Analysis of the role of Arabidopsis class I TCP genes AtTCP7, AtTCP8, AtTCP22, and AtTCP23 in leaf development

Abstract

TCP family of plant-specific transcription factors regulates plant form through control of cell proliferation and differentiation. This gene family is comprised of two groups, class I and class II. While the role of class II TCP genes in plant development is well known, data about the function of some class I TCP genes is lacking. We studied a group of phylogenetically related class I TCP genes: AtTCP7, AtTCP8, AtTCP22, and AtTCP23. The similar expression pattern in young growing leaves found for this group suggests similarity in gene function. Gene redundancy is characteristic in this group, as also seen in the class II TCP genes. We generated a pentuple mutant tcp8 tcp15 tcp21 tcp22 tcp23 and show that loss of function of these genes results in changes in leaf developmental traits. We also determined that these factors are able to mutually interact in a yeast two-hybrid assay and regulate the expression of KNOX1 genes. To circumvent the issue of genetic redundancy, dominant negative forms with SRDX repressor domain were used. Analysis of transgenic plants expressing AtTCP7-SRDX and AtTCP23-SRDX indicate a role of these factors in the control of cell proliferation.

Keywords: Arabidopsis; SRDX construct; TCP; leaf development; transcription factor.

Figure 1

Phylogenetic tree of class I TCP genes. Phylogenetic tree of class I TCP proteins from Arabidopsis, tomato, and rice. Protein sequences were analyzed with the maximum-likelihood method by MEGA 5 software. Bootstrap values after 1000 replicates higher than 50% are shown. The tree was rooted using the translated sequence of the maize teosinte-branched1 (tb1) gene as outgroup. In bold letters are the genes analyzed in this study.

Figure 2

Expression pattern of class I TCP genes. (A) RT-PCR analysis of the tissue expression of class I TCP genes. ACTIN8 was used as control gene. (B–I) Analysis of the expression pattern using the reporter marker uidA fused to the promoters of the genes in 12 days-old T3 transgenic plants. (B) ProAtTCP7:GUS; (C) ProAtTCP8:GUS; (D) ProAtTCP22:GUS; (E) ProAtTCP23:GUS. Inset, shoot apex, dashed lines indicate leaf primordia or leaf petiole margins. Arrow indicates the localization of the GUS signal. (F–I) Cross-sections of 12 days-old T3 lines. (F) ProAtTCP7:GUS; (G) ProAtTCP8:GUS; (H) ProAtTCP22:GUS; (I) ProAtTCP23:GUS. Dashed lines indicate leaf primordia. Bars = 100 μm.

Figure 3

Subcellular localization of class I TCP factors in root hair cells and characterization of class I TCP alleles. (A–C) AtTCP7, (D–F) AtTCP15, (G–I) AtTCP22. (A,D,G) The GFP signal is located at the nuclei (B,E,H) Bright-field images. (C,F,I) Merged images. Arrows indicate nuclear location. (J) Scheme of the genes and position of the insertions. Gray boxes indicate the coding sequence of the genes. Triangles indicate the insertion lines. Double triangles indicate a tandem insertion. The fragment used for RNAi lines for tcp7-RNAi is indicated. Numbers indicate the position of the insertions or the length of the coding sequences respect to the start codon. (K) RT-PCR analysis of the alleles. ACTIN8 was used as internal control. Number of cycles is indicated.

Figure 4

Phenotypic characterization of class I TCP gene mutants. (A) Rosettes at bolting stage of Col wt, the single mutants tcp8-1, tcp15-1, tcp22-1, tcp23-1, and tcp21-1, the quadruple mutant tcp8-1 tcp15-1 tcp22-1 tcp23-1 (tcp8, 15, 22, 23) and the pentuple mutant tcp8-1 tcp15-1 tcp22-1 tcp23-1 tcp21-1 (tcp8, 15, 21, 22, 23). Bars are 1 cm. (B) Leaf series of plants at bolting stage of Col wt and the mutants tcp8-1, tcp15-1, tcp22-1, tcp23-1, tcp21-1, tcp8, 15, 22, 23, and tcp8, 15, 22, 23, 21. (C–H) Characterization of leaf traits in Col wt and class I TCP gene mutants. Number of rosette leaves (C), petiole length (D), blade length (E), blade width (F), blade perimeter (G), and blade area (H). (I) Number of rosette leaves in AS1 allele as1-1 and in the triple mutant as1-1 tcp8-1 tcp22-1. n = 13 to 28 in (C–H); n = 30 to 33 in (I). Asterisks (*) indicate statistically significant differences determined by Student's t-test, p < 0.05.

Figure 5

Class I TCP factors regulate KNOX1 genes and are capable of protein–protein interaction. (A) Gene expression analysis by qPCR in Col wt and the pentuple mutant tcp8, 15, 21, 22, 23. Error bars are SD of three biological replicates. (B) EMSA of the K-box region of the STM promoter. Tested were factors AtTCP7, AtTCP22 and AtTCP23 in combinations indicated. Arrow indicates shifted bands. (C) Yeast-two hybrid assay to test for TCP protein–protein interactions. Left panel, co-transformed yeast grown in media without aminoacids Leucine and Tryptophan (-L-T media); right panel, β –Gal filter assay: co-transformed yeast were replica-plated in media without Leucine, Tryptophan, Histidine and Adenine and plus X-gal (-L-T-H-A+X-gal). The factors assayed were class I TCP factors AtTCP7, AtTCP8, AtTCP15, AtTCP21 and AtTCP23 and the class II factors AtTCP2 and AtTCP3. Interactions with empty vectors were used as negative controls.

Figure 6

Characterization of transgenic plants harboring class I TCP genes AtTCP7 and AtTCP23 fused to the repressor domain SRDX. (A–C) Phenotype of 10 days-old seedlings of TCP7-SRDX and TCP23-SRDX transgenic lines. (A) Col wt. (B) TCP7-SRDX. (C) TCP23-SRDX. (D) Plants at bolting stage of Col wt; (E) of TCP7-SRDX and (F) of TCP23-SRDX. (G–L) Leaf epidermis nail polish impressions in Col wt (G,J), TCP7-SRDX (H,K) and TCP23-SRDX (I,L) plants. (G–I) Adaxial side; (J–L) abaxial side. (M) Inflorescence and (N) isolated flower of a Col wt plant. (O) Inflorescence and (P) flower of a TCP7-SRDX transgenic plant. (Q) Inflorescence and (R) flower in a TCP23-SRDX transgenic plant. (S) qPCR expression analysis of some gene markers comparing Col wt plants with TCP7-SRDX plants and (T) with TCP23-SRDX plants. Error bars are SD of three biological replicates. Scale bars are: (D), 1 cm; (E), (F), 0.5 cm; (G–L), 50 μm.