Biochemical properties and pigment contents of Satureja genotypes affected by plant growth regulators and temperature stress

Abstract

There is little data, to our knowledge, on the biochemical properties of different Satureja sp. genotypes affected by plant growth regulators (PGR) under temperature stress. A split plot research on the basis of a complete randomized block design with three replicates examining temperature stress (planting dates, 8th of April, May and June) (main factor), and the factorial combination of plant growth regulators (PGR, control (CO), gibberellic acid (GA), fertilization (MI), and amino acid (A)), and genotypes (Khuzestani, Mutika, and Bakhtiari) on plant biochemical properties, was conducted. Plant pigment contents (chlorophyll a, and b and carotenoids (car)), antioxidant activity (catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GP)), and leaf protein were determined. Treatments significantly and differently affected the genotypes performance. PD3 and PD1resulted in significantly higher activity of APX (0.059 U. mg⁻¹) and GP (0.190 U. mg⁻¹), respectively (P ≤ 0.05). Temperature stress significantly affected plant CAT activity (U. mg⁻¹) at PD1 (0.084) and PD3 (0.820). Higher temperature significantly enhanced leaf Pro, MI increased plant APX (0.054) and CAT activities (0.111 U. mg⁻¹) significantly, and GA resulted in the highest and significantly different GP activity (0.186 U. mL⁻¹). Treatments T1 and T3 significantly enhanced Chla and Car content, and MI resulted in significantly higher Chlb content (0.085 mg g⁻¹ leaf fresh weight). Car and CAT are the two most sensitive biochemical traits under temperature stress and can more effectively regulate Satureja growth and activity. It is possible to alleviate temperature stress on Satureja biochemical properties by the tested PGR.

Introduction

Due to the worldwide use of medicinal plants for human health, they have become among the most important and useful plant species (Mostafavi et al. 2019; Tahamolkonan et al. 2022). Since Satureja species contain different metabolites including tannin, fatty acids, antioxidants, sugars, and essential oils, they can be favorably used for treating different diseases (Khalediyan et al. 2021). However, different parameters such as stress may decrease the growth of medicinal plants and affect their biochemical properties (Bagheri et al. 2021). Temperature stress (fluctuations), resulted by different planting dates, may significantly affect the growth and biochemical properties of Satureja species (Mirjani et al. 2019).

Temperature is among the most important environmental parameters determining the activity and distribution of organisms, worldwide (Mohammadi and Asadi-Gharneh 2018; Bakhshian et al. 2022). Temperature fluctuations, resulting in cold and heat stresses, may unfavorably affect plant growth and biochemical properties. Plants may react to temperature stress by the alteration of their morphological and physiological properties (Challinor et al. 2016; Nievola et al. 2017).

Different methods have been used to alleviate the effects of stress on plant growth, among which the use of plant growth regulators (PGR) may be the most effective one (Miransari et al. 2022a, b). Plant genotype is the other important factor affecting the growth and activity of Satureja species in different conditions including stress (Shafeiee and Ehsanzadeh 2019). Since macro- and micro-nutrients are essential for regulating different plant activities and biochemical properties, under nutrient deficient conditions, plant growth decreases (Sabet and Mortazaeinezhad 2018; Alavi et al. 2021). Researchers have also indicated the use of plant hormones and amino acids can enhance plant growth and physiology in different conditions including stress (Mirbolook et al. 2021; Bakhshian et al. 2022).

With respect to the above-mentioned details, it was hypothesized the use of PGR including plant hormones, nutrients and amino acids can alleviate temperature stress on Satureja sp. physiology and pigment content as there is little data, to our knowledge, in this respect. The objective was to determine the biochemical properties (antioxidant activities) and pigment contents of different Satureja genotypes affected by PGR under temperature stress.

Materials and methods

Experimental location

The experiment was conducted in the research field of Isfahan Islamic Azad University, Iran, with the northern latitude of 32° 26′ and eastern longitude of 51° 42′, and the altitude of 1550 m. The climate of the region (according to Koppen) is dry with dry and warm summers. The rainfall and temperature averages are 120 mm and 16 °C, respectively (Table 1).

Table 1.

