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CO2-enrichment and nutrient availability alter ectomycorrhizal fungal communities.

Ectomycorrhizal fungi (EMF), a phylogenetically and physiologically diverse guild, form symbiotic associations with many trees and greatly enhance their uptake of nutrients and water. Elevated CO2, which increases plant carbon supply and demand for mineral nutrients, may change the composition of the EMF community, possibly altering nutrient uptake and ultimately forest productivity. To assess CO2 effects on EMF communities, we sampled mycorrhizae from the FACTS-I (Forest-Atmosphere Carbon Transfer and Storage) research site in Duke Forest, Orange County, North Carolina, USA, where Pinus taeda forest plots are maintained at either ambient or elevated CO2 (200 ppm above ambient) concentrations. Mycorrhizae were identified by DNA sequence similarity of the internal transcribed spacer ribosomal RNA gene region. EMF richness was very high; 72 distinct phylotypes were detected from 411 mycorrhizal samples. Overall EMF richness and diversity were not affected by elevated CO2, but increased CO2 concentrations altered the relative abundances of particular EMF taxa colonizing fine roots, increased prevalence of unique EMF species, and led to greater EMF community dissimilarity among individual study plots. Natural variation among plots in mean potential net nitrogen (N) mineralization rates was a key determinant of EMF community structure; increasing net N mineralization rate was negatively correlated with EMF richness and had differential effects on the abundance of particular EMF taxa. Our results predict that, at CO2 concentrations comparable to that predicted for the year 2050, EMF community composition and structure will change, but diversity will be maintained. In contrast, high soil N concentrations can negatively affect EMF diversity; this underscores the importance of considering CO2 effects on forest ecosystems in the context of background soil chemical parameters and other environmental perturbations such as acid deposition or fertilizer runoff.

Biodiversity↗

Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase.

Cytochrome P450 monooxygenases (P450s) are important enzymes for generating some of the enormous structural diversity of plant terpenoid secondary metabolites. In conifers, P450s are involved in the formation of a suite of diterpene resin acids (DRAs). Despite their important role in constitutive and induced oleoresin defense, a P450 gene of DRA formation has not yet been identified. By using phylogenetic cluster analysis of P450-like ESTs from loblolly pine (Pinus taeda), functional cDNA screening in yeast (Saccharomyces cerevisiae), and in vitro enzyme characterization, we cloned and identified a multifunctional and multisubstrate cytochrome P450 enzyme, CYP720B1 [abietadienol/abietadienal oxidase (PtAO)]. PtAO catalyzes an array of consecutive oxidation steps with several different diterpenol and diterpenal intermediates in loblolly pine DRA biosynthesis. Recombinant PtAO oxidized the respective carbon 18 of abietadienol, abietadienal, levopimaradienol, isopimara-7,15-dienol, isopimara-7,15-dienal, dehydroabietadienol, and dehydroabietadienal with apparent Michaelis-Menten (K(m)) values of 0.5-5.3 muM. PtAO expressed in yeast also catalyzed in vivo oxidation of abietadiene to abietic acid, but with activity much lower than with abietadienol or abietadienal. Consistent with a role of DRAs in conifer defense, PtAO transcript levels increased upon simulated insect attack using methyl jasmonate treatment of loblolly pine. The multisubstrate, multifunctional P450 diterpene oxidase PtAO, in concert with expression of a family of single-product and multiproduct diterpene synthases, allows for formation of a diverse suite of DRA defense metabolites in long-lived conifers.

Abietanes↗

Possible explanation of the disparity between the in vitro and in vivo measurements of Rubisco activity: a study in loblolly pine grown in elevated pCO2.

