Effects of plant growth regulators and sucrose on proliferation and quality of embryogenic tissue in Picea pungens.

Embryogenic tissue (ET) is important for genetic modification and plant re-generation. The proliferation ability and vigor of ET are crucial for plant propagation via somatic embryogenesis. In this study, ET was induced from mature zygotic embryos in blue spruce (Picea pungens Engelm.). There were significant differences in ET induction between two provenances, i.e. 78.8 ± 12.5% and 62.50 ± 12.8% respectively. Effects of 2,4-Dichlorophenoxy acetic acid (2,4-D), 6-Benzyl amino-purine (6-BA) and/or sucrose on ET proliferation and somatic embryo (SE) maturation were further investigated with four cell lines. The highest ET proliferation rate reached 1473.7 ± 556.0% biweekly. Concentrations of 2,4-D or 6-BA applied at tissue proliferation stage impacted SE maturation among the cell lines, whereas sucrose showed less effects. The highest rate, 408 ± 230 mature SEs/g FW, was achieved in SE maturation cultures. This research demonstrated that the culture conditions, i.e. the specific concentrations of 2,4-D and BA, at ET proliferation stage affected not only ET growth, but also the quality of ET for SE maturation. This study revealed the necessity and benefit in developing both the general and the genotype-specific protocols for efficient production of mature SEs, or somatic plants in blue spruce.

Analysis of polycomb repressive complex 2 (PRC2) subunits in Picea abies with a focus on embryo development.

BACKGROUND: Conserved polycomb repressive complex 2 (PRC2) mediates H3K27me3 to direct transcriptional repression and has a key role in cell fate determination and cell differentiation in both animals and plants. PRC2 subunits have undergone independent multiplication and functional divergence in higher plants. However, relevant information is still absent in gymnosperms. RESULTS: To launch gymnosperm PRC2 research, we identified and cloned the PRC2 core component genes in the conifer model species Picea abies, including one Esc/FIE homolog PaFIE, two p55/MSI homologs PaMSI1a and PaMSI1b, two E(z) homologs PaKMT6A2 and PaKMT6A4, a Su(z)12 homolog PaEMF2 and a PaEMF2-like fragment. Phylogenetic and protein domain analyses were conducted. The Esc/FIE homologs were highly conserved in the land plant, except the monocots. The other gymnospermous PRC2 subunits underwent independent evolution with angiospermous species to different extents. The relative transcript levels of these genes were measured in endosperm and zygotic and somatic embryos at different developmental stages. The obtained results proposed the involvement of PaMSI1b and PaKMT6A4 in embryogenesis and PaKMT6A2 and PaEMF2 in the transition from embryos to seedlings. The PaEMF2-like fragment was predominantly expressed in the endosperm but not in the embryo. In addition, immunohistochemistry assay showed that H3K27me3 deposits were generally enriched at meristem regions during seed development in P. abies. CONCLUSIONS: This study reports the first characterization of the PRC2 core component genes in the coniferous species P. abies. Our work may enable a deeper understanding of the cell reprogramming process during seed and embryo development and may guide further research on embryonic potential and development in conifers.

Integrated Lipidomic and Transcriptomic Analysis Reveals Phospholipid Changes in Somatic Embryos of Picea asperata in Response to Partial Desiccation.

Partial desiccation treatment (PDT) is an effective technology for promoting the germination and conversion of conifer somatic embryos (SEs). PDT, as a drought stress, induces intensive physiological responses in phospholipid metabolism, which are not well understood in the conifer SEs. Here, we integrated lipidomics, transcriptomics and proteomics analyses to reveal the molecular basis of lipid remodeling under PDT in Picea asperata SEs. Among the 82 lipid molecular species determined by mass spectrometry, phosphatidic acid (PA) had a significant effect after PDT and was the most critical lipid in the response to PDT. The transcriptomics results showed that multiple transcripts in the glycerolipid and glycerophospholipid metabolism pathways were differentially expressed, and these included five PLDα1 transcripts that catalyze the conversion of phosphatidylcholine (PC) to PA. Furthermore, the enzyme activity of this phospholipase D (PLD) was significantly enhanced in response to PDT, and PDT also significantly increased the protein level of PLDα1 (MA_10436582g0020). In addition, PA is a key factor in gibberellin, abscisic acid and ethylene signal transduction. One GDI1, one DELLA, three ABI1s, two SnRK2s, one CTR and 12 ERFs showed significantly differential expression between SEs before and after PDT in this study. Our data suggest that the observed increases in the PA contents might result from the activation of PLDα by PDT. PA not only affects the physical and chemical properties of the cell membrane but also participates in plant hormone signal transduction. Our work provides novel insight into the molecular mechanism through which PDT promotes the germination of SEs of coniferous tree species and fills the gap in the understanding of the mechanism of somatic embryo lipid remodeling in response to PDT.

