Nitrogen Fixing Bacteria Reduces Drought Stress and Improve Molecular and Biochemical Response in the Garden Thyme

doi:10.21203/rs.3.rs-4726162/v1

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Nitrogen Fixing Bacteria Reduces Drought Stress and Improve Molecular and Biochemical Response in the Garden Thyme

https://doi.org/10.21203/rs.3.rs-4726162/v1

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Abiotic stresses such as water-deficient are the most important factor that could impress the agricultural process causing a reduction of crop yield. Use of Bio-fertilizers could improve growth condition of drought stressed plant to reduce the damage of water deficit. The objective of this work is to assay comparative effect of some organic and non-organic fertilizers on the flowering branches essential and some antioxidant activity (APX, PO, CAT), then expression analysis of two thymus and carvacrol bio-synthesis related genes(CYP171 d178 and CYP171D180 ) in Thymus vulgaris under water deficit stress in a field trial was investigated using qRT-PCR method. The treatments were arranged in a completely randomized design with a total of 42 experimental separate blocks. The highest and lowest yield of flowering branches was observed in NPK and mycorrhizae treatments Approximately, Azospirillum bio-fertilizer showed high effect on enzyme activity in treated plants. Unlike, chemical fertilizer showed minimum efficiency on enzyme activity. A high expression level (approximately, 6-fold change) of both genes was observed in treated plants with Azospirillum bio-fertilizer while the lowest expression level (1.3-fold change) was observed in chemical fertilizer application when compared to other fertilizers. According to the obtained results of this work, we conclude that yearly fertilizer application could have a significant effect on molecular and biochemical traits and resulted in increase of yield component via decrease of drought stress.

Plant medicinal

Essentials oil

Gene expression

Thymol

Carvacrol

Thymus vulgaris belonging to the Lamiaceae family is originally cultivated in Southern Europe and has a worldwide distribution. In the field of plant medicine, this species is known as thyme. The use of thyme has been recommended in traditional medicine as an infusion to treat cough, decrease blood insulin levels, control infectious diseases, and for digestive conditions in a syrup form (Ekoh et al, 2014). The antiseptic, antibiotic, and antifungal properties of thyme have been demonstrated by several studies (Dauqan and Abdullah 2017; Hosseinzadeh et al, 2015; Javed et al, 2013). Under water stress conditions, reactive species oxygen (ROS) is necessary to retain energy balance in influenced cells. The key role of Ascorbate peroxidase, peroxidase, and catalase as an antioxidant enzyme in free radical scavenging to water stress responses has been well demonstrated (Caverzan et al, 2012; Faize et al, 2011; Zhang and Kirkham 1994). Drought stress in soybean leads to a clear increase in the activity of antioxidant enzymes (Caverzan et al, 2012; Faize et al, 2011; Zhang and Kirkham 1994). Abiotic stresses such as water-deficient are the most important factor that could impress the agricultural process causing a reduction of crop yield. The physiology and growth of plants under drought stress can be influenced in many ways (Waraich et al, 2011). Soil moisture plays an important role in the mineral nutrient transport from the soil solution to the roots. Thus, any decrease of soil moisture due to water deficiency stress can affect nutrient transport from the roots to the shoots (da Silva et al, 2011). The use of bio-fertilizers in higher plants under drought stress can ameliorate the drought effect and enhance the soil’s water retention capacity and increase the root growth plus yield (Li and Ni 1996). Thymol and its isomer carvacrol are the main constituents of essential oil in thyme species. The biological and pharmacological properties of thymol and carvacrol as biocidal and antiseptic effects have been well documented (Ahmad et al, 2011; Gursul et al, 2019; Lambert et al, 2001).

The objective of this study was to investigate the effect of bio-fertilizers on the change of antioxidant enzymes and expression level of two CYP171 d178 and CYP171D180 genes involved in the biosynthesis of thymol and carvacrol and also two SOD and APX genes involve in plant drought resistance in thymus vulgaris under water deficit stress.

2.1 Plant materials and culture condition

This experimental field was performed during 2018–2019 at the Yazd province, Iran (Longitude 51° 17', Latitude 31° 53', Height 1230.2 m). At the first step, prior to thyme seedling transportation, chemical analysis of cultivation soil was performed on the soil sample (upper 25-cm layer) to determine soil texture and characteristics (Table 1).

