Analysis of needle proteins and N-terminal amino acid sequences of two photosystem II proteins of western white pine (Pinus monticola D. Don)

A method is described for extracting needle proteins from mature western white pine (Pinus monticola D. Don) trees. Extracted proteins were separated into 26 components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The predominant protein component (32-39%) had a molecular weight of 57 kDa. The second most predominant protein component (14-25%) had a molecular weight of 16 kDa. The amino acid composition of the twenty-six protein components was determined and a few selected proteins were subjected to further amino acid sequence analysis. Of these, two proteins, designated as Pin m I and Pin m II, were identified by sequence homology with other Photosystem II proteins. The Pin m I and Pin m II proteins had molecular weights of 22.6 kDa and 23.4 kDa, respectively. Pin m I had a much higher proline content than Pin m II.     

Family differences in height growth and photosynthetic traits in three conifers

We investigated variation in height growth, gas exchange, chlorophyll fluorescence and leaf stable carbon isotope ratio among wind-pollinated progenies of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. glauca), ponderosa pine (Pinus ponderosa Dougl. ex Laws.) and western white pine (Pinus monticola Dougl. ex D. Don) from a small group of contiguous stands on the Priest River Experimental Forest in northern Idaho. Photosynthetic variables differed between height classes in the pines, but not in Douglas-fir. Among species and families, tall families of ponderosa pine regained photosynthetic capacity earliest in the spring and maintained it latest in the growing season. Tall families of western white pine had higher instantaneous water-use efficiencies and lower photosynthetic rates than short families on warm days in August.     

Cold acclimation of Pinus contorta and Pinus sylvestris assessed by chlorophyll fluorescence

Needle samples of six provenances each of lodgepole pine (Pinus contorta Dougl. var. latifolia) and Scots pine (Pinus sylvestris L.), originating from latitudes 55 to 68 degrees N in western Canada and northern Sweden, were collected during the autumn and subjected to freezing temperatures in the range of -8 to -29 degrees C on three occasions in September and October. Needle injury was assessed by two different methods: visual assessment and chlorophyll a fluorescence. Chlorophyll a fluorescence data showed a highly significant correlation with the visual assessments of injury, indicating that the technique can be used as a simple, non-destructive and objective measure for rapid detection of freezing injury and for ranking of needle materials with respect to development of cold acclimation. The analyses showed that, during the autumn, lodgepole pine needles were more hardy and acclimated to low temperatures earlier than Scots pine needles.     

Effects of pocket gophers,bracken fern,and western coneflower on planted conifers in northern Idaho – an update and two more species

Grand Fir Mosaic forests innorthern Idaho are difficult to regenerate afternatural or human-caused disturbances. Sparseconifer regeneration appears to be associated withhigh populations of northern pocket gophers (Thomomys talpoides) plus expansion by bracken fern(Pteridium aquilinum (L.) Kuhn) and invasionof western coneflower (Rudbeckia occidentalisNutt.). This report updates an earlier study(Ferguson and Adams 1994) that quantified theeffects of four treatments on survival and growth ofplanted conifers: unweeded with gophers, weeded withgophers, unweeded without gophers, and weededwithout gophers. Weeding removed only bracken fernand western coneflower. Subalpine fir (Abieslasiocarpa (Hook.) Nutt.), grand fir (Abiesgrandis (Dougl.) Lindl.), and western larch (Larix occidentalis Nutt.) had over 80% mortalityfrom pocket gophers and other causes. Lodgepolepine (Pinus contorta var. latifoliaEngelm.) had 49.2% mortality from pocket gophersand 6.8% mortality from other causes, but snowdamage is a problem for lodgepole pine. Engelmannspruce (Picea engelmannii Parry ex Engelm.)had 42.2% gopher-caused mortality and 8.4%mortality from other causes. Spruce suffered littletop damage from snowpacks and was stout enough towithstand senescing bracken fern fronds. Westernwhite pine (Pinus monticola Dougl.) had only24.1% mortality from pocket gophers after 4 years,and only 11.8% of the white pine died from causesother than gophers. Of the mortality caused bypocket gophers, 76.8% occurred the first summer andnext two winters after planting. The recommendedspecies for reforestation are Engelmann spruce andwhite pine.     

