Abstract
Chloroplasts have a critical role in plant immunity as a site for the production for salicylic acid and jasmonic acid, important mediators of plant immunity. However, the molecular link between chloroplasts and the cytoplasmic-nuclear immune system remains largely unknown. Here we show that pathogen-associated molecular pattern (PAMP) signals are quickly relayed to chloroplasts and evoke specific Ca2+ signatures in the stroma. We further demonstrate that a chloroplast-localized protein, named calcium-sensing receptor (CAS), is involved in stromal Ca2+ transients and responsible for both PAMP-induced basal resistance and R gene-mediated hypersensitive cell death. CAS acts upstream of salicylic acid accumulation. Transcriptome analysis demonstrates that CAS is involved in PAMP-induced expression of defence genes and suppression of chloroplast gene expression possibly through 1O2-mediated retrograde signalling, allowing chloroplast-mediated transcriptional reprogramming during plant immune responses. The present study reveals a previously unknown chloroplast-mediated signalling pathway linking chloroplasts to cytoplasmic-nuclear immune responses.
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Introduction
Plants are continuously exposed to a variety of biotic and abiotic stresses, and have evolved an intricate network of signal transduction pathways leading to transcriptome and metabolic reprogramming. Pathogen-associated molecular patterns (PAMPs) are recognized by pattern-recognition receptors in the plasma membrane and activate an array of basal defence responses (PAMP-triggered immunity, PTI), whereas pathogen-derived effector proteins trigger hypersensitive response (HR) cell death at the site of pathogen infection and confine the spread of pathogens (effector-triggered immunity, ETI)1. Plant immunity activates two parallel signal transduction pathways: cytoplasmic mitogen-activated protein kinase (MAPK) and Ca2+ signalling pathways leading to transcriptional reprogramming, and a chloroplast-mediated pathway leading to the generation of chloroplast-derived reactive oxygen species (ROS) and the production of defence-related hormones, such as salicylic acid (SA) and jasmonic acid (JA)2. Possible involvement of chloroplasts in plant innate immunity has been discussed3. However, the molecular link between the chloroplast and cytoplasmic-nuclear immune signalling pathways remains largely unknown. In particular, it is not clear how immune signals are relayed to chloroplasts and how chloroplasts control the expression of nuclear-encoded defence genes.
Extracellular stimuli evoke Ca2+ signals in not only the cytoplasm, but also organelles such as mitochondria, peroxisomes and nuclei4,5. For example, the massive accumulation of Ca2+ in mitochondria leads to the dysfunction of mitochondria and induction of apoptosis of mammalian cells6. However, very few studies have focused on Ca2+ signals in chloroplasts. Calcium-sensing receptor (CAS)7 is a thylakoid membrane-associated Ca2+-binding protein involved in the regulation of cytoplasmic Ca2+ oscillations and extracellular Ca2+-induced stomatal closure8,9,10.
Here we show that PAMP signals evoke a CAS-dependent transient Ca2+ signal in chloroplasts, and that CAS is involved in transcriptional reprogramming through 1O2-mediated retrograde signalling during plant innate immunity. This study is an important advancement in the understanding of cross talk between chloroplasts and the plant immune system.
Results
Biotic and abiotic stress-induced chloroplast Ca2+ signals
To measure the free Ca2+ concentration in the stroma, the calcium-sensitive bioluminescent protein aequorin was targeted to the chloroplast stroma using an ribulose-1,5-bisphosphate carboxylase (RBCS) signal peptide (Supplementary Fig. S1). As reported previously, stromal Ca2+ was maintained at a very low concentration, as low as that in the cytoplasm (Fig. 1a–f), suggesting that Ca2+ in chloroplasts is sequestered in the thylakoid lumen or by Ca2+-binding proteins11. We found that the bacterial PAMP flagellin peptide (flg22) induces a rapid Ca2+ transient in the cytoplasm, followed by a long-lasting increase in the stromal free Ca2+ level (Fig. 1a). A similar stromal Ca2+ transient was also induced by the fungal PAMP chitin (Fig. 1b). On the other hand, control peptides induced no Ca2+ signal in chloroplasts (Supplementary Fig. S2a). Furthermore, a flg22-induced stromal Ca2+ transient was observed in transplastomic tobacco plants in which the aequorin gene had been integrated into the chloroplast genome (Supplementary Fig. S2b), confirming the occurrence of Ca2+ dynamics in the chloroplast stroma. These findings demonstrate that extracellular PAMP signals are relayed rapidly to chloroplasts and evoke intrachloroplastic Ca2+ transients.
We further examined the effects of abiotic stresses on cytoplasmic and stromal Ca2+ signals. Light-to-dark transition evoked a slow Ca2+ transient in the stroma, but not the cytoplasm (Fig. 1c)11. We also found that other abiotic stresses induced stromal Ca2+ transients in a stress-dependent manner. Cold and salt stresses elicited rapid stromal Ca2+ spikes within a minute, whereas hyperosmotic stress induced a biphasic long-lasting Ca2+ transient in the stroma (Fig. 1d–f). Stress-specific Ca2+ signatures in the stroma may be involved in the regulation of photosynthesis and metabolic processes in response to the given stresses.
