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. 2017 Mar 1;173(4):2294–2307. doi: 10.1104/pp.16.00008

The Arabidopsis Mitochondrial Protease FtSH4 Is Involved in Leaf Senescence via Regulation of WRKY-Dependent Salicylic Acid Accumulation and Signaling1

Shengchun Zhang 1,2, Cui Li 1,2, Rui Wang 1,2, Yaxue Chen 1,2, Si Shu 1,2, Ruihua Huang 1,2, Daowei Zhang 1,2, Jian Li 1,2, Shi Xiao 1,2, Nan Yao 1,2, Chengwei Yang 1,2,*
PMCID: PMC5373041  PMID: 28250067

The mitochondria-localized protein FtSH4 regulates leaf senescence and modulates the cross talk of ROS, SA, and WRKY signaling pathways.

Abstract

Mitochondria and autophagy play important roles in the networks that regulate plant leaf senescence and cell death. However, the molecular mechanisms underlying the interactions between mitochondrial signaling and autophagy are currently not well understood. This study characterized the function of the Arabidopsis (Arabidopsis thaliana) mitochondrial AAA-protease gene FtSH4 in regulating autophagy and senescence, finding that FtSH4 mediates WRKY-dependent salicylic acid (SA) accumulation and signaling. Knockout of FtSH4 in the ftsh4-4 mutant resulted in severe leaf senescence, cell death, and high autophagy levels. The level of SA increased dramatically in the ftsh4-4 mutant. Expression of nahG in the ftsh4-4 mutant led to decreased SA levels and suppressed the leaf senescence and cell death phenotypes. The transcript levels of several SA synthesis and signaling genes, including SALICYLIC ACID INDUCTION DEFICIENT2 (SID2), NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), and NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1), increased significantly in the ftsh4-4 mutants compared with the wild type. Loss of function of SID2, NDR1, or NPR1 in the ftsh4-4 mutant reversed the ftsh4-4 senescence and autophagy phenotypes. Furthermore, ftsh4-4 mutants had elevated levels of transcripts of several WRKY genes, including WRKY40, WRKY46, WRKY51, WRKY60, WRKY63, and WRKY75; all of these WRKY proteins can bind to the promoter of SID2. Loss of function of WRKY75 in the ftsh4-4 mutants decreased the levels of SA and reversed the senescence phenotype. Taken together, these results suggest that the mitochondrial ATP-dependent protease FtSH4 may regulate the expression of WRKY genes by modifying the level of reactive oxygen species and the WRKY transcription factors that control SA synthesis and signaling in autophagy and senescence.

Leaf senescence is a complex and highly regulated developmental process of tissue degeneration and nutrient recycling. It is characterized by the loss of chlorophyll and the degradation of proteins, nucleic acids, and lipids as well as by nutrient remobilization (Lim et al., 2007). The programmed cell death and macromolecular degradation in senescence allow plants to remobilize nutrients from senescing cells into seeds, storage organs, or actively growing tissues (Gan and Amasino, 1997).

Reactive oxygen species (ROS) are key signaling molecules that regulate growth and development and coordinate responses to biotic and abiotic stresses in plants (Apel and Hirt, 2004). Aging and cell death are influenced by ROS and oxidative damage (Overmyer et al., 2003; Jing et al., 2008). Mitochondria play an important role in the production of ROS (Moller, 2001; Tiwari et al., 2002; Overmyer et al., 2003; Mittler et al., 2004) and in regulating aging and cell death (Laloi et al., 2004). Although some links have been identified between aging, ROS production, oxidative damage, and mitochondrial metabolism (Tiwari et al., 2002), the signaling pathways and detailed mechanisms behind the regulation of senescence and cell death by mitochondrial ROS have not yet been determined.

WRKY transcription factors (TFs) play a central role in the regulation of leaf senescence. Expression profiling in Arabidopsis (Arabidopsis thaliana) revealed that WRKY TFs are the second largest family of TFs involved in senescence (Guo et al., 2004). Several WRKY genes, such as WRKY22, WRKY30, WRKY53, WRKY54, WRKY70, and WRKY75, have important functions in senescence (Miao and Zentgraf, 2007; Zhou et al., 2011; Besseau et al., 2012; Li et al., 2012). The relationship between WRKYs and ROS during senescence has been studied intensively. The expression of several WRKY genes is up-regulated in response to ROS or ROS-generating stimuli, imbalances in redox homeostasis, and endogenous ROS-dependent processes such as senescence (Ulker and Somssich, 2004). Arabidopsis WRKY22 and WRKY53 are up-regulated in response to hydrogen peroxide (H2O2) treatment and play important roles in senescence (Miao and Zentgraf, 2007; Zhou et al., 2011); however, it is unknown if ROS are the immediate trigger for increased WRKY expression. Moreover, information is lacking on how many WRKY genes are related to mitochondria-triggered plant senescence and how WRKY genes receive and transmit ROS signaling to downstream components during this process.

Salicylic acid (SA) is involved in disease resistance, leaf senescence, flowering, and thermogenesis (Vlot et al., 2009; Zhang et al., 2013). Substantial work has examined the roles of SA in plant defense and programmed cell death (Vlot et al., 2009). A few SA signaling genes, such as NPR1 and PHYTOALEXIN DEFICIENT4, have been found to play roles in leaf senescence (Morris et al., 2000; Zhao et al., 2016), but the underlying molecular mechanisms behind this process are not well understood.

Mitochondria contain three main types of ATP-dependent proteases: Lon, Clp, and FtSH (for filamentation temperature sensitive). To date, four mitochondrial FtSH genes have been identified in Arabidopsis. FtSH3 and FtSH10 are considered to be mitochondrial-AAA (m-AAA) proteases (Janska et al., 2010); FtSH4 and FtSH11 show characteristics of mitochondrial inner membrane-AAA proteases (Urantowka et al., 2005). The function of plant m-AAA proteases is still unclear, but complementation experiments have shown that the functions of m-AAA proteases in fungi and plants are conserved (Kolodziejczak et al., 2002). With the exception of AtFtSH11, the Arabidopsis m-AAA proteases are related to the plant oxidative phosphorylation (OXPHOS) system (Kolodziejczak et al., 2007). FtSH4 influences the formation of late rosette leaves in Arabidopsis under short-day (SD) conditions by preventing the accumulation of oxidized proteins (Gibala et al., 2009). The loss-of-function ftsh4 mutation causes reduced cardiolipin contents in the mitochondria. This leads to perturbations within the OXPHOS complexes, which then generate more ROS and less ATP, leading to the deregulation of mitochondrial dynamics and the accumulation of oxidative damage (Smakowska et al., 2016). The induced ROS in the ftsh4 mutants interact with the phytohormone auxin to affect plant architecture (Zhang et al., 2014) and cause the premature termination of shoot meristems at 30°C under long-day (LD) conditions (Dolzblasz et al., 2016). These results imply that mitochondria-located FtSH4 plays important roles in regulating phytohormone signaling and TF genes through ROS-dependent mechanisms.

In this study, we investigated the function of mitochondrial FtSH4 in leaf senescence and plant cell death through effects on SA-mediated autophagy and WRKY gene transcript levels. Collectively, our data indicated that the mitochondrial ATP-dependent protease FtSH4 regulates leaf senescence and cell death by modifying ROS levels and SA homeostasis.

