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Anchored reference loci in loblolly pine (Pinus taeda L.) for integrating pine genomics.

Anchored reference loci provide a framework for comparative mapping. They are landmarks to denote conserved chromosomal segments, allowing the synthesis of genetic maps from multiple sources. We evaluated 90 expressed sequence tag polymorphisms (ESTPs) from loblolly pine (Pinus taeda L.) for this function. Primer sets were assayed for amplification and polymorphism in six pedigrees, representing two subgenera of Pinus and a distant member of the Pinaceae, Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). On average, 89% of primer sets amplified in four species of subgenus Pinus, 49% in one species of subgenus Strobus, and 22% in Douglas-fir. Polymorphisms were detected for 37-61% of the ESTPs within each pedigree. Comparative mapping in loblolly and slash pine (P. elliottii Englm.) revealed that ESTPs mapped to the same location. Disrupted synteny or significant disruptions in colinearity were not detected. Thirty-five ESTPs met criteria established for anchor loci. The majority of those that did not meet these criteria were excluded when map location was known in only a single species. Anchor loci provide a unifying tool for the community, facilitating the creation of a "generic" pine map and serving as a foundation for studies on genome organization and evolution.

Base Sequence↗

Characterization of two laccases of loblolly pine (Pinus taeda) expressed in tobacco BY-2 cells.

We previously showed that eight laccase genes (Lac 1-Lac 8) are preferentially expressed in differentiating xylem and are associated with lignification in loblolly pine (Pinus taeda) [Sato et al. (2001) J Plant Res 114:147-155]. In this study we generated transgenic tobacco suspension cell cultures that express the pine Lac 1 and Lac 2 proteins, and characterized the abilities of these proteins to oxidize monolignols. Lac 1 and Lac 2 enzymatic activities were detected only in the cell walls of transgenic tobacco cells, and could be extracted with high salt. The optimum pH for laccase activity with coniferyl alcohol as substrate was 5.0 for Lac 1 and between 5.0 and 6.0 for Lac 2. The activities of Lac 1 and Lac 2 increased as the concentration of CuSO(4) in the reaction mixtures increased in the range from 1 to 100 microM. Both enzymes were able to oxidize coniferyl alcohol and to produce dimers of coniferyl alcohol. These results are consistent with the hypothesis that Lac 1 and Lac 2 are involved in lignification in differentiating xylem of loblolly pine.

Cell Line, Transformed↗

Construction of an AFLP genetic map with nearly complete genome coverage in Pinus taeda.

De novo construction of complete genetic linkage maps requires large mapping populations, large numbers of genetic markers, and efficient algorithms for ordering markers and evaluating order confidence. We constructed a complete genetic map of an individual loblolly pine (Pinus taeda L.) using amplified fragment length polymorphism (AFLP) markers segregating in haploid megagametophytes and PGRI mapping software. We generated 521 polymorphic fragments from 21 AFLP primer pairs. A total of 508 fragments mapped to 12 linkage groups, which is equal to the Pinus haploid chromosome number. Bootstrap locus order matrices and recombination matrices generated by PGRI were used to select 184 framework markers that could be ordered confidently. Order support was also evaluated using log likelihood criteria in MAPMAKER. Optimal marker orders from PGRI and MAPMAKER were identical, but the implied reliability of orders differed greatly. The framework map provides nearly complete coverage of the genome, estimated at approximately 1700 cM in length using a modified estimator. This map should provide a useful framework for merging existing loblolly pine maps and adding multiallelic markers as they become available. Map coverage with dominant markers in both linkage phases will make the map useful for subsequent quantitative trait locus mapping in families derived by self-pollination.

Chromosome Mapping↗

Evaluation of major genetic loci contributing to inbreeding depression for survival and early growth in a selfed family of Pinus taeda.

