Nitrogen loading increases the ozone sensitivity of larch seedlings with higher sensitivity to nitrogen loading.

Larch (Larix sp.) tree is a critical species for the future afforestation in Northeast Asia. The impacts of elevated concentrations of ground-level ozone (O3) and nitrogen (N) deposition are raising concerns. However, knowledge of the combined effects of elevated O3 and N loading are still limited. We investigated whether nitrogen loading mitigates the negative impacts of ozone on two larch species: the Japanese larch (L. kaempferi) and its hybrid larch F1 (L. gmelinii var. japonica × L. kaempferi) or not. We used open-top cambers and compared responses of the larch seedlings. Results showed the N loading mitigated the negative effects of O3 on Japanese larch. However, in hybrid larch F1, N loading did not mitigate O3-induced inhibition of growth and photosynthetic capacity. Mitigation effect of N loading on negative O3 impacts may vary between the two Larix spp., Hybrid larch F1 could be more affected by the combined effects of O3 and N loading due to its higher growth response to N loading. Elevated O3 also reduced leaf nitrogen/phosphorus (N/P) ratio by elevated O3, with significant effects in hybrid larch F1, particularly under N loading. In the present study, leaf N/P ratio was utilized to validate the hypothesis that a positive effect of N loading may be observed if O3 does not induce P limitation in Larix spp. We demonstrated a potential leaf N/P ratio function, which could reflect responses to O3 and N loading in hybrid larch F1.

Ozone risk assessment for an Alpine larch forest in two vegetative seasons with different approaches: comparison of POD1 and AOT40.

The upper vegetation belts like larch forests are supposed to be under great pressure because of climate change in the next decades. For this reason, the evaluation of the risks due to abiotic stressors like ozone is a key step. Two different approaches were used here: mapping AOT40 index by means of passive samplers and direct measurements of ozone deposition.Measurements of ozone fluxes using the eddy-correlation technique were carried out for the first time over a larch forest in Paspardo (I) at 1750 m a.s.l. Two field campaigns were run: the first one in 2010 from July to October and the second one in the following year from June to September. Vertical exchange of ozone, energy, and momentum were measured on a tower platform at 26 m above ground level to study fluxes dynamics over this ecosystem. Since the tower was located on a gentle slope, an "ad hoc" methodology was developed to minimize the effects of the terrain inclination. The larch forest uptake was estimated by means of a two-layer model to separate the understorey uptake from the larch one. Even if the total ozone fluxes were generally high, up to 30-40 nmol O3 m-2 s-1 in both years, the stomatal uptake by the larch forest was relatively low (around 15% of the total deposition).Ozone risk was assessed considering the POD1 received by the larch forest and the exposure index AOT40 estimated with both local data and data from the map obtained by the passive samplers monitoring.

Stem and crown growth of Japanese larch and its hybrid F1 grown in two soils and exposed to two free-air O3 regimes.

Ozone (O3) pollution and soil infertility may negatively affect boreal forests across the Northern Hemisphere. Impacts to economically and ecologically important larches (Larix sp., Pinacaeae) are particularly concerning. Using a free air O3 enrichment (FACE) system, we investigated the effect of 2-year elevated O3 exposure (≈66 nmol mol-1) on Japanese larch (L. kaempferi) and its hybrid larch F1 (L. gmelinii var. japonica × L. kaempferi) planted directly into either fertile brown forest soil (BF) or BF mixed with infertile volcanic ash soil (VA). Overall, photosynthetic pigmentation and the growth performance of the stem and crown were reduced in both taxa exposed to elevated O3. Furthermore, hybrid larch, in both O3 treatments, performed better than Japanese larch. This finding contradicts findings of prior experiments with potential experimental artifacts of O3 exposure facilities and root restrictions. Elevated O3 also disproportionately inhibited stem diameter growth and caused an imbalance in chlorophylls a/b and chlorophyll/carotenoid ratios. Hybrid and Japanese larches grown in BF and VA had a significantly lower drop of stem diameter over the run of stem height (from base to top) when exposed to elevated O3, compared to ambient O3. This finding indicates altered stem shape under elevated O3. Among 11 response variables, there were no significant interactions between O3 treatment and taxa. There was also no significant interaction of soil condition and taxa, suggesting that the two larches shared a similar response to O3 and soil type. Understanding the performance of hybrid larch in relation to its parent species has ramifications for breeding success in a soil-degraded and O3-polluted environment.

