Proteomic insights into adventitious root formation in Larix kaempferi.

The adventitious root formaton (ARF) in excised plant parts is essential for the survival of isolated plant fragments. In this study, we explored the complex mechanisms of ARF in Larix kaempferi by conducting a comprehensive proteomic analysis across three distinct stages: the induction of adventitious root primordia (C1, 0-25 d), the formation of adventitious root primordia (C2, 25-35 d), and the elongation of adventitious roots (C3, 35-45 d). We identified 1976 proteins, with 263 and 156 proteins exhibiting increased abundance in the C2/C1 and C3/C2 transitions, respectively. In contrast, a decrease in the abundance of 106 and 132 proteins suggests a significant demand for metabolic processes during the C2/C1 phase. The abundance of IAA-amino acid hydrolase and S-adenosylmethionine synthase were increased in the C2/C1 phase, underscoring the role of auxin in adventitious root induction. The decrease in abundance of photosynthesis-related proteins during the C2/C1 phase highlights the significance of initial light conditions in adventitious root induction. Moreover, variation in cell wall synthesis and metabolic proteins in the C2/C1 and C3/C2 stages suggests that cell wall metabolism is integral to adventitious root regeneration. Gene Ontology enrichment analysis revealed pathways related to protein modification enzymes, including deubiquitinases and kinases, which are crucial for modulating protein modifications to promote ARF. Furthermore, the increased abundance of antioxidant enzymes, such as peroxidases and glutathione peroxidases, indicates a potential approach for enhancing ARF by supplementing the culture medium with antioxidants. Our study provides insights into metabolic changes during ARF in L. kaempferi, offering strategies to enhance adventitious root regeneration. These findings have the potential to refine plant propagation techniques and expedite breeding processes. SIGNFICANCE: The main challenge in the asexual reproduction technology of Larix kaempferi lies in adventitious root formation (ARF). While numerous studies have concentrated on the efficiency of ARF, proteomic data are currently scarce. In this study, we collected samples from three stages of ARF in L. kaempferi and subsequently performed proteomic analysis. The data generated not only reveal changes in protein abundance but also elucidate key metabolic processes involved in ARF. These insights offer a novel perspective on addressing the challenge of adventurous root regeneration.

Cavitation fatigue in conifers: a study on eight European species.

After drought-induced embolism and repair, tree xylem may be weakened against future drought events (cavitation fatigue). As there are few data on cavitation fatigue in conifers available, we quantified vulnerability curves (VCs) after embolism/repair cycles on eight European conifer species. We induced 50% and 100% loss of conductivity (LC) with a cavitron, and analyzed VCs. Embolism repair was obtained by vacuum infiltration. All species demonstrated complete embolism repair and a lack of any cavitation fatigue after 50% LC . After 100% LC, European larch (Larix decidua), stone pine (Pinus cembra), Norway spruce (Picea abies), and silver fir (Abies alba) remained unaffected, while mountain pine (Pinus mugo), yew (Taxus baccata), and common juniper (Juniperus communis) exhibited 0.4-0.9 MPa higher vulnerability to embolism. A small cavitation fatigue observed in Scots pine (Pinus sylvestris) was probably biased by incomplete embolism repair, as indicated by a correlation of vulnerability shifts and conductivity restoration. Our data demonstrate that cavitation fatigue in conifers is species-specific and depends on the intensity of preceding LC. The lack of fatigue effects after moderate LC, and relevant effects in only three species after high LC, indicate that conifers are relatively resistant against cavitation fatigue. This is remarkable considering the complex and delicate conifer pit architecture and may be important considering climate change projections.

Couplings in cell differentiation kinetics mitigate air temperature influence on conifer wood anatomy.

