Genomic survey and expression analysis of LcARFs reveal multiple functions to somatic embryogenesis in Liriodendron.

BACKGROUND: Auxin response factors (ARFs) are critical transcription factors that mediate the auxin signaling pathway and are essential for regulating plant growth. However, there is a lack of understanding regarding the ARF gene family in Liriodendron chinense, a vital species in landscaping and economics. Thus, further research is needed to explore the roles of ARFs in L. chinense and their potential applications in plant development. RESULT: In this study, we have identified 20 LcARF genes that belong to three subfamilies in the genome of L. chinense. The analysis of their conserved domains, gene structure, and phylogeny suggests that LcARFs may be evolutionarily conserved and functionally similar to other plant ARFs. The expression of LcARFs varies in different tissues. Additionally, they are also involved in different developmental stages of somatic embryogenesis. Overexpression of LcARF1, LcARF2a, and LcARF5 led to increased activity within callus. Additionally, our promoter-GFP fusion study indicated that LcARF1 may play a role in embryogenesis. Overall, this study provides insights into the functions of LcARFs in plant development and embryogenesis, which could facilitate the improvement of somatic embryogenesis in L. chinense. CONCLUSION: The research findings presented in this study shed light on the regulatory roles of LcARFs in somatic embryogenesis in L. chinense and may aid in accelerating the breeding process of this tree species. By identifying the specific LcARFs involved in different stages of somatic embryogenesis, this study provides a basis for developing targeted breeding strategies aimed at optimizing somatic embryogenesis in L. chinense, which holds great potential for improving the growth and productivity of this economically important species.

Genome-Wide Analysis of the Liriodendron chinense Hsf Gene Family under Abiotic Stress and Characterization of the LcHsfA2a Gene.

Heat shock factors (Hsfs) play a crucial role in plant defense processes. However, the distribution and functional characteristics of Hsf genes in the relict plant Liriodendron chinense are still unclear. In this study, a total of 19 LcHsfs were identified and divided into three separate subgroups, comprising 10 LcHsfA, 7 LcHsfB, and 2 LcHsfC genes, respectively, based on their phylogenetic tree and the presence/absence of conserved protein domains. Whole-genome duplication and segmental duplication led to an expansion of the LhHsf gene family. The promoters of LcHsf genes are enriched for different types of cis-acting elements, including hormone responsive and abiotic-stress-responsive elements. The expression of LcHsfA3, LcHsfA4b, LcHsfA5, LcHsfB1b, and LcHsfB2b increased significantly as a result of both cold and drought treatments. LcHsfA2a, LcHsfA2b, and LcHsfA7 act as important genes whose expression levels correlate strongly with the expression of the LcHsp70, LcHsp110, and LcAPX genes under heat stress. In addition, we found that transiently transformed 35S:LcHsfA2a seedlings showed significantly lower levels of hydrogen peroxide (H2O2) after heat stress and showed a stronger thermotolerance. This study sheds light on the possible functions of LcHsf genes under abiotic stress and identifies potentially useful genes to target for molecular breeding, in order to develop more stress-resistant varieties.

Overexpression of Liriodendron Hybrid LhGLK1 in Arabidopsis Leads to Excessive Chlorophyll Synthesis and Improved Growth.

Chloroplasts is the site for photosynthesis, which is the main primary source of energy for plants. Golden2-like (GLK) is a key transcription factor that regulates chloroplast development and chlorophyll synthesis. However, most studies on GLK genes are performed in crops and model plants with less attention to woody plants. In this study, we identified the LhGLK1 and LhGLK2 genes in the woody plant Liriodendron hybrid, and they are specifically expressed in green tissues. We showed that overexpression of the LhGLK1 gene improves rosette leaf chlorophyll content and induces ectopic chlorophyll biogenesis in primary root and petal vascular tissue in Arabidopsis. Although these exhibit a late-flowering phenotype, transgenic lines accumulate more biomass in vegetative growth with improved photochemical quenching (qP) and efficiency of photosystem II. Taken together, we verified a conserved and ancient mechanism for regulating chloroplast biogenesis in Liriodendron hybrid and evaluated its effect on photosynthesis and rosette biomass accumulation in the model plant Arabidopsis.

