Janice E. K. Cooke

Mountain pine beetle host‐range expansion threatens the boreal forest

The current epidemic of the mountain pine beetle (MPB), an indigenous pest of western North American pine, has resulted in significant losses of lodgepole pine. The leading edge has reached Alberta where forest composition shifts from lodgepole to jack pine through a hybrid zone. The susceptibility of jack pine to MPB is a major concern, but there has been no evidence of host‐range expansion, in part due to the difficulty in distinguishing the parentals and their hybrids. We tested the utility of a panel of microsatellite loci optimized for both species to classify lodgepole pine, jack pine and their hybrids using simulated data. We were able to accurately classify simulated individuals, and hence applied these markers to identify the ancestry of attacked trees. Here we show for the first time successful MPB attack in natural jack pine stands at the leading edge of the epidemic. This once unsuitable habitat is now a novel environment for MPB to exploit, a potential risk which could be exacerbated by further climate change. The consequences of host‐range expansion for the vast boreal ecosystem could be significant.

The dynamic nature of bud dormancy in trees: environmental control and molecular mechanisms.

In tree species native to temperate and boreal regions, the activity-dormancy cycle is an important adaptive trait both for survival and growth. We discuss recent research on mechanisms controlling the overlapping developmental processes that define the activity-dormancy cycle, including cessation of apical growth, bud development, induction, maintenance and release of dormancy, and bud burst. The cycle involves an extensive reconfiguration of metabolism. Environmental control of the activity-dormancy cycle is based on perception of photoperiodic and temperature signals, reflecting adaptation to prevailing climatic conditions. Several molecular actors for control of growth cessation have been identified, with the CO/FT regulatory network and circadian clock having important coordinating roles in control of growth and dormancy. Other candidate regulators of bud set, dormancy and bud burst have been identified, such as dormancy-associated MADS-box factors, but their exact roles remain to be discovered. Epigenetic mechanisms also appear to factor in control of the activity-dormancy cycle. Despite evidence for gibberellins as negative regulators in growth cessation, and ABA and ethylene in bud formation, understanding of the roles that plant growth regulators play in controlling the activity-dormancy cycle is still very fragmentary. Finally, some of the challenges for further research in bud dormancy are discussed.

Involvement of Pinus taeda MYB1 and MYB8 in phenylpropanoid metabolism and secondary cell wall biogenesis: a comparative in planta analysis

The involvement of two R2R3-MYB genes from Pinus taeda L., PtMYB1 and PtMYB8, in phenylpropanoid metabolism and secondary cell wall biogenesis was investigated in planta. These pine MYBs were constitutively overexpressed (OE) in Picea glauca (Moench) Voss, used as a heterologous conifer expression system. Morphological, histological, chemical (lignin and soluble phenols), and transcriptional analyses, i.e. microarray and reverse transcription quantitative PCR (RT-qPCR) were used for extensive phenotyping of MYB-overexpressing spruce plantlets. Upon germination of somatic embryos, root growth was reduced in both transgenics. Enhanced lignin deposition was also a common feature but ectopic secondary cell wall deposition was more strongly associated with PtMYB8-OE. Microarray and RT-qPCR data showed that overexpression of each MYB led to an overlapping up-regulation of many genes encoding phenylpropanoid enzymes involved in lignin monomer synthesis, while misregulation of several cell wall-related genes and other MYB transcription factors was specifically associated with PtMYB8-OE. Together, the results suggest that MYB1 and MYB8 may be part of a conserved transcriptional network involved in secondary cell wall deposition in conifers.

