Chloroplast-genome-wide maps of small RNAs in Chinese cabbage (Brassica rapa) and Arabidopsis thaliana (Ler).
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ABSTRACT: Deep sequencing provided evidence that a novel subset of small RNAs were derived from the chloroplast genome of Chinese cabbage (Brassica rapa) and Arabidopsis (Ler). The chloroplast small RNAs (csRNAs) include those derived from mRNA, rRNA, tRNA and intergenic RNA. The rRNA-derived csRNA were preferentially located at the 3M-CM-"M-BM-^@M-BM-^Y-ends of the rRNAs, while the tRNA-derived csRNAs were mainly located at 5M-CM-"M-BM-^@M-BM-^Y-termini of the tRNAs. After heat treatment, the abundance of csRNAs decreased in chinese cabbage seedlings, except those of 24 nt in length. The novel heat-responsive csRNAs and their locations in the chloroplast were verified by Northern blotting. The regulation of some csRNAs to the putative target genes were identified by real-time PCR. Our results indicated that high temperature regulated the production of some csRNAs, which may have potential roles in transcriptional or post-transcriptional regulation, and affected putative target genes expression in chloroplast. Examination of two replicates of heat treated (HT) and control (MT) Chinese cabbage sample respectively, and one Arabidopsis (Ler) RNA sample.
Project description:Deep sequencing provided evidence that a novel subset of small RNAs were derived from the chloroplast genome of Chinese cabbage (Brassica rapa) and Arabidopsis (Ler). The chloroplast small RNAs (csRNAs) include those derived from mRNA, rRNA, tRNA and intergenic RNA. The rRNA-derived csRNA were preferentially located at the 3â-ends of the rRNAs, while the tRNA-derived csRNAs were mainly located at 5â-termini of the tRNAs. After heat treatment, the abundance of csRNAs decreased in chinese cabbage seedlings, except those of 24 nt in length. The novel heat-responsive csRNAs and their locations in the chloroplast were verified by Northern blotting. The regulation of some csRNAs to the putative target genes were identified by real-time PCR. Our results indicated that high temperature regulated the production of some csRNAs, which may have potential roles in transcriptional or post-transcriptional regulation, and affected putative target genes expression in chloroplast.
Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line âFTâ using a combination of isolated microspore culture and radiation treatment (60Co γ-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line âFTâ, the drmâs chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between âFTâ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between âFTâ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage. The RNA from the third true leaves (day 15 to day 24 after the appearance of the third true leaves) of a developmentally retarded mutant (drm) and its wild type âFTâ in Chinese cabbage were sequenced by RNA-Seq, in triplicate.
Project description:GUN1 proteins controls protein homeostasis in chloroplast development in cotyledons of the model plant Arabidopsis thaliana, via coordination of nuclear encoded polymerase (NEP)-dependent chloroplast genes expression with plastid encoded polymerase (PEP)-dependent chloroplast genes expression. Lack of GUN1 leads to development of abnormal plastids and, consequently, accumulation of nuclear-encoded chloroplast-targeted (NECT) proteins which in many cases have been found still in their precursor form. Data dependent acquisition (DDA) mass spectrometry analysis of cotyledons soluble fractions, as well as targeted proteomics analysis of specific FtsH protease forms recognized by FtsH antibodies in western blotting, have been performed to identify peptides from the chloroplast transit peptide (cTP) of NECT in cotyledons extracts of wild type and GUN1 knocked-out mutant plants. The aim was to compare the number of cTPs found in 6 days after sowing (DAS) seedlings grown on plates with or without the inhibitor of plastid translation lincomycin.
