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Effects of ABA, auxin, and gibberellin on the expression of genes for vacuolar H+ -inorganic pyrophosphatase, H+ -ATPase subunit A, and Na+/H+ antiporter in barley.

We analyzed the effects of ABA, auxin, and gibberellin on the expression of two genes (HVP1 and HVP10) for vacuolar H(+)-inorganic pyrophosphatase (EC 3.6.1.1) and one (HvVHA-A) for the catalytic subunit (subunit A) of vacuolar H(+)-ATPase (EC 3.6.1.3) by quantification of the transcript levels, to identify the hormones responsible for regulating the expression of these genes in barley (Hordeum vulgare L.) in response to environmental changes such as salt stress. ABA markedly induced the expression of HVP1 and slightly increased the expression of HVP10 and HvVHA-A. In contrast, 2,4-D only increased the expression of HVP1, and GA(3) had no significant effects on any gene. The maximum level of HVP1 transcripts in response to these hormones was also much higher than the levels of HVP10 and HvVHA-A transcripts. In addition, we also analyzed the expression of one gene (HvNHX1) for vacuolar Na(+)/H(+) antiporter, and HvNHX1 expression changed in a pattern similar to that of the HVP1 expression. Furthermore, treatment with ABA and 2,4-D increased Na(+)/H(+) antiport activity and proton-translocating activities by H(+)-PPase and H(+)-ATPase in tonoplast vesicles, and treatment with ABA also increased the amount of V-PPase protein of tonoplast vesicles. These results suggest that the hormones ABA and 2,4-D regulate the expression of the H(+)-pump and Na(+)/H(+) antiporter genes and are thus important effectors that regulate the expression of HVP1and HvNHX1.

2,4-Dichlorophenoxyacetic Acid↗

The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement.

In the present study, we investigated a new member of the ABC transporter superfamily of Arabidopsis thaliana, AtMRP5. AtMRP5 encodes a 167 kDa protein and exhibits low glutathione conjugate and glucuronide conjugate transport activity. Promotor- beta-glucuronidase fusion constructs showed that AtMRP5 is expressed mainly in the vascular bundle and in the epidermis, especially guard cells. Using reverse genetics, we identified a plant with a T-DNA insertion in AtMRP5 (mrp5-1). mrp5-1 exhibited decreased root growth and increased lateral root formation. Auxin levels in the roots of mrp5-1 plants were increased. This observation may indicate that AtMRP5 works as an auxin conjugate transporter or that mutant plants are affected in ion uptake, which may lead to changes in auxin concentrations. Experiments on epidermal strips showed that in contrast to wild type, the sulfonylurea glibenclamide had no effect on stomatal opening in mrp5-1 plants. This result strongly suggests that AtMRP5 may also function as an ion channel regulator.

ATP-Binding Cassette Transporters↗

Ethylene and phosphorus availability have interacting yet distinct effects on root hair development.

The hypothesis that ethylene participates in the regulation of root hair development by phosphorus availability in Arabidopsis thaliana was tested by chemically manipulating ethylene synthesis and response and with ethylene-insensitive mutants. Low phosphorus-induced root hair development could be mimicked by adding the ethylene precursor, 1-aminocyclopropane-1-carboxylate (ACC), to high phosphorus media, and inhibited by adding ethylene inhibitors to low phosphorus media. Ethylene-insensitive mutants showed a reduced response to low phosphorus, indicating ethylene involvement in root hair responses to phosphorus deficiency. To dissect the nature of this involvement, the morphological and anatomical changes associated with increased root hair density were investigated. Growth in low phosphorus resulted in smaller, more numerous cortical cells, resulting in a larger number of root hair-bearing epidermal cell files. Cortical cell number was not affected by ethylene inhibitors, ACC, or mutations reducing ethylene sensitivity in roots grown with low phosphorus, indicating that ethylene does not participate in this response. The exception was the eir1 mutation, which strongly reduced this change in radial anatomy, supporting a role for polar auxin transport in this process. Trichoblast cell length was reduced by low phosphorus availability in all genotypes, but even more so for ein2-1 and ein4. The proportion of epidermal cells forming hairs and root hair length were reduced in ethylene-insensitive mutants, especially in the presence of low phosphorus. These results demonstrate multiple effects of low phosphorus from the earliest stages of root hair development, and cross-talk between ethylene and phosphorus in the control of a subset of the low phosphorus effects, concentrating on those later in development.

