Transcriptome analysis of two structurally related flavonoids; Apigenin and Chrysin revealed hypocholesterolemic and ketogenic effects in mouse embryonic fibroblasts.

There is no known single therapeutic drug for treating hypercholesterolemia that comes with negligible systemic side effects. In the current study, using next generation RNA sequencing approach in mouse embryonic fibroblasts we discovered that two structurally related flavonoid compounds. Apigenin and Chrysin exhibited moderate blocking ability of multiple transcripts that regulate rate limiting enzymes in the cholesterol biosynthesis pathway. The observed decrease in cholesterol biosynthesis pathway correlated well with an increase in transcripts involved in generation and trafficking of ketone bodies as evident by the upregulation of Bdh1 and Slc16a6 transcripts. The hypocholesterolemic potential of Apigenin and Chrysin at higher concentrations along with their ability to generate ketogenic substrate especially during embryonic stage is useful or detrimental for embryonic health is not clear and still debatable. Our study will serve as a steppingstone to further the investigation in whole animal studies and also in translating this knowledge to human studies.

Seasonal nitrogen remobilization and the role of auxin transport in poplar trees.

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.

Auxin regulates adventitious root formation in tomato cuttings.

BACKGROUND: Adventitious root (AR) formation is a critical developmental process in cutting propagation for the horticultural industry. While auxin has been shown to regulate this process, the exact mechanism and details preceding AR formation remain unclear. Even though AR and lateral root (LR) formation share common developmental processes, there are exist some differences that need to be closely examined at the cytological level. Tomato stem cuttings, which readily form adventitious roots, represent the perfect system to study the influence of auxin on AR formation and to compare AR and LR organogenesis. RESULTS: Here we show the progression by which AR form from founder cells in the basal pericycle cell layers in tomato stem cuttings. The first disordered clumps of cells assumed a dome shape that later differentiated into functional AR cell layers. Further growth resulted in emergence of mature AR through the epidermis following programmed cell death of epidermal cells. Auxin and ethylene levels increased in the basal stem cutting within 1 h. Tomato lines expressing the auxin response element DR5pro:YFP showed an increase in auxin distribution during the AR initiation phase, and was mainly concentrated in the meristematic cells of the developing AR. Treatment of stem cuttings with auxin, increased the number of AR primordia and the length of AR, while stem cuttings treated with the pre-emergent herbicide/auxin transport inhibitor N-1-naphthylphthalamic acid (NPA) occasionally developed thick, agravitropic AR. Hormone profile analyses showed that auxin positively regulated AR formation, whereas perturbations to zeatin, salicylic acid, and abscisic acid homeostasis suggested minor roles during tomato stem rooting. The gene expression of specific auxin transporters increased during specific developmental phases of AR formation. CONCLUSION: These data show that AR formation in tomato stems is a complex process. Upon perception of a wounding stimulus, expression of auxin transporter genes and accumulation of auxin at founder cell initiation sites in pericycle cell layers and later in the meristematic cells of the AR primordia were observed. A clear understanding and documentation of these events in tomato is critical to resolve AR formation in recalcitrant species like hardwoods and improve stem cutting propagation efficiency and effectiveness.

Auxin homeostasis: the DAO of catabolism.

Nearly all programmed and plastic plant growth responses are at least partially regulated by auxins, such as indole-3-acetic acid (IAA). Although vectorial, long distance auxin transport is essential to its regulatory function, all auxin responses are ultimately localized in individual target cells. As a consequence, cellular auxin concentrations are tightly regulated via coordinated biosynthesis, transport, conjugation, and oxidation. The primary auxin oxidative product across species is 2-oxindole-3-acetic acid (oxIAA), followed by glucose and amino acid conjugation to oxIAA. Recently, the enzymes catalyzing the oxidative reaction were characterized in Arabidopsis thaliana. DIOXYGENASE OF AUXIN OXIDATION (DAO) comprises a small subfamily of the 2-oxoglutarate and Fe(II) [2-OG Fe(II)] dependent dioxygenase superfamily. Biochemical and genetic studies have revealed critical physiological functions of DAO during plant growth and development. Thus far, DAO has been identified in three species by homology. Here, we review historical and recent studies and discuss future perspectives regarding DAO and IAA oxidation.

Directed plant cell-wall accumulation of iron: embedding co-catalyst for efficient biomass conversion.

