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Publications - Cell and Metabolic Biology

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Darwish, E.; Ghosh, R.; Bentzer, J.; Tsardakas Renhuldt, N.; Proux‐Wera, E.; Kamal, N.; Spannagl, M.; Hause, B.; Sirijovski, N.; Van Aken, O.; The dynamics of touch‐responsive gene expression in cereals Plant J. (2023) DOI: 10.1111/tpj.16269

Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steerplant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of signifi-cant research interest, yet a molecular understanding of transcriptional responses to touch is largely absentin cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation ofwheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred 25 minafter touching, with most of the genes being upregulated. While most genes returned to basal expressionlevel by 1–2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat.Functional categories such as transcription factors, kinases, phytohormones, and Ca2+regulation wereaffected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosyn-thesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wallcomposition. Furthermore, several cereal-specific transcriptomic footprints were identified that were notobserved in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced sig-nalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and markergenes for further study of (a)biotic stress responses in cereals.

Ninck, S.; Halder, V.; Krahn, J. H.; Beisser, D.; Resch, S.; Dodds, I.; Scholtysik, R.; Bormann, J.; Sewald, L.; Gupta, M. D.; Heilmann, G.; Bhandari, D. D.; Morimoto, K.; Buscaill, P.; Hause, B.; van der Hoorn, R. A. L.; Kaschani, F.; Kaiser, M.; Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Arabidopsis Epoxide Hydrolase Related to Phytohormone Signaling ACS Chem. Biol. 18, 1076-1088, (2023) DOI: 10.1021/acschembio.2c00322

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in Arabidopsis thaliana that revealed Neratinib (Ner), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that Ner covalently modifies a surface-exposed cysteine residue of Arabidopsis epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition. Physiologically, the Ner application induces jasmonate metabolism in an AtEH7-dependent manner as an early response. In addition, it modulates PATHOGENESIS RELATED 1 (PR1) expression as a hallmark of SA signaling activation as a later effect. AtEH7, however, is not the exclusive target for this physiological readout induced by Ner. Although the underlying molecular mechanisms of AtEH7-dependent modulation of jasmonate signaling and Ner-induced PR1-dependent activation of SA signaling and thus defense response regulation remain unknown, our present work illustrates the powerful combination of forward chemical genetics and chemical proteomics for identifying novel phytohormone signaling modulatory factors. It also suggests that marginally explored metabolic enzymes such as epoxide hydrolases may have further physiological roles in modulating signaling.

Manh, M. B.; Ost, C.; Peiter, E.; Hause, B.; Krupinska, K.; Humbeck, K.; WHIRLY1 acts upstream of ABA-related reprogramming of drought-induced gene expression in Barley and affects stress-related histone modifications Int. J. Mol. Sci. 24, 6326, (2023) DOI: 10.3390/ijms24076326

WHIRLY1, a small plant-specific ssDNA-binding protein, dually located in chloroplasts and the nucleus, is discussed to act as a retrograde signal transmitting a stress signal from the chloroplast to the nucleus and triggering there a stress-related gene expression. In this work, we investigated the function of WHIRLY1 in the drought stress response of barley, employing two overexpression lines (oeW1-2 and oeW1-15). The overexpression of WHIRLY1 delayed the drought-stress-related onset of senescence in primary leaves. Two abscisic acid (ABA)-dependent marker genes of drought stress, HvNCED1 and HvS40, whose expression in the wild type was induced during drought treatment, were not induced in overexpression lines. In addition, a drought-related increase in ABA concentration in the leaves was suppressed in WHIRLY1 overexpression lines. To analyze the impact of the gain-of-function of WHIRLY1 on the drought-related reprogramming of nuclear gene expression, RNAseq was performed comparing the wild type and an overexpression line. Cluster analyses revealed a set of genes highly up-regulated in response to drought in the wild type but not in the WHIRLY1 overexpression lines. Among these genes were many stress- and abscisic acid (ABA)-related ones. Another cluster comprised genes up-regulated in the oeW1 lines compared to the wild type. These were related to primary metabolism, chloroplast function and growth. Our results indicate that WHIRLY1 acts as a hub, balancing trade-off between stress-related and developmental pathways. To test whether the gain-of-function of WHIRLY1 affects the epigenetic control of stress-related gene expression, we analyzed drought-related histone modifications in different regions of the promoter and at the transcriptional start sites of HvNCED1 and HvS40. Interestingly, the level of euchromatic marks (H3K4me3 and H3K9ac) was clearly decreased in both genes in a WHIRLY1 overexpression line. Our results indicate that WHIRLY1, which is discussed to act as a retrograde signal, affects the ABA-related reprogramming of nuclear gene expression during drought via differential histone modifications.

