ABSTRACT - We all know there are a number of different types of roots and they undergo periods of episodic growth, but do we know what we are looking at when we look at a root? I will use white spruce as a model to outline differences in root physiology related to morphology and to season. Respiration is the best measure of metabolic activity. In other systems, such as seeds, high respiration rates are indicative of rapid growth and breakdown of storage reserves. Thus we would expect the same relationship to hold true in conifers. But, what is growth? Growth can be measured as cell divisions or as increased cell size, with both contributing to visible growth. Cell expansion seems to require the breakdown of storage products both for energy and as an osmoticum. So we see a loss of starch as growth is about to begin and this is associated with increased respiration. Within an individual root there are regions that are important for root growth, for uptake of nutrients, for initiation of lateral roots, for transport of nutrients and for storage. Each of these regions show differences in metabolic activity, permeability, surface area and morphology.

Introduction:

Many of you are very familiar with root morphology. There is the tap root with many laterals branching off. They are primary, secondary, tertiary and quaternary. Some are very short, some are long. Any roots that reach the bottom of the plug are air pruned. This means that instead of having a regular root tip as is shown in this slide, the tip has been killed, and lateral roots arise very close to the dead tip.

The root tip is the region of the root giving rise to most of the root tissue. The important cell layers are the meristems, where cell division occurs, giving rise to the root cap and the rest of the root. Proximal (or up the root) from this, is the region where cell expansion takes place and cell differentiation. We can see the cortex and the vascular tissue (which is responsible for transport of nutrients up and down the roots). The outer layer of the cortex may have root hairs. These are very important for absorption, as they increase the surface area of the root greatly. Not all roots have these, and some roots have a tremendous number of them. In this region, there is a specialized cell layer called the endodermis that all the water and nutrients must pass through. Further up the root (more proximal), this endodermis will become impermeable, and then it is referred to as the secondary state of the endodermis. The cortex will die and a necrotic layer forms (necrotic simply means dead).

The cell layer that lies just inside of the endodermis is called the pericycle. The pericycle is an important cell layer in the root for two reasons: It is very important for storage of starch during periods of inactivity; and it is the tissue that gives rise to all the lateral roots. This is an important point, because in the shoot, we know that lateral branches are laid out in the apical meristem. In the root, this does not happen. This explains why, when a root is air pruned, new laterals are produced. If the root was like the shoot, once the meristem was killed, laterals could not be formed.

There are various stages of root growth. The elongating root is actively growing. Absorbing roots are also actively growing but are characterized by determinate growth, no secondary thickenings and a length of a few centimeters. They are responsible for most nutrient uptake. As such, the seedling relies upon a large population of absorbing roots for proper growth. Absorbing roots often die after one growth cycle.

When the root stops growing, the secondary state of the endodermis moves down the root. At the same time, the cells around the root tip form a specialized layer called a metacutization layer. These two layers eventually meet and form what has been called a dormancy layer. This root is therefore a brown root, with a complete dormancy layer. When growth begins again, the dormancy layer is broken and growth begins.

Electron micrographs show the dormancy layer very well. It is obvious that this layer prevents the movement of materials in and out of the root.

Plant respiration takes the sugars and starch that are created from photosynthesis and changes them into a useable form of energy. In doing so plants use oxygen. Because respiration is providing a useable energy source, it is very active during periods of active growth. As such, it is a good measure of metabolic activity.

The first figure shows respiration rates in spruce roots over the year (figure 1p). These data were collected from brown roots of seedlings that were grown on the coast and spent the winter in a lathehouse. Respiration rates increased in the spring as root growth increased, and then continued to increase after root growth had stopped. This indicates that the roots were very busy doing something even though they were brown. If we look at the other two lines, we can get some insight into what is going on. The lower line is a type of respiration that is associated with high utilization of starch and sugars. You can see that it is high during root growth, then decreases and increases again during shoot growth. I suspect that it would continue to be high during the period of active shoot elongation. The decrease is associated with a period of storage.

If we look at the different types of roots, we can see that they all are active (figure 2p). The most active are the elongating roots. These are definitely growing more quickly than the absorbing roots. Again, looking at the respiration that is associated with utilization of sugars, we can see that the elongating roots seem to be using up alot of reserves, relative to the other roots types. This means that they are relying alot on reserves and not on the carbohydrates produced from photosynthesis at the time.

We can also measure starch and bound carbohydrate changes directly and see if our predictions are correct (figure 3p). The total carbohydrate decreases as the root starts to grow, with the greatest change in starch content. You may also want to note that the swollen root tip has far more starch than does the brown root.

Now what is going on in the different root segments of the actively growing roots (figure 4p)? They are all quite active, with the apical segment (the regions containing the apical meristem) and the zones of elongation and differentiation being higher than the suberizing segment (this is the region that has the necrotic tissue and is above the region where the absorption takes place). The contribution of respiration that is associated with mobilization of reserves decreases as you move up the root.

So from the respiration work we can see that all the root types are actively respiring, the respiration rate in brown roots increases during periods of active root and shoot growth, the high respiratory activity at these times is caused by a type of respiration that is involved in mobilization of stored carbohydrates, and respiration at other times (i.e. between the period of root and shoot growth) is associated with renewed storage of reserves.

There are a few questions that arise from this: Is it the root morphology that is causing respiration rates to be lower in the brown root and in the suberized regions? Does the root have to bulk up on stored reserves before it can grow? If so, are the reserves simply acting as an energy source, or do they play another role?