Table 1

Analysis of soil texture and physical and chemicals characteristics of sample cultivation soil
N (%)	P (mg/Kg)	K (mg/kg)	CO (%)	Loam (%)	Clay (%)	Sand (%)	pH	EC (ds/m)	Soil Texture
0.029	14.8	190.8	0.339	15	10	75	7.66	2.05	Sandy loam

Meteorological and climate data were obtained from nearest weather station at Yazd province, Iran.

Meteorological and climate data were obtained from nearest weather station at Yazd province, Iran. Average annual of atmospheric characteristics of weather are summarized in Table 2.

Table 2

The annual average of climate data in Yazd province
Number of sunny hours	annual evaporation (mm)	Total annual rainfall (mm)	Average maximum annual relative humidity (%)	Average minimum annual relative humidity (%)	Average annual relative humidity (%)	Average maximum annual temperature (°C)	Average minimum annual temperature (°C)	Average annual temperature (°C)	Station
3409.9	3063.8	71.6	40.7	15.2	27.9	27.7	14.4	21	Yazd

Seeds of Thymus vulgaris were surface sterilized by 1% chloramine T (Sigma) and then were rinsed three times with tap water. Disinfected seeds were planted in Gifi pot containing peat moss and were maintained in the nursery. Seedlings were transported (50 plantlet/ plot) from the nursery to the main field designed ( October) to blocking plots with determined size (4 × 3.5 m). The first irrigation after transplanting was performed by the flooding method. After installing plants in the soil, plants were irrigated by the drip irrigation system. The volume of consumed water was the same for all treatments. The periods of irrigation were continued until sampling time at the flowering stage.

2.3 The treatments

The treatments consisted of: drought stress: three irrigation intervals (W₁ = 7day, W₂ = 12 day, and W₃ = 17d), Bio-fertilizer treatments (B₁ = control, B₂ = Mycorrhiza (Rhizophagus irregular fungi; 5%,150gr/plant), B₃ = Azospirillum brasilense;200gr/10lit water, B₄ = Pseudomonas fluorescens bacterial; 200gr/10lit water and B5 = NKP (20:20:20) fertilizer;200kg/ha). Controls were drought-stressed plants without bio-fertilizer application. Three independent replicates were done for all treatments.The treatments were arranged in a completely randomized design with a total of 45 experimental units.

2.2 Flowering branches yield and essential oil measurement

During the experiment from Two months after the date of transplanting to the field and at the flowering stage for a week, Thymus flowers were counted and removed three times, and the individual counts were combined. At the end of the experiment (at the flowering stage ), shoots were separated from stems, and shoot fresh weight (FW) was recorded. Flowers were then dried at 80°C for 2 days, and dry weight (DW) was recorded. Clevenger apparatus was used to extract the essential oil in treated plants with bio-fertilizer under drought stress. Essential oils were stored in dark glass containers and in the refrigerator for further analysis.

2.4 Plant extraction and enzyme activity assay

Plant materials were frozen in liquid nitrogen and then were grinded into a fine powder with a mortar and pestle. Sodium phosphate buffer (50mM, pH 7.0) containing 0.1 mM EDTA and polyvinyl pyrrolidone (1% w/v) was used as extraction buffer. The homogenate was filtered through cheesecloth and the filtrate was centrifuged at 14 000 g for 25 min at 4º C. Enzyme activity was determined using aliquots of the supernatant as a crude extract. Catalase activity (µmol(H2O2) g- /min) was done by the method of Goth (Goth 1991). One unit of CAT activity was defined as the absorbance change of 0.01 per min. Guaiacol is a hydrogen donor was used to determine peroxidase activity (Fielding and Hall 1978) Nitroblue tetrazolium (NBT) reagent was used to measure Superoxide dismutase (SOD) activity. SOS can inhibit photochemical reduction of NBT (Kjari et al, 2000). Ascorbate peroxidase activity was determined by the Nakano and Asada Method (Nakano and Asada 1981) based on the oxidation of ascorbate.

