Class I TCP transcription factors TCP14 and TCP15 promote axillary branching in Arabidopsis by counteracting the action of Class II TCP BRANCHED1.

Shoot branching is determined by a balance between factors that promote axillary bud dormancy and factors that release buds from the quiescent state. The TCP family of transcription factors is classified into two classes, Class I and Class II, which usually play different roles. While the role of the Class II TCP BRANCHED1 (BRC1) in suppressing axillary bud development in Arabidopsis thaliana has been widely explored, the function of Class I TCPs in this process remains unknown. We analyzed the role of Class I TCP14 and TCP15 in axillary branch development in Arabidopsis through a series of genetic and molecular studies. In contrast to the increased branch number shown by brc1 mutants, tcp14 tcp15 plants exhibit a reduced number of branches compared with wild-type. Our findings provide evidence that TCP14 and TCP15 act by counteracting BRC1 function through two distinct mechanisms. First, they indirectly reduce BRC1 expression levels. Additionally, TCP15 directly interacts with BRC1 decoying it from chromatin and thereby preventing the transcriptional activation of a set of BRC1-dependent genes. We describe a molecular mechanism by which Class I TCPs physically antagonize the action of the Class II TCP BRC1, aligning with their opposite roles in axillary bud development.

Cytochrome c levels affect the TOR pathway to regulate growth and metabolism under energy-deficient conditions.

Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.

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.

Cytochrome c and the transcription factor ABI4 establish a molecular link between mitochondria and ABA-dependent seed germination.

During germination, seed reserves are mobilised to sustain the metabolic and energetic demands of plant growth. Mitochondrial respiration is presumably required to drive germination in several species, but only recently its role in this process has begun to be elucidated. Using Arabidopsis thaliana lines with changes in the levels of the respiratory chain component cytochrome c (CYTc), we investigated the role of this protein in germination and its relationship with hormonal pathways. Cytochrome c deficiency causes delayed seed germination, which correlates with decreased cyanide-sensitive respiration and ATP production at the onset of germination. In addition, CYTc affects the sensitivity of germination to abscisic acid (ABA), which negatively regulates the expression of CYTC-2, one of two CYTc-encoding genes in Arabidopsis. CYTC-2 acts downstream of the transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), which binds to a region of the CYTC-2 promoter required for repression by ABA and regulates its expression. The results show that CYTc is a main player during seed germination through its role in respiratory metabolism and energy production. In addition, the direct regulation of CYTC-2 by ABI4 and its effect on ABA-responsive germination establishes a link between mitochondrial and hormonal functions during this process.

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.

Breaking boundaries: exploring short- and long-distance mitochondrial signalling in plants.

Communication of mitochondria with other cell compartments is essential for the coordination of cellular functions. Mitochondria send retrograde signals through metabolites, redox changes, direct organelle contacts and protein trafficking. Accumulating evidence indicates that, in animal systems, changes in mitochondrial function also trigger responses in other, either neighbouring or distantly located, cells. Although not clearly established, there are indications that this type of communication may also be operative in plants. Grafting experiments suggested that the translocation of entire mitochondria or submitochondrial vesicles between neighbouring cells is possible in plants, as already documented in animals. Changes in mitochondrial function also regulate cell-to-cell communication via plasmodesmata and may be transmitted over long distances through plant hormones acting as mitokines to relay mitochondrial signals to distant tissues. Long-distance movement of transcripts encoding mitochondrial proteins involved in crucial aspects of metabolism and retrograde signalling was also described. Finally, changes in mitochondrial reactive species (ROS) production may affect the 'ROS wave' that triggers systemic acquired acclimation throughout the plant. In this review, we summarise available evidence suggesting that mitochondria establish sophisticated communications not only within the cell but also with neighbouring cells and distant tissues to coordinate plant growth and stress responses in a cell nonautonomous manner.

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.

Cytochrome c Deficiency Differentially Affects the In Vivo Mitochondrial Electron Partitioning and Primary Metabolism Depending on the Photoperiod.

Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

Cross-talk between mitochondrial function, growth, and stress signalling pathways in plants.

