Monitoring the in vivo redox state of plant mitochondria: Effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge

In animals, the impact of ROS production by mitochondria on cell physiology, death, disease and ageing is well recognised. In photosynthetic organisms such as higher plants, however, the chloroplast and peroxisomes are the major sources of ROS during normal metabolism and the importance of mitochondria in oxidative stress and redox signalling is less well established. To address this, the in vivo oxidation state of a mitochondrially-targeted redox-sensitive GFP (mt-roGFP2) was investigated in Arabidopsis leaves. Classical ROS-generating inhibitors of mitochondrial electron transport (rotenone, antimycin A and SHAM) had no effect on mt-roGFP oxidation when used singly, but combined inhibition of complex III and alternative oxidase by antimycin A and SHAM did cause significant oxidation. Inhibitors of complex IV and aconitase also caused oxidation of mt-roGFP2. This oxidation was not apparent in the cytosol whereas antimycin A + SHAM also caused oxidation of cytosolic roGFP2. Menadione had a much greater effect than the inhibitors, causing nearly complete oxidation of roGFP2 in both mitochondria and cytosol. A range of severe abiotic stress treatments (heat, salt, and heavy metal stress) led to oxidation of mt-roGFP2 while hyperosmotic stress had no effect and low temperature caused a slight but significant decrease in oxidation. Similar changes were observed for cytosolic roGFP2. Finally, the recovery of oxidation state of roGFP in mitochondria after oxidation by H₂O₂ treatment was dramatically slower than that of either the cytosol or chloroplast. Together, the results highlight the sensitivity of the mitochondrion to redox perturbation and suggest a potential role in sensing and signalling cellular redox challenge.     

死亡信号传递: 叶绿体与线粒体间信号交流调控植物程序性细胞死亡

程序性细胞死亡(PCD)是生物体受遗传调控的自主细胞死亡现象, 在植物生长发育和抵抗环境胁迫中起重要作用。PCD的发生可受线粒体中活性氧(ROS)诱导。中国科学院遗传与发育生物学研究所李家洋研究组早期的研究发现了1个拟南芥(Arabidopsis thaliana)细胞死亡突变体mod1, 并暗示植物细胞中存在叶绿体与线粒体之间的信号交流调控PCD, 但其中的具体作用机制尚不清楚。最近, 他们通过大规模筛选mod1突变体的抑制突变体, 克隆了3个新的抑制基因plNAD- MDH、DiT1和mMDH1。此3个基因分别编码质体定位的NAD依赖的苹果酸脱氢酶、叶绿体被膜定位的二羧酸转运蛋白1和线粒体定位的苹果酸脱氢酶1, 突变后都可抑制mod1中ROS的积累及PCD的发生。通过对这些基因进行深入的功能分析, 他们论证了苹果酸从叶绿体到线粒体的转运对线粒体中ROS的产生及随后PCD的诱导起重要作用。该研究拓展了我们对植物细胞中细胞器间交流的认识, 为我们深入理解植物PCD发生机制提供了新线索, 是该领域的一项突破性进展。     

Arabidopsis ATG11, a scaffold that links the ATG1-ATG13 kinase complex to general autophagy and selective mitophagy

Autophagy is essential for nutrient recycling and intracellular housekeeping in plants by removing unwanted cytoplasmic constituents, aggregated polypeptides, and damaged organelles. The autophagy-related (ATG)1-ATG13 kinase complex is an upstream regulator that integrates metabolic and environmental cues into a coherent autophagic response directed by other ATG components. Our recent studies with Arabidopsis thaliana revealed that ATG11, an accessory protein of the ATG1-ATG13 complex, acts as a scaffold that connects the complex to autophagic membranes. We showed that ATG11 encourages proper behavior of the ATG1-ATG13 complex and faithful delivery of autophagic vesicles to the vacuole, likely through its interaction with ATG8. In addition, we demonstrated that Arabidopsis mitochondria are degraded during senescence via an autophagic route that requires ATG11 and other ATG components. Together, ATG11 appears to be an important modulator of the ATG1-ATG13 complex and a multifunctional scaffold required for bulk autophagy and the selective clearance of mitochondria.     

Metabolite regulation of the interaction between Arabidopsis thaliana PII and N-acetyl-l-glutamate kinase

The metabolic control of the interaction between ArabidopsisN-acetyl-l-glutamate kinase (NAGK) and the PII protein has been studied. Both gel exclusion and affinity chromatography analyses of recombinant, affinity-purified PII (trimeric complex) and NAGK (hexameric complex) showed that NAGK strongly interacted with PII only in the presence of Mg-ATP, and that this process was reversed by 2-oxoglutarate (2-OG). Furthermore, metabolites such as arginine, glutamate, citrate, and oxalacetate also exerted a negative effect on the PII-NAGK complex formation in the presence of Mg-ATP. Using chloroplast protein extracts and PII affinity chromatography, NAGK interacted with PII only in the presence of ATP-Mg²⁺, and this process was antagonized by 2-OG. These results reveal a complex metabolic control of the PII interaction with NAGK in the chloroplast stroma of higher plants.     