Heat-shock response of Pinus and Picea seedlings

The effect of a short period of elevated temperature, or heat shock, on protein synthesis was investigated in 2-day-old seedlings of jack pine (Pinus banksiana Lamb.), loblolly pine (P. taeda L.), lodgepole pine (P. contorta Dougl.), black spruce (Picea mariana (Mill.) BSP), and white spruce (P. glauca (Moench) Voss.). In all species, heat shock led to increased [(35)S]methionine incorporation into heat-shock proteins (hsp's) with molecular masses of 83 and 72 kDA. Heat shock also induced synthesis of several low molecular weight proteins that were absent from control seedlings. The low molecular weight hsp's produced by pine seedlings had molecular masses of 27, 24.6, 20.5 and 17.5 kDa, whereas those produced by spruce seedlings had molecular masses of 27.2, 19.8, 18.4, 17.2 and 16.0 kDa. All of the low molecular weight hsp's showed isoelectric variants. Heat shock led to increased [(35)S]methionine incorporation into a group of low molecular weight hsp's that were also present in control seedlings.     

Winter water relations of New England conifers and factors influencing their upper elevational limits. I. Measurements

The upper elevational limits of tree species are thought to be controlled by abiotic factors such as temperature and the soil and atmospheric conditions affecting plant water status. We measured relative water contents (RWC), water potentials (Psi) and cuticular conductances (g(c)) of shoots of four conifer species-eastern hemlock (Tsuga canadensis (L.) Carr.), eastern white pine (Pinus strobus L.), red pine (P. resinosa Ait.) and red spruce (Picea rubens Sarg.)-during two winters on Mt. Ascutney, Vermont, USA. Some micrometeorological measurements are also reported. Eastern hemlock and white pine were studied near their upper elevational limits at a 640-m site, and red pine was studied near its upper elevational limit at 715 m. Red spruce was also studied at the 715-m site, which is in the middle of its elevational range on this mountain. There was no evidence of winter desiccation stress in any species. The observed distribution of seedlings suggested that the upper elevational limits on shade-intolerant eastern white pine and red pine are set by the absence of suitable seed beds after 100 years without fire. Eastern hemlock is able to reproduce in deep shade on organic substrates, but germination at high elevations may be restricted by low temperatures.     

Seasonal nutrient dynamics in white pine and white spruce in response to environmental manipulation

Seasonal retranslocation in white pine (Pinus strobus L.) and white spruce (Picea glauca (Moench) Voss) was examined in response to silvicultural treatments (scarification, annual fertilization application, and annual control of competing vegetation with herbicide) that changed both environmental conditions and the growth rate of the trees. Four years after plantation establishment and initial treatment, nutrient accumulation in current-year needles of white pine and retranslocation from 1-year-old needles were increased following the vegetation control treatment, which increased resource availability (nutrients, water and light) and, hence, growth rate. Nutrient accumulation also increased in current-year white spruce needles following the same treatment, whereas retranslocation decreased in 1-year-old white spruce needles. Correlations of retranslocation (N, P and K) with growth rate (shoot biomass increment) showed a strong positive relationship for white pine and a negative relationship for white spruce. Retranslocation of K was correlated with foliar and soil K concentrations; the availability of this nutrient was also significantly reduced by vegetation control. A general theory for the control of nutrient retranslocation in conifers, which is not based exclusively on either sink strength or soil nutrient availability, is proposed. We conclude that retranslocation response is species specific and related to the potential phenotypic growth response to changing environmental conditions and to short-term imbalance in the supply versus the demand for nutrients.     

