Klaus Apel

Transcriptomic Footprints Disclose Specificity of Reactive Oxygen Species Signaling in Arabidopsis

Reactive oxygen species (ROS) are key players in the regulation of plant development, stress responses, and programmed cell death. Previous studies indicated that depending on the type of ROS (hydrogen peroxide, superoxide, or singlet oxygen) or its subcellular production site (plastidic, cytosolic, peroxisomal, or apoplastic), a different physiological, biochemical, and molecular response is provoked. We used transcriptome data generated from ROS-related microarray experiments to assess the specificity of ROS-driven transcript expression. Data sets obtained by exogenous application of oxidative stress-causing agents (methyl viologen, Alternaria alternata toxin, 3-aminotriazole, and ozone) and from a mutant (fluorescent) and transgenic plants, in which the activity of an individual antioxidant enzyme was perturbed (catalase, cytosolic ascorbate peroxidase, and copper/zinc superoxide dismutase), were compared. In total, the abundance of nearly 26,000 transcripts of Arabidopsis (Arabidopsis thaliana) was monitored in response to different ROS. Overall, 8,056, 5,312, and 3,925 transcripts showed at least a 3-, 4-, or 5-fold change in expression, respectively. In addition to marker transcripts that were specifically regulated by hydrogen peroxide, superoxide, or singlet oxygen, several transcripts were identified as general oxidative stress response markers because their steady-state levels were at least 5-fold elevated in most experiments. We also assessed the expression characteristics of all annotated transcription factors and inferred new candidate regulatory transcripts that could be responsible for orchestrating the specific transcriptomic signatures triggered by different ROS. Our analysis provides a framework that will assist future efforts to address the impact of ROS signals within environmental stress conditions and elucidate the molecular mechanisms of the oxidative stress response in plants.

FLU: A negative regulator of chlorophyll biosynthesis in Arabidopsis thaliana

Tetrapyrroles such as chlorophylls and bacteriochlorophylls play a fundamental role in the energy absorption and transduction activities of photosynthetic organisms. Because of these molecules, however, photosynthetic organisms are also prone to photooxidative damage. They had to evolve highly efficient strategies to control tetrapyrrole biosynthesis and to prevent the accumulation of free intermediates that potentially are extremely destructive when illuminated. In higher plants, the metabolic flow of tetrapyrrole biosynthesis is regulated at the step of δ-aminolevulinic acid synthesis. This regulation previously has been attributed to feedback control of Glu tRNA reductase, the first enzyme committed to tetrapyrrole biosynthesis, by heme. With the recent discovery of chlorophyll intermediates acting as signals that control both nuclear gene activities and tetrapyrrole biosynthesis, it seems likely that heme is not the only regulator of this pathway. A genetic approach was used to identify additional factors involved in the control of tetrapyrrole biosynthesis. In Arabidopsis thaliana, we have found a negative regulator of tetrapyrrole biosynthesis, FLU, which operates independently of heme and seems to selectively affect only the Mg2+ branch of tetrapyrrole biosynthesis. The identity of this protein was established by map-based cloning and sequencing the FLU gene. FLU is a nuclear-encoded plastid protein that, after import and processing, becomes tightly associated with plastid membranes. It is unrelated to any of the enzymes known to be involved in tetrapyrrole biosynthesis. Its predicted features suggest that FLU mediates its regulatory effect through interaction with enzymes involved in chlorophyll synthesis.

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Shortly after the release of singlet oxygen (1O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1O2-responsive genes. Retrograde control of 1O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana

Upon a dark-to-light shift, the conditional fluorescent (flu) mutant of Arabidopsis releases singlet oxygen (1O2) within the plastid compartment. Distinct sets of nuclear genes are activated that are different from those induced by superoxide (O2•−) and/or hydrogen peroxide (H2O2), suggesting that different types of reactive oxygen species activate distinct signaling pathways. It is not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX). The overexpression of the H2O2-specific scavenger reduced strongly the activation of nuclear genes in plants treated with the herbicide paraquat that in the light leads to the enhanced generation of O2•− and H2O2. In the flu mutant overexpressing tAPX, the intensity of 1O2-mediated cell death and growth inhibition was increased when compared with the flu parental line. Also, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX did not lead to visible stress responses and had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions.

