Last October, the journal Developmental Cell published this article about tissue growth control in Arabidopsis thaliana, examining the effects of mutating a particular gene thought to influence plant growth.
The title of the article buries the lead a little though, as the method they developed to assess the effects of modifying this particular gene could potentially be put to good use in systems engineering studies.
The article starts with the researchers noting the importance of the apical meristems in plants, being the source of stem cells which differentiate to allow plant growth at both the root and shoot ends of the plant. Root apical meristems are well studied and while the effects of different treatments on shoot apical meristems and consequent growth of the stem, leaves and flowers have been studied, how these tissues are initiated within the apical shoot apical meristem is still a bit of mystery.
The cause of the mystery is not a result of under-appreciation of the importance of this part of the plant, but due to the complexity of the structure and growth characteristics plus the difficulty of studying the inaccessible region of the plant in real time.
Within the introduction the researchers underpin the importance of the shoot apical meristem with a description of its structure. It has distinct zones with distinct functions, including a peripheral zone from which come leaves and flowers and a central zone which replenishes the peripheral zone cells. Underlying these zones is the rib zone from which stem growth originates. The rib is named as it is due to it being made up of a cells which divide in a different orientation to the overlying layers.
Fig 1 from article. Figure 1F (bottom left) shows the locations of the peripheral (PZ), central (CZ) and rib zones (RZ) within the stem apical meristem.
As can be made out from the coloured lines indicating the orientation of cell division in figure 1G above, cells within the peripheral and central zones divide horizontally (or perpendicular to the surface of the meristem), while cells in the rib zone divide so that the daughter cells are orientated vertically, causing upward stem growth.
These cell divisions are controlled and vary depending on the stage of plant growth. During the transition to the vegetative stage of growth the outer zones are active while the rib zone is restrained from cell division. During the flowering stage the rib zone is active and stem elongation rates are increased.
Therefore, knowing how the rates of cell division are controlled could lead to advances in crop growth and production, and the method of visualising the effects of manipulating genes could be invaluable to food production.
The 3D imaging method
Imaging the division of the cells within the shoot apical meristem in real time is something that has not previously been achieved but is a technical advance required to understand the underlying basis of plant growth. So, these clever researchers at the Norwich Research Park invented a way to do it.
Based on the fact that the cell wall is extended perpendicular to the mitotic spindle during cell division, the researchers thought it likely that the new wall will be thinner than the existing walls. To utilise this characteristic of cell wall extension and the information it provides about the orientation of cell division, the researchers cross-linked polysaccharides in the cell wall with fluorescing propidium iodide. The thinner, newer cell wall should fluoresce at a lower intensity compared the thicker, existing wall and therefore the orientation of cell division within the meristem can be discovered and imaged.
It was using this method that the great images shown in figure 1 were obtained.
Using the new imaging technique to test the effects of gene modifications
The researchers examined the role of REPLUMLESS (RPL), a gene which transcribes a transcription factor known to regulate stem growth. The method by which RPL controls stem elongation and the transitions between different growth stages is unknown however. Therefore, using the new imaging method, the study looked at the differences in cell division between a wild-type A. thaliana and an rpl mutant.
Imaging showed that the RZ of the wild-type was well-defined when the orientations of the cell divisions were analysed while cell divisions in the rpl mutant were less well organised, indicating that the differentiation between the different boundaries may be controlled by the gene.
Testing these findings, the researchers labeled cells with a Green Fluorescence Protein to track cells as they divided and the directions of tissue growth, finding that the outer regions of the shoot apical meristem grew laterally in relation to the stem. Conversely, the central region of the meristem divided and grew vertically and this was different between the wild-type and rpl mutant.
Delving further in the function of RPL, chromatin immunoprecipitation was used to detect regions of the genome bound by the transcription factor, finding that RPL interacts with a large number of genes that control stem development. Particularly, differential gene expression of a gene LSH4, a gene that controls organ boundary development, was detected.
Therefore, using their new technique, the researchers found the RPL affects the organised division of cells in the shoot apical meristem which in turn affects the control of growth during the various stages of the growth cycle. The result of mutating the RPL gene on plant growth can be seen in figure 6G and 6H from the article.
Figure 6 from article. 6G is the wild-type and 6H is the rpl mutant, demonstrating the effect of the disorientated meristem divisions when RPL was not controlling stem elongation. Growth rates are shown in figure 6K while the orientation of cell divisions are demonstrated in the top left of the figure.
The researchers have developed a method of imaging cell divisions within the stem apical meristem which was used to study the method of growth control exerted by a gene known to play a role in stem morphogenesis.
Critically, the method described can be used by other researchers to research and test genetic modifications that could help increase crop productivity or adapt crops for better or more efficient growth in differing conditions.