Effects of Heavy Metals on the Metabolome of Pinus sylvestris (Pinaceae).

The effects of Cu, Ni, and Cd on the Pinus sylvestris metabolome was studied in experimental conditions by gas chromatography-mass spectrometry (GC-MS). Structural changes in plant metabolite network became detectable on day 6 of exposure to the metals, 3-6 days earlier than visual signs of toxicity developed. Differences at the metabolome level arose earlier in a control group of plants, and specific effects of particular metals on the plant metabolome became distinct on day 9. Both nature and concentration of a metal equally contributed to the plant metabolome clustering. Plant responses (changes in concentrations of individual metabolites) to metal exposure substantially differed depending on the metal concentration (1 or 5 mM) and nature. The effects of Cd and Cu were generally similar, while the effect of Ni was often different. Dynamic changes visualized in plant metabolite matrix reflected the changes in its correlation structure, rather than depending on the set of particular compounds.

Forest biomass carbon pool dynamics in Tibet Autonomous Region of China: Inventory data 1999-2019.

According to the forest resources inventory data for different periods and the latest estimation parameters of forest carbon reserves in China, the carbon reserves and carbon density of forest biomass in the Tibet Autonomous Region from 1999 to 2019 were estimated using the IPCC international carbon reserves estimation model. The results showed that, during the past 20 years, the forest area, forest stock, and biomass carbon storage in Tibet have been steadily increasing, with an average annual increase of 1.85×104 hm2, 0.033×107 m3, and 0.22×107 t, respectively. Influenced by geographical conditions and the natural environment, the forest area and biomass carbon storage gradually increased from the northwest to the southeast, particularly in Linzhi and Changdu, where there are many primitive forests, which serve as important carbon sinks in Tibet. In terms of the composition of tree species, coniferous forests are dominant in Tibet, particularly those containing Abies fabri, Picea asperata, and Pinus densata, which comprise approximately 45% of the total forest area in Tibet. The ecological location of Tibet has resulted in the area being dominated by shelter forest, comprising 68.76% of the total area, 64.72% of the total forest stock, and 66.34% of the total biomass carbon reserves. The biomass carbon storage was observed to first increase and then decrease with increasing forest age, which is primarily caused by tree growth characteristics. In over-mature forests, trees' photosynthesis decreases along with their accumulation of organic matter, and the trees can die. In addition, this study also observed that the proportion of mature and over-mature forest in Tibet is excessively large, which is not conducive to the sustainable development of forestry in the region. This problem should be addressed in future management and utilization activities.

Sustaining the growth of Pinaceae trees under nutrient-limited edaphic conditions via plant-beneficial bacteria.

Lodgepole pine, a prominent Pinaceae tree species native to western North America, is well-known for its ability to thrive in highly disturbed and degraded areas. One such area is the Sub-Boreal Pine-Spruce xeric-cold (SBPSxc) region in British Columbia, Canada, which is characterized by weakly-developed, parched soils that lack an organic forest floor and essential plant-available nutrients. We hypothesized that plant growth-promoting bacteria could play a significant role in sustaining the growth of lodgepole pine trees in the SBPSxc region. Testing this hypothesis, we evaluated plant growth-promoting abilities of six endophytic bacterial strains previously isolated from lodgepole pine trees growing in this region. These bacterial strains significantly enhanced the length and biomass of their natural host (lodgepole pine) as well as a foreign host (hybrid white spruce) in a 540-day long greenhouse trial. This growth stimulation could be linked to the diverse plant growth-promoting (PGP) abilities detected in these strains using in vitro assays for inorganic/organic phosphate-solubilization, siderophore production IAA production, ACC deaminase activity, lytic enzymes (chitinase, ß-1,3-glucanase, protease, and cellulase) activity, ammonia production and catalase activity. ACC deaminase activity was also detected in vivo for all strains using ethylene-sensitive plants-canola and tomato. Notably, strains belonging to the Burkholderiaceae family (HP-S1r, LP-R1r and LP-R2r) showed the greatest potential in all PGP assays and enhanced pine and spruce seedling length and biomass by up to 1.5-fold and 4-fold, respectively. Therefore, such bacterial strains with multifarious PGP abilities could be crucial for survival and growth of lodgepole pine trees in the SBPSxc region and could potentially be utilized as bioinoculant for Pinaceae trees in highly disturbed and nutrient-poor ecosystems.