RESULTS

The ftsh4-4 Mutants Showed an Early Leaf Senescence Phenotype

FtSH4, an AAA+ (for ATPase associated with diverse cellular activities) protease-type metalloproteinase, has protease activity and chaperone activity and can mediate the repair and degradation of membrane proteins in bacteria, mitochondria, and chloroplasts. The rosette leaves of the ftsh4-1 mutants have a premature senescence phenotype under SD conditions (Gibala et al., 2009). Another mutant allele of FtSH4, ftsh4-4 (Zhang et al., 2014), also showed a leaf senescence phenotype under SD conditions (Supplemental Fig. S1) and LD conditions (Fig. 1A). And the ftsh4-4 complemented plants (FtSH4-ftsh4-4) lacked the leaf senescence phenotype (Fig. 1, A and B). To understand whether the leaf senescence phenotype was caused by nutrient limitations, ftsh4-4 seedlings also were cultured on medium with high levels of nutrients for 3 weeks. The leaves of the ftsh4-4 mutants showed premature senescence on high-nutrient medium compared with the wild-type plants (Supplemental Fig. S2). To analyze the leaf senescence phenotype, different FtSH4 genotype plants were observed at different growth stages (Supplemental Fig. S3). The senescence phenotype of ftsh4-4 appeared at 3 weeks old (Supplemental Fig. S3). The chlorophyll contents in the 5-week-old FtSH4 genotype plants were measured, and its level in the ftsh4-4 mutant was decreased significantly compared with the wild-type plant (Supplemental Fig. S4).

Figure 1.

Figure 1.

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Deficiency of FtSH4 causes an early senescence phenotype. A, Five-week-old ftsh4-4 mutants display the early leaf senescence phenotype under LD conditions. OE, Overexpression. Bar = 1 cm. B, Rosette leaves of 5-week-old wild-type Columbia-0 (Col-0), ftsh4-4, FtSH4-ftsh4-4, and 35S::FtSH4 plants under LD conditions. Bar = 1 cm. C, Up-regulated leaf senescence-associated genes (SAG) and down-regulated leaf photosynthesis genes in the ftsh4-4 mutants at different growth stages. CAB1, Chlorophyll a/b-binding protein1; LHCA1, light-harvesting complex associated1; RBCS, ribulose bisphosphate carboxylase small chain 1A. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s honestly significant difference (HSD) test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter.

To characterize the senescence phenotype at the molecular level, we measured the transcript levels of senescence-associated genes and photosynthesis-related genes. As expected, the transcript levels of the senescence-associated genes increased and the transcript levels of the photosynthesis-related genes decreased substantially in 3-week-old ftsh4-4 plants compared with wild-type plants. These genes remained at normal levels in the FtSH4-ftsh4-4 complemented plants (Fig. 1C). These results indicate that FtSH4 is involved in senescence in Arabidopsis.

FtSH4 Is Involved in Preventing Cell Death and Autophagy

To verify whether FtSH4-related leaf senescence is involved in plant cell death, the leaves of wild-type, ftsh4-4 mutant, and FtSH4-ftsh4-4 complemented plants were stained with Lactophenol-Trypan Blue, which only stains dead cells (van Wees, 2008). The cells in the ftsh4-4 leaves showed substantially more staining than those of wild-type and FtSH4-ftsh4-4 leaves (Fig. 2A), indicating that FtSH4 is involved in plant cell death.

Figure 2.

Figure 2.

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FtSH4 is involved in cell death and autophagy. A, The fifth leaf of 4-week-old wild-type Col-0, ftsh4-4, and FtSH4-ftsh4-4 plants under LD conditions were stained with Lactophenol-Trypan Blue. Com stands for complemented plant FtSH4-ftsh4-4. The top image shows whole leaves. Bar = 1 cm. The bottom image shows magnified views. Bars = 2 mm. B, The protoplasts of 4-week-old wild-type Col-0, ftsh4-4, and FtSH4-ftsh4-4 plants were stained with LTG. Bars = 100 μm. C, Statistical results of the relative autophagic activity. Over 1,000 protoplast cells for each genotype plant were used for quantification. Five technical replicates for one sample and three samples for every genotype plant were performed for each of three biological replicates. Error bars indicate se. According to Tukey’s HSD test (P = 0.05), means of autophagic cell ratio do not differ significantly if they are indicated with the same letter. D, Higher autophagosome accumulation level in the protoplasts of 3-week-old ftsh4-4 mutants compared with the wild type detected by ATG8a fluorescence observation. Five technical replicates for one sample and three samples for every genotype plant were performed for each of three biological replicates. Bars = 50 μm.

To test whether FtSH4-mediated cell death involves autophagy, the fluorescent dye LysoTracker Green (LTG), which is widely used to detect autophagic structures in plants and animals (Klionsky et al., 2008), was used to stain the cells of the different FtSH4 genotypes. After growing the plants for 4 weeks under LD conditions, we prepared protoplasts of wild-type, ftsh4-4, and FtSH4-ftsh4-4 leaves and stained them with LTG. The cells of the ftsh4-4 mutant showed substantially more staining than those of wild-type and FtSH4-ftsh4-4 plants (Fig. 2B). The percentage of autophagic cells in the ftsh4-4 mutant was 70.15%, but only 0.47% was found in the wild-type and 21.91% was found in the FtSH4-ftsh4-4 plants (Fig. 2C). In order to confirm further that FtSH4 loss of function is involved in autophagy, the GFP-ATG8a marker for the autophagosomal structures was observed in 3-week-old plants. The result showed that the fluorescent signal of autophagosomes was stronger in the ftsh4-4 protoplast compared with the wild-type protoplast (Fig. 2D). These results suggest that the FtSH4 gene plays a role in autophagy-mediated cell death and senescence.

FtSH4 Plays a Role in the ATG5- and ATG8-Mediated Cell Autophagy Pathway

The formation of autophagic vesicles requires two ubiquitin pathways: the ATG8-phosphatidylethanolamine pathway and the ATG5-ATG12-binding pathway (Klionsky, 2005). To confirm which pathway is involved in FtSH4-regulated plant autophagy, we constructed double mutants of ftsh4-4 with atg8a and atg5. The rosette leaves of the ftsh4-4atg5 double mutant showed an enhanced premature senescence phenotype compared with wild-type, ftsh4-4, atg5, and atg8a plants; however, the ftsh4-4atg8a double mutant showed the same senescence phenotype as the atg8a mutant, and the senescence degree was much lower than in the ftsh4-4 mutant under the normal condition (Fig. 3A) or the temperature stress condition (Supplemental Fig. S5). The chlorophyll content measurement results were consistent with the phenotype observation (Supplemental Fig. S4). Furthermore, atg8e loss of function also could rescue the senescence phenotype of ftsh4-4 mutant plants (Supplemental Fig. S6). These results suggest that FtSH4-regulated leaf senescence may be related to the ATG5 and ATG8 genes. To further confirm this, we measured the transcript levels of ATG8 and ATG5 genes in the ftsh4-4 mutant. The transcript levels of ATG8a, ATG8c, ATG8e, ATG8h, and ATG5 were increased in 3-week-old ftsh4-4 mutants compared with the wild type (Fig. 3B; Supplemental Fig. S7). However, the transcript levels of ATG8a, ATG8c, and ATG8e were down-regulated (Supplemental Fig. S8), and ATG5 was not affected by the absence of FtSH4 in 4-week-old ftsh4-4 mutants (Supplemental Fig. S7). Interestingly, the ATG8h gene expression level was increased in different aged ftsh4-4 plants compared with wild-type plants (Fig. 3B; Supplemental Fig. S8). These results imply that ATG5 and several ATG8 genes responded variously to different ftsh4 growth stage-induced signaling.

