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Background Larch ( Larix gmelinii (Rupr.) Kuzen.) is an important timber and ecological tree species in northern China. Excellent germplasm resources have been acquired through time-consuming traditional breeding. Molecular breeding offers a promising approach to shorten the breeding cycle and achieve genetic improvements more efficiently. MicroRNAs (miRNAs) are non-coding, single-stranded small RNAs that primarily affect plant growth and stress resistance, including drought stress. However, the study of miRNAs in larch under drought stress has not been well explored. Results In this study, to investigate the function of Lol-miR11467 under PEG osmotic stress in larch, embryogenic callus tissue of Larix kaempferi 3 × L. gmelinii 9 was employed as the experimental material, serving as the explants for this study. Lol-miR11467 was transferred into the explants using an Agrobacterium-mediated method to determine the physiological changes and survey gene expression changes in overexpressing Lol-miR11467 cell lines. The results showed that the fresh weight, peroxidase (POD), soluble protein and soluble sugar content of the overexpressing Lol-miR11467 were lower than that of the wild-type, while malondialdehyde (MDA) content increased under PEG osmotic stress. Transcriptome analysis showed that genes associated with phenylpropanoid metabolism, transcription factors, oxidoreductase, plant hormone signal transduction, glucose metabolism and bioprotective macromolecules were mainly downregulated in Lol-miR11467 cell lines. Conclusions Overall, these results indicated that the drought resistance of the overexpressing Lol-miR11467 cell lines was reduced. This study’s findings might provide a foundation for understanding the molecular mechanisms of miRNAs under PEG osmotic stress in larch, potentially contributing to the development of strategies for improving plant resilience to environmental stresses.

Purpose In plant-soil systems, phosphorus partitioning during the annual cycle related to nitrogen partitioning remains largely unknown. The present study aims at assessing the soil-plant P allocation patterns of four tree species along four phenological stages and its relationship with tissues and soil N concentrations. Methods Cryptomeria japonica, Larix kaempferi, Fagus crenata and Robinia pseudoacacia trees were selected to sample coarse roots, sapwood, foliage, litter and soil during four phenological stages where total and Olsen extractable P and nitrogen content were measured respectively. Results Intra- and inter plant tissue nitrogen correlated well with phosphorus during the four phenological stages, especially root nitrogen. Fagus and Robinia were phosphorus limited, Larix was nitrogen limited and Cryptomeria co-limited. All species reabsorbed phosphorus and nitrogen from foliage prior to leaf abscission and stored nitrogen in roots and sapwood. Phosphorus storage was solely found in sapwood of Robinia . Soil dissolved ammonium correlated positively with nitrogen reabsorption efficiency during the green leaf stage, while single soil nutrient variables did not correlate with phosphorus reabsorption efficiency. Conclusions Plant tissues nitrogen partitioning correlated well with their respective phosphorus partitioning and the increase of soil NH 4 + correlated positively with nitrogen reabsorption efficiency, regardless of tree species during the green leaf stage. The results of this study show the intricate relationship that exists between nitrogen and phosphorus in the soil-plant continuum as well as the tree species specific internal cycling of these nutrients.

Drought stress critically impacts plant growth and productivity. The bZIP transcription factor family is crucial for abiotic stress responses, yet its role in larch drought tolerance remains unclear. This study identified 19 bZIP genes in Larix kaempferi (Lamb.) Carr. and characterized LkbZIP4. Bioinformatics analysis classified it into the A subgroup. Subcellular localization and yeast two-hybrid assays confirmed that it is a nucleus-localized transactivator. Expression pattern analysis revealed that LkbZIP4 was highly specifically expressed in roots and was significantly induced by drought stress. A series of transgenic overexpression lines was successfully established through Agrobacterium tumefaciens-mediated method, using embryogenic callus of hybrid larch (L. kaempferi × L. gmelinii). Under 7% PEG-induced drought stress, LkbZIP4-overexpressing transgenic calli displayed enhanced drought tolerance relative to wild-type. This was evidenced by better growth, higher biomass, and reduced membrane damage, indicated by lower malondialdehyde content and relative electrolyte leakage. Meanwhile, these transgenic calli accumulated higher levels of osmoregulatory substances, including proline and soluble sugars, along with enhanced activities of antioxidant enzymes including superoxide dismutase and peroxidase. Our results indicate that LkbZIP4 functions to promote drought tolerance in larch, likely through the enhancement of osmotic adjustment and oxidative defense mechanisms.

