Function of MYB8 in larch under PEG simulated drought stress.

Larch, a prominent afforestation, and timber species in northeastern China, faces growth limitations due to drought. To further investigate the mechanism of larch's drought resistance, we conducted full-length sequencing on embryonic callus subjected to PEG-simulated drought stress. The sequencing results revealed that the differentially expressed genes (DEGs) primarily played roles in cellular activities and cell components, with molecular functions such as binding, catalytic activity, and transport activity. Furthermore, the DEGs showed significant enrichment in pathways related to protein processing, starch and sucrose metabolism, benzose-glucuronic acid interconversion, phenylpropyl biology, flavonoid biosynthesis, as well as nitrogen metabolism and alanine, aspartic acid, and glutamic acid metabolism. Consequently, the transcription factor T_transcript_77027, which is involved in multiple pathways, was selected as a candidate gene for subsequent drought stress resistance tests. Under PEG-simulated drought stress, the LoMYB8 gene was induced and showed significantly upregulated expression compared to the control. Physiological indices demonstrated an improved drought resistance in the transgenic plants. After 48 h of PEG stress, the transcriptome sequencing results of the transiently transformed LoMYB8 plants and control plants exhibited that genes were significantly enriched in biological process, cellular component and molecular function. Function analyses indicated for the enrichment of multiple KEGG pathways, including energy synthesis, metabolic pathways, antioxidant pathways, and other relevant processes. The pathways annotated by the differential metabolites mainly encompassed signal transduction, carbohydrate metabolism, amino acid metabolism, and flavonoid metabolism.

Phylogeny and expression patterns of ERF genes that are potential reproductive inducers in hybrid larch.

BACKGROUND: Larch is an important component of northern forests and a major cultivated tree species in restoration of forest cover using improved seed material. In recent years, the continuous low seed production has severely affected the production of improved variety seedlings and natural regeneration. However, research on the reproductive growth of gymnosperms is extremely scarce. RESULTS: In this study, based on differential transcriptome analysis of two asexual reproductive phases, namely high-yield and low-yield, we further screened 5 ERF family genes that may affect the reproductive development of larch. We analyzed their genetic relationships and predicted their physicochemical properties. The expression patterns of these genes were analyzed in different tissues, developmental stages, hormone treatments, and environmental conditions in hybrid larch. CONCLUSION: The results showed that all 5 genes were induced by low temperature and ABA, and their expression patterns in different tissues suggested a suppressive role in the development of female cones in larch. Among them, LkoERF3-like1 and LkoERF071 may be involved in the flowering age pathway. This study enriches the scarce research on reproductive development in gymnosperms and provides a theoretical basis and research direction for regulating the reproductive development of larch in seed orchards.

Analysis of oxidase activity and transcriptomic changes related to cutting propagation of hybrid larch.

Hybrid larch is the main timber and afforestation tree species in Northeast China. To solve the problem of rooting difficulties in larch cutting propagation, enzyme activity determination and transcriptome sequencing were carried out on the rooting tissues at five timepoints after cutting. peroxidase (POD), indole acetic acid oxidase (IAAO) and polyphenol oxidase (PPO) play important roles in the larch rooting process after cutting. A total of 101.20 Gb of clean data was obtained by transcriptome sequencing, and 43,246 unigenes were obtained after further screening and assembly. According to GO analysis and KEGG enrichment analysis, we think that plant hormones play an important role in the rooting process of larch stem cuttings. in the plant hormone signal transduction pathway, a larch gene c141104.graph_c0 that is homologous to the Arabidopsis AUX1 was found to be significantly up-regulated. We suggest that AUX1 may promote IAA transport in larch, thus affecting adventitious root development. According to the results of POD, PPO IAAO indexes and GO analysis, we think s1 and s2 periods may be important periods in the rooting process of larch stem cuttings, so we built a gene regulatory network, a total of 14genes, including LBD, NAC, AP2/ERF, bHLH and etc., may be important in different stages of cutting propagation. As the rooting rate after cutting inhibits the development of larch clone propagation, identifying the genes that regulate rooting could help us to preliminarily understand the molecular mechanism of adventitious root formation and select a better treatment method for cutting propagation.

Anatomical observation and transcriptome analysis of buds reveal the association between the AP2 gene family and reproductive induction in hybrid larch (Larix kaempferi × Larix olgensis).

