Evolution of the WRKY Family in Angiosperms and Functional Diversity under Environmental Stress.

The transcription factor is an essential factor for regulating the responses of plants to external stimuli. The WRKY protein is a superfamily of plant transcription factors involved in response to various stresses (e.g., cold, heat, salt, drought, ions, pathogens, and insects). During angiosperm evolution, the number and function of WRKY transcription factors constantly change. After suffering from long-term environmental battering, plants of different evolutionary statuses ultimately retained different numbers of WRKY family members. The WRKY family of proteins is generally divided into three large categories of angiosperms, owing to their conserved domain and three-dimensional structures. The WRKY transcription factors mediate plant adaptation to various environments via participating in various biological pathways, such as ROS (reactive oxygen species) and hormone signaling pathways, further regulating plant enzyme systems, stomatal closure, and leaf shrinkage physiological responses. This article analyzed the evolution of the WRKY family in angiosperms and its functions in responding to various external environments, especially the function and evolution in Magnoliaceae plants. It helps to gain a deeper understanding of the evolution and functional diversity of the WRKY family and provides theoretical and experimental references for studying the molecular mechanisms of environmental stress.

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.

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.

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 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.

Genome-wide analyses of calmodulin and calmodulin-like proteins in the halophyte Nitraria sibirica reveal their involvement in response to salinity, drought and cold stress.

The calmodulin (CaM) and calmodulin-like (CML) proteins are major calcium sensors that play a critical role in environmental stimulus response in plants. Nevertheless, the CaM/CML proteins from the specific plants with extreme tolerance to abiotic stresses remained so far uncharacterized. In this study, 66 candidate proteins (three NsCaMs and sixty-three NsCMLs) were identified from the halophyte Nitraria sibirica, which can withstand an extreme salinity. Bioinformatic analysis of upstream cis-acting elements predicted the potential involvement of NsCaM/CMLs in abiotic stress responses and various hormone responses. Additionally, the Nitraria sibirica transcriptome revealed that 17 and 7 NsCMLs were significantly upregulated under 100 mM or 400 mM NaCl treatment. Transcription of most salt-responsive genes was similarly upregulated under cold stress, yet downregulated under drought treatment. Moreover, predictive subcellular localization analysis suggested that the stress-responsive NsCML proteins mainly localize at the cellular membrane and within the nucleus. Furthermore, transgenic overexpression of two NsCMLs (NISI03G1136 and NISI01G1645) was found to mitigate H2O2 accumulation caused by salt stress. These results provide insights into the potential function of Nitraria sibirica CaM/CML proteins, which could aid the investigation of molecular mechanisms of extreme tolerance to abiotic stresses in halophytes.

Genome-wide analysis of the GRAS gene family in Liriodendron chinense reveals the putative function in abiotic stress and plant development.

Introduction: GRAS genes encode plant-specific transcription factors that play essential roles in plant growth and development. However, the members and the function of the GRAS gene family have not been reported in Liriodendron chinense. L. chinense, a tree species in the Magnolia family that produces excellent timber for daily life and industry. In addition, it is a good relict species for plant evolution research. Methods: Therefore, we conducted a genome-wide study of the LcGRAS gene family and identified 49 LcGRAS genes in L. chinense. Results: We found that LcGRAS could be divided into 13 sub-groups, among which there is a unique branch named HAM-t. We carried out RNA sequencing analysis of the somatic embryos from L. chinense and found that LcGRAS genes are mainly expressed after heart-stage embryo development, suggesting that LcGRAS may have a function during somatic embryogenesis. We also investigated whether GRAS genes are responsive to stress by carrying out RNA sequencing (RNA-seq) analysis, and we found that the genes in the PAT subfamily were activated upon stress treatment, suggesting that these genes may help plants survive stressful environments. We found that PIF was downregulated and COR was upregulated after the transient overexpression of PATs, suggesting that PAT may be upstream regulators of cold stress. Discussion: Collectively, LcGRAS genes are conserved and play essential roles in plant development and adaptation to abiotic stress.

