The L. album is a well-known rich source of SMs, particularly lignans. Several studies have been carried out on their physiological traits, biochemical properties, and expression of some key genes involved in the biosynthesis of lignans in vitro cultures so far 3,9,20. This study aimed to recognize a common base of transcriptional changes occurring in different tissues during multiple stresses in vivo cultures and genes of PTOX.
First, 20 commonly regulated genes responsive to biotic and abiotic stresses and 38 commonly regulated genes responsive to abiotic stresses were found using transcriptome data analyses in the shoot and root, respectively. Then, only one commonly regulated gene, endochitinase EP3, with a significant increase, was identified in both tissues. The EP3, catalyzing the hydrolysis of chitin, is involved in the plant development processes, generation of signal molecules, plant defense responses, and programmed cell death 21,22. The expression of the EP3 gene increased in response to drought, salinity treatments of UV light, exogenous elicitor treatment, wounding, and pathogen attack 11,21,22. Moreover, the β-1,3-glucanase gene increased in our results associated with the shoot that previously was demonstrated as crucial for flax resistance to Fusarium spp. 23. Flax plants with β-1,3-glucanase overexpression, generating pectinase inhibitors, and employing chitinases and peroxidases have increased the accumulation of SMs and changes in cell wall composition, leading to building a barrier to fungal growth 24.
Biological processes, including secondary metabolism, stress, development, cell wall, lipid metabolism, and protein degradation, were commonly altered by all stress. Such common regulation reflects the flexibility of biological systems through the high flexibility of their metabolism in response to stimulants during the evolution of the plant's immobile life 11.
The stress-responsive common GO terms consisted of AP2/ERF, HB-7, and NAC transcription factor families that significantly upregulated in ABA treatment and drought stress and downregulated in Fusarium treatment and K+ deficiency. Individual study of flax under repeated drought has identified 10 top TFs, including bHLH (Basic helix-loop-helix), C2H2, NAC, MYB, ERF, bZIP, WRKY, and MYB, as well as DREB, HSF, and NFYA10 as known main regulators of abiotic resistance pathways 10. NAC TFs also contribute to stress response to maize, rice, and flax tolerance to aluminum. These TFs, along with MADS-box, adjust plant growth and development and involve cell wall alteration leading to tolerance to aluminum 13. Most of the TFs related to potassium starvation have been demonstrated to belong to MYB, bHLH, NAC, B3, bZIP, WRKY, and AP2/ERF, participating in physiological plant processes, stress resistance, and secondary metabolism 18. The stresses signaling pathways share common components comprising ROS, calcium ions, hormones, TFs, and mitogen-activated protein kinase (MAPK) cascades 25. The root GO terms showed that biological processes, namely TCA / org transformation, oxidoreductase activity, cell wall, transport, hormone metabolism, and receptor kinase signaling, are specifically modulated under abiotic stress. In an individual study of flax under unfavorable pH and Zn deficiency, GO terms of oxidoreductase activity, particularly peroxidases, cell wall, ion homeostasis and response to stress were most changed 12.
The total number of DEGs observed for drought stress was more than the number of DEGs for other stresses. Under drought stress, the differences between tolerance and susceptible genotypes arise in not only gene expression patterns but also phenotypes 10. Concerning the fact that the expression level of stress-responsive genes is typically higher in tolerance genotypes 13. Therefore, these results corroborate the concept that flax has better drought tolerance than other stresses 10.
As already stated, some of our results agreed with the individual studies, while some opposed them. These individual stresses might cause plants a different adjustment response that is shared or different components. When plants simultaneously encounter stress combinations, it could require similar or opposing molecular, physiological, and metabolic responses. The precise choice of which adjustment strategy during multiple stress is presumably to be affected by factors like the intensity of each individual stress, the time course of stress, plant age, and genotype (tolerant or susceptible to any one of the individual stress) 26. This choice may influence the accumulation of SMs, causing spatial and temporal modulation of the biosynthetic pathways, subsequently, survival improvement under long-term stressful environments 27.
MapMan analysis categorized SMs into 16 groups. Phenylpropanoid and flavonoid pathways were highly affected by all of the stresses. The highest accumulation in transcript abundance was for flavonoids and phenylpropanoids under drought stress. The increase of these compounds is extremely related to the balance of carbohydrates between sources and sinks. Under severe drought stress, the accumulation of flavonoids and phenolics is increased, resulting from the transport of soluble sugars due to decrease water potentials 28,29.
In contrast, outcomes related to drought stress in this work showed a reduced five-fold (-5) transcript abundance in the glucosinolate pathway. Similar to the results of this work, drought stress significantly reduced glucosinolate accumulation in Boechera holboellii Hornem. Á.Löve & D.Löve and some Brassica carinata A.Braun cultivars, whereas some B. carinata cultivars reported a significant increase in glucosinolate accumulation 30,31. Treatment of potassium sulfate during drought conditions on canola declined glucosinolate accumulation compared to the untreated plants 32. Root tissue showed a few changes in the transcript abundance of SMs. However, mostly stress-responsive transcripts are altered under adverse conditions. Interestingly, SMs, such as sinapic acid, lignin, and flavanols, which are proposed to have defensive roles, increased in Sinopodophyllum hexandrum Royle under water deficit 33.
Several phenolic compounds originating from phenylalanine, including flavonoids, monolignols, lignans, lignins, coumarins, and hydrolyzable tannins, are formed through the phenylpropanoid pathway 34. The Linum genus, exclusively L. album, contains the highest levels of lignans, especially PTOX. In contrast to the notable amount of experience in the lignans pathway, the pathway of this compound until the end product PTOX has not been completely clarified.
