Author response: A chloroplast retrograde signal, 3’-phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination (2017)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 Ca2+; 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. https://doi.org/10.7554/eLife.23361.001 Introduction Organelles such as chloroplasts and mitochondria can regulate nuclear gene transcription via several signaling pathways in a process called retrograde signaling (Chi et al., 2013; Chan et al., 2016a). While this has been conventionally viewed as a bilateral communication to optimize organelle function and/or repair, there is emerging evidence that retrograde signaling contributes to multiple cellular processes and complex whole-plant traits including programmed cell death, drought tolerance, biotic stress tolerance, light-regulated seedling development and flowering (Chi et al., 2013; Chan et al., 2016a; Feng et al., 2016; Kleine and Leister, 2016). Interestingly, specific role(s) have never been examined for any chloroplast retrograde signal identified to date in relation to drought tolerance and abscisic acid (ABA) -mediated signaling in specialized cells such as guard cells surrounding stomata. The hormone ABA mediates signaling pathways that regulate stomatal closure and seed germination. The timing of seed germination needs to be coordinated with favorable environmental conditions to ensure seedling viability, while stomata are the gateways for gas exchange and water loss in leaves and thus closure mediated by guard cells is one of the most important and immediate avoidance responses to drought stress in plants (Murata et al., 2015). Intriguingly, although regulation of stomatal closure by ABA directly impacts on photosynthesis and chloroplast function (Yamburenko et al., 2015), how and to what extent signals emanating from oxidatively-stressed chloroplasts may be integrated with ABA signaling in guard cells have remained largely enigmatic. The metabolite 3’-phosphoadenosine 5’-phosphate (PAP) acts as a retrograde signal during oxidative stress. PAP accumulates during high light exposure and drought via redox inactivation of its catabolic phosphatase SAL1, and moves from chloroplasts to the nucleus via a transporter (Estavillo et al., 2011; Gigolashvili et al., 2012; Chan et al., 2016b). PAP is perceived by and inhibits exoribonuclease (XRN)-mediated RNA metabolism as evidenced in xrn double and triple mutants phenocopying sal1 mutants; resulting in drought tolerance and activation of 25% of the high light stress transcriptome. Mutant alleles lacking SAL1 catabolic activity, such as altered ascorbate peroxidase2 expression 8 (alx8, hereafter termed sal1-8) and fiery1-6 (fry1-6, hereafter termed sal1-6), constitutively accumulate PAP and are consequently drought tolerant (Rossel et al., 2006; Wilson et al., 2009; Estavillo et al., 2011). PAP was initially proposed to act largely via ABA-independent pathways as the drought tolerance in sal1 correlated with accumulation of osmoprotectants, and there were conflicting reports on the impacts of sal1 mutations on stomatal conductance: an earlier study suggested that SAL1 was not involved in stomatal regulation, whereas we found markedly decreased stomatal conductance in sal1 with elevated PAP (Xiong et al., 2001; Rossel et al., 2006; Wilson et al., 2009; Estavillo et al., 2011). Additionally, a subset of ABA-responsive genes are misregulated in sal1 mutants (Wilson et al., 2009), raising the question as to whether PAP can participate in ABA-mediated processes such as stomatal closure and seed germination. Binding of ABA to its receptors (RCAR/PYR1/PYL) (Ma et al., 2009; Park et al., 2009) leads to inactivation of the group A Protein Phosphatase 2C (PP2C) proteins such as ABI1 and activation of SNF1-Related Kinases 2.2, 2.3 and 2.6/OST1 (SnRK2.2, SnRK 2.3, SnRK2.6/OST1) (Koornneef et al., 1984; Leung et al., 1994; Meyer et al., 1994; Mustilli et al., 2002). The central role of PP2Cs and SnRKs in ABA signaling are demonstrated by the reduced sensitivity to ABA-mediated germination inhibition and stomatal closure in abi1-1 which is insensitive to ABA-PYR/PYL-mediated inhibition, and stomatal closure in ost1-2 (Koornneef et al., 1984; Leung et al., 1994; Meyer et al., 1994; Mustilli et al., 2002; Umezawa et al., 2009). Indeed, both seeds and guard cells of the triple mutant of ABA-activated SnRKs are almost completely ABA-insensitive (Fujii and Zhu, 2009; Nakashima et al., 2009). In guard cells active OST1 phosphorylates Slow Anion Channel-Associated 1 (SLAC1) allowing anion release, as well as facilitating potassium efflux by