2.5 mRNA extraction and cDNA synthesis

Total RNA of 50mg of leaf tissue from treated and non-treated thyme plants was extracted by the Pure Plant RNA Purification Kit (TopazGene, Iran) according to the manufacturer’s instructions. RNase-free DNase I (Sinaclon, Iran) was used to eliminate genomic DNA contamination. Total RNA was quantified using a Nanodrop ND-100 spectrophotometer and RNA quality was assessed by 1% agarose gel electrophoresis stained by Ethidium bromide (Sabbagh, et al, 2012). Purified RNA was subjected to first-strand cDNA synthesis using a superscript reverse transcriptase kit (Sinaclon, Iran). The Actin gene was used as a reference gene. Sequences of CYP71D178 and CYP71D180 genes were provided from Crocoll (Crocoll 2011). The primers for SOD, APX genes were selected from Baek and Mehrabadi pour-began (Baek and Skinner 2003; Mehrabad Pour-Benab et al, 2019). The character of primer sequences for the selected genes is summarized in Table 3.

Table 3

Nucleotide sequences of primers used in PCR.
gene	Forward primer 5′ -3′	Reverse primer 5′ -3	Reference
CYP178	TGGCCTTTGGAAGCGTCG	TCAGGCTCATTCCAATAGAGG	Majidi
CYP180	GGTAAACTGGCGGACTTGGT	CGAACGGGATTAACTCGAAA	Majidi
actin	ATTCTTACCCTCAAGTAT	CATGATCTGAGTCATCTT

2.6 Relative gene expression analysis

Real-time PCR (qRT-PCR) was done in a Rotor gene Real-Time PCR System (Corbett 3000, Australia) a volume of 25 µL containing 8 µL of Eva Green qPCR Mix (5x Hot FIREPOL, ROX, Ampliqon), 3 µL of diluted cDNA, 2 µL of forward and reverse primers ( 0.5 µmol/L) of, and 10 µL of RNase-free water. The PCR temperature cycling conditions were as follows: a first initial denaturation in 95°C for 4 min followed by: 95°C for 20 seconds, 20-second primer annealing at optimum degree for each primer, and elongation at 72°C for 30 seconds in 40 cycles, with a five-minute extension at 72°C after the final cycle. The reactions were repeated two times (as two technical replications) for each of two biological samples The Ct values of the calibrator and the samples were normalized to the endogenous housekeeping gene. Relative gene expressions were determined according to the ∆∆Ct method, using the formula: 2-∆∆Ct where, ∆∆Ct= [∆] Ct sample -[∆]Ct reference where, [∆] Ct sample was the Ct value for any sample normalized to the endogenous actin gene as reference gene, and -[∆]Ct reference value for the reference sample normalized to the endogenous housekeeping gene (Livak and Schmittgen 2001).

2.6. Data analysis

Enzymes activity was measured on three independent experiments. One-way analysis of variance (ANOVA) with three replications was carried out. If significant differences were found, pairs of samples were compared by Tukey's test. Statistical analysis was performed with SAS software SAS 9.1 software package. The relative expression level of targets genes was analyzed with ∆∆Ct method and was normalized to the expression level of the actin gene. SPSS (v. 22) software was used to the analysis of gene expression data obtained using the qRT-PCR method. All data were statistically analyzed using LSD 0.05.

3.1. Flowering branches and Essential oil

The results of analysis of variance of flowering branch trait showed that the interaction effect of irrigation periods and fertilizers on the yield of flowering branches was significant at the 1% probability level (Table 4). The highest yield of flowering branches was observed during the irrigation period of 7d (W1) and in NPK fertilizer treatment (30.2 g/plantlet), while the lowest amount of this trait was related to the irrigation period of 17 days with mycorrhizae application (11.7 g/plantlet). The reduction of this trait (61%) showed a close relation between drought stress and flowering branch production (Fig. 1a).

Table 4

Analysis of variance of flowering branches yield, essential oil yield and activity of antioxidant enzymes in treated Thymus plant with fertilizer under drought stress.
			Mean square
Source of Variance	df	Catalase	Peroxidase	Ascorbate peroxidase (APX)	Superoxide dismutase(SOD)
Repetition(R)	2	0.004^n.s	0.001 ^n.s	63.82 ^n.s	0.00008^*
Water deficit Stress(W)	2	0.38^**	0.012^*	5305.87^**	0.0004^*
Error a(R*A)	4	0.0006	0.0007	27.99	0.00004
Fertilizers(B)	4	1.56^**	0.035^**	3803.93^**	0.00020^**
W*B	8	0.45^**	0.047^**	3907.58^**	0.00012^**
Error b	24	0.0003	0.0003	5.23	0.00002
C.V. (%)		1.77	8.59	6.2	16.38

ns, *, **: non-significantly difference and significantly differences at 5 and 1% of probability levels, respectively.

