Jasmonates, gibberellins, and powdery mildew modify cell cycle progression and evoke differential spatiotemporal responses along the barley leaf.

Barley (Hordeum vulgare) is an important cereal crop, and its development, defence, and stress responses are modulated by different hormones including jasmonates (JAs) and the antagonistic gibberellins (GAs). Barley productivity is severely affected by the foliar biotrophic fungal pathogen Blumeria hordei. In this study, primary leaves were used to examine the molecular processes regulating responses to methyl-jasmonate (MeJA) and GA to B. hordei infection along the leaf axis. Flow cytometry, microscopy, and spatiotemporal expression patterns of genes associated with JA, GA, defence, and the cell cycle provided insights on cell cycle progression and on the gradient of susceptibility to B. hordei observed along the leaf. Notably, the combination of B. hordei with MeJA or GA pre-treatment had a different effect on the expression patterns of the analysed genes compared to individual treatments. MeJA reduced susceptibility to B. hordei in the proximal part of the leaf blade. Overall, distinctive spatiotemporal gene expression patterns correlated with different degrees of cell proliferation, growth capacity, responses to hormones, and B. hordei infection along the leaf. Our results highlight the need to further investigate differential spatial and temporal responses to pathogens at the organ, tissue, and cell levels in order to devise effective disease control strategies in crops.

Stable Transformation of Arabidopsis thaliana Cell Suspension Cultures: A Case Study for The Overexpression of the COI1 Receptor.

Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities. In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long term maintenance of transformed cells. Details on culture maintenance, both in liquid and solid media are provided, alongside with evidence of protein expression to confirm transformation. The system described provides a powerful tool for synthetic biology to study signaling independent of developmental control and to obtain metabolites of interest for the biotechnological and medical sectors.

Molecular genetic and biochemical characterization of a putative family of zinc metalloproteins in Caenorhabditis elegans.

Four highly similar genes (W08E12.2, W08E12.3, W08E12.4 and W08E12.5) which are consecutively aligned on chromosome IV of the C. elegans genome are predicted to code for small (120-141aa) yet cysteine rich (18-19 cysteines) proteins. Cloning and sequencing of the genomic regions of the isoforms confirmed the presence and order of all genes. The generation of transgenic worms strains with an integrated single copy or extrachromosomal multi-copy PW08E12.3;W08E12.4::GFP uncovered that W08E12.3 and W08E12.4 are constitutively expressed in the pharynx and significantly induced in worms exposed to 100 µM Zn. Knockdown by RNAi did not have a marked consequence on reproductive performance nor was a Zn-dependent effect on nematode growth observed. However, RNAi of these genes led to an accumulation of Zn in the intestinal cells. W08E12.3 was recombinantly expressed in E. coli and the purified protein was shown to be able to bind up to 6.5 Zn molecules at neutral pH. Zn-binding was acid-labile and the apo protein was observed at pH < 4.3. This characterization suggests W08E12.2, W08E12.3, W08E12.4 and W08E12.5 belong to a family of putative Metalloproteins which, akin to metallothioneins, may play an important role in Zn-sensing, homeostasis and/or detoxification.

COI1-dependent jasmonate signalling affects growth, metabolite production and cell wall protein composition in arabidopsis.

Background and Aims: Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described. Methods: Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels. Key Results: Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as ß-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses. Conclusions: This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.