TCP Transcription Factors in Plant Reproductive Development: Juggling Multiple Roles.

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs) are plant-specific transcriptional regulators exerting multiple functions in plant growth and development. Ever since one of the founding members of the family was described, encoded by the CYCLOIDEA (CYC) gene from Antirrhinum majus and involved in the regulation of floral symmetry, the role of these TFs in reproductive development was established. Subsequent studies indicated that members of the CYC clade of TCP TFs were important for the evolutionary diversification of flower form in a multitude of species. In addition, more detailed studies of the function of TCPs from other clades revealed roles in different processes related to plant reproductive development, such as the regulation of flowering time, the growth of the inflorescence stem, and the correct growth and development of flower organs. In this review, we summarize the different roles of members of the TCP family during plant reproductive development as well as the molecular networks involved in their action.

Physiological Roles and Mechanisms of Action of Class I TCP Transcription Factors.

TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 and 2 (TCP) proteins constitute a plant-specific transcription factors family exerting effects on multiple aspects of plant development, such as germination, embryogenesis, leaf and flower morphogenesis, and pollen development, through the recruitment of other factors and the modulation of different hormonal pathways. They are divided into two main classes, I and II. This review focuses on the function and regulation of class I TCP proteins (TCPs). We describe the role of class I TCPs in cell growth and proliferation and summarize recent progresses in understanding the function of class I TCPs in diverse developmental processes, defense, and abiotic stress responses. In addition, their function in redox signaling and the interplay between class I TCPs and proteins involved in immunity and transcriptional and posttranslational regulation is discussed.

The N-terminal region located upstream of the TCP domain is responsible for the antagonistic action of the Arabidopsis thaliana TCP8 and TCP23 transcription factors on flowering time.

TCP proteins (TCPs) are plant-exclusive transcription factors that exert effects on multiple aspects of plant development, from germination to flower and fruit formation. TCPs are divided into two main classes, I and II. In this study, we found that the Arabidopsis thaliana class I TCP transcription factor TCP8 is a positive regulator of flowering time. TCP8 mutation and constitutive expression delayed and accelerated flowering, respectively. Accordingly, TCP8 mutant plants showed a delay in the maximum expression of FT and reduced SOC1 transcript levels, while plants overexpressing TCP8 presented increased transcript levels of both genes. Notably, the related class I protein TCP23 showed the opposite behavior, since TCP23 mutation and overexpression accelerated and retarded flowering, respectively. To elucidate the molecular basis of these differences, we analyzed TCP8 and TCP23 comparatively. We found that both proteins are able to physically interact and bind class I TCP motifs, but only TCP8 shows transcriptional activation activity when expressed in plants, which is negatively affected by TCP23. From the analysis of plants expressing different chimeras between the TCPs, we found that the N-terminal region located upstream of the TCP domain is responsible for the opposite effect that TCP8 and TCP23 exert over flowering time and regulation of FT and SOC1 expression. These results suggest that structural features outside the TCP domain modulate the specificity of action of class I TCPs.

TCP15 interacts with GOLDEN2-LIKE 1 to control cotyledon opening in Arabidopsis.

After germination, exposure to light promotes the opening and expansion of the cotyledons and the development of the photosynthetic apparatus in a process called de-etiolation. This process is crucial for seedling establishment and photoautotrophic growth. TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) transcription factors are important developmental regulators of plant responses to internal and external signals that are grouped into two main classes. In this study, we identified GOLDEN2-LIKE 1 (GLK1), a key transcriptional regulator of photomorphogenesis, as a protein partner of class I TCPs during light-induced cotyledon opening and expansion in Arabidopsis. The class I TCP TCP15 and GLK1 are mutually required for cotyledon opening and the induction of SAUR and EXPANSIN genes, involved in cell expansion. TCP15 also participates in the expression of photosynthesis-associated genes regulated by GLK1, like LHCB1.4 and LHCB2.2. Furthermore, GLK1 and TCP15 bind to the same promoter regions of different target genes containing either GLK or TCP binding motifs and binding of TCP15 is affected in a GLK1-deficient background, suggesting that a complex between TCP15 and GLK1 participates in the induction of these genes. We postulate that GLK1 helps to recruit TCP15 for the modulation of cell expansion genes in cotyledons and that the functional interaction between these transcription factors may serve to coordinate the expression of cell expansion genes with that of genes involved in the development of the photosynthetic apparatus.

