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.

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.

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.

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.

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.

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.

Genome-wide identification of the Liriodendron chinense WRKY gene family and its diverse roles in response to multiple abiotic stress.

BACKGROUND: Liriodendron chinense (Lchi) is a tree species within the Magnoliaceae family and is considered a basal angiosperm. The too low or high temperature or soil drought will restrict its growth as the adverse environmental conditions, thus improving L. chinense abiotic tolerance was the key issues to study. WRKYs are a major family of plant transcription factors known to often be involved in biotic and abiotic stress responses. So far, it is still largely unknown if and how the LchiWRKY gene family is tied to regulating L. chinense stress responses. Therefore, studying the involvement of the WRKY gene family in abiotic stress regulation in L. chinense could be very informative in showing how this tree deals with such stressful conditions. RESULTS: In this research, we performed a genome-wide analysis of the Liriodendron chinense (Lchi) WRKY gene family, studying their classification relationships, gene structure, chromosomal locations, gene duplication, cis-element, and response to abiotic stress. The 44 members of the LchiWRKY gene family contain a significant amount of sequence diversity, with their lengths ranging from 525 bp to 40,981 bp. Using classification analysis, we divided the 44 LchiWRKY genes into three phylogenetic groups (I, II, II), with group II then being further divided into five subgroups (IIa, IIb, IIc, IId, IIe). Comparative phylogenetic analysis including the WRKY families from 17 plant species suggested that LchiWRKYs are closely related to the Magnolia Cinnamomum kanehirae WRKY family, and has fewer family members than higher plants. We found the LchiWRKYs to be evenly distributed across 15 chromosomes, with their duplication events suggesting that tandem duplication may have played a major role in LchiWRKY gene expansion model. A Ka/Ks analysis indicated that they mainly underwent purifying selection and distributed in the group IId. Motif analysis showed that LchiWRKYs contained 20 motifs, and different phylogenetic groups contained conserved motif. Gene ontology (GO) analysis showed that LchiWRKYs were mainly enriched in two categories, i.e., biological process and molecular function. Two group IIc members (LchiWRKY10 and LchiWRKY37) contain unique WRKY element sequence variants (WRKYGKK and WRKYGKS). Gene structure analysis showed that most LchiWRKYs possess 3 exons and two different types of introns: the R- and V-type which are both contained within the WRKY domain (WD). Additional promoter cis-element analysis indicated that 12 cis-elements that play different functions in environmental adaptability occur across all LchiWRKY groups. Heat, cold, and drought stress mainly induced the expression of group II and I LchiWRKYs, some of which had undergone gene duplication during evolution, and more than half of which had three exons. LchiWRKY33 mainly responded to cold stress and LchiWRKY25 mainly responded to heat stress, and LchiWRKY18 mainly responded to drought stress, which was almost 4-fold highly expressed, while 5 LchiWRKYs (LchiWRKY5, LchiWRKY23, LchiWRKY14, LchiWRKY27, and LchiWRKY36) responded equally three stresses with more than 6-fold expression. Subcellular localization analysis showed that all LchiWRKYs were localized in the nucleus, and subcellular localization experiments of LchiWRKY18 and 36 also showed that these two transcription factors were expressed in the nucleus. CONCLUSIONS: This study shows that in Liriodendron chinense, several WRKY genes like LchiWRKY33, LchiWRKY25, and LchiWRKY18, respond to cold or heat or drought stress, suggesting that they may indeed play a role in regulating the tree's response to such conditions. This information will prove a pivotal role in directing further studies on the function of the LchiWRKY gene family in abiotic stress response and provides a theoretical basis for popularizing afforestation in different regions of China.

The Transcriptional Landscape of Polyploid Wheats and Their Diploid Ancestors during Embryogenesis and Grain Development.

Modern wheat production comes from two polyploid species, Triticum aestivum and Triticum turgidum (var durum), which putatively arose from diploid ancestors Triticum urartu, Aegilops speltoides, and Aegilops tauschii How gene expression during embryogenesis and grain development in wheats has been shaped by the differing contributions of diploid genomes through hybridization, polyploidization, and breeding selection is not well understood. This study describes the global landscape of gene activities during wheat embryogenesis and grain development. Using comprehensive transcriptomic analyses of two wheat cultivars and three diploid grasses, we investigated gene expression at seven stages of embryo development, two endosperm stages, and one pericarp stage. We identified transcriptional signatures and developmental similarities and differences among the five species, revealing the evolutionary divergence of gene expression programs and the contributions of A, B, and D subgenomes to grain development in polyploid wheats. The characterization of embryonic transcriptional programming in hexaploid wheat, tetraploid wheat, and diploid grass species provides insight into the landscape of gene expression in modern wheat and its ancestral species. This study presents a framework for understanding the evolution of domesticated wheat and the selective pressures placed on grain production, with important implications for future performance and yield improvements.plantcell;31/12/2888/FX1F1fx1.