The magnitude of fitness effects at genetic loci causing inbreeding depression at various life stages has been an important question in plant evolution. We used genetic mapping in a selfed family of loblolly pine (Pinus taeda L.) to gain insights on inbreeding depression for early growth and viability. Two quantitative trait loci (QTLs) were identified that explain much of the phenotypic variation in height growth through age 3 and may account for more than 13% inbreeding depression in this family. One of these QTLs maps to the location of cad-nl, a lignin biosynthesis mutation. Both QTLs show evidence of overdominance, although evidence for true versus pseudo-overdominance is inconclusive. Evidence of directional dominance for height growth was noted throughout the genome, suggesting that additional loci may contribute to inbreeding depression. A chlorophyll-deficiency mutation, spf did not appear to be associated with growth effects, but had significant effects on survival through age 3. Previously identified embryonic viability loci had little or no overall effect on germination, survival, or growth. Our results challenge, at least in part, the prevailing hypothesis that inbreeding depression for growth is due to alleles of small effect. However, our data support predictions that loci affecting inbreeding depression are largely stage specific.

Biological Evolution↗

Movement of respiratory CO(2) in stems of loblolly pine (Pinus taeda L.) seedlings.

Temperature-independent fluctuations in stem CO(2) efflux were measured in Pinus taeda L. seedlings. Stem CO(2) efflux was measured during high and low transpiration rates, high and low net photosynthesis rates, and normal and interrupted substrate supply conditions. Stem CO(2) efflux rates were an average of 6.7% lower during periods of high transpiration compared to periods of low transpiration. This difference in stem CO(2) efflux rates was not due to water stress. The most likely cause was movement of respiratory CO(2) in the transpiration stream. Interruption of substrate supply to the stem by phloem girdling reduced stem CO(2) efflux rates. Increasing net photosynthesis rates from low to high had no effect on stem CO(2) efflux, but decreasing net photosynthesis from high to low caused relatively small reductions in stem CO(2) efflux. These results indicate that diurnal changes in net photosynthesis rate may play a small role in temperature-independent afternoon depressions of stem CO(2) efflux. The transport of respiratory CO(2) by the transpiration stream compromises measurements of woody tissue respiration obtained by commonly accepted gas exchange techniques. This phenomenon could also affect measurement of leaf net photosynthesis and branch woody tissue respiration.

Journal Article↗

Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified delta-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud.

A synthetic version of the CRY1Ac gene of Bacillus thuringiensis has been used for the transformation of loblolly pine (Pinus taeda L.) using particle bombardment. Mature zygotic embryos were used to be bombarded and to generate organogenic callus and transgenic regenerated plants. Expression vector pB48.215 DNA contained a synthetic Bacillus thuringiensis (B.t.) CRY1Ac coding sequence flanked by the double cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator sequences, and the neomycin phosphotransferase II (NPTII) gene controlled by the promoter of the nopaline synthase gene was introduced into loblolly pine tissues by particle bombardment. The transformed tissues were proliferated and selected on media with kanamycin. Shoot regeneration was induced from the kanamycin-resistant calli, and transgenic plantlets were then produced. More than 60 transformed plants from independent transformation events were obtained for each loblolly pine genotype tested. The integration and expression of the introduced genes in the transgenic loblolly pine plants was confirmed by polymerase chain reactions (PCR) analysis, by Southern hybridization, by Northern blot analysis, and by Western blot analysis. Effective resistance of transgenic plants against Dendrolimus punctatus Walker and Crypyothelea formosicola Staud was verified in feeding bioassays with the insects. The transgenic plants recovered could represent a good opportunity to analyse the impact of genetic engineering of pine for sustainable resistance to pests using a B. thuringiensis insecticidal protein. This protocol enabled the routine transformation of loblolly pine plants that were previously difficult to transform.

Amino Acid Oxidoreductases↗

Mild ozone exposure alters (14)C dynamics in foliage of Pinus taeda L.