Arabidopsis thaliana TCP15 interacts with the MIXTA-like transcription factor MYB106/NOECK.

MYB106 and MYB16 are MIXTA-like transcription factors that control trichome maturation and cuticle formation in Arabidopsis. In a recent study, we found that the TEOSINTE BRANCHED 1, CYCLOIDEA and PROLIFERATING CELL FACTORS (TCP) transcription factor TCP15 also acts as an important regulator of aerial epidermis specialization in Arabidopsis through the control of trichome development and cuticle formation. TCP15 and MYB106 regulate the expression of common groups of genes, including genes coding for transcription factors and enzymes of the cuticle biosynthesis pathway. In this study, we report that TCP15 physically interacts with MYB106 when both proteins are expressed in yeast cells or Nicotiana bentamiana leaves. Furthermore, we also observed interaction in leaves of Arabidopsis thaliana. Altogether, our findings raise the possibility that TCP15 and MYB106 bind together to the promoters of target genes to exert their action. Our data provide a base to investigate the role of TCP-MIXTA complexes in the context of cuticle development in Arabidopsis thaliana.

Class I TCP proteins TCP14 and TCP15 are required for elongation and gene expression responses to auxin.

KEY MESSAGE: Two class I TCP transcription factors are required for an efficient elongation of hypocotyls in response to auxin and for the correct expression of a subset of auxin-inducible genes In this work, we analyzed the response to auxin of plants with altered function of the class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15. Several SMALL AUXIN UP RNA (SAUR) genes showed decreased expression in mutant plants defective in these TCPs after an increase in ambient temperature to 29 °C, a condition that causes an increase in endogenous auxin levels. Overexpression of SAUR63 caused a more pronounced elongation response in the mutant than in the wild-type at 29 °C, suggesting that the decreased expression of SAUR genes is partly responsible for the defective elongation at warm temperature. Notably, several SAUR genes and the auxin response gene IAA19 also showed reduced expression in the mutant after auxin treatment, while the expression of other SAUR genes and of IAA29 was not affected or was even higher. Expression of the auxin reporter DR5::GUS was also higher in a tcp15 mutant than in a wild-type background after auxin treatment. However, the elongation of hypocotyls in response to auxin was impaired in the mutant. Remarkably, a significant proportion of auxin inducible genes and of targets of the AUXIN RESPONSE FACTOR 6 are regulated by TCP15 and often contain putative TCP recognition motifs in their promoters. Furthermore, we demonstrated that several among them are recognized by TCP15 in vivo. Our results indicate that TCP14 and TCP15 are required for an efficient elongation response to auxin, most likely by regulating a subset of auxin inducible genes related to cell expansion.

Class I TCP transcription factors regulate trichome branching and cuticle development in Arabidopsis.

Trichomes and the cuticle are two specialized structures of the aerial epidermis that are important for plant organ development and interaction with the environment. In this study, we report that Arabidopsis thaliana plants affected in the function of the class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 show overbranched trichomes in leaves and stems and increased cuticle permeability. We found that TCP15 regulates the expression of MYB106, a MIXTA-like transcription factor involved in epidermal cell and cuticle development, and overexpression of MYB106 in a tcp14 tcp15 mutant reduces trichome branch number. TCP14 and TCP15 are also required for the expression of the cuticle biosynthesis genes CYP86A4, GPAT6, and CUS2, and of SHN1 and SHN2, two AP2/EREBP transcription factors required for cutin and wax biosynthesis. SHN1 and CUS2 are also targets of TCP15, indicating that class I TCPs influence cuticle formation acting at different levels, through the regulation of MIXTA-like and SHN transcription factors and of cuticle biosynthesis genes. Our study indicates that class I TCPs are coordinators of the regulatory network involved in trichome and cuticle development.

