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Comprehensive RNA-Seq-based study and metabolite profiling to identify genes involved in podophyllotoxin biosynthesis in Linum album Kotschy ex Boiss. (Linaceae) – Scientific Reports

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The L. album is a well-known source of lignans. Several studies have been carried out on their physiological and biochemical properties and expression of some key genes involved in the biosynthesis of lignans in vitro cultures techniques3,9,21. This study aimed to recognize a common base of transcriptional changes, especially changes associated with PTOX biosynthesis occurring in different organs during multiple stresses in vivo cultures.

The expression profiles showed obviously differences in the number of up- and down-regulated DEGs under multiple stresses. Fewer DEGs were observed in this plant’s stresses related to the roots (such as Al toxicity) compared to the shoots. These differences are driven by differences between datasets that can have various sources, such as biological or technical variability.

Identification of a large number of DEGs under drought stress indicated that a considerable portion of the transcriptome was engaged by this stress22. Under drought stress, the differences between tolerance and susceptible genotypes arise in gene expression patterns, and phenotypes10. Also, it has been observed that the expression level of stress genes responsive is usually higher in tolerance genotypes13. Therefore, these results corroborate the concept that flax has better drought tolerance than other stresses10.

According to the identified DEGs, 20 commonly regulated genes responsive to biotic and abiotic stresses and 38 commonly regulated genes responsive to abiotic stresses were overlapped using Venn diagram analyses in the shoot and root, respectively. Then, only one commonly regulated gene, endochitinase EP3, was identified with a significant increase in both organs. The EP3, catalyzing the hydrolysis of chitin, is involved in the plant development processes, generation of signal molecules, plant defense responses, and programmed cell death23,24. The expression of the EP3 gene increased in response to drought, salinity, treatments of UV light, exogenous elicitor treatment, wounding, and pathogen attack11,23,24. Moreover, the β-1,3-glucanase gene increased in results related to the shoot, which was previously demonstrated to be crucial for flax resistance to Fusarium spp.25. Flax plants with β-1,3-glucanase overexpression, generating pectinase inhibitors, and employing chitinases and peroxidases increased the production of SMs and changed cell wall compositions, leading to the construction a barrier to fungal growth26.

Biological processes, including secondary metabolism, stress, development, cell wall, lipid metabolism, and protein degradation, were commonly altered by all stresses. Such common regulation reflects the flexibility of biological systems through the adjustment of complex metabolism networks in response to stimulants during the evolution of the plant’s immobile life11.

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. The study of individual stress has identified 11 top TFs, including bHLH (Basic helix-loop-helix), C2H2, NAC, MYB, ERF, bZIP, WRKY, MYB, DREB, HSF, and NFYA10 as known main regulators of abiotic resistance pathways under repeated drought in flax10. NAC TFs also contributed to stress response to maize, rice, and flax tolerance to aluminum stress. These TFs, along with MADS-box, adjust plant growth and development and involve cell wall alteration leading to tolerance to aluminum13. Most 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 metabolism18. The stress signaling pathways share common components comprising ROS, calcium ions, hormones, TFs, and mitogen-activated protein kinase (MAPK) cascades27.

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 a study of flax under individual stress, GO terms of oxidoreductase activity, particularly peroxidases, cell wall, ion homeostasis, and stress response were most changed under unfavorable conditions of the pH and the Zn deficiency12.

Some of our results agreed with the individual studies, while some opposed them. These individual stressors might cause different adjustment responses, which comprise various or shared components in plants. 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)28. The type of adjustment can influence the accumulation of SMs, causing spatial and temporal modulation of the biosynthetic pathways, improving the chance of survival under long-term stressful environments29.

MapMan analysis categorized SMs into 16 groups. Phenylpropanoid and flavonoid pathways were highly affected by all of the stresses. The highest transcript abundance was related to flavonoid and phenylpropanoid pathways under drought stress. The increase of these compounds was extremely related to the balance of carbohydrates between sources and sinks. Also, it has been reported that water potentials were decreased in the plant under severe drought stress, leading to the transport of soluble sugars. Therefore the accumulation of flavonoids and phenolics increased30,31. Outcomes related to drought stress in this work showed a reduced five-fold (− 5) transcript abundance in the glucosinolate pathway. According to these results, drought stress significantly reduced glucosinolate content in Boechera holboellii Hornem. Á.Löve & D.Löve and some Brassica carinata A.Braun cultivars, whereas some B. carinata cultivars showed a significant increase in glucosinolate32,33. Treatment of potassium sulfate declined glucosinolate content during drought conditions on canola compared to the untreated plants34. Roots showed a few changes in the transcript abundance of SMs. However, stress-responsive transcripts are mostly altered under adverse conditions. SMs, such as sinapic acid, lignin, and flavanols, with defensive roles, increased in Sinopodophyllum hexandrum Royle under water deficit35.

