Structure-guided engineering of a receptor-agonist pair for inducible activation of the ABA adaptive response to drought

Strategies to activate abscisic acid (ABA) receptors and boost ABA signaling by small molecules that act as ABA receptor agonists are promising biotechnological tools to enhance plant drought tolerance. Protein structures of crop ABA receptors might require modifications to improve recognition of chemical ligands, which in turn can be optimized by structural information. Through structure-based targeted design, we have combined chemical and genetic approaches to generate an ABA receptor agonist molecule (iSB09) and engineer a CsPYL1 ABA receptor, named CsPYL15m, which efficiently binds iSB09. This optimized receptor-agonist pair leads to activation of ABA signaling and marked drought tolerance. No constitutive activation of ABA signaling and hence growth penalty was observed in transformed Arabidopsis thaliana plants. Therefore, conditional and efficient activation of ABA signaling was achieved through a chemical-genetic orthogonal approach based on iterative cycles of ligand and receptor optimization driven by the structure of ternary receptor-ligand-phosphatase complexes

Mg-chelatase H subunit affects ABA signaling in stomatal guard cells, but is not an ABA receptor in Arabidopsis thaliana

Mg-chelatase H subunit (CHLH) is a multifunctional protein involved in chlorophyll synthesis, plastid-to-nucleus retrograde signaling, and ABA perception. However, whether CHLH acts as an actual ABA receptor remains controversial. Here we present evidence that CHLH affects ABA signaling in stomatal guard cells but is not itself an ABA receptor. We screened ethyl methanesulfonate-treated Arabidopsis thaliana plants with a focus on stomatal aperture-dependent water loss in detached leaves and isolated a rapid transpiration in detached leaves 1 (rtl1) mutant that we identified as a novel missense mutant of CHLH. The rtl1 and CHLH RNAi plants showed phenotypes in which stomatal movements were insensitive to ABA, while the rtl1 phenotype showed normal sensitivity to ABA with respect to seed germination and root growth. ABA-binding analyses using 3H-labeled ABA revealed that recombinant CHLH did not bind ABA, but recombinant pyrabactin resistance 1, a reliable ABA receptor used as a control, showed specific binding. Moreover, we found that the rtl1 mutant showed ABA-induced stomatal closure when a high concentration of extracellular Ca2+ was present and that a knockout mutant of Mg-chelatase I subunit (chli1) showed the same ABA-insensitive phenotype as rtl1. These results suggest that the Mg-chelatase complex as a whole affects the ABA-signaling pathway for stomatal movements

Characterization of a putative Triticum aestivum abscisic acid receptor and its role in fungal pathogen resistance

Abscisic acid (ABA) has been well defined as an important stress hormone in plants. The signaling pathway of ABA involves a family of pyrabactin resistant-like-1 PYR/PYL/RCAR receptors (PYL receptors) that bind ABA and form a complex with a protein phosphatase 2C (PP2C) family member resulting in downstream signaling events. The ABA receptor family has been well characterized in the model dicot Arabidopsis thaliana and more recently this characterization has branched out into cereals Oryza sativa (rice) and Hordeum vulgare (barley), as well as the monocot model plant Brachypodium distachyon and Fragaria vesca (strawberry). The analysis of these characterized ABA receptors and the use of online databases has allowed the identification of multiple putative ABA receptors in Triticum aestivum (wheat). ABA has been historically called a positive effector. Overexpression of proteins in the ABA signalling pathway or exogenous application of ABA is known to cause an increase in drought, cold, and salt tolerance. More recently ABA has been linked to increased fungal susceptibility in several plants. The role ABA plays in the biotic stress response is still largely unexplored. The focus of this project was to identify and characterize a putative wheat ABA receptor through bioinformatics and an in vitro enzyme activity assay, and use virus induced gene silencing (VIGS) to test what role this receptor plays in fugal susceptibility. A total of 13 putative ABA receptors were located, nine of which are unique between the wheat subgenomes. One receptor TaPYL5.1 was recombinantly expressed, purified, and confirmed as an ABA receptor through a phosphatase based enzyme activity assay. A receptor with high sequence identity to TaPYL5.1, TaPYL5.2A, was targeted for plant trials because the TaPYL5.1 plasmid sequence was codon optimized. A VIGS approach was used to knock down TaPYL5.2A in planta. The TaPYL5.2A knockdown plants were found to have an increased resistance to Fusarium Head Blight progression in the early stages of the disease. In conclusion, wheat ABA receptors were successfully identified and an important correlation between decreased receptor levels and increased early Fusarium Head Blight resistance was found. This correlation however was not easily reproducible due to the severity of coupling VIGS with Fusarium Head Blight, and should be followed up with additional studies looking at the broader family of wheat ABA receptors

