ABSTRACT
Carotenoids are yellow, orange, and red pigments commonly found in plants. In leaves, these molecules are essential for photosynthesis, but they also play a major role in plant growth and development. Efficiently monitoring concentrations of specific carotenoids in plant tissues could help to explain plant responses to environmental stressors, infection and disease, fertilization, and other conditions. Previously, Raman methods have been used to demonstrate a correlation between plant fitness and the carotenoid content of leaves. Due to solvatochromatic effects and structural similarities within the carotenoid family, current Raman spectroscopy techniques struggle to assign signals to specific carotenoids with certainty, complicating the determination of amounts of individual carotenoids present in a sample. In this work, we use thin layer chromatography-Raman spectroscopy, or TLC-Raman, to identify and quantify carotenoids extracted from tomato leaves. These quick and accurate methods could be applied to study the relationship between pigment content and a number of factors affecting plant health.
Subject(s)
Carotenoids , Plant Leaves , Solanum lycopersicum , Spectrum Analysis, Raman , Plant Leaves/chemistry , Spectrum Analysis, Raman/methods , Chromatography, Thin Layer/methods , Carotenoids/analysis , Carotenoids/chemistry , Solanum lycopersicum/chemistry , Solanum lycopersicum/metabolism , Pigments, Biological/analysis , Pigments, Biological/chemistryABSTRACT
BACKGROUND: Non-caloric artificial sweeteners (NCAS) are widely used as a substitute for dietary sugars to control body weight or glycemia. Paradoxically, some interventional studies in humans and rodents have shown unfavorable changes in glucose homeostasis in response to NCAS consumption. The causative mechanisms are largely unknown, but adverse changes in gut microbiota have been proposed to mediate these effects. These findings have raised concerns about NCAS safety and called into question their broad use, but further physiological and dietary considerations must be first addressed before these results are generalized. We also reasoned that, since NCAS are bona fide ligands for sweet taste receptors (STRs) expressed in the intestine, some metabolic effects associated with NCAS use could be attributed to a common mechanism involving the host. RESULTS: We conducted a double-blind, placebo-controlled, parallel arm study exploring the effects of pure saccharin compound on gut microbiota and glucose tolerance in healthy men and women. Participants were randomized to placebo, saccharin, lactisole (STR inhibitor), or saccharin with lactisole administered in capsules twice daily to achieve the maximum acceptable daily intake for 2 weeks. In parallel, we performed a 10-week study administering pure saccharin at a high dose in the drinking water of chow-fed mice with genetic ablation of STRs (T1R2-KO) and wild-type (WT) littermate controls. In humans and mice, none of the interventions affected glucose or hormonal responses to an oral glucose tolerance test (OGTT) or glucose absorption in mice. Similarly, pure saccharin supplementation did not alter microbial diversity or composition at any taxonomic level in humans and mice alike. No treatment effects were also noted in readouts of microbial activity such as fecal metabolites or short-chain fatty acids (SCFA). However, compared to WT, T1R2-KO mice were protected from age-dependent increases in fecal SCFA and the development of glucose intolerance. CONCLUSIONS: Short-term saccharin consumption at maximum acceptable levels is not sufficient to alter gut microbiota or induce glucose intolerance in apparently healthy humans and mice. TRIAL REGISTRATION: Trial registration number NCT03032640 , registered on January 26, 2017. Video abstract.
Subject(s)
Gastrointestinal Microbiome , Glucose Intolerance , Healthy Volunteers , Saccharin/administration & dosage , Saccharin/pharmacology , Adult , Animals , Double-Blind Method , Female , Gastrointestinal Microbiome/drug effects , Gastrointestinal Microbiome/genetics , Glucose Intolerance/chemically induced , Humans , Male , Mice , Young AdultABSTRACT
Host-plant resistance (HPR) is an important tool for pest management, affording both economic and environmental benefits. The mechanisms of aphid resistance in soybean are not well understood, but likely involve the induction of the jasmonic acid (JA) pathway, and possibly other phytohormone signals involved in plant defense responses. Despite the efficacy of aphid resistance in soybean, virulent aphids have overcome this resistance through mostly unknown mechanisms. Here, we have used metabolomic tools to define the role of plant phytohormones, especially the JA pathway, in regulating interactions between aphid-resistant soybean and virulent aphids. We hypothesized that virulent aphids avoid or suppress the JA pathway to overcome aphid resistance. Our results suggested that aphid-resistant soybean increased accumulation of JA-isoleucine (JA-Ile) only when infested with avirulent aphids; virulent aphids did not cause induction of JA-Ile. Further, applying JA-Ile to aphid-resistant soybean reduced subsequent virulent aphid populations. The concentrations of other phytohormones remained unchanged due to aphid feeding, highlighting the importance of JA-Ile in this interaction. These results increase our knowledge of soybean resistance mechanisms against soybean aphids and contribute to our understanding of aphid virulence mechanisms, which will in turn promote the durability of HPR.
