Two-wave induction of the phosphate starvation response.

<p>(A) Schematic illustration of the phosphate starvation response: Reduction in internal phosphate leads to the nuclear localization of the transcription factor Pho4 and the induction of its target genes. Genes induced by Pho4 include high-affinity transporters (e.g., <i>PHO84</i>) and an inhibitor of low-affinity transporters (<i>SPL2</i>), genes involved in phosphate mobilization in and out of vacuole storage as PolyP (e.g., <i>PHM3</i>), and phosphatases, which scavenge phosphate from molecules inside or outside the cell. (B) Cells grow for a limited number of generations following transfer to media containing different low phosphate levels: Shown is the temporal increase in cellular density (measured by optical density [OD]) following transfer to low-phosphate media, as indicated (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a>). The data are the mean and standard error of 2 replicates. (C–D) Sequential induction of the phosphate transcription response: Wild-type (WT) cells were transferred from rich medium into media containing the indicated low Pi level and followed for 24.7 hours (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a>). Samples for RNA sequencing were taken at the indicated time points. Shown in (C) is the log₂(expression) change in Pho4-target genes, stress response genes (Stress), and genes coding for ribosomal proteins (Protein Synthesis), as defined in [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref020" target="_blank">20</a>] (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2 Data</a>). Data were normalized as described in the Materials and methods, and values under detectable levels (not a number [NaN]) are depicted in grey. Vertical lines indicate the second transcription wave, as defined by clustering (using k-means) of the Pearson correlation matrix, shown in (D). Note that when transferred to 0.06 mM Pi, cells transiently induced the second wave approximately 3–4.5 hours following the transfer, followed by a stable induction at approximately 7 hours after the transfer. This transient induction is likely due to the double-feedback design of the system, as we showed [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref021" target="_blank">21</a>]. The reduction in growth rate (see E–F below) is observed already with first induction of the second transcription wave. The data in (C–D) are from 1 replicate. Additional biological (and experimental) replicates for growth in 0.06 mM and 0.2 mM Pi are found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g003" target="_blank">Fig 3C</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1C Fig</a>, respectively, and for 0 mM Pi, see [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref021" target="_blank">21</a>]. For further supporting data for (C), see (E). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1A and S1B Fig</a> for definition of Pho4-target genes. (E–F) Phosphate becomes growth limiting concomitant with the induction of the second transcription wave: The PHO84p-Venus reporter is up-regulated following transfer of cells to low-phosphate media, as indicated (E). The cell growth rate was calculated by the logarithmic slope of the OD curve, shown in (B), and is plotted as a function of reporter expression (F). To control for density-dependent effects, the growth rate was normalized by that of cells transferred to rich phosphate medium (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#sec010" target="_blank">Materials and methods</a>). The data in (E–F) are the mean and standard error of 2 replicates. Note that growth rate begins to decrease when the PHO84p-Venus reporter crosses a given threshold, independently of the incubating conditions. This crossing of the activation threshold coincides with the time at which the second transcription wave is induced, as defined by clustering (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1E Fig</a>). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1D Fig</a> for the normalized growth rate as a function of time. The raw data for (B, E–F) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s008" target="_blank">S1 Data</a>, and those for (C–D) are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2 Data</a>. Pi, inorganic phosphate; WT, wild type.</p

Lab evolution selects for <i>PHO84</i> mutations that rescue the recovery phenotype of PHO84^C.