Identification, Phylogenetic and Expression Analyses of the AAAP Gene Family in Liriodendron chinense Reveal Their Putative Functions in Response to Organ and Multiple Abiotic Stresses.

In this study, 52 AAAP genes were identified in the L. chinense genome and divided into eight subgroups based on phylogenetic relationships, gene structure, and conserved motif. A total of 48 LcAAAP genes were located on the 14 chromosomes, and the remaining four genes were mapped in the contigs. Multispecies phylogenetic tree and codon usage bias analysis show that the LcAAAP gene family is closer to the AAAP of Amborella trichopoda, indicating that the LcAAAP gene family is relatively primitive in angiosperms. Gene duplication events revealed six pairs of segmental duplications and one pair of tandem duplications, in which many paralogous genes diverged in function before monocotyledonous and dicotyledonous plants differentiation and were strongly purification selected. Gene expression pattern analysis showed that the LcAAAP gene plays a certain role in the development of Liriodendron nectary and somatic embryogenesis. Low temperature, drought, and heat stresses may activate some WRKY/MYB transcription factors to positively regulate the expression of LcAAAP genes to achieve long-distance transport of amino acids in plants to resist the unfavorable external environment. In addition, the GAT and PorT subgroups could involve gamma-aminobutyric acid (GABA) transport under aluminum poisoning. These findings could lay a solid foundation for further study of the biological role of LcAAAP and improvement of the stress resistance of Liriodendron.

ICE-CBF-COR Signaling Cascade and Its Regulation in Plants Responding to Cold Stress.

Cold stress limits plant geographical distribution and influences plant growth, development, and yields. Plants as sessile organisms have evolved complex biochemical and physiological mechanisms to adapt to cold stress. These mechanisms are regulated by a series of transcription factors and proteins for efficient cold stress acclimation. It has been established that the ICE-CBF-COR signaling pathway in plants regulates how plants acclimatize to cold stress. Cold stress is perceived by receptor proteins, triggering signal transduction, and Inducer of CBF Expression (ICE) genes are activated and regulated, consequently upregulating the transcription and expression of the C-repeat Binding Factor (CBF) genes. The CBF protein binds to the C-repeat/Dehydration Responsive Element (CRT/DRE), a homeopathic element of the Cold Regulated genes (COR gene) promoter, activating their transcription. Transcriptional regulations and post-translational modifications regulate and modify these entities at different response levels by altering their expression or activities in the signaling cascade. These activities then lead to efficient cold stress tolerance. This paper contains a concise summary of the ICE-CBF-COR pathway elucidating on the cross interconnections with other repressors, inhibitors, and activators to induce cold stress acclimation in plants.

The Identification and Expression Analysis of the Nitraria sibirica Pall. Auxin-Response Factor (ARF) Gene Family.

Nitraria sibirica is a shrub that can survive in extreme drought environments. The auxin-response factors (ARFs) are a class of transcription factors that are widely involved in plant growth and development, as well as in the regulation of stress resistance. However, the genome-wide identification of the ARF gene family and its responses to environmental stresses, especially drought stress, in N. sibirica has not yet been reported. Here, we identified a total of 12 ARF genes in the genome of N. sibirica, which were distributed over 10 chromosomes and divided into three clades. Intragenome synteny analysis revealed one collinear gene pair in the ARF gene family, i.e., NsARF9a and NsARF9b. Cis-acting element analysis showed that multiple hormones and stress-responsive cis-acting elements were found in the promoters of NsARFs, suggesting that NsARFs may be involved in multiple biological processes. Quantitative real-time PCR (qRT-PCR) showed that many NsARFs had tissue-specific expression patterns, with the highest expression of NsARF16 in the seedlings of N. sibirica. In addition, most of the NsARFs that were upregulated under drought were independent of endogenous ABA biosynthesis, whereas the response of NsARF5 and NsARF7a to drought was disrupted by the ABA-biosynthesis inhibitor fluridone. These studies provide a basis for further research into how NsARFs in N. sibirica respond to hormonal signaling and environmental stresses.

Integrative analysis of transcriptome and proteome revealed nectary and nectar traits in the plant-pollinator interaction of Nitraria tangutorum Bobrov.