H2O2 Significantly Affects Larix kaempferi × Larix olgensis Somatic Embryogenesis.

Larch is widely distributed throughout the world and is an important species for timber supply and the extraction of industrial raw materials. In recent years, the hybrid breeding of Larix kaempferi and Larix olgensis has shown obvious heterosis in quick-growth, stress resistance and wood properties. However, its growth and development cycle is too long to meet general production needs. In order to shorten the breeding cycle, we have for the first time successfully established and optimized a somatic embryogenesis system for Larix kaempferi × Larix olgensis. We found that the highest rate of embryonal-suspensor mass (ESM) induction was observed when late cotyledonary embryos were used as explants. The induced ESMs were subjected to stable proliferation, after which abscisic acid (ABA) and polyethylene glycol (PEG) were added to successfully induce somatic embryos. Treatment with PEG and ABA was of great importance to somatic embryo formation and complemented each other's effect. ABA assisted embryo growth, whereas PEG facilitated the formation of proembryo-like structures. On top of this, we studied in more detail the relationship between redox homeostasis and the efficiency of somatic embryogenesis (frequency of ESM induction). During subculture, we observed the gradual formation of three distinct types of ESM. The Type I ESM is readily able to form somatic embryos. In contrast to type I, the type III ESM suffers from severe browning, contains a higher level of hydrogen peroxide (H2O2) and demonstrates a decreased ability to form somatic embryos. External treatment with H2O2 decreased the somatic embryogenesis efficiency of Type I and type III ESMs, or the higher the exogenous H2O2 content, the lower the resulting somatic embryogenesis efficiency. We found that treatment with the H2O2 scavenger DMTU (dimethylthiourea) could significantly increase the somatic embryogenesis efficiency of the type III ESM, as a result of a decline in endogenous H2O2 content. Overall, these findings have contributed to setting up a successful somatic embryogenesis system for larch production.

The Identification and Expression Analysis of the Liriodendron chinense F-Box Gene Family.

The F-box gene family is one of the largest gene families in plants, and it plays a crucial role in regulating plant development, reproduction, cellular protein degradation, and response to biotic and abiotic stresses. Despite their significance, a comprehensive analysis of the F-box gene family in Liriodendron chinense and other magnoliaceae species has not been reported. In this study, we report for the first time the identification of 144 full-length F-box genes in L. chinense. Based on specific domains and phylogenetic analyses, these genes were divided into 10 distinct subfamilies. We further analyzed their gene structure, conserved domain and chromosome distribution, genome-wide replication events, and collinearity. Additionally, based on GO analysis, we found that F-box genes exhibit functional specificity, with a significant proportion of them being involved in protein binding (GO:0005515), suggesting that F-box genes may play an important role in gene regulation in L. chinense. Transcriptome data and q-PCR results also showed that F-box genes are involved in the development of multiple tissues in L. chinense, regulate the somatic embryogenesis of Liriodendron hybrids, and play a pivotal role in abiotic stress. Altogether, these findings provide a foundation for understanding the biological function of F-box genes in L. chinense and other plant species.

Physiological and transcriptomic analysis revealed that the accumulation of reactive oxygen species caused the low temperature sensitivity of Liriodendron × sinoamericanum.