Conifer trees possess a typical anatomical tree-ring structure characterized by a transition from large and thin-walled earlywood tracheids to narrow and thick-walled latewood tracheids. However, little is known on how this characteristic structure is maintained across contrasting environmental conditions, due to its crucial role to ensure sap ascent and mechanical support. In this study, we monitored weekly wood cell formation for up to 7 years in two temperate conifer species (i.e., Picea abies (L.) Karst and Larix decidua Mill.) across an 8°C thermal gradient from 800 to 2,200 m a.s.l. in central Europe to investigate the impact of air temperature on rate and duration of wood cell formation. Results indicated that towards colder sites, forming tracheids compensate a decreased rate of differentiation (cell enlarging and wall thickening) by an extended duration, except for the last cells of the latewood in the wall-thickening phase. This compensation allows conifer trees to mitigate the influence of air temperature on the final tree-ring structure, with important implications for the functioning and resilience of the xylem to varying environmental conditions. The disappearing compensation in the thickening latewood cells might also explain the higher climatic sensitivity usually found in maximum latewood density.

Root order-based traits of Manchurian walnut & larch and their plasticity under interspecific competition.

Manchurian walnut and larch are key timber species of northeast China but information on (fine) root traits of both species is scarce. Plasticity of root traits in mixed plantations has been studied rarely although this could give important insights into mechanisms of root competition. This study examined root traits by branching order in 30-yr-old monocultures and their plasticity in mixed plantations. In monocultures, Manchurian walnut and larch differed in key fine root traits. Larch roots hold more absorptive root orders, larger diameter and lower specific root length/area. Walnut root orders featured greater cortex:stele ratios, N-concentrations and respiration rates. Under interspecific competition, the proportion of walnut root tips increased, the biomass/length of larch root orders 1-3 decreased. Larch possessed a greater morphological and anatomical plasticity of terminal root orders than walnut. Mycorrhizal colonization rates of walnut were reduced. Both species differed fundamentally in their fine root properties. Absorptive fine root orders reacted plastic under interspecific competition while traits of higher root orders remained unchanged. In mixture, larch roots possessed a greater plasticity in traits related to resource uptake (efficiency) than walnut roots whose reaction norm is suggested to be predominantly based on interference competition via juglone exudation.

Mating patterns and pollen dispersal in a Japanese larch (Larix kaempferi) clonal seed orchard: a case study.

Pollination dynamics highly determines the genetic quality of seed orchard crops. However, there is less research about the effect of mating patterns on seed productivity of orchard crops. So far, clonal seed orchards have been producing genetically improved seedlings used for most Japanese larch (Larix kaempferi (Lamb.) Carr.) plantations in China. In the present study, a total of 17 highly variable simple sequence repeat (SSR) markers were used for genotyping a progeny trial population consisting of 647 open-pollinated progenies germinated from seeds which were collected from 63 maternal clones with 140 potential paternal clones in a Japanese larch clonal seed orchard in China. Paternity analysis was used in the present case study in order to evaluate the level of paternal gametic contribution, estimate pollen contamination and selfing rates, and investigate pollination patterns, pollen dispersal patterns and the impact of mating patterns on seed productivity of orchard crops. We observed 93.7% of the success rate of the parental assignment, unequal paternal gametic contribution (0-12.4%) with 6.3% of the progenies derived from pollen contamination or unsampled pollen donors, and absence of evidence for selfing. We also found that pollination rate highly depended on the distance between pollen donors and maternal parents, the majority of the identified crossing (65.7%) occurred between clones within a 150-m radius, and large variations in growth performance existed among the paternal half-siblings. Progeny growth performance (diameter at breast (DBH) and height (HGT)) was measured at Age-20 in order to investigate the impact of mating patterns on timber production of orchard crops. As either the paternal or maternal, two clones (i. e., clones Z38 and Z62) were identified to have produced progenies with higher average stem volume breeding values than that of all of the progenies. Specifically, the genetic gains for volume were 3.53% for the two clones as paternal parents, and 8.26% as the maternal parents at Age-20. Thus, both elite clones were ideal candidates for the construction of next-generation clonal seed orchards due to their synchronous reproductive phenology with greater crossing rate and higher genetic gain. These results improved the pedigree information to provide solid evidence of mating patterns for future design and effective management of seed orchards and for the development of viable long-term breeding strategies for other coniferous species.