Antioxidative and antifungal response of woody species to environmental conditions in the urban area.

The complexity of ecological conditions in urban areas imposes the plant species need for the development of various biochemical and physiological adaptive strategies. The aim of our research was to examine the antioxidative and antifungal metabolism of species Pinus nigra, Picea omorika, Tilia cordata and Betula pendula from the area of Banja Luka City (urban area) during two vegetation seasons (spring and autumn) and compared with the same species from forest habitats. Changes in the protein concentration, activity and isoenzyme profiles of peroxidases (POD, EC 1.11.1.7), content and antioxidative activity of total phenols and antifungal activity in leaves and needles of the plants from the urban area and forest habitats were monitored. The obtained results indicate that urban areas induce changes in antioxidative metabolism in all examined species, but that the response is species specific. The most sensitive parameter that indicates different adaptation strategy of Pinus nigra, Picea omorika, Tilia cordata and Betula pendula to environment conditions in the urban area were peroxidase isoenzyme patterns. Less specific parameter was phenol content even though there are some indications for role of their antioxidative capacity in the adjustment to specific habitat. In addition, each species had different metabolic strategy to cope with the changes caused by the urban environment.

Polyamine- and Amino Acid-Related Metabolism: The Roles of Arginine and Ornithine are Associated with the Embryogenic Potential.

The mechanisms that control polyamine (PA) metabolism in plant cell lines with different embryogenic potential are not well understood. This study involved the use of two Araucaria angustifolia cell lines, one of which was defined as being blocked, in that the cells were incapable of developing somatic embryos, and the other as being responsive, as the cells could generate somatic embryos. Cellular PA metabolism was modulated by using 5 mM arginine (Arg) or ornithine (Orn) at two time points during cell growth. Two days after subculturing with Arg, an increase in citrulline (Cit) content was observed, followed by a higher expression of genes related to PA catabolism in the responsive cell line; whereas, in the blocked cell line, we only observed an accumulation of PAs. After 14 d, metabolism was directed towards putrescine accumulation in both cell lines. Exogenous Arg and Orn not only caused a change in cellular contents of PAs, but also altered the abundance of a broader spectrum of amino acids. Specifically, Cit was the predominant amino acid. We also noted changes in the expression of genes related to PA biosynthesis and catabolism. These results indicate that Arg and Orn act as regulators of both biosynthetic and catabolic PA metabolites; however, we suggest that they have distinct roles associated with embryogenic potential of the cells.

Evolution of the biosynthesis of two hydroxyacetophenones in plants.

Acetophenones are phenolic metabolites of plant species. A metabolic route for the biosynthesis and release of 2 defence-related hydroxyacetophenones in white spruce (Picea glauca) was recently proposed to involve 3 phases: (a) biosynthesis of the acetophenone aglycons catalysed by a currently unknown set of enzymes, (b) formation and accumulation of the corresponding glycosides catalysed by a glucosyltransferase, and (c) release of the aglycons catalysed by a glucosylhydrolase (PgßGLU-1). We tested if this biosynthetic model is conserved across Pinaceae and land plant species. We assayed and surveyed the literature and sequence databases for possible patterns of the presence of the acetophenone aglycons piceol and pungenol and their glucosides, as well as sequences and expression of Pgßglu-1 orthologues. In the Pinaceae, the 3 phases of the biosynthetic model are present and differences in expression of Pgßglu-1 gene orthologues explain some of the interspecific variation in hydroxyacetophenones. The phylogenetic signal in the metabolite phenotypes was low across species of 6 plant divisions. Putative orthologues of PgßGLU-1 do not form a monophyletic group in species producing hydroxyacetophenones. The biosynthetic model for acetophenones appears to be conserved across Pinaceae, whereas convergent evolution has led to the production of acetophenone glucosides across land plants.

The Douglas-Fir Genome Sequence Reveals Specialization of the Photosynthetic Apparatus in Pinaceae.

