Unknown,Transcriptomics,Genomics,Proteomics

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Uncovering small RNA-mediated responses to phosphate-deficiency in Arabidopsis by deep sequencing

ABSTRACT: Deep sequencing of Arabidopsis thaliana small RNAs was conducted to reveal microRNAs (miRNAs) and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6-1.2 million unique sequence tags from each Pi-sufficient or -deficient root or shoot sample were mapped to the Arabidopsis genome. We showed that upon Pi deprivation, the expression of miR156, miR399, miR778 and miR827 was induced, whereas the expression of miR169, miR395 and miR398 was repressed. We found crosstalks coordinated by these miRNAs under different nutrient deficiencies. Moreover, we found a new miRNA family upregulated specifically by Pi deficiency. In addition to miRNAs, we identified one Pi starvation-induced DCL1-dependent small RNA derived from the long terminal repeat of a retrotransposon and a group of 19-nucleotide small RNAs corresponding to the 5' end of tRNA and expressed at a high level in Pi-starved roots. Interestingly, we observed an increased abundance of TAS4-derived trans-acting siRNAs (ta-siRNAs) in Pi-deficient shoots and uncovered an autoregulatory mechanism of PAP1/MYB75 via miR828 and TAS4-siR81(-) that regulates the biosynthesis of anthocyanin. This finding sheds light on the regulatory network between miRNA/ta-siRNA and its target gene. Of note, a substantial amount of miR399* accumulated under Pi deficiency. Like miR399, miR399* can move across the graft junction, implying a potential biological role for miR399*. This study represents a comprehensive expression profiling of Pi-responsive small RNAs and advances our understanding of the regulation of Pi homeostasis mediated by small RNAs. Keywords: Transcriptome analysis Examination of 4 samples from root and shoot tissues of Arabidopsis in either phosphate-sufficient or phosphate-deficient condition

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Li-Ching Hsieh

PROVIDER: E-GEOD-17741 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.

Hsieh Li-Ching LC Lin Shu-I SI Shih Arthur Chun-Chieh AC Chen June-Wei JW Lin Wei-Yi WY Tseng Ching-Ying CY Li Wen-Hsiung WH Chiou Tzyy-Jen TJ

Plant physiology 20091023 4

Recent studies have demonstrated the important role of plant microRNAs (miRNAs) under nutrient deficiencies. In this study, deep sequencing of Arabidopsis (Arabidopsis thaliana) small RNAs was conducted to reveal miRNAs and other small RNAs that were differentially expressed in response to phosphate (Pi) deficiency. About 3.5 million sequence reads corresponding to 0.6 to 1.2 million unique sequence tags from each Pi-sufficient or Pi-deficient root or shoot sample were mapped to the Arabidopsis ...[more]

PMID: 19854858

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Project description:MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHO2 is crucial for phosphate (Pi) acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ-based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis roots by mass spectrometry, 35.2 % of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, 5 were significantly differentially expressed between the wild-type and pho2 mutant under 2 growth conditions. Using immunoblot analysis, we validated the upregulation of several PHT1 Pi transporters and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests their roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the post-endoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research. Spectra Processing: The MS/MS raw spectra generated from the Waters Q-TOF Premier (the first replicate) and the AB SCIEX TripleTOF 5600 (the second and third replicates) instruments were processed to detect MS/MS peaks using UniQua (Chang et al., 2013). For the UniQua processing, the spectra were converted into mzXML format using massWolf (version 4.3.1) and ProteoWizard (version 3.0.4623) for the raw data generated from the Waters and AB SCIEX instruments, respectively. The UniQua parameters for Waters Q-TOF Premier were: smoothing = 9, centroiding high = 80%, maximum resolution = 16,000, baseline cutoff = 1.5 counts, reporter m/z range = 114-117, and reporter dynamic range = 5-1,000. The UniQua parameters for AB SCIEX TripleTOF 5600 were: smoothing = 11, centroiding high = 80%, maximum resolution = 25,000, baseline cutoff = 4 counts, reporter m/z range= 114-117 and reporter dynamic range = 5-100,000. The processed MS/MS spectra were then converted into Mascot generic format (.mgf) using mzXML2Search in Trans Proteomics Pipeline (TPP)1 (Deutsch et al., 2010) version 4.4 rev. 1. To obtain better quantification results, the MS/MS spectra with peak number less than 10, average intensity of the top 10 abundant peaks less than 15 counts and iTRAQ average intensity less than 30 counts were further removed from the peaklist file. Protein Qualification and Quantitation: The processed spectra were searched against the TAIR10 database (December 2011, 27,416 sequences) using the Mascot version 2.3 (Matrix Science, London, UK). For the MASCOT search, the mass tolerance for peptide precursor and MS/MS fragments was 0.1 Da. Only one missed cleavage was allowed for tryptic peptides. The methylthiolation (C), iTRAQ4plex (N-term) and iTRAQ4plex (K) were set as fixed modification. The Oxidation (M) and iTRAQ4plex (Y) were set as variable modification. The quantitation option was enabled and set to the iTRAQ4-plex approach. The peptide was considered to be identified if the MASCOT ion score greater than 27 (P less than 0.05). In addition, the protein was considered to be identified if the MASCOT protein score was greater than or equal to 27 with at least one unique and two different peptides identified. For the protein quantitation, only unique peptide was quantified in MASCOT and used to determine the protein ratio in MASCOT. To minimize the systematic ratio error due to sample preparation, the protein ratios were normalized by the mode of the overall protein ratios. For the data acquired by the Q-TOF Premier, 72,817 peptides and 3107 proteins were quantified. For the data acquired by the TripleTOF 5600, 41,885 peptides and 2893 proteins were quantified in the first replicate; 82,397 peptides and 4939 proteins were quantified in the second replicate.