Rubisco activity can be measured using gas exchange (in vivo) or using in vitro methods. Commonly in vitro methods yield activities that are less than those obtained in vivo. Rubisco activity was measured both in vivo and in vitro using a spectrophotometric technique in mature Pinus taeda L. (loblolly pine) trees grown using free-air CO2 enrichment in elevated (56 Pa) and current (36 Pa) pCO2. In addition, for studies where both in vivo and in vitro values of Rubisco activity were reported net CO2 uptake rate (A) was modelled based on the in vivo and in vitro values of Rubisco activity reported in the literature. Both the modelling exercise and the experimental data showed that the in vitro values of Rubisco activity were insufficient to account for the observed values of A. A trichloroacetic acid (TCA) precipitation of the protein from samples taken in parallel with those used for activity analysis was co-electrophoresed with the extract used for determining in vitro Rubisco activity. There was significantly more Rubisco present in the TCA precipitated samples, suggesting that the underestimation of Rubisco activity in vitro was attributable to an insufficient extraction of Rubisco protein prior to activity analysis. Correction of in vitro values to account for the under-represented Rubisco yielded mechanistically valid values for Rubisco activity. However, despite the low absolute values for Rubisco activity determined in vitro, the trends reported with CO2 treatment concurred with, and were of equal magnitude to, those observed in Rubisco activity measured in vivo.

Carbon Dioxide↗

Xylem-specific gene expression in loblolly pine.

Two genes preferentially expressed in differentiating xylem of loblolly pine (Pinus taeda L.) were cloned from cDNA and genomic libraries and designated PtX3H6 and PtX14A9. Transcripts of PtX3H6 and PtX14A9 are very abundant in differentiating xylem, less abundant in needles, and very low or non-detectable in embryos and megagametophytes. PtX3H6 contains a putative signal peptide, a threonine-rich region, a proline-rich region, and a hydrophobic tail. Repeats of Pro-Pro-Pro-Val-X-X are similar to repeats found in proline-rich cell wall proteins. The amino acid compositions of PtX3H6 and PtX14A9 are similar to those of arabinogalactan proteins (AGPs). PtX14A9 contains an 8 amino acid sequence similar to amino terminal sequences of ryegrass, carrot and rose AGPs. Upstream sequences have been determined from genomic clones encoding PtX3H6 and PtX14A9. A 7 bp sequence found in the 5' flanking regions of both genes has previously been shown to be involved in the vascular-specific expression of GRP 1.8, a glycine-rich protein found in bean. The sequence is also present upstream of another glycine-rich protein from bean, GRP 1.0, and may be partially responsible for the xylem-specific expression of pTx3H6 and PtX14A9.

Amino Acid Sequence↗

Aluminum fractions in root tips of slash pine and loblolly pine families differing in Al resistance.

Aluminum (Al) distribution among several cellular fractions was investigated in root tips of seedlings of one Al-resistant and one Al-sensitive family of slash pine (Pinus elliottii Engelm.) and loblolly pine (Pinus taeda L.) grown in nutrient solution containing 100 microM AlCl3 (pH 4) for 167 h. Aluminum present in 5-mm-long root tips was fractionated into cell-wall-labile (desorbed in 0.5 mM citric acid), cell-wall-bound (retained after filtering disrupted cells through 20-microm mesh) and symplasmic (filtrate following cell disruption) fractions. When averaged across both species, 12% of Al absorbed by root tips appeared in the symplasmic fraction and 88% in the apoplasmic fraction (55% as cell-wall-labile, and 33% as cell-wall-bound). On a fresh mass basis, total Al in root tips was lower in loblolly pine than in slash pine, lower in the Al-resistant slash pine family than in the Al-sensitive slash pine family, and lower in the Al-resistant families than in the Al-sensitive families across species. Although the data support the hypothesis that Al-resistant plants limit Al uptake to root apices, they do not exclude other mechanisms of Al resistance. Differential Al resistance between the species and between slash pine families may also be associated with the size of the total non-labile and cell-wall-labile Al fractions, respectively. We were unable to identify the basis for differential Al resistance in loblolly pine.

Aluminum↗

Functional genomics and cell wall biosynthesis in loblolly pine.

