The study of the diverse physiological and molecular mechanisms of plant seedlings under various abiotic stresses could be an appealing issue for scientists looking to generate resistant lines. These abiotic stress-resistant plant seedlings will considerably outperform future abiotic stress challenges in orchard preparation and forestation. Plants underwent morphological, physiological, and cellular changes as a result of abiotic stress. We investigated the impact of abiotic stress on three key physiological characteristics that determine plant growth, survival, and productivity in this experiment. The expression of stress sensitive genes was also examined in order to confirm their expression pattern under stress.
Physiological Analysis:
When compared to control seedlings, salt treated seedlings had lower chlorophyll concentration followed by osmotic stress. Lu et al. (2021) found a similar finding in three woody plant species (Tamarix ramosissima, Populus euphratica, and Haloxylon ammodendron) cultivated under various levels of saline stress. P. euphratica reduced Chl more than H. ammodendron and T. ramosissima at lower NaCl concentrations. Under BS cultivars, chl a, chl b, and Tchl were considerably reduced in moderate and high salinity stress (Rahneshan, et al., 2018) also in the natural forest trees of Eastern China's North-South Transect (Li et al., 2018). With increasing levels of PEG-induced water stress, photosynthetic pigment was shown to be significantly reduced in the fig genotypes at 15% PEG treatment (Abdolinejad & Shekafandeh, 2022) and in leaves of the four tree species B. variegate, C. fistula, D. regia and P. pterocarpum (Sinhababu & Banerjee, 2013). Photo-oxidation, chlorophyll breakdown by the enzyme chlorophyllase, and reduced chlorophyll production are the main causes (Santos, 2004 & Jafarnia et al., 2017). High levels of sodium (Na+) and chloride (Cl−) ions in chloroplasts cause considerable damage to the thylakoid membrane, (Wu and Zou, 2009) resulting in chlorophyll leaching and thylakoid degeneration (Sneha et al., 2012). The pigment-protein-lipid complex may be weakened in high-saline conditions (Levitt 1980), and salinity may promote the activity of the Chl-degrading enzyme chlorophyllase (Reddy and Vora 1986). Salinity stress reduces chlorophyll content by depleting Mg2 + in plants, which is a critical component of chlorophyll (Khan et al. 2000). Furthermore, ROS are abundantly formed in plants due to irreversible photophosphorylation inactivation and electron transport obstruction in the thylakoid membrane. Degrading the chloroplast and activating anti-oxidative scavenging systems to safeguard cell structure can reduce ROS generation (Veiga et al., 2007 & Mittal et al., 2012). Plant species, duration, severity, and amount of stress all influence chlorophyll decrease under stress.
Membrane stability index is a measurement of membrane integrity and permeability. Cell membrane integrity or electrolyte leakage reflects cell health in relation to external conditions and is extensively used as a reliable indication of plants under abiotic stress (Fan & Blake, 1997 & Tafeshri et al., 2021). MSI was dramatically reduced in salt and osmotic stress seedlings at 12 DAT when compared to control irrigated seedlings. When comparing control and enhanced salt stress, our findings show lower membrane stability in Acacia auriculiformis (Rahman et al., 2016). Yue et al. (2018) found that salinity stress reduced MSI by 31.7 percent to 54.1 percent at 100 and 200 mM in black locusts (Robinia pseudoacacia). Pistachio rootstock treated with 100 to 200 mM NaCl showed a considerable reduction in MSI (Rad et al., 2021). The findings are similarly consistent with those of PEG-treated almonds (Prunus dulcis (Mill.)), which had lower MSI as PEG concentration increased (Karimi et al., 2012). At 10% PEG, fig (Ficus carica) explants showed increased ion leakage, but tetraploid explants showed larger ion leakage at 20–25% PEG treatment (Abdolinejad & Shekafandeh, 2022). In cells, a lack of water causes an imbalance in metabolism and the generation of reactive oxygen species (ROS) (Karimi et al., 2012). These free radicals are the primary cause of cell membrane damage. All components bound in the cell and organelles are released when the cell membrane is disrupted (Abdolinejad & Shekafandeh, 2022). Ions primarily flow out of the cell, and the number of ions released is a measure of the cell membrane's integrity. Ion leakage is associated with decreased cell membrane stability. As a result, under drought stress, membrane stability is compromised by numerous defects (Cao et al., 2017 & Jafarnia et al., 2017), and electrolytes are lost to the environment.
