Posttranslational regulation of photosynthetic activity via the TOR kinase in plants.

Chloroplasts are the powerhouse of the plant cell, and their activity must be matched to plant growth to avoid photooxidative damage. We have identified a posttranslational mechanism linking the eukaryotic target of rapamycin (TOR) kinase that promotes growth and the guanosine tetraphosphate (ppGpp) signaling pathway of prokaryotic origins that regulates chloroplast activity and photosynthesis in particular. We find that RelA SpoT homolog 3 (RSH3), a nuclear-encoded enzyme responsible for ppGpp biosynthesis, interacts directly with the TOR complex via a plant-specific amino-terminal region which is phosphorylated in a TOR-dependent manner. Down-regulating TOR activity causes a rapid increase in ppGpp synthesis in RSH3 overexpressors and reduces photosynthetic capacity in an RSH-dependent manner in wild-type plants. The TOR-RSH3 signaling axis therefore regulates the equilibrium between chloroplast activity and plant growth, setting a precedent for the regulation of organellar function by TOR.

Insights into the function of the chloroplastic ribosome-associated GTPase high frequency of lysogenization X in Arabidopsis thaliana.

Ribosome-associated GTPases are conserved enzymes that participate in ribosome biogenesis and ribosome function. In bacteria, recent studies have identified HflX as a ribosome-associated GTPase that is involved in both ribosome biogenesis and recycling under stress conditions. Plants possess a chloroplastic HflX homolog, but its function remains unknown. Here, we characterized the role of HflX in the plant Arabidopsis thaliana. Our findings show that HflX does not affect normal plant growth, nor does it play an essential role in acclimation to several different stresses, including heat, manganese, cold, and salt stress under the conditions tested. However, we found that HflX is required for plant resistance to chloroplast translational stress mediated by the antibiotic lincomycin. Our results suggest that HflX is a chloroplast ribosome-associated protein that may play a role in the surveillance of translation. These findings provide new insight into the function of HflX as a ribosome-associated GTPase in plants and highlight the importance of investigating conserved proteins in different organisms to gain a comprehensive understanding of their biological roles.

New perspectives on the molecular mechanisms of stress signalling by the nucleotide guanosine tetraphosphate (ppGpp), an emerging regulator of photosynthesis in plants and algae.

The nucleotides guanosine tetraphosphate and guanosine pentaphosphate (together (p)ppGpp) are found in a wide range of prokaryotic and eukaryotic organisms where they are associated with stress signalling. In this review, we will discuss recent research highlighting the role of (p)ppGpp signalling as a conserved regulator of photosynthetic activity in the chloroplasts of plants and algae, and the latest discoveries that open up new perspectives on the emerging roles of (p)ppGpp in acclimation to environmental stress. We explore how rapid advances in the study of (p)ppGpp signalling in prokaryotes are now revealing large gaps in our understanding of the molecular mechanisms of signalling by (p)ppGpp and related nucleotides in plants and algae. Filling in these gaps is likely to lead to the discovery of conserved as well as new plant- and algal-specific (p)ppGpp signalling mechanisms that will offer new insights into the taming of the chloroplast and the regulation of stress tolerance.

MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein-protein interactions.

BACKGROUND: The bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein-protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed. RESULTS: Here we describe the development of a flexible modular cloning-based toolkit for BiFC (MoBiFC) and proximity labelling in the chloroplast and other cellular compartments using synthetic biology principles. We used pairs of chloroplast proteins previously shown to interact (HSP21/HSP21 and HSP21/PTAC5) and a negative control (HSP21/ΔPTAC5) to develop standardised Goldengate-compatible modules for the assembly of protein fusions with fluorescent protein (FP) fragments for BiFC expressed from a single multigenic T-DNA. Using synthetic biology principles and transient expression in Nicotiana benthamiana, we iteratively improved the approach by testing different FP fragments, promoters, reference FPs for ratiometric quantification, and cell types. A generic negative control (mCHERRY) was also tested, and modules for the identification of proximal proteins by Turbo-ID labelling were developed and validated. CONCLUSIONS: MoBiFC facilitates the cloning process for organelle-targeted proteins, allows robust ratiometric quantification, and makes available model positive and negative controls. Development of MoBiFC underlines how Goldengate cloning approaches accelerate the development and enrichment of new toolsets, and highlights several potential pitfalls in designing BiFC experiments including the choice of FP split, negative controls, cell type, and reference FP. We discuss how MoBiFC could be further improved and extended to other compartments of the plant cell and to high throughput cloning approaches.

Tomato contrasting genotypes responses under combined salinity and viral stresses.

Tomato yellow leaf curl disease (TYLCD) and salinity stress adversely affect tomato production worldwide by causing extensive damages. In Tunisia, identifying TYLCD resistant cultivars selected in different environments is useful to devise counter-measures. To this end, 20 tomato commercial cultivars were screened for different Ty gene alleles' combinations and evaluated either for TYLCD incidence or salinity constraint. We built a biological multi-layer network for integrating, visualizing and modelling generated data. It is a simple representation view linking allelic combinations to tomato cultivars behaviour under viral and salt stresses. In addition, we analyzed differential expression of transcriptions factors (TFs) belonging to WRKY and ERF families in selected resistant (R) and susceptible (S) tomato cultivars. Gene expression was evaluated for short- and long stress exposure to either TYLCSV infection or to both viral and salinity stresses. Evidence is that TFs promote resistance to abiotic and biotic stresses through a complex regulatory network.