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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (1): 63-75.doi: 10.3724/SP.J.1006.2022.01100

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning of Ta4CL1 and its function in promoting plant growth and lignin deposition in transgenic Arabidopsis plants

MENG Ying1(), XING Lei-Lei1, CAO Xiao-Hong1, GUO Guang-Yan1, CHAI Jian-Fang2,*(), BEI Cai-Li1,*()   

  1. 1Life Science College, Hebei Normal University, Shijiazhuang 050024, Hebei, China
    2Institute of Genetics and Physiology, Hebei Academy of Agriculture and Forest Sciences, Plant Genetic Engineering Center of Hebei Province, Shijiazhuang 050051, Hebei, China
  • Received:2020-12-23 Accepted:2021-04-14 Online:2022-01-12 Published:2021-06-02
  • Contact: CHAI Jian-Fang,BEI Cai-Li E-mail:mengying941027@tom.com;chai87652130@163.com;beicaili@sina.com
  • Supported by:
    Natural Science Foundation of Hebei Province(C2019205102);Key Research and Development Projects of Hebei Province(20326348D);Key Foundation of Hebei Normal University(L2019Z05);Doctoral Foundation of Hebei Normal University(L2018B14)

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Abstract:

4-Coumarate:coenzyme A ligase (4CL; EC 6.2.1.12) acts upstream of the branch point of phenylpropanoid, which is a key enzyme in the phenylpropanoid metabolic pathways for monolignol and flavonoid biosynthesis, and these compounds play important roles in plant growth and development as well as stress adaptability. Many 4CLs had been extensively studied in dicotyledons, but their function in monocotyledons, especially in crops, was relatively poorly understood. In this study, Ta4CL1, which encoded 4-Coumarate:coenzyme A ligase, was isolated from common wheat by RACE technique. Polygenetic analysis revealed that Ta4CL1 could be clustered to the same group of 4CLs from rice, maize, and sorghum, which was mainly involved in lignin biosynthesis. Ta4CL1-overexpressed Arabidopsis lines, at4cl1, at4cl3, and at4cl14cl3 as well as their corresponding functional recovery lines were used to elucidate the function of Ta4CL1 in the phenylpropanoid metabolic pathway. The results suggested that Ta4CL1 had similar function with At4CL1 in regulating lignin biosynthesis but it had no effect on flavonoid biosynthesis. Ta4CL was the major contributor of 4CL enzyme activity in transgenic Arabidopsis plants. Overexpression of Ta4CL led to enlarged leaves and thickened stems in transgenic Arabidopsis seedlings, the expression of Ta4CL was also affected by MeJA, GA, and IAA treatments. These results provide the theoretical basis for improving the utilizing efficiency of plant straws using Ta4CL1 by genetic engineering.

Key words: 4-Coumaric coenzyme A ligase, Ta4CL1, lignin deposition, promote growth, wheat, Arabidopsis thaliana L.

Table 1

Primers used in this study"

引物Primer 序列Sequence (5′-3′) 用途Usage
Ta4CL1 F1 CCGCGGGGAGCAGATCATGAAAGGTTAC 3′RACE第1轮PCR 1st run PCR of 3′RACE
Ta4CL1 F2 ACATCAAGAAATTCGTCGCAAAGGAGGTT 3′RACE第2轮PCR 2nd run PCR of 3′RACE
Ta4CL1 R1/R2 AAGCAGTGGTATCAACGCAGAGTAC(T)30N-1N (N=A, C, G or T; N-1=A, G or C) 3′RACE
Ta4CL1 F3/F4 (T25)N-1N (N=A, C, G or T; N-1=A, G or C) 5′RACE
Ta4CL1 R3 AACCTCCTTTGCGACGAATTTCTTGATGT 5′RACE第1轮PCR 1st run PCR of 5′RACE
Ta4CL1 R4 F: TGTCTCCGGTGTGCAGCCATCCAT 5′RACE第2轮PCR 2nd run PCR of 5′RACE
Ta4CL1 F5/R5 F: CGCACGCACGCACACGCACAA
R: ATCAACATTACACAAGCAGGAAGAACCA		 全长cDNA克隆
Isolation of full-length cDNA
Ta4CL1 F6/R6 F: TCTAGAATGGGGTCTGTGCCGGAG (Xba I)
R: ggTACCCTAGCTTGGGATGCCGGC (Kpn I)		 Ta4CL1过表达载体构建
Construction of Ta4CL1-overexpression vector
Ta4CL1pro F1/R1 F:CCAGTGCCCTCCATCTCT
R:CGCCGCCGCAACCGACTC		 克隆Ta4CL1启动子
Cloning of Ta4CL1 promoter
Ta4CL1pro F2/R3 F: AAGCTTCCAGTGCCCTCCATCTCT (Hind III)
R: GAATTCTTCAACGATCGTGGTAGA (EcoR I)		 Ta4CL1pro:Gus载体构建
Construction of Ta4CL1 pro:Gus vector
GUS F: GGGCAGGCCAGCGTATCG
R: GTCCCGCTAGTGCCTTGTC		 鉴定Ta4CL1pro:Gus载体
Identification of Ta4CL1pro:Gus vector
Actin2
(At3g18780)		 F: GCTGAGAGATTCAGATGCCC
R: CTCGGCCTTGGAGATCCACA		 RT-PCR
Actin2
(At3g18780)		 F: TCGCTGACCGTATGAGCAAAG
R: TGTGAACGATTCCTGGACCTG		 qRT-PCR
AtC3H
(At2g40890)		 F: GTTGGACTTGACCGGATCTT
R: ATTAGAGGCGTTGGAGGATG		 qRT-PCR
AtCOMT1-1
(At5g54160)		 F: TTCCATTGCTGCTCTTTGTC
R: CATGGTGATTGTGGAATGGT		 qRT-PCR
AtCCoAOMT1
(At4g34050)		 F: CTCAGGGAAGTGACAGCAAA
R: GTGGCGAGAAGAGAGTAGCC		 qRT-PCR
AtCAD5
(At4g34230)		 F: TTGGCTGATTCGTTGGATTA
R: ATCACTTTCCTCCCAAGCAT		 qRT-PCR
AtF5H
(At4g36220)		 F: CTTCAACGTAGCGGATTTCA
R: AGATCATTACGGGCCTTCAC		 qRT-PCR
AtHCT
(At5g48930)		 F: GCCTGCACCAAGTATGAAGA
R: GACAGTGTTCCCATCCTCCT		 qRT-PCR
AtCCR1
(At1g15950)		 F: GTGCAAAGCAGATCTTCAGG
R: GCCGCAGCATTAATTACAAA		 qRT-PCR
Ta4CL1 F7/R7 F: TCGTCGCAAAGGAGGTTGTT
R: GCGGCCGAGTCTGGCTCT		 qRT-PCR
TaEF 1a
(M90077)		 F: CAGATTGGCAACGGCTACG
R: CGGACAGCAAAACGACCAAG		 qRT-PCR