The statistical variables of the climatic data during the season

Variable	N	Mean	Std dev	Minimum	Maximum
Tmin	246	11.00	5.56	− 3.40	22.60
Tmax	246	29.90	7.57	12.10	40.50
Rain	246	0.27	1.22	0.00	11.30
Evapo	246	8.26	4.05	0.00	17.40
Sun hours	246	9.56	2.99	0.00	13.20
Tave	246	21.33	6.95	4.60	33.20
RM	246	31.17	18.91	10.50	83.38

Tmin minimum temperature; Tmax maximum temperature; Evapo evaporation; Tave average temperature; RM relative moisture; Std Dev Standard deviation

Soil physicochemical properties

The physicochemical properties of the experimental soil (0–30 cm) were determined by collecting random soil samples, which were analysed in Research & Education Center for Agriculture and Natural Resources (Isfahan Branch) using standard methods (Miransari et al. 2008) (Table 2).

Table 2.

Soil physicochemical properties

EC	pH	TN	OC	Avail. P	Avail. K	Avail. Cu	Avail. Zn	Avail. Mn	Avail. Fe	Sand	Silt	Clay	Tex.
dS/m		%		mg/kg						%
3.8	7.8	0.10	1.05	24.4	600	0.76	0.36	6.6	0.38	19	36	45	clayey

EC salinity; TN total N; OC organic carbon; Avail. Available; Tex. texture

Experimental design

The experiment was split plot on the basis of complete randomized block design with three replicates. The experimental treatments of temperature (planting date, PD), as the main factor, and the factorial combination of the subfactors including plant growth regulators and genotypes were tested. The seeds were planted on the 8th of April, May and June. Plant growth regulators (PGR) including control (CO, water), gibberellic acid (GA, 2 mM) fertilization (2 g L⁻¹), and amino acid (2 g L⁻¹) were applied four times (15-day interval). The genotypes used for the experiment were Khuzestani, Mutika, and Bakhtiari.

Plant growth regulators

1. Gibberellic acid (Merck, Germany, CAS 77-06-5)

2. Microbiomin fertilization (S 464) containing different macro- and micro-nutrients (on a weight basis) including organic N (6%), soluble sulfur (S, 10%), iron (Fe, 4.5%), manganese (Mn, 6%), zinc (Zn, 4.5%), copper (Cu, 2%), boron (B, 0.8%), molybdenum (Mo, 0.1%), and cobalt (Co, 0.0005%), in the form of glycine, mineral sulfate, and the chelates of Fe, Mn, Zn, Cu, B, and Mo (JHBiotech, USA) were used by fertigation of 1 kg.

3. The amino acid (soluble in water), used at 1 kg for the experiment, contained 45% (weight percentage) free amino acids of 4% aspartic acid, 9% glutamic acid, 1% histidine, 5% serine, 2% glycine, 3% threonine, 5% arginine, 3% alanine, 1% tyrosine, 2% valine, 1% phenylalanine, 5% isoleucine, 1% leucine, 2% lysine, and 4% proline, and 18% total N, 8% organic N, and 10% NH₄-N, with the pH of 4.5 (diluted at 1%) (Future Co., Spain), and was verified by the Iranian Institute of Soil and Water.

Plant genotypes

The seeds of Satureja species (with the germination rate of more than 80%) were collected from the Research & Education Center for Agriculture and Natural Resources (Isfahan Branch). The seeds were planted in seedling trays using 100% pitmass with the brand of Domoflor Mix and were planted in the main fields under three different temperatures. The fields were prepared by cultivating, disking and fertilizing according to the soil test. The fields measuring 20 × 65 m (total area of 2000 m²) were plotted by 60-cm row spacing and planted by 40-cm seed spacing.

The plots measured 3 × 2.5 m and each of three replicates (with a one- meter interspacing) consisted of three main plots and 12 sub plots making to the total of nine main plots and 36 subplots under each temperature. Plants were sprayed under no rainy and windy conditions early morning so that all leaves were covered with the solutions. The plants were surface irrigated, according to plant water requirement, after each seven days. Thin-leaf weeds were controlled using gallant (Haloxyfop-R-methyl, 1.5 g L⁻¹) herbicide during the V2-V4 growth stages, and Cyperus rotundus was controlled by hand.

Sampling and measurements

The two middle rows of each plot were used for sampling ignoring a 0.5-m distance from the sides of each plot at the time of 50% flowering for one week. Plant samples were analysed for pigment contents including chlorophyll a, b and total (chla, b and chlt) and carotenoids (car), and biochemical properties including the antioxidant enzymes (catalase, ascorbate peroxidase and guaiacol peroxidase).

