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Involvement of calcium-dependent protein kinase in rice (Oryza sativa L.) lamina inclination caused by brassinolide.

Promotive effect of brassinolide (BL) on green lamina inclination was concentration-dependent when excised rice (Oryza sativa L.) lamina was floated on BL solution under continuous light conditions. Protein kinase inhibitor staurosporine and Ca2+ channel blocker LaCl3 could completely, while Ca2+ chelator EGTA could partially inhibit the lamina inclination caused by BL. Two protein kinases with apparent molecular masses of 45 and 54 kDa were detected using an in-gel kinase assay with histone III-S as a substrate. In particular, the changes in 45 kDa protein kinase activity correlated with lamina inclination caused by BL. The 45 kDa kinase activity was inhibited by Ca2+ chelator EGTA, protein kinase inhibitor, staurosporine and calmodulin antagonist W-7. Therefore, this 45 kDa protein kinase was identified as a Ca2+ -dependent protein kinase (CDPK). Patterns of 2-dimensional PAGE after in vitro phosphorylation of crude extracts showed that the phosphorylation of 56 and 41 kDa proteins, which was Ca2+ -dependent, was strongly increased by BL treatment. These results suggested that CDPK and Ca2+ -dependent protein phosphorylation are involved in BL-induced rice lamina inclination.

Brassinosteroids↗

Aldehyde oxidase (AO) in the root nodules of Lupinus albus and Medicago truncatula: identification of AO in meristematic and infection zones.

Phytohormones are involved in the organogenesis of legume root nodules. The source of the auxin indole-3-acetic acid (IAA) in nodules has not been clearly determined. We studied the enzyme aldehyde oxidase (AO; EC 1.2.3.1), that catalyzes the last step of IAA biosynthesis in plants, in the nodules of Lupinus albus and Medicago truncatula. Primordia and young lupin nodules and mature M. truncatula nodules showed AO activity bands after native polyacrylamide gel electrophoresis. Gel activity analyses using indole-3-aldehyde as substrate indicated that the nodules of white lupin and M. truncatula have the capability to synthesize IAA via the indole-3-pyruvic acid pathway. Immunolocalization and in situ hybridization experiments revealed that AO is preferentially expressed in the meristematic and the invasion zones in Medicago nodules and in the lateral meristematic zone of Lupinus nodules. High IAA immunolabeling was also detected in the meristematic and invasion zones. Low expression levels and no AO activity were detected in lupin Fix- nodules that displayed restricted growth and early senescence. We propose that local synthesis of IAA in the root nodule meristem and modulation of AO expression and activity are involved in regulation of nodule development.

Abscisic Acid↗

Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings.

Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified.

Amino Acids↗

IAR4, a gene required for auxin conjugate sensitivity in Arabidopsis, encodes a pyruvate dehydrogenase E1alpha homolog.

The formation and hydrolysis of indole-3-acetic acid (IAA) conjugates represent a potentially important means for plants to regulate IAA levels and thereby auxin responses. The identification and characterization of mutants defective in these processes is advancing the understanding of auxin regulation and response. Here we report the isolation and characterization of the Arabidopsis iar4 mutant, which has reduced sensitivity to several IAA-amino acid conjugates. iar4 is less sensitive to a synthetic auxin and low concentrations of an ethylene precursor but responds to free IAA and other hormones tested similarly to wild type. The gene defective in iar4 encodes a homolog of the E1alpha-subunit of mitochondrial pyruvate dehydrogenase, which converts pyruvate to acetyl-coenzyme A. We did not detect glycolysis or Krebs-cycle-related defects in the iar4 mutant, and a T-DNA insertion in the IAR4 coding sequence conferred similar phenotypes as the originally identified missense allele. In contrast, we found that disruption of the previously described mitochondrial pyruvate dehydrogenase E1alpha-subunit does not alter IAA-Ala responsiveness or confer any obvious phenotypes. It is possible that IAR4 acts in the conversion of indole-3-pyruvate to indole-3-acetyl-coenzyme A, which is a potential precursor of IAA and IAA conjugates.

