The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development

Abstract

The TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa). Phylogenic analysis of this plant-specific protein family unveils a conserved bHLH-containing DNA-binding motif known as the TCP domain. In accordance with the structure of this shared domain, TCP proteins are grouped into class I (TCP-P) and class II (TCP-C), which are suggested to antagonistically modulate plant growth and development via competitively binding similar cis-regulatory modules called site II elements. Over the last decades, TCPs across the plant kingdom have been demonstrated to control a plethora of plant processes. Notably, TCPs also regulate plant development and defense responses via stimulating the biosynthetic pathways of bioactive metabolites, such as brassinosteroid (BR), jasmonic acid (JA) and flavonoids. Besides, mutagenesis analysis coupled with biochemical experiments identifies several crucial amino acids located within the TCP domain, which confer the redox sensitivity of class I TCPs and determine the distinct DNA-binding properties of TCPs. In this review, developmental functions of TCPs in various biological pathways are briefly described with an emphasis on their involvement in the synthesis of bioactive substances. Furthermore, novel biochemical aspects of TCPs with respect to redox regulation and DNA-binding preferences are elaborated. In addition, the unexpected participation of TCPs in effector-triggered immunity (ETI) and defense against insects indicates that the widely recognized developmental regulators are capable of fine-tuning defense signaling and thereby enable plants to evade deleterious developmental phenotypes. Altogether, these recent impressive breakthroughs remarkably advance our understanding as to how TCPs integrate internal developmental cues with external environmental stimuli to orchestrate plant development.

Introduction

TCP transcription factors constitute a plant-specific family of developmental regulators and share a conserved region that is predicted to form a non-canonical basic helix-loop-helix (bHLH) DNA-binding domain called the TCP domain.^1–3 This protein family is represented by 4 founding members, including TEOSINTE BRANCHED1 (TB1), CYCLOIDEA (CYC), PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2, which are identified on the basis of either their functions in plant development or their DNA binding capacities.^4-8 Whereas TB1 suppresses lateral branching in maize (Zea mays) and CYC controls floral dorsoventral asymmetry in snapdragon (Antirrhinum majus), PCF1 and PCF2 promote cell proliferation and organ growth in rice (Oryza sativa).^4-8 According to the differential features within the plant-specific TCP domain, TCP proteins can be distinguished into 2 divergent types, including the class I (TCP-P) and class II (TCP-C), and the latter is further divided into 2 subgroups, namely ubiquitous CINCINNATA (CIN) and angiosperm-specific CYC/TB1.^2,9 Interestingly, TCP proteins have in common a short α-helical L**LL motif located in the second helix of the DNA-binding domain.¹ In both animals and plants, the hydrophobic face formed by the conserved leucine residues in the L**LL motif has been demonstrated to mediate protein interactions.^10,11 In Arabidopsis (Arabidopsis thaliana), this short leucine-rich motif exists in the C-termini of the land plant-specific CC-type glutaredoxins (ROXYs) and is essential for the molecular interactions between ROXYs and TGA transcription factors.^11,12 Outside of the TCP domain, some of the class II TCPs possess a functionally unknown arginine-rich motif (the R-domain), which is predicted to form a coiled coil and likely mediates protein interactions.^1,13 Additionally, most members of the CYC/TB1 subclass contain a conserved ECE (glutamic acid-cysteine-glutamic acid) motif that is functionally uncharacterized and resides between their TCP and R domains.^14,15 Electrophoretic mobility shift assays (EMSAs) reveals the capacity of the TCP domain to associate specifically with the promoter element of the rice proliferating cell nuclear antigen (PCNA) gene.^8,16 These cis-regulatory modules known as site II elements are indispensable for the transcriptional activation of the PCNA gene in rice meristematic tissues.⁸ As the cis-acting elements recognized by the 2 types of TCPs are not mutually exclusive,¹⁶ it is suggested that both classes of TCPs share common target genes and act antagonistically to regulate plant growth and development via competitively associating with similar site II elements.¹⁷ TCPs are able to form not only homodimers but also heterodimers preferentially between specific members of the same class.^16,18-21 Apart from binding site II elements more efficiently,^16,18 heterodimers exhibit a different sequence preference than do the respective homodimers.^18,19 Therefore, different homodimeric and heterodimeric combinations interact with slightly divergent cis-regulatory elements to recognize target genes with differential affinity, thus contributing to functional diversity and specificity.