Pigment contents

Plant chlorophyll contents were measured using the method of Lichtenthaler and Buschmann (2001). Accordingly, 0.5 g plant leaf, while treating with acetone 80%, was smashed using a crucible, and the solution was then filtrated with a Whatman filter paper. The extract was centrifuged for 10 min in a refrigerated centrifuge (Eppendorf 5810R), at 3000 g. The contents of each tube were brought up to volume with acetone 80%, and light absorption was measured at the wavelengths of 646, 663, and 470 nm, respectively using spectrophotometer (Model Unico 2100). The instrument was calibrated by acetone 80% without plant extract. The concentrations of plant pigments were determined using the following formulas:

$$Chlorophylla=\frac{\left[\left(12.21\ast \text{Abs}663\right)-2.82\ast \text{Abs}646\right)\ast \text{Aceton}\text{mL})}{\text{Leaf}(\text{mg})}$$

$$Chorophyllb=\frac{\left[\left(20.13\ast \text{Abs}646\right)-\left(5.03\ast \text{Abs}6.63\right)\ast \text{Acetone}(\text{mL})\right]}{\text{Leaf}(\text{mg})}$$

$$\text{Carotenoids}=\frac{\left[1000\ast \text{Abs}470-3.27\left(\text{chla}\right)-104\frac{\left(\text{chlb}\right)}{227}\right]\ast Acetone(mL)}{\text{Leaf}(\text{mg})}$$

Antioxidant enzymes

The activity of the antioxidant enzymes including ascorbate peroxidase (APX), guaiacol peroxidase (GP), and catalase (CAT) were determined according to the following. The leaf sample (0.1 g), which had been preserved in liquid nitrogen (− 80 °C), was homogenized with 1 mL extraction buffer containing polyvinylpyrrolidone (1%), triton X-100, and buffer of K₃(PO₄)₂ (100 mM, pH = 7), in a cooled crucible. The extract was centrifuged at 15,000 g and 4 °C for 20 min. The upper clear part of the extract was used for enzymatic analysis.

APX

The activity of APX was measured according to Nakano and Asada (1981) with some modification using spectrophotometer and the reduction of absorption at the wavelength of 290 nm. The reaction was initiated, by mixing 2.95 mL assay buffer including the buffer of K₃(PO₄)₂ at 50 mM (pH = 7) and hydrogen peroxide (0.5 mM), and the activity of APX was calculated using the following equation.

$$\text{Protein}\text{concentration}\left(\frac{\text{U}}{\text{mg}}\right)=\frac{\text{Apx}\left(\frac{\text{U}}{\text{mL}}\right)}{\text{Protein}\text{concentration}\left(\frac{\text{mg}}{\text{mL}}\right)}$$

“U” is one unit of APX, which is equal to the amount of enzyme, catalyzing one micromole H₂O₂ to O₂ and H₂O in one minute. ΔA = the absorption difference of 290 nm wavelength in one minute, VT = total volume (assay buffer and extract, 3 mL), VE = extract volume (0.05 mL), ϵ = extinction coefficient (2.8 Mm⁻¹ cm⁻¹), and D is dilution coefficient.

GP

The activity of GP enzyme was determined according to Herzog and Fahimi (1973), with some modification using spectrophotometer at the wavelength of 470 nm. The Na₃PO₄ buffer (3 mL), hydrogen peroxide (4.51 µL), guaiacol (3.35 µL), and the extract (50 µL) were mixed in a 3-mL cuvette, and after two minutes the absorption was measured. The calibrating solution contained the above solutions excluding the extract. The activity of GP was determined using the following equations:

$$\text{Gp}(\text{U})=\frac{\mathrm{\Delta }\text{A}\ast \text{TV}\ast \text{D}}{\epsilon \ast \text{EV}}$$

$$\text{Activity}\text{of}\text{Gp}\left(\frac{\text{U}}{\text{mL}}\right)=\frac{\text{Activity}\text{of}\text{Gp}\left(\text{U}\right)}{\text{volume}\left(\text{mL}\right)}$$

$$\text{Protein}\left(\frac{\text{U}}{\text{mg}}\right)=\frac{\text{Activity}\text{of}\text{Gp}\left(\frac{\text{U}}{\text{mL}}\right)}{\text{Protein}\text{concentration}\left(\frac{\text{mg}}{\text{mL}}\right)}$$

“U” is one unit of enzyme activity, which is equal to the amount of enzyme catalyzing one micro mole of H₂O₂ to O₂ and H₂O in one minute. ΔA is the absorption difference of 470 nm in one minute. VT is the total volume (assay buffer and extract, 3 mL), VE is the extract volume (0.05 mL), and ϵ is the extinction coefficient of the enzyme (26.6 Mm⁻¹ cm⁻¹).