Subject(s)
Aphids , Animals , Cyclopentanes , Isoleucine , Oxylipins , Plant Defense Against Herbivory , Glycine maxABSTRACT
Auxin (indole-3-acetic acid, IAA) has been implicated as a susceptibility factor in both beneficial and pathogenic molecular plant-microbe interactions. Previous studies have identified a large number of auxin-related genes underlying quantitative disease resistance loci (QDRLs) for Phytophthora sojae. Thus, we hypothesized that auxin may be involved the P. sojae-soybean interaction. The levels of IAA and related metabolites were measured in mycelia and media supernatant as well as in mock and inoculated soybean roots in a time course assay. The expression of 11 soybean Pin-formed (GmPIN) auxin efflux transporter genes was also examined. Tryptophan, an auxin precursor, was detected in the P. sojae mycelia and media supernatant. During colonization of roots, levels of IAA and related metabolites were significantly higher in both moderately resistant Conrad and moderately susceptible Sloan inoculated roots compared with mock controls at 48 h postinoculation (hpi) in one experiment and at 72 hpi in a second, with Sloan accumulating higher levels of the auxin catabolite IAA-Ala than Conrad. Additionally, one GmPIN at 24 hpi, one at 48 hpi, and three at 72 hpi had higher expression in inoculated compared with the mock control roots in Conrad. The ability of resistant cultivars to cope with auxin accumulation may play an important role in quantitative disease resistance. Levels of jasmonic acid (JA), another plant hormone associated with defense responses, were also higher in inoculated roots at these same time points, suggesting that JA also plays a role during the later stages of infection.
Subject(s)
Phytophthora , Gene Expression Regulation, Plant , Indoleacetic Acids , Plant Diseases , Plant Roots , Glycine maxABSTRACT
The phenotype of modern commercial turkeys is substantially different than that of unselected, heritage turkey lines. These phenotypic changes have arisen from alterations in the genome/transcriptome, as well as the influence of many external factors on growth performance including nutrition, environment, and management. To investigate the phenotypic changes resulting from genetic selection for increased body weight, The Ohio State University maintains 2 unique genetic turkey lines: the randombred control (RBC2) line, which is comprised of genetics from 1960 era commercial turkeys and has been maintained without conscious selection for any trait; and the F line, which was originally selected from the RBC2 line and has been selected for increased 16 wk body weight for over 50 generations. This study used broad-spectrum mass-spectrometry profiling techniques to identify and quantify differences in the metabolome of the serum of F and RBC2 turkey lines. Serum samples from both F and RBC2 turkeys were subject to quantitative time of flight liquid chromatography tandem mass spectrometry analyses. Principle component analyses showed distinct populations of metabolites in the F vs. RBC2 serum, suggesting that increased body weight is associated with the accumulation of several metabolites. Comparing the spectral features to online databases resulted in the selection of 104 features with potentially identifiable chemical structures. Of these 104 features, 25 were found at higher levels in the serum of the RBC2 line turkeys, while 79 were found at a greater abundance in the F line turkeys. A more detailed analysis of these 104 features allowed for the putative identification of 49 compounds, which were clustered into 6 functional groups: 1) energy metabolism; 2) vitamins; 3) hormones and signaling molecules; 4) lipid derivatives, fatty acid metabolites, and membrane components; 5) amino acid/protein metabolism; and 6) microbial metabolites. Further validation and experimentation is needed to confirm the identity of these metabolites and understand their biological relevance and association with selection for increased body weight.