TCP proteins have been functionally characterized so far only in angiosperms, exerting a regulatory role in shaping plant morphology. Intriguingly, TCP genes also occur in gymnosperms and non-seed plants, such as pteridophytes, lycophytes, mosses, and algae.⁹ Molecular phylogenetic studies reveal that TCP proteins likely emerged first in the Streptophyta lineage prior to the divergence of the Zygnemophyta between 650 and 800 million years ago, as both classes of TCP genes are detectable in Cosmarium sp. (Zygnemophyta), but not in Klebsormidium flaccidum (Klebsormidiophyta), Chlorokybus atmophyticus (Chlorokybophyta) or Mesostigma viride (Mesostygmatophyta).⁹ In non-seed plants, TCP genes constitute a small family of 5 to 6 members.⁹ During the evolution of land plants, subfunctionalization of duplicated genes, followed by neofunctionalization, expands the TCP gene family to comprise a multitude of members in gymnosperms and angiosperms.^2,9 Duplication and subsequent diversification occurring in the ECE (CYC/TB1) clade of TCP proteins exemplify the evolution of this ancient family.¹⁵ Phylogenetic analysis reveals that 3 major copies of CYC have arisen from duplication events predating the core eudicots.¹⁵ A genome-wide extensive survey identifies a complement of more than 20 TCPs in Arabidopsis, rice, poplar (Populus trichocarpa), grapevine (Vitis vinifera), and tomato (Solanum lycopersicum)^2,22 (Fig. 1). TCP proteins from various species throughout the plant kingdom have been demonstrated to regulate a large variety of biological processes, such as floral asymmetry,^23-34 plant architectures including branching,^35-39 lateral organ development,^40-55 seed germination,^56,57 gametophyte development,^58,59 leaf senescence,^44,60 circadian rhythms,^61,62 and defense responses.^63-65 Noteworthily, several aspects of these plant processes involve a regulatory role for TCPs in the biosynthetic pathways of bioactive compounds.^19,60,66-69 In this review, developmental functions of TCP proteins are synoptically summarized and their participation in the biosynthetic processes impacting both plant development and defense against leafhoppers are emphasized (Table A1). Furthermore, 2 novel biochemical aspects, with respect to the distinct DNA-binding properties of TCP factors conferred by several key amino acid residues within the TCP domain as well as the redox-dependent modulation of DNA-binding activities via a conserved cysteine of class I TCP proteins, are also presented in detail. Besides, the demonstrated involvement of TCP proteins in effector-triggered innate immunity (ETI) and defense against insect vectors indicates that these classic developmental regulators can also fine-tune defense signaling pathways and thereby enable plants to evade deleterious developmental phenotypes. Taken together, recent advances in elucidating the physiological and biochemical functions of TCP factors significantly contribute to our understanding of how TCP proteins coordinate external environmental inputs with internal developmental signals to modulate plant development.

Figure 1.

A phylogenetic tree of the Arabidopsis TCP protein family with different classes (I and II) and subgroups (CYC/TB1 and CIN). Amino acid sequences of TCP proteins were aligned with ClustalW2 using default parameters (http://www.ebi.ac.uk.tools/clustalw2) and displayed as a cladogram (Neighbor Joining with 1000 bootstrap replicates) using Phylodendron (http://iubio.bio.indiana.edu/soft/molbio/java/apps/trees). The four founding members of the TCP protein family, including maize TB1, snapdragon CYC, rice PCF1 and PCF2, are used as internal references.

Developmental Functions of TCP Proteins

Out of the 24 TCPs encoded by the Arabidopsis genome, 13 are classified into class I and the rest fall into class II.^15,16 Functional analysis of the Arabidopsis TCP gene family has allocated distinct functions to all of the class II members but only to some members of the more numerous class I subfamily⁹ (Table A1). Given the similar cis-elements bound by both classes of TCP factors, it has been postulated that TCP proteins participate in a diverse spectrum of both antagonistic and synergistic biological interactions.^17,60,70,71 The antagonistic relationship between the 2 classes of TCP factors is known to balance contrasting activities of class I members, which promote cell proliferation in leaves, and class II CIN-TCPs, which act as negative regulators of leaf growth and positive modulators of senescence.^17,60,70,71