CAT

CAT activity was measured according to Aebi (1984) with some modification. Accordingly, 2.95 mL buffer assay including K₃ (PO4)₂ buffer (50 mM, pH = 7), hydrogen peroxide (15 mM) and 0.05 mL enzyme extract were mixed. The specific activity of catalase enzyme (ΔAbs min⁻¹ g⁻¹ pro) was determined by dividing the volumetric activity of the enzyme by the extract protein according to the method of Bradford (1976).

$$\text{Cat}=\frac{\mathrm{\Delta }\text{A}\ast \text{TV}\ast \text{D}}{\epsilon \ast \text{EV}}$$

$$\text{Cat}\left(\frac{\text{U}}{\text{mL}}\right)=\frac{\text{Cat}(\text{U})}{\text{Unit}\text{volume}(\text{mL})}$$

$$\text{Protein}\left(\frac{\text{U}}{\text{mg}}\right)=\frac{\text{Cat}\left(\frac{\text{U}}{\text{ml}}\right)}{\text{Protein}\text{concentration}\left(\frac{\text{mg}}{\text{ml}}\right)}$$

“U” is one unit of CAT activity, which is equal to the amount of enzyme, which catalyses one micromole H₂O₂ to O₂ and H₂O in one minute. ΔA = the absorption difference at the wavelength of 240 nm in one minute, VT = total volume of assay buffer and extract (3 mL), VE = extract volume (0.05 mL), ϵ = CAT extinction coefficient (39.4 Mm⁻¹ cm⁻¹), and D is the dilution coefficient.

Protein (pro) concentration

Leaf protein concentration (Pro) was measured according to the method of Bradford (1976) using bovine serum albumin (BSA) as the standard (in the range of 0–50 µg. L⁻¹).

Statistical analysis

Data were subjected to the analysis of variance using SAS (Ver 9.3). The statistical variables of the climatic data during the season were also calculated using SAS. The significant differences among the means were determined by least significant difference (LSD) at P ≤ 0.05. The graphs were plotted by SAS Proc Plot.

Results

Analysis of variance

According to the analysis of variance, genotype and temperature significantly affected plant Chla. However, plant Chlb and Car were significantly affected by temperature. The interaction effects were also significant on Chla and b and Car (Table 3). The single and the interaction effects significantly affected the activity of the measured antioxidant enzymes including APX, GP and CAT (Table 4).

Table 3.

Analyses of variance indicating plant pigment contents affected by the experimental treatments

			a				b				c
Source	DF	Type I SS	Mean square	F value	Pr > F	Type I SS	Mean square	F value	Pr > F	Type I SS	Mean square	F value	Pr > F
PGR	3	0.08	0.03	2.02	0.111	0.04	0.01	1.96	0.1197	0.09	0.03	0.82	0.4812
G	2	0.19	0.09	6.65	0.0015**	0.01	0.00	0.48	0.6223	0.16	0.08	2.27	0.1049
T	2	0.28	0.14	9.98	< .0001**	0.14	0.07	9.19	0.0001**	0.51	0.26	7.28	0.0008**
PGR * G	6	0.17	0.03	2.04	0.0599	0.09	0.02	2.01	0.0642	0.48	0.08	2.28	0.0361*
PGR * T	6	0.06	0.01	0.67	0.6771	0.05	0.01	1.16	0.3283	0.15	0.02	0.7	0.6477
G * T	4	0.35	0.09	6.22	< .0001**	0.09	0.02	2.89	0.0228*	0.68	0.17	4.86	0.0008**
PGR * G * T	12	0.27	0.02	1.61	0.0887	0.26	0.02	2.87	0.0009**	0.95	0.08	2.25	0.01**

*Significant at P ≤ 0.05; **Significant at P ≤ 0.01

a: chlorophyll a, b: chlorophyll b, c: carotenoids

PGR plant growth regulators; G genotype; T temperature (PD); n.s. Not significant

Table 4.