Israeli, A.; Schubert, R.; Man, N.; Teboul, N.; Serrani Yarce, J. C.; Rosowski, E. E.; Wu, M.-F.; Levy, M.; Efroni, I.; Ljung, K.; Hause, B.; Reed, J. W.; Ori, N.; Modulating auxin response stabilizes tomato fruit set Plant Physiol. 192, 2336-2355, (2023) DOI: 10.1093/plphys/kiad205

Fruit formation depends on successful fertilization and is highly sensitive to weather fluctuations that affect pollination. Auxin promotes fruit initiation and growth following fertilization. Class A auxin response factors (Class A ARFs) repress transcription in the absence of auxin and activate transcription in its presence. Here, we explore how multiple members of the ARF family regulate fruit set and fruit growth in tomato (Solanum lycopersicum) and Arabidopsis thaliana, and test whether reduction of SlARF activity improves yield stability in fluctuating temperatures. We found that several tomato Slarf mutant combinations produced seedless parthenocarpic fruits, most notably mutants deficient in SlARF8A and SlARF8B genes. Arabidopsis Atarf8 mutants deficient in the orthologous gene had less complete parthenocarpy than did tomato Slarf8a Slarf8b mutants. Conversely, Atarf6 Atarf8 double mutants had reduced fruit growth after fertilization. AtARF6 and AtARF8 likely switch from repression to activation of fruit growth in response to a fertilization-induced auxin increase in gynoecia. Tomato plants with reduced SlARF8A and SlARF8B gene dosage had substantially higher yield than the wild type under controlled or ambient hot and cold growth conditions. In field trials, partial reduction in the SlARF8 dose increased yield under extreme temperature with minimal pleiotropic effects. The stable yield of the mutant plants resulted from a combination of early onset of fruit set, more fruit-bearing branches and more flowers setting fruits. Thus, ARF8 proteins mediate the control of fruit set, and relieving this control with Slarf8 mutations may be utilized in breeding to increase yield stability in tomato and other crops.

Zeng, M.; Dam, N. M.; Hause, B.; MtEIN2 affects nitrate uptake and accumulation of photosynthetic pigments under phosphate and nitrate deficiency in Medicago truncatula Physiol. Plant. 175, e13899, (2023) DOI: 10.1111/ppl.13899

Ethylene (ET) controls many facets of plant growth and development under abiotic and biotic stresses. MtEIN2, as a critical element of the ET signaling pathway, is essential in biotic interactions. However, the role of MtEIN2 in responding to abiotic stress, such as combined nutrient deficiency, is less known. To assess the role of ethylene signaling in nutrient uptake, we manipulated nitrate (NO3−) and phosphate (Pi) availability for wild-type (WT) and the ethylene-insensitive (MtEIN2-defective) mutant, sickle, in Medicago truncatula. We measured leaf biomass and photosynthetic pigments in WT and sickle to identify conditions leading to different responses in both genotypes. Under combined NO3− and Pi deficiency, sickle plants had higher chlorophyll and carotenoid contents than WT plants. Under these conditions, nitrate content and gene expression levels of nitrate transporters were higher in the sickle mutant than in the WT. This led to the conclusion that MtEIN2 is associated with nitrate uptake and the content of photosynthetic pigments under combined Pi and NO3−deficiency in M. truncatula. We conclude that ethylene perception plays a critical role in regulating the nutrient status of plants.

Schindele, P.; Merker, L.; Schreiber, T.; Prange, A.; Tissier, A.; Puchta, H.; Enhancing gene editing and gene targeting efficiencies in Arabidopsis thaliana by using an intron‐containing version of ttLbCas12a Plant Biotechnol. J. 21, 457-459, (2023) DOI: 10.1111/pbi.13964


Cankar, K.; Hakkert, J. C.; Sevenier, R.; Papastolopoulou, C.; Schipper, B.; Baixinho, J. P.; Fernández, N.; Matos, M. S.; Serra, A. T.; Santos, C. N.; Vahabi, K.; Tissier, A.; Bundock, P.; Bosch, D.; Lactucin synthase inactivation boosts the accumulation of anti-inflammatory 8-deoxylactucin and its derivatives in Chicory (Cichorium intybus L.) J. Agr. Food Chem. 71, 6061-6072, (2023) DOI: 10.1021/acs.jafc.2c08959

For several sesquiterpene lactones (STLs) found in Asteraceae plants, very interesting biomedical activities have been demonstrated. Chicory roots accumulate the guaianolide STLs 8-deoxylactucin, lactucin, and lactucopicrin predominantly in oxalated forms in the latex. In this work, a supercritical fluid extract fraction of chicory STLs containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin was shown to have anti-inflammatory activity in an inflamed intestinal mucosa model. To increase the accumulation of these two compounds in chicory taproots, the lactucin synthase that takes 8-deoxylactucin as the substrate for the regiospecific hydroxylation to generate lactucin needs to be inactivated. Three candidate cytochrome P450 enzymes of the CYP71 clan were identified in chicory. Their targeted inactivation using the CRISPR/Cas9 approach identified CYP71DD33 to have lactucin synthase activity. The analysis of the terpene profile of the taproots of plants with edits in CYP71DD33 revealed a nearly complete elimination of the endogenous chicory STLs lactucin and lactucopicrin and their corresponding oxalates. Indeed, in the same lines, the interruption of biosynthesis resulted in a strong increase of 8-deoxylactucin and its derivatives. The enzyme activity of CYP71DD33 to convert 8-deoxylactucin to lactucin was additionally demonstrated in vitro using yeast microsome assays. The identified chicory lactucin synthase gene is predominantly expressed in the chicory latex, indicating that the late steps in the STL biosynthesis take place in the latex. This study contributes to further elucidation of the STL pathway in chicory and shows that root chicory can be positioned as a crop from which different health products can be extracted.