So far, TCP proteins have been demonstrated to be involved in a large variety of developmental processes (Table A1). Several independent studies have shed light on a redundant role in lateral organ organogenesis for 8 CIN-like TCP genes, including miRJAW–targeted TCP2–4, TCP10 and TCP24 as well as miRJAW–resistant TCP5, TCP13 and TCP17. ^41,42,72,76 These CIN-like TCP factors seem to crosstalk with multiple different cellular pathways to control leaf development.^41,42,72,76 The Arabidopsis CYC/TB1 subgroup is less numerous and consists of only 3 members, including TCP1, TCP12 (BRANCHED2) and TCP18 (BRANCHED1) (Fig. 1). TCP18 interacts with the florigen proteins FLOWRING LOCUS T (FT) and TWIN SISTER OF FT (TSF) and modulates their activity in the axillary buds to repress the premature floral transition of axillary meristems.⁷⁷ In addition, TCP18 acts redundantly with TCP12 as integrators of branching signals within axillary buds to control branch outgrowth.^35,78,79 Comparatively, less is currently known with respect to the developmental roles of class I TCPs probably owing to the genetic redundancies among its members.^21,70 A functional genomics approach reveals TCP21/CCA1 HIKING EXPEDITION (CHE) as an integral component of the circadian clock.⁶¹ This TCP protein interacts with TIMING OF CAB EXPRESSION 1 (TOC1) and suppresses the transcription of CIRCADIAN AND CLOCK ASSOCIATED1 (CCA1) via associating with the CCA1 promoter, thus establishing a molecular link between the 2 core elements of the clock oscillator.⁶¹ The circadian clock cross-talks with a diverse range of plant physiological processes, including stress acclimatization, hormone signaling, photomorphogenesis and defense signaling,⁸⁰ suggestive of the functional diversity of TCP proteins. Genetic dissection of the tcp14 single mutant unravels the involvement of TCP14 in the activation of embryonic growth potential during seed germination.⁵⁶ Further characterization of the tcp14 tcp15 double mutant demonstrates a redundant function for TCP14 and TCP15 in the regulation of cell proliferation in young stem internodes, developing leaf blades and floral tissues.⁵¹ Very recently, yeast one-hybrid assays have identified a crucial role for TCP20 in the systemic signaling pathway that directs nitrate foraging by Arabidopsis roots.⁸¹ Strikingly, some effects of TCP proteins on plant growth and development are mediated by their participation in the biosynthesis of bioactive metabolites, including brassinosteroids (BRs), jasmonic acid (JA) and flavonoids.^19,60,66-68 As the natural polyhydroxy steroidal phytohormones, brassinosteroids (BRs) play crucial roles in multiple physiological processes ranging from seed germination to leaf senescence. Perturbations in BR biosynthesis and signaling give rise to characteristic phenotypic alterations, such as reduced plant statures, shortened leaf petioles, and rounded leaves.⁸² A gain-of-function genetic approach identifies TCP1 as a genetic suppressor of the weak BR receptor mutant brassinosteroid insensitive1–5 (bri1–5), as the activation-tagged locus tcp1–1D is able to partially inhibit the defective phenotypes of bri1–5.^66,83 On the contrary, overexpression of the TCP1SRDX chimeric repressor gene in wild type plants leads to dwarfed transgenic plants resembling typical BR-deficient or –insensitive mutants.^66,84 DWARF4 (DWF4) codes for a 22-hydroxylase and is responsible for multiple 22-hydroxylation steps during BR biosynthesis.⁸⁵ Combined with real-time RT-PCR analysis and chromatin immunoprecipitation experiments, quantitative analysis of BR biosynthetic intermediates reveals that TCP1 promotes BR biosynthesis by directly stimulating the expression of DWF4, possibly thereby leading to the longitudinal elongation of petioles, rosette leaves and inflorescent stems.^66,86 Apart from TCP1 playing a role in BR metabolism, both classes of TCP proteins participate in jasmonic acid (JA) biosynthesis and thus affect leaf senescence in an antagonistic fashion.^19,60 It is well known that exogenously applied methyl jasmonate (MeJA) can accelerate leaf aging and that several JA biosynthetic genes, including LIPOXYGENASE2 (LOX2), are transiently inducible during leaf developmental senescence.^87,88 LOX2 encodes a chloroplast-localized lipoxygenase that catalyzes the conversion of α-linolenic acid (18:3) into 13(S)-hydroperoxylinolenic acid, the first dedicated step for the biosynthesis of the oxylipin JA.⁸⁹ miRJAW-controlled CIN-like TCPs directly activate LOX2 transcription to promote JA synthesis, thus redundantly contributing to leaf aging.⁶⁰ Interestingly, LOX2 and class I TCP9 genes are identified as direct targets of the class I TCP20 protein ¹⁹. Further analysis of senescence phenotypes reveals an earlier onset of leaf aging in the tcp9 tcp20 double mutant but not in the tcp9 or tcp20 single mutant ¹⁹, indicating an opposite role played by class I TCPs in the control of leaf senescence via the JA signaling pathway. In addition, combinatorial analysis of transgenic plants expressing either the miRJAW-resistant mTCP3 or the dominant-negative TCP3SRDX unveils a novel regulatory function for TCP3 in flavonoid synthesis.⁶⁸ Seedlings and seeds of mTCP3 plants are found to hyperproduce the 3 end products of the flavonoid pathway, including flavonols, anthocyanins and proanthocyanidins.⁶⁸ Protein interaction experiments demonstrate that TCP3 associates with R2R3-MYBs and strengthens the transcriptional activation capacity of TT8-bound R2R3-MYBs.⁶⁸ R2R3-MYBs control not only the early flavonoid biosynthetic steps but also activate the late flavonoid biosynthetic genes by forming a ternary R2R3-MYB/bHLH/WD40 (MBW) complex. Transcriptome analysis of mTCP3 and TCP3SRDX plants reveals many deregulated genes involved in flavonoid biosynthesis,⁶⁸ further underlining a role for TCP3 in the activation of the flavonoid biosynthetic pathway. Moreover, several auxin-related developmental defects, such as altered leaf phyllotaxy, abnormal vasculature patterning, reduced apical dominance, and impaired root development, are observed in mTCP3 plants.⁶⁸ Genetic experiments unravels that the chalcone synthase mutant tt4–11, which fails to produce any class of flavonoids, abolishes auxin-related defects caused by the expression of mTCP3.⁶⁸ Taken together, these observations suggest that TCP3 interacts with R2R3-MYBs to enhance flavonoid production, which further negatively modulates auxin response. Apart from directly contributing to the biosynthesis of bioactive metabolites, TCP proteins are also known to regulate plant development via participating in plant hormone signaling. TCP14 and TCP15 interact physically and genetically with the O-linked N-acetylglucosamine transferase (OGT) SPINDLY (SPY) to promote cytokinin (CK) responses in leaves and flowers, thereby affecting leaf shape and trichome development.⁹⁰ Molecular characterization of the indole-3-acetic acid (IAA) carboxyl methyltransferase dominant mutant iamt1-D suggests that downregulation of 4 miRJAW–targeted TCP genes partially accounts for its leaf curling phenotype.⁷² Although the exact mechanism by which IAMT1 affects the expression of TCP genes is still obscure, it appears likely that perturbation of IAA homeostasis caused by altered expression of IAMT1 may be involved in this cross-communication. Since IAA3/SHORT HYPOCOTYL2 (SHY2) functions to suppress auxin response and a rice SMALL AUXIN UP RNA (SAUR) gene acts as a negative regulator of auxin synthesis and transport,^91,92 the direct activation of IAA3/SHY2 and a SAUR gene (At1g29460) by CIN-like TCPs indicates that these functionally redundant TCPs control lateral organ morphogenesis via exerting a negative effect on auxin response.^42,75

Despite the above-described roles of class II TCP genes in diverse developmental pathways, experimental evidence with respect to the developmental functions of class I TCP genes is still lacking. Functional redundancies among family members and posttranscriptional downregulation by miRJAW of several TCP genes complicate and hamper further functional elucidation of TCP proteins. Coupled with a combination of multiple tcp mutants, engineering miRJAW-resistant versions of mTCP and dominant-negative forms of TCPSRDX has successfully circumvented these obstacles. Alternatively, identification of target genes and further characterization of their functions will offer further insights into the signaling pathways that TCP proteins participate in.

