Small molecules: from structural diversity to signalling and regulatory roles

For many years, the role of low molecular weight chemicals in plant signaling and regulation was restricted to a small set of molecules, largely those considered phytohormones including auxins, cytokinines, abscisates, gibberellins, and more. Yet, small molecules belonging to diverse chemical classes that are not defined as hormones have been demonstrated to act as mediators of signaling and regulatory cascades. While it is currently difficult to estimate how many such molecules function in plants, it might be that those identified to date are only a small portion of the ones functioning in a single plant. Small molecules could be acting not only at the level of proteins and membranes, but could also mediate mechanisms associated with DNA, mRNA, and small and long non-coding RNAs. Identifying such molecules is a difficult task partially due to the limited availability of methodologies to conduct large-scale in vivo detection of direct interactions with small molecules as well as the classically acknowledged low abundance of these molecules. At the same time, in contrast to what is generally recognized, a certain portion of signaling and regulatory small molecules accumulate to significant levels rather than at low levels, particularly lipid and carbohydrates derivatives. Even in cases when a certain molecule is associated with a particular process, the in vivo activity and role of its derivatives, conjugates and catabolites typically remain unresolved. Adding to the complexity of the role of small molecules in signaling and regulation is their intra-organismal transport that can take place in the same cell, between connected cells, in between nearby unconnected cells and distant cells. It may also be extraorganismal as in the case of volatiles and secreted chemicals. Despite the many challenges in detecting and functionally characterizing small molecules, genomics advances paired with increasingly sophisticated metabolomics approaches have led to a steady increase in the discovery of metabolites of diverse structure and roles in signaling and regulatory processes. This special issue will highlight recent advances and discoveries in the field of small molecule research ranging from signaling metabolites in primary metabolism to molecules with roles in inter- or intra-specific interactions in plants and algae. The reader will find traditional functional classifications of small molecules to become increasingly blurred since many chemicals act as exogenous signals and simultaneously serve important endogenous functions in plant physiology and development. In the first paper of this special issue, Lunn et al. (2014) describe particular sugars, namely trehalose and its phosphorylated intermediate trehalose 6-phosphate (Tre6P), as potential signaling metabolites. The non-reducing disaccharide trehalose is renown in many organisms as an osmoprotectant while in plants it is currently implicated in signaling associated with abiotic stress response as well as below and above ground interactions. Reports on a tight correlation between levels of Tre6P and sucrose suggested together with additional evidence that Tre6P is a signal of sucrose availability and a regulator of its levels in the plant. This homeostatic mechanism is somewhat analogous to the control of animal blood glucose levels by glucagon and insulin. Apparently, Tre6P does not only impact metabolism but Tre6P mutant analysis points to a major influence on plant growth and development. The N-Acylethanolamines (NAEs) consist of a fatty acid linked by an amide bond and have been studied extensively since the discovery that ethanolamine of arachidonic acid (N-arachidonylethanolamine) plays a role as an endogenous ligand of the plasma-membrane cannabinoid receptor. In this special issue, Blancaflor et al. (2014) describe the metabolism including hydrolysis and oxidation as well as functions of NAEs in higher plants. Recent work provides evidence that NAEs act as endogenous signals that modulate seed germination, seedling establishment, plant pathogen interactions, chloroplast development and flowering. Very little is known with respect to the molecular mechanisms accompanying the action of this understudied group of metabolites, particularly the molecular targets of NAEs metabolites. Understanding these processes may provide insights to the evolutionary relations between NAE mediated signaling in plants and diverse organisms. Okazaki and Saito (2014) provide a more extensive overview of various lipid molecules acting as signaling metabolites during stress response in plants. Lysophospholipids, free fatty acids, phosphatidic acid, inositol phosphates and diacylglycerols, oxylipins and sphingolipids are reviewed in detail. This review also covers the role of lipids species as mitigators to reduce the impact of stressors. Several classes of plant hormones including abscisates, gibberellins, brassinosteroids and the recently identified strigolactones belong to the isoprenoid group. The large isoprenoids class also includes carotenoids, most of them derived from the linear tetraterpene phytoene. While carotenoids are well known for their function in plants as antioxidants, accessory light harvesting pigments and attractants for pollinators and seed dispersers, their 40-carbon carotenoid cleavage products (i.e. apocarotenoids) have recently emerged as stress signaling molecules. Such carotenoid derivatives could be a result of enzymatic cleavage as in the case of strigolactone biosynthesis. Here, Havaux (2014) reviews the action of apocarotenoids formed by oxidation through reactive oxygen species. Some of these derivatives such as β-cyclocitral are bioactive species produced in plastids that can induce nuclear gene expression leading to acclimation to stress conditions. The discovery of strigolactones has been one of the most exciting findings of carotenoid-derived small molecules with multiple functions in plant development and organismal interactions. Despite numerous reviews in the fast moving field of strigolactone research, this special issue would have been incomplete without an update on strigolactone biology. Waldie et al. (2014) focus their review on recent advances in understanding the biosynthesis and role of strigolactones as endogenous signals in the regulation of shoot branching. The authors discuss the mechanisms of strigolactone perception, critically review the findings on downstream targets, and present the current models for how strigolactones are believed to act in regulating