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1.
Practical optimization of liquid chromatography/mass spectrometry conditions and pretreatment methods toward the sensitive quantification of auxin in plants.
Sugahara, K, Kitao, K, Yamagaki, T, Koyama, T
Rapid communications in mass spectrometry : RCM. 2020;(7):e8625
Abstract
RATIONALE The plant hormone auxin, indole-3-acetic acid, regulates many aspects of plant growth and development. Auxin quantification should offer broad insights into its mechanistic action in plants. However, limited auxin content in plant tissues hampers the establishment of quantification methods without the highest graded instruments or deeply specialized experimental techniques. METHODS In this study, we detailed optimized conditions for high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry (LC/MS). We compared LC/MS conditions, such as columns, mobile phases, parameters of acquisition methods (selective or multiple ion monitoring), dwell times (DTs), and channel numbers, using differentially mixed authentic auxin and its related compounds. We further investigated pretreatment methods through the optimization of auxin recovery and irrelative compound removal from plant tissues prior to the LC/MS analysis. RESULTS Our LC/MS analysis demonstrated the particular importance of the column, DTs, and channel numbers on detection sensitivity. Our comparative analysis developed optimal pretreatment methods, including the pulverization of plants, concentration of extract through centrifugal evaporation, and removal of irrelative metabolites using liquid-liquid extraction and a spin filter. We injected plant samples into our LC/MS system, quantified auxin and eight related compounds in a single measurement, and determined the auxin increase in an auxin over-producing mutant. CONCLUSIONS Our practical optimization of LC/MS conditions and pretreatment methods provides detailed experimental processes toward the sensitive quantification of auxin from 10 mg of plant tissue. These processes have not always been clearly documented; therefore, our protocol could broadly contribute to technical advances in plant growth and development research.
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New insights into gibberellin signaling in regulating flowering in Arabidopsis.
Bao, S, Hua, C, Shen, L, Yu, H
Journal of integrative plant biology. 2020;(1):118-131
Abstract
In angiosperms, floral transition is a key developmental transition from the vegetative to reproductive growth, and requires precise regulation to maximize the reproductive success. A complex regulatory network governs this transition through integrating flowering pathways in response to multiple exogenous and endogenous cues. Phytohormones are essential for proper plant developmental regulation and have been extensively studied for their involvement in the floral transition. Among various phytohormones, gibberellin (GA) plays a major role in affecting flowering in the model plant Arabidopsis thaliana. The GA pathway interact with other flowering genetic pathways and phytohormone signaling pathways through either DELLA proteins or mediating GA homeostasis. In this review, we summarize the recent advances in understanding the mechanisms of DELLA-mediated GA pathway in flowering time control in Arabidopsis, and discuss its possible link with other phytohormone pathways during the floral transition.
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3.
The lipid code-dependent phosphoswitch PDK1-D6PK activates PIN-mediated auxin efflux in Arabidopsis.
Tan, S, Zhang, X, Kong, W, Yang, XL, Molnár, G, Vondráková, Z, Filepová, R, Petrášek, J, Friml, J, Xue, HW
Nature plants. 2020;(5):556-569
Abstract
Directional intercellular transport of the phytohormone auxin mediated by PIN-FORMED (PIN) efflux carriers has essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. PIN activity is therefore regulated by multiple internal and external cues, for which the underlying molecular mechanisms are not fully elucidated. Here, we demonstrate that 3'-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub that perceives upstream lipid signalling and modulates downstream substrate activity through phosphorylation. Using genetic analysis, we show that the loss-of-function Arabidopsis pdk1.1 pdk1.2 mutant exhibits a plethora of abnormalities in organogenesis and growth due to defective polar auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 protein kinase, a well-known upstream activator of PIN proteins. We uncover a lipid-dependent phosphorylation cascade that connects membrane-composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.
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4.
Auxin-Abscisic Acid Interactions in Plant Growth and Development.
Emenecker, RJ, Strader, LC
Biomolecules. 2020;(2)
Abstract
Plant hormones regulate many aspects of plant growth, development, and response to biotic and abiotic stress. Much research has gone into our understanding of individual plant hormones, focusing primarily on their mechanisms of action and the processes that they regulate. However, recent research has begun to focus on a more complex problem; how various plant hormones work together to regulate growth and developmental processes. In this review, we focus on two phytohormones, abscisic acid (ABA) and auxin. We begin with brief overviews of the hormones individually, followed by in depth analyses of interactions between auxin and ABA, focusing on interactions in individual tissues and how these interactions are occurring where possible. Finally, we end with a brief discussion and future prospects for the field.
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5.
Theoretical and experimental approaches to understand the biosynthesis of starch granules in a physiological context.
Pfister, B, Zeeman, SC, Rugen, MD, Field, RA, Ebenhöh, O, Raguin, A
Photosynthesis research. 2020;(1):55-70
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Abstract
Starch, a plant-derived insoluble carbohydrate composed of glucose polymers, is the principal carbohydrate in our diet and a valuable raw material for industry. The properties of starch depend on the arrangement of glucose units within the constituent polymers. However, key aspects of starch structure and the underlying biosynthetic processes are not well understood, limiting progress towards targeted improvement of our starch crops. In particular, the major component of starch, amylopectin, has a complex three-dimensional, branched architecture. This architecture stems from the combined actions of a multitude of enzymes, each having broad specificities that are difficult to capture experimentally. In this review, we reflect on experimental approaches and limitations to decipher the enzymes' specificities and explore possibilities for in silico simulations of these activities. We believe that the synergy between experimentation and simulation is needed for the correct interpretation of experimental data and holds the potential to greatly advance our understanding of the overall starch biosynthetic process. We furthermore propose that the formation of glucan secondary structures, concomitant with its synthesis, is a previously overlooked factor that directly affects amylopectin architecture through its impact on enzyme function.
