Assuming that there is no stem water available through winter, mathematical models predict that cuticular transpiration alone may account for expected water loss of needles at some timberlines (Sowell 1982). However, for trees at timberline in the Gunnison Basin the assumption that water is unavailable to desiccating needles during winter is erroneous; stem water is available to Engelmann spruce (Picea engelmannii (Parry) Engelm.) needles during winter (Sowell, McNulty, and Schilling 1996). With this additional water, stomatal transpiration may indeed be occurring though undetected to date.

Sowell et al. (1982) examined needle transpiration rates in excised shoots of Picea albicaulis. During the initial 24 h, a high rate of transpiration was observed followed by a steep decline after which transpiration remained constant for the remaining 90 h. The initially high transpiration rate has been attributed to stomatal opening, and this seems reasonable since the specimens were collected during the summer when photosynthesis is at its zenith. However, Baig et al. (1974) observed similar transpiration curves for excised shoots of Picea abies that were collected November through March. This stomatal activity of excised shoots may suggest a propensity for stomata to open during favorable winter conditions in situ. The question remains as to the extent and causes of such stomatal opening in winter.

Grace (1990) hypothesized that abrasion by wind and ice particles may cause mechanical damage to the leaf epidermis and stomatal dysfunction at timberline in Scotland. This damage, it is conjectured, could prevent the stomata from closing and result in the excessive desiccation observed at timberline. Schaub and Sowell (1994, unpublished data) viewed first-year, third-year, and fifth-year needles using scanning electron microscopy. Micrographs showed that the stomatal antechambers, or crypts, of first-year needles were plugged with cuticular wax while third- and fifth-year needles showed significantly less plugging of stomatal crypts. This loss of stomatal plugging was greater for needles located in exposed sites than for needles in more protected forest sites. Similar loss of stomatal plugging on wind-exposed Engelmann spruce needles has been observed by Hadley and Smith (1986). Though the influence of stomatal plugs on stomatal transpiration is not known, it is likely that the existence of stomatal plugs in first-year needles decreases stomatal conductance. If stomata do indeed open during winter, Grace's (1990) inferred stomatal dysfunction at maritime treeline might simply be a result of less stomatal plugging of older, exposed needles.

If indeed the initial high transpiration rate of excised needles is due to stomatal opening then transpiration should be influenced by environmental factors known to promote stomatal activity. The purpose of this study was to investigate the influence of light and temperature on the possible stomatal opening of excised shoots of Engelmann spruce and bristlecone pine collected throughout the winter season.

Twenty Engelmann spruce and 16 bristlecone pine trees were chosen and a branch large enough to provide four sample shoot tips was selected from the southern aspect of each tree on each sample date. Engelmann spruce collections took place on four dates: 8 January, 14 February, 26 March, and 12 May. Bristlecone pine samples were collected three times throughout the season: 14 February, 26 March, and 12 May. Specimens were treated in the laboratory within 5 h of collection. Four 5 cm shoot tips were excised from each branch, glued into sample cups with a low temperature glue gun and randomly assigned one of four treatments: (1) 4^oC and dark, (2) 4^oC and light, (3) 20^oC and dark, or (4) 20^oC and light. Regardless of treatment, all shoot tips were contained within an airtight chamber in which humidity was maintained using anhydrous calcium. The tops of the containers were glass, either clear or spray painted black, and fluorescent bulbs installed 6 cm above the containers provided a PPF of 41 umol photons m^-2 s^-1. Dark containers were spray painted black on all sides and did not admit light. Samples were weighed prior to placement in containers (fresh mass) and to generate desiccation curves, again at approximately 16 h and then 24 h intervals for six consecutive days. Dry mass was measured after needles were dried for 72 h at 70^oC.

Leaf conductance (g_l) was calculated using g_l = E_l/, where E_l is the leaf transpiration rate in g H₂O m^-2 s^-1 and is the vapor concentration gradient between leaf and air in g H₂O m^-2. E_l was calculated for each time interval on a leaf area basis. Leaf area was estimated using specific leaf areas of 0.009593376 m²/g dry mass and 0.0119791 m²/g dry mass for Engelmann spruce and bristlecone pine, respectively (McGinnis, 1998 unpublished data). The vapor concentration gradient assumed 100% r.h. within the leaf and 1.05% and 3.34% r.h. in the 4^oC and 20^oC chambers, respectively. The conductance of the needle boundary layer was considered to be negligible compared to leaf conductance and was not used in the calculations (Sowell 1985).

For each species, a repeated measures 3-way MANOVA with factorial design was used to test the effects of collection date, temperature of chamber, and light level within the chamber on leaf conductance. The significance of all interactions on leaf conductance was also tested.

 

If stomata are open after excision, they do exhibit effective closing. Thus, unlike Grace's (1980) hypothesis that stomatal dysfunction is common at maritime timberlines in Scotland, Engelmann spruce and bristlecone pine stomata studied here appear functional.

Holmgren et al. (1965) found cuticular conductance to decrease with decreasing temperature. Similarly, the g_l for Engelmann spruce was significantly lower at 4^oC as compared to 20^oC during the cuticular conductance phase (Figure 1). Interestingly this temperature effect was also observed during the presumed stomatal conductance phase. Scanning electron micrographs by Schaub and Sowell (unpublished data 1990) illustrated the presence of cuticular wax plugs within the stomatal crypts of Engelmann spruce needles. Therefore, temperature's effects on stomatal conductance may be two-fold. While guard cell activity and thus stomatal aperture may be influenced, temperature may also affect the permeability of wax plugs thus affecting stomatal conductance.

The environmental and physiological factors that cause stomatal opening in excised shoots during an obviously stressful period is unknown. However, such stomatal activity in excised shoots does demonstrate a propensity for stomatal opening during winter, thus bringing into question the assumption that stomata remain closed through this harsh season.

Grace, J. 1990. Cuticular water loss unlikely to explain tree-line in Scotland. Oecologia 84:64-68

Hadley, J.L. and W.K. Smith. 1983. Influence of wind exposure on needle desiccation and mortality for timberline conifers in Wyoming, U.S.A. Arctic and Alpine Research 15:127-135

Hadley, J.L. and W.K. Smith. 1989. Wind erosion on leaf surface wax in alpine timberline conifers. Arctic and Alpine Research 21:392-398.

Holmgren, P., Jarvis, P.G., and Jarvis, M.S. 1965. Resistances to carbon dioxide and water vapour transfer in leaves of different plant species. Physiologia Plantarum 18:557-573.

Sowell, J.B. 1985. Winter water relations of trees at alpine timberline. Eidgenössische Anstalt für das forstliche Versuchswesen, Berichte 270:71-77.

Sowell, J.B., and D.L. Koutnik, and A.J. Lansing. 1982. Cuticular transpiration of whitebark pine (Pinus albicaulis) within a Sierra Nevadan timberline ecotone, U.S.A. Arctic and Alpine Research 14:97-103.

Sowell, J.B., S.P. McNulty, and B.K. Schilling. 1996. The role of stem recharge in reducing the winter desiccation of Picea engelmannii (Pinaceae) needles at the alpine timberline. American Journal of Botany 83:1351-1355.

Tranquillini, W. 1979. Physiological ecology of the alpine timberline. Springer-Verlag, Berlin.

1 July 1998