Gene expression profile of the heat shock protein Hsp 23.6 in wheat leaves (Triticum aestivum L.) subjected to NaCl stress

Introduction

Approximately 20% of soils worldwide suffer from salinity problems, which are more pronounced in arid and semi-arid regions, in addition to other human and climatic factors (Lopez et al., 2002; Munns & Tester, 2008; Ding et al.2021) [16,18,5] . In response to the rapid growth of the world ' s population, agricultural production must increase by 70% by 2050 (Hassan et al., 2020) [14] . Abiotic stressors result in a 50% decrease in agricultural yield, posing a significant danger to world food security (Seleiman et al., 2019) [21] . Plant response to high-salt environments has become a major topics of interest to researchers in the field of agriculture and corp production due to its close connection to the source to the human food supply (El Sayed, 2011) [7] . Wheat is the world's leading grain producer and is a major food source for about 36% of the world's population, containing 20% of calories and 55% of carbohydrates (El Sayed, 2011; Hasanuzzaman et al., 2017) [13,7] . Many stressors exist that directly harm plants, such as drought, salinity, extreme temperatures, and exposure to chemicals, causing secondary damage such as oxidative stress and salt stress. Plant exposure to these factors is inevitable. Plants have developed a range of mechanisms that enable them to resist these stresses, whether morphological or physiological. Exposure to environmental stress stimulates the formation of a group of heat shock proteins, a phenomenon common in many living organisms. Heat shock proteins are a widespread protein group that forms as a rapid response to oxidative and salt stress, especially at high temperatures. These proteins are divided into five types depending to their molecular weights, including those with low molecular weights, which are then called low heat shock proteins (Al-Whaibi, 2011; Park and Seo, 2015) [3,20] . Low molecular weight protein(LMWPs) including heat shock proteins (HSPs) perform many vital roles, such as maintaining cellular homeostasis and as molecular companion proteins that enable the plant to adapt to different stresses and prevent and maintain protein structure variation (Huttner & Strasser, 2012; Swindell et al., 2007) [15,25] . Minimal heat shock proteins Have molecular weights ranging from 15-42 kDa and have a conserved region at the carboxyl end (Sun et al., 2002) [24] . Structural alignments, cross-reactivity and cellular location are employed to classify LMWPs. This classification consists of six classes. Three of these classes are resides within the nucleus or cytoplasm, and the remaining three exist in the endoplasmic reticulum, mitochondria, and chloroplasts (Vierling, 1991; Sun et al., 2001) [26,23] . These proteins (LMW-HSPs) in various environmental stresses was protein intermediates-saving. At very early stages of the unfolding, thereby averting their irreversible aggregation. Organellar (LMW-HSPs) represent a distinctive characteristic of plant cells (Escobar et al., 2021) [8] . These proteins serve as molecular chaperones, performing essential functions throughout plant growth stages, development processes, and stress response (Wang et al., 2024) [28] . In addition to elevated temperatures, low molecular weight proteins can be induced by many stressors, including water stress, salinity, and cold exposed. They may also be generated during other developmental phases, such as germination of seeds, embryogenesis, and the development of the fruit (Sun et al., 2002) [24] . Plantstress responses and the elimination of reactive oxygen species (ROS) generated by the electron transport chain depend heavily on mitochondria. A variety of abiotic stimuli cause the up-regulation of genes expression that encoding proteins related stress, such as mitochondrial small HSPs (M-sHSPs) (Escobar et al., 2021) [8] . Nuclear genes that encode mitochondria-localized low molecular weight proteins have been found and examined in numerous plant species. Two mitochondrial small heat shock protein genes (TaHSP23.5 and TaHSP23.6) were identified as being expressed in wheat during heat shock conditions (Basha et al., 1999) [4] . Showed increased expression of the genes for the low molecular weight proteins HSP17.4, HSP17.7, and HSP19.1in wheat grown under influence salt stress (Muthusamy et al., 2017) [19] . A recent work indicated that the HSP23.9 protein is crucial for the wheat plant's response to salinity and heat stress. Moreover, Wang et al. (2020) [29] documented a significant rise in HSP23.9 expression in response to these stressors. Another study conducted on durum wheat grown under the influence of salinity also indicated an increase in the concentrations of a number of LMWPs proteins known as HSPs with different molecular weights (17.8, 26.3,70 and 101kDa) (Al Khateeb et al., 2020) [2] . The development of LMW-HSPs has been recorded in many plants are stressed by various types of stressors such as elevated temperatures, drought, and salinity (Ahn & Zimmerman, 2006; Volkov et al., 2006; Zahur et al., 2009) [1, 27, 30] . These works demonstrate that LMWPs (HSPs) can significantly contribute to a plant's empowered to endure environmental constraints. The aimed of our study was to study the gene expression profile of the HSP 23.6 protein in the leaves of a local wheat variety grown under influence NaCl salinity stress.