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.¹ The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.     

Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC·G, but not for T·G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.     

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).     

我国科学家在程序性细胞死亡机制研究领域取得重大突破

程序性细胞死亡在调控植物发育和胁迫响应中具有重要作用, 而活性氧是导致程序性细胞死亡的关键因子。日前, 中科院遗传与发育所李家洋研究组对活性氧调控程序性细胞死亡的分子机制进行了深度解析, 首次阐明了苹果酸作为信号分子, 经由叶绿体-线粒体穿梭途径而引发活性氧产生, 继而导致细胞死亡。该研究成果是程序性细胞死亡调控机制领域的重大突破。     

Glutathione and tryptophan metabolites are key players in Arabidopsis nonhost resistance against Colletotrichum gloeosporioides

The nonhost resistance of Arabidopsis against hemibiotrophic fungi in the genus Colletotrichum consists of pre- and post-invasive immune responses. Previously, we reported EDR1 and PEN2 as important components of Arabidopsis pre-invasive resistance toward non-adapted Colletotrichum gloeosporioides (Cg). However, despite their defect in entry control pen2 and edr1 mutants terminated further growth of this pathogen by activating the post-invasive hypersensitive response (HR) accompanied by plant cell death. In the present study, we showed that γ-glutamylcysteine synthetase (GSH1), which is required for glutathione biosynthesis, and tryptophan (Trp) metabolism contribute to pre- and post-invasive non-host resistance against Cg. We found GSH1 to be involved in the PEN2-dependent entry control of Cg. Opposite to pen2 and edr1, gsh1 mutants failed to restrict the invasive growth of the pathogen, which demonstrated the requirement for GSH1 during post-invasive non-host resistance. Based on the infection and metabolic phenotypes of Arabidopsis mutants defective in Trp metabolism, we showed that the biosynthesis of Trp-derived phytochemicals is also essential for resistance to Cg during the post-invasive HR. By contrast, GSH1 and these metabolites are dispensable for the induction of HR cell death, which is triggered in the non-invaded mesophyll cells adjacent to the Cg-invaded epidermal cells.     

Arabidopsis Zinc Ribbon 3 is the ortholog of yeast mitochondrial HSP70 escort protein HEP1 and belongs to an ancient protein family in mitochondria and plastids

ZR proteins belong to a phylogenetically conserved family of small zinc-ribbon proteins in plastids and mitochondria of higher plants. The function of these proteins is so far unclear. The mitochondrial proteins share sequence similarities with mitochondrial Hsp70 escort proteins (HEP) from Saccharomyces cerevisiae (HEP1) and human. Expression of the mitochondrial ZR protein from Arabidopsis, ZR3, rescued a hep1 knockout mutant from yeast. Accordingly, ZR3 was found to physically interact with mitochondrial Hsp70 from Arabidopsis. Our findings support the idea that mitochondrial and plastidic ZR proteins from higher plants are orthologs of HEP proteins.

Structured summary of protein interactions

ZR3physically interacts with mtHSC70-2 by pull down (View interaction)ZR3physically interacts with mtHSC70-1 by pull down (View interaction)     

Implication of reactive oxygen species and mitochondrial dysfunction in the early stages of plant programmed cell death induced by ultraviolet-C overexposure

Recent studies have suggested that ultraviolet-C (UV-C) overexposure induces programmed cell death (PCD) in Arabidopsis thaliana (L.) Heynh, and this process includes participation of caspase-like proteases, DNA laddering as well as fragmentation of the nucleus. To investigate possible early signal events, we used microscopic observations to monitor in vivo the behaviour of mitochondria, as well as the production and localization of reactive oxygen species (ROS) during protoplast PCD induced by UV-C. A quick burst of ROS was detected when the protoplasts were kept in continuous light after UV-C exposure, which was restricted in chloroplasts and the adjacent mitochondria. Pre-incubation with ascorbic acid (AsA, antioxidant molecule) or 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU, an inhibitor of photosynthetic electron transport) decreased the ROS production and partially protected protoplasts from PCD. A mitochondrial transmembrane potential (MTP) loss occurred prior to cell death; thereafter, the mitochondria irregularly clumped around chloroplasts or aggregated in other places within the cytoplasm, and the movement of mitochondria was concomitantly blocked. Pre-treatment with an inhibitor of mitochondrial permeability transition pores (MPTP), cyclosporine (CsA), effectively retarded the decrease of MTP and reduced the percentage of protoplasts undergoing PCD after UV-C overexposure. Our results suggest that the MTP loss and the changes in distribution and mobility of mitochondria, as well as the production of ROS play important roles during UV-induced plant PCD, which is in good accordance with what has been reported in many types of apoptotic cell death, both in animals and plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Several MAMPs,including chitin fragments,enhance AtPep-triggered oxidative burst independently of wounding

