[Show abstract][Hide abstract] ABSTRACT: Background
Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT¿s dynamic metabolic activities which will help towards pathway engineering.ResultsSpearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases.Conclusions
The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint.
[Show abstract][Hide abstract] ABSTRACT: Recent research on long noncoding RNAs (lncRNAs) has expanded our understanding of gene transcription regulation and the generation of cellular complexity. Depending on their genomic origins, lncRNAs can be transcribed from intergenic or intragenic regions or from introns of protein coding genes. We have recently reported more than 6,000 intergenic lncRNAs in Arabidopsis. Here, we systematically identified long noncoding natural antisense transcripts (lncNATs), defined as lncRNAs transcribed from the opposite DNA strand of coding or noncoding genes. We found a total of 37,238 sense-antisense transcript pairs and 70% of annotated mRNAs to be associated with antisense transcripts in Arabidopsis. These lncNATs could be reproducibly detected by different technical platforms, including strand-specific tiling arrays, Agilent custom expression arrays, strand-specific RNA-seq and qRT-PCR experiments. Moreover, we investigated the expression profiles of sense-antisense pairs in response to light and observed spatial and developmental-specific light effects on 626 concordant and 766 discordant NAT pairs. Genes for a large number of the light-responsive NAT pairs are associated with histone modification peaks, and histone acetylation is dynamically correlated with light-responsive expression changes of NATs.
Genome Research 01/2014; 24(3). DOI:10.1101/gr.165555.113 · 13.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Among signaling components downstream of phytochrome A, HY5, HFR1, and LAF1 are transcription factors that regulate expression of phyA responsive genes. Previous work has shown that FHY1/FHL distribute phyA signals directly to HFR1 and LAF1, both of which regulate largely independent pathways, but the relationship of HY5 to these two factors was unclear. Here, we investigated the genetic relationship among the genes encoding these 3 transcription factors, HY5, HFR1, and LAF1. Analyses of double and triple mutants showed that HY5, a bZIP factor, HFR1, a bHLH factor, and LAF1, a Myb factor, independently transmit phyA signals downstream. We showed that HY5 but not its homolog, HYH, could interact with HFR1 and LAF1; on the other hand, FHY1 and its homolog, FHL did not interact with HY5 nor HYH. Together, our results suggest that HY5 transmits phyA signals through an FHY1/FHL-independent pathway but it may also modulate FHY1/FHL signal through its interaction with HFR1 and LAF1.
[Show abstract][Hide abstract] ABSTRACT: Circadian clocks enable organisms to adapt to a 24-h diurnal cycle and anticipate rhythmic changes in the environment. The Arabidopsis central oscillator contains 3 genes encoding core clock components. CCA1/LHY and TOC1 reciprocally repress genes encoding each other and are critical for the generation of circadian rhythms controlling many clock outputs. A precise regulation of transcriptional events is, therefore, essential for proper circadian function. Here, we investigated histone 3 (H3) tail modifications of CCA1, LHY and TOC1 under various conditions. We found specific association of only H3K4Me3 and H3K9/14Ac with the translational start site of these 3 genes. These H3 marks were enriched at circadian time points of their increased transcription at different photoperiods and under free-running conditions, suggesting circadian regulation of H3 modifications. Analysis of clock-compromised CCA1-OX lines provided evidence that light/dark photoperiods signal the establishment of these chromatin changes which are gated by the clock.
[Show abstract][Hide abstract] ABSTRACT: The final expression level of a transgene-derived protein in transgenic plants depends on transcriptional and post-transcriptional processes. Here, we focus on methods to improve protein stability without comprising biological function. We found that the four isoforms of the Arabidopsis RAD23 protein family are relatively stable. The UBA2 domain derived from RAD23a can be used as a portable stabilizing signal to prolong the half-life of two unstable transcription factors (TFs), HFR1 and PIF3. The increased stability of the TF-UBA2 fusion proteins results in an enhanced phenotype in transgenic plants compared to expression of the TF alone. Similar results were obtained for the RAD23a UBA1 domain. In addition to UBA1/2 of RAD23a, the UBA domain from the Arabidopsis DDI1 protein also increased the half-life of the unstable protein JAZ10.1, which is involved in jasmonate signaling. Taken together, our results suggest that UBA fusions can be used to increase the stability of unstable proteins for basic plant biology research as well as crop improvement.