LANCL1 binds abscisic acid and stimulates glucose transport and mitochondrial respiration in muscle cells via the AMPK/PGC-1α/Sirt1 pathway

Objective: Abscisic acid (ABA) is a plant hormone also present and active in animals. In mammals, ABA regulates blood glucose levels by stimulating insulin-independent glucose uptake and metabolism in adipocytes and myocytes through its receptor LANCL2. The objective of this study was to investigate whether another member of the LANCL protein family, LANCL1, also behaves as an ABA receptor and, if so, which functional effects are mediated by LANCL1. Methods: ABA binding to human recombinant LANCL1 was explored by equilibrium-binding experiments with [3H]ABA, circular dichroism, and surface plasmon resonance. Rat L6 myoblasts overexpressing either LANCL1 or LANCL2, or silenced for the expression of both proteins, were used to investigate the basal and ABA-stimulated transport of a fluorescent glucose analog (NBDG) and the signaling pathway downstream of the LANCL proteins using Western blot and qPCR analysis. Finally, glucose tolerance and sensitivity to ABA were compared in LANCL2−/− and wild-type (WT) siblings. Results: Human recombinant LANCL1 binds ABA with a Kd between 1 and 10 μM, depending on the assay (i.e., in a concentration range that lies between the low and high-affinity ABA binding sites of LANCL2). In L6 myoblasts, LANCL1 and LANCL2 similarly, i) stimulate both basal and ABA-triggered NBDG uptake (4-fold), ii) activate the transcription and protein expression of the glucose transporters GLUT4 and GLUT1 (4-6-fold) and the signaling proteins AMPK/PGC-1α/Sirt1 (2-fold), iii) stimulate mitochondrial respiration (5-fold) and the expression of the skeletal muscle (SM) uncoupling proteins sarcolipin (3-fold) and UCP3 (12-fold). LANCL2−/− mice have a reduced glucose tolerance compared to WT. They spontaneously overexpress LANCL1 in the SM and respond to chronic ABA treatment (1 μg/kg body weight/day) with an improved glycemia response to glucose load and an increased SM transcription of GLUT4 and GLUT1 (20-fold) of the AMPK/PGC-1α/Sirt1 pathway and sarcolipin, UCP3, and NAMPT (4- to 6-fold). Conclusions: LANCL1 behaves as an ABA receptor with a somewhat lower affinity for ABA than LANCL2 but with overlapping effector functions: stimulating glucose uptake and the expression of muscle glucose transporters and mitochondrial uncoupling and respiration via the AMPK/PGC-1α/Sirt1 pathway. Receptor redundancy may have been advantageous in animal evolution, given the role of the ABA/LANCL system in the insulin-independent stimulation of cell glucose uptake and energy metabolism

Role of the ABA/LANCL system in the insulin-independent stimulation of cell glucose uptake and energy metabolism in myoblasts and adipocytes through an AMPK-mediated mechanism