Materials and Methods

Plant Growth Conditions and NaCl Treatment

The current study utilized the bread wheat (Triticum aestivum L.) cultivar Ebba 99. The seeds were surface sterilization with 70% ethanol.They were rinsed three times with sterile distilled water, then surface sterilized using sodium hypochlorite 5% for 30 minutes. Subsequently, they were rinsed three times with sterile distilled water, each rinse lasting 30 seconds. Following that, the seeds were cultured in five aseptic Petri dishes containing twenty seeds to evaluate seed viability and determine the germination percentage. Seeds were incubated in a growth chamber at 25 °C ± 2 °C, under to a 16/8 hour light/dark photoperiod. Seeds were planted in plastic containers supplied with a 2:1 mixture of river sand and peat moss. For the initial 10 days, all of the pots were watered with distilled water. The pots were classified into four categories: the first was administered distilled water (control), the second was irrigated with 50 mM NaCl, the third with 100 mM NaCl, and the fourth with 150 mM NaCl. The plants were grown in a controlled growth chamber, where the temperature was maintained at 25 °C ± 2 °C, and the photoperiod consisted of 16 hours of light and 8 hours of darkness.

Gene expression analysis

The effect of sodium chloride treatment on the gene expression pattern of the nuclear gene encoding a small mitochondrial protein with a molecular weight of 23.6 kDa was studied using a semi-quantitative method. Four-weekold plant leaves, grown under controlled conditions, were used and then exposed to different levels of sodium chloride (50, 100, and 150 mM). Subsequently, RNA was extracted, and semi-quantitative polymerase chain reaction (PCR) analysis was performed to determine gene expression levels.

Total RNA isolation from leaves

Using an RNA extraction kit (Z3100; Bromage, USA), RNA was isolated from plant leaves. RNA quality was assessed using an ethidium bromide-stained agarose gel, and its concentration was measured using a Nano drop spectrophotometer.

Complementary DNA (cDNA) synthesis by reverse transcription

After total RNA extraction, complementary DNA (cDNA) was generated by reverse transcription using the GoScript™ Reverse Transcription System (A5000; Promega, USA) according to the manufacturer's protocol, with 1 μL of total RNA and the Oligo(dT)15 primer. Incubation conditions included 5 minutes at 25°C, 60 minutes at 40°C, and 15 minutes at 70°C. The cDNA was amplified using specific primers for the target gene and a primer for the α-tubulin gene as a reference gene (housekeeping gene) Table1. PCR was conducted with the following program using Master Mix (Geneaid) Table 2. 1 cycle perdenaturation step at 95 C˚ for 5 min; 30 cycles of denaturation/ annealing/elongation at 95 C˚ for 30 s/ 60 C˚ for 30 s/72C˚ for 1 min followed by one cycle final extension at 72 C˚ for 7 min. resulting PCR amplicons were analyzed, and the reactions were loaded on ethidium bromide stained 1% agarose gels for visualization. The quantification of the band was conducted utilizing scanning density measurement with BIO-RAD (Image Lab 6.0.1). The measurement yielded band intensity as the unit of expression. The equation established the relative expression of target genes (RE) (Martiansyah et al., 2018) [17] . Equation RE = expression level of target gene/expression level of reference gene.

Data analysis

Use analysis of variance (ANOVA) to data analyzed, and mean differences were compared by the Least Significant Difference (LSD0.05) test by GenStat program.