In mammals, signal molecules such as staurosporine initially stimulate an increase in the cytoplasmic Ca2+ concentration, and subsequently induce a mitochondrial Ca2+ increase before apoptosis6. Similarly, PAMP-induced stromal Ca2+ transients were always preceded by cytoplasmic Ca2+ changes. We found that both chloroplastic and cytoplasmic Ca2+ changes were markedly decreased by the extracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid (BAPTA; Fig. 1g). Furthermore, the Ca2+ ionophore ionomycin induced a biphasic Ca2+ increase in both the cytoplasm and the stroma (Fig. 1h). Interestingly, the second ionomycin-induced stromal Ca2+ increase was preceded by the cytoplasmic Ca2+ transient. From these results, we presume that PAMPs first induce Ca2+ entry into the cytoplasm and, subsequently, the cytoplasmic Ca2+ triggers a transient increase in the stromal Ca2+ concentration. In addition, both the cytoplasmic and stromal Ca2+ oscillations were partially reduced by inhibitors of serine/threonine protein kinase (K252a) and MAPKKs (U-0126), but not by an NADPH oxidase inhibitor (DPI; Fig. 1g), suggesting the possible involvement of a MAP kinase cascade in the generation of cytoplasmic and stromal Ca2+ signals.
CAS is involved in chloroplast Ca2+ signals
CAS is a plant-specific thylakoid-associated Ca2+-binding protein8,9,10,12. To examine the possible role of CAS in the generation of stromal Ca2+ transients, we measured flg22-induced stromal Ca2+ transients in aequorin-expressing plants with a CAS knockout background (cas-1). The aequorin-expressing cas-1 plants accumulated the same amount of aequorin protein as wild-type plants (Supplementary Fig. S1a). We found that flg22- and dark-induced long-lasting stromal Ca2+ transients were markedly reduced in cas-1 (Fig. 1i,j), whereas cold-induced rapid stromal Ca2+ spikes were not impaired in cas-1 (Supplementary Fig. S3). Considering the localization of CAS in thylakoid membranes, CAS may be involved in Ca2+ release from thylakoid membranes and the generation of a long-lasting stromal Ca2+ signal.
CAS is responsible for both PTI and ETI
The presence of PAMP-induced Ca2+ transients in the stroma underscores the importance of chloroplasts in plant immune signalling. We found that cas-1 plants exhibited severely impaired resistance to the virulent bacterium Pseudomonas syringae pv. tomato (Pst) strain DC3000 and to avirulent Pst DC3000 varieties (Fig. 2a). Surprisingly, PAMP (flg22)-induced defence-related events, such as stomatal closure (Fig. 2b), callose deposition (Fig. 2c) and accumulation of defence-related phenylpropanoids/flavonoids and their precursors (Supplementary Fig. S4), were markedly compromised in cas-1. Furthermore, flg22-induced PR1 and PR2 expression and stomatal closure were suppressed in two independent homozygous T-DNA insertion lines, cas-1 and cas-2 (ref. 8), whereas expression of CAS cDNA at wild-type levels in cas-1 restored flg22-induced PR1 and PR2 expression and stomatal closure (Fig. 2d, Supplementary Fig. S5). These results demonstrate that the chloroplast protein CAS is involved in a range of basal defence responses. We further found that flg22-induced stomatal closure was impaired by the NADPH oxidase inhibitor diphenyleneiodonium (DPI) and by the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) in wild-type plants (Fig. 2e). On the other hand, exogenous application of hydrogen peroxide (H2O2) or the NO donor sodium nitroprusside (SNP) could induce stomatal closure in cas-1 (Fig. 2f), suggesting that CAS acts upstream of ROS and NO signals in flg22-induced stomatal closure.
Some pathogens secret effector proteins (Avr gene products) that suppress PTI. Conversely, effectors are recognized by plant proteins, which activate ETI leading to hypersensitive cell death at the infection site. We found that the cas-1 plants exhibited delayed and suppressed HR cell death compared with the wild type after inoculation with Pst DC3000 (avrRpt2; Fig. 3a,b). Interestingly, the Pst DC3000 (avrRpt2)-induced accumulation of ROS (Supplementary Fig. S6a,b) and NO (Supplementary Fig. S6c,d) was significantly reduced in cas-1 plants. Functional proof that CAS is a critical component in plant cell death was further demonstrated using virus-induced gene silencing (VIGS) in Nicotiana benthamiana plants. We cloned NbCAS from N. benthamiana (Supplementary Fig. S7a) and used VIGS to reduce its expression to <1% of the wild-type level (Supplementary Fig. S7b). NbCAS silencing resulted in a clear delay in the elicitor protein INF1-, the cf9/Avr9 elicitor/receptor- and the constitutively active N. tabacum MAP kinase kinase (NtMEK2DD)-dependent cell death (Fig. 3c,d, Supplementary Fig. S7c), suggesting that CAS has a conserved role in plant cell death and may act downstream of the MAP kinase cascade in ETI. Considering a chloroplast localization of CAS, the above results suggest that chloroplasts might have critical roles in the positive regulation of plant immunity.