Phytosulfokine contributes to suspension culture of Cunninghamia lanceolata through its impact on redox homeostasis.

BACKGROUND: Suspension culture is widely used in the establishment of efficient plant regeneration systems, as well as in the mass production of plant secondary metabolites. However, the establishment of a suspension culture system of Cunninghamia lanceolata is genotype-dependent given that proembryogenic masses (PEMs) are prone to browning during this process in recalcitrant genotypes. Previously, we reported that the plant peptide hormone phytosulfokine (PSK) can tremendously decrease the hydrogen peroxide (H2O2) level and help to initiate somatic embryogenesis (SE) in recalcitrant C. lanceolata genotypes. However, to date, no studies have revealed whether or how PSK may contribute to the establishment of a suspension culture system in these recalcitrant genotypes. RESULTS: Here, we demonstrated that exogenous application of PSK effectively inhibited PEM browning during suspension culture in a recalcitrant genotype of C. lanceolata. Comparative time-series transcriptome profiling showed that redox homeostasis underwent drastic fluctuations when PEMs were cultured in liquid medium, while additional PSK treatment helped to maintain a relatively stable redox homeostasis. Interestingly, PSK seemed to have a dual effect on peroxidases (PRXs), with PSK simultaneously transcriptionally repressing ROS-producing PRXs and activating ROS-scavenging PRXs. Furthermore, determination of H2O2 and MDA content, as well as cell viability, showed that exogenous PSK treatment inhibited PEM browning and safeguarded PEM suspension culture by decreasing the H2O2 level and increasing PEM activity. CONCLUSIONS: Collectively, these findings provide a valuable tool for the future establishment of large-scale C. lanceolata PEM suspension culture without genotype limitations.

Genome-wide identification of the NAC gene family and its functional analysis in Liriodendron.

As one of the largest plant specific transcription factor families, NAC family members play an important role in plant growth, development and stress resistance. To investigate the function of NAC transcription factors during abiotic stress, as well as during somatic embryogenesis, we identified and characterized the NAC gene family in Liriodendron chinense. We found that most LcNAC members contain more than three exons, with a relatively conserved gene and motif structure, especially at the N-terminus. Interspecies collinearity analysis revealed a closer relationship between the L. chinense NACs and the P. trichocarpa NACs. We analyzed the expression of LcNAC in different tissues and under three abiotic stresses. We found that 12 genes were highly expressed during the ES3 and ES4 stages of somatic embryos, suggesting that they are involved in the development of somatic embryos. 6 LcNAC genes are highly expressed in flower organs. The expression pattern analysis of LcNACs based on transcriptome data and RT-qPCR obtained from L. chinense leaves indicated differential expression responses to drought, cold, and heat stress. Genes in the NAM subfamily expressed differently during abiotic stress, and LcNAC6/18/41/65 might be the key genes in response to abiotic stress. LcNAC6/18/41/65 were cloned and transiently transformed into Liriodendron protoplasts, where LcNAC18/65 was localized in cytoplasm and nucleus, and LcNAC6/41 was localized only in nucleus. Overall, our findings suggest a role of the NAC gene family during environmental stresses in L. chinense. This research provides a basis for further study of NAC genes in Liriodendron chinense.

Genome-Wide Identification and Expression Analysis of CCO Gene Family in Liriodendron chinense.

Carotenoid cleavage oxygenase (CCO) is an enzyme that can catalyze carotenoids to volatile aromatic substances and participate in the biosynthesis of two important phytohormones, i.e., abscisic acid (ABA) and strigolactone (SL). However, the genome-wide identification and analysis of the CCO gene family in the rare and endangered woody plant Liriodendron chinense has not been reported. Here, we performed a genome-wide analysis of the CCO gene family in the L. chinense genome and examined its expression pattern during different developmental processes and in response to various abiotic stresses. A total of 10 LcCCO genes were identified and divided into 6 subfamilies according to the phylogenetic analysis. Subcellular localization prediction showed that most of the LcCCO proteins were located in the cytoplasm. Gene replication analysis showed that segmental and tandem duplication contributed to the expansion of this gene family in the L. chinense genome. Cis-element prediction showed that cis-elements related to plant hormones, stress and light response were widely distributed in the promoter regions of LcCCO genes. Gene expression profile analysis showed that LcNCED3b was extensively involved in somatic embryogenesis, especially the somatic embryo maturation, as well as in response to heat and cold stress in leaves. Furthermore, qRT-PCR analysis showed that LcNCED3b obviously responded to drought stress in roots and leaves. This study provides a comprehensive overview of the LcCCO gene family and a potential gene target for the optimization of the somatic embryogenesis system and resistance breeding in the valuable forest tree L. chinense.