BACKGROUND: Plant lignocellulosic biomass is an abundant, renewable feedstock for the production of biobased fuels and chemicals. Previously, we showed that iron can act as a co-catalyst to improve the deconstruction of lignocellulosic biomass. However, directly adding iron catalysts into biomass prior to pretreatment is diffusion limited, and increases the cost of biorefinery operations. Recently, we developed a new strategy for expressing iron-storage protein ferritin intracellularly to accumulate iron as a catalyst for the downstream deconstruction of lignocellulosic biomass. In this study, we extend this approach by fusing the heterologous ferritin gene with a signal peptide for secretion into Arabidopsis cell walls (referred to here as FerEX). RESULTS: The transgenic Arabidopsis plants. FerEX. accumulated iron under both normal and iron-fertilized growth conditions; under the latter (iron-fertilized) condition, FerEX transgenic plants showed an increase in plant height and dry weight by 12 and 18 %, respectively, compared with the empty vector control plants. The SDS- and native-PAGE separation of cell-wall protein extracts followed by Western blot analyses confirmed the extracellular expression of ferritin in FerEX plants. Meanwhile, Perls' Prussian blue staining and X-ray fluorescence microscopy (XFM) maps revealed iron depositions in both the secondary and compound middle lamellae cell-wall layers, as well as in some of the corner compound middle lamella in FerEX. Remarkably, their harvested biomasses showed enhanced pretreatability and digestibility, releasing, respectively, 21 % more glucose and 34 % more xylose than the empty vector control plants. These values are significantly higher than those of our recently obtained ferritin intracellularly expressed plants. CONCLUSIONS: This study demonstrated that extracellular expression of ferritin in Arabidopsis can produce plants with increased growth and iron accumulation, and reduced thermal and enzymatic recalcitrance. The results are attributed to the intimate colocation of the iron co-catalyst and the cellulose and hemicellulose within the plant cell-wall region, supporting the genetic modification strategy for incorporating conversion catalysts into energy crops prior to harvesting or processing at the biorefinery.

DAO1 catalyzes temporal and tissue-specific oxidative inactivation of auxin in Arabidopsis thaliana.

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.

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice.

Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusion polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.

Immunolocalization of PIN and ABCB Transporters in Plants.

PIN auxin efflux carriers and ABCB auxin transporters are important for polar auxin transport, organogenesis and long distance auxin transport. Along with the auxin influx symporter AUX1, they are essential for tropic responses such as gravitropism and phototropism where lateral redistribution of auxin is required for the tropic response to occur. Immunolocalization of plant membrane transporters is an effective technique to determine the transporters' subcellular localization patterns in the tissues of interest, especially when fluorescent protein fusions of the protein of interest are not available. Immunolocalization is also a valuable tool for validation of the localization of fluorescent protein fusions when the fusions are available. Here we describe the procedures to prepare plant tissue samples and fix them for whole mount or embedding and sectioning. We focus on immunolocalizations of PINs and ABCBs in Arabidopsis and maize tissues. In addition, we describe treatments of roots with inhibitors of cellular trafficking: brefeldin A (BFA), a fungal compound that blocks exocytosis; wortmannin, a fungal compound that inhibits phosphatidylinositol 3-kinase and induces fusion of pre-vacuolar compartments and multi-vascular bodies; and oryzalin, a fungal compound that depolymerizes microtubules. Inhibitor treatments are performed prior to fixation and affect the localization patterns of PINs and ABCBs, giving insight into cell type -specific trafficking mechanisms. The procedures described for Arabidopsis and maize can be easily adapted for other herbaceous plants.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase.

The presence of a large central vacuole is one of the hallmarks of a prototypical plant cell, and the multiple functions of this compartment require massive fluxes of molecules across its limiting membrane, the tonoplast. Transport is assumed to be energized by the membrane potential and the proton gradient established by the combined activity of two proton pumps, the vacuolar H(+)-pyrophosphatase (V-PPase) and the vacuolar H(+)-ATPase (V-ATPase). Exactly how labor is divided between these two enzymes has remained elusive. Here, we provide evidence using gain- and loss-of-function approaches that lack of the V-ATPase cannot be compensated for by increased V-PPase activity. Moreover, we show that increased V-ATPase activity during cold acclimation requires the presence of the V-PPase. Most importantly, we demonstrate that a mutant lacking both of these proton pumps is conditionally viable and retains significant vacuolar acidification, pointing to a so far undetected contribution of the trans-Golgi network/early endosome-localized V-ATPase to vacuolar pH.