TCP Proteins Participate in Defense Responses

Plants usually utilize cell surface-located pattern-recognization receptors (PRRs) to perceive conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs), leading to MAMP/PAMP-triggered immunity (MTI), which is sufficient to fend off most microbial pathogens.⁹³ To counteract MTI, evolutionarily diverse microbes independently evolved mechanisms to secrete and deliver effector proteins into the plant extracellular space (apoplastic effectors) or inside the plant cell (cytoplasmic effectors), where pathogen effectors associate with cellular host targets to regulate MTI and/or interfere with metabolism in a fashion conducive to pathogen infection. As a countermeasure, plants recognize specific pathogen effectors via deploying a set of polymorphic intracellular immune receptors, also referred to as disease resistance (R) proteins.^94,95 The majority of plant R proteins share both nucleotide-binding (NB) and leucine-rich repeat (LRR) domains with either coiled coil or Toll/interleukin 1 receptor (TIR) domains at their N-terminus. Perception of pathogen effectors or their activities by R proteins results in effector-triggered immunity (ETI) that often includes hypersensitive responses (HR) and systemic acquired resistance (SAR).

Transcriptional reprogramming is a crucial step of plant defense in response to pathogen recognition.^96,97 Transcription regulators, including transcription factors and their regulatory proteins, have been proven to fine-tune the plant defense transcriptome.^96,97 Well-known examples include WRKY and TGA transcription factors, which participate in the regulation of basal resistance and ETI.^98-105 Several recent studies have demonstrated that TCP transcription factors also function as a cellular hub in plant defense signaling.^63-65 Based on the systematic analyses of binary protein-protein interactions between Arabidopsis immune proteins, ca. 8000 full-length Arabidopsis proteins and effector proteins of 2 evolutionarily divergent pathogens, including the Gram-negative bacterium Pseudomonas syringae (Psy) and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa), a plant-pathogen protein-protein interactome network was created, which contain 1358 interactions among 926 proteins, including 83 pathogen effectors, 170 immune proteins and 673 immune interactors.⁶³ Remarkably, 3 of the identified immune interactors, including TCP13, TCP14, and TCP19, are found to be directly targeted by effectors from both pathogens.⁶³ Further functional validation reveals that the tcp13, tcp14 and tcp19 single mutants exhibit enhanced disease susceptibility to 2 different avirulent Hpa isolates (Emwa1 and Emoy2), indicating that each of these 3 TCPs is required for a full immune system function.⁶³ However, the tcp15 mutant displays improved disease resistance to the virulent Hpa isolate Noco2, underpinning a role for TCP15 to act as a disease susceptibility gene that is required for maximal pathogen colonization.⁶³ Therefore, TCP proteins that are targeted by pathogen effectors probably fine-tune cellular signaling networks and thus integrate appropriate immune responses with developmental processes. A second vivid example supporting the direct participation of TCP proteins in the plant immune system arises from the identification of SUPPRESSOR OF rps4-RLD1 (SRFR1) as a specific negative regulator of ETI.⁶⁴ SRFR1 exists as an adaptor protein in cytoplasmic microsomal and nuclear protein complexes containing the defense regulator ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and resistance proteins of the TIR-NB-LRR class.^106,107 The Arabidopsis accession RLD carrying the srfr1–1 allele exhibits enhanced resistance to DC3000 (avrRps4) but not to virulent DC30000, and excluding SRFR1 from the nucleus disables the complementation of the srfr1–1 mutant.¹⁰⁸ This observation suggests that a nuclear activity of SRFR is required for its function in suppressing resistance to DC30000 (avrRps4) in RLD plants.⁶⁴ To identify nuclear proteins that associate specifically with SRFR1, yeast 2-hybrid assays are performed, leading to the isolation of 6 class I members of the TCP transcription factor family. Of these SRFR1-interacting TCP proteins, TCP8, TCP14, and TCP15 interact most strongly with SRFR1, whereas TCP20, TCP22, and TCP23 display weak interactions.⁶⁴ The availability of the single, double and triple mutants of TCP8, TCP14, and TCP15 makes it feasible to explore genetic interactions between SRFR1 and TCP genes.⁶⁴ In planta bacterial growth experiments reveal that the growth of DC30000 (avrRps4) in single and double mutants does not significantly differ from that observed in the wild type Col-0.⁶⁴ By contrast, the tcp8 tcp14 tcp15 triple mutant displays reduced ETI that is mediated not only by the resistance gene RPS4 but also by RPS2, RPS6 or RPM1.⁶⁴ Furthermore, the expression of PATHOGENESIS-RELATED PROTEIN2 is impaired in the quadruple srfr1–4 tcp8–1 tcp14–5 tcp 15–3 mutant in comparison to the single srfr1–4 mutant.⁶⁴ These genetic data collectively indicate that a subset of TCP proteins modulates defense gene expression antagonistic to SRFR1, thus balancing plant development and immunity. Although TCP proteins were found to interact with pathogen effectors,⁶³ whether TCP factors directly or indirectly regulate levels of immunity still remains an open question. It seems likely that TCP proteins determine a general immune threshold by regulating gene expression, which in turn explains why they are targeted by pathogen effectors.⁶³ Apart from playing a pivotal role in plant ETI as described above, TCP proteins also participate in defense against insect vectors in phytoplasm-infected plants.⁶⁵ Phytoplasms belong to insect-transmitted phytopathogenic bacteria, which can secrete pathogen effectors to modulate specific plant targets, thus resulting in changes in plant development and insect performance. Mining genomic sequence data leads to the identification of 56 candidate effector proteins encoded by the genome of the Aster Yellows phytoplasma strain Witches´ Broom (AY-WB).⁶⁵ Of these potential effectors, the secreted AY-WB protein 11 (SAP11) is able to bind and destabilize all of the 8 Arabidopsis CIN-related TCP proteins,⁶⁵ which operate redundantly to promote the expression of LOX2 that is involved in JA synthesis.⁶⁵ Both SAP11-overexpressing lines and AY-WB-infected Arabidopsis plants accumulate less JA on wounding.⁶⁵ Moreover, the AY-WB insect vector produces more progeny on SAP11 transgenic lines, AY-WB-infected plants, octuple cin-tcp plants as well as LOX2-silenced lines.⁶⁵ Altogether, SAP11-mediated destabilization of CIN-TCPs compromises LOX2 expression, reduces JA synthesis and thus heightens insect fecundity, which highlights that pathogen effectors can reach beyond a plant-pathogen interface to modulate a third organism in biological interactions. It is also intriguing that SAP11 only targets class II CIN-TCP proteins for posttranslational degradation. Down-regulation of CIN-TCPs leads to the production of large and crinkly leaves, which mature slowly and contain a large population of small and immature cells.⁷⁶ As phytoplasms belong to biotrophs and are obligate inhabitants of plant hosts and insect vectors, a maturation delay of developing vegetative organs in phytoplasm-infected plants is in favor of phytoplasm survival and offers an increased opportunity to be acquired by insect vectors.