Amino Acid Sequence↗

Auxin is required for leaf vein pattern in Arabidopsis.

To investigate possible roles of polar auxin transport in vein patterning, cotyledon and leaf vein patterns were compared for plants grown in medium containing polar auxin transport inhibitors (N-1-naphthylphthalamic acid, 9-hydroxyfluorene-9-carboxylic acid, and 2,3,5-triiodobenzoic acid) and in medium containing a less well-characterized inhibitor of auxin-mediated processes, 2-(p-chlorophynoxy)-2-methylpropionic acid. Cotyledon vein pattern was not affected by any inhibitor treatments, although vein morphology was altered. In contrast, leaf vein pattern was affected by inhibitor treatments. Growth in polar auxin transport inhibitors resulted in leaves that lacked vascular continuity through the petiole and had broad, loosely organized midveins, an increased number of secondary veins, and a dense band of misshapen tracheary elements adjacent to the leaf margin. Analysis of leaf vein pattern developmental time courses suggested that the primary vein did not develop in polar auxin transport inhibitor-grown plants, and that the broad midvein observed in these seedlings resulted from the coalescence of proximal regions of secondary veins. Possible models for leaf vein patterning that could account for these observations are discussed.

Arabidopsis↗

Different pathways are involved in phosphate and iron stress-induced alterations of root epidermal cell development.

Low bioavailability of phosphorus (P) and iron (Fe) induces morphogenetic changes in roots that lead to a higher surface-to-volume ratio. In Arabidopsis, an enlargement in the absorptive surface area is achieved by an increase in the length and frequency of hairs in roots of Fe- and P-deficient plants. The extra root hairs are often located in positions that are occupied with non-hair cells under normal conditions, i.e. over a tangential wall of underlying cortical cells. An involvement of auxin and ethylene in root epidermis cell development of Fe- and P-deficient plants was inferred from phenotypical analysis of hormone-related Arabidopsis mutants and from the application of substances that interfere with either synthesis, transport, or perception of the hormones. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or the auxin analog 2,4-D caused a marked increase in root hair density in plants of all growth types and confers a phenotype characteristic of ethylene-overproducing mutants. Hormone insensitivity and application of hormone antagonists inhibited the initiation of extranumerary root hairs induced by Fe deficiency, but did not counteract the formation of extra hairs in response to P deprivation. A model is presented summarizing putative pathways for alterations in root epidermal cell patterning induced by environmental stress.

2,4-Dichlorophenoxyacetic Acid↗

Gravitropism in higher plant shoots. IV. Further studies on participation of ethylene.

Ethylene at 1.0 and 10.0 cubic centimeters per cubic meter decreased the rate of gravitropic bending in stems of cocklebur (Xanthium strumarium L.) and tomato (Lycopersicon esculentum Mill), but 0.1 cubic centimeter per cubic meter ethylene had little effect. Treating cocklebur plants with 1.0 millimolar aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) delayed stem bending compared with controls, but adding 0.1 cubic centimeter per cubic meter ethylene in the surrounding atmosphere (or applying 0.1% ethephon solution) partially restored the rate of bending of AVG-treated plants. Ethylene increases in bending stems, and AVG inhibits this. Virtually all newly synthesized ethylene appeared in bottom halves of horizontal stems, where ethylene concentrations were as much as 100 times those in upright stems or in top halves of horizontal stems. This was especially true when horizontal stems were physically restrained from bending. Ethylene might promote cell elongation in bottom tissues of a horizontal stem or indicate other factors there (e.g. a large amount of 'functioning' auxin). Or top and bottom tissues may become differentially sensitive to ethylene. Auxin applied to one side of a vertical stem caused extreme bending away from that side; gibberellic acid, kinetin, and abscisic acid were without effect. Acidic ethephon solutions applied to one side of young seedlings of cocklebur, tomato, sunflower (Helianthus annuus L.), and soybean (Glycine max [L.] Merr.) caused bending away from that side, but neutral ethephon solutions did not cause bending. Buffered or unbuffered acid (HCl) caused similar bending. Neutral ethephon solutions produced typical ethylene symptoms (i.e. epinasty, inhibition of stem elongation). HCl or acidic ethephon applied to the top of horizontal stems caused downward bending, but these substances applied to the bottom of such stems inhibited growth and upward bending--an unexpected result.