Figure 3.

Figure 3.

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FtSH4 plays a role in ATG5- and ATG8-mediated plant cell autophagy. A, Leaf senescence phenotypes of 5-week-old wild-type Col-0, ftsh4-4, atg5, ftsh4-4atg5, atg8a, and ftsh4-4atg8a plants under LD and 22°C conditions. Bars = 1 cm. B, Expression levels of ATG8 genes in 3-week-old FtSH4 loss-of-function mutants. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s HSD test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter. C, The protoplasts of 4-week-old wild-type Col-0, ftsh4-4, atg5, ftsh4-4atg5, atg8a, and ftsh4-4atg8a plants were stained with LTG. Bars = 100 μm. D, Statistical results of the relative autophagic activity. Over 1,000 protoplast cells for each genotype plant were used for quantification. Five technical replicates for one sample and three samples for every genotype plant were performed for each of three biological replicates. Error bars indicate se. According to Tukey’s HSD test (P = 0.05), means of autophagic cell ratio do not differ significantly if they are indicated with the same letter.

Next, the leaves of 4-week-old wild-type, ftsh4-4, atg5, atg8a, ftsh4-4atg5, and ftsh4-4atg8a plants were stained with Lactophenol-Trypan Blue dye. The cell death of the ftsh4-4atg8a double mutant was less than that of the ftsh4-4 mutant and similar to that of the atg8a mutant (Supplemental Fig. S9). Furthermore, the cell death extent in the ftsh4-4atg5 double mutant was intermediate between that of ftsh4-4 and atg5 (Supplemental Fig. S9). Thus, the Lactophenol-Trypan Blue staining results were consistent with the plant phenotypes. To further confirm that FtSH4 is involved in the autophagy pathway, the protoplasts of 4-week-old wild-type, ftsh4-4, atg5, atg8a, ftsh4-4atg5, and ftsh4-4atg8a leaves were stained with LTG. The degrees of autophagy in the ftsh4-4atg5 and ftsh4-4atg8a double mutants were decreased by the loss function of ATG5 or ATG8a (Fig. 3, C and D). And the extent of autophagy in the ftsh4-4 mutant was about 2.2 times that in the ftsh4-4atg5 double mutant and 4 times that in the ftsh4-4atg8a double mutant (Fig. 3D). These results indicate that the FtSH4 metalloproteinase gene is involved in the ATG5- and ATG8-mediated plant cell autophagy pathway.

FtSH4 Is Involved in SA-Mediated Plant Senescence and Autophagy

Plant autophagy can be regulated by SA (Hofius et al., 2009; Yoshimoto et al., 2009). To test whether SA also regulates FtSH4-mediated cell death and autophagy, we measured the SA levels in plants with different FtSH4 genotypes. Whole wild-type, ftsh4-4, and FtSH4-ftsh4-4 plants were harvested after growing under LD conditions for 3 weeks, and their SA contents were determined by gas chromatography-mass spectrometry. The free SA content in the ftsh4-4 mutant was about 2 times that in the wild-type and FtSH4-ftsh4-4 plants, and the total SA content in the ftsh4-4 mutant was about 4.5 times that in the wild-type and FtSH4-ftsh4-4 plants (Table I), indicating that the loss of function of FtSH4 results in increased SA levels in Arabidopsis.

Table I. SA levels increased in the 3-week-old ftsh4-4 mutant.

Values shown are means ± se. According to Tukey’s HSD test (P = 0.05), means of SA levels do not differ significantly if they are indicated with the same letter.

Genotype Free SA Total SA
μg g−1 fresh wt
Wild type 0.33 ± 0.041 a 0.37 ± 0.051 a
ftsh4-4 0.63 ± 0.149 b 1.66 ± 0.525 b
FtSH4-ftsh4-4 0.28 ± 0.049 a 0.38 ± 0.048 a
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To further confirm that FtSH4 is involved in SA-mediated plant senescence, the ftsh4-4 mutant was crossed with two different lines that have altered SA contents, the nahG transgenic line and the cpr5 mutants. The nahG transgenic plants show a small amount of accumulation of salicylate hydroxylase or no accumulation of SA (Gaffney et al., 1993), and the loss-of-function cpr5 mutants show a high level of SA (Bowling et al., 1997). The premature aging phenotype of the ftsh4-4 mutant was reversed by the expression of nahG. Under LD conditions, 4-week-old ftsh4-4nahG plants displayed the same phenotype as wild-type and nahG plants and much lower senescence degree than ftsh4-4; however, the premature aging phenotype of the ftsh4-4 mutant was enhanced by the loss of function of cpr5 (Fig. 4, A and B). The chlorophyll content measurement results were consistent with the phenotype observation (Supplemental Fig. S4). Furthermore, under LD conditions, the 3-week-old ftsh4-4 mutant did not yet show the premature aging phenotype, while at the same stage, ftsh4-4cpr5 plants showed an advanced premature aging phenotype compared with the ftsh4-4 and cpr5 mutants (Supplemental Fig. S10). Next, 3-week-old leaves of wild-type, cpr5, ftsh4-4, and ftsh4-4cpr5 plants were stained with Lactophenol-Trypan Blue. The presence of the cpr5 mutation increased the amount of cell death in the ftsh4-4 mutants (Fig. 4C), consistent with the plant senescence phenotypes. When 4-week-old leaves of wild-type, nahG, ftsh4-4, and ftsh4-4nahG plants were stained with Lactophenol-Trypan Blue, the amount of cell death in the ftsh4-4 mutant was decreased by the expression of nahG, consistent with the plant senescence phenotypes (Fig. 4D). These results show that FtSH4-mediated plant senescence is related to SA contents.

Figure 4.

Figure 4.

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The leaf senescence phenotype of the ftsh4-4 mutant is associated with the SA level. A, The early leaf senescence phenotype of the ftsh4-4 mutants is accelerated by the loss-of-function cpr5 mutation and delayed by the nahG gene. Four-week-old plants are shown. Bars = 1 cm. B, Rosette leaves of 4-week-old wild-type Col-0, ftsh4-4, cpr5, ftsh4-4 cpr5, ftsh4-4nahG, and nahG plants under LD conditions. Bars = 1 cm. C, The cell death of the ftsh4-4 leaf is accelerated by the cpr5 mutation. The fifth leaves of 3-week-old wild-type Col-0, ftsh4-4, ftsh4-4cpr5, and cpr5 plants under LD conditions were stained with Lactophenol-Trypan Blue. Bars = 2 mm. D, Cell death is delayed by the nahG gene in ftsh4-4nahG plants. The fifth leaves of 4-week-old wild-type Col-0, ftsh4-4, ftsh4-4nahG, and nahG plants under LD conditions were stained with Lactophenol-Trypan Blue. Bars = 2 mm.