CAS regulates SA biosynthesis
It is unlikely that the compromised immunity of CAS knockout plants is caused by limited cellular resources, as cas-1 plants are photosynthetically competent8 and do not show severe growth retardation (Supplementary Fig. S8). Chloroplasts are involved in the biosynthesis of plant hormones such as SA, JA, abscisic acid (ABA), indole-3-acetic acid (IAA) and cytokinins (CKs). Thus, we examined the profiles of a set of plant hormones (SA, JA, ABA, IAA and CKs) during flg22-induced PTI. The accumulation of free SA was transiently induced by flg22 treatment within 6 h in wild-type plants, whereas JA and other hormones showed no significant change in levels (Fig. 4a–e), confirming a crucial role of SA in PTI signalling. Importantly, we found that CAS deficiency prevented flg22-induced accumulation of free SA (Fig. 4a) as well as glycosylated SA (Supplementary Fig. S9). As the basal SA levels before flg22 treatment were normal in cas-1 plants, CAS is likely involved in the regulation of SA biosynthesis rather than the biosynthesis itself. SA is synthesized in chloroplasts via the chorismate pathway in Arabidopsis13. It has been shown that PAMPs induce SA biosynthesis in an ICS1-dependent manner14,15. Interestingly, the flg22-induced early expression of PAD4, EDS5, PBS3 and ICS1, all of which are essential for SA biosynthesis, was significantly reduced in cas-1 plants before SA accumulation (~1 h; Fig. 4f). On the other hand, PR1 expression was induced significantly by SA in cas-1 plants similarly to in wild-type plants (Supplementary Fig. S10), indicating that the signal transduction pathways downstream of SA are independent of CAS. These results demonstrate that CAS is involved in flg22-induced SA biosynthesis through promoting the expression of nuclear-encoded SA biosynthesis genes.
CAS-dependent induction of nuclear-encoded defence genes
These findings led us to hypothesize that CAS may be involved in chloroplast-to-nucleus retrograde signalling to control the expression of nuclear-encoded defence genes, including SA biosynthesis genes. Flg22 induces the expression of numerous defence-related genes in the nucleus15,16. To identify flg22-induced genes that are under the control of CAS, an Arabidopsis gene expression microarray (v4) was used to compare the flg22-induced transcription profiles of wild-type and cas-1 plants. We identified 687 upregulated and 1,235 downregulated genes in cas-1 plants treated with flg22 for 2 h (Fig. 5a and Supplementary Data 1 and 2). By contrast, only 7 genes were upregulated and 197 genes were downregulated in non-treated cas-1 plants (Fig. 5a and Supplementary Data 3 and 4). In all, 121 of the 197 downregulated and 6 of the 7 upregulated genes in non-treated cas-1 plants were also down- and upregulated in flg22-treated cas-1 plants, respectively. Quantitative reverse transcription–PCR (qRT–PCR) confirmed the array results for selected targets (Figs 4f and 5c). Interestingly, the expressions of one-third (827 genes) of the flg22-induced early genes (within 3 h)17 were suppressed in cas-1 (Fig. 5a,b, Supplementary Fig. S11). Similarly, two-thirds of genes downregulated in flg22-treated cas-1 were flg22-induced in wild type. Furthermore, the 403 genes upregulated in flg22-treated cas-1 were enriched for genes that were suppressed by flg22 in wild type (58.7%). Gene expression analysis using the Expression Browser at Bio-Array Resource (BAR)18 showed that genes downregulated in flg22-treated cas-1 were also enriched in NPP1- and HrpZ-responsive genes, as well flg22-responsive genes (Supplementary Fig. S12a). CAS, therefore, likely has a universal role in PAMP-induced gene expression.
Gene ontology analysis using the Arabidopsis Classification Super Viewer demonstrated a significant over-representation of 'response to stress' (3.04-fold, P=5.33E−50), 'response to abiotic and biotic stimulus' (2.49-fold, P=2.83e–28) and signal transduction (2.26-fold, P=7.17e–14) categories among the flg22-treated cas-1 downregulated genes (Supplementary Fig. S12b–e). The downregulated genes included a number of GTPase, Ca2+ signalling and SA signalling genes (Supplementary Data 5). CAS was also required for flg22-induced expression of CYP79B2, CYP79B3 and PAD3 genes for cytochrome P450 that are essential for the production of camalexin, the charateristic phytoalexin of Arabidopsis. On the other hand, the flg22-treated cas-1 upregulated genes were enriched for those involved in 'electron transport or energy pathways' (2.34-fold, P=1.34e–3). Interestingly, genes for plastid (1.93-fold, P=1.46e–5) and chloroplast (1.86-fold, P=5.29e–12) proteins were significantly enriched among the upregulated genes in flg22-treated cas-1. These results suggest that chloroplasts may promote defence gene expression and suppressing chloroplast (photosynthesis) gene expression through CAS during PTI.