The Plant Journal 05/2012; 72(2):345-54. DOI:10.1111/j.1365-313X.2012.05060.x · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent genome-wide surveys showed that acetylation of H3K9 and H3K27 is correlated with gene activation during deetiolation of Arabidopsis thaliana seedlings, but less is known regarding changes in the histone status of repressed genes. Phytochrome A (phyA) is the major photoreceptor of deetiolation, and phyA expression is reversibly repressed by light. We found that in adult Arabidopsis plants, phyA activation in darkness was accompanied by a significant enrichment in the phyA transcription and translation start sites of not only H3K9/14ac and H3K27ac but also H3K4me3, and there was also moderate enrichment of H4K5ac, H4K8ac, H4K12ac, and H4K16ac. Conversely, when phyA expression was repressed by light, H3K27me3 was enriched with a corresponding decline in H3K27ac; moreover, demethylation of H3K4me3 and deacetylation of H3K9/14 were also seen. These histone modifications, which were focused around the phyA transcription/translation start sites, were detected within 1 h of deetiolation. Mutant analysis showed that HDA19/HD1 mediated deacetylation of H3K9/14 and uncovered possible histone crosstalk between H3K9/14ac and H3K4me3. Neither small RNA pathways nor the circadian clock affected H3 modification status of the phyA locus, and DNA methylation was unchanged by light. The presence of activating and repressive histone marks suggests a mechanism for the rapid and reversible regulation of phyA by dark and light.
The Plant Cell 02/2011; 23(2):459-70. DOI:10.1105/tpc.110.080481 · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ubiquitin is a small polypeptide and ubiquitination is the post-translational modification by ubiquitin protein, resulting in degradation of target proteins by the 26S proteasome complex. Here, we found that E3 ubiquitin ligase SINAT5, an Arabidopsis homologue of the Drosophila SINA RING-finger protein, interacts directly with LHY, a component of the circadian oscillator, and DET1, a negative regulator of light-regulated gene expression. We also found that SINAT5 has E3 ubiquitination activity for LHY but not for DET1. Interestingly, LHY ubiquitination by SINAT5 was inhibited by DET1. Late flowering of sinat5 mutants indicates that flowering time can be controlled by DET1 through regulation of LHY stability by SINAT5.
Biochemical and Biophysical Research Communications 07/2010; 398(2):242-6. DOI:10.1016/j.bbrc.2010.06.067 · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many plant photoresponses from germination to shade avoidance are mediated by phytochrome B (phyB). In darkness, phyB exists as the inactive Pr in the cytosol but upon red (R) light treatment, the active Pfr translocates into nuclei to initiate signaling. Degradation of phyB Pfr likely regulates signal termination, but the mechanism is not understood. Here, we show that phyB is stable in darkness, but in R, a fraction of phyB translocates into nuclei and becomes degraded by 26S proteasomes. Nuclear phyB degradation is mediated by COP1 E3 ligase, which preferentially interacts with the PhyB N-terminal region (PhyB-N). PhyB-N polyubiquitination by CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in vitro can be enhanced by different PHYTOCHROME INTERACTING FACTOR (PIF) proteins that promote COP1/PhyB interaction. Consistent with these results, nuclear phyB accumulates to higher levels in pif single and double mutants and in cop1-4. Our results identify COP1 as an E3 ligase for phyB and other stable phytochromes and uncover the mechanism by which PIFs negatively regulate phyB levels.
The Plant Cell 07/2010; 22(7):2370-83. DOI:10.1105/tpc.109.072520 · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It was known that long after far-red light 1 (LAF1), a positive phytochrome A (phyA) signal transducer, is ubiquitinated by constitutive photomorphogenic 1 (COP1) in vitro and stabilizes long hypocotyl in far-red 1 (HFR1), another phyA signal transducer, through direct interaction. Our results imply that LAF1 stabilizes phyA by inhibiting or blocking COP1 activity, or by directly interacting with phyA, resulting in the stimulation of phyA signaling.
