Temporal pattern of jasmonate-induced alterations in gene expression of barley leaves

Leaf tissues of barley (Hordeum vulgare L. cv. Salome) respond to methyl jasmonate (JaMe) treatment with a characteristic pattern of gene expression. Jasmonate-induced proteins (JIPs), such as leaf thionins (jip15 gene product) and ribosome-inactivating proteins (jip60 gene product), rapidly accumulate. Their genes are transiently transcriptionally activated, as shown here by the determination of in-vitro transcription rates in run-off assays. In contrast to jip genes, expression of photosynthetic genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS gene product) and a type III light-harvesting chlorophyll-a/b-binding protein (LHCP; lhbC1 gene product), for example, was rapidly down-regulated in JaMe-treated barley leaves. Despite decreasing rates of rbcS and lhbC1 gene transcription, their transcripts were maintained in JaMe-treated leaf tissues for at least 36 h. Only at a later stage, was there a decline in the levels of rbcS and lhbC1, but not jip, transcripts, suggesting a selective destabilization of photosynthetic mRNAs in JaMe-treated leaf tissue

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:proto¬chlorophyllide oxidoreductase (POR) A

NADPH: protochlorophyllide oxido reductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Amongthem are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radio labeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of35S-Oep16/Ptc16 is stimulated by GTP.35S-Oep16/Ptc16forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photo oxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import

An Ethylene-Protected Achilles' Heel of Etiolated Seedlings for Arthropod Deterrence

A small family of Kunitz protease inhibitors exists in Arabidopsis thaliana, a member of which (encoded by At1g72290) accomplishes highly specific roles during plant development. Arabidopsis Kunitz-protease inhibitor 1 (Kunitz-PI;1), as we dubbed this protein here, is operative as cysteine PI. Activity measurements revealed that despite the presence of the conserved Kunitz-motif the bacterially expressed Kunitz-PI;1 was unable to inhibit serine proteases such as trypsin and chymotrypsin, but very efficiently inhibited the cysteine protease RESPONSIVE TO DESICCATION 21. Western blotting and cytolocalization studies using mono-specific antibodies recalled Kunitz-PI;1 protein expression in flowers, young siliques and etiolated seedlings. In dark-grown seedlings, maximum Kunitz-PI;1 promoter activity was detected in the apical hook region and apical parts of the hypocotyls. Immunolocalization confirmed Kunitz-PI;1 expression in these organs and tissues. No transmitting tract (NTT) and HECATE 1 (HEC1), two transcription factors previously implicated in the formation of the female reproductive tract in flowers of Arabidopsis, were identified to regulate Kunitz-PI;1 expression in the dark and during greening, with NTT acting negatively and HEC1 acting positively. Laboratory feeding experiments with isopod crustaceans such as Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug) pinpointed the apical hook as ethylene-protected Achilles? heel of etiolated seedlings. Because exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and mechanical stress (wounding) strongly up-regulated HEC1-dependent Kunitz-PI;1 gene expression, our results identify a new circuit controlling herbivore deterrence of etiolated plants in which Kunitz-PI;1 is involved

The Outer Chloroplast Envelope Protein OEP16-1 for Plastid Import of NADPH:Protochlorophyllide Oxidoreductase A in Arabidopsis thaliana

The outer plastid envelope protein OEP16-1 was previously identified as an amino acid-selective channel protein and translocation pore for NADPH:protochlorophyllide oxidoreductase A (PORA). Reverse genetic approaches used to dissect these mutually not exclusive functions of OEP16-1 in planta have led to descriptions of different phenotypes resulting from the presence of several mutant lines in the SALK_024018 seed stock. In addition to the T-DNA insertion in the AtOEP16-1 gene, lines were purified that contain two additional T-DNA insertions and as yet unidentified point mutations. In a first attempt to resolve the genetic basis of four different lines in the SALK_024018 seed stock, we used genetic transformation with the OEP16-1 cDNA and segregation analyses after crossing out presumed point mutations. We show that AtOEP16-1 is involved in PORA precursor import and by virtue of this activity confers photoprotection onto etiolated seedlings during greenin

Implication of the oep16-1 mutation in a flu-independent, singlet oxygen-regulated cell death pathway in Arabidopsis thaliana

Singlet oxygen is a prominent form of reactive oxygen species in higher plants. It is easily formed from molecular oxygen by triplet–triplet interchange with excited porphyrin species. Evidence has been obtained from studies on the flu mutant of Arabidopsis thaliana of a genetically determined cell death pathway that involves differential changes at the transcriptome level. Here we report on a different cell death pathway that can be deduced from the analysis of oep16 mutants of A. thaliana. Pure lines of four independent OEP16-deficient mutants with different cell death properties were isolated. Two of the mutants overproduced free protochlorophyllide (Pchlide) in the dark because of defects in import of NADPH:Pchlide oxidoreductase A (pPORA) and died after illumination. The other two mutants avoided excess Pchlide accumulation. Using pulse labeling and polysome profiling studies we show that translation is a major site of cell death regulation in flu and oep16 plants. flu plants respond to photooxidative stress triggered by singlet oxygen by reprogramming their translation toward synthesis of key enzymes involved in jasmonic acid synthesis and stress proteins. In contrast, those oep16 mutants that were prone to photooxidative damage were unable to respond in this way. Together, our results show that translation is differentially affected in the flu and oep16 mutants in response to singlet oxygen

Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX)

Mammals including humans use highly specific pathways for tissue differentiation. One such pathway is operative in reticulocytes and involves the programmed destruction of the cell?s organellar complement by 15-lipoxygenase (15-LOX), which oxygenates polyunsaturated membrane fatty acids and provokes organelle leakage. As we report here, plants make use of a similar LOX pathway to degrade their chloroplasts during leaf senescence. The enzyme involved is a 13-LOX with unique positional specificity and molecular terms. Because 15-LOX and 13-LOX pathway products likewise operate in biological defense, a mechanism of cross-kingdom conservation of pathway regulation and function was uncovered for multicellular eukaryotes

Analyse fonctionnelle de la protéine WSCP chez Arabidopsis thaliana

Les protéines WSCP (Water-soluble {chlorophyll binding proteins) sont des protéines solubles capables de fixer la chlorophylle et ses dérivés chez les Brassicaceae. Ces protéines font partie de la famille des inhibiteurs de protéases. Leurs fonctions in planta sont à ce jour très peu documentées. Cette thèse présente l'étude de la fonction physiologique d'une WSCP chez la plante modèle Arabidopsis thaliana. Nous avons montré dans un premier temps que la protéine WSCP est exprimée dans les plantules à l'obscurité (étiolées) et dans le tissu de transmission des fleurs, lieu de passage des tubes polliniques. Dans la fleur, nous avons mis en évidence la régulation de WSCP par les facteurs de transcriptions HEC et NTT impliqués dans le développement floral. Par une approche de génique inverse, nous avons montré un rôle de WSCP dans le contrôle de la mort cellulaire du tissu de transmission. En conséquence, la progression des tubes polliniques est altérée et la mort cellulaire non spécifique influence le développement des graines. Chez les plantules étiolées, le transcrit WSCP est exprimé au niveau de la crosse apicale, structure dont la fonction est de protéger les plantules au cours de l'émergence hors du sol. De manière intéressante, les facteurs NTT et HEC sont également exprimés dans les plantules étiolées mais régulent l'expression du gène WSCP de façon différente. De plus, la protéine WSCP s'accumule en présence de l'hormone de stress éthylène dans la crosse apicale et l'activité du promoteur du gène WSCP augmente en conditions de stress mécaniques. Prises ensembles, ces expériences laissent présager d'un rôle protecteur de la protéine WSCP au cours de la skotomorphogénèse.Class II WSCP proteins (Water-~oluble {;.hlorophyll binding froteins) are soluble proteins that interact with chlorophyll and its derivatives in Brassicaceae. These proteins belong to the protease inhibitor family and they are induced by abiotic stress under light conditions. To date, their in planta functions are not weil documented to date. The aim ofthis study is to characterize the physiological function of a WSCP protein in model plants Arabidopsis thaliana. We demonstrated initially that the WSCP protein is not induced under stress conditions during photomorphogenesis. However, the protein is expressed in dark grown (etiolated) seedlings as weil as in the flower transmitting tract, the path of pollen tubes growth. ln the flower, we have demonstrated a WSCP regulation by HEC and NTT transcription factors involved in floral development. By a reverse genetic approach, we have highlighted a regularory WSCP function in transmitting tract cell death. Consequently, the pollen tube growth is impaired and the resulting nonspecific cell death influence seed development. ln etiolated seedlings, WSCP transcript is expressed in the apical hook, a structure which protects seedlings during emergence trom soil. Interestingly, HEC and NTT transcription factors are also expressed in etiolated seedlings and regulate WSCP gene expression differently. Furthermore, WSCP protein accumulates in the presence of the stress hormone ethylene in apical hook and WSCP gene promoter activity increases during mechanical wounding. Taken together, these experiments suggest a Drotective role ofWSCP Droteins during skotomomhogenesis.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

In situ conversion of protochlorophyllide b to protochlorophyllide a in barley. Evidence for a novel role of 7-formyl reductase in the prolamellar body of etioplasts

We recently put forth a model of a protochlorophyllide (Pchlide) light-harvesting complex operative during angiosperm seedling de-etiolation (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999) Nature 397, 80–84). This model, which was based on in vitro reconstitution experiments with zinc analogs of Pchlide a and Pchlide b and the two NADPH:protochlorophyllide oxidoreductases (PORs), PORA and PORB, of barley, predicted a 5-fold excess of Pchlide b, relative to Pchlide a, in the prolamellar body of etioplasts. Recent work (Scheumann, V., Klement, H., Helfrich, M., Oster, U., Schoch, S., and Rüdiger, W. (1999) FEBS Lett. 445, 445–448), however, contradicted this model and reported that Pchlide b would not be present in etiolated plants. Here we demonstrate that Pchlide b is an abundant pigment in barley etioplasts but is rather metabolically unstable. It is rapidly converted to Pchlide a by virtue of 7-formyl reductase activity, an enzyme that had previously been implicated in the chlorophyll (Chl) b to Chl a reaction cycle. Our findings suggest that etiolated plants make use of 7-formyl reductase to fine tune the levels of Pchlide b and Pchlidea and thereby may regulate the steady-state level of light-harvesting POR-Pchlide compl