The SA signaling network consists of SA biosynthesis, SA accumulation, and SA signaling. To further study whether the SA signal is altered in the ftsh4-4 mutant, the SA-response marker gene PR1, the SA biosynthesis gene SID2, the SA signaling gene NPR1, and the SA accumulation genes NDR1, PAD4, and EDS1 were measured by quantitative reverse transcription (RT)-PCR in the wild type and the ftsh4-4 mutant. All of the tested transcripts increased substantially in the ftsh4-4 mutant compared with the wild-type plant. The expression levels of PR1, SID2, NPR1, NDR1, PAD4, and EDS1 in the 3-week-old ftsh4-4 mutant were approximately 3.5, 4.3, 3.2, 2.6, 2.8, and 3.2 times that in the wild-type plants, respectively (Fig. 5). These results suggest that the SA biosynthesis and signaling genes are inhibited by FtSH4 expression.

Figure 5.

Figure 5.

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Transcript levels of the SA biosynthesis, accumulation, signaling, and response genes were increased in the ftsh4-4 mutants. The SA response marker gene PR1, SA biosynthesis gene SID2, SA signaling gene NPR1, and SA accumulation genes NDR1, PAD4, and EDS1 were detected by quantitative RT-PCR in 3-week-old wild-type Col-0, ftsh4-4, and FtSH4-ftsh4-4 plants. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s HSD test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter.

To further investigate the molecular mechanism of FtSH4 in SA signaling-mediated autophagy, we generated a series of double mutants between ftsh4-4 and several SA loss-of-function mutants: the SA biosynthesis mutant sid2, the SA signaling mutant npr1, and the SA accumulation mutant ndr1. The senescence phenotype of the ftsh4-4 mutant was delayed by the ndr1 or the npr1 mutation and was similar to that of ndr1 or npr1 (Fig. 6, A and B; Supplemental Fig. S3). The SA biosynthesis mutant sid2 suppressed the ftsh4-4 premature aging phenotype in the double mutant to the same degree as sid2 (Fig. 6C; Supplemental Fig. S3). The chlorophyll content measurement results were consistent with the phenotype observation (Supplemental Fig. S4). Next, leaves of 4-week-old wild-type, ftsh4-4, ndr1, ftsh4-4ndr1, ftsh4-4npr1, npr1, ftsh4-4sid2, and sid2 plants grown under LD conditions were stained with Lactophenol-Trypan Blue. Consistent with the phenotypic changes, there were fewer dead cells in the ftsh4-4ndr1, ftsh4-4npr1, and ftsh4-4sid2 double mutants compared with those in the ftsh4-4 mutant, and dead cell levels were similar to the ndr1, npr1, and sid2 single mutants (Fig. 6D). These results indicate that FtSH4-mediated senescence requires SID2, NPR1, and NDR1.

Figure 6.

Figure 6.

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The leaf senescence phenotype of the ftsh4-4 mutant is regulated by SA biosynthesis, accumulation, and signaling genes. A, The early leaf senescence phenotype of the ftsh4-4 mutants is reversed by the loss-of-function ndr1 and npr1 mutations. Four-week-old plants are shown. Bars = 1 cm. B, Rosette leaves of 4-week-old wild-type Col-0, ftsh4-4, ftsh4-4ndr1, ndr1, ftsh4-4npr1, and npr1 plants under LD conditions. Bars = 1 cm. C, The early leaf senescence phenotype of the ftsh4-4 mutants is reversed by the loss-of-function sid2 mutation. Four-week-old plants and rosette leaves are shown. Bars = 1 cm. D, The cell death phenotype of the ftsh4-4 leaf is reversed by the ndr1, npr1, and sid2 mutations. The fifth leaves of 4-week-old wild-type Col-0, ftsh4-4, ftsh4-4ndr1, ndr1, ftsh4-4npr1, npr1, ftsh4-4sid2, and sid2 plants under LD conditions were stained with Lactophenol-Trypan Blue. Bars = 2 mm.

To further test whether FtSH4-mediated autophagy also requires these SA signaling pathway components, protoplasts of 4-week-old wild-type, ftsh4-4, ftsh4-4ndr1, ftsh4-4npr1, and ftsh4-4sid2 plants were stained with LTG. The extent of autophagy in the ftsh4-4 mutants was much greater than in wild-type, ftsh4-4ndr1, ftsh4-4npr1, and ftsh4-4sid2 plants (Fig. 7A). The extent of autophagy in the ftsh4-4 mutant was 3.88 times that in ftsh4-4sid2, 2.88 times that in ftsh4-4ndr1, and 3.95 times that in ftsh4-4npr1 (Fig. 7B). Taken together, these results indicate that FtSH4-mediated autophagy, similar to FtSH4-mediated senescence, depends on SID2-mediated SA biosynthesis, NDR1-mediated SA accumulation, and NPR1-mediated signaling pathways.

Figure 7.

Figure 7.

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The increased autophagy phenotype of the ftsh4-4 mutant is suppressed by loss of function of SA biosynthesis, accumulation, and signaling genes. A, The protoplasts of 4-week-old wild-type Col-0, ftsh4-4, ftsh4-4ndr1, ftsh4-4npr1, ftsh4-4sid2, ndr1, npr1, and sid2 plants were stained with LTG. Bars = 100 μm. B, Statistical results of the relative autophagic activity. Over 1,000 protoplast cells for each genotype plant were used for quantification. Five technical replicates for one sample and three samples for every genotype plant were performed for each of three biological replicates. Error bars indicate se. According to Tukey’s HSD test (P = 0.05), means of autophagic cell ratio do not differ significantly if they are indicated with the same letter.

FtSH4 Regulates SID2 through WRKYs

How does the mitochondrial FtSH4 regulate cytoplasmic or nuclear events? It is reported that WRKYs play important roles in transmitting signaling from organelles to the cytoplasm to the nucleus (Agarwal et al., 2011). The microarray data showed that several WRKY family transcripts, including WRKY75, WRKY60, WRKY51, WRKY46, WRKY45, and WRKY25, were increased noticeably in the ftsh4-4 mutant (Zhang et al., 2014). Thus, we predicted that the elevated level of ROS in the ftsh4-4 mutants may cause the increase of SA through positive regulation of WRKY genes. Therefore, the transcript levels of four WRKY genes (WRKY46, WRKY51, WRKY60, and WRKY75) from the previous microarray data, and two other WRKY genes (WRKY40 and WRKY63) not detected in the microarray experiments but that function in communication between organelles and nucleus (Van Aken et al., 2013), were measured by real-time PCR. The transcript levels of WRKY40, WRKY46, WRKY51, WRKY60, WRKY63, and WRKY75 were increased significantly in the ftsh4-4 mutant (Fig. 8A). To determine whether the increased expression of WRKY genes can elevate SA levels via the regulation of SID2, a yeast one-hybrid assay was performed to detect whether the WRKYs can bind to the promoter of SID2. The results indicate that WRKY46, WRKY51, WRKY63, and WRKY75 could bind to the WRKY-binding motifs of the SID2 promoter region (Fig. 8, B and C; Supplemental Fig. S11). This result implies that the WRKY genes may regulate the expression of SID2 to control the level of SA. Since WRKY75 has the highest expression level among the WRKY genes in ftsh4-4 (Fig. 8A) and WRKY75 has been reported to be a positive regulator of leaf senescence, the down-regulation of WRKY75 can delay plant aging (Li et al., 2012). Thus, we predicted that WRYK75 could regulate leaf senescence through regulation of the SA level by affecting SID2 transcript levels. Electrophoretic mobility shift assay (EMSA) results showed that WRKY75 binds directly to the SID2 promoter region (Fig. 8D). When combined with the yeast one-hybrid assay result, this suggests that WRKY75 regulates the transcript levels of SID2. Next, we detected SID2 transcript levels in the loss-of-function wrky75 mutants and the WRKY75 overexpression plants. The SID2 expression level increased by almost 2-fold in the WRKY75 overexpression plants and decreased by 60% in the wrky75 mutants compared with the wild-type plants (Fig. 8E). SA content also changed with the changes in SID2 transcript level in both the WRKY75 overexpression and knockout plants (Fig. 8E; Table II). These results indicate that WRKY75 can control SA levels through the direct regulation of SID2 expression.