Amino Acids, Cyclic↗

ABP1 expression regulated by IAA and ABA is associated with the cambium periodicity in Eucommia ulmoides Oliv.

A cDNA clone of Eucommia ulmoides Oliv. encoding auxin binding protein 1 (ABP1), one of the putative receptors of auxin, was isolated, and the seasonal expression of ABP1 in relation to IAA and ABA annual variation was investigated by different technical approaches including RT-PCR, real-time PCR, northern blotting, western blotting, and immunolocalization. In the cambial region, ABP1 expression at both the protein and the mRNA level was found to be high, low, and remarkably scarce in the active, quiescent, and resting stages, respectively, during cambium periodicity. The signal abundance of ABP1 follows the opposite pattern to ABA accumulation and correlates with auxin responsiveness of the cambial tissues, suggesting a role for ABP1 in mediating auxin-dependent regulation of cambial activation in the activity-dormancy cycle. This paper attempts to explain why IAA would 'boost' the reactivation of a quiescent cambium, and not that of a resting cambium. Results also show that ABP1 expression is improved by IAA, while inhibited by ABA.

Abscisic Acid↗

Production of homo- and hetero-dimeric isozymes from two aldehyde oxidase genes of Arabidopsis thaliana.

Polyclonal antibodies were raised against synthetic peptides or recombinant polypeptides encoded by Arabidopsis atAO-1 and atAO-2 cDNAs, which have sequences similar to maize and animal aldehyde oxidase (AO) cDNAs. Anti-atAO-1 antibodies recognized AOalpha and AObeta among the three isoforms, AOalpha, AObeta, and AOgamma, detected in Arabidopsis seedlings after native PAGE, while anti-atAO-2 antibodies reacted with AObeta and AOgamma. The polypeptide specifically recognized by each antibody was collected as the Protein-A/IgG/antigen complex. The 150- and 145-kDa polypeptides were purified by SDS-PAGE and digested with Achromobacter Protease I. From the amino acid sequences and molecular masses of the derivative peptides, it was revealed that the 150- and 145-kDa polypeptides were the products of atAO-1 and atAO-2, respectively. Molecular masses of the native forms of AOalpha, AObeta, and AOgamma were estimated as approximately 290-300 kDa. These results suggest that AOalpha and AOgamma are homodimers consisting of atAO-1 and atAO-2 products, respectively, and that AObeta is a heterodimer of the atAO-1 and atAO-2 products.

Aldehyde Oxidase↗

Seasonal changes of plasma membrane H(+)-ATPase and endogenous ion current during cambial growth in poplar plants.

The plasma membrane H(+)-ATPase (PM H(+)-ATPase), potassium ions, and endogenous ion currents might play a fundamental role in the physiology of cambial growth. Seasonal changes of these parameters were studied in twigs of Populus nigra and Populus trichocarpa. Monoclonal and polyclonal antibodies against the PM H(+)-ATPase, x-ray analysis for K(+) localization and a vibrating electrode for measurement of endogenous ion currents were used as probes. In dormant plants during autumn and winter, only a slight immunoreactivity against the PM H(+)-ATPase was found in cross sections and tissue homogenates, K(+) was distributed evenly, and the density of endogenous current was low. In spring during cambial growth, strong immunoreactivity against a PM H(+)-ATPase was observed in cambial cells and expanding xylem cells using the monoclonal antibody 46 E5 B11 F6 for fluorescence microscopy and transmission electron microscopy. At the same time, K(+) accumulated in cells of the cambial region, and strong endogenous current was measured in the cambial and immature xylem zone. Addition of auxin to dormant twigs induced the formation of this PM H(+)-ATPase in the dormant cambial region within a few days and an increase in density of endogenous current in shoot cuttings within a few hours. The increase in PM H(+)-ATPase abundance and in current density by auxin indicates that auxin mediates a rise in number and activity of an H(+)-ATPase in the plasma membrane of cambial cells and their derivatives. This PM H(+)-ATPase generates the necessary H(+)-gradient (proton-motive force) for the uptake of K(+) and nutrients into cambial and expanding xylem cells.

