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The methylerythritol phosphate (MEP) pathway is responsible for producing isoprenoids, metabolites with essential functions in the bacterial kingdom and plastid-bearing organisms including plants and Apicomplexa. Additionally, the MEP-pathway intermediate methylerythritol cyclodiphosphate (MEcPP) serves as a plastid-to-nucleus retrograde signal. A suppressor screen of the high MEcPP accumulating mutant plant (ceh1) led to the isolation of 3 revertants (designated Rceh1-3) resulting from independent intragenic substitutions of conserved amino acids in the penultimate MEP-pathway enzyme, hydroxymethylbutenyl diphosphate synthase (HDS). The revertants accumulate varying MEcPP levels, lower than that of ceh1, and exhibit partial or full recovery of MEcPP-mediated phenotypes, including stunted growth and induced expression of stress response genes and the corresponding metabolites. Structural modeling of HDS and ligand docking spatially position the substituted residues at the MEcPP binding pocket and cofactor binding domain of the enzyme. Complementation assays confirm the role of these residues in suppressing the ceh1 mutant phenotypes, albeit to different degrees. In vitro enzyme assays of wild type and HDS variants exhibit differential activities and reveal an unanticipated mismatch between enzyme kinetics and the in vivo MEcPP levels in the corresponding Rceh lines. Additional analyses attribute the mismatch, in part, to the abundance of the first and rate-limiting MEP-pathway enzyme, DXS, and further suggest MEcPP as a rheostat for abundance of the upstream enzyme instrumental in fine-tuning of the pathway flux. Collectively, this study identifies critical residues of a key MEP-pathway enzyme, HDS, valuable for synthetic engineering of isoprenoids, and as potential targets for rational design of antiinfective drugs.

Cellular adaptive responses arise from an array of spatially and temporally distinct biochemical interactions that modulate biological processes and reorganize subcellular structures tailored to the nature of stimulus. As such, cells have evolved elegantly and tightly regulated mechanisms to enable interorganellar communication in part through the dynamic readjustment of physical distance enabling the tethering between two closely apposed membranous organelles and thus formation of Membrane Contact Sites (MCSs). MCSs are dynamic and ubiquitous interorganellar structures that serve as regulatory interfaces to facilitate transmission of signals and to integrate synthesis of metabolic pathways such as lipids required for upholding cellular homeostasis in response to environmental and developmental inputs. Endoplasmic reticulum (ER) is the most copious endomembrane system that extend throughout the cell, and functions in production, processing, and transport of proteins and lipids, as well as in intracellular signaling. Reminiscent of the ancient Silk Road, ER connection to other membranous organelles via MCSs alters cellular landscape and serves as nexus for coordinating exchange of metabolites such as lipids, ions such as Ca2+, and other small molecules involved in maintaining cellular integrity under prevailing conditions. Delineating the molecular organization of the tethering complexes, molecular action of exchanged molecules and hence the nature of information transmitted will afford insight into underlying basis of interorganellar communication and shed light on the evolutionarily conserved function of ER as the ancient trans-kingdom Silk Road trafficking vital metabolites via the non-vesicular pathway.

The ancient morphoregulatory hormone auxin dynamically realigns dedicated cellular processes that shape plant growth under prevailing environmental conditions. However, the nature of the stress-responsive signal altering auxin homeostasis remains elusive. Here we establish that the evolutionarily conserved plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) controls adaptive growth by dual transcriptional and post-translational regulatory inputs that modulate auxin levels and distribution patterns in response to stress. We demonstrate that in vivo accumulation or exogenous application of MEcPP alters the expression of two auxin reporters, DR5:GFP and DII-VENUS, and reduces the abundance of the auxin-efflux carrier PIN-FORMED1 (PIN1) at the plasma membrane. However, pharmacological intervention with clathrin-mediated endocytosis blocks the PIN1 reduction. This study provides insight into the interplay between these two indispensable signaling metabolites by establishing the mode of MEcPP action in altering auxin homeostasis, and as such, positioning plastidial function as the primary driver of adaptive growth.

Plants have evolved tightly regulated signaling networks to respond and adapt to environmental perturbations, but the nature of the signaling hub(s) involved have remained an enigma. We have previously established that methylerythritol cyclodiphosphate (MEcPP), a precursor of plastidial isoprenoids and a stress-specific retrograde signaling metabolite, enables cellular readjustments for high-order adaptive functions. Here, we specifically show that MEcPP promotes two Brassicaceae-specific traits, namely endoplasmic reticulum (ER) body formation and induction of indole glucosinolate (IGs) metabolism selectively, via transcriptional regulation of key regulators NAI1 for ER body formation and MYB51/122 for IGs biosynthesis). The specificity of MEcPP is further confirmed by the lack of induction of wound-inducible ER body genes as well as IGs by other altered methylerythritol phosphate pathway enzymes. Genetic analyses revealed MEcPP-mediated COI1-dependent induction of these traits. Moreover, MEcPP signaling integrates the biosynthesis and hydrolysis of IGs through induction of nitrile-specifier protein1 and reduction of the suppressor, ESM1, and production of simple nitriles as the bioactive end product. The findings position the plastidial metabolite, MEcPP, as the initiation hub, transducing signals to adjust the activity of hard-wired gene circuitry to expand phytochemical diversity and alter the associated subcellular structure required for functionality of the secondary metabolites, thereby tailoring plant stress responses.