In plants, relative water content (RWC) is an important and dependable indicator of a cell's ability to retain water under unfavorable conditions. It establishes a balance between the water supply to the plant system and the rate of transpiration through the stomata (Sinhababu & Banerjee, 2013). When compared to control, RWC was dramatically lowered in 150 mM NaCl followed by 15% PEG at 7 DAT. Yue et al. (2013) found similar low RWC data in Robinia pseudoacacia under 100 and 200 mM NaCl stress, as well as in Pistacia vera var. Ghermez-Pesteh under 200 mM NaCl stress (Rad et al., 2021). Tafeshri et al. (2021) found that 3% PEG and 6% PEG reduced RWC in Myrtus communis shoots. RWC in almond explant leaves was reduced when the PEG concentration in the media was increased (Karimi et al., 2012). Sinhababu & Banerjee (2013) found that PEG treatment reduced RWC in C. fistula and B. variegate compared to control. RWC is involved in absorbing more water from the soil and/or controlling water loss through the stomata (Xiao et al., 2009 & Cao et al., 2017). The characteristics and structure of cells are altered under abiotic drought stress, and they are unable to maintain turgor and osmotic pressure (Bolat et al., 2014). The osmotic pressure of soil water is higher than that of plant root cells due to the larger concentration of dissolved salts. As a result, plant root cells are unable to receive water from the soil via osmosis (Nejadsahebi et al., 2010 & Zarafshar et al., 2014). Furthermore, there is no other available type of water for absorption by plant root cells when water is scarce (Ying et al., 2015 & Haider et al., 2018).
Gene Expression Analysis:
MYB transcription factors are found throughout higher plants and are involved in abiotic stress responses (Liu et al., 2017). MYB-3 was shown to be considerably up-regulated by salt stress as compared to control. Cao et al. (2013) found that genes including MdoMYB22, 121, 146, 148, 155, and 206 displayed up-regulations in Arabidopsis under NaCl and PEG stress conditions. Under salt and PEG treatment, the MYB genes Aco001113 and Aco007733 were likewise shown to be up-regulated in pineapple (Liu et al., 2017). Similarly, during NaCl and PEG treatments, the expression of MYB genes was up-regulated in Tamarix hispida, a woody plant (Zhang et al., 2018). Through RT-PCR, eight MYB genes were up-regulated for NaCl stress and eight MYB genes were down-regulated for PEG6000 stress in T. mongolica. Their findings varied depending on the duration of stress and the tissue (Chen et al., 2019). Under 300 mmol/l NaCl treatment, oil palm revealed greater EgMYB gene expression at various time intervals (Zhou, et al., 2020). JrMYB73 expression was up-regulated in walnut following 20% polyethylene glycol stress, but JrMYB44 expression was down-regulated. When comparing JrMYB44 3 h to control, the transcription value decreased (Li et al., 2021). Based on our findings and previous research, we believe that the teak MYB-3 gene regulates secondary cell wall deposition, cell cycle control, hormone signaling, secondary metabolism, meristem development, and cellular morphogenesis under abiotic stress conditions (Zhang et al., 2018 & Yang et al., 2019). As a result, increased expression of multiple MYB genes improves many species' ability to withstand abiotic stress (Li et al., 2021). Furthermore, the structure and function of MYB TF family genes vary substantially between species (Yang et al., 2019).
In the current study, 15% PEG stress increased HSP expression. According to Robinet et al. (2010), expression of HSPs in loblolly pine was co-regulated with photosynthetic acclimation under mild drought and was differently regulated based on different water regimes. The divergent expression patterns of Cqhsp70s in quinoa during drought were also reported by Liu et al. (2018), showing the various functions of HSPs genes in drought tolerance and the functional diversity of Hsp70. HSPs are also known to operate as molecular chaperones, limiting the aggregation formation of other proteins, as well as participating in protein folding and mending misfolded conformers in stressed or unstressed cells (Robinet et al., 2010; Zhang et al., 2015; Lui et al., 2018 & Yer et al., 2018). SHSP expression has risen slightly in response to heat, salt, oxidative, and water stress. As a result, HSP protein expression is one of the most important indicators of persistent stress (Sun et al. 2002 & Zhai et al., 2016).
The importance of BAX inihibitor-1 (BI-1) during cell death under various stresses was studied by Duan et al., 2010; Wang et al., 2012; Ramiro et al., 2016 & Lu et al., 2018. The expression of the BI-1 gene was up-regulated in the presence of 15% PEG, which is consistent with a previous report by Duan et al. (2010), who found that AtBI1 suppressed ER stress-induced PCD and assisted the cell in recovering from water stress damage in Arabidopsis. Wang et al. (2012) discovered that over-expression of TaBI-1 in wheat significantly reduces cell mortality in response to abiotic stress. Over-expression of the AtBI-1 increased sugarcane's water deficit tolerance capabilities, according to Ramiro et al. (2016). BI-1 levels in plants rose during leaf senescence (Duan et al., 2010), drought (Ramiro et al., 2016), and heat stress (Ramiro et al., 2016) (Lu et al., 2018). Over-expression of BI can thereby inhibit cell death activation (Duan et al., 2010). Senescence, as well as a variety of biotic and abiotic factors, influence the activation of programmed cell death (Duan et al., 2010). BI-1 gene expression is increased in stressful situations to prevent cell death and protect cells from the negative effects of stress (Watanabe and Lam, 2009). The BI-1 gene is over-expressed in plants under diverse biotic and abiotic stressors (Kawai et al., 1999; Yamada et al., 2001 & Watanabe & Lam, 2006).
