PRJNA215013: Spatio-Temporal Transcript Profiling of Rice Roots and Shoots in Response to Phosphate Starvation and Recovery
Summary: Using rice (Oryza sativa) as a model crop species, we performed an in-depth temporal transcriptome analysis, covering the early and late stages of Pi deprivation as well as Pi recovery in roots and shoots, using next-generation sequencing. Analyses of 126 paired-end RNA sequencing libraries, spanning nine time points, provided a comprehensive overview of the dynamic responses of rice to Pi stress. Differentially expressed genes were grouped into eight sets based on their responses to Pi starvation and recovery, enabling the complex signaling pathways involved in Pi homeostasis to be untangled. A reference annotation-based transcript assembly was also generated, identifying 438 unannotated loci that were differentially expressed under Pi starvation. Several genes also showed induction of unannotated splice isoforms under Pi starvation. Among these, PHOSPHATE2 (PHO2), a key regulator of Pi homeostasis, displayed a Pi starvation–induced isoform, which was associated with increased translation activity. In addition, microRNA (miRNA) expression profiles after long-term Pi starvation in roots and shoots were assessed, identifying 20 miRNA families that were not previously associated with Pi starvation, such as miR6250. In this article, we present a comprehensive spatio-temporal transcriptome analysis of plant responses to Pi stress, revealing a large number of potential key regulators of Pi homeostasis in plants.
Overall Design: To better understand the complex mechanisms regulating Pi homeostasis in rice (Oryza sativa L. cv. Nipponbare), a time course experiment was performed, where pre-germinated seedlings were grown hydroponically for two weeks on Pi-sufficient medium (0.32 mM Pi), before transferring half of the plants to Pi-deficient solution (0 mM Pi) for 21 days (d) . After three weeks of Pi-starvation treatment, half of these plants where then re-supplied with Pi sufficient media for up to 24 hours (h). In total, nine time points were selected in order to cover short and long term responses to Pi starvation as well as the effects of Pi re-supply on Pi starved plants.
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Hydroponic experiments were performed under controlled conditions (day/night temperature of 30/22°C and a 12-h photoperiod, 200 µmol photons m-2 s-1), allowing 0.5 liters of hydroponic solution per plant. The hydroponic solution consisted of a modified solution as described by Yoshida et al. (1976), containing 1.425 mM NH4NO3, 0.513 mM K2SO4, 0.998 mM CaCl2, 1.643 mM MgSO4, 0.075 µM (NH4)6Mo7O24, 0.25 mM NaSiO3, 0.009 mM MnCl2, 0.019 µM H3BO3, 0.155 µM CuSO4, 0.152 µM ZnSO4, and 0.125 mM EDTA-Fe, with or without 0.323 mM NaH2PO4, resulting in the +Pi and -Pi conditions. The pH of the solution was adjusted to 5.5, and the solution was renewed every 3 d. |
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Rice seeds were first pregerminated in tap water for 2 d before being transferred into the hydroponic solution, containing 0.323 mM Pi (+Pi) for 2 weeks. Half of the seedlings were then transferred to a solution lacking Pi (0 mM Pi) for 21 d before being resupplemented with 0.323 mM Pi for 24 h, while the other half of the seedlings continuously remained in +Pi conditions (control). During the resupply experiment, some rice seedlings were left in Pi-deficient media to serve as control. Roots and shoots were harvested separately at the following time points: 1 h, 6 h, 24 h, 3 d, 7 d, and 21 d after transfer to –Pi conditions, as well as 1, 6, and 24 h after Pi resupply. Furthermore, all experiment procedures such as media replacement and sample collection were performed at a similar time of the day (2 h after light) to minimize possible circadian effects. |
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The total RNA from the roots and shoots tissues was extracted using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. |
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For RNAseq library synthesis (three biological replicates per condition and one plant per replicate), total RNA was first depleted of rRNA using the Ribo-Zero rRNA removal kit (Plant Leaf and Plant Seed/Root kits; Epicentre). To do so, 1 µg of total RNA from root samples was used as input for rRNA removal, while 2 µg of total RNA was used for shoot samples. Sequencing libraries were generated using the TruSeq RNA sample prep kit (Illumina). An average of eight libraries were multiplexed and loaded on each lane of the Illumina Hiseq flow cell v3. Sequencing was then performed on a Hisequation 1000, as a 2 × 101 paired-end run, according to the manufacturer’s instruction (Illumina). |
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Unspecific |
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Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery.
The Plant cell . 2013-11-18 [PMID:
24249833]