<p>(A) Experimental scheme: 16 independent lines expressing constitutively high levels of <i>PHO84</i> were subjected to 10 cycles of alternated growth in high and low phosphate for a total of 300 generations. (B) <i>PHO84</i> mutations identified in selected lines: Single-cell isolates (evolved for approximately 150 and approximately 300 generations) were selected from the 10 cultures showing improved recovery and were sent for whole-genome sequencing. All isolates showing an adaptive phenotype contained mutations in the Pho84 open reading frame (ORF), with 56 out of the 60 identified mutations mapped to the same amino-acid substitution (L74F). (C–E) Selected <i>PHO84</i> mutation rescues the recovery phenotype: The <i>PHO84</i> mutations identified in the selected stains were introduced into the wild-type allele and their effect on the growth in low Pi and on the recovery phenotype was examined when <i>PHO84</i> was expressed at wild-type levels or constitutively (both the constitutive overexpression and the evolved mutations were introduced into <i>PHO84</i> endogenous site). Shown is the recovery of the constitutive strain with and without the amino-acid substitution (Pho84-L74F(ttG/ttC)) following 24.7 hours incubation in 0.2 mM Pi (C, D) and the corresponding pho4-target gene expression during recovery (E). Note the rescue of the recovery phenotype and the immediate down-regulation of pho4-target genes. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s006" target="_blank">S6B–SBJ Fig</a> for the growth in low-phosphate media and recovery phenotypes of the selected mutations: Pho84-L74F(ttG/ttC), Pho84-L259P, and Pho84-V383L. The data in (C) are from 1 replicate; see (D) for additional supporting data. The data in (D) are the mean and standard error of 3 replicates. The data in (E) are from 1 replicate. Additional data as in (E) for recovery (from 0.06 mM Pi) are found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s006" target="_blank">S6E Fig</a>. The conclusions of (E) are further supported by experiments with the Venus reporter (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>). The raw data for (C–D) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>. The raw data for (E) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2 Data</a>. Gen, generations; NaN, not a number; Pi, inorganic phosphate; WT, wild type.</p

Dual role of starvation signaling in promoting growth and recovery

<div><p>Growing cells are subject to cycles of nutrient depletion and repletion. A shortage of nutrients activates a starvation program that promotes growth in limiting conditions. To examine whether nutrient-deprived cells prepare also for their subsequent recovery, we followed the transcription program activated in budding yeast transferred to low-phosphate media and defined its contribution to cell growth during phosphate limitation and upon recovery. An initial transcription wave was induced by moderate phosphate depletion that did not affect cell growth. A second transcription wave followed when phosphate became growth limiting. The starvation program contributed to growth only in the second, growth-limiting phase. Notably, the early response, activated at moderate depletion, promoted recovery from starvation by increasing phosphate influx upon transfer to rich medium. Our results suggest that cells subject to nutrient depletion prepare not only for growth in the limiting conditions but also for their predicted recovery once nutrients are replenished.</p></div

Constitutive activation of Pho4-target genes impacts cell growth.

<p>(A–D) Constitutive expression Pho4-dependent genes promote growth at low phosphate: Δ<i>pho80</i> cells and cells expressing the constitutive PHO4^SA12346 allele were mixed with wild-type cells (either wild-type or mutant cells were mCherry labeled, respectively) and incubated in media containing different low-phosphate levels, as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g002" target="_blank">Fig 2A</a>. The (log₂) relative abundance of Δ<i>pho80</i> cells in the mixture at different times following the incubation is shown in (A) as a function of time (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s003" target="_blank">S3D and S3E Fig</a> for PHO4^SA12346 and PHO4^SA1234 cells). (B) and (D) depict the growth rate of Δ<i>pho80</i> and PHO4^SA12346 cells as a function of reporter expression (PHO84p-Venus) in the wild-type cells, respectively. Note that, independently of the incubation conditions, the mutant cells begin to outcompete the wild-type cells when reporter activation in wild-type cells crosses the same threshold value at which wild-type cells become limiting for growth (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g001" target="_blank">Fig 1F</a>), Δ<i>pho4</i> deleted cells begin to be outcompeted by wild-type cells (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g002" target="_blank">Fig 2C</a>), and wild-type cells activate the second wave of Pho4-target gene expression (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1E Fig</a>). The data in (A–B) are the mean and standard error of 3 replicates. (C) Temporal gene expression in cells expressing a constitutive Pho4 allele (PHO4^SA12346), as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g001" target="_blank">Fig 1C</a>. Note that PHO4^SA12346 strongly expresses the Pho4-target genes even in high Pi levels, which is consistent with previous reports [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.ref019" target="_blank">19</a>]. The data in (C) are from 1 replicate, but see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s003" target="_blank">S3B and S3C Fig</a> for related data showing equivalent behavior upon transfer into low Pi of 0.2 mM and 0.5 mM, respectively. (E–G) The growth phenotype of the Pho4-constitute strain depends on the mobilization of phosphate into storage: (E–F) same as (D), for the indicated strains. The data in (D–F) display 1 replicate; see (G) for additional supporting data. (G) The experiments in (D–F) were repeated using a variety of starvation media, and the fitness difference of mutant and wild-type cells was measured following approximately 24 hours incubation in low-Pi media. This fitness difference is shown as a function of the initial phosphate level in the starvation media. The data in (G) are the mean and standard error of 3 replicates. As described, Phm3 is a subunit of the vacuolar transporter chaperone complex, required for the storage of phosphate as polyP in the vacuoles. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4E–S4I Fig</a> for recovery of the above mutants from low Pi. The raw data for (A–B and D–F) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>. The raw data for (C) and (G) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s012" target="_blank">S5</a> Data, respectively. NaN, not a number; Pi, inorganic phosphate; WT, wild type.</p

