Original link:
https://onlinelibrary.wiley.com/doi/full/10.1111/tpj.16847
Outline of Research
In a recent study published in The Plant Journal, a breakthrough discovery has unraveled the unique two-enzyme mechanism of pyrrolizidine alkaloids (PAs) biosynthesis in Symphytum officinale, which not only reveals the complexity of secondary metabolic pathways in plants, but also opens up new avenues for agricultural crop improvement and plant toxin reduction.
Background:
Pyrrolizidine alkaloids (PAs) are a class of nitrogenous secondary metabolites synthesized by specific plants, known for their unique pyrroline ring structure, which plays a vital role in the phytochemical defense system, helping to defend against herbivores and pathogens. However, they are also significantly toxic and can cause harm to humans and animals, especially when ingested through the food chain, which can lead to serious health problems. Therefore, the study of pyrrolizidine alkaloids not only helps to reveal the self-protection mechanism of plants, but also has important practical significance for ensuring the safety of food and drugs.
Fig.1 Puthalizidine alkaloid (PAs) biosynthesis pathway
Key results
1. Unique dual-enzyme mechanism
The researchers identified two high-spermidine oxidase (HSO) homologous genes, SoCuAO1 and SoCuAO5, which are independently involved in the biosynthesis of PAs in different organs of the aggregate grass. Using transcriptome sequencing technology (RNAseq), they identified and confirmed two homologous genes, SoCuAO1 and SoCuAO5, as candidate genes for high spermidine oxidase (HSO). SoCuAO1 and SoCuAO5 have a consistent co-expression pattern with HSS, a key enzyme in PAs biosynthesis (SoHSS) in polymeric grass: SoCuAO1 is expressed in young leaves, while SoCuAO5 is expressed in roots, while PAs are mainly produced in roots and young leaves, suggesting that they may play a key role in PAs biosynthesis (Figure 2). In addition, phylogenetic analysis showed that SoCuAO1 and SoCuAO5 were clustered with known HiHSOs, providing strong support for their potential functions in PAs biosynthesis.
Figure 2: Relative expression levels of HSS and CuAOs genes in PAs-producing and non-producing tissues analyzed by RT-qPCR
2. SoCuAO1和SoCuAO5的体冂縰驸证
Through RISPR/Cas9 experiments, the key role of these genes in the synthesis of PAs was confirmed, and a potential molecular target was provided for manipulating the PAs content in medicinal plants. The researchers found that the knockout pairs of SoCuAO5 genes resulted in a significant accumulation of Hspd in these hairy root lines, while PAs were completely undetectable. This significant substrate accumulation and product deletion provides direct evidence for a critical step in the conversion of Hspd to PAs, confirming the irreplaceable role of SoCuAO5 in the PAs biosynthesis pathway (Figure 4). In addition, the metabolic analysis of these lines by gas chromatography-mass spectrometry (GC-MS) further confirmed the unique function of SoCuAO5 in catalyzing the formation of PAs from Hspd. PAs were not detected in the non-flowering stage in regenerated plants after SoCuAO5 gene knockout, but the synthesis of PAs was observed in flowers and young leaves during the flowering stage, and the overexpression of SoCuAO1 led to the production of PAs with different stereochemical properties (Figure 5), suggesting that SoCuAO1 may have complementary functions in specific tissues, providing valuable insights into the complex mechanisms of PAs biosynthesis in herbaceous aggregate grasses.
Fig.4 Levels of Hspd and PAs in hairy roots of SoCuAO5 gene knockout
Fig.5 Analysis of PAs during plant regeneration after SoCuAO5 gene knockout
3. In vitro enzyme activity validation of SoCuAOs
Through detailed enzyme kinetic analysis, the researchers revealed that these two enzymes have a high affinity and catalytic efficiency for specific substrate Hspd, especially SoCuAO5 exhibits higher catalytic activity than SoCuAO1, in addition to being regulated by feedback of substrate concentration (Figure 6). Using gas chromatography-mass spectrometry (GC-MS), the researchers further identified the reaction products produced by SoCuAO1 and SoCuAO5 in response to Hspd, confirming that these two enzymes can effectively catalyze the formation of the key intermediates of the pyrroline alkaloid backbone, the two stereoisomers of monocyclic N-(4-aminobutyl) pyrroline ion and 1-formylpyrroline (Figure 7). In contrast, SoCuAO2 is not involved in PAs biosynthesis, which further highlights the unique role of SoCuAO1 and SoCuAO5 in PAs biosynthesis and their differences in biochemical properties from other CuAOs (Figure 7). These findings deepen our understanding of the key enzyme roles in the PAs biosynthesis pathway of Polymer Grass.
SoCuAO5SoCuAO2与不同底底物 (Hspd㈕(S) (S)
Fig. 7 GC-MS analysis shows the reaction products after incubation of SoCuAO1 and SoCuAO5
conclusion
PAs are an important part of the phytochemical defense system, helping to defend against herbivores and pathogens, but their toxicity also poses a potential threat to humans and animals. In this study, we found that two different high spermidine oxidase (HSO) homologous genes, SoCuAO1 and SoCuAO5, were independently involved in the biosynthesis of pyrrolizidine alkaloids (PAs) in different organs, which not only enriched our understanding of plant self-protection mechanism, but also had important practical significance for ensuring the safety of food and drugs. By gaining a deeper understanding of the PAs biosynthesis pathway, we can make better use of plant resources while avoiding their potential harms.
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