Molecular characterization of complement regulatory factor CD46 in Trachinotus ovatus and its role in the antimicrobial immune responses and complement regulation.

CD46, as a cofactor of complement I factor, not only regulates the complement system but also functions as a pathogen receptor and is involved in controlling early pathogen infection through autophagy. In this study, a new CD46 gene (ToCD46) was identified from golden pompano (Trachinotus ovatus), which showed higher sequence homology with other teleosts CD46. Homology comparison showed that ToCD46 had higher sequence homology (46.95-52.85%) with other teleosts CD46 and lower homology with mammal. Tissue expression profile analysis showed that ToCD46 was generally expressed in all tissues with the highest expression level in liver, followed by head kidney, and showed different patterns of up-regulation in immune-related tissues after stimulation by Streptococcus agalactiae and Vibrio alginolyticus. The hemolytic activity analysis and apoptosis assay showed that rToCD46 decreased the hemolytic activity of serum of golden pompano and effectively inhibited the damage of A549 cells, suggesting that ToCD46 might be involved in the regulation of complement activation of golden pompano. In vitro antibacterial experiments showed that rToCD46 had antibacterial activity against gram negative bacteria V. alginolyticus but no effect on positive bacteria S. agalactiae. These results suggest that ToCD46 may be involved in the immune response of golden pompano to pathogens, which will provide important basic information for elucidating the evolutionary history of the complement system of golden pompano.

Identification and functional characterization of complement regulatory protein CD59 in golden pompano (Trachinotus ovatus).

CD59, one of the essential inhibitors of the complement membrane attack complex (MAC), plays a crucial role in regulation of complement activation. In this study, we cloned and identified the CD59 gene (named ToCD59) of golden pompano (Trachinotus ovatus). The ORF sequence of ToCD59 is 357 bp long encoding 118 amino acids with a molecular weight of 13.09 kDa. Prediction of protein domains showed that ToCD59 contained an Lu domain and a C-terminal glycosylphosphatidylinositol (GPI) partial anchor. Homology comparisons indicated that ToCD59 shared the high sequence similarity with other fish CD59. RT-qPCR analysis showed that ToCD59 was expressed in all tested healthy tissues of golden pompano, with the highest level of expression in the brain. After stimulation with bacteria, ToCD59 expression levels were significantly up-regulated in head kidney, liver, gill and brain, but down-regulated in spleen. Subcellular localization results showed that ToCD59 localized to the cytoplasm of A549 cells. The hemolytic activity analysis showed that rToCD59 might have complement inhibitory activity through the alternative complement pathway. In addition, antibacterial test showed that rToCD59 had antibacterial ability against S. agalactiae and V. alginolyticus in vitro. These results suggest that ToCD59 might play an important role in the immune response against pathogens, which would provide basic information for elucidating the functional evolutionary history of complement system in teleost.

The highly continuous reference genome of a leaf-chimeric red pineapple (Ananas comosus var. bracteatus f. tricolor) provides insights into elaboration of leaf color.

Ananas comosus var. bracteatus f. tricolor (GL1) is a red pineapple accession whose mostly green leaves with chimeric white leaf margins turn red in spring and autumn and during flowering. It is an important ornamental plant and ideal plant research model for anthocyanin metabolism, chimeric leaf development, and photosynthesis. Here, we generated a highly contiguous chromosome-scale genome assembly for GL1 and compared it with other 3 published pineapple assemblies (var. comosus accessions MD2 and F153, and var. bracteatus accession CB5). The GL1 assembly has a total size of ∼461 Mb, with a contig N50 of ∼2.97 Mb and Benchmarking Universal Single-Copy Ortholog score of 97.3%. More than 99% of the contigs are anchored to 25 pseudochromosomes. Compared with the other 3 published pineapple assemblies, the GL1 assembly was confirmed to be more continuous. Our evolutionary analysis showed that the Bromeliaceae and Poaceae diverged from their nearest common ancestor ∼82.36 million years ago (MYA). Population structure analysis showed that while GL1 has not undergone admixture, bracteatus accession CB5 has resulted from admixture of 3 species of Ananas. Through classification of orthogroups, analysis of genes under positive selection, and analysis of presence/absence variants, we identified a series of genes related to anthocyanin metabolism and development of chimeric leaves. The structure and evolution of these genes were compared among the published pineapple assemblies with reveal candidate genes for these traits. The GL1 genome assembly and its comparisons with other 3 pineapple genome assemblies provide a valuable resource for the genetic improvement of pineapple and serve as a model for understanding the genomic basis of important traits in different pineapple varieties and other pan-cereal crops.

Validation of Reference Genes for Quantitative Real-Time PCR Normalization in Ananas comosus var. bracteatus During Chimeric Leaf Development and Response to Hormone Stimuli.