Transcriptome Analysis of mRNA and miRNA in Somatic Embryos of Larix leptolepis Subjected to Hydrogen Treatment.

Hydrogen is a therapeutic antioxidant that has been used extensively in clinical trials. It also acts as a bioactive molecule that can alleviate abiotic stress in plants. However, the biological effects of hydrogen in somatic embryos and the underlying molecular basis remain largely unknown. In this study, the morphological and physiological influence of exogenous H2 treatment during somatic embryogenesis was characterized in Larix leptolepis Gordon. The results showed that exposure to hydrogen increased the proportions of active pro-embryogenic cells and normal somatic embryos. We sequenced mRNA and microRNA (miRNA) libraries to identify global transcriptome changes at different time points during H2 treatment of larch pro-embryogenic masses (PEMs). A total of 45,393 mRNAs and 315 miRNAs were obtained. Among them, 4253 genes and 96 miRNAs were differentially expressed in the hydrogen-treated libraries compared with the control. Further, a large number of the differentially expressed mRNAs and miRNAs were related to reactive oxygen species (ROS) homeostasis and cell cycle regulation. We also identified 4399 potential target genes for 285 of the miRNAs. The differential expression data and the mRNA-miRNA interaction network described here provide new insights into the molecular mechanisms that determine the performance of PEMs exposed to H2 during somatic embryogenesis.

Physiological responses of the hybrid larch (Larix × eurolepis Henry) to cadmium exposure and distribution of cadmium in plantlets.

Phytoextraction of Cd is a growing biotechnology although we currently know few Cd hyperaccumulators, i.e., plant species able to accumulate at least 0.1 mg Cd g(-1) dry weight in aerial organs. Owing their deep root system and high biomass, trees are more and more preferred to herbaceous species for phytoextraction. Assuming that conifers could be relevant models under cold climates, we investigated cadmium tolerance of the hybrid larch Larix × eurolepis Henry (Larix decidua × Larix kaempferi) and the efficiency of this species to store this metal. In vitro grown larches were chosen in order to reduce time of exposure and to more rapidly evaluate their potential efficiency to accumulate Cd. One-month-old plantlets were exposed for 2 and 4 weeks to 250 and 500 µM Cd. Results showed that they tolerated a 4-week exposure to 250 µM Cd, whereas the content of photosynthetic pigment strongly dropped in plantlets growing in the presence of 500 µM Cd. In the presence of 250 µM Cd, shoot growth slightly decreased but photosynthetic pigment and total soluble carbohydrate contents were not modified and no lipid peroxidation was detected. In addition, these plantlets accumulated proline, particularly in shoots (two to three times more than control). In roots, Cd concentration in the intracellular fraction was always higher than in the cell wall fraction contrary to shoots where Cd concentration in the cell wall fraction increased with time and Cd concentration in the medium. In shoots, Cd concentration was lower than in roots with a ratio of 0.2 after 4 weeks of exposure but stayed around 0.2 mg g(-1) dry weight, thus a value higher than the threshold requested for Cd hyperaccumulators. Hybrid larch would thus be a relevant candidate for field test of Cd phytoextraction.

Transcriptomic Response to Nitric Oxide Treatment in Larix olgensis Henry.

