Elevated temperature is the primary abiotic stress for plants as a result of climate change and future major plant disease epidemics are anticipated to develop more frequent and larger, according to several consequences. In nature, temperature-sensitive resistance in plants has been regularly recognized and is universal26. There are metabolic pathways that are down/up regulated under pathogen attack to reinforce the defence mechanisms and elevated temperatures can neutralize defence through altering these regulations. Engineering new crop varieties and plant improvement tactics involves an understanding of how climate change affects innate immunity in plants and the discovery of elite genes that confer disease resistance27. How molecular pathways affect plant defense against infections and how climate change may influence them is mostly unknown26–27. To better comprehend the transcriptional responses arbitrated by the host canola Rlm7 genotype when confronted with the pathogen L. maculans and virulence genes that are involved in host defense under incompatible and compatible interactions, a complete transcriptomic study was accomplished24. Our study is the first to examine the effect of this gene at elevated temperatures using metabolomic techniques.
To determine the susceptibility of canola cultivars to blackleg, a 0–9 ordinal scale was used to rate blackleg symptoms. Plants with symptoms such as the production of pycnidia, lack of dark margins around the wounds, and rapid infestation of infected tissue by ten days28,29 are considered susceptible. In the present study, these symptoms were well observed on the Drakkar at both 21ºC and 28 ºC (although no sporulation was observed at 28ºC). Inspection of 30 plants for these symptoms showed that compared to plants kept at optimum temperatures (21/16ºC), in the susceptible cultivar, lesion size decreased by half when plants were exposed to elevated temperatures (28/23ºC).
Conversely, the lesion size on the resistant cultivar was tripled when incubated at 28ºC compared to those at 21ºC. The reduction of the lesion size on the susceptible cultivar may result from lower pathogen virulence, enhanced physiological vigor of the plant (eg., leaf area), and stomatal closure, which reduces pathogen penetration 26,27. Furthermore, the impact of temperature on the resistance gene and its diminished efficacy might be cited as a potential cause for the larger spot size realized at 28ºC compared to 21ºC. Notwithstanding decreased fungal ability and increased plant vigor, the host plant was susceptible, presumably owing to suppressed resistance pathways at 28 ºC in the resistant cultivar. Since genetic background is critical for single Rlm gene-mediated resistance expression12, 17, this investigation confirmed again that the Rlm7 resistance gene, exclusive to LM in the Excel cultivar, exhibited temperature sensitivity. Several studies indicate that Rlm7 is a promising candidate for inclusion in the canola host gene pool. Genes Rlm930, Rlm4, and Rlm731 encode wall-associated kinase-like (WAKL) proteins. Cloning these genes has shown them to be allelic variants. WAKLs are a recently discovered set of proteins that induce a particular immunological response in plants known as effector-triggered immunity7.
LM is a prevalent hemibiotrophic pathogen that infects a wide variety of hosts. The findings of our study demonstrate that metabolic profiling represents a highly effective approach for investigating the underlying mechanisms of the pathogenesis of the hemibiotrophic pathogen and the mechanisms of the susceptibility of canola. We also found out that LM could cause up-regulation of a range of metabolic pathways. Various crucial metabolites associated with many pathways were likely to have role in canola's resistance to LM infection. Metabolites linked to the successful infection of LM were also shown. The findings of our study also indicate a hierarchical structure in the control of multi-dimensional metabolic networks when responding to the presence of numerous simultaneous stressors, such as pathogen-induced (biotic) and heat-induced (abiotic) pressures. The observed activation may exhibit a correlation with the rapid inhibition of the fungal infection, as has been shown in other instances of plant-pathogen interactions32.
Metabolic pathways for fatty acids, alpha-Linolenic acid, nicotinate and nicotinamide, galactose, histidine, porphyrin and chlorophyll, fatty acid elongation in mitochondria, and aminoacyl tRNA biosynthesis were considered as resistance-related (RR-) specific pathways or pathogen stress-related specific pathway as well.