BACKGROUND: Nitraria tangutorum is an important desert shrub that shows resistance to drought, salt and wind erosion stresses. It is a central ecological species in its area. Here, we have studied how N. tangutorum has adapted to achieve a successful reproduction strategy. RESULTS: We found that N. tangutorum is mainly pollinated by insects of the Hymenoptera, Diptera and Coleoptera orders. Nitraria tangutorum has very small flowers, with the nectary composed of secretive epidermal cells from which nectar is secreted, located within the inner petals. In addition, analyzing the transcriptome of four successive flower developmental stages revealed that mainly differentially expressed genes associated with flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction show dynamic expression. From the nectar, we could identify seven important proteins, of which the L-ascorbate oxidase protein was first found in plant nectar. Based on the physiological functions of these proteins, we predict that floral nectar proteins of N. tangutorum play an important role in defending against microbial infestation and scavenging active oxygen. CONCLUSIONS: This study revealed that N. tangutorum is an insect-pollinated plant and its nectary is composed of secretive epidermal cells that specialized into secretive trichomes. We identified a large number of differentially expressed genes controlling flower and nectary development, nectar biosynthesis and secretion, flavonoid biosynthesis, plant hormone signal transduction and plant-pathogen interaction. We suggest that proteins present in N. tangutorum nectar may have both an antibacterial and oxygen scavenging effect. These results provide a scientific basis for exploring how the reproductive system of N. tangutorum and other arid-desert plants functions.

Gibberellin Oxidase Gene Family in L. chinense: Genome-Wide Identification and Gene Expression Analysis.

GAox is a key enzyme for the transformation of gibberellins, and belongs to the 2-ketoglutarate dependent dioxygenase gene family (2ODD). However, a systematic analysis of GAox in the angiosperm L. chinense has not yet been reported. Here, we identified all LcGAox gene family members in L. chinense, which were classified into the three subgroups of GA20ox, C19GA2ox, and C20GA2ox. Comparison of the gene structure, conserve motifs, phylogenetic relationships, and syntenic relationships of gibberellin oxidase gene families in different species indicated that the gene functional differences may be due to the partial deletion of their domains during evolution. Furthermore, evidence for purifying selection was detected between orthologous GAox genes in rice, grape, Arabidopsis, and L. chinense. Analysis of the codon usage patterns showed that mutation pressure and natural selection might have induced codon usage bias in angiosperms; however, the LcGAox genes in mosses, lycophytes, and ambarella plants exhibited no obvious codon usage preference. These results suggested that the gibberellin oxidase genes were more primitive. The gene expression pattern was analyzed in different organs subjected to multiple abiotic stresses, including GA, abscisic acid (ABA), and chlormequat (CCC) treatment, by RNA-seq and qRT-PCR, and the stress- and phytohormone-responsive cis-elements were counted. The results showed that the synthesis and decomposition of GA were regulated by different LcGAox genes in the vegetative and reproductive organs of L. chinense, and only LcGA2ox1,4, and 7 responded to the NaCl, polyethylene glycol, 4 °C, GA, ABA, and CCC treatment in the roots, stems, and leaves of seedlings at different time periods, revealing the potential role of LcGAox in stress resistance.

The Transcriptome of Cunninghamia lanceolata male/female cone reveal the association between MIKC MADS-box genes and reproductive organs development.

BACKGROUND: Cunninghamia lanceolata (Chinese fir), a member of the conifer family Cupressaceae, is one of the most popular cultivated trees for wood production in China. Continuous research is being performed to improve C. lanceolata breeding values. Given the high rate of seed abortion (one of the reasons being the failure of ovule and pollen development) in C. lanceolata, the proper formation of female/male cones could theoretically increase the number of offspring in future generations. MIKC MADS-box genes are well-known for their roles in the flower/cone development and comprise the typical/atypical floral development model for both angiosperms and gymnosperms. RESULTS: We performed a transcriptomic analysis to find genes differentially expressed between female and male cones at a single, carefully determined developmental stage, focusing on the MIKC MADS-box genes. We finally obtained 47 unique MIKC MADS-box genes from C. lanceolata and divided these genes into separate branches. 27 out of the 47 MIKC MADS-box genes showed differential expression between female and male cones, and most of them were not expressed in leaves. Out of these 27 genes, most B-class genes (AP3/PI) were up-regulated in the male cone, while TM8 genes were up-regulated in the female cone. Then, with no obvious overall preference for AG (class C + D) genes in female/male cones, it seems likely that these genes are involved in the development of both cones. Finally, a small number of genes such as GGM7, SVP, AGL15, that were specifically expressed in female/male cones, making them candidate genes for sex-specific cone development. CONCLUSIONS: Our study identified a number of MIKC MADS-box genes showing differential expression between female and male cones in C. lanceolata, illustrating a potential link of these genes with C. lanceolata cone development. On the basis of this, we postulated a possible cone development model for C. lanceolata. The gene expression library showing differential expression between female and male cones shown here, can be used to discover unknown regulatory networks related to sex-specific cone development in the future.