Identification of NADPH:Protochlorophyllide Oxidoreductases A and B: A Branched Pathway for Light-Dependent Chlorophyll Biosynthesis in Arabidopsis thaliana

Illumination releases the arrest in chlorophyll (Chl) biosynthesis in etiolated angiosperm seedlings through the enzymatic photoreduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), the first light-dependent step in chloroplast biogenesis. NADPH: Pchlide oxidoreductase (POR, EC 1.3.1.33), a nuclear-encoded plastid-localized enzyme, mediates this unique photoreduction. Paradoxically, light also triggers a drastic decrease in the amounts of POR activity and protein before the Chl accumulation rate reaches its maximum during greening. While investigating this seeming contradiction, we identified two distinct Arabidopsis thaliana genes encoding POR, in contrast to previous reports of only one gene in angiosperms. The genes, designated PorA and PorB, by analogy to the principal members of the phytochrome photoreceptor gene family, display dramatically different patterns of light and developmental regulation. PorA mRNA disappears within the first 4 h of greening, whereas PorB mRNA persists even after 16 h of illumination, mirroring the behavior of two distinct POR protein species. Experiments designed to help define the functions of POR A and POR B demonstrate exclusive expression of PorA in young seedlings and of PorB both in seedlings and in adult plants. Accordingly, we propose the existence of a branched light-dependent Chl biosynthesis pathway in which POR A performs a specialized function restricted to the initial stages of greening and POR B maintains Chl levels throughout angiosperm development.

Differences in gene expression between natural and artificially induced leaf senescence

Gene expression during artificially induced senescence of barley (Hordeum vulgare L.) leaves was examined by in-vitro translation and mRNA hybridization with several copy-DNA (cDNA) clones for newly induced transcripts. When detached barley leaves were incubated in darkness, senescence symptoms as indicated by chlorophyll loss were rapidly induced. By in-vitro translation, concomitant changes in translatable mRNA levels were shown to occur with some translation products decreasing and others increasing in abundance. For closer analysis, cDNA clones for newly induced transcripts were isolated by differential screening. Six cDNA clones, derived from three different transcripts were identified and classified according to the expression of the respective mRNAs. Two of the three transcripts showed very similar expression patterns: in detached leaves they were induced by abscisic acid and inhibited by kinetin. They were also induced by wounding and osmotic stress, but could not be detected in naturally senescing leaves. The third mRNA, represented by only one of the six cDNA clones, behaved differently. There was no significant effect of hormone application, wounding or drought conditions, but the transcript accumulated during natural senescence of barley flag leaves. We conclude that only a minor part of the mRNA changes observed during dark incubation of detached leaves is connected with leaf senescence, whereas stress-related transcripts appear to predominate quantitatively.

The identification of leaf thionin as one of the main jasmonate-induced proteins of barley (Hordeum vulgare)

Jasmonic acid (JA) and its methyl ester (JA-Me) are able to introduce the accumulation of several specific polypeptides in cut leaf segments of barley. Two of the most prominent JA-induced proteins of Mr 15 000 and 23 000 have been characterized by isolating and sequencing complete cDNA sequences. While the sequence of the Mr 23 000 polypeptide shows no similarity to published sequences, the sequence of the Mr 15 000 polypeptide corresponds to the higher-molecular-weight precursor of a leaf thionin previously characterized.Transcripts for the Mr 23 000 and Mr 15 000 polypeptides accumulate in leaf segments shortly after the beginning of JA treatment. JA and JA-Me induce the appearance of the two proteins not only in leaf segments but also in intact barley seedlings. However, in seedlings the accumulation of JA-induced proteins occurs much more slowly and requires high concentrations of volatile JA-Me. Thus, in barley it seems unlikely that volatile JA-Me is involved in the interaction between different members of this species, as has been proposed recently for tomato seedlings.

