Environmental factors and stresses cause plants to respond by inducing several morphological, physiological, and metabolic responses. Included in these responses are the changes in metabolic pathways resulting in altered production and accumulation of secondary metabolites (Mareri et al. 2022), such as withanolides. Withanolide biosynthesis is therefore dependent on the growing conditions of the plants, with soil salinization being a major environmental factor to consider if large-scale, formal cultivation of W. somnifera is needed to meet the growing global requirement for withanolides. Yet, the cultivation of this plant may be a viable strategy for exploiting saline soils, provided that an increase in the production of withanolides in response to high soil salinity is observed. The response by plants to salt stress is a complex mechanism, involving various physiological and biochemical processes. However, there is a lack of molecular characterization of W. somnifera in response to salt treatment, and therefore this study serves as an initial investigation to address this imperative approach.
Germination data
Plant species exhibit varying degrees of sensitivity to salt stress across different growth stages, with the seed germination period being the most vulnerable to salt stress. During this phase, exposure to salt stress results in a decrease in germination percentage, a delay in the germination rate, and an inhibition of plant tissue elongation (Tarchoun et al. 2022). This is attributed to ion toxicity, oxidative stress and increased external osmotic potential, resulting in the reduction of water uptake during imbibition (Shahid et al. 2020). Salt treatment of W. somnifera seeds significantly reduced their germination rate compared to untreated control groups (Fig. 1a). This is in agreement with previous studies, showing a reduction in the germination rate of W. somnifera seeds exposed to as little as 10 mM NaCl (Jaleel et al. 2008; 2009). Furthermore, salt stress appeared to induce damage to the root surface and prevent the colonization of the root by rhizospheric microbiota in comparison to untreated controls (Fig. 1b-g). While the mechanisms and implications of these induced changes on W. somnifera roots are yet to be investigated, damage to or changes in root structure in other plants species have been observed following salinity stress (Hasan et al. 2018, Silva et al. 2021, Tan et al. 2020).
Differentially expressed genes and gene ontology analysis
Salt stress resulted in the significant regulation of a large number of genes in (Online Resource 3) W. somnifera leaf tissue. However, of those genes submitted for GO analysis, few were returned with descriptions (Figs. 3 and 4), highlighting the lack of annotated genes for this medicinally important plant species in the publicly available database. In response to salt stress, significantly upregulated genes with annotated descriptions indicate molecular-level adaptations aimed at mitigating stress. Increased detoxification pathways facilitate the removal of toxic ions (Puccio et al. 2023), while the upregulation of genes related to transmembrane, vesicle-mediated transport, and specific components like plasma membrane and Golgi apparatus point toward adjustments in compartmentalization in order to discharge or isolate salt ions (Heydarian et al. 2018; Sun et al. 2021). Additionally, increased expression of genes related to DNA integration, RNA binding, and diverse molecular functions (enzymes, binding proteins, regulatory activities) suggests adaptations to manage protein synthesis, gene expression, and overall cellular processes under stress (Diray-Arce et al. 2015).
The significant downregulation of genes related to anatomical structure development, cell differentiation, cell wall organization and mitotic nuclear division suggests the plant prioritizes immediate survival over growth and expansion under stress. This is supported by the observed decrease in activity within carbohydrate and lipid metabolic processes, DNA replication, repair, recombination, and chromatin organization, indicating a reallocation of resources toward stress response mechanisms and a reduction in cell division (Kitavi et al. 2023). Furthermore, reductions in intracellular protein transport and protein glycosylation as well as large reductions in transferase activity imply adjustments in protein movement and modifications and metabolic pathways, thereby conserving resources (Han et al. 2020).
Overall, analysis of the highly differentially expressed genes in W. somnifera under salt stress reveals an upregulation of detoxification pathways and an adjustment in transmembrane transport mechanisms to manage toxic ion concentrations. Concurrently, a notable downregulation of energy-related processes, growth factors, and protein modification pathways underscores a prioritization of internal cellular maintenance over growth. These findings underscore the intricate molecular adaptations employed by W. somnifera to navigate salt stress conditions, highlighting a strategic balance between cellular homeostasis and environmental adaptation.