A reference genome sequence for Pseudotsuga menziesii var. menziesii (Mirb.) Franco (Coastal Douglas-fir) is reported, thus providing a reference sequence for a third genus of the family Pinaceae. The contiguity and quality of the genome assembly far exceeds that of other conifer reference genome sequences (contig N50 = 44,136 bp and scaffold N50 = 340,704 bp). Incremental improvements in sequencing and assembly technologies are in part responsible for the higher quality reference genome, but it may also be due to a slightly lower exact repeat content in Douglas-fir vs. pine and spruce. Comparative genome annotation with angiosperm species reveals gene-family expansion and contraction in Douglas-fir and other conifers which may account for some of the major morphological and physiological differences between the two major plant groups. Notable differences in the size of the NDH-complex gene family and genes underlying the functional basis of shade tolerance/intolerance were observed. This reference genome sequence not only provides an important resource for Douglas-fir breeders and geneticists but also sheds additional light on the evolutionary processes that have led to the divergence of modern angiosperms from the more ancient gymnosperms.

Scaling of phloem structure and optimality of photoassimilate transport in conifer needles.

The phloem vascular system facilitates transport of energy-rich sugar and signalling molecules in plants, thus permitting long-range communication within the organism and growth of non-photosynthesizing organs such as roots and fruits. The flow is driven by osmotic pressure, generated by differences in sugar concentration between distal parts of the plant. The phloem is an intricate distribution system, and many questions about its regulation and structural diversity remain unanswered. Here, we investigate the phloem structure in the simplest possible geometry: a linear leaf, found, for example, in the needles of conifer trees. We measure the phloem structure in four tree species representing a diverse set of habitats and needle sizes, from 1 (Picea omorika) to 35 cm (Pinus palustris). We show that the phloem shares common traits across these four species and find that the size of its conductive elements obeys a power law. We present a minimal model that accounts for these common traits and takes into account the transport strategy and natural constraints. This minimal model predicts a power law phloem distribution consistent with transport energy minimization, suggesting that energetics are more important than translocation speed at the leaf level.

Nitrogen uptake over entire root systems of tree seedlings.

Uptake of nitrogen (N) by sequential root regions in six tree species was measured in roots of 16- to 26-month-old seedlings at 50 and 1500 µM NH4NO3 concentration, at the cell level using oscillating microelectrodes and at the root region level using enriched (15)N application. Our objective was to determine the root regions making the greatest contribution to total N uptake in each species as measured by the two contrasting techniques. White and condensed tannin zones were the regions with the smallest surface area in all species, but these zones often had the highest net flux of NH4(+) and NO3(-). For most species, little variation was found among root regions in N flux calculated using a (15)N mass balance approach, but where significant differences existed, high N flux was observed in white, cork or woody zones. When N fluxes measured by each of the two methods were multiplied by the estimated surface area or biomass of each root region, the effect of root region size had the greatest influence on regional N uptake. Root regions of greatest overall N uptake were the cork and woody zones, on average. Total N uptake may thus be greatest in older regions of tree seedling roots, despite low rates of uptake per unit area.

Bioinformatic and phylogenetic analysis of the CLAVATA3/EMBRYO-SURROUNDING REGION (CLE) and the CLE-LIKE signal peptide genes in the Pinophyta.

BACKGROUND: There is a rapidly growing awareness that plant peptide signalling molecules are numerous and varied and they are known to play fundamental roles in angiosperm plant growth and development. Two closely related peptide signalling molecule families are the CLAVATA3-EMBRYO-SURROUNDING REGION (CLE) and CLE-LIKE (CLEL) genes, which encode precursors of secreted peptide ligands that have roles in meristem maintenance and root gravitropism. Progress in peptide signalling molecule research in gymnosperms has lagged behind that of angiosperms. We therefore sought to identify CLE and CLEL genes in gymnosperms and conduct a comparative analysis of these gene families with angiosperms. RESULTS: We undertook a meta-analysis of the GenBank/EMBL/DDBJ gymnosperm EST database and the Picea abies and P. glauca genomes and identified 93 putative CLE genes and 11 CLEL genes among eight Pinophyta species, in the genera Cryptomeria, Pinus and Picea. The predicted conifer CLE and CLEL protein sequences had close phylogenetic relationships with their homologues in Arabidopsis. Notably, perfect conservation of the active CLE dodecapeptide in presumed orthologues of the Arabidopsis CLE41/44-TRACHEARY ELEMENT DIFFERENTIATION (TDIF) protein, an inhibitor of tracheary element (xylem) differentiation, was seen in all eight conifer species. We cloned the Pinus radiata CLE41/44-TDIF orthologues. These genes were preferentially expressed in phloem in planta as expected, but unexpectedly, also in differentiating tracheary element (TE) cultures. Surprisingly, transcript abundances of these TE differentiation-inhibitors sharply increased during early TE differentiation, suggesting that some cells differentiate into phloem cells in addition to TEs in these cultures. Applied CLE13 and CLE41/44 peptides inhibited root elongation in Pinus radiata seedlings. We show evidence that two CLEL genes are alternatively spliced via 3'-terminal acceptor exons encoding separate CLEL peptides. CONCLUSIONS: The CLE and CLEL genes are found in conifers and they exhibit at least as much sequence diversity in these species as they do in other plant species. Only one CLE peptide sequence has been 100% conserved between gymnosperms and angiosperms over 300 million years of evolutionary history, the CLE41/44-TDIF peptide and its likely conifer orthologues. The preferential expression of these vascular development-regulating genes in phloem in conifers, as they are in dicot species, suggests close parallels in the regulation of secondary growth and wood formation in gymnosperm and dicot plants. Based on our bioinformatic analysis, we predict a novel mechanism of regulation of the expression of several conifer CLEL peptides, via alternative splicing resulting in the selection of alternative C-terminal exons encoding separate CLEL peptides.

