Plant Chemical Biology

Plant science has a high affinity with chemical biology. The major plant hormones are small organic compounds and, historically, chemical syntheses of plant hormone analogs have accelerated our understanding of hormone physiology and signaling in plants. In fact, by exploiting the advantages of chemical biology, many breakthroughs have been brought to the field of plant science. Notable examples include the identification of receptors for the plant hormone ABA (Park et al. 2009), and a recent study focused on elucidating the molecular mechanism of strigolactone (SL) signaling by imparting functions to molecules using a SL hormone mimic turn-on fluorescent probe (Tsuchiya et al. 2015). In addition to understanding processes, chemical biology has also been applied to synthetic biology. Orthogonal plant hormone–receptor pairs have been developed recently to induce ABA and auxin responses specifically and selectively (Park et al. 2015, Uchida et al. 2018). This artificial hormone–receptor system provides a powerful tool to control hormone signaling in any specific cells or tissues of interest, and future design and manipulation of hormone-mediated plant responses. Thus, the impact of chemical biology on hormone research is now self-evident. This in turn raises the question ‘can chemical-based approaches be applied to understanding other aspects of plant growth and development?’ Chemical biology can be harnessed to overcome obstacles of gene essentiality or high redundancy in gene families, which are critical problems in classical genetics approaches. Therefore, chemical biology should have important uses in many areas of plant biology. With this in mind, in this Special Issue we not only focus on applications of chemical biology to classical plant hormone signaling, but also highlight new research progress in areas ranging from peptide–receptor pairing, to stomatal responses and immune responses. Hopefully these papers will stimulate the broader plant research community to integrate chemical biological approaches into their programs.

Chemical Biology for Understanding Hormone Action

Small molecule probes of ABA biosynthesis and signaling

The Cutler lab was the first to elucidate the role of ABA receptors and the early ABA signaling pathway using an elegant chemical biology approach (Park et al. 2009) and they also developed a specific and selective system for ABA-induced responses using orthogonal plant hormone–receptor pairs (Park et al. 2015). ABA mediates multiple physiological processes including embryo maturation, induction of seed dormancy, plant growth regulation, stomatal closure, and biotic and abiotic stress responses. In their seminal review, Dejonghe et al. (2018) summarize key small molecules, which have helped to better understand both ABA biosynthesis and the ABA signaling pathway. The authors also discuss how we can further improve and utilize small molecules in ABA research and agriculture.

Manipulation and sensing of auxin actions

Another plant hormone, auxin, regulates virtually every aspect of plant growth and development. For example, auxin functions as a master regulator in embryogenesis, apical dominance, lateral root formation, hypocotyl elongation, tropic responses to light and gravity, vascular tissue differentiation and lateral branching of shoots (Woodward and Bartel 2005, Enders and Strader 2015). As a wag once said: ‘all plant researchers are working on auxin but they just don’t know it yet’ Current research using chemical biology has greatly contributed to identifying important genes in auxin biosynthesis, transport and signaling. Moreover, molecular genetic technologies and structural information for auxin regulatory components have accelerated the identification and characterization of many novel small molecule modulators in auxin biology. In this issue, Fukui and Hayashi (2018) summarize natural and synthetic molecules that function as auxins. They also review research progress of the synthetic and endogenous small molecule modulators in auxin metabolism, auxin transport and auxin signaling, and introduce the application of these modulators in plant biology.

Auxin probes for artificial ligand–receptor pairs

The artificial control of endogenous auxin signaling should, in principle, enable the precise delineation of auxin-mediated biological events as well as the agricultural application of auxin. To this end, the recent report of a synthetic auxin–receptor pair that works orthogonally to natural auxin signaling to evoke auxin-mediated developmental and physiological responses is an impressive step forward (Uchida et al. 2018). In this issue, Yamada et al. (2018) further describe improved versions of synthetic auxin–receptor pairs that can now act at much lower concentrations—in some cases 10,000-fold lower than their prototype version. The improved synthetic auxin–receptor pair with such high affinity will not only benefit fundamental research but should also have applications in agriculture/horticulture.

Small molecule probes for strigolactones

Plants emit small molecule signals to inform surrounding organisms in the biosphere of their physiological status. For example, many plants deprived of phosphate exude the plant hormone SL into the soil to attract symbiotic arbuscular mycorrhizal fungi that provide inorganic phosphate to the plants. Intriguingly, SLs also act as an endogenous hormone to direct plant development both below and above ground. Unfortunately, the diverse ecological/hormonal functions of SLs have been co-opted as a germination cue by parasitic plants from the family Orobanchaceae to allow the parasite to co-ordinate its life cycle with that of the host. The past decade has been very fruitful for the field of SL research, and the mechanisms for the biosynthesis and signaling of SLs have been revealed from genetic studies using a variety of model systems such as Arabidopsis and rice. However, understanding parasitic plant SL signaling is difficult as these plants are not lab friendly. In this issue, Tsuchiya (2018) discuss the possible utility of small molecule probes as a platform to address the roles of SLs in parasitic plants and how this information can be used to understand the evolution of SL signaling between model and non-model systems.