Element accumulation patterns of deciduous and evergreen tree seedlings on acid soils: implications for sensitivity to manganese toxicity

Foliar nutrient imbalances, including the hyperaccumulation of manganese (Mn), are correlated with symptoms of declining health in sensitive tree species growing on acidic forest soils. The objectives of this study were to: (1) compare foliar nutrient accumulation patterns of six deciduous (sugar maple (Acer saccharum Marsh.), red maple (Acer rubrum L.), red oak (Quercus rubra L.), white oak (Quercus alba L.), black cherry (Prunus serotina Ehrh.) and white ash (Fraxinus americana L.)) and three evergreen (eastern hemlock (Tsuga canadensis L.), white pine (Pinus strobus L.) and white spruce (Picea glauca (Moench) Voss.)) tree species growing on acidic forest soils; and (2) examine how leaf phenology and other traits that distinguish evergreen and deciduous tree species influence foliar Mn accumulation rates and sensitivity to excess Mn. For the first objective, leaf samples of seedlings from five acidic, non-glaciated field sites on Pennsylvania's Allegheny Plateau were collected and analyzed for leaf element concentrations. In a second study, we examined growth and photosynthetic responses of seedlings exposed to excess Mn in sand culture. In field samples, Mn in deciduous foliage hyperaccumulated to concentrations more than twice as high as those found in evergreen needles. Among species, sugar maple was the most sensitive to excess Mn based on growth and photosynthetic measurements. Photosynthesis in red maple and red oak was also sensitive to excess Mn, whereas white oak, black cherry, white ash and the three evergreen species were tolerant of excess Mn. Among the nine species, relative rates of photosynthesis were negatively correlated with foliar Mn concentrations, suggesting that photosynthetic sensitivity to Mn is a function of its rate of accumulation in seedling foliage.     

Influences of Thinning,Chipping, and Fire on Understory Vegetation in a Sierran Mixed Conifer Stand

Thinning implemented with a cut-to-length harvesting system coupled with on-site slash chipping and redistribution and followed by prescribed underburning were assessed for their impacts on a shrub understory in an uneven-aged Sierra Nevada mixed conifer stand. Overstory species consisted of California white fir (Abies concolor var. lowiana [Gord.] Lemm.), Jeffrey pine (Pinus jeffreyi Grev. & Balf.), sugar pine (Pinus lambertiana Dougl.), incense-cedar (Libocedrus decurrens Torr.), and red fir (Abies magnifica A. Murr.), while huckleberry oak (Quercus vacciniifolia Kellogg) was predominant among 10 understory shrubs. Herbaceous species were entirely absent from the site for the 4-yr duration of the study. The mechanized treatments exerted minimal detriment effects on overall understory cover and weight, and for prostrate ceanothus (Ceanothus prostratus Benth.) and creeping snowberry (Symphoricarpos mollis Nutt.)—two of the lesser shrubs—were stimulatory. In contrast, losses to the total understory from the underburn amounted to two-thirds of cover and weight in the absence of the mechanized treatments and more than three-quarters where they had been implemented, with huckleberry oak prevalence especially diminished. For almost all of the understory species individually as well as for the total, greater pretreatment abundance predisposed greater posttreatment prevalence. Results of this study provide insight into the understory impacts of restoration treatments that are deemed especially appropriate for sensitive sites in western U.S. forests.     

Transient gene expression in western white pine using agroinfiltration

A method for transient gene expression was developed for western white pine(WWP, Pinus monticola Dougl. ex D.Don) using reporter gene uid A encoding bglucuronidase(GUS). GUS was transiently expressed in cross sections of primary and secondary needles, cotyledons, and current and second year stems of WWP via vacuum-infiltration with Agrobacterium tumefaciens. Histochemical assays of cross sections of secondary needles showed stronger blue color indicating GUS expression at day 1 and 2 than on other days post agroinfiltration(dpa).GUS activity expressed inside WWP cells was confirmed using light microscopy. In fluorometric assays, GUS expression was high at 1 dpa and lasted until 4 dpa in detached secondary needles, while similarly high expression levels only lasted until 2 dpa in attached secondary needles then dropped significantly. Although the length of GUS-staining zones varied among different WWP organs and between growth and dormant seasons, all tested WWP tissues using the protocol had high levels of transient GUS expression. Thus, heterologous candidate genes or endogenous silencing can be expressed in various WWP tissues or organs using this agroinfiltration approach. The current protocol for efficient transient gene expression willaid functional genomics study of WWP and its pathogens and related conifer species.     