stimulating potassium efflux channels and inhibiting the inward potassium channels (KAT1 and KAT2), respectively (Geiger et al., 2009; Sato et al., 2009; Brandt et al., 2012). These anion and cation fluxes are necessary to close stomata (Schroeder and Hagiwara, 1989; Vahisalu et al., 2008; Geiger et al., 2009). OST1 activation also triggers gene expression changes, production of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) via NADPH oxidases, and interacts with intracellular Ca2+ signaling which involves cytosolic fluctuations in Ca2+ levels termed Ca2+ transients (Murata et al., 2015). The ABA-induced intracellular Ca2+ transients activate Calcium Dependent Protein Kinases (CDPKs) (Mori et al., 2006). There are at least 34 CDPKs in Arabidopsis thaliana, of which at least eight function in ABA signaling and ROS homeostasis in guard cells (Boudsocq and Sheen, 2013; Zou et al., 2015; Simeunovic et al., 2016). The subgroup II CDPKs are particularly important in guard cell signaling because three members (CDPK3, 21 and 23) can regulate SLAC1 and KAT channel activities (Cheng et al., 2002; Geiger et al., 2010; Brandt et al., 2012, 2015). With respect to SLAC1; CDPK3, 21 and 23 preferentially phosphorylate a residue different from that targeted by OST1 (Geiger et al., 2010; Brandt et al., 2012, 2015). Thus, SLAC1 channel activity is controlled by the joint action of OST1 and three CDPKs in counterbalance with competitive dephosphorylation by PP2Cs (Brandt et al., 2015). The understanding of guard cell regulation is far from complete, in part due to the complex interaction between ABA and secondary messengers such as Ca2+ and ROS. Notably, Ca2+-activation of CDPKs and ROS production by NADPH oxidases are interdependent processes with multiple layers of feedback regulation. Furthermore, the extent to which SnRK2-independent ABA/Ca2+signaling contributes to stomatal closure in whole plants under drought stress has not been thoroughly investigated, as various reports to date have largely utilized epidermal peels of unstressed plants or heterologous systems (Geiger et al., 2010; Brandt et al., 2012, 2015). In this regard, how ABA, Ca2+ and ROS in guard cells may interact with chloroplast signaling during drought stress also remains to be elucidated. Herein we present the unexpected finding that a chloroplast-mediated retrograde signaling pathway can bypass the canonical ABA pathway described above, which closes stomata and restores responsiveness in abi1-1 and ost1-2 mutants to ABA thereby conferring drought tolerance to these hitherto drought-sensitive mutants. The novel roles of the nucleotide phosphatase SAL1 and its associated phosphoadenosine signal, PAP, in guard cell regulation are investigated in the context of known and uncharacterized ABA signaling components and secondary messengers. Finally, the extent to which the interaction between ABA and PAP signaling occurs in other tissues and processes, such as seed germination, was also investigated. Results PAP restores guard cell responsiveness in ABA-insensitive plants We investigated whether SAL1-PAP can interact with ABA signaling by crossing the sal1 mutant, sal1-8, with the ABA insensitive mutants abi1-1 and ost1-2 (Koornneef et al., 1984; Leung et al., 1994; Mustilli et al., 2002). Elevated levels of PAP, a lack of SAL1 protein and many of the visible phenotypes of sal1-8 were still present in the double mutants (Figure 1A, Figure 1—figure supplement 1A). When challenged by drought stress, the single mutants, abi1-1 and ost1-2, displayed the expected widespread wilting and death before wild type. Conversely and unexpectedly, the ABA signaling double mutants containing the sal1-8 lesion were green, turgid and photosynthetically viable after ten days of drought (Figure 1A). Figure 1 with 3 supplements see all Download asset Open asset PAP restores drought tolerance and ABA-responsive stomatal closure in ABA signaling mutants. (A) Representative photos of two plants per genotype subjected to 10 days of drought. Statistically significant differences in survival during drought between genotypes are indicated (n = 4 per genotype per experiment, three independent experiments). (B) Effect of 20 µM ABA on stomatal conductance (gs) after 2 hr feeding through the roots of hydroponically-grown plants. The data is the average of two independent experiments (n = 3 plants per genotype per experiment) ± SEM. (C) The effect of 50 µM ABA treatment for 2 hr on stomatal aperture of leaf peels from five to six-week old plants. https://doi.org/10.7554/eLife.23361.002 We then studied whether the drought tolerance in