Shoot Responses to Root Stress - A Resource Gathering Point of View

Plant Material

Young ramets of a Populus trichocarpa x P. deltoides hybrid obtained from a clone bank (6) were propagated from greenwood cuttings and grown in a greenhouse as previously described (22). Plants were used 5 to 8 weeks after propagation, when they were approximately 30 cm in height.

Root Treatments

For the root hypoxia experiments, the plants were grown in aerated or hypoxic solution cultures as previously described (23). For the root restriction experiments, ramets were transferred to small (0.5 L) and large (3.5 L) pots. Plants were carefully watered at all times to prevent water deficits, particularly in the smaller pots. All measurements were taken after two weeks.

Physiological Measurements

Growth was measured with a ruler (tip to petiole) on leaves in the linear phase of the sigmoidal growth curve at the same time each day. Leaf water potential measurements were made with a Scholander pressure chamber (Soil Moisture Corp., Model 3005, Santa Barbara, California, USA). Osmotic potential was estimated using sap expressed from frozen-thawed tissue with a thermocouple psychrometer (Decagon, Model SC-10, Pullman, WA), and turgor pressure was calculated from the difference.

Wall extensibility of growing leaves was measured using a CAWL (capacity for acid-induced wall loosening) assay. Estimates of CAWL of leaf strips were made using a constant stress creep apparatus using a 10 g load (18). Three to four strips (2.2 mm x 5 mm) from growing leaves (2-3 plants) were frozen, thawed and extended while bathed in neutral pH solution (50 mM Na acetate buffer, pH 6.0). After 25 min, the pH was changed to 4.5 and the rate of extension was recorded for the next 6 h.

For hormone measurements, abscisic acid (ABA) and zeatin riboside (ZR) concentrations in extracts of xylem sap were determined by immunoassay after partial purification as previously described (23). Measured hormone values were corrected for losses during handling by counting an internal standard of [3H]-(±)-ABA or [³H]-dihydrozeatin at the beginning and end of the purification procedure.

For carbohydrate extraction and analysis, mature source leaves were dried and ground in a mortar. Starch concentration was determined as described by Robbins and Pharr (19).

In all whole plant experiments, a random design with three to six replicates were used in each treatment. All experiments were repeated at least three times with similar results.

Leaf Disk Growth Assay

The residual effects of root stress on leaf growth were measured using an in vitro growth assay of leaf disks. Five or six leaf disks (7.2 mm diameter) were excised from leaves in the linear stage of growth on plants with non-restricted and restricted roots. Disks were incubated on control media (KCI plus sucrose, 10mM each), with or without added hormones, at a range of concentrations (1 mM to 100 pM) and on two-fold concentrated expressed xylem sap collected from roots of non-restricted or restricted plants as previously described (14). The disks were grown in vitrounder lights (250 μM/m²s PAR) for 24 h. Growth rates were determined by measuring the increase in disk diameter after 24 h.

Effects of Root Hypoxia and Root Restriction on Leaf Growth and Water Relations

Experiments were aimed at describing and understanding the effects of root stresses on leaf expansion. Both root restriction and root hypoxia decreased the rate of leaf growth (Table 1) resulting in smaller leaves on stressed plants than leaves on control plants (Figure 1). To determine whether changes in turgor were associated with the measured changes in leaf growth, bulk leaf water potential was measured by pressure chamber on the same samples that osmotic potential was determined using sap expressed from frozen-thawed leaves. Values obtained on leaves of control and root-stressed plants showed no decrease in bulk leaf water potential or turgor in either root restricted or in plants with hypoxic roots (Table 1).

Effects of Root Stress on Cell Walls

Since cell growth is controlled by turgor and wall characteristics, the reduction in leaf growth could be related to changes in wall rheology. Root restriction and root hypoxia caused a reduction in the capacity for acid-induced wall loosening (Table 1).

In vitro Growth Measurements

To study the residual effects of root restriction on leaves, leaf disks were excised from the growing leaves of plants exposed to root restriction for two weeks. Leaf disks from control plants grew less on twofold concentrated xylem sap from plants with restricted roots than on sap collected from plants with non-restricted roots (Table 2). Disks treated with xylem sap from restricted roots grew less than disks incubated on sap collected from nonrestricted roots, abscisic acid caused a reduction in the in vitro growth of leaf disks from both plants with restricted and non-restricted roots. Zeatin riboside increased the in vitro growth of all leaf disks.

Endogenous Plant Hormone Measurements

The concentration of abscisic acid and zeatin riboside-type cytokinins in xylem sap was reduced in roots of restricted plants (Table 1).

