Environmental and developmental effects on the biosynthesis of UV-B screening pigments in Scots pine (Pinus sylvestris L.) needles

The major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles are flavonol 3-o-glycosides (F3Gs) esterified with hydroxycinnamic acids at positions 3" and 6". Acylation is the last step in biosynthesis and is catalysed by position-specific hydroxycinnamoyl transferases (3" and 6"HCT). The UV-B dependence of these enzyme activities was studied in primary needles of Scots pine seedlings grown under different UV-B conditions in environmentally controlled sun simulators. 6"HCT activity was induced upon UV-B irradiation while 3"HCT activity was not induced but showed high constitutive values. To investigate the biosynthesis of diacylated F3Gs during needle development under natural conditions, the HCT activities and metabolite contents were analysed in needles of field-grown mature pine trees. Accumulation of diacylated compounds as well as of 6"HCT activity occurred transiently in the first year of needle development only. In contrast, 3"HCT activity exhibited broad maxima in two consecutive years during needle growth. The data suggest that acylated F3Gs are first formed as soluble compounds which are then translocated into the cell wall to be bound by their hydroxycinnamoyl residues.     

Short-term effects of fertilization on loblolly pine (Pinus taeda L.) physiology

Fertilization commonly increases biomass production in loblolly pine (Pinus taeda L.). However, the sequence of short‐term physiological adjustments allowing for the establishment of leaf area and enhanced growth is not well understood. The effects of fertilization on photosynthetic parameters, root respiration, and growth for over 200 d following the application of diammonium phosphate were intensively investigated in an effort to establish a relative sequence of events associated with improved growth. Root respiration, foliar nitrogen concentration [N]_f, and light‐saturated net photosynthesis (A_sat) temporarily increased following fertilization. A_sat was correlated positively with [N]_f when non‐fertilized and fertilized treatments were pooled (R² = 0.47). Increased photosynthetic capacity following fertilization was due to both improved photochemical efficiency and capacity and enhanced carboxylation capacity of Rubisco. Positive effects of fertilization on growth were observed shortly after A_sat increased. Fertilized seedlings had 36.5% more leaf area and 36.5% greater total dry weight biomass at 211 d following fertilization. It is concluded that fertilization temporarily increased photosynthetic capacity, which resulted in a pool of photo‐assimilate used to build leaf area. The N from fertilizer initially invested in photosynthetic structures and enzymes probably re‐translocated to newly developing foliage, explaining the reduction in [N]_f and A_sat that was observed after peak levels were achieved following fertilization.     

The influence of elevated ultraviolet-B radiation (UV-B) on tissue quality and decomposition of loblolly pine (Pinus taeda L.) needles

Stratospheric ozone depletion is expected to elevate the influx of ultraviolet-B radiation (UV-B) to the biosphere. Increased levels of UV-B may, in turn, alter important ecosystem processes such as decomposition. Previous studies have shown that growth under elevated UV-B can alter leaf quality in angiosperm species and thereby indirectly change subsequent rates of leaf decay. In this experiment, we determined if elevated UV-B would alter the chemical composition and decay of needle tissue from two seed sources of the gymnosperm Pinus taeda L. Maryland and Virginia seed sources of P. taeda were grown in the field for 3 years beneath lampbanks supplying either ambient, low elevated or high elevated UV-B. These levels of UV-B corresponded to 0, 16 and 25% stratospheric ozone depletion at the experimental site in Beltsville, MD (39 degrees N). Needles were collected from six randomly chosen plants for each combination of seed source and UV-B level. The needle samples were analyzed for total C and N, UV-B absorbing compounds, and carbon fractions. Decay rates were also determined by measuring rates of CO(2) evolution from needle material decomposed under laboratory conditions. UV-B did not significantly alter the chemical composition of needles from the Virginia seed source. In contrast, needles from the Maryland seed source tended to have elevated lignin/N ratios and a lower holocellulose content when grown under the highest level of UV-B. Furthermore, while needles from the Virginia pines did not have UV-B altered decay rates, Maryland needles grown under low elevated UV-B conditions decomposed 36% more rapidly than needles from other treatments. Results from this experiment illustrate at least three characteristics about the indirect effect of UV-B on decomposition, (1) UV-B can modify decomposition of tissue from gymnosperms as well as angiosperms; (2) UV-B effects on tissue chemistry and decay may not only be species-specific but also seed-source specific; and (3) UV-B effects on decomposition may not increase with increasing UV-B dose.     

Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

Summary Several factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory. Published with approval of the Director of Arkansas Agricultural Experimental Station.     