Class I TCP Transcription Factors Target the Gibberellin Biosynthesis Gene GA20ox1 and the Growth-Promoting Genes HBI1 and PRE6 during Thermomorphogenic Growth in Arabidopsis.

Plants respond to a rise in ambient temperature by increasing the growth of petioles and hypocotyls. In this work, we show that Arabidopsis thaliana class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 are required for optimal petiole and hypocotyl elongation under high ambient temperature. These TCPs influence the levels of the DELLA protein RGA and the expression of growth-related genes, which are induced in response to an increase in temperature. However, the class I TCPs are not required for the induction of the auxin biosynthesis gene YUCCA8 or for auxin-dependent gene expression responses. TCP15 directly targets the gibberellin biosynthesis gene GA20ox1 and the growth regulatory genes HBI1 and PRE6. Several of the genes regulated by TCP15 are also targets of the growth regulator PIF4 and show an enrichment of PIF4- and TCP-binding motifs in their promoters. PIF4 binding to GA20ox1 and HBI1 is enhanced in the presence of the TCPs, indicating that TCP14 and TCP15 directly participate in the induction of genes involved in gibberellin biosynthesis and cell expansion by high temperature functionally interacting with PIF4. In addition, overexpression of HBI1 rescues the growth defects of tcp14 tcp15 double mutants, suggesting that this gene is a major outcome of regulation by both class I TCPs during thermomorphogenesis.

Redox-Dependent Modulation of Anthocyanin Biosynthesis by the TCP Transcription Factor TCP15 during Exposure to High Light Intensity Conditions in Arabidopsis.

TCP proteins integrate a family of transcription factors involved in the regulation of developmental processes and hormone responses. It has been shown that most members of class I, one of the two classes in which the TCP family is divided, contain a conserved Cys that leads to inhibition of DNA binding when oxidized. In this work, we describe that the class-I TCP protein TCP15 inhibits anthocyanin accumulation during exposure of plants to high light intensity by modulating the expression of transcription factors involved in the induction of anthocyanin biosynthesis genes, as suggested by the study of plants that express TCP15 from the 35SCaMV promoter and mutants in TCP15 and the related gene TCP14. In addition, the effect of TCP15 on anthocyanin accumulation is lost after prolonged incubation under high light intensity conditions. We provide evidence that this is due to inactivation of TCP15 by oxidation of Cys-20 of the TCP domain. Thus, redox modulation of TCP15 activity in vivo by high light intensity may serve to adjust anthocyanin accumulation to the duration of exposure to high irradiation conditions.

Redox modulation of plant developmental regulators from the class I TCP transcription factor family.

TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR1 (TCP) transcription factors participate in plant developmental processes associated with cell proliferation and growth. Most members of class I, one of the two classes that compose the family, have a conserved cysteine at position 20 (Cys-20) of the TCP DNA-binding and dimerization domain. We show that Arabidopsis (Arabidopsis thaliana) class I proteins with Cys-20 are sensitive to redox conditions, since their DNA-binding activity is inhibited after incubation with the oxidants diamide, oxidized glutathione, or hydrogen peroxide or with nitric oxide-producing agents. Inhibition can be reversed by treatment with the reductants dithiothreitol or reduced glutathione or by incubation with the thioredoxin/thioredoxin reductase system. Mutation of Cys-20 in the class I protein TCP15 abolished its redox sensitivity. Under oxidizing conditions, covalently linked dimers were formed, suggesting that inactivation is associated with the formation of intermolecular disulfide bonds. Inhibition of class I TCP protein activity was also observed in vivo, in yeast (Saccharomyces cerevisiae) cells expressing TCP proteins and in plants after treatment with redox agents. This inhibition was correlated with modifications in the expression of the downstream CUC1 gene in plants. Modeling studies indicated that Cys-20 is located at the dimer interface near the DNA-binding surface. This places this residue in the correct orientation for intermolecular disulfide bond formation and explains the sensitivity of DNA binding to the oxidation of Cys-20. The redox properties of Cys-20 and the observed effects of cellular redox agents both in vitro and in vivo suggest that class I TCP protein action is under redox control in plants.