Variation, coordination, and trade-offs between needle structures and photosynthetic-related traits across five Picea species: consequences on plant growth.

BACKGROUND: Picea species are distributed and planted world-wide due to their great ecological and economic values. It has been reported that Picea species vary widely in growth traits in a given environment, which reflects genetic and phenotypic differences among species. However, key physiological processes underlying tree growth and the influencing factors on them are still unknown. RESULTS: Here, we examined needle structures, needle chemical components, physiological characteristics and growth traits across five Picea species in a common garden in Tianshui, Gansu province in China: Picea glauca, P. mariana, P. likiangensis, P. koraiensis, and P. crassifolia, among which P. glauca and P. mariana were introduced from North America, P. likiangensis was from Lijiang, Yunan province in China, P. koraiensis was from Yichun, Heilongjiang province in China, and P. crassifolia was native to the experimental site. It was found that nearly all traits varied significantly among species. Tissue-level anatomical characteristics and leaf mass per area (LMA) were affected by needle size, but the variations of them were not associated with the variations in photosynthetic and biochemical capacity among species. Variations in area-based maximum photosynthesis (Pnmax) were affected by stomatal conductance (gs), mesophyll conductance (gm) and biochemical parameters including maximum carboxylation rate (Vcmax), and maximum electron transport rate (Jmax). The fraction of N allocated to different photosynthetic apparatus displayed contrasting values among species, which contributed to the species variations in photosynthetic nitrogen use efficiency (PNUE) and Pnmax. Additionally, all growth traits were positively correlated with Pnmax and PNUE. CONCLUSION: Needle structures are less important than needle biochemical parameters in determining the variations in photosynthetic capacity across the five Picea species. Pnmax and PNUE are closedly associated with the fraction of N allocated to photosynthetic apparatus (Pphoto) compared with leaf N content per area (Narea). The tremendous growth differences among the five Picea species were substantially related to the interspecies variation in Pnmax and PNUE.

Cryo-Treatment Enhances the Embryogenicity of Mature Somatic Embryos via the lncRNA-miRNA-mRNA Network in White Spruce.

In conifers, somatic embryogenesis is uniquely initiated from immature embryos in a narrow time window, which is considerably hindered by the difficulty to induce embryogenic tissue (ET) from other tissues, including mature somatic embryos. In this study, the embryogenic ability of newly induced ET and DNA methylation levels was detected, and whole-transcriptome sequencing analyses were carried out. The results showed that ultra-low temperature treatment significantly enhanced ET induction from mature somatic embryos, with the induction rate from 0.4% to 15.5%, but the underlying mechanisms remain unclear. The newly induced ET showed higher capability in generating mature embryos than the original ET. DNA methylation levels fluctuated during the ET induction process. Here, WGCNA analysis revealed that OPT4, TIP1-1, Chi I, GASA5, GST, LAX3, WRKY7, MYBS3, LRR-RLK, PBL7, and WIN1 genes are involved in stress response and auxin signal transduction. Through co-expression analysis, lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might bind to pre-novel_miR_339 to promote the expression of WRKY7 genes for stress response; LAX3 could be protected by lncRNAs MSTRG.1070680.1 and MSTRG.33602.1 via serving as sponges for novel_miR_495 to initiate auxin signal transduction; lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might serve as sponges for novel_miR_527 to enhance the expression of Chi I for early somatic embryo development. This study provides new insight into the area of stress-enhanced early somatic embryogenesis in conifers, which is also attributable to practical applications.

Transcriptomic and physiological analysis identifies a gene network module highly associated with brassinosteroid regulation in hybrid sweetgum tissues differing in the capability of somatic embryogenesis.

