Morphologic and anatomic response, catalase gene expression in drought varieties of tomato and bioinformatics analyses of microarray studies of the catalase gene

Crops in arid and semi-arid regions are exposed to adverse environmental factors such as drought. Experiments were conducted to determine the morphologic and anatomic response of drought-susceptible and tolerant varieties of tomato (Solanum lycopersicum L.) under drought conditions (100%, 75%, 50%, 25% of eld capacity). To investigate the role of antioxidant enzyme, catalase gene expression was examined by real-time RT-qPCR and microarray studies of the catalase gene in tomatoes under stress examined utilizing bioinformatics. The results showed signicant morphological changes under drought conditions. Anatomical studies revealed that CaljN3 is more resistant than SuperstrainB varieties under drought stress. Relative expression of the CAT1 gene did not show any signicant difference in both Caljn3 and SuperstrainB varieties based on quantitative Real-Time PCR, under drought stress. The bioinformatics results from microarray analysis revealed that this gene did not show a signicant difference in expression in any of the cultivars and under any of the stresses. This gene is in the conserve cluster, a cluster with 118 members and a z score of 14.26148. This showed that this cluster is fully protected between two susceptible and tolerant varieties. The enrichment gene of this cluster did not show any signicant intracellular pathways. It appears that in response to stress, an activating mechanism other than catalase is necessary. The ght against oxidative stress may begin one step before that of the enzymes and seeks to combat the stressor by activating proteins, especially channels, pumps and some cellular messengers.


Introduction
Water de cit stress is considered one of the main barriers to the production of crops in many places around the world, especially in arid and semi-arid areas such as Iran. Aridity is the most prevalent environmental stress, which constricts almost 25 percent of the land on earth. More than half of the area of Iran is considered a part of semi-arid areas of the earth with rainfall of around 250 mm or less. Aridity is one of the most critical environmental stresses which affects morphological, physiological, and molecular processes, causing a lack of growth in plants (Mesgaran et al., 2017).
Tomato (Solanum lycopersicum L.) is the chief agricultural produce in many countries and is an essential contributor to human health. The plant is rich in vitamin A, C, and ber and is cholesterol-free (Honson and Davies, 1971). It also has a considerable amount of lycopene, which is an essential carotenoid antioxidant protecting the cell from deleterious free radicals and preventing cancer (Gerster 1997).
Plants responded to water de cit by making morphological, physiological, and metabolic changes in all their organs. Some studies have shown that stress due to water de cit leads to a lack of growth in various parts of the plant, including roots, shoots, leaf area, height, and dry weight. A decrease in stomata closure during photosynthesis, and a decline in the levels of chlorophyll have also been observed (Hung et al., 2005). Plants make certain morphological and physiological changes to minimize damages caused by stress, and there is a remarkable diversity between the cultivated species and even members of the same species (Zhang et al., 2020).
Drought changes the metabolic process and function of some enzymes in plants and make some changes on the anatomical and morphological levels. One of the biochemical changes which occur due to the placement of plants in drought conditions is an increase in the production of free radicals of oxygen (ROS). Their toxic effects are neutralized by the plant's antioxidant immune system (enzymatic and non-enzymatic). The degree of sensitivity to oxidative stress relies on the proportion of agents producing ROS and production of antioxidants in the plants (Nadarajah, 2020). ROS is very reactive and would destroy the natural metabolism of the plants in the absence of any defensive mechanism by oxidative damage to lipid, protein, and other macromolecules (Rout and Shaw 2001).
The structure of catalase (CAT) includes a tetrameric protein, porphyrin iron, and is considered one of the most important antioxidant enzymes. It prevents the harmful effects of hydrogen peroxide through degradation to water and oxygen. Catalase is found in all living organisms, including plant cells, animal cells, and aerobic microorganisms. In plants, catalase exists in peroxisomes, glyoxisomes, and mitochondria but does not exists in chloroplasts (Sarker and Oba, 2018).
Catalase performs a vital function in neutralizing H 2 O 2 , which is produced as a result of various processes such as electron ow in the mitochondrial electron transport chain, beta-oxidation of fatty acids and oxidation during photorespiration. Furthermore, the role of catalase in the immune system and the senescence phenomenon in plants has been proven (Mura et al. 2007). Catalase in animals is only coded by one particular gene, whereas in plants, a small gene family code the enzyme catalase. In Arabidopsis, a small family of proteins including CAT1, CAT2, CAT3 is coded by the catalase gene (CAT).
These proteins have a vital function in neutralizing reactive oxygen species (Du et al. 2008). Based on studies performed on the Arabidopsis plant, it was shown that the expression of CAT2 and CAT3 is controlled by circadian rhythm with CAT2 being expressed in the morning and CAT3 in the evening (Micheal and McClung, 2002).
Selection of drought-tolerant plants and nding mechanisms that increase plant tolerance to drought are essential. The purpose of the current study is to measure changes in the morphological and anatomical characteristics of two varieties of tomato, one drought-susceptible and one drought-tolerant. The expression of catalase genes involved in the production of antioxidant enzymes was measured utilizing Quantitative real-time-PCR. Morphological and anatomical changes due to stress and the way in which the genes were expressed in different varieties was evaluated.
Microarray analysis is utilized in biology as a predictable tool for analyzing the number of transcripts (Trevino et al. 2007). One of the most useful and informative ways for analyzing different conditions of stress is to study transcripts (Shaik and Ramakrishna 2014). Microarray studies of plant genomes gives essential data to test the expression of some genes. This research seeks to study the effects of drought stress on the expression of catalase through real-time PCR. Bioinformatics data obtained from the tomato microarray was also analyzed.