Ai, H.; Bellstaedt, J.; Bartusch, K. S.; Eschen‐Lippold, L.; Babben, S.; Balcke, G. U.; Tissier, A.; Hause, B.; Andersen, T. G.; Delker, C.; Quint, M.; Auxin‐dependent regulation of cell division rates governs root thermomorphogenesis EMBO J. 42, e111926, (2023) DOI: 10.15252/embj.2022111926

Roots are highly plastic organs enabling plants to adapt to a changing below-ground environment. In addition to abiotic factors like nutrients or mechanical resistance, plant roots also respond to temperature variation. Below the heat stress threshold, Arabidopsis thaliana seedlings react to elevated temperature by promoting primary root growth, possibly to reach deeper soil regions with potentially better water saturation. While above-ground thermomorphogenesis is enabled by thermo-sensitive cell elongation, it was unknown how temperature modulates root growth. We here show that roots are able to sense and respond to elevated temperature independently of shoot-derived signals. This response is mediated by a yet unknown root thermosensor that employs auxin as a messenger to relay temperature signals to the cell cycle. Growth promotion is achieved primarily by increasing cell division rates in the root apical meristem, depending on de novo local auxin biosynthesis and temperature-sensitive organization of the polar auxin transport system. Hence, the primary cellular target of elevated ambient temperature differs fundamentally between root and shoot tissues, while the messenger auxin remains the same.

Schreiber, T.; Tripathee, S.; Iwen, T.; Prange, A.; Vahabi, K.; Grützner, R.; Horn, C.; Marillonnet, S.; Tissier, A.; DNA double strand breaks lead to de novo transcription and translation of damage-induced long RNAs in planta bioRxiv (2022) DOI: 10.1101/2022.05.11.491484

DNA double strand breaks (DSBs) are lethal threats that need to be repaired. Although many of the proteins involved in the early steps of DSB repair have been characterized, recent reports indicate that damage induced long and small RNAs also play an important role in DSB repair. Here, using a Nicotiana benthamiana transgenic line originally designed as a reporter for targeted knock-ins, we show that DSBs generated by Cas9 induce the transcription of long stable RNAs (damage-induced long RNAs - dilRNAs) that are translated into proteins. Using an array of single guide RNAs we show that the initiation of transcription takes place in the vicinity of the DSB. Single strand DNA nicks are not able to induce transcription, showing that cis DNA damage-induced transcription is specific for DSBs. Our results support a model in which a default and early event in the processing of DSBs is transcription into RNA which, depending on the genomic and genic context, can undergo distinct fates, including translation into protein, degradation or production of small RNAs. Our results have general implications for understanding the role of transcription in the repair of DSBs and, reciprocally, reveal DSBs as yet another way to regulate gene expression.

Zeng, M.; Hause, B.; van Dam, N. M.; Uthe, H.; Hoffmann, P.; Krajinski, F.; Martínez‐Medina, A.; The mycorrhizal symbiosis alters the plant defence strategy in a model legume plant Plant Cell Environ. 45, 3412-3428, (2022) DOI: 10.1111/pce.14421

Arbuscular mycorrhizal (AM) symbiosis modulates plant‐herbivore interactions. Still, how it shapes the overall plant defence strategy and the mechanisms involved remain unclear. We investigated how AM symbiosis simultaneously modulates plant resistance and tolerance to a shoot herbivore, and explored the underlying mechanisms. Bioassays with Medicago truncatula plants were used to study the effect of the AM fungus Rhizophagus irregularis on plant resistance and tolerance to Spodoptera exigua herbivory. By performing molecular and chemical analyses, we assessed the impact of AM symbiosis on herbivore‐triggered phosphate (Pi)‐ and jasmonate (JA)‐related responses. Upon herbivory, AM symbiosis led to an increased leaf Pi content by boosting the mycorrhizal Pi‐uptake pathway. This enhanced both plant tolerance and herbivore performance. AM symbiosis counteracted the herbivore‐triggered JA burst, reducing plant resistance. To disentangle the role of the mycorrhizal Pi‐uptake pathway in the plant\'s response to herbivory, we used the mutant line ha1‐2, impaired in the H+‐ATPase gene HA1, which is essential for Pi‐uptake via the mycorrhizal pathway. We found that mycorrhiza‐triggered enhancement of herbivore performance was compromised in ha1‐2 plants. AM symbiosis thus affects the defence pattern of M. truncatula by altering resistance and tolerance simultaneously. We propose that the mycorrhizal Pi‐uptake pathway is involved in the modulation of the plant defence strategy.
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