A White Spruce Gene Catalog for Conifer Genome Analyses

Several angiosperm plant genomes, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), poplar (Populus trichocarpa), and grapevine (Vitis vinifera), have been sequenced, but the lack of reference genomes in gymnosperm phyla reduces our understanding of plant evolution and restricts the potential impacts of genomics research. A gene catalog was developed for the conifer tree Picea glauca (white spruce) through large-scale expressed sequence tag sequencing and full-length cDNA sequencing to facilitate genome characterizations, comparative genomics, and gene mapping. The resource incorporates new and publicly available sequences into 27,720 cDNA clusters, 23,589 of which are represented by full-length insert cDNAs. Expressed sequence tags, mate-pair cDNA clone analysis, and custom sequencing were integrated through an iterative process to improve the accuracy of clustering outcomes. The entire catalog spans 30 Mb of unique transcribed sequence. We estimated that the P. glauca nuclear genome contains up to 32,520 transcribed genes owing to incomplete, partially sequenced, and unsampled transcripts and that its transcriptome could span up to 47 Mb. These estimates are in the same range as the Arabidopsis and rice transcriptomes. Next-generation methods confirmed and enhanced the catalog by providing deeper coverage for rare transcripts, by extending many incomplete clusters, and by augmenting the overall transcriptome coverage to 38 Mb of unique sequence. Genomic sample sequencing at 8.5% of the 19.8-Gb P. glauca genome identified 1,495 clusters representing highly repeated sequences among the cDNA clusters. With a conifer transcriptome in full view, functional and protein domain annotations clearly highlighted the divergences between conifers and angiosperms, likely reflecting their respective evolutionary paths.

Pathogen challenge, salicylic acid, and jasmonic acid regulate expression of chitinase gene homologs in pine.

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.

Influence of nitrogen fertilization on xylem traits and aquaporin expression in stems of hybrid poplar

We studied the influence of nitrogen (N) on hydraulic traits and aquaporin (AQP) expression in the stem xylem of hybrid poplar saplings (Populus trichocarpa (Torr. & Gray) x deltoides Bartr. ex Marsh clone H11-11). Plants were grown in a controlled environment and were kept well watered throughout the experiments. Hydraulic measurements were done on basal and distal stem segments of plants receiving high N fertilization (high N plants) versus plants receiving only adequate N fertilization (adequate N plants). High N plants grew faster and exhibited more leaf area than adequate N controls. These morphological differences were paralleled by wider vessels and higher specific conductivities (K(S)) in high N plants. However, stems of high N plants were more vulnerable to xylem cavitation, at least in one of two experiments, and showed lower wood densities than stems of adequate N plants. Leaf area was strongly correlated with cross-sectional xylem area in both plant groups. Since higher K(S) in high N plants was accompanied by concomitant increases in leaf area, leaf-specific conductivities were similar in both plant groups. Influences of N on hydraulic traits were paralleled by changes in AQP expression. Seven AQPs were upregulated in the stem xylem of high N plants, five of which have been identified recently as water transporters. The enhanced growth of secondary xylem of high N plants has been shown to result from both increased cambial activity as well as increased cell size. We suggest that some of these water-transporting AQPs could play a role in xylogenesis, facilitating the influx of water into the zone of differentiating and maturing cells in secondary xylem, including expanding vessels.

Identification of conserved core xylem gene sets: conifer cDNA microarray development, transcript profiling and computational analyses

One approach for investigating the molecular basis of wood formation is to integrate microarray profiling data sets and sequence analyses, comparing tree species with model plants such as Arabidopsis. Conifers may be included in comparative studies thanks to large-scale expressed sequence tag (EST) analyses, which enable the development of cDNA microarrays with very significant genome coverage. A microarray of 10,400 low-redundancy sequences was designed starting from white spruce (Picea glauca (Moench.) Voss) cDNAs. Computational procedures that were developed to ensure broad transcriptome coverage and efficient PCR amplification were used to select cDNA clones, which were re-sequenced in the microarray manufacture process. White spruce transcript profiling experiments that compared secondary xylem to phloem and needles identified 360 xylem-preferential gene sequences. The functional annotations of all differentially expressed sequences were highly consistent with the results of similar analyses carried out in angiosperm trees and herbaceous plants. Computational analyses comparing the spruce microarray sequences and core xylem gene sets from Arabidopsis identified 31 transcripts that were highly conserved in angiosperms and gymnosperms, in terms of both sequence and xylem expression. Several other spruce sequences have not previously been linked to xylem differentiation (including genes encoding TUBBY-like domain proteins (TLPs) and a gibberellin insensitive (gai) gene sequence) or were shown to encode proteins of unknown function encompassing diverse conserved domains of unknown function.