Results and Discussion

The statistical analysis results in Figures 1 and2 showed a significant decrease in α-tubulin gene bundle intensity with an increase in NaCl concentrations, specifically at 100 and 150 mM concentrations, compared to the control treatment. The results shown in Figures 3 and4 illustrated that elevated NaCl concentrations caused a significant enhancement in gene bundle intensity of HSP 23.6 at concentrations of 100 and 150 mM, relative to the control treatment. In comparison, the 50 mM treatment did not cause any significant increase compared to the control. Gene expression of stress-related proteins, including M-sHSPs, is changed by many abiotic stimuli, and mitochondria are key components of the plant's stress response and free radical detoxification, as they generate reactive oxygen species during electron transport.

(Escobar et al., 2021) [8] . The study showed that treatment with 100 and 150 mM concentrations of sodium chloride increased gene expression of the 23.6HSP protein compared with the control group. Gene expression of sHSP 23.6 was observed in all treatments, whether in sodium chloride-treated or untreated wheat plants. When plants are subjected to salt stress, many transcription factors and genes can be upregulated or downregulated to produce specific gene products, such as proteins or RNA(Gupta & Huang, 2014) [10] .

The synthesis of various protein assemblies is induced when organisms are exposed to stressors; these are known as stress-induced proteins, including heat shock proteins (HSPs), and are common phenomena in all living organisms that rely on them as molecular cofactors (Al-Whaibi, 2011; Gupta et al., 2010) [3,11] . Protein stability depends on a set of these companion proteins that contribute to the assembly of polypeptide chains, ensure their proper folding, and transport within the cell. Exposure to stress causes damage to protein structure, and this is where the role of molecular cofactors comes into play, with increased production to prevent incorrect protein folding, as well as to facilitate the breakdown and rebuilding of damaged proteins (Finka et al., 2016) [9] . Previous studies suggested that sHsps could potentially exert key role in plant adaptation to environmental stresses. MsHSPs exhibit increased expression in the salttreated maize plants, to improve the mitochondrial electron transport effectiveness (Hamilton & Heckathorn, 2001) [12] . The results are consistent with those Al-Kutaibi et al., (2021) in durum wheat plants under salt stress (200 mmol NaCl). The authors found that under salt stress the genes are likely up regulated such as family of HSPs including HSP101, HSP70 and HSP26. 3, and HSP17. 8. Particularly, the M23 and T11 highest increase of protein contents compared to the other varieties which clearly indicates enhanced resistance to salt stress. The results that gained by Subba et al., (2013) [22] on chickpea plants treated under osmotic stress, showed the accumulation of HSPs. sHSP in drought resistant cultivars against susceptible ones at the initial stage of growth period. The expression of heat shock genes in wheat leaves at either 0 mM or 50 mM concentration in control treatment can be attributed the sHSP effect during growth and development under both normal and stress conditions and processes such as photosynthesis, cell membrane stability, and the plant's response to heat stress. Escobar et al. (2021) [8] demonstrated that Arabidopsis thaliana plant with decreased expression of M-sHSPs showed significant growth disturbances, leading to reduced plant size and adverse effects on physiological performance, including photosynthetic efficiency and heat stress response, while the formation of ROS and accumulation causes increased permeability of cellular membranes, emphasis the cellular damage resulting from the decline of M-sHSPs. The reduced gene expression following treatment with 50 mM NaCl may result from this stress level not being insufficient to stimulate an increase in gene expression. The reaction of HSPs is dependent upon nature of stress, whether biotic or abiotic, the plant genotype, the duration of exposure, and the developmental stage of the plant (Dodds & Rathjen, 2010) [6] .

Conclusion

The findings of this study demonstrated that HSP 23.6 gene regulation in wheat leaves under normal growth conditions. Gene expression also increased in response to salt stress conditions generated by sodium chloride (NaCl). This illustrates the significance of this protein's expression in regularly growing plants and its role in aiding plants to cope with salinity stress.

Acknowledgement

AcknowledgementsWe extend our sincere thanks and appreciation to the Dean of the College of Education -Department of Biology at the University of Basra for their acceptance and support in conducting the required measurements in the college laboratories.

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