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.

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.

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.

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.

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.

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].

The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis.

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4(pro) :ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4(pro) :ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-ß-D-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.

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.

The catalytic and protein-protein interaction domains are required for APM1 function.

Aminopeptidase M1 (APM1) is essential for embryonic, vegetative, and reproductive development in Arabidopsis (Arabidopsis thaliana). Here, we show that, like mammalian M1 proteases, APM1 appears to have distinct enzymatic and protein-protein interaction domains and functions as a homodimer. Arabidopsis seedlings treated with ezetimibe, an inhibitor of M1 protein-protein interactions, mimicked a subset of apm1 phenotypes distinct from those resulting from treatment with PAQ-22, an inhibitor of M1 catalytic activity, suggesting that the APM1 catalytic and interaction domains can function independently. apm1-1 knockdown mutants transformed with catalytically inactive APM1 did not prevent seedling lethality. However, apm1-2 has a functional enzymatic domain but lacks the carboxyl (C) terminus, and transformation with catalytically inactive APM1 rescued the mutant. Overexpression of human insulin-responsive aminopeptidase/oxytocinase rescued all apm1 phenotypes, suggesting that the catalytic activity was sufficient to compensate for loss of APM1 function, while overexpression of catalytically inactive insulin-responsive aminopeptidase/oxytocinase only rescued apm1-2. Increased catalytic activity alone is not sufficient to compensate for loss of APM1 function, as overexpression of another Arabidopsis M1 family member lacking an extended C terminus did not rescue apm1-1. The protein interactions facilitating enzymatic activity appear to be dependent on the C terminus of APM1, as transformation with an open reading frame containing an internal deletion of a portion of the C terminus or a point mutation in a dileucine motif did not rescue the mutant. These results suggest that both the catalytic and interaction domains are necessary for APM1 function but that APM1 function and dimerization do not require these domains to be present in the same linear molecule.

The role of multifunctional M1 metallopeptidases in cell cycle progression.

BACKGROUND: Metallopeptidases of the M1 family are found in all phyla (except viruses) and are important in the cell cycle and normal growth and development. M1s often have spatiotemporal expression patterns which allow for strict regulation of activity. Mutations in the genes encoding M1s result in disease and are often lethal. This family of zinc metallopeptidases all share the catalytic region containing a signature amino acid exopeptidase (GXMXN) and a zinc binding (HEXXH[18X]E) motif. In addition, M1 aminopeptidases often also contain additional membrane association and/or protein interaction motifs. These protein interaction domains may function independently of M1 enzymatic activity and can contribute to multifunctionality of the proteins. SCOPE: A brief review of M1 metalloproteases in plants and animals and their roles in the cell cycle is presented. In animals, human puromycin-sensitive aminopeptidase (PSA) acts during mitosis and perhaps meiosis, while the insect homologue puromycin-sensitive aminopeptidase (PAM-1) is required for meiotic and mitotic exit; the remaining human M1 family members appear to play a direct or indirect role in mitosis/cell proliferation. In plants, meiotic prophase aminopeptidase 1 (MPA1) is essential for the first steps in meiosis, and aminopeptidase M1 (APM1) appears to be important in mitosis and cell division. CONCLUSIONS: M1 metalloprotease activity in the cell cycle is conserved across phyla. The activities of the multifunctional M1s, processing small peptides and peptide hormones and contributing to protein trafficking and signal transduction processes, either directly or indirectly impact on the cell cycle. Identification of peptide substrates and interacting protein partners is required to understand M1 function in fertility and normal growth and development in plants.

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.

ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis.

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.

Brachytic2/ZmABCB1 functions in IAA export from intercalary meristems.

Dwarfism traits in Zea mays are regulated by multiple factors including the hormone auxin. Dwarf brachytic2 (br2) mutants harbour lesions in the gene encoding an orthologue of Arabidopsis thaliana ABCB1 which functions in auxin efflux out of meristematic regions in the shoot and root. br2 mesocotyls and coleoptiles exhibit reduced auxin transport. However, the dwarf stature of br2 derives from shortened lower internodes whilst the upper portion of the plant is completely normal. As such, it is counter-intuitive to attribute br2 dwarfism exclusively to reduced auxin export out of the shoot apex. Arabidopsis abcb1 mutants exhibit only minor reductions in auxin transport and plant height unless combined with mutations in the ABCB19 auxin transporter. Phylogenetic modelling analysis excludes the possibility that BR2 is more closely related to ABCB19 which has three more closely related orthologues in maize. BR2 is expressed in nodal meristems, and analyses of auxin transport and content indicate that BR2 function in these grass-specific tissues is analogous to ABCB1 function in the shoot and root apex of Arabidopsis. These results indicate that ABCB1/BR2 function is conserved between dicots and monocots, but also suggests that this function must be understood in the context of the segmental organization of grass plants.

Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1.

Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

Flavonoids and auxin transport: modulators or regulators?

Flavonoids are polyphenolic compounds found in all vascular and non-vascular plants. Although nonessential for plant growth and development, flavonoids have species-specific roles in nodulation, fertility, defense and UV protection. Flavonoids have been shown to modulate transport of the phytohormone auxin in addition to auxin-dependent tropic responses. However, flavonoids are not essential regulators of these processes because transport and tropic responses occur in their absence. Flavonoids modulate the activity of auxin-transporting P-glycoproteins and seem to modulate the activity of regulatory proteins such as phosphatases and kinases. Phylogenetic analysis suggests that auxin transport mechanisms evolved in the presence of flavonoid compounds produced for the scavenging of reactive oxygen species and defense from herbivores and pathogens.

Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+-pyrophosphatase.

Plants challenged by limited phosphorus undergo dramatic morphological and architectural changes in their root systems in order to increase their absorptive surface area. In this paper, it is shown that phosphorus deficiency results in increased expression of the type I H+-pyrophosphatase AVP1 (AVP, Arabidopsis vacuolar pyrophosphatase), subsequent increased P-type adenosine triphosphatase (P-ATPase)-mediated rhizosphere acidification and root proliferation. Molecular genetic manipulation of AVP1 expression in Arabidopsis, tomato and rice results in plants that outperform controls when challenged with limited phosphorus. However, AVP1 over-expression and the resulting rhizosphere acidification do not result in increased sensitivity to AlPO4, apparently because of the enhancement of potassium uptake and the release of organic acids. Thus, the over-expression of type I H+-pyrophosphatases appears to be a generally applicable technology to help alleviate agricultural losses in low-phosphorus tropical/subtropical soils and to reduce phosphorus runoff pollution of aquatic and marine environments resulting from fertilizer application.