To maintain homeostasis in the face of intrinsic and extrinsic insults, cells have evolved elaborate quality control networks to resolve damage at multiple levels. Interorganellar communication is a key requirement for this maintenance, however the underlying mechanisms of this communication have remained an enigma. Here we integrate the outcome of transcriptomic, proteomic, and metabolomics analyses of genotypes including ceh1, a mutant with constitutively elevated levels of both the stress-specific plastidial retrograde signaling metabolite methyl-erythritol cyclodiphosphate (MEcPP) and the defense hormone salicylic acid (SA), as well as the high MEcPP but SA deficient genotype ceh1/eds16, along with corresponding controls. Integration of multi-omic analyses enabled us to delineate the function of MEcPP from SA, and expose the compartmentalized role of this retrograde signaling metabolite in induction of distinct but interdependent signaling cascades instrumental in adaptive responses. Specifically, here we identify strata of MEcPP-sensitive stress-response cascades, among which we focus on selected pathways including organelle-specific regulation of jasmonate biosynthesis; simultaneous induction of synthesis and breakdown of SA; and MEcPP-mediated alteration of cellular redox status in particular glutathione redox balance. Collectively, these integrated multi-omic analyses provided a vehicle to gain an in-depth knowledge of genome-metabolism interactions, and to further probe the extent of these interactions and delineate their functional contributions. Through this approach we were able to pinpoint stress-mediated transcriptional and metabolic signatures and identify the downstream processes modulated by the independent or overlapping functions of MEcPP and SA in adaptive responses.

The exquisite harmony between hormones and their corresponding signaling pathways is central to prioritizing plant responses to simultaneous and/or successive environmental trepidations. The crosstalk between jasmonic acid (JA) and salicylic acid (SA) is an established effective mechanism that optimizes and tailors plant adaptive responses. However, the underlying regulatory modules of this crosstalk are largely unknown. Global transcriptomic analyses of mutant plants (ceh1) with elevated levels of the stress-induced plastidial retrograde signaling metabolite 2-C-methyl-D-erythritol cyclopyrophosphate (MEcPP) revealed robustly induced JA marker genes, expected to be suppressed by the presence of constitutively high SA levels in the mutant background. Analyses of a range of genotypes with varying SA and MEcPP levels established the selective role of MEcPP-mediated signal(s) in induction of JA-responsive genes in the presence of elevated SA. Metabolic profiling revealed the presence of high levels of the JA precursor 12-oxo-phytodienoic acid (OPDA), but near wild type levels of JA in the ceh1 mutant plants. Analyses of coronatine-insensitive 1 (coi1)/ceh1 double mutant plants confirmed that the MEcPP-mediated induction is JA receptor COI1 dependent, potentially through elevated OPDA. These findings identify MEcPP as a previously unrecognized central regulatory module that induces JA-responsive genes in the presence of high SA, thereby staging a multifaceted plant response within the environmental context.

Cellular homeostasis in response to internal and external stimuli requires a tightly coordinated interorgannellar communication network. We recently identified methylerythritol cyclodiphosphate (MEcPP) as a novel stress-specific retrograde signaling metabolite that accumulates in response to environmental perturbations to relay information from plastids to the nucleus. We now demonstrate, using a combination of transcriptome and proteome profiling approaches, that mutant plants (ceh1) with high endogenous levels of MEcPP display increased transcript and protein levels for a subset of the core unfolded protein response (UPR) genes. The UPR is an adaptive cellular response conserved throughout eukaryotes to stress conditions that perturb the endoplasmic reticulum (ER) homeostasis. Our results suggest that MEcPP directly triggers the UPR. Exogenous treatment with MEcPP induces the rapid and transient induction of both the unspliced and spliced forms of the UPR gene bZIP60. Moreover, compared with the parent background (P), ceh1 mutants are less sensitive to the ER-stress-inducing agent tunicamycin (Tm). P and ceh1 plants treated with Tm display similar UPR transcript profiles, suggesting that although MEcPP accumulation causes partial induction of selected UPR genes, full induction is triggered by accumulation of misfolded proteins. This finding refines our perspective of interorgannellar communication by providing a link between a plastidial retrograde signaling molecule and its targeted ensemble of UPR components in ER.