Loblolly pine (Pinus taeda L.) is the most widely planted tree species in the USA and an important tree in commercial forestry world-wide. The large genome size and long generation time of this species present obstacles to both breeding and molecular genetic analysis. Gene discovery by partial DNA sequence determination of cDNA clones is an effective means of building a knowledge base for molecular investigations of mechanisms governing aspects of pine growth and development, including the commercially relevant properties of secondary cell walls in wood. Microarray experiments utilizing pine cDNA clones can be used to gain additional information about the potential roles of expressed genes in wood formation. Different methods have been used to analyze data from first-generation pine microarrays, with differing degrees of success. Disparities in predictions of differential gene expression between cDNA sequencing experiments and microarray experiments arise from differences in the nature of the respective analyses, but both approaches provide lists of candidate genes which should be further investigated for potential roles in cell wall formation in differentiating pine secondary xylem. Some of these genes seem to be specific to pine, while others also occur in model plants such as Arabidopsis, where they could be more efficiently investigated.

Arabidopsis↗

Comparison of standard exponential and linear techniques to amplify small cDNA samples for microarrays.

BACKGROUND: The need to perform microarray experiments with small amounts of tissue has led to the development of several protocols for amplifying the target transcripts. The use of different amplification protocols could affect the comparability of microarray experiments. RESULTS: Here we compare expression data from Pinus taeda cDNA microarrays using transcripts amplified either exponentially by PCR or linearly by T7 transcription. The amplified transcripts vary significantly in estimated length, GC content and expression depending on amplification technique. Amplification by T7 RNA polymerase gives transcripts with a greater range of lengths, greater estimated mean length, and greater variation of expression levels, but lower average GC content, than those from PCR amplification. For genes with significantly higher expression after T7 transcription than after PCR, the transcripts were 27% longer and had about 2 percentage units lower GC content. The correlation of expression intensities between technical repeats was high for both methods (R2 = 0.98) whereas the correlation of expression intensities using the different methods was considerably lower (R2 = 0.52). Correlation of expression intensities between amplified and unamplified transcripts were intermediate (R2 = 0.68-0.77). CONCLUSION: Amplification with T7 transcription better reflects the variation of the unamplified transcriptome than PCR based methods owing to the better representation of long transcripts. If transcripts of particular interest are known to have high GC content and are of limited length, however, PCR-based methods may be preferable.

Arabidopsis↗

Additional virulence genes and sonication enhance Agrobacterium tumefaciens-mediated loblolly pine transformation.

Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.). Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A. tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47. pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene. Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days. The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter. Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA. Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines. Stably transformed cultures and transgenic plants were produced from embryos transformed with A. tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine. The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses.

Agrobacterium tumefaciens↗

Improving loblolly pine somatic embryo maturation: comparison of somatic and zygotic embryo morphology, germination, and gene expression.

Clonal production of loblolly pine ( Pinus taeda L.) through somatic embryogenesis has the potential to meet the increasing industrial demands for high-quality uniform raw materials. A major barrier to the commercialization of this technology is the low quality of the resulting embryos. Twenty-five newly initiated loblolly pine genotypes were followed through the process of liquid culture establishment, embryo maturation, germination, and retrieval from cryogenic storage. A maturation medium, capable of promoting the development of loblolly pine somatic embryos that can germinate, is presented that combines 1/2 P6 modified salts, 2% maltose, 13% polyethylene glycol 8000 (PEG), 5 mg/l abscisic acid (ABA), and 2.5 g/l Gelrite. A procedure for converting and acclimating germinants to growth in soil and greenhouse conditions is also described. A set of somatic seedlings, produced from the maturation medium, showed 100% survival when planted in a field setting. Somatic seedlings showed normal yearly growth relative to standard seedlings from natural seed. The quality of the resulting embryos was examined and compared to that of zygotic embryos using such parameters as morphology, dry weight, germination performance, and gene expression. All of the observations that were made support the conclusion that even with the new maturation medium somatic embryos grow approximately only halfway through the normal sequence of development and then prematurely discontinue growth.

Abscisic Acid↗

Identification of quantitative trait loci influencing wood specific gravity in an outbred pedigree of loblolly pine.