abi1-1 sal1-8 and ost1-2 sal1-8 was due to restoration of guard cell ABA responsiveness. The SAL1 protein is present in epidermal peels and localized to chloroplasts of guard cells (Figure 1—figure supplement 1B,C). Significantly, while the stomatal conductance (gs) in ost1-2 sal1-8 and abi1-1 sal1-8 remained high under control conditions, the double mutant guard cells exhibited restored ABA sensitivity, with their gs and stomatal aperture decreasing in response to ABA (Figure 1B,C). Consistent with this finding, higher PAP content in well-watered sal1-8 plants conferred constitutively decreased gs and elevated leaf temperature which are indicative of enhanced stomatal closure [Figure 1B, Figure 1—figure supplement 2B and Rossel et al. (2006)]. Both epidermal peels and leaves of intact sal1-8 plants also exhibited enhanced sensitivity to ABA and closed stomata to a greater extent than wild type (Figure 1—figure supplement 2). Complementation in ABA insensitive mutants by PAP is not due to changes in stomatal index, genetic background or ABA content We investigated whether the complementation in abi1-1 sal1-8 and ost1-2 sal1-8 is driven by restoration of ABA signaling by PAP as opposed to contribution from other non-specific or pleiotropic effects. We previously showed that drought tolerance in sal1-8 is not due to slower water loss from soil (Wilson et al., 2009) and the tolerance is conferred by PAP in shoots, not roots (Hirsch et al., 2011). Therefore we tested whether PAP in leaves of the double mutants decreased constitutive gs or stomatal density, which can enhance drought tolerance (Doheny-Adams et al., 2012; Hepworth et al., 2015). The gs in well-watered double mutants were still as high as those in the parental abi1-1 and ost1-2 (Figure 1B). CryoSEM measurements of stomatal morphology and density revealed that the significantly higher total stomatal opening area per leaf area in the ABA signaling single mutants (abi1-1, ost1-2) remained high in the drought tolerant double mutants and were not decreased to wild type levels by sal1-8, which itself has wild type-like stomatal opening area per leaf area (Figure 1—figure supplement 3A). Next, since ost1-2 and abi1-1 are in Ler and sal1-8 in Col-0, we analyzed Col-0, Ler, ColLer F1 hybrids and segregating F2 and F3 plants of the crosses; no ecotype effects that could account for the drought tolerance independent of the sal1-8 mutation were observed (Figure 1—figure supplement 3A,B). We also generated double mutants containing both lesions in the Col-0 background using T-DNA mutants of ost1 (salk_008068) and sal1-6. The ost1 (Col-0 background) mutant was similar to ost1-2 (Ler background), being ABA-insensitive and failing to close stomata after four days of drought stress (Figure 1—figure supplement 3C,D). Significantly, the ost1 sal1-6 (Col-0 background) mutant had restored stomatal closure under drought stress (Figure 1—figure supplement 3D), ruling out ecotype effects as the major driver for the drought tolerance in ost1-2 sal1-8. We then tested whether the complementation could be explained by differences in ABA content. We previously reported that ABA levels are increased in sal1-8 (Rossel et al., 2006) and herein observed that ABA content was indeed slightly higher in sal1-8 and ost1-2 sal1-8, but this elevation was not significantly different (ANOVA, p=0.09) (Figure 1—figure supplement 3E). However, the marginally higher ABA content did not decrease gs values in the well-watered double mutants, which were still as high as those in the parental abi1-1 and ost1-2 (Figure 1B). Furthermore, when ABA-deficient aba2-3 [a leaky allele (Léon-Kloosterziel et al., 1996; Laby et al., 2000; Barrero et al., 2005)] was crossed to sal1-8, the double mutant showed no significant change in relative water content after eight days of water stress (WW 0.81 ± 0.11 vs WS 0.84 ± 0.06), as opposed to a significant decline for aba2-3 (WW 0.78 ± 0.02 vs WS 0.61 ± 0.08, p<0.05). Correspondingly, aba2-3 sal1-8 survived significantly longer than aba2-3 (16 days vs 11 days, p<0.005) as assayed by chlorophyll fluorescence (Woo et al., 2008). Therefore the restoration of ABA responsiveness and drought tolerance in abi1-1 sal1-8 and ost1-2 sal1-8 by PAP did not appear to primarily proceed via enhanced ABA synthesis, nor is it likely to, given the extensively reported insensitivity of abi1-1 and ost1-2 to ABA. Biochemical manipulation of PAP signaling induces stomatal closure, and PAP-mediated stomatal closure can be enhanced by ABA, but does not act via flg-22 mediated pathogen signaling We hypothesized that if PAP is a genuine