Larix olgensis Henry is an important coniferous species found in plantation forests in northeastern China, but it is vulnerable to pathogens. Nitric oxide (NO) is an important molecule involved in plant resistance to pathogens. To study the regulatory role of NO at the transcriptional level, we characterized the transcriptomic response of L. olgensis seedlings to sodium nitroprusside (SNP, NO donor) using Illumina sequencing and de novo transcriptome assembly. A significant number of putative metabolic pathways and functions associated with the unique sequences were identified. Genes related to plant pathogen infection (FLS2, WRKY33, MAPKKK, and PR1) were upregulated with SNP treatment. This report describes the potential contribution of NO to disease resistance in L. olgensis as induced by biotic stress. Our results provide a substantial contribution to the genomic and transcriptomic resources for L. olgensis, as well as expanding our understanding of the involvement of NO in defense responses at the transcriptional level.

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii).

BACKGROUND AND AIMS: In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question. METHODS: Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis. KEY RESULTS: Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg(-1) f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g(-1) d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap. CONCLUSIONS: In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.

Ectomycorrhizal colonization and growth of the hybrid larch F1 under elevated CO2 and O3.

We studied the colonization of ectomycorrhizal fungi and species abundance of a hybrid larch (F1) under elevated CO2 and O3. Two-year-old seedlings were planted in an Open-Top-Chamber system with treatments: Control (O3 < 6 nmol/mol), O3 (60 nmol/mol), CO2 (600 µmol/mol), and CO2 + O3. After two growing seasons, ectomycorrhiza (ECM) colonization and root biomass increased under elevated CO2. Additionally, O3 impaired ECM colonization and species richness, and reduced stem biomass. However, there was no clear inhibition of photosynthetic capacity by O3. Concentrations of Al, Fe, Mo, and P in needles were reduced by O3, while K and Mg in the roots increased. This might explain the distinct change in ECM colonization rate and diversity. No effects of combined fumigation were observed in any parameters except the P concentration in needles. The tolerance of F1 to O3 might potentially be related to a shift in ECM community structure.

CO2 enrichment alters diurnal stem radius fluctuations of 36-yr-old Larix decidua growing at the alpine tree line.

To understand how trees at high elevations might use water differently in the future, we investigated the effects of CO2 enrichment and soil warming (separately and combined) on the water relations of Larix decidua growing at the tree line in the Swiss Alps. We assessed diurnal stem radius fluctuations using point dendrometers and applied a hydraulic plant model using microclimate and soil water potential data as inputs. Trees exposed to CO2 enrichment for 9 yr showed smaller diurnal stem radius contractions (by 46 ± 16%) and expansions (42 ± 16%) compared with trees exposed to ambient CO2 . Additionally, there was a delay in the timing of daily maximum (40 ± 12 min) and minimum (63 ± 14 min) radius values for trees growing under elevated CO2 . Parameters optimized with the hydraulic model suggested that CO2 -enriched trees had an increased flow resistance between the xylem and bark, representing a more buffered water supply system. Soil warming did not alter diurnal fluctuation dynamics or the CO2 response. Elevated CO2 altered the hydraulic water flow and storage system within L. decidua trees, which might have contributed to enhanced growth during 9 yr of CO2 enrichment and could ultimately influence the future competitive ability of this key tree-line species.

Effect of nursery nitrogen application of bare-root Larix olgensis seedlings on growth, nitrogen uptake and initial field performance.