Journal of the Korean Society for Applied Biological Chemistry 01/2010; 53(1). DOI:10.3839/jksabc.2010.020 · 0.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Among the five phytochromes in Arabidopsis thaliana, phytochrome A (phyA) plays a major role in seedling deetiolation. Mutant analyses have identified more than 10 positive components acting downstream of phyA to inhibit hypocotyl elongation. However, their sites of action and their hierarchical relationships are poorly understood. Here, we investigated the genetic and molecular relationship between two homologous proteins, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and FHY1-LIKE (FHL), and two transcription factors, LONG AFTER FAR-RED LIGHT1 (LAF1) and LONG HYPOCOTYL IN FAR-RED1 (HFR1). Analyses of double and triple mutants showed that LAF1, a myb factor, and HFR1, a basic helix-loop-helix factor, independently transmit phyA signals downstream of FHY1 and FHL. Coimmunoprecipitation experiments showed that phyA, FHY1, FHL, LAF1, and HFR1 are components of protein complexes in vivo. In vitro pull-down assays demonstrated direct interactions between partner proteins with the N-terminal region of FHY1, as well as that of FHL, interacting with the LAF1 N-terminal portion and the HFR1 C-terminal region. These results suggest that, in addition to assisting phyA nuclear accumulation, FHY1 and FHL are required to assemble photoreceptor/transcription factor complexes for phyA signaling.
The Plant Cell 06/2009; 21(5):1341-59. DOI:10.1105/tpc.109.067215 · 9.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Post-translational modification is an important mechanism to determine protein levels and/or activities in cells. The process of conjugation of ubiquitin units to particular proteins, ubiquitination, usually leads to proteasomal degradation. During the past several years considerable work has been done to reveal the role of ubiquitination in the regulation of plant signaling and development. This article focuses on recent advances made on the study of ubiquitin-mediated proteolysis of several light-related signaling pathways, such as photomorphogenesis, circadian clock function, and photoperiodic flowering.
Current opinion in plant biology 02/2009; 12(1):49-56. DOI:10.1016/j.pbi.2008.10.009 · 9.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ubiquitination plays important roles in plant development, including programmed cell death. Here, we characterize a novel membrane-bound RING motif protein, encoded by RING1, that is expressed at a low level in all Arabidopsis tissues but can be upregulated by fumonisin B1 (FB1) treatment and pathogen infection. RING1 displays E3 ubiquitin ligase activity in vitro, which is dependent on the integrity of the RING motif. GFP fusion protein localization and cell fractionation experiments show that this E3 ligase is associated with the lipid rafts of plasma membranes. Knock-down of RING1 transcripts using artificial microRNA (amiR-R1(159)) leads to FB1 hyposensitivity, but overexpression of RING1 confers hypersensitivity. Additionally, expression of the pathogenesis-related 1 (PR-1) gene is lower and delayed in amiR-R1(159) plants compared with wild-type and RING1-overexpressing plants. The FB1 hyposensitivity of amiR-R1(159) plants can be rescued by expression of cleavage-resistant RING1mut transcripts. Our results suggest that RING1 acts as a signal from the plasma membrane lipid rafts to trigger the FB1-induced plant programmed cell death pathway.
The Plant Journal 11/2008; 56(4):550-61. DOI:10.1111/j.1365-313X.2008.03625.x · 6.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are 21 nt RNAs that regulate many biological processes in plants by mediating translational inhibition or cleavage of target transcripts. Arabidopsis mutants defective in miRNA biogenesis have overlapping and highly pleiotropic phenotypes including serrated leaves and ABA hypersensitivity. Recent evidence indicates that miRNA genes are transcribed by RNA polymerase II (Pol II). Since Pol II transcripts are capped, we hypothesized that CBP (cap-binding protein) 20 and 80 may bind to capped primary miRNA (pri-miRNA) transcripts and play a role in their processing. Here, we show that cbp20 and cbp80 mutants have reduced miRNA levels and increased pri-miRNA levels. Co-immunoprecipitation experiments revealed that pri-miRNAs 159, 166, 168 and 172 could be associated with CBP20 and CBP80. We found that CBP20 and CBP80 are stabilized by ABA by a post-translational mechanism, and these proteins are needed for ABA induction of miR159 during seed germination. The lack of miR159 accumulation in ABA-treated seeds of cbp20/80 mutants leads to increased MYB33 and MYB101 transcript levels, and presumably higher levels of these positive regulators result in ABA hypersensitivity. Genetic and molecular analyses show that CBP20 and 80 have overlapping function in the same developmental pathway as SE and HYL1. Our results identify new components in miRNA biogenesis.
