Final Report Summary - DAMOKLUS (Dynamics and mechanism of KLU-signalling in the control of plant organ size)
Background
Growth of plant organs is tightly regulated, ensuring that they reach their appropriate species-specific size and shape. However, the molecular and genetic regulation of organ growth is only poorly understood. In previous work the Lenhard group had isolated a gene encoding a cytochrome P450 (CYP) enzyme that promotes growth. This CYP gene is thought to contribute to the synthesis of a signal at the margins of organs, raising the question of how far this signal can act. Previous work had also identified a number of additional genes encoding CYP enzymes that may act in a reaction cascade with the CYP enzyme described above.
Objectives
The three major objectives of this fellowship were:
(1) to determine the range of action of the growth signal,
(2) to test whether the additional enzymes act together with the original CYP to control growth,
(3) to identify additional components involved in generating and perceiving the growth signal.
Results
1. Range of action
To study the range of action of the presumed growth signal, chimaeric plants were generated consisting of wild-type tissue with and mutant tissue without the activity of the CYP enzyme. This was achieved by changing the genotype of the stem cells at the growing tip of the plant, from which all the other cells in the shoot are derived. After eliminating the CYP gene from all or some of these stem cells, shoots are formed that are composed entirely or partly of mutant tissue. If in such chimaeric plants the mutant tissue grows more than it would in a completely mutant plant, this indicates that the activity of the gene in the neighbouring wild-type tissue can promote growth of the mutant tissue.
Using this strategy, it was shown that the range of the signal extends beyond individual organs and even individual flowers. This long range of the signal suggests that in addition to promoting growth, it also coordinates the growth of the different organs within one flower and of the different flowers within one inflorescence (flower head) to ensure they all reach their appropriate relative size. As a result, the flowers and inflorescences would be more symmetrical, which is known to be important for pollinator attraction.
2. The role of additional CYP genes
The function of the additional identified CYP genes was analysed by reducing their activity in plants. In several cases this led to plants with smaller leaves, thinner stems and smaller flowers, resembling plants lacking the original CYP gene. Thus, it is very likely that these CYP genes act together to ultimately generate the mobile growth signal.
3. Identification of additional genes involved in generating or perceiving the signal
Plants lacking the function of the CYP gene, but with the possibility of switching it back on, and also having a reporter gene that is subsequently activated in response to the CYP gene's activity were mutagenised. This identified one mutant line that is no longer able to activate the reporter and forms even smaller organs than plants lacking the CYP gene. This line is currently being analysed further.
Conclusions
This fellowship has demonstrated that the growth signal is active throughout an entire inflorescence, suggesting that it acts to coordinate growth of the individual organs to ensure the symmetry of flowers. By confirming the role of additional CYP genes in growth control and identifying a mutant that appears to disrupt signalling output from the CYP gene, it has also laid an important foundation for dissecting this signalling pathway and its role in growth control further.
Impact
The described system for generating chimaeric plants is likely to be of general use for the plant research community. Also, the knowledge gained about the range of the signal improves our understanding about the coordination of growth in plants. Ultimately, this is expected to lead to more rational approaches for manipulating organ growth in economically relevant crops.
Growth of plant organs is tightly regulated, ensuring that they reach their appropriate species-specific size and shape. However, the molecular and genetic regulation of organ growth is only poorly understood. In previous work the Lenhard group had isolated a gene encoding a cytochrome P450 (CYP) enzyme that promotes growth. This CYP gene is thought to contribute to the synthesis of a signal at the margins of organs, raising the question of how far this signal can act. Previous work had also identified a number of additional genes encoding CYP enzymes that may act in a reaction cascade with the CYP enzyme described above.
Objectives
The three major objectives of this fellowship were:
(1) to determine the range of action of the growth signal,
(2) to test whether the additional enzymes act together with the original CYP to control growth,
(3) to identify additional components involved in generating and perceiving the growth signal.
Results
1. Range of action
To study the range of action of the presumed growth signal, chimaeric plants were generated consisting of wild-type tissue with and mutant tissue without the activity of the CYP enzyme. This was achieved by changing the genotype of the stem cells at the growing tip of the plant, from which all the other cells in the shoot are derived. After eliminating the CYP gene from all or some of these stem cells, shoots are formed that are composed entirely or partly of mutant tissue. If in such chimaeric plants the mutant tissue grows more than it would in a completely mutant plant, this indicates that the activity of the gene in the neighbouring wild-type tissue can promote growth of the mutant tissue.
Using this strategy, it was shown that the range of the signal extends beyond individual organs and even individual flowers. This long range of the signal suggests that in addition to promoting growth, it also coordinates the growth of the different organs within one flower and of the different flowers within one inflorescence (flower head) to ensure they all reach their appropriate relative size. As a result, the flowers and inflorescences would be more symmetrical, which is known to be important for pollinator attraction.
2. The role of additional CYP genes
The function of the additional identified CYP genes was analysed by reducing their activity in plants. In several cases this led to plants with smaller leaves, thinner stems and smaller flowers, resembling plants lacking the original CYP gene. Thus, it is very likely that these CYP genes act together to ultimately generate the mobile growth signal.
3. Identification of additional genes involved in generating or perceiving the signal
Plants lacking the function of the CYP gene, but with the possibility of switching it back on, and also having a reporter gene that is subsequently activated in response to the CYP gene's activity were mutagenised. This identified one mutant line that is no longer able to activate the reporter and forms even smaller organs than plants lacking the CYP gene. This line is currently being analysed further.
Conclusions
This fellowship has demonstrated that the growth signal is active throughout an entire inflorescence, suggesting that it acts to coordinate growth of the individual organs to ensure the symmetry of flowers. By confirming the role of additional CYP genes in growth control and identifying a mutant that appears to disrupt signalling output from the CYP gene, it has also laid an important foundation for dissecting this signalling pathway and its role in growth control further.
Impact
The described system for generating chimaeric plants is likely to be of general use for the plant research community. Also, the knowledge gained about the range of the signal improves our understanding about the coordination of growth in plants. Ultimately, this is expected to lead to more rational approaches for manipulating organ growth in economically relevant crops.