The starvation program promotes growth in low phosphate.

<p>(A) Experimental scheme: Cells deleted of <i>PHO4</i> were mixed with fluorescently labeled wild-type cells and were coincubated in different low-Pi media. Samples were taken at different times following the incubation and analyzed using flow cytometry to define the relative fraction of Δ<i>pho4</i> cells (cells not expressing mCherry) and the activation of the starvation program reporter PHO84p-Venus in individual cells. (B–C) The early phase of Pho4-target gene induction does not contribute to cell growth: The relative abundance of Δ<i>pho4</i> cells in the mixed culture is shown as a function of time (B) and as a function of the PHO84p-Venus reporter induction in wild-type cells (C). The data in (B–C) are the mean and standard error of 2 replicates. The raw data are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>. Note that, regardless of the inoculation conditions, Δ<i>pho4</i> cells begin to reduce in frequency when the reporter level increases beyond the same activation threshold at which wild-type cells begin to reduce their growth (compare <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g001" target="_blank">Fig 1F</a>) and induce the second wave of Pho4-target gene expression (compare <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s001" target="_blank">S1E Fig</a>). See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s002" target="_blank">S2C–S2E Fig</a> for the temporal induction of the reporter and a similar analysis of <i>Δpho81</i> cells. Pi, inorganic phosphate; WT, wild type.</p

The starvation program promotes recovery once phosphate is replenished.