Dose-Response Relationship, Drug↗

Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme.

The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.

2,4-Dichlorophenoxyacetic Acid↗

Springback in root gravitropism.

Conditions under which a gravistimulus of Merit corn roots (Zea mays L.) is withdrawn result in a subsequent loss of gravitropic curvature, an effect which we refer to as springback.' This loss of curvature begins within 1 to 10 minutes after removal of the gravistimulus. It occurs regardless of the presence or absence of the root cap. It is insensitive to inhibitors of auxin transport (2,3,5-triiodobenzoic acid, naphthylphthalamic [correction of naphthylphthalmaic] acid) or to added auxin (2,4-dichlorophenoxyacetic acid). Springback is prevented if a clinostat treatment is interjected to neutralize gravistimulation during germination, which suggests that the change in curvature is a response to a memory' effect carried over from a prior gravistimulation.

2,4-Dichlorophenoxyacetic Acid↗

The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness.

Rapid, auxin-responsive degradation of multiple auxin/indole-3-acetic acid (Aux/IAA) proteins is essential for plant growth and development. Domain II residues were previously shown to be required for the degradation of several Arabidopsis thaliana Aux/IAA proteins. We examined the degradation of additional full-length family members and the proteolytic importance of N-terminal residues outside domain II using luciferase (LUC) fusions. Elimination of domain I did not affect degradation. However, substituting an Arg for a conserved Lys between domains I and II specifically impaired basal degradation without compromising the auxin-mediated acceleration of degradation. IAA8, IAA9, and IAA28 contain domain II and a conserved Lys, but they were degraded more slowly than previously characterized family members when expressed as LUC fusions, suggesting that sequences outside domain II influence proteolysis. We analyzed the degradation of IAA31, with a region somewhat similar to domain II but without the conserved Lys, and of IAA20, which lacks domain II and the conserved Lys. Both IAA20:LUC and epitope-tagged IAA20 were long-lived, and their longevity was not influenced by auxin. Epitope-tagged IAA31 was long-lived, like IAA20, but by contrast, it showed accelerated degradation in response to auxin. The existence of long-lived and auxin-insensitive Aux/IAA proteins suggeststhat they may play a novel role in auxin signaling.

Amino Acid Motifs↗

IAR3 encodes an auxin conjugate hydrolase from Arabidopsis.

Amide-linked conjugates of indole-3-acetic acid (IAA) are putative storage or inactivation forms of the growth hormone auxin. Here, we describe the Arabidopsis iar3 mutant that displays reduced sensitivity to IAA-Ala. IAR3 is a member of a family of Arabidopsis genes related to the previously isolated ILR1 gene, which encodes an IAA-amino acid hydrolase selective for IAA-Leu and IAA-Phe. IAR3 and the very similar ILL5 gene are closely linked on chromosome 1 and comprise a subfamily of the six Arabidopsis IAA-conjugate hydrolases. The purified IAR3 enzyme hydrolyzes IAA-Ala in vitro. iar 3 ilr1 double mutants are more resistant than either single mutant to IAA-amino acid conjugates, and plants overexpressing IAR3 or ILR1 are more sensitive than is the wild type to certain IAA-amino acid conjugates, reflecting the overlapping substrate specificities of the corresponding enzymes. The IAR3 gene is expressed most strongly in roots, stems, and flowers, suggesting roles for IAA-conjugate hydrolysis in those tissues.

Alleles↗

The stimulating effects of ethylene and auxin on petiole elongation and on hyponastic curvature are independent processes in submerged Rumex palustris.