Abscisic acid (ABA) is a hormone with a very long evolutionary history, dating back to the earliest living organisms, of which modern (ABA-producing) cyanobacteria are likely the descendants, well before separation of the plant and animal kingdoms, with a conserved role as a signal regulating cell responses to environmental challenge. Nanomolar ABA is also present and active in mammals where it controls the metabolic response to glucose availability by stimulating glucose uptake in skeletal muscle and adipose tissue with an insulin-independent mechanism and increasing brown adipose tissue activity and browning through its receptor LANCL2. Previous data showed that, in LANCL2-/- mice, the genetic ablation of the receptor doesn\u2019t modify fasting glycemia values but results in the reduction of glucose tolerance compared with wild-type siblings. Animals are still responsive to exogenous ABA. The result that the effect of ABA was not completely abrogated by LANCL2 silencing, despite the very high percentage of reduction of protein and mRNA expression, suggests that other ABA receptors might be partly involved in the effect of ABA on muscle. LANCL1, a LANCL2 homolog, is indeed expressed in murine skeletal muscle at levels like those of LANCL2. Thus, I focused my attention on the metabolic function of the ABA/LANCL1 system. Recombinant human LANCL1 protein binds ABA with a Kd in a concentration range that lies between the low and high-affinity ABA binding sites of LANCL2. In rat L6 myoblasts, LANCL receptors similarly stimulate both basal and ABA-triggered fluorescently labeled deoxyglucose analog 2-NBDG uptake, activate mRNA levels and protein expression of the glucose transporters GLUT1 and GLUT4 and the signaling proteins AMPK/PGC-1\u3b1/Sirt1, stimulate mitochondrial respiration and the expression of the skeletal muscle (SM) uncoupling proteins sarcolipin and UCP3. Moreover, LANCL2-/- mice overexpress LANCL1 in the SM and respond to chronic ABA treatment (1 \ub5g/kg body weight/day) with an improved glycemia response to glucose load and increased SM transcription of GLUTs, of the AMPK/PGC-1\u3b1/Sirt1 pathway and of sarcolipin, UCP3 and NAMPT. LANCL1 behaves as an ABA receptor with a somewhat lower affinity for ABA than LANCL2 but with overlapping effector functions: stimulating glucose uptake and the expression of muscle glucose transporters and mitochondrial uncoupling and respiration via the AMPK/PGC-1\u3b1/Sirt1 pathway. Furthermore, since adipose tissue seems to be a direct target of ABA, along with skeletal muscle, I investigated the browning effects of ABA in brown and white human adipocytes. ABA increases AMPK phosphorylation and AMPK and PGC-1\u3b1 protein levels in pre-adipocytes overexpressing LANCL1 and LANCL2. The expression of genes related to browning, oxidative consumption, glucose transport, mitochondrial biogenesis, respiratory uncoupling and AMPK/PGC-1\u3b1/Sirt1 pathway is increased after adipocyte differentiation and further increases upon ABA treatment. Chronic ABA treatment stimulates mitochondrial DNA content in the brown adipose tissue of mice. These results suggest that ABA may be used as an anti-obesity molecule. Concluding, receptor redundancy may have been advantageous in animal evolution, given the role of the ABA/LANCL1-2 system in the insulin-independent stimulation of cell glucose uptake and energy metabolism

G-protein coupling and nuclear translocation of the human abscisic acid receptor LANCL2

Abscisic acid (ABA), a long known phytohormone, has been recently demonstrated to be present also in humans, where it targets cells of the innate immune response, mesenchymal and hemopoietic stem cells and cells involved in the regulation of systemic glucose homeostasis. LANCL2, a peripheral membrane protein, is the mammalian ABA receptor. We show that N-terminal glycine myristoylation causes LANCL2 localization to the plasmamembrane and to cytoplasmic membrane vesicles, where it interacts with the \u3b1 subunit of a Gi protein and starts the ABA signaling pathway via activation of adenylate cyclase. Demyristoylation of LANCL2 by chemical or genetic means triggers its nuclear translocation. Nuclear enrichment of native LANCL2 is also induced by ABA treatment. Therefore human LANCL2 is a non-transmembrane G protein-coupled receptor susceptible to hormone-induced nuclear translocation

“Role of LANCL1 and LANCL2 in the physiology and biochemistry of myocytes and cardiomyocytes.”