The answer to the first question is "No"-root morphology is not causing the respiration rates to be lower. Although we saw that the dormancy layer would be a very good barrier to the movement of materials in and out of the root, what we didn’t see is that the actively growing roots also have barriers to movement. Sealing the cut end of the roots decreased respiration rates by 17 percent in white roots and 14 percent in brown roots This indicates that morphology is not causing the brown roots to have lower respiration rates. Of greater significance is the fact that the movement of a dye between the cells is restricted in white (elongating and absorbing) and brown roots. This means that neither root can freely take up nutrients. We looked for a barrier in the white roots and found that they had a polysaccharide layer surrounding the root tip. I will come back to this point later.

The answer to the next question appears to be "Yes", the root tip appears to need a buildup of reserves in the tip before growth can occur. Histochemical analysis of the root tip showed a large increase in starch content in the root tip just before growth began and then the starch was rapidly degraded as the root tip began to swell. This indicates that the starch reserves are needed for renewed root growth. As most of the growth is from cell expansion at this time, it is apparent that the breakdown of starch is leading to an increased osmotic potential in the cells, causing water to flow into the cell and making them swell. This means that the starch reserves are acting both as a energy source and as a biophysical means of causing cell expansion.

The relationship between root growth and starch reserves brings me to an important observation. It has been widely assumed that root growth stops once shoot growth begins because the shoot takes the carbohydrates away from the root. I found that once the root had depleted its supply of stored carbohydrates, growth stopped, regardless of whether the shoot was growing or not. Before the root can grow again, the carbohydrate reserves have to be replenished. At this time, the root relies on the shoot for carbohydrates. So, roots will not grow if the shoot is growing and using up all the carbohydrates, but shoot growth will not stop root growth. Other workers have noted that the absorbing roots meet growth maintenance requirements strictly from reserves and that a root dies when its starch and sugar reserves are depleted.

Until now, I have referred to roots as being either white or brown. This is an oversimplification. Brown roots can be divided into dormant and quiescent roots. So how does one tell if a root is dormant or quiescent? These can be differentiated on the basis of cell division, respiratory activity and RGC. Cell division increase in the spring prior to the increase in visible growth. In other words, a seemingly dormant root is preparing to grow.  From this graph, you can see the increase in mitotic activity, followed by growth (figure 7p). Also, you can note that the metacutization layer may or may not be intact. If we were to superimpose the respiration data on this, we would see that it too began to increase before the main burst in visible growth. Both respiration and mitotic activity are not easily measured in a nursery setting, so from your perspective, root growth capacity tests are the best. If you compare the root growth capacity with the cell division, you see that they correlate well. RGC is very low or nonexistent in dormant roots and is high in quiescent roots. This slide shows the normal cycle of root growth.

The final point that I would like to return to pertains to the white roots. The white roots are classified as elongating and absorbing. The elongating roots function to expand the root system, while the absorbing roots are very important for uptake. Earlier I said that white roots have a polysaccharide layer around the root that prevents movement of materials and water in and out of the root. They also have a suberized layer further up the root that prevents movement in and out. This leaves a very small region for uptake, which is why the roots that are most effective at uptake and transport have a high number of root hairs. These root hairs increase the surface area of the root in a region of the root that does not have a polysaccharide layer, a suberized layer or a dormancy layer. Next time you look at the root system, notice how small a region in the root that is actually responsible for uptake. This is the most metabolically active region of the root. The importance of the root hairs is evident from the fact that survival is highest when seedlings are outplanted during the time frame of rapid production of absorbing roots.

To summarize, as roots stop elongating, they start to brown. This signifies the beginning of the formation of the dormancy layer. Respiration rates are decreasing, carbohydrates are being stored and RGC is low. The root then becomes dormant. At this time, respiration rates are low, as is mitotic activity. RGC is very low. Quiescence follows, characterized by increasing RGC and respiration. Prior to renewed visible growth, RGC is high, mitotic activity has increased and there is a surge in stored carbohydrates. As the root starts to elongate, mitotic activity is high, respiration rates rapidly increase and RGC is high. Active elongation follows and mitotic activity decreases, RGC decreases, respiration rates are high, there is little stored carbohydrate and the dormancy layer is absent.

In conclusion, there is alot that we don't know about root physiology, and of course, many aspects of physiology that I did not touch on. I focused on respiration, stored carbohydrates, mitotic activity and RGC. I hope that I have been able to give you a little insight into what is going on in the root at various times of the year and in various root types. We are continuing our studies on seedling root physiology by studying chemical and morphological parameters of seedling roots in relation to superior tree production, and I hope to be able to share the results from this work in the future.

Figure 7

Figure 3  Changes in starch and bound carbohydrate (milligrams per gram fresh weight) accompanying the transformation from brown to elongating roots in white spruce seedlings
	Developmental Stage
Carbohydrate	Brown	Swollen	Newly Elongating	Elongating
Starch	75.9	104.7	93.2	49.4
Bound	72.3	41.4	34.3	42.6
Total storage carbohydrate	148.2	146.1	127.5	92.0

Figure 4 Respiratory Parameters or Morphological Segments from White Spruce Seedling White Roots
Root Class	Root Morphology nmol/min-g fresh wt	02 Uptake	p	valt	valt X p	vyct	vres
Elongation	Apical segment	238 +/- 17	0.78	42	33	50	17
	Zones of elongation & differentiation	229 +/- 33	0.71	38	27	45	27
	Suberizing segment	158 +/- 10	0.15	22	33	59	38
Absorbing	Apical segment	213 +/- 28	0.73	53	39	50	11
	Zones of elongation & differentiation	239 +/- 26	0.95	36.5	35	51	14
	Suberizing segment	180 +/- 33	1.01	28	28	37	35

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