Analysis of variance indicating plant enzymatic activities and protein concentration affected by the experimental treatments

			Apx				Gp
Source	DF	Type I SS	Mean square	F value	Pr > F	Type I SS	Mean square	F value	Pr > F
PGR	3	0.002	0.001	1.66	0.1824	0.327	0.109	142.43	< .0001**
G	2	0.004	0.002	3.87	0.0254*	0.112	0.056	72.85	< .0001**
T	2	0.007	0.004	7.63	0.0010**	0.502	0.251	327.41	< .0001**
PGR * G	6	0.032	0.005	11.67	< .0001**	0.371	0.062	80.58	< .0001**
PGR * T	6	0.045	0.007	16.25	< .0001**	0.781	0.130	169.95	< .0001**
G * T	4	0.025	0.006	13.87	< .0001**	0.257	0.064	83.81	< .0001**
PGR * G * T	12	0.053	0.004	9.70	< .0001**	0.441	0.037	47.95	< .0001**
			Cat				Pro
PGR	3	0.074	0.025	74.01	< .0001**	0.0002	0.0001	6.55	0.0006**
G	2	0.007	0.003	10.23	0.0001**	0.0001	0.0001	6.28	0.0031**
T	2	0.020	0.010	29.47	< .0001**	0.0002	0.0001	12.53	< .0001**
PGR * G	6	0.070	0.012	34.8	< .0001**	0.0010	0.0002	17.75	< .0001**
PGR * T	6	0.012	0.002	6.16	< .0001**	0.0015	0.0002	25.89	< .0001**
G * T	4	0.079	0.020	59.22	< .0001**	0.0011	0.0003	27.62	< .0001**
PGR * G * T	12	0.228	0.019	57.01	< .0001**	0.0009	0.0001	7.86	< .0001**

*Significant at P ≤ 0.05; **Significant at P ≤ 0.01

Apx ascorbate peroxidase; Gp guaiacol peroxidase; Cat catalase; Pro protein concentration; PGR plant growth regulators; G genotype; T temperature (PD); n.s. Not significant

The effects of temperature (PD) on biochemical properties

The results indicated the significant effects of temperature (planting date, PD) on the activity of APX (U. mg⁻¹) as the highest activity of enzyme was resulted by PD3 (0.059) significantly higher than PD1 (0.044) and PD2 (0.040). However, PD1 (0.190) resulted in the highest GP (U. mg⁻¹) activity significantly higher than PD3 (0.052) and PD2 (0.039). Plant CAT activity (U. mg⁻¹) was significantly affected by temperature as the highest ones were resulted by PD1 (0.084) and PD3 (0.820), significantly higher than PD2 (0.056). With increasing temperature, leaf Pro (U. mg⁻¹) increased as PD3 (0.229) and PD2 (0.228) resulted in the highest value, significantly higher than PD1 (0.225) (Fig. 1).

Fig. 1.

Plant enzymatic activities including Apx (ascorbate peroxidase), Gp (guaiacol peroxidase), and Cat (catalase), and protein concentration (Pro) affected by temperature and plant growth regulators (PGR) including A (amino acid), CO (Control), GA (gibberellic acid), and MI (fertilization). APX (ascorbate peroxidase), CAT (catalase), GA (gibberellic acid), GP (guaiacol peroxidase), PGR (plant growth regulators), Pro (protein)

The effects of PGR on biochemical properties

Plant APX (U. mg⁻¹) was the highest by MI (0.054) followed by GA (0.047) and CO (0.048), which were higher than A (0.041). However, GA resulted in the highest GP activity (0.186 U. mg⁻¹), significantly higher than A (0.085), MI (0.059), and CO (0.045). MI (0.111) significantly enhanced CAT activity, related to A (0.083), CO (0.061) and GA (0.041). Leaf Pro (U. mg⁻¹) was the highest by A (0.229), MI (0.228) and GA (0.227), significantly higher than the control treatment (0.225 U. mg⁻¹) (Fig. 1).

The effects of temperature (PD) on pigment contents

The highest Chla content (mg g⁻¹ leaf fresh weight) was resulted by T3 (0.231) and T1 (0.205) significantly higher than T2 (0.160), indicating the effects of temperature stress on plant chla content. However, T1 resulted in the highest plant Chlb content (0.093) significantly higher than T3 (0.054) and T2 (0.045). T1 (0.298) and T3 (0.279) treatments resulted in the highest plant Car (mg g⁻¹ leaf fresh weight), significantly different from T2 (0.206) (Fig. 2).

Fig. 2.