We report the identification of quantitative trait loci (QTL) influencing wood specific gravity (WSG) in an outbred pedigree of loblolly pine (Pinus taeda L.). QTL mapping in an outcrossing species is complicated by the presence of multiple alleles (> 2) at QTL and marker loci. Multiple alleles at QTL allow the examination of interaction among alleles at QTL (deviation from additive gene action). Restriction fragment length polymorphism (RFLP) marker genotypes and wood specific gravity phenotypes were determined for 177 progeny. Two RFLP linkage maps were constructed, representing maternal and paternal parent gamete segregations as inferred from diploid progeny RFLP genotypes. RFLP loci segregating for multiple alleles were vital for aligning the two maps. Each RFLP locus was assayed for cosegregation with WSG QTL using analysis of variance (ANOVA). Five regions of the genome contained one or more RFLP loci showing differences in mean WSG at or below the P = 0.05 level for progeny as grouped by RFLP genotype. One region contained a marker locus (S6a) whose QTL-associated effects were highly significant (P > 0.0002). Marker S6a segregated for multiple alleles, a prerequisite for determining the number of alleles segregating at the linked QTL and analyzing the interactions among QTL alleles. The QTL associated with marker S6a appeared to be segregating for multiple alleles which interacted with each other and with environments. No evidence for digenic epistasis was found among the five QTL.

Alleles↗

Semiochemicals from fungal associates of bark beetles may mediate host location behavior of parasitoids.

In laboratory olfactometer bioassays, females of two hymenopteran parasitoid species, Roptrocerus xylophagorum (Ratzeburg) (Hymenoptera: Pteromalidae) and Spathius pallidus (Ashmead) (Hymenoptera: Braconidae), were attracted to odors from bark or bolts of loblolly pine, Pinus taeda L., colonized by bluestain fungi (genus Ophiostoma) associated with the parasitoids' bark beetle hosts. Mock-inoculated bolts and bark were less attractive or unattractive in these bioassays. Bark infested with host larvae that lacked their fungal and other normal microbial associates was attractive to R. xylophagorum females, but was less so than bark infested with larvae possessing their normal complement of associated microbes. In contrast, in oviposition bioassays, R. xylophagorum females spent approximately equal time searching, made similar numbers of oviposition attempts, parasitized similar percentages of hosts, and laid similar numbers of eggs in bark fragments infested with either associate-free or associate-bearing host larvae. Furthermore, in field bioassays using bluestain-inoculated or mock-inoculated loblolly pine bolts as sources of attractants, the numbers of parasitoids attracted by the two treatments did not differ significantly and the two treatments were less attractive than bolts naturally infested with bark beetle larvae. Whereas our laboratory olfactometer data suggest that bark beetle fungal associates may enhance attraction of some parasitoids, our bioassays with associate-free hosts indicate that associate-produced are not required for short-range host location and parasitization. In addition, our field trials indicated that long-range attraction of parasitoids to the host-fungi-tree complex is not caused simply by an interaction between bluestain fungi and tree tissues.

Animals↗

An arabinogalactan protein associated with secondary cell wall formation in differentiating xylem of loblolly pine.

Arabinogalactan proteins (AGPs) are abundant plant proteoglycans implicated in plant growth and development. Here, we report the genetic characterization, partial purification and immunolocalization of a classical AGP (PtaAGP6, accession number AF101785) in loblolly pine (Pinus taeda L.). A PtaAGP6 full-length cDNA clone was expressed in bacteria. PtaAGP6 resembles tomato LeAGP-1 and Arabidopsis AtAGP17-19 in that they all possess a subdomain composed of basic amino acids. The accessibility of this domain in the glycoprotein makes it possible to label the PtaAGP6 epitopes on the cell surface or in the cell wall with polyclonal antibodies raised against this subdomain. The antibodies recognize the peptide of the basic subdomain and bind to the intact protein molecule. A soluble protein-containing fraction was purified from the differentiating xylem of pine trees by using beta-glucosyl Yariv reagent (beta-glcY) and was recognized by antibodies against the basic subdomain. Immunolocalization studies showed that the PtaAGP6 epitopes are restricted to a file of cells that just precede secondary cell wall thickening, suggesting roles in xylem differentiation and wood formation. The location of apparent labeling of the PtaAGP6 epitopes is separated from the location of lignin deposition. Multiple single nucleotide polymorphisms (SNPs) were detected in EST variants. Denaturing HPLC analysis of PCR products suggests that PtaAGP6 is encoded by a single gene. Mobility variation in denaturing gel electrophoresis was used to map PtaAGP6 SNPs to a site on linkage group 5.