A novel insight into the regulation of light-independent chlorophyll biosynthesis in Larix decidua and Picea abies seedlings.

Light-independent chlorophyll (Chl) biosynthesis is a prerequisite for the assembly of photosynthetic pigment-protein complexes in the dark. Dark-grown Larix decidua Mill. seedlings synthesize Chl only in the early developmental stages and their Chl level rapidly declines during the subsequent development. Our analysis of the key regulatory steps in Chl biosynthesis revealed that etiolation of initially green dark-grown larch cotyledons is connected with decreasing content of glutamyl-tRNA reductase and reduced 5-aminolevulinic acid synthesizing capacity. The level of the Chl precursor protochlorophyllide also declined in the developing larch cotyledons. Although the genes chlL, chlN and chlB encoding subunits of the light-independent protochlorophyllide oxidoreductase were constitutively expressed in the larch seedlings, the accumulation of the ChlB subunit was developmentally regulated and ChlB content decreased in the fully developed cotyledons. The efficiency of chlB RNA-editing was also reduced in the mature dark-grown larch seedlings. In contrast to larch, dark-grown seedlings of Picea abies (L.) Karst. accumulate Chl throughout their whole development and show a different control of ChlB expression. Analysis of the plastid ultrastructure, photosynthetic proteins by Western blotting and photosynthetic parameters by gas exchange and Chl fluorescence measurements provide additional experimental proofs for differences between dark and light Chl biosynthesis in spruce and larch seedlings.

Dynamics of low-temperature acclimation in temperate and boreal conifer foliage in a mild winter climate.

To provide baseline data for physiological studies of extreme low-temperature (LT) tolerance in boreal conifers, we profiled LT stress responses, liquid nitrogen (LN(2))-quench tolerance, and sugar concentrations in foliage of boreal-temperate species pairs in the genera Abies, Picea and Pinus, growing in an arboretum in a temperate oceanic climate from August 2006 through April 2007. The boreal species acclimated more rapidly and deeply than the temperate species, acquiring LN(2)-quench tolerance by late November, despite unusually warm conditions throughout the autumn and early winter. Maximum LT tolerance in the temperate species was in the -25 to -35 degrees C range, and was reached only after a period of freezing temperatures in late January and February. During LT acclimation in the temperate species, sigmoid temperature-relative electrolyte leakage (REL) curves shifted toward lower temperatures, whereas in boreal species there was both a temperature shift and a lowering of the maximum REL until it fell below a threshold associated with irreversible injury. These differences may reflect differences in mechanisms of LT acclimation and LT tolerance. The concentrations of total and individual sugars did not show a clear pattern that could differentiate the boreal and temperate groups. Raffinose and, in three of the six species, stachyose showed the closest association with LT tolerance. Sugar concentrations, principally sucrose, decreased during mild weather, perhaps because of respiratory losses or phloem export, and increased after periods of freezing temperatures. Low-temperature acclimation in boreal species appears to follow a rigid program that may affect their ability to avoid excessive respiratory losses in the event of continued climate warming in boreal regions.

Age-dependent xylogenesis in timberline conifers.

