Plant Biology
The ferredoxin/thioredoxin pathway constitutes an indispensable redox-signaling cascade for light-dependent reduction of chloroplast stromal proteins
To ensure efficient photosynthesis, chloroplast proteins need to be flexibly regulated under fluctuating light conditions. Thiol-based redox regulation plays a key role in reductively activating several chloroplast proteins in a light-dependent manner. The ferredoxin (Fd)/thioredoxin (Trx) pathway has long been recognized as the machinery that transfers reducing power generated by photosynthetic electron transport reactions to redox-sensitive target proteins; however, its biological importance remains unclear, because the complete disruption of the Fd/Trx pathway in plants has been unsuccessful to date.A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase
The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation.Intrinsically disordered plant protein PARCL colocalizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD
In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins facilitate RNA translocation.Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins
Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔμH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔμH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions.The major trimeric antenna complexes serve as a site for qH-energy dissipation in plants
Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants’ survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state.Evolution and molecular basis of substrate specificity in a 3-ketoacyl-CoA synthase gene cluster from Populus trichocarpa
Very long chain fatty acids (VLCFAs) are precursors to sphingolipids, glycerophospholipids, and plant cuticular waxes. In plants, members of a large 3-ketoacyl-CoA synthase (KCS) gene family catalyze the substrate-specific elongation of VLCFAs. Although it is well understood that KCSs have evolved to use diverse substrates, the underlying molecular determinants of their specificity are still unclear. In this study, we exploited the sequence similarity of a KCS gene cluster from Populus trichocarpa to examine the evolution and molecular determinants of KCS substrate specificity.Chloroplast envelope ATPase PGA1/AtFtsH12 is required for chloroplast protein accumulation and cytosol-chloroplast protein homeostasis in Arabidopsis
The establishment of photosynthetic protein complexes during chloroplast development requires the influx of a large number of chloroplast proteins that are encoded by the nuclear genome, which is critical for cytosol and chloroplast protein homeostasis and chloroplast development. However, the mechanisms regulating this process are still not well understood in higher plants. Here, we report the isolation and characterization of the pale green Arabidopsis pga1-1 mutant, which is defective in chloroplast development and chloroplast protein accumulation.Ubiquitin-conjugating activity by PEX4 is required for efficient protein transport to peroxisomes in Arabidopsis thaliana
Protein transport to peroxisomes requires various proteins, such as receptors in the cytosol and components of the transport machinery on peroxisomal membranes. The Arabidopsis apem (aberrant peroxisome morphology) mutant apem7 shows decreased efficiency of peroxisome targeting signal 1–dependent protein transport to peroxisomes. In apem7 mutants, peroxisome targeting signal 2–dependent protein transport is also disturbed, and plant growth is repressed. The APEM7 gene encodes a protein homologous to peroxin 4 (PEX4), which belongs to the ubiquitin-conjugating (UBC) protein family; however, the UBC activity of Arabidopsis PEX4 remains to be investigated.Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin β-ODAP in grass pea
Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified.Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition
Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3′-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin.The plastoglobule-localized protein AtABC1K6 is a Mn2+-dependent kinase necessary for timely transition to reproductive growth
The Absence of bc1 Complex (ABC1) is an ancient, atypical protein kinase family that emerged prior to the archaeal-eubacterial divergence. Loss-of-function mutants in ABC1 genes are linked to respiratory defects in microbes and humans and to compromised photosynthetic performance and stress tolerance in plants. However, demonstration of protein kinase activity remains elusive, hampering their study. Here, we investigate a homolog from Arabidopsis thaliana, AtABC1K6, and demonstrate in vitro autophosphorylation activity, which we replicate with a human ABC1 ortholog.The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18
The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain–containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins.A novel strigolactone receptor antagonist provides insights into the structural inhibition, conditioning, and germination of the crop parasite Striga
Crop parasites of the Striga genera are a major biological deterrent to food security in Africa and are one of the largest obstacles to poverty alleviation on the continent. Striga seeds germinate by sensing small-molecule hormones, strigolactones (SLs), that emanate from host roots. Although SL receptors (Striga hermonthica HYPOSENSITIVE TO LIGHT [ShHTL]) have been identified, discerning their function has been difficult because these parasites cannot be easily grown under laboratory conditions.Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system