Hybrid larch is an excellent afforestation species in northern China. The instability of seed yield is an urgent problem to be solved. The biological characteristics related to seed setting in larch are different from those in angiosperms and other gymnosperms. Studying the developmental mechanism of the larch sporophyll can deepen our understanding of conifer reproductive development and help to ensure an adequate supply of seeds in the seed orchard. The results showed that the formation of microstrobilus primordia in hybrid larch could be observed in anatomical sections collected in the middle of July. The contents of endogenous gibberellin 3 (GA3) and abscisic acid (ABA) were higher and the contents of GA4, GA7, jasmonic acid and salicylic acid were lower in multiseeded larch. Transcriptome analysis showed that transcription factors were significantly enriched in the AP2 family. There were 23 differentially expressed genes in the buds of the multiseeded and less-seeded types, and the expression of most of these genes was higher in the buds than in the needles. We conclude that mid-July is the early stage of reproductive organ development in hybrid larch and is suitable for the study of reproductive development. GA3 and ABA may be helpful for improving seed setting in larch, and 23 AP2/EREBP family genes are involved in the regulation of reproductive development in larch.

Genetic transformation of LoHDZ2 and analysis of its function to enhance stress resistance in Larix olgensis.

To study the function of LoHDZ2 in larch, we first constructed a VB191103-LoHDZ2::GUS overexpression vector. Through Agrobacterium-mediated infection, the expression vector was transferred into a larch embryogenic cell line. A stable resistant cell line was subsequently screened, and mature embryos were induced to grow until they developed into seedlings. Antagonistic cell lines were identified at both the DNA and RNA levels. The transgenic cell lines were then subjected to GUS staining, and transgenic cell lines were ultimately identified and obtained. These transgenic cell lines were sequenced to identify differentially expressed genes, and a cluster analysis was performed. The resistant cell lines were cultured under stress conditions involving 20% PEG6000 and 200 mM NaCl proliferation media (1/10-BM). After the stress treatment, the contents of peroxidase (POD), malondialdehyde (MDA) and superoxide dismutase (SOD) in both wild-type and transgenic cell lines were measured. The results are summarized below: (1) When the specific fragment of the target gene in the genome of the resistant cell line was amplified. At the RNA level, the expression of the fragment in four resistant lines increased. In addition, GUS staining showed a blue reaction, indicating that LoHDZ2 was successfully integrated into the larch embryonic cell lines. (2) To verify the accuracy and reliability of the transcriptome data, 10 differentially expressed genes (5 upregulated and 5 down regulated genes) were subjected to qRT-PCR verification. The results showed that the expression trend of the 10 differentially expressed genes was the same as that revealed by RNA-Seq, indicating that the transcriptome data were reliable. (3) The transcriptome sequencing showed that 176 genes were upregulated and that 140 genes were down regulated. Through GO enrichment analysis and KEGG metabolic pathway analysis, the screened differentially expressed genes were related to biological processes such as larch metabolism and response to stimuli, indicating that these genes may be closely involved in the regulation of the larch response to external stimuli, including heat stress, drought stress, metal ion stress and bacterial infection, and may participate in the growth process. (4) After 20% PEG6000 treatment, the POD enzyme activity of the transgenic cell line was greater than that of the wild-type; this activity could effectively remove the amount of peroxide produced. The MDA content of the transgenic cell lines was lower than that of the wild-type cell lines, and the accumulation degree of harmful substances was low, indicating that the degree of oxidative damage of the transgenic cell lines was lower than that of the wild-type cell lines. The SOD content of the transgenic cell lines was lower than that of the wild-type cell lines, indicating that the drought resistance of the transgenic cell lines was enhanced. After 200 mM NaCl treatment, although the increase in SOD content was not obvious, the same trend was detected, indicating that the resistance of the transgenic cell lines was indeed stronger than that of the wild-type cell lines. According to the results of previous experiments, after this gene was overexpressed in tobacco, the transformed plants showed obvious dwarfing, which may indicate that the stress resistance of the plant was enhanced. In conclusion, a transgenic larch cell line was successfully obtained, and transgenic larch seedlings were successfully induced. LoHDZ2 may participate in the response of plants to the external environment, and may participate in the growth and development of Larix olgensis by affecting plant metabolic pathways.

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.

Diversity in Fruit Morphology and Nutritional Composition of Juglans mandshurica Maxim in Northeast China.

Although Manchurian walnut (Juglans mandshurica Maxim) is widely distributed in northeast China, very few studies had been reported on its diversity among different populations. We surveyed 12 J. mandshurica populations in their native habitats across the northeast region of China and profiled 13 fruit morphological traits. We found a large degree of variations for these traits, especially for fruit weight (coefficient of variation, or CV of 22.00%), nut weight (CV of 19.42%), and kernel weight (CV of 19.89%). Statistical analysis showed that a large portion of the total variation can be attributed to within-population variation (66.64%), followed by random error (20.96%). We also comprehensively quantified the nutritional composition including fatty acids, amino acids, vitamins, and micronutrients. Similar to fruit morphological traits, we found large variation for most kernel components, which mostly can be explained by within-population variation. Further correlation analysis revealed the dependence of some morphological and nutritional traits on key geographical and ecological factors such as latitude, accumulated temperature, and day length. For instance, a significant positive correlation was found between fruit dimensions and equivalent latitude and precipitation, indicating that such factors should be considered for breeding. Taken together, our data provided a rich dataset for characterizing the variation among J. mandshurica populations and a foundation for selective breeding.