Comparative mapping in Pinus: sugar pine (Pinus lambertiana Dougl.) and loblolly pine (Pinus taeda L.)

The majority of genomic research in conifers has been conducted in the Pinus subgenus Pinus mostly due to the high economic importance of the species within this taxon. Genetic maps have been constructed for several of these pines and comparative mapping analyses have consistently revealed notable synteny. In contrast, little genomic research has been conducted on the Pinus subgenus Strobus, even though these pines have strong ecological relevance. We report a consensus genetic linkage map for sugar pine (Pinus lambertiana Dougl.) constructed with 399 single nucleotide polymorphisms markers derived from annotated genes. The map is 1,231 cM in length and organized into 19 linkage groups. Two of the mapping populations were derived from trees that were segregating for the major gene of resistance (Cr1) to Cronartium ribicola, the fungal pathogen responsible for white pine blister rust. The third mapping population was derived from a full-sib cross segregating for partial resistance to white pine blister rust. In addition, we report the first comparative mapping study between subgenera Strobus and Pinus. Sixty mapped markers were found in common between sugar pine and the loblolly pine reference map with 56 of them (93%) showing collinearity. All 19 linkage groups of the sugar pine consensus map coaligned to the 12 linkage groups of the loblolly pine reference map. The syntenic relationship observed between these two clades of pines provides a foundation for advancing genomic research and genetic resources in subgenus Strobus.     

Food reserves of Scots pine (Pinus sylvestris L.)

Summary The amounts of starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were studied in Scots pine (Pinus sylvestris L.) during an annual cycle in current-year needles and in 1-, 2- and 3-year-old needles collected shortly after bud break. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. Newly emerging needles contained relatively large amounts of starch, but only trace amounts of fat. During autumn and winter, fat content rose, while starch content decreased; amounts of both these reserve materials were very high the next spring shortly before bud break and decreased again during shoot elongation. Concentration of intermediates in triacylglycerol biosynthesis (diacylglycerols and free fatty acids), were low in summer and high in winter. The same pattern was observed for fructose and glucose (the predominant soluble sugars), galactose/arabinose and raffinose/melibiose. In contrast, sucrose concentrations were highest in spring and in autumn. Mature needles of different ages collected in May showed significant differences only in their triacylglycerol and starch content. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance and the possibility of carbohydrate-fat interconversions.     

Water status and growth of loblolly pine (Pinus taeda L.) callus

Water status of Pinus taeda L. callus supported on Murashige and Skoog (MS) liquid medium was characterized over an 8 week period using thermocouple psychrometry. Medium with 30 gl⁻¹ sucrose was used to produce a high water potential (Ψ_w) of −0.4 MPa (H), and the same medium was used to create a moderate Ψ_w of −0.7 MPa (M) by the addition of 10% polyethylene glycol (PEG, w/v, MW=8000). Calli were produced from cotyledon explants on H medium for 2 weeks and then transferred to either M or H medium. Callus absorption of PEG accounted for 40% of the callus dry weight and less than 7% of the callus fresh weight. Callus dry weight (without the PEG fraction) on M medium was 40% of that observed on H medium. Fresh weight on M medium was only 15% of that observed on H medium. The Ψ_w of both H and M media remained constant throughout the culture period. On H medium, callus Ψ_w and osmotic potential (Ψ_s) both increased 0.05 MPa/week with the callus Ψ_w approaching that of the external medium. On M medium, callus Ψ_w and Ψ_s both decreased more than 0.1 MPa/week with the callus Ψ_w decreasing greatly below that of the external medium. The latter was attributed to a rapidly produced osmotic shock induced upon callus transfer and/or PEG which caused less callus hydration and resulted in reduced growth. Callus turgor potential (Ψ_p) was estimated to be +0.02 to +0.09 MPa and turgor was maintained as callus Ψ_w increased or decreased. After 8 weeks, cell volumes from callus on M medium were 50 to 60% less than on H medium, suggesting that reduced cell volumes were related to turgor maintenance.     

Anatomy of seedling tap roots of loblolly pine (Pinus taeda L.)