AtPeps are a family of small peptides in Arabidopsis that are believed to act as endogenous amplifiers of the plant’s innate immune response. In our recent study,¹⁰ we showed that in Arabidopsis leaf disks, bacterial MAMPs (microbe-associated molecular patterns) such as the flagellin derived elicitor flg22, greatly enhanced the release of reactive oxygen species (ROS) triggered by a subsequent AtPep-perception. This enhanced ROS production could be a hallmark either of improved local defense or of the initiation of ROS-based systemic signaling. Here, we established a superior ROS detection system based on a new derivative of luminol (L-012). With this sensitive system we were able to show that chitin, too, acts as an enhancer of AtPep-triggered ROS, linking this specific defense response amplification also to the recognition of fungal pathogens. In addition we used the more sensitive ROS assay to transfer the experimental setup from cut leaf disks to unwounded seedlings. Thereby we revealed that wounding is not a prerequisite to enable the MAMP-induced enhancement of the AtPep-triggered ROS response.     

Natural variation in phosphorylation of photosystem II proteins in Arabidopsis thaliana: is it caused by genetic variation in the STN kinases?

Reversible phosphorylation of photosystem II (PSII) proteins is an important regulatory mechanism that can protect plants from changes in ambient light intensity and quality. We hypothesized that there is natural variation in this process in Arabidopsis (Arabidopsis thaliana), and that this results from genetic variation in the STN7 and STN8 kinase genes. To test this, Arabidopsis accessions of diverse geographical origins were exposed to two light regimes, and the levels of phospho-D1 and phospho-light harvesting complex II (LHCII) proteins were quantified by western blotting with anti-phosphothreonine antibodies. Accessions were classified as having high, moderate or low phosphorylation relative to Col-0. This variation could not be explained by the abundance of the substrates in thylakoid membranes. In genotypes with atrazine-resistant forms of the D1 protein, low D1 and LHCII protein phosphorylation was observed, which may be due to low PSII efficiency, resulting in reduced activation of the STN kinases. In the remaining genotypes, phospho-D1 levels correlated with STN8 protein abundance in high-light conditions. In growth light, D1 and LHCII phosphorylation correlated with longitude and in the case of LHCII phosphorylation also with temperature variability. This suggests a possible role of natural variation in PSII protein phosphorylation in the adaptation of Arabidopsis to diverse environments.     

A protein containing an XYPPX repeat and a C2 domain is associated with virally induced hypersensitive cell death in plants

In this study, we characterized a Capsicum hypersensitive response (HR)-associated gene, SS52, which encodes a protein that contains an N-terminal C2 domain and a C-terminal XYPPX repeat. Expression analyses revealed that SS52 and its homologue in Arabidopsis were induced by infection with incompatible viruses, indicating the conserved function of this gene. SS52 was not induced by treatment with defense-related hormones, but was induced by abiotic stresses, including wounding. Overexpression of SS52 in tobacco plants suppressed the spread of HR cell death and restricted the spread of an incompatible virus from local lesions. Collectively, the results suggest that SS52 negatively regulates plant HR cell death.     

Characterization of harpin<Subscript>Xoo</Subscript> induced hypersensitive responses in non host plant,tobacco

Harpin proteins encoded by hrp genes are bacterial protein elicitors that can stimulate hypersensitive response (HR) in non-host plants. HR-related pathogen resistance involves a complex form of programmed cell death (PCD). It is increasingly viewed as a key component of the hypersensitive disease response of plants. Currently, the evidence of harpin proteins-induced PCD is deficient though it exhibits phenotypic parallels with HR, and the mechanism of harpin proteins-induced PCD is not well understood. In this study, we demonstrate that harpin_Xoo protein from Xanthomonas oryzae pv. oryzae of rice bacterial blight expressed and isolated from bacterial cells acted as an agent to induce PCD in infiltrated tobacco plants. Treatment of tobacco leaves with harpin_Xoo induced typical PCD-related morphological and biochemical changes including cell shrinkage and nuclear DNA degradation. We further analyzed the expression of several genes in signal transduction pathway of PCD in tobacco plants by real-time qRT-PCR analysis using EF-1α as an endogenous control. Our results showed that the expression of NtDAD1 was down-regulated and the expression of BI-1, tpa1 and aox1 was up-regulated following the infiltration of harpin_Xoo into tobacco leaves. Our data suggest that harpin_Xoo can induce PCD with the coordination of PCD-related genes in infiltrated tobacco leaves, providing evidence to further investigate the signal transduction pathways of HR and PCD.     

Dimeric H-ATP synthase in the chloroplast of Chlamydomonas reinhardtii

H⁺-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane.By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF₁ part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.