Heterologous Expression of GbTCP4, a Class II TCP Transcription Factor, Regulates Trichome Formation and Root Hair Development in Arabidopsis

Abstract

Two class I family teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins from allotetraploid cotton are involved in cotton fiber cell differentiation and elongation and root hair development. However, the biological function of most class II TCP proteins is unclear. This study sought to reveal the characteristics and functions of the sea-island cotton class II TCP gene GbTCP4 by biochemical, genetic, and molecular biology methods. GbTCP4 protein localizes to nuclei, binding two types of TCP-binding cis-acting elements, including the one in its promoter. Expression pattern analysis revealed that GbTCP4 is widely expressed in tissues, with the highest level in flowers. GbTCP4 is expressed at different fiber development stages and has high transcription in fibers beginning at 5 days post anthesis (DPA). GbTCP4 overexpression increases primary root hair length and density and leaf and stem trichomes in transgenic Arabidopsis relative to wild-type plants (WT). GbTCP4 binds directly to the CAPRICE (CPC) promoter, increasing CPC transcript levels in roots and reducing them in leaves. Compared with WT plants, lignin content in the stems of transgenic Arabidopsis overexpressing GbTCP4 increased, and AtCAD5 gene transcript levels increased. These results suggest that GbTCP4 regulates trichome formation and root hair development in Arabidopsis and may be a candidate gene for regulating cotton fiber elongation.

Keywords: TCP transcription factor; root hair development; sea-island cotton; trichome formation.

Figure 1

Phylogenetic tree analysis and sequence alignment of GbTCP4. (A) The phylogenetic tree of GbTCP4 and Arabidopsis TCP family proteins. The accession numbers of the proteins are as follows: AtTCP1, AT1G67260.1; AtTCP2, AT4G18390.1; AtTCP3, AT1G53230.1; AtTCP4, AT3G15030.1; AtTCP5, AT5G60970.1; AtTCP6, AT5G41030.1; AtTCP7, AT5G23280.1; AtTCP8, AT1G58100.1; AtTCP9, AT2G45680.1; AtTCP10, AT2G31070.1; AtTCP11, AT2G37000.1; AtTCP12, AT1G68800.1; AtTCP13, AT3G02150.2; AtTCP14, AT3G47620.1; AtTCP15, AT1G69690.1; AtTCP16, AT3G45150.1; AtTCP17, AT5G08070.1; AtTCP18, AT3G18550.1; AtTCP19, AT5G51910.1; AtTCP20, AT3G27010.1; AtTCP21, AT5G08330.1; AtTCP22, AT1G72010.1; AtTCP23, AT1G35560.1; AtTCP24, AT1G30210.1. (B) Comparison of amino acid sequences of GbTCP4 and AtTCP4. Black indicates identical amino acids. The horizontal line indicates the conserved teosinte branched1/cycloidea/proliferating cell factor1 (TCP) domain.

Figure 2

Molecular characterization of the GbTCP4 transcription factor. (A) Subcellular localization analysis of the GbTCP4 transcription factor. 35S:GFP is an empty vector control. The red arrow indicates the nucleus. (B) Analysis of the transcriptional activation activity of GbTCP4. pGBKT7 is an empty vector control. (C) Analyses of the binding of GbTCP4 to TCP I (TGGGTCCCACAT), TCP II (TTGTGGGCCCCT), mTCP I (TAGATTCTAAAG), and mTCP II (TCGGGAGACTCG). (D) Analyses of the binding of GbTCP4 to the TCP-binding element in its promoter.

Figure 3

The expression pattern of the GbTCP4 gene in different tissues and fibers at different developmental stages of cotton. (A) Transcriptional levels of GbTCP4 in the cotton main root, fibrous root, stem, hypocotyl, leaf, receptacle, calyx, and flower. The transcriptional levels were calibrated by the control (hypocotyl) (B) Transcriptional levels of GbTCP4 in developing cotton fibers collected at 0 (ovule), 5, 10, 15, 20, and 25 d post anthesis (DPA). The transcriptional levels were calibrated by the control (0 d days post anthesis (DPA)). GbUBQ7 is used as an internal reference gene. Values represent the means of three biological replicates, and error bars represent standard deviations. Asterisks indicate a significant difference (*p < 0.05) relative to the corresponding controls.

Figure 4

Constitutive overexpression of GbTCP4 in Arabidopsis increases root hair length. (A) Transcriptional levels of the GbTCP4 gene in three 35S:GbTCP4 transgenic Arabidopsis lines. The transcriptional levels were calibrated by the control (Col-0). AtUBQ3 was used as an internal reference gene. Values represent the means of three biological replicates, and error bars represent standard deviations. (B) Root hair phenotype from 7-d-old vertically grown seedlings of Col-0 and 35S:GbTCP4 transgenic lines. Scale bars: 100 µm. (C) Comparison of the root hair length on the roots from the Col-0 and 35S:GbTCP4 transgenic lines. (D) Comparison of the root hair density on the roots from the Col-0 and 35S:GbTCP4 transgenic lines. Values are means (±SE) of 30 seedlings for each of three independent experiments. Asterisks indicate significant differences (*p < 0.05; **p < 0.01) relative to Col-0.

Figure 5

Constitutive overexpression of GbTCP4 in Arabidopsis affects trichome formation. (A) Leaf trichome phenotype from the first two rosette leaves of 2-week-old soil-grown seedlings of the Col-0 and 35S:GbTCP4 transgenic lines. Scale bars: 500 µm. (B) Comparison of the trichome number on the first two rosette leaves from Col-0 and 35S:GbTCP4 transgenic lines. (C) Stem trichome phenotype from the main stem internodes from the bottom of 1-month-old soil-grown seedlings of Col-0 and 35S:GbTCP4 transgenic lines. Scale bars: 500 µm. (D) Comparison of the trichome density on the main stem internodes from the bottom of Col-0 and 35S:GbTCP4 transgenic lines. Values are means (±SE) of 30 seedlings for each of three independent experiments. Asterisks indicate significant differences (*p < 0.05) relative to Col-0.

Figure 6

Constitutive overexpression of the GbTCP4 gene increases lignin content in transgenic Arabidopsis stems. (A) Phloroglucinol staining of stem cross-sections of the Col-0 and 35S:GbTCP4 transgenic lines. xy: xylem; if: interfascicular fibers; co: cortex; ep: epidermis; bars: 100 µm. (B) Determination of lignin content in stems of the Col-0 and 35S:GbTCP4 transgenic lines. (C) Quantitative PCR (qPCR) analysis of transcriptional levels of the AtCAD5 gene in the Col-0 and 35S:GbTCP4 transgenic lines. The transcriptional levels were calibrated by the control (Col-0). AtUBQ3 was used as an internal reference gene. Values represent the means of three biological replicates, and error bars represent standard deviations. Asterisks indicate significant differences (*p < 0.05) relative to Col-0. The accession number of the gene is as follows: AtCAD5:At4g34230. (D) Analyses of the binding of GbTCP4 to the TCP-binding cis-element in the promoter of AtCAD5.

Figure 7

CAPRICE (CPC) is a target gene of GbTCP4. (A) Analyses of the binding of GbTCP4 to the TCP-binding cis-element in the promoter of CPC. (B) Detection of the transcription levels of CPC gene in the roots and leaves of Col-0 and 35S:GbTCP4 seedlings by qPCR. The transcriptional levels were calibrated by the control (Col-0). AtUBQ3 expression was used as a control. Values represent the means of three biological replicates, and error bars represent standard deviations. Asterisks indicate significant differences (*p < 0.05) relative to Col-0.