Starch Partitioning

The concentration of starch was increased in source leaves by both root restriction and root hypoxia (Table 1).

Why is Leaf Growth Inhibited in Plants with Stressed Roots?

The development of leaf area is related to the rate of cell expansion, which is determined by cell turgor and characteristics of the cell walls. On one hand, it is possible that leaf growth is inhibited due to altered supply of water. In some cases root stresses are correlated with substantial reduction in water uptake (22). It is easy to imagine some type of hydraulic signaling in which the water supply to leaves is reduced and acts as a source of information on the status of the roots. In the leaves of poplars with hypoxic or restricted roots, however, leaf growth is not limited by reduced turgor (Table 1). In poplar plants with hypoxic roots, the reduction in leaf growth may be partially explained by reduced cell wall extensibility. Wall extensibility can be expressed as the capacity for acid induced wall loosening (CAWL). CAWL is a biologically mediated response, correlated with growth rate (26,15). Reduced cell wall extensibility has also been demonstrated in leaves of plants with dehydrated roots (26). How the root environment alters leaf cell wall extensibility is currently unknown.

How Does the Plant “Know” That Its Roots are Stressed and “Tell” the Shoot to Slow Growth?

Evidence shows that the development of both roots and shoots depends upon their complementary functions such that carbon from leaves is necessary for root growth and chemical signals from roots may regulate leaf functions (3,4).

Inadditionto water, roots supply both nutritional and hormonal factors necessary for leaf function. Studies have shown that changes in the root environment will affect active ion uptake and ion concentration in the xylem (21). However, instead of being a direct signaling mechanism in the growth response of root-stressed plants, it is possiblethatthe nutritional statusof plants modifies plant responses to other factors (17,27). Nutrient delivery itself may be unreliable as an indicator of root zone conditons because delivery occurs through the transpiration stream, which is subject to many aerial factors resulting in fluctuations in the rate of water movement through the plant (7).

Blackman and Davies (1) suggested that roots and shoots are integrated through changes in the flux of hormones in response to soil perturbations. Results from several laboratories suggest that ABA is exported at high levels from stressed roots and has been shown to be important in leaf responses of flooded and drought-stressed plants (10,13,28,29). Others have suggested that suboptimal soil conditions which reduce growth and root meristematic activity also reduce the production of growth enhancing compounds, such ascytokinins, by roots (8). A numberof experments using hybrid poplars have been performed to describe the nature of the root-sourced factors generated by changes in the root environment (14,23,24). These studies found: 1) that the growth of poplar leaf disks was decreased when disks were grown on sap expressed from hypoxic roots, which suggests that the xylem sap may contain or be lacking a compound which influences leaf growth, 2) a correlation between reduced leaf growth and decreased cytokinin transport in root stressed plants, and 3) that cytokinins increased the growth rate of leaf disks in vitro. It was also shown that although ABA decreased the rate of growth of leaf disks in vitro, there was no increase of ABA in xylem sap of poplars with hypoxic roots. Experiments of this sort suggest that poplar roots may normally supply factors to leaves which stimulate growth, and when root growth is restricted, the leaves no longer receive as much of the growth stimulating compound.

Is the Production of Leaf Area Related to the Sink Strength of the Roots?

Leaves store starch during the day, which they break down and send to the roots as sugar during the night for growth. Roots are sinks for photoassimilates, they influence carbon partitioning in woody plants, and are particularly responsive to changes in carbon allocation within shoots (4). In herbaceous plants, the removal of reproductive sinks alters photosynthetic rates and assimilate partitioning (5,16). There may be similar effects on woody plants when the sink strength of the roots is limited by the rooting environment. This effect can be shown with results fromthe studies presented herewith plantsgrown in small (0.5 L) pots and in large (3.5 L) pots. Starch concentrations were highest in the leaves of plants with stressed roots suggesting that growth does not appear to be limited by photosynthates (Table 1). Instead, it appears that it is the utilization of photoassimilates ratherthan the production of photoassimilates which is most sensitive to root stress. It is possible that the decline in leaf growth reduces the demand for carbon resulting in accumulation of carbohydrates in source leaves. Root stresses may, therefore, simply result in fewer growing roots resulting in a lower demand for assimilates (reduced sink strength) which is sensed in leaves as the build-up of assimilates and subsequently slows leaf growth. It is possible that a change in the balance of carbohydrates would have the effect of acting as a “warning system” that results in reduced growth before there is a severe imbalance in the resource gathering capacity of the plant. It is also possible that leaves respond to subtle changes in the flux of carbon instead of accumulated starch or through the ratio of carbon to some other resource (e.g. nitrogen) which is somehow “sensed” by leaves resulting in reduced leaf growth (4).