Introduction

Programmed cell death (PCD) is a sequence of genetically regulated events resulting in the elimination of specific cells, tissues, or whole organs1, which is required both for normal development and to face stress conditions2,3,4. In plants, one well-characterized example of PCD is hypersensitive response taking place on incompatible plant–pathogen interactions3, which leads to cell death and then forms visible lesions at the site infected by an avirulent pathogen, as a result, limits the pathogen spread4. Phytohormones including salicylic acid (SA) and jasmonate (JA) appear to be key players for hypersensitive response regulation5.

To date, a large number of mutants displaying spontaneous cell death lesions have been identified in plants including Arabidopsis, rice, barley, maize, and so on6,7,8,9. These mutants have been named as lesion-mimic mutants (LMM) because of the form of lesions in the absence of pathogen infection10. In some of LMM, the SA or JA signaling has been activated9,11. By isolating LMM’s genes, many of regulators that play important roles in PCD and SA or JA signal defense responses have been identified, including ACCELERATED CELL DEATH11, LESION SIMULATING DISEASE1, and NICOTIANA BENTHAMIANA HOMEOBO112,13,14.

SA is involved in plant defense and cell death15,16. The level of SA correlates with the expression of PATHOGENESIS-RELATED1 (PR1) gene and resistance to pathogen attack17,18. The NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) gene is required for SA-induced expression of PR1 gene and resistance in Arabidopsis19,20.

Jasmonates (JAs) including jasmonic acid, methyl jasmonate (MeJA), and other derivatives, are a basic class of plant hormones involved in different processes, including plant growth, development, and responses to biotic and abiotic stresses21,22,23. JA signaling pathway is closely involved in plant PCD24,25. The F-box protein CORONATINE INSENSITIVE1 (COI1) has been found to be an indispensable component of the JA signaling pathway26,27,28. JA induces expression of many genes including those for vegetative storage proteins (VSPs), a thionin (THI2.1), and a plant defensin (PDF1.2), which is abolished in the coi1 mutant28,29.

In plants, PCD also correlates to reactive oxygen species (ROS), which produces in plants as byproducts of aerobic metabolism and controls a variety of physiological functions including responses to abiotic and biotic stress and plant growth and development30,31. The generation of ROS is one of the most normal responses to PCD32,33,34 and the genes associated with oxidative stress are up-regulated during PCD35,36,37,38. For example, the expression of ascorbate peroxidase 2 (APX2) is rapidly induced by oxidative stress37. Oxidative signal inducible 1 (OXI1) is regulated by ROS and the OXI1 expression is specifically induced by stress conditions that cause cell death38,39. The expressions of bonzai1-associated protein1 (BAP1) and a putative c2h2 zinc finger transcription factor (ZP) are induced specifically by singlet oxygen, one form of ROS40.

In addition, plant PCD is resulted from blockage of some metabolic pathways such as Tyr degradation41, an essential pathway to animals42. The Tyr degradation pathway includes five-step enzymatic reactions42. First, Tyr aminotransferase (TAT) catalyzes the conversion of Tyr into 4-hydroxyphenylpyruvate, which is then converted into homogentisate by 4-hydroxyphenylpyruvate dioxygenase. Next, homogentisate dioxygenase (HGO) catalyzes homogentisate to yield maleylacetoacetate that is isomerized by maleylacetoacetate isomerase (MAAI) to fumarylacetoacetate, and finally fumarylacetoacetate hydrolase (FAH) hydrolyses fumarylacetoacetate to fumarate and acetoacetate42. Loss of FAH results in the accumulation of fumarylacetoacetate and maleylacetoacetate, both of which would undergo spontaneous reduction to succinylacetoacetate that is converted to succinylacetone (SUAC) by spontaneous nonenzymatic decarboxylation42. SUAC is toxic to cells and tissues resulting in severe metabolic disorder diseases in mammals42,43,44. In Arabidopsis, we have identified one LMM named as short-day sensitive cell death1 (sscd1) displaying spontaneous cell death lesions under short day (SD) conditions, and isolated the SSCD1 gene encoding the Arabidopsis putative FAH, which uncovered the role of Tyr degradation pathway in plant41.

