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 CuSO₄ in the reaction mixtures increased in the range from 1 to 100 μM. 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.     

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

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

Association genetics of the loblolly pine (Pinus taeda, Pinaceae) metabolome

The metabolome of a plant comprises all small molecule metabolites, which are produced during cellular processes. The genetic basis for metabolites in nonmodel plants is unknown, despite frequently observed correlations between metabolite concentrations and stress responses. A quantitative genetic analysis of metabolites in a nonmodel plant species is thus warranted. Here, we use standard association genetic methods to correlate 3563 single nucleotide polymorphisms (SNPs) to concentrations of 292 metabolites measured in a single loblolly pine (Pinus taeda) association population. A total of 28 single locus associations were detected, representing 24 and 20 unique SNPs and metabolites, respectively. Multilocus Bayesian mixed linear models identified 2998 additional associations for a total of 1617 unique SNPs associated to 255 metabolites. These SNPs explained sizeable fractions of metabolite heritabilities when considered jointly (56.6% on average) and had lower minor allele frequencies and magnitudes of population structure as compared with random SNPs. Modest sets of SNPs (n = 1-23) explained sizeable portions of genetic effects for many metabolites, thus highlighting the importance of multi-SNP models to association mapping, and exhibited patterns of polymorphism consistent with being linked to targets of natural selection. The implications for association mapping in forest trees are discussed.     

Monoterpene synthases of loblolly pine (Pinus taeda) produce pinene isomers and enantiomers

The turpentine fraction of conifer oleoresin is a complex mixture of monoterpene olefins and plays important roles in defense and in the mediation of chemical communication between conifer hosts and insect predators. The stereochemistry of the turpentine monoterpenes is critical in these interactions, influencing host recognition, toxicity, and potency of derived pheromones, and the stereochemical composition of these compounds lends insight into their biogenetic origin, with implications for the numbers and types of enzymes responsible and their corresponding genes. Analysis of the oleoresin from several tissues of loblolly pine (Pinus taeda) showed the derived turpentine to consist mainly of (+)-(3R:5R)-alpha-pinene and (-)-(3S:5S)-beta-pinene. Cell-free extracts from xylem tissue yielded three monoterpene synthases which together account for the monoterpene isomer and enantiomer content of the turpentine of this tissue. The major products of these enzymes, produced from the universal precursor of monoterpenes, geranyl diphosphate, were shown to be (+)-alpha-pinene, (-)-alpha-pinene, and (-)-beta-pinene, respectively. In most properties (molecular mass of approximately 60 kDa, K(m) for geranyl diphosphate of 3 microM, requirement for monovalent and divalent cations), these enzymes resemble other monoterpene synthases from conifer species.     

Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine)

Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell‐wall xyloglucans and heteromannans. The full‐length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem‐specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)‐α‐xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum‐graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell‐culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP‐xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP‐galactose and the same cello‐oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α‐linkages. Analysis of the products of PrGT34B by MALDI‐TOF MS showed that up to three xylosyl residues were transferred from UDP‐xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.     

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

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

Intergenotypic interactions among families of loblolly pine (Pinus taeda L.)

Summary The effects of competition on the growth of families of loblolly pine (Pinus taeda, L.) seedlings were investigated. The experimental design made it possible to evaluate the effects of crowding on growth and to determine the types and magnitudes of intergenotypic interactions among pairs of families. The results showed that intergenotypic interactions were both highly variable and pronounced in their effect on early growth. Evidence was also found for precompetition cooperating interactions occurring among seedlings surrounded by neighbors of the same family.     

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.     

Reference karyotype and cytomolecular map for loblolly pine (Pinus taeda L.).