Neither anatomical change nor physiological abnormalities have been observed in the cambia of older trees. However, different sensitivity and period of significant responses to climate suggest the existence of some age-related change in the patterns of cambial activity and/or wood cell formation. Here, weekly cambial activity and timing and duration of xylem cell enlargement and wall thickening were compared in adult (50-80 yr) and old (200-350 yr) trees of Larix decidua, Pinus cembra and Picea abies at the Alpine timberline during 2004 and 2005. Timings and durations of xylogenesis differed between adult and old trees, with 2-3 wk shorter cambial activity found in the latter. The delayed onset of cambium division and lower cell production in old trees, with respect to adult trees, led to reductions of 15-20% in the overall duration of xylem differentiation. These results demonstrate that cambial dynamics change during the tree lifespan and that the time window of tree-ring production shortens with age. Variations in the period of xylem growth may be the cause of age-dependent responses to climate. The observed shorter xylogenesis in older plants at the Alpine timberline could be related to a size effect and not just to age per se.

Rising atmospheric CO2 reduces sequestration of root-derived soil carbon.

Forests have a key role as carbon sinks, which could potentially mitigate the continuing increase in atmospheric carbon dioxide concentration and associated climate change. We show that carbon dioxide enrichment, although causing short-term growth stimulation in a range of European tree species, also leads to an increase in soil microbial respiration and a marked decline in sequestration of root-derived carbon in the soil. These findings indicate that, should similar processes operate in forest ecosystems, the size of the annual terrestrial carbon sink may be substantially reduced, resulting in a positive feedback on the rate of increase in atmospheric carbon dioxide concentration.

Trichloroacetic acid in Norway spruce/soil-system. II. Distribution and degradation in the plant.

Independently from its origin, trichloroacetic acid (TCA) as a phytotoxic substance affects coniferous trees. Its uptake, distribution and degradation were thus investigated in the Norway spruce/soil-system using 14C labeling. TCA is distributed in the tree mainly by the transpiration stream. As in soil, TCA seems to be degraded microbially, presumably by phyllosphere microorganisms in spruce needles. Indication of TCA biodegradation in trees is shown using both antibiotics and axenic plants.

Winter water relations of New England conifers and factors influencing their upper elevational limits. I. Measurements.

The upper elevational limits of tree species are thought to be controlled by abiotic factors such as temperature and the soil and atmospheric conditions affecting plant water status. We measured relative water contents (RWC), water potentials (Psi) and cuticular conductances (g(c)) of shoots of four conifer species-eastern hemlock (Tsuga canadensis (L.) Carr.), eastern white pine (Pinus strobus L.), red pine (P. resinosa Ait.) and red spruce (Picea rubens Sarg.)-during two winters on Mt. Ascutney, Vermont, USA. Some micrometeorological measurements are also reported. Eastern hemlock and white pine were studied near their upper elevational limits at a 640-m site, and red pine was studied near its upper elevational limit at 715 m. Red spruce was also studied at the 715-m site, which is in the middle of its elevational range on this mountain. There was no evidence of winter desiccation stress in any species. The observed distribution of seedlings suggested that the upper elevational limits on shade-intolerant eastern white pine and red pine are set by the absence of suitable seed beds after 100 years without fire. Eastern hemlock is able to reproduce in deep shade on organic substrates, but germination at high elevations may be restricted by low temperatures.

Differences in nitrogen economy of temperate trees.

Twenty-four temperate tree species were classified into three groups based on cluster analysis of relative growth rate, nitrogen concentration, nitrogen-production efficiency, nitrogen-distribution ratio and nitrogen-use efficiency as follows: Group I (Asteridae and Rosidae), Group II (Dilleniidae and Hamamelidae) and Group III (Coniferopsidae). Relative growth rate (RGR) was high in Group II, moderate in Group I and low in Group III. The regression coefficient for the relationship between RGR and leaf nitrogen concentration was higher in Group II than in Group I, and no relationship was observed in Group III. Parameter analysis of RGR indicated that RGR per unit leaf nitrogen was important for all three groups, but that the allocation of nitrogen to leaves was particularly important in Groups I and II. The ratio of dark respiratory rate (R) to net photosynthetic rate (A) was higher in Group I than in Group II. Neither A nor R was measured in the Group III species. A linear relationship was observed between leaf nitrogen concentration and A in Group II, but this relationship was not evident in Group I.