The mean comparison of obtained data revealed a high level of essential oil production (7.15 g/plantlet) during the irrigation period of 12 days and with Pseudomonas bio-fertilizer application. This amount was reduced (3.15 g/plantlet) in the irrigation period of 7d with Azospirillum bio-fertilizer application. The effect of fertilizers on the essential oil production indicated that the Pseudomonas treatment had a significant effect on this parameter when compared to the control treatment (Fig. 1b).

Figure 1 Weight of flowering branches (a) and essential oil (b) in Thymus plants, 7, 12 and 17days after irrigation in the field condition. Plants were either non-inoculated with any fertilizer or were inoculated with one of three mycorrhiza, Azospirillum and Pseudomonas bio- fertilizers Inocula, and NKP fertilizer. Different letters denote significant differences between means of control and fertilizers treatments determined by a t test (P < 0.05)

3.2. Enzyme activity

The results of the analysis of variance indicated that the interaction effect of drought stress and fertilizers treatments on the activity of tested enzymes was significant at the 1% probability level (Table 4). Catalase activity assay showed an enhancement of catalase activity upon prolongation of the drought period. In all irrigation intervals, the maximum activity of the catalase enzyme occurred in treated plants with Pseudomonas bio-fertilizer. During irrigation intervals of 12 days, mycorrhizae and Azospirillum application did not affect the enzyme activity when compared to control plants (Fig. 2). The maximum amount of peroxidase enzyme (0.043 u/g Fw) occurred during an irrigation interval of 12 days in plants treated with Azospirillum bio-fertilizer. The lowest rate of enzyme expression (0.015 u/gFw) was detected in plants treated with NPK fertilizer under water stress during an irrigation period of 7 days 12 days. Further, all bio-fertilizers caused increased peroxidase activity water stress caused in all treatments (Fig. 2). The greatest change in SOD enzyme activity occurred during the irrigation period of 12 days in plants treated with Azospirillum; as with other enzymes, the lowest enzyme activity was observed in the control plant (7d) for NPK application. Within 17-day irrigation, Pseudomonas bio-fertilizer caused high activity of SOD enzyme (Fig. 2). Unlike other enzymes, the use of fertilizer did not show any effect on APX enzyme activity. Almost in all three irrigation intervals, the expression of the APX enzyme was reduced.

Table 4 Analysis of variance of flowering branches yield, essential oil yield and activity of antioxidant enzymes in treated Thymus plant with fertilizer under drought stress

Figure 2 Antioxidant Enzyme Changes in treated Thymus plants with fertilizer in response to drought stress. Vertical bars indicate SE of three replicates for each treatment. Values are means with standard error of the means ± SE and different letters indicate a significant difference at p < 0.01

3.3 Differential expression of thymus bio-synthesis genes in response to fertilizers application

The relative gene expression levels between different samples were captured with the qRT-PCR method at three irrigation intervals. The plants treated with different fertilizers under water deficit stress were subjected to this investigation. The results of gene expression analysis showed that both investigated genes were differentially affected by fertilizer application. The higher transcription level of two tested genes was observed within 12-day irrigation as compared to other water-stress time periods (17 days). At this time period, the transcription level of CYP71d176 and CYP71D180 was significantly elevated for Azospirillum application. A 6- and 5.8-fold increase for CYP71d176 and CYP71D180 genes was observed in plants treated with Azospirillum bio-fertilizer during 12 and 17 days, respectively (Fig. 3). The lowest transcription level of both genes was observed in plants treated with NKP fertilizer when compared to bio-fertilizer treatments. A significant reduction of gene expression occurred during 17 days with different intensities for all genes. An insignificant difference was observed when NPK fertilizer was applied in both drought stresses.

