Liwang Qi

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.

Somatic embryogenesis is valuable for clonal propagation and genetic improvement, and it also serves as an ideal system for studying plant development mechanisms. In Larix kaempferi, microRNA171 and its target gene L. kaempferi SCARECROW-LIKE6 (LaSCL6), which has two alternative splicing variants, can regulate somatic embryogenesis; however, the underlying molecular mechanism is still unknown. In this study, we overexpressed these two LaSCL6 variants in Oryza sativa and Arabidopsis thaliana and then used the RNA-Seq method to screen genes from O. sativa and A. thaliana, whose expression patterns are related to those of LaSCL6 variants. The screened genes were then used to search L. kaempferi proteins to identify the candidate target genes of LaSCL6. After yeast one-hybrid and dual- luciferase transcriptional activity assays, cytochrome P450, family 89, subfamily A, polypeptide 5 (CYP89A5), and wall-associated receptor kinase-like 20 (WAKL20) were confirmed to be the target genes of LaSCL6-var1; in addition, WAKL20 and UDP-glycosyltransferase 85A3 (UGT85A3) were confirmed to be the target genes of LaSCL6-var2. Moreover, APETALA2-like protein 2, a transcription factor from the AP2/ERF family, was shown to interact with LaSCL6-var1 and LaSCL6-var2. Taken together, our results suggest a regulatory network of miR171-LaSCL6. The findings presented here not only provide novel insights into the regulation of the miR171-LaSCL6 module but also explain the mechanism underlying larch somatic embryogenesis and other biological processes.

Promoters are powerful tools for breeding new varieties using transgenic technology. However, the low and unstable expression of target genes is still a limiting factor in Larix kaempferi (Lamb.) Carr (Japanese larch) genetic transformation. In this study, we analyzed L. kaempferi transcriptome data, screened out highly expressed genes, cloned their promoters, and constructed plant expression vectors containing the β-glucuronidase (GUS) reporter gene driven by these promoters. Recombinant vectors were introduced into the L. kaempferi embryogenic callus by means of the Agrobacterium-mediated transient or stable genetic transformation method, and the promoter activity was then determined by measuring GUS expression and its enzyme activity in the transformed materials. Four highly expressed genes were identified: L. kaempferi Zhang Chen Yi-1 (LaZCY-1), Zhang Chen Yi-2 (LaZCY-2), Translationally Controlled Tumor Protein (LaTCTP), and ubiquitin (LaUBQ). The 2000 bp fragments upstream of ATG in these sequences were cloned as promoters and named pLaZCY-1, pLaZCY-2, pLaTCTP, and pLaUBQ. Semi-quantitative and quantitative RT-PCR analyses of transient genetic transformation materials showed that all four promoters could drive GUS expression, indicating that they have promoter activities. Semi-quantitative and quantitative RT-PCR analyses and the histochemical staining of stable genetic transformation materials showed that the pLaUBQ promoter had higher activity than the other three L. kaempferi promoters and the CaMV35S promoter. Thus, the pLaUBQ promoter was suggested to be used in larch genetic transformation.

SCARECROW-LIKE6 (SCL6) plays a role in the formation and maintenance of the meristem. In Larix kaempferi (Lamb.) Carr., an important afforestation tree species in China, SCL6 (LaSCL6) has two alternative splicing variants—LaSCL6-var1 and LaSCL6-var2—which are regulated by microRNA171. However, their roles are still unclear. In this study, LaSCL6-var1 and LaSCL6-var2 were transformed into the Arabidopsis thaliana (L.) Heynh. genome, and the phenotypic characteristics of transgenic A. thaliana, including the germination percentage, root length, bolting time, flower and silique formation times, inflorescence axis length, and branch and silique numbers, were analyzed to reveal their functions. It was found that LaSCL6-var1 and LaSCL6-var2 overexpression shortened the root length by 41% and 31%, respectively, and increased the inflorescence axis length. Compared with the wild type, the bolting time in transgenic plants was delayed by approximately 2–3 days, the first flower and silique formation times were delayed by approximately 3–4 days, and the last flower and silique formation times were delayed by about 5 days. Overall, the life cycle in transgenic plants was prolonged by approximately 5 days. These results show that LaSCL6 overexpression inhibited the transitions from the vegetative meristem to inflorescence meristem and from the flower meristem to meristem arrest in A. thaliana, revealing the roles of LaSCL6-var1 and LaSCL6-var2 in the fate transition and maintenance of the meristem.

To reveal the regulatory mechanisms underlying age-dependent expression patterns, we characterized seven age-related genes, LaDAL1, LaAGL2-2, LaAGL2-3, LaAGL11, LaSOC1-1, LaAP2-1, and LaAP2-2 in terms of transcription and intron splicing in Larix kaempferi. Based on the exon–intron structures, we quantified the pre-mRNA levels and mature mRNA levels of these seven genes using quantitative reverse transcription polymerase chain reaction experiments. We found that the pre-mRNA levels manifested age-related patterns, indicating that their transcription was primarily regulated by age. By comparing the increasing or decreasing rates of the pre-mRNA and spliced mRNA levels, we found that their splicing efficiencies also changed with age. These results clearly show that both pre-mRNA transcription and splicing of five age-related genes are regulated by age, indicating that age-dependent expression patterns are controlled at both transcriptional and post-transcriptional levels, and unveiling the underlying regulatory molecular mechanisms should focus on the transcription factors, epigenetic regulation, and RNA splicing. These data provide new insights into the age-mediated regulation of gene expression in woody perennials in terms of longevity.

