Publikation
Anwer, M. U.; Davis, A.; Davis, S. J.; Quint, M.; Photoperiod sensing of the circadian clock is controlled by EARLY FLOWERING 3 and GIGANTEA Plant J. 101, 1397-1410, (2020) DOI: 10.1111/tpj.14604
ELF3 and GI are two important components of the Arabidopsis circadian clock. They are not only essential for the oscillator function but are also pivotal in mediating light inputs to the oscillator. Lack of either results in a defective oscillator causing severely compromised output pathways, such as photoperiodic flowering and hypocotyl elongation. Although single loss of function mutants of ELF3 and GI have been well‐studied, their genetic interaction remains unclear. We generated an elf3 gi double mutant to study their genetic relationship in clock‐controlled growth and phase transition phenotypes. We found that ELF3 and GI repress growth differentially during the night and the day, respectively. Circadian clock assays revealed that ELF3 and GI are essential Zeitnehmers that enable the oscillator to synchronize the endogenous cellular mechanisms to external environmental signals. In their absence, the circadian oscillator fails to synchronize to the light‐dark cycles even under diurnal conditions. Consequently, clock‐mediated photoperiod‐responsive growth and development are completely lost in plants lacking both genes, suggesting that ELF3 and GI together convey photoperiod sensing to the central oscillator. Since ELF3 and GI are conserved across flowering plants and represent important breeding and domestication targets, our data highlight the possibility of developing photoperiod‐insensitive crops by adjusting the allelic combination of these two key genes.
Publikation
Grubb, C. D.; Zipp, B. J.; Kopycki, J.; Schubert, M.; Quint, M.; Lim, E.-K.; Bowles, D. J.; Pedras, M. S. C.; Abel, S.; Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis Plant J. 79, 92-105, (2014) DOI: 10.1111/tpj.12541
The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S‐glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP‐glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1‐2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge.
Publikation
Brandt, R.; Salla-Martret, M.; Bou-Torrent, J.; Musielak, T.; Stahl, M.; Lanz, C.; Ott, F.; Schmid, M.; Greb, T.; Schwarz, M.; Choi, S.-B.; Barton, M. K.; Reinhart, B. J.; Liu, T.; Quint, M.; Palauqui, J.-C.; Martínez-García, J. F.; Wenkel, S.; Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses Plant J. 72, 31-42, (2012) DOI: 10.1111/j.1365-313X.2012.05049.x
Unlike the situation in animals, the final morphology of the plant body is highly modulated by the environment. During Arabidopsis development, intrinsic factors provide the framework for basic patterning processes. CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD‐ZIPIII) transcription factors are involved in embryo, shoot and root patterning. During vegetative growth HD‐ZIPIII proteins control several polarity set‐up processes such as in leaves and the vascular system. We have identified several direct target genes of the HD‐ZIPIII transcription factor REVOLUTA (REV) using a chromatin immunoprecipitation/DNA sequencing (ChIP‐Seq) approach. This analysis revealed that REV acts upstream of auxin biosynthesis and affects directly the expression of several class II HD‐ZIP transcription factors that have been shown to act in the shade‐avoidance response pathway. We show that, as well as involvement in basic patterning, HD‐ZIPIII transcription factors have a critical role in the control of the elongation growth that is induced when plants experience shade. Leaf polarity is established by the opposed actions of HD‐ZIPIII and KANADI transcription factors. Finally, our study reveals that the module that consists of HD‐ZIPIII/KANADI transcription factors controls shade growth antagonistically and that this antagonism is manifested in the opposed regulation of shared target genes.
Publikation
Sreenivasulu, N.; Radchuk, V.; Alawady, A.; Borisjuk, L.; Weier, D.; Staroske, N.; Fuchs, J.; Miersch, O.; Strickert, M.; Usadel, B.; Wobus, U.; Grimm, B.; Weber, H.; Weschke, W.; De-regulation of abscisic acid contents causes abnormal endosperm development in the barley mutant seg8 Plant J. 64, 589-603, (2010) DOI: 10.1111/j.1365-313X.2010.04350.x
Grain development of the maternal effect shrunken endosperm mutant seg8 was analysed by comprehensive molecular, biochemical and histological methods. The most obvious finding was de‐regulation of ABA levels, which were lower compared to wild‐type during the pre‐storage phase but higher during the transition from cell division/differentiation to accumulation of storage products. Ploidy levels and ABA amounts were inversely correlated in the developing endosperms of both mutant and wild‐type, suggesting an influence of ABA on cell‐cycle regulation. The low ABA levels found in seg8 grains between anthesis and beginning endosperm cellularization may result from a gene dosage effect in the syncytial endosperm that causes impaired transfer of ABA synthesized in vegetative tissues into filial grain parts. Increased ABA levels during the transition phase are accompanied by higher chlorophyll and carotenoid/xanthophyll contents. The data suggest a disturbed ABA‐releasing biosynthetic pathway. This is indicated by up‐regulation of expression of the geranylgeranyl reductase (GGR) gene, which may be induced by ABA deficiency during the pre‐storage phase. Abnormal cellularization/differentiation of the developing seg8 endosperm and reduced accumulation of starch are phenotypic characteristics that reflect these disturbances. The present study did not reveal the primary gene defect causing the seg8 phenotype, but presents new insights into the maternal/filial relationships regulating barley endosperm development.