No single way to understand singlet oxygen signalling in plants.

When plant cells are under environmental stress, several chemically distinct reactive oxygen species (ROS) are generated simultaneously in various intracellular compartments and these can cause oxidative damage or act as signals. The conditional flu mutant of Arabidopsis, which generates singlet oxygen in plastids during a dark‐to‐light transition, has allowed the biological activity of singlet oxygen to be determined, and the criteria to distinguish between cytotoxicity and signalling of this particular ROS to be defined. The genetic basis of singlet‐oxygen‐mediated signalling has been revealed by the mutation of two nuclear genes encoding the plastid proteins EXECUTER (EX)1 and EX2, which are sufficient to abrogate singlet‐oxygen‐dependent stress responses. Conversely, responses due to higher cytotoxic levels of singlet oxygen are not suppressed in the ex1/ex2 background. Whether singlet oxygen levels lower than those that trigger genetically controlled cell death activate acclimation is now under investigation.

Chloroplasts of Arabidopsis Are the Source and a Primary Target of a Plant-Specific Programmed Cell Death Signaling Pathway

Under mild light stress, plants enhance the production of singlet oxygen that acts as a signal. Singlet oxygen–mediated signaling forms an integral part of photosynthesis that translates environmental variability affecting photosynthetic electron transport into signals that regulate the readjustment of the plant to environmental changes. Enhanced levels of singlet oxygen (1O2) in chloroplasts trigger programmed cell death. The impact of 1O2 production in chloroplasts was monitored first in the conditional fluorescent (flu) mutant of Arabidopsis thaliana that accumulates 1O2 upon a dark/light shift. The onset of 1O2 production is rapidly followed by a loss of chloroplast integrity that precedes the rupture of the central vacuole and the final collapse of the cell. Inactivation of the two plastid proteins EXECUTER (EX1) and EX2 in the flu mutant abrogates these responses, indicating that disintegration of chloroplasts is due to EX-dependent signaling rather than 1O2 directly. In flu seedlings, 1O2-mediated cell death signaling operates as a default pathway that results in seedlings committing suicide. By contrast, EX-dependent signaling in the wild type induces the formation of microlesions without decreasing the viability of seedlings. 1O2-mediated and EX-dependent loss of plastid integrity and cell death in these plants occurs only in cells containing fully developed chloroplasts. Our findings support an as yet unreported signaling role of 1O2 in the wild type exposed to mild light stress that invokes photoinhibition of photosystem II without causing photooxidative damage of the plant.