The development of tap root anatomical features was investigated in seedlings of loblolly pine (Pinus taeda L.) under both pot and pouch growth regimes. The roots possessed the three anatomical zones previously observed in jack pine (Pinus banksiana Lamb) and Eucalyptus pilularis Sm. - white, condensed tannin (CT), and cork - suggesting that this developmental sequence is preserved over species and growth conditions. Xylem development was centripetal and similar to that found earlier in P. sylvestris. Tracheids with lignified, secondary walls were detected distal to the point of endodermal Casparian band deposition. However, tests for ability to conduct fluid indicated that the protoxylem was capable of transport only proximal to the Casparian bands. Detailed examination of suberin lamella deposition in the endodermis demonstrated that passage cells were present through the white and CT zones. Progressive, centripetal cortical death in the CT zone did not include the endodermis, which remained alive until the cork layer formed, at which point the endodermis was crushed. Therefore, passage cells remain as functional portals for nutrient and water uptake in the CT zone even though the central cortex is dead. Tracer tests indicated that the endodermis provides an apoplastic barrier to tracer diffusion into the stele and that this function was taken over by the young cork layers. Results of this study point to a strong role for the endodermis in the regulation of nutrient and water uptake until the maturation of the first cork layer.     

Seasonal ectomycorrhizal fungal biomass development on loblolly pine (Pinus taeda L.) seedlings

Ergosterol, a membrane sterol found in fungi but not in plants, was used to estimate live mycelial biomass in ectomycorrhizae. Loblolly pine (Pinus taeda L.) seeds were sown in April 1993 and grown with standard nursery culture practices. Correlations between total seedling ergosterol and visual assessment of mycorrhizal colonization were high during July and August but low as ectomycorrhizal development continued into the growing season. Percentages of mycelial dry weight over lateral roots decreased from 9% in July to 2.5% in November because seedling lateral root dry weight accumulated faster than mycelial dry weight. Total ergosterol per seedling increased from July through February. As lateral root dry weight ceased to increase during winter months, ectomycorrhizal mycelia became the major carbohydrate sink of pine seedlings. No distinctive seasonal pattern of soil ergosterol content was observed. The impact of ectomycorrhizal fungi on plant carbohydrate source-sink dynamics can be quantitatively estimated with ergosterol analysis but not with conventional visual determination.     

Polyethylene glycol-promoted development of somatic embryos in loblolly pine (Pinus taeda L.)

Summary The effect of polyethylene glycol (PEG) combined with abscisic acid (ABA) and KCl on somatic embryo development in loblolly pine was investigated. Two embryogenic cell lines, which had not produced cotyledonary stage embryos using previously published methods, were employed in this study. As a maturation medium, basal medium was supplemented with 0 to 10% PEG (MW 3350), 10 to 40 mg/l (37.8 to 151.3 μM) ABA, 0 or 10 mM KCl, 1.5 g/l activated charcoal, 30 g/l sucrose, and 6 g/l agar. Without PEG in the maturation medium, most somatic embryos at stage 1 could not mature further. Embryogenic tissues on the maturation medium with 5 to 7.5% PEG consistently produced stage 2 and stage 3 somatic embryos. PEG at 10% significantly decreased the number of stage 2 and 3 embryos compared to PEG at 5 to 7.5% ABA at 40 mg/l (151.3 μM), combined with 7.5% PEG and 10 mM KCl, gave the maximum number of stage 3 embryos in both cell lines. ABA at 10 mg/l (37.8 μM) induced an over-proliferation of embryogenic tissues and generally failed to produce mature embryos. KCl also significantly enhanced initial stage embryo formation and subsequent embryo maturation.     

Natural hybridization within seed sources of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf and loblolly pine trees (n = 93 and 102, respectively) from 22 seed sources of the Southwide Southern Pine Seed Source Study plantings or equivalent origin were evaluated for amplified fragment length polymorphism (AFLP) variation. These sampled trees represent shortleaf pine and loblolly pine, as they existed across their native geographic ranges before intensive forest management. Using 17 primer pairs, a total of 96 AFLPs between shortleaf pine and loblolly pine were produced and scored on the sample trees and two control-pollinated F1 interspecies hybrids and their parents. In addition, the well known isocitrate dehydrogenase (IDH) isozyme marker was scored for all trees. IDH detected two putative hybrids among the loblolly pine samples and two among the shortleaf pine samples, while either 13 or 12 putative hybrids were detected using all AFLP markers and IDH and either NewHybrids or Structure software, respectively. Results of this study show that later generation hybrids can be reliably identified using AFLP markers and confirmed that IDH is not a definitive marker for detecting hybrids; that is, at least in some seed sources, the alternative species’ IDH allele resides in the source species. Based on all the markers, hybridization frequency varied geographically, ranging from 30% in an Arkansas seed source to 0% in several other seed sources. The hybridization level was higher in populations west of the Mississippi River than in populations east of the river; the shortleaf pine hybridization rates were 16.3% and 2.4% and the loblolly pine rates were 4.5% and 3.3%, west and east of the river, respectively. The results suggest that hybridization between these two species is significant but varies by seed source and species, and the potential for the unintended creation of hybrids should be considered in forest management decisions regarding both natural and artificial regeneration.     