Materials And Method
Page 4/23 2.1. Primary tomato seed cultures Seeds from tolerant and susceptible varieties of tomato (CaljN3 and SuperstrainB) were germinated in pots containing sterilized sand. The sand was hydrated with distilled water every few days to prevent dehydration. After the emergence of early lea ets (20 days), germinated seeds were transferred to pots containing coco peat and perlite mix (70%-30%) which were washed with distilled water and wholly dried at ambient temperature before plant transfer.

Exposure To Salinity Stress
Lea ets were illuminated with a light (1 h, 21°C)/darkness (8 h, 18°C) cycle. For cultures, the Hoagland diet formula (pH 7 − 6) was utilized. After two weeks, salinity was applied (0, 200, 250, 300 mM NaCl). At the end of each week, the pots were utterly rinsed with distilled water, and saline treatments again applied. The plants were harvested after approximately four weeks of salinity stress and utilized for the various studies. Experiments were performed utilizing a completely random design with three repetitions.

Morphological And Anatomical Studies
Morphological parameters such as plant height, root length, fresh & dry weight of root and shoot and leaf area were measured. Different parts of the plant such as the internodes, roots and leaves (sixth internode, middle or apical lea et in the seventh leaf, for roots two centimeters from the root cap) after xing and sectioning (or cutting) were stained with safranin-fast green (Berlyn and Miksche 1976), and evaluated. The xylem was red and the rest of the cell content green.

RNA expression analysis by real-time RT-qPCR
Total RNA was extracted from frozen samples utilizing YTzol (Pure RNA isolation reagent) (Yekta Tajhiz Azuma Co., Iran) and stored at − 80°C. RNA was quanti ed at a wavelength of 260nm utilizing a Nanodrop spectrophotometer (NanoDrop 2000™, USA). RNA purity was determined from the Optical Density ratio (260/280 nm), which should be between 1.6 and 1.8.
Reactions were performed in duplicate. Reactions without cDNA were performed in parallel as a negative control. qRT-PCR results were analyzed based on the ΔΔCt method, utilizing the Step One software 2.1. Relative quanti cation was performed according to the comparative 2ΔΔCt method as described previously (Pfa , 2001). To check the validity of the primer for the housekeeping gene (GAPDH) and PCS, ampli cation e ciencies were performed (Paff, 2001).

Statistical analysis
Data analyses were performed using the SPSS 20.0 software package (SPSS Inc., Chicago, IL, USA). All experimental data were presented as the mean ± SD. One-way ANOVA was used to test differences between various means, followed by the post hoc Tukey test. The level of signi cance was set at P < 0.05 for all tests.
2.6. Microarray Study 2.6.1. Collection of data GPL4741 was obtained from the geodatabase containing 47 series and 744 samples. This particular GPL belongs to the [Tomato] Affymetrix Tomato Genome Array. Within the 47 series, four were associated with salinity, drought and heat stressors on the tomato plant. Samples were further subdivided based on plant sensitivity, tolerance or applied stressor, which resulted in the creation of 10 datasets (GSE16401-salt susceptible, GSE16401-salt-tolerant, GSE22304-SENSITIVE WATER, GSE22304-SUCEPTIBL HEAT, GSE22304-TOLERANT HEAT, GSE22304-TOLERANT WATER, GSE39894 susceptible water, GSE39894 tolerance water, GSE97045 WATER SENSITIVE, GSE97045 WATER tolerance).

Expression analysis
Each of the selected GSE (Gene Expression Omnibus (GEO) Series (GSE), was analyzed utilizing the ReadAffy function from the affy package followed by normalization with the RMA function. Probes identi ed with different expression patterns when exposed to stress were analyzed with the Limma package with signi cant probes having adjusted p values of less than 0.05 and a log fold change of > 2 or <-2.