TCP transcription factors: architectures of plant form.

After its initial definition in 1999, the TCP family of transcription factors has become the focus of a multiplicity of studies related with plant development at the cellular, organ, and tissue levels. Evidence has accumulated indicating that TCP transcription factors are the main regulators of plant form and architecture and constitute a tool through which evolution shapes plant diversity. The TCP transcription factors act in a multiplicity of pathways related with cell proliferation and hormone responses. In recent years, the molecular pathways of TCP protein action and biochemical studies on their mode of interaction with DNA have begun to shed light on their mechanism of action. However, the available information is fragmented and a unifying view of TCP protein action is lacking, as well as detailed structural studies of the TCP-DNA complex. Also important, the possible role of TCP proteins as integrators of plant developmental responses to the environment has deserved little attention. In this review, we summarize the current knowledge about the structure and functions of TCP transcription factors and analyze future perspectives for the study of the role of these proteins and their use to modify plant development.

The impact of the long-distance transport of a BEL1-like messenger RNA on development.

BEL1- and KNOTTED1-type proteins are transcription factors from the three-amino-loop-extension superclass that interact in a tandem complex to regulate the expression of target genes. In potato (Solanum tuberosum), StBEL5 and its Knox protein partner regulate tuberization by targeting genes that control growth. RNA movement assays demonstrated that StBEL5 transcripts move through the phloem to stolon tips, the site of tuber induction. StBEL5 messenger RNA originates in the leaf, and its movement to stolons is induced by a short-day photoperiod. Here, we report the movement of StBEL5 RNA to roots correlated with increased growth, changes in morphology, and accumulation of GA2-oxidase1, YUCCA1a, and ISOPENTENYL TRANSFERASE transcripts. Transcription of StBEL5 in leaves is induced by light but insensitive to photoperiod, whereas in stolon tips growing in the dark, promoter activity is enhanced by short days. The heterodimer of StBEL5 and POTH1, a KNOTTED1-type transcription factor, binds to a tandem TTGAC-TTGAC motif that is essential for regulating transcription. The discovery of an inverted tandem motif in the StBEL5 promoter with TTGAC motifs on opposite strands may explain the induction of StBEL5 promoter activity in stolon tips under short days. Using transgenic potato lines, deletion of one of the TTGAC motifs from the StBEL5 promoter results in the reduction of GUS activity in new tubers and roots. Gel-shift assays demonstrate BEL5/POTH1 binding specificity to the motifs present in the StBEL5 promoter and a double tandem motif present in the StGA2-oxidase1 promoter. These results suggest that, in addition to tuberization, the movement of StBEL5 messenger RNA regulates other aspects of vegetative development.

Determinants of the DNA binding specificity of class I and class II TCP transcription factors.