Transcription factor genes responding to salt stress
Salt-tolerance mechanisms strongly rely on transcription factors (TFs) to link salt-sensory pathways to stress-responsive gene expression. As such, the expression levels of these various TFs and the genes they regulate may impact the degree of salt tolerance of plants. In this study, the expression levels of 60 TF genes, from 26 TF families, were markedly altered concomitant with salt stress (Fig. 5 and Online Resource 4). Several of the upregulated TF families have been intensively studied, such as WRKY, MYB and C2H2 Zinc finger proteins (reviewed in Baillo et al. 2019; Liu et al. 2022) and are associated with enhanced stress tolerance in plants. In salt stressed W. somnifera leaf tissue, the TF family with the greatest number of altered members was the AP2/ERF-ERF family (Online Resource 4). The AP2/ERF TF family comprises a family of plant-specific transcription factors extensively engaged in diverse biological processes, including growth, development, as well as responses to both biotic and abiotic stresses (Licausi et al. 2013). Transcriptome analysis of other Solanaceous species, namely tomato and pepper, indicate that AP2/ERF are involved in stress response mechanisms, including salt-stress. In particular, the AP2/ERF-ERF subgroup members were enriched in differentially expressed genes, both up- and down-regulated, in Solanaceous species indicating their key role in salt-stress tolerance (Choi et al. 2023). Six members of the NAC transcription factor family were also upregulated in W. somnifera following salt stress (Online Resource 4). NAC TFs are a class of stress-related TFs that directly regulate the expression of abiotic stresses related genes and have received much attention as they have been found to play a positive role in drought and salt stress tolerance through modulating an ABA-mediated pathway (Hong et al. 2016; Nuruzzaman et al. 2013; Su et al. 2023). The transcript exhibiting the highest level of induction following salt stress in this study (log2FC = 2.75, Online Resource 4) with similarity to a TF belonged to the GRAS family. GRAS TF members have been found to participate in the response to abiotic stress, including salt stress, where their overexpression or silencing generated gain- and loss- of salt tolerant phenotypes in Betula platyphylla (He et al. 2022). The most strongly downregulated transcript with similarity to a TF in response to salt stress belonged to the HB-WOX family (log2FC = 3.94, Online Resource 4). While typically associated with plant developmental processes, recent studies have revealed their importance in regulating responses to salt stress (Hao et al. 2019; Li et al., 2022), with several members observed to be downregulated (Akbulut et al, 2022).
When considering the significant regulation of multiple transcription factors from diverse families, many of which have previously been associated with salt-stress resistance in various plant species, it strongly suggests their pivotal role in W. somnifera's salt stress response mechanism. The alteration in TF expression may cause extensive changes in the regulation of gene expression (particularly stress-related genes), signal transduction pathways, biochemical modulation and protein profile accumulation which remains to be explored. Furthermore, this work contributes to the identification of novel regulators involved in the abiotic stress responses in W. somnifera, which may represent future targets for engineering improved salt-tolerance mechanisms.
Withanolide biosynthesis pathway
Withanolides, the signature and major active secondary metabolites of W. somnifera, are synthesized via the triterpenoid pathway. As such, transcripts of genes involved in the isoprenogenesis precursor pathway were investigated for altered expression in salt stressed plant leaf tissue. The transcript of nine and eleven genes implicated in the MEP and MVA pathways respectively of isoprenogenesis were examined (Online Resource 3). The transcript levels of the cytosolic MVA pathway gene 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) were significantly reduced (log2FC = -1.4, Fig. 6, Online Resource 3) in salt stressed leaf tissue compared to control plants. This may indicate a reduced HMG-CoA pool for the downstream conversion into mevalonic acid, a key regulatory step of isoprenogenesis (Akhtar et al. 2013). However, no other statistically significant differences in the transcript levels of MEP or MVP pathway genes were observed between control and salt stressed leaf tissue. These results suggest that the salt treatment applied in this study has little effect on isoprenogenesis, implying that the metabolic flux of precursor molecules towards withanolide biosynthesis was unaltered.
Additional key regulatory genes implicated in withanolide biosynthesis were investigated in this study for altered expression following salt stress. Within the withanolide biosynthesis pathway, genes such as farnesyl pyrophosphate synthase (FPPS), squalene synthase (SQS), cycloartenol synthase (CAS), and sterol methyl transferase (SMT) lie at critical branch-points of several pathways leading to the synthesis of compounds essential for plant growth and development as well as withanolides (Singh et al. 2014). Changes in the expression of these genes caused by salt stress may result in a deregulation of downstream metabolic flux towards 24-methylene cholesterol and subsequent withanolides.