To investigate whether the appearance of spontaneous cell death lesions in the sscd1 mutant is related to SA and JA, in this study, we first analyzed expression of some SA- and JA-inducible genes and then generated double mutants of sscd1 with npr1 and coi1, respectively, and found that cell death in sscd1 is accompanied by JA accumulation and repressed by mutation of COI1, however, it is unrelated to SA although it is accompanied by up-regulation of SA-inducible PR1. Furthermore, we found that the up-regulation of ROS marker genes such as APX2, OXI1, BAP1, and ZP in the sscd1 mutant is also repressed by mutation of COI1. In addition, we found that treatment of Arabidopsis seedlings with SUAC induces expression of JA-inducible genes. However, treatment with JA induces expression of some Tyr degradation pathway genes including TAT3 encoding an Arabidopsis putative TAT45, HGO, and MAAI, which is dependence of COI1. Our work uncovered a crosstalk between JA signaling and Tyr degradation pathway in the regulation of sscd1 cell death, i.e. JA signaling positively regulates sscd1 cell death by up-regulating Tyr degradation.

Results

Cell death in sscd1 is uncorrelated to SA signaling although it is accompanied by up-regulation of SA-inducible PR1

The sscd1 mutant grows normally under long day (LD), but displays obvious cell death symptoms after transferred to SD for 3 days41. To investigate whether cell death in sscd1 is related to SA, we first analyzed expression of PR1, one of SA-inducible genes, in wild-type and sscd1 seedlings transferred from LD to SD for 1, 2 and 3 days by quantitative real-time polymerase chain reaction (RT-qPCR). As shown in Fig. 1a, no significant difference in the expression level of PR1 between wild type and sscd1 was observed before seedlings were transferred to SD or after they were transferred to SD for 1 day, however, the expression level of PR1 was significantly increased in sscd1 compared to wild type when seedlings were transferred to SD for 2 days and that this increase was much more obvious after seedlings were transferred to SD for 3 days.

Figure 1
figure 1

Cell death in sscd1 is uncorrelated to SA signaling although it is accompanied by the up-regulation of SA-inducible PR1. (a) Relative expression level of SA-inducible genes PR1 in wild-type (WT) and sscd1 seedlings that were grown under LD for 3 weeks, and then transferred to SD for 0, 1, 2 and 3 days. (b) The content of SA in wild-type (WT) and sscd1 seedlings that were grown under LD for 3 weeks, and then transferred to SD for 0, 2 and 3 days. (c) Relative expression level of SA-inducible genes PR1 in wild-type (WT), npr1-1, sscd1 and sscd1npr1 seedlings that were grown under LD for 3 weeks, and then transferred to SD for 3 days. (d) The rate of seedlings death in sscd1 and sscd1npr1 seedlings grown on MS under SD for 6–9 days. LD, long day; SD, short day. The expression of gene was analyzed by RT-qPCR, relative expression level was normalized to those of ACTIN2 and the control (in wild type) was set to 1. Mean ± SE from three biological replicates. Asterisk ** represents the significance of differences (two-tailed Student’s t-test) at the level of P < 0.01.

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Since SA-inducible gene PR1 was significantly up-regulated in the sscd1 mutant compared to wild type when seedlings were transferred from LD to SD for 2–3 days (Fig. 1a), we next measured the content of SA to investigate whether up-regulation of PR1 is resulted from accumulation of SA in the sscd1 mutant. Unexpected, the content of SA was not significantly increased in sscd1 compared to wild type before seedlings were transferred to SD or after they were transferred to SD for 2 or 3 days (Fig. 1b). Therefore, the up-regulation of PR1 in sscd1 was not related to SA.

NPR1 is a SA receptor in SA signaling46 and expression of SA-inducible PR1 is abolished in the npr1 mutant19. To investigate whether loss of NPR1 influences the up-regulation of PR1 as well as the cell death in sscd1, a double mutant of sscd1 and npr1-1 was generated and then expression of PR1 was analyzed as well as the seedlings phenotype was observed. As shown in Fig. 1c, expression of PR1 was almost undetected in the npr1-1 mutant whereas it was significantly induced in the sscd1npr1 double mutant although its level was much lower than that in the sscd1 single mutant after seedlings were transferred to SD for 3 days. Furthermore, the rate of seedlings death in sscd1npr1 was similar to that in sscd1 (Fig. 1d). These results demonstrated that both up-regulation of PR1 and cell death in sscd1 are independent of NPR1 and that cell death in sscd1 is uncorrelated to SA signaling although it is accompanied by the up-regulation of SA-inducible PR1.