Force-displacement measurements of earlywood bordered pits using a mesomechanical tester.

The elastic properties of pit membranes are reported to have important implications in understanding air-seeding phenomena in gymnosperms, and pit aspiration plays a large role in wood technological applications such as wood drying and preservative treatment. Here we present force-displacement measurements for pit membranes of circular bordered pits, collected on a mesomechanical testing system. The system consists of a quartz microprobe attached to a microforce sensor that is positioned and advanced with a micromanipulator mounted on an inverted microscope. Membrane displacement is measured from digital image analysis. Unaspirated pits from earlywood of never-dried wood of Larix and Pinus and aspirated pits from earlywood of dried wood of Larix were tested to generate force-displacement curves up to the point of membrane failure. Two failure modes were observed: rupture or tearing of the pit membrane by the microprobe tip, and the stretching of the pit membrane until the torus was forced out of the pit chamber through the pit aperture without rupture, a condition we refer to as torus prolapse.

Resin duct size and density as ecophysiological traits in fire scars of Pseudotsuga menziesii and Larix occidentalis.

BACKGROUND AND AIMS: Resin ducts (RDs) are features present in most conifer species as defence structures against pests and pathogens; however, little is known about RD expression in trees following fire injury. This study investigates changes in RD size and density in fire scars of Douglas fir (Pseudotsuga menziesii) and western larch (Larix occidentalis) as a means to evaluate the ecophysiological significance of traumatic resinosis for tree defence and survival. METHODS: Transverse and tangential microsections were prepared for light microscopy and image analysis in order to analyse axial and radial RDs, respectively. Epithelial cells of RDs and fusiform rays associated with radial RDs were also examined. RDs were compared between normal xylem and wound xylem at four different section heights along the fire-injured stem. KEY RESULTS: Following fire injury, P. menziesii axial RDs narrowed by 38-43 % in the first year after injury, and the magnitude of this change increased with stem height. Larix occidentalis axial RDs widened by 46-50 % in the second year after injury. Radial RDs were of equivalent size in P. menziesii, but widened by 162-214 % in L. occidentalis. Fusiform rays were larger following fire injury, by 4-14 % in P. menziesii and by 23-38 % in L. occidentalis. Furthermore, axial RD density increased in both species due to the formation of tangential rows of traumatic RDs, especially in the first and second years after injury. However, radial RD density did not change significantly. CONCLUSIONS: These results highlight traumatic resinosis as a species-specific response. Pseudotsuga menziesii produce RDs of equivalent or reduced size, whereas L. occidentalis produce wider RDs in both the axial and radial duct system, thereby increasing resin biosynthesis and accumulation within the whole tree. Larix occidentalis thus appears to allocate more energy to defence than P. menziesii.

[Interannual variation patterns of heavy metals concentrations in tree rings of Larix gmelinii near Xilin Lead-zinc Mine, Yichun of Northeast China].

By using dendro-environmental methods, this paper measured and analyzed the variations of five heavy metals (Pb, Cd, Zn, Cu, and Mn) concentrations in the tree rings of Larix gmelinii near Xilin Lead-zinc Mine, Yichun of Northeast China. Among the test heavy metals, the Mn concentration in the tree rings was the highest, while the Cd concentration was the lowest. The Cd, Zn, and Cu concentrations in the tree rings near the ground (0.3 m high from the ground, D0.3) were significantly higher than those at breast height (1.3 m high from the ground, D1.3), while the Pb and Mn concentrations at the two heights had less difference. In 1987-2010, the Pb concentration in the tree rings had a slight increase, but the Cd, Zn, Cu, and Mn concentrations presented a decreasing trend. The Cd concentration decreased most obviously, while the Zn, Cu, and Mn concentrations decreased after an initial increase. With the increase of tree ring width, the Pb concentration decreased, while the Cd, Zn, Cu, and Mn concentrations were in adverse. The relationships between the Pb and other four heavy metals concentrations in the tree rings near the ground and at breast height had definite differences. Near the ground, the Pb concentration showed a significant positive correlation with the other four heavy metals concentrations, but at breast height, less correlation was observed, and even, the Cd concentration decreased significantly with increasing Pb concentration. The variations of the heavy metals concentrations in the L. gmelinii tree rings could be affected by the production and mining activities of Xilin Lead-zinc Mine, an thus, it would be possible to use the Pb concentration in the tree rings to reconstruct the mining his tory of the study area. At present, the Pb concentration in the tailing wastes has polluted the surrounding environments near Xilin Lead-zinc Mine. Therefore, countermeasures should be adopted to manage the heavy metals in tailing wastes if the Mine would be continued to be mined.