The circadian clock rephases during lateral root organ initiation in Arabidopsis thaliana.

The endogenous circadian clock enables organisms to adapt their growth and development to environmental changes. Here we describe how the circadian clock is employed to coordinate responses to the key signal auxin during lateral root (LR) emergence. In the model plant, Arabidopsis thaliana, LRs originate from a group of stem cells deep within the root, necessitating that new organs emerge through overlying root tissues. We report that the circadian clock is rephased during LR development. Metabolite and transcript profiling revealed that the circadian clock controls the levels of auxin and auxin-related genes including the auxin response repressor IAA14 and auxin oxidase AtDAO2. Plants lacking or overexpressing core clock components exhibit LR emergence defects. We conclude that the circadian clock acts to gate auxin signalling during LR development to facilitate organ emergence.

Over-expression of the Arabidopsis proton-pyrophosphatase AVP1 enhances transplant survival, root mass, and fruit development under limiting phosphorus conditions.

Phosphorus (P), an element required for plant growth, fruit set, fruit development, and fruit ripening, can be deficient or unavailable in agricultural soils. Previously, it was shown that over-expression of a proton-pyrophosphatase gene AVP1/AVP1D (AVP1DOX) in Arabidopsis, rice, and tomato resulted in the enhancement of root branching and overall mass with the result of increased mineral P acquisition. However, although AVP1 over-expression also increased shoot biomass in Arabidopsis, this effect was not observed in tomato under phosphate-sufficient conditions. AVP1DOX tomato plants exhibited increased rootward auxin transport and root acidification compared with control plants. AVP1DOX tomato plants were analysed in detail under limiting P conditions in greenhouse and field trials. AVP1DOX plants produced 25% (P=0.001) more marketable ripened fruit per plant under P-deficient conditions compared with the controls. Further, under low phosphate conditions, AVP1DOX plants displayed increased phosphate transport from leaf (source) to fruit (sink) compared to controls. AVP1DOX plants also showed an 11% increase in transplant survival (P<0.01) in both greenhouse and field trials compared with the control plants. These results suggest that selection of tomato cultivars for increased proton pyrophosphatase gene expression could be useful when selecting for cultivars to be grown on marginal soils.

Measure for measure: determining, inferring and guessing auxin gradients at the root tip.

The plant hormone auxin is transported from sites of synthesis to sites of action. Auxin responses are mediated by fast (non-transcriptional) and slow (transcriptional; ubiquitinylation) responses, which affect physiological changes at cellular and organismal scales. As such, auxin transport vectors regulate programmed and plastic growth responses to optimize growth and development. Here we address some common problems in extrapolating 'universal' understanding of auxin transport streams from analyses of loss-of-function mutants and auxin transport inhibitors. We also discuss the analytical methods and tools used to directly quantify, measure and infer auxin gradients within the plant [DR5:GUS/GFP (beta-glucuronidase/green fluorescent protein), DII-VENUS; surface electrodes, direct quantification]. We discuss the assumptions and limitations of each of these analyses, present comparative summaries of auxin transport methods and assay conditions (diffusion, non-specific transport and relevant assay conditions), and consider what is actually being transported and measured [labeled-indole-3-acetic acid (IAA), IAA metabolites].

From perception to attenuation: auxin signalling and responses.

The plant hormone auxin is essential for growth, development, and responses to environmental factors. Recently, Auxin Binding Protein 1 was shown to mediate non-transcriptional auxin signalling at the cell periphery. This has provoked reexamination of the paradigm that all auxin perception is intracellular and is mediated by the TIR1/AFB-Aux/IAA co-receptors for which auxin functions as a concentration-dependent molecular glue. Further, another F-box protein, SKP2a, was shown to bind auxin in the same way as TIR1/AFB, which provides a link to the role of auxin in the cell cycle. New work on auxin signalling and homeostasis include D6 PROTEIN KINASE activation of PINFORMED (PIN) auxin carriers, ROP-GTPase mediation of PIN localization, endoplasmic reticulum localization PIN and PIN-LIKES auxin carriers, and auxin biosynthesis and metabolism.

Evidence of oxidative attenuation of auxin signalling.