Cell death in sscd1 is accompanied by up-regulation of JA-inducible genes and accumulation of jasmonic acid

Since cell death in sscd1 is uncorrelated to SA signaling (Fig. 1), we next investigated whether it is related to JA signaling. We first analyzed the expression of JA-inducible genes including VSP2, PDF1.2, and THI2.1 in wild-type and sscd1 seedlings which were transferred from LD to SD for 1, 2 and 3 days. The results showed that the expression level of these genes was similar in wild type and sscd1 before seedlings were transferred to SD or after they were transferred to SD for 1 day, however, it was significantly increased in sscd1 compared to wild type after seedlings were transferred to SD for 2 days and that this increase was much more obvious after seedlings were transferred to SD for 3 days (Fig. 2a–c). Then, we measured the content of jasmonic acid in wild type and sscd1 before seedlings were transferred to SD or after they were transferred to SD for 2 and 3 days to investigate whether the up-regulation of these genes is resulted from the accumulation of jasmonic acid. The result showed that there was no significant difference in the content of jasmonic acid between wild type and sscd1 before seedlings were transferred to SD, however, the content of jasmonic acid was significantly increased in sscd1 compared to that in wild type after seedlings were transferred to SD for 2 days and that this increase was much more distinct after seedlings were transferred to SD for 3 days (Fig. 2d). These results indicated that the cell death in sscd1 is accompanied by both up-regulation of JA-inducible genes and accumulation of jasmonic acid and suggested that the up-regulation of JA-inducible genes is caused by the accumulation of jasmonic acid.

Figure 2
figure 2

Cell death in sscd1 is accompanied by the up-regulation of JA-inducible genes and the accumulation of jasmonic acid. (a–c) Relative expression level of JA -inducible genes VSP2 (a), PDF1.2 (b), THI2.1 (c) in wild-type (WT) and sscd1 seedlings that were grown under LD for 3 weeks, and then transferred to SD for 0, 1, 2 and 3 days. (d) The content of jasmonic acid in wild-type (WT) and sscd1 seedlings that were grown under LD for 3 weeks, and then transferred to SD for 0, 2 and 3 days. LD, long day; SD, short day. The expression of genes was analyzed by RT-qPCR, relative expression level was normalized to those of ACTIN2 and the control (in wild type) was set to 1. Mean ± SE from three biological replicates. Asterisk * and ** represent the significance of differences (two-tailed Student’s t-test) at the levels of P < 0.05 and P < 0.01, respectively.

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Cell death in sscd1 is repressed by mutation of COI1

COI1 is a JA receptor in JA signaling47. To investigate whether JA signaling mediates the sscd1 cell death, we generated the sscd1coi1 double mutant through a cross of sscd1 with coi1-228 to break the JA signaling, and then observed the phenotype of seedlings. It was interesting that the phenotype of seedlings death was obviously rescued in sscd1coi1 compared to sscd1 (Fig. 3a). For example, 65% of 7-old sscd1 seedlings grown under SD were dead whereas the rate of sscd1coi1 seedlings death was only 43% (Fig. 3b). This result suggested that the cell death in sscd1 is repressed by breakdown of JA signaling through mutation of COI1 and that JA signaling positively regulates the sscd1 cell death.

Figure 3
figure 3

Cell death in sscd1 is reduced by mutation of COI1. (a) The phenotype of wild-type (WT), sscd1, coi1-2 and sscd1coi1 seedlings grown on MS under SD for 7 days. (b) The rate of seedlings death in coi1-2 and sscd1coi1 seedlings grown on MS under SD for 7 days. SD, short day. Mean ± SE from three biological replicates. Asterisk ** represents the significance of differences (two-tailed student’s t-test) at the level of P < 0.01.

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Mutation of COI1 suppresses the up-regulation of ROS marker genes in sscd1

Previously, we found that ROS marker genes such as APX2, OXI1, BAP1 and ZP were up-regulated before an occurrence of cell death in the sscd1 mutant48, so, we next investigated whether the repression of cell death in sscd1 by mutation of COI1 is correlated with the expression of these genes. Since the cell death phenotype of sscd1 seedlings that were grown under SD appeared on the 6th day41,49, therefore, we tested the expression of APX2, OXI1, BAP1 and ZP in seedlings grown under SD for 5 days. As shown in Fig. 4, the expression pattern of APX2, OXI1, BAP1 and ZP was similar in both WT and coi1-2, however, the up-regulation of these genes in sscd1 was significantly suppressed in sscd1coi1 (Fig. 4), which indicated that the up-regulation of ROS marker genes in sscd1 could be suppressed by the mutation of COI1.