Co-expression Data Analysis
Weighted gene co-expression network analysis (WGCNA) were utilized in deriving co-expression networks, followed by implementation in the R WGCNA package. The power of beta = 12 was chosen based on the scale-free topology criterion. The modules resulted in possessed genes that were signi cantly interconnected, thereby allowing the construction of two distinct networks, one that utilized tolerant samples, with the other only utilizing susceptible samples.

Module Preservation
Module preservation statistics in WGCNA was utilized to predict whether a particular module as de ned in the reference data set (tolerant network) was also a part of the test data set (susceptible network). The signi cance of the preservation statistics as ascertained from summary Eq. (2) takes into account several preservation statistics.
Based on the proposed thresholds from the rst methodology proposal (Goldberg and Roth 2003), a summary < two indicates no preservation, two < summary < ten suggests weak to moderate preservation, with Page 7/23 a summary > ten suggesting vital module preservation. Intramodule connectivity or Kin is a measure given to each gene in a module. It shows how much the gene has relationship with other genes in the same module. Catalse's Kins in both tolerant and susceptible groups was calculated to determine the difference.

Effects of drought-stress on morphometric features of varieties of tomato
Morphological results from the application of drought-stress to different varieties of tomato showed an effect on plant height, fresh & dry weight of tomato shoots. Plant height decreased substantially due to drought-stress. An increase in the level of drought decreased the height of both varieties. The most decrease was observed at the highest level of drought treatment. Of the two varieties, CaljN3 showed more tolerance than SuperstrainB at all levels of drought treatment. SuperstrainB is therefore the susceptible variety. In most cases, drought stress resulted in a decrease in leaf surface area. Decreasing the leaf surface area reduces transpiration and increases plant tolerance to osmotic stress conditions. Drought resulted in a decrease in leaf surface area for both varieties (Table 1).  that the length of palisade parenchyma cells decreased, but their diameter increased. Empty intercellular space within the spongy parenchyma cells decreased in treated plants relative to the control. The difference in growth and development between that of the control and treated plants is well visible, and in all cases, the control plants showed more growth in comparison to the treated plants (Fig. 1).

Effect of drought-stress on CAT1 gene expression in tomato varieties
A study of the relative expression of the CAT1 gene in two varieties of tomato in conditions of drought was conducted (Fig. 2). As is seen, in Fig. 2 the relative expression of the gene CAT1 did not show any considerable difference as the level of stress increased for both Caljn3 and SuperstrainB varieties. A comparison of the level of expression of the CAT1 gene in the two varieties revealed that expression of the gene CAT1 in Caljn3 and Superstrain3 in control conditions are similar. Likewise, for the treated samples expression of the gene CAT1 did not show considerable differences in both varieties (Fig. 2).

Bioinformatics Study Of Cat1 Gene Utilizing Microarray Analysis
In this study, Prob Id named Les.3098.1.S1_at was selected indicative of Cat1 in Solanum Lycopersicum with Gene ID 543990. Of the studied groups which were divided based on varieties and type of stress, the prob did not show any signi cant log fold change ( Table 2). The gene located in the Salmon module, which is a cluster with 118 genes had a z score equal to 14.26. The Z score showed that this cluster was conserved between the susceptible and tolerant cultivars. Gene enrichment performed utilizing Kobas3 also did not show any pathway with a signi cant p value for the cluster. Table 3 shows that in both groups the average expression was just around 13, and the Kin did not differ. Genes representing the hub gene changed between the two tolerant and susceptible states in different clusters with a speci c color. The Kin parameter was derived from the amount of hub gene and gene descriptions. Kin CI and kin MS are related to the tolerant and susceptible varieties, respectively.

