Identification and Functional Analysis of an Agropyron mongolicum Caffeic Acid 3-O-methyltransferase Gene AmCOMT1

doi:10.11983/CBB24013

Abstract

Abstract: Agropyron mongolicum is one of northern China’s most representative perennial forage grasses, showing strong tolerance to cold and drought. In plants, caffeic acid O-methyltransferase (COMT) is a key gene involved in the biosynthesis of lignin and melatonin, and plays an important role in regulating plant growth, biomass quality, and stress tolerance. In this study, through the analysis of the full-length transcriptome data of A. mongolicum, the COMT candidate gene AmCOMT1 was cloned. AmCOMT1 is highly expressed in tissues with high lignin content, such as stem and root, and its expression is induced by a variety of abiotic stresses, including drought and salt. Overexpression of AmCOMT1 in Arabidopsis wild type (Col-0) and mutant (omt1-2) significantly promoted the synthesis of lignin in transgenic Arabidopsis, restoring the lignin content and composition of the mutant to wild type level and the lignin content in Col-0/35S:AmCOMT1 was increased by 11%. In addition, overexpression of AmCOMT1 increased the melatonin content in Col-0/35S:AmCOMT1 transgenic Arabidopsis. Under salt stress conditions, the average root length of this transgenic line increased by 20.3% compared to the wild type, showing higher stress tolerance. In this study, we identified AmCOMT1 from A. mongolicum as a key gene regulating both lignin biosynthesis and melatonin biosynthesis, improving the stress tolerance of transgenic Arabidopsis. Our results highlighted the application potential of AmCOMT1 in genetic improvement of forage grasses through molecular breeding.

Key words: Agropyron mongolicum, caffeic acid 3-O-methyltransferase, lignin, melatonin, salt stress

Jinyu Du, Zhen Sun, Yanlong Su, Heping Wang, Yaling Liu, Zhenying Wu, Feng He, Yan Zhao, Chunxiang Fu. Identification and Functional Analysis of an Agropyron mongolicum Caffeic Acid 3-O-methyltransferase Gene AmCOMT1[J]. Chinese Bulletin of Botany, 2024, 59(3): 383-396.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24013

https://www.chinbullbotany.com/EN/Y2024/V59/I3/383

Figures/Tables 7

Table 1 Primers used in this study

Purpose	Primer name	Sequence (5′-3′)
Gene clone	AmCOMT1-CDS-F	ATGGGCTCCATCGCCGCCGG
	AmCOMT1-CDS-R	CTACTTAGTGAACTCGATGGCCC
	AmCOMT1-pMDC32-F	CTAGAGGATCCCCGGATGGGCTCCATCGCCG
	AmCOMT1-pMDC32-R	GATCGGGGAAATTCGCTACTTAGTGAACTCGATGGCC
	AmCOMT1-pGWC-F	AGCAGGCTTTGACTTTATGGGCTCCATCGCCGCCGG
	AmCOMT1-pGWC-R	TGGGTCTAGAGACTTCTACTTAGTGAACTCGATGGCCC
Positive detection	M13F	ACTGGCCGTCGTTTTAC
	M13R	GTCATAGCTGTTTCCTG
	pMDC32-JC-F	CACTATCCTTCGCAAGACCCTTC
	pMDC32-JC-R	TTGAACGATCGGGGAAATTCGAG
	PGWC-JC-F	TAATACGACTCACTATAGGG
	PGWC-JC-R	ATTTAGGTGACACTATAG
qRT-PCR	AmCOMT1-qRT-F	GACGCTGCTCAAGAACTGCT
	AmCOMT1-qRT-R	CCATGCGTTGGCGTAGATGT
	18s-qRT-F	CAATGGGAAGCAAGGCTGTAA
	18s-qRT-R	AACAATCCGAACTGAGGCAATC
	AtACTIN-F	CATCAGGAAGGACTTGTACGG
	AtACTIN-R	GATGGACCTGACTCGTCATAC
Identification of Arabidopsis mutant	LP	TCCGGTTTGCAAGTATTTGAC
	BP	ATTTTGCCGATTTCGGAAC
	RP	CTAGGGTCAGTCCCGTGGTAC

Figure 1 Phylogenetic tree of COMT proteins in different species COMT protein sequences from different species and CCoAOMT sequence from Arabidopsis were obtained from NCBI database, and the phylogenetic tree (Bootstrap=1 000) was constructed by Neighbor-Joining (NJ) method with MEGA with AmCOMT1.

Figure 2 Alignment of AmCOMT1 with COMT sequences from different species

Figure 3 Expression of AmCOMT1 in different tissue of Agropyron mongolicum and under different treatments (A) The expression of AmCOMT1 in different tissues of A. mongolicum (R: Root; S: Stem; LB: Leaf blade; LS: Leaf sheath; I: Inflorescence); (B), (C) The expression of AmCOMT1 under 50 and 150 mmol·L-1 NaCl treatments; (D), (E) The expression of AmCOMT1 under 75 and 150 mmol·L-1 mannitol treatments. Different lowercase letters in the bar chart indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3), NS indicate not significant, ** P<0.01.

Figure 4 Phenotypic and lignin staining analysis of transgenic Arabidopsis (A), (B) Expression level of AmCOMT1 in transgenic Arabidopsis; (C) 8-week-old transgenic Arabidopsis plants comparing with Col-0 and omt1-2; (D), (E) Lignin staining of stem cross section of different Arabidopsis lines with phloroglucinol staining method (D) and Mäule staining method (E) (bars=100 μm).

Figure 5 Effects of AmCOMT1 overexpression on lignin content (A), composition (B), and S/G ratio (C) of Arabidopsis Col-0 and omt1-2 Different lowercase letters in the bar chart indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3).

Figure 6 Overexpression of AmCOMT1 improved melatonin synthesis and salt tolerance in transgenic Arabidopsis (A) Melatonin content in different Arabidopsis lines; (B), (C) Root length and growth phenotype of different Arabidopsis lines growing on plates after NaCl treatment (bars=1 cm). Different lowercase letters in the bar chart indicate significant differences (P<0.05) among the data according to the results of ANOVA analysis (n=3).

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