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
10
|
α-Amylcinnamaldehyde
|
C14H18O
|
202.29
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
-
|
11
|
α-Phellandrene
|
C10H16
|
136.23
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
-
|
12
|
Thujopsene-(I2)
|
C15H24
|
204.35
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
+
|
-
|
-
|
-
|
S1M1: control, S1M2: melatonin 5 μM, S1M3: melatonin 15 μM, S1M4: melatonin 25 μM, S2M1: NaCl 50 mM, S3M1: NaCl 100 mM, S2M2: NaCl 50 mM and melatonin 5 μM, S2M3: NaCl 50 mM and melatonin 15 μM, S2M4: NaCl 50 mM and melatonin 25 μM, S3M2: NaCl 100 mM and melatonin 5 μM, S3M3: NaCl 100 mM and melatonin 15 μM, S3M4: NaCl 100 mM and melatonin 25 μM
Growth parameters
The results showed that adding NaCl to the culture medium at all levels inhibited shoot and plant biomass growth and development. In addition, plant samples grown in the NaCl medium exhibited necrosis symptoms and died. NaCl toxicity symptoms intensified as the NaCl levels rose to 100 mM (Fig. 1d). The highest biomass (0.22 g) and SL (25.38 cm) were recorded for 5 μM melatonin under no NaCl application, while higher concentrations of melatonin (15 and 25 M) reduced both plant growth parameters (Table 3). Under all levels of NaCl, exogenously applied melatonin (5 μM and 15 μM) significantly reduced the plant growth inhibition caused by NaCl deficiency compared to that in the plant samples exposed only to NaCl (Fig. 1b and c).
Biochemical features and antioxidant activity
Although increasing the level of NaCl to 100 mM led to the lowest content of chlorophyll (0.75 mg.g-1 FW), the effect of 50 mM NaCl treatment was not statistically significant. Application of melatonin at levels of 5, 15, and 25 μM increased chlorophyll concentration by 44%, 57%, and 48%, respectively, compared to that in plant samples exposed only to 100 mM NaCl (Fig. 2a). With an increase in the NaCl level to 50 mM, carotenoid content slightly increased (by 18.07%) compared to that in the control group. At the higher NaCl concentration (100 mM), carotenoid content decreased by 15.66% and reached its lowest level (0.07 mg.g-1 FW). However, these changes were not significant. Melatonin treatments led to enhanced carotenoid content. The highest carotenoid concentration (0.19 mg.g-1 FW) was observed in plants treated with 5 μM of melatonin without NaCl application. Combined application of NaCl and melatonin resulted in higher carotenoid values than in control plants under salt stress (Fig. 2b).
We evaluated the effect of melatonin in the amelioration of the adverse effects of salt stress-induced oxidative stress under both 50 and 100 mM NaCl treatments. The results showed that melatonin-treated plants (without NaCl application), indicated no significant effects on lipid peroxidation or H2O2 content as compared to that in the control treatment (Table 3). The presence of NaCl in the medium at both levels of 50 and 100 mM significantly increased the concentration of H2O2 by 2.09 and 2.38 times and MDA content by 2.96 and 3.05 times, respectively, compared to those in the control conditions.
