Karin Groten

Redox regulation of peroxiredoxin and proteinases by ascorbate and thiols during pea root nodule senescence

Redox factors contributing to nodule senescence were studied in pea. The abundance of the nodule cytosolic peroxiredoxin but not the mitochondrial peroxiredoxin protein was modulated by ascorbate. In contrast to redox‐active antioxidants such as ascorbate and cytosolic peroxiredoxin that decreased during nodule development, maximal extractable nodule proteinase activity increased progressively as the nodules aged. Cathepsin‐like activities were constant throughout development but serine and cysteine proteinase activities increased during senescence. Senescence‐induced cysteine proteinase activity was inhibited by cysteine, dithiotreitol, or E‐64. Senescence‐dependent decreases in redox‐active factors, particularly ascorbate and peroxiredoxin favour decreased redox‐mediated inactivation of cysteine proteinases.

Symbiosis between Nicotiana attenuata and Glomus intraradices: ethylene plays a role, jasmonic acid does not

Phytohormones are thought to mediate plant-arbuscular mycorrhizal (AM) interactions. To explore the role of phytohormones in the interaction between Nicotiana attenuata and Glomus intraradices, we analysed levels of jasmonic acid (JA) and its amino acid conjugate JA-isoleucine/JA-leucine (JA-Ile), salicylic acid (SA) and ethylene in either infected or non-infected N. attenuata wild-type (WT) plants growing in soils that mimic the nutrient supply rates found in the plants native environment. Under these conditions, the infection decreases plant growth and reproductive performance. Levels of JA, JA-Ile and SA did not change upon infection, but ethylene release was slightly decreased. Transgenic N. attenuata plants defective in JA signalling (aslox3 and ircoi1) did not differ significantly in growth or reproductive performance compared with infected WT. Furthermore, no difference in infection rates could be observed. Transgenic plants unable to produce (iraco) or perceive (etr1) ethylene showed significantly larger decreases in growth and number of seed capsules produced between infected and non-infected plants compared with WT plants. We conclude that ethylene, rather than JA, signalling plays a role in the interaction between N. attenuata and AM, from which the plant does not realize a fitness benefit.

Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris.

Mitochondrial electron transport pathways exert effects on carbon–nitrogen (C/N) relationships. To examine whether mitochondria–N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered ‘nitrate-sensing’ phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.

Quantification of growth–defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹⁵N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹⁵N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.

Nicotiana tabacum agglutinin is active against Lepidopteran pest insects

A jasmonate-inducible lectin called Nicotiana tabacum agglutinin or NICTABA was found in tobacco (Nicotiana tabacum cv Samsun) leaves. Since NICTABA expression is also induced after insect herbivory, a role in the defence response of tobacco was suggested. In this report, a detailed analysis was made of the entomotoxic properties of NICTABA using different transgenic approaches. First, purified NICTABA was shown to be strongly resistant to proteolytic degradation by enzymes present in the Lepidopteran midgut. To address the question of whether NICTABA is also active against Lepidopteran larvae, transgenic N. tabacum plants that silence endogenous NICTABA expression were constructed using RNA interference. Feeding experiments with these transgenic N. tabacum plants demonstrated that silencing of NICTABA expression enhances the larval performance of the generalist pest insect Spodoptera littoralis. In a second transgenic approach, NICTABA was ectopically expressed in the wild diploid tobacco Nicotiana attenuata, a species that lacks a functional NICTABA gene. When these transgenic N. attenuata plants were used in feeding experiments with S. littoralis larvae, a clear reduction in mass gain and significantly slower development were observed. In addition, feeding experiments with the Solanaceae specialist, Manduca sexta, provided further evidence that NICTABA exerts clear entomotoxic effects on Lepidopteran larvae.

Analysis of plant-bacteria interactions in their native habitat: bacterial communities associated with wild tobacco are independent of endogenous jasmonic acid levels and developmental stages

Jasmonic acid (JA) mediates defense responses against herbivores and necrotrophic pathogens but does it influence the recruitment of bacterial communities in the field? We conducted field and laboratory experiments with transformed Nicotiana attenuata plants deficient in jasmonate biosynthesis (irAOC) and empty vector controls (EV) to answer this question. Using both culture-dependent and independent techniques, we characterized root and leaf-associated bacterial communities over five developmental stages, from rosette through flowering of plants grown in their natural habitat. Based on the pyrosequencing results, alpha and beta diversity did not differ among EV and irAOC plants or over ontogeny, but some genera were more abundant in one of the genotypes. Furthermore, bacterial communities were significantly different among leaves and roots. Taxa isolated only from one or both plant genotypes and hence classified as ‘specialists’ and ‘generalists’ were used in laboratory tests to further evaluate the patterns observed from the field. The putative specialist taxa did not preferentially colonize the jasmonate-deficient genotype, or alter the plants elicited phytohormone signaling. We conclude that in N. attenuata, JA signaling does not have a major effect on structuring the bacterial communities and infer that colonization of plant tissues is mainly shaped by the local soil community in which the plant grows.

