After a transient inhibition of shoot growth, photosynthesis and the increase in needle necrosis, the recovery and resumption of shoot growth in flooded tamarack coincided with the initiation of adventitious roots. Since the existing younger roots disintegrated over time, there were only old woody roots and new adventitious roots that were present in seedlings at the end of the flooding treatment. Even though needle chlorophyll concentrations were reduced by the flooding treatment, transpiration rates, net photosynthesis, and shoot water potentials were similar in flooded and non-flooded plants. These results suggest that, despite some hypoxia-induced toxicity symptoms, adventitious roots maintained adequate water supply to flooded seedlings. Flooding commonly triggers reductions in leaf chlorophyll concentrations in plants [4
]. The lack of an effect of reduced needle chlorophyll concentrations on net photosynthesis in flooded tamarack suggests that stomatal factors are likely to override photosynthetis responses to flooding under the study conditions.
The initial responses of plants to flooding include an inhibition of gas exchange [18
]. However, transpiration and photosynthesis have been often reported to recover in flooding-tolerant plants [1
] suggesting that the tolerant plants can restore their water balance over time. The recovery of root hydraulic conductance in flooded tamarack seedlings coincided with the emergence of adventitious roots suggesting that adventitious roots were more flooding-tolerant compared with non-adventitious roots [16
]. This recovery is usually related with the production of ethylene within the roots that will induce an increase on root water transport [11
] and hence an increase on the phosphorylation of aquaporins. Hypoxia, which is the main consequence of flooding, inhibits root water uptake through its effect on the aquaporin-mediated water transport due to low cytosolic pH and inhibition of respiration [11
]. Therefore, adaptations of the root system to flooding may be expected to include the structural and functional modifications which decrease the dependence of the root system on the aquaporin-mediated water transport.
In the present study, a large part of the root system in flooded tamarack seedlings consisted of long, non-woody, adventitious roots with a large unsuberized absorption surface. As part of the adaptations to flooding, the adventitious roots contained greatly reduced vascular bundles when examined as far as 3
cm from the root apex. Also, their endodermal layer was poorly developed and often not apparent when examined at the same distance from the root apex as the roots of non-flooded plants. The fact that the cortex of adventitious roots contained abundant starch grains suggests that they were the sink for carbohydrates. Starch is a primary energy storage compound and its allocation pattern and translocation may be critical for growth and hypoxia tolerance [22
]. It has been suggested that plant survival in wetland habitats may depend on root carbohydrate reserves [23
] and starch abundance is considered to be among the principal characteristics of flooding-tolerant tree species [24
]. High accumulation of starch in the rooting regions has been also associated with adventitious root formation [26
]. It is plausible that large amounts of starch are needed to supply adventitious roots with sufficient energy required to support their growth and basic metabolic functions under hypoxic conditions. However, a possible effect of starch accumulation on matric potential of the root cortex and its consequences for root water relations also deserve further attention.
Both KTOT and LTOT of the whole root systems were similar in flooded and non-flooded plants (Study 1). When measured in individual roots, KIND and LIND were several-fold lower in flooded adventitious roots compared with non-flooded control roots (Study 2). Therefore, there were likely water entry points through the parts of the old, partly disintegrated root system. Regardless of the differences in the hydraulic conductance of flooded and non-flooded plants, growth, gas exchange, and shoot water potentials suggest that water transport was not the limiting factor to the flooded plants with established adventitious roots. This was likely facilitated by the greater water absorption area of adventitious roots and structural modifications increasing the apoplastic bypass.
The results of PTS3
and immunostaining with the PIP1 and PIP2 antibodies support the notion of the reduced role of cell-to-cell water transport in adventitious flooded roots. The concentration of PTS3
in the xylem sap expressed from the individual flooded adventitious roots was almost two-fold higher compared with control non-flooded roots. PTS3
is a water-soluble fluorescent, non-toxic dye that does not cross cell membranes or adhere to cell walls [28
]. Although the exact concentrations of apoplastic flow calculated from the fluorescent tracer dye concentrations are not precise estimates of the actual apoplastic flow rates, they have been used to estimate relative changes in water flow pathways [10
]. Increases in PTS3
concentrations in the xylem sap have been also correlated with the inhibition of aquaporin-mediated transport in plants exposed to drought [30
], mercury [31
], and metabolic inhibitors [10
Higher activation energy for water flow in flooded adventitious roots (4.95
) compared with non-flooded control roots (3.25
) suggests that water transport in flooded adventitious roots is more sensitive to temperature compared with the non-flooded roots. For the transmembrane transport, Ea
increases with increasing restriction of water movement through aquaporins [34
] and the overexpression of PIP1 and PIP2 aquaporins in Arabidopsis
abolishes temperature sensitivity of root cell hydraulic conductivity over the range of 10 to 25
°C (unpublished results). However, at the whole root level, hydraulic responses to temperature are confounded by the effects of apoplastic pathway [36
The immunolocalization of PIP aquaporins showed that in addition to the overall reduced intensity, immunofluorescence was concentrated in the outer parts of the cortex and epidermis of adventitious flooded roots. This suggests their relatively greater role in the root surface water uptake as demonstrated for PIP2;5 in maize [38
]. A more uniform distribution and higher intensity of immunostaining throughout the root cortex of non-adventitious non-flooded roots combined with the presence of larger vascular bundles and well-developed endodermis suggests a greater functional importance of radial water transport in this root zone.
The ability to produce adventitious roots offers an opportunity to tamarack to develop the features that allow trees to survive flooding conditions. It remains to be determined whether the modifications in water transport properties constitute a general flooding tolerance mechanism or are unique to this tree species.