Liriodendron × sinoamericanum is widely cultivated in southern China as an excellent wood and garden ornamental trees. However, its intolerance to low temperature limits its application to high latitudes. Understanding the molecular mechanism of low temperature sensitivity of Liriodendron × sinoamericanum is very important for its further application. In this study, combined with physiological and transcriptomic analysis, it was revealed that low temperature stress can lead to water loss and decreased photosynthetic capacity of Liriodendron × sinoamericanum leaves. The accelerated accumulation of reactive oxygen species (ROS) caused by the imbalance of cell REDOX homeostasis is one of the important reasons for the low temperature sensitivity. Further analysis showed that several transcription factors could be involved in regulating the synthesis and degradation of ROS, among which LsNAC72 and LsNAC73a could regulate the accumulation of O2- and H2O2 in leaves by affecting the expression level of LsAPX, LsSOD, LsPAO, and LsPOD.

The Role of Liriodendron Dof Gene Family in Abiotic Stress Response.

The DOF (DNA-binding with one finger) transcription factors are exclusive to plants and play crucial roles in plant growth, development, and environmental adaptation. Although extensive research has been conducted on the Dof gene family in Arabidopsis, maize, and Solanum, investigations concerning the role of this gene family in Liriodendron remain unreported, leaving its biological function largely unknown. In this study, we performed a comprehensive genome-wide identification of the Dof gene family based on the Liriodendron genome, resulting in the discovery of a total of 17 LcDof gene members. Based on the results of phylogenetic analysis, the 17 LcDof proteins were classified into eight subfamilies. The motif analysis revealed the diverse nature of motifs within the D1 subfamily, which includes a distinct type of Dof transcription factor known as CDF (Cycling Dof Factor). We further characterized the chromosomal distribution, gene structure, conserved protein motifs, and cis-elements in the promoter regions. Additionally, utilizing transcriptome data from Liriodendron hybrids and conducting RT-qPCR experiments, we investigated the expression patterns of LhDofs under various abiotic stresses such as drought, cold, and heat stress. Notably, we found that several LhDofs, particularly LhDof4 and LhDof6, were significantly upregulated in response to abiotic stress. Furthermore, we cloned LhDof4 and LhDof6 genes and found that its encoding protein was mainly located in the nucleus by transient transformation in Liriodendron hybrids protoplast. Subsequently, we used LhDof6-overexpressing Liriodendron hybrid seedlings. We found that overexpression of LhDof6 enhanced the cold tolerance of the plants, increasing their survival rate at -20 °C. This result was further validated by changes in physiological indicators.

Unraveling the Role of the Liriodendron Thioredoxin (TRX) Gene Family in an Abiotic Stress Response.

Thioredoxin (TRX) is a small protein with REDOX activity that plays a crucial role in a plant's growth, development, and stress resistance. The TRX family has been extensively studied in Arabidopsis, rice, and wheat, and so it is likely that its members have similar biological functions in Liriodendron that have not been reported in Liriodendron. In this study, we performed the genome-wide identification of the TRX gene family based on the Liriodendron chinense genome, leading to a total of 42 LcTRX gene members. A phylogenetic analysis categorized these 42 LcTRX proteins into 13 subfamilies. We further characterized their chromosome distributions, gene structures, conserved protein motifs, and cis-elements in the promoter regions. In addition, based on the publicly available transcriptome data for Liriodendron hybrid and following RT-qPCR experiments, we explored the expression patterns of LhTRXs to different abiotic stressors, i.e., drought, cold, and heat stress. Notably, we found that several LhTRXs, especially LhTRX-h3, were significantly upregulated in response to abiotic stress. In addition, the subcellular localization assay showed that LhTRX-h3 was mainly distributed in the cytoplasm. Subsequently, we obtained LhTRX-h3 overexpression (OE) and knockout (KO) callus lines in Liriodendron hybrid. Compared to the wild type (WT) and LhTRX-h3-KO callus proliferation of LhTRX-h3-OE lines was significantly enhanced with reduced reactive oxygen species (ROS) accumulation under drought stress. Our findings that LhTRX-h3 is sufficient to improve drought tolerance. and underscore the significance of the TRX gene family in environmental stress responses in Liriodendron.