Fatty acids (FA) metabolic pathways have significant roles in plant defense against pathogens. Previous research on FAs indicates that they have passive functions in plant defense, such as acting as biosynthetic precursors for cuticular components or the phytohormone jasmonic acid (JA). Studies of FA metabolic mutants also demonstrated an active signaling role for the cuticle in plant defense33. In this study, alpha-Linolenic acid metabolism was shown to have a significant effect in both positive and negative ionization modes (Figs. 7 and 8).
Alpha-linolenic acid, acting as a phytohormone, reacts to pathogen infections in plants, collaborates with other plant hormones to promote growth, cope with stress, and control metabolic processes34. It improves the function of many antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase, NADH peroxidase, and glutathione reductase35. Oxidation control of unsaturated fatty acids may facilitate their long-distance transport in plants36.The metabolisms of 12-OPDA, and 13(S)-HPOT, alpha-linolenic acids, were up-regulated in the LM-canola interaction at 120 hpi in the R cultivar under 21ºC. 12-OPDA is a precursor for synthesizing jasmonic acid (JA). 12-Oxo-9(Z)-dodecenoic acid strongly inhibited mycelial growth and spore germination of eukaryotic microbes37. Functions of oxylipins include direct antimicrobial effect, stimulation of plant defense gene expression, and regulation of plant cell death38. Increasing JA in the fifth-day post-infection is probably in response to the fungal necrotrophic stage.
In this study, the abundance of palmitic acid (hexadecanoic acid) a precursor for JA39, was significantly reduced in both R and S plants treated with the pathogen compared to their control at 48 hpi under 21ºC. This reduction at 48 hpi is probably related to suppressing the production of JA and increasing the expression of salicylic acid synthesis. An increase of palmitic acid at 120 hpi may represent higher demands for the production of JA. Palmitic acid also affects the pathways of Fatty acid biosynthesis, Fatty acid elongation in mitochondria, and Fatty acid metabolism. The study conducted by Sa_sek et al. (2012) reported the expression of multiple marker genes linked to different plant hormones elevated in resistant and susceptible B. napus plants infected with LM40. Additionally, the study revealed that the expression of genes related to the ET pathway increased in later stages of the infection (8–10 dai), while genes related to SA were induced earlier (4–6 dpi) 40. In a similar vein, a recent study41 demonstrated that the marker genes for SA and ABA were up-regulated at 7- and 14-days post-inoculation (dpi) of B. napus inoculation with LM. Haddadi and co-workers42 provide a more detailed snapshot of the global transcriptome that is active in B. napus and LM during cotyledon infection. The gene expression profile associated with different plant hormones revealed the importance of SA at the earlier stages of infection (until 4 dpi) and the up-regulation of genes related to the JA pathway at the later stages (6–8 dpi). Studies have shown co-expression of components that react to SA/ET/JA throughout the late stage of infection, along with the important significance of early SA activation in providing long-lasting resistance. SA is better at protecting against diseases that rely on living host cells (i.e., biotrophic and hemibiotrophic stages), whereas ET/JA signaling is more effective at combating necrotrophic pathogens that kill host cells as well as herbivorous insects. Research on gene-for-gene interactions in the B. napus–L.maculans pathosystem revealed the early activation of SA/JA responsive factors, highlighting the significance of these genes in the incompatible connection. Early activation of SA-responsive factors in resistant B. napus genotypes ensures a strong defense against pathogens by enhancing plant immunity via systemic acquired resistance. The delayed activation of defense genes might be a result of extensive colonization of plant tissues in the necrotrophic stage, making it difficult for the host to halt the infection owing to the high fungal burden (mycelia)43. Each hormone has certain responsive factors and signaling pathways, where the responsive pathways of different hormones also have different potential connections, building up an integrated and systemic signaling network in order to cope with various challenges43. SA–JA relationship is not always antagonistic. Tamaoki et al. (2013) elucidated that the defense system is triggered by the interaction of both SA and JA signaling interaction during the initiation of defense response. A partially common signal transduction pathway should be used for the signaling of both JA and SA44.