Evolution of Chlorophyll Biosynthesis—The Challenge to Survive Photooxidation

The capability to perform protochlorophyllide reduction under aerobic conditions and thereby to avoid the danger of photooxidation was certainly a landmark in the evolution of porphyrin biosynthesis. Such a scenario, however, rests on the assumption that POR already occurred at the roots of all plant organisms. In fact, por-related gene sequences have been shown to occur in cyanobacteria (Suzuki and Bauer 1995xSuzuki, J.Y and Bauer, C.E. Proc. Natl. Acad. Sci. USA. 1995; 92: 3749–3753Crossref | PubMedSee all ReferencesSuzuki and Bauer 1995) and thus can be traced back to the presumed endosymbiotic origin of chloroplasts.In ancestral cyanobacteria and presumably also in early forms of vascular plants, such as the rhyniophytes, both types of protochlorophyllide-reducing enzymes, BCHLNB and POR, were likely to coexist. However, during the evolution of angiosperms, bchL, bchB, and bchN were lost.Molecular evidence based on the analysis of chloroplast DNA structure and organization of the major extant lineages of vascular land plants suggests that presumably as a result of a DNA inversion in a particular 30-kb region, the chlB, chlL, and chlN genes, which are the counterparts of bchB, bchL, and bchN of Rhodobacter and chlB, chlL, and chlN of extant cyanobacteria, were lost at the divergence point between gymnosperms and angiosperms (Raubeson and Jansen 1992xRaubeson, L.A and Jansen, R.K. Science. 1992; 255: 1697–1699Crossref | PubMedSee all ReferencesRaubeson and Jansen 1992). By contrast, the gene encoding POR was preserved and relocated to the nucleus, where it duplicated and diverged into two distinct por genes, termed porA and porB (3xArmstrong, G.A, Runge, S, Frick, G, Sperling, U, and Apel, K. Plant Physiol. 1995; 108: 1505–1517Crossref | PubMedSee all References, 7xHoltorf, H, Reinbothe, S, Reinbothe, C, Bereza, B, and Apel, K. Proc. Natl. Acad. Sci. USA. 1995; 92: 3254–3258Crossref | PubMedSee all References). This diversification is likely to have occurred even before speciation of gymnosperms and angiosperms.Reasons that evolution invented and maintained two POR enzymes may be deduced from our studies of the expression patterns of the PORA and PORB polypeptides (3xArmstrong, G.A, Runge, S, Frick, G, Sperling, U, and Apel, K. Plant Physiol. 1995; 108: 1505–1517Crossref | PubMedSee all References, 7xHoltorf, H, Reinbothe, S, Reinbothe, C, Bereza, B, and Apel, K. Proc. Natl. Acad. Sci. USA. 1995; 92: 3254–3258Crossref | PubMedSee all References). In both gymnosperms and angiosperms, PORA and possibly PORB are active during the transitory stage from dark to light growth (Lebedev et al. 1995xLebedev, N, van Cleve, B, Armstrong, G.A, and Apel, K. Plant Cell. 1995; 7: 2081–2090PubMedSee all ReferencesLebedev et al. 1995) when protochlorophyllide and chlorin must be shielded from interacting with O2 in the atmosphere. PORB is present also in light-adapted plants, where it may serve housekeeping functions in chlorophyll synthesis (3xArmstrong, G.A, Runge, S, Frick, G, Sperling, U, and Apel, K. Plant Physiol. 1995; 108: 1505–1517Crossref | PubMedSee all References, 7xHoltorf, H, Reinbothe, S, Reinbothe, C, Bereza, B, and Apel, K. Proc. Natl. Acad. Sci. USA. 1995; 92: 3254–3258Crossref | PubMedSee all References). Due to the operation of PORA and PORB, the risk is kept very low that porphyrins, such as protochlorophyllide and chlorin, will cause photooxidative damage to cellular and subcellular structures during the entire plant life cycle.The proposed rooting of POR is based on the hidden assumption that the last common ancestor of all photosynthetic eubacteria contained bacteriochlorophyll, not chlorophyll, in its reaction center. In such a “bacteriochlorophyll-first” scenario, chlorophyll-based photosynthesis would be a late invention unique to the cyanobacteria/chloroplast lineage. In the alternative, “chlorophyll-first” scenario, however, extant bacteriochlorophyll-containing purple bacteria would represent “deposed monarchs,” waiting, in specialized environments such as thermoclines of lakes, until “aerobic republicans” have gone out of fashion (Allen 1995xAllen, J.F. Nature. 1995; 376: 26CrossrefSee all ReferencesAllen 1995). The lower number of steps required for chlorophyll biosynthesis, as compared to bacteriochlorophyll synthesis, would be consistent with such a model. However, the “chlorophyll-first” hypothesis does not account for the fact that primitive cyanobacteria, by analogy to those existing today, already contained both, the oxygen-insensitive POR and the bchLNB-encoded protochlorophyllide reductase. We therefore prefer the “bacteriochlorophyll-first” hypothesis, which could explain why anoxygenic purple bacteria, which synthesize a photosystem only under anaerobic conditions, contain only a light-independent form and why cyanobacteria, which produce oxygen as a consequence of photosynthesis, have evolved an additional protochlorophyllide-reducing enzyme. Presumably the need to cope with the problem of photooxidation in the O2-rich atmosphere led to the evolution of POR.