Figure 3 Gene expression analysis of CYP71d176 and CYP71D180 genes in treated Thymus plant with fertilizer under drought stress

Drought stress negatively affects crop growth and is the most important factor-limiting yield in agricultural systems around the world, especially in arid and semi- arid. Little is known about the efficiency of fertilizer application to reduce the water drought stress damages in plant farming under drought conditions. In medicinal plants, water deficit can reduce the quality and quantity of aromatic compounds such as terpenoids, thymol, and carvacrol. The glandular trichomes located on the aerial part of thyme species are the local production of essential oil in plants belonging to Lamiaceae. The maximum amount of essential oil secretion would occur at the flowering time. Our results indicated that some fertilizers in the single form including nitrogen-fixing bacterial Azospirillum and Rh. irregular fungi at irrigation intervals of 7days positively affected flowering branches but water overtime period led to a reduction of flowering. The maximum effect of fertilizer treatment on the flowering branch occurred with Azospirillum. It showed a direct relationship between flowering branch traits and bio-fertilizer treatments. The use of different elicitors should not have any physical damage to terpene structures (Martin et al, 2003). Exogenous application of MeJA and MeSA increased total terpene and linalool emission in the leaf of Norway Spruce plant (Picea abies) without any inflicting physical damage to these compounds. Thus, we suggest the use of nutritional supplements to induce plants to increase terpenoid compounds and elevate the thymol and carvacrol bio-synthesis in T. vulgaris. The application of biochemical and molecular methods can illustrate the mechanisms involved in the biosynthesis of phenolic monoterpenes and understanding the regulatory mechanisms by which secondary metabolic are modulated (Iijima 2014).

The maximum effect of fertilizer treatment on the flowering branch occurred with Azospirillum. It showed a direct relationship between the flowering branch and activity of all investigated enzymes. The use of different elicitors does not have any physical damage to terpene structures (Martin et al, 2003). Exogenous application of MeJA and MeSA increased total terpene and linalool emission in the leaf of Norway Spruce plants (Picea abies) without any inflicting physical damage in response to these compounds. Thus, we suggest the use of nutritional supplements as plant inducers to increase terpenoid compounds in T. vulgaris.

The application of biochemical and molecular methods can illustrate the mechanisms involved in the biosynthesis of phenolic monoterpenes and understanding the regulatory mechanisms by which secondary metabolic are modulated (Iijima 2014). Nitrogen resources in the rhizosphere can play a key role in plant tolerance in response to drought stress (Marino et al, 2007; Pimratch et al, 2008). Change in roots system which is influenced by the complementary treatment of organic and inorganic fertilizers could improve plant growth and response to water deficit stress. In this study, the impact of drought stress and fertilizers application on recuperating of some antioxidant enzymes, and expression level of some related genes to thymol bio-synthesis and also involved in drought stress resistance was investigated.

Treatment of drought-stressed thyme plants with fertilizers led to a significant increase in the number and weight of flowering branches. Application of fertilizers in a single form also leads to changes in antioxidant enzyme activity. Antioxidant enzymes and reactive oxygen species (ROS) are involved in plant tolerance (Caverzan et al, 2016). Similar studies have shown that water and salinity stress may increase some antioxidant enzymes (Pan et al, 2006). In this research, all investigated enzymes showed higher activity when the thymus plant was irrigated for a period of 12 days. Our results are in accordance with previous results that have shown high antioxidant enzymes activity of maize plants in response to drought stress (Ali et al, 2010; Boaretto et al, 2014). Mohammadi and co-workers found that the use of mycorrhizal fungi can alleviate water deficit stress more than nitrogen-fixing bacteria and chemical fertilizers in the Oenothera biennis plant (Mohammadi et al, 2019). Unlike these results, our observation indicates reduce of enzyme activity in the mycorrhizal application under drought stress while enzyme activity was positively increased in treated plants with nitrogen-fixing bacteria. It can be due to an increase of electron donors in plant-bacteria association causing the increase in metabolic transport across cell membranes (Udvardi and Day 1997). Furthermore, researchers have reported that mycorrhizal symbiosis in the biotic zone of the rhizosphere may lead to increases in leaf water potential transpiration rate, reduce leaf temperature, and restrain the decomposition of chlorophyll content under water deficit stress. Mycorrhizal fungi with a mild form of parasitism cause high osmotic adjustment by increasing the accumulation of inorganic ions (Na, Ca2+, and Mg2+) (Wu and Xia 2006). A combination of different organic and non-organic fertilizers can cause synergistic effects on water-deficit stress response (Abbaspour 2010). Nodulated soybean plants under water deficit stress showed less sensitivity to drought stress when compared to treated nitrate feed plants individually (Kirova et al, 2008). It seems that photo-respiratory flux under water stress can activate the root system to uptake soil nitrogen by nitrogen-fixing bacteria. The use of nitrogen and phosphorus with an irrigation frequency in Salvia officinalis L. plants showed no effect on essential oil (Rioba et al, 2015). But, in this work fertilizer treatments caused an increase in thymol and carvacrol-related gene expression. Thymol and carvacrol are two main components of some plant families such as Lamiaceae and Ranunculaceae. The potential of anti-stress effects of these compounds against various biotic and abiotic stresses have been demonstrated. The application of animal fertilizers leads to an increase of thymol percentage in the essential oil while the percentages of other terpenoid compounds were not affected (Naghdi Badi et al, 2017). The effect of some abiotic elicitors such as methyl jasmonate and salicylic on the relative expression gene related to thymol-biosynthesis showed that these factors affected the biosynthesis of these terpenoid compounds (Majdi et al, 2017). Bio- Fertilizer treatment could lead to an increase of pharmaceutically active compounds (PhACs) in the Lamiaceae family.