Fig. 1

Cloning of full-length cDNA of Ta4CL1 by RACE technique Amplifying the fragments of 5′ RACE (A) and 3′ RACE (B) of Ta4CL1. 1: the 1st run PCR; 2: the 2nd run PCR; C: amplifying the full-length cDNA of Ta4CL. 1: the full-length cDNA of Ta4CL. M: marker; the arrows point to the target bands."

Fig. 2

Phylogeneic tree of Ta4CL1 and its homologues from other plants species Unrooted phylogenetic tree of 4CL isoforms from plant species is constructed (Bootstrap value: 1000 replicates). The scale bar equals to 0.05 amino acid substitutions per position in the sequence."

Fig. 3

Tissue-specific expression of Ta4CL1 in Arabidopsis seedlings carrying Ta4CL1pro:Gus A: the leaves of 3-week-old Arabidopsis seedlings harbouring Ta4CL1pro:Gus; B: enlarged picture of the leave with box in figure A; C: the roots of 2-week-old Arabidopsis seedlings harbouring Ta4CL1pro:Gus; D: the cross section of inflorescence stem of 6-week-old Arabidopsis seedlings harbouring Ta4CL1pro:Gus. Bar: 1 mm (A-C) and 0.18 mm (D)."

Fig. 4

Relative expression levels of Ta4CL1 on GA, IAA, and MeJA treatments"

Fig. 5

Identification of homozygous lines harbouring Ta4CL1 in transgenic Arabidopsis seedlings on RNA level"

Fig. 6

Ectopic expression of Ta4CL1 affects plant growth in transgenic Arabidopsis seedlings A: two-week-old Arabidopsis seedlings; B: six-week-old Arabidopsis seedlings; the measurement of plant height (C) and diameter (D) of the inflorescence stem of the eight-week-old Arabidopsis seedlings, One-way ANOVA followed by Duncan’s test, columns with different letters indicate significant differences, dataset share same letter means no significant differences at P < 0.05. Col-0, wild-type Arabidopsis plants; OX-1, OX-2, and OX-3: Ta4CL1-overexpressing Arabidopsis lines; Ta4CL1/at4cl1 L3, L9, and L11: Arabidopsis lines overexpressing Ta4CL1 in the at4cl1 background; Ta4CL1/at4cl3 L10, L12, and L13: Arabidopsis lines overexpressing Ta4CL1 in the at4cl3 background; Ta4CL1/at4cl1 4cl3 L6, L10, and L11: Arabidopsis lines overexpressing Ta4CL1 in the at4cl1 4cl3 background."

Fig. 7

Determination of total lignin content in the Arabidopsis lines ectopic expressing Ta4CL1 Measurement of total lignin content in stems of 6-week-old (A) and 8-week-old (B) Arabidopsis lines (P < 0.05)."

Fig. 8

Determination of flavonoid and anthocyanin content in the Arabidopsis lines ectopic expressing Ta4CL1 Measurement of flavonoid content (A) and anthocyanin content (B) of the one-month-old Arabidopsis seedlings (P < 0.05)."

Fig. 9

Activity of 4CL enzyme in the Arabidopsis lines ectopic expressing Ta4CL1 Measurement of 4CL enzyme activity in rosette leaves (A) and inflorescent stems (B) of 4-week-old Arabidopsis lines ectopic expressing Ta4CL1 (P < 0.05)."

Fig. 10

Relative expression levels of genes involving in lignin biosynthesis downstream of 4CL in Ta4CL1-overexpressed Arabidopsis lines Col-0: wild-type Arabidopsis plants; OX-1, OX-2, and OX-3: Ta4CL1-overexpressing Arabidopsis lines. Student’s t-test, *P<0.05, **P < 0.01, and ***P < 0.001."

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