Multilocus species identification and fungal DNA barcoding: insights from blue stain fungal symbionts of the mountain pine beetle

There is strong community‐wide interest in applying molecular techniques to fungal species delimitation and identification, but selection of a standardized region or regions of the genome has not been finalized. A single marker, the ribosomal DNA internal transcribed spacer region, has frequently been suggested as the standard for fungi. We used a group of closely related blue stain fungi associated with the mountain pine beetle (Dendroctonus ponderosae Hopkins) to examine the success of such single‐locus species identification, comparing the internal transcribed spacer with four other nuclear markers. We demonstrate that single loci varied in their utility for identifying the six fungal species examined, while use of multiple loci was consistently successful. In a literature survey of 21 similar studies, individual loci were also highly variable in their ability to provide consistent species identifications and were less successful than multilocus diagnostics. Accurate species identification is the essence of any molecular diagnostic system, and this consideration should be central to locus selection. Moreover, our study and the literature survey demonstrate the value of using closely related species as the proving ground for developing a molecular identification system. We advocate use of a multilocus barcode approach that is similar to the practice employed by the plant barcode community, rather than reliance on a single locus.

Functional characterization of drought-responsive aquaporins in Populus balsamifera and Populus simonii×balsamifera clones with different drought resistance strategies

We have characterized poplar aquaporins (AQPs) to investigate their possible functions in differential drought responses of Populus balsamifera and Populus simonii×balsamifera leaves. Plants were exposed to mild and severe levels of drought stress and to drought stress recovery treatment, and their responses were compared with well-watered controls. Compared with P. balsamifera, P. simonii×balsamifera used drought avoidance as the main drought resistance strategy, and rapidly reduced stomatal conductance in response to stress. This strategy is correlated with growth rate reductions. Eleven AQPs were transcriptionally profiled in leaves from these experiments and five were functionally characterized for water channel activity. PIP1;3 and PIP2;5 were among the most highly expressed leaf AQPs that were responsive to drought. Expression of PIP1;3 and five other AQPs increased in response to drought in the leaves of P. simonii×balsamifera but not in P. balsamifera, suggesting a possible role of these AQPs in water redistribution in the leaf tissues. PIP2;5 was upregulated in P. balsamifera, but not in P. simonii×balsamifera, suggesting that this AQP supports the transpiration-driven water flow. Functional characterization of five drought-responsive plasma membrane intrinsic proteins (PIPs) demonstrated that three PIP2 AQPs (PIP2;2, PIP2;5, PIP2;7) functioned as water transporters in Xenopus laevis oocytes, while the two PIP1 AQPs (PIP1;2 and PIP1;3) did not, consistent with the notion that they may be functional only as heterotetramers.

Molecular events of apical bud formation in white spruce, Picea glauca.

Bud formation is an adaptive trait that temperate forest trees have acquired to facilitate seasonal synchronization. We have characterized transcriptome-level changes that occur during bud formation of white spruce [Picea glauca (Moench) Voss], a primarily determinate species in which preformed stem units contained within the apical bud constitute most of next seasons growth. Microarray analysis identified 4460 differentially expressed sequences in shoot tips during short day-induced bud formation. Cluster analysis revealed distinct temporal patterns of expression, and functional classification of genes in these clusters implied molecular processes that coincide with anatomical changes occurring in the developing bud. Comparing expression profiles in developing buds under long day and short day conditions identified possible photoperiod-responsive genes that may not be essential for bud development. Several genes putatively associated with hormone signalling were identified, and hormone quantification revealed distinct profiles for abscisic acid (ABA), cytokinins, auxin and their metabolites that can be related to morphological changes to the bud. Comparison of gene expression profiles during bud formation in different tissues revealed 108 genes that are differentially expressed only in developing buds and show greater transcript abundance in developing buds than other tissues. These findings provide a temporal roadmap of bud formation in white spruce.