AT-hook motif nuclear localized (AHL) is a transcription factor that can directly induce plant somatic embryogenesis without adding exogenous hormones. One of its functional domains, the AT-hook motif, has a chromatin-modifying function and participates in various cellular processes, including DNA replication and repair and gene transcription leading to cell growth. Liriodendron chinense (Hemsl.) Sargent is an important ornamental and timber tree in China. However, its low drought-resistant ability further leads to a low natural growth rate of its population. Based on bioinformatics analysis, this study identified a total of 21 LcAHLs in L. chinense. To explore the expression pattern of the AHL gene family under drought and somatic embryogenesis, we performed a systematic analysis including basic characteristics, gene structure, chromosome localization, replication event, cis-acting elements and phylogenetic analyses. According to the phylogenetic tree, the 21 LcAHL genes are divided into three separate clades (Clade I, II, and III). Cis-acting element analysis indicated the involvement of the LcAHL genes in drought, cold, light, and auxin regulation. In the generated drought stress transcriptome, a total of eight LcAHL genes showed increased expression levels, with their expression peaking at 3 h and leveling off after 1 d. Nearly all LcAHL genes were highly expressed in the process of somatic embryogenesis. In this study, we performed a genome-wide analysis of the LcAHL gene family and found that LcAHLs take part in resistance to drought stress and the development of somatic embryos. These findings will provide an important theoretical basis for understanding of the LcAHL gene function.

To adapt and sense environmental perturbations, including a variety of biotic and abiotic stress conditions, plants have developed disparate regulatory pathways. Mitogen-activated protein kinase (MAPK or MPK) signaling cascades are found widespread across the eukaryotic kingdoms of life. In plants, they may regulate signaling pathways aimed at resisting the stressful effects of low temperature, salt damage, drought, touch, and mechanical damage. To date, no conclusive studies into Liriodendron chinense (Hemsl.) Sarg MPK-related stress resistance signaling have been performed. In our study, we cloned three homologous L. chinense MAP kinase kinase family genes: LcMKK2, LcMKK4, and LcMKK6. LcMKK2 and LcMKK6 have their highest expression level in the root, while LcMKK4 is highly expressed in the stem. LcMKK2 showed upregulation in response to salt and cold stress conditions in L. chinense. To further analyze its gene function, we overexpressed LcMKK2 in wild-type Arabidopsis thaliana (L.) Heynh. and obtained transgenic plants. Overexpression of LcMKK2 caused a significant reduction in plant mortality (from 96% to 70%) in response to a 7-day 200 mM NaCl treatment. Therefore, we conclude that LcMKK2 is involved in a signaling response to salt stress, and it could thus prove an effective target gene for breeding strategies to improve Liriodendron salt tolerance.

The precise expression of transcription factors (TFs) is crucial for plant growth and development, especially during somatic embryogenesis. However, conventional overexpression approaches, commonly used for functional genetics, can lead to deleterious effects. Therefore, it is imperative to ensure that TFs are expressed in a controlled and timely manner when aiming to enhance the efficiency of somatic embryogenesis. In this study, a dexamethasone/glucocorticoid receptor (DEX/GR) inducible expression system was employed to modulate the protein expression levels of target TFs within the nucleus during somatic embryogenesis in Liriodendron hybrids. We selected the WUSCHEL (WUS) gene, a well-established functional TF known for its vital role in somatic embryogenesis, as a model to assess the effectiveness of this system. Through DEX treatment, we induced the translocation of LhWUS-GR/LhWUS-GFP-GR fusion proteins from the cytoplasm to the nucleus, consequently leading to WUS-driven somatic embryogenesis. As the DEX concentration increased, there was a corresponding increase in the migration of the LhWUS-GFP-GR fusion protein into the nucleus. Additionally, we observed a higher proliferation ratio of callus expressing LhWUS-GR when exposed to varying DEX concentrations. Notably, the efficiency of somatic embryogenesis exhibited significant improvement under optimal DEX concentration. In conclusion, our study successfully utilizes the DEX/GR inducible system in Liriodendron hybrids, offering a valuable tool for the precise control and utilization of TFs at the desired levels. This innovative approach holds promise for advancing our understanding of TF function and enhancing plant development through the regulated manipulation of TF expression.

The phytohormone abscisic acid (ABA) plays important roles in response to abiotic and biotic stresses in plants. Pyrabactin resistance 1-like (PYR/PYL) proteins are well-known as ABA receptors, which are responsible for ABA signal transduction. Nevertheless, the characteristics of PYL genes from Liriodendron chinense, an endangered timber tree, remain unclear in coping with various stresses. In this study, five PYLs were identified from the genome of Liriodendron chinense by sequence alignment and conserved motif analysis, which revealed that these LcPYLs contain a conserved gate and latch motif for ABA binding. The LcPYL promoters possess a series of cis-acting elements involved in response to various hormone and abiotic stresses. Moreover, the transcriptome data of Liriodendron hybrid leaves reveal that LcPYL genes specifically transcript under different abiotic stresses; Lchi11622 transcription was induced by drought and cold treatment, and Lchi01385 and Lchi16997 transcription was upregulated under cold and hot stress, respectively. Meanwhile, the LcPYLs with high expression levels shown in the transcriptomes were also found to be upregulated in whole plants treated with the same stresses tested by qPCR. Moreover, under biotic stress caused by scale insect and whitefly, Liriodendron hybrid leaves exhibited a distinct phenotype including disease spots that are dark green in the middle and yellow on the margin; the qPCR results showed that the relative expression levels of Lchi13641 and Lchi11622 in infected leaves were upregulated by 1.76 and 3.75 folds relative to normal leaves, respectively. The subcellular localizations of these stress-responsive LcPYLs were also identified in protoplasts of Liriodendron hybrid. These results provide a foundation to elucidate the function of PYLs from this elite tree species and assist in understanding the molecular mechanism of Liriodendron hybrid in dealing with abiotic and biotic stresses. In future research, the detailed biological function of LcPYLs and the genetic redundancy between LcPYLs can be explored by gene overexpression and knockout based on this study.