Plant mitochondria harbour complex metabolic routes that are interconnected with those of other cell compartments, and changes in mitochondrial function remotely influence processes in different parts of the cell. This implies the existence of signals that convey information about mitochondrial function to the rest of the cell. Increasing evidence indicates that metabolic and redox signals are important for this process, but changes in ion fluxes, protein relocalization, and physical contacts with other organelles are probably also involved. Besides possible direct effects of these signalling molecules on cellular functions, changes in mitochondrial physiology also affect the activity of different signalling pathways that modulate plant growth and stress responses. As a consequence, mitochondria influence the responses to internal and external factors that modify the activity of these pathways and associated biological processes. Acting through the activity of hormonal signalling pathways, mitochondria may also exert remote control over distant organs or plant tissues. In addition, an intimate cross-talk of mitochondria with energy signalling pathways, such as those represented by TARGET OF RAPAMYCIN and SUCROSE NON-FERMENTING1-RELATED PROTEIN KINASE 1, can be envisaged. This review discusses available evidence on the role of mitochondria in shaping plant growth and stress responses through various signalling pathways.

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.

The sunflower TLDc-containing protein HaOXR2 confers tolerance to oxidative stress and waterlogging when expressed in maize plants.

The sunflower (Helianthus annuus L.) genome encodes six proteins containing a TLDc domain, typical of the eukaryotic OXidation Resistance (OXR) protein family. Expression of sunflower HaOXR2 in Arabidopsis generated plants with increased rosette diameter, higher number of leaves and increased seed production. Maize inbred lines expressing HaOXR2 also showed increased total leaf area per plant. In addition, heterologous expression of HaOXR2 induced an increase in the oxidative stress tolerance in Arabidopsis and maize. Maize transgenic plants expressing HaOXR2 experienced less oxidative damage and exhibited increased photosynthetic performance and efficiency than non-transgenic segregant plants after treatment of leaves with the reactive oxygen species generating compound Paraquat. Expression of HaOXR2 in maize also improved tolerance to waterlogging. The number of expanded leaves, aerial biomass, and stem height and cross-section area were less affected by waterlogging in HaOXR2 expressing plants, which also displayed less aerial tissue damage under these conditions. Transgenic plants also showed an increased production of roots, a typical adaptive stress response. The results show the existence of functional conservation of OXR proteins in dicot and monocot plants and indicate that HaOXR2 could be useful to improve plant performance under conditions that increase oxidative stress.

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.

Arabidopsis thaliana SURFEIT1-like genes link mitochondrial function to early plant development and hormonal growth responses.

Mutations in SURFEIT1 (SURF1) genes affect cytochrome c oxidase (COX) levels in different prokaryotic and eukaryotic organisms. In this work, we report that Arabidopsis thaliana has two genes that potentially encode SURF1 proteins, as a result of a duplication that took place in Brassicaceae. Both genes encode mitochondrial proteins and mutation in AtSURF1a causes embryonic lethality. Mutation in AtSURF1b, instead, causes defects in hypocotyl elongation under growth-stimulating conditions, such as low light intensity, increased ambient temperature and incubation with glucose. Mutants in AtSURF1b show reduced expression of the auxin reporter DR5:GUS and increased levels of the gibberellin reporter GFP-RGA, suggesting that auxin and gibberellin homeostasis are affected. In agreement, growth defects caused by AtSURF1b mutation can be overcome by treatment with indole-3-acetic acid and gibberellin A3 , and also by increasing expression of the auxin biosynthesis gene YUC8 or the transcription factor PIF4, which shows lower abundance in AtSURF1b-deficient plants. Mutants in AtSURF1b display lower COX levels, higher alternative oxidase and superoxide levels, and increased expression of genes that respond to mitochondrial dysfunction. Decreased hypocotyl growth and DR5:GUS expression can be reversed by treatment with reduced glutathione, suggesting that redox changes, probably related to mitochondrial dysfunction, are responsible for the effect of AtSURF1b deficiency on hormone responses. The results indicate that changes in AtSURF1b affect mitochondrial function and the production of reactive oxygen species, which, in turn, impinges on a growth regulatory circuit that involves auxin, gibberellins and the transcription factor PIF4.