Reverse transcription quantitative real-time PCR (RT-qPCR) is a common way to study gene regulation at the transcriptional level due to its sensibility and specificity, but it needs appropriate reference genes to normalize data. Ananas comosus var. bracteatus, with white-green chimeric leaves, is an important pantropical ornamental plant. Up to date, no reference genes have been evaluated in Ananas comosus var. bracteatus. In this work, we used five common statistics tools (geNorm, NormFinder, BestKeeper, ΔCt method, RefFinder) to evaluate 10 candidate reference genes. The results showed that Unigene.16454 and Unigene.16459 were the optimal reference genes for different tissues, Unigene.16454 and zinc finger ran-binding domain-containing protein 2 (ZRANB2) for chimeric leaf at different developmental stages, isocitrate dehydrogenase [NADP] (IDH) and triacylglycerol lipase SDP1-like (SDP) for seedlings under different hormone treatments. The comprehensive results showed IDH, pentatricopeptide repeat-containing protein (PPRC), Unigene.16454, and caffeoyl-CoA O methyltransferase 5-like (CCOAOMT) are the top-ranked stable genes across all the samples. The stability of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the least during all experiments. Furthermore, the reliability of recommended reference gene was validated by the detection of porphobilinogen deaminase (HEMC) expression levels in chimeric leaves. Overall, this study provides appropriate reference genes under three specific experimental conditions and will be useful for future research on spatial and temporal regulation of gene expression and multiple hormone regulation pathways in Ananas comosus var. bracteatus.

Adventitious root primordia formation and development in the stem of Ananas comosus var. bracteatus slip.

There are about 4-6 slips on a fruit, and they are good materials for effective regeneration of Ananas comosus var. bracteatus. Adventitious root (AR) induction is essential for the propagation of Ananas comosus var. bracteatus slips. Growth regulator treatment, and culture medium are imperative factors that affect slip growth and rooting. In order to screen the optimal methods for slips rooting and reveal the anatomic procedure of slip rooting, this study induced slip rooting by different treatment of growth regulator, culture medium, observed the slip stem structure, AR origination and formation procedure through paraffin sections. The results showed that, slip cuttings treated with 100 mg/L of Aminobenzotriazole (ABT) for 6 hrs, cultured in river sand: coconut chaff: garden soil 2:2:1 medium is the optimal method for rooting. The proper supplementary of ABT can enhance the soluble sugar content, soluble protein content, polyphenol oxidase (PPO) activity and peroxidase (POD) enzyme activity, which resulted in the improvement of rooting. The slip stem structure is quite different from other monocots, which consists of epidermis, cortex, and stele with vascular tissues distributed in the cortex and stele. The AR primordia originates from the parenchyma cells located on the borderline between the cortex and stele. The vascular tissues in the AR develop and are connected with vascular tissue of the stem before the AR grew out the stem. The number of primary xylem poles in AR is about 30.

Metabolome and transcriptome profiling reveals anthocyanin contents and anthocyanin-related genes of chimeric leaves in Ananas comosus var. bracteatus.

BACKGROUND: Ananas comosus var. bracteatus is a colorful plant used as a cut flower or landscape ornamental. The unique foliage color of this plant includes both green and red leaves and, as a trait of interest, deserves investigation. In order to explore the pigments behind the red section of the chimeric leaves, the green and red parts of chimeric leaves of Ananas comosus var. bracteatus were sampled and analyzed at phenotypic, cellular and molecular levels in this study. RESULTS: The CIELAB results indicated that the a* values and L* values samples had significant differences between two parts. Freehand sections showed that anthocyanin presented limited accumulation in the green leaf tissues but obviously accumulation in the epidermal cells of red tissues. Transcriptomic and metabolomic analyses were performed by RNA-seq and LC-ESI-MS/MS. Among the 508 identified metabolites, 10 kinds of anthocyanins were detected, with 6 significantly different between the two samples. The cyanidin-3,5-O-diglucoside content that accounts for nearly 95.6% in red samples was significantly higher than green samples. RNA-Seq analyses showed that 11 out of 40 anthocyanin-related genes were differentially expressed between the green and red samples. Transcriptome and metabolome correlations were determined by nine quadrant analyses, and 9 anthocyanin-related genes, including MYB5 and MYB82, were correlated with 7 anthocyanin-related metabolites in the third quadrant in which genes and metabolites showing consistent change. Particularly, the PCCs between these two MYB genes and cyanidin-3,5-O-diglucoside were above 0.95. CONCLUSION: Phenotypic colors are closely related to the tissue structures of different leaf parts of Ananas comosus var. bracteatus, and two MYB transcription factors might contribute to differences of anthocyanin accumulation in two parts of Ananas comosus var. bracteatus chimeric leaves. This study lay a foundation for further researches on functions of MYBs in Ananas comosus var. bracteatus and provides new insights to anthocyanin accumulation in different parts of chimeric leaves.