Efficient asexual reproduction techniques are crucial for the expansion of larch; however, the process of adventitious roots (ARs) regeneration has hindered its development. Through comprehensive root development proteomics and transcriptomics analysis, we have identified key epigenetic modifying enzymes involved in the organogenesis of Larix kaempferi. Subsequently, we cloned the enhancer of zeste homolog CURLY LEAF (CLF) from L. kaempferi and performed molecular modeling. The molecular docking results between LkCLF and the histone H3 lysine 27 trimethylation (H3K27me3) inhibitor GSK126 revealed the affinity value is –9 kcal/mol, indicating strong binding interaction between the two. Adding inhibitor GSK126 to the ARs induction medium, morphologically clear primordia appeared between 20 and 25 days after cutting, which was 7–10 d earlier than in the control group, accompanied by an 18.17% increase in rooting rate. Besides, western blot analysis demonstrated the effective inhibition of H3K27me3 levels in stem bases treated with GSK126. Real-time quantitative reverse transcription polymerase chain reaction results showed a significantly elevated expression of BABY BOOM2 compared with 0 μM GSK126 or dimethyl sulfoxide treated groups. Our findings suggest that treating stem bases with 0.01 μM GSK126 during early-stage AR regeneration expedites the developmental process and enhances the rooting rate. This study lays the foundation for a deeper understanding of the roles by H3K27me3 and polycomb repressive complex 2 in the AR regeneration of larch cuttings.Key messageThe inhibitor GSK126 effectively reduces H3K27me3 levels and enhances the expression of LkBBM2, thereby expediting the AR regeneration process and improving rooting rate in Larix. kaempferi.

Heat treatment improves the dimensional stability and hydrophobicity of wood, and heat-treated wood is currently attracting attention as a new interior material. However, there are few evaluations where the acoustic properties of heat-treated wood are reported when such wood is used as an interior material. In this study, Larix kaempferi wood, typically used as a building material, was heat-treated at 200, 220, and 240 °C for 9, 12, 15, and 18 h. The sound absorption coefficients of the treated wood samples were measured at 250, 500, 1000, 2000, and 4000 Hz in a reverberation room. The sound absorption coefficient increased with the treatment temperature and the treatment time. The results of this study showed that the high-frequency band range sound absorption coefficient of wood can be increased dramatically by heat treatment.

Somatic embryogenesis is an ideal model process for studying early plant development. Embryonic cell lines of Larix kaempferi (Lamb.) Carr overexpressing LaMIR166a were obtained in our previous study. Here, a combination of de novo transcriptomics and extensively targeted metabolomics was used to study the transcriptional profiles and metabolic changes in wild-type and LaMIR166a-overexpressed embryonic cell lines. A total of 459 metabolites were found in the wild-type and transgenic cell lines. Compared to those in the wild-type cell lines, transcripts and metabolites were significantly altered in the LaMIR166a-overexpressed cell lines. Among differentially expressed genes (DEGs), phenylalanine and flavonoid synthesis genes were significantly enriched, and among differentially accumulated metabolites (DAMs), phenolic acids and flavonoids accumulated in particularly high amounts. Thus, the flavonoid biosynthetic pathway seems to be the most abundant pathway in response to LaMIR166a overexpression. Based on the Kyoto Encyclopedia of Genes and Genomes database, the association analysis of metabolome and transcriptome data showed that flavonoid biosynthesis and plant hormone signal transduction processes were significantly changed in miR166a-overexpression lines, suggesting that miR166 might be involved in these processes. The present study identified a number of potential metabolites associated with LaMIR166a overexpression, providing a significant foundation for a better understanding of the regulatory mechanisms underlying miR166.

