From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants.

The study of protein-protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.

Cold sensitivity of mitochondrial ATP synthase restricts oxidative phosphorylation in Arabidopsis thaliana.

The combined action of the electron transport chain (ETC) and ATP synthase is essential in determining energy efficiency in plants, and so is important for cellular biosynthesis, growth and development. Owing to the sessile nature of plants, mitochondria must operate over a wide temperature range in the environment, necessitating a broad temperature tolerance of their biochemical reactions. We investigated the temperature response of mitochondrial respiratory processes in isolated mitochondria and intact plants of Arabidopsis thaliana and considered the effect of instantaneous responses to temperature and acclimation responses to low temperatures. We show that at 4°C the plant mitochondrial ATP synthase is differentially inhibited compared with other elements of the respiratory pathway, leading to decreased ADP : oxygen ratios and a limitation to the rate of ATP synthesis. This effect persists in vivo and cannot be overcome by cold-temperature acclimation of plants. This mechanism adds a new element to the respiratory acclimation model and provides a direct means of temperature perception by plant mitochondria. This also provides an alternative explanation for non-phosphorylating ETC bypass mechanisms, like the alternative oxidase to maintain respiratory rates, albeit at lower ATP synthesis efficiency, in response to the sensitivity of ATP synthase to the prevailing temperature.

Temperature Sensing in Plants.

Temperature is a key environmental cue that influences the distribution and behavior of plants globally. Understanding how plants sense temperature and integrate this information into their development is important to determine how plants adapt to climate change and to apply this knowledge to the breeding of climate-resilient crops. The mechanisms of temperature perception in eukaryotes are only just beginning to be understood, with multiple molecular phenomena with inherent temperature dependencies, such as RNA melting, phytochrome dark reversion, and protein phase change, being exploited by nature to create thermosensory signaling networks. Here, we review recent progress in understanding how temperature sensing in four major pathways in Arabidopsis thaliana occurs: vernalization, cold stress, thermomorphogenesis, and heat stress. We discuss outstanding questions in the field and the importance of these mechanisms in the context of breeding climate-resilient crops.

Plant cell cultures as heterologous bio-factories for secondary metabolite production.

Synthetic biology has been developing rapidly in the last decade and is attracting increasing attention from many plant biologists. The production of high-value plant-specific secondary metabolites is, however, limited mostly to microbes. This is potentially problematic because of incorrect post-translational modification of proteins and differences in protein micro-compartmentalization, substrate availability, chaperone availability, product toxicity, and cytochrome p450 reductase enzymes. Unlike other heterologous systems, plant cells may be a promising alternative for the production of high-value metabolites. Several commercial plant suspension cell cultures from different plant species have been used successfully to produce valuable metabolites in a safe, low cost, and environmentally friendly manner. However, few metabolites are currently being biosynthesized using plant platforms, with the exception of the natural pigment anthocyanin. Both Arabidopsis thaliana and Nicotiana tabacum cell cultures can be developed by multiple gene transformations and CRISPR-Cas9 genome editing. Given that the introduction of heterologous biosynthetic pathways into Arabidopsis and N. tabacum is not widely used, the biosynthesis of foreign metabolites is currently limited; however, therein lies great potential. Here, we discuss the exemplary use of plant cell cultures and prospects for using A. thaliana and N. tabacum cell cultures to produce valuable plant-specific metabolites.

Two mitochondrial phosphatases, PP2c63 and Sal2, are required for posttranslational regulation of the TCA cycle in Arabidopsis.

Protein phosphorylation is a well-established post-translational mechanism that regulates protein functions and metabolic pathways. It is known that several plant mitochondrial proteins are phosphorylated in a reversible manner. However, the identities of the protein kinases/phosphatases involved in this mechanism and their roles in the regulation of the tricarboxylic acid (TCA) cycle remain unclear. In this study, we isolated and characterized plants lacking two mitochondrially targeted phosphatases (Sal2 and PP2c63) along with pyruvate dehydrogenase kinase (PDK). Protein-protein interaction analysis, quantitative phosphoproteomics, and enzymatic analyses revealed that PDK specifically regulates pyruvate dehydrogenase complex (PDC), while PP2c63 nonspecifically regulates PDC. When recombinant PP2c63 and Sal2 proteins were added to mitochondria isolated from mutant plants, protein-protein interaction and enzymatic analyses showed that PP2c63 directly phosphorylates and modulates the activity of PDC, while Sal2 only indirectly affects TCA cycle enzymes. Characterization of steady-state metabolite levels and fluxes in the mutant lines further revealed that these phosphatases regulate flux through the TCA cycle, and that altered metabolism in the sal2 pp2c63 double mutant compromises plant growth. These results are discussed in the context of current models of the control of respiration in plants.

Isolation of Mitochondria from Model and Crop Plants.

The ability to isolate intact and functional mitochondria has greatly deepened our understanding of mitochondrial structure and function. With the advancement of molecular biology techniques and progression into omics-based research over recent decades, mitochondrial research has shifted from crop species such as wheat, pea, and potato to genetically sequenced models such as Arabidopsis thaliana and rice. Although there are many attributes that make model species particularly appealing for plant research, they are often less than ideal for conducting biochemical investigations and as such, considerable modification to mitochondrial isolation methods has been made.As the cost of genome sequencing continues to decrease however, an increasing number of crop species are now being sequenced and with these new resources it appears that the research community is turning back toward crop research. In this chapter we present mitochondrial isolation methods using density gradient centrifugation for both model species such as Arabidopsis thaliana, rice, and Medicago and crop species including wheat, potato, and pea. In addition, we present a number of marker enzyme assays to confirm mitochondrial purity as well as respiratory assays to determine outer membrane integrity and respiratory function of isolated mitochondria.