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Background Plant mitochondrial genomes (mitogenomes) can be structurally complex while their size can vary from ~ 222 Kbp in Brassica napus to 11.3 Mbp in Silene conica . To date, in comparison with the number of plant species, only a few plant mitogenomes have been sequenced and released, particularly for conifers (the Pinaceae family). Conifers cover an ancient group of land plants that includes about 600 species, and which are of great ecological and economical value. Among them, Siberian larch ( Larix sibirica Ledeb.) represents one of the keystone species in Siberian boreal forests. Yet, despite its importance for evolutionary and population studies, the mitogenome of Siberian larch has not yet been assembled and studied. Results Two sources of DNA sequences were used to search for mitochondrial DNA (mtDNA) sequences: mtDNA enriched samples and nucleotide reads generated in the de novo whole genome sequencing project, respectively. The assembly of the Siberian larch mitogenome contained nine contigs, with the shortest and the largest contigs being 24,767 bp and 4,008,762 bp, respectively. The total size of the genome was estimated at 11.7 Mbp. In total, 40 protein-coding, 34 tRNA, and 3 rRNA genes and numerous repetitive elements (REs) were annotated in this mitogenome. In total, 864 C-to-U RNA editing sites were found for 38 out of 40 protein-coding genes. The immense size of this genome, currently the largest reported, can be partly explained by variable numbers of mobile genetic elements, and introns, but unlikely by plasmid-related sequences. We found few plasmid-like insertions representing only 0.11% of the entire Siberian larch mitogenome. Conclusions Our study showed that the size of the Siberian larch mitogenome is much larger than in other so far studied Gymnosperms, and in the same range as for the annual flowering plant Silene conica (11.3 Mbp). Similar to other species, the Siberian larch mitogenome contains relatively few genes, and despite its huge size, the repeated and low complexity regions cover only 14.46% of the mitogenome sequence.

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.

Background Larix gmelinii is an excellent stress resistant coniferous tree species with a wide distribution and important economic and ecological value. However, at seedling stage, L. gmelinii is extremely susceptible to abiotic stresses, and systematic research on the adaptation mechanisms of L. gmelinii seedlings to abiotic stress is still lacking. Results Phenotypic observation and physiological index detection showed that L. gmelinii seedlings wilted with needles withered and yellowish at later stages of drought and salt stress; Under low temperature, the seedlings grew slowly and turned red at later stage. Under all 3 abiotic stresses, the chlorophyll content in seedlings significantly decreased, while the MDA content significantly increased; The activity of SOD and CAT showed a trend of increasing first and then decreasing. Transcriptome analysis revealed that DEGs were mainly involved in carbohydrate and amino acid metabolism, phenylpropanoid biosynthesis, and flavonoid synthesis metabolism. Metabolomic analysis found unique DAMs under 3 stress treatments. The combined analysis of transcriptome and metabolome showed that the changing patterns of DEGs and DAMs in primary and secondary metabolism were consistent: carbohydrate were significantly accumulated under low temperature stress; amino acids showed the most significant changes under salt stress. The variation pattern of secondary metabolism was similar under both drought and salt stress, while anthocyanin accumulation was the most obvious only under low temperature stress. Conclusion Our study provides insightful information about the different mechanisms that L. gmelinii seedlings employ in response to drought, low temperature or salt stress.

Microbial community and diversity in the rhizosphere is strongly influenced by biotic and/or abiotic factors, like root exudates, nutrient availability, edaphon and climate. Here we report on the microbial diversity within the rhizosphere of Larix decidua , a dominant tree species in the Alps, as compared with the microbiome within the surrounding soil. We describe how increased light intensity influenced the rhizobiome and put emphasize on methane cycling microorganisms. Microbial taxa were classified into 26 bacterial, 4 archaeal and 6 fungal phyla revealing significant differences between bulk and rhizosphere soils. The dominant prokaryotic phyla were Proteobacteria, Acidobacteria, Actinobacteria (both, rhizosphere and bulk soil) and Bacteroidetes (rhizosphere soil only) and dominant fungal phyla in both fractions included Ascomycota and Basidiomycota. The rhizosphere community was indicated by Suillus sp., plant growth-promoting bacteria and Candidatus Saccharibacteria. Predicted genes in membrane transport and carbohydrate metabolism were significantly more abundant in rhizosphere soils while genes connected with energy metabolisms and cell motility increased in bulk soils. Dominant methanotrophic microorganisms were Upland Soil Cluster (USC) α methanotrophs, Methylogaea spp. and Methylosinus spp., while most methanogens belonged to Methanomassiliicoccales. The overall abundance of methanotrophs distinctly increased in the rhizosphere but to a very different species-specific extent. The increased light intensity only led to minor changes in the rhizobiome, nevertheless a couple of indicator species (e.g. Pseudomonas sp.) for intensified light conditions were established.