Plant pigment contents including Chla (chlorophyll a), Chlb (chlorophyll b), and Car (carotenoids), affected by temperature and plant growth regulators (PGR) including A (amino acid), CO (Control), GA (gibberellic acid), and MI (fertilization)

The effects of PGR on pigment contents

Plant chla content (mg g⁻¹ leaf fresh weight) was the highest by the control treatment (0.224) significantly different from GA (0.201), MI (0.187), and A (0.183). MI resulted in the highest chlb content (0.085 mg g⁻¹ leaf fresh weight) significantly higher than A (0.057) and GA (0.0.56). There were not any significant differences among the PGR treatments affecting plant Car ranging from 0.281 (CO) to 0.237 mg g⁻¹ leaf fresh weight (A) (Fig. 2).

The interaction of temperature and PGR affecting biochemical properties

According to the general trend of the plant antioxidant activity, with increasing temperature PGR significantly increased APX activity ranging from 0.021 to 0.103 U. mg⁻¹. However, with increasing temperature, GP activity, ranging from 0.024 to 0.480 U. mg⁻¹ was significantly decreased by PGR except GA, which resulted in the highest enzyme activity (0.480 U. mg⁻¹) at T1. The response of CAT activity to PGR, ranging from 0.030 to 0.140 U. mg⁻¹, did not follow a clear trend, and the highest value (0.140 U. mg⁻1) was resulted by MI at T1. Interestingly, with increasing temperature the tested PGR were able to significantly increase leaf Pro ranging from 0.219 to 0.234 U. mg⁻¹ (Fig. 3).

Fig. 3.

Plant enzymatic activities including Apx (ascorbate peroxidase), Gp (guaiacol peroxidase), and Cat (catalase), and protein concentration (Pro) affected by the interaction of temperature (T) and genotype (G) with plant growth regulators (PGR) including A (amino acid), CO (Control), GA (gibberellic acid), and MI (fertilization), APX (ascorbate peroxidase), CAT (catalase), GA (gibberellic acid), GP (guaiacol peroxidase), PGR (plant growth regulators), Pro (protein)

The interaction of genotype and PGR affecting biochemical properties

The APX activity in B genotype was the most responsive to PGR (MI), ranging from 0.33 to 0.098 U. mg⁻¹. However, M genotype in response to GA (0.165 U. mg⁻¹), resulted in the highest GP activity followed by B genotype (0.160 U. mg⁻¹) in response to A. Although there was not a clear trend of genotype response to the tested PGR, MI resulted in the highest CAT activity in M and KH genotypes ranging from 0.037 to 0.172 U. mg⁻¹. The Pro concentration was the highest in KH genotype (0.233 U. mg⁻¹) in response to GA, followed by M genotype in response to A (Fig. 3).

The interaction of temperature and PGR affecting pigment contents

The interaction of T3 and control (0.275) increased Chla to the highest, followed by the interaction of T3 and Ga (0.230). However, the interaction of T2 and MI (0.129) resulted in the least Chla. Plant Chlb was the highest by T1 and MI (0.134), followed by T1 and GA (0.090). The interactions of T2 and A (0.037) and T2 and GA (0.039) resulted in the least Chlb (Fig. 4).

Fig. 4.

Plant pigment contents including Chla (chlorophyll a), Chlb (chlorophyll b), and Car (carotenoids), affected by the interaction of temperature (T) and genotype (G) with plant growth regulators (PGR) inducing A (amino acid), CO (Control), GA (gibberellic acid), and MI (fertilization)

The interaction of genotype and PGR affecting pigment contents

The interaction of M genotype and MI resulted in the highest Chla (0.251) content followed by the interaction of M genotype and control (0.250). The B Genotype treated with MI and M genotype treated with Mi resulted in the highest Chlb content (0.107 and 0.098). Plant Car was the highest by the M genotype treated with MI (0.343) and, B genotype treated with MI resulted in the second highest Car content (Fig. 4).

Discussion

The results indicated it is possible to alleviate cool (early planting) and warm (late planting) temperature stresses on the biochemical properties and pigment contents of different Satureja genotypes using the tested PGR treatments including GA, M and A. Plant responds to temperature stress by altering its biochemistry and physiology at the cellular and molecular level, resulting in the production of different compounds, which may alleviate the stress (Hasanuzzaman et al. 2013; Bakhshian et al. 2022). According to the results, while increasing temperature enhanced APX activity, it decreased GP activity, and CAT activity increased by decreasing or increasing temperature (Fig. 5). Such results indicate CAT is a more sensitive antioxidant enzyme under stress conditions and can more effectively regulate plant growth and activity compared with APX and GP (Nievola et al. 2017).

