Transcriptome-wide isolation and expression of NF-Y gene family in male cone development and hormonal treatment of Pinus tabuliformis.

It is known that nuclear factor Y (NF-Y) transcription factors play an important role in flowering time regulation and hormone response (ABA, GA) in angiosperms, but, little known in conifers. Moreover, the NF-Y gene family has not been comprehensively reported in conifers. Here, we identified 9 NF-YA, 9 NF-YB and 10 NF-YC genes in Pinus tabuliformis using Arabidopsis NF-Y protein sequences as queries. Additionally, by comparing conserved regions and phylogenetic relationships of the PtNF-Ys, we found that NF-Ys were both conserved and altered during evolution. PtTFL2, PtCO, PtNF-YC1 and PtNF-YC4 were exploited by expression profile in male cone development and correlation analysis. Furthermore, NF-YC1/4 and DPL (DELLA protein of P. tabuliformis) were interacted by yeast two-hybrid and BiFC assays, which suggested that NF-YC1/4 may be involved in gibberellins signaling pathway. Moreover, the multiple types of phytohormones-responsive cis-elements (ABA, JA, IAA, SA) have been found, and gene expression profile analysis showed that many NF-Y genes responded positively to SA and as opposed to IAA and JA, revealing the potential role of NF-Ys in conifers resistance. In summary, this study provided the basis for further investigation of the function of NF-Y genes in conifers.

Genome-wide identification of the Pinus tabuliformis CONSTANS-like gene family and their potential roles in reproductive cone development.

The CONSTANS-like (COL) genes, as a core transcription factor in the photoperiod regulation pathway, play a key role in plant reproduction development. However, their molecular characterization has rarely been studied in Pinus tabuliformis. Here, 10 PtCOL genes were identified in the P. tabuliformis genome and multiple sequence alignments have indicated that the PtCOL proteins contained highly conserved B-BOX1 and CCT domains. Sequence similarity analysis showed that PtCOL1 and PtCOL3 had the higher similarity with Norway spruce COLs (PaCOL2 and PaCOL1) and Arabidopsis COLs (AtCOL3, 4 and 5), respectively. Phylogeny and gene structure analyses revealed that PtCOLs were divided into three subgroups, each with identical or similar distributions of exons, introns, and motifs. Moreover, 10 PtCOLs were distributed on 6 chromosomes and PtCOL9 has syntenic gene pairs in both Ginkgo biloba and Sequoiadendron giganteum. Interestingly, in transcriptome profiles, most PtCOLs exhibited a diurnal oscillation pattern under both long (LD) and short (SD) day conditions. Additionally, PtCOLs were highly expressed in needles and female cones, and showed different spatial expression patterns. Among the ten PtCOLs, PtCOL1/3 heterologous overexpression Arabidopsis displayed a delayed-flowering phenotype under SD, indicating that they are likely to play a crucial role in the reproductive development. Additionally, PtCOL1 and PtCOL3 were not only capable of interacting with each other, but they were each capable of interacting with themselves. Furthermore, PtCOL1 and PtCOL3 were also involved in the MADS-box protein-protein interaction (PPI) network in P. tabuliformis cone development. Direct interactions of PtDAL11 with PtCOL1/3 impeded PtCOL1/3 translocation into the nucleus. In summary, this study provided comprehensive understanding for the functions of the PtCOL gene family and revealed their biological roles in the photoperiod-dependent P. tabuliformis cone development.

Dark septate endophyte Anteaglonium sp. T010 promotes biomass accumulation in poplar by regulating sucrose metabolism and hormones.