[Show abstract][Hide abstract] ABSTRACT: Several positive regulators of phytochrome A signaling--e.g., LAF1, HFR1, and HY5--operate downstream from the photoreceptor, but their relative sites of action in the transduction pathway are unknown. Here, we show that HFR1RNAi/laf1 or hfr1-201/LAF1RNAi generated by RNA interference (RNAi) has an additive phenotype under FR light compared with the single mutants, hfr1-201 or laf1. This result indicates that LAF1 and HFR1 function in largely independent pathways. LAF1, an R2R3-MYB factor, interacts with HFR1, a basic helix-loop-helix (bHLH) factor, and this interaction is abolished by the R97A mutation in the LAF1 R2R3 domain. Polyubiquitinations of LAF1 and HFR1 by the COP1 E3 ligase in vitro are inhibited by LAF1/HFR1 association. Consistent with this result, endogenous HFR1 is less stable in laf1 compared with wild type, and similarly, LAF1-3HA expressed from a transgene is also less stable in hfr1-201 than wild type. In transgenic plants, HFR1 levels are significantly elevated upon induced expression of LAF1 but not LAF1(R97A). Moreover, induced expression of LAF1 but not LAF1(R97A) delays post-translational HFR1 degradation in FR light. Constitutive coexpression of HFR1 and LAF1 but not HFR1 and LAF1 (R97A) confers FR hypersensitivity in double transgenic plants. Our results show that in addition to their independent functions in phyA signaling, LAF1 and HFR1 also cooperate post-translationally to stabilize each other through inhibition of ubiquitination by COP1, thereby enhancing phyA photoresponses.
Genes & Development 09/2007; 21(16):2100-11. DOI:10.1101/gad.1568207 · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Upon activation by far-red light, phytochrome A signals are transduced through several pathways to promote photomorphogenesis. The COP1 E3 ligase represses photomorphogenesis in part by targeting transcription activators such as LAF1 and HY5 for destruction. Another positive regulator of photomorphogenesis is HFR1, a basic helix-loop-helix (bHLH) transcription factor. Here, we show that HFR1 colocalizes with COP1 in nuclear bodies, and that the HFR1 N-terminal region (amino acids 1-131) interacts with the COP1 WD40 domain. HFR1(DeltaN), an HFR1 mutant lacking the two N-terminal, COP1-interacting motifs, still localizes in nuclear bodies and retains weak affinity for COP1. Both HFR1 and HFR1(DeltaN) can be ubiquitinated by COP1, although with different efficiencies. Expression of 35S-HFR1(DeltaN) in wild-type plants confers greater hypersensitivity to FR than 35S-HFR1 expression, and only seedlings expressing 35S-HFR1(DeltaN) display constitutive photomorphogenesis. These phenotypic differences can be attributed to the instability of HFR1 compared with HFR1(DeltaN). In transgenic plants, HFR1 levels are significantly elevated upon induced expression of a dominant-negative COP1 mutant that interferes with endogenous COP1 E3 activity. Moreover, induced expression of wild-type COP1 in transgenic plants accelerates post-translational degradation of HFR1 under FR light. Taken together, our results show that HFR1 is ubiquitinated by COP1 E3 ligase and marked for post-translational degradation during photomorphogenesis.
Genes & Development 04/2005; 19(5):593-602. DOI:10.1101/gad.1247205 · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The composition and distribution of seed storage proteins are important factors for eating quality such as flavor and texture in rice. The levels of rice protein disulfide isomerase and rice chaperone binding protein proteins were examined in rice cultivars which have different eating qualities. Results showed that their levels have no direct correlation with eating quality.
Journal of the Korean Society for Applied Biological Chemistry 53(4). DOI:10.3839/jksabc.2010.077 · 0.54 Impact Factor