XB-ART-53580Elife. September 25, 2017; 6
A chloroplast retrograde signal, 3''-phosphoadenosine 5''-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination.
Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3''-phosphoadenosine 5''- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca(2+); up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.
PubMed ID: 28323614
PMC ID: PMC5406205
Article link: Elife.
Grant support: R01 GM060396 NIGMS NIH HHS
Genes referenced: aadat abi1 kyat1 rpn1 shroom1
Article Images: [+] show captions
|Figure 1—figure supplement 2. Enhanced ABA sensitivity in guard cells of sal1-8.(A) Stomatal aperture, calculated using measurements of pore width and length, in leaf peels of wild type and sal1-8 plants treated with 0, 1, 10, 50 and 100 µM for 1 hr. Values are means, expressed as a percentage compared to time 0, of 12–16 stomata ± SEM. Significant differences between genotypes and treatments are shown by a,b,c (2-way ANOVA and Tukey’s HSD, p<0.05). (B) Thermography of 35-day old wild type and sal1-8 intact plants sprayed with 0, 2.5, 10 or 100 µM ABA. Mean and SEM of leaf temperature from four individual plants per genotype are shown. Plants were returned to growth chamber and temperature measured after 2 hr. Significant differences between genotypes are treatments are shown by a,b (2-way ANOVA and Tukey’s HSD, p<0.05).DOI: http://dx.doi.org/10.7554/eLife.23361.004|
|Figure 2. Exogenous PAP interacts with ABA signaling and acts in stomatal closure in both Arabidopsis and barley.(A) Stomatal aperture, calculated using measurements of pore width and length, in leaf peels of wild type (ColLer) plants treated with either 100 µM PAP or 100 µM ABA over a period of 1 hr. Values are means, expressed as a percentage compared to t = 0 min, of at least 20 stomata ± SEM. Rates of closure were compared by modelling the closure between 10–25 min (log-transformed data), significant difference groups (p<0.05) are denoted by #, *. Final level of closure was also considered by ANOVA across the final 30 min; significant difference (p<0.05) denoted a, b, c. (B) Stomatal aperture in leaf epidermal peels of three-week old barley plants in measuring buffer (Control) for 10 min before adding 100 µM ABA or 100 µM PAP for another 50 min. Values are means ± SEM (n = 17–20 stomata of four plants). Significant difference (p<0.05) is denoted a, b. (C) Stomatal aperture as in (A) but treated with either 100 µM PAP or 1 mM ATP alone or in combination, in measuring buffer. Values are means of at least eight stomata ± SEM. The control treatment for (A), (B) and (C) was 1 hr of measuring buffer. (D) Thermography of 35-day old wild type leaves petiole fed with 250 µL of different combinations of 20 µM ABA, 100 mM LiCl, 1 mM PAP and 10 mM ATP in infiltration buffer or buffer alone (Control). Mean and SEM of leaf temperature from three leaves from three plants per genotype are shown. Leaves in solution were returned to growth chamber and temperature measured at indicated timepoints. Significant differences to control are shown (*p<0.05; **p<0.01). Also see Figure 2—figure supplement 1.DOI: http://dx.doi.org/10.7554/eLife.23361.006Figure 2—figure supplement 1. Exogenous PAP feeding to plant leaves via epidermal leaf peels or petiole feeding.Petiole feeding of PAP for 1 hr results in accumulation of PAP in leaves. Levels were significantly enhanced by co-application with LiCl, an inhibitor of the PAP catabolic enzyme SAL1, or with ATP, which outcompetes PAP for transport into plastids where PAP is degraded. ATP also allows PAP to be localized to its sites of action, the nucleus/cytoplasm. Results averaged from three individual plants ± SEM. a, b and c represent significant differences (p<0.05).DOI: http://dx.doi.org/10.7554/eLife.23361.007|