The beginning of this pathway starts with the deamination of phenylalanine and synthesizes 4-coumaroyl-CoA by 4CL. Then, 4-coumaroyl-CoA is converted to caffeoyl-CoA through several reactions by HCT, which catalyzes two different steps, followed by methylation via CCoAOMT and synthesizes feruloyl-CoA 1,35. Feruloyl-CoA is converted to coniferyl alcohol through two reduction reactions by CCR and CAD 8,36. Since coniferyl alcohol has been known as a critical precursor in the biosynthesis of PTOX, the above-stated steps were considered upstream 37. In the present study, CAD contains the highest number of transcripts in upstream steps showing spatial expression patterns and may have different functional roles in specified tissues; for example, OsCCR10 involve in response to drought in rice root tissues 38. knockout of OsCCR10 with the CRISPR/Cas9 system revealed reduced drought tolerance due to a decline in lignin accumulation in root 39. The later specified steps of PTOX and its derivative synthesis after coniferyl alcohol were considered downstream. Downstream steps begin with coupling two molecules of coniferyl alcohol to get pinoresinol enantiomers by an oxidase (LAC11) or peroxidase (POD) with the aid of dirigent proteins, depending on plant species 40–42. L. usitatissimum generates both enantiomers (-)- and (+)-pinoresinol, followed by the stepwise reductive conversion to lariciresinol and then SECO through PLR (1–4) 43. (-) SECO, resulting from (+)-pinoresinol, is catalyzed to matairesinol by the action of SDH. Subsequently, matairesinol is converted to deoxypodophyllotoxin by several enzymatic reactions in P. hexandrum, such as PhOMT3, CYP71CU1, PhOMT1, and 2-ODD. Although the genes encoding enzymes associated with steps between matairesinol to deoxypodophyllotoxin are not yet identified in Linum, This study realized some of these genes 8,44,45. The transcriptomic analysis of L. usitatissimum revealed that the expression level of LAC11, POD, 4CL, and SDH genes was highest under drought stress. In contrast, root tissues subjected to abiotic stress demonstrated the downregulation of the expression level of these enzymes. Conversely, quantitative expression with qRT-PCR of SDH genes in L. album was an increasing trend in root tissues subjected to stress and differing in shoot tissues. Furthermore, 2-ODD excluding drought stress and OMT1 showed a rising trend in L. album, conforming to previously reported studies in P. hexandrum, but this pathway evolved independently in the two species 19. Quantitative expression of genes related to PTOX biosynthesis, studying in different tissues of P. hexandrum exhibited that SDH, CAD, CCR and cinnamate 4-hydroxylase genes increase in rhizomes more than roots 35. A study reported increased gene expressions of growth and development and PTOX biosynthesis at 15°C, while decreased gene expressions and content accumulation of PTOX and dominated genes responsive to stress at 25°C 1. In the study carried out by Kumari et al. (2022), genes of phenylalanine ammonia lyase (ShPAL), Sh4CL, ShC3H, ShCCoAOMT, ShCOMT, ShCAD, ShDPO, ShPLR, and ShSDH demonstrated upregulation, as well as, an increase in PTOX accumulation content in root subjected drought stress 33. however, PTOX was not evident in the leaf.
High-performance liquid chromatography (HPLC) analysis, consistent with qRT-PCR results, demonstrated the combination of both conditions in the root with increased SECO content and upregulation of three selected genes (SDH, OMT1, and 2-ODD) led to producing the highest PTOX at 48h. At the same time, shoot tissues subjected to such conditions formed PTOX by consuming the precursor of SECO. Under drought stress in the root at 12h, a copious amount of SECO and overexpression of 2-ODD caused the conversion of 6-MPTOX and PTOX during stepwise reactions 1. However, PTOX content declined because of the downregulation of 2-ODD after 48h, according to qRT-PCR. Probability involving other enzymes related to lignans biosynthesis toward 6-MPTOX generation, the PTOX subject to drought stress decreased. Despite the high SECO amount and overexpression of 2-ODD (in the shoot after 48h) significantly decreased PTOX because of the downregulation of SDH and OMT1. The K+ deficiency and drought stress often have opposite accumulation patterns of three selected lignans. The differences in response to individual and combined stresses in L. album suggest the complex regulatory mechanism of lignans biosynthesis, which needs further investigation. The expression of PLR in S. hexandrum contradicts the PTOX content in the different tissues, proposing that the PTOX-producing tissue cannot necessarily be its pool 46.
Application of abiotic and biotic elicitors, including chitosan, methyl jasmonate, salicylic acid, yeast extract, and Ag+, has mostly established that the expression of genes associated with lignan biosynthesis and lignan content enhanced in Linum spp. in vitro 47–50. Also, a study carried out on different accessions of L. album under drought stress presented different patterns based on physiological and biochemical responses 51.
In general, the results from qRT-PCR and HPLC analysis and transcriptomic analysis are consistent, corroborating with previous studies, although they may be different in some cases due to species or genotype differences, type of culture, and stress intensity. The K+ deficiency and drought stress often had opposite up/down-regulation patterns, which with the synergistic negative effects, put a lot of expenses on the plant. While fusarium's up/down-regulation pattern was similar to K+ deficiency, drought stress was similar to ABA treatment.
Therefore, strategies based on transcriptome for species that likely accumulate lignans would aid in identifying common features between species and environmental cues to clarify the PTOX biosynthesis pathway.