FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4249762111> ?p ?o ?g. }

• W4249762111 abstract "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), suggesting synergistic action of PAP accumulation and ABA. A theoretical explanation for our observation that exogenous PAP closes stomata is that it is perceived apoplastically and then stimulates a chloroplast calcium response that can close stomata, as is the case for the flg-22 Pathogen Associated Molecular Pattern (PAMP) pathogen response pathway mediated by the chloroplastic Calcium-Sensing Receptor (CAS) (Nomura et al., 2012; Guzel Deger et al., 2015). However, we found that PAP-mediated stomatal closure does not require functional CAS protein (Figure 1—figure supplement 3F), and, in agreement with the published data neither does ABA (Han et al., 2003; Nomura et al., 2008). We then tested whether PAP elicits an extracellular PAMP response by feeding PAP, ABA or the flg22 PAMP receptor elicitor to leaf disks. Luminol-based assays of leaf disks revealed that the magnitude and kinetics of ROS burst in the ABA- or PAP-treated leaf disks were 40-fold lower than that induced by flg22 (data not shown). PAP restores multiple ABA signaling outputs in ABA-insensitive plants Given the connections between PAP and ABA responses we systematically tested for the complementation of key outputs of ABA signaling, namely ion channel fluxes, ROS bursts and gene expression, in wild type, ost1-2 sal1-8 plants and in ost1-2 plants treated with exogenous PAP. First, K+ and Cl- fluxes which are indicators of ion transport into or out of guard cells that enable stomatal closure were measured. Experiments with ion-selective microelectrodes revealed that ion fluxes in ost1-2 were not responsive to ABA treatment, but ion fluxes of ost1-2 sal1-8 were restored to wild type and sal1-8 levels, with K+ and Cl- efflux both stimulated upon ABA treatment (Figure 3A). This indicates complementation of the ost1-2 phenotype in ost1-2 sal1-8, most likely through genetically accumulated PAP (Figure 1—figure supplement 1A). Exogenous PAP also stimulated the ion flux responses from guard cells similar to that of ABA and Ca2+ in all genotypes tested (Figure 3A,B; Figure 3—figure supplement 1A). However, exogenous PAP does not affect the activity of the key ion channels SLAC1, KAT1 and KAT2 when supplemented into oocytes (Figure 3—figure supplement 1B); suggesting that PAP influences the ion fluxes indirectly through restoration of ABA signaling. Figure 3 with 2 supplements see all Download asset Open asset PAP restores guard cell ion fluxes, ROS burst and global transcriptional response to ABA when accumulated genetically in ost1-2 sal1-8 and when applied exogenously to ost1-2 and wild type. Effects of (A) 500 µM ABA or (B) 500 µM PAP on combined net flux of each of the ion transporters for K+ and Cl− from guard cells in leaf epidermal peels of four-week old Arabidopsis plants. Average net ion fluxes ± SEM (n = 5–7 plants) are shown for control, 10 min and 50 min after ABA or PAP treatment. Asterisk shows statistically significant difference to 0 min (p<0.05, ANOVA). (C) Mean corrected total cell fluorescence of ROS in the presence of 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA), a ROS probe that detects primarily H2O2 and to a lesser extent hydroxyl radicals (Wojtala et al., 2014), in guard cells before and after 10 min of 100 µM ABA or 100 µM PAP. Means ± SEM of 45–54 (+ABA) or 73–92 (+PAP) stomata per genotype is shown with significant differences denoted (t-test, p<0.05). (D–E) Hierarchical clustering comparing ABA transcriptional response in wild type (WT) and mutants for (D) transcripts responsive to ABA in WT in this study; and (E) transcripts known to respond to ABA in guard cells (Wang et al., 2011). The blue (down)-red (up) scale is log2 fold change for each genotype +/− ABA, respectively. 