PYL Family Genes from Liriodendron chinense Positively Respond to Multiple Stresses.

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.

Genome-Wide Identification and Abiotic-Stress-Responsive Expression of CKX Gene Family in Liriodendron chinense.

Liriodendron chinense is a tree species of the Magnoliaceae family, an ancient relict plant mainly used for landscaping and timber production due to its excellent material properties and ornamental value. The cytokinin oxidase/dehydrogenase (CKX) enzyme regulates cytokinin levels and plays an important role in plant growth, development, and resistance. However, too-high or too-low temperatures or soil drought can limit the growth of L. chinense, representing a key issue for research. Here, we identified the CKX gene family in the L. chinense genome and examined its transcriptional responses to cold, drought, and heat stresses. A total of five LcCKX genes, distributed on four chromosomes and divided into three phylogenetic groups, were identified across the whole L. chinense genome. Further analysis showed that multiple hormone- and stress-responsive cis-acting elements are located in the promoter regions of LcCKXs, indicating a potential role of these LcCKXs in plant growth, development, and response to environmental stresses. Based on existing transcriptome data, LcCKXs, especially LcCKX5, were found to transcriptionally respond to cold, heat, and drought stresses. Furthermore, quantitative reverse-transcription PCR (qRT-PCR) showed that LcCKX5 responds to drought stress in an ABA-dependent manner in stems and leaves and in an ABA-independent manner in roots. These results lay a foundation for functional research on LcCKX genes in the resistance breeding of the rare and endangered tree species of L. chinense.

Characterization of the Liriodendron chinense Pentatricopeptide Repeat (PPR) Gene Family and Its Role in Osmotic Stress Response.

The Pentatricopeptide repeat (PPR) superfamily is a large gene family in plants that regulates organelle RNA metabolism, which is important for plant growth and development. However, a genome-wide analysis of the PPR gene family and its response to abiotic stress has not been reported for the relict woody plant Liriodendron chinense. In this paper, we identified 650 PPR genes from the L. chinense genome. A phylogenetic analysis showed that the LcPPR genes could roughly be divided into the P and PLS subfamilies. We found that 598 LcPPR genes were widely distributed across 19 chromosomes. An intraspecies synteny analysis indicated that duplicated genes from segmental duplication contributed to the expansion of the LcPPR gene family in the L. chinense genome. In addition, we verified the relative expression of Lchi03277, Lchi06624, Lchi18566, and Lchi23489 in the roots, stems, and leaves and found that all four genes had the highest expression in the leaves. By simulating a drought treatment and quantitative reverse transcription PCR (qRT-PCR) analysis, we confirmed the drought-responsive transcriptional changes in four LcPPR genes, two of which responded to drought stress independent of endogenous ABA biosynthesis. Thus, our study provides a comprehensive analysis of the L. chinense PPR gene family. It contributes to research into their roles in this valuable tree species' growth, development, and stress resistance.

Genome-Wide Analysis and Abiotic Stress-Responsive Patterns of COBRA-like Gene Family in Liriodendron chinense.

