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The distribution of genetic parameter estimates and confidence intervals from small disconnected diallels.

The distributions of genetic variance components and their ratios (heritability and type-B genetic correlation) from 105 pairs of six-parent disconnected half-diallels of a breeding population of loblolly pine (Pinus taeda L.) were examined. A series of simulations based on these estimates were carried out to study the coverage accuracy of confidence intervals based on the usual t-method and several other alternative methods. Genetic variance estimates fluctuated greatly from one experiment to another. Both general combining ability variance (sigma(2) (g)) and specific combining ability variance (sigma(2) (s)) had a large positive skewness. For sigma(2) (g) and sigma(2) (s), a skewness-adjusted t-method proposed by Boos and Hughes-Oliver (Am Stat 54:121-128, 2000) provided better upper endpoint confidence intervals than t-intervals, whereas they were similar for the lower endpoint. Bootstrap BCa-intervals (Efron and Tibshirani, An introduction to the bootstrap. Chapman & Hall, London 436 p, 1993) and Hall's transformation methods (Zhou and Gao, Am Stat 54:100-104, 2000) had poor coverages. Coverage accuracy of Fieller's interval endpoint(J R Stat Soc Ser B 16:175-185, 1954) and t-interval endpoint were similar for both h(2) and r(B) for sample sizes n</=10, but for n=30 the Fieller's method is much better.

Analysis of Variance↗

Comparison of REML and Gibbs sampling estimates of multi-trait genetic parameters in Scots pine.

Multi-trait (co)variance estimation is an important topic in plant and animal breeding. In this study we compare estimates obtained with restricted maximum likelihood (REML) and Bayesian Gibbs sampling of simulated data and of three traits (diameter, height and branch angle) from a 26-year-old partial diallel progeny test of Scots pine (Pinus sylvestris L.). Based on the results from the simulated data we can conclude that the REML estimates are accurate but the mode of posterior distributions from the Gibbs sampling can be overestimated depending on the level of the heritability. The mean and median of the posteriors were considerably higher than the expected values of the heritabilities. The confidence intervals calculated with the delta method were biased downwardly. The highest probability density (HPD) interval provides a better interval estimate, but could be slightly biased at the lower level. Similar differences between REML and Gibbs sampling estimates were found for the Scots pine data. We conclude that further simulation studies are needed in order to evaluate the effect of different priors on (co)variance components in the genetic individual model.

Analysis of Variance↗

Low nitrification rates in acid Scots pine forest soils are due to pH-related factors.

In a previous study, ammonia-oxidizing bacteria (AOB)-like sequences were detected in the fragmentation layer of acid Scots pine (Pinus sylvestris L.) forest soils (pH 2.9-3.4) with high nitrification rates (>11.0 microg g-1 dry soil week-1), but were not detected in soils with low nitrification rates (<0.5 microg g-1 dry soil week-1). In the present study, we investigated whether this low nitrification rate has a biotic cause (complete absence of AOB) or an abiotic cause (unfavorable environmental conditions). Therefore, two soils strongly differing in net nitrification were compared: one soil with a low nitrification rate (location Schoorl) and another soil with a high nitrification rate (location Wekerom) were subjected to liming and/or ammonium amendment treatments. Nitrification was assessed by analysis of dynamics in NH4+-N and NO3- -N concentrations, whereas the presence and composition of AOB communities were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis and sequencing of the ammonia monooxygenase (amoA) gene. Liming, rather than ammonium amendment, stimulated the growth of AOB and their nitrifying activity in Schoorl soil. The retrieved amoA sequences from limed (without and with N amendment) Schoorl and Wekerom soils exclusively belong to Nitrosospira cluster 2. Our study suggests that low nitrification rates in acidic Scots pine forest soils are due to pH-related factors. Nitrosospira cluster 2 detected in these soils is presumably a urease-positive cluster type of AOB.

Bacteria↗

Managing plantation forests to provide short- to long-term supplies of wood to streams: a simulation study using New Zealand's pine plantations.