Nursery nitrogen application has been used to improve seedling quality. The technique has received little attention with bare-root seedlings and their subsequent field performance on weed competition sites. Our research objective was to examine responses of one- and two- year-old bare-root Olga Bay larch (Larix olgensis Henry) seedlings to nursery nitrogen supplements and subsequent one-year field performance on a competitive site. The fertilizer levels (kg N ha(-1)) were 0 (control), 60 (conventional fertilization, 60 C), 120 (additional nitrogen applied two times, 120 L), 180 (additional nitrogen applied three times, 180 L) and N were applied in increments of 30 kg ha(-1 )at 15-day interval to maintain a base nutrient level Although pre-planting morphological attributes and nitrogen status of one-year-old (la) seedlings were more sensitive to 60 C than for two-year-old (2a) seedlings, the conventional application failed to enhance their field survival (15.6% vs 17.8%), relative height growth (89.0% vs 79.6%), and relative diameter growth (17.0% vs 22.9%). The la seedlings' field survival (15.6% for 0, 17.8% for 60 C) and 2a seedlings' relative height growth rate (11.0% for 0, 8.9% for 60 C) were not increased significantly until they were provided the 120 L (survival of 23.3% for la, relative height growth rate of 15.0% for 2a). According to pre-planting attributes and field performance, optimum nursery nitrogen application was 120 L for the 2a seedlings and 180 L for la seedlings. Except for component nitrogen concentration, pre-planting morphological attributes and component N content for the 2a seedlings were as much 3.3 to 37.7 times that of la seedlings. In conclusion, the contrasting survival of poor (15.6%-28.9%) for la seedlings and high (84.4%-91.1%) for 2a seedlings indicated that additional nitrogen fertilizer would not equal the benefits of an another year's growth in the nursery. Successful reforestation could not be fulfilled by la seedlings regardless of their prenutrients. An alternative technique for sites with competing vegetation was to apply 120 kg N ha(-1) in the nursery during July and August on 2a seedlings.

Hybrid larch (Larix x eurolepis Henry): a good candidate for cadmium phytoremediation?

Studies related to phytoremediation by conifers are still at their beginning. Thus, we investigated the ability of a hybrid larch (Larix x eurolepis) to accumulate cadmium (Cd). One-month-old clonal plantlets grown in vitro were exposed for 1 week to a high Cd concentration (1.5 mM). No significant effect was observed on root and shoot biomass, root length, and needle number as a result of Cd treatment. Leaf photosynthetic pigment content and total soluble protein concentration in roots and shoots remained unchanged compared to control plantlets. Taken together, these results suggested that hybrid larch tolerated Cd in our conditions. The high Cd concentration in shoots (200 µg Cd gram(-1) dry weight) showed the good capacity of larch to translocate Cd and thus a potential use of this species for phytoremediation. Furthermore, under our conditions, phytochelatin biosynthesis pathway was slightly stimulated, suggesting that this pathway did not reach the threshold and/or another mechanism of Cd storage may be involved to explain larch tolerance to Cd.

[Somatic embryogenesis in in vitro culture of three larch species].

Embryogenic callus formation in different larch species from Siberia (Larix sibirica, L. gmelinii, and L. sukaczewii) was carried out on MSGm medium supplemented with growth regulators (2.4-D and BAP) and followed one and the same scheme: elongation of somatic cells and their asymmetric division with formation of initial and tube cells. The cells of embryo initial underwent sequential divisions and formed embryonic globules which caused the formation of somatic embryos. Somatic embryos became mature and germinated by addition of ABA and PEG into the medium. Long-term proliferating cell lines and regenerant plants were obtained in Sukachev larch and its hybrid with Siberian larch. The success of somatic embryogenesis depended on the genotype of the donor tree.

[Effects of nitrogen fertilization on ectomycorrhizal infection of first order roots and root morphology of Larix gmelinii plantation].

In this paper, the first order roots of Larix gmelinii plantation under N fertilization were sampled from different soil depths in different seasons to study their morphology under effects of ectomycorrhizal fungi. The results showed that the infection rate of ectomycorrhizal fungi on the first order roots was significantly affected by soil N availability, soil depth, and season. N fertilization induced a decrease of the infection rate, and the decrement varied with soil depth and season. In comparing with the control, the infected first order roots had an obvious variation of their morphology, e. g., averagely, root diameter increased by 18.7%, root length decreased by 23.7%, and specific root length decreased by 16.3%, which differed significantly with N application rate, soil depth, and season. The infection of ectomycorrhizal fungi changed the first order root morphology of L. gmelinii, which might substantially affect the physiological and ecological processes of host plant fine roots.

Exemplifying whole-plant ozone uptake in adult forest trees of contrasting species and site conditions.