The COBRA gene encodes a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein (GAP), which plays an important role in cell wall cellulose deposition. In this study, a total of 7 COBRA-like (COBL) genes were identified in the genome of the rare and endangered woody plant Liriodendron chinense (L. chinense). Phylogenetic analysis showed that these LcCOBL genes can be divided into two subfamilies, i.e., SF I and II. In the conserved motif analysis of two subfamilies, SF I contained 10 predicted motifs, while SF II contained 4-6 motifs. The tissue-specific expression patterns showed that LcCOBL5 was highly expressed in the phloem and xylem, indicating its potential role in cellulose biosynthesis. In addition, the cis-element analysis and abiotic stress transcriptomes showed that three LcCOBLs, LcCOBL3, LcCOBL4 and LcCOBL5, transcriptionally responded to abiotic stresses, including cold, drought and heat stress. In particular, the quantitative reverse-transcription PCR (qRT-PCR) analysis further confirmed that the LcCOBL3 gene was significantly upregulated in response to cold stress and peaked at 24-48 h, hinting at its potential role in the mechanism of cold resistance in L. chinense. Moreover, GFP-fused LcCOBL2, LcCOBL4 and LcCOBL5 were found to be localized in the cytomembrane. In summary, we expect these results to be beneficial for research on both the functions of LcCOBL genes and resistance breeding in L. chinense.

The Glutathione Peroxidase Gene Family in Nitraria sibirica: Genome-Wide Identification, Classification, and Gene Expression Analysis under Stress Conditions.

Plant glutathione peroxidases (GPXs) are the main enzymes in the antioxidant defense system that sustain H2O2 homeostasis and normalize plant reaction to abiotic stress conditions. However, the genome-wide identification of the GPX gene family and its responses to environmental stresses, especially salt stress, in Nitraria sibirica, which is a shrub that can survive in saline environments, has not yet been reported. Here, we first report the genome-wide analysis of the GPX gene family in N. sibirica, leading to a total of seven NsGPX genes that are distributed on six of the twelve chromosomes. Phylogenetic analysis showed that NsGPX genes were grouped into four major groups (Group I-IV). Three types of cis-acting elements were identified in the NsGPX promoters, mainly related to hormones and stress response. The quantitative real-time PCR (qRT-PCR) analysis indicated that NsGPX1 and NsGPX3 were significantly up-regulated in stem and leaf, while NsGPX7 transcriptionally in root in response to salt stress. The current study identified a total seven NsGPX genes in N. sibirica via genome-wide analysis, and discovered that NsGPXs may play an important role in response to salt stress. Taken together, our findings provide a basis for further functional studies of NsGPX genes, especially in regarding to the resistance to salt stress of this halophyte plant N. sibirica, eventually aid in the discovery of new methods to restore overtly saline soil.

Genome-Wide Identification and Expression Analysis of TPS Gene Family in Liriodendron chinense.

Terpenoids play a key role in plant growth and development, supporting resistance regulation and terpene synthase (TPS), which is the last link in the synthesis process of terpenoids. Liriodendron chinense, commonly called the Chinese tulip tree, is a rare and endangered tree species of the family Magnoliaceae. However, the genome-wide identification of the TPS gene family and its transcriptional responses to development and abiotic stress are still unclear. In the present study, we identified a total of 58 TPS genes throughout the L. chinense genome. A phylogenetic tree analysis showed that they were clustered into five subfamilies and unevenly distributed across six chromosomes. A cis-acting element analysis indicated that LcTPSs were assumed to be highly responsive to stress hormones, such as methyl jasmonate (MeJA) and abscisic acid (ABA). Consistent with this, transcriptome data showed that most LcTPS genes responded to abiotic stress, such as cold, drought, and hot stress, at the transcriptional level. Further analysis showed that LcTPS genes were expressed in a tissue-dependent manner, especially in buds, leaves, and bark. Quantitative reverse transcription PCR (qRT-PCR) analysis confirmed that LcTPS expression was significantly higher in mature leaves compared to young leaves. These results provide a reference for understanding the function and role of the TPS family, laying a foundation for further study of the regulation of TPS in terpenoid biosynthesis in L. chinense.

Genetic diversity and structure of the 4th cycle breeding population of Chinese fir (Cunninghamia lanceolata (lamb.) hook).