Somatic embryogenesis is a preferred method for large-scale production of forest trees due to its high propagation efficiency. In this study, hybrid sweetgum leaves with phase changes from mature to embryogenic state were selected as experimental material to study somatic embryo initiation. Embryogenicity ranged from high to low, i.e. from 45%, 25%, and 12.5% to 0, with the samples of embryogenic callus (EC), whiten leaf edge (WLI), whiten leaf (WLII), and green leaf (GL) respectively. High correlations existed between embryogenicity and endogenous brassinosteroids (BRs) (r = 0.95, p < 0.05). Similarly, concentrations of endogenous BRs of the sample set correlated positively (r = 0.93, 0.99, 0.87, 0.99, 0.96 respectively, P < 0.05) to expression of somatic embryo (SE)-related genes, i.e. BBM, LEC2, ABI3, PLT2, and WOX2. Hierarchical cluster and weighted gene coexpression network analysis identified modules of coexpressed genes and network in 4820 differentially expressed genes (DEGs) from All-BR-Regulated Genes (ABRG). Moreover, exogenously-supplemented epiBR, together with 2,4-D and 6-BA, increased embryogenicity of GL-sourced callus, and expression of SE- and auxin-related genes, while brassinazole (BRZ), a BR biosynthesis inhibitor, reduced embryogenicity. Evidences obtained in this study revealed that BRs involved in phase change of leaf explants and may function in regulating gene expression and enhancing auxin effects. This study successfully established protocols for inducing somatic embryogenesis from leaf explants in hybrid sweetgum, which could facilitate the propagation process greatly, and provide theoretical basis for manipulating SE competence of explants in ornamental woody plants.

Global Transcriptome and Coexpression Network Analyses Reveal New Insights Into Somatic Embryogenesis in Hybrid Sweetgum (Liquidambar styraciflua × Liquidambar formosana).

Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Despite recent scientific headway in deciphering the difficulties of somatic embryogenesis, the overall picture of key genes, pathways, and co-expression networks regulating SE is still fragmented. Therefore, deciphering the molecular basis of somatic embryogenesis of hybrid sweetgum remains pertinent. In the present study, we analyzed the transcriptome profiles and gene expression regulation changes via RNA sequencing from three distinct developmental stages of hybrid sweetgum: non-embryogenic callus (NEC), embryogenic callus (EC), and redifferentiation. Comparative transcriptome analysis showed that 19,957 genes were differentially expressed in ten pairwise comparisons of SE. Among these, plant hormone signaling-related genes, especially the auxin and cytokinin signaling components, were significantly enriched in NEC and EC early. The K-means method was used to identify multiple transcription factors, including HB-WOX, B3-ARF, AP2/ERF, and GRFs (growth regulating factors). These transcription factors showed distinct stage- or tissue-specific expression patterns mirroring each of the 12 superclusters to which they belonged. For example, the WOX transcription factor family was expressed only at NEC and EC stages, ARF transcription factor was expressed in EC early, and GRFs was expressed in late SE. It was noteworthy that the AP2/ERF transcription factor family was expressed during the whole SE process, but almost not in roots, stems and leaves. A weighted gene co-expression network analysis (WGCNA) was used in conjunction with the gene expression profiles to recognize the genes and modules that may associate with specific tissues and stages. We constructed co-expression networks and revealed 22 gene modules. Four of these modules with properties relating to embryonic potential, early somatic embryogenesis, and somatic embryo development, as well as some hub genes, were identified for further functional studied. Through a combination analysis of WGCNA and K-means, SE-related genes including AUX22, ABI3, ARF3, ARF5, AIL1, AIL5, AGL15, WOX11, WOX9, IAA29, BBM1, MYB36, LEA6, SMR4 and others were obtained, indicating that these genes play an important role in the processes underlying the progression from EC to somatic embryos (SEs) morphogenesis. The transcriptome information provided here will form the foundation for future research on genetic transformation and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; Key genes obtained from coexpression network analysis at each critical stage of somatic embryo can be considered as potential candidate genes to verify these networks.

Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca.