TCP proteins constitute a family of plant transcription factors with more than 20 members in angiosperms. They can be divided in two classes based on sequence homology and the presence of an insertion within the basic region of the TCP DNA binding and dimerization domain. Here, we describe binding site selection studies with the class I protein TCP16, showing that its DNA binding preferences are similar to those of class II proteins. Through sequence comparison and the analysis of mutants and chimeras of TCP16, TCP20 (class I), and TCP4 (class II), we established that the identity of residue 11 of the class I TCP domain or the equivalent residue 15 of the class II domain, whether it is Gly or Asp, determines a preference for a class I or a class II sequence, respectively. Footprinting analysis indicated that specific DNA contacts related to these preferences are established with one of the strands of DNA. The dimerization motif also influences the selectivity of the proteins toward class I and class II sequences and determines a requirement of an extended basic region in proteins with Asp-15. We postulate that differences in orientation of base-contacting residues brought about by the presence of either Gly or Asp are responsible for the binding site preferences of TCP proteins. Expression of repressor forms of TCP16 with Asp-11 or Gly-11 differently affects leaf development. TCP16-like proteins with Asp-11 in the TCP domain arose in rosids and may be related to developmental characteristics of this lineage of eudicots.

The class I protein AtTCP15 modulates plant development through a pathway that overlaps with the one affected by CIN-like TCP proteins.

The function of the class I TCP transcription factor TCP15 from Arabidopsis thaliana has been studied through the analysis of plants that express a fusion of this protein to the EAR repressor domain. Constitutive expression of TCP15-EAR produces growth arrest at the seedling stage, before leaf emergence. Expression of the repressor fusion from the AtTCP15 promoter produces small plants with leaves whose margins progressively curve upwards, starting from the basal part of the lamina. Leaves contain smaller and less differentiated cells, both on the adaxial and abaxial sides. The abaxial domain is relatively enlarged, with disorganized cells separated by empty spaces. TCP15-EAR also affects the growth of leaf petioles, flower pedicels, and anther filaments. Flowers show reduced elongation of the three outer whorls and altered gynoecia with irregular carpel surfaces and enlarged repla. Ectopic stigma-like structures develop from medial and basal parts of the replum. TCP15-EAR produces an increase in expression of the boundary-specific genes LOB, CUC1, and CUC2. Changes in CUC1 and CUC2 expression can be explained by the existence of lower levels of miR164 in leaves and the repression of IAA3/SHY2 and the SAUR-like gene At1g29460 in leaves and flowers. TCP15 binds to the promoter regions of IAA3/SHY2 and At1g29460, suggesting that these genes may be direct targets of the transcription factor. The results indicate that TCP15 regulates the expression of boundary-specific genes through a pathway that affects auxin homeostasis and partially overlaps with the one modulated by class II CIN-like TCP proteins.

Footprinting and missing nucleoside analysis of transcription factor-DNA complexes.

In the following chapter we describe methods and protocols to analyze the interaction of proteins with DNA using footprinting and related techniques based on the modification of DNA with either hydroxyl radicals or methylating agents. Footprinting, based on the protection from chemical modification of DNA through the specific binding of a protein, gives information about the nucleotides that are in close contact with the protein upon binding. The derived missing nucleoside and interference techniques identify nucleotides that are energetically important for protein binding. These methods are highly valuable to study in detail the interaction of a transcription factor with nucleotides on both strands of its target DNA sequence.

A mechanistic link between STM and CUC1 during Arabidopsis development.

The KNOXI transcription factor SHOOT MERISTEMLESS (STM) is required to establish and maintain the Arabidopsis (Arabidopsis thaliana) apical meristem, yet little is known about its direct targets. Using different approaches we demonstrate that the induction of STM causes a significant up-regulation of the organ boundary gene CUP SHAPED COTYLEDON1 (CUC1), which is specific and independent of other meristem regulators. We further show that the regulation of CUC1 by STM is direct and identify putative binding sites in its promoter. Continuous expression of STM in Arabidopsis leaf primordia also causes the activation of CUC2-3, as well as microRNA MIR164a, which provides a negative feedback loop by posttranscriptionally regulating CUC1 and CUC2. The results bring new insights into the mechanistic links between KNOXI and CUC transcription factors and contribute to the understanding of the regulatory network controlled by STM.