Subject(s)
Metabolome , Selection, Genetic , Turkeys/genetics , Turkeys/metabolism , Animals , Avian Proteins/analysis , Blood Chemical Analysis/veterinary , Male , Turkeys/bloodABSTRACT
Seasonal nitrogen (N) cycling in Populus, involves bark storage proteins (BSPs) that accumulate in bark phloem parenchyma in the autumn and decline when shoot growth resumes in the spring. Little is known about the contribution of BSPs to growth or the signals regulating N remobilization from BSPs. Knockdown of BSP accumulation via RNAi and N sink manipulations were used to understand how BSP storage influences shoot growth. Reduced accumulation of BSPs delayed bud break and reduced shoot growth following dormancy. Further, 13N tracer studies also showed that BSP accumulation is an important factor in N partitioning from senescing leaves to bark. Thus, BSP accumulation has a role in N remobilization during N partitioning both from senescing leaves to bark and from bark to expanding shoots once growth commences following dormancy. The bark transcriptome during BSP catabolism and N remobilization was enriched in genes associated with auxin transport and signaling, and manipulation of the source of auxin or auxin transport revealed a role for auxin in regulating BSP catabolism and N remobilization. Therefore, N remobilization appears to be regulated by auxin produced in expanding buds and shoots that is transported to bark where it regulates protease gene expression and BSP catabolism.
Subject(s)
Populus , Indoleacetic Acids , Nitrogen , Nitrogen Radioisotopes , Plant Proteins/genetics , Plant Shoots , Populus/genetics , Seasons , TreesABSTRACT
Differences in glucose uptake in peripheral and neural tissues account for the reduced efficacy of insulin in nervous tissues. Herein, we report the design of short peptides, referred as amino acid compounds (AAC) with and without a modified side chain moiety. At nanomolar concentrations, a candidate therapeutic molecule, AAC2, containing a 7-(diethylamino) coumarin-3-carboxamide side-chain improved glucose control in human peripheral adipocytes and the endothelial brain barrier cells by activation of insulin-insensitive glucose transporter 1 (GLUT1). AAC2 interacted specifically with the leptin receptor (LepR) and activated atypical protein kinase C zeta (PKCς) to increase glucose uptake. The effects induced by AAC2 were absent in leptin receptor-deficient predipocytes and in Leprdb mice. In contrast, AAC2 established glycemic control altering food intake in leptin-deficient Lepob mice. Therefore, AAC2 activated the LepR and acted in a cytokine-like manner distinct from leptin. In a monogenic Ins2Akita mouse model for the phenotypes associated with type 1 diabetes, AAC2 rescued systemic glucose uptake in these mice without an increase in insulin levels and adiposity, as seen in insulin-treated Ins2Akita mice. In contrast to insulin, AAC2 treatment increased brain mass and reduced anxiety-related behavior in Ins2Akita mice. Our data suggests that the unique mechanism of action for AAC2, activating LepR/PKCς/GLUT1 axis, offers an effective strategy to broaden glycemic control for the prevention of diabetic complications of the nervous system and, possibly, other insulin insensitive or resistant tissues.
Subject(s)
Blood Glucose , Diabetes Mellitus, Experimental , Amino Acids , Animals , Anxiety , Diabetes Mellitus, Experimental/drug therapy , Insulin , Mice , Mice, Inbred C57BL , Receptors, LeptinABSTRACT
Previous studies have demonstrated that the freezing tolerance (FT) of grapevine was enhanced by foliar application of exogenous abscisic acid (exo-ABA), a treatment which might be incorporated into cultural practices to mitigate cold damage in vineyards. To investigate the underlying mechanisms of this response, a two-year (2017 and 2018) study was conducted to characterize the effects of exo-ABA on greenhouse-grown 'Cabernet franc' grapevine. In control grapevines, both physiological (deeper dormancy) and biochemical (sugar accumulation in buds) changes occurred, indicating that grapevines initiated cold acclimation in the greenhouse. Compared to control, exo-ABA decreased stomatal conductance 2 h after application. Two weeks post application, exo-ABA treated grapevines showed accelerated transition of grapevine physiology during cold acclimation (increased depth of dormancy, decreased bud water content and enhanced bud FT), relative to control. Exo-ABA induced the accumulation of several sugars in buds including the raffinose family oligosaccharides (RFOs), and the RFO precursor, galactinol. The expression of raffinose and galactinol synthase genes was higher in exo-ABA treated grapevine buds, compared to control. The new findings from this study have advanced our understanding of the role of ABA in grapevine FT, which will be useful to develop future strategies to protect grapevines from cold damage.