Conifers are the world's major source of timber and pulpwood and have great economic and ecological value. Currently, little research on the application of CRISPR/Cas9, the commonly used genome-editing tool in angiosperms, has been reported in coniferous species. An efficient CRISPR/Cas9 system based on somatic embryogenesis (SEis) suitable for conifers could benefit both fundamental and applied research in these species. In this study, the SpCas9 gene was optimized based on codon bias in white spruce, and a spruce U6 promoter was cloned and function-validated for use in a conifer specific CRISPR/Cas9 toolbox, i.e., PgCas9/PaU6. With this toolbox, a genome-editing vector was constructed to target the DXS1 gene of white spruce. By Agrobacterium-mediated transformation, the genome-editing vector was then transferred into embryogenic tissue of white spruce. Three resistant embryogenic tissues were obtained and used for regenerating plants via SEis. Albino somatic embryo (SE) plants with mutations in DXS1 were obtained in all of the three events, and the ratios of the homozygous and biallelic mutants in the 18 albino mutants detected were 22.2% in both cases. Green plants with mutations in DXS1 were also produced, and the ratios of the DXS1 mutants to the total green plants were 7.9, 28, and 13.5%, respectively, among the three events. Since 22.7% of the total 44 mutants were edited at both of the target sites 1 and 2, the CRISPR/Cas9 toolbox in this research could be used for multi-sites genome editing. More than 2,000 SE plants were regenerated in vitro after genome editing, and part of them showed differences in plant development. Both chimerism and mosaicism were found in the SE plants of white spruce after genome editing with the CRISPR/Cas9 toolbox. The conifer-specific CRISPR/Cas9 system developed in this research could be valuable in gene function research and trait improvement.

Transcriptomic, Metabolomic, and Physiological Analyses Reveal That the Culture Temperatures Modulate the Cryotolerance and Embryogenicity of Developing Somatic Embryos in Picea glauca.

Cryopreservation is one of the key technologies for the mass propagation of conifers via somatic embryogenesis. Cryotolerance and embryogenecity of conifer somatic embryos (SEs) could be affected by different temperature treatments, for which the underlying mechanisms were unknown. In this study, the developing SEs of Picea glauca obtained their cryotolerance with a survival rate of 100% when cultured on maturation medium at either 23°C for 4 weeks or 4°C for 10 weeks. However, only the embryos that underwent 4°C acclimation remained high embryogenicity, i.e., 91.7% based on cryovials or 29.3% on the plant tissue. Analysis of differentially expressed genes (DEGs) revealed that both 23 and 4°C treatments led to drastic changes in the gene expression, i.e., 21,621 and 14,906 genes, respectively, and the general increase in many oligosaccharides and flavonoids, in addition to the content change of proline (1.9- and 2.3-fold at 23 or 4°C) and gallic acid (6,963- and 22,053-fold). There were 249 significantly different metabolites between the samples of 23 and 4°C treatments and the changing trend of the sorbitol, fatty acids, and monosaccharides differed between these samples. During 4°C-acclimation, the metabolites of the arginine biosynthesis pathway increased between 2.4- and 8.1-fold, and the expression of antioxidant genes was up-regulated significantly. At 4°C, the up-regulated genes were for germ-like proteins, instead of seed storage proteins at 23°C. Concentrations of abscisic acid and jasmonic acid increased up to 2- and 1.5-fold, respectively, in the cold-acclimated embryos. After 10 weeks at 4°C, the embryos stayed at pre-cotyledonary stage with 17.1% less DNA methylation and fewer storage substances than those at 23°C for 4 weeks, which developed cotyledons. This research provides new insights into mechanisms underlying the response of SEs to different culture temperatures and benefits method development for germplasm conservation in conifers.

Multi-omics sequencing provides insight into floral transition in Catalpa bungei. C.A. Mey.

BACKGROUND: Floral transition plays an important role in development, and proper time is necessary to improve the value of valuable ornamental trees. The molecular mechanisms of floral transition remain unknown in perennial woody plants. "Bairihua" is a type of C. bungei that can undergo floral transition in the first planting year. RESULTS: Here, we combined short-read next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to provide a more complete view of transcriptome regulation during floral transition in C. bungei. The circadian rhythm-plant pathway may be the critical pathway during floral transition in early flowering (EF) C. bungei, according to horizontal and vertical analysis in EF and normal flowering (NF) C. bungei. SBP and MIKC-MADS-box were seemingly involved in EF during floral transition. A total of 61 hub genes were associated with floral transition in the MEturquoise model with Weighted Gene Co-expression Network Analysis (WGCNA). The results reveal that ten hub genes had a close connection with the GASA homologue gene (Cbu.gene.18280), and the ten co-expressed genes belong to five flowering-related pathways. Furthermore, our study provides new insights into the complexity and regulation of alternative splicing (AS). The ratio or number of isoforms of some floral transition-related genes is different in different periods or in different sub-genomes. CONCLUSIONS: Our results will be a useful reference for the study of floral transition in other perennial woody plants. Further molecular investigations are needed to verify our sequencing data.