The deciduous conifer larch has been widely distributed around the world, which is a high-quality wood species and is also used to extract industrial raw materials and medicines. In this study, we developed an organogenesis protocol for Larix olgensis from both mature zygotic embryos and needles, and analyzed the content of taxifolin in different tissues. The highest callus induction (96.8%) from mature zygotic embryo was got in the Douglas-fir Cotyledon Revised (DCR) medium augmented with 2.0 mg/L 6-Benzylaminopurine (6-BA) and 0.2 mg/L α-Naphthaleneacetic acid (NAA), while from needles the highest callus induction (92.03%) was got in the Murashige and Skoog (MS) medium augmented with 3 mg/L 6-BA and 0.3 mg/L NAA. The best shoot regeneration capacity from zygotic embryo-derived calli (83.3%) was obtained in DCR medium augmented with 1.0 mg/L 6-BA and 0.01 mg/L NAA, and needle-derived calli were 77.3%. The shoots achieved the highest elongation (75.6%) in the DCR medium supplemented with 0.5 mg/L 6-BA, 0.05 mg/L NAA and 2g/L activated charcoal (AC). The rooting rate was 62.8% in DCR medium augmented with 3 mg/L Indole-3-butyric acid (IBA) and 100 mg/L phloroglucinol (PG). The accumulation of the taxifolin in elongation shoots and lignified elongation shoots have greatly improved along with the development process, were 28.6 µg/g, and 53 µg/g respectively. The content of the taxifolin in callus was 1.99-5.26 µg/g, adventitious shoots were 4.8µg/g, and adventitious roots were 2.86 µg/g. To date, we reported an efficient organogenesis and taxifolin production protocol in larch for the first time.

Perennial woody plants are long-lived, and their life-cycle events occur in order in each generation, but what drives the occurrence and restart of these events in their offspring is unknown. Based on its age-dependent expression pattern and function, Larix kaempferi DEFICIENS-AGAMOUS-LIKE 1 (LaDAL1), a MADS transcription factor has been suggested to be a time recorder and life-cycle event coordinator. Here, we studied the dynamic spatiotemporal expression pattern of LaDAL1 in the life cycle of L. kaempferi to analyze the molecular mechanism of life-cycle progression. In full view of the life cycle, LaDAL1 transcription was related with life-cycle progression, and its transcript level increased sharply from age 3 to 5 years, which might be the molecular characteristic of the vegetative phase change, and then stayed at a high level. During sexual reproduction, LaDAL1 transcript level decreased sequentially during meiosis and embryogenesis, suggesting that meiosis rapidly lowers the age signal, and after fertilization, the age signal was reset to “0” with the embryogenesis. When a seed germinates, the next generation restarts, and the age is re-counted. Altogether, these results not only provide important and novel insights into the life-cycle progression and transgeneration in perennial woody plants, but also advance our understanding of age recording.

A simple and efficient protocol for the genetic transformation of Japanese larch (Larix kaempferi (Lamb.) Carr.) was developed by altering the infection method and duration and the bacterial removal process. More than 600 hygromycin-resistant embryonal masses with the vector pCAMBIA1301 were obtained, with an average of 20 transgenic lines per gram of fresh weight. Nine hygromycin-resistant transformation events (designated P1–P9) were analyzed using PCR, quantitative real-time PCR, and histochemical β-glucuronidase (GUS) assays. The GUS transcript abundance in each transformed cell line ranged from 10¹ to 10³ magnitudes, with a maximum abundance of 2.89 × 10³. In addition, the pLaTCTP::GUS vector, which contains GUS under the control of the L. kaempferi LaTCTP promoter, led to constitutive expression of GUS in embryonal-suspensor mass and somatic embryos. The transcript abundance of the exogenous genes HPT and GUS, driven by the CaMV 35S or LaTCTP promoter, ranged from 10¹ to 10⁴, which was equivalent to genes with moderate and low abundances in Japanese larch. The relatively low expression levels of exogenous genes in transformants might reflect the large genome of Japanese larch. Additional transgenic cell lines need to be screened to obtain transformants with higher expression levels of foreign genes for further functional research in Japanese larch.

Homologs of Larix kaempferiDEFICIENS-AGAMOUS-LIKE 1 (LaDAL1) promote flowering in Arabidopsis. However, their functional role in the whole life-cycle is limited. Here, we analyzed the phenotypes and transcriptomes of Arabidopsis plants over-expressing LaDAL1. With respect to the defined life-cycle stage of Arabidopsis based on the meristem state, the results showed that LaDAL1 promoted seed germination, bolting, flower initiation, and global proliferative arrest, indicating that LaDAL1 accelerates the meristem reactivation, the transitions of vegetative meristem to inflorescence and flower meristem, and meristem arrest. As a marker gene of meristem, TERMINAL FLOWER 1 was down-regulated after LaDAL1 over-expression. These results reveal that LaDAL1 accelerates the life-cycle progression in Arabidopsis by promoting the transition of meristem fate, providing more and novel functional information about the conifer age-related gene DAL1.

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L. kaempferi cyclin-dependent kinase B 1;2 (LaCDKB1;2) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material.