Seasonal differences in freezing stress resistance of needles of Pinus nigra and Pinus resinosa: evaluation of the electrolyte leakage method

Seasonal changes in freezing stress resistance of needles of red pine (Pinus resinosa Ait.) and Austrian pine (Pinus nigra Arnold) trees were measured by an electrolyte leakage method and by visual observation. During most of the year, freezing stress resistance determined by the two methods gave similar results. The electrolyte leakage method provided a good estimate of seasonal changes in freezing stress resistance except for red pine needles in their most winter-hardy state. To obtain a reliable estimate of freezing stress resistance in winter-hardy red pine needles it was necessary to combine the electrolyte leakage method with visual observations. When red pine needles survived exposure to -80 degrees C or lower, electrolyte leakage was never more than 30% even when the needles were exposed to a slow freeze-thaw stress of -196 degrees C. However, rapid freezing of red pine needles to -196 degrees C resulted in electrolyte leakage of over 80%. Red pine needles attained a much higher freezing stress resistance during the winter than Austrian pine. Red pine needles also acclimated and deacclimated faster than Austrian pine needles. An index of injury was developed based on the electrolyte leakage method ((R(2) + R(1))/2, where R(1) is the minimum % electrolyte leakage from noninjured tissue and R(2) is the maximum % electrolyte leakage at the highest injury) that reliably predicted freezing stress resistance of pine needles for most of the year. Important aspects for developing a successful index of injury for pine needles are: use of cut needles, vacuum infiltration and shaking during incubation in water.We conclude that: (1) during cold acclimation the cell wall properties of the pine needles changed and these changes, which appeared to differ in the two species, might explain the very low leakage of electrolytes from winter-hardy needles of red pine; (2) pine needles survive winter by developing the ability to tolerate extracellular ice formation, because after rapid freezing the needles were severely injured; and (3) red pine is adapted to a shorter growing season and colder winters than Austrian pine.     

Defoliation by the Needle-shortening Pine Gall Midge,Thecodiplosis brachyntera,on Pines in Central Sweden

An outbreak of the needle-shortening pine gall midge, Thecodiplosis brachyntera , occurred on Scots pine, Pinus sylvestris (L.) and lodgepole pine, P. contorta (Dougl.) in central Sweden during 1995-1998. The larval feeding of T. brachyntera kills current-year needles and heavy infestation causes severe defoliation. A pattern of fluctuating defoliation among years and sites was found on both pine species. Defoliation was evenly distributed in the crown of P. sylvestris during years of high as well as low infestation; apical whorls had as much defoliation as basal whorls. In P. contorta , however, there was a tendency for defoliation to be concentrated on branches in the most apical whorls. Growth ring increments on P. sylvestris with high levels of defoliation (71%) did not differ from trees with moderate levels (26%) of defoliation. Shoot lengths were significantly shorter, however, on heavily infested trees the year after defoliation.     

Microculture of western white pine (Pinus monticola) by induction of shoots on bud explants from 1- to 7-year-old trees

We developed a protocol for the production of shoots from bud explants from 1- to 7-year-old trees of western white pine (Pinus monticola Dougl.). The best explant was a 2-mm-thick cross-sectional slice of the early winter bud. Genotype of the donor tree was a significant factor affecting shoot production, but more than 80% of the genotypes tested produced shoots. Of the media tested, bud slices from 1- to 3-year-old trees grew best in Litvay's medium containing N(6)-benzyladenine in the range of 1 to 30 micro M, whereas bud slices from older trees grew best in Gupta and Durzan's DCR medium with zeatin riboside. Up to 400 shoots more than 3 mm in height were obtained from 100 bud-slice explants taken from 7-year-old western white pine trees.     

Light quality treatments enhance somatic seedling production in three southern pine species