To explore the physiological mechanisms underlying ozone-induced growth reductions in loblolly pine (Pinus taeda L.), seedlings were exposed to sub-ambient (charcoal-filtered), ambient or twice-ambient ozone in open-top chambers for three growing seasons. In the final year of exposure, current-year needle fascicles were labeled with (14)CO(2) and the incorporation of (14)C into biochemical fractions was followed for 48 hours. Irrespective of ozone treatment, losses of (14)C-assimilates from foliage to respiration and translocation were minimal during the first 3 hours, whereas more than 60% of the label was lost during the next 45 hours. Radiolabel in sugar decreased rapidly after a lag period, roughly paralleling the pattern of total (14)C loss. The amount of (14)C label in starch and lipids plus pigments remained constant throughout the 48-hour chase period, whereas the amount of (14)C label in other fractions showed a net decrease over the 48-hour chase period. Ozone treatments altered foliar carbon dynamics in two ways: (1) ozone exposure increased foliar (14)C retention up to 21% for the first 5 hours after labeling, but not thereafter, and (2) ozone exposure decreased partitioning of (14)C into starch and increased partitioning of (14)C into organic acids, residue, and lipids plus pigments, indicating an intensified partitioning of carbon to injury and repair processes. Both short-term carbon retention and diversion of carbon from storage compounds to repair processes are foliar mechanisms by which ozone exposure could decrease growth in loblolly pine seedlings.

Journal Article↗

Utilization of polar metabolite profiling in the comparison of juvenile wood and compression wood in loblolly pine (Pinus taeda).

Juvenile wood (JW) of conifers is often associated with compression wood (CW), with which it is sometimes believed to be identical. To determine whether JW and CW can be distinguished metabolically, we compared gas chromatographic profiles of 25 polar metabolites from rooted cuttings of a single loblolly pine (Pinus taeda L.) clone raised in controlled environment chambers and subject to three treatments: (1) grown erect with minimal wind sway (control); (2) swayed by wind from oscillating fans; and (3) with 30-cm growth increments successively bent at an angle of 45 degrees to the vertical. Profiles were compared by principal component analysis. Substantial increases in abundances of coniferin and p-glucocoumaryl alcohol separated immature JW-forming xylem tissues of the control trees from the CW-forming xylem of the bent and swayed trees.

Carbohydrates↗

Regulation of loblolly pine (Pinus taeda L.) arginase in developing seedling tissue during germination and post-germinative growth.

After seed germination, hydrolysis of storage proteins provides a nitrogen source for the developing seedling. In conifers the majority of these reserves are located in the living haploid megagametophyte tissue. In the developing loblolly pine (Pinus taeda L.) seedling an influx of free amino acids from the megagametophyte accompanies germination and early seedling growth. The major component of this amino acid pool is arginine, which is transported rapidly and efficiently to the seedling without prior conversion. This arginine accounts for nearly half of the total nitrogen entering the cotyledons and is likely a defining factor in early seedling nitrogen metabolism. In the seedling, the enzyme arginase is responsible for liberating nitrogen, in the form of ornithine and urea, from free arginine supplied by the megagametophyte. In this report we investigate how the seedling uses arginase to cope with the large arginine influx. As part of this work we have cloned an arginase cDNA from a loblolly pine expression library. Analysis of enzyme activity data, accumulation of arginase protein and mRNA abundance indicates that increased arginase activity after seed germination is due to de novo synthesis of the enzyme. Our results suggest that arginase is primarily regulated at the RNA level during loblolly pine seed germination and post-germinative growth.

Amino Acid Sequence↗

Pinus taeda phenylpropenal double-bond reductase: purification, cDNA cloning, heterologous expression in Escherichia coli, and subcellular localization in P. taeda.