Increased activity of nicotinate and nicotinamide metabolic pathway in the pathogen inoculated R cultivar compared to control at 21ºC/48 hpi might be considered as an indication of greater supply of these two key precursors for the generation of coenzymes, NAD + and NADP+, which are crucial for redox reactions and electron transportation from one reaction to another. These coenzymes are essential for many metabolic pathways including TCA cycle, glycolysis, fatty acid biosynthesis, pentose phosphate cycle, and many other metabolic pathways. NAD is a main redox carrier in cellular oxidation during catabolism and vital for plant growth and development. NAD serves as a signal molecule primary and secondary carbon metabolism, in addition to its redox activity. Recent studies using integrated omics methods in conjunction with molecular plant pathology has shown that manipulating NAD biosynthesis and recycling leads to an alteration of metabolite pools and developmental processes and changes in the resistance to various pathogens. NAD levels could be considered a possible target to improve tolerance of crops to biotic stress 45.
An increase in the abundance of Galactose metabolism at 48 hpi compared to control in the R cultivar at 21ºC showed the importance of the central carbohydrate metabolism as one of the most key sources for protecting cells from stresses48. Galactose has a defensive function and is involved in synthesizing hemicellulose49. Sugars play a central role in the interaction between plants and pathogens, serving as a vital energy source for defense mechanisms and functioning as signaling molecules for the regulation of defense genes50, leading to the invent of the concept of Sweet Immunity and sugar-enhanced defense relating the potential involvement of certain sugars in plant immunity49. Small sugars, mono, di, and oligosaccharides, activate plant defense responses and increase the resistance of plants to pathogens51. Based on network analysis and Enrichment analysis, it was also identified as an important pathway.
Phenylalanine metabolism, Tryptophan metabolism, beta-Alanine metabolism, Isoquinoline alkaloid biosynthesis, Tyrosine metabolism, Pantothenate and CoA biosynthesis, Sphingolipid metabolism, Glyoxylate, and dicarboxylate metabolism with the highest effect in positive and negative ion modes (Figs. 7 and 8) were categorized as general or non-specific stress-related pathways. Through the use of network analysis and enrichment analysis, these pathways were also recognized as significant. KEGG mapping also displayed enriched biosynthetic pathways for the biosynthesis of phenylalanine, tyrosine, tryptophan, pantothenate and CoA, sphingolipid, valine, leucine, isoleucine, arginine, proline, caffeine, biotin, glycan, starch and sucrose, galactose, fructose and mannose, ubiquinone and terpenoids-quinones, as well as selenocompound (Figs. 7 and 8). A significant number of these pathways are related with the amino acid metabolism, which serves as a common source for secondary metabolites such phenolics and alkaloids72. Enrichment of amino acid metabolism is essential for influencing metabolic processes after pathogen contact. These pathways shield the plants from a variety of abiotic stresses73, have the ability to act as electron transporters in the process of photosynthesis74, and the ability to protect the young and soft tissues from infections caused by the pathogens75 as well as functioning as signaling molecules in stress and plant defense responses, and contribution in the energy metabolism of a plant76.
Biosynthesis of many essential natural plant defense-related metabolites require amino acid metabolism and consequently affects the plant's immune system. Glycosinolates, secondary metabolites in the Brassicaceae family are based on amino acid precursors53. Most phenolic groups in plants are derived from phenylalanine or tyrosine. An increase of tyrosine in wheat-resistant cultivars against Indian smut after inoculation with Neovossia indica was reported. Phenolic metabolites appear to accelerate the lignification process in wheat-resistant genotypes compared to the susceptible plant. The two said amino acids are precursors of the benzoylic glycosinolate group with antifungal and antibacterial properties53. In this study, tyrosine & phenylalanine metabolism as general or non-specific stress-related pathways was increased in all treatments compared to control at 21ºC and in resistance cultivar at 48 hpi under the elevated temperature regime. Tyrosine metabolism was up-regulated in soybean at 1 and 2 dpi in response to R. solani, which is consistent with our findings54, 56. Moreover, tyrosine has the capability to undergo catabolism, resulting in the production of diverse precursors for the synthesis of hydroxycinnamic acid amides that are linked to the cell wall55. It has been hypothesized that tyrosine decarboxylated precursors (tyramine and dopamine) are used in the synthesis of isoquinolines in plants due to the up-regulation of the isoquinoline alkaloids pathway. Historically, the physiological activity of these metabolites had a role in plant defense systems, either by a direct toxic impact or by suppressing certain processes of invading pathogens57, 58.