A common downstream enzyme of precursor biosynthesis, FPPS catalyzes the formation of farnesyl diphosphate (FPP), a short-chain prenyl diphosphate important in various branched pathways (Senthil et al. 2015). As such, FPPS is considered a branch-point enzyme capable of directing carbon flow away from central isoprenoid biosynthesis (Dhar et al. 2013). This implies that changes in the levels of FPPS would alter the flux of isoprenoids towards various branches of this pathway (Dhar et al., 2012). Furthermore, FPPS acts as a key enzyme in isoprenoid biosynthesis, particularly in its role in initiating the triterpenoid precursor synthesis that contributes to withanolide biosynthesis (Thirugnanasambantham and Senthil, 2016). In this study, two isoforms of FPPS were detected in the transcriptome data. While one isoform displayed no significant differential regulation, the other isoform exhibited significant downregulation in response to salt stress compared to control tissues (log2FC = -1.14, Fig. 6, Online Resource 3) This may indicate a perturbation in downstream withanolide biosynthesis, however, as FPPS is positioned at the intersection of different sterol biosynthesis pathways and is involved in the synthesis of sesquiterpene precursors for several classes of essential metabolites, changes in FPPS expression may not directly modulate withanolide content.
This study identified three separate transcripts sharing a strong similarity to WsCYP98A, each exhibiting distinct regulation profiles. These findings potentially indicate the existence of multiple isoforms for WsCYP98A within W. somnifera. However, only a single transcript was significantly downregulated in salt stressed tissues (TRINITY_DN14649_c0_g1, log2FC = -1.00, Fig. 6, Online Resource 3). Transcript abundance of WsCYP98A, which plays a role in biotic and abiotic stress adaptation of W. somnifera through the synthesis of defense molecules, has been found to correlate with the accumulation of withanolides (Shilpashree et al., 2022). Therefore, a decrease in WsCYP98A expression in response to salt stress as observed in this study may indicate a reduction in withanolide content. While some studies have demonstrated a clear linear correlation between the expression patterns of FPPS, WsCYP89A, and the accumulation of major withanolides (Rana et al. 2014; Senthil et al. 2015), others have only identified a weak association between the expression of FPPS and withanolide accumulation (Gupta et al. 2011; Pandey et al. 2018). As such, direct measurement of the leaf withanolide content would be necessary to determine if downregulation of these genes in response to salt stress resulted in reduced withanolide concentrations.
Contribution of LEA proteins in W. somnifera in response to salt stress
LEA proteins are low molecular weight, hydrophilic proteins that accumulate in plant cells as a functional adaptation to stress, primarily in response to drought conditions, but also in reaction to salinity and low temperatures (Hong-Bo et al. 2005; Tunnacliffe and Wise 2007). While LEA proteins have been identified from various plant species, no inventory of LEA proteins in W. somnifera has as yet been performed. In this study, W. somnifera transcriptome sequences with similarity to potato LEA gene families resulted in the identification of 77 transcripts with similarity to LEA genes, where the majority of transcripts were classified as part of the LEA-2 family (Online Resource 5). Through genome-wide analysis studies, the LEA-2 group has been identified as the largest LEA family in several plant species (Huang et al. 2022; Ibrahime et al. 2019; Liu et al. 2023; Magwangwa et al. 2018; Nagaraju et al. 2019), including the halophyte E. salsugineum. (Li et al. 2021). While transcriptome studies such as this may not comprehensively capture the entire LEA gene repertoire of W. somnifera, the substantial identification of LEA-2 members aligns with these findings. In Sorghum bicolor, LEA-2 proteins have been found to contain a high proportion of salt-responsive cis-regulatory elements in their gene promoters (Nagaraju et al. 2019) which may account for their high expression during salt-stress responses. Among the LEA-2 protein sub-family, dehydrins were the most upregulated LEA family members, with the expression of four DHN transcripts markedly induced in response to salt stress (Online Resource 5). Dehydrins, which accumulate late in embryogenesis and in nearly all vegetative tissues during normal growth conditions and in response to stress-induced cellular dehydration, have their expression influenced by various abiotic factors and phytohormones. Notably, certain dehydrins, known as Response to ABA (RAB) proteins, exhibit responsiveness to abscisic acid (ABA). This association is significant in the context of ABA’s pivotal role as a plant hormone, regulating salt stress responses. Exposure to stressors such as salinity, induces ABA accumulation and therefore dehydrins (Allagulova et al. 2003; Hundertmark and Hincha 2008; Rorat 2006), thereby enhancing the plant’s salt tolerance by stabilizing membranes, enzymes, and nucleotides in cells under abiotic stresses (Yu et al. 2018). The increased expression of dehydrins and other members of LEA family members observed in this study suggests that the accumulation of LEA proteins may represent a significant strategy employed by W. somnifera to mitigate the effects of salt stress.