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Plant fertility defects induced by the enhanced expression of microRNA167.

The plant hormone auxin plays a critical role in regulating plant growth and development. Recent advances have been made in the understanding of auxin response pathways, primarily by the characterization of auxin response mutants in Arabidopsis. In addition, microRNAs (miRNAs) have been shown to be critical regulators of genes important for normal plant development and physiology. However, little is known about possible interactions between miRNAs and hormonal signaling during normal development. Here we show that an Arabidopsis microRNA, miR167, which has a complementary sequence to a portion of the AUXIN RESPONSE FACTOR6 (ARF6) and ARF8 mRNAs, can cause transcript degradation for ARF8, but not for ARF6. We report phenotypic characterizations of 35S::MIR167b transgenic lines, and show that severe 35S::MIR167b transgenic lines had phenotypes similar to those of an arf6 arf8 double mutant. The transgenic phenotypes suggest that miR167 may repress ARF6 at the level of translation. We demonstrate that the transgenic plants are defective in all four whorls of floral organs. In the transgenic flowers, filaments were abnormally short, anthers could not properly release pollen, and pollen grains did not germinate. Our results provide an important link between the miRNA-mediated regulatory pathway of gene expression and the auxin signaling network promoting plant reproductive development.

Arabidopsis↗

Deuterium in vivo labelling of cytokinins in Arabidopsis thaliana analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry.

A method was developed for analysing the biosynthetic rate of the cytokinin class of plant hormones. Transgenic, cytokinin-overproducing Arabidopsis thaliana plants were incubated in liquid culture media enriched with 30% deuterium oxide, and incorporation into the different parts of the cytokinin molecule was analysed by capillary liquid chromatography/frit-fast atom bombardment mass spectrometry after precolumn propionylation. The sugar moieties of the cytokinins generally showed a high and independent incorporation, so the analysis in this study focused on the cytokinin base moieties. It was observed that during a 24 h incubation period almost all labelling was incorporated into the side-chain, rather than the adenine moiety. The incorporation dynamics of isopentenyladenosine-5'-monophosphate, zeatinriboside-5'-monophosphate (ZRMP) and zeatin-9-glucoside were investigated through analysis of the cytokinin base fragments in high-resolution selective ion monitoring mode. Using a fractional synthetic rate approach