Potential function of CbuSPL and gene encoding its interacting protein during flowering in Catalpa bungei.

BACKGROUND: "Bairihua", a variety of the Catalpa bungei, has a large amount of flowers and a long flowering period which make it an excellent material for flowering researches in trees. SPL is one of the hub genes that regulate both flowering transition and development. RESULTS: SPL homologues CbuSPL9 was cloned using degenerate primers with RACE. Expression studies during flowering transition in "Bairihua" and ectopic expression in Arabidopsis showed that CbuSPL9 was functional similarly with its Arabidopsis homologues. In the next step, we used Y2H to identify the proteins that could interact with CbuSPL9. HMGA, an architectural transcriptional factor, was identified and cloned for further research. BiFC and BLI showed that CbuSPL9 could form a heterodimer with CbuHMGA in the nucleus. The expression analysis showed that CbuHMGA had a similar expression trend to that of CbuSPL9 during flowering in "Bairihua". Intriguingly, ectopic expression of CbuHMGA in Arabidopsis would lead to aberrant flowers, but did not effect flowering time. CONCLUSIONS: Our results implied a novel pathway that CbuSPL9 regulated flowering development, but not flowering transition, with the participation of CbuHMGA. Further investments need to be done to verify the details of this pathway.

Transcriptomics and Proteomics Reveal the Cellulose and Pectin Metabolic Processes in the Tension Wood (Non-G-Layer) of Catalpa bungei.

: Catalpa bungei is an economically important tree with high-quality wood and highly valuable to the study of wood formation. In this work, the xylem microstructure of C. bungei tension wood (TW) was observed, and we performed transcriptomics, proteomics and Raman spectroscopy of TW, opposite wood (OW) and normal wood (NW). The results showed that there was no obvious gelatinous layer (G-layer) in the TW of C. bungei and that the secondary wall deposition in the TW was reduced compared with that in the OW and NW. We found that most of the differentially expressed mRNAs and proteins were involved in carbohydrate polysaccharide synthesis. Raman spectroscopy results indicated that the cellulose and pectin content and pectin methylation in the TW were lower than those in the OW and NW, and many genes and proteins involved in the metabolic pathways of cellulose and pectin, such as galacturonosyltransferase (GAUT), polygalacturonase (PG), endoglucanase (CLE) and ß-glucosidase (BGLU) genes, were significantly upregulated in TW. In addition, we found that the MYB2 transcription factor may regulate the pectin degradation genes PG1 and PG3, and ARF, ERF, SBP and MYB1 may be the key transcription factors regulating the synthesis and decomposition of cellulose. In contrast to previous studies on TW with a G-layer, our results revealed a change in metabolism in TW without a G-layer, and we inferred that the change in the pectin type, esterification and cellulose characteristics in the TW of C. bungei may contribute to high tensile stress. These results will enrich the understanding of the mechanism of TW formation.

PICEAdatabase: a web database for Picea omics and phenotypic information.

Picea belongs to the Pinaceae family and is a famous commercial tree species because of its straight trunk and excellent timber traits. Recently, omics have been widely used for fundamental and mechanism studies on Picea plants. To improve the accessibility to omics and phenotypic data and facilitate further studies, we compiled the sequences of 2 chloroplast genomes (Picea crassifolia and Picea asperata) and 32 complete omics data sets, including 20 transcriptomes, 4 proteomes, 2 degradomes and 6 microRNAs from P. crassifolia, P. asperata, Picea balfouriana and Picea abies tissues under different treatments, in PICEAdatabase. In addition, phenotypic data on plant growth and wood property traits were collected from two field trials of P. crassifolia. PICEAdatabase also includes useful analysis tools, such as BLAST, DESeq2 and JBrowse, to assist with analyses.

Quantitative Phosphoproteomic and Physiological Analyses Provide Insights into the Formation of the Variegated Leaf in Catalpa fargesii.