Indole-3-acetic acid (IAA) is the principle auxin in Arabidopsis and is synthesized primarily in meristems and nodes. Auxin is transported to distal parts of the plant in response to developmental programming or environmental stimuli to activate cell-specific responses. As with any signalling event, the signal must be attenuated to allow the system to reset. Local auxin accumulations are thus reduced by conjugation or catabolism when downstream responses have reached their optima. In most cell types, localized auxin accumulation increases both reactive oxygen species (ROS) and an irreversible catabolic product 2-oxindole-3-acid acid (oxIAA). oxIAA is inactive and does not induce expression of the auxin-responsive reporters DR5 or 2XD0. Here it is shown that oxIAA is not transported from cell to cell, although it appears to be a substrate for the ATP-binding cassette subfamily G (ABCG) transporters that are positioned primarily on the outer lateral surface of the root epidermis. However, oxIAA and oxIAA-Glc levels are higher in ABCB mutants that accumulate auxin due to defective cellular export. Auxin-induced ROS production appears to be at least partially mediated by the NAD(P)H oxidase RbohD. oxIAA levels are higher in mutants that lack ROS-scavenging flavonoids (tt4) and are lower in mutants that accumulate excess flavonols (tt3). These data suggest a model where IAA signalling is attenuated by IAA catabolism to oxIAA. Flavonoids appear to buffer ROS accumulations that occur with localized increases in IAA. This buffering of IAA oxidation would explain some growth responses observed in flavonoid-deficient mutants that cannot be explained by their established role in partially inhibiting auxin transport.

ROOT ULTRAVIOLET B-SENSITIVE1/weak auxin response3 is essential for polar auxin transport in Arabidopsis.

The phytohormone auxin regulates virtually every aspect of plant development. To identify new genes involved in auxin activity, a genetic screen was performed for Arabidopsis (Arabidopsis thaliana) mutants with altered expression of the auxin-responsive reporter DR5rev:GFP. One of the mutants recovered in the screen, designated as weak auxin response3 (wxr3), exhibits much lower DR5rev:GFP expression when treated with the synthetic auxin 2,4-dichlorophenoxyacetic acid and displays severe defects in root development. The wxr3 mutant decreases polar auxin transport and results in a disruption of the asymmetric auxin distribution. The levels of the auxin transporters AUXIN1 and PIN-FORMED are dramatically reduced in the wxr3 root tip. Molecular analyses demonstrate that WXR3 is ROOT ULTRAVIOLET B-SENSITIVE1 (RUS1), a member of the conserved Domain of Unknown Function647 protein family found in diverse eukaryotic organisms. Our data suggest that RUS1/WXR3 plays an essential role in the regulation of polar auxin transport by maintaining the proper level of auxin transporters on the plasma membrane.

Detection of herbicides in the urine of pet dogs following home lawn chemical application.

Exposure to herbicide-treated lawns has been associated with significantly higher bladder cancer risk in dogs. This work was performed to further characterize lawn chemical exposures in dogs, and to determine environmental factors associated with chemical residence time on grass. In addition to concern for canine health, a strong justification for the work was that dogs may serve as sentinels for potentially harmful environmental exposures in humans. Experimentally, herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxypropionic acid (MCPP), dicamba] were applied to grass plots under different conditions (e.g., green, dry brown, wet, and recently mowed grass). Chemicals in dislodgeable residues were measured by LC-MS at 0.17, 1, 24, 48, 72 h post treatment. In a separate study, 2,4-D, MCPP, and dithiopyr concentrations were measured in the urine of dogs and in dislodgeable grass residues in households that applied or did not apply chemicals in the preceding 48 h. Chemicals were measured at 0, 24, and 48 h post application in treated households and at time 0 in untreated control households. Residence times of 2,4-D, MCPP, and dicamba were significantly prolonged (P<0.05) on dry brown grass compared to green grass. Chemicals were detected in the urine of dogs in 14 of 25 households before lawn treatment, in 19 of 25 households after lawn treatment, and in 4 of 8 untreated households. Chemicals were commonly detected in grass residues from treated lawns, and from untreated lawns suggesting chemical drift from nearby treated areas. Thus dogs could be exposed to chemicals through contact with their own lawn (treated or contaminated through drift) or through contact with other grassy areas if they travel. The length of time to restrict a dog's access to treated lawns following treatment remains to be defined. Further study is indicated to assess the risks of herbicide exposure in humans and dogs.