Sculpting a plant: TCP transcription factors as key regulators of plant growth and development

Sam W. Es

Research output: Thesisinternal PhD, WU

Abstract

Several members of the Arabidopsis thaliana TCP transcription factor (TF) family affect plant growth and development at different moments during the plant life cycle. In this study, we investigated possible associations between single or multiple TCP functions and phenotypic characteristics, such as vegetative growth parameters and several yield aspects, including total seed yield, seed number and seed weight. We showed that mutations in particular members of the TCP  TF family resulted in an altered branching phenotype and that this coincides with a reduction in seed yield under the applied environmental conditions. Previously, it has been proposed that class I and class II TCP TFs fulfil opposite functions in plant growth and development, but here we reveal that this hypothesis needs revision, as mutation in members from both classes showed similar developmental effects. Additionally, the supposed functional redundancy within particular sub-clades of the TCP TF family was analysed, revealing that stacking of mutations in several TCPs led in some cases to less severe phenotypes in comparison with single mutant phenotypes instead of the expected increased phenotypic effects. Furthermore, for some genes supposed to act redundantly, such as BRANCHED1 (BRC1) and BRC2, contrasting phenotypes were found for particular traits in the single mutants. Altogether our analyses showed the importance of comprehensive and comparative phenotyping of mutants and of detailed quantitative analyses in order to get a full understanding of the contribution of individual members of the TCP TF family to particular biological functions. 

Flowers of most dicotyledons have petals that, together with the sepals, initially protect the reproductive organs. Later during development petals are required to open the flower and to attract pollinators. These diverse set of functions demand a tight temporal and spatial regulation of petal development. We studied the functioning of the Arabidopsis thaliana TCP5-like transcription factors (TFs) in petals. Overexpression of TCP5 in petal epidermal cells results in smaller petals, whereas tcp5 tcp13 tcp17 triple knockout lines have wider petals with an increased surface area. Comprehensive expression studies revealed effects of TCP5-like TFs on the expression of genes related to cell cycle, growth regulation, and organ growth. Additionally, the ethylene biosynthesis genes 1-amino-cyclopropane-1-carboxylate (ACC) synthase 2 (ACS2) and ACC oxidase 2 (ACO2) and several ETHYLENE RESPONSE FACTORS (ERFs) are found to be differentially expressed in TCP5 mutant and overexpression lines. Chromatin immunoprecipitation–quantitative PCR showed direct binding of TCP5 to the ACS2 locus in vivo. Ethylene is known to influence cell elongation and the petal phenotype of the tcp5 tcp13 tcp17 mutant could be complemented by treatment of the plants with an ethylene pathway inhibitor. Altogether, this reveals a novel role for TCP5-like TFs in the regulation of ethylene-mediated petal development and growth.

The Arabidopsis transcription factor BRC1 plays an important role in the inhibition of axillary bud outgrowth and as such determines plant architecture. We used chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) to determine direct genome-wide targets of BRC1 and to elucidate the molecular mode of action of BRC1 during axillary bud dormancy. The search for a putative consensus binding site that is centrally enriched in the BRC1 ChIP-seq peaks, resulted in the identification of the cis-element ‘GDCCCA’, which is close to previously observed consensus TCP binding sites. Additionally, we identified enrichment of the ‘G-box’ either up- or downstream of the centrally enriched consensus TCP binding site. Subsequently, we linked the identified BRC1 binding peaks to the nearest gene, revealing a potential role for BRC1 in ABA biosynthesis. Several ABA biosynthesis genes, including NCED3, NCED9 and ABA2, were bound by BRC1 and showing differential expression upon induction of BRC1. Furthermore, we provide evidence for a regulatory role of BRC1 in repressing cytokinin (CK) levels in dormant buds by activating two CK oxidases and repressing several CK signalling genes. These direct effects on ABA and CK pathways, together with the binding of several other gene loci, including genes involved in cell wall composition and genes with a potential role in symplastic intercellular connectivity, provide a molecular and potential mechanistic basis for the functioning of BRC1 in the repression of axillary bud outgrowth.

Floral organ identity specification and growth are two very distinct but intertwined processes. In this study we describe the physical interaction between the floral organ identity specifying MADS domain protein APETALA1 (AP1) and a member of the TCP transcription factor (TF) family, TCP5, previously described to be involved in petal growth. This interaction and other potentially interesting protein-interactions were identified by immunoprecipitation-based native protein complex isolation, using TCP5 as bait and followed by liquid chromatography-tandem MS (LC-MS/MS). Yeast two hybrid assays (Y2H) confirmed the identified AP1-TCP5 interaction. To determine the regions within the respective proteins and the exact amino acids vital for such an interaction, we created libraries of randomly mutagenized alleles for both AP1 and TCP5, by error prone PCR. Subsequently, the Y2H assay was performed to find ‘edgetic’ alleles in which only the interaction of AP1-TCP5 was disturbed, while the well-known heterodimeric interactions of AP1 with other MADS domain proteins were maintained. We show by screening six mutated AP1 alleles the potential of this approach. Finally, we describe the possibilities of this technique and we provide suggestions for several strategies to scale-up this assay and to continue it by further functional characterisation in planta.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • Angenent, Gerco, Promotor
  • Immink, Richard, Promotor
Award date18 Sep 2018
Place of PublicationWageningen
Publisher
Print ISBNs9789463434898
DOIs
Publication statusPublished - 2018

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