Plants must fine-tune defense responses to avoid deleterious effects on growth and development. The direct participation of TCP proteins in ETI and defense against insect vectors lends strong support to the intimate link between plant development and defense responses. Thus, plants might have evolved an elaborate mechanism to perceive and respond to biotic stresses by adjusting development processes.

DNA-Binding Preferences of TCP Proteins

Across the plant kingdom, members of the TCP transcription factor family share a ca. 60 amino acid residue-long TCP domain¹⁰⁹ (Fig. 2). This plant-specific DNA-binding region is predicted to form a non-canonical bHLH structure and exhibits low sequence homology to the canonical bHLH domain occurring in both plants and animals.^110,111 Sequence alignment of the conserved TCP domain from 57 representative TCP proteins reveals a 3-residue insertion in the basic region of the class II but not the class I TCP domain¹⁰⁹ (Fig. 2). Thus, the TCP domain might have evolved from the canonical bHLH domain with or without a short insertion in the basic region during the evolution of land plants.⁹ Up to date, still less has been known regarding how the TCP domain interacts with its target DNA sequence. Electrophoretic mobility shift assays (EMSA) of the Arabidopsis TCP4 protein unveil that a 58-residue-long TCP domain is essential and sufficient for interacting with DNA and possesses DNA-binding parameters comparable to those of canonical bHLH proteins.¹⁰⁹ Furthermore, the residues that are crucial for DNA binding and homodimerization are identified using site-directed mutagenesis combined with a yeast-based functional screening of random point mutations.¹⁰⁹ These resultant mutant TCP4 proteins with defects in DNA binding or homodimerization lose their capacity to rescue the strong epinastic phenotype of tcp4 cotyledons,¹⁰⁹ indicating that they are nonfunctional in vivo. Structural prediction and molecular homology modeling coupled with functional characterization of mutant proteins lead to the proposition of a possible DNA-binding mechanism for class II TCP proteins.¹⁰⁹ Specifically, the C-terminal HLH fold of the TCP domain mediates the formation of a stable homodimer and only the dimeric form of TCP4 is able to associate with DNA. On the other hand, the basic region of the TCP domain harbors 2 helical stretches separated by a loop consisting of 3 inserted residues, and interacts with the major groove of the DNA double helix. Whether this class II-specific short insertion in the basic region contributes to differential DNA-binding properties and thus functional divergence for both classes of TCP factors still remains unresolved. Biochemical assays of their interactions with DNA and structural insights into the TCP-DNA complexes might be helpful to validate this possibility and shed light on the distinct DNA-binding properties of both classes of TCP proteins.

Figure 2.

Sequence alignment of the TCP domain for 57 representative TCP proteins from different plant species. Amino acid sequences of the TCP domain were aligned with ClustalW2 using the default parameters (http://www.ebi.ac.uk.tools/clustalw2). The class I TCP domain possesses a conserved cysteine residue (Cys20) indicated by a star. The class II TCP domain has an exclusive 3-residue insertion in the basic domain. Modified from¹⁰⁹ (www.plantcell.org; Copyright American Society of Plant Biologists).

Several key amino acid residues in the basic region of the TCP domain have been recently identified as crucial determinants of differential DNA-binding specificities for both classes of TCP factors.^18,112 TCP11, TCP15, TCP16 and TCP20 are all categorized into class I TCP proteins. Whereas TCP15 and TCP20 display similar DNA-binding preferences and interact with non-palindromic sites (GTGGGGNCCNN), TCP11 shows a different DNA-binding feature with a preference for the sequence GTGGGCCNNN.¹⁸ Distinct DNA-binding properties of TCP11 are attributed to a threonine residue at position 15 of the TCP domain, which is occupied by an arginine residue in all the analyzed TCP proteins¹⁸ (Fig. 2). Surprisingly, TCP16 exhibits a DNA-binding preference similar to that of class II TCP proteins such as TCP4.¹¹² Sequence comparison combined with characterization of mutant proteins reveal that the identity of the residue 11 of the class I TCP domain or the equivalent residue 15 of the class II TCP domain, whether it is Gly or Asp, determines a preference for a class I or a class II sequence, respectively¹¹² (Fig. 2). Conceivably, modifications of DNA-binding preferences maybe one mechanism whereby class I TCP proteins achieve their functional specificities.

