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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (11): 2720-2730.doi: 10.3724/SP.J.1006.2024.44019

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

Functional identification of MeTCP3a transcription factor in cassava leaf development

WANG Lian-Nan1(), LI Yuan-Chao3, YU Nai-Tong2, MAI Wei-Tao1, LI Ya-Jun2,*(), CHEN Xin2,*()   

  1. 1College of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, Hainan, China
    2Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, Hainan, China
    3College of Life Science and Technology, Guangxi University, Nanning 530004, Guangxi, China
  • Received:2024-01-31 Accepted:2024-06-20 Online:2024-11-12 Published:2024-07-11
  • Contact: *E-mail: liyajun@itbb.org.cn; E-mail: chenxin@itbb.org.cn
  • Supported by:
    Chinese Academy of Tropical Agricultural Sciences for Science and Technology Innovation Team of National Tropical Agricultural Science Center(CATASCXTD202301);China Agriculture Research System of MOF and MARA (Cassava, CARS11-HNCX)

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Abstract:

Leaves are the primary organs of photosynthesis in green plants, and their development indirectly affects the yield and starch content of cassava storage roots. However, the regulatory mechanisms governing cassava leaf development remain unclear. In this study, we explored the function of the MeTCP3a transcription factor in cassava leaf development. Amino acid sequence alignment and phylogenetic analysis showed that MeTCP3a was closely related to the TCP4 protein in rubber (Hevea brasiliensis Muell. Arg.). Subcellular localization and protein interaction assays demonstrated that MeTCP3a was localized in the nucleus, possessed transcriptional activation ability, and could form both homodimers and heterodimers in the nucleus, indicating its properties as a transcription factor. Expression analysis showed that the relative expression level of MeTCP3a was significantly higher in mature leaves compared to other tissues. When MeTCP3a expression was suppressed using VIGS (virus-induced gene silencing) technology, cassava heart leaves exhibited curled edges and a wrinkled appearance. Further real-time quantitative PCR analysis revealed that the expression levels of downstream genes related to leaf morphological development, such as MeCUC1/2/3 and MeIAA, were also down-regulated. This result suggested that MeTCP3a might influence leaf morphological development by regulating the expression of these downstream genes. This study provides a theoretical basis for further exploring the regulatory mechanisms of TCP-like transcription factors associated with leaf growth and development in cassava.

Key words: cassava, leaf development, MeTCP3a, protein interaction, VIGS

Table 1

Primers used in this study"

基因名称
Gene name		 上游引物
Forward primer (5'-3')		 下游引物
Reverse primer (5'-3')
MeTCP3a-pG1300-GFP GTTGATACATATGCCCGTCGACATGAAAGGAGCTGGAGGAG GTGGTGGAGCTCGGATCCGGTACCAGGAGTGGAGGAGGATGG
MeTCP3a-pGADT7 ATGATCGATGAATTCGTCGACATGAAAGGAGCTGGAGGAG CATCTGCAGCTCGATGGATCCAGGAGTGGAGGAGGATGG
MeTCP3a-pGBKT7 AGGACCTGCATATGATCGATATGAAAGGAGCTGGAGGAG TAGTTATGCGGCCGCTGCATAGGAGTGGAGGAGGATGG
MeTCP3a-BiFC AGTGGTCTCTGTCCAGTCCTATGAAAGGAGCTGGAGGAG GGTCTCAGCAGACCACAAGTAGGAGTGGAGGAGGATGG
MeTCP3a-nLUC GGACGAGCTCGGTACCCGGGATCCATGAAAGGAGCTGGAGGAG CCACGCGTACGAGATCTGGTCGACAGGAGTGGAGGAGGATGG
MeTCP3a-cLUC CCCGGGGCGGTACCCGGGATCCATGAAAGGAGCTGGAGGAG AACGAAAGCTCTGCAGGTCGACAGGAGTGGAGGAGGATGG
MeTCP3a-pCsCMV AGTGGTCTCTGTCCAGTCCTGTATAGTTTTCAGCTCCACAG GGTCTCAGCAGACCACAAGTATTGAAGCTGAGCCTGCAAA
qMeActin TCTTCTCAACTGAGGAGCTGCT CCTTCGTCTGGACCTTGCTG
qCP TTGTAGCTGCCGTCCTAACTTGG ACCAAATTGGAGGCTGGCTTCA
qMeTCP3a TGCCACAGCCACAATCAATG CCTCTGTTGCAAACCTGCTG
qMeCUC1 CTGCTGAGTTCCCTAGTCTTCC GCAATCTTGATGCGAGACGG
qMeCUC2 TCTCAACCTCATGTACTGTCTGG TCTGTCTTGCACTGTTTGGGA
qMeCUC3 TACCTCCTGGCTTTAGGTTTCAC GGCTCTATTAGTTCTCAGCCCA
qMeIAA CAAGACTGCTGTGCTGGAGA TTGTTGGGTTAGGTCGAAGGAA

Fig. 1

Amino acid sequence alignment (A) and phylogenetic tree (B) analysis of MeTCP3a with its homologs in other plants The bHLH motifs are in red, blue, yellow, and green boxes in panel A. The scale in panel B represents genetic similarity."

Fig. 2

Subcellular localization and transcriptional activation ability of MeTCP3a protein A: subcellular localization of MeTCP3a protein in tobacco (bar is 30 μm); B: detecting the transcriptional activation ability of MeTCP3a in yeast."

Fig. 3

Verifying the protein interaction by using Yeast two-hybridization, LCI, and BiFC assays A: MeTCP3a could form homologous dimer by itself by Yeast two-hybrid assay; B: MeTCP3a could form homologous dimers by LCI assay; C: BiFC assay showing MeTCP3a could form homologous and heterodimers in the nucleus. Bar is 30 μm."

Fig. 4

Expression analysis of MeTCP3a in root, stem, leaf, and axillary bud of cassava SC8 R: storage root; Tl: tender leaf; Ts: tender stem; Tsc: tender stem cortex; Ml: mature leaf; P: petiole; Lb: lateral bud; S: stem; Sc: stem cortex; Sx: stem xylem. Different lowercase letters represented significant difference (P < 0.05)."

Fig. 5

Detection of virus accumulation and MeTCP3a gene expression and the identification of phenotypic in gene silenced plants A: the virus accumulation in MeTCP3a-VIGS plants and control plants (CsCMV-NC) leaves; B: the expression level of MeTCP3a in MeTCP3a-VIGS plants leaves; C: after 36 days of inoculation, the phenotypes of new leaves in MeTCP3a-VIGS plants and control plants (CsCMV-NC). Mock is an uninoculated plant, MeChlI-VIGS is the positive control. * represents significant correlation (P < 0.05); ** represents extremely significant correlation (P < 0.01)."

Fig. 6

Relative expression of candidate target genes in CsCMV-NC and MeTCP3a-VIGS plants A: the relative expression of candidate target genes in MeTCP3a-VIGS plants at seven days after inoculation; B: the relative expression of candidate target gene in MeTCP3a-VIGS plants at 36 days after inoculation. * represents significant correlation (P < 0.05); ** represents extremely significant correlation (P < 0.01)."

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