Figure 8.

Figure 8.

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FtSH4 regulates SID2 expression through WRKY genes. A, Several WRKY gene transcript levels were up-regulated by the loss-of-function ftsh4 mutation. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s HSD test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter. B, Diagram of the WRKY-binding cis-elements (W-box and W-box-like motifs) in the promoter region of the SID2 gene. W1, W2, W3, W4, and W5 indicate different WRKY-binding sites. ATG represents the start codon of the SID2 gene. Numbers indicate the positions upstream of the ATG, and letters in each box indicate the W-box motif sequence. C, WRKY75 can bind to the WRKY-binding cis-elements of the SID2 promoter in vivo. The ability of WRKY75 to bind to the five different W-boxes of the SID2 promoter was detected by the yeast one-hybrid assay. −1 and −2 indicate the yeast concentration diluted by 0.1× and 0.01×. AbA, Aureobasidin A; SD, synthetic dextrose. Three biological replicates each with three technical replicates were performed. D, WRKY75 can bind to the WRKY-binding cis-elements of the SID2 promoter in vitro. The ability of WRKY75 to bind to the five different W-boxes of the SID2 promoter was detected by the EMSA experiment. Three biological replicates each with three technical replicates were performed. E, The transcript level of SID2 was up-regulated by overexpression of WRKY75 and down-regulated by loss of function of WRKY75. The transcript levels of SID2 were detected in 3-week-old wrky75-25, wrky75-RNAi, and 35S::WRKY75 plants by quantitative RT-PCR. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s HSD test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter.

Table II. SA levels in the 4-week-old ftsh4-4 mutant were reduced by loss of function of WRKY75.

Values shown are means ± se. According to Tukey’s HSD test (P = 0.05), means of SA levels do not differ significantly if they are indicated with the same letter.

Genotype Free SA Total SA
μg g−1 fresh wt
Wild type 0.36 ± 0.045 a 0.44 ± 0.048 a
ftsh4-4 1.09 ± 0.090 b 3.21 ± 0.310 b
FtSH4-ftsh4-4 0.40 ± 0.044 a 0.48 ± 0.047 a
wrky75-25 0.23 ± 0.016 c 0.31 ± 0.028 c
35S::WRKY75 0.74 ± 0.081 d 1.31 ± 0.093 d
ftsh4-4wrky75-25 0.41 ± 0.031 a 0.47 ± 0.039 a
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Based on these results, we speculated that FtSH4 regulates SID2 expression and SA levels, as well as leaf senescence, through WRKY75. Thus, we constructed ftsh4-4wrky75-25 and ftsh4-4WRKY75-RNAi plants and detected the SA level in the ftsh4-4wrky75-25 double mutant. The leaf senescence and cell death phenotypes of the ftsh4wrky75 double mutant showed that the early senescence phenotype can be delayed by the loss of function of WRKY75 and is similar to that of the wrky75 mutant (Fig. 9A; Supplemental Fig. S3). The chlorophyll content measurement results were consistent with the phenotype observation (Supplemental Fig. S4). Compared with the ftsh4-4 mutant, the SA level in the ftsh4-4wrky75-25 double mutant decreased to a level similar to that in the wild-type plants (Table II). Similarly, compared with the ftsh4-4 mutant, the expression levels of SID2 and NPR1 in the ftsh4wrky75 double mutant decreased to the level of the wild-type plants (Fig. 9, B and C). These results suggest that the loss of function of the WRKY75 gene in the ftsh4wrky75 double mutant is sufficient to restore the SA level to the level of the wild-type plants. These results imply that WRKY75 may control the SA level in the ftsh4-4 mutants by controlling the expression of SID2.

Figure 9.

Figure 9.

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The loss of function of WRKY75 reverses the leaf senescence phenotype of the ftsh4-4 mutants. A, The early leaf senescence phenotype of the ftsh4-4 mutants is reversed by the loss-of-function wrky75 mutation. Left, Four-week-old plants are shown. Bar = 1 cm. Right, Rosette leaves of the 4-week-old wild-type Col-0, ftsh4-4, ftsh4-4wrky75-25, wrky75-25, ftsh4-4wrky75-RNAi, and wrky75-RNAi plants under LD conditions. Bar = 1 cm. B, Loss of function of WRKY75 reduces the expression of SID2 in the ftsh4-4wrky75-25 double mutant. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s HSD test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter. C, Loss of function of WRKY75 reduces the expression of NPR1 in the ftsh4-4wrky75-25 double mutant. Error bars indicate se. Three technical replicates were performed for each of three biological replicates. According to Tukey’s HSD test (P = 0.05), means of relative transcript levels do not differ significantly if they are indicated with the same letter.

DISCUSSION

During the past decade, our knowledge of leaf senescence has been expanded by the identification of numerous senescence-associated genes (Kim et al., 2016). However, the signals and the signaling pathways that integrate senescence-associated exogenous and endogenous factors are not well understood. In particular, it would be interesting to study how the communication between the nucleus and organelles coordinately regulates gene expression to initiate, execute, and complete leaf senescence. The ROS that originate in the mitochondria can initiate cell death and senescence through protein oxidation, and mitochondrial function is very important in stress responses and aging (Yao et al., 2004). However, the function of mitochondrial ROS as the signal for communicating with the nucleus and regulating transcript levels during senescence is still not well understood. FtSH4 is an inner mitochondrial membrane-embedded metalloprotease that maintains the proper function of the OXPHOS complexes, protects the leaf and shoot apical meristems, and limits the effects of ROS by removing oxidatively damaged proteins (Gibala et al., 2009; Kicia et al., 2010; Smakowska et al., 2014, 2016). Until now, the downstream events (e.g. the cellular and molecular mechanisms of ftsh4-triggered ROS) in premature senescence remained unknown. In this study, we propose one pathway by which the FtSH4 gene regulates leaf senescence through the WRKY TF genes that regulate SA biosynthesis and signaling pathways. These results provide insight into the relationship between ROS and SA in regulating cell death, senescence, and responses to biotic or abiotic stress.