The chloroplast chaperone CLPC1 unfolds and delivers substrates to the stromal CLPPRT protease complex for degradation. We previously used an in vivo trapping approach to identify interactors with CLPC1 in Arabidopsis thaliana by expressing a STREPII-tagged copy of CLPC1 mutated in its Walker B domains (CLPC1-TRAP) followed by affinity purification and mass spectrometry. To create a larger pool of candidate substrates, adaptors, or regulators, we carried out a far more sensitive and comprehensive in vivo protein trapping analysis.Protein–protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain
A network of protein–protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression.A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (coenzyme Q) in Arabidopsis
Ubiquinone (Coenzyme Q) is a vital respiratory cofactor and liposoluble antioxidant. In plants, it is not known how the C-6 hydroxylation of demethoxyubiquinone, the penultimate step in ubiquinone biosynthesis, is catalyzed. The combination of cross-species gene network modeling along with mining of embryo-defective mutant databases of Arabidopsis thaliana identified the embryo lethal locus EMB2421 (At1g24340) as a top candidate for the missing plant demethoxyubiquinone hydroxylase. In marked contrast with prototypical eukaryotic demethoxyubiquinone hydroxylases, the catalytic mechanism of which depends on a carboxylate-bridged di-iron domain, At1g24340 is homologous to FAD-dependent oxidoreductases that instead use NAD(P)H as an electron donor.UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis
Glycosyltransferases constitute a large family of enzymes across all domains of life, but knowledge of their biochemical function remains largely incomplete, particularly in the context of plant specialized metabolism. The labdane diterpenes represent a large class of phytochemicals with many pharmacological benefits, such as anti-inflammatory, hepatoprotective, and anticarcinogenic. The medicinal plant kalmegh (Andrographis paniculata) produces bioactive labdane diterpenes; notably, the C19-hydroxyl diterpene (andrograpanin) is predominantly found as C19-O-glucoside (neoandrographolide), whereas diterpenes having additional hydroxylation(s) at C3 (14-deoxy-11,12-didehydroandrographolide) or C3 and C14 (andrographolide) are primarily detected as aglycones, signifying scaffold-selective C19-O-glucosylation of diterpenes in planta.Nanoscale dynamics of cellulose digestion by the cellobiohydrolase TrCel7A
Understanding the mechanism by which cellulases from bacteria, fungi, and protozoans catalyze the digestion of lignocellulose is important for developing cost-effective strategies for bioethanol production. Cel7A from the fungus Trichoderma reesei is a model exoglucanase that degrades cellulose strands from their reducing ends by processively cleaving individual cellobiose units. Despite being one of the most studied cellulases, the binding and hydrolysis mechanisms of Cel7A are still debated. Here, we used single-molecule tracking to analyze the dynamics of 11,116 quantum dot-labeled TrCel7A molecules binding to and moving processively along immobilized cellulose.The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding
The dynamic responses of microtubules (MTs) to internal and external signals are modulated by a plethora of microtubule-associated proteins (MAPs). In higher plants, many plant-specific MAPs have emerged during evolution as advantageous to their sessile lifestyle. Some members of the IQ67 domain (IQD) protein family have been shown to be plant-specific MAPs. However, the mechanisms of interaction between IQD proteins and MTs remain elusive. Here we demonstrate that the domain of unknown function 4005 (DUF4005) of the Arabidopsis IQD family protein ABS6/AtIQD16 is a novel MT-binding domain.Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defense
Microbial plant pathogens secrete effector proteins, which manipulate the host to promote infection. Effectors can be recognized by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognized by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a noncanonical integrated heavy-metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defenses.The acyltransferase PMAT1 malonylates brassinolide glucoside
Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown.Arabidopsis thaliana 3-mercaptopyruvate sulfurtransferases interact with and are protected by reducing systems
The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems.Phytol derived from chlorophyll hydrolysis in plants is metabolized via phytenal
Phytol is the isoprenoid alcohol bound in ester linkage to chlorophyll, the most abundant photosynthetic pigment in plants. During leaf senescence, large amounts of phytol are released by chlorophyll degradation. However, the pathway of phytol catabolism in plants is unknown. We hypothesized that phytol degradation in plants might involve its oxidation into the long-chain aldehyde phytenal. Using GC-MS for aldehyde quantification after derivatization with methylhydroxylamine, phytenal was identified in leaves, whereas other long-chain aldehydes (phytanal and pristanal) were barely detectable.Multiple copies of a novel amphipathic α-helix forming segment in Physcomitrella patens dehydrin play a key role in abiotic stress mitigation
Plants use a diverse set of proteins to mitigate various abiotic stresses. The intrinsically disordered protein dehydrin is an important member of this repertoire of proteins, characterized by a canonical amphipathic K-segment. It can also contain other stress-mitigating noncanonical segments—a likely reflection of the extremely diverse nature of abiotic stress encountered by plants. Among plants, the poikilohydric mosses have no inbuilt mechanism to prevent desiccation and therefore are likely to contain unique noncanonical stress-responsive motifs in their dehydrins.Chemokine-like MDL proteins modulate flowering time and innate immunity in plants
Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes.