signal regulating stomatal closure and it interacts with ABA signaling, then application of exogenous PAP to leaves should elicit similar responses as other known guard cell regulators such as ABA, ROS and Ca2+. Therefore we established and validated protocols for direct PAP application to leaves either via petioles or application to epidermal leaf peels; and evaluated effectiveness, uptake, transport and degradation of the fed PAP. In our system both barley and Arabidopsis leaf peels responded to the positive control, ABA, to a degree expected for each species compared to the mock measuring buffer containing Ca2+[which is known to promote certain levels of stomatal closure (Blatt et al., 1990)]. We then tested 10, 50 and 100 µM exogenous PAP. The PAP-induced closure, shown for 100 µM (Figure 2A,B) was significantly greater than the mock. Both 10 and 50 µM PAP were capable of causing a similar degree of closure to 100 µM PAP (10 µM PAP: 59 ± 5% closure, 50 µM PAP: 52 ± 7%, 100 µM PAP: 46 ± 8%; p=0.4 by ANOVA), albeit at a slower rate as expected for a physiological dose-dependent response. Significantly, both the rate and extent of closure of Arabidopsis and barley leaf peels with 100 µM PAP was comparable to the respective ABA responses (Figure 2A,B). We then tested whether exogenous PAP could induce stomatal closure in ost1-2, and observed significant PAP-induced closure (72 ± 2% closure in +PAP vs 90 ± 1% in control, p<0.001). Figure 2 with 1 supplement see all Download asset Open asset 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. https://doi.org/10.7554/eLife.23361.006 Next we investigated the uptake, transport and degradation of exogenous PAP in guard cells by biochemically manipulating its transport and degradation in leaf peels and in petiole-fed leaves. Exogenous ATP is a known co-substrate for the PAP transporter (Gigolashvili et al., 2012), therefore we used 10-fold higher ATP as this is predicted to outcompete PAP for import into chloroplasts, thus preventing the degradation of exogenous PAP. In leaf peels, applying ATP and PAP simultaneously increased the rate and magnitude of stomatal closure (Figure 2C). We then measured and observed significantly elevated PAP content in petiole-fed leaves co-treated with PAP+ATP compared to +PAP or +ATP alone (Figure 2—figure supplement 1). The elevated PAP in PAP+ATP leaves was associated with an increase in leaf temperature (Figure 2D), a typical consequence of stomatal closure. We then tested for PAP degradation in petiole-fed leaves by using LiCl, a SAL1 inhibitor (Quintero et al., 1996), and observed elevated PAP with a similarly increased leaf temperature (Figure 2D, Figure 2—figure supplement 1). Leaf temperature by PAP+LiCl could be further enhanced by co-treatment with ABA (Figure 2D), action of PAP accumulation and ABA. A for our that exogenous PAP closes stomata is that it is perceived and then a chloroplast response that can close as is the for the flg-22 pathogen response pathway mediated by the et al., 2012; et al., 2015). However, we found that PAP-mediated stomatal closure does not protein (Figure 1—figure supplement in with the data does ABA et al., et al., 2008). We then tested whether PAP an response by feeding PAP, ABA or the to leaf of leaf revealed that the magnitude and of ROS in the or leaf were than that by not PAP restores multiple ABA signaling in ABA-insensitive plants the between PAP and ABA responses we tested for the complementation of of ABA signaling, ion channel ROS and gene expression, in wild ost1-2 sal1-8 plants and in ost1-2 plants treated with exogenous PAP. and fluxes which are of ion transport into or out of guard cells that stomatal closure were with revealed that ion fluxes in ost1-2 were not to ABA but ion fluxes of ost1-2 sal1-8 were restored to wild type and sal1-8 with and efflux both ABA treatment (Figure 3A). complementation of the ost1-2 in ost1-2 sal1-8, most likely through PAP (Figure 1—figure supplement 1A). Exogenous PAP also the ion responses from guard cells similar to that of ABA and Ca2+ in all genotypes tested (Figure Figure supplement 1A). However, exogenous PAP does not the activity of the ion channels and when into (Figure supplement that PAP the ion fluxes through restoration of ABA Figure 3 with 2 supplements see all Download asset Open asset PAP restores guard cell ion ROS and response to ABA when in ost1-2 sal1-8 and when exogenously to ost1-2 and wild type. of (A) µM ABA or (B) µM PAP on of each of the ion for and from guard cells in leaf epidermal peels of old Arabidopsis plants. ion fluxes ± SEM (n = are shown for control, 10 min