Plant biomass is a highly promising renewable feedstock for the production of biofuels, chemicals and materials. Enhancing the content of plant biomass through endophyte symbiosis can effectively reduce economic and technological barriers in industrial production. In this study, we found that symbiosis with the dark septate endophyte (DSE) Anteaglonium sp. T010 significantly promoted the growth of poplar trees and increased plant biomass, including cellulose, lignin and starch. To further investigate whether plant biomass was related to sucrose metabolism, we analyzed the levels of relevant sugars and enzyme activities. During the symbiosis of Anteaglonium sp. T010, sucrose, fructose and glucose levels in the stem of poplar decreased, while the content of intermediates such as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and UDP-glucose (UDPG), and the activity of enzymes related to sucrose metabolism, including sucrose synthase (SUSY), cell wall invertase (CWINV), fructokinase (FRK) and hexokinase, increased. In addition, the contents of glucose, fructose, starch, and their intermediates G6P, F6P and UDPG, as well as the enzyme activities of SUSY, CWINV, neutral invertase and FRK in roots were increased, which ultimately led to the increase of root biomass. Besides that, during the symbiotic process of Anteaglonium sp. T010, there were significant changes in the expression levels of root-related hormones, which may promote changes in sucrose metabolism and consequently increase the plant biomass. Therefore, this study suggested that DSE fungi can increase the plant biomass synthesis capacity by regulating the carbohydrate allocation and sink strength in poplar.

Involvement of PtCOL5-PtNF-YC4 in reproductive cone development and gibberellin signaling in Chinese pine.

It is well documented that the CO/NF-YB/NF-YC trimer (NF-Y-CO) binds and regulates the FT promoter. However, the FT/TFL1-like (FLOWERING LOCUS T/TERMINALFLOWER1-like) genes in gymnosperms are all flowering suppressors, and the regulation model of NF-Y in gymnosperms is different from that in angiosperms. Here, using Chinese pine (Pinus tabuliformis), we identified a CONSTANS-LIKE gene, PtCOL5, the expression of which was strongly induced during cones development and it functioned as a repressor of flowering. PtNF-YC4, which interacted with PtCOL5, was highly correlated with PtCOL5 during growth and development, has been demonstrated. Moreover, PtNF-YC4 and PtCOL5 can bind to PtTFL2 promoter, and their interaction can enhance PtTFL2 expression. Interestingly, we found PtNF-YC4 and PtCOL5 were involved in gibberellin signaling and their interaction was inhibited by PtDELLA protein, thus affecting PtTFL2 expression. Collectively, PtCOL5-PtNF-YC4 was involved in reproductive cone development and gibberellin signaling in Chinese pine. Our findings uncovered reproductive cone development and signal transduction mechanism of COL-NF-Y in gymnosperms.

Complex Physical Structure of Complete Mitochondrial Genome of Quercus acutissima (Fagaceae): A Significant Energy Plant.

Quercus acutissima Carruth. is a Chinese important energy plant with high ecological and economic values. While the species chloroplast genome has been reported, its mitochondrial genome (mitogenome) is still unexplored. Here, we assembled and annotated the Q. acutissima mitogenome, and we compared its characteristic differences with several closely related species. The Q. acutissima mitogenome's main structure is branched with three distinguished contigs (linear molecule 1, circular molecule 2, and circular molecule 3) with 448,982 bp total length and 45.72% GC content. The mitogenome contained 51 genes, including 32 protein-coding, 16 tRNA and 3 rRNA genes. We examined codon usage, repeated sequences, genome recombination, chloroplast to mitochondrion DNA transformation, RNA editing, and synteny in the Q. acutissima mitogenome. Phylogenetic trees based on 29 species mitogenomes clarified the species classification. Our results provided comprehensive information of Q. acutissima mitogenome, and they are expected to provide valuable information for Fagaceae evolutionary biology and to promote the species germplasm utilization.

An efficient system for Agrobacterium-mediated transient transformation in Pinus tabuliformis.