Table 3 Effect of application of melatonin and NaCl on physiological and biochemical parameters of H. longiforus plants grown in in vitro conditions
Treatments
|
Parameters
|
NaCl (mM)
|
Melatonin (µM)
|
Fresh weight (g)
|
Shoot length (cm)
|
H2O2 (μmol g-1 FW)
|
MDA (μmol g-1 FW)
|
RWC (%)
|
0
|
0
|
0.20±0.00b
|
14.31±0.19b
|
2.05±0.16e
|
1.54±0.13d
|
76.73±0.81ab
|
5
|
0.22±0.01a
|
25.38±0.18a
|
2.10±0.13e
|
1.46±0.07d
|
79.14±1.05a
|
15
|
0.10±0.01d
|
7.84±0.15d
|
2.21±0.16e
|
1.49±0.27d
|
78.14±0.50ab
|
25
|
0.10±0.00de
|
7.99±0.09d
|
2.30±0.15e
|
1.63±0.03d
|
75.56±0.46bc
|
50
|
0
|
0.04±0.00fg
|
1.83±0.04hi
|
4.29±0.35bc
|
4.56±0.18ab
|
75.48±2.41bc
|
5
|
0.16±0.01c
|
9.21±0.30c
|
3.41±0.23d
|
2.09±0.42d
|
74.96±0.14bc
|
15
|
0.10±0.00de
|
4.64±0.14f
|
3.96±0.28cd
|
3.82±0.32bc
|
73.08±0.62cd
|
25
|
0.03±0.00gh
|
2.06±0.11h
|
3.40±0.38d
|
4.98±0.29a
|
71.62±2.09de
|
100
|
0
|
0.03±0.00gh
|
2.28±0.09h
|
4.87±0.18ab
|
4.70±0.36a
|
56.86±0.48g
|
5
|
0.09±0.00e
|
6.60±0.14e
|
4.06±0.17c
|
3.35±0.53c
|
70.33±1.17de
|
15
|
0.04±0.00f
|
3.97±0.09g
|
4.52±0.09bc
|
4.45±0.32ab
|
69.30±0.47e
|
25
|
0.02±0.00h
|
1.52±0.05i
|
5.40±0.18a
|
5.22±0.16a
|
62.20±0.66f
|
The values indicated by different letters (means ± SE) within each column show a significant difference (p < 0.05)
The production of H2O2 showed significant decrease under salt stress with all levels of melatonin addition to the medium, except when 25 μM melatonin was applied along with 100 mM NaCl. The application of 5 μM melatonin showed the highest decrease in H2O2 and MDA levels (by 16.63% and 28.72%, respectively) in the medium where NaCl was used. However, the application of 25 μM melatonin raised the amount of MDA in plants exposed to salt stress (Table 3). To further evaluate the protective role of melatonin against NaCl-induced damage, leaf RWC was measured. There was no significant difference in RWC between the 50 mM NaCl and control treatments. However, a noticeable decline in RWC (by 25.90%) was evident in plants subjected to 100 mM NaCl, compared to that in the control treatment. Under normal conditions, RWC remained relatively constant in H. longiflorus plants, regardless of melatonin treatment, while the application of melatonin (at 5-25 μM) resulted in notable increases by 23.69%, 21.88%, and 9.39%, respectively, in RWC compared to that in untreated plants under 100 mM NaCl conditions (Table 3).
The activities of SOD, CAT, and APX changed in response to different melatonin and NaCl concentrations in H. longiflorus plants (Fig. 3). Regardless of the melatonin application, higher NaCl concentrations notably stimulated enzyme activities. No significant change was observed in the activities of antioxidant enzymes in H. longiflorus plants grown under media containing only melatonin compared to those in the control treatment. The highest activities of CAT and SOD were observed in plants grown under 100 mM NaCl plus 5 and 25 μM melatonin applications, respectively (Fig. 3a and b). The 50 mM NaCl and melatonin (5 μM) treatment exhibited the highest APX activity (Fig. 3c).
Total phenolic compounds and phenylalanine ammonia-lyase (PAL) activity
The results showed that the phenolic compounds were significantly increased by 23.81% in plants as the NaCl level rose to 100 mM. In contrast, the PAL enzyme activity did not significantly change compared to that in untreated plants under any level of NaCl alone (Fig. 4). Although applying melatonin at 15 μM slightly elevated phenolic compounds (17.48%) compared to those in non-treated control plants, its other levels alone had no significant effect on total phenol or PAL enzyme activity. There was no significant difference in total phenolic content or PAL enzyme activity between the plants grown in the medium containing 50 mM NaCl plus 25 µM melatonin and those grown in the medium with 100 mM NaCl and 25 µM melatonin. However, H. longiflorus plants grown under a combination of salt stress conditions (50 and 100 mM NaCl) and 25 μM of melatonin showed not only the highest phenolic content but the highest PAL enzyme activity as compared to those in the other treatments (Fig. 4a and b).