and 50 min after ABA or PAP significant difference to 0 min (C) Mean total cell fluorescence of ROS in the of a ROS that primarily and to a extent et al., in guard cells before and after 10 min of 100 µM ABA or 100 µM PAP. ± SEM of or stomata per genotype is shown with significant differences denoted p<0.05). ABA response in wild type and mutants for (D) to ABA in in this and known to to ABA in guard cells et al., 2011). The is change for each genotype ABA, The has been such that the and at the of the all greater or to or than or to in and ost1-2 sal1-8 are Also see 1. the ROS in guard cells measured with the was observed in response to ABA treatment in wild type and was as expected in ost1-2 (Figure The primarily and to a extent et al., significant of was observed for the control measuring buffer treatment the of the fluorescence of ± at 0 min vs ± at 10 min, Significantly, the ABA-mediated ROS was restored in ost1-2 sal1-8, to a similar degree to that of wild type. Furthermore, the timing and extent of the ABA-induced ROS was by exogenous PAP in wild ost1-2 and ost1-2 sal1-8 (Figure we of well-watered whole leaves of wild ost1-2, sal1-8, and ost1-2 sal1-8 or ABA (Figure Figure supplement 2 1). ost1-2 gene expression were largely to ABA compared to wild type leaves. Significantly, the response to ABA was restored in ost1-2 sal1-8 although the magnitude was (Figure 1). for a subset of genes that responded to ABA in wild type compared to ost1-2, the expression of genes was largely restored in the ost1-2 sal1-8 ABA, such that were no longer significantly different to wild type ABA p<0.05). the was on whole we whether complementation was also observed in ost1-2 sal1-8 for ABA-responsive genes expressed in guard cells et al., 2011). Indeed, a of these guard genes that were by ABA in wild type were not in ost1-2, but were significantly in ost1-2 sal1-8 Figure 3E). We the using the genes known to be involved in ABA signaling et al., 2011). revealed in all sal1-8 and compared to wild type and a genes by ABA in sal1-8 and ost1-2 sal1-8, but not in ost1-2 (Figure Significantly, we identified several genes involved in but of ABA signaling that were expressed in ost1-2 sal1-8 under constitutive and/or two transcription Ca2+ signaling proteins and and genes regulating ROS homeostasis as well as to 2). Furthermore, many of these genes in ost1-2 sal1-8 have been shown to have the to regulate the activities of inward channels and anion channels for stomatal closure. signaling is for regulation of potassium inward by ABA and Ca2+ et al., 2015), directly the channel et al., while various CDPKs are to the group II CDPKs (CDPK3, 21 and 23) known to directly regulate SLAC1 and activity (Geiger et al., 2010; Brandt et al., 2012). transport and also have and close with known to regulate channels and stomatal closure et al., 2001; et al., 2012). these that PAP-mediated signaling restores ABA sensitivity at multiple levels for stomatal closure in plants lacking wild type OST1 or how is PAP perceived in the cell during ABA signaling and how does it of by which PAP-mediated retrograde signaling with ABA signaling to regulate stomatal closure the that PAP and signaling in ABA responses act via its established retrograde signaling pathway we crossed ost1-2 to the drought tolerant double mutant, which PAP inhibition of nuclear and sal1 et al., Estavillo et al., 2011). to for ost1-2 sal1-8, ABA responsiveness and stomatal closure were restored in both intact plants and epidermal leaf peels of ost1-2 (Figure ABA-responsive guard cell ion fluxes were restored to wild type levels in ost1-2 whereas ost1-2 remained insensitive as expected (Figure We also observed complementation of ABA-responsive ROS in this triple mutant to the extent as that in ost1-2 sal1-8 (Figure our data that PAP in ABA signaling through its established retrograde communication Figure 4 Download asset Open asset of ABA-responsive stomatal closure, guard cell ion fluxes and ROS production in ost1-2 (A) Leaf a of stomatal closure, in leaves of intact plants of wild ost1-2, sal1-8, ost1-2 sal1-8, and ost1-2 after treatment with 20 µM ABA for 2 hr. Significant differences relative to control are indicated by Values shown are means ± SEM of per The ABA responses of ost1-2 sal1-8 and associated were in an independent compared to ost1-2 (B) Stomatal aperture in leaf peels of wild ost1-2, and ost1-2 after treatment with 50 µM ABA for 2 hr stomata per genotype ± Significant difference at is shown for The was observed in two independent (C) of µM ABA on of each of the ion for and from guard cells in leaf epidermal peels of old