Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

Modulation of the Poly(ADP-ribosyl)ation Reaction via the Arabidopsis ADP-Ribose/NADH Pyrophosphohydrolase,AtNUDX7, Is Involved in the Response to Oxidative Stress

Here, we assessed modulation of the poly(ADP-ribosyl)ation (PAR) reaction by an Arabidopsis (Arabidopsis thaliana) ADP-ribose (Rib)/NADH pyrophosphohydrolase, AtNUDX7 (for Arabidopsis Nudix hydrolase 7), in AtNUDX7-overexpressed (Pro_35S:AtNUDX7) or AtNUDX7-disrupted (KO-nudx7) plants under normal conditions and oxidative stress caused by paraquat treatment. Levels of NADH and ADP-Rib were decreased in the Pro_35S:AtNUDX7 plants but increased in the KO-nudx7 plants under normal conditions and oxidative stress compared with the control plants, indicating that AtNUDX7 hydrolyzes both ADP-Rib and NADH as physiological substrates. The Pro_35S:AtNUDX7 and KO-nudx7 plants showed increased and decreased tolerance, respectively, to oxidative stress compared with the control plants. Levels of poly(ADP-Rib) in the Pro_35S:AtNUDX7 and KO-nudx7 plants were markedly higher and lower, respectively, than those in the control plants. Depletion of NAD⁺ and ATP resulting from the activation of the PAR reaction under oxidative stress was completely suppressed in the Pro_35S:AtNUDX7 plants. Accumulation of NAD⁺ and ATP was observed in the KO-nudx7- and 3-aminobenzamide-treated plants, in which the PAR reaction was suppressed. The expression levels of DNA repair factors, AtXRCC1 and AtXRCC2 (for x-ray repair cross-complementing factors 1 and 2), paralleled that of AtNUDX7 under both normal conditions and oxidative stress, although an inverse correlation was observed between the levels of AtXRCC3, AtRAD51 (for Escherichia coli RecA homolog), AtDMC1 (for disrupted meiotic cDNA), and AtMND1 (for meiotic nuclear divisions) and AtNUDX7. These findings suggest that AtNUDX7 controls the balance between NADH and NAD⁺ by NADH turnover under normal conditions. Under oxidative stress, AtNUDX7 serves to maintain NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and thus regulates the defense mechanisms against oxidative DNA damage via modulation of the PAR reaction.Reactive oxygen species (ROS) are by-products of normal metabolic processes, including chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems, in all aerobic organisms (Gutteridge and Halliwell, 1989). Although the production and destruction of ROS are in balance, the imposition of biotic and abiotic stressful conditions can give rise to excess concentrations of ROS, leading to an imbalance of production and scavenging mechanisms (Mittler, 2002; Mullineaux and Karpinski, 2002; Kroj et al., 2003; Mahalingam et al., 2003). Excess ROS, leading to oxidative stress, can damage organelles, oxidize proteins, nick DNA (single-base DNA damage), deplete antioxidant levels, and ultimately trigger cell death (Gutteridge and Halliwell, 1989). Recently, ROS have been recognized as important signaling molecules that control diverse signaling pathways involved in a variety of cellular responses such as programmed cell death, pathogen defense, and hormone signaling (Foyer and Noctor, 2005; Kwak et al., 2006; Torres et al., 2006). In addition, oxidative stress causes dramatic inhibition of the tricarboxylic acid cycle and large sectors of amino acid metabolism followed by backing up of glycolysis and diversion of carbon into the oxidative pentose phosphate pathway (Baxter et al., 2007). Therefore, organisms have developed efficient systems to keep ROS levels in check and repair damage from attack by ROS.Among various defense systems against attack by ROS, the poly(ADP-ribosyl)ation (PAR) of proteins by poly(ADP-Rib)polymerase (PARP), by which branched polymers of ADP-Rib are attached using β-NAD⁺ to a specific amino acid residue of an acceptor protein, is a posttranslational modification for responding early to DNA damage, such as single-strand DNA break and resealing, caused by oxidative stress and, thus, is crucial for genomic integrity and cell survival (Qin et al., 2008). PARP detects DNA strand breaks and converts the damage into intracellular signals that can activate DNA repair programs or cell death, according to the severity of the injury, via the PAR reaction of nuclear proteins involved in chromatin architecture and DNA metabolism and interacts with the x-ray repair cross complementing factor 1 (XRCC1), an adaptor protein that also has two interfaces with two important single-strand DNA break (SSB) repair (SSBR)/base excision repair (BER) enzymes: DNA ligase and DNA polymerase β (Caldecott et al., 1995, 1996; Kubota et al., 1996; Masson et al., 1998). DNA polymerase β fills the single nucleotide gap, preparing the strand for ligation by a complex of DNA ligase III and XRCC1 (Winters et al., 1999; Thompson and West, 2000). Thereby, the fast recruitment of SSBR/BER factors is archived in the site of the lesion. Modifications of proteins with poly(ADP-Rib) are reversed by poly(ADP-Rib) glycohydrolase (PARG), by which ADP-Rib polymers are hydrolyzed to free ADP-Rib, since incorrect signal transduction is caused by excessive accumulation of poly(ADP-Rib) modification (Davidovic et al., 2001). However, it has been reported that a massive PAR reaction results in the overconsumption of NAD⁺ and ATP and, ultimately, in energy depletion causing necrotic cell death (Ha and Snyder, 1999; Virág and Szabó, 2002; De Block et al., 2005).Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases catalyze the hydrolysis of intact and oxidatively damaged nucleoside diphosphates and triphosphates, nucleotide sugars, coenzymes, dinucleoside polyphosphates, and RNA caps in various organisms such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). We have previously reported the characteristics of cytosolic Nudix hydrolases (AtNUDX1–AtNUDX11) in Arabidopsis (Arabidopsis thaliana; Ogawa et al., 2005). Among them, the recombinant AtNUDX7 showed high affinity for ADP-Rib and NADH as substrates in vitro, converting NADH to a reduced form of nicotinamide mononucleotide (NMNH) plus AMP and ADP-Rib to AMP plus Rib 5-P (Ogawa et al., 2005). AtNUDX7 was expressed more strongly in leaf than in stem and root. Therefore, the enzyme might be involved in nucleotide recycling relating to the metabolism of NADH and/or poly(ADP-Rib).Recent studies revealed that the actions of AtNUDX7 (At4g12720) are closely related to immune responses to pathogens. Knockout of AtNUDX7 (KO-nudx7) in Arabidopsis plants led to deleterious inference for cells, such as microscopic cell death, constitutive expression of pathogenesis-related genes, resistance to bacterial pathogens, and accumulation of NADH (Jambunathan and Mahalingam, 2006). Furthermore, AtNUDX7 exerted a negative regulatory effect on EDS1 signaling, which controls the activation of defenses and programmed cell death conditioned by intracellular Toll-related immune receptors that recognized specific pathogen effectors (Bartsch et al., 2006). More recently, Ge et al. (2007) reported that KO-nudx7 plants show heightened defense responses, which are both dependent on and independent of the accumulation of NPR1 and salicylic acid, to pathogenic attack. On the other hand, Adams-Phillips et al. (2008) reported that KO-nudx7 plants exhibit a reduced hypersensitive-response phenotype, although the growth of both virulent and avirulent pathogens is suppressed in the plants. These findings support the hypothesis that regulation of the metabolism of NADH and/or ADP-Rib by Nudix hydrolases is important for stress-related defense systems in higher plants. However, the direct actions of the enzymes on stress responses are not established yet.In this study, to assess the functions of Arabidopsis Nudix hydrolases having ADP-Rib and NADH pyrophosphohydrolase activities under normal conditions and oxidative stress, we analyzed the effect of the overexpression or disruption of AtNUDX7 on levels of ADP-Rib, NAD(H), and ATP as well as PAR activity and oxidative stress tolerance in Arabidopsis. The evidence presented here suggests that AtNUDX7 serves to balance between NADH and NAD⁺ by NADH turnover under normal conditions. In addition, AtNUDX7 functions in the maintenance of NAD⁺ levels by supplying ATP via nucleotide recycling from free ADP-Rib molecules and the modulation of the PAR reaction, thereby regulating the DNA repair pathways, in response to oxidative stress.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