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. Clusters showing co-expression in WT and ost1-2 sal1-8 are marked (I). Also see Supplementary file 1. https://doi.org/10.7554/eLife.23361.008 Second, the ROS burst in guard cells measured with the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) dye was observed in response to ABA treatment in wild type and was attenuated as expected in ost1-2 (Figure 3C). The H2DCFDA dye primarily detects H2O2 and to a lesser extent hydroxyl radicals (Wojtala et al., 2014). No significant photooxidation of H2DCFDA was observed for the control measuring buffer treatment within the timeframe of the experiment (mean fluorescence of 1.2 ± 0.4×105 at 0 min vs 1.5 ± 0.5 × 105 at 10 min, p=0.37). Significantly, the ABA-mediated ROS burst was restored in ABA-treated ost1-2 sal1-8, to a similar degree to that of wild type. Furthermore, the timing and extent of the ABA-induced ROS burst was phenocopied by exogenous PAP in wild type, ost1-2 and ost1-2 sal1-8 (Figure 3C). Third, we performed transcriptome analyses of well-watered whole leaves of wild type, ost1-2, sal1-8, and ost1-2 sal1-8 plus or minus ABA (Figure 3D–E, Figure 3—figure supplement 2 ,Supplementary file 1). As expected, ost1-2 gene expression profiles were largely unresponsive to ABA compared to wild type leaves. Significantly, the transcriptional response to ABA was substantially restored in ost1-2 sal1-8 +ABA although the magnitude was attenuated (Figure 3D, Supplementary file 1). Specifically, for a subset of 1723 genes that responded differently to ABA in wild type compared to ost1-2, the expression of 1705 (99%) genes was largely restored in the ost1-2 sal1-8 plus ABA, such that they were no longer significantly different to wild type plus ABA (Supplementary file 1, FDR adjusted p<0.05). Because the transcriptome was performed on whole leaves, we verified whether complementation was also observed in ost1-2 sal1-8 for 1173 ABA-responsive genes expressed in guard cells (Wang et al., 2011). Indeed, a large proportion of these guard cell-expressed genes that were induced by ABA in wild type were not induced in ost1-2, but were significantly induced in ost1-2 sal1-8 (gene cluster I, Figure 3E). We re-analyzed the transcriptome using the 132 genes known to be involved in ABA signaling (Hauser et al., 2011). Hierarchical clustering revealed some subsets differentially down-regulated in all sal1-8 backgrounds and treatments compared to wild type +ABA, and a few genes more highly induced by ABA in sal1-8 and ost1-2 sal1-8, but not in ost1-2 (Figure 3D). Significantly, we identified several genes involved in diverse, but interlinked, aspects of ABA signaling that were differentially expressed in ost1-2 sal1-8 under constitutive and/or +ABA conditions. This included two transcription factors, 16 Ca2+ signaling proteins [CDPKs, CRKs, CBLs and CIPKs], and genes regulating ROS homeostasis as well as vesicle trafficking to plasma membranes (Supplementary file 2). Furthermore, many of these genes up-regulated in ost1-2 sal1-8 have been shown to have the ability to regulate the activities of inward rectifying channels (KATs) and slow anion channels (SLAC1/SLAHs) for stomatal closure. For instance, CDPK19-mediated signaling is required for regulation of potassium inward currents by ABA and Ca2+ (Zou et al., 2015), CIPK6 directly activates the key K+ channel AKT1 (Lan et al., 2011), while various up-regulated CDPKs are closely related to the group II CDPKs (CDPK3, 21 and 23) known to directly regulate SLAC1 and SLAH3 activity (Geiger et al., 2010; Brandt et al., 2012). Two up-regulated vesicle transport syntaxin genes, SYP41 and SYP124, also have protein-protein interactions and close sequence relationships with syntaxins known to regulate K+ channels and stomatal closure (Sanderfoot et al., 2001; Eisenach et al., 2012). Collectively, these results indicate that PAP-mediated signaling restores ABA sensitivity at multiple levels for stomatal closure in plants lacking wild type OST1 or ABI1. If so, how is PAP perceived in the cell during ABA signaling and how does it act? Investigation of mechanisms by which PAP-mediated