Subject(s)
Abscisic Acid/metabolism , Acclimatization/physiology , Cold Temperature , Vitis/metabolism , Acclimatization/genetics , Carbohydrate Metabolism , Freezing , Galactosyltransferases/genetics , Galactosyltransferases/metabolism , Gene Expression Regulation, Plant , Plant Leaves/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Stomata/metabolism , Raffinose/metabolism , Sugars/metabolism , Vitis/genetics , Water/metabolismABSTRACT
The plant hormone auxin is essential for plant growth and development, controlling both organ development and overall plant architecture. Auxin homeostasis is regulated by coordination of biosynthesis, transport, conjugation, sequestration/storage, and catabolism to optimize concentration-dependent growth responses and adaptive responses to temperature, water stress, herbivory, and pathogens. At present, the best defined pathway of auxin biosynthesis is the TAA/YUC route, in which the tryptophan aminotransferases TAA and TAR and YUCCA flavin-dependent monooxygenases produce the auxin indole-3-acetic acid from tryptophan. This review highlights recent advances in our knowledge of TAA/YUC-dependent auxin biosynthesis focusing on membrane localization of auxin biosynthetic enzymes, differential regulation in root and shoot tissue, and auxin biosynthesis during abiotic stress.
Subject(s)
Acclimatization , Adaptation, Physiological , Indoleacetic Acids/metabolism , Plant Growth Regulators/biosynthesis , Plant Proteins/metabolism , Plants/enzymology , Mixed Function Oxygenases/metabolism , Stress, Physiological , Tryptophan Transaminase/metabolismABSTRACT
Plants use a tightly regulated immune system to fight off various pathogens. Phospholipase D (PLD) and its product, phosphatidic acid, have been shown to influence plant immunity; however, the underlying mechanisms remain unclear. Here, we show that the Arabidopsis mutants pldα1 and pldδ, respectively, exhibited enhanced resistance and enhanced susceptibility to both well-adapted and poorly adapted powdery mildew pathogens, and a virulent oomycete pathogen, indicating that PLDα1 negatively while PLDδ positively modulates post-penetration resistance. The pldα1δ double mutant showed a similar infection phenotype to pldα1, genetically placing PLDα1 downstream of PLDδ. Detailed genetic analyses of pldδ with mutations in genes for salicylic acid (SA) synthesis (SID2) and/or signaling (EDS1 and PAD4), measurement of SA and jasmonic acid (JA) levels, and expression of their respective reporter genes indicate that PLDδ contributes to basal resistance independent of EDS1/PAD4, SA, and JAsignaling. Interestingly, while PLDα1-enhanced green fluorescent protein (eGFP) was mainly found in the tonoplast before and after haustorium invasion, PLDδ-eGFP's focal accumulation to the plasma membrane around the fungal penetration site appeared to be suppressed by adapted powdery mildew. Together, our results demonstrate that PLDα1 and PLDδ oppositely modulate basal, post-penetration resistance against powdery mildew through a non-canonical mechanism that is independent of EDS1/PAD4, SA, and JA.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Ascomycota/physiology , Phospholipase D/metabolism , Plant Diseases/immunology , Salicylic Acid/metabolism , Arabidopsis/genetics , Arabidopsis/immunology , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Plant , Phospholipase D/economics , Phospholipase D/genetics , Plant Diseases/microbiology , Plant ImmunityABSTRACT
Ferredoxins, the major distributors for electrons to various acceptor systems in plastids, contribute to redox regulation and antioxidant defence in plants. However, their function in plant immunity is not fully understood. In this study, we show that the expression of the major leaf ferredoxin gene Fd2 is suppressed by Pseudomonas syringae pv. tomato (Pst) DC3000 infection, and that knockout of Fd2 (Fd2-KO) in Arabidopsis increases the plant's susceptibility to both Pst DC3000 and Golovinomyces cichoracearum. On Pst DC3000 infection, the Fd2-KO mutant accumulates increased levels of jasmonic acid and displays compromised salicylic acid-related immune responses. Fd2-KO also shows defects in the accumulation of reactive oxygen species induced by pathogen-associated molecular pattern-triggered immunity. However, Fd2-KO shows enhanced R-protein-mediated resistance to Pst DC3000/AvrRpt2 infection, suggesting that Fd2 plays a negative role in effector-triggered immunity. Furthermore, Fd2 interacts with FIBRILLIN4 (FIB4), a harpin-binding protein localized in chloroplasts. Interestingly, Fd2, but not FIB4, localizes to stromules that extend from chloroplasts. Taken together, our results demonstrate that Fd2 plays an important role in plant immunity.