Histidine metabolism was increased in the R cultivar compared to control at 21ºC/48 hpi. Histidine biosynthesis pathway is integrated with several metabolic pathways, including tryptophan. Tryptophan plays critical role in regulating plant development and defense responses and is the precursor for indoleacetic acid, a plant hormone that promotes cell expansion46. A liquid fertilizer derived from a yeast cell extract with histidine showed inhibitory effects on the growth of bacterial wilt disease in tobacco plants caused by the soil-borne bacterial pathogen Ralstonia solanacearum47.
Lysine biosynthesis was increased 48 and 120 hpi compared to the control in both R and S cultivars under elevated temperature regimes. An important precursor for synthesis of glutamate is lysine, which is an essential final energy source for the production of carbohydrates, lipids, peptides, and secondary metabolites. Lysine levels increase under abiotic stress, such as salt, water, heavy metals, and nutritive stress69. Endogenous lysine metabolism or its exogenous application helps reduce stress70. Recently, it has been proposed that lysine metabolism induces the jasmonate signaling pathway and tryptophan metabolism in response to stress71.
Pantothenate (vitamin B5) is the precursor to coenzyme A (CoA), which in turn, plays an essential role in fatty acid and pyruvate metabolism59. The main function of vitamins is to act as a cofactor in diverse metabolic pathways, facilitate the production of essential compounds for plants, induce resistance against pathogens, directly promote plant growth and participate in energy conservation in the plant from stored compounds59. In this study, sphingolipid metabolism was increased at 120 hpi compared to control in the R cultivar under both optimum/elevated temperature regimes. Sphingolipids could act as second messengers with effects on cellular homeostasis60.
Carotenoid biosynthesis, pyrimidine metabolism, and lysine biosynthesis were classified as heat stress-specific related pathways. In this study, carotenoid biosynthesis pathway was up-regulated in pathogen inoculated plants of the resistant cultivar compared to those of mock at 28°C /120 hpi. The functional metabolism of carotenoids encompasses various processes, such as the stability of membrane lipid bilayers, the elimination of free radicals induced by reactive oxygen species, and the safeguarding of membrane lipid integrity61, 62. The plant hormones abscisic acid and strigolactones as well as other apocarotenoids that are involved in a variety of developmental processes and stress responses, are all synthesized from carotenoids63. Quan et al. (2022) found that Glutamic acid and Poly-γ-glutamic acid enhanced the heat tolerance of Chinese cabbage (Brassica rapa L. Ssp. Pekinensis) by refining carotenoid production, photosynthesis, and ROS Signaling64. Foliar application of biostimulants like carotenoids may reduce the damage caused by high temperatures in canola and enhance growth and physiological traits.
Nucleotide metabolism is an important component of plant metabolism and development, and affects many metabolic pathways. Specifically, pyrimidine nucleotides participate directly in nucleic acid synthesis54, 65. The present research observed an up-regulation of the pyrimidine metabolism route at a temperature of 28°C. Notably, the rise in this pathway was shown to be more pronounced in the S cultivar compared to the R cultivar. Recently, genes of the pyrimidine catabolic pathway have emerged as potential participants in abiotic stress responses of plants. The role of pyrimidine catabolism in stress tolerance may be related to the production of proline biosynthesis precursors65. In plants, a complete characterization of the pathway is lacking66, 67. Purine and pyrimidine base accumulation in R cultivars at 28°C/48 hpi might be interpreted as the plant's reaction to a mix of pathogen and heat stress. Furthermore, Pyrimidine derivatives exhibit a wide range of biological activities, such as antibacterial, antifungal, insecticidal, herbicidal, and antiviral activities 68. Also elevated levels of Aminoacyl-tRNA biosynthesis in the R cultivar compared to control at 21ºC/48 hpi was observed. Aminoacyl-tRNAs are essential for translation and act in decoding of the genetic information into amino acids. Aminoacyl-tRNA synthesis functions to accurately pair amino acids with tRNAs that have the matching anticodon52.