Background. Abscisic acid (ABA) is an isoprenoid hormone present in unicellular organisms and conserved across kingdoms in modern plants and animals. In mammals, ABA is produced by different cell types and is involved in tissue-specific physiological functions, such as inflammation, hemopoietic stem cell regeneration and control of blood glucose levels. The latter occurs through the insulin-independent stimulation of glucose uptake and metabolism in adipose tissue and skeletal muscle through its receptor LANCL2. The aim of this study was to investigate whether: • LANCL1, another member of the LANCL protein family, also behaves as an ABA receptor, sharing signalling pathway and functional properties with LANCL2 in the control of muscle glucose uptake; • ABA could improve glycemic control in a murine model of (insulin-deficient) type 1 diabetes. Methods. Rat L6 myoblasts overexpressing or silenced for LANCL1 and LANCL2, treated or not with ABA, were used to investigate the signalling pathway downstream of LANCL1 by Western Blot and qPCR experiments. Energy metabolism was studied in H9C2 cardiomyoblasts overexpressing or silenced for LANCL1/2 by monitoring the mitochondrial proton gradient. Finally, experiments were performed on LANCL2 knock-out mice, which overexpress LANCL1 in skeletal muscle, to investigate whether ABA could improve glucose tolerance after induction of insulin deficiency with streptozotocin. Results. In L6 cells, both LANCL1 and LANCL2 mediate basal and ABA-stimulated glucose uptake (4-fold), activate transcription and protein expression of the glucose transporters GLUT4 and GLUT1 (4-6-fold) and the AMPK/PGC-1a/Sirt1 signalling proteins (2-fold). In H9c2, overexpression of LANCL1 and LANCL2 increases, while their combined silencing almost abrogates, the mitochondrial proton gradient. LANCL2 knock-out mice have a reduced glucose tolerance compared to WT, but they do respond to chronic ABA treatment (1 µg/kg BW/day) with an improved glycemia response to glucose load and an increased skeletal muscle transcription of GLUT4, GLUT1 (20-fold) and of the AMPK/PGC-1a/Sirt1 axis. In mice rendered diabetic with low dose streptozotocin, chronic treatment with ABA improves glycemia control. In mice treated with high-dose streptozotocin (causing complete insulin deficiency) treatment with ABA improves the effect of low-dose insulin on glycemia control. In diabetic mice, ABA treatment increases skeletal muscle expression of the AMPK/PGC-1a/Sirt1 axis, of glucose transporters GLUT1/4, of the insulin receptor and of glycolytic enzymes. Conclusions. Having regard to the results of this study, the conclusions are: • LANCL1 shares with LANCL2 similar properties as an ABA receptor, stimulating glucose transport in skeletal muscle, mitochondrial function in cardiomyoblasts and whole-body glucose disposal. • ABA supplementation in conjunction with insulin holds promise as a means to reduce the dose of insulin required for glycemia control, reducing the risk of hypoglycaemia, and improving skeletal muscle insulin sensitivity and glucose consumption as well as cardiomyocyte mitochondrial function

Anaplastic Lymphoma Kinase Receptor: Possible Involvement in Anorexia Nervosa

The pathophysiology of Anorexia Nervosa (AN) has not been fully elucidated. Anaplastic lymphoma kinase (ALK) receptor is a protein-tyrosine kinase mainly known as a key oncogenic driver. Recently, a genetic deletion of ALK in mice has been found to increase energy expenditure and confers resistance to obesity in these animals, suggesting its role in the regulation of thinness. Here, we investigated the expression of ALK and the downstream intracellular pathways in female rats subjected to the activity-based anorexia (ABA) model, which reproduces important features of human AN. In the hypothalamic lysates of ABA rats, we found a reduction in ALK receptor expression, a downregulation of Akt phosphorylation, and no change in the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation. After the recovery from body weight loss, ALK receptor expression returned to the control baseline values, while it was again suppressed during a second cycle of ABA induction. Overall, this evidence suggests a possible involvement of the ALK receptor in the pathophysiology of AN, that may be implicated in its stabilization, resistance, and/or its exacerbation