Transcription factors are able to recognize and associate with specific cognate cis-regulatory modules and fulfill their transcriptional regulatory functions. Although members of the same TF family have a consensus recognition sequence in common, DNA-binding preferences occur for different members of each TF family or even each subfamily. These differential DNA-binding properties might have evolved from evolutionary forces driven by nucleotide mutations in the DNA-binding domain and eventually contribute to the functional specificities and diversities for members of each TF family.

Redox Regulation of TCP Proteins

In response to developmental cues and environmental stimuli, free and accessible protein thiols can be oxidized into multiple states, such as intra-/intermolecular disulfide bridges, S-glutathionylation, S-nitrosylation, sulfenic acid (SOH), sulfinic acid (SO₂H) and sulfonic acid (SO₃H). With the exception of SO₃H, all the other oxidized forms of cysteine residues can be reversibly reduced by glutaredoxins (GRXs), thioredoxins (TRXs) or sulfiredoxins (SRXs).^113-116 A large body of evidence indicates that plant transcription factors involved in stress acclimation and development can operate as direct targets of these posttranslational modifications. ^98,117,118 Several different mechanisms are employed by plants to achieve the redox control of transcription factor activities, which encompass redox-modulated subcellular compartmentation, redox-regulated conformational changes, redox-controlled assembly with coregulators, oxidative disassembly of metal-sulfur clusters, indirect redox effects on posttranslational modifications, redox-triggered proteolytic processing, and direct control of DNA binding.¹¹⁷

As discussed above, several key residues in the the TCP domain can confer differential DNA-binding specificities for both classes of TCP factors.^18,112 More interestingly, sequence alignment of the TCP domain unveils that most members of class I TCP proteins from several plant species share a conserved cysteine residue at position 20 (Cys-20) (Fig. 2), which however lacks in the class II TCP domain.¹⁰⁹ EMSA experiments show that Arabidopsis class I TCP factors with Cys-20 are sensitive to redox conditions, as their DNA-binding activity is inhibited by different types of oxidants examined.¹¹⁹ Nevertheless, this inhibition can be reversed by treatment with reductants or by incubation with a TRX/TRX reductase system,¹¹⁹ suggesting that a reduced state is required for class I TCP proteins to interact with their cognate DNA sequences. Mutagenesis of Cys-20 in class I TCP proteins renders their DNA-binding activity insensitive to changes in redox status.¹¹⁹ Under oxidative conditions, class I TCP proteins harboring Cys-20 are found to exist as covalently linked homodimers,¹¹⁹ hinting at the formation of intermolecular disulfide bridges in vitro. Furthermore, the DNA-binding activity of class I TCP proteins with Cys-20 is also observed to be inhibited in vivo after pretreatment with redox agents.¹¹⁹ Homology modeling using eukaryotic bHLH proteins as a template reveals that Cys-20 resides at the dimer interface near the DNA-binding surface.¹¹⁹ This spatial orientation allows for the formation of intermolecular disulfide bonds and accounts for the sensitivity of DNA binding to the oxidation of Cys-20. Taken together, these observations suggest that the conserved Cys-20 acts as a redox switch, enabling a subset of TCP proteins with such a residue to participate in redox-dependent developmental processes. Given the reversible formation of an intermolecular disulfide bridge via Cys-20, we cannot rule out a possible role for the redox property of Cys-20 to protect these TCP proteins from irreversible oxidation.

The redox regulation of DNA-binding activities via a conserved cysteine makes class I TCP proteins ideal candidates for mediating the effects of changing redox conditions on plant development and stress responses. This layer of regulation of transcription factor activities can be achieved by redox modifications of cysteine residues in the DNA-binding domain, thus influencing the binding of transcription factors to their cognate cis elements. TGA transcription factors can serve as an attractive model to exemplify the redox-modulation of transcription factor activities. Accumulating evidence suggests that the land plant-specific CC-type GRXs (ROXYs) are involved in the posttranslational modifications of both floral and stress-related TGA proteins.^{11,113,114,120-123} However, it still remains unresolved whether TCP transcription factors associate with ROXYs and are thus subjected to the ROXY-mediated redox modulation. Posttranslational regulation of DNA-binding and/or transcriptional activities in a redox-dependent fashion seems to operate as a key mechanism by which plants sense redox status to trigger transcriptional reprogramming during plant normal development and under stress conditions.

Conclusions and Perspectives

TCP transcription factors can integrate hormonal, environmental and developmental signals to modulate numerous biological processes. Notably, TCP proteins have been recruited into 2 seemingly unrelated processes, namely plant development and defense responses. This unexpected finding suggests a crucial role for TCP proteins to balance development and defense, thus enabling plants to achieve maximum fitness under biotic stresses. To accelerate functional clarification and assignment, several approaches have been successfully used to dissect gene redundancies within the members of the TCP gene family. In spite of considerable functional redundancies, specific functions for some members of the TCP protein family have been clarified (Table A1). The molecular basis behind functional specificities may lie in DNA-binding preferences, differential expression patterns and protein interactions. Identification and characterization of target genes, interaction partners and upstream regulatory proteins will favor the elucidation of the complex modulatory network associated with TCP proteins.