In summary, the enrichment analysis showed a significant presence of metabolite hits in many pathways. The prevalence of these hits suggests that plant responses are being influenced in terms of defense, resistance, and/or survival. Network analysis provides a clear depiction of the pathways in which the annotated metabolites are engaged, enhancing our knowledge of their significance in these processes77. Cellular overview of the different pathways in canola cotyledons in response Leptosphaeria maculans is shown in Fig. 9. The majority of the 15 significant pathways shown in Table 2 are mostly associated with the production of amino acids and subsequently carbohydrates. Cardoso and colleagues performed a meta-analysis on a wide range of species and various stress circumstances. Metabolite profiling data from plants suffering from water deficit, cold, salt, and nitrogen deprivation was employed. Their results show that amino acid metabolism is central to plant stress adaptation78. In addition to providing carbon and nitrogen for various pathways, amino acids are effective stress signal messengers that help the plant adapt to stress78.
The specific consequences of heat stressed diseased plants depend on several factors, such as the host plant's and the pathogen's genetic background, among many others. Most studies have described negatively impacted resistances under elevated temperature, while others noticed positive or neutral effects of elevated temperature on resistance. Embracing the complexity of the phenomenon is necessary to understand how plants cope with abiotic and biotic stimuli at the same time, both positively and negatively, in order to develop solutions to safeguard food security in the face of rising temperatures79. According to the results obtained in the study of Canola-LM interaction, the elevated temperature might negatively affect the resistance of the R cultivar to LM. The capacity of many pathogens to tolerate and adapt rapidly to circumstances beyond the optimum growth temperature of their host plants may account for the large numbers of adversely affected resistances seen at increased temperatures24. Under high-temperature growth conditions, the resistant genotypes of B. napus achieve a state of homeostasis, wherein both the activation and suppression of defensive mechanisms take place. When there aren't many harmful pathogens in the environment, plants may also reduce their defense mechanisms. Since the R gene-related defense is harmful to plants, they may evolve defensive mechanisms like a guard model to activate their resistance when facing a high concentration of pathogen inoculum. However, research has shown that B. napus exhibits incompatible interactions while simultaneously balancing its defense and development mechanisms at higher temperatures77. One of the most effective approaches to developing crops that are adaptable to climate change is to understand the diverse plant immune mechanisms impacted by this phenomenon and identify key genes that can enhance disease resistance through genome editing techniques25. In the present study, a detailed dynamic metabolite investigation of compatible and incompatible LM-B. napus interaction at 21 and 28°C was carried out. We showed important alterations in the diversity of metabolite classes that proposed an infection-related reprogramming of particular pathways. These modifications point to a particular coordinated control of the metabolic network involved in defense, a key step in the canola defense responses to LM. Moreover, our results point to a hierarchical regulation of multidimensional metabolic networks in the face of several concurrent challenges, such as pathogen and heat stress. Under heat stress, LM-infected canola is expected to exhibit resistance breakdown at 28ºC because the plant defense mechanisms controlled by its R genes are either not started or start too slowly to guarantee an incompatible interaction. It is anticipated that many cellular processes are involved in development of temperature-sensitive resistance in interactions between plants and fungi, bacteria, viruses, and nematodes, among other plant pathogens. Scientific advancements in RNA sequencing, proteomics, and metabolomics using different platforms are essential for determining the other underlying mechanisms of physiological processes.