Several phenolic compounds including flavonoids, monolignols, lignans, lignins, coumarins, and hydrolysable tannins, are formed through the phenylpropanoid pathway36. The Linum genus, particularly L. album, contains the highest levels of lignans, especially PTOX. However, many studies have investigated the lignan biosynthesis pathway, the lignan pathway until the end product PTOX has not been completely clarified8,20.

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-CoA1,37. Feruloyl-CoA is converted to coniferyl alcohol through two reduction reactions by CCR and CAD8,38. Since coniferyl alcohol has been known as a critical precursor in the biosynthesis of PTOX, the above-stated steps were considered upstream39. In the present study, CAD contains the highest number of transcripts in the upstream steps showing spatial expression patterns and might have different functional roles in specific organs. For example, OsCCR10 involves in response to drought in rice root40. Knockout of OsCCR10 with the CRISPR/Cas9 system revealed that drought tolerance reduced rice due to a decline in lignin content in the root41. After coniferyl alcohol, the later specified steps of PTOX and its derivative biosynthesis were considered as a downstream. These 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 species42,43,44. L. usitatissimum generates both enantiomers (−)- and ( +)-pinoresinol, followed by the stepwise reductive conversion to lariciresinol and then SECO through PLR (1–4)45. (−) SECO, resulting from ( +)-pinoresinol, is catalyzed by the action of SDH to matairesinol. Subsequently, matairesinol is converted to deoxypodophyllotoxin by several enzymatic reactions in P. hexandrum, such as PhOMT3, CYP71CU1, PhOMT1, and 2-ODD. However, the genes encoding enzymes associated with steps between matairesinol to deoxypodophyllotoxin are not yet identified in Linum8,46,47. This study realized some of these genes. The transcriptomic analysis of L. usitatissimum revealed that the highest expression levels of LAC11, POD, 4CL, and SDH genes were under drought stress. The roots subjected to abiotic stress demonstrated the downregulation of the expression level of these enzymes. Conversely, quantitative expression of SDH using qRT-PCR showed an increasing trend in L. album roots and differed in its shoots under all treatment. Furthermore, 2-ODD, excluding drought stress, and OMT1 showed a rising trend in L. album under all stress, conforming to previously reported studies in P. hexandrum. However this pathway evolved independently in the two species20. Quantitative expression of genes related to PTOX biosynthesis, studied in different organs of P. hexandrum, exhibited that SDH, CAD, CCR, and cinnamate 4-hydroxylase genes increased in rhizomes more than roots37. The increased gene expressions related to growth and development and PTOX biosynthesis were also reported at 15 °C in S. hexandrum. While gene expressions and content of PTOX decreased and genes responsive to stress dominated at 25 °C in this plant1. In the study conducted by Kumari et al. (2022), genes of phenylalanine ammonia-lyase (ShPAL), Sh4CL, ShC3H, ShCCoAOMT, ShCOMT, ShCAD, ShDPO, ShPLR, and ShSDH upregulated, as well as, increased PTOX content in root under drought stress. However, there was no evidence of PTOX in the leaf35.

High-performance liquid chromatography (HPLC) analysis, consistent with qRT-PCR results, demonstrated an increase of SECO content and upregulation of three selected genes (SDH, OMT1, and 2-ODD) led to producing the highest PTOX in the roots after 48 h combination treatment. At the same time, shoots produced PTOX by consuming the precursor of SECO. Under drought stress in the root at 12 h, a copious amount of SECO and overexpression of 2-ODD caused the conversion of 6-MPTOX and PTOX during stepwise reactions1. However, PTOX content declined because of the downregulation of 2-ODD after 48 h, according to qRT-PCR. Other enzymes related to lignan biosynthesis are probability involved in the 6-MPTOX generation, so PTOX decreased under drought stress. Despite the high SECO amount and overexpression of 2-ODD (in the shoot after 48 h), PTOX significantly decreased because of the downregulation of SDH and OMT1. The K+ deficiency and drought stress often had opposite accumulation patterns of three selected lignans. The different responses to individual and combined stresses in L. album are suggested the complex regulatory mechanism of lignans biosynthesis, which requires further investigation. The expression of PLR in S. hexandrum contradicts the PTOX content in the different organs, proposing that the PTOX-producing tissue cannot necessarily be its pool48.

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 vitro49,50,51,52. Also, a study conducted on different accessions of L. album under drought stress presented different patterns based on physiological and biochemical responses53.

Generally, the results from qRT-PCR and HPLC analysis with transcriptomic analysis are consistent, corroborating 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, with the negative synergistic effects putting 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.

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