Chloroplast regulation of nuclear transcriptional programmes
We identified 99 and 78 transcription factor (TF) genes among the downregulated and upregulated genes in cas-1, respectively. WRKYs are a large family of plant-specific TFs (with 72 genes in Arabidopsis) that act mainly in the defence response network of plant cells19,20. We identified one-third of WRKY genes under the control of CAS (25 downregulated and 1 upregulated; Supplementary Data 1), suggesting an important role for WRKYs in CAS-dependent PTI. In fact, the most represented motif in the promoters (500-bp upstream regions) of the flg22-treated cas-1 downregulated genes was TTGAC (Supplementary Table S1). TTGAC is part of the W-box consensus sequence (TTGAC[C/T]) of the WRKY-binding site19. The W-box motif was found in the promoters of 539 of the 1,235 genes downregulated in flg22-treated cas-1 (44%). The W-box sequences were enriched in the promoter region at 200–400 bp upstream of the translation start site (Supplementary Fig. S13), confirming the W-box as a cis-element of the flg22-treated cas-1 downregulated (CAS-dependent) genes. The flg22-treated cas-1 downregulated TFs included several key factors, such as WRKY33 relaying the MAPK signal to the transcription network in PTI19, and WRKY28/WRKY46 (ref. 21) and CBP60g/SARD1 (ref. 22), essential for the activation of ICS1 and SA synthesis. Taken together, WRKY TFs might have a critical role in PAMP (flg22)-induced chloroplast-mediated transcriptional reprogramming in plant innate immunity.
By contrast, the upregulated TF genes included AtGLK2 (ref. 23), CIA2 (ref. 24), GATA21(GNC)25 and a set of chloroplast sigma factors (AtSIG1, AtSIG3, AtSIG4 and AtSIG6), which are required for chloroplast development26. Interestingly, the I-box motif involved in light-responsive photosynthetic gene expression27 was found in the promoters of 149 of the 687 genes upregulated in flg22-treated cas-1 plants. Thus, it seems that chloroplasts (CAS) regulate plant immune responses through activation of defence-related TFs and suppression of chloroplast function/development by inactivating TFs required for chloroplast development.
Chloroplast-derived ROS and CAS-dependent gene expression
To examine the role of flg22-induced TFs during early PTI, we performed a time course expression analysis using qRT–PCR. PAMP (flg22) triggered a rapid (within 15 min) and transient increase in the expression of TF genes such as CBP60g, WRKY28, WRKY33 and MYB122 (Fig. 5c), suggesting a role for them in early transcriptional reprogramming during PTI. On the other hand, the expression of WRKY75 (Fig. 5c) and SA biosynthesis-related genes (Fig. 4f) peaked at 1–2 h, and PR1 gene expression was induced after 12 h. Interestingly, flg22-induced early expression of TF genes (within 30 min) was not suppressed in cas-1 mutants, but CAS was needed for the middle phase (1–2 h) expression of TF (Fig. 5c) and SA biosynthesis-related genes (Fig. 4f), and PR1 gene expression at the late phase (24 h). On the other hand, overexpression of CAS hastened and enhanced early expression of a set of TF genes such as CBP60g, WRKY33 and MYB122 (Fig. 5c), suggesting a possible activating role of CAS in defence-related TF gene expression. These results imply that after a ~30-min lag chloroplasts may generate CAS-dependent retrograde signals, which is required for prolonged expression of the early defence-related TFs and promotes subsequent expression of defence-related genes. It has been proposed that chloroplast-derived ROS signals, such as 1O2 and H2O2, may activate the expression of nuclear-encoded defence genes28,29. Notably, we found that 427 of 1,235 genes downregulated in flg22-treated cas-1 overlapped with genes that were 1O2-responsive in the flu mutant (fold change >3.0), including several 1O2 marker genes, EDS5 and ERD1 (Fig. 5b and Supplementary Data 1)29. By contrast, only 153 genes overlapped with methyl viologen (MV)-induced O2−/H2O2-responsive genes (fold change >3.0; Supplementary Data 1)30. These results suggest a role for CAS in 1O2 signalling to control nuclear-encoded defence gene expression.
Discussion
CAS is a plant-specific low-affinity/high-capacity non-EF-hand Ca2+-binding protein that is associated with thylakoid membranes of chloroplasts (Supplementary Fig. S14)8,9,10,12. This study demonstrates that the chloroplast protein CAS is required for both PTI and R gene-mediated ETI. PTI and ETI are controlled by complex interconnected signalling networks including the MAP kinase cascade, ROS signalling and the plant hormones SA, JA and ethylene1,2. We found that CAS may act upstream of ROS signalling in PAMP-induced stomatal closure, and downstream of the MAP kinase cascade and upstream of ROS/NO in ETI (Figs 2f, 3c and Supplementary Fig. S6). Furthermore, we demonstrated that CAS is required for the flg22-induced accumulation of SA (Fig. 4). Considering the chloroplast localization of CAS, these findings suggest that chloroplasts have a critical role in both PTI and ETI. CAS may be involved in the coupling of chloroplasts and cellular immune responses and may function upstream of ROS signalling and SA biosynthesis.
