TCP14 AND TCP15 Affect Internode Length and Leaf Shape in Arabidopsis

             The Plant Journal (2011) 68, 147-158

         Author: Martin Kieffer, Vera Master, Richard Waites, and Brendan Davies

The TCP family of plant-specific, non-canonical bHLH transcription factors has been specifically linked to regulation of cell proliferation during development. Understanding of this family is essential for establishment of a genetic model to describe the interaction between cell proliferation and spatial development. Representatives of the two distinct TCP sub-families found in Arabidopsis and known as class I (TCPp) and class II (TCPc).
These two sub families are also found in algae Cosmarium spp. There has been many research about the TCP family for both class and how they work together in plant. According to those researches, TCP factors have been suggested to act antagonistically on plant cell division and growth, with class I promoting growth, and class II factors inhibiting it. This journal describe the developmental roles of two closely related Arabidopsis class I TCP factors, TCP14 (At3g47620) and TCP15 (At1g69690). The research shows that these factors act redundantly to regulate plant stature by promoting cell proliferation in young internodes.

The plant material is consist of  tcp 15-3, tcp15-1, tcp 14-6, tcp 14-4, tcp 14-5, and tcp 15-2 isolated by screening T-DNA alpha population of Arabidopsis. Before double mutant construction, tcp 14-4 was backcrossed four times. All mutants were genotyped by PCR using specific primer for each sample. Plant were grown at 20^oC under long-day conditions with 60% humidity. There’s a stage for vector construction where the mutant carrying the gene reporter (using GUS assay). The RNA is analyzed by Real-time PCR machine using specific primers. Leaves were collected from WT and mutants and scanned at resolution of 300 dpi then processed using LEAFANALYSER software.  Scanning electron microscopy samples were also analyzed in this research.

The results show that TCP14 and TCP15 redundantly regulate internode elongation. Three independent TCP14 T-DNA insertion lines were studied in detail and the Northern blot analysis showed that the T-DNA insertions in the three mutant lines caused a reduction in transcript level and resulted in production of truncated transcripts in all cases. While for the TCP15, northern blot analysis shows that it was disrupted. Biometric analysis of inflorescence stems revealed that internode elongation was significantly reduced in the double mutants. The scanning micrographs of shoot apices also showed that internode elongation is reduced in the double mutant from early stages. The expression of five effectors of cell division analyzed by quantitative real-time PCR also showed that the five gene expressing in cell cycle was reduced in mutant apices relative to the wild type. These mutants also showed  leaf curling in mature leaves of plants grown under short-day condition. The double mutant leaves were upwardly curled at their margins but only found in mature leaves. These suggested that TCP14 and TCP15 also involved in regulating cell proliferation within the leaf.

The results also showed that TCP14 and TCP15 are dynamically expressed in young proliferating tissues. As expected from the mutant analysis, both genes are expressed in all internodes of the young inflorescence stems and in young flower pedicles, and their expression gradually ceases as the tissues mature. There is a spatial regulation for these two genes along the leaves maturation. These genes are also expressed in flower, particularly in sepals. In older flowers, they are expressed in young stamens and carpel valves. As the gynecium matures, expression in the valves is reduced.

The quantitative imaging shows that TCP14 and TCP15 modulate leaf blade expansion. According to the result of LEAFANALYSER software, it consistent with their dynamic expression patterns during leaf development, TCP14 and TCP15 modulate overall leaf shape. These findings suggest that organ shape is defined by rigorous control of cell proliferation in specific cells and tissues, mediated by expression of a combination of TCP factors. It can be also concluded that TCP14/15 genes repress cell proliferation in leaf and floral tissues. It can be observed from the double mutant phenotype where the plants showing reduced cell proliferation in internodes and enhanced cell proliferation in leaves and floral tissues. So that the influence of TCP factors on cell proliferation is context dependent.