Variegated plants are valuable materials for investigating leaf color regulated mechanisms. To unveil the role of posttranslational modification in the variegated phenotype, we conducted global quantitative phosphoproteomic analysis on different leaf color sectors of Maiyuanjinqiu and the corresponding of Catalpa fargesii using Ti4+-IMAC phosphopeptide enrichment. A total of 3778 phosphorylated sites assigned to 1646 phosphoproteins were identified, and 3221 in 1434 proteins were quantified. Differential phosphoproteins (above 1.5 or below 1/1.5) in various leaf color sectors were selected for functional enrichment analyses. Gene ontology (GO) enrichment revealed that processes of photosynthesis, regulation of the generation of precursor metabolites, response to stress, homeostasis, amino acid metabolism, transport-related processes, and most of the energy metabolisms might contribute to leaf color. KEGG pathway enrichment analysis was performed based on differential phosphoproteins (DPs) in different organelles. The result showed that most enriched pathways were located in the chloroplasts and cytosol. The phosphorylation levels of glycometabolism enzymes might greatly affect leaf variegation. Measurements of fluorescence parameters and enzyme activities confirmed that protein phosphorylation could affect plant physiology by regulating enzyme activity. These results provide new clues for further study the formation mechanisms of naturally variegated phenotype.

Single-nucleotide polymorphisms(SNPs) in a sucrose synthase gene are associated with wood properties in Catalpa fargesii bur.

BACKGROUND: Association study is a powerful means for identifying molecular markers, such as single-nucleotide polymorphisms (SNPs) associated with important traits in forest trees. Catalpa fargesii Bur is a valuable commercial tree in China and identifying SNPs that associate with wood property would make a foundation of the marker-assisted breeding in the future. However, related work has not been reported yet. RESULTS: We cloned a 2887 bp long sucrose synthase (SUS) gene from the genome of C. fargesii, which is a key enzyme in sucrose metabolism and also associated to wood formation in trees, coding 806 amino acids that expressed mainly in young branches, xylem, and leaves according to real-time quantitative PCR. Then we identified allelic variations of CfSUS associated with nine wood quality associated traits in Catalpa fargesii Bur. Totally, 135 SNPs were identified through cloning and sequencing the CfSUS locus from a mapping population (including 93 unrelated individuals) and 47 of which were genotyped as common SNPs (minor allele frequency > 5%) in the association population that comprised of 125 unrelated individuals collected from main distribution area. Nucleotide diversity and linkage disequilibrium (LD) analysis showed CfSUS has a relative low SNP diversity (πT = 0.0034) and low LD (r2 dropped below 0.1 within 1600 bp). Using the association analysis, we found 11 common SNPs and 14 haplotypes were significantly associated with the traits (false discovery rate, Q<0.1), explaining 3.21-12.41% of the phenotypic variance. These results provide molecular markers above associated with wood basic density, pore rate, and six other traits of wood, which have potential applications in breeding of Catalpa fargesii Bur. CONCLUSION: We first cloned a SUS gene in C. fargesii, then identified several SNPs and haplotypes that associated with wood properties within this gene, suggesting CfSUS participates in the wood formation of C. fargesii. Moreover, molecular markers we identified in this study may be applied into marker-assisted breeding of C. fargesii in the future.

Identification of novel miRNAs and miRNA expression profiling in embryogenic tissues of Picea balfouriana treated by 6-benzylaminopurine.

Here, we compared miRNA expression profiles in embryonic cell cultures of the conifer Picea balfouriana following application of the synthetic cytokinin 6-benzylaminopurine (6-BAP). We used next-generation sequencing to analyze three libraries of small RNAs from the treated embryogenic cell cultures and generated 24,000,000 raw reads from each of the libraries. Over 70 differentially regulated micro RNA (miRNA) families (≥2 fold change in expression) were identified between pairs of treatments. A quantitative analysis showed that miR3633 and miR1026 were upregulated in tissues with the highest embryogenic ability. These two miRNAs were predicted to target genes encoding receptor-like protein kinase and GAMYB transcription factors, respectively. In one library, miR1160, miR5638, miR1315, and miR5225 were downregulated. These four miRNAs were predicted to target genes encoding APETALA2, calmodulin-binding protein, and calcium-dependent protein kinase transcription factors. The expression patterns of the miRNAs and their targets were negatively correlated. Approximately 181 potentially novel P. balfouriana miRNAs were predicted from the three libraries, and seven were validated during the quantitative analysis. This study is the first report of differential miRNA regulation in tissues treated with 6-BAP during somatic embryogenesis. The differentially expressed miRNAs will be of value for investigating the mechanisms of embryogenic processes that are responsive to 6-BAP in P. balfouriana.

