The relict woody plant genus Liriodendron contains two endangered species, Liriodendron chinense and Liriodendron tulipifera. Understanding the molecular mechanisms involved in early embryo development is important for horticultural and ecological research, particularly for the development of improved somatic embryogenesis systems. However, the specific molecular processes underlying embryogenesis in these species remain largely unexplored. To address this, we investigated expression of the WOX (WUSCHEL-related homeobox) gene family of transcription factors throughout somatic embryogenesis. We confirmed expression of eight out of 11 novel candidate LcWOX genes in L.chinense using qRT-PCR and examined spatiotemporal expression patterns of the expressed genes using stable reporter lines that had been transformed with different LcWOX promoters driving GUS expression. We observed embryo developmental stages and expression patterns that broadly correlated with those reported for Arabidopsis somatic embryogenesis. LcWUS was weakly expressed during the transition stage and was predominantly restricted to the apical meristem. LcWOX5 was specifically expressed in the root meristem and restricted to the cotyledons thereafter, and LcWOX4 expression was restricted to the vascular tissue of cotyledonary embryos. In contrast, LcWOX9 was expressed in the embryonic callus and the entire embryonic cell mass, then became restricted to the basal cells, indicating a potential role in regulating embryonic maintenance. Our findings provide insights into spatiotemporally specific WOX transcription and shed new light on potential functions of WOX genes during Liriodendron somatic embryogenesis.

The auxin efflux carrier PIN-FORMED (PIN) proteins are required for the polar transport of auxin between cells through their asymmetric distribution on the plasma membrane, thus mediating the differential distribution of auxin in plants, finally, affecting plant growth and developmental processes. In this study, 11 LcPIN genes were identified. The structural characteristics and evolutionary status of LcPIN genes were thoroughly investigated and interpreted combining physicochemical property analysis, evolutionary analysis, gene structure analysis, chromosomal localization, etc. Multi-species protein sequence analysis showed that angiosperm PIN genes have strong purification options and some functional sites were predicted about PIN protein polarity, trafficking and activity in L. chinense. Further qRT-PCR and transcriptome data analysis indicated that the long LcPINs have highly expressed from globular embryo to plantlet, and the LcPIN6a started upregulated in cotyledon embryo. The LcPIN3 and LcPIN6a are both highly expressed during the development of stamens and petals and the expression of LcPIN2 is related to root elongation, suggesting that they may play an important role in these processes. Experiment data indicates that LcPIN5 and LcPIN8 might play a key role in auxin transport in Liriodendron stems and leaves under abiotic stress. Analyzed the response of LcPIN genes to abiotic stress and as a basis for uncovering the biological role of LcPIN genes in development and adaption to adverse environments. This study provides a foundation for further genetic and functional analyses.

Surviving relict populations of species that were more widespread in ancient times can teach us a lot, such as evolution and genetic differentiation. One such relict plant is Liriodendron, of which populations remain in China (L. chinense (Hemsl.) Sarg.) and the USA (L. tulipifera L.). Studying the genetic structure of these populations would give insight into the genetic differentiation and the breeding strategy. In this work, we developed and characterized 29 novel simple sequence repeat (SSR) markers based on expressed sequence tags (ESTs) from hybrid Liriodendron (Liriodendron chinense × tulipifera) callus. In total, 29 SSRs with perfect primer-designed were used to assess genetic diversity and differentiation. The set of polymorphic EST-SSR loci was identified in 48 Liriodendron individuals, represented by 35 individuals sampled from 14 provenances of L. chinense and 13 individuals sampled from 5 provenances of L. tulipifera. Our results indicated that L. chinense populations possess slightly higher genetic diversity than L. tulipifera populations. Based on genetic distances, 48 Liriodendron individuals clustered into three groups (the eastern China L. chinense, the western China L. chinense and L. tulipifera), although the STRUCTURE analysis of the Liriodendron populations revealed just two clear genetic clusters (L. chinense and L. tulipifera). Among these 29 novel markers, ESSR119 showed an obvious species-specific characteristic which can be very useful in marker-assisted selection (MAS). In general, all these EST-SSR markers may have agronomic potential and constitute a basis for future studies on the identification, innovation, and even preservation of Liriodendron germplasms.

Roots are essential for plant growth, and studies on root-related genes, exemplified by WUSCHEL-RELATED HOMEOBOX5 (WOX5), have mainly concentrated on model organisms with less emphasis on the function of these genes in woody plants. Here, we report that overexpression of the WOX5 gene from Liriodendron hybrid (LhWOX5) in Arabidopsis leads to significant morphological changes in both the aerial and subterranean organs. In the Arabidopsis aerial parts, overexpression of LhWOX5 results in the production of ectopic floral meristems and leaves, possibly via the ectopic activation of CLV3 and LFY. In addition, in the Arabidopsis root, overexpression of LhWOX5 alters root apical meristem morphology, leading to a curled and shortened primary root. Importantly, these abnormal phenotypes in the aerial and subterranean organs caused by constitutive ectopic expression of LhWOX5 mimic the observed phenotypes when overexpressing AtWUS and AtWOX5 in Arabidopsis, respectively. Taken together, we propose that the LhWOX5 gene, originating from the Magnoliaceae plant Liriodendron, is a functional homolog of the AtWUS gene from Arabidopsis, while showing the highest degree of sequence similarity with its ortholog, AtWOX5. Our study provides insight into the potential role of LhWOX5 in the development of both the shoot and root.

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.

Clerodendrum japonicum (Thunb.) sweet, a member of Verbenaceae, is a traditional Chinese medicinal plant mainly distributed in tropical and subtropical Asia. Herein, we reported the complete chloroplast genome sequence of C. japonicum. The size of the chloroplast genome is 152,171 bp in length, including a large single-copy region (LSC) of 83,415 bp, a small single-copy region (SSC) of 17,318 bp, which was separated by a pair of inverted repeated regions of 25,719 bp. The C. japonicum chloroplast genome encodes 133 genes, including 88 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The phylogenetic tree showed that C. japonicum is closely related to C. mandarinorum and C. yunnanense.