Markus Hartl

Specificity in Ecological Interactions. Attack from the Same Lepidopteran Herbivore Results in Species-Specific Transcriptional Responses in Two Solanaceous Host Plants

Model systems have proven enormously useful in elucidating the biochemical function of plant genes. However their ecological function, having been sculpted by evolutionary forces specific to a species, may be less conserved across taxa. Responses to wounding and herbivore attack differ among plant families and are known to be mediated by oxylipin, ethylene, and systemin-signaling networks. We analyzed transcriptional responses of two native Solanaceous species to the attack of an herbivore whose elicitors are known not to be influenced by diet. With The Institute for Genomic Research 10k-cDNA potato (Solanum tuberosum) microarray, we compared the transcriptional responses of Nicotiana attenuata with those of black nightshade (Solanum nigrum) when both were attacked by the Solanaceous generalist herbivore, Manduca sexta. Based on an NADH dehydrogenase subunit F phylogeny, S. nigrum is more closely related to potato than N. attenuata but responded significantly less to M. sexta attack. Apart from transcriptional differences anticipated from their differences in secondary metabolism, both species showed distinct transcriptional patterns (with only 10% overlap in significantly regulated genes), which point to fundamental differences in the signaling cascades and downstream genes mediating herbivore resistance. The lackluster transcriptional response of S. nigrum could not be attributed to its inability to respond to elicitation, because methyl jasmonate elicitation of S. nigrum resulted in a strong transcriptional response. Given that attack from the same herbivore elicits profoundly different responses in two Solanaceaous taxa, we conclude that blueprints for commonly regulated responses to plant-herbivore interactions appear unlikely.

Serine Protease Inhibitors Specifically Defend Solanum nigrum against Generalist Herbivores but Do Not Influence Plant Growth and Development

Serine protease inhibitors (SPIs) are antidigestive proteins that presumably defend plants against herbivore attack. This work identifies and characterizes SPIs in the wild plant Solanum nigrum and evaluates the consequences of SPI silencing on plant defense, growth, and development. Solanaceaeous taxa produce diverse peptide serine proteinase inhibitors (SPIs), known antidigestive defenses that might also control endogenous plant proteases. If and how a plant coordinates and combines its different SPIs for the defense against herbivores and if these SPIs simultaneously serve developmental functions is unknown. We examine Solanum nigrum’s SPI profile, comprising four different active inhibitors, of which the most abundant proved to be novel, to understand their functional specialization in an ecological context. Transcript and activity characterization revealed tissue-specific and insect-elicited accumulation patterns. Stable and transient gene silencing of all four SPIs revealed different specificities for target proteinases: the novel SPI2c displayed high specificity for trypsin and chymotrypsin, while two other SPI2 homologs were highly active against subtilisin. In field and lab experiments, we found all four SPIs to display herbivore- and gene-specific defensive properties, with dissimilar effects on closely related species. However, we did not observe any clear developmental phenotype in SPI-silenced plants, suggesting that SPIs do not play a major role in regulating endogenous proteases under the conditions studied. In summary, specific single SPIs or their combinations defend S. nigrum against generalist herbivores, while the defense against herbivores specialized on SPI-rich diets requires other unknown defense mechanisms.

The Arabidopsis Class II Sirtuin Is a Lysine Deacetylase and Interacts with Mitochondrial Energy Metabolism

The mitochondrial deacetylase targets protein complexes involved in energy metabolism and regulates the activity of the ATP/ADP carrier. The posttranslational regulation of proteins by lysine (Lys) acetylation has recently emerged to occur not only on histones, but also on organellar proteins in plants and animals. In particular, the catalytic activities of metabolic enzymes have been shown to be regulated by Lys acetylation. The Arabidopsis (Arabidopsis thaliana) genome encodes two predicted sirtuin-type Lys deacetylases, of which only Silent Information Regulator2 homolog (SRT2) contains a predicted presequence for mitochondrial targeting. Here, we have investigated the function of SRT2 in Arabidopsis. We demonstrate that SRT2 functions as a Lys deacetylase in vitro and in vivo. We show that SRT2 resides predominantly at the inner mitochondrial membrane and interacts with a small number of protein complexes mainly involved in energy metabolism and metabolite transport. Several of these protein complexes, such as the ATP synthase and the ATP/ADP carriers, show an increase in Lys acetylation in srt2 loss-of-function mutants. The srt2 plants display no growth phenotype but rather a metabolic phenotype with altered levels in sugars, amino acids, and ADP contents. Furthermore, coupling of respiration to ATP synthesis is decreased in these lines, while the ADP uptake into mitochondria is significantly increased. Our results indicate that SRT2 is important in fine-tuning mitochondrial energy metabolism.

The mitochondrial lysine acetylome of Arabidopsis

Posttranslational modifications are essential regulators of protein functions as they can modify enzyme activities or protein-molecule interactions by changing the charge state or chemical properties of their target amino acid. The acetyl moiety of the central energy metabolite acetyl-CoA can be transferred to the ε-amino group of lysine, a process known as lysine acetylation which is implicated in the regulation of key metabolic enzymes in various organisms. Since plant mitochondria are of great importance for plant growth and development and as they house key enzymes of oxidative phosphorylation and photorespiration, it is essential to investigate the occurrence of lysine acetylation in this organelle. Here we characterised the plant mitochondrial acetylome of Arabidopsis mitochondria by LC-MS/MS analysis. In total 120 lysine-acetylated mitochondrial proteins containing 243 acetylated sites were identified. These proteins were mapped into functional categories showing that many proteins with essential functions from the tricaboxylic cycle and the respiratory chain are lysine-acetylated, as well as proteins involved in photorespiration, amino acid and protein metabolism, and redox regulation. Immuno-detection of mitochondrial proteins revealed that many lysine-acetylated proteins reside in native protein complexes. Furthermore, in vitro experiments demonstrated that lysine acetylation can occur non-enzymatically in Arabidopsis mitochondria at physiological matrix pH.

