Growth and Development of higher plants

It has long been established that the rate at which a plant organ like a root grows is a function of only two processes, cell division and cell expansion. It has proven difficult to determine how these processes mediate growth differences in response to environmental conditions and/or genetic predisposition. The first difficulty, to define a mathematical framework to capture the numerical aspects of this analysis were worked out in the middle of the last century. This kinematic analysis was developed on the basis of principles from fluid dynamics. The root tip proved an ideal system for this analysis due to its essentially linear structure, cells in organized in discrete files with dividing cells directly below the root cap and elongating cells in the zone right below it.
The analysis involves the measurement of two parameters in function of position along the root axis. The first parameter is the velocity at which cells are moving away from the quiescent center (QC) at tip of the root (Note that although the tip of a growing root is moving, it is taken as the reference point. Inversely, the mature part of the root from this perspective is seen as moving away from the QC instead of being stationary. This framework allows that a root growing steady-state has a constant growth distribution rather than one that continuously moves in space and therefore much more difficult to interpret). In root tips velocity profile is typically measured by using time-lapse imaging: In images taken at known time intervals, the distance of recognizable structures (cell walls or artificial marks such as graphite or toner particles) from the root tip or QC is measured. The increase in this distance divided by the time interval is a measure for the average velocity that a structure at a particular position is moving. By doing this for a large number of such structures, a more or less continuous velocity function (v(x)) can be determined. It can be easily understood that an increase in velocity in a particular section of the root is due to expansion occurring in that section. Therefore, taking the derivative of this function (dv/dx) yields the local relative rate of cell expansion can be determined for each position along the root. On its own, this expansion or strain rate profile is extremely useful to identify the position where differences in growth are occurring.
To expand these analyses to include cell division, additional measurements of cell length need to be made in function of position along the root. The Arabidopsis root is superbly suited for this because its limited diameter allows DIC imaging of most cell layers on whole mounted roots. The obtained cell length profile typically shows small cells at the tip of the root followed by a region of increasing cell length and a constant cell length in the mature part of the root. To determine cell division parameters, velocity and cell length data need to be combined to calculate the fluxrate (F(x)), the number of cells passing at any position along the root per unit time. This rate can be calculated by dividing velocity by cell length for each position (v(x)/l(x)). Analogous to the increase in velocity it can be seen that an increase in cell flux over a particular interval must be due to cell production taking place in this interval. Consequently, relative cell production rates (P(x)) can be calculated as the derivative of the fluxrate function (dF(x)/dx). To calculate cell division rates (D(x)) cell production rates need to be scaled for differences in cell density, which comes down to multiplication by local cell length: D(x) = P(X)*l(x).
This way both cell division and cell expansion rates are known for each position along the root and a full analysis of the cellular basis of growth rate differences resulting from experimental treatments can be made.
Until recently, the time-lapse and cell length measurements for such analyses were done manually. This was laborious work that could only be executed by skilled microscopists. However, the increasing power of computers and imaging technology has allowed for automation of the velocity and growth rate measurements (Walter et al., 2002; van der Weele et al., 2003). Recent collaborative experiments of P1A and P1C enabled time-lapse imaging using confocal microscopy. This allows in one image sequence to measure the distribution of velocity and cell length and thereby a full analysis of cell expansion and division rates. For this we are now collaborating with EU1 and P4C to develop the software to automate such measurements. Developing such a platform will increase the capacity of the kinematic analysis platform within this project and clearly demonstrates the added value of the combined expertise of the different partners involved.