Distinct regulation of Arabidopsis ADP-ribose/NADH pyrophosphohydrolases,AtNUDX6 and 7, in biotic and abiotic stress responses

Among Arabidopsis Nudix hydrolases (AtNUDX1∼27), AtNUDX6 and AtNUDX7 having ADP-ribose/NADH pyrophosphohydrolase activities have been found to contribute to keeping the energy and redox homeostasis, and/or modulating defense responses against biotic and abiotic stress. Interestingly, AtNUDX6 had an opposite effect to AtNUDX7 on the regulation of immune responses. A comparison of the activities of ADP-ribose/NADH pyrophosphohydrolase among wild-type, knockout (KO)-nudx6, and KO-nudx7 plants revealed AtNUDX7 to contribute more than AtNUDX6 to the total pyrophosphohydrolase activity toward both ADP-ribose and NADH under normal conditions and oxidative stress, while AtNUDX6 accounted for the majority of total NADH pyrophosphohydrolase activity under salicylic acid treatment. These results support the idea that the metabolism of ADP-ribose and/or NADH needs to be finely tuned for accurate regulation of cellular responses to biotic and abiotic stress.Key words: nudix hydrolase, ADP-ribose/NADH pyrophosphohydrolases, biotic and abiotic stress responseNudix (nucleoside diphosphates linked to some moiety X) hydrolases distributed among all classes of organisms from archaea to vertebrates have the potential to hydrolyze a wide range of substrates such as dinucleoside polyphosphates, various coenzymes, nucleotide sugars, ribo- and deoxynucleoside triphophates, and alcohols.^1–3 Recently, Nudix hydrolases having hydrolysis activity toward other compounds containing pyrophosphate bounds, such as nucleoside diphophates, the mRNA cap, 5′triphosphorylated RNA, and guanosine 3′,5′-bispyrophosphate, and non-nucleoside substrates such as diphosphoinositol polyphosphates, 5-phosphoribosyl 1-diphosphate, thiamine pyrophosphate, and dihydroneopterin triphosphate, have been identified.³ Several of these substrates are potentially toxic compounds, cell signaling molecules, metabolic intermediates, or coenzymes. Nudix hydrolases are thus considered to be associated with various cellular processes by hydrolytically removing these substrates.Arabidopsis thaliana has 27 genes encoding Nudix hydrolase (AtNUDX1-27), which can be classified into three types by their predicted subcellular localization, the cytosol (AtNUDX1∼11 and 25), mitochondria (AtNUDX12∼18), or chloroplasts (AtNUDX19∼24, 26 and 27).^4,5 It is remarkable that there are a large number of AtNUDXs having ADP-ribose or NADH pyrophosphohydrolase activity (AtNUDX2, 6, 7, 10, 14, 19 and 23); the number (7) of enzymes in the subfamily is greater than that in humans, which have 5 genes encoding the putative ADP-ribose or NADH pyrophosphohydrolase. Recombinant forms of AtNUDX2, 6 and 7 have showed the pyrophosphohydrolase activity toward both ADP-ribose and NADH with high affinity in vitro.⁴ Recent studies have demonstrated that the modulation of ADP-ribose and/or NADH levels through the hydrolysis by AtNUDX2, 6 and 7 contributes to keeping the energy and redox homeostasis, and/or modulating defense responses to both biotic and abiotic stress,^6–10 indicating the diverse roles of Nudix hydrolases in plants. AtNUDX2 might not function physiologically, because of its low levels even under stressful conditions.⁶ It should be noted that the physiological role of AtNUDX6 differs considerably from that of AtNUDX7, although their enzymatic properties in vivo are partly the same: we previously demonstrated that AtNUDX7 acts in the hydrolysis of both ADP-ribose and NADH in cells, while AtNUDX6 acts only on NADH.^7,8It was demonstrated that AtNUDX7 acts as a negative regulator to prevent excessive stimulation of the defense response, which is dependent on and independent of Nonexpresser of Pathogenesis-Related genes 1, a master regulator of salicylic acid (SA)-induced defense genes, and SA accumulation,¹⁰ while AtNUDX6 acts as a positive regulator through NPR1-dependent SA signaling pathways.⁸ In addition, AtNUDX7, but not AtNUDX6, modulated the poly(ADP-ribosyl)ation reaction, which is one of the early responses to DNA damage caused by oxidative stress.