Relationships between root respiration rate and root morphology, chemistry and anatomy in Larix gmelinii and Fraxinus mandshurica.

Tree roots are highly heterogeneous in form and function. Previous studies revealed that fine root respiration was related to root morphology, tissue nitrogen (N) concentration and temperature, and varied with both soil depth and season. The underlying mechanisms governing the relationship between root respiration and root morphology, chemistry and anatomy along the root branch order have not been addressed. Here, we examined these relationships of the first- to fifth-order roots for near surface roots (0-10 cm) of 22-year-old larch (Larix gmelinii L.) and ash (Fraxinus mandshurica L.) plantations. Root respiration rate at 18 °C was measured by gas phase O2 electrodes across the first five branching order roots (the distal roots numbered as first order) at three times of the year. Root parameters of root diameter, specific root length (SRL), tissue N concentration, total non-structural carbohydrates (starch and soluble sugar) concentration (TNC), cortical thickness and stele diameter were also measured concurrently. With increasing root order, root diameter, TNC and the ratio of root TNC to tissue N concentration increased, while the SRL, tissue N concentration and cortical proportion decreased. Root respiration rate also monotonically decreased with increasing root order in both species. Cortical tissue (including exodermis, cortical parenchyma and endodermis) was present in the first three order roots, and cross sections of the cortex for the first-order root accounted for 68% (larch) and 86% (ash) of the total cross section of the root. Root respiration was closely related to root traits such as diameter, SRL, tissue N concentration, root TNC : tissue N ratio and stele-to-root diameter proportion among the first five orders, which explained up to 81-94% of variation in the rate of root respiration for larch and up to 83-93% for ash. These results suggest that the systematic variations of root respiration rate within tree fine root system are possibly due to the changes of tissue N concentration and anatomical structure along root branch orders in both tree species, which provide deeper understanding in the mechanism of how root traits affect root respiration in woody plants.

Temperature-induced responses of xylem structure of Larix sibirica (Pinaceae) from the Russian Altay.

PREMISE OF THE STUDY: Xylem structure determines the hydraulic and mechanical properties of a stem, and its plasticity is fundamental for maintaining tree performance under changing conditions. Unveiling the mechanism and the range of xylem adjustment is thus necessary to anticipate climate change impacts on vegetation. METHODS: To understand the mechanistic process and the functional impact of xylem responses to warming in a cold-limited environment, we investigated the relationship between temperature and tracheid anatomy along a 312-yr tree-ring chronology of Larix sibirica trees from the Altay Mountains in Russia. KEY RESULTS: Climate-growth analyses indicated that warming favors wider earlywood cell lumen, thicker latewood walls, denser maximum latewood, and wider rings. The temperature signal of the latewood was stronger (r > 0.7) and covered a longer and more stable period (from June to August) than that of earlywood and tree-ring width. Long-term analyses indicated a diverging trend between lumen and cell wall of early- and latewood. CONCLUSIONS: Xylem anatomy appears to respond to warming temperatures. A warmer early-growing season raises water conduction capacity by increasing the number and size of earlywood tracheids. The higher-performing earlywood tracheids promote more carbon fixation of the latewood cells by incrementing the rate of assimilation when summer conditions are favorable for growth. The diverging long-term variation of lumen and cell wall in earlywood vs. latewood suggests that xylem adjustments in latewood increase mechanical integrity and support increasing tree size under the ameliorated growing conditions.