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6.
Nitrogen-phosphorus interplay: old story with molecular tale.
Hu, B, Chu, C
The New phytologist. 2020;(4):1455-1460
Abstract
Nitrogen (N) and phosphorus (P) are the two most abundant mineral nutrients used by plants, and are also the mostly widely used fertilizer elements driving crop yield improvement in agricultural production. The coordinated utilization of N and P is essential to maintain optimal plant growth and achieve maximal crop yield. The signaling pathways of N and P are generally studied separately, so our understanding of N-P interactions is very limited. A series of recent studies have revealed the critical components regulating N-P interactions in both Arabidopsis thaliana and rice (Oryza sativa), and have shed light on our in-depth understanding of the network integrating N and P signaling pathways. Here, we summarize recent progress on N-P interaction and propose possible working mechanisms integrating these N-P interactive regulation pathways. We further discuss future work that might reveal the N-P interactive regulation network in plants.
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ARABIDOMICS-A new experimental platform for molecular analyses of plants in drop towers, on parabolic flights, and sounding rockets.
Hauslage, J, Görög, M, Krause, L, Schüler, O, Schäfer, M, Witten, A, Kesseler, L, Böhmer, M, Hemmersbach, R
The Review of scientific instruments. 2020;(3):034504
Abstract
Plants represent an essential part of future life support systems that will enable human space travel to distant planets and their colonization. Therefore, insights into changes and adaptations of plants in microgravity are of great importance. Despite considerable efforts, we still know very little about how plants respond to microgravity environments on the molecular level, partly due to a lack of sufficient hardware and flight opportunities. The plant Arabidopsis thaliana, the subject of this study, represents a well-studied model organism in gravitational biology, particularly for the analysis of transcriptional and metabolic changes. To overcome the limitations of previous plant hardware that often led to secondary effects and to allow for the extraction not only of RNA but also of phytohormones and proteins, we developed a new experimental platform, called ARABIDOMICS, for exposure and fixation under altered gravity conditions. Arabidopsis seedlings were exposed to hypergravity during launch and microgravity during the free-fall period of the MAPHEUS 5 sounding rocket. Seedlings were chemically fixed inflight at defined time points, and RNA and phytohormones were subsequently analyzed in the laboratory. RNA and phytohormones extracted from the fixed biological samples were of excellent quality. Changes in the phytohormone content of jasmonate, auxin, and several cytokinins were observed in response to hypergravity and microgravity.
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Deciphering the Binding of Salicylic Acid to Arabidopsis thaliana Chloroplastic GAPDH-A1.
Pokotylo, I, Hellal, D, Bouceba, T, Hernandez-Martinez, M, Kravets, V, Leitao, L, Espinasse, C, Kleiner, I, Ruelland, E
International journal of molecular sciences. 2020;(13)
Abstract
Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA's ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein-ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket-a surface cavity around Asn35-would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.
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Mechanisms regulating PIF transcription factor activity at the protein level.
Favero, DS
Physiologia plantarum. 2020;(3):325-335
Abstract
Plant growth and development are precisely regulated by transcription factors (TFs) such as the PHYTOCHROME INTERACTING FACTORs (PIFs). PIFs regulate growth in response to various internal and external stimuli, and, accordingly, are regulated by a variety of different factors and mechanisms. Canonically, PIF activity is inhibited by light through the sequential phosphorylation, ubiquitination and degradation of these TFs in a manner dependent on their interaction with active phytochrome photoreceptors. However, work in more recent years has revealed that phytochromes also antagonize PIF activity by inhibiting their ability to bind DNA and, at least in the case of PIF7, by causing it to be retained in the cytoplasm. Recent work has also revealed specific kinases, phosphatases and E3 ubiquitin ligases which alter PIFs at the post-translational level. In a few cases, these studies have gone as far as identifying potential kinases responsible for phosphorylating PIFs in response to light. Moreover, additional factors have been identified that positively or negatively affect PIF binding to DNA or bind directly to PIF-DNA complexes and affect the transcriptional activation of target genes by these TFs. This review summarizes the variety of different mechanisms involved in PIF regulation and discusses some of the major unanswered questions in this area of research.
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10.
Low endogenous NO levels in roots and antioxidant systems are determinants for the resistance of Arabidopsis seedlings grown in Cd.
Terrón-Camero, LC, Del Val, C, Sandalio, LM, Romero-Puertas, MC
Environmental pollution (Barking, Essex : 1987). 2020;:113411
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Abstract
Cadmium (Cd), which is a toxic non-essential heavy metal capable of entering plants and thus the food chain, constitutes a major environmental and health concern worldwide. An understanding of the tools used by plants to overcome Cd stress could lead to the production of food crops with lower Cd uptake capacity and of plants with greater Cd uptake potential for phytoremediation purposes in order to restore soil efficiency in self-sustaining ecosystems. The signalling molecule nitric oxide (NO), whose function remains unclear, has recently been involved in responses to Cd stress. Using different mutants, such as nia1nia2, nox1, argh1-1 and Atnoa1, which were altered in NO metabolism, we analysed various parameters related to reactive oxygen and nitrogen species (ROS/RNS) metabolism and seedling fitness following germination and growth under Cd treatment conditions for seven days. Seedling roots were the most affected, with an increase in ROS and RNS observed in wild type (WT) seedling roots, leading to increased oxidative damage and fitness loss. Mutants that showed lower NO levels in seedling roots under Cd stress were more resistant than WT seedlings due to the maintenance of antioxidant systems which protect against oxidative damage.