Amino Acid Sequence↗

Photosynthetic acclimation is reflected in specific patterns of gene expression in drought-stressed loblolly pine.

Because the product of a single gene can influence many aspects of plant growth and development, it is necessary to understand how gene products act in concert and upon each other to effect adaptive changes to stressful conditions. We conducted experiments to improve our understanding of the responses of loblolly pine (Pinus taeda) to drought stress. Water was withheld from rooted plantlets of to a measured water potential of -1 MPa for mild stress and -1.5 MPa for severe stress. Net photosynthesis was measured for each level of stress. RNA was isolated from needles and used in hybridizations against a microarray consisting of 2173 cDNA clones from five pine expressed sequence tag libraries. Gene expression was estimated using a two-stage mixed linear model. Subsequently, data mining via inductive logic programming identified rules (relationships) among gene expression, treatments, and functional categories. Changes in RNA transcript profiles of loblolly pine due to drought stress were correlated with physiological data reflecting photosynthetic acclimation to mild stress or photosynthetic failure during severe stress. Analysis of transcript profiles indicated that there are distinct patterns of expression related to the two levels of stress. Genes encoding heat shock proteins, late embryogenic-abundant proteins, enzymes from the aromatic acid and flavonoid biosynthetic pathways, and from carbon metabolism showed distinctive responses associated with acclimation. Five genes shown to have different transcript levels in response to either mild or severe stress were chosen for further analysis using real-time polymerase chain reaction. The real-time polymerase chain reaction results were in good agreement with those obtained on microarrays.

Acclimatization↗

Brassinolide improves embryogenic tissue initiation in conifers and rice.

Somatic embryogenesis (SE), the most promising technology for the large-scale production of high-value coniferous trees from advanced breeding and genetic engineering programs, is expected to play an important role in increasing productivity, sustainability, and the uniformity of future U.S. forests. To be successful for commercial use, SE technology must work with a variety of genetically diverse trees. Initiation in loblolly pine ( Pinus taeda L.), our main focus species, is often recalcitrant for desirable genotypes. Initiation percentages of loblolly pine, Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco], and Norway spruce ( Picea abies L., Karst.) were improved through the use of brassinolide. Brassinosteroids, which include brassinolide, are a relatively new group of natural plant growth regulators that are found in many plant species. They have been shown to have diverse, tissue-specific, and species-specific effects, including the stimulation of cell elongation and ethylene production and increasing resistance to abiotic stress. In our media, brassinolide was effective at concentrations ranging from 0.005-0.25 micro M. Using control medium (no brassinolide) and brassinolide-supplemented (0.1 micro M) medium, we achieved improved initiation percentages in loblolly pine, Douglas-fir, Norway spruce, and rice-15.0% to 30.1%, 16.1% to 36.3%, 34.6% to 47.4%, and 10%, respectively. Brassinolide increased the weight of loblolly pine embryogenic tissue by 66% and stimulated initiation in the more recalcitrant families of loblolly pine and Douglas-fir, thus compensating somewhat for genotypic differences in initiation. Initiation percentages in loblolly pine were improved through the combination of modified 1/2-P6 salts, 50 mg/l activated carbon (AC), adjusted levels of Cu and Zn (to compensate for adsorption by AC), 1.5% maltose, 2% myo-inositol (to raise the osmotic level, partially simulating the megagametophyte environment), 500 mg/l casamino acids, 450 mg/l glutamine, 2 mg/l alpha-naphthaleneacetic acid, 0.63 mg/l 6-benzylaminopurine, 0.61 mg/l kinetin, 3.4 mg/l silver nitrate, 10 micro M cGMP, 0.1 micro M brassinolide, and 2 g/l Gelrite. Across 12 open-pollinated families of loblolly pine, initiation percentages ranged from 2.5% to 50.7%, averaging 22.5%.

Brassinosteroids↗

Enzymatic properties of two beta-glucosidases from Ceriporiopsis subvermispora produced in biopulping conditions.

