Functional and expression studies of plant transcription factors of the TCP family, AtTCP15 and AtTCP16
Creators: name: Coscueta, Ezequiel R.
Publisher: name: Zenodo
Year: 2013
Subjects: Arabidopsis thaliana
EAR domain
Ectopic meristem
Plant development
TCP domain
Transcription factor

description: The shape and architecture of plants are dictated by the various processes that modulate growth and differentiation of organs. The size and shape of these organs, in turn, depends on the number, size and type of cells that constitute them. Therefore, maintaining a delicate regulation of these processes is essential to produce the correct structure of the plants. In general, the main regulators of the processes of cell division, growth and differentiation are transcription factors. Likewise, the action of hormones is usually related to developmental modulation processes, acting both upstream and downstream of transcription factors. TCP proteins (TCPs) are transcription factors involved in various growth-related processes, such as leaf development, lateral branching, floral morphogenesis, pollen development, germination, senescence, circadian rhythm, regulation of the cell cycle and hormonal signalling. Based on the characteristics present both inside and outside the TCP domain, the family is divided into two sub-families or classes: class I (also called PCF or TCP-P) and class II (CYC / TB1 or TCP-C). Little is known about the function of class I proteins, although there is evidence that also links some of them with the regulation of cell proliferation and growth. The general objective of this thesis was to carry out functional studies of plant transcription factors of the TCP family and determine their specific role in the regulation of plant development, using <em>Arabidopsis thaliana</em> as a model. The ultimate goal was to obtain information on the molecular mechanisms involved in the action of this group of proteins and on the interaction networks through which they fulfil their function. Following this objective, on the one hand, the regulation of the gene encoding AtTCP15 was studied through the expression pattern conferred by the promoter region. The strategy used consisted in obtaining Arabidopsis thaliana plants containing fusions of different portions of the AtTCP15 promoter (300,500 and 1100 bp upstream of the translation start site) to the uidA reporter gene (gus), which were analyzed patterns and levels of expression. For this, a series of successive deletions were made to delimit important regions and in them to be able to identify possible regulatory elements. In the promoter regions important for expression, the possible existence of known regulatory elements and that may be related to the observed expression patterns were analyzed. The study of the expression pattern of AtTCP15, made it possible to determine that the cis-regulatory elements necessary to promote the expression of AtTCP15 are found mostly in the 1100 bp region upstream of the translation initiation site. However, the presence of activating regulatory elements above 1100 bp is clear, since the removal of 400 bp from the 5'end of the promoter leads to loss of expression in embryos and anther filaments, and a decrease in expression in flower stalks, gynoecia and siliques. The 300 and 500 bp fragments of the promoter were unable to direct the expression of the gus gene. This could be because the activating elements are upstream of these regions, or because these regions contain cis-elements capable of inhibiting gene expression. On the other hand, throughout the 1500 bp of the complete promoter elements recognized by type B ARR factors, activators of the CK response, were found. The important amount of these elements, as well as their wide distribution along the promoter sequence, propose a relevant function in the regulation of AtTCP15. On the other hand, the morphological and developmental changes observed in the germinative and post germinative stages of Arabidopsis thaliana plants that ectopically express the AtTCP16 protein fused to a dominant repressor domain (called AtTCP16-EAR) were studied, using vertical plate assays. The stage to be evaluated was defined based on previous phenotypic studies carried out with the same plants grown on land. Vertical plate analysis included germination and growth in standard culture medium (agarized MS) and/or supplemented with various sugars and plant hormones. It was possible to conclude that the AtTCP16-EAR plants, which present a slight delay in post-germination development when grown in soil, which is visibly exacerbated when growing the plants in vertical plates. Likewise, the germination tests in the dark show a shortening of the hypocotyl of the plants expressing the fusion, which can be reversed with the addition of exogenous sugars. These results indicate a possible alteration in the mobilization pathway of reserve nutrients, which indicates that AtTCP16 could be participating in the modulation of the pathways that regulate post-germination processes.