Publikation
Halim, V. A.; Altmann, S.; Ellinger, D.; Eschen-Lippold, L.; Miersch, O.; Scheel, D.; Rosahl, S.; PAMP-induced defense responses in potato require both salicylic acid and jasmonic acid Plant J. 57, 230-242, (2009) DOI: 10.1111/j.1365-313X.2008.03688.x
To elucidate the molecular mechanisms underlying pathogen‐associated molecular pattern (PAMP)‐induced defense responses in potato (Solanum tuberosum ), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep‐13, a PAMP from Phytophthora , induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive‐like cell death. We have previously shown that SA is required for Pep‐13‐induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12‐oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F‐box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep‐13 treatment, neither did they contain significant amounts of JA after elicitation. In response to infiltration of Pep‐13, the transgenic plants exhibited a highly reduced accumulation of reactive oxygen species as well as reduced hypersensitive cell death. The ability of the JA‐deficient plants to accumulate SA suggests that SA accumulation is independent or upstream of JA accumulation. These data show that PAMP responses in potato require both SA and JA and that, in contrast to Arabidopsis, these compounds act in the same signal transduction pathway. Despite their inability to fully respond to PAMP treatment, the transgenic RNA interference plants are not altered in their basal defense against Phytophthora infestans .
Publikation
Ziegler, J.; Voigtländer, S.; Schmidt, J.; Kramell, R.; Miersch, O.; Ammer, C.; Gesell, A.; Kutchan, T. M.; Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis Plant J. 48, 177-192, (2006) DOI: 10.1111/j.1365-313X.2006.02860.x
Plants of the order Ranunculales, especially members of the species Papaver , accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum ) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum , 69 show increased expression in morphinan alkaloid‐containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7‐epi ‐salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo‐keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.
Publikation
Sreenivasulu, N.; Radchuk, V.; Strickert, M.; Miersch, O.; Weschke, W.; Wobus, U.; Gene expression patterns reveal tissue-specific signaling networks controlling programmed cell death and ABA- regulated maturation in developing barley seeds Plant J. 47, 310-327, (2006) DOI: 10.1111/j.1365-313X.2006.02789.x
Gene expression patterns covering over 10 000 seed‐expressed sequences were analyzed by macroarray technology in maternal tissue (mainly pericarp) and filial endosperm and embryo during barley seed development from anthesis until late maturation. Defined sets of genes showing distinct expression patterns characterized both tissue type and major developmental phases. The analysis focused on regulatory networks involved in programmed cell death (PCD) and abscisic acid (ABA)‐mediated maturation. These processes were similar in the different tissues, but typically involved the expression of alternative members of a common gene family. The analysis of co‐expressed gene sets and the identification of cis regulatory elements in orthologous rice gene ‘promoter’ regions suggest that PCD in the pericarp is mediated by distinct classes of proteases and is under the hormonal control of both jasmonic acid (JA) and ethylene via ethylene‐responsive element binding protein (EREBP) transcription factors (TFs). On the other hand, PCD in endosperm apparently involves only the ethylene pathway, but employs distinct gene family members from those active in the pericarp, and a different set of proteases and TFs. JA biosynthetic genes are hardly activated. Accordingly, JA levels are high in the pericarp but low in the endosperm during middle and late developmental stages. Similarly, genes acting in the deduced ABA biosynthetic pathway and signaling network differ between endosperm and embryo. ABA in the endosperm appears to exert an influence over storage product synthesis via SNF1 kinase. In the embryo, ABA seems to influence the acquisition of desiccation tolerance via ABA response element binding factors, but the data also suggest the existence of an ABA‐independent but interactive pathway acting via the dehydration‐responsive element binding (DREB) 2A TF.
