Arabidopsis miR156 Regulates Tolerance to Recurring Environmental Stress through SPL Transcription Factors

Abstract

Plants are sessile organisms that gauge stressful conditions to ensure survival and reproductive success. While plants in nature often encounter chronic or recurring stressful conditions, the strategies to cope with those are poorly understood. Here, we demonstrate the involvement of ARGONAUTE1 and the microRNA pathway in the adaptation to recurring heat stress (HS memory) at the physiological and molecular level. We show that miR156 isoforms are highly induced after HS and are functionally important for HS memory. miR156 promotes sustained expression of HS-responsive genes and is critical only after HS, demonstrating that the effects of modulating miR156 on HS memory do not reflect preexisting developmental alterations. miR156 targets SPL transcription factor genes that are master regulators of developmental transitions. SPL genes are posttranscriptionally downregulated by miR156 after HS, and this is critical for HS memory. Altogether, the miR156-SPL module mediates the response to recurring HS in Arabidopsis thaliana and thus may serve to integrate stress responses with development.

Figure 1.

Representation of Different Types of Thermotolerance, Their Assay Conditions, and Schematic Representation of the Thermotolerance Profile of a Mutant with a Specific Defect in the Maintenance of Acquired Thermotolerance (i.e., HS Memory). (A) Basal thermotolerance. ‘, minutes; t, time. (B) Acquisition of thermotolerance. (C) Maintenance of acquired thermotolerance (HS memory) is assayed by a tester HS of 80 to 120 min 2 or 3 d after a priming HS (ACC), which induced thermotolerance. (D) Schematic representation of the temporal profile of acquired thermotolerance in the wild type and a mutant with a specific defect in its maintenance (such as hsfa2). There is no defect in the acquisition of thermotolerance (b), but a clear difference (see double-pointed arrow) in its maintenance (c).

Figure 2.

The miRNA Pathway Is Required for the Maintenance of Acquired Thermotolerance. (A) ago1-25 and ago1-27 mutants were assayed for the maintenance of acquired thermotolerance by applying a tester HS two (+2, 110 min) or three (+3, 90 min) days after a priming HS (ACC); hsfa2 was included as a control. Photographs were taken 14 d after ACC. All plants of one treatment were grown on the same plate. One representative replicate of at least three independent biological replicates is shown. For quantification, see (B) and (C). ACC, priming HS. (B) Quantification of data shown in (A); average fresh weight from 59 ± 14 seedlings was determined 14 d after ACC. Due to very low weight, d+3 samples were not analyzed. Instead, ACC + HS d+2 90 min samples were included, which were omitted in (A) due to space constraints. (C) Quantification of data shown in (A); the percentage of seedlings in different phenotypic classes was determined for different genotypes and heat treatments. Gray arrows indicate the corresponding plates from (A). Panels are (top to bottom): ACC, ACC + HS d+2 110’, ACC + HS d+3 90’. Two photographs with representative seedlings of each class are shown to illustrate the damage classes. Asterisk, significantly different distribution compared with Col-0 (Fisher’s exact test, *P < 0.05; **P < 0.005). (D) dcl1-9 mutants show reduced maintenance of acquired thermotolerance (Fisher’s exact test, P < 0.001). Individual seedlings were phenotypically categorized 14 d after a ACC + HS d+2 90’ treatment as green, weak (bleached out cotyledons but green hypocotyl and meristem), and dead and genotyped for dcl1-9 (n = 78).

Figure 3.

The Effect of ago1-25 on the Transcriptome Is Most Pronounced during the Late Stages of the HS Response. (A) Venn diagram representation of ATH1 GeneChip transcriptome profiling of Col-0 samples after a priming HS (ACC). Samples were harvested 4 or 52 h after the end of the ACC; the number of genes that are up- or downregulated at either 4 or 52 h or at both time points relative to a 4 h ntc are shown. (B) Experimental validation of the indicated HS memory–related genes in Col-0 (black bars) and ago1-25 (white bars) over a detailed time course after ACC or mock treatment using qRT-PCR. Expression values were normalized to TUB6 and to Col-0 0 h ntc [(GENE OF INTEREST/TUB6)_x/(GENE OF INTEREST/TUB6)_{Col0 h ntc}]. Error bars are se of the mean of three replicates. One representative of at least three independent experiments is shown. (C) Percentage of genes whose induction or repression differed at least 2-fold in ago1-25 compared with Col-0 in the classes described in (A) or among all remaining genes on the microarray (all others), respectively.