We found that CAS is required for transcriptional activation of SA biosynthesis genes, such as ICS1, EDS5, PBS3 and PAD4, whereas the SA signalling pathway downstream of SA is not impaired in cas-1 plants (Fig. 4 and Supplementary Fig. S10). Furthermore, microarray analysis revealed that the expression of a set of TF genes (WRKY28/WRKY46 and CBP60g/SARD1)21,22, essential for activation of SA biosynthesis genes, was greatly reduced in cas-1 plants (Supplementary Data 1). These results suggest that CAS mediates the chloroplast control of SA biosynthesis through these TFs, and the resultant SA subsequently induces a range of basal defence responses. On the other hand, considering the co-localization of CAS and SA biosynthesis in chloroplasts, there remains a possibility that CAS might directly modulate ICS1 activity within chloroplasts.
Recognition of pathogens results in a massive reprogramming of plant cells to activate defence responses and reduce other cellular activities, such as cellular development/organization and photosynthesis31. Microarray analysis demonstrates that CAS controls transcriptional reprogramming during PTI through activation of defence-related TFs including a number of WRKYs and suppression of TFs involved in chloroplast development. We found that a short DNA motif, W-box (TTGAC[T/C]), is over-represented in the promoter sequences of flg22-treated cas-1 downregulated (CAS-dependent flg22-induced) genes (52.6%). The WRKY family TFs regulate biotic and abiotic stress responses and share the WRKY domain that recognizes the W-box19. Interestingly, the W-box is over-represented not only in the promoter regions of defence-related genes, but also in O3-responsive32 and superoxide-responsive promoters30. On the other hand, some promoters of CAS-dependent flg22-suppressed genes contain an I-box motif, a common cis-element characteristic of photosynthesis genes (Supplementary Table S1)27. Thus, these results suggest that CAS-dependent defence gene expression might be mediated by WRKY TFs that target W-box-containing promoters of plant defence genes in a ROS-dependent manner, whereas CAS may control PAMP-induced suppression of photosynthesis gene expression via an I-box-binding protein/I-box-mediated transcription system. Interestingly, some TF genes occur more rapidly in CAS-overexpressing plants, suggesting a direct role for CAS in the promotion of defence-related TF gene expression. However, the molecular function of CAS remains largely unknown. CAS deficiency may disrupt unidentified chloroplast function related to defence responses and indirectly affect the expression pattern of flg22-induced defence genes. Nevertheless, these findings strongly suggest that chloroplasts have a critical role in the transcriptional regulation of plant immune signalling.
It has been proposed that chloroplast-derived retrograde signals, such as chlorophyll synthesis intermediates and chloroplast-derived ROS, control the expression of nuclear-encoded genes for photosynthesis and defence responses33, respectively. When the cell is unable to dissipate excess excitation energy, chloroplasts generate ROS, such as singlet oxygen (1O2) or hydrogen peroxide (H2O2), which activate distinct sets of genes. Interestingly, 1O2 triggers a cell death programme that resembles the effector-triggered HR34. Here, we found that almost one-third of CAS-dependent flg22-induced genes are also induced by 1O2 in the flu mutant (Fig. 5b), but not by MV-induced H2O2. Furthermore, CAS-dependent flg22-induced genes also overlap with the light-induced genes in flu mutants overexpressing thylakoid-localized ascorbate peroxidase, which is expected to compensate for chloroplast-derived O2− and H2O2, but not 1O2 (ref. 29). These results suggest a role for chloroplast-derived 1O2 signalling in CAS-dependent transcriptional control of nuclear-encoded defence genes. Moreover, the flg22-induced extracellular ROS burst was not impaired in cas-1 plants (Supplementary Fig. S15), suggesting a limited role of NADPH oxidase-derived O2− and H2O2 in CAS-dependent immune responses. In addition, it has been recently shown by another group that extracellular Ca2+-induced generation of chloroplast NO and ROS is dependent on CAS, but not plasma membrane-localized NADPH oxidase35, providing further confirmation of the role of CAS in ROS production in chloroplasts. Thus, we assume that CAS may be involved in the chloroplast-derived 1O2-mediated retrograde signalling that regulates the expression of nuclear-encoded defence-related genes at an early stage in PTI. In fact, the release of 1O2 in the flu mutant triggers the expression of WRKY33 and WRKY46 TFs during the first 30 min of reillumination36, a subsequent drastic increase in free SA levels, and the expression of PR1 and PR5 genes37. Time course qRT–PCR analysis revealed that chloroplasts generate signal(s) after a 30-min lag time. Interestingly, it has been reported that Cle elicitor induces 1O2 in cultured cells after a 10-min lag period, and peaks at 30 min after the treatment38. This time course coincides well with those of flg22-induced defence-related gene expression and subsequent SA biosynthesis. We propose that a CAS-dependent chloroplast-derived signal (possibly 1O2) may modulate the defence responses through transcriptional reprogramming of defence-related genes.