Redox regulation of transcription factor activities has recently attracted much attention and its significance in developmental processes and defense responses against biotic and abiotic stresses is becoming a focal issue. Posttranslational modifications of protein activities in a redox-regulated manner seem to constitute a commonly encountered mechanism that governs the association of transcription factors with their respective cognate cis elements. Single residues in the TCP domain not only operate as determinants of DNA-binding specificities for both classes of TCP factors but also confer redox modulation of DNA-binding activity for class I TCP proteins.^18,112,119 The conserved Cys-20 localized in the TCP domain of class I TCP proteins functions as a redox switch to trigger transcriptional reprogramming in response to cellular redox changes, thus adding another layer of complexity to the signaling pathways related to class I TCP factors. Further identification of TCP target genes, which are involved in redox metabolism or whose expression is affected by redox conditions, will favor the dissection of the molecular link between redox regulation, developmental events and defense signaling.

In the near future, the molecular mechanisms by which TCP proteins act to regulate development and stress responses will come to light. ChIP-Seq experiments with different TCP proteins will help define the complex regulatory networks through which TCP factors exert their action. Protein interaction studies can be employed to explore TCP-interacting proteins and further investigate their functions. Increasing knowledge obtained from these studies can be used to not only generate novel morphologies of agronomical interest but also engineer genetically modified plants with elevated resistance against pathogens and insects. Besides, comparative functional studies of TCP proteins across the plant kingdom will offer deep insights into the sub- and neofunctionalization of duplicated TCP genes that might have occurred during the evolution of land plants.

Appendix

Table A1.

Roles of Arabidopsis TCP proteins.