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic (TCA) cycle of mitochondria of all organisms, as it catalyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intra- as well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.

Jasmonoyl-l-Isoleucine Coordinates Metabolic Networks Required for Anthesis and Floral Attractant Emission in Wild Tobacco (Nicotiana attenuata)

This study demonstrates that jasmonoyl-l-isoleucine synchronizes the corolla limb-opening process and metabolic pathways for the modulation of floral turgidity with the CORONATINE INSENSITIVE1-based expression of floral attractants (benzylacetone) and rewards (nectar production). Jasmonic acid and its derivatives (jasmonates [JAs]) play central roles in floral development and maturation. The binding of jasmonoyl-l-isoleucine (JA-Ile) to the F-box of CORONATINE INSENSITIVE1 (COI1) is required for many JA-dependent physiological responses, but its role in anthesis and pollinator attraction traits remains largely unexplored. Here, we used the wild tobacco Nicotiana attenuata, which develops sympetalous flowers with complex pollination biology, to examine the coordinating function of JA homeostasis in the distinct metabolic processes that underlie flower maturation, opening, and advertisement to pollinators. From combined transcriptomic, targeted metabolic, and allometric analyses of transgenic N. attenuata plants for which signaling deficiencies were complemented with methyl jasmonate, JA-Ile, and its functional homolog, coronatine (COR), we demonstrate that (1) JA-Ile/COR-based signaling regulates corolla limb opening and a JA-negative feedback loop; (2) production of floral volatiles (night emissions of benzylacetone) and nectar requires JA-Ile/COR perception through COI1; and (3) limb expansion involves JA-Ile-induced changes in limb fresh mass and carbohydrate metabolism. These findings demonstrate a master regulatory function of the JA-Ile/COI1 duet for the main function of a sympetalous corolla, that of advertising for and rewarding pollinator services. Flower opening, by contrast, requires JA-Ile signaling-dependent changes in primary metabolism, which are not compromised in the COI1-silenced RNA interference line used in this study.

The multiple functions of plant serine protease inhibitors: defense against herbivores and beyond.

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development.

Plant mitochondrial retrograde signaling: post-translational modifications enter the stage

Beside their central function in respiration plant mitochondria play important roles in diverse processes such as redox homeostasis, provision of precursor molecules for essential biosynthetic pathways, and programmed cell death. These different functions require the organelle to communicate with the rest of the cell by perceiving, transducing, and emitting signals. As the vast majority of mitochondrial proteins are encoded in the nuclear genome, changes in mitochondrial status must be fed back to the nucleus to coordinate gene expression accordingly, a process termed retrograde signaling. However, the nature of these signaling pathways in plants and their underlying signaling molecules – or indirect metabolite or redox signals – are not completely resolved. We explore the potential of different post-translational modifications (PTMs) to contribute to mitochondrial retrograde signaling. Remarkably, the substrates used for modifying proteins in many major PTMs are either central metabolites or redox-active compounds, as for example ATP, acetyl-CoA, NAD+, and glutathione. This suggests that the metabolic status of organelles and of the cell in general could be indirectly gaged by the enzymes catalyzing the various PTMs. We examine the evidence supporting this hypothesis with regard to three major PTMs, namely phosphorylation, lysine acetylation, and glutathionylation and assess their potential to regulate not only organellar processes by modifying metabolic enzymes but also to influence nuclear gene expression.

Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis

Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome‐wide signatures of the RPD3/HDA1 class of histone deacetylases in Arabidopsis. Relative quantification of the changes in the lysine acetylation levels was determined on a proteome‐wide scale after treatment of Arabidopsis leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1‐like histone deacetylases in Arabidopsis, of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar‐localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss‐of‐function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low‐light conditions.

The use of VIGS technology to study plant-herbivore interactions

Plants employ a large variety of defense strategies to resist herbivores, which require transcriptional reprogramming of cells and profound changes in plant metabolism. Due to the large number of genes involved in defense processes, rapid screening strategies are essential for elucidating the contributions of individual genes in the responses of plants to herbivory. However, databases and seed banks of mutant plants which allow rapid retrieval of mutant genotypes are limited to a few model plant species, namely, Arabidopsis thaliana and Oryza sativa (rice). In other plants, virus-induced gene silencing (VIGS) offers an efficient alternative for screening the functions of individual genes in order to prioritize the allocations of the large time investments required to establish stably transformed RNAi-silenced lines. With VIGS, it is usually possible to achieve strong, specific silencing of target genes in the ecological models Nicotiana attenuata and Solanum nigrum, allowing the rapid assessment of gene silencing effects on phytohormone accumulation, signal transduction and accumulation of defense metabolites. VIGS plants are also useful in bioassays with specialist and generalist herbivores, allowing direct verification of gene function in plant resistance to herbivores.