Genome Sequence and Comparative Analysis of Colletotrichum gloeosporioides Isolated from Liriodendron Leaves.

Colletotrichum gloeosporioides is a hemibiotrophic pathogen causing significant losses to economically important crops and forest trees, including Liriodendron. To explore the interaction between C. gloeosporioides and Liriodendron and to identify the candidate genes determining the pathogenesis, we sequenced and assembled the whole genome of C. gloeosporioides Lc1 (CgLc1) using PacBio and Illumina next generation sequencing and performed a comparative genomic analysis between CgLc1 and Cg01, the latter being a described endophytic species of the C. gloeosporioides complex. Gene structure prediction identified 15,744 protein-coding genes and 837 noncoding RNAs. Species-specific genes were characterized using an ortholog analysis followed by a pathway enrichment analysis, which showed that genes specific to CgLc1 were enriched for the arginine biosynthetic process. Furthermore, genome synteny analysis revealed that most of the protein-coding genes fell into collinear blocks. However, two clusters of polyketide synthase genes were identified to be specific for CgLc1, suggesting that they might have an important role in virulence control. Transcriptional regulators coexpressed with polyketide synthase genes were detected through a Weighted Correlation Network Analysis. Taken together, this work provides new insight into the virulence- and pathogenesis-associated genes present in C. gloeosporioides and its possible lifestyle.

Complete chloroplast genome sequence of a major economic species, Ziziphus jujuba (Rhamnaceae).

Ziziphus jujuba is an important woody plant with high economic and medicinal value. Here, we analyzed and characterized the complete chloroplast (cp) genome of Z. jujuba, the first member of the Rhamnaceae family for which the chloroplast genome sequence has been reported. We also built a web browser for navigating the cp genome of Z. jujuba ( http://bio.njfu.edu.cn/gb2/gbrowse/Ziziphus_jujuba_cp/ ). Sequence analysis showed that this cp genome is 161,466 bp long and has a typical quadripartite structure of large (LSC, 89,120 bp) and small (SSC, 19,348 bp) single-copy regions separated by a pair of inverted repeats (IRs, 26,499 bp). The sequence contained 112 unique genes, including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The genome structure, gene order, GC content, and codon usage are similar to other typical angiosperm cp genomes. A total of 38 tandem repeats, two forward repeats, and three palindromic repeats were detected in the Z. jujuba cp genome. Simple sequence repeat (SSR) analysis revealed that most SSRs were AT-rich. The homopolymer regions in the cp genome of Z. jujuba were verified and manually corrected by Sanger sequencing. One-third of mononucleotide repeats were found to be erroneously sequenced by the 454 pyrosequencing, which resulted in sequences of 1-4 bases shorter than that by the Sanger sequencing. Analyzing the cp genome of Z. jujuba revealed that the IR contraction and expansion events resulted in ycf1 and rps19 pseudogenes. A phylogenetic analysis based on 64 protein-coding genes showed that Z. jujuba was closely related to members of the Elaeagnaceae family, which will be helpful for phylogenetic studies of other Rosales species. The complete cp genome sequence of Z. jujuba will facilitate population, phylogenetic, and cp genetic engineering studies of this economic plant.

Comparative Analysis of the Chloroplast Genomic Information of Cunninghamia lanceolata (Lamb.) Hook with Sibling Species from the Genera Cryptomeria D. Don, Taiwania Hayata, and Calocedrus Kurz.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) is an important coniferous tree species for timber production, which accounts for ~40% of log supply from plantations in southern China. Chloroplast genetic engineering is an exciting field to engineer several valuable tree traits. In this study, we revisited the published complete Chinese fir (NC_021437) and four other coniferous species chloroplast genome sequence in Taxodiaceae. Comparison of their chloroplast genomes revealed three unique inversions found in the downstream of the gene clusters and evolutionary divergence were found, although overall the chloroplast genomic structure of the Cupressaceae linage was conserved. We also investigated the phylogenetic position of Chinese fir among conifers by examining gene functions, selection forces, substitution rates, and the full chloroplast genome sequence. Consistent with previous molecular systematics analysis, the results provided a well-supported phylogeny framework for the Cupressaceae that strongly confirms the "basal" position of Cunninghamia lanceolata. The structure of the Cunninghamia lanceolata chloroplast genome showed a partial lack of one IR copy, rearrangements clearly occurred and slight evolutionary divergence appeared among the cp genome of C. lanceolata, Taiwania cryptomerioides, Taiwania flousiana, Calocedrus formosana and Cryptomeria japonica. The information from sequence divergence and length variation of genes could be further considered for bioengineering research.

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.