Project description:Maize is a globally important food and feed crop, and a low-phosphate (Pi) supply in the soil frequently limits maize yield in many areas. MicroRNAs (miRNAs) play important roles in the development and adaptation of plants to the environment. In this study, the spatio-temporal miRNA transcript profiling of the maize inbred line Q319 root and leaf in response to low Pi was analyzed with high-throughput sequencing technologies, and the expression patterns of certain target genes were detected by real-time RT-PCR. Complex small RNA populations were detected after low-Pi culture and displayed different patterns in the root and leaf. miRNAs identified as responding to Pi deficiency can be grouped into ‘early’ miRNAs that respond rapidly, and often non-specifically, to Pi deficiency, and ‘late’ miRNAs that alter the morphology, physiology or metabolism of plants upon prolonged Pi deficiency. The miR827-Nitrogen limitation adaptation (NLA)-mediated post-transcriptional pathway was conserved in response to Pi availability of maize, but the miR399-mediated post-transcriptional pathway was different from Arabidopsis. Abiotic stress-related miRNAs engaged in interactions of different signaling and/or metabolic pathways. Auxin-related miRNAs (zma-miR393, zma-miR160a/b/c, zma-miR160d/e/g, zma-miR167a/b/c/d and zma-miR164a/b/c/d/g) and their targets play important roles in promoting primary root growth, inhibiting lateral root development and retarding upland growth of maize when subjected to low Pi. The changes in expression of miRNAs and their target genes suggest that the miRNA regulation/alterations compose an important mechanism in the adaptation of maize to a low-Pi environment; certain miRNAs participate in root architecture modification via the regulation of auxin signaling. A complex regulatory mechanism of miRNAs in response to a low-Pi environment exists in maize, revealing obvious differences from that in Arabidopsis. Maize (Zea mays L.) inbred line Q319 was used in this study. Seeds of the maize inbred line Q319 were surface sterilized and held at 28°C in darkness. Seedlings (4 days old) were transferred to a sufficient phosphate (SP, 1,000 μM KH2PO4) solution (Ca(NO3)2.4H2O 2 mM, NH4NO3 1.25 mM, KCl 0.1 mM, K2SO4 0.65 mM, MgSO4 0.65 mM, H3BO3 10.0 mM, (NH4)6Mo7O24 0.5 mM, MnSO4 1.0 mM, CuSO4.5H2O 0.1 mM, ZnSO4.7H2O 1.0 mM, Fe-EDTA 0.1 mM), allowed to grow for 4 days (plants with 2–3 leaves). After 2-3 days of re-culturing in SP nutrient solutions, half of the seedlings were transplanted into a low phosphate (LP, same composition as the SP solution, except that 5 μM KH2PO4 and 1 mM KH2PO4 were replaced with 1 mM KCl) nutrient solution. The plants were grown under a 32°C/25°C (day/night) temperature regime at a photon flux density (PFD) of 700 μmol m-2 s-1 with a 14 h/10 h light/dark cycle in a greenhouse with approximately 65% relative humidity. The roots and leaves were then harvested at 0, 1, 2, 4, 8 days and 8 days and cultured in SP solution (as a normal growth control) for small RNA analysis. The samples were frozen in liquid nitrogen immediately and stored at -80℃ for further analysis. Each biological repeat contains segments from 15~20 plants. Total RNA was extracted as previously described in Molecular Cloning (Sambrook and Russell David, 1989) and was then subjected to two additional chloroform washes prior to nucleic acid precipitation. The small RNA digitalization analysis based on HiSeq high-throughput sequencing takes the SBS-sequencing by synthesis. Then the 50nt sequence tags from HiSeq sequencing will go through the data cleaning first, which includes getting rid of the low quality tags and several kinds of contaminants from the 50nt tags. Length distribution of clean tags are then summarized. Afterwards, the standard bioinformatics analysis will annotate the clean tags into different categories and take those which can not be annotated to any category to predict the novel miRNA and base edit of potential known miRNA. Compare the known miRNA expression between two samples to find out the differentially expressed miRNA. The procedures are shown as below: (1)Normalize the expression of miRNA in two samples (control and treatment) to get the expression of transcript per million (TPM). Normalization formula: Normalized expression = Actual miRNA count/Total count of clean reads*1000000 (2)Calculate fold-change and P-value from the normalized expression. Then generate the log2ratio plot and scatter plot.

Dataset Information

Uncovering small RNA-mediated responses to phosphate-deficiency in Arabidopsis by deep sequencing

Publications

Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.

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