A reference karyotype is presented for loblolly pine (Pinus taeda L., subgenus Pinus, section Pinus, subsection Australes), based on fluorescent in situ hybridization (FISH), using 18S-28S rDNA, 5S rDNA, and an Arabidopsis-type telomere repeat sequence (A-type TRS). Well separated somatic chromosomes were prepared from colchicine-treated root meristems, using an enzymatic digestion technique. Statistical analyses performed on chromosome-arm lengths, centromeric indices, and interstitial rDNA and telomeric positions were based on observations from 6 well-separated metaphase cells from each of 3 unrelated trees. Statistically, 7 of the 12 loblolly pine chromosomes could be distinguished by their relative lengths. Centromeric indices were unable to distinguish additional chromosomes. However, the position and relative strength of the rDNA and telomeric sites made it possible to uniquely identify all of the chromosomes, providing a reference karyotype for use in comparative genome analyses. A dichotomous key was developed to aid in the identification of loblolly pine chromosomes and their comparison to chromosomes of other Pinus spp. A cytomolecular map was developed using the interstitial 18S-28S rDNA and A-type TRS signals. A total of 54 bins were assigned, ranging from 3 to 5 bins per chromosome. This is the first report of a chromosome-anchored physical map for a conifer that includes a dichotomous key for accurate and consistent identification of the P. taeda chromosomes.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.     

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

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

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

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

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

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

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

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

High-throughput genotyping and mapping of single nucleotide polymorphisms in loblolly pine (Pinus taeda L.)

The development and application of genomic tools to loblolly pine (Pinus taeda L.) offer promising insights into the organization and structure of conifer genomes. The application of a high-throughput genotyping assay across diverse forest tree species, however, is currently limited taxonomically. This is despite the ongoing development of genome-scale projects aiming at the construction of expressed sequence tag (EST) libraries and the resequencing of EST-derived unigenes for a diverse array of forest tree species. In this paper, we report on the application of Illumina’s high-throughput GoldenGate™ SNP genotyping assay to a loblolly pine mapping population. Single nucleotide polymorphisms (SNPs) were identified through resequencing of previously identified wood quality, drought tolerance, and disease resistance candidate genes prior to genotyping. From that effort, a 384 multiplexed SNP assay was developed for high-throughput genotyping. Approximately 67% of the 384 SNPs queried converted into high-quality genotypes for the 48 progeny samples. Of those 257 successfully genotyped SNPs, 70 were segregating within the mapping population. A total of 27 candidate genes were subsequently mapped onto the existing loblolly pine consensus map, which consists of 12 linkage groups spanning a total map distance of 1,227.6 cM. The ability of SNPs to be mapped to the same position as fragment-based markers previously developed within the same candidate genes, as well as the pivotal role that SNPs currently play in the dissection of complex phenotypic traits, illustrate the usefulness of high-throughput SNP genotyping technologies to the continued development of pine genomics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in mRNA populations during loblolly pine (Pinus taeda) seed stratification, germination and post-germinative growth

Changes in the patterns of gene expression were examined during loblolly pine ( Pinus taeda L.) seed stratification, germination, and post-germinative growth. In both the megagametophyte and the embryo, DNA contents remained relatively constant at all stages examined. RNA contents, however, increased in both tissues following seed germination, particularly in the embryo where a 7-fold increase in the RNA content was observed 5 days after germination. Poly(A)⁺ RNA, extracted from megagametophytes and embryos, was translated in vitro in a rabbit reticulocyte lysate cell-free system. Analysis of [³⁵S]-methionine-labelled translation products by two-dimensional electrophoresis/fluorography indicated that there were changes in the populations of mRNAs during all developmental stages examined. In both the megagametophyte and the embryo several distinct mRNA populations, including one constitutively present at all stages examined, were identified. One mRNA population, present in the mature seed, decreased during seed stratification. Another population, not present in the mature seed, rose during the period of stratification that coincided with an increase in seed germinability. A third population, which appeared during seed germination, increased steadily during post-germinative growth. Besides these similarities, specific differences between megagametophyte and embryo were noted. For example, one mRNA population, which was present in the megagametophyte of the mature seed and remained constant during the stratification period, disappeared immediately following seed germination. In the embryo, one set of messages was germination specific. In total, these results show that mRNA populations change in a temporal fashion that is consistent with the patterns of de novo protein synthesis known to occur in loblolly pine during the same developmental periods.     

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

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