Riparian functions such as the recruitment of wood to streams take decades to recover after a clear-fell harvest to the stream edge. The implications of two sets of riparian management scenarios on the short- and long-term recruitment of wood to a hypothetical stream (central North Island, New Zealand) were compared through simulation modeling. In the first set (native forest buffer), a designated tree-less riparian buffer was colonized by native forest species after a pine crop (Pinus radiata) had been harvested to the stream bank. In the second set (pine to native forest buffer), native forest species were allowed to establish under the pine canopy in a designated riparian buffer. In general, the volume of wood was greater in streams with wider buffers (5-m to 50-m) and this effect increased with forest age (800 years). The pine to native forest buffer supplied more wood to the stream more quickly, and matched the long-term supply to the stream from the native forest buffer. For the native forest buffer, total wood volume was minimal for the first 70 years and then increased uniformly for the remainder of the simulation. In contrast, the pine to native forest buffer produced a bimodal response in total wood volume with the initial sharp peak at year 100 attributed to pine recruitment and a second more gradual peak lasting for the rest of the simulation, which was similar to levels in the native forest simulations. These results suggest that existing plantations could be an important source of wood to the stream during the first 100+ years of native forest development.

Computer Simulation↗

Effects of management on understory diversity in the forest ecosystems of northern Spain.

Pine plantations are an alternative to marginal agriculture in many countries, and are often presented as an option that improves biodiversity. However, these plantations can have adverse environmental effects if improperly managed. To evaluate the effect of forest management practices on biodiversity, the diversity, species richness, dominance and frequency of understory woody plant species in different forests of the Basque Country (northern Spain) were compared. Plantations of exotic conifers (Pinus radiata [D.] Don) of different ages were compared with deciduous forests of Quercus robur L. and Fagus sylvatica L. The effects of different types and intensities of management were taken into account. The differences observed were mainly conditioned by the intensity of forestry management, although the response varied according to forest type and age. In unmanaged pine plantations, the diversity and species richness of the understory increased rapidly after planting (while dominance decreased), remained stable in the intermediate age range, and reached a maximum in plantations more than 25 years of age. Management practices resulted in decreased understory diversity and species richness, as well as greater dominance. This was more pronounced in younger than in older stands. Moderate management, however, favored a greater diversity of the understory in deciduous forests. The species composition of the plantations and deciduous forests were different, the latter having a wider range of characteristic species. Knowledge of how forestry practices influence biodiversity (in terms of diversity, richness, dominance, and species composition) may allow predictions to be made about the diversity achievable with different management systems.

Ecosystem↗

Stricter ozone ambient air quality standard has beneficial effect on ponderosa pine in California.

Ambient air quality standards and control strategies are implemented to protect humans and vegetation from adverse effects. We used a process-based tree-growth model (TREGRO) to show that over the past 37 years, changes in O(3) exposure, with accompanying variation in climate, are reflected in changes in the growth of Pinus ponderosa Dougl. ex Laws. in the San Bernardino Mountains near Los Angeles, California, USA. Despite variation in temperature and precipitation over the study period (1963-1999), O(3) exposure consistently reduced simulated tree growth. Simulated growth reductions increased concurrent with increasing O(3) exposure. The maximum growth reduction occurred in 1979. As O(3) exposures decreased during the 1980s and 1990s, effects on growth also decreased. This implies that emission control strategies taken to reduce exposures to attain O(3) standards benefited P. ponderosa growth in the San Bernardino Mountains. This modeling approach provides a powerful tool for solving the difficult problem of evaluating regulatory effectiveness by simulating plant response using long-term climate and air pollution exposure records for a given region.

Air Pollutants↗

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↗

High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine.

Inducible gene expression systems are needed in functional genomics of tree species. A glucocorticoid-inducible gene expression system was established in a gymnosperm species Virginia pine (Pinus virginiana Mill.) through Agrobacterium tumefaciens-mediated genetic transformation. The results demonstrate that expression of the m-gfp5-ER reporter gene was tightly controlled and 0.1 microM of the glucocorticoid hormone triamcinolone was able to induce m-gfp5-ER expression in transgenic cells. Differential expression of gfp in transgenic cells induced by different concentrations of triamcinolone was observed and confirmed by Northern Blot analysis and by quantitative green fluorescence analyses with Laser Scanning Microscopy. In transgenic plantlets, triamcinolone was taken up efficiently by roots. Triamcinolone was able to induce m-gfp5-ER activity throughout the whole plant. The phenotype of transgenic plantlets was not affected 6 weeks after treatment with 0.1-10 microM triamcinolone. However, 6-week inductions with 100 microM triamcinolone caused growth retardation and developmental defects, as well as inhibition of root formation and elongation. With careful selection of transgenic lines, the inducible gene expression presented in this study could be a very valuable alternative for functional identification of novel genes in plants, especially in pine.

