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This study was conducted in six plots along an elevation gradient in the Qinghai spruce (Picea crassifolia Kom.) forest ecosystem of the Qilian Mountains, northwest China. Soil CO sub(2) efflux over bare soil (R sub(s)) and moss covered soil (R sub(s+m)) were investigated from June to September in 2010 and 2011 by means of an automated soil CO sub(2) flux system (LI-8100). The results showed that R sub(s) ranged from 1.51 to 3.96 (mean 2.64 plus or minus 0.72) mu mol m super(-2) s super(-1) for 2010, and from 1.41 to 4.09 (mean 2.55 plus or minus 0.70) mu mol m super(-2) s super(-1) for 2011. The daily change trend of R sub(s) resembled that of air temperature (T sub(a)), and there was a hysteresis between R sub(s) and soil temperature (T sub(s)). The seasonal variations of R sub(s) at lowlands (i.e., Plot 1, Plot 2 and Plot 3) were driven by soil moisture and temperature (T sub(a) and T sub(s)), while that at highlands (i.e., Plot 4, Plot 5 and Plot 6) were obviously affected by temperature. There were higher values at Plot 2 and Plot 6, which were caused by the interaction between soil moisture and temperature. In addition, soil CO sub(2) efflux over moss covered soil (R sub(s+m)) was 8.83 % less than that over bare soil (R sub(s)), indicating that moss was another factor affecting R sub(s). It was concluded that R sub(s) had temporal and spatial variations and was mainly controlled by temperature and soil moisture; the main determinants differed at different elevations; moss could reduce R sub(s).

Repeated homoploid hybrid speciation (HHS) events with the same parental species have rarely been reported. In this study, we used population transcriptome data to test paraphyly and HHS events in the conifer Picea brachytyla. Our analyses revealed non-sister relationships for two lineages of P. brachytyla, with the southern lineage being placed within the re-circumscribed P. likiangensis species complex (PLSC) and P. brachytyla sensu stricto (s.s.) consisted solely of the northern lineage, forming a distinct clade that is paratactic to both the PLSC and P. wilsonii. Our phylogenetic and coalescent analyses suggested that P. brachytyla s.s. arose from HHS between the ancestor of the PLSC before its diversification and P. wilsonii through an intermediate hybrid lineage at an early stage and backcrossing to the ancestral PLSC. Additionally, P. purpurea shares the same parents and an extinct lineage with P. brachytyla s.s. but backcrossing to the other parent, P. wilsonii at a later stage. We reveal the first case that backcrossing to different parents of the same extinct hybrid lineage produced two different hybrid species. Our results highlight the existence of more reticulate evolution during species diversification in the spruce genus and more complex homoploid hybrid events than previously identified.

Stilbenes are dibenzyl polyphenolic compounds produced in several unrelated plant families that appear to protect against various biotic and abiotic stresses. Stilbene biosynthesis has been well described in economically important plants, such as grape (Vitis vinifera), peanut (Arachis hypogaea), and pine (Pinus species). However, very little is known about the biosynthesis and ecological role of stilbenes in spruce (Picea), an important gymnosperm tree genus in temperate and boreal forests. To investigate the biosynthesis of stilbenes in spruce, we identified two similar stilbene synthase (STS) genes in Norway spruce (Picea abies), PaSTSl and PaSTS2, which had orthologs with high sequence identity in sitka (Picea sitchensis) and white (Picea glauca) spruce. Despite the conservation of STS sequences in these three spruce species, they differed substantially from angiosperm STSs. Several types of in vitro and in vivo assays revealed that the P. abies STSs catalyze the condensation of p-coumaroyl-coenzyme A and three molecules of malonyl-coenzyme A to yield the trihydroxystilbene resveratrol but do not directly form the dominant spruce stilbenes, which are tetrahydroxylated. However, in transgenic Norway spruce overexpressing PaSTS1, significantly higher amounts of the tetrahydroxystilbene glycosides, astringin and isorhapontin, were produced. This result suggests that the first step of stilbene biosynthesis in spruce is the formation of resveratrol, which is further modified by hydroxylation, O-methylation, and O-glucosylation to yield astringin and isorhapontin. Inoculating spruce with fungal mycelium increased STS transcript abundance and tetrahydroxystilbene glycoside production. Extracts from STSoverexpressing lines significantly inhibited fungal growth in vitro compared with extracts from control lines, suggesting that spruce stilbenes have a role in antifungal defense.

Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of enf-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ercf-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism.

Outbreaks of the Eurasian spruce bark beetle ( Ips typographus ) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I . typographus can distinguish fungal symbionts of the genera Grosmannia , Endoconidiophora , and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle’s host tree, Norway spruce ( Picea abies ), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and β-pinene to trans -4-thujanol and other oxygenated products. Electrophysiological measurements showed that I . typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans -4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus ( Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I . typographus . Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.

Background Picea species are distributed and planted world-wide due to their great ecological and economic values. It has been reported that Picea species vary widely in growth traits in a given environment, which reflects genetic and phenotypic differences among species. However, key physiological processes underlying tree growth and the influencing factors on them are still unknown. Results Here, we examined needle structures, needle chemical components, physiological characteristics and growth traits across five Picea species in a common garden in Tianshui, Gansu province in China: Picea glauca , P. mariana , P. likiangensis , P. koraiensis , and P. crassifolia , among which P. glauca and P. mariana were introduced from North America, P. likiangensis was from Lijiang, Yunan province in China, P. koraiensis was from Yichun, Heilongjiang province in China, and P. crassifolia was native to the experimental site. It was found that nearly all traits varied significantly among species. Tissue-level anatomical characteristics and leaf mass per area (LMA) were affected by needle size, but the variations of them were not associated with the variations in photosynthetic and biochemical capacity among species. Variations in area-based maximum photosynthesis (P nmax ) were affected by stomatal conductance (g s ), mesophyll conductance (g m ) and biochemical parameters including maximum carboxylation rate (V cmax ), and maximum electron transport rate (J max ). The fraction of N allocated to different photosynthetic apparatus displayed contrasting values among species, which contributed to the species variations in photosynthetic nitrogen use efficiency (PNUE) and P nmax . Additionally, all growth traits were positively correlated with P nmax and PNUE. Conclusion Needle structures are less important than needle biochemical parameters in determining the variations in photosynthetic capacity across the five Picea species. P nmax and PNUE are closedly associated with the fraction of N allocated to photosynthetic apparatus (P photo ) compared with leaf N content per area (N area ). The tremendous growth differences among the five Picea species were substantially related to the interspecies variation in P nmax and PNUE.

Carbon (C) allocation plays a central role in tree responses to environmental changes. Yet, fundamental questions remain about how trees allocate C to different sinks, for example, growth vs storage and defense. In order to elucidate allocation priorities, we manipulated the whole‐tree C balance by modifying atmospheric CO2 concentrations [CO2] to create two distinct gradients of declining C availability, and compared how C was allocated among fluxes (respiration and volatile monoterpenes) and biomass C pools (total biomass, nonstructural carbohydrates (NSC) and secondary metabolites (SM)) in well‐watered Norway spruce (Picea abies) saplings. Continuous isotope labelling was used to trace the fate of newly‐assimilated C. Reducing [CO2] to 120 ppm caused an aboveground C compensation point (i.e. net C balance was zero) and resulted in decreases in growth and respiration. By contrast, soluble sugars and SM remained relatively constant in aboveground young organs and were partially maintained with a constant allocation of newly‐assimilated C, even at expense of root death from C exhaustion. We conclude that spruce trees have a conservative allocation strategy under source limitation: growth and respiration can be downregulated to maintain ‘operational’ concentrations of NSC while investing newly‐assimilated C into future survival by producing SM.

Norway spruce is one of the most important commercial forestry species in Europe, and is commonly infected by the bark beetle-vectored necrotrophic fungus, Endoconidiophora polonica. Spruce trees display a restricted capacity to respond to environmental perturbations, and we hypothesized that water limitation will increase disease severity in this pathosystem. To test this prediction, 737 seedlings were randomized to high (W+) or low (W−) water availability treatment groups, and experimentally inoculated with one of three E. polonica strains or mock-inoculated. Seedling mortality was monitored throughout an annual growing season, and total seedling growth and lesion length indices were measured at the experiment conclusion. Seedling growth was greater in the W+ than W− treatment group, demonstrating limitation due to water availability. For seedlings infected with two of the fungal strains, no differences in disease severity occurred in response to water availability. For the third fungal strain, however, greater disease severity (mortality and lesion lengths) occurred in W− than W+ seedlings. While the co-circulation in nature of multiple E. polonica strains of varying virulence is known, this is the first experimental evidence that water availability can alter strain-specific disease severity.

