Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes1–5. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. Upon JA-Ile perception, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated6,7. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL)8 and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel INteractor of JAZ (NINJA). NINJA acts as a transcriptional repressor of which the activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress- and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

SUMMARYTranscription factors of the plant-specific apetala2/ethylene response factor (AP2/ERF) family control plant secondary metabolism, often as part of signalling cascades induced by jasmonate (JA) or other elicitors. Here, we functionally characterized the JA-inducible tobacco (Nicotiana tabacum) AP2/ERF factor ORC1, one of the members of the NIC2-locus ERFs that control nicotine biosynthesis and a close homologue of ORCA3, a transcriptional activator of alkaloid biosynthesis in Catharanthus roseus. ORC1 positively regulated the transcription of several structural genes coding for the enzymes involved in nicotine biosynthesis. Accordingly, overexpression of ORC1 was sufficient to stimulate alkaloid biosynthesis in tobacco plants and tree tobacco (Nicotiana glauca) root cultures. In contrast to ORCA3 in C. roseus, which needs only the GCC motif in the promoters of the alkaloid synthesis genes to induce their expression, ORC1 required the presence of both GCC-motif and G-box elements in the promoters of the tobacco nicotine biosynthesis genes for maximum transactivation. Correspondingly, combined application with the JA-inducible Nicotiana basic helix-loop-helix (bHLH) factors that bind the G-box element in these promoters enhanced ORC1 action. Conversely, overaccumulation of JAZ repressor proteins that block bHLH activity reduced ORC1 functionality. Finally, the activity of both ORC1 and bHLH proteins was post-translationally upregulated by a JA-modulated phosphorylation cascade, in which a specific mitogen-activated protein kinase kinase, JA-factor stimulating MAPKK1 (JAM1), was identified. This study highlights the complexity of the molecular machinery involved in the regulation of tobacco alkaloid biosynthesis and provides mechanistic insights about its transcriptional regulators.

The phytohormones jasmonates (JAs) constitute an important class of elicitors for many plant secondary metabolic pathways. However, JAs do not act independently but operate in complex networks with crosstalk to several other phytohormonal signaling pathways. Here, crosstalk was detected between the JA and abscisic acid (ABA) signaling pathways in the regulation of tobacco (Nicotiana tabacum) alkaloid biosynthesis. A tobacco gene from the PYR/PYL/RCAR family, NtPYL4, the expression of which is regulated by JAs, was found to encode a functional ABA receptor. NtPYL4 inhibited the type-2C protein phosphatases known to be key negative regulators of ABA signaling in an ABA-dependent manner. Overexpression of NtPYL4 in tobacco hairy roots caused a reprogramming of the cellular metabolism that resulted in a decreased alkaloid accumulation and conferred ABA sensitivity to the production of alkaloids. In contrast, the alkaloid biosynthetic pathway was not responsive to ABA in control tobacco roots. Functional analysis of the Arabidopsis (Arabidopsis thaliana) homologs of NtPYL4, PYL4 and PYL5, indicated that also in Arabidopsis altered PYL expression affected the JA response, both in terms of biomass and anthocyanin production. These findings define a connection between a component of the core ABA signaling pathway and the JA responses and contribute to the understanding of the role of JAs in balancing tradeoffs between growth and defense.nicotine | phenylpropanoid | primary metabolism | secondary metabolism | stress response