Subject(s)
Arabidopsis/metabolism , Ferredoxins/metabolism , Plant Leaves/metabolism , Cyclopentanes/metabolism , Disease Resistance , Oxylipins/metabolism , Plant Diseases/microbiology , Plant Immunity/physiology , Reactive Oxygen Species/metabolism , Salicylic Acid/metabolismABSTRACT
Salt stress causes worldwide reductions in agricultural yields, a problem that is exacerbated by the depletion of global freshwater reserves and the use of contaminated or recycled water (i.e. effluent water). Additionally, salt stress can occur as cultivated areas are subjected to frequent rounds of irrigation followed by periods of moderate to severe evapotranspiration, which can result in the heterogeneous aggregation of salts in agricultural soils. Our understanding of the later stages of salt stress and the mechanisms by which salt is transported out of cells and roots has greatly improved over the last decade. The precise mechanisms by which plant roots perceive salt stress and translate this perception into adaptive, directional growth away from increased salt concentrations (i.e. halotropism), however, are not well understood. Here, we provide a review of the current knowledge surrounding the early responses to salt stress and the initiation of halotropism, including lipid signaling, protein phosphorylation cascades, and changes in auxin metabolism and/or transport. Current models of halotropism have focused on the role of PIN2- and PIN1-mediated auxin efflux in initiating and controlling halotropism. Recent studies, however, suggest that additional factors such as ABCB transporters, protein phosphatase 2A activity, and auxin metabolism should be included in the model of halotropic growth.
Subject(s)
Indoleacetic Acids/metabolism , Lipid Metabolism , Phosphorylation , Plant Growth Regulators/metabolism , Plant Physiological Phenomena , Plant Proteins/metabolism , Salt Tolerance , Signal Transduction , Stress, PhysiologicalABSTRACT
Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.
Subject(s)
Cinnamates/metabolism , Indoleacetic Acids/metabolism , Plant Roots/drug effects , Plant Roots/growth & development , Arabidopsis/drug effects , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Bryopsida/drug effects , Bryopsida/growth & development , Cinnamates/chemistry , Cinnamates/pharmacology , Cyclin B/genetics , Cyclin B/metabolism , Gene Expression Regulation, Plant , Isomerism , Plant Roots/metabolism , Plants, Genetically Modified , Qa-SNARE Proteins/genetics , Qa-SNARE Proteins/metabolism , Selaginellaceae/drug effects , Selaginellaceae/growth & development , Signal TransductionABSTRACT
Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or "acid" taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.
Subject(s)
Electrophoresis, Capillary , Plant Structures , Coffea , Organic Chemicals , Plants , SeedsABSTRACT
Tight homeostatic regulation of the phytohormone auxin [indole-3-acetic acid (IAA)] is essential to plant growth. Auxin biosynthetic pathways and the processes that inactivate auxin by conjugation to amino acids and sugars have been thoroughly characterized. However, the enzyme that catalyzes oxidation of IAA to its primary catabolite 2-oxindole-3-acetic acid (oxIAA) remains uncharacterized. Here, we show that DIOXYGENASE FOR AUXIN OXIDATION 1 (DAO1) catalyzes formation of oxIAA in vitro and in vivo and that this mechanism regulates auxin homeostasis and plant growth. Null dao1-1 mutants contain 95% less oxIAA compared with wild type, and complementation of dao1 restores wild-type oxIAA levels, indicating that DAO1 is the primary IAA oxidase in seedlings. Furthermore, dao1 loss of function plants have altered morphology, including larger cotyledons, increased lateral root density, delayed sepal opening, elongated pistils, and reduced fertility in the primary inflorescence stem. These phenotypes are tightly correlated with DAO1 spatiotemporal expression patterns as shown by DAO1pro:ß-glucuronidase (GUS) activity and DAO1pro:YFP-DAO1 signals, and transformation with DAO1pro:YFP-DAO1 complemented the mutant phenotypes. The dominant dao1-2D mutant has increased oxIAA levels and decreased stature with shorter leaves and inflorescence stems, thus supporting DAO1 IAA oxidase function in vivo. A second isoform, DAO2, is very weakly expressed in seedling root apices. Together, these data confirm that IAA oxidation by DAO1 is the principal auxin catabolic process in Arabidopsis and that localized IAA oxidation plays a role in plant morphogenesis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Biocatalysis , Indoleacetic Acids/metabolism , Organ Specificity , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis Proteins/chemistry , DNA, Bacterial/genetics , Flowers/metabolism , Gene Expression Regulation, Plant , Gene Knockdown Techniques , Genetic Complementation Test , Metabolome , Mutation/genetics , Oxidation-Reduction , Phenotype , Phylogeny , Plant Roots/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Sequence Alignment , Tandem Mass Spectrometry , Time FactorsABSTRACT