The flooding-tolerant plant species Rumex palustris (Sm.) responds to complete submergence with stimulation of petiole elongation mediated by the gaseous hormone ethylene. We examined the involvement of auxin in petiole elongation. The manipulation of petiolar auxin levels by removing the leaf blade, or by addition of synthetic auxins or auxin transport inhibitors, led to the finding that auxin plays an important role in submergence-induced petiole elongation in R. palustris. A detailed kinetic analysis revealed a transient effect of removing the auxin source (leaf blade), explaining why earlier studies in which less frequent measurements were taken failed to identify any role for auxin in petiole elongation. We previously showed that the onset of stimulated petiole elongation depends on a more upright petiole angle being reached by means of hyponastic (upward) curvature, a differential growth process that is also regulated by ethylene and auxin. This raised the possibility that both ethylene and auxin stimulate elongation only indirectly by influencing hyponastic growth. We show here that the action of ethylene and auxin in promoting petiole elongation in submerged R. palustris is independent of the promoting effect that these hormones also exert on the hyponastic curvature of the same petiole.

Amino Acids, Cyclic↗

Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology.

To investigate the physiological basis of salt adaptation in poplar, we compared the effect of salt stress on wood anatomy and auxin physiology of the salt-resistant Populus euphratica and salt-sensitive Populus x canescens. Both poplar species showed decreases in vessel lumina associated with increases in wall strength in response to salt, however, in P. euphratica at three-fold higher salt concentrations than in P. x canescens. The predicted hydraulic conductivity of the wood formed under salt stress decreased in P. x canescens, while in P. euphratica, no significant effects of salt on conductivity and transpiration were observed. The concentration of free indole-3-acetic acid (IAA) decreased under salt stress in the xylem of both poplar species, but to a larger extent in P. x canescens than in P. euphratica. Only salt-treated P. euphratica exhibited an increase in IAA-conjugates in the xylem. Genes homologous to the auxin-amidohydrolase ILL3 were isolated from the xylems of P. euphratica and P. x canescens. For functional analysis, the auxin-amidohydrolase from P. x canescens was overexpressed in Arabidopsis. Transgenic Arabidopsis plants were more resistant to salt stress than the wild-type plants. Increased sensitivity of the transgenic Arabidopsis to IAA-Leu showed that the encoded hydrolase used IAA-Leu as a substrate. These results suggest that poplar can use IAA-amidoconjugates in the stem as a source of auxin to balance the effects of salt stress on auxin physiology.

Adaptation, Physiological↗

Mutations in Arabidopsis acyl-CoA oxidase genes reveal distinct and overlapping roles in beta-oxidation.

Indole-3-butyric acid (IBA) is an endogenous auxin used to enhance rooting during propagation. To better understand the role of IBA, we isolated Arabidopsis IBA-response (ibr) mutants that display enhanced root elongation on inhibitory IBA concentrations but maintain wild-type responses to indole-3-acetic acid, the principle active auxin. A subset of ibr mutants remains sensitive to the stimulatory effects of IBA on lateral root initiation. These mutants are not sucrose dependent during early seedling development, indicating that peroxisomal beta-oxidation of seed storage fatty acids is occurring. We used positional cloning to determine that one mutant is defective in ACX1 and two are defective in ACX3, two of the six Arabidopsis fatty acyl-CoA oxidase (ACX) genes. Characterization of T-DNA insertion mutants defective in the other ACX genes revealed reduced IBA responses in a third gene, ACX4. Activity assays demonstrated that mutants defective in ACX1, ACX3, or ACX4 have reduced fatty acyl-CoA oxidase activity on specific substrates. Moreover, acx1 acx2 double mutants display enhanced IBA resistance and are sucrose dependent during seedling development, whereas acx1 acx3 and acx1 acx5 double mutants display enhanced IBA resistance but remain sucrose independent. The inability of ACX1, ACX3, and ACX4 to fully compensate for one another in IBA-mediated root elongation inhibition and the ability of ACX2 and ACX5 to contribute to IBA response suggests that IBA-response defects in acx mutants may reflect indirect blocks in peroxisomal metabolism and IBA beta-oxidation, rather than direct enzymatic activity of ACX isozymes on IBA-CoA.