SummaryAlthough sequence information and genome annotation are improving at an impressive pace, functional ontology is still non-existent or rudimentary for most genes. In this regard, transient expression assays are very valuable for identification of short functional segments in particular pathways, because they can be performed rapidly and at a scale unattainable in stably transformed tissues. Vectors were constructed and protocols developed for systematic transient assays in plant protoplasts. To enhance throughput and reproducibility, protoplast treatments were performed entirely by a liquid-handling robot in multiwell plates, including polyethylene glycol/Ca 2þ cell transfection with plasmid mixtures, washes and lysis. All transcriptional readouts were measured using a dual firefly/Renilla luciferase assay, in which the former was controlled by a reporter promoter and the latter by the 35S CaMV promoter, which served as internal normalization standard. The automated protocols were suitable for transient assays in protoplasts prepared from cell cultures of Nicotiana tabacum Bright Yellow-2 and Arabidopsis thaliana. They were implemented in a screen to discover potential regulators of genes coding for key enzymes in nicotine biosynthesis. Two novel tobacco transcription factors were found, NtORC1 and NtJAP1, that positively regulate the putrescine N-methyltransferase (PMT) promoter. In addition, combinatorial tests showed that these two factors act synergistically to induce PMT transcriptional activity. The development and use of high-throughput plant transient expression assays are discussed.

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In recent reports, granulocyte PRV-1 mRNA has been shown to be over-expressed in the majority of polycythemia vera (PV) patients and with a variable frequency in essential thrombocythemia (ET) patients. Here we evaluate PRV-1 mRNA quantification in the diagnosis of PV and ET. Following patients were included in the study: 23 PV patients; 5 secondary erythrocytosis (SE) cases, 4 by lung disease and 1 by a hemoglobinopathy (Hb San Diego); 4 relative erythrocytosis (RE) cases; and 13 ET patients. PV and ET diagnosis was made according to the WHO criteria. At time of sampling 43% of PV patients and 54% of ET patients were receiving myelosuppressive therapy. To establish a cut-off value 28 healthy persons (15 female, 13 male) were also analysed. RNA was extracted from whole-blood leucocytes and quantified by real-time TaqMan PCR. ABL was used to control the mRNA quality (Ct ABL < 27.0 – mean 24.7) and to normalise the PRV-1 values. PRV-1 primer and probe sequences originated from Brohée D et al, Blood, 2002, 100:3146a and ABL primer and probe sequences from Beillard E et al, Leukemia, 2003, 17:2474. The results were expressed as a ratio PRV-1/ABL which could be calculated using the ΔΔCt method as the efficiencies of both PCR reactions were almost equal (for PRV-1 93% and ABL 94%). Within-run and between-run reproducibility of a normal sample had a CV of 10.1 % and 28.6% and of a highly expressing sample a CV of 9.5% and 21.6%. The values of the healthy controls had a normal distribution and a mean of 0.28 with a SD of 0.35. A cut-off level was determined as the upper 95% percentile and was 0.98. Results of the different patient groups are summarised in the table. The differences between PV vs healthy controls, PV vs SE/RE and ET vs healthy controls were significant with a p-value of respectively <.001, <.001 and =.006. Three of the 23 PV patients had a PRV-1 result below the cut-off level. Thus we found a diagnostic sensitivity of 87%. Two of the 3 negative patients had no detectable PRV-1 expression, we hypothise resulting from a congenital deficiency. None of the 9 SE and RE patients had a PRV-1 expression above the cut-off level. Thus in our small series of polycythemic patients the specificity of PRV-1 over-expression for PV is 100%. Five of the 13 (38%) ET patients had a PRV-1 expression above the cut-off level. Patients with secondary thrombocythemia have to be studied to establish the specificity of PRV-1 over-expression for ET. The over-expressing ET patients had a significantly lower level of PRV-1 than the PV patients (p =.001). None of the 5 positive (as neither the other 8) ET had thrombotic complications nor developed PV. In conclusion PRV-1 mRNA quantification on whole-blood leucocytes is easy, reproducible and highly sensitive and specific for PV. The test should facilitate the diagnosis of this disease. For ET more cases of secondary thrombocythemia have to be studied to establish the diagnostic specificity of the assay. PRV-1/ABL mRNA ratio in healthy controls, PV, SE/RE and ET Normal PV SE/RE ET Median/mean 0.28 21.3 0.18 0.62 Range <0.01 – 1.39 <0.01 – 450 <0.01– 0.61 0.03 – 87.4 Frequency of positivity 20/23 (87%) 0/9 5/13 (38%)