Rationally designed strigolactone antagonists for strigolactone receptors

As mentioned, SLs act as signals in many aspects of plant development. The most studied of these is the ability of SLs to inhibit shoot branching through the SL receptor class called DWARF14-type receptors (D14) (Umehara et al. 2008, Brewer et al. 2013). D14 receptors are also α/β-hydrolases that not only signal SL but also cleave the four member ring of the hormone into an ABC tricyclic lactone and a butenolide group called the D-ring. However, the functional implication of receptor-mediated SL hydrolysis and the active forms of SLs remain controversial (Carlsson et al. 2018). In this issue, Takeuchi et al. (2018) designed novel SL receptor antagonists, carba-SL compounds based on the structural requirement for SL bioactivity and the mechanism for activating the receptor. They conclude that the hydrolysis of the D-ring of SLs may be insufficient for activating the receptor, which begs the question as to why D14 receptors have hydrolase activity. Furthermore, this study provides data relevant to designing SL receptor antagonists.

Chemical inducer of dedifferentiation and callus formation

A remarkable ability of plants is their capacity for dedifferentiation and regeneration. It is widely known that treatments with auxin and cytokinins—in the appropriate combination—could induce uncontrolled cell proliferation of plant explants in vitro, resulting in the formation of a massive ‘tumor’ known as a callus (Ikeuchi et al. 2013). Roots, shoots and whole plantlets can be subsequently regenerated from the callus and, for this reason, a precise control of callus formation and regeneration is critical for tissue culture and transformation technologies. Through a chemical screen, Nakano et al. (2018) identified fipexide (FPX) as a potent inducer of dedifferentiation and callus formation. A transcriptomic analysis of FPX-induced/repressed genes revealed shared and unique effects of FPX and auxin/cytokinin treatments. FPX can promote callus formation in a wide variety of plant species, including Arabidopsis, rice, poplar, soybean, tomato and cucumber. Thus, FPX could be used as a potential tool for understanding the mechanism of plant regeneration and for the transformation of economically important plant species.

Chemical Biology for Understanding Cell Signaling

Probing plant receptor kinase functions with labeled ligands

Plants possess a large number of receptor kinases (RKs) to recognize self from non-self, to co-ordinate developmental programs and to respond to arrays of environmental stresses as well as pathogens and symbionts. Accordingly, plant genomes also encode an enormous number of small open reading frames, which are annotated as small secreted peptides. Despite decades of studies, the overwhelming majority of RKs and peptides remain as ‘orphans’. In this issue, Sharma and Russinova (2018) summarize an updated catalog of labeled ligands that have been successfully applied to identify ligand–receptor pairs, to monitor ligand perception and to visualize further the subcellular dynamics of ligand-activated receptors. Ligand labeling techniques include photoaffinity labeling, chemiluminescence and fluorescence (e.g. Stenvik et al. 2008, Irani et al. 2012, Nakayama et al. 2017). The authors discuss how combinations of chemical tools and large-scale interactome analyses/bioinformatics could narrow the gap in understanding the functions and regulation of peptide–RK complexes.

Chemical screening for stomatal movements

Stomata in the plant epidermis play a pivotal role in regulating gas exchange between leaves and the atmosphere. Stomata open in response to light to facilitate gas exchange. This is a key response for higher plant life as gas exchange is necessary not only for photosynthesis but also for nutrient uptake in roots. Although the molecular mechanisms of stomatal movements are not fully understood, we know that the plant hormone ABA promotes stomatal closure to prevent water loss (Inoue and Kinoshita 2017). Since ABA is a small molecule, it would be intriguing to know if other small molecules can control this system. Here, Toh et al. (2018) report a comprehensive screening of chemical libraries to identify compounds that affect stomatal movements and the underlying molecular mechanisms. They showed that stomatal closing compounds (SCLs) suppress light-induced stomatal opening, and that SCL1 and SCL2 suppress light-induced stomatal opening at least in part by inhibiting blue light-induced activation of plasma membrane H⁺-ATPase, which is a key enzyme for stomatal opening. Interestingly, SCL1 and SCL2 do not impinge on the ABA signaling pathway. They also found that temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), and CP-100356, a high-affinity P-glycoprotein/MDR-1 inhibitor, induced stomatal opening even in the dark, suggesting the involvement of Arabidopsis target of rapamycin (AtTOR) and ABC transporters in the regulation of stomatal aperture. The analysis of these compounds provides novel insight into the regulation of stomatal movements and should lead to new avenues of research for the Ag-biotech community.