Project description:PURPOSE:To clarify the mechanism of the wax deficiency, the wax-less flowering Chinese cabbage doubled-haploid (DH) line ‘CX001’ and Chinese cabbage DH line ‘FT’, obtained from isolated microspore culture, were used in the experiments. Transcriptome analysis indicated that BraA09g066480.3C was expressed in ‘FT’ but not in ‘CX001’.The work presented herein demonstrated that BraA09g066480.3C was the causal gene for wax-less flowering Chinese cabbage ‘CX001’
Project description:The leaf of Chinese cabbage is the major place of photosynthesis, the mutation of leaf may directly affect the rate of plant growth and development and the formation of leafy head, and ultimately influence the yield and quality of Chinese cabbage. We identified a developmentally retarded mutant (drm) exhibiting stable inheritance, which was derived from Chinese cabbage DH line ‘FT’ using a combination of isolated microspore culture and radiation treatment (60Co γ-rays). The drm exhibited slow growth and development at the seedling and heading stages, leading to the production of a tiny, leafy head, as well as chlorophyll-deficient leaves, especially in seedlings. Genetic analysis indicated that the phenotype of drm was controlled by a single recessive nuclear gene. Compared with wild-type line ‘FT’, the drm’s chlorophyll content was significantly reduced and its chloroplast structure was abnormal. Moreover, the photosynthetic efficiency and chlorophyll fluorescence parameters were significantly decreased. The changes in leaf color, combined with these altered physiological characters may influence the growth and development of plant, ultimately resulting in the developmentally retarded phenotype of drm. To further understand the molecular regulatory mechanisms of phenotypic differences between ‘FT’ and drm, comparative transcriptome analysis were performed using RNA-Seq, a total of 338 differentially expressed genes (DEGs) were detected between ‘FT’ and drm. According to GO and KEGG pathway analysis, a number of DEGs which involved in the chlorophyll degradation and photosynthesis were identified, such as chlorophyllase and ribulose-1,5-bisphosphate carboxylase/oxygenase. In addition, the expression patterns of 12 DEGs, including three chlorophyll degradation- and photosynthesis-related genes and nine randomly selected genes, were confirmed by qRT-PCR. Numerous single nucleotide polymorphisms were also identified, providing a valuable resource for research and molecular marker-assistant breeding in Chinese cabbage. These results contribute to our understanding of the molecular regulatory mechanisms underlying growth and development and lay the foundation for future genetic and functional genomics studies in Chinese cabbage.
Project description:Chloroplast, the energy organelle unique to plants and green algae, performs a wide range of functions including photosynthesis and biosynthesis of metabolites. However, as the most important tuber crop worldwide, the potato (Solanum tuberosum) chloroplast proteome has not been explored. Here, we use Percoll density gradient centrifugation to isolate intact chloroplasts from leaves of potato cultivar E3 and establish a reference proteome map of potato chloroplast by bottom-up proteomics. A total of 1834 non-redundant proteins, including 51 proteins encoded by the chloroplast genome, were identified in the chloroplast proteome. Extensive sequence-based localization prediction revealed over 62% of proteins to be chloroplast resident by at least one algorithm. A total of 16 proteins were selected for evaluating the prediction result by transient fluorescence assay and confirmed that 14 of them were distributed on distinct internal compartments of the chloroplast. In addition, 136 phosphorylation sites were identified in 61 proteins encoded by chloroplast proteome. Furthermore, by a comparative analysis between chloroplast and previously reported amyloplast proteomes, we reconstruct the starch metabolic pathways in the two different types of plastids. Altogether, our results establish a comprehensive proteome map with post-translationally modified sites of potato chloroplast, which would provide the theoretical principle for the research of photosynthesis pathway and starch metabolism.
Project description:The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco containing compartments known as pyrenoids in diverse algal lineages and carboxysomes in prokaryotes. Inside these compartments actively transported bicarbonate is converted into CO2 gas, which saturates the carboxylase with its substrate. Using co-immunoprecipitation experiments in Phaeodactylum tricornutum we have identified the Rubisco linker protein PYCO1. Similar to the green algal Rubisco linker protein EPYC1, PYCO1 is intrinsically disordered, possesses repeats and is positively charged at physiological pH. However, it possesses no sequence similarity to EPYC1, as expected for convergent evolution of a red Rubisco containing pyrenoid. Fluorescent PYCO1 fusion proteins localize as a rod shaped structure in the diatom chloroplast, consistent with the shape of the pyrenoid defined by transmission electron microscopy. To test the hypothesis that PYCO1 is the diatom pyrenoid scaffold we produced pure protein in Escherichia coli. Recombinant PYCO1 protein undergoes homotypic liquid liquid phase separation in a salt dependent manner. Diatom Rubisco specifically partitions into PYCO1 condensates. Heterotypic PYCO1-Rubisco condensates can bind up to three Rubisco hexadecamers per PYCO1 protein. Rubisco carboxylase function is unaffected in the condensates. PYCO1 is highly mobile in homotypic condensates. In contrast PYCO1 condensates saturated with diatom Rubisco have greatly reduced dynamics, with both PYCO1 and Rubisco becoming immobile. Consistently, FRAP experiments indicate that PYCO1 is not mobile in vivo. A combination of Cryo-electron microscopy and site-directed mutagenesis data show that the KWSP motif found in PYCO1 repeats binds to small subunits at the entrance of the Rubisco hexadecamer’s solvent channel. Analysis of mutant PYCO1 proteins show that both the “KWSP” tryptophan and another repeating tyrosine are essential for homotypic phase separation. We speculate that the unusual material properties of the PYCO1-Rubisco condensate are necessary to support the unusual non-spherical shape of the Phaeodactylum pyrenoid. Careful characterization of multiple diverse Rubisco condensates will strengthen translational approaches aiming to introduce pyrenoids and other metabolic condensates into new host organisms.