Fig. 5.

The regulating functions and variation of antioxidant enzymes including ascorbate peroxidase (APX), guaiacol peroxidase (GP), and catalase (CAT) on Satureja sp. physiology during warm and cool temperature stresses

According to our results, high temperature stress increased APX activities. Similarly, Shen et al. (2024) investigated the effects of high temperature on APX activity, as a regulator of plant physiological activities (by removing H₂O₂) in different conditions including stress. They found the up-regulating effects of high temperature on the activity of APX genes (SmAPXs), especially SmAPX2 localized in cytoplasm and cytomembrane. SmAPX2 is able to act as an enzyme resulting the oxidation of ascorbic acid by H₂O₂ and subsequent alleviation of H₂O₂ accumulation. Interestingly, the silencing of SmAPX2 decreased plant tolerance under high temperature stress, indicating the positive effects of SmAPX2 on plant biochemical properties in stress conditions (Shen et al. 2024).

Our results indicated the higher activity of GP in cool temperature stress, which is similar to the results of a recent research. Khan et al. (2023) determined the effects of cool temperature on the oxidative stress of Himalayan herbs (accumulation of reactive oxygen species). They found the upregulating effects of the stress genes on the activities of different antioxidant enzymes including GP in correlation with the m-RNA levels. The conclusion was that the cool temperature stress induces controlling physiological mechanisms mainly by inducing the activities of antioxidant enzymes including GP, which eventually may result in the complete or partial alleviation of cool temperature stress on plant performance (Khan et al. 2023).

The results of the present research indicated the induced activities of CAT in both cool and warm temperature stresses. The enzyme is able to alleviate different stresses including temperature stress by scavenging reactive oxygen species. Accordingly, Ghorbel et al. (2023) performed a wide range of gene analyses to indicate the activities of CAT genes induced in wheat in different stress conditions, and found six TdCAT as the stress regulating genes. The authors indicated the induction of TdCAT2 and TdCAT3 in different conditions including cool and warm temperature stresses. Such results indicate the significance of CAT enzyme for regulating plant growth and development in different conditions including temperature stress.

According to the analysis of variance, among the experimental treatments, PGR was not significant on plant pigment contents. Similar to our results, Gong et al. (2021) found the high concentrations of gibberellic acid (more than 1 mM = 40 mg L⁻¹) did not increase or decrease the chlorophyll contents in spinach (Spinacia oleracea L.). The important parameters, which influence the effects of PGR on plant pigment contents are plant species, plant growth stage, soil and climate properties and PGR type and concentration (Radowski 2018). The authors found a high variation of plant chlorophyll content affected by the use of amino acids. For example, depending on plant growth stage and amino acid concentrations, chlorophyll content was differently affected by PGR (amnio acid). This may explain the non-significant effects of fertilization, gibberellic acid and amino acids on plant pigment contents in temperature stress conditions.

According to our results, among the measured parameters, Car is also a sensitive physiological factor (Bayat et al. 2022), under temperature stress, as it also increased in response to cool and warm temperatures. Plant may alleviate temperature stress by inducing the production of non-enzymatic products including carotenoids, and enzymatic products including APX, GP and CAT enzymes (Apel and Hirt 2004; Gill and Tuteja 2010; Fortunato et al. 2023). The first step in the detoxification of reactive oxygen species is the conversion of superoxide dismutase superoxide radicals into H₂O₂, followed by the conversion of H₂O₂ by CAT, APX and GP into H₂O (Praveen et al. 2023). Another important role of APX is to act in the cycle of ascorabte-glutathione with glutathione reductase and other enzymes, resulting in the production of ascorbate and reduced glutathione (Anjum et al. 2010). Temperature stress imbalances the production and removal of reactive oxygen species and so increases the amount of reactive oxygen species. The level of temperature stress determines plant response as at higher stress levels, plant may not be able to resist the stress by detoxifying reactive oxygen species resulting in oxidative stress and subsequent damage to the plant including the photosynthetic apparatus. Accordingly, plant ability to scavenge the reactive oxygen species and control redox homeostasis determines plant tolerance under temperature stress. The production of antioxidant enzymes including APX, GP and CAT and plant genotype are among the most important factors determining plant response under temperature stress (de Pinto et al. 2015; Zou et al. 2017).