branching. Moreover, a scenario for the evolution of strigolactone signaling is discussed assuming an expansion from exogenous effects in the rhizosphere of early land plants to the function as phytohormones in higher land plants. Karrikins are similar to strigolactones in structure as both contain a 3-methyl-butenolide ring. They were discovered as chemicals present in charred plant material and smoke that could stimulate germination of seeds of fire-following plant species. Waters et al. (2014) report on research, mostly through studies in Arabidopsis that indicate the similarity and differences in the biosynthesis and perception mechanisms of karrikins and strigolactones. These authors review the signaling role of karrikins in stimulation of seed germination, seedling establishment and leaf morphogenesis. Three articles in this special issue focus on the role and metabolism of small molecules as ‘specialized’ metabolites in plant biotic stress protection. Chemical defenses of crops have been studied for decades, but only recent systems-based approaches provide insight to the full complexity of defense metabolites and their biochemical and regulatory networks. Schmelz et al. (2014) give an update on terpenoid phytoalexins as important chemical resistance traits of cereal crops, specifically rice and maize. Remarkably, several pathogen-induced terpenoid metabolites and their corresponding biosynthetic enzymes have been discovered only recently and provide, in the case of diterpenoid phytoalexin biosynthesis, insight in the assembly of metabolic gene clusters at the evolutionary branch point to gibberellin biosynthesis. Schmelz et al. (2014) also present a comprehensive overview of the biotic elicitors and complex signaling pathways that regulate the coordinate expression of the specific terpenoid biosynthetic steps. Gaquerel et al. (2014) further emphasize the time- and tissue-specific metabolic coordination of small molecules as a key factor in herbivore-induced plant defense. Using phenolamides, which are common insect-induced phenylpropanoid derivatives, as a study system, the authors have developed a sophisticated systems toolkit of metabolomics and multifactorial gene expression analysis in the wild tobacco, Nicotiana attenuata, to elucidate stress-induced metabolic reorganization in space and time. Using these tools helps define tissue- and stress specific gene expression clusters in local and systemic response and thus aids in the discovery of stress-induced biosynthetic pathways of small defense molecules. As in the case of strigolactones, it is becoming increasingly clear that small molecules involved in environmental interactions often serve more than a single function. Gaquerel et al. (2014) point out the presumed role of phenolamides in developmental homeostasis, especially in flower development. Schmelz et al. (2014) elaborate on allelopathic and anti-herbivore activities of rice and maize labdane diterpenoids beyond their role as phytoalexins and discuss possible functions of labdane diterpenoid biosynthetic intermediates as volatile signals or in maintaining gibberellin phytohormone homeostatis. In line with these findings, the discovery of small molecules with previously unknown functions in systemic defense signaling is reviewed by Shah et al. (2014). The molecules described in this article in association with systemic acquired resistance (SAR) are of diverse metabolic origin and function at picomolar concentrations as direct mobile or indirect/priming signals that act upstream or affect the accumulation of salicylic acid. Interestingly, there is growing evidence that SAR-associated signaling molecules such as the diterpenoid dehydroabietinal involve epigenetic changes that also affect flowering, thus suggesting that these molecules act at the interface of defense and developmental transition. The reason for plants to employ several different metabolites in systemic defense remains to be determined, but Shah et al. argue that different molecules could be necessary to optimally regulate SAR under different environmental conditions. Besides plants, microalgae have emerged as a source for renewable fuels and value products. Due to the growing industrial importance of algae and also their crucial role in aquatic ecosystems, the function of signaling molecules in algal cultures is of increased interest. Besides the use of interspecific signals in competition and defense, algae produce pheromones as chemical signals for sexual reproduction. Frenkel et al. (2014) provide a fascinating, up-to-date insight to the chemical diversity of the pheromones and signaling systems employed by different algal models. Although experimentally challenging, analysis of these intraspecific signals bears significant potential in regulating the life cycle and controlling sexual reproduction in algal cultures. Finally, small molecules could also act in the direct regulation of RNA as exemplified by Bocobza and Aharoni (2014) that review metabolite-dependent gene regulation through riboswitches; RNA sensors that affect posttranscriptional process through their capacity to bind small molecules. These authors describe the structure and mode of action of the single known plant riboswitch that binds thiamine pyrophosphate. They further discuss the significance of this non-coding RNA element in maintaining optimal thiamine levels as well as homeostasis in plant central metabolism. Riboswitches also provide new possibilities for engineering metabolite sensors and molecular platforms for gene control. It is currently unknown how many similar elements are active in plants and new riboswitches still await discovery. To conclude, the contributions to this special issue clearly demonstrate that plants employ many more small molecules in signaling and regulatory processes than originally expected. Systems biology approaches will keep pushing the limits in discovering new functional molecules and lead to new challenges in elucidating mechanisms of their perception and downstream signaling cascades and targets. In light of the extraordinary chemical diversity in plants and other photosynthetic organisms, more information will be needed as to what extent the diverse array of small molecules is required not only for inter-specific interactions but also for the fine-tuning and selective adaptation of endogenous signaling systems. With plants being one of the largest producers of chemicals among living creatures, it is likely that our current knowledge on mechanisms by which small molecules mediate signaling and regulatory networks is just the tip of the iceberg.