MicroRNAs Roles in Plants Secondary Metabolism.

Plant growth and development is dependent on the regulation of classes of microRNAs (miRNAs) that have emerged as important gene regulators. These miRNAs can regulate plant gene expression to function. They play an important roles in biological homeostasis and environmental response controls. A wide range of plant biological and metabolic processes, including developmental timing, tissues specific development, and differentiation, depends on miRNAs. They perpetually regulate secondary metabolite functions in different plant family lines. Mapping of molecular phylogenies shows the distribution of secondary metabolism in the plant territory. More importantly, a lot of information related to miRNA regulatory processes in plants is revealed, but the role of miRNAs in secondary metabolism regulation and functions of the metabolites are still unclear. In this review, we pinnacle some potential miRNAs regulating the secondary metabolite biosynthesis activities in plants. This will provide an alternative knowledge for functional studies of secondary metabolism.

AbhemC encoding porphobilinogen deaminase plays an important role in chlorophyll biosynthesis and function in albino Ananas comosus var. bracteatus leaves.

BACKGROUND: The chimeric leaves of Ananas comosus var. bracteatus are composed of normal green parts (Grs) and albino white parts (Whs). Although the underlying mechanism of albinism in A. comosus var. bracteatus leaves is not fully understood, it is likely associated with the chlorophyll (Chl) biosynthesis. In this biosynthetic process, porphobilinogen deaminase (PBGD) plays a crucial role by catalyzing the conversion of porphobilinogen (PBG) to uroporphyrinogen III (Urogen III). Therefore, its encoding gene AbhemC was investigated here in association with Chl biosynthesis and albinism in chimeric A. comosus var. bracteatus leaves. METHODS: The Chl content, main Chl biosynthesis precursor content, and main enzyme activity were determined and compared between the Whs and Grs of A. comosus var. bracteatus leaves. In addition, AbhemC was cloned and its transcriptional expression and prokaryotic protein expression were analyzed. Furthermore, RNAi-mediated silencing of AbhemC was produced and assessed in tobacco plants. RESULTS: The concentration of Chl a and Chl b in the Grs was significantly higher than that in the Whs, respectively. Additionally, the content of the Chl biosynthesis precursor Urogen III decreased significantly in the Whs compared with the Grs. Thus, the transition of PBG to Urogen III may be the first rate-limiting step leading to albinism in the chimeric leaves of A. comosus var. bracteatus. The gene AbhemC comprised 1,135 bp and was encoded into a protein with 371 amino acids; phylogenetically, AbhemC was most closely related to hemC of pineapple. Prokaryotic expression and in vitro enzyme activity analysis showed that the cloned mRNA sequence of AbhemC was successfully integrated and had PBGD activity. Compared with control plants, transgenic tobacco leaves with pFGC5941-AbhemC-RNAi vector were substantially less green with significantly reduced hemC expression and Chl content, as well as reduced PBGD enzyme activity and significantly decreased content of Chl biosynthesis precursors from Urogen III onwards. Our results suggest that the absence of hemC expression reduces the enzyme activity of PBGD, which blocks the transition of PBG to Urogen III, and in turn suppresses Chl synthesis leading to the pale-green leaf color. Therefore, we suggest that AbhemC plays an important role in Chl synthesis and may be an important factor in the albinism of A. comosus var. bracteatus leaves.

Systematic identification and comparative analysis of lysine succinylation between the green and white parts of chimeric leaves of Ananas comosus var. bracteatus.

BACKGROUND: Lysine succinylation, an important protein posttranslational modification (PTM), is widespread and conservative. The regulatory functions of succinylation in leaf color has been reported. The chimeric leaves of Ananas comosus var. bracteatus are composed of normal green parts and albino white parts. However, the extent and function of lysine succinylation in chimeric leaves of Ananas comosus var. bracteatus has yet to be investigated. RESULTS: Compared to the green (Gr) parts, the global succinylation level was increased in the white (Wh) parts of chimeric leaves according to the Western blot and immunohistochemistry analysis. Furthermore, we quantitated the change in the succinylation profiles between the Wh and Gr parts of chimeric leaves using label-free LFQ intensity. In total, 855 succinylated sites in 335 proteins were identified, and 593 succinylated sites in 237 proteins were quantified. Compared to the Gr parts, 232 (61.1%) sites in 128 proteins were quantified as upregulated targets, and 148 (38.9%) sites in 70 proteins were quantified as downregulated targets in the Wh parts of chimeric leaves using a 1.5-fold threshold (P < 0.05). These proteins with altered succinylation level were mainly involved in crassulacean acid metabolism (CAM) photosynthesis, photorespiration, glycolysis, the citric acid cycle (CAC) and pyruvate metabolism. CONCLUSIONS: Our results suggested that the changed succinylation level in proteins might function in the main energy metabolism pathways-photosynthesis and respiration. Succinylation might provide a significant effect in the growth of chimeric leaves and the relationship between the Wh and Gr parts of chimeric leaves. This study not only provided a basis for further characterization on the function of succinylated proteins in chimeric leaves of Ananas comosus var. bracteatus but also provided a new insight into molecular breeding for leaf color chimera.