Figure 4
figure 4

Mutation of COI1 suppresses the up-regulation of ROS marker genes in sscd1. (a–d) Analysis of the relative expression levels of APX2 (a), OXI1 (b), BAP1 (c) and ZP (d) in wild-type (WT), coi1-2, sscd1 and sscd1coi1 seedlings that were grown under SD for 5 days. ROS, Reactive Oxygen Species; SD, short day. The expression of genes was analyzed by RT-qPCR, relative expression level was normalized to those of ACTIN2 and the control (in wild type) was set to 1. Mean ± SE from three biological replicates. Asterisk * represents the significance of differences (two-tailed Student’s t-test) at the level of P < 0.05.

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SUAC treatment activates the expression of JA-inducible genes

Previously, we speculated that the cell death in sscd1 is resulted from the accumulation of SUAC and also found that treatment of Arabidopsis wild-type seedlings with SUAC mimicked the cell death phenotype of sscd141. We next investigated whether SUAC treatment activates the expression of JA-inducible genes. To this end, we analyzed the expression of VSP2 and THI2.1 in wild-type seedlings treated with SUAC, in which some leaves started wilting. The result showed the expression of both VSP2 and THI2.1 was significantly increased upon SUAC treatment (Fig. 5), indicating that SUAC treatment could activate the expression of JA-inducible genes.

Figure 5
figure 5

Exogenous SUAC up-regulates the expression of JA-inducible genes VSP2 and THI2.1. (a,b) Relative expression level of JA-inducible genes VSP2 (a) and THI2.1 (b) in wild-type seedlings that were grown under LD for 3 weeks, and then transferred to SD and treated with ddH20 or 1,280 μg/mL SUAC for 3 days. SUAC, succinylacetone; LD, long day; SD, short day. The expression of genes was analyzed by RT-qPCR, relative expression level was normalized to those of ACTIN2 and the control (without SUAC treatment) was set to 1. Mean ± SE from three biological replicates. Asterisk * and ** represent the significance of differences (two-tailed Student’s t-test) at the levels of P < 0.05 and P < 0.01, respectively.

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Treatment with MeJA causes the COI1-dependent up-regulation of some Tyr degradation pathway genes

Since the cell death in sscd1 is accompanied by the accumulation of jasmonic acid (Fig. 2d) and could be repressed by breakdown of JA signaling through mutation of COI1 (Fig. 3), we next investigated whether treatment of Arabidopsis wild-type and coi1-2 seedlings with MeJA influences the Tyr degradation pathway by analyzing the expression of Tyr degradation pathway genes including TAT3, HGO, MAAI, and SSCD1. The results showed that the expression level of TAT3, HGO, and MAAI except SSCD1 was significantly increased in wild type upon MeJA treatment (Fig. 6), especially, an increase of TAT3 expression level in wild type treated with MeJA was much more significant compared with HGO and MAAI (Fig. 6a–c). However, it was interesting that the expression level of these genes was not significantly increased in the coi1-2 mutant upon MeJA treatment (Fig. 6). These results suggested that MeJA up-regulates the expression of some Tyr degradation pathway genes, which would promote Tyr degradation, however, the breakdown of JA signaling through mutation of COI1 could eliminate an effect of JA on Tyr degradation pathway.

Figure 6
figure 6

Exogenous MeJA up-regulates the expression of Tyr degradation pathway genes TAT3, HGO, and MAAI and this up-regulation is dependent on COI1. (a–d) Relative expression level of Tyr degradation pathway genes TAT3 (a), HGO (b), MAAI (c) and SSCD1 (d) in wild-type (WT) and coi1-2 seedlings that were grown under LD for 7 days, and then transferred to SD and treated with ddH20 or 100 μM MeJA for 3 days. MeJA, methyl jasmonate; LD, long day; SD, short day. The expression of genes was analyzed by RT-qPCR, relative expression level was normalized to those of ACTIN2 and the control (in wild type without MeJA treatment) was set to 1. Mean ± SE from three biological replicates. Asterisk * and ** represent the significance of differences (two-tailed Student’s t-test) at the levels of P < 0.05 and P < 0.01, respectively.