Abscisic Acid↗

Stunted plant 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis.

Plants control organ growth rate by adjusting the rate and duration of cell division and expansion. Surprisingly, there have been few studies where both parameters have been measured in the same material, and thus we have little understanding of how division and expansion are regulated interdependently. We have investigated this regulation in the root meristem of the stunted plant 1 (stp1) mutation of Arabidopsis, the roots of which elongate more slowly than those of the wild type and fail to accelerate. We used a kinematic method to quantify the spatial distribution of the rate and extent of cell division and expansion, and we compared stp1 with wild type and with wild type treated with exogenous cytokinin (1 microM zeatin) or auxin (30 nM 2,4-dichlorophenoxyacetic acid). All treatments reduced average cell division rates, which reduced cell production by the meristem. Auxin lowered root elongation by narrowing the elongation zone and reducing the time spent by a cell in this zone, but did not decrease maximal strain rate. In addition, auxin increased the length of the meristem. In contrast, cytokinin reduced root elongation by lowering maximal strain rate, but did not change the time spent by a cell within the elongation zone; also, cytokinin blocked the increase in length and cell number of the meristem and elongation zone. The cytokinin-treated wild type phenocopied stp1 in nearly every detail, supporting the hypothesis that cytokinin affects root growth via STP1. The opposite effects of auxin and cytokinin suggest that the balance of these hormones may control the size of the meristem.

2,4-Dichlorophenoxyacetic Acid↗

Reduction of benzenoid synthesis in petunia flowers reveals multiple pathways to benzoic acid and enhancement in auxin transport.

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a beta-oxidative or non-beta-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between L-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

Acyltransferases↗

The etr1-2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist-chilling and germination.

In Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moist-chilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moist-chilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.

Abscisic Acid↗

Hormone profiling in Arabidopsis.

A highly sensitive and accurate multiplex gas chromatography-mass spectrometry (GC-MS/MS)-technique is reported for the quantitative analysis of acidic phytohormones in Arabidopsis thaliana and other plant species. The optimized setup allows the routine processing and analysis of between 20 mg and 5 g of tissue. The protocol was designed and the equipment used was chosen to facilitate implementation of the method into other laboratories and to provide access to state-of-the-art analytical tools for the acidic phytohormones and related signaling molecules.

Abscisic Acid↗

Pathways of the abscisic acid hormonal signal transduction across the plant cell plasma membrane.

Signal transduction pathways of a phytohormone abscisic acid (ABA) in the plant cell plasma membrane are reviewed. The mechanisms of ABA perception; ABA-induced alterations of Ca2+-, K+-, and anion channel activity, the ABA effects on intracellular pH and membrane-bound enzyme activity, as well as the phytohormone interaction with the lipid phase of plasma membrane are considered, and the role of the membrane protein phosphorylation is discussed. The available data suggest that the regulatory signal of ABA can be transmitted via a ramified chain of reactions. In different tissues, functioning of various branches of this chain can be independent or interrelated.

Abscisic Acid↗

In vitro uptake and processing of maize auxin-binding proteins by ER-derived microsomes.

We have analyzed auxin-binding proteins from maize encoded by the Zm-ERabp gene family. Open reading frames of cDNA clones predict proteins containing N-terminal hydrophobic signal sequences. In vitro studies show that the Zm-ERabp1 protein can be translocated into ER-derived microsomes where it is processed and glycosylated. A cDNA clone encoding the Zm-ERabp4 protein predicts an open reading frame with a signal sequence that shows striking differences in charge distribution, in comparison to the signal sequence of Zm-ERabp1. Two translation products are synthesized from the Zm-ERabp4 transcript in the in vitro system, but only one of them is translocated into maize endosperm microsomes, indicating that specific cotranslational modifications in the primary sequence remaining after processing may play a role in the cellular trafficking of the Zm-ERabp4 protein.

Amino Acid Sequence↗