In the current study, the expression of CESs was reduced in both treatments as compared to the control. This could be due to the fact that the expression of this gene has only been detected in response to biotic stress (Islam and Yun, 2016), where transcripts of the VfCXE gene showed active responses in grapevines against the pathogens E. ampelina, R. vitis, and B. cinerea. Under cadmium stress, Li et al. (2019) found that the esteras genes of the SaGELP gene family were expressed differently in different tissues and at different times in S. alfredii. Carboxylesterases catalyse the hydrolysis of carboxylic esters of biomolecules and control the synthesis and release of bioactive metabolites like hormones, which accumulate in plants as carboxylesters and amides (Gershater and Edwards, 2007). As a result, these enzymes are implicated in the systemic acquired immunity signaling pathway and are up-regulated in plants following pathogen infection (Chen et al., 2017). Carboxylesters also control cell death during HR, which is necessary to prevent plant disease (Marshall et al., 2003; Gershater and Edwards, 2007; Wheelock et al., 2008 & Chen et al., 2017). After 6 hours of cadmium exposure, the Esteras/lipase gene family was enhanced in Sedum alfredii (Li et al., 2019). During bioitc stress, CXE expression was elevated in the xylem of the vascular bundle of leaf tissue, and they were involved in defense-related signal transduction cascades (An et al., 2008). Even though carboxylesterase activity has been identified in various investigations, the actual mechanism of CXEs in plants is unknown.
The function of PAL is to aid in the manufacture of various secondary metabolites in order to combat abiotic stress, as previously stated (Fossdal et al., 2007; Jeong et al., 2012; Kelij et al., 2013; Khakdana et al., 2018 & Liu et al., 2019). The PAL is vital for plant development, structural support, defence responses, and abiotic stimulant endurance, as well as for plant cell wall lignification. The current finding of a down-regulated PAL gene is consistent with Khakdana et al. (2018), who found that the expression of PALs in basil was either down- or up-regulated depending on the degree and duration of abiotic stress. In numerous tissues, the PAL gene family encodes several defensive chemicals such as flavanoids, phytoalexins, furanocoumarin, and cell wall components (Jahnen and Hahlbrock, 1988).
Plants' cellulose production is controlled by cellulose synthases (CSs) genes (Burton et al., 2006). Many scientists have investigated the mechanism of cell wall production during abiotic stress (Guerriero et al., 2014; Behr et al., 2015; Kestena et al., 2017 & Goncalves et al., 2019). The CSs gene was up-regulated in seedlings treated with 15% PEG and down-regulated in seedlings treated with 150 mM NaCl. This study supported by Li et al(2019)'s findings, which looked at the expression profiles of ZmCsl genes in maize during PEG-induced drought stress. After 60 hours of treatment, they discovered that more ZmCsl genes were up-regulated. PEG treatment increased the expression of four genes: ZmCslF6-1, ZmCslD1, ZmCslC2-1, and ZmCslC2-2. Under PEG-induced drought stress, Zhao et al. (2022) confirmed that the GhMCesA35 gene was involved in cellulose production in cotton. GhMCesA35 gene expression was up-regulated in Tm-1 and Hai-7124 during fibre secondary wall biosynthesis development. The findings revealed that this gene could boost the strength of the cell wall, which keeps the cell in shape and can withstand abiotic challenges like salt and dehydration. Under adverse conditions, it also modulates cell wall integrity (CWI) by inducing changes in lignin production and cellulose deposition to strengthen the cell wall (Delgado et al., 2003). The cell wall synthesis associated genes were dramatically up regulated in sweet orange grafted on the drought tolerant ('Rangpur') in drought condition (Goncalves et al., 2019), and the CesA6F gene was up regulated in salt situation in alfalfa plants (Behr et al., 2015), up regulation of MsCesA6-B gene after 96 h of heat and salt stress in alfalfa by Guerriero et al. (2014). Plants have a huge number of cellulose synthase genes, which are members of the glycosyltransferase family and are responsible for cellulose synthesis (Richmond, & Somerville, 2000 & Somerville, 2006).