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Discussion

Tyr degradation pathway is essential to animals42 but it is not well understood in plants. Previously, we found that mutation of SSCD1 encoding Arabidopsis FAH, an enzyme catalyzing the final step of Tyr degradation pathway, results in spontaneous cell death under SD, which uncovered an important role of Tyr degradation pathway in plants41. Afterwards, we found that sugar suppresses cell death caused by disruption of FAH in Arabidopsis, indicating that Tyr degradation is regulated by sugar in plants49. Recently, we found that cell death resulted from loss of FAH in sscd1 is related to chlorophyll (Chl) biosynthesis, suggesting a crosstalk between Tyr degradation and Chl biosynthetic pathways in mediating the sscd1 cell death48. Phytohormones such as SA and JA are involved in PCD13,14,15,16,24,25,50. In this study, the investigation whether cell death resulted from loss of FAH in Arabidopsis is related to SA and JA would expand our understandings on the regulation of Tyr degradation pathway in plants.

Through testing expression of SA-inducible PR1 and content of SA, we found that cell death in sscd1 was accompanied by the up-regulation of SA-inducible PR1 (Fig. 1a), however, the content of SA was not significantly altered between in sscd1 and wild type (Fig. 1b), which indicated that the up-regulation of PR1 in sscd1 is independent of SA. Similarly, an increase of PR1 expression in the loh1 mutant displaying spontaneous cell death phenotype is also independent of SA51. Breakdown of SA signaling by mutation of NPR1 that encodes a receptor of SA46 represses expression of PR119. In our study, the expression of PR1 was also repressed in sscd1npr1 compared to sscd1 (Fig. 1c), however, the rate of seedlings death was similar in sscd1npr1 and sscd1 (Fig. 1d), suggesting that the cell death in sscd1 is uncorrelated to both SA signaling and the up-regulation of PR1. In addition, we also generated the sscd1nahG double mutant by crossing sscd1 with nahG harboring a bacterial gene encoding salicylate hydroxylase that catalyzes the decarboxylation of SA52,53 and found that the degree of cell death was similar between sscd1nahG and sscd1 (data not shown), indicating that the degradation of SA would not affect the cell death in sscd1, which further confirmed the sscd1 cell death is not related to SA.

However, cell death in sscd1 was accompanied by the up-regulation of JA-inducible genes as well as the accumulation of jasmonic acid (Fig. 2). The up-regulation of JA-inducible genes in sscd1 should be resulted from the accumulation of jasmonic acid, but why the cell death of sscd1 is accompanied by the accumulation of jasmonic acid? In animals, loss of FAH results in the accumulation of Tyr degradation pathway’s abnormal metabolite SUAC that is toxic to cells and tissues resulting in severe metabolic disorder diseases42. In plants, we have found that treatment of Arabidopsis wild-type seedlings with SUAC mimicked the sscd1 cell death phenotype41 and demonstrated that the cell death of sscd1 seedlings correlates with the accumulation of SUAC54. Recently, we found that SUAC affects Chl biosynthesis, resulting in the generation of ROS and then inducing cell death48. Some researcher’s work has shown that JA could be synthesized in response to singlet oxygen that is one form of ROS25,55. Singlet oxygen is very unstable and difficult to detect within a cell55, however, some genes were specifically induced by singlet oxygen40. Recently, we found that the genes induced specifically by singlet oxygen40 were up-regulated in sscd148, suggesting that an effect of SUAC on Chl biosynthesis results in the generation of singlet oxygen in the sscd1 mutant. Furthermore, we found that treatment of Arabidopsis wild-type seedlings with SUAC activated the expression of JA-inducible genes (Fig. 5). Taken together, we concluded that cell death in sscd1 was accompanied by the accumulation of JA (Fig. 2d) is due to the synthesis of JA in response to singlet oxygen.