Agrobacterium tumefaciens↗

Enhanced stress tolerance in transgenic pine expressing the pepper CaPF1 gene is associated with the polyamine biosynthesis.

ERF/AP2 transcription factors play an important role in plant stress tolerance. However, little is known about the functional significance of ERF/AP2 genes in pine, compared to the model plant species Arabidopsis. Capsicum annuum pathogen and freezing tolerance-related protein 1 (CaPF1) is an ERF/AP2 transcription factor. We show here that overexpression of CaPF1 resulted in a dramatic increase in tolerance to drought, freezing, and salt stress in a gymnosperm species, eastern white pine (Pinus strobus L.). Measurement of polyamines demonstrated that the levels of putrescine (Put), spermidine (Spd), and spermine (Spm) did not increase but remain constant in CaPF1-overexpressed eastern white pine, whereas the levels decreased in the controls, probably increasing the ability of transgenic callus cultures and plants to stress tolerance. These results demonstrated that enhanced stress tolerance in transgenic pine expressing the pepper CaPF1 gene is associated with the polyamine biosynthesis and this pepper transcription factor may be used to engineer pine species for multiple stress tolerance.

Adaptation, Physiological↗

Loblolly pine arginase responds to arginine in vitro.

Following germination of loblolly pine (Pinus taeda L.) seeds, storage proteins in the embryo and megagametophyte are broken down to provide nitrogen, in the form of amino acids, to the developing seedling. A substantial portion of the free amino acids released in this process is arginine. Arginine is hydrolyzed in the cotyledons of the seedling by the enzyme arginase (EC 3.5.3.1), which is under developmental control. It has been shown previously that the seedling is able to initiate arginase gene expression in vitro in the absence of the megagametophyte, however, presence of the megagametophyte causes a greater accumulation of arginase protein and mRNA. Using an in vitro culture system we show that arginine itself may be responsible for up-regulating arginase activity. Application of exogenous arginine to cotyledons of seedlings germinated in the absence of the megagametophyte caused an increase in total shoot pole arginase activity as well as arginase specific activity. Arginine was also able to induce arginase mRNA accumulation in the same tissue.

Arginase↗

Systemic induction of traumatic resin ducts and resin flow in Austrian pine by wounding and inoculation with Sphaeropsis sapinea and Diplodia scrobiculata.

The potential role of the resin system in the response of Austrian pine (Pinus nigra Arn.) seedlings to mechanical injury and fungal infection was studied in greenhouse experiments. Anatomical observations were performed on 2-year-old plants wounded at collar level and inoculated with Sphaeropsis sapinea (Fr.: Fr.) Dyko & Sutton in Sutton or Diplodia scrobiculata (J. de Wet, B. Slippers & M. J. Wingfield, sp. nov.; sensu de Wet et al. 2003), two fungal pathogens that cause shoot blight and canker on conifers, and that are characterized by different levels of aggressiveness. Histological examination of host tissue taken from the stem at 0, 8, and 12 cm above the treatment site revealed significant treatment- and time-dependent effects on the course of locally and systemically induced traumatic resin duct (TRD) development. Occurrence of TRDs was observed after 4 days only in seedlings inoculated with D. scrobiculata. At 12 days, TRDs were present also in mock-inoculated controls. No TRDs appeared in seedlings inoculated with S. sapinea. However, S. sapinea caused loss of vacuolar phenolics, severe disruption of cambial tissue and invaded the host xylem quickly and apparently unimpeded, whereas D. scrobiculata was never detected in the host xylem. Five-year-old Austrian pines subjected to the same stem base treatments were used to determine the resin mass flowing from the stem 30 cm above the treatment sites. Wounding and/or inoculation induced a significant, 8.3-fold average increase in systemic resin flow over the untreated trees 3 weeks after basal treatment, suggesting that wounding is the sole prerequisite for systemic induction of resin flow. The results are discussed in the context of current disease resistance models.

Ascomycota↗

Characterization of the photosynthetic apparatus in cortical bark chlorenchyma of Scots pine.