Novel method for improving ardicrenin content in hairy roots of Ardisia crenata Sims plants.

To develop an alternative medicine related with taxol/camptothecin, a hairy roots induction system for measuring triterpenoid saponin ardicrenin was established. In the current study, mature and healthy seeds of Ardisia crenata plants were selected for obtaining aseptic seedlings. Two Agrobacterium rhizogenes strains ATCC 15834 and A4 were used to infect aseptic euphylla for inducing hairy roots of A. crenata plants. For the best combination of seeds germination, a Murashige-Skoog medium containing 1.0 mg L-1 6-benzylaminopurine and 1.0 mg L-1 naphthalene acetic acid was made, which reached a rate of 92.4 %. Results showed that ATCC 15834 and A4 both induced hairy roots of A. crenata for improving ardicrenin production. The PCR analysis demonstrated that ATCC 15834 and A4 Ri plasmid T-DNA had been successfully transferred and integrated into the genome of leaf cell nuclei, however the Vir region was not. Further, ardicrenin content in hairy roots ACHR 15834 8.2 %) induced by ATCC 15834 was quantified by the RP-HPLC, which was also 1.8-, 2.7-, 9.4- and 2.6-fold greater than those of ACHR 4 induced by A4 (4.5 %), ACR C formed by tissue culture (3.1 %), euphylla (0.8 %) and NR C formed nature (3.2 %), respectively. Taken together, hairy root lines of A. crenata obtained were able to express naturally more ardicrenin than natural plants.

Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves.

Long noncoding RNAs (lncRNAs) have been reported to play key regulatory roles in plant growth, development, and biotic and abiotic stress physiology. Revealing the mechanism of lncRNA regulation in the albino portions of leaves is important for understanding the development of chimeric leaves in Ananas comosus var. bracteatus. In this study, a total of 3,543 candidate lncRNAs were identified, among which 1,451 were differentially expressed between completely green (CGr) and completely white (CWh) leaves. LncRNAs tend to have shorter transcripts, lower expression levels, and greater expression specificity than protein-coding genes. Predicted lncRNA targets were functionally annotated by the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A lncRNA-mRNA interaction network was constructed, and 36 target mRNAs related to chlorophyll metabolism were predicted to interact with 86 lncRNAs. Among these, 25 significantly differentially expressed lncRNAs putatively interacted with 16 target mRNAs. Based on an expression pattern analysis of the lncRNAs and their target mRNAs, the lncRNAs targeting magnesium chelatase subunit H (ChlH), protochlorophyllide oxidoreductase (POR), and heme o synthase (COX10) were suggested as key regulators of chlorophyll metabolism. This study provides the first lncRNA database for A. comosus var. bracteatus and contributes greatly to understanding the mechanism of epigenetic regulation of leaf albinism.

SMRT sequencing analysis reveals the full-length transcripts and alternative splicing patterns in Ananas comosus var. bracteatus.

BACKGROUND: Ananas comosus var. bracteatus is an herbaceous perennial monocot cultivated as an ornamental plant for its chimeric leaves. Because of its genomic complexity, and because no genomic information is available in the public GenBank database, the complete structure of the mRNA transcript is unclear and there are limited molecular mechanism studies for Ananas comosus var. bracteatus. METHODS: Three size fractionated full-length cDNA libraries (1-2 kb, 2-3 kb, and 3-6 kb) were constructed and subsequently sequenced in five single-molecule real-time (SMRT) cells (2 cells, 2 cells, and 1 cell, respectively). RESULTS: In total, 19,838 transcripts were identified for alternative splicing (AS) analysis. Among them, 19,185 (96.7%) transcripts were functionally annotated. A total of 9,921 genes were identified by mapping the non-redundant isoforms to the reference genome. A total of 10,649 AS events were identified, the majority of which were intron retention events. The alternatively spliced genes had functions in the basic metabolism processes of the plant such as carbon metabolism, amino acid biosynthesis, and glycolysis. Fourteen genes related to chlorophyll biosynthesis were identified as having AS events. The distribution of the splicing sites and the percentage of conventional and non-canonical AS sites of the genes categorized in pathways related to the albino leaf phenotype (ko00860, ko00195, ko00196, and ko00710) varied greatly. The present results showed that there were 8,316 genes carrying at least one poly (A) site, which generated 21,873 poly (A) sites. These findings indicated that the quality of the gene structure and functional information of the obtained genome was greatly improved, which may facilitate further genetic study of Ananas comosus var. bracteatus.