A phenylpropenal double-bond reductase (PPDBR) was obtained from cell suspension cultures of loblolly pine (Pinus taeda L.). Following trypsin digestion and amino acid sequencing, the cDNA encoding this protein was subsequently cloned, with the functional recombinant protein expressed in Escherichia coli and characterized. PPDBR readily converted both dehydrodiconiferyl and coniferyl aldehydes into dihydrodehydrodiconiferyl and dihydroconiferyl aldehydes, when NADPH was added as cofactor. However, it was unable to reduce directly either the double bond of dehydrodiconiferyl or coniferyl alcohols in the presence of NADPH. During this reductive step, the corresponding 4-proR hydrogen was abstracted from [4R-3H]-NADPH during hydride transfer. This is thus the first report of a double-bond reductase involved in phenylpropanoid metabolism, and which is presumed to be involved in plant defense. In situ mRNA hybridization indicated that the PPDBR transcripts in P. taeda stem sections were localized to the vascular cambium, as well as to radial and axial parenchyma cell types. Additionally, using P. taeda cell suspension culture crude protein extracts, dehydrodiconiferyl and coniferyl alcohols could be dehydrogenated to afford dehydrodiconiferyl and coniferyl aldehydes. Furthermore, these same extracts were able to convert dihydrodehydrodiconiferyl and dihydroconiferyl aldehydes into the corresponding alcohols. Taken together, these results indicate that in the crude extracts dehydrodiconiferyl and coniferyl alcohols can be converted to dihydrodehydrodiconiferyl and dihydroconiferyl alcohols through a three-step process, i.e. by initial phenylpropenol oxidation, then sequential PPDBR and phenylpropanal reductions, respectively.

Aldehyde Reductase↗

Stomatal and nonstomatal limitations to net photosynthesis in Pinus taeda L. under different environmental conditions.

Net photosynthesis, transpiration and stomatal conductance of two-year-old Pinus taeda L. seedlings were compared under various environmental conditions. Responses to air temperature, irradiance, ambient CO(2) concentration, absolute humidity deficit and xylem pressure potential were examined. The seedlings exhibited little response to a wide range of absolute humidity deficits (7 to 16 g m(-3)) and temperatures (20 to 35 degrees C), but were sensitive to changes in water deficit, irradiance and CO(2) concentration. Net photosynthesis and stomatal conductance were linearly related under all of the environmental conditions measured. However, the gas phase limitation to photosynthesis was generally small (20 to 30%). It was concluded that although the stomatal response was closely coupled to changes in photosynthesis, internal limitations, rather than the rate of gaseous diffusion of CO(2), were primarily responsible for limiting photosynthesis.

Journal Article↗

Ozone, acidic rain and soil magnesium effects on growth and foliar pigments of Pinus taeda L.

Height and diameter growth, biomass accumulation and leaf pigment concentrations were measured in loblolly pine (Pinus taeda L.) seedlings grown in soil containing 12 or 35 microg Mg g(-1) and exposed from May to October to subambient, ambient, or twice-ambient ozone (O(3)), and to simulated acidic rain with a pH of either 4.0 or 5.3. At the end of one growing season, height and diameter growth of seedlings exposed to twice-ambient O(3) were not statistically different from those of seedlings exposed to subambient O(3). Biomass of all plant parts was reduced by 7 to 16% in response to increasing O(3) concentration. No statistically significant growth responses to rain chemistry or soil magnesium status were observed, and there were no statistically significant interactive treatment effects. Needle pigment concentrations were not significantly affected by rain chemistry or soil Mg status and there were no visible signs of injury to needles that could be attributed to O(3) stress or Mg deficiency. Concentrations of chlorophyll a and b, and carotenes were 23, 30 and 21% higher (P </= 0.05) respectively, in seedlings exposed to twice-ambient O(3) than in seedlings from the other ozone treatments. There were no interactive treatment effects on needle pigment concentrations.

Journal Article↗

Modeling seed dispersal distances: implications for transgenic Pinus taeda.

Predicting forest-tree seed dispersal across a landscape is useful for estimating gene flow from genetically engineered (GE) or transgenic trees. The question of biocontainment has yet to be resolved, although field-trial permits for transgenic forest trees are on the rise. Most current field trials in the United States occur in the Southeast where Pinus taeda L., an indigenous species, is the major timber commodity. Seed dispersal distances were simulated using a model where the major determinants were: (1) forest canopy height at seed release, (2) terminal velocity of the seeds, (3) absolute seed release, and (4) turbulent-flow statistics, all of which were measured or determined within a P. taeda plantation established from seeds collected from wild forest-tree stands at the Duke Forest near Durham, North Carolina, USA. In plantations aged 16 and 25 years our model results showed that most of the seeds fell within local-neighborhood dispersal distances, with estimates ranging from 0.05 to 0.14 km from the source. A fraction of seeds was uplifted above the forest canopy and moved via the long-distance dispersal (LDD) process as far as 11.9-33.7 km. Out of 10(5) seeds produced per hectare per year, roughly 440 seeds were predicted to be uplifted by vertical eddies above the forest canopy and transported via LDD. Of these, 70 seeds/ha traveled distances in excess of 1 km from the source, a distance too great to serve as a biocontainment zone. The probability of LDD occurrence of transgenic conifer seeds at distances exceeding 1 km approached 100%.