Acyl-CoA Oxidase↗

Thigmomorphogenesis: the relationship of mechanical perturbation to elicitor-like activity and ethylene production.

An extracellular solution obtained from bean (Phaseolus vulgaris L. cv. Resistant Cherokee Wax) stems induced phytoalexin-like substance and ethylene production in a soybean [Glycine max (L.) Merr. cv. Wayne] cotyledon bioassay. The elicitor-like activity for phytoalexin formation and ethylene production was increased by mechanical perturbation of bean stems. Moreover, the application of extracted or known elicitors to bean plants mimicked the effect of mechanical perturbation (i.e., inhibition of stem elongation and enhancement of radial growth). The effects of extract when applied exogenously, on elicitor-like activity in the bioassay as well as stem thickening were decreased by aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis. These results suggest that elicitor-like substances which are formed in response to mechanical perturbation contribute to the thigmomorphogenesis.

Amino Acids↗

Root gravitropism.

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Abscisic Acid↗

Automorphosis of etiolated pea seedlings in space is simulated by a three-dimensional clinostat and the application of inhibitors of auxin polar transport.

Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under microgravity conditions in space show automorphosis: bending of epicotyls, inhibition of hook formation and changes in root growth direction. In order to determine the mechanisms of microgravity conditions that induce automorphosis, we used a three-dimensional clinostat and obtained the successful induction of automorphosis-like growth of etiolated pea seedlings. Kinetic studies revealed that epicotyls bent at their basal region towards the clockwise direction far from the cotyledons from the vertical line (0 degrees) at approximately 40 degrees in seedlings grown both at 1 g and in the clinostat within 48 h after watering. Thereafter, epicotyls retained this orientation during growth in the clinostat, whereas those at 1 g changed their growth direction against the gravity vector and exhibited a negative gravitropic response. On the other hand, the plumular hook that had already formed in the embryo axis tended to open continuously by growth at the inner basal portion of the elbow; thus, the plumular hook angle initially increased; this was followed by equal growth on the convex and concave sides at 1 g, resulting in normal hook formation; in contrast, hook formation was inhibited on the clinostat. The automorphosis-like growth and development of etiolated pea seedlings was induced by auxin polar transport inhibitors (9-hydroxyfluorene-9-carboxylic acid, N-(1-naphthyl)phthalamic acid and 2,3,5-triiodobenzoic acid), but not by anti-auxin (p-chlorophenoxyisobutyric acid) at 1 g. An ethylene biosynthesis inhibitor, 1-aminooxyacetic acid, inhibited hook formation at 1 g, and ethylene production of etiolated seedlings was suppressed on the clinostat. Clinorotation on the clinostat strongly reduced the activity of auxin polar transport of epicotyls in etiolated pea seedlings, similar to that observed in space experiments (Ueda J, Miyamoto K, Yuda T, Hoshino T, Fujii S, Mukai C, Kamigaichi S, Aizawa S, Yoshizaki I, Shimazu T, Fukui K (1999) Growth and development, and auxin polar transport in higher plants under microgravity conditions in space: BRIC-AUX on STS-95 space experiment. J Plant Res 112: 487492). These results suggest that clinorotation on a three-dimensional clinostat is a valuable tool for simulating microgravity conditions, and that automorphosis of etiolated pea seedlings is induced by the inhibition of auxin polar transport and ethylene biosynthesis.

Aminooxyacetic Acid↗

Cross-talk in abscisic acid signaling.

"Cross-talk" in hormone signaling reflects an organism's ability to integrate different inputs and respond appropriately, a crucial function at the heart of signaling network operation. Abscisic acid (ABA) is a plant hormone involved in bud and seed dormancy, growth regulation, leaf senescence and abscission, stomatal opening, and a variety of plant stress responses. This review summarizes what is known about ABA signaling in the control of stomatal opening and seed dormancy and provides an overview of emerging knowledge about connections between ABA, ethylene, sugar, and auxin synthesis and signaling.

Abscisic Acid↗