Whole-tree O3 uptake was exemplified for Picea abies, Fagus sylvatica and Larix decidua in stands at high and low altitude and contrasting water availability through sap flow measurement in tree trunks, intrinsically accounting for drought and boundary layer effects on O3 flux. O3 uptake of evergreen spruce per unit foliage area was enhanced by 100% at high relative to low elevation, whereas deciduous beech and larch showed similar uptake regardless of altitude. The responsiveness of the canopy conductance to water vapor and, as a consequence, O3 uptake to soil moisture and air humidity did not differ between species. Unifying findings at the whole-tree level will promote cause-effect based O3 risk assessment and modeling.

Effects of elevated atmospheric CO2 concentration on the nutrient uptake characteristics of Japanese larch (Larix kaempferi).

We evaluated the response of Japanese larch (Larix kaempferi Sieb. & Zucc.) to elevated atmospheric CO(2) concentration ([CO(2)]) (689 +/- 75 ppm in 2002 and 697 +/- 90 ppm in 2003) over 2 years in a field experiment with open-top chambers. Root activity was assessed as nitrogen, phosphorus and potassium uptake rates estimated from successive measurements of absorbed amounts. Dry matter production of whole plants was unaffected by elevated [CO(2)] in the first year of treatment, but increased significantly in response to elevated [CO(2)] in the second year. In contrast, elevated [CO(2)] increased the root to shoot ratio and fine root dry mass in the first year, but not in the second year. Elevated [CO(2)] had no effect on tissue N, P and K concentrations. Uptake rates of N, P and K correlated with whole-plant relative growth rates, but were unaffected by growth [CO(2)], as was ectomycorrhizal colonization, a factor assumed to be important for nutrient uptake in trees. We conclude that improved growth of Larix kaempferi in response to elevated [CO(2)] is accompanied by increased root biomass, but not by increased root activity.

Effects of exogenous mevalonic acid on sterol lipid classes in Larix kaempferi callus.

In Japanese larch (Larix kaempferi (Lamb.) Carr.) calli, free sterol (FS), acylsterol (AS) and glycosylsterol, including the acylated type, were found in the proportion of 1.0:0.1:0.8. When the calli were cultured in the presence of 10 mM mevalonic acid (MVA), the content of AS, but not FS and glycosylsterol, was increased remarkably. The major component sterol in each sterol lipid class was usually sitosterol (more than 90%) with campesterol as a minor one. There were no differences on the sterol compositions between the calli cultured with or without MVA. When the calli cultured with 10 mM MVA for 6 weeks were transferred to the control medium without exogenous MVA, AS contents decreased to the level of the control calli. Thus, it was shown that sterol lipids, such as FS and glycosylsterols, with the structural functions was maintained in the constant content and the excess sterol biosynthesized from exogenous MVA was esterified to form AS for storage of sterol components.

Shoot water status and ABA responses of transgenic hybrid larch Larix kaempferi x L. decidua to ectomycorrhizal fungi and osmotic stress.

It has been postulated that osmotic effects on plant tissue are mediated by abscisic acid (ABA). Hybrid larch (Larix kaempferi (Lambert) Carr. x L. decidua Mill.) plantlets, transformed with the ABA-inducible wheat Em promoter associated with the Gus reporter gene, were axenically inoculated with two ectomycorrhizal fungi: Cenococcum geophilum Fr., considered tolerant to water stress, and Laccaria bicolor (Marie) Orton, considered less tolerant to drought. The mycorrhizal and non-mycorrhizal transgenic plantlets were subjected to osmotic stress by adding polyethylene glycol (PEG) to the culture medium. In the presence of PEG, L. bicolor and C. geophilum reduced shoot water potential and turgor potential, but increased host osmotic potential. Treatment of plantlets with PEG induced a significant increase in endogenous ABA concentrations. Laccaria bicolor and C. geophilum behaved similarly and significantly decreased the ABA response of plantlets to PEG treatment. Moreover, inoculation with either fungus regulated the ABA response of the plantlets even when the fungus was separated from the host by a cellophane sheet that prevented mycorrhiza formation. Although the wheat Em promoter was inducible in larch plantlets, it was not regulated by endogenous ABA. Induction of the wheat Em promoter in larch plantlets depended on organ type, with maximum induction in the root apex. Induction of the Em promoter was significantly decreased by mycorrhizal inoculation.