Winter-induced inhibition of photosynthesis in Scots pine (Pinus sylvestris L.) needles is accompanied by a 65% reduction of the maximum photochemical efficiency of photosystem II (PSII), measured as Fv/Fm, but relatively stable photosystem I (PSI) activity. In contrast, the photochemical efficiency of PSII in bark chlorenchyma of Scots pine twigs was shown to be well preserved, while PSI capacity was severely decreased. Low-temperature (77 K) chlorophyll fluorescence measurements also revealed lower relative fluorescence intensity emitted from PSI in bark chlorenchyma compared to needles regardless of the growing season. Nondenaturating SDS-PAGE analysis of the chlorophyll-protein complexes also revealed much lower abundance of LHCI and the CPI band related to light harvesting and the core complex of PSI, respectively, in bark chlorenchyma. These changes were associated with a 38% reduction in the total amount of chlorophyll in the bark chlorenchyma relative to winter needles, but the Chl a/b ratio and carotenoid composition were similar in the two tissues. As distinct from winter pine needles exhibiting ATP/ADP ratio of 11.3, the total adenylate content in winter bark chlorenchyma was 2.5-fold higher and the estimated ATP/ADP ratio was 20.7. The photochemical efficiency of PSII in needles attached to the twig recovered significantly faster (28-30 h) then in detached needles. Fluorescence quenching analysis revealed a high reduction state of Q(A) and the PQ-pool in the green bark tissue. The role of bark chlorenchyma and its photochemical performance during the recovery of photosynthesis from winter stress in Scots pine is discussed.

Adenosine Diphosphate↗

Differential gene silencing induced by short interfering RNA in cultured pine cells associates with the cell cycle phase.

The double-stranded short interfering RNA (siRNA) molecules can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript. The rapid adoption of technologies based on this siRNA interference mechanism has been a widely used method to analyze gene function in plants, invertebrates, and mammalian systems. In order to understand the dynamics of siRNA-mediated gene inactivation during cell division, we have investigated the relationship between the cell cycle phase and the post-transcriptional gene silencing mediated by siRNA in gfp transgenic Virginia pine (Pinus virginiana Mill.) cells. Among the different phases of the cell cycle, transgenic cells at the M phase gave 2-3 times lower gfp silencing than those at the G1, S, and G2 phases. The similar results of the siRNA-mediated gfp silencing were obtained in three transgenic cell lines. Differential gfp silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that siRNA-mediated gene inactivation is associated with the cell cycle phase in Virginia pine.

Cell Cycle↗

High levels of asparagine synthetase in hypocotyls of pine seedlings suggest a role of the enzyme in re-allocation of seed-stored nitrogen.

A pine asparagine synthetase gene expressed in developing seedlings has been identified by cloning its cDNA (PsAS1) from Scots pine (Pinus sylvestris L.). Genomic DNA analysis with PsAS1 probes and a sequence-based phylogenetic tree are consistent with the possibility of more than one gene encoding asparagine synthetase in pine. However, the parallel patterns of free asparagine content and PsAS1 products indicate that the protein encoded by this gene is mainly responsible for the accumulation of this amino acid during germination and early seedling development. The temporal and spatial patterns of PsAS1 expression together with the spatial distribution of asparagine content suggest that, early after germination, part of the nitrogen mobilized from the megagametophyte is diverted toward the hypocotyl to produce high levels of asparagine as a reservoir of nitrogen to meet later specific demands of development. Furthermore, the transcript and protein analyses in seedlings germinated and growth for extended periods under continuous light or dark suggest that the spatial expression pattern of PsAS1 is largely determined by a developmental program. Therefore, our results suggest that the spatial and temporal control of PsAS1 expression determines the re-allocation of an important amount of seed-stored nitrogen during pine germination.

Amino Acid Sequence↗

Coordination of PsAS1 and PsASPG expression controls timing of re-allocated N utilization in hypocotyls of pine seedlings.

During pine seed germination, a large amount of N mobilized from the storage proteins is re-allocated in the hypocotyl as free asparagine, as a result of the high levels of asparagine synthetase (AS) encoded by the PsAS1 gene. To determine the role of this re-allocated N reserve, a full-length cDNA encoding L: -asparaginase (ASPG) has been cloned from Scots pine (Pinus sylvestris L.) seedlings and characterized. Like other N-terminal nucleophile hydrolases, pine ASPG requires a post-translational processing to exhibit enzymatic activity. However, in contrast to previous reports on other plant ASPGs, purified recombinant pine ASPG does not undergo autoproteolytic cleavage in vitro. Our results suggest that the processing requires accessory proteins to assist in the proteolysis or in the proper folding before autocleavage in a divalent cation-dependent manner. Sequence comparison analysis revealed that the pine protein is included in the K+-dependent subfamily of plant ASPGs. The expression of the ASPG-encoding gene (PsASPG) was higher in organs with extensive secondary development of the vascular system. The increase in transcript abundance observed at advanced stages of hypocotyl development was concomitant with a decrease of PsAS1 transcript abundance and a remarkable increase in the number of xylem elements and highly lignified cell walls. These results, together with the precise localization of PsASPG transcripts in cells of the cambial region, suggest that the expression of PsAS1 and PsASPG is temporally coordinated, to control the re-allocation of N from seed storage proteins toward the hypocotyl to be later used during early development of secondary vascular system.