Studying population genetic structure and diversity is crucial for the marker-assisted selection and breeding of coniferous tree species. In this study, using RAD-seq technology, we developed 343,644 high-quality single nucleotide polymorphism (SNP) markers to resolve the genetic diversity and population genetic structure of 233 Chinese fir selected individuals from the 4th cycle breeding program, representing different breeding generations and provenances. The genetic diversity of the 4th cycle breeding population was high with nucleotide diversity (Pi ) of 0.003, and Ho and He of 0.215 and 0.233, respectively, indicating that the breeding population has a broad genetic base. The genetic differentiation level between the different breeding generations and different provenances was low (Fst < 0.05), with population structure analysis results dividing the 233 individuals into four subgroups. Each subgroup has a mixed branch with interpenetration and weak population structure, which might be related to breeding rather than provenance, with aggregation from the same source only being in the local branches. Our results provide a reference for further research on the marker-assisted selective breeding of Chinese fir and other coniferous trees.

miR394 enhances WUSCHEL-induced somatic embryogenesis in Arabidopsis thaliana.

Many plant species can give rise to embryos from somatic cells after a simple hormone treatment, illustrating the remarkable developmental plasticity of differentiated plant cells. However, many species are recalcitrant to somatic embryo formation for unknown reasons, which poses a significant challenge to agriculture, where somatic embryogenesis is an important tool to propagate desired genotypes. The micro-RNA394 (miR394) promotes shoot meristem maintenance in Arabidopsis thaliana, but the underlying mechanisms have remained elusive. We analyzed whether miR394 affects indirect somatic embryogenesis and determined the transcriptome of embryogenic callus upon miR394-enhanced somatic embryogenesis. We show that ectopic miR394 expression enhances somatic embryogenesis in the recalcitrant Ler accession when co-expressed with the transcription factor WUSCHEL (WUS) and that miR394 acts in this process through silencing the target LEAF CURLING RESPONSIVENESS (LCR). Furthermore, we show that higher endogenous miR394 levels are required for the elevated embryogenic potential of the Columbia accession compared with Ler, providing a mechanistic explanation for this natural variation. Our transcriptional analysis provides a framework for miR394 function in regulating pluripotency by expanding WUS-mediated direct transcriptional repression.

Overexpression of Liriodenron WOX5 in Arabidopsis Leads to Ectopic Flower Formation and Altered Root Morphology

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.

The plant peptide hormone phytosulfokine promotes somatic embryogenesis by maintaining redox homeostasis in Cunninghamia lanceolata.

Somatic embryogenesis (SE) is widely used for studying the mechanisms of embryo development. However, little is known about the underlying mechanisms, especially in woody plants. Previous studies have established an SE system for Chinese fir (Cunninghamia lanceolata), but this system is genotype-dependent, which limits its application in practice. Here, we found that phytosulfokine (PSK), a plant peptide hormone, can not only increase SE efficiency, but also establish SE in recalcitrant genotypes of C. lanceolata. Proembryogenic mass (PEM) browning and determination of hydrogen peroxide (H2 O2 ) content by 2',7'-dichlorofluorescein staining indicated that a reactive oxygen species (ROS) burst occurred rapidly after PEMs were transferred to SE induction medium. Transcriptome analysis and quantitative reverse transcriptase-PCR validation showed that PSK treatment helped to maintain ROS homeostasis by decreasing the activity of peroxidases in early SE induction. This PSK-regulated redox microenvironment might be helpful to induce expression of SE-related genes like WOX2 in early SE induction. Further analyses suggested that PSK promotes SE induction in C. lanceolata partially through decreasing H2 O2 levels, which is necessary but not sufficient for SE induction in recalcitrant genotypes of C. lanceolata. Furthermore, heterologous overexpression of ClPSK in Arabidopsis led to enhanced SE induction and resistance to H2 O2 stress. Taken together, our study reveals a biological function for the plant peptide hormone PSK, extends our knowledge about SE in woody trees, and provides a valuable tool for establishing an efficient and genotype-independent SE system in C. lanceolata and other coniferous trees.