BACKGROUND: Functional genomic studies using genetics approaches of conifers are hampered by the complex and enormous genome, long vegetative growth period, and exertion in genetic transformation. Thus, the research carried out on gene function in Pinus tabuliformis is typically performed by heterologous expression based on the model plant Arabidopsis. However, due to the evolutionary and vast diversification from non-flowering (gymnosperms) to flowering (angiosperms) plants, several key differences may alter the underlying genetic concerns and the analysis of variants. Therefore, it is essential to develop an efficient genetic transformation and gene function identification protocol for P. tabuliformis. RESULTS: In the present study we established a highly efficient transgene Agrobacterium-mediated transient expression system for P. tabuliformis. Using a ß-glucuronidase gene (GUS) as a reporter gene expression, the highest transformation efficiency (70.1%) was obtained by co-cultivation with Agrobacterium strain GV3101 at an optical density at 600 nm of 0.8, with 150 µM acetosyringone for 30 min followed by 3 days in the dark at 23 ± 1 °C. This protocol would be applied to other conifers; GUS staining was observed 24 h post-infection. CONCLUSIONS: We report a simple, fast, and resilient system for transient Agrobacterium-mediated transformation high-level expression of target genes in P. tabuliformis, which will also improve transformation efficiency in other conifer species.

Proteomic analysis of Potentilla fruticosa L. leaves by iTRAQ reveals responses to heat stress.

High temperature is an important environmental factor that affects plant growth and crop yield. Potentilla fruticosa L. has a developed root system and characteristics of resistance to several stresses (e.g., high temperature, cold, drought) that are shared by native shrubs in the north and west of China. To investigate thermotolerance mechanisms in P. fruticosa, 3-year-old plants were subjected to a high temperature of 42°C for 1, 2, and 3 days respectively before analysis. Then, we studied changes in cell ultrastructure using electron microscopy and investigated physiological changes in the leaves of P. fruticosa. Additionally, we used isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to study proteomic changes in P. fruticosa leaves after 3 d of 42°C heat stress. we found that the cell membrane and structure of chloroplasts, especially the thylakoids in P. fruticosa leaves, was destroyed by a high temperature stress, which might affect the photosynthesis in this species. We identified 35 up-regulated and 23 down-regulated proteins after the heat treatment. Gene Ontology (GO) analysis indicated that these 58 differentially abundant proteins were involved mainly in protein synthesis, protein folding and degradation, abiotic stress defense, photosynthesis, RNA process, signal transduction, and other functions. The 58 proteins fell into different categories based on their subcellular localization mainly in the chloroplast envelope, cytoplasm, nucleus, cytosol, chloroplast, mitochondrion and cell membrane. Five proteins were selected for analysis at the mRNA level; this analysis showed that gene transcription levels were not completely consistent with protein abundance. These results provide valuable information for Potentilla thermotolerance breeding.

Transcriptome Analysis of Syringa oblata Lindl. Inflorescence Identifies Genes Associated with Pigment Biosynthesis and Scent Metabolism.

Syringa oblata Lindl. is a woody ornamental plant with high economic value and characteristics that include early flowering, multiple flower colors, and strong fragrance. Despite a long history of cultivation, the genetics and molecular biology of S. oblata are poorly understood. Transcriptome and expression profiling data are needed to identify genes and to better understand the biological mechanisms of floral pigments and scents in this species. Nine cDNA libraries were obtained from three replicates of three developmental stages: inflorescence with enlarged flower buds not protruded, inflorescence with corolla lobes not displayed, and inflorescence with flowers fully opened and emitting strong fragrance. Using the Illumina RNA-Seq technique, 319,425,972 clean reads were obtained and were assembled into 104,691 final unigenes (average length of 853 bp), 41.75% of which were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 36,967 were assigned to gene ontology categories and 19,956 were assigned to eukaryoticorthologous groups. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, 12,388 unigenes were sorted into 286 pathways. Based on these transcriptomic data, we obtained a large number of candidate genes that were differentially expressed at different flower stages and that were related to floral pigment biosynthesis and fragrance metabolism. This comprehensive transcriptomic analysis provides fundamental information on the genes and pathways involved in flower secondary metabolism and development in S. oblata, providing a useful database for further research on S. oblata and other plants of genus Syringa.