⁷ These observations raise the question of how AtNUDXs control such different processes.To evaluate the physiological importance of each AtNUDX, here we compared AtNUDX6 and 7 in ADP-ribose and/or NADH pyrophosphohydrolase activity in Arabidopsis cells under various conditions. From the difference in activity of extracts prepared from the leaves of wild-type, knockout (KO)-nudx6, and KO-nudx7 plants grown under normal conditions for 2 weeks, it was estimated that AtNUDX7 accounts for 23% of the total ADP-ribose pyrophosphohydrolase activity, but AtNUDX6 barely contributes to the activity (Fig. 1). Oxidative stress caused by 3 µM paraquat (PQ) for 7 days caused an increase in the total ADP-ribose pyrophosphohydrolase activity. Under oxidative stress, the contribution of AtNUDX7 to the activity increased to 34%. Treatment with 0.5 mM SA, a signaling molecule necessary for the onset of systemic acquired resistance, had no effect on the activity.Open in a separate windowFigure 1Changes in the ADP-ribose/NADH pyrophosphohydrolase activity of AtNUDX6 and 7 in Arabidopsis leaves under treatment with PQ or SA. The activities of pyrophosphohydrolase toward ADP-ribose (A) and NADH (B) in the leaves of wild-type plants grown on MS medium for 2 weeks under long-day conditions [16 h of light (100 µmol photons m⁻² s⁻¹), 25°C/8 h of dark, 22°C] are shown as Control. PQ treatment was imposed by growing 2-week-old plants in MS medium containing the agent at 3 µm for 7 days under long-day conditions (PQ). SA treatment was imposed by growing 2-week-old plants in MS medium containing 0.5 mM SA for 24 h under long-day conditions (SA). The ADP-ribose and NADH pyrophosphohydrolase activities were measured as described previously.⁸ The contributions (%) of AtNUDX6 and AtNUDX7 to total ADP-ribose/NADH pyrophosphohydrolase activity under treatment with PQ and SA were estimated from the decrease in activity in the respective knockout mutants (KO-nudx6 and KO-nudx7)^7,8 and are indicated in parentheses. Data are the mean ± SD for three individual experiments (n = 3) using plants grown independently. Different letters indicate significant differences (p < 0.05).AtNUDX6 and 7 accounted for 25 and 53%, respectively, of the total pyrophosphohydrolase activity toward NADH under normal conditions (Fig. 1). The activity was increased by oxidative stress, with AtNUDX7 contributing 57%. On the other hand, under treatment with SA, the total NADH pyrophosphohydrolase activity was increased and AtNUDX6 accounted for 53% of the activity. These results indicated that AtNUDX7 contributed more than AtNUDX6 to the total pyrophosphohydrolase activity toward both ADP-ribose and NADH under normal conditions and oxidative stress.⁷ On the other hand, AtNUDX6 accounted for the majority of the total NADH pyrophosphohydrolase activity under SA treatment.⁸Plants are simultaneously exposed to abiotic and biotic hazards in nature. There is increasing evidence of crosstalk among the signaling pathways for biotic and abiotic stress.^12,13 It is worth noting that the expression of AtNUDX7, but not AtNUDX6, is regulated by intracellular levels of reactive oxygen species (ROS), since it is induced by not only pathogen infections but also oxidative stress including PQ treatment, all of which are known to cause the production of ROS in the cells.^8–11 On the other hand, the expression of AtNUDX6 was induced only by the application of SA and its analogues, 2,6-dichloroisonicotinic acid or acibenzolar-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, and not by oxidative stress.^8,14,15 Therefore, the expression of AtNUDX6 was thought to be regulated by intracellular SA levels, although it was also induced by pathogenic attacks causing local excessive production of both ROS and SA.⁹ The differences in the regulation of AtNUDX6 and 7 and timing of production of ROS and SA in response to biotic stress raise the possibility that the total activity of ADP-ribose/NADH pyrophosphohydrolase and subsequent metabolism of ADP-ribose and/or NADH must be finely tuned for accurate regulation of such cellular responses. The activation of metabolism caused by the accumulation of either ROS or SA at specific phases in plant cells might have different effects on cellular responses through cooperation with other factors.     