<p>(A) Recovery time depends on the conditions leading to starvation: Cells were incubated in media containing different low phosphate levels and maintained in the starvation conditions for 21 hours before being transferred back into a rich medium. The number of generations that cells underwent 3 or 5.7 hours after the transfer to rich medium is shown as a function of the phosphate level in the incubating media. The data in (A) are the mean and standard error of 2 replicates. (B) Pho4-target genes are rapidly down-regulated when phosphate is replenished: Recovery expression profiles of cells grown for 24.7 hours in low-Pi media (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g001" target="_blank">Fig 1C</a>) and recovered in rich medium. The data in (B) are from 1 replicate. Additional replicates for recovery from 0.2 mM and 0.06 mM Pi are found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g006" target="_blank">Fig 6E</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s006" target="_blank">S6E Fig</a>, respectively. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a> for related experiments using a reporter gene. (C) The starvation program promotes recovery: Competition experiments, as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g002" target="_blank">Fig 2</a>, were performed to compare the growth of Δ<i>pho4</i> and wild-type cells following recovery in rich medium. Wild-type and Δ<i>pho4</i> cells were coincubated in media containing different levels of low phosphate, grown to saturation, and maintained in the starvation conditions for 24.7 hours before being transferred back into rich medium. The (log₂) relative abundance of Δ<i>pho4</i> cells at different times following return to rich medium is shown. Note that Δ<i>pho4</i> cells are outcompeted by wild-type cells during recovery from starvation that was induced by incubation in media containing intermediate phosphate (e.g., 0.2 mM Pi). These conditions introduce the longest time gap between the first and second wave of Pho4-target gene induction (“preparation” time). The data in (C) are the mean and standard error of 2 replicates. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4A Fig</a> for similar experiments using Δ<i>pho81</i> deleted cells. (D, E) Constitutive expression of PHO84 partially rescues the recovery phenotype of Δpho4 cells: Experiments as shown in (C) were repeated for cells that constitutively express the PHO84 transporter (PHO84^C) in a wild-type or Δ<i>pho4</i> background. The (log₂) relative abundance of mutant cells during recovery from starvation induced by incubating in media containing 0.2 mM Pi is shown in (D). In (D), the mean of 2 replicates is shown for Δ<i>pho4</i>, and 1 replicate is shown for PHO84^C with and without Δ<i>pho4</i>. These data are supported by the experiments shown in (E) and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.g005" target="_blank">Fig 5B</a>. (E) The experiments in (D) were repeated using a variety of starvation media, and the fitness difference of mutant and wild-type cells was measured following 24.7 hours of incubation in rich medium. This fitness difference is shown as a function of the initial phosphate level in the starvation media. Note that deletion of <i>PHO4</i> impaired recovery when cells were first incubated at intermediate phosphate levels (around 0.2 mM Pi), allowing sufficient preparation time, and that constitutive expression of <i>PHO84</i> partially rescued this phenotype. The data in (E) are the mean and standard error of 3 replicates. See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4B Fig</a> for similar experiments using Δ<i>pho81</i> cells. (F–I) Phosphate influx limits recovery from starvation: Competition experiments as in (C) were repeated for cells of the indicated genotype (F–I). Shown are the (log₂) relative abundance of the indicated mutant strains (F–G) and the level of the PHO84p-Venus reporter in the mutants and wild-type cells (H–I) during competitive growth. In (F, H) are shown several time points within the first 10 hours of recovery (into 20 mM Pi), and those after 9 hours of recovery into 20 mM Pi are shown in (G, I). The data in (F, H) are from 1 replicate and are supported by data in (G, I), which show the mean and standard error of 2 replicates. See (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s004" target="_blank">S4C and S4D Fig</a>) for recovery into a high Pi of 7.3 mM Pi. The raw data for (C, D, and F–I) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s011" target="_blank">S4 Data</a>. The raw data for (A, B, and E) are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s008" target="_blank">S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s009" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002039#pbio.2002039.s012" target="_blank">S5</a> Data, respectively. NaN, not a number; Pi, inorganic phosphate; WT, wild type.</p

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in <i>Arabidopsis thaliana</i> with Tiling Microarray

<div><p>Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix <i>Arabidopsis</i> tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression.</p></div

Flowchart of the algorithm used for detecting stress-regulated genes and alternative splicing (see text).

<p>Flowchart of the algorithm used for detecting stress-regulated genes and alternative splicing (see text).</p

Defining splicing type according to probes expression level.

<p>The common forms of alternative splicing represented here are; exon skipping, intron retention, alternative 3' acceptor site and alternative 5' donor site. Boxes joined by lines represent the exons and introns, respectively, of immature transcripts; diagonal lines indicate splicing patterns. Highly expressed probes are depicted as short thick bars while probes with low expression are depicted as short thin bars below the transcript. The splicing variant (right side) is defined as intron retention when all probes in an intron are significantly highly expressed, i.e., have near exon level expression. All other cases of altered intron probes expression are defined as unknown, as the splicing variant can include exon skipping, intron retention or alternative 5' or 3'.</p

A sample list of detected transcripts with putative stress-regulated alternative splicing.

<p>The listed genes were used for validation of predicted cold-regulated alternative splicing events by RT-PCR. All genes, except AT1G47530, were not defined as cold responsive based on showing less than 2 and more than -2 fold change (FDR <0.05).</p>a<p>Example of cold responsive gene.</p>b<p>Alternative splicing types are intron retention (r) or unknown (u, possible exon skipping, alternative 5'/3' or intron retention).</p>c<p>The alternatively spliced intron is not completely excised in either cold treatment or control (ctrl).</p>d<p>Predictions that were confirmed by RT-PCR.</p