|Figure 3—figure supplement 2. Changes in gene expression of ABA receptors, PP2Cs and SnRKs in response to ABA.Hierarchical clustering of the expression of (A) ABA signalling gene sets in wild type, ost1-2, sal1-8 and ost1-2 sal1-8 ± ABA; (B) seven PYLs; (B) 52 PP2C genes; and (C) three SnRK2 kinases. For all hierarchical clustering analyses performed, gene expression was compared to wild type untreated of the four genotypes ± ABA. Scale = log2, blue lower and red higher expression. The scale has been condensed such that the red and blue colours at the end of the scale encompass all fold-changes greater or equal to 2, or less than or equal to 0.5, respectively.DOI: http://dx.doi.org/10.7554/eLife.23361.010|
|Figure 6. Exogenous PAP does not stimulate cytosolic Ca2+ transients in guard cells.Three representative Ca2+ oscillation patterns obtained from time-resolved Ca2+ imaging experiments with PAP treatment. Numbers of observed cells in each group are labeled above each graph (measurements were obtained from 10 different plants). Guard cells of YC3.6 plants were monitored for their FRET emission at 535 nm and 480 nm. FRET ratio increases after the addition of 10 mM Ca2+ suggesting the cells are capable of sensing intracellular Ca2+ level changes.DOI: http://dx.doi.org/10.7554/eLife.23361.014|
|Figure 7. Interaction between PAP-mediated signaling with Ca2+.(A) Wild type stomatal aperture with or without PAP in the presence of low (50 µM) Ca2+ or high (1 mM) Ca2+ in the measuring buffer. Means ± SEM for 9–10 stomata per treatment are shown. Significant differences between treatments at t = 60 min (ANOVA, p<0.05) are indicated by a, b. (B) Stomatal aperture in leaf peels treated with PAP and an intracellular calcium chelator (BAPTA-AM) or an extracellular calcium chelator (EGTA).Values are relative to control (measuring buffer). Means ± SEM for 18 stomata from four plants per treatment are shown. Significant differences between treatments at t = 60 min (ANOVA, p<0.05) are indicated by a, b. (C) Stomatal aperture in leaf peels treated with control (ethanol), 100 µM PAP or 10 µM ABA in the presence of low Ca2+ (50 µM). Means and SEM for four plants with >28 stomata per plant are shown. Significant differences between treatments (ANOVA, p<0.05) are indicated by a, b. (D) Stomatal aperture in leaf peels that were pretreated with 20 µM diphenyleneiodonium (DPI) prior to treatment with either 100 µM PAP or 100 µM ABA, in measuring buffer. The control treatment was leaf peels which were not pretreated with DPI and were treated with measuring buffer. Values are means ± SEM for 41–51 stomata per treatment. Final level of closure was considered by ANOVA after treatment; significant difference (p<0.05) denoted a, b.DOI: http://dx.doi.org/10.7554/eLife.23361.015|
|Figure 9. Model for fine-tuning of stomatal closure by PAP retrograde signaling.Proposed intersection between PAP and ABA signaling during drought stress, or in ost1-2 sal1-8 / abi1-1 sal1-8 treated with ABA. Binding of ABA to its receptors (PYR/PYLs) inactivates the inhibitory PP2Cs, thus allowing activation of OST1 for phosphorylation of proteins such as transcription factors and SLAC1. This is the major pathway for stomatal closure. Additionally, ABA signaling results in Ca2+ release which could activate multiple Ca2+ signaling proteins including CDPKs and CBLs/CIPKs thus allowing phosphorylation and activation of SLAC1. Many of these ABA and Ca2+ signaling proteins can be regulated by PAP predominantly via PAP-XRN-mediated retrograde signaling. The CDPK activation of SLAC1 can occur in parallel with Ca2+-independent OST1, allowing a convergence between chloroplast and ABA signaling at this key anion channel for stomatal closure. It is possible that PAP can also regulate stomatal closure through other proteins, though there is as yet no evidence for this in plants. The relative contributions of each pathway towards control of stomatal closure are indicated by the thickness of the arrows and lines. Solid lines and arrows indicate characterized pathways. Signaling pathways which have not been fully studied are indicated with dashed lines and ‘?'.DOI: http://dx.doi.org/10.7554/eLife.23361.018|