Background The NAC family of transcription factors is one of the largest gene families of transcription factors in plants and the conifer NAC gene family is at least as large, or possibly larger, as in Arabidopsis . These transcription factors control both developmental and stress induced processes in plants. Yet, conifer NACs controlling stress induced processes has received relatively little attention. This study investigates NAC family transcription factors involved in the responses to the pathogen Heterobasidion annosum (Fr.) Bref. sensu lato. Results The phylogeny and domain structure in the NAC proteins can be used to organize functional specificities, several well characterized stress-related NAC proteins are found in III-3 in Arabidopsis (Jensen et al. Biochem J 426:183–196, 2010). The Norway spruce genome contain seven genes with similarity to subgroup III-3 NACs. Based on the expression pattern PaNAC03 was selected for detailed analyses. Norway spruce lines overexpressing PaNAC03 exhibited aberrant embryo development in response to maturation initiation and 482 misregulated genes were identified in proliferating cultures. Three key genes in the flavonoid biosynthesis pathway: a CHS, a F3’H and PaLAR3 were consistently down regulated in the overexpression lines . In accordance, the overexpression lines showed reduced levels of specific flavonoids, suggesting that PaNAC03 act as a repressor of this pathway, possibly by directly interacting with the promoter of the repressed genes. However, transactivation studies of Pa NAC03 and PaLAR3 in Nicotiana benthamiana showed that Pa NAC03 activated PaLAR3A, suggesting that PaNAC03 does not act as an independent negative regulator of flavan-3-ol production through direct interaction with the target flavonoid biosynthetic genes. Conclusions PaNAC03 and its orthologs form a sister group to well characterized stress-related angiosperm NAC genes and at least PaNAC03 is responsive to biotic stress and appear to act in the control of defence associated secondary metabolite production.

Proanthocyanidins (PAs) are common polyphenolic polymers of plants found in foliage, fruit, bark, roots, rhizomes, and seed coats that consist of flavan-3-ol units such as 2,3-trans-(+)-catechin and 2,3-cis-(-)-epicatechin. Although the biosynthesis of flavan-3-ols has been studied in angiosperms, little is known about their biosynthesis and ecological roles in gymnosperms. In this study, the genes encoding leucoanthocyanidin reducíase, a branch point enzyme involved in the biosynthesis of 2,3-trans-(+)-flavan-3-ols, were identified and functionally characterized in Norway spruce (Picea abies), the most widespread and economically important conifer in Europe. In addition, the accumulation of flavan-3-ols and PAs was investigated in Norway spruce saplings after wounding or inoculation with the fungal pathogen Ceratocystis polonica, which is vectored by bark beetles (Ips typographus) and is usually present during fatal beetle attacks. Monomeric and dimeric flavan-3-ols were analyzed by reverse-phase high-pressure liquid chromatography, while the size and subunit composition of larger PAs were characterized using a novel acid hydrolysis method and normal phase chromatography. Only flavan-3-ol monomers with 2,3-trans stereochemistry were detected in spruce bark; dimeric and larger PAs contained flavan-3-ols with both 2,3-trans and 2,3-cis stereochemistry. Levels of monomers as well as PAs with a higher degree of polymerization increased dramatically in spruce bark after infection by C. polonica. In accordance with their role in the biosynthesis of 2,3-trans-(+)-flavan-3-ols, transcript 'abundance of Norway spruce LEUCOANTHOCYANIDIN REDUCTASE genes also increased significantly during fungal infection. Bioassays with C. polonica revealed that the levels of 2,3-trans-(+)-catechin and PAs that are produced in the tree in response to fungal infection inhibit C. polonica growth and can therefore be considered chemical defense compounds.