ABSTRACT
Tryptophan metabolism pathways are important components of the plant immune system; for example, serotonin is derived from tryptophan, and plays a vital role in rice (Oryza sativa) innate immunity. Recently, we isolated a rice mutant, early lesion leaf 1 (ell1), which exhibits lesions. RNA-seq analysis revealed that KEGG pathways related to amino acid metabolism were significantly enriched in the transcripts differentially expressed in this mutant. Furthermore, measurements of free amino acid contents revealed the accumulated tryptophan of ell1 mutant. In addition, the transcript levels of genes related to tryptophan biosynthesis were significantly enhanced in the ell1 mutant. These results revealed that ELL1 plays a critical role in tryptophan metabolism. Based on these findings, it is revealed that loss of ELL1 function may disrupt tryptophan metabolism, thereby inducing cell death and forming lesions in rice.
KEYWORDS: RNA-seq analysis, tryptophan metabolism, cell death
Introduction
To fight against pathogens, plants induce cell death, strengthen their cell walls, and produce an arsenal of chemicals with antimicrobial or toxic activities, such as the antibacterial compounds termed phytoalexins.1,2 Some of these defense molecules originate from amino acid precursors, indicating that amino acid metabolism plays an important role in plant defenses. For example, tryptophan (Trp) is a precursor of serotonin, which is involved in the physical defense system in rice (Oryza sativa).3 Serotonin acts as an effective internal ROS scavenger and serotonin polymerization and incorporation into cell walls forms a physical barrier in rice.2–4 In plants, serotonin is biosynthesized from Trp by L-tryptophan decarboxylase, which forms tryptamine; tryptamine is then converted to serotonin by tryptamine 5-hydroxylase, which is encoded by ELL1/SL.3,5
Studies of lesion mimic mutants have revealed key aspects of plant metabolism, cell death, and disease resistance. Here, we characterized the rice lesion mimic mutant early lesion leaf 1 (ell1), and revealed a potential link between Trp metabolism and the cell death phenotype.6
Results and discussions
Recently, we isolated a lesion mimic mutant termed early lesion leaf 1 (ell1).6 At the seedling stage, small, sporadic reddish-brown lesions appear in ell1 leaves (Figure 1(a,d)). At the heading stage, the lesions become larger and more distinctive, and both new and old leaves develop lesions, although the younger leaves have smaller and fewer lesions than the older leaves (Figure 1(b,c,e)).
Figure 1.
Leaf phenotype of wild-type and ell1 mutant. (a) Plants at the seedling stage. (b) Plants at the tillering stage. (c) Plants at the heading stage. (d) Phenotype of leaf at the seedling stage, corresponding to (A). From a to d, flag leaf, inverted two leaf, inverted three leaf, inverted fourth leaf. (e) Phenotype of the first, second, third, and fourth fully expanded leaves from the tip to the base of the main tiller of plants at the tillering stage, corresponding to (B). Scale bar = 5 cm in A, B, C, E; 1 cm in D
We next conducted an RNA-seq analysis in the ell1 mutant and the wild type. Gene ontology (GO) analysis assigned most of the differentially expressed genes (DEGs) to the following terms: ‘emergency response to cell wall’, ‘external encapsulating structure’, ‘metabolic process’, and ‘secondary metabolic process’.6 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified 29 significantly enriched pathways in the DEGs, including phenylpropanoid biosynthesis; tyrosine metabolism; glutathione metabolism; cysteine and methionine metabolism; glycine, serine, and threonine metabolism; alanine, aspartate, and glutamate metabolism; and nitrogen metabolism (Figure 2). Interestingly, most of these pathways are related to amino acid metabolism, suggesting that ELL1 is involved in amino acid metabolism.
Figure 2.
Kyoto encyclopedia of genes and genomes (KEGG) functional classification chart of differentially expressed proteins
ELL1, a cytochrome P450 monooxygenas, participated in synthesis of serotonin, which is a Trp-derived secondary metabolites.3,6 To explore the effect of ELL1 on tryptophan metabolism, we measured free amino acid contents in the wild type and the ell1 mutant at the heading stage. The contents of many amino acids, such as Lys, His, Gly, Cys, Arg, Leu, Val, Asn, Thr, Ser, Ala, Phe, Gln, and Ile, dramatically increased, reflecting a general accumulation of free amino acids in the ell1 mutant (Figure 3). In addition, the Trp content in the ell1 mutant was 7.12-fold higher than that in the wild type (Figure 3). These results showed that Trp metabolism was disturbed in the ell1 mutant.
Figure 3.
Free amino acid contents in the wild type and ell1 leaves. Data are presented as means ± SD. Three independent plants were measured to calculate the means value. Statistically significant differences from the respective wild type at *P < .05 and **P < .01 were detected using t-tests
Furthermore, we examined the expression of genes related to Trp biosynthesis (OASA1, OASA2, OASB1, and OASB2) in the wild type and the ell1 mutant.7,8 Quantitative real-time PCR revealed that the OASA1, OASA2, OASB1, and OASB2 transcript levels were higher in the ell1 mutant compared to the wild type (Figure 4). These results demonstrated that loss of function of ELL1 affects the expressions of genes related to Trp biosynthesis.
Figure 4.
Expression of genes associated tryptophan synthesis. **, P < .01 (Student’s t-test); Error bars, ±SD (n = 3)
Overall, we propose three theories to explain the lesion phenotype of ell1 mutants, based on our previous and present studies: (1) When ELL1 is mutated, ell1 mutant fails to synthetize serotonin, which acts as an effective internal ROS scavenger, resulting in high endogenous oxidation level so that produce excessive ROS.4,6,9,10 (2) In addition, the failure biosynthesis of serotonin induces the accumulation of tryptamine. Abundant tryptamine is oxidized by monoamine oxidase to generate H2O2, thereby causing cell death.11 (3) The accumulation of Trp leads to the increase of tryptamine, which produces excessive H2O2, thereby resulting in cell death and forming lesion in leaves.11
Acknowledgments
This work was supported by the National Natural Science Foundation of China (32071993, 91735304) .
Funding Statement
This work was supported by the National Natural Science Foundation of China [32071993, 91735304]; Central Public-interest Scientific Institution Basal Research Fund [NO.CPSIBRF-CNRRI-202111].
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
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