The FtSH4 Loss-of-Function Mutation Activates SA Biosynthesis and Signaling to Promote Cell Death and Senescence

FtSH4 showed a high expression level in the rosette leaves, and its transcript levels were stable during different growth stages (Zhang et al., 2014). These results, combined with the premature senescence phenotype of the ftsh4-4 mutant, indicate that FtSH4 may be an upstream regulator of senescence. Although FtSH4 plays important roles in regulating premature senescence by altering the levels of ROS (Gibala et al., 2009; Kicia et al., 2010; Smakowska et al., 2014, 2016), the detailed mechanism behind this process is not clear. ROS are important multifaceted signaling molecules that can regulate a number of cellular pathways and, thus, play critical roles in plant development (Foyer and Noctor, 2013). ROS and autophagy are associated with cell death, and more recent evidence indicates that both ROS and autophagy play important roles in signaling and cellular adaptation to stress (Wang et al., 2011). Mitochondria are known to play key roles in triggering cell death via altering cellular redox to release or activate autophagy-related proteins (Heo and Rutter, 2011). The loss of function of FtSH4 increased cell death and autophagy in the ftsh4-4 mutant, and these changes were complemented by the expression of FtSH4 (Fig. 2). This result indicated that the loss of function of FtSH4 triggered the autophagy pathway to adapt to endogenous oxidative stress. The important role of autophagy in the aging process of cells was exemplified by the Arabidopsis knockout mutants of autophagy-related ATG genes (ATG4, ATG5, ATG7, ATG9, ATG10, and ATG18a), as these mutants show premature aging phenotypes under nitrogen-sufficient conditions (Bassham et al., 2006).

Two ubiquitin pathways are required in the formation of autophagic vesicles: one is the ATG8-phosphatidylethanolamine pathway and the other is the ATG5-ATG12-binding pathway (Klionsky, 2005). The expression levels of ATG5 and several ATG8 genes were increased in 3-week-old ftsh4-4 mutants but were not changed or decreased in 4-week-old ftsh4-4 mutants (Fig. 3; Supplemental Figs. S5 and S6), and the autophagosomal structures in the ftsh4-4 mutants were decreased by atg5 or atg8e (Fig. 7), implying that the increased autophagy may be regulated by the ATG5- and ATG8-mediated pathway. There should exist some unknown transcript feedback regulation mechanism, such as high-level autophagy-related proteins, that can inhibit the gene expression of ATG8 homologs after senescence initiation in the ftsh4-4 mutant. ATG8 plays an important role in autophagy in Arabidopsis in a variety of biological processes, but the single atg8 loss-of-function mutant has not shown an obvious phenotype (Sláviková et al., 2005; Kim et al., 2012). Studies of the ftsh4-4 and atg8 mutants and their double mutant showed that the atg8a or atg8e mutation can reduce the autophagy level in the ftsh4-4 mutant (Fig. 7; Supplemental Fig. S6), and atg8a loss of function could reduce the H2O2 level in the ftsh4-4 mutant (Supplemental Fig. S12), implying that the loss of function of ATG8 genes may activate or inhibit some unknown factors to eliminate the ROS level of the ftsh4-4 mutant to rescue the senescence phenotype. The intriguing relationship among ROS, autophagy, and ATG8 proteins in ftsh4-4 need more experiments to elucidate.

Autophagy plays an important role in plant growth, especially in the aging and defense responses. The premature aging phenotype of the atg5 mutant is related to the SA content, as the SA content of the atg5 mutant is 3 times that in the wild type (Yoshimoto et al., 2009), indicating that autophagy is an SA-dependent process. Developmental and environmental cues trigger phytohormones (SA, jasmonates, and ethylene), which play key roles in promoting leaf senescence (Jibran et al., 2013). The SA content in 3-week-old ftsh4-4 mutants was increased dramatically compared with the wild type (Table I), implying that FtSH4-mediated senescence may be dependent on SA contents. This SA dependence was verified by genetic analysis. After elevating the SA level in the ftsh4-4 mutants by crossing with the cpr5 mutant or decreasing it by crossing with the nahG transgenic plants, the cell death and senescence phenotypes were exaggerated or returned to normal, respectively (Fig. 4). Moreover, the transcript levels of the SA biosynthesis, signaling, and response genes, such as SID2, NPR1, NDR1, and PR1, increased significantly in the ftsh4-4 mutants (Fig. 5). After blocking the SID2, NPR1, and NDR1 genes in the ftsh4-4 mutants, the cell death, autophagy, and senescence phenotypes were reversed, indicating that FtSH4-mediated senescence is dependent on SA biosynthesis and signaling and that FtSH4 mediates SA biosynthesis and signaling in the SID2-, NPR1-, and NDR1-dependent pathways. Previous transcriptomic and genetic studies suggested that SA signaling is specifically involved in natural, but not starvation-induced, leaf senescence (van der Graaff et al., 2006). Thus, NPR1- and NDR1-dependent SA signaling, as well as SID2-dependent SA biosynthesis, explain the phenotypes of the FtSH4 loss-of-function mutants.

FtSH4 Regulates SA Biosynthesis through WRKY Genes

A number of studies have examined the effect of mitochondrial ROS on plant development. However, the mechanisms of ROS signal transduction to the nucleus remain unknown. WRKYs act as activators or repressors in a TF network that participates in many cytoplasmic and nuclear processes, including signaling from organelles to the cytoplasm and the nucleus (Agarwal et al., 2011). Interestingly, WRKY15 participates in mitochondria-nucleus signaling (Vanderauwera et al., 2012). WRKY40 and WRKY63 are involved in stress responses by modifying the expression of stress-responsive nuclear genes encoding mitochondrial and chloroplast proteins (Van Aken et al., 2013). This provides additional evidence for the involvement of WRKYs in communication between organelles.

Several ROS-dependent responses are controlled by WRKYs, including the progression of senescence (Zentgraf et al., 2010), but it remains to be studied whether ROS are the immediate triggers that regulate WRKY gene expression. The transcripts of several WRKYs increased significantly in the ftsh4-4 mutant, and their transcript levels were suppressed by the expression of FtSH4 (Fig. 8). Increased ROS is one of the main phenotypes of the FtSH4 loss-of-function mutation, so it is plausible that mitochondrial ROS act as one of the immediate triggers of WRKY TFs. Several WRKYs, such as WRKY40, are involved in abiotic and biotic stress responses that induce ROS or cause imbalances in ROS homeostasis (Van Aken et al., 2013). Intriguingly, WRKY75 also plays important roles in H2O2 homeostasis in both wild-type and ftsh4-4 plants (Supplemental Fig. S12). Stress responses often involve hormone signaling; therefore, WRKYs are likely also part of a complex hormone signaling network. WRKYs can function upstream and downstream of hormones, are involved in the antagonistic functions of SA and jasmonic acid/ethylene, and control developmental processes via auxins, cytokinins, and brassinosteroids (Guo and Gan, 2005). These phytohormones have different effects on WRKY TFs (e.g. WRKY70 acts as an activator of SA-induced genes and as a repressor of jasmonic acid-responsive genes), thereby integrating signals from these mutually antagonistic pathways (Li et al., 2004). WRKY28 can activate the SA biosynthesis gene ICS1 (van Verk et al., 2011). This suggests that the promoted expression of the SA biosynthesis gene SID2 is induced by the elevated expression of WRKY genes in the ftsh4-4 mutants. Several studies on the DNA-binding characteristics of WRKY TFs have led to the generally accepted consensus-binding sequence TTGAC[C/T] or TGAC[C/T], commonly referred to as the W-box or W-box-like, respectively (Ciolkowski et al., 2008). About five W-boxes and W-box-like can be found in the 2,000-bp region upstream of the SID2 coding sequence, and almost all of the WRKYs that were induced by the ftsh4 mutation can bind to the W-box of SID2 (Fig. 8; Supplemental Fig. S11). The binding of the WRKYs to the W-boxes of SID2 likely induced the expression of SID2, because the overexpression of WRKY75 elevated the expression of SID2 and increased the SA level, but loss of function of WRKY75 had the opposite effects on SID2 expression and SA levels (Fig. 8). Consequently, the loss of function of WRKY75 can reverse the senescence phenotypes of the ftsh4-4 mutants by down-regulating the SA level (Table II). Therefore, it is of interest to study the functions of other FtSH4-related WRKYs in regulating cell death and senescence, as well as other developmental aspects, through regulating SA biosynthesis and signaling. These studies will help to elucidate a variety of functions of FtSH4 in growth and development and in communication between plant organs such as the mitochondria and nucleus.