Therefore, it is speculated that pathogen signals are transmitted to chloroplasts before the production of chloroplast-derived ROS signal(s). Sai and Johnson11 previously reported that light-to-dark transition triggers a transient rise of stromal free Ca2+ levels in tobacco chloroplasts. Here, we show that PAMPs evoke a long-lasting stromal Ca2+ increase following a rapid cytoplasmic Ca2+ transient (Fig. 1a,b). It is known that the thylakoid lumen accumulates Ca2+ at a high level. CAS is responsible for the full activation of long-lasting stromal Ca2+ transients induced by flg22 and darkness (Fig. 1i,j). Thus, it is plausible that CAS regulates the release and/or uptake of Ca2+ through thylakoid membranes to evoke long-lasting stromal Ca2+ signals. We further demonstrate that several abiotic stresses also induce stress-specific stromal Ca2+ signals (Fig. 1d–f), suggesting that stromal Ca2+ signatures may be involved in stress-specific responses, including rapid inhibition of photosynthesis and accumulation of JA39,40. Interestingly, chloroplasts contain several EF-hand Ca2+ sensor proteins and other types of Ca2+-binding proteins41.
PAMP-induced stromal Ca2+ transients precede CAS-dependent defence gene expression and SA biosynthesis, suggesting a role for chloroplast Ca2+ signatures in the activation of defence gene expression. Interestingly, genes induced by some abiotic stimuli such as light-to-dark transition and osmotic stress, which evoke long-lasting stromal Ca2+ signals, were enriched among genes downregulated in flg22-treated cas-1, but cold and salt-induced genes were not (Supplementary Fig. S16). The W-box sequences were also over-represented in the promoter regions of genes upregulated by osmotic stress, but not of cold and salt stimili-induced genes (Supplementary Tables S1 and S2). Thus, chloroplast Ca2+ signatures are likely involved in stress-specific gene expression. It has been shown that Ca2+ regulates FBPase42,43, and the stability of the photosystem II and thylakoid O2 evolving complexes44. The stromal Ca2+ transients coincide with the stress-induced rapid downregulation of photosynthesis39,45,46. Importantly, photosynthesis collapse may lead to photo-induced production of ROS including 1O2 (ref. 47). Taken together, long-lasting stromal Ca2+ signals may induce the downregulation of photosynthetic electron flow, leading to the production of ROS. Interestingly, cas-1 mutants show higher values of non-photochemical chlorophyll fluorescence quenching than do wild-type plants8. Non-photochemical chlorophyll fluorescence quenching has a major role in the protection of the photosynthetic apparatus against damage by excess light and the production of ROS48.
Herein, we demonstrated a novel chloroplast-dependent pathway that controls plant innate immunity through stromal Ca2+ signalling and chloroplast-to-nuclei retrograde ROS signalling (Fig. 6). Recent studies have shown that plant innate immune responses are modified by light and intrinsic circadian cues49,50. Chloroplasts may act as an environmental sensor in plants51. Interestingly, CAS is required for photoacclimation in Chlamydomonas12. Furthermore, CAS has been shown to be involved in the coupling of diurnal extracellular Ca2+ oscillations to cytosolic Ca2+ oscillations52. Characterization of the molecular function of CAS and chloroplast Ca2+ signalling would provide insight into the role of chloroplasts in cross talk among environmental regulation pathways and plant immunity.
Methods
Plant materials and growth conditions
Arabidopsis thaliana wild-type Columbia ecotype, CAS knockout mutants (cas-1 and cas-2)8, CAS-overexpressing plants8 and apoaequorin transgenic plants were germinated and grown on one-half Murashige and Skoog (MS) medium containing 0.8% (w/v) agar at 22 °C with 16-h light (80–100 μmol m−2 s−1)/8-h dark cycles for 2–4 weeks. For pathogen inoculation experiments, 4- to 5-week-old wild-type and cas-1 plants grown on soil with 10-h light (140–160 μmol m−2 s−1)/14-h dark cycles were used.
Measurements of cytosolic and stromal Ca2+ concentrations
Ca2+ measurements were performed as previously described, with some modifications8. Detached rosette leaves from plate-grown transgenic wild-type and cas-1 Arabidopsis plants expressing aequorin were floated on one-half MS liquid medium containing 5 μM coelenterazine, then kept overnight at 22 °C in the dark. Aequorin luminescence was measured with Lumi Counter 2500 (Microtec). The amount of reconstituted aequorin in the plants was determined by the addition of an equal volume of 2 M CaCl2 in 20% ethanol at the end of the measurements. Aequorin luminescence calibration was performed as previously described53.
Elicitor peptide flg22 was synthesized by Biologica(Nagoya). The chitin suspension was prepared as previously described54. Chitin (Wako) was hydrolysed with concentrated HCl, and left for 2 h at room temperature with occasional mixing. The hydrolysed chitin was then washed with water twice, and dissolved in one-half MS medium to achieve a final chitin concentration of 1.0 mg ml−1. Cold shock stress was applied to the plants by adding ice-cold one-half MS medium to a leaf floated in a cuvette. The measurement of dark-stimulated Ca2+ changes was performed as described11. Ten-day-old plants were incubated at 22 °C under 24-h light conditions (22 μmol m−2 s−1), and then transferred to the dark. In pharmacological experiments, BAPTA (Wako), K-252a (Wako), U-0126 (Calbiochem) or DPI (Calbiochem) were dissolved in ethanol or DMSO and applied to plants in a cuvette at the appropriate concentration and left for 30 min before treatment with flg22.