Publikation
Danon, A.; Miersch, O.; Felix, G.; op den Camp, R. G. L.; Apel, K.; Concurrent activation of cell death-regulating signaling pathways by singlet oxygen in Arabidopsis thaliana Plant J. 41, 68-80, (2005) DOI: 10.1111/j.1365-313X.2004.02276.x
Upon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen (1O2), a non‐radical reactive oxygen species that is restricted to the plastid compartment. Immediately after the shift, plants stop growing and develop necrotic lesions. We have established a protoplast system, which allows detection and characterization of the death response in flu induced by the release of 1O2. Vitamin B6 that quenches 1O2 in fungi was able to protect flu protoplasts from cell death. Blocking ethylene production was sufficient to partially inhibit the death reaction. Similarly, flu mutant seedlings expressing transgenic NahG were partially protected from the death provoked by the release of 1O2, indicating a requirement for salicylic acid (SA) in this process, whereas in cells depleted of both, ethylene and SA, the extent of cell death was reduced to the wild‐type level. The flu mutant was also crossed with the jasmonic acid (JA)‐depleted mutant opr3 , and with the JA, OPDA and dinor OPDA (dnOPDA)‐depleted dde2‐2 mutant. Analysis of the resulting double mutants revealed that in contrast to the JA‐induced suppression of H2O2/superoxide‐dependent cell death reported earlier, JA promotes singlet oxygen‐mediated cell death in flu , whereas other oxylipins such as OPDA and dnOPDA antagonize this death‐inducing activity of JA.
Publikation
Quint, M.; Ito, H.; Zhang, W.; Gray, W. M.; Characterization of a novel temperature-sensitive allele of the CUL1/AXR6 subunit of SCF ubiquitin-ligases Plant J. 43, 371-383, (2005) DOI: 10.1111/j.1365-313X.2005.02449.x
Selective protein degradation by the ubiquitin‐proteasome pathway has emerged as a key regulatory mechanism in a wide variety of cellular processes. The selective components of this pathway are the E3 ubiquitin‐ligases which act downstream of the ubiquitin‐activating and ‐conjugating enzymes to identify specific substrates for ubiquitinylation. SCF‐type ubiquitin‐ligases are the most abundant class of E3 enzymes in Arabidopsis. In a genetic screen for enhancers of the tir1‐1 auxin response defect, we identified eta1 /axr6‐3 , a recessive and temperature‐sensitive mutation in the CUL1 core component of the SCFTIR1 complex. The axr6‐3 mutation interferes with Skp1 binding, thus preventing SCF complex assembly. axr6‐3 displays a pleiotropic phenotype with defects in numerous SCF‐regulated pathways including auxin signaling, jasmonate signaling, flower development, and photomorphogenesis. We used axr6‐3 as a tool for identifying pathways likely to be regulated by SCF‐mediated proteolysis and propose new roles for SCF regulation of the far‐red light/phyA and sugar signaling pathways. The recessive inheritance and the temperature‐sensitive nature of the pleiotropically acting axr6‐3 mutation opens promising possibilities for the identification and investigation of SCF‐regulated pathways in Arabidopsis.
Publikation
Stenzel, I.; Hause, B.; Maucher, H.; Pitzschke, A.; Miersch, O.; Ziegler, J.; Ryan, C. A.; Wasternack, C.; Allene oxide cyclase dependence of the wound response and vascular bundle-specific generation of jasmonates in tomato - amplification in wound signalling Plant J. 33, 577-589, (2003) DOI: 10.1046/j.1365-313X.2003.01647.x
The allene oxide cyclase (AOC)‐catalyzed step in jasmonate (JA) biosynthesis is important in the wound response of tomato. As shown by treatments with systemin and its inactive analog, and by analysis of 35S::prosysteminsense and 35S::prosysteminantisense plants, the AOC seems to be activated by systemin (and JA) leading to elevated formation of JA. Data are presented on the local wound response following activation of AOC and generation of JA, both in vascular bundles. The tissue‐specific occurrence of AOC protein and generation of JA is kept upon wounding or other stresses, but is compromised in 35S::AOCsense plants, whereas 35S::AOCantisense plants exhibited residual AOC expression, a less than 10% rise in JA, and no detectable expression of wound response genes. The (i) activation of systemin‐dependent AOC and JA biosynthesis occurring only upon substrate generation, (ii) the tissue‐specific occurrence of AOC in vascular bundles, where the prosystemin gene is expressed, and (iii) the tissue‐specific generation of JA suggest an amplification in the wound response of tomato leaves allowing local and rapid defense responses.