Embryogenic cultures of loblolly pine (Pinus taeda L.), slash pine (Pinus elliottii Engelm.), longleaf pine (Pinus palustris Mill.) and slash pine x longleaf pine hybrids were initiated from immature seeds on an initiation medium containing 13.57 microM 2,4-dichlorophenoxyacetic acid and 2.22 microM benzylaminopurine. Embryogenic cultures proliferated and somatic embryos developed, matured and germinated following a modified protocol and media originally developed for radiata pine (Pinus radiata D. Don.) somatic seedling production. A discrete, light-sensitive pre-germination stage and a later germination (radicle emergence) stage were identified by the differential response of somatic embryos to light of different wavelengths. Different light quality treatments were applied during the pre-germination and germination steps, using cool white fluorescent bulbs or light-emitting diodes (LEDs), or both. In general, red wavelengths provided by LEDs during these steps resulted in higher frequencies of somatic embryo germination (up to 64%) and conversion (up to 50%), longer tap roots and more first-order lateral roots than the standard cool white fluorescent treatments or treatment with blue wavelengths from LEDs. In addition, exposure to red light allowed germination of somatic embryos of some clones that failed to produce germinants under fluorescent light. Germination and conversion were further enhanced by sequential application of cool white fluorescent light and red light, resulting in up to 100% germination and conversion in one experiment. Longleaf pine somatic embryos were especially responsive to the light quality treatments, resulting in the first report of somatic seedling production for this species.     

The effect of weed control and fertilization on survival and growth of four pine species in the Virginia Piedmont

The growth response of loblolly pine (Pinus taeda L.), shortleaf pine (Pinus echinata Mill.), Virginia pine (Pinus virginiana Mill.), and white pine (Pinus strobus L.) to weed control and fertilization in the Piedmont of Virginia was assessed. Four different silvicultural treatments were evaluated: (1) check (no treatment); (2) weed control; (3) fertilization; (4) weed control plus fertilization. The weed control treatment included a series of herbicide and mechanical treatments to eliminate competing hardwoods. The fertilizer treatments added N, P, K, and S. Survival and growth was measured annually through age 5. There were significant differences in survival and growth among species. Survival was greatest for loblolly pine, lower in shortleaf and Virginia pine, and lowest in white pine. Fertilization without controlling the competing hardwoods decreased survival in all planted pines due to the increased hardwood competition. Loblolly pine was tallest through the 5-year period, shortleaf and Virginia pine were shorter and white pine was shortest. Silvicultural treatments had no impact on tree height but significantly affected DBH. Weed control increased DBH while fertilization did not. When applied in combination with weed control, there was no additional increase in growth of the pines due to fertilization beyond that from weed control only. Fertilization stimulated the growth of the competing hardwoods which were significantly taller in the fertilized plots.     

Needle polyamine concentrations and potassium nutrition in Scots pine

The response of free polyamines (putrescine, spermidine and spermine) in needles of Scots pine (Pinus sylvestris L.) to varying needle potassium concentrations was investigated in two potassium fertilization experiments on drained peatlands. A significant negative correlation was observed between putrescine and potassium concentrations in needles. Putrescine responded more sensitively to decreasing needle K concentrations during the growing season than during the winter. Putrescine accumulation started when needle potassium concentrations were above 5.5 mg g(dw) (-1) in summer and above 5.0 mg g(dw) (-1) in winter. A decrease in needle potassium concentrations below 4.3 mg g(dw) (-1) in summer and below 3.5 mg g(dw) (-1) in winter resulted in an exponential increase in putrescine concentrations. Putrescine accumulation was initiated well above the needle potassium concentrations generally considered indicative of deficiency. The exponential increase in putrescine concentrations in winter occurred within the range of needle potassium concentrations that has been identified as indicating severe potassium deficiency.     

Winter water relations of New England conifers and factors influencing their upper elevational limits. II. Modeling

We used the water relations model, WINWAT, to model winter water relations of three conifer species-eastern hemlock (Tsuga canadensis (L.) Carr.), eastern white pine (Pinus strobus L.) and red pine (P. resinosa Ait.)-growing at their upper elevational limits on Mt. Ascutney, Vermont, USA, in the winters of 1997 and 1999. Modeled relative water contents remained above 60% in the two youngest foliar age classes of all three species during both winters, indicating that desiccation stress in winter is not responsible for setting the upper elevational limits of these species at this site under present climatic conditions. WINWAT indicated that winter water relations of these low-elevational species were sensitive to low relative humidity, which increased transpiration rates, and low temperatures, which inhibited recharge, but are much less sensitive to summer climate than in the case of subalpine conifers in Colorado. Our results indicate that summer and winter temperatures and relative humidities (or precipitation/potential evapotranspiration ratios) should be incorporated into climate change models designed to simulate future tree distributions.