Plant inoculation and quantification of bacteria
Pathogen inoculations, bacterial growth counts and ion conductance measurements were performed as previously described, with some modifications55. The virulent strain of Pst DC3000 and avirulent strains of Pst DC3000 (carrying avrRpt2 or avrRpm1) were cultured at 28 °C in King's B medium containing 25 μg ml−1 kanamycin, 100 μg ml−1 rifampicin and 50 μg ml−1 cycloheximide, washed once in 10 mM MgCl2 and resuspended in 10 mM MgCl2. Needleless syringe infiltration was used to inoculate plants with pathogen in all cases. Immediately after the infiltration, the plants were allowed to dry and were then kept under 100% humidity for the remainder of the experiment. At indicated time points, leaf discs were collected, ground to homogeneity in water and the titre was determined by serial dilution and plating.
For measurements of ion leakage, 6 leaf discs (8-mm diameter) were removed immediately following infiltration (t=0) and floated in 25 ml of water. After 30 min, the water was removed and replaced with 10 ml of new water. The conductance of this water was measured at the indicated time points.
Stomatal aperture measurements
Stomatal aperture measurements were performed as previously described8. Rosette leaves from 3- to 4-week-old plate-grown plants were detached and floated on the incubation buffer (10 mM 2-(N-morpholino)ethanesulfonic acid (MES)–KOH, pH 6.15; 10 mM KCl; 50 μM CaCl2) for 2 h at 22 °C, in 80–100 μmol m−2 s−1 light. After 2 h, the leaves in the incubation buffer were treated with or without 5 μM flg22, 10 μM H2O2 or 50 μM SNP and then incubated for an additional 2 h. For inhibitor experiments, the leaves were pre-incubated with 50 μM DPI or 200 μM cPTIO for 30 min before treatment with flg22. After the incubation period, epidermal strips of the leaves were observed by microscopy. The stomatal aperture was calculated as the ratio of the pore width/guard cell length. A guard cell length of 22–28 μm was used in the experiments.
Callose deposition
Rosette leaves of 4- to 5-week-old soil grown wild-type and cas-1 plants were infiltrated with 10 mM Tris–HCl (pH 7.2; mock) or the same solution containing 5 μM flg22. After 24 h, the leaves were detached, cleared with ethanol and stained with 10 mM Tris–HCl (pH 7.2) containing 0.01% aniline blue for 30 min. Leaves were stored in 50% glycerol and observed via epifluorescence microscopy under UV illumination with a broadband DAPI filter set. Measurements of callose deposition were made using the image-processing software IMAGEJ.
Metabolite analysis by nano-LC/MS/MS and UPLC/TOFMS
Levels of SA, JA, ABA, IAA, zeatin and zeatin riboside were analysed by nano-liquid chromatography ?" tandem mass spectrometry (LC/MS/MS) with a modification of the methods described by Izumi et al.56. Briefly, 100 mg of leaf samples were homogenized using a ballmill and extracted with methanol/H2O/formic acid (75:20:5) solution containing internal standards (40 pmol (d7-DHZOG, d3-DHZR, d6-iP, d6-iPR), 200 pmol (d6-ABA), 400 pmol (d3-DHZ), 1,000 pmol (d2-GA1, d5-IAA, d2-GA7), 2 μmol lidocaine and 10 μmol (+)-10-camphorsulfonic acid). After centrifugation, half of the supernatant was used for analyses of SA, JA and small charged molecules by ultra-performance liquid chromatography - time-of-flight mass spectrometry (UPLC/TOFMS) as described previously56,57. The remaining supernatant was extracted with water and dried. The dried samples were dissolved in 1 M formic acid and applied to the OASIS MCX cartridge. The fraction containing ABA and IAA was eluted with methanol and, subsequently, the CK fraction was eluted with 60% methanol solution containing 0.35 M NH4. Eluted fractions were further purified and analysed by nano-LC/MSMS.
SA was also measured using a conventional high-performance liquid chromatography system. A total of 200 mg of seedling samples without roots was homogenized and extracted with 100% methanol containing the internal standard anisic acid. Free and glycosylated SA were separated and analysed by high-performance liquid chromatography.
VIGS and Agrobacterium-mediated transient expression
VIGS was done as described by Ratcliff et al.58. pBINTRA6 (RNA1) and the pTV00 containing the NbCAS cDNA fragment (RNA2) were transformed by electroporation separately into Agrobacterium tumefaciens strain GV3101 (pSoup). A mixture of equal parts of Agrobacterium suspensions of RNA1 and RNA2 was infiltrated into 2- to 3-week-old N. benthamiana leaves.