AIMS: Ceriporiopsis subvermispora produces endoglucanase and beta-glucosidase when cultivated on cellulose or wood, but biodegradation of cellulose during biopulping by C. subvermispora is low even after long periods. To resolve this discrepancy, we grew C. subvermispora on Pinus taeda wood chips and purified the major beta-glucosidases it produced. Kinetic parameters were determined to clear if this fungus produces enzymes capable of yielding assimilable glucose from wood. METHODS AND RESULTS: Ceriporiopsis subvermispora was grown on P. taeda wood chips under solid-state fermentation. After 30 days, the crude extract obtained from enzyme extraction with sodium acetate buffer 50 mmol l(-1), pH 5.4, was filtrated in membranes with a molecular mass exclusion limit of 100 kDa. Enzyme purification was carried out using successively Sephacryl S-300 gel filtration. The retained fraction attained 76% of beta-glucosidase activity with 3.7-fold purification. Two beta-glucosidases were detected with molecular mass of 110 and 53 kDa. We have performed a characterization of the enzymatic properties of the beta-glucosidase of 110 kDa. The optimum pH and temperature were 3.5 and 60 degrees C, respectively. The K(m) and V(max) values were respectively 3.29 mmol l(-1) and 0.113 micromol min(-1) for the hydrolysis of p-nitrophenyl-beta-glucopyranoside (pNPG) and 2.63 mmol l(-1) and 0.103 micromol min(-1), towards cellobiose. beta-Glucosidase activity was strongly increased by Mn(2+) and Fe(3+), while Cu(2+) severely inhibited it. CONCLUSIONS: Ceriporiopsis subvermispora produces small amounts of beta-glucosidase when grown on wood. The gel filtration and polyacrylamide gel electrophoresis data revealed the existence of two beta-glucosidases with 110 and 53 kDa. The 110 kDa beta-glucosidase from C. subvermispora can be efficiently purified in a single step by gel filtration chromatography. The enzyme has an acid pH optimum with similar activity on pNPG and cellobiose and is thus typical beta-glucosidase. SIGNIFICANCE AND IMPACT OF THE STUDY: Ceriporiopsis subvermispora produces beta-glucosidase with limited action during wood decay making able its use for the production of biomechanical and biochemical pulps. The results presented in this paper show the importance of studying the behaviour of beta-glucosidases during biopulping.

Basidiomycota↗

Growth and differentiation of transgenic callus regulated by phytohormones and antibiotics in transformation of loblolly pine.

Mature zygotic embryos of loblolly pine (Pinus taeda L.) were transformed by Agrobacterium tumefaciens strain LBA 4404 harbouring the plasmid pBI121 which carried the selectable marker gene, neomycin phosphotransferase II (npt II) controlled by the promoter of the nopaline synthase gene, and the uidA reporter gene, encoding beta-glucuronidase (GUS) driven by the cauliflower mosaic virus 35S promoter. Organogenic transgenic calli and transgenic regenerated plantlets were produced on selection medium containing 15 mg/L kanamycin, and confirmed by GUS histochemical staining, polymerase chain reaction (PCR), and southern blot analysis. Influences of phytohormone (BA/IBA) and antibiotics on growth and differentiation of organogenic transgenic calli were investigated. Of the phytohormone (BA/IBA) and antibiotics administered, 500 mg/L carbenicillin combined with 2 mg/L BA and 0.5 mg/L IBA (BA/IBA = 4) resulted in a 54.2% higher increase in the growth of transgenic calli as well as in the differentiation of transgenic calli, which was 45.7% more than that of control on the 6th week of culture. Claforan at 500 mg/L combined with 2 mg/L BA and 0.5 mg/L IBA resulted in a 40.8% increase in the growth of transgenic calli, and 38.7% increase in the frequency of transgenic calli forming adventitious shoots compared with the control. The growth and differentiation of transgenic calli of loblolly pine was reduced preferentially by higher BA/IBA (BA/IBA = 8), as well as high concentration of antibiotics (carbenicillin and claforan, 550 mg/L each). But it was observed that 450 mg/L and 500 mg/L carbenicillin and claforan caused an increase in growth and differentiation of transgenic calli. These results suggested that the establishment of an efficient Agrobacterium tumefaciens-mediated transformation protocol for stable integration of foreign genes into loblolly pine was also dependent on the regulation of phytohormone and antibiotic on growth and differentiation of transgenic calli. This work could be useful for the future studies of genetic transformation of conifers.