CONCLUSION

In conclusion, our results suggest a role for FtSH4 as a negative regulator of leaf senescence, and we propose a model (Fig. 10). The mutation of the mitochondrial AAA-protease FtSH4 gene, which leads to obviously increased H2O2 (Zhang et al., 2014), can result in the increased expression of several WRKY genes (e.g. WRKY46, WRKY63, and WRKY75) as well as SA biosynthesis and signaling genes including SID2, NPR1, and NDR1. The FtSH4 mutation-induced WRKY proteins can bind to the SA biosynthesis gene SID2 promoter to elevate the SA content in the ftsh4-4 mutant. Loss of function of WRKY75 results in decreased SA levels in ftsh4-4 mutants by down-regulating SID2 expression. However, the direct target of FtSH4 in regulating ROS production, and the working relationship between FtSH4 and WRKY75 as well as the other WRKYs, must be elucidated in the future.

Figure 10.

Figure 10.

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Schematic representation for FtSH4 control of leaf senescence through the WRKY-regulated SA pathway. Loss of function of FtSH4 induces the high level of expression of WRKY genes through the increase in ROS in an unknown way. Then, the WRKY proteins, especially WRKY75, bind to the SID2 promoter and result in elevated SA biosynthesis. Finally, the high level of SA and activated SA signaling promote cell death, autophagy, and senescence of plant cells. Meanwhile, ROS have a direct function in regulating plant autophagy and senescence.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Seeds of Arabidopsis (Arabidopsis thaliana) in the Col-0 ecotype background were sown in a petri dish containing sterile solid medium consisting of 1× Murashige and Skoog salts, 1.5% Suc, and 0.8% Phytagel (Sigma-Aldrich) at pH 5.7. Seeds were surface sterilized, arranged on the surface of the solid medium, and given a cold treatment at 4°C for 72 h. Seeds were germinated and seedlings were grown on a light shelf under a 16-h/8-h light/dark cycle for 12 d and then transferred into a well-watered potting mix (Faerdigblanding Substrate; Pindstrup). Light was supplied by cool- and warm-white fluorescent bulbs reaching an intensity of approximately 100 µmol m−2 s−1 on the surface of the shelf.

The double mutant lines in this study were created by cross-pollination between the relevant mutants. The mutant lines ndr1-1npr1-2 (CS6355), ndr1-1 (CS6358), and sid2-2 (CS16438) were obtained from the Arabidopsis Biological Resource Center. The atg5 (CS39993), atg8e (SALK_126394C), and atg8a (SALK_045344C) mutants were provided by Shi Xiao. All of them were crossed with the ftsh4-4 mutant (Zhang et al., 2014) to obtain different single, double, or triple mutants, and their genotypes were verified by resistance, PCR, or restriction endonuclease digestion (for site mutation). The primers for mutant verification are listed in Supplemental Table S1.

Gene Expression Analysis

Quantitative RT-PCR was performed as described previously (Zhang et al., 2014), and the transcript data were normalized using the UBQ10 gene as an internal control. The leaves used to detect gene expressions were the fifth and sixth rosette leaves with no senescence symptoms from 3-week-old plants. Error bars in the figures indicate se. All experiments were performed with three biological replicates. For each biological replicate, three technical replicates were performed for each of three samples. The primers used for quantitative RT-PCR are listed in Supplemental Table S1.

Trypan Blue Staining

Lactophenol-Trypan Blue staining was performed to visualize dying cells as described previously (Kim et al., 2009) with slight modifications. The fourth or fifth rosette leaves of 3- or 4-week-old plants were detached and submerged in Lactophenol-Trypan Blue solution (10 mL of lactic acid, 10 mL of glycerol, 10 g of phenol, and 20 mg of Trypan Blue dissolved in 10 mL of distilled water). The samples were boiled for approximately 100 s in the staining solution and then washed in chloral hydrate solution (2.5 g of chloral hydrate dissolved in 1 mL of distilled water) to reduce background staining, then photographed using a BX51 stereomicroscope (Olympus). Three biological replicates each with three technical replicates were used for Trypan Blue staining.

LTG Staining and Confocal Microscopy

Rosette leaves were used for protoplast extraction following the protocol from Jen Sheen’s laboratory (Yoo et al., 2007), and the fifth and sixth leaves with no senescence symptoms were used for protoplast extraction. LTG DND-26 (Invitrogen; catalog no. L7526) was dissolved in dimethyl sulfoxide to the concentration of 100 μm. Three microliters of 100 μm LTG was added to 500 μL of Arabidopsis protoplast solution and then stained for 20 min at room temperature. The same volume of stained protoplasts was added to the recess slides. The LTG fluorescence signal was detected using a laser scanning confocal microscope (Zeiss LSM710) with 405 nm for excitation and 470 to 600 nm for emission. Chlorophyll autofluorescence also was observed with 488 nm for excitation and 580 to 695 nm for emission. All cells with autophagosomes with green fluorescent signals were counted. For every LTG staining experiment, four views for one sample and three samples for every genotype were observed, and almost 1,000 protoplasts were used for autophagosome analysis and statistics. These experiments were performed with three biological repeats. And the data were analyzed using SPSS software. The GFP-ATG8a fluorescence signal of the protoplasts also was detected using a laser scanning confocal microscope (Zeiss LSM710) with 488 nm for excitation and 505 to 530 nm for emission.

Yeast One-Hybrid Assay

Yeast one-hybrid assays were performed with the kit provided by Clontech (Matchmaker Gold Yeast One-Hybrid Library Screening System; catalog no. 630491) using the Saccharomyces cerevisiae Y1H Gold yeast strain according to the user’s manual. The primers used to clone the related cDNAs or promoter DNAs are listed in Supplemental Table S2. The promoter DNA fragments were subcloned into the SacI/XhoI sites of the pAbAi vector. First, the pAbAi bait vector harboring the target gene promoter was transformed into the Y1H Gold yeast genome. The resulting yeast cells were first grown in SD-uracil medium to ensure that the yeast cells were transformed successfully. Then, the pGADT7 prey vector harboring the WRKY open reading frame was integrated into Y1H Gold [pBait-AbAi] yeast strain, and the yeast cells were grown on SD-Leu medium supplemented with 500 ng mL−1 aureobasidin A (Clontech; catalog no. 630466) to identify the interaction. Aureobasidin A is toxic to S. cerevisiae at low concentrations, and expression of a mutant gene, AUR1-C, in transformed yeast confers resistance to the drug. The plates were incubated for 3 d at 30°C. Three biological replicates each with three technical replicates were performed.