Ecology↗

Mineral Nutrient Requirements of a Loblolly Pine (Pinus taeda) Cell Suspension Culture : Evaluation of a Medium Formulated from Seed Composition Data.

The mineral nutrient requirements of Pinus taeda cells were explored using quantitative cell culture growth measurements. An appraisal was thereby made of the critical features of a novel and successful medium which was developed specifically for this gymnosperm using chemical composition data for developing seeds, and characterized by generally high concentration of all micronutrients, high magnesium, and low calcium. The high magnesium concentration was found not to be detrimental and possibly beneficial whereas the calcium level bordered on a deficiency threshold. Within the microelements high iodide was found to be essential, as was a higher borate level than is present in media developed for angiosperms. High zinc concentrations were also beneficial, with normal levels permitting slower but nevertheless healthy growth. An improved medium was thereby formulated which was stress-free and exhibited broader genotype specificity. This new formulation has proved very successful in maintaining long-term growth of highly uniform and apparently meristematic suspension cultures of Pinus radiata.

Journal Article↗

Seasonal variation in gene expression for loblolly pines (Pinus taeda) from different geographical regions.

In developing xylem, gene expression levels vary in different genotypes, at different stages of development, throughout a growing season, and in response to stresses. Commercially important characteristics such as wood-specific gravity are known to differ with seed source. For example, when grown on a common site, the specific gravity of Arkansas loblolly pine (Pinus taeda L.) trees is greater than that of Louisiana loblolly pine, and Texas loblolly pines have a greater specific gravity than loblolly pines from the Atlantic coast. A microarray analysis was performed to examine variation in gene expression among trees from different geographical sources when grown on a common site, and seasonal variation in gene expression in each seed source. We used microarrays containing 2171 expressed sequence tags (ESTs) with putative functions of interest, selected from several loblolly pine xylem partial cDNA libraries and a shoot tip library. Genes with significant variation in expression for each factor were identified. Many genes preferentially expressed in latewood compared with earlywood were for proteins involved in cell wall biosynthesis. Variation in gene expression among trees from the two seed sources in each growing season suggests that there may be more differences between South Arkansas trees and South Louisiana trees in latewood than in earlywood. Variation in gene expression among trees from different regions may reflect adaptation to different environments.

Gene Expression Profiling↗

Purification and Characterization of Catalase from Loblolly Pine (Pinus taeda L.) Megagametophytes.

Catalase (EC 1.11.1.6) was purified to near homogeneity from isolated megagametophytes of germinated loblolly pine (Pinus taeda L.) seeds, and monospecific antibodies were elicited in rabbits. Following a procedure that involved acetone extraction, (NH4)2SO4 fractionation, and four chromatographic steps (i.e. DE-52 cellulose, Superdex-200, hydroxylapatite, and phenyl-Sepharose CL-4B), catalase was purified about 140-fold to a final specific activity of 2215 mmol min-1 mg-1 of protein. Cotton isocitrate lyase antibodies were used, and protein immunoblots revealed that the resolution on hydroxylapatite and phenyl-Sepharose allowed for the complete separation of catalase from contaminating isocitrate lyase. The molecular masses of the native enzyme and its subunit are 235 and 59 kD, respectively, indicating that the pine holoenzyme is a homotetramer. Loblolly pine catalase exists as multiple isoforms. When megagametophytes taken 7 d after imbibition at 30[deg]C were extracted, subjected to nondenaturing isoelectric focusing, and stained for catalase activity, at least four catalase isoforms were observed, including one dominant form with an isoelectric point of 6.87. Purified pine catalase is not a glycoprotein and has a ratio of absorbance at 208 nm to absorbance at 405 nm of 1.5. When probed with loblolly pine catalase antibodies, protein blots of cell-free extracts from megagametophytes of mature, stratified, and germinated loblolly pine seeds, the megagametophyte glyoxysomal fraction, and purified loblolly pine catalase all revealed one immunoreactive 59-kD polypeptide. This indicates that no detectable change in the enzyme's monomeric molecular mass occurs during seed stratification and germination, early seedling growth, and purification.