Free radical generation in Pinus sylvestris and Larix decidua seeds primed with polyethylene glycol or potassium salt solutions.

Electron paramagnetic resonance (EPR) spectra of Pinus sylvestris and Larix decidua seeds show that priming with PEG+200 mg kg(-1) gibberelic acid (GA(3)) results in appreciably higher free radical contents than in unprimed control samples. Only relatively minor changes in the free radical levels were observed in seeds primed with K(+) salts. However, both priming treatments have been reported previously to result in faster germination rates compared to controls without changing the germination percentage. In measurements on individual seeds of L. decidua, there were no significant differences between the mean free radical levels in viable and non-viable seeds within each treatment group. Thus, the elevation in free radical levels in the PEG+GA(3) treatments appear to be a direct consequence of the priming treatment and do not correspond to the initiation of germination.

Identification of a novel factor, vanillyl benzyl ether, which inhibits somatic embryogenesis of Japanese larch (Larix leptolepis Gordon).

In contrast to angiosperms, some gymnosperms form well-developed suspensors in somatic embryogenesis. This characteristic makes it easy to study suspensor biology. In cultures with high cell densities, somatic embryogenesis of Japanese larch, especially the suspensor development, is strongly inhibited due to factor(s) that are released by the cells into the culture medium. In this study, we purified and identified one of the inhibitory factors present in high-cell-density conditioned medium (HCM) of larch cells. The factor with the strongest inhibitory activity was purified by dialysis, extraction by ethyl acetate, octadecylsilyl (ODS) column chromatography and high-performance liquid chromatography (HPLC). The inhibitory factor was identified as vanillyl benzyl ether (VBE) by physicochemical analysis. This compound was first isolated from natural resources. Authentic VBE inhibited somatic embryo formation in Japanese larch, and the inhibitory effect in the suspensor was stronger than in the embryo proper. Furthermore, quantification of VBE by HPLC demonstrated that VBE accumulates at high concentrations in HCM. These results suggest that VBE is a novel negative regulator of somatic embryogenesis.

Responses of black spruce (Picea mariana) and tamarack (Larix laricina) to flooding and ethylene.

Black spruce (Picea mariana (Mill.) BSP) and tamarack (Larix laricina (Du Roi) K. Koch) are the predominant tree species in the boreal peatlands of Alberta, Canada, where low nutrient availability, low soil temperature and a high water table limit their growth. Effects of flooding for 28 days on morphological and physiological responses were investigated in greenhouse-grown black spruce and tamarack seedlings in a growth chamber. Flooding reduced root hydraulic conductance, net assimilation rate and stomatal conductance, and increased water-use efficiency (WUE) and needle electrolyte leakage in both species. Although flooded black spruce seedlings maintained higher net assimilation rates and stomatal conductance than flooded tamarack seedlings, flooded tamarack seedlings were able to maintain higher root hydraulic conductance than flooded black spruce seedlings. Needles of flooded black spruce developed tip necrosis and electrolyte leakage after 14 days of flooding, and these symptoms were subsequently more prominent than in needles of flooded tamarack seedlings. Flooded tamarack seedlings exhibited no visible injury symptoms and developed hypertrophied lenticels at their stem base. Application of exogenous ethylene resulted in a significant reduction in net assimilation, stomatal conductance and root respiration, whereas root hydraulic conductivity increased in both species. Thus, although flooded black spruce seedlings maintained a higher stomatal conductance and net assimilation rate than tamarack seedlings, black spruce did not cope with the deleterious effects of prolonged soil flooding and exogenous ethylene as well as tamarack.