Figure 4.

Primary MIR Profiling and Transcript Levels of HS-Responsive miRNAs during Maintenance of Acquired Thermotolerance. (A) Venn diagram representation of primary MIR transcript levels after a priming HS (ACC); samples were taken 4 or 52 h after the end of ACC. Up- or downregulated pri-miRNAs at either 4 or 52 h or at both time points are displayed. In blue are the miRNAs further analyzed in (B) and (C). (B) qRT-PCR of the indicated primary MIR transcripts. (C) qRT-PCR of the indicated mature miRNAs. Expression values were normalized to TUB6 and the respective 4 h ntc [(GENE OF INTEREST/TUB6)_x/(GENE OF INTEREST/TUB6)_{4 h ntc}]. Black and white bars indicate HS-treated (ACC) and control (ntc) samples, respectively. Error bars are se of the mean of three replicates. One representative of at least three independent experiments is shown. [See online article for color version of this figure.]

Figure 5.

Levels of Mature (Spliced) and Unspliced SPL Transcripts during the Maintenance of Acquired Thermotolerance and Their Dependency on Functional AGO1 and the miR156 Binding Site. (A) Mature (top panel) and unspliced (middle panel) SPL2, SPL9, and SPL11 transcript levels relative to TUB6 (SPLn/TUB6)_x in Col-0 and ago1-25 at the indicated times after a priming HS. Transcript level for the mature but not the unspliced SPL2, 9, and 11 was increased in ago1-25. Bottom panel: fold reduction of SPL transcript levels relative to the 0 h control. The repression depends on functional AGO1 and is evident for the mature mRNAs, but not the unspliced mRNAs, indicating that regulation occurs at the posttranscriptional level. The color code below the third panel applies to all panels in (A). Error bars are se of the mean of three replicates. The experiment was repeated three times independently, and one representative is shown. (B) Reduction of SPL2 and SPL11 mRNA levels after HS is dependent on an intact miR156 binding site. ProSPL2:rSPL2 and ProSPL11:rSPL11 plants were subjected to a priming HS or no treatment (ntc) and harvested directly after the end of the treatment. Levels of the indicated endogenous SPL or rSPL transcripts were determined and the ACC/ntc ratio calculated. Error bars are se of the mean of three replicates. The experiment was repeated three times independently, and one representative is shown. (C) GUS protein activity after a priming HS in ProSPL9:SPL9-GUS and ProSPL9:rSPL9-GUS as determined by MUG activity assay. Activity relative to the respective ntc and normalized to the 0-h time point averaged over three independent experiments ± se indicates that miR156 represses SPL9-GUS protein levels after HS.

Figure 6.

miR156 Regulates the Maintenance of Acquired Thermotolerance. (A) Knockdown of miR156a-f in Pro35S:MIM156 impairs HS memory, assayed by applying a tester HS two (+2) or three (+3) days after a priming HS (ACC). Photographs were taken 14 d after ACC. (B) Quantification of data shown in (A); the percentage of seedlings in different phenotypic classes was determined for different genotypes and heat treatments. Gray arrows indicate the corresponding plates from (A). Panels are (top to bottom): ACC, ACC + HS d+2 110’, and ACC + HS d+3 90’. Damage classes are described in (D) and Figure 2C. (C) Overexpression of miR156h enhances HS memory, assayed by applying a tester HS two (+2) or three (+3) days after a short ACC (37°C, 60’). Photographs were taken 14 d after ACC. (D) Quantification of data shown in (C); the percentage of seedlings in different phenotypic classes was determined for different genotypes and heat treatments. Gray arrows indicate the corresponding plates from (C). Panels are (top to bottom): short ACC, short ACC + HS d+2 60’, and short ACC + HS d+3 45’. Damage classes are described below panels and in Figure 2C. (E) Quantification of (C); average seedling fresh weight was determined from 27 ± 1 seedlings per genotype and treatment. All plants of one treatment were grown on the same plate. The experiments were repeated at least five times with at least two independent transgenic lines with similar results. Asterisks indicate significantly different distribution compared with Col-0 (Fisher’s exact test, *P < 0.05; **P < 0.005).