Effects of abscisic acid on ethylene biosynthesis and perception in Hibiscus rosa-sinensis L. flower development

The effect of the complex relationship between ethylene and abscisic acid (ABA) on flower development and senescence in Hibiscus rosa-sinensis L. was investigated. Ethylene biosynthetic (HrsACS and HrsACO) and receptor (HrsETR and HrsERS) genes were isolated and their expression evaluated in three different floral tissues (petals, style\u2013stigma plus stamens, and ovaries) of detached buds and open flowers. This was achieved through treatment with 0.1 mM 1-aminocyclopropane-1-carboxylic acid (ACC) solution, 500 nl l21 methylcyclopropene (1-MCP), and 0.1 mM ABA solution. Treatment with ACC and 1-MCP confirmed that flower senescence in hibiscus is ethylene dependent, and treatment with exogenous ABA suggested that ABA may play a role in this process. The 1-MCP impeded petal in-rolling and decreased ABA content in detached open flowers after 9 h. This was preceded by an earlier and sequential increase in ABA content in 1-MCP-treated petals and style\u2013stigma plus stamens between 1 h and 6 h. ACC treatment markedly accelerated flower senescence and increased ethylene production after 6 h and 9 h, particularly in style\u2013stigma plus stamens. Ethylene evolution was positively correlated in these floral tissues with the induction of the gene expression of ethylene biosynthetic and receptor genes. Finally, ABA negatively affected the ethylene biosynthetic pathway and tissue sensitivity in all flower tissues. Transcript abundance of HrsACS, HrsACO, HrsETR, and HrsERS was reduced by exogenous ABA treatment. This research underlines the regulatory effect of ABA on the ethylene biosynthetic and perception machinery at a physiological and molecular level when inhibitors or promoters of senescence are exogenously applied

The GCR2 Gene Family Is Not Required for ABA Control of Seed Germination and Early Seedling Development in Arabidopsis

BACKGROUND: The plant hormone abscisic acid (ABA) regulates diverse processes of plant growth and development. It has recently been proposed that GCR2 functions as a G-protein-coupled receptor (GPCR) for ABA. However, the structural relationships and functionality of GCR2 have been challenged by several independent studies. A central question in this controversy is whether gcr2 mutants are insensitive to ABA, because gcr2 mutants were shown to display reduced sensitivity to ABA under one experimental condition (e.g. 22 degrees C, continuous white light with 150 micromol m(-2) s(-1)) but were shown to display wild-type sensitivity under another slightly different condition (e.g. 23 degrees C, 14/10 hr photoperiod with 120 micromol m(-2) s(-1)). It has been hypothesized that gcr2 appears only weakly insensitive to ABA because two other GCR2-like genes in Arabidopsis, GCL1 and GCL2, compensate for the loss of function of GCR2. PRINCIPAL FINDINGS: In order to test this hypothesis, we isolated a putative loss-of-function allele of GCL2, and then generated all possible combinations of mutations in each member of the GCR2 gene family. We found that all double mutants, including gcr2 gcl1, gcr2 gcl2, gcl1 gcl2, as well as the gcr2 gcl1 gcl2 triple mutant displayed wild-type sensitivity to ABA in seed germination and early seedling development assays, demonstrating that the GCR2 gene family is not required for ABA responses in these processes. CONCLUSION: These results provide compelling genetic evidence that GCR2 is unlikely to act as a receptor for ABA in the context of either seed germination or early seedling development