BackgroundLysine 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.ResultsCompared 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.ConclusionsOur 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.

BackgroundLysine 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.ResultsCompared 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.ConclusionsOur 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.

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.

Background. Ananas comosus var. bracteatus has high ornamental value due to its chimeric leaves. However, the chimeric trait is very unstable in red pineapple plants, and transcriptional variation between the two types of cells (white/green cells) and the molecular mechanism responsible for their albino phenotype remain poorly understood.Methods. Comparative transcriptomic and proteomic analyses of the white parts (Whs) and green parts (Grs) of chimeric leaves were performed.Results. In total, 1,685 differentially expressed genes (DEGs) (712 upregulated and 973 downregulated) and 1,813 differentially abundant proteins (DAPs) (1,018 with low abundance and 795 with high abundance) were identified. Based on Gene Ontology (Go) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the DEGs were mostly involved in carbon fixation in photosynthetic organisms, porphyrin and chlorophyll metabolism and oxidative phosphorylation, while proteomic analysis revealed that DAPs were mostly related to ribosomes, photosynthesis, photosynthesis antennas, and porphyrin and chlorophyll metabolism. Combined analysis showed increased mRNA levels but low abundance of nine proteins level in Whs/Grs related to photosynthetic pigment and photosynthesis. Transcriptional changes, posttranscriptional regulation and translational alterations of key enzymes involved in chlorophyll biosynthesis and photosynthesis may play important roles in the albino parts of chimeric 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.

Studies of the molecular mechanisms involved in the formation of the albino leaf cells are important for understanding the development of chimera leaves in Ananas comosus var. bracteatus. In this study, we identified a total of 163 novel miRNAs involved in the development of complete white (CWh) and complete green (CGr) leaves using high-throughput sequencing method. The potential miRNA target genes were predicted and annotated using the NR, Swiss-Prot, GO, COG, KEGG, KOG and Pfam databases. The main biological processes regulated by miRNAs were revealed. The miRNAs which potentially play important roles in the development of the leaves and the albino of the CWh leaf cells were selected and their expression patterns were analyzed. The expression levels of nine miRNAs and their potential target genes were studied using qRT-PCR. These results will help to elucidate the functional and regulatory roles of miRNAs in the formation of the albino cells and the development of the leaves of A. comosus var. bracteatus. These data may also be helpful as a resource for studies of small RNA in other leaf color chimeric plant species.

Leaf coloration is one of the most important and attractive characteristics of Ananas comosus var. bracteatus. The chimeric character is not stable during the in vitro tissue culturing. Many regenerated plants lost economic values for the loss of the chimeric character of leaves. In order to reveal the molecular mechanisms involved in the albino phenotype of the leaf cells, the physiological and transcriptional differences between complete white (CWh) and green (CGr) leaf cells of A. comosus var. bracteatus were analyzed. A total of 1,431 differentially expressed unigenes (DEGs) in CGr and CWh leaves were identified using RNA-seq. A comparison to the COG, GO and KEGG annotations revealed DEGs involved in chlorophyll biosynthesis, chloroplast development and photosynthesis. Furthermore, the measurement of main precursors of chlorophyll in the CWh leaves confirmed that the rate limiting step in chlorophyll biosynthesis, and thus the cause of the albino phenotype of the white cells, was the conversion of pyrrole porphobilinogen (PBG) to uroporphyrinogen III (Uro III). The enzyme activity of porphobilinogen deaminase (PBGD) and uroporporphyrinogn III synthase (UROS), which catalyze the transition of PBG to Uro III, was significantly decreased in the CWh leaves. Our data showed the transcriptional differences between the CWh and CGr plants and characterized key steps in chlorophyll biosynthesis of the CWh leaves. These results contribute to our understanding of the mechanisms and regulation of pigment biosynthesis in the CWh leaf cells of A. comosus var. bracteatus.

为筛选表达稳定的内参基因,以红苞凤梨（Ananas comosus var.bracteatus）不同发育时期的全绿、全白苗为材料,对10个组成型表达基因EF1、UBQ、ACT、GADPH、Histone、TUA、TUB、18S、elf-5A、α-tubulin进行筛选,并分析PetF基因的表达模式。结果表明,10个候选内参基因在红苞凤梨全绿、全白苗不同生长阶段中的表达稳定性不同。红苞凤梨不同生长时期以Histone和α-tubulin为最适内参基因,而全绿苗和全白苗的对比分析以18S、EF1和α-tubulin为最理想的内参组合。PetF基因在红苞凤梨发育过程及绿、白苗对比分析中的表达水平变化趋势一致,因此,所选的内参基因是合适的。

BackgroundAnanas comosus var. bracteatus (Red Pineapple) is an important ornamental plant for its colorful leaves and decorative red fruits. Because of its complex genome, it is difficult to understand the molecular mechanisms involved in the growth and development. Thus high-throughput transcriptome sequencing of Ananas comosus var. bracteatus is necessary to generate large quantities of transcript sequences for the purpose of gene discovery and functional genomic studies.ResultsThe Ananas comosus var. bracteatus transcriptome was sequenced by the Illumina paired-end sequencing technology. We obtained a total of 23.5 million high quality sequencing reads, 1,555,808 contigs and 41,052 unigenes. In total 41,052 unigenes of Ananas comosus var. bracteatus, 23,275 unigenes were annotated in the NCBI non-redundant protein database and 23,134 unigenes were annotated in the Swiss-Port database. Out of these, 17,748 and 8,505 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. Functional annotation against Kyoto Encyclopedia of Genes and Genomes Pathway database identified 5,825 unigenes which were mapped to 117 pathways. The assembly predicted many unigenes that were previously unknown. The annotated unigenes were compared against pineapple, rice, maize, Arabidopsis, and sorghum. Unigenes that did not match any of those five sequence datasets are considered to be Ananas comosus var. bracteatus unique. We predicted unigenes encoding enzymes involved in terpenoid and phenylpropanoid biosynthesis.ConclusionThe sequence data provide the most comprehensive transcriptomic resource currently available for Ananas comosus var. bracteatus. To our knowledge; this is the first report on the de novo transcriptome sequencing of the Ananas comosus var. bracteatus. Unigenes obtained in this study, may help improve future gene expression, genetic and genomics studies in Ananas comosus var. bracteatus.