The study of somatic embryogenesis can provide insight into early plant development. We previously obtained LaMIR166a-overexpressing embryonic cell lines of Larix kaempferi (Lamb.) Carr. To further elucidate the molecular mechanisms associated with miR166 in this species, the transcriptional profiles of wild-type (WT) and three LaMIR166a-overexpressing transgenic cell lines were subjected to RNA sequencing using the Illumina NovaSeq 6000 system. In total, 203,256 unigenes were generated using Trinity de novo assembly, and 2467 differentially expressed genes were obtained by comparing transgenic and WT lines. In addition, we analyzed the cleaved degree of LaMIR166a target genes LaHDZ31–34 in different transgenic cell lines by detecting the expression pattern of LaHdZ31–34, and their cleaved degree in transgenic cell lines was higher than that in WT. The downstream genes of LaHDZ31–34 were identified using Pearson correlation coefficients. Yeast one-hybrid and dual-luciferase report assays revealed that the transcription factors LaHDZ31–34 could bind to the promoters of LaPAP, LaPP1, LaZFP5, and LaPHO1. This is the first report of gene expression changes caused by LaMIR166a overexpression in Japanese larch. These findings lay a foundation for future studies on the regulatory mechanism of miR166.

During the process of subculture of embryogenic cultures, sometimes they may become non-embryogenic, which is not desirable. However, this offers an opportunity to explore the mechanisms underlying cell fate determination and the maintenance of embryogenic potential of explants during the process of somatic embryogenesis. In a previous study, differential expression of microRNAs (miRNAs) has been detected between embryogenic and non-embryogenic cultures as well as during somatic embryo maturation of Larix kaempferi (Lamb.) Carr. However, little is known about the target genes of these miRNAs during these cellular differentiation processes. In this study, full-length cDNA of the MYB homologue from L. kaempferi, LaMYB33, was cloned. Sequence analysis showed that the miR159 target sequence is present in LaMYB33. The isolation of the miRNA-guided cleavage products of LaMYB33 further suggested that this gene is regulated by miRNA. LaMYB33 transcript levels between embryogenic and non-embryogenic cultures and during the late stage of somatic embryo maturation were measured and the results showed opposite patterns in the expression of LaMYB33 and mature miR159. Based on the relationships between the expression patterns of LaMYB33 and mature miR159, we concluded that the post-transcriptional regulation of LaMYB33 by miR159 participates in the maintenance of embryogenic or non-embryogenic potential and somatic embryo maturation, providing new insights into the regulatory mechanisms of somatic embryogenesis.

Somatic embryogenesis provides an opportunity to explore the mechanisms underlying the determination of cell fate. In a previous study, differential levels of mature miR171 were found between embryogenic and non-embryogenic cultures and during somatic embryo maturation in Larix kaempferi (Lamb.) Carr. However, little is known about its target genes in these processes. Here, a full-length cDNA for the SCARECROW-LIKE 6 (SCL6) homolog from L. kaempferi, LaSCL6, was cloned. Sequence analysis showed that the miR171 target sequence was present in the LaSCL6 transcript. Isolation of the miRNA-guided cleavage products of LaSCL6 further suggested that this gene was being regulated by miR171. LaSCL6 transcript levels in embryogenic and non-embryogenic cultures and during the late stage of somatic embryo maturation were measured, and the results showed that cleavage of the LaSCL6 mRNAs occurred actively in embryogenic cultures. Based on the relationships between the expression patterns of LaSCL6 and mature miR171, we concluded that the post-transcriptional regulation of LaSCL6 by miR171 might participate in the maintenance of embryogenic potential, providing new insights into the regulatory mechanisms of somatic embryogenesis.

Japanese larch (Larix kaempferi = L. leptolepis) is often characterized by its high growth rate with heterophyllous shoots, but the functional differences of heterophyllous shoots still remain unclear. Recently, abrupt high temperature and drought during spring induced high photosynthetic rate via change in leaf morphology of the deciduous habit. In order to reveal the photosynthetic characteristics of both short and long-shoot needles of sunny canopy of the larch trees using a canopy tower, we calculated the seasonal change of gas exchange characters and leaf mass per area (LMA) and foliar nitrogen content (N) of heterophyllous needles: short and long-shoot needles over 3 years. No marked difference in light-saturated photosynthetic rates (Psat) was observed between short and long shoots after leaf maturation to yellowing, although the difference was obvious in a specific year, which only shows that seasonal change in temperature and soil moisture determines the in situ photosynthetic capacity of needles. The large annual and seasonal variations in Psat in both shoots were found to be mainly determined by climatic variations, while shoot types determined the strategy of their photosynthetic N utilization as well as the stomatal regulation.