Chemical dissection of thigmomorphogenesis

Thigmomorphogenesis, a plant growth syndrome caused by mechanical sensation, such as touch, has fascinated horticulturists and botanists. The identification of ‘touch induced’ (TCH) genes (Braam and Davis 1990) unraveled the critical roles of Ca²⁺ signaling in thigmomorphogenesis, as TCH genes turned out to encode calmodulin and other predicted Ca²⁺ binding proteins. Okamoto et al. (2018) report that omeprazole, a known proton-pump inhibitor used as medication for treatments of gastroesophageal reflux disease, enhances mechanical stress-induced root growth reduction in Arabidopsis. The application of omeprazole to Arabidopsis seedlings induced TCH genes. Genetic- and pharmacological studies further revealed that the enhanced root growth reduction is pronounced in low pH (acidic conditions), and requires proper ethylene signaling as well as cellular Ca²⁺. Taken together, Okamoto et al. suggest that omeprazole enhances thigmomorpohgenesis by inhibiting subcellular Ca²⁺ flux. Because omeprazole is a known inhibitor of mammalian P-type H⁺/K⁺⁻ATPase, it would be fascinating to determine whether omeprazole also targets Ca²⁺ pumps and proton pumps (H⁺⁻ ATPases) in plants, which are known drivers of cell elongation.

Chemical Biology for Understanding Pathogen Defense/Plant Immunity

Multiple functions of benzoxazinoids

Benzoxazinoids are a class of indole-derived plant metabolites that function in defense against numerous pests and pathogens. In plants such as maize, the predominant benzoxazinoids can account for 0.1–0.3% of fresh weight in some tissues. Although the biosynthetic pathways for most maize benzoxazinoids have been identified, unanswered questions remain about the developmental and defense-induced regulation of benzoxazinoid metabolism. Recent research shows that, in addition to their central role in the maize chemical defense repertoire, benzoxazinoids may have important functions in regulating other defense responses, flowering time, auxin metabolism, iron uptake and perhaps aluminum tolerance. In this issue, Zhou et al. (2018) provide an updated review of benzoxazinoid biosynthesis and function, with an emphasis on describing new developments from the past 3 years.

Chemical biology in plant immune responses

Plants utilize multilayered defense strategies consisting of pre-formed and inducible mechanisms to resist pathogen infection. The Arabidopsis protein ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), together with its direct signaling partners, PHYTOALEXIN DEFICIENT4 (PAD4) and SENESCENCE ASSOCIATED GENE101 (SAG101), regulates plant basal immunity against virulent biotrophic pathogens and effector-triggered immunity (ETI), mediated by intracellular TOLL/INTERLEUKIN-1 RECEPTOR–NUCLEOTIDE BINDING–LEUCINE RICH REPEAT (TNL) receptors, against avirulent pathogenic strains (Rietz et al. 2011, Wagner et al. 2013). In their study, Joglekar et al. (2018) performed a chemical biology approach to dissect signaling pathways controlling FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) expression, which is a marker gene of the EDS1- and PAD4-controlled, salicylic acid-independent signaling branch, and identified thaxtomin A (TXA), a phytotoxin from plant pathogenic Streptomyces scabies. Further analysis shows that TXA is a potent chemical activator of EDS1- and PAD4-regulated defense outputs. Thus, TXA is a promising new tool to activate conditionally and dissect further EDS1/PAD4-regulated plant immune responses.

We hope that this Special Focus Issue will provide a useful overview of the field of plant chemical biology and inspire new ideas and directions for future investigations.

Funding

This work is supported by the National Science and Engineering Research Council of Canada [to P.M.]; Technology of Japan; the Japan Science and Technology Agency [the Advanced Low Carbon Technology Research and Development Program (JPMJAL1011 to T.K.)]; and the Japan Society for the Promotion of Science [Grant-in-Aid for Scientific Research on Innovative Areas (JP15H05956 to T.K., JP16H01237 and JP17H06476 to K.U.T.)]. K.U.T. is an HHMI Investigator.

Acknowledgments

We thank Professor Hitoshi Sakakibara, Editor-in-Chief, Plant and Cell Physiology, for providing the opportunity for this Special Focus Issue on Plant Chemical Biology, and Dr. Liliana M. Costa, Managing Editor, for helpful advice and assistance. We wish to acknowledge the authors and reviewers who have contributed to this issue.

Disclosures

The authors have no conflicts of interest to declare.

References