Proteomic analysis of stress-related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation.

Partial desiccation treatment (PDT) stimulates germination and enhances the conversion of conifer somatic embryos. To better understand the mechanisms underlying the responses of somatic embryos to PDT, we used proteomic and physiological analyses to investigate these responses during PDT in Picea asperata. Comparative proteomic analysis revealed that, during PDT, stress-related proteins were mainly involved in osmosis, endogenous hormones, antioxidative proteins, molecular chaperones and defence-related proteins. Compared with those in cotyledonary embryos before PDT, these stress-related proteins remained at high levels on days 7 (D7) and 14 (D14) of PDT. The proteins that differentially accumulated in the somatic embryos on D7 were mapped to stress and/or stimuli. They may also be involved in the glyoxylate cycle and the chitin metabolic process. The most significant difference in the differentially accumulated proteins occurred in the metabolic pathways of photosynthesis on D14. Furthermore, in accordance with the changes in stress-related proteins, analyses of changes in water content, abscisic acid, indoleacetic acid and H2 O2 levels in the embryos indicated that PDT is involved in water-deficit tolerance and affects endogenous hormones. Our results provide insight into the mechanisms responsible for the transition from morphologically mature to physiologically mature somatic embryos during the PDT process in P. asperata.

Global Lysine Acetylome Analysis of Desiccated Somatic Embryos of Picea asperata.

Partial desiccation treatment (PDT) promotes the germination capacity of conifer somatic embryos. Lysine acetylation (LysAc) is a dynamic and reversible post-translational modification that plays a key role in many biological processes including metabolic pathways and stress response. To investigate the functional impact of LysAc in the response of Picea asperata somatic embryos to PDT, we performed a global lysine acetylome analysis. Here, combining antibody-based affinity enrichment and high-resolution mass spectrometry, we identified and validated 1079 acetylation sites in 556 acetylated proteins from P. asperata somatic embryos during PDT. These data represent a novel large-scale dataset of lysine-acetylated proteins from the conifer family. Intensive bioinformatics analysis of the Gene Ontology of molecular functions demonstrated that lysine-acetylated proteins were mainly associated with binding, catalytic activities, and structural molecular activities. Functional characterization of the acetylated proteins revealed that in the desiccated somatic embryos, LysAc is mainly involved in the response to stress and central metabolism. Accordingly, the majority of these interacting proteins were also highly enriched in ribosome, proteasome, spliceosome, and carbon metabolism clusters. This work provides the most comprehensive profile of LysAc for a coniferous species obtained to date and facilitates the systematic study of the physiological role of LysAc in desiccated somatic embryos of P. asperata.

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii).

BACKGROUND AND AIMS: In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question. METHODS: Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis. KEY RESULTS: Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg(-1) f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g(-1) d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap. CONCLUSIONS: In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.

Isolation and characterization of a novel cDNA encoding methyl jasmonate-responsive transcription factor TcAP2 from Taxus cuspidata.

A novel transcription factor, TcAP2, was isolated from Taxus cuspidata by yeast one-hybrid strategy. This factor interacts with jasmonate- and elicitor-responsive element. Analysis of the deduced TcAP2 amino acid sequence revealed that TcAP2 contained a conserved AP2/ethylene-responsive element binding protein domain that consisted of 268 amino acids in a potential nuclear localization sequence. The factor of TcAP2 had a high homology, in its AP2 domain, to other AP2 family members. Based on phylogenetic analysis, it was different from other five DRE-binding proteins in their evolutionary relationship. The transcription of TcAP2 gene in yew accumulated primarily in young organs, such as young stems. Quantitative real-time RT-PCR analysis indicated that TcAP2 gene was inducible to express by treatments with methyl jasmonate plus salicylic acid, high salinity, and cold. This gene showed no response to either abscisic acid or drought treatment.