EMSA

EMSA was performed via the Light Shift Chemiluminescent EMSA Kit (Thermo Scientific). The DNA sequence for the WRKY75 (GST-WRKY75) protein was cloned in frame to pGEX-4T-1 using the following primers: forward primer 5′-GGATCCATGGAGGGATATGATAATGGGT-3′ and reverse primer 5′-CTCGAGCTAGAAAGAAGAGTAGATTTGCAT-3′. The purified, bacterially expressed GST-WRKY75 protein was used to test the binding ability to the WRKY-binding motif of SID2 (SID2W2) with purified GST protein as a negative control.

For binding assays, the double-stranded oligonucleotides from the SID2 promoter (shown below) were used for EMSA analysis: 5′-AATAACAATTTGACTTCTAAAGTCT-3′ and 5′-AGACTTTAGAAGTCAAATTGTTATT-3′. All of the oligonucleotides were labeled with biotin using the DNA 3′ End Biotinylation Kit (Thermo Scientific) and then annealed to form double-stranded oligonucleotides. The unlabeled oligonucleotides were used as the specific competitor. The binding reaction and detection were carried out as follows: 10 fmol of labeled double-stranded probe DNA was incubated with recombinant proteins (1–5 mg) in binding buffer [10 mm Tris-HCl, pH 8, 10 mm MgCl2, 5 mm DTT, 10% glycerol, and 50 ng mL−1 poly(dI·dC) as the nonspecific competitor]. After the reaction at room temperature for 30 min, samples were resolved on a 4% native acrylamide gel in 0.5× Tris-borate/EDTA and then transferred to nylon membranes for chemiluminescence detection (Thermo Scientific). Three biological replicates each with three technical replicates were performed.

SA Measurement

SA was extracted and measured as described previously (Pan et al., 2010). For the extraction of free SA and SA glucosides, powdered leaf tissue was weighed into a 2-mL centrifuge tube and extracted with 1 mL of extraction buffer (2-propanol:water:concentrated HCl, 2:1:0.002, v/v/v) with 50 ng of D6-SA (Sigma-Aldrich) as an internal standard. The mixtures were agitated at 100 rpm for 30 min at 4°C followed by the addition of 1 mL of dichloromethane and agitation for another 30 min at 4°C. The samples were then centrifuged at 13,000g for 10 min. The 900 mL of solvent from the lower phase was collected and concentrated using an evaporator with nitrogen flow. The samples were dissolved in 100 mL of solution with 60 mL of methanol and 40 mL of distilled water. Chromatographic analysis was performed on the Shimadzu UFLCXR System equipped with an LC-20AD-XR binary pump, an SIL-20AD-XR autosampler, and a CTO-20A column oven. The Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 m; Agilent Technologies) was maintained at 40°C. The mobile phase was composed of methanol with 0.1% formic acid (A) and water with 0.1% formic acid (B) using a multistep linear gradient elution: 40% A at 0 to 5 min, 40% to 95% A at 5 to 13 min, and 40% A at 13 to 18 min with the flow rate kept at 0.3 mL min−1. Five microliters of each sample was injected. Mass spectrometry was performed on the Triple TOF 5600 device (AB Sciex), and the mass range was set at m/z 100 to 1,000. The conditions of the tandem mass spectrometry detector were set as follows: ion spray voltage, 1,500 V; ion source gas, 150 p.s.i.; ion source gas, 260 p.s.i.; temperature, 500°C; curtain gas, 15 p.s.i.; collision gas pressure, 8 p.s.i.; entrance potential, 10 V. Nitrogen was used as a nebulizer and auxiliary gas. The acquisition and analysis of data were controlled by PeakView Software TMV.1.1 (AB Sciex). Three biological replicates each with three technical replicates were performed.

Chlorophyll Content Measurement

The total chlorophyll content in the plants was determined by spectrophotometer. Leaf samples (300 mg) were ground to a powder in liquid nitrogen and then transferred to a 15-mL Falcon tube. Next, 5 mL of 80% acetone was added to the tube, and the contents were mixed thoroughly before being allowed to stand overnight in the dark. Centrifugation was then performed at 4°C for 15 min (3,000 rpm). The supernatant from each tube was then transferred to a clean tube, and the chlorophyll content was determined based on the absorption spectrum (hereafter termed A) by spectrophotometry according to the following equation (using 80% acetone as a blank control): Ca+b (mg/g) = [8.026A663 + 20.206A645] × V/1,000 × W, where V is the volume of the extract (mL) and W is the weight of the fresh leaves (g). Three biological replicates each with three technical replicates were performed.

H2O2 Content Measurement

H2O2 contents were determined by the POD-coupled assay protocols. About 0.1 g of Arabidopsis leaves was ground in liquid N2, and the powder was extracted in 2 mL of 1 m HClO4 in the presence of 5% insoluble polyvinylpyrrolidone. The homogenate was centrifuged at 12,000g for 10 min, and the supernatant was neutralized with 5 m K2CO3 to pH 5.6 in the presence of 100 μL of 0.3 m phosphate buffer, pH 5.6. The solution was centrifuged at 12,000g for 1 min, and the sample was incubated for 10 min with 1 unit of ascorbate oxidase to oxidize ascorbate prior to assay. The reaction mixture was composed of 0.1 m phosphate buffer, pH 6.5, 3.3 mm 3-(dimethylamino)benzoic acid, 0.07 mm 3-methyl-2-benzothiazoline hydrazone, and 0.3 units of peroxidase. The reaction was initiated by the addition of 200 μL of sample. The absorbance change at 590 nm was monitored at 25°C. Three biological replicates each with three technical replicates were performed.

Statistical Analysis

A one-way ANOVA was performed on the data. Differences between means were evaluated for significance using Tukey’s HSD test (P < 0.05); means with the same letter in the figures are not significantly different. Statistical analyses were performed with the statistical software SPSS 10.0 (SPSS).

Accession Numbers

Sequence data from this article can be found in The Arabidopsis Information Resource database under the following accession numbers: FtSH4 (At2g26140), WRKY75 (At5g13080), SID2 (At1g74710), NDR1 (At3g20600), NPR1 (At1g64280), and ATG8A (At4g21980). Gene identifiers for genes used for quantitative RT-PCR can be found in Supplemental Table S1.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_173_4_2294__index.html (1.4KB, html)

Acknowledgments

We thank Shi Xiao and Nan Yao from Sun Yat-Sen University for help with SA measurement, Shi Xiao for providing the atg5 (CS39993), atg8e (SALK_126394C) and atg8a (SALK_045344C) mutant seeds, and Faqiang Li from South China Agricultural University for offering some suggestions.

Glossary

ROS

reactive oxygen species

TF

transcription factor

SA

salicylic acid

OXPHOS

oxidative phosphorylation

LD

long-day

SD

short-day

LTG

LysoTracker Green

EMSA

electrophoretic mobility shift assay

Col-0

Columbia-0

RT

reverse transcription

HSD

honestly significant difference

Footnotes

1

This work was supported by the National Natural Science Foundation of China (grant nos. 31370350 and 31271471), the Natural Science Foundation of Guangdong Province (grant no. 2014A030313443), the Guangzhou Pearl River New Star Project for Science and Technology (grant no. 2012J2200033), the Education Department of Guangdong Province (grant no. 2012CXZD0019), and the Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (grant no. 2016 to S.Z.).

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