Genetic diversity within and among populations of shortleaf pine (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.)

Shortleaf pine (n = 93) and loblolly pine (n = 112) trees representing 22 seed sources or 16 physiographic populations were sampled from Southwide Southern Pine Seed Source Study plantings located in Oklahoma, Arkansas, and Mississippi. The sampled trees were grown from shortleaf pine and loblolly pine seeds formed in 1951 and 1952, prior to the start of intensive forest management across their native ranges. Amplification fragment length polymorphism (AFLP) markers were developed and used to study genetic diversity and its structure in these pine species. After screening 48 primer pairs, 17 and 21 pairs were selected that produced 794 and 647 AFLPs in shortleaf pine and loblolly pine, respectively. High-AFLP-based genetic diversity exists within shortleaf pine and loblolly pine, and most (84.73% in shortleaf pine; 87.69% in loblolly pine) of this diversity is maintained within physiographic populations. The high value of unbiased measures of genetic identity and low value of genetic distance for all pairwise comparisons indicates that the populations have similar genetic structures. For shortleaf pine, there was no significant correlation between geographic distance and genetic distance (r = 0.28), while for loblolly pine there was a weak but significant correlation (r = 0.51).     

Changes in leaf expansion and epidermal screening effectiveness in Liquidambar styraciflua and Pinus taeda in response to UV-B radiation

Absorption or screening of ultraviolet-B (UV-B) radiation by the epidermis may be an important protective method by which plants avoid damage upon exposure to potentially harmful UV-B radiation. In the present study we examined the relationships among epidermal screening effectiveness, concentration of UV-absorbing compounds, epidermal anatomy and growth responses in seedlings of loblolly pine (Pinus taeda L.) and sweetgum (Liquidambar styraciflua L.). Seedlings of each species were grown in a greenhouse at the University of Maryland under either no UV-B radiation or daily supplemental UV-B radiation levels of 4, 8 or 11 kJ m^?2 of biologically effective UV-B (UV-B_BE) radiation. Loblolly pine seedlings were subsequently grown in the field under either ambient or supplemental levels of UV-B radiation. At the conclusion of the growing season, measurements of epidermal UV-B screening effectiveness were made with a fiber-optic microprobe. In loblolly pine, less than 0.5% of incident UV-B radiation was transmitted through the epidermis of fascicle needles and about 1% was transmitted in primary needles. In contrast, epidermal transmittance in sweetgum ranged from about 20% in leaves not preconditioned to UV-B exposure, to about 10% in leaves grown under UV-B radiation. The concentration of UV-absorbing compounds was unaffected by UV-B exposure, but generally increased with leaf age. Increases in epidermal thickness were observed in response to UV-B treatment in loblolly pine, and this accounted for over half of the variability in UV-B screening effectiveness. In spite of the low levels of UV-B penetration into the mesophyll, delays in leaf development (both species) and final needle size (loblolly pine) were observed. Seedling biomass was reduced by supplemental UV-B radiation in loblolly pine. We hypothesize that the UV-induced growth reductions were manifested by changes in either epidermal anatomy or epidermal secondary chemistry that might negatively impact cell elongation.     

The effects of drought and disturbance on the growth and developmental instability of loblolly pine (Pinus taeda L.)

Ecological indicators provide early warning of adverse environmental change, helping land managers adaptively manage their resources while minimizing costly remediation. In 1999 and 2000, we studied two such indicators, growth and developmental instability, of loblolly pine (Pinus taeda L.) influenced by mechanized infantry training at Fort Benning, Georgia. Disturbed areas were used for military training; tracked and wheeled vehicles damaged vegetation and soils. Highly disturbed sites had fewer trees, diminished ground cover, warmer soils in the summer, and more compacted soils with a shallower A-horizon. We hypothesized that disturbance would decrease the growth of needles, branches, and tree rings, increase the complexity of tree rings, and increase the developmental instability of needles. Contrary to our expectations, however, disturbance enhanced growth in the first year of the study, possibly by reducing competition. In the second year, a drought reduced growth of branches and needles, eliminating the stimulatory effect of disturbance. Growth-ring widths increased with growing-season precipitation, and decreased with growing-season temperature over the last 40 years. Disturbance had no effect on tree-ring complexity, as measured by the Hurst exponent. Within-fascicle variation of current-year needle length, a measure of developmental instability, differed among the study populations, but appeared unrelated to mechanical disturbance or drought.