In this research, the relative expression of two CYP71d176 and CYP71D180 genes in response to plant nutrition supplementation under water stress showed that the expression level of both tested genes could be affected by fertilizer application. The measure of the transcriptional level of CYP71d176 and CYP71D180 genes in thyme leaf at two irrigation periods of 12day and 17day and compared to irrigation period of 7d showed an increase in the gene expression in all treatments. On the other hand, the expression pattern of gene expression varied between treatments.

Based on these results, we can conclude that this molecular marker will be useful in understanding the expression of drought-responsive genes in T. vulgaris when plant growth inducers are used for the increase drought resistance.

In this work, we attempted to investigate the effect of nutritional supplements such as fertilizers in water-stressed T. vulgaris plants on changes of some antioxidant enzymes and relative expression of four genes involved in thymol biosynthesis and drought resistance. Our results revealed that the application of fertilizers can affect all tested enzymes. The change of enzyme activity was gradually variable depending on the type of bio-fertilizer. The maximum tolerance to water stress was determined during the 12-day irrigation period. An increase in the irrigation time period could reduce the adaptation of plants to water deficit. Based on these results, we suggest that at moderate water stress, the application of fertilizers can display a serial molecular and biochemical mechanism to tolerate drought stress. To elucidate the regulatory mechanism involved in the biosynthesis pathway of torpedoed compounds and drought resistance, transcriptional analysis through microarray is recommended.

Author Contributions Statemen•F. G. Participated in designing the evaluation, performed parts of the statistical analysis and helped to draft the manuscript S.K.S conceived and designed the evaluation and drafted the manuscript, M. H. re-evaluated the acquired data, revised the manuscript and performed the statistical analysis and revised the manuscript, M.M. critical revision of the manuscript for important intellectual content. All authors read and approved the final manuscript.

Acknowledgments

We would like to thank the member of Department of biology at Yazd University to access of laboratory equipment's. We also thank Dr. Mohammad Reza Sarafraz Ardakani to help us in data analysis and Dr. Mahta Mazaheri for qRT-PCR data analysis. This study was supported by vice research of Yazd University (Grant No. 99/P/2728).

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interests

The authors declare no conflict of interest.

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Nitrogen Fixing Bacteria Reduces Drought Stress and Improve Molecular and Biochemical Response in the Garden Thyme

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Plant materials and culture condition

2.3 The treatments

2.2 Flowering branches yield and essential oil measurement

2.4 Plant extraction and enzyme activity assay

2.5 mRNA extraction and cDNA synthesis

2.6 Relative gene expression analysis

2.6. Data analysis

3. Results

3.1. Flowering branches and Essential oil

3.2. Enzyme activity

3.3 Differential expression of thymus bio-synthesis genes in response to fertilizers application

4. Discussion

5. Conclusions

Declarations

References

Supplementary Files

Status:

Version 1