Soluble sugars play an important role in freezing tolerance in both herbaceous and woody plants, functioning in both the reduction of freezing-induced dehydration and the cryoprotection of cellular constituents. The quantification of soluble sugars in plant tissues is, therefore, essential in understanding freezing tolerance. While a number of analytical techniques and methods have been used to quantify sugars, most of these are expensive and time-consuming due to complex sample preparation procedures which require the derivatization of the carbohydrates being analyzed. Analysis of soluble sugars using capillary zone electrophoresis (CZE) under alkaline conditions with direct UV detection has previously been used to quantify simple sugars in fruit juices. However, it was unclear whether CZE-based methods could be successfully used to quantify the broader range of sugars present in complex plant extracts. Here, we present the development of an optimized CZE method capable of separating and quantifying mono-, di-, and tri-saccharides isolated from plant tissues. This optimized CZE method employs a column electrolyte buffer containing 130 mM NaOH, pH 13.0, creating a current of 185 µA when a separation voltage of 10 kV is employed. The optimized CZE method provides limits-of-detection (an average of 1.5 ng/µL) for individual carbohydrates comparable or superior to those obtained using gas chromatography-mass spectrometry, and allows resolution of non-structural sugars and cell wall components (structural sugars). The optimized CZE method was successfully used to quantify sugars from grape leaves and buds, and is a robust tool for the quantification of plant sugars found in vegetative and woody tissues. The increased analytical efficiency of this CZE method makes it ideal for use in high-throughput metabolomics studies designed to quantify plant sugars.
ABSTRACT
Auxins are a particularly notable class of phytohormones in that they regulate plant growth and development at sites of synthesis, and via a regulated polar transport system comprising PIN, ABCB, and AUX/LAX transport proteins. In order to fully understand auxin-regulated physiological processes, it is therefore essential to be able to determine where indole-3-acetic acid and related compounds are being synthesized, where they are transported to, and how much IAA is accumulating in any given tissue. Auxin may be visualized either indirectly, through the use of auxin responsive promoters; directly, through the use of radiolabelled auxin or fluorescent auxin analogs; or biochemically through extraction and mass-spectrometric quantification of auxin and auxin metabolites from target cells or tissues. Here we focus on the use of the DR5::GUS synthetic auxin promoter reporter construct, fluorescent auxin analogs, and confirmatory biochemical (high-pressure liquid chromatography tandem mass-spectrometry) visualization of auxin and auxin metabolites.
Subject(s)
Indoleacetic Acids/metabolism , Gene Expression Regulation, Plant/genetics , Gene Expression Regulation, Plant/physiology , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolismABSTRACT
Within the cultivated tomato germplasm, sun, ovate and fs8.1 are the three predominant QTLs controlling fruit elongation. Although SUN and OVATE have been cloned, their role in plant growth and development are not well understood. To compare and contrast the effects of the three QTLs in a homogeneous background, we developed near isogenic lines (NILs) in the wild species Solanum pimpinellifolium LA1589 background. We carried out detailed morphological characterization of reproductive and vegetative organs in the single, double and triple NILs and determined the epistatic interactions of the three loci affecting fruit shape. The phenotypic evaluations demonstrated that the three loci regulate unique aspects of ovary and fruit elongation and in different temporal manners. The strongest effect on organ shape was caused by sun. In addition to fruit shape, sun also affected leaf and sepal elongation and stem thickness. The synergistic interaction between sun and ovate or fs8.1 suggested that the pathways involving SUN, OVATE and the gene(s) underlying fs8.1 may converge at a common node. The results of an extensive profiling analysis suggested that the degree of fruit elongation was not related to the accumulation of any of the classical hormones.