The Arabidopsis class I TCP transcription factor AtTCP11 is a developmental regulator with distinct DNA-binding properties due to the presence of a threonine residue at position 15 of the TCP domain.

The TCP domain is a DNA-binding domain present in plant transcription factors that modulate different processes. In the present study, we show that Arabidopsis class I TCP proteins are able to interact with a dyad-symmetric sequence composed of two GTGGG half-sites. TCP20 establishes symmetric interactions with the 5' half of each strand, whereas TCP11 interacts mainly with the 3' half. SELEX (systematic evolution of ligands by exponential enrichment) experiments with TCP15 and TCP20 indicated that these proteins have similar, although not identical, DNA-binding preferences and are able to interact with non-palindromic binding sites of the type GTGGGNCCNN. TCP11 shows a different DNA-binding specificity, with a preference for the sequence GTGGGCCNNN. The distinct DNA-binding properties of TCP11 are due to the presence of a threonine residue at position 15 of the TCP domain, a position that is occupied by an arginine residue in most TCP proteins. TCP11 also forms heterodimers with TCP15 that have increased DNA-binding efficiency. The expression in plants of a repressor form of TCP11 demonstrated that this protein is a developmental regulator that influences the growth of leaves, stems and petioles, and pollen development. The results suggest that changes in DNA-binding preferences may be one of the mechanisms through which class I TCP proteins achieve functional specificity.

Binding properties of the complex formed by the Arabidopsis TALE homeodomain proteins STM and BLH3 to DNA containing single and double target sites.

We have analyzed the DNA-binding properties of the complex formed by the Arabidopsis TALE homeodomain (HD) proteins STM and BLH3 in comparison with those of the individual proteins. In vitro DNA-binding assays indicated that complex formation increases binding affinity for sequences carrying either a single target site or two such sites arranged in tandem. Complex formation is not correlated with the establishment of new detectable contacts as deduced from missing-nucleoside experiments. Increased binding was also observed when using BLH3 with a mutation that renders the HD unable to bind DNA, suggesting that only the STM functional HD is necessary for tight binding by the complex. Yeast one-hybrid assays using single or double target sites showed that the effect of complex formation is more dramatic for the double target site and that under these conditions competition for binding by the individual proteins is reduced. The results indicate that even if complex formation produces an increase in binding to DNA sequences containing either one or two target sites, the relative increase in binding produced after complex formation is dependent on the type of target sequence that is considered. This differential effect of complex formation on binding may have implications in the regulatory properties of these transcription factors within the cell.

Characterization of promoter elements required for expression and induction by sucrose of the Arabidopsis COX5b-1 nuclear gene, encoding the zinc-binding subunit of cytochrome c oxidase.

Arabidopsis COX5b-1 encodes an isoform of the zinc binding subunit 5b of mitochondrial cytochrome c oxidase. A promoter region required for expression and induction by sucrose of this gene was analyzed using plants stably transformed with mutagenized promoter fragments fused to the gus reporter gene. Promoter dependent expression is absolutely dependent on a G-box present at -228 from the translation start site. This element interacts in vitro and in vivo with transcription factors from the bZip family, preferentially with the abscisic acid-responsive element binding factor AREB2/ABF4. A region located upstream of the G-box (-333/-259) contains elements with the core sequence ATCATT and distalB-like sequences (CCACTTG) that are required for expression in vegetative tissues. These sequences bind different sets of proteins present in plant nuclear extracts and participate in induction by sucrose (ATCATT) and abscisic acid (distalB) of the COX5b-1 promoter. We propose that the COX5b-1 promoter has acquired novel regulatory mechanisms during evolution after gene duplication. These novel mechanisms have allowed the diversification of expression patterns, but also the conservation of some responses that, as induction by sucrose, are shared by COX5b-1 and other genes encoding components of the mitochondrial respiratory chain. Conservation of these responses may be a pre-requisite for the successful incorporation of new regulatory elements in this class of genes.