Environment and genetics combine to influence tree growth and should therefore be jointly considered when evaluating forest responses in a warming climate. Here, we combine dendroclimatology and population genetic approaches with the aim of attributing climatic influences on growth of European larch (Larix decidua) and Norway spruce (Picea abies). Increment cores and genomic DNA samples were collected from populations along a ~900-m elevational transect where the air temperature gradient encompasses a ~4 °C temperature difference. We found that low genetic differentiation among populations indicates gene flow is high, suggesting that migration rate is high enough to counteract the selective pressures of local environmental variation. We observed lower growth rates towards higher elevations and a transition from negative to positive correlations with growing season temperature upward along the elevational transect. With increasing elevation there was also a clear increase in the explained variance of growth due to summer temperatures. Comparisons between climate sensitivity patterns observed along this elevational transect with those from Larix and Picea sites distributed across the Alps reveal good agreement, and suggest that tree-ring width (TRW) variations are more climate-driven than genetics-driven at regional and larger scales. We conclude that elevational transects are an extremely valuable platform for understanding climatic-driven changes over time and can be especially powerful when working within an assessed genetic framework.

The miR156 family, crucial for phase transition and stress responses in plants, remains functionally uncharacterized in the ecologically and commercially important gymnosperm Larix kaempferi. This study systematically investigated L. kaempferi miR156 through phylogenetic analysis, structural prediction, expression profiling during somatic embryogenesis, and heterologous functional validation in Arabidopsis. Four MIR156 family members (LkMIR156s) were identified in Larix kaempferi, each with a characteristic stem-loop structure and highly conserved mature sequences. Computational predictions indicated that these LkMIR156s target four LkSPL family genes (LkSPL1, LkSPL2, LkSPL3, and LkSPL9). qRT-PCR analysis showed that mature LkmiR156s expression remained relatively low during early embryonic development but was significantly upregulated at the cotyledonary stage (21–42 days). Precursor transcript levels peaked earlier (around 28 days) than those of the mature LkmiR156, which remained highly expressed throughout cotyledonary embryo development. This sustained high expression coincided with cotyledon morphogenesis and embryonic dormancy. Functional validation via heterologous overexpression of LkMIR156b1 in Arabidopsis resulted in increased rosette leaf numbers (42.86% ± 6.19%) and individual leaf area (54.90% ± 6.86%), phenotypically consistent with the established role of miR156 in growth regulation. This study reveals the temporal expression dynamics of LkmiR156s during L. kaempferi somatic embryogenesis and its coordinated expression patterns with cotyledon development and embryonic dormancy. The functional conservation of the miR156-SPL module was confirmed in a model plant, providing key molecular insights into the developmental regulatory network of conifers. These findings offer potential strategies for optimizing somatic embryogenesis techniques in conifer species.

Global forests face increasingly severe drought events, which suppress the growth of larch. Transcription factors (TFs), acting as regulators, can increase plant stress tolerance by modulating gene expression networks. This study integrated RNA-seq, WGCNA and physiology to systematically analyze the adaptation mechanisms of Larix olgensis under drought stress. Among the 50,110 unigenes identified via RNA-seq, 1,391 unigenes were annotated as TFs. These TFs were clustered into four distinct clusters based on expression profiles. TFs within Cluster 1 exhibited significant differential expression during visible wilting of L. olgensis (72 h and 96 h). Ten TFs were selected as candidate TFs from Cluster 1 based on fold-change rank ( p  < 0.05). Subsequent WGCNA clustered the 50,110 unigenes into 19 co-expression modules. Notably, the firebrick4 module displayed a strong positive correlation with hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) contents, whereas the darkolivegreen module showed a strong positive correlation with peroxidase (POD) activity. Three TFs in the firebrick4 module (TRINITY_DN13923_c0_g2, TRINITY_DN3004_c0_g1 and TRINITY_DN1230_c0_g1) and two TFs in the darkolivegreen module (TRINITY_DN1156_c1_g3 and TRINITY_DN1156_c1_g1) were identified by co-expression network analysis. The findings of this study contribute to the knowledge of drought-responsive TFs in L. olgensis , establishing a foundational resource for future functional studies and molecular breeding.

MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm-breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated 9 of them by 5' RACE. 11 conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stagespecific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stages 5-8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cotyledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.

Background Larch is an important timber tree species. The traditional methods of tree genetic breeding have been progressing slowly. It is necessary to carry out gene function analysis and genetically modified breeding research. The NAC transcription factor family is a plant-specific transcription factor family with various biological functions, as shown in recent research. However, there are few studies on the NAC gene among gymnosperm coniferous species. Results LoNAC3 with complete cds was identified and isolated from the cDNA of Larix olgensis based on transcriptome data. The cDNA length of LoNAC3 is 1185 bp, encoding 394 amino acids, with a conserved NAM domain located at the N-terminus, and subcellular localization in the nucleus. The results of real-time quantitative PCR analysis showed that at different growth stages and in different tissues of L. olgensis, the relative expression level of LoNAC3 was highest in the needles. After drought, salt, alkali stress and hormone treatment, expression was induced to different degrees. The expression level of LoNAC3 was significantly increased under drought and salt conditions. The relative expression level changed under methyl jasmonate (MeJA) and abscisic acid (ABA) treatment. By observing the phenotype of overexpressed LoNAC3 tobacco, it was found that overexpressed tobacco is shorter and blooms earlier than wild-type tobacco. Under abiotic stress, LoNAC3 overexpressed tobacco has lower germination rates and poorer growth status. Transgenic tobacco under stress treatment has a higher malondialdehyde (MDA) content than wild-type tobacco, while peroxidase (POD) activity is lower than wild-type tobacco. Conclusions Through the analysis of LoNAC3 sequence and promoter expression, it can be concluded that LoNAC3 is involved in the drought and salt stress response processes of L. olgensis , and is induced by ABA and MeJA expression. Overexpression of LoNAC3 leads to stunted tobacco growth and negatively regulates its tolerance to drought and salt stress through the reactive oxygen species pathway. The preliminary analysis of the expression pattern and function of the LoNAC3 can provide a theoretical basis and high-quality materials for genetic improvement of larch in later stages.

The rapid development of the CRISPR–Cas9, –Cas12a and –Cas12b genome editing systems has greatly fuelled basic and translational plant research 1 , 2 , 3 , 4 , 5 – 6 . DNA targeting by these Cas nucleases is restricted by their preferred protospacer adjacent motifs (PAMs). The PAM requirement for the most popular Streptococcus pyogenes Cas9 (SpCas9) is NGG (N = A, T, C, G) 7 , limiting its targeting scope to GC-rich regions. Here, we demonstrate genome editing at relaxed PAM sites in rice (a monocot) and the Dahurian larch (a coniferous tree), using an engineered SpRY Cas9 variant 8 . Highly efficient targeted mutagenesis can be readily achieved by SpRY at relaxed PAM sites in the Dahurian larch protoplasts and in rice transgenic lines through non-homologous end joining (NHEJ). Furthermore, an SpRY-based cytosine base editor was developed and demonstrated by directed evolution of new herbicide resistant OsALS alleles in rice. Similarly, a highly active SpRY adenine base editor was developed based on ABE8e (ref. 9 ) and SpRY-ABE8e was able to target relaxed PAM sites in rice plants, achieving up to 79% editing efficiency with high product purity. Thus, the SpRY toolbox breaks a PAM restriction barrier in plant genome engineering by enabling DNA editing in a PAM-less fashion. Evidence was also provided for secondary off-target effects by de novo generated single guide RNAs (sgRNAs) due to SpRY-mediated transfer DNA self-editing, which calls for more sophisticated programmes for designing highly specific sgRNAs when implementing the SpRY genome editing toolbox. An engineered SpRY Cas9 variant enables efficient gene editing without PAM requirement in rice transgenic lines and Dahurian larch protoplasts, and its derived base editors can edit the rice genome efficiently in a PAM-less fashion too.