Salicylic Acid Regulates Plasmodesmata Closure during Innate Immune Responses in Arabidopsis

In plants, mounting an effective innate immune strategy against microbial pathogens involves triggering local cell death within infected cells as well as boosting the immunity of the uninfected neighboring and systemically located cells. Although not much is known about this, it is evident that well-coordinated cell–cell signaling is critical in this process to confine infection to local tissue while allowing for the spread of systemic immune signals throughout the whole plant. In support of this notion, direct cell-to-cell communication was recently found to play a crucial role in plant defense. Here, we provide experimental evidence that salicylic acid (SA) is a critical hormonal signal that regulates cell-to-cell permeability during innate immune responses elicited by virulent bacterial infection in Arabidopsis thaliana. We show that direct exogenous application of SA or bacterial infection suppresses cell–cell coupling and that SA pathway mutants are impaired in this response. The SA- or infection-induced suppression of cell–cell coupling requires an ENHANCED DESEASE RESISTANCE1– and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1–dependent SA pathway in conjunction with the regulator of plasmodesmal gating PLASMODESMATA-LOCATED PROTEIN5. We discuss a model wherein the SA signaling pathway and plasmodesmata-mediated cell-to-cell communication converge under an intricate regulatory loop.     

The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in Arabidopsis thaliana

Ubiquitination is an important post-translational protein modification that is known to play critical roles in diverse biological processes in eukaryotes. The RING E3 ligases function in ubiquitination pathways, and are involved in a large diversity of physiological processes in higher plants. The RING domain-containing E3 ligase AtRDUF1 was previously identified as a positive regulator of ABA-mediated dehydration stress response in Arabidopsis. In this study, we report that AtRDUF1 is involved in plant responses to salt stress. AtRDUF1 expression is upregulated by salt treatment. Overexpression of AtRDUF1 in Arabidopsis results in an insensitivity to salt and osmotic stresses during germination and seedling growth. A double knock-out mutant of AtRDUF1 and its close homolog AtRDUF2 (atrduf1atrduf2) was hypersensitive to salt treatment. The expression levels of the stress-response genes RD29B, RD22, and KIN1 are more sensitive to salt treatment in AtRDUF1 overexpression plants. In summary, our data show that AtRDUF1 positively regulates responses to salt stress in Arabidopsis.