Amino Acid Sequence↗

Purification and cloning of an arabinogalactan-protein from xylem of loblolly pine.

An arabinogalactan-protein (AGP) was purified from differentiating xylem of loblolly pine (Pinus taeda L.) and the N-terminal sequence used to identify a cDNA clone. The protein, PtaAGP3, was not coded for by any previously identified AGP-like genes. Moreover, PtaAGP3 was abundantly and preferentially expressed in differentiating xylem. The encoded protein contains four domains, a signal peptide, a cleaved hydrophilic region, a region rich in serine, alanine, and proline/hydroxyproline, and a hydrophobic C-terminus. It is postulated to contain a GPI (glycosylphosphatidylinositol) anchor site. If the protein is cleaved at the putative GPI anchor site, as has been observed in other classical AGPs, all but the Ser-Ala-Pro/Hyp-rich domain may be missing from the mature protein. Xylem-specific AGPs are hypothesized to be involved in xylem development.

Amino Acid Sequence↗

Expression of a transcription factor from Capsicum annuum in pine calli counteracts the inhibitory effects of salt stress on adventitious shoot formation.

Transcription factors regulating the stress-responsive gene expression play an important role in plant stress adaptation. In this study, we examined the salt stress tolerance of transgenic Virginia pine (Pinus virginiana Mill.) overexpressing a Capsicum annuum ERF/AP2-type transcription factor (CaPF1), which may enhance the ability of transgenic plants to tolerate various kinds of stresses during vegetative growth. CaPF1 transgene increased the salt and oxidative stress tolerances of pine tissues and counteracted the inhibitory effects of salt stress on the growth of transgenic Virginia pine calli, shoots, and plants. To our surprise, the ability of shoot formation was enhanced in three CaPF1 transgenic Virginia pine cell lines under stress of different NaCl concentrations. NaCl at 200 mM significantly increased the frequency of adventitious shoot formation and the number of shoots per gram calli. Measurement of plant hormone demonstrated that the levels of cytokinin was altered in CaPF1-overexpressed Virginia pine calli, compared to the control. Based on our results, we speculate that the altered level of cytokinin may result in enhancing adventitious shoot formation of transgenic calli exposed to salt for 1 week via an unknown mechanism.

Adaptation, Physiological↗

Controls of fine root dynamics across a gradient of gap sizes in a pine woodland.

Controls of fine dynamics were investigated in a Pinus palustris Mill. (longleaf pine) woodland subjected to two understory vegetation treatments (control versus removed) and four overstory treatments (no gap control, and canopy gaps of three sizes with constant total gap area per stand). Fine root (<2 mm diameter) dynamics were measured over 11 months using ingrowth cores (all treatments) and minirhizotrons (understory removed in no gap control and large gap treatments only). At the fine (microsite) spatial scale, pine and non-pine root mass production responded negatively to each other (P=0.033). Each life form was significantly (P< or =0.028) related to nearby overstory density, and pine root production compensated for reductions in non-pine roots if understory vegetation was removed. Soil moisture and NO(3) mineralization rate were negatively related to pine root mass production (ingrowth cores; P<0.001 and P=0.052) and positively related to pine root length production, mortality and turnover (minirhizotrons; P from <0.001 to 0.078). Temperature variance was negatively related to pine root lifespan P<0.001) and positively related to pine root turnover (P=0.003). At the ecosystem scale, pattern of overstory disturbance (gap size and number) had no significant effect on non-pine, pine, or total root production. However, the presence of gaps (versus the no-gap control) increased non-pine root mass production (ANOVA, P=0.055) in natural understory conditions, and reduced pine root mass production (P=0.035) where the understory was removed. Ecosystem-wide pine root length production, mortality and turnover were positively related to weekly soil temperature (P< or =0.02). In natural systems, fine root dynamics are highly variable and strongly affected by biotic factors. Roots quickly close belowground gaps because one life form (pine or non-pine) compensates for the absence of the other. When understory vegetation is removed, however, pine roots respond to the local abiotic environment, particularly moisture and NO(3).

Biomass↗