F-box proteins everywhere.

The ubiquitin proteasome system is a key regulator of many biological processes in all eukaryotes. This mechanism employs several types of enzymes, the most important of which are the ubiquitin E3 ligases that catalyse the attachment of polyubiquitin chains to target proteins for their subsequent degradation by the 26S proteasome. Among the E3 families, the SCF is the best understood; it consists of a multi-protein complex in which the F-box protein plays a crucial role by recruiting the target substrate. Strikingly, nearly 700 F-box proteins have been predicted in Arabidopsis, suggesting that plants have the capacity to assemble a multitude of SCF complexes, possibly controlling the stability of hundreds of substrates involved in a plethora of biological processes. Interestingly, viruses and even pathogenic bacteria have also found ways to hijack the plant SCF and to reprogram it for their own purposes.

Histidine-containing phosphotransfer domain extinction by RNA interference turns off a cytokinin signalling circuitry in Catharanthus roseus suspension cells.

We previously reported that cytokinins (CK) induce the fast and specific transcription of CrRR1, a gene encoding a type A response regulator in Catharanthus roseus cell cultures. Here, we characterized the CrHPt1 gene that encodes a histidine-containing phosphotransfer domain. CrHPt1 was silenced through RNA interference (RNAi) to test its possible implication in the CK signalling pathway. In transgenic lines stably transformed with an intron-spliced construct, the degradation of CrHPt1 transcripts abolishes the CK inductive effect on CrRR1 transcription. These result give a new in vivo functional argument for the crucial role of HPt proteins in the CK signalling pathway leading to the expression of the genes encoding type A response regulators. They also show that RNAi is a powerful strategy to turn off the CK signalling circuitry.

ePathBrick: a synthetic biology platform for engineering metabolic pathways in E. coli.

Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we report the development of a set of vectors compatible with BioBrick standards and its application in metabolic engineering. The engineered ePathBrick vectors comprise four compatible restriction enzyme sites allocated on strategic positions so that different regulatory control signals can be reused and manipulation of expression cassette can be streamlined. Specifically, these vectors allow for fine-tuning gene expression by integrating multiple transcriptional activation or repression signals into the operator region. At the same time, ePathBrick vectors support the modular assembly of pathway components and combinatorial generation of pathway diversities with three distinct configurations. We also demonstrated the functionality of a seven-gene pathway (~9 Kb) assembled on one single ePathBrick vector. The ePathBrick vectors presented here provide a versatile platform for rapid design and optimization of metabolic pathways in E. coli.

The exoribonuclease XRN4 is a component of the ethylene response pathway in Arabidopsis.

EXORIBONUCLEASE4 (XRN4), the Arabidopsis thaliana homolog of yeast XRN1, is involved in the degradation of several unstable mRNAs. Although a role for XRN4 in RNA silencing of certain transgenes has been reported, xrn4 mutant plants were found to lack any apparent visible phenotype. Here, we show that XRN4 is allelic to the unidentified components of the ethylene response pathway ETHYLENE-INSENSITIVE5/ACC-INSENSITIVE1 (EIN5/AIN1) and EIN7. xrn4 mutant seedlings are ethylene-insensitive as a consequence of the upregulation of EIN3 BINDING F-BOX PROTEIN1 (EBF1) and EBF2 mRNA levels, which encode related F-box proteins involved in the turnover of EIN3 protein, a crucial transcriptional regulator of the ethylene response pathway. Epistasis analysis placed XRN4/EIN5/AIN1 downstream of CTR1 and upstream of EBF1/2. XRN4 does not appear to regulate ethylene signaling via an RNA-INDUCED SILENCING COMPLEX-based RNA silencing mechanism but acts by independent means. The identification of XRN4 as an integral new component in ethylene signaling adds RNA degradation as another posttranscriptional process that modulates the perception of this plant hormone.

Cytokinin and ethylene control indole alkaloid production at the level of the MEP/terpenoid pathway in Catharanthus roseus suspension cells.

The Madagascar periwinkle Catharanthus roseus accumulates a number of terpenoid indole alkaloids, some of which have high therapeutic interest. The biotechnological approach with cells in vitro remains an alternative to the field culture of periwinkle for the production of such compounds. We previously reported that two phytohormones, cytokinin and ethylene, remarkably enhanced the accumulation of alkaloids in periwinkle cell suspensions. In this work, we investigated the effects of these hormones on the regulation of several genes of the indole alkaloid biosynthetic pathway. We show that cytokinin and/or ethylene greatly enhanced the expression of the geraniol 10-hydroxylase gene. When given together, these hormones also increased the expression of three genes belonging to the methyl-erythritol pathway. These results make it possible to consider elements of cytokinin and ethylene signalling pathways as tools for improving terpenoid indole alkaloid production through metabolic engineering.

Differential expression of two type-A response regulators in plants and cell cultures of Catharanthus roseus (L.) G. Don.

Two full-length cDNAs named CrRR2 and CrRR3 have been isolated by PCR from a C. roseus cDNA library. The first one encodes a 154 amino acid putative protein with a high percentage of identity with the Arabidopsis thaliana response regulators ARR16 and ARR17, and is not expressed in C. roseus organs and cell cultures. The second one encodes a 188 amino acid ORF sharing the highest homologies with the A. thaliana ARR8 and ARR9 response regulators. Its expression is root-specific and the transcripts are transiently up-regulated after trans-zeatin treatment in C. roseus suspension cells. CrRR3 protein might be involved in the cytokinin-enhanced alkaloid production in C. roseus cell cultures.