Project description:The transition from vegetative growth to reproductive growth involves many pathways. Vernalization is crucial to the formation of floral organs, the regulation of flowering time and plant breeding. The purpose of this study was to identify the mRNA, microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) related to vernalization of Chinese cabbage, and to construct a competitive endogenous RNA (ceRNA) network, so as to provide valuable information for exploring the molecular mechanism of vernalization of Chinese cabbage. Results: The results of whole-transcriptome sequencing showed that 2702 mRNAs, 151 lncRNAs, 16 circRNA, and 233 miRNAs were differentially expressed in vernalized (‘Ver’) and non-vernalized (‘Nor’) seeds of Chinese cabbage. Some transcription factors and regulatory proteins that play important roles in vernalization pathway have been identified, such as the transcription factors of WRKY, MYB, NAC, bHLH, and MADS-box, zinc finger protein CONSTANS like gene and B3 domain protein. We constructed vernalization-related ceRNA-miRNA-target gene network and obtained 199 pairs of ceRNA relationships, including 108 DEmiRNA-DEmRNA, 67 DEmiRNA-DElncRNA, and 12 DEmiRNA-DEcircRNA interactions in Chinese cabbage. Meanwhile, several important vernalization-related genes and their interacting lncRNAs, circRNAs, and miRNAs were identified, which were involved in the regulation of flowering time, floral organ formation, bolting and flowering. Conclusions: The candidate differentially expressed mRNA, miRNA, lncRNA and circRNA for vernalization of Chinese cabbage were identified by the whole-transcriptome sequencing, and the ceRNA network was constructed. This study laid a foundation for further study on the molecular mechanism of vernalization in Chinese cabbage.
Project description:The transition from vegetative growth to reproductive growth involves many pathways. Vernalization is crucial to the formation of floral organs, the regulation of flowering time and plant breeding. The purpose of this study was to identify the mRNA, microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA) related to vernalization of Chinese cabbage, and to construct a competitive endogenous RNA (ceRNA) network, so as to provide valuable information for exploring the molecular mechanism of vernalization of Chinese cabbage. Results: The results of whole-transcriptome sequencing showed that 2702 mRNAs, 151 lncRNAs, 16 circRNA, and 233 miRNAs were differentially expressed in vernalized (‘Ver’) and non-vernalized (‘Nor’) seeds of Chinese cabbage. Some transcription factors and regulatory proteins that play important roles in vernalization pathway have been identified, such as the transcription factors of WRKY, MYB, NAC, bHLH, and MADS-box, zinc finger protein CONSTANS like gene and B3 domain protein. We constructed vernalization-related ceRNA-miRNA-target gene network and obtained 199 pairs of ceRNA relationships, including 108 DEmiRNA-DEmRNA, 67 DEmiRNA-DElncRNA, and 12 DEmiRNA-DEcircRNA interactions in Chinese cabbage. Meanwhile, several important vernalization-related genes and their interacting lncRNAs, circRNAs, and miRNAs were identified, which were involved in the regulation of flowering time, floral organ formation, bolting and flowering. Conclusions: The candidate differentially expressed mRNA, miRNA, lncRNA and circRNA for vernalization of Chinese cabbage were identified by the whole-transcriptome sequencing, and the ceRNA network was constructed. This study laid a foundation for further study on the molecular mechanism of vernalization in Chinese cabbage.