Figure 7.

Downregulation of SPL2 Is Required for the Maintenance of Acquired Thermotolerance and Sustained Expression of HS Memory–Related Genes. (A) ProSPL2:rSPL2 seedlings were assayed for the maintenance of acquired thermotolerance by applying a tester HS (HS) two (+2) or three (+3) days after a priming HS (ACC). Pictures were taken 14 d after ACC. All plants of one treatment were grown on the same plate. The experiments were repeated at least three times with two independent transgenic lines with similar results. (B) Quantification of data shown in (A); the percentage of seedlings in different phenotypic classes was determined for different genotypes and heat treatments. Gray arrows indicate the corresponding plates from (A). Panels are (top to bottom): ACC, ACC + HS d+2 115’, and ACC + HS d+3 80’. Damage classes are illustrated in Figure 2C. Asterisks indicate significantly different distribution compared with Col-0 (Fisher’s exact test, **P < 0.005). (C) Expression of selected HS memory–related genes during maintenance of acquired thermotolerance determined by qRT-PCR in Col-0 (black) and ProSPL2:rSPL2 (gray). Expression values were normalized to TUB6 and 48 h ntc [(GENE OF INTEREST/TUB6)_x/(GENE OF INTEREST/TUB6)_{48 h ntc}] and are displayed on a log₁₀ axis for better resolution. Error bars are se of the mean of three technical and two biological replicates.

Figure 8.

miR156 Is Required after HS to Regulate the Maintenance of Acquired Thermotolerance. All plants of one treatment were grown on the same plate. The experiments were repeated at least three times with two independent transgenic lines with similar results. Asterisks indicate significantly different distribution compared with Col-0 (Fisher’s exact test, *P < 0.05; **P < 0.005). (A) Heat-inducible knockdown of miR156h (ProHSP21:MIM156h) impairs the HS memory, assayed by applying a tester HS (HS) two (+2) or three (+3) days after a priming HS (ACC). Photographs were taken 14 d after ACC. For quantification, see (B) and (C). (B) Quantification of data shown in (A); the percentage of seedlings in different phenotypic classes was determined for different genotypes and heat treatments. Gray arrows indicate the corresponding plates from (A). Panels are (top to bottom): ACC, ACC + HS d+2 110’, and ACC + HS d+3 80’. Damage classes are illustrated in Figure 2C. (C) Quantification of (A); average seedling fresh weight was determined from 32 ± 3 seedlings per genotype and treatment. (D) Heat-inducible overexpression of MIR156h (ProHSP21:MIR156h) enhances the HS memory, assayed by applying a tester HS two (+2) or three (+3) days after a priming HS (ACC). Photographs were taken 14 d after ACC. For quantification, see (E) and (F). (E) Quantification of data shown in (D); the percentage of seedlings in different phenotypic classes was determined for different genotypes and heat treatments. Gray arrows indicate the corresponding plates from (D). Panels are (top to bottom): ACC, ACC + HS d+2 110’, and short ACC + HS d+3 80’. Damage classes are illustrated in Figure 2C. (F) Quantification of (D); average seedling fresh weight was determined from 24 ± 3 seedlings per genotype and treatment. (G) Mature miRNA levels of miR156a-f and miR156h in ProHSP:MIR156h and ProHSP:MIM156h determined by qRT-PCR relative to TUB6 (miR156h/TUB6)_x. A several hundred fold induction of miR156h is observed in ProHSP:MIR156h plants after ACC, while no or only a very slight effect is observed for miR156a-f. Error bars are se of the mean of three replicates. Three independent experiments were performed with one representative shown. (H) Expression of HS memory–related genes determined by qRT-PCR is increased after a priming HS in ProHSP:MIR156h. Expression values were normalized to TUB6 and 48 h ntc [(GENE OF INTEREST/TUB6)_x/(GENE OF INTEREST/TUB6)_{48 h ntc}]. Error bars are se of the mean of three replicates. Three independent experiments were performed with one representative shown.