Silencing a key gene of the common symbiosis pathway in Nicotiana attenuata specifically impairs arbuscular mycorrhizal infection without influencing the root‐associated microbiome or plant growth

While the biochemical function of calcium and calmodulin-dependent protein kinase (CCaMK) is well studied, and plants impaired in the expression of CCaMK are known not to be infected by arbuscular mycorrhizal fungi (AMF) in glasshouse studies, the whole-plant and ecological consequences of CCaMK silencing are not well understood. Here we show that three independently transformed lines of Nicotiana attenuata plants silenced in CCaMK (irCCaMK) are neither infected by Rhizophagus irregularis in the glasshouse nor by native fungal inoculum in the field. The overall fungal community of field-grown roots did not differ significantly among empty vector (EV) and the transgenic lines, and the bacterial communities only showed minor differences, as revealed by the alpha-diversity parameters of bacterial OTUs, which were higher in EV plants compared with two of the three transformed lines, while beta-diversity parameters did not differ. Furthermore, growth and fitness parameters were similar in the glasshouse and field. Herbivory-inducible and basal levels of salicylic acid, jasmonic acid and abscisic acid did not differ among the genotypes, suggesting that activation of the classical defence pathways are not affected by CCaMK silencing. Based on these results, we conclude that silencing of CCaMK has few, if any, non-target effects.

Determination of 15N-incorporation into plant proteins and their absolute quantitation: a new tool to study nitrogen flux dynamics and protein pool sizes elicited by plant-herbivore interactions

Herbivory leads to changes in the allocation of nitrogen among different pools and tissues; however, a detailed quantitative analysis of these changes has been lacking. Here, we demonstrate that a mass spectrometric data-independent acquisition approach known as LC-MS(E), combined with a novel algorithm to quantify heavy atom enrichment in peptides, is able to quantify elicited changes in protein amounts and (15)N flux in a high throughput manner. The reliable identification/quantitation of rabbit phosphorylase b protein spiked into leaf protein extract was achieved. The linear dynamic range, reproducibility of technical and biological replicates, and differences between measured and expected (15)N-incorporation into the small (SSU) and large (LSU) subunits of ribulose-1,5-bisphosphate-carboxylase/oxygenase (RuBisCO) and RuBisCO activase 2 (RCA2) of Nicotiana attenuata plants grown in hydroponic culture at different known concentrations of (15)N-labeled nitrate were used to further evaluate the procedure. The utility of the method for whole-plant studies in ecologically realistic contexts was demonstrated by using (15)N-pulse protocols on plants growing in soil under unknown (15)N-incorporation levels. Additionally, we quantified the amounts of lipoxygenase 2 (LOX2) protein, an enzyme important in antiherbivore defense responses, demonstrating that the approach allows for in-depth quantitative proteomics and (15)N flux analyses of the metabolic dynamics elicited during plant-herbivore interactions.

Specificity of root microbiomes in native-grown Nicotiana attenuata and plant responses to UVB increase Deinococcus colonization

Plants recruit microbial communities from the soil in which they germinate. Our understanding of the recruitment process and the factors affecting it is still limited for most microbial taxa. We analysed several factors potentially affecting root microbiome structure – the importance of geographic location of natural populations, the microbiome of native seeds as putative source of colonization and the effect of a plants response to UVB exposure on root colonization of highly abundant species. The microbiome of Nicotiana attenuata seeds was determined by a culture‐dependent and culture‐independent approach, and the root microbiome of natural N. attenuata populations from five different locations was analysed using 454‐pyrosequencing. To specifically address the influence of UVB light on root colonization by Deinococcus, a genus abundant and consistently present in N. attenuata roots, transgenic lines impaired in UVB perception (irUVR8) and response (irCHAL) were investigated in a microcosm experiment with/without UVB supplementation using a synthetic bacterial community. The seed microbiome analysis indicated that N. attenuata seeds are sterile. Alpha and beta diversities of native root bacterial communities differed significantly between soil and root, while location had only a significant effect on the fungal but not the bacterial root communities. With UVB supplementation, root colonization of Deinococcus increased in wild type, but decreased in irUVR8 and irCHAL plants compared to nontreated plants. Our results suggest that N. attenuata recruits a core root microbiome exclusively from soil, with fungal root colonization being less selective than bacterial colonization. Root colonization by Deinococcus depends on the plants response to UVB.

Catechol, a major component of smoke, influences primary root growth and root hair elongation through reactive oxygen species-mediated redox signaling

Nicotiana attenuata germinates from long-lived seedbanks in native soils after fires. Although smoke signals have been known to break seed dormancy, whether they also affect seedling establishment and root development remains unclear. In order to test this, seedlings were treated with smoke solutions. Seedlings responded in a dose-dependent manner with significantly increased primary root lengths, due mainly to longitudinal cell elongation, increased numbers of lateral roots and impaired root hair development. Bioassay-driven fractionations and NMR were used to identify catechol as the main active compound for the smoke-induced root phenotype. The transcriptome analysis revealed that mainly genes related to auxin biosynthesis and redox homeostasis were altered after catechol treatment. However, histochemical analyses of reactive oxygen species (ROS) and the inability of auxin applications to rescue the phenotype clearly indicated that highly localized changes in the roots redox-status, rather than in levels of auxin, are the primary effector. Moreover, H2 O2 application rescued the phenotype in a dose-dependent manner. Chemical cues in smoke not only initiate seed germination, but also influence seedling root growth; understanding how these cues work provides new insights into the molecular mechanisms by which plants adapt to post-fire environments.