The increased production of antioxidant enzymes under different stresses including temperature stress are among the mechanism used by plants to tolerate the stress (Anjum et al. 2016). According to our results, the responses of the tested antioxidant enzymes to temperature stress were different. The results indicated that the tested PGR are able to enhance plant growth and activity under temperature stress by affecting the activities of antioxidant enzymes. The plant was able to synthesize more protein with increasing temperature, which is due to enhanced photosynthesis resulting in the production of more photosynthates. The MI treatment significantly increased the activity of APX and CAT, which indicates the presence of macro- and micro-nutrients are essential for the activity of such enzymes by acting as cofactor in the structure of such enzymes. However, the GA treatment was the most effective one significantly increasing GP activity, and the A treatment had the highest impact on plant Pro indicating the role of the tested amino acids for protein production (Al-Harthi et al. 2021). Research has indicated the positive effects of GA on the induction of antioxidant activities, and subsequent scavenging of reactive oxygen species in stress conditions (Zhang et al. 2023).

Soengas et al. (2018) investigated the effects of temperature stress (cold and heat) on the photosynthesis and yield of cabbage (Brassica oleracea var. capitata L.) and kale (B. oleracea var. capitata L.) as biennial plants. They found under temperature stress, due to the increased production of reactive oxygen species, the activity of antioxidant enzymes increased. The production of reactive oxygen species, decreased plant chlorophyll content, and plant dry weight. The effect of cold stress was more pronounced than heat stress on the increase of antioxidant activities and reduction of chlorophyll content. However, heat stress decreased plant dry weight more severely than cold stress, probably due to the inactivation of Rubisco and the related enzymes (Mohan et al. 2023). Although, high level of temperature stress may decrease plant pigment contents, under milder stress, such as the one tested in the present research, the increase of plant pigment contents may be one of the mechanisms by which plant tolerates the stress and increases its photosynthesis rate and production of photosynthates (Zahra et al. 2023).

Clemente‐Moreno et al. (2020) investigated the effects of cold stress on the growth of Deschampsi antarctica (DA), as one of the native vascular species of Antarctica, and compared it with a non-Antarctica species (Triticum aestivum) (TA) from the same family (Poaceae). The cold temperature (4 °C) decreased the photosynthetic activities of DA and TA by 70 and 80%, respectively, and the symptoms of oxidative stress appeared in both plant species. However, the activities of antioxidant enzymes increased in both plants under the stress. The analyses indicated, in DA under stress, the mechanisms of osmoprotection, stabilization of membrane, remodeling of cell wall, and production of antioxidants (phenylpropanoids and flavonols), along with mobilization of nutrients, help the plant to tolerate the stress compared with TA species.

Another important result of the present research is the significant interactions of the tested treatments on the measured parameters. This indicates depending on the combination of the treatments tested, plat response (antioxidant activity and pigment content) may differ in cool and warm temperature stresses. It is accordingly important to find the most suitable combination of treatments, which best alleviates temperature stress on Saturega species physiology and pigment content.

Conclusion

According to the results, plant catalase and carotenoids were the two most sensitive indicators of plant response under temperature stress (cool, mild, and warm), as they increased under both cool and warm temperature. However, plant APX increased in warm temperature stress and plant GP increased in cool warm temperature stress. Accordingly, the results indicated the significance of CAT for regulating plant growth and development by scavenging the reactive oxygen species and controlling redox homeostasis under temperature stress conditions. The tested PGR were able to significantly affect plant response (the production of antioxidant enzymes and plant pigment contents) under the stress and in most cases the fertilization treatment was the most effective one followed by amino acids and gibberellic acid. Another important finding of the present research is the significant interaction among the tested treatments on the measured parameters. Accordingly, it is important to determine the most suitable combination of the tested treatments for the alleviation of temperature stress on Satureja species. It is possible to improve Satureja sp. biochemical properties, in temperature stress, using the tested PGR, however, plant genotype can also determine plant response under the stress. The presented results are of environmental, economic, and health significance.

Abbreviations

APX

Ascorbate peroxidase

Car

Carotenoids

CAT

Catalase

GA

Gibberellic acid

GP

Guaiacol peroxidase

PGR

Plant growth regulators

Author contribution

MB conducted the experiments, collected and analysed data, MRN supervised the research, HRJ co-supervised the research, BB co-supervised the research.

Funding

There was not any funding for the research.

Data availability

The authors selected not to share data.

Declarations

Conflict of interest

The authors declare they do not have any conflict of interest.