Anti-Bacterial Agents↗

Effects of phenology, water availability and seed source on loblolly pine biomass partitioning and transpiration.

One-year-old loblolly pine (Pinus taeda L.) seedlings from four seed sources (Arkansas, Georgia, Texas and Virginia) grown in 1-m-deep sand-filled pits in two water regimes (well-watered and drought) were studied, to gain insight into the process of seedling establishment. Whole-plant transpiration was measured biweekly from July to December. Whole-plant harvests were conducted at 6-week intervals from April to December. Whole-plant transpiration and transpiration per unit leaf and root area were affected by treatment, seedlot and phenology. Seedlings of the Arkansas seedlot maintained significantly higher transpiration rates per unit leaf and root area during drought than seedlings of the Virginia, Georgia or Texas seedlots, but did not accumulate greater biomass. The high transpiration rates of the Arkansas seedlings were attributed to their deep root systems. Allometric relationships indicated that, relative to the whole plant, biomass allocation to needles of drought-treated seedlings was enhanced during the summer (allometric ratio 1.09), whereas allocation to roots was enhanced in the spring and fall (allometric ratios of 1.13 and 1.09, respectively). Relative to the whole plant, biomass allocation to needles of well-watered seedlings was enhanced throughout the experiment (allometric ratio of 1.16 declining to 1.05), whereas the allometric ratio of root to total biomass was 0.89 or less throughout. Allometric relationships also indicated variation in biomass partitioning to roots in three soil layers (0-30, 30-60 and 60-100 cm), which differed among harvests in each soil layer. Root growth in both well-watered and drought-treated seedlings was concentrated in the top soil layer in the spring, shifted to the middle and bottom soil layers in the summer, and then increased in the top soil layer in the fall. Compared with well-watered seedlings, drought-treated seedlings had higher rates of root growth in the bottom soil layer in the fall, a characteristic that would confer tolerance to future periods of limited soil water availability.

Biomass↗

Stem growth and respiration in loblolly pine plantations differing in soil resource availability.

Stem respiration and growth in 10-year-old loblolly pine (Pinus taeda L.) plantations were measured monthly during the third year of fertilization and irrigation treatments to determine whether soil resource availability differentially altered growth and respiration in stem tissue. Fertilized trees had significantly greater stem biomass, stem nitrogen concentration ([N]) and growth rate than unfertilized trees. Stem respiration (Rt) was significantly greater in fertilized trees when expressed on a per unit surface area (Rt,a, micromol CO2 m-2 s-1), sapwood volume (Rt,v, micromol CO2 m-3 s-1), or mass (Rt,w, nmol CO2 g-1 s-1) basis; however, there was no difference between treatments when expressed as a function of stem N content (Rt,n, micromol CO2 (mol N)-1 s-1). Irrigation had no significant effect on Rt or annual stem growth. Daily total respiration (Rd, mol CO2 m-2 day-1) and stem diameter growth both had a seasonal bimodal pattern with peaks in early spring and midsummer. Stem [N] declined significantly during the growing season. Stem growth rate and [N] explained 75% of the seasonal variation in temperature-normalized Rt,a. The mature tissue method was used to partition total stem respiration (Rt) into maintenance (Rm) and growth (Rg) components. There was a linear correlation between winter Rt,v, a measure of basal Rm, and sapwood N content; however, Rt,v per unit N was greater in January before diameter growth started than in the following December after growth ceased, indicating that Rt,v declined as stem diameter increased. Consequently, estimates of annual maintenance respiration (RM) based on January data were 44% higher than estimates based on December data. Growth respiration was correlated with stem growth rate (r2 = 0.55). The growth respiration coefficient (rg)-the slope of the relationship between Rg and stem growth rate-was 0.24. Respiration accounted for 37% of annual stem carbon budget. Stem carbon-use efficiency (CUE)-the ratio of stem growth to stem growth plus respiration-averaged 0.63 and was unaffected by fertilization.

Cell Respiration↗