Discussion
The study revealed that under drought stress there was a substantial decrease in length, fresh weight and dry weight of the roots for both varieties. Research has shown that with su cient moisture root growth increased and that by going away from the optimal amount of moisture root growth decreased. Asang et al. (1998) reported a decrease in root growth and growth limitations in upper layers of the soil due to water de cit. In low irrigation, less moisture is around the root. This results in mechanical resistance of the soil against root development and, as a result, a reduction in the length and density of the root in common irrigation treatments. With su cient irrigation, water is more reserved in the root area and the plant by condensing its roots make better use of water. As a result, acceptable use of water in this treatment was increased relative to low irrigation treatments. Plant growth is dependent on the supply of essential carbohydrates to stems and shoot. Factors limiting photosynthesis like light and water, in addition to decreasing plant function also decreases root growth. This is the main reason for the observed difference in the dry weight of the root in different treatments. Plants in dry environment prefer to deposit its photosynthetic production in the root and not in the stems and shoots as the plant can preserve its ability to absorb more amounts of soil water (Assenge et al. 1998). Tomato is susceptible to drought stress, and therefore, when applying stress, its vegetative growth and function decrease. Miguel and Francisco (2007) also reported a reduction in root growth, fresh weight and dry weight in tomatoes, which is comparable to the results of this study.
Plant growth under stress usually depends on roots ability to absorb water from the soil and transferring it to stems (Navarro et al. 2008). The intensity of root growth affects the shoot of a plant. Root length is an index for absorbing water from deep layers of soil (Franco et al. 2011).
Based on this study's results, drought stress caused a reduction in leaf surface in both CaljN3 and On the other hand, in the control and under stress conditions, shoot dry weight of in susceptible varieties was lower relative to tolerant varieties, which can be used as an index for selection of susceptible and tolerant varieties. The typical reaction of a plant to drought stress includes reducing stem growth and the size of the whole plant (Munns 2002). A decrease in leaf area causes a reduction in the receival of light and photosynthesis (Ourcut and Nilsen, 2000). Researchers also reported that water stress causes a reduction of shoot dry weight in plants and is reported in Vigna radiate, Cicer arietinum, Glycine max, Medicago sativa and Trifolium subterraneum (Mrema et al. 1997).
A reduction in photosynthesis, increased production of inhibitory substances, and a reduction in the levels of hormones during drought stress are possibly some of the reasons for decreased growth and shoot weight (Hayat & Ahmad, 2007). It is expected that under water de cit conditions, the absorption of nutritional substances decreases, transpiration is reduced which closes the stomata thereby preventing the entrance of carbon dioxide and reducing photosynthesis. These events cause a reduction in the growth and expansion of shoots in the plants. Water de cit causes a reduction of root and shoot growth.
The reduction for the shoot is however greater than that of the root (KirnaK 2001).
The level of production of fresh and dry matter of plants has a strong co relation with leaf area and Tissues placed in water de cit conditions usually demonstrate a decrease in cell size and amount of vascular tissues. Cell wall thickness also increased. Under these conditions, processes corresponding to cell elongation are more vulnerable compared to processes related to cell division (Claudio et al. 1998).
In this study the number of parenchyma cells under the midrib decreased in both tomato varieties. The length of palisade parenchyma cells also decreased, which is comparable with the results of Eydie Najaf Abadi & Enteshari (1389). The space between spongy parenchyma cells seems to be bene cial for the prevention of water wastage. Blade thickness did not show any change in the tomato varieties studied. In a study of the effect of water stress on some varieties of avocado (Americana Persea), it was shown that palisade parenchyma and the total leaf thickness was lower than that of the control plants with an observable 35-45 percent reduction in intercellular space (Chartozoulakis et al. 1999). Reduction in blade thickness, palisade and spongy parenchyma in some species of Acacia auriculiformis under water de cit stress was reported by Liu et al. (2004). A leaf is considered a responsive organ to environmental conditions (Nevo et al. 2000) and among environmental factors that could potentially affect the structure of a leaf, certainly drought stress is one of the most important ones (Nardini et al. 2005). Changes in leaf anatomy in plants under stress could be related to reduced transportation via the stomata. A reduction of leaf expansion could be related to different mechanisms such as reduction in cell division (Granier et al. 2000), hardening of cell wall (Neumann 1995), or reduction of turgor pressure (Bouchabke et al. 2002).
Based on the results of this study, drought stress did not have a signi cant effect on the expression of the  and salinity treatments, rather than activating the catalase pathway, the cell process activates the SOS pathway of cells, pumps, carriers, and cellular messengers until they have an enzymatic response.
Tomatoes seem to go one step further in response to stress oxidation and increased oxygen free radicals, activating enzymes other than catalase. Apparently, in this plant, the ght against oxidative stress begins one step before the enzymes and seeks to expel the stressor by activating proteins, especially channels, pumps and cellular messengers.

Conclusion
Anatomic observations showed that drought stress causes a reduction in the diameter of vessels and WGCNA results showed that this gene is in a fully conserved cluster without any variance between susceptible and tolerant cultivars and lacking any signi cant cell pathways in enrichment gene study. Gene hub studies on the catalase gene also indicated that this gene is a non-hub from microarray studies. It seems that in some cases tomatoes undergoing abiotic stress instead of activating the catalase pathway, the cell process activates other pathways such as SOS, pumps, carriers, and cellular messengers. Tomatoes seem to go one step further in responding to stress oxidation and increased oxygen free radicals, by activating mechanisms other than catalase. Apparently, in this plant, the ght against oxidative stress begins one step before the enzymes and seeks to expel the stressor by activating proteins, especially channels, pumps and cellular messengers.  Comparison of relative expression of CAT1 gene in CaljN3 and SuperstrainB varieties.