Journal Article↗

Influence of elevated CO2 and mycorrhizae on nitrogen acquisition: contrasting responses in Pinus taeda and Liquidambar styraciflua.

An understanding of root system capacity to acquire nitrogen (N) is critical in assessing the long-term growth impact of rising atmospheric CO2 concentration ([CO2]) on trees and forest ecosystems. We examined the effects of mycorrhizal inoculation and elevated [CO2] on root ammonium (NH4+) and nitrate (NO3-) uptake capacity in sweetgum (Liquidambar styraciflua L.) and loblolly pine (Pinus taeda L.). Mycorrhizal treatments included inoculation of seedlings with the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith in sweetgum and the ectomycorrhizal (EM) fungus Laccaria bicolor (Maire) Orton in loblolly pine. These plants were then equally divided between ambient and elevated [CO2] treatments. After 6 months of treatment, root systems of both species exhibited a greater uptake capacity for NH4+ than for NO3-. In both species, mycorrhizal inoculation significantly increased uptake capacity for NO3-, but not for NH4+. In sweetgum, the mycorrhizal effect on NO3- and NH4+ uptake capacity depended on growth [C02]. Similarly, in loblolly pine, the mycorrhizal effect on NO3- uptake capacity depended on growth [CO2], but the effect on NH4+ uptake capacity did not. Mycorrhizal inoculation significantly enhanced root nitrate reductase activity (NRA) in both species, but elevated [CO2] increased root NRA only in sweetgum. Leaf NRA in sweetgum did not change significantly with mycorrhizal inoculation, but increased in response to [CO2]. Leaf NRA in loblolly pine was unaffected by either treatment. The results indicate that the mycorrhizal effect on specific root N uptake in these species depends on both the form of inorganic N and the mycorrhizal type. However, our data show that in addressing N status of plants under high [CO2], reliable prediction is possible only when information about other root system adjustments (e.g., biomass allocation to fine roots) is simultaneously considered.

Basidiomycota↗

Loblolly pine (Pinus taeda L.) contains multiple expressed genes encoding light-dependent NADPH:protochlorophyllide oxidoreductase (POR).

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key regulatory step in chlorophyll biosynthesis. In most angiosperms, POR is encoded by a small nuclear gene family, containing at least two differentially-expressed genes designated porA and porB. We have demonstrated that the PORs of loblolly pine (Pinus taeda L.), a gymnosperm, are encoded by a large multigene family, composed of two distinct subfamilies encoding porA and porB genes similar to those previously described in angiosperms. DNA gel blot analysis of genomic DNA showed that the two por subfamilies of loblolly pine have duplicated at different rates, with the porA subfamily containing two members, and the porB subfamily containing at least 11 potential members. DNA sequence analysis and gel blot hybridization studies also showed that a subset of the por genes present in the loblolly pine genome are pseudogenes. Based on the results of 5'- and 3'-RACE analysis, it appears that multiple porA and porB genes are expressed in loblolly pine cotyledons and stems during development. Using gene specific probes, no difference was observed in the steady-state levels of the different porA and porB transcripts in cotyledons of dark-grown seedlings before and following illumination. However, the steady state levels of the porA and porB transcripts were found to increase at different rates in the stems of dark-grown seedlings following illumination. The phylogenetic relationship between the por gene family members in P. taeda and other pine species and the potential significance of the two por subfamilies to the evolution of por gene function are discussed.

Amino Acid Sequence↗