TCP	Description [Reference]
TCP1	Overexpression causes longitudial elongation of petioles, rosette leaves, and inflorescent stems; Participating in brassinosteroid (BR) biosynthesis through directly activating DWARF4 (DWF4) expression.66,86
TCP2/4/10	Triple mutants display epinastic cotyledons and slightly enlarged leaves.60
TCP3/4/5/9	TCP9 is upregulated by light specifically in the shoot apex. TCP3/4 exhibits cotyledon-specific expression, TCP5 displays downregulation of expression by light in shoot apices.124
TCP3/4/10/24	Four TCP genes involved in the regulation of leaf development are down-regulated in the iamt1-D line, resulting in crinkled leaf phenotypes.72
TCP2/3/4/10/24	Overexpressing miRJAW reduces the expression of these 5 TCP genes, leading to larger crinkly leaves, epinastic cotyledons, crinkled fruits, and a modest delay in flowering.41
	Jasmonate biosynthesis and senescence.60
	Interact with the TCP interactor containing EAR motif protein1 (TIE1), a transcriptional repressor as a major modulator of TCP activities during leaf development. TIEl regulates leaf size and morphology by inhibiting the activities of CIN-like TCPs through the recruitment of the TPL/TPR corepressors to form a tertiary complex at early stages of leaf development.73
	miRJAW-targeted TCPs interact with ASYMMETRIC LEAVES2 (AS2) to form a protein complex, which associats with the promoter regions of the class-1 KNOTTED 1-like homeobox (KNOX) genes, including BREVIPEDICELLUS (BP) and KNAT2, to repress their expression required for normal leaf development.74
	TCP3 binds to the promoters of AS1, miR164A, IAA3/SHY2 and Atlg29460 encoding SAUR, activates their expression and negatively regulates the transcription of CUC genes. In addition, TCP3 regulates auxin response, as genes downstream of TCP3 overlaps to a considerable extent a group of genes encoding auxin-inducible proteins, including SMALL AUXIN UP RNA (SAUR) and IAA3/SHY2.75
	In early leaves, miR319-targeted TCP transcription factors interfere with the function of miR164-dependent and miR164-independent CUC proteins, preventing the formation of serrations in A. thaliana. As plants age, accumulation of miR156-regulated SPLs acts as a timing cue that destabilizes TCP-CUC interactions. The destabilization licenses activation of CUC protein complexes and thereby the gradual increase of leaf complexity in the newly formed organs.125
TCP2/3/4/5/10/13/17/24	SRDX fusions reveal a redundant role in the regulation of lateral organ morphogenesis via negative modulation of expression of boundary-specific genes, includimg CUCs.42
	Downregulation of TCP5/13/17 in 35S::miR-3TCP plants results in larger leaves and proximal expansion of the blade into the petiole domain. MiR319-overexpressing lines coupled with downregulated TCP5/13/17 using miR-3TCP, show dark green, deeply lobed, and serrated leaves, exceeding in size both parental lines.76
	SAP11 interacts with each of the 8 CIN-TCPs, causing their destabilization and down-regulation of LOX2 expression and JA synthesis as well as increased fecundity of the insect vector.65
TCP2	Plants transformed with miRJAW-resistant mTCP2 are smaller and greener, have longer hypocotyls, and display reduced apical dominance.39
TCP4	Accelerating multiple aspects of plant maturation.44
	The tcp4 mutant produces more leaves before flowering and displays an maternal effect causing endosperm development arrested.60
	Proper regulation by miR319a of TCP4 is pivotal for petal and stamen development.43
	Expression of mTCP4 causes an arrest at the seedling stage, such as the fusion of cotyledons and the lack of a shoot apical meristem. Surviving transgenics have bushy rosette leaves and abnormal inflorescences.41
TCP4/7/9/14/15/17/21/22/23	Interacting specifically with FT but not TFL1 and functioning as candidate factors mediating differential activity of FT and TERMINAL FLOWER1 (TFL1)126
TCP7/8/22/23	The similar expression pattern of TCP7/8/22/23 in young growing leaves suggests similarity in gene function. The pentuple mutant tcp8 tcp15 tcp21 tcp22 tcp23 shows changes in leaf developmental traits. Analysis of transgenic plants expressing TCP7SRDX and TCP23SRDX indicate a role of these factors in the control of cell proliferation.70
TCP9/20	The LIPOXYGENASE2 (LOX2) and class I TCP9 genes were identified as direct targets of TCP20. The tcp9 and tcp20 single mutants display increased pavement cell sizes during early leaf developmental stages. Analysis of senescence showed an earlier onset of this process only in the double mutant. Both the cell size and senescence phenotypes are opposite to those of the known class II TCP mutants and in miRJAW-overexpressing plants. These results point to an antagonistic function of class I and class II TCP proteins in the control of leaf development via the jasmonate signaling pathway.19
TCP10	Interacting with histidine-containing phosphotransmitters (AHPs), suggesting a possible link between His-to-Asp phosphorelay and plant development.48
TCP11/15/2/3	Interacting with various components of the core circadian clock in both Y2H and direct protein-protein interaction assays. tcp11 and tcp15 plants showed altered transcript profiles for a number of the core clock components, including LATE ELONGATED HYPOCOTYL and PSEUDO RESPONSE REGULATOR5.62
TCP12	TCP12 (BRC2) controls shoot branching. [3579]
TCP13/14/15/19	Experimental validation of host proteins targeted by multiple pathogen effectors reveals enhanced disease susceptibility to 2 different avirluent Hpa isolates Emwa1 and Emoy2 for tcp13/14/19 single mutants and enhanced disease resistance to the virulent Hpa isolate Noco2 for tcp15 mutants.63
TCP13	Binding to the chloroplast psbD light-responsive promoter (LRP) and acting as a trans-acting factor of the psbD LRP.127
	Interacts with AHPs in Y2H, raising the possibility that TCP13 is regulated by CK signal.48
TCP14	Activating embryonic growth potential during seed germination. The tcp14 mutant shows delayed germination, indicating a role in the GA regulation of embryo growth during seed germination. In addition, the tcp14 mutant ishypersensitive to exogenously aplied abscisic acid and paclobutrazol.56
TCP15	Modulating plant development through a pathway overlapping with the one affected by 8 CIN-like TCPs.47
	Interacting with the core circadian clock component PRR5, and the diurnal oscillations in PRR5 transcript abundance are slightly affected in tcp15 mutants.62
	Directly regulating the expression of key cell cycle genes and inhibiting endoreduplication.71
TCP14/15	Influencing plant stature, internode and pedicle length via redundantly promoting cell proliferation in young bolting stem internodes; Repressing cell division in developing leave blades and floral tissues; Affect leaf shape.51
	Interacting physically and genetically with SPY and act together to promote CK responses in leaves and flowers; these CK responses affect leaf shape and trichome development.90
TCP16	The RNAi approach reveals a role in early processes of pollen development, RNAi plants shows the abortion of early pollen development.59
TCP18	TCP18 is also named BRC1. Controlling shoot branching and interacting with FLOWERING LOCUS T to repress the floral transition of the axillary meristems.35,77,79
TCP19/20	TCP19 plays a role in the control of leaf senescence in a redundant fashion with TCP20. tcp19 tcp20 double mutants show a greatly enhanced senescence phenotype.21
TCP20	Involved in the regulation of cell division, cell expansion as well as cell growth and differentiation as revealed by inducing expression of TCP20 fused with the repressor domain SRDX and the transcriptional activator domain VP16.50
	Transcriptome analysis of axillary shoot induction shows that the promoters of upregulated genes are enriched for GGCCCAWW (Up1, which has a putative TCP20 binding function). Up1-mediated transcription of protein synthesis genes and cell cycle genes is important during the initiation of axillary shoot outgrowth.49
	TCP20 binds to the cognate GCCCR motif in the promoters of cyclin CYCB1;1 and ribosomal protein genes (such as S27 and L24 ribosomal subunit genes) that are required for ribosomal biogenesis, translation and protein synthesis. TCP6 and TCP11 also bind this motif present in the CYCB1;1 promoter.17
	This TCP-binding elements are also implicated in the meristem- and anther-specific expression of several nuclear-encoded components of the mitochondrial respiratory chain, indicating a role in mitochondrial development.128
	Site II motifs bound by TCPs, such as TCP20, are observed in the promoters of numerous genes expressed in cycling/dividing cells, including several cell cycle-related genes and 153 Arabidopsis genes encoding ribosomal proteins.129
	Functioning in determining leaf pavement cell sizes and in controlling the onset of senescence.19 Mediating nitrate foraging by Arabidopsis roots through the systemic signaling pathway.81
TCP21	TCP21 is also named CHE (CCA1 HIKING EXPEDITION). Involved in the regulation of the circadian clock activity. TCP21 is a clock component partially redundant with LHY in the repression of CCA1. The tcp21 mutant shows enhanced activity of the circadian clock promoter of CCA1 (CIRCADIAN CLOCK ASSOCIATED1). TCP21 interacts with the core element of the clock oscillator, PSEUDO-RESPONSE REGULATOR1 (PRR1)/TIMING OF CAB EXPRESSION1 (TOC1), and binds specifically to the CCA1 promoter to suppress its transcription.61

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