(1/54) Whole-genome characterization of embryonic stage inbreeding depression in a selfed loblolly pine family.

Inbreeding depression is important in the evolution of plant populations and mating systems. Previous studies have suggested that early-acting inbreeding depression in plants is primarily due to lethal alleles and possibly epistatic interactions. Recent advances in molecular markers now make genetic mapping a powerful tool to study the genetic architecture of inbreeding depression. We describe a genome-wide evaluation of embryonic viability loci in a selfed family of loblolly pine (Pinus taeda L.), using data from AFLP markers from an essentially complete genome map. Locus positions and effects were estimated from segregation ratios using a maximum-likelihood interval mapping procedure. We identified 19 loci showing moderately deleterious to lethal embryonic effects. These loci account for >13 lethal equivalents, greater than the average of 8.5 lethal equivalents reported for loblolly pine. Viability alleles show predominantly recessive action, although potential overdominance occurs at 3 loci. We found no evidence for epistasis in the distribution of pairwise marker correlations or in the regression of fitness on the number of markers linked to deleterious alleles. The predominant role of semilethal alleles in embryonic inbreeding depression has implications for the evolution of isolated populations and for genetic conservation and breeding programs in conifers.  (+info)

(2/54) Effect of different daytime and night-time temperature regimes on the foliar respiration of Pinus taeda: predicting the effect of variable temperature on acclimation.

The objectives of this study were to determine the acclimation of loblolly pine (Pinus taeda L.) foliar respiration to different night-time low temperatures, daytime high temperatures, and daily mean temperatures, and then to use the responses of temperature acclimation to various temperature regimes to predict acclimation under fluctuating temperatures. Experiments were conducted on two-year-old seedlings in growth chambers using different combinations of day and night-time temperatures. The first experiment exposed trees to 22/22, 29/22, 22/15, and 29/15 degrees C day/night (d/n). When measured at a common temperature (15, 22 or 29 degrees C), respiration rates were lower for trees exposed to higher treatment temperatures and acclimation was influenced by both day and night-time temperature. However, the extent of acclimation did not relate to mean temperature, i.e. respiration rates measured at a common temperature ranked as follows for seedlings exposed to different temperature regimes, 22/15>22/22>29/15 congruent with29/22 degrees C d/n. Rather, acclimation of foliar respiration was linearly related to mean daily respiration rate, where mean daily respiration rate is the average of the respiration rates measured at the day and night-time treatment temperatures. The discrepancy between mean daily respiration rate and mean daily temperature occurred because respiration increased exponentially with increasing temperature. In a second experiment, the same seedlings were exposed to 22/22, 15/15, 25.5/18.5, and 25.5/15 degrees C d/n to test the relationship between mean daily respiration rate and acclimation. As in the first experiment, acclimation was linearly related to mean daily respiration rate. The concept of effective acclimation temperature, which is the temperature at which the mean daily respiration rate occurs, was derived from these results as a means to predict the extent that foliar respiration acclimates to treatment temperature.  (+info)

(3/54) 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.  (+info)

(4/54) 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.  (+info)

(5/54) Agrobacterium-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.).

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for beta-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.  (+info)

(6/54) An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of loblolly pine.

In contrast to angiosperms, pines and other gymnosperms form well-developed suspensors in somatic embryogenic cultures. This creates a useful system to study suspensor biology. In a study of gene expression during the early stages of conifer embryogenesis, we identified a transcript, PtNIP1;1, that is abundant in immature loblolly pine (Pinus taeda) zygotic and somatic embryos, but is undetectable in later-stage embryos, megagametophytes, and roots, stems, and needles from 1 year-old seedlings. Analysis of PtNIP1;1 transcript in embryo proper and suspensor tissues by reverse transcription-polymerase chain reaction suggests preferential expression in the suspensor. Based on comparisons of derived amino acid sequences, PtNIP1;1 belongs to the nodulin-like members of the major intrinsic protein superfamily branch of the aquaporin (major intrinsic protein) superfamily. Through heterologous expression in Xenopus laevis oocytes and the yeast (Saccharomyces cerevisiae) fps1(-) mutant, PtNIP1;1 has been shown to be an active aquaglyceroporin.  (+info)

(7/54) Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism.

Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading to coniferyl alcohol, was not up-regulated under any of the conditions employed, suggesting that it is not, in fact, involved in monolignol biosynthesis.  (+info)

(8/54) 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.  (+info)