TAT catalyzes the first step in Tyr degradation pathway56. For the first time, Titarenko et al.57 reported that TAT could be induced by wounding as well as by JA. The gene for the F-box protein COI1 was identified for its irreplaceable role in JA signal transduction26,27,28. Mutations in the COI1 gene result in plants compromised in all known JA responses: defense against biotic and abiotic stresses, growth inhibition, and fertility26,27,28. Titarenko et al.57 reported that wounding induced TAT in wild type but not in the coi1 mutant, suggesting that wound-induced TAT is dependent on JA signaling. Brosché and Kangasjärvi58 reported that expression of TAT3 encoding Arabidopsis putative TAT45 was induced by JA. In this study, we not only confirmed that expression of TAT3 was induced by JA (Fig. 6a) but also found that expression of some of Tyr degradation pathway’s genes including HGO and MAAI was also induced by JA (Fig. 6b,c), however, the expression of these genes in the coi1-2 mutant was not significantly induced by JA (Fig. 6a–c), which suggested that JA signaling up-regulates Tyr degradation in plants.

JA plays an important role in cell death regulation. Singlet oxygen- and JA-mediated cell death in irradiated flu plants is likely to be a form of PCD59. Inactivation of the EXECUTER1 protein abrogates not only singlet oxygen-mediated cell death of flu plants but also accumulation of JA, however, inactivation of JA biosynthesis in the aos/flu double mutant does not affect singlet oxygen-mediated cell death55, hence, JA does not act as second messengers during singlet oxygen-mediated cell death but forms an integral part of a stress-related signaling cascade activated by singlet oxygen that encompasses several signaling pathways known to be activated by abiotic and biotic stressors55. In our study, the cell death of sscd1 seedlings was repressed by mutation of COI1 (Fig. 3). Accordingly, the up-regulation of ROS-inducible genes APX2 and OXI1, as well as singlet oxygen specifically induced genes BAP1 and ZP was also repressed by mutation of COI1 (Fig. 4), suggesting that the breakdown of JA signaling reduces the generation of ROS in the sscd1 mutant. We have just discussed above that JA signaling up-regulates Tyr degradation. Therefore, the accumulation of JA in sscd1 would promote cell death by up-regulating Tyr degradation producing more SUAC. However, blockage of JA signaling by mutation of COI1 breaks the action of JA in Tyr degradation in sscd1, resulting in repression of cell death.

Taken all above together, we concluded that cell death resulted from loss of FAH in Arabidopsis is related to JA but not SA, and proposed a model for the relationship between JA and Tyr degradation pathway in mediating the sscd1 cell death. In the sscd1 mutant, the accumulation of SUAC results in the generation of singlet oxygen, which induces cell death as well as JA synthesis. The accumulation of JA in sscd1 accelerates Tyr degradation by up-regulating Tyr degradation pathway, producing more SUAC, which promotes cell death. Once JA signaling is broken by mutation of COI1, the up-regulation of Tyr degradation by JA in sscd1 is eliminated, reducing production of SUAC, as a result, the sscd1 cell death is repressed.

Methods

Plant material and growth conditions

The sscd1 mutant was isolated previously in our laboratory41. The coi1-2 mutant28 was kindly provided by Professor Xie (Tsinghua University). The npr1-1 mutant19 was obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, Columbus, OH, USA).

Seeds were surfaced sterilized and plated on Murashige & Skoog (MS) medium in which 1% sucrose was added. Plates were chilled at 4 °C in darkness for 3 days and then transferred to a growth chamber with LD (16 h of light/8 h of dark) or SD (8 h of light/16 h of dark) under 150 μmol photons m−2 s−1, controlled temperature (22 ± 2 °C).

For RT-qPCR analysis and determination of SA and jasmonic acid in Figs. 1 and 2, the seeds were germinated on MS medium and grown under LD for 1 week and then the seedlings were transplanted to a new MS medium for additional 2 weeks’ growth under LD, and then transferred to SD.

Construction of double mutants

The sscd1coi1 double mutant was created by first selecting F2 individuals from a cross between sscd1 and coi1-2 on plates containing 25 mM MeJA by screening for decreased sensitivity to JA28, and then F3 lines were selected by sequencing the SSCD1 gene41. The primers for sequencing the SSCD1 gene are as follows: forward primer is 5′-CCTCGTCCTGCCGTCGCTAT-3′ and reverse primer is 5′-CTTGTGGATGGCCCTGACCT-3′.