[Larix gmelinii tree-ring width chronology and its responses to climate change in Kuduer, Great Xing' an Mountains].

Based on the established tree-ring width chronology of Larix gmelinii in Kuduer, Great Xing' an Mountains, this paper analyzed the relationships between L. gmelinii tree-ring width chronology and related climatic variables, including air temperature, precipitation, and PDSI. In the study area, the L. gmelinii tree-ring width chronology was significantly negatively correlated with the air temperature in May and July (P<0.01), had no significant correlation with precipitation, but significantly positively correlated with the PDSI in June-August (P<0.05), suggesting that hydro-thermal variables had significant coupling effects to the radial growth of L. gmelinii in its growth season, particularly in May and July. The L. gmelinii tree-ring width chronology had significant correlations with large scale climatic regimes such as the Pacific Decadal Oscillation (PDO) in both low and high frequency variations, illustrating the significant effects of the remote oceanic climatic regimes to the local tree growth.

Three-dimensional analysis of the anatomical growth response of European conifers to mechanical disturbance.

Studies on tree reaction after wounding were so far based on artificial wounding or chemical treatment. For the first time, type, spread and intensity of anatomical responses were analyzed and quantified in naturally disturbed Larix decidua Mill., Picea abies (L.) Karst. and Abies alba Mill. trees. The consequences of rockfall impacts on increment growth were assessed at the height of the wounds, as well as above and below the injuries. A total of 16 trees were selected on rockfall slopes, and growth responses following 54 wounding events were analyzed on 820 cross-sections. Anatomical analysis focused on the occurrence of tangential rows of traumatic resin ducts (TRD) and on the formation of reaction wood. Following mechanical disturbance, TRD production was observed in 100% of L. decidua and P. abies wounds. The radial extension of TRD was largest at wound height, and they occurred more commonly above, rather than below, the wounds. For all species, an intra-annual radial shift of TRD was observed with increasing axial distance from wounds. Reaction wood was formed in 87.5% of A. alba following wounding, but such cases occurred only in 7.7% of L. decidua. The results demonstrate that anatomical growth responses following natural mechanical disturbance differ significantly from the reactions induced by artificial stimuli or by decapitation. While the types of reactions remain comparable between the species, their intensity, spread and persistence disagree considerably. We also illustrate that the external appearance of wounds does not reflect an internal response intensity. This study reveals that disturbance induced under natural conditions triggers more intense and more widespread anatomical responses than that induced under artificial stimuli, and that experimental laboratory tests considerably underestimate tree response.

Do centennial tree-ring and stable isotope trends of Larix gmelinii (Rupr.) Rupr. indicate increasing water shortage in the Siberian north?

Tree-ring width of Larix gmelinii (Rupr.) Rupr., ratios of stable isotopes of C (delta(13)C) and O (delta(18)O) of whole wood and cellulose chronologies were obtained for the northern part of central Siberia (Tura, Russia) for the period 1864-2006. A strong decrease in the isotope ratios of O and C (after atmospheric delta(13)C corrections) and tree-ring width was observed for the period 1967-2005, while weather station data show a decrease in July precipitation, along with increasing July air temperature and vapor pressure deficit (VPD). Temperature at the end of May and the whole month of June mainly determines tree radial growth and marks the beginning of the vegetation period in this region. A positive correlation between tree-ring width and July precipitation was found for the calibration period 1929-2005. Positive significant correlations between C isotope chronologies and temperatures of June and July were found for whole wood and cellulose and negative relationships with July precipitation. These relationships are strengthened when the likely physiological response of trees to increased CO(2) is taken into account (by applying a recently developed delta(13)C correction). For the O isotope ratios, positive relationships with annual temperature, VPD of July and a negative correlation with annual precipitation were observed. The delta(18)O in tree rings may reflect annual rather than summer temperatures, due to the late melting of the winter snow and its contribution to the tree water supply in summer. We observed a clear change in the isotope and climate trends after the 1960s, resulting in a drastic change in the relationship between C and O isotope ratios from a negative to a positive correlation. According to isotope fractionation models, this indicates reduced stomatal conductance at a relatively constant photosynthetic rate, as a response of trees to water deficit for the last half century in this permafrost region.