Cross-associations of the treatments and studied parameters
Fig. 5 presents the results of the linear correlation between the measured parameters. According to the results, 1,7-di-epi-β-cedrene and copaene did not correlate significantly with any of the physiological and biochemical characteristics, while ar-curcumene showed significant positive correlations only with RWC and 5,15-rosadiene, and cadalene, abietatriene, cadine-1,4-diene, α-cadinene, and pimaradiene exhibited positive correlations only with APX. FW and SL showed significant positive correlations with amorpha-4,11-diene, terpinen-4-ol, and β-eudesmol, whereas FW exhibited negative correlations with ylangene, γ-terpinene, α-cedrene, α-thujene, and d-limonene. Moreover, a highly negative correlation with α-cedrene was observed for SL. Additionally, there were positive correlations between H2O2 and MDA and ylangene, γ-terpinene, α-cedrene, α-thujene, and d-limonene. However, H2O2 exhibited inverse correlations with epicubenol, epi-γ-eudesmol, α-calacorene, and eremophila ketone, and both H2O2 and MDA showed inverse correlations with amorpha-4,11-diene, terpinen-4-ol, and β-eudesmol. The correlations of total chlorophyll with epicubenol, α-calacorene, amorpha-4,11-diene, terpinen-4-ol, β-eudesmol, and eremophila ketone were significantly positive, while those with ylangene, γ-terpinene, α-thujene, and d-limonene were inverse. Furthermore, significant positive correlations were observed between carotenoid content and epicubenol, α-calacorene, and terpinen-4-ol. We also observed significant positive correlations between RWC and epicubenol, epi-γ-eudesmol, ar-curcumene, α-calacorene, amorpha-4,11-diene, terpinen-4-ol, β-eudesmol, and eremophila ketone, while the correlations between RWC and ylangene and d-limonene were negative. Total phenol had significant positive correlations with metabolites such as ylangene, α-cedrene, and α-pinene, whereas it exhibited negative correlations with terpinen-4-ol and β-eudesmol. The PAL enzyme had positive correlations with ylangene, γ-terpinene, d-limonene, and α-pinene. The CAT and SOD enzymes also exhibited positive correlations with ylangene, γ-terpinene, α-thujene, d-limonene, and α-pinene and negative correlations with epicubenol, epi-γ-eudesmol, α-calacorene, amorpha-4,11-diene, terpinen-4-ol, β-eudesmol, and eremophila ketone. APX showed a negative correlation only with terpinen-4-ol.
Heatmap cluster analysis was performed for the studied traits based on the salinity and melatonin treatments. The analyses revealed that the applied treatments (12 combinations) were clustered into four distinct groups (Fig. 6). The control plants (S1M1) and those grown in the salt-free medium containing different melatonin levels were grouped in the same cluster. Severe salinity stress (100 mM NaCl) was contained in one cluster. The moderate salt stress (50 mM) plus various melatonin levels were placed in a distinguished cluster. The heatmap cluster graph also showed that the treatments containing severe salinity (100-mM NaCl) plus melatonin (5-25 μM) were separated into another cluster. In addition, this analysis indicated that the control treatment and those containing melatonin without any NaCl, especially S1M2, improved plant growth parameters more than the other treatments. Moreover, the treatments containing moderate and severe salinity plus melatonin (depending on the concentration) effectively changed the phytochemical parameters. We also noticed that the application of NaCl alone mitigated both the growth and the synthesis of some essential oil components (Fig. 6).
In the current study, the results of heatmap analysis and cross-correlation between treatments with biochemical and phytochemical characteristics were confirmed by principal component analysis (PCA) (Fig. 7). As visualized by the biplot in Fig. 7, some physiological characteristics of H. longiflorus, such as FW, SL, Total-Chlorophyll, Carotenoids, and RWC, were placed at short distances in the same quarter but at long distances from the oxidative stress indicator groups (H2O2 and MDA) and from total phenol content and the PAL, SOD, CAT, and APX enzymes, indicating negative relationships for the mentioned characteristics. The biplot diagram revealed that most of the phytochemical compounds, including abietatriene, pimaradiene, cadalene, 5,15-rosadiene, cadine-1,4-diene, α-cadinene, α-calacorene, epicubenol, eremophila ketone, amorpha-4,11-diene, epi-γ-eudesmol, and ar-curcumene, were placed in the first quarter, close to the 50 mM NaCl treatments containing melatonin (5-25 μM). β-eudesmol, terpinen-4-ol, and 1,7-di-epi-β-cedrene showed positive correlations with FW, SL, RWC, and photosynthetic pigments and were placed in the second quarter of the biplot, close to the control treatment and different levels of melatonin treatment without NaCl application. Many of the main constituents of the essential oil, such as d-limonene, α-pinene, α-thujene, γ-terpinene, copaene, and ylangene were located in the third quarter and the vicinity of 100 mM NaCl plus melatonin (5-25 μM). Meanwhile, α-cedrene was observed, along with total phenol, in the fourth quarter of the graph, which contained only the application of NaCl (50-100 mM).