The sscd1npr1 double mutant was created by selecting F2 individuals from a cross between sscd1 and npr1-1 (a recessive mutation with a single base mutation in NPR119) by sequencing SSCD1 and NPR1, respectively. The primers for sequencing the NPR1 gene are as follows: forward primer is 5′-GTGTGCTCTTCATTTCGCTGTTG-3′ and reverse primer is 5′-ACCCGGTGATGTTCTCTTCGTA-3′.

RT-qPCR analysis

RT-qPCR analysis were performed as described48. Total RNA was isolated using TRIZOL reagent (LIFE TECHNOLOGIES, https://www.thermofisher.com/us/en/home/brands/life-technologies.html). After incubation with DNase I (RNase Free, THERMO FISHER SCIENTIFIC, https://www.thermofisher.com/) at 37 °C for 30 min and then at 65 °C for 10 min to remove genomic DNA, RNA concentrations and purities were measured spectrophotometrically using OD260/OD280 and OD260/OD230 ratios (ND-1000, NanoDrop, THERMO FISHER SCIENTIFIC). Complementary DNA was synthesized from the mixture of oligo-dT primers and random primers using a ReverTraAce qPCR RT kit (perfect real time) according to the manufacturer’s instructions (TOYOBO, https://www.toyobo-global.com/).

RT-qPCR was performed in 96-well blocks using a SYBR qPCR mix (ROCHE, https://lifescience.roche.com/) with a BIO-RAD CFX CONNECT Real-Time PCR detection system (https://www.biorad.com/) following the manufacturer’s instructions. The RT-qPCR amplifications were performed under the following conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The primers of genes tested by RT-qPCR are listed in Table 1, and ACTIN2 was used as an internal control. The gene expression for each sample was calculated on three analytical replicates, and the relative expression was quantified using the 2−ΔΔCt method. The experiment was performed in three independent biological repeats. The significance of differences between datasets was evaluated using the two-tailed Student’ t-test.

Table 1 Primers of genes tested by RT-qPCR.
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Determination of the dead seedlings

Seedlings of sscd1 and sscd1npr1 were grown under SD and the number of dead seedling (all leaves were completely bleached) was counted from day 6 to 9. Seedlings of sscd1 and sscd1coi1 were grown under SD for 7 days and the number of dead seedlings was counted. The rate of seedling death was calculated as the percentage of dead seedlings from 250 to 300 seedlings. At least three independent biological repeats were performed.

Detection of jasmonic acid and SA

0.5 g of leaves from WT and sscd1 seedlings that were grown under LD for 3 weeks and then transferred to SD for 0, 2 and 3 days was harvested for jasmonic acid and SA extraction. The harvested tissues were immediately ground to a fine powder in liquid N2, and then exposed to extraction buffer (1.0 mL of 80% methanol) at 4 °C overnight. The samples were centrifuged at 10,000g for 5 min, and the residues were re-extracted with 0.6 mL of 80% methanol (HPLC grade methanol, Merck, Germany). The supernatants were vacuum freeze dried to dryness at − 60 °C, then dissolved in 200 μL of 0.1 M sodium phosphate buffer (pH 7.8), and extracted with 200 μL of petroleum ether. The aqueous phase was purified using a Waters Sep-Pak C18 cartridge (Waters, USA). The cartridge was washed with 200 μL of ddH2O and then eluted with 1.5 mL of 80% methanol. The eluate with 80% methanol was vacuum freeze dried. The dried extract was dissolved in 40 μL of 50% methanol and used for LC/MS assay in a WATERS ACQUITY SQD (LC/MS) system according to Liu et al.60.

MeJA treatments

For MeJA treatment, the seedlings of WT and coi1-2 were first grown under LD for 7 days, and then transferred to SD for 3 days. Once transferred to SD, plants were sprayed with 100 μM MeJA or ddH2O (as a control) under light once per day for 3 days. After treatment for 3 days, the plants were harvested and used for RT-qPCR analysis. The experiment was performed in three independent biological repeats.

Treatment with SUAC

The seeds were germinated on MS medium and grown under LD for 1 week and then the seedlings were transplanted to a new MS medium for additional 2 weeks’ growth under LD, and then transferred to SD and sprayed with 1,280 μg mL−1 SUAC (SIGMA) or ddH2O (as a control) twice per day for 3 days. After treatment for 3 days, the plants were harvested and used for RT-qPCR analysis. The concentration of SUAC treatment was determined following our previous work49. The experiment was performed in three independent biological repeats.