retrograde signaling might intersect with ABA signaling to regulate stomatal closure To investigate the possibility that PAP perception and signaling in ABA responses act via its established retrograde signaling pathway we crossed ost1-2 to the drought tolerant xrn2-1 xrn3-3 double mutant, which genetically phenocopies PAP inhibition of nuclear XRNs and sal1 (Dichtl et al., 1997; Estavillo et al., 2011). Similar to observations obtained for ost1-2 sal1-8, ABA responsiveness and stomatal closure were restored in both intact plants and epidermal leaf peels of ost1-2 xrn2-1 xrn3-3 (Figure 4A,B). ABA-responsive guard cell ion fluxes were restored to wild type levels in ost1-2 xrn2-1 xrn3-3, whereas ost1-2 remained insensitive as expected (Figure 4C). We also observed complementation of ABA-responsive ROS burst in this triple mutant to the same extent as that seen in ost1-2 sal1-8 (Figure 4D). Therefore, our data indicate that PAP participates in ABA signaling through its established SAL1-PAP-XRN retrograde communication pathway. Figure 4 Download asset Open asset Restoration of ABA-responsive stomatal closure, guard cell ion fluxes and ROS production in ost1-2 xrn2-1 xrn3-3. (A) Leaf temperature, a proxy of stomatal closure, in leaves of intact plants of wild type, ost1-2, sal1-8, ost1-2 sal1-8, xrn2-1 xrn3-3 and ost1-2 xrn2-1 xrn3-3 after treatment with 20 µM ABA for 2 hr. Significant differences relative to control are indicated by asterisk (*p<0.05). Values shown are means ± SEM of 3–9 biological replicates per treatment. The ABA responses of ost1-2 sal1-8 and associated controls were performed in an earlier, independent experiment compared to ost1-2 xrn2-1 xrn3-3. (B) Stomatal aperture in leaf peels of wild type, ost1-2, xrn2-1 xrn3-3 and ost1-2 xrn2-1 xrn3-3 after treatment with 50 µM ABA for 2 hr (40–60 stomata per genotype ± SD). Significant difference (*) at p<0.05 is shown for +ABA. The same trend was observed in two independent experiments. (C) Effects of 500 µM ABA on combined net flux of each of the ion transporters for K+ and Cl− from guard cells in leaf epidermal peels of four-week old Arabido" @default.
• W4249762111 created "2022-05-12" @default.
• W4249762111 creator A5002273022 @default.
• W4249762111 creator A5008465182 @default.
• W4249762111 creator A5014096625 @default.
• W4249762111 creator A5016350697 @default.
• W4249762111 creator A5019922163 @default.
• W4249762111 creator A5032099774 @default.
• W4249762111 creator A5034071966 @default.
• W4249762111 creator A5036427066 @default.
• W4249762111 creator A5037451186 @default.
• W4249762111 creator A5038013995 @default.
• W4249762111 creator A5049776569 @default.
• W4249762111 creator A5049780967 @default.
• W4249762111 creator A5057094394 @default.
• W4249762111 creator A5058487113 @default.
• W4249762111 creator A5060711830 @default.
• W4249762111 creator A5069873305 @default.
• W4249762111 creator A5070501215 @default.
• W4249762111 creator A5074382169 @default.
• W4249762111 creator A5082208168 @default.
• W4249762111 creator A5090562123 @default.
• W4249762111 date "2017-03-11" @default.
• W4249762111 modified "2023-10-09" @default.
• W4249762111 title "Author response: A chloroplast retrograde signal, 3’-phosphoadenosine 5’-phosphate, acts as a secondary messenger in abscisic acid signaling in stomatal closure and germination" @default.
• W4249762111 doi "https://doi.org/10.7554/elife.23361.026" @default.
• W4249762111 hasPublicationYear "2017" @default.
• W4249762111 type Work @default.
• W4249762111 citedByCount "2" @default.
• W4249762111 countsByYear W42497621112022 @default.
• W4249762111 countsByYear W42497621112023 @default.
• W4249762111 crossrefType "peer-review" @default.
• W4249762111 hasAuthorship W4249762111A5002273022 @default.
• W4249762111 hasAuthorship W4249762111A5008465182 @default.
• W4249762111 hasAuthorship W4249762111A5014096625 @default.
• W4249762111 hasAuthorship W4249762111A5016350697 @default.
• W4249762111 hasAuthorship W4249762111A5019922163 @default.