Genetic diversity analysis and fingerprint construction of Korean pine (Pinus koraiensis) clonal seed orchard.

Korean pine is a native tree species in Northeast China. In order to meet the needs of germplasm resource evaluation and molecular marker-assisted breeding of Korean pine, we collected Korean pine clones from 7 populations in Northeast China, analyzed the genetic diversity and genetic structure by SSR molecular marker technology and clustered them to revealed the inter- and intrapopulation differentiation characteristics of each clone. The fingerprint profiles of 161 Korean pine clones were also constructed. 77 alleles were detected for 11 markers, and 18 genotypes were identified on average for each marker. The PIC of the different markers ranged from 0.155-0.855, and the combination of PI and PIsibs for the 11 markers was 3.1 × 10-8 and 1.14 × 10-3, respectively. MANOVA showed that genetic variation existed mainly within populations, accounting for 98% of the total variation. The level of genetic differentiation among populations was low, with an average Nm between populations of 11.036. Genetic diversity is lower in the Lushuihe population and higher in the Tieli population. The 161 Korean pine clones were divided into 4 or 7 populations, and the 7 populations were not clearly distinguished from each other, with only the Lushuihe population showing partial differentiation. There is no significant correlation between the genetic distance of Korean pine populations and the geographical distance of their superior tree sources. This result can provide recommendations for future Korean pine breeding programs. The combination of 11 markers could completely distinguish 161 clones and establish the fingerprint. Genetic diversity of Korean pine clones from the 7 populations was abundant, and the genetic distances of individuals and populations were evenly dispersed. The fingerprint map can be used for the identification of Korean pine clones.

Glutathione, carbohydrate and other metabolites of Larix olgensis A. Henry reponse to polyethylene glycol-simulated drought stress.

Drought stress in trees limits their growth, survival, and productivity and it negatively affects the afforestation survival rate. Our study focused on the molecular responses to drought stress in a coniferous species Larix olgensis A. Henry. Drought stress was simulated in one-year-old seedlings using 25% polyethylene glycol 6000. The drought stress response in these seedlings was assessed by analyzing select biochemical parameters, along with gene expression and metabolite profiles. The soluble protein content, peroxidase activity, and malondialdehyde content of L. olgensis were significantly changed during drought stress. Quantitative gene expression analysis identified a total of 8172 differentially expressed genes in seedlings processed after 24 h, 48 h, and 96 h of drought stress treatment. Compared with the gene expression profile of the untreated control, the number of up-regulated genes was higher than that of down-regulated genes, indicating that L. olgensis mainly responded to drought stress through positive regulation. Metabolite analysis of the control and stress-treated samples showed that under drought stress, the increased abundance of linoleic acid was the highest among up-regulated metabolites, which also included some saccharides. A combined analysis of the transcriptome and metabolome revealed that genes dominating the differential expression profile were involved in glutathione metabolism, galactose metabolism, and starch and sucrose metabolism. Moreover, the relative abundance of specific metabolites of these pathways was also altered. Thus, our results indicated that L. olgensis prevented free radical-induced damage through glutathione metabolism and responded to drought through sugar accumulation.

Genetic transformation and growth index determination of the Larix olgensis LoHDZ2 transcription factor gene in tobacco.

Homeodomain-leucine zippers (HD-Zip) are plant-specific transcription factors that participate in different plant development processes and differentially regulate metabolic processes. LoHDZ2 is an HD-ZipII subfamily transcription factor gene that we identified from a transcriptomic analysis of Larix olgensis. To understand its function, we built a LoHDZ2 expression vector and then inserted it into tobacco by genetic transformation. Transgenic plants were identified at the DNA and RNA levels. Phenotypic index analysis of transgenic tobacco showed dwarfed growth with larger leaves and earlier flowering than the wild type. LoHDZ2 was expressed differently after hormone treatment with IAA, MeJA and 2,4-D. The results suggested that LoHDZ2 may respond to hormones and be involved in regulating growth and metabolism. These results helped us better understand the function of LoHDZ2 and provided a candidate gene for Larix olgensis molecular breeding.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9.

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

Stable and Efficient Agrobacterium-Mediated Genetic Transformation of Larch Using Embryogenic Callus.