For cell death assay, Agrobacterium strain GV3101 (pSoup) carrying NtMEK2DD, INF1, cf9/Avr9 or GUS (pGreen binary vectors) was infiltrated into the upper leaves of 4- to 5-week-old NbCAS-silenced N. benthamiana plants.
Microarray experiments and data analysis
The genome-wide microarray analyses were performed using the Arabidopsis v4 2 colour microarray (Agilent Technologies). Light grown, 2-week-old, wild-type and cas-1 mutant plants were floated on one-half MS medium for 1 day (0 h), and then treated with 1 μM flg22 for 2 h. Total RNA was isolated with the Qiagen RNeasy plant mini kit following the manufacturer's instructions. Two independent biological replicates were performed for each sample and control. Each 200-ng total RNA sample was used to prepare Cy3- or Cy5-labelled target cRNA with the Low Input Quick Amp Labeling Kit (Agilent Technologies) and used in dual colour microarray hybridization with the Agilent Arabidopsis v4 oligo microarray slide. A dye-swap experiment was performed with two different RNA populations to eliminate the signal variation caused by the differential labelling efficiency of Cy3 and Cy5 dyes. The microarray data were normalized by the LOWESS method using the Feature Extraction software v. 10.7 (Agilent Technologies) and the expression ratios were analysed (Non-Uniformity Outlier and Feature Population Outlier). Data with a P-value of >0.01 were eliminated. The genes that showed a consistent expression pattern in cas-1 and at least a twofold difference in the expression level in both slides are listed in Supplementary Data 1.
The genes induced by flg22 (ref. 17), by illumination of the flu mutant (chloroplast-derived 1O2)29 and the flu mutant overexpressing thylakoid-localized ascorbate peroxidase29 and by MV30 were obtained from publications. To extract flu-regulated genes, raw signal intensity data (GSE10812) were obtained from the NCBI GEO database29. The genes that revealed a consistent expression pattern at 2 h after a dark-to-light shift in the flu mutant compared with wild type and exhibited at least a threefold difference in the expression level were used for this analysis.
Gene ontology and promoter sequence analyses
Gene ontology analysis was performed with the Arabidopsis Classification SuperViewer at the BAR of the University of Toronto. We compared the gene expression profiles of our microarray experiments with available expression data via the expression browser at BAR. We also searched for over-represented cis-elements in the 500-bp upstream regions of the down- and upregulated genes in flg22-treated cas-1 plants using the Regulatory Sequence Analysis tool (RSAT; http://rsat.ulb.ac.be/rsat).
Additional information
Accession codes: The NbCAS sequence has been deposited in the DNA Data Bank of Japan under accession code AB720027.
How to cite this article: Nomura, H. et al. Chloroplast-mediated activation of plant immunity signalling in Arabidopsis. Nat. Commun. 3:926 doi: 10.1038/ncomms1926 (2012).
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Acknowledgements
We thank M. Isobe and M. Kuse for providing coelenterazine, D.C. Baulcombe for pTV00 vector, Y. Ohashi and I. Mitsuhara for the NtMEK2DD clone, P. Mullineaux and R. Hellens for pGreen binary vector, S. Kamoun for INF1, J.D.G. Jones for cf9/Avr9 and the Leaf Tobacco Research Center, Japan Tobacco, for N. benthamiana seeds. We also thank Y. Kubo, M.H. Sato, Y. Yagi and Y. Ishizaki for discussions, and T. Kimura and K. Yamasaki for technical assistance. This work was supported by Grants-in-Aid (No. 20200060 to Y.N and 22370022 to T.S.), Private University Strategic Research Foundation Support Program (S0801060) and Joint Research Program implemented at the Institute of Plant Science and Resources, Okayama University from MEXT, and this work was also supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
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H.N., H.Y. and T. Shiina designed the research plan; H.N., T.K., S.U., Y.K., K.S., K.N., T.F., S.S., I.N.S., H.Y., N.Y. and T. Shiina performed experiments; K.T., T. Sugimoto and E.F. carried out metabolome and hormone analyses; H.N. and T. Shiina wrote the manuscript.
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Supplementary information
Supplementary Figures, Tables, Methods and References.
Supplementary Figures S1-S16, Supplementary Tables S1-S2, Supplementary Methods and Supplementary References. (PDF 3013 kb)
Supplementary Data 1
List of down-regulated genes (1,235) in cas-1 plants treated with flg22 for 2 h. (XLS 327 kb)
Supplementary Data 2
List of up-regulated genes (687) in cas-1 plants treated with flg22 for 2 h. (XLS 183 kb)
Supplementary Data 3
List of down-regulated genes (197) in cas-1 plants without treatment. (XLS 72 kb)
Supplementary Data 4
List of up-regulated genes (7) in cas-1 plants without treatment. (XLS 29 kb)
Supplementary Data 5
Functional classification of down-regulated genes in flg22-treated cas-1. (XLS 137 kb)
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Nomura, H., Komori, T., Uemura, S. et al. Chloroplast-mediated activation of plant immune signalling in Arabidopsis. Nat Commun 3, 926 (2012). https://doi.org/10.1038/ncomms1926
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DOI: https://doi.org/10.1038/ncomms1926
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