• W4249762111 hasAuthorship W4249762111A5032099774 @default.
• W4249762111 hasAuthorship W4249762111A5034071966 @default.
• W4249762111 hasAuthorship W4249762111A5036427066 @default.
• W4249762111 hasAuthorship W4249762111A5037451186 @default.
• W4249762111 hasAuthorship W4249762111A5038013995 @default.
• W4249762111 hasAuthorship W4249762111A5049776569 @default.
• W4249762111 hasAuthorship W4249762111A5049780967 @default.
• W4249762111 hasAuthorship W4249762111A5057094394 @default.
• W4249762111 hasAuthorship W4249762111A5058487113 @default.
• W4249762111 hasAuthorship W4249762111A5060711830 @default.
• W4249762111 hasAuthorship W4249762111A5069873305 @default.
• W4249762111 hasAuthorship W4249762111A5070501215 @default.
• W4249762111 hasAuthorship W4249762111A5074382169 @default.
• W4249762111 hasAuthorship W4249762111A5082208168 @default.
• W4249762111 hasAuthorship W4249762111A5090562123 @default.
• W4249762111 hasBestOaLocation W42497621111 @default.
• W4249762111 hasConcept C100701293 @default.
• W4249762111 hasConcept C104317684 @default.
• W4249762111 hasConcept C146834321 @default.
• W4249762111 hasConcept C163235415 @default.
• W4249762111 hasConcept C17744445 @default.
• W4249762111 hasConcept C185592680 @default.
• W4249762111 hasConcept C194082555 @default.
• W4249762111 hasConcept C199360897 @default.
• W4249762111 hasConcept C199539241 @default.
• W4249762111 hasConcept C2779591172 @default.
• W4249762111 hasConcept C2779843651 @default.
• W4249762111 hasConcept C41008148 @default.
• W4249762111 hasConcept C55493867 @default.
• W4249762111 hasConcept C59822182 @default.
• W4249762111 hasConcept C62478195 @default.
• W4249762111 hasConcept C69305403 @default.
• W4249762111 hasConcept C86803240 @default.
• W4249762111 hasConcept C95444343 @default.
• W4249762111 hasConceptScore W4249762111C100701293 @default.
• W4249762111 hasConceptScore W4249762111C104317684 @default.
• W4249762111 hasConceptScore W4249762111C146834321 @default.
• W4249762111 hasConceptScore W4249762111C163235415 @default.
• W4249762111 hasConceptScore W4249762111C17744445 @default.
• W4249762111 hasConceptScore W4249762111C185592680 @default.
• W4249762111 hasConceptScore W4249762111C194082555 @default.
• W4249762111 hasConceptScore W4249762111C199360897 @default.
• W4249762111 hasConceptScore W4249762111C199539241 @default.
• W4249762111 hasConceptScore W4249762111C2779591172 @default.
• W4249762111 hasConceptScore W4249762111C2779843651 @default.
• W4249762111 hasConceptScore W4249762111C41008148 @default.
• W4249762111 hasConceptScore W4249762111C55493867 @default.
• W4249762111 hasConceptScore W4249762111C59822182 @default.
• W4249762111 hasConceptScore W4249762111C62478195 @default.
• W4249762111 hasConceptScore W4249762111C69305403 @default.
• W4249762111 hasConceptScore W4249762111C86803240 @default.
• W4249762111 hasConceptScore W4249762111C95444343 @default.
• W4249762111 hasLocation W42497621111 @default.
• W4249762111 hasOpenAccess W4249762111 @default.
• W4249762111 hasPrimaryLocation W42497621111 @default.
• W4249762111 hasRelatedWork W1993095251 @default.
• W4249762111 hasRelatedWork W2093676869 @default.
• W4249762111 hasRelatedWork W2136727913 @default.
• W4249762111 hasRelatedWork W2169979796 @default.
• W4249762111 hasRelatedWork W2361660894 @default.
• W4249762111 hasRelatedWork W2374980232 @default.
• W4249762111 hasRelatedWork W2376142978 @default.
• W4249762111 hasRelatedWork W2604050389 @default.

• next