Class-I TCP Transcription Factors Activate the SAUR63 Gene Subfamily in Gibberellin-Dependent Stamen Filament Elongation.

In autogamous plants like Arabidopsis (Arabidopsis thaliana), stamen filament elongation must be finely regulated to ensure that anthers reach the pistil at the correct developmental stage. In this work, we studied the roles of Arabidopsis TEOSINTE BRANCHED1, CYCLOIDEA, PCF15 (TCP15), and related class-I TCP transcription factors in stamen filament elongation. Plants with decreased expression of class-I TCPs and plants that express a fusion of TCP15 to a repressor domain (pTCP15::TCP15-EAR) had shorter stamens, indicating that class-I TCPs stimulate filament growth. These plants also showed reduced expression of several SMALL AUXIN UP RNA (SAUR)63 subfamily genes, which contain TCP target motifs in their promoters. Mutational analysis indicated that the TCP target motif in the SAUR63 promoter is required for expression of SAUR63 in stamen filaments. Moreover, TCP15 directly binds to the SAUR63 promoter region that contains the TCP target motif in vivo, highlighting the role of the TCPs in this process. Class-I TCPs are also required for the induction of SAUR63 subfamily genes by gibberellins (GAs). In addition, overexpression of SAUR63 restores filament growth in pTCP15::TCP15-EAR plants, whereas overexpression of TCP15 rescues the short stamen phenotype of GA-deficient plants. The results indicate that TCP15 and related class-I TCPs modulate GA-dependent stamen filament elongation by direct activation of SAUR63 subfamily genes through conserved target sites in their promoters. This work provides insight into GA-dependent stamen filament elongation.

Arabidopsis SCO Proteins Oppositely Influence Cytochrome c Oxidase Levels and Gene Expression during Salinity Stress.

SCO (synthesis of cytochrome c oxidase) proteins are involved in the insertion of copper during the assembly of cytochrome c oxidase (COX), the final enzyme of the mitochondrial respiratory chain. Two SCO proteins, namely, homolog of copper chaperone 1 and 2 (HCC1 and HCC2) are present in seed plants, but HCC2 lacks the residues involved in copper binding, leading to uncertainties about its function. In this study, we performed a transcriptomic and phenotypic analysis of Arabidopsis thaliana plants with reduced expression of HCC1 or HCC2. We observed that a deficiency in HCC1 causes a decrease in the expression of several stress-responsive genes, both under basal growth conditions and after applying a short-term high salinity treatment. In addition, HCC1 deficient plants show a faster decrease in chlorophyll content, photosystem II quantum efficiency and COX levels after salinity stress, as well as a faster increase in alternative oxidase capacity. Notably, HCC2 deficiency causes opposite changes in most of these parameters. Bimolecular fluorescence complementation analysis indicated that both proteins are able to interact. We postulate that HCC1 is a limiting factor for COX assembly during high salinity conditions and that HCC2 probably acts as a negative modulator of HCC1 activity through protein-protein interactions. In addition, a direct or indirect role of HCC1 and HCC2 in the gene expression response to stress is proposed.

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.

The mitochondrial oxidation resistance protein AtOXR2 increases plant biomass and tolerance to oxidative stress.

This study demonstrates the existence of the oxidation resistance (OXR) protein family in plants. There are six OXR members in Arabidopsis that contain the highly conserved TLDc domain that is characteristic of this eukaryotic protein family. AtOXR2 is a mitochondrial protein able to alleviate the stress sensitivity of a yeast oxr1 mutant. It was induced by oxidative stress and its overexpression in Arabidopsis (oeOXR2) increased leaf ascorbate, photosynthesis, biomass, and seed production, as well as conferring tolerance to methyl viologen, antimycin A, and high light intensities. The oeOXR2 plants also showed higher ABA content, changes in ABA sensitivity, and modified expression of ABA- and stress-regulated genes. While the oxr2 mutants had a similar shoot phenotype to the wild-type, they exhibited increased sensitivity to stress. We propose that by influencing the levels of reactive oxygen species (ROS), AtOXR2 improves the efficiency of photosynthesis and elicits basal tolerance to environmental challenges that increase oxidative stress, allowing improved plant growth and biomass production.