Larix olgensis or larch is an economically important coniferous tree species with rapid growth in the early stages, strong adaptability, and a short time to harvest. The genetic improvement of larch has garnered considerable attention in recent years for reclaiming timber forests. However, traditional breeding methods are largely ineffective for achieving rapid genetic improvement of L. olgensis. Studies show that the efficiency of plant regeneration can be improved by optimizing somatic embryogenesis. On this basis, we devised a stable, fast and efficient Agrobacterium-mediated genetic transformation method using suspended embryogenic calluses as explants and ß-glucuronidase as the reporter. We evaluated the effects of the Agrobacterium load, co-culture period, and addition of acetosyringone and transformant screening antibiotic on the transformation efficiency. In addition, we tested the pCAMBIA 1300-PtHCA 2-1 promoter-GUS binary expression vector, which contains the GUS gene ORF under the control of Populus trichocarpa high cambial activity PtHCA 2-1 promoter, and observed the tissue-specific expression of the GUS gene in the somatic embryos of transgenic larch. This novel technique can not only accelerate the generation of superior transgenic strains of L. olgensis but also aid in future gene functional studies.

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.

Identification of miRNAs and their target genes in Larix olgensis and verified of differential expression miRNAs.

BACKGROUND: MiRNAs (microRNA) are 18-24 nt endogenous noncoding RNAs that regulate gene expression at the post-transcriptional level, including tissue-specific, developmental timing and evolutionary conservation gene expression. RESULTS: This study used high-throughput sequencing technology for the first time in Larix olgensis, predicted 78 miRNAs, including 12,229,003 reads sRNA, screened differentially expressed miRNAs. Predicting target genes was helpful for understanding the miRNA regulation function and obtained 333 corresponding target genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were analysed, mostly including nucleic acid binding, plant hormone signal transduction, pantothenate and CoA biosynthesis, and cellulose synthase. This study will lay the foundation for clarifying the complex miRNA-mediated regulatory network for growth and development. In view of this, spatio-temporal expression of miR396, miR950, miR164, miR166 and miR160 were analysed in Larix olgensis during the growth stages of not lignified, beginning of lignification, and completely lignified in different tissues (root, stem, and leaf) by quantitative real-time PCR (qRT-PCR). There were differences in the expression of miRNAs in roots, stems and leaves in the same growth period. At 60 days, miR160, miR166 and miR396-2 exhibited the highest expression in leaves. At 120 days, most miRNAs in roots and stems decreased significantly. At 180 days, miRNAs were abundantly expressed in roots and stems. Meanwhile, analysis of the expression of miRNAs in leaves revealed that miR396-2 was reduced as time went on, whereas other miRNAs increased initially and then decreased. On the other hand, in the stems, miR166-1 was increase, whereas other miRNAs, especially miR160, miR164, miR396 and miR950-1, first decreased and then increased. Similarly, in the roots, miR950-2 first decreased and then increased, whereas other miRNAs exhibited a trend of continuous increase. CONCLUSIONS: The present investigation included rapid isolation and identification of miRNAs in Larix olgensis through construction of a sRNA library using Solexa and predicted 78 novel miRNAs, which showed differential expression levels in different tissues and stages. These results provided a theoretical basis for further revealing the genetic regulation mechanism of miRNA in the growth and development of conifers and the verification of function in target genes.

Complete plastome sequences of Picea asperata and P. crassifolia and comparative analyses with P. abies and P. morrisonicola.

Picea asperata and P. crassifolia have sympatric ranges and are closely related, but the differences between these species at the plastome level are unknown. To better understand the patterns of variation among Picea plastomes, the complete plastomes of P. asperata and P. crassifolia were sequenced. Then, the plastomes were compared with the complete plastomes of P. abies and P. morrisonicola, which are closely and distantly related to the focal species, respectively. We also used these sequences to construct phylogenetic trees to determine the relationships among and between the four species as well as additional taxa from Pinaceae and other gymnosperms. Analysis of our sequencing data allowed us to identify 438 single nucleotide polymorphism (SNPs) point mutation events, 95 indel events, four inversion events, and seven highly variable regions, including six gene spacer regions (psbJ-petA, trnT-psaM, trnS-trnD, trnL-rps4, psaC-ccsA, and rps7-trnL) and one gene (ycf1). The highly variable regions are appropriate targets for future use in the phylogenetic reconstructions of closely related, sympatric species of Picea as well as Pinaceae in general.

ConTEdb: a comprehensive database of transposable elements in conifers.

Conifers are the largest and most ubiquitous group of gymnosperms and have significant ecological significance and economic importance. However, the huge and complex genomes have hindered the sequencing and mining of conifer genomes. In this study, we identified 413 423 transposable elements (TEs) from Picea abies, Picea glauca and Pinus taeda using a combination of multiple approaches and classified them into 11 133 families. A comprehensive web-based database, ConTEdb, was constructed and served for researchers. ConTEdb enables users to browse, retrieve and download the TE sequences from the database. Several analysis tools are integrated into ConTEdb to help users mine the TE data easily and effectively. In summary, ConTEdb provides a platform to study TE biology and functional genomics in conifers.

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.