Rockfall and snow avalanche impacts leave different anatomical signatures in tree rings of juvenile Larix decidua.

Rockfall and snow avalanche events often cause injury to European larch (Larix decidua Mill.) trees, giving rise to the formation of callus tissue and tangential rows of traumatic resin ducts (TRDs). We analyzed and quantified anatomical reactions of juvenile trees injured before the start of the growing season by snow avalanches (15 trees, 324 cross sections) or rockfalls (18 trees, 270 cross sections). Traumatic resin ducts were observed in the growth ring formed following injury in 94.3% of the rockfall samples and 87.3% of the snow avalanche samples. Traumatic resin ducts were formed at the beginning of the new annual ring around wounds caused by rockfalls. In contrast, in trees injured by snow avalanches, TRDs were not formed until after the formation of several rows of early earlywood (EE) tracheids (mean +/- SD = 4.19 +/- 2.56 rows). The dimensions of the EE tracheids observed in the snow avalanche samples were greatly reduced in the tissues bordering the wound, with radial width reaching an average of only 50% and lumen cross-sectional area an average of only 46% of pre-event values. It is therefore possible to differentiate injuries due to past snow avalanches from injuries due to rockfall based on anatomical growth reactions in the tissues bordering scars.

Isolation and characterization of the Larix gmelinii ANGUSTIFOLIA (LgAN) gene.

ANGUSTIFOLIA (AN), a plant homolog of C-terminal binding protein, controls the polar elongation of leaf cells and the trichome-branching pattern in Arabidopsis thaliana. In the present study, degenerate PCR was used to isolate an ortholog of AN, referred to as LgAN, from larch (Larix gmelinii). The LgAN cDNA is predicted to encode a protein of 646 amino acids that shows striking sequence similarity to AN proteins from other plants. The predicted amino acid sequence has a conserved NAD-dependent 2-hydroxy acid dehydrogenase (D2-HDH) motif and a plant AN-specific LxCxE/D motif at its N-terminus, as well as a plant-specific long C-terminal region. The LgAN gene is a single-copy gene that is expressed in all larch tissues. Expression of the LgAN cDNA rescued the leaf width and trichome-branching pattern defects in the angustifolia-1 (an-1) mutant of Arabidopsis, showing that the LgAN gene has effects complementary to those of AN. These results suggest that the LgAN gene has the same function as the AN gene.

Long-term change in the sensitivity of tree-ring growth to climate forcing in Larix decidua.

Tree rings are widely used long-term proxy data which, if combined with long-term instrumental climate records, can provide excellent information on global climate variability. This research aimed to determine whether interannual climate-growth responses in Alpine treeline forests are stationary over time. We used tree-ring width chronologies of Larix decidua (European larch) from 17 sites and monthly temperatures and precipitation data for the period 1800-1999. Climate-growth relationships were assessed with correlation and response functions, and their stationarity and consistency over time were measured using moving correlation. Tree-ring chronologies showed similar interannual variations over the last two centuries, suggesting that the same climatic factors synchronously limited growth at most sites. The most sensitive variables showed significant transient responses varying within the time period, indicating a possible deviation from the uniformitarian principle applied to dendroclimatology. If these findings are confirmed in future studies on other species and in other regions, we suggest that time-dependent variables should be taken into account to avoid overestimation of treeline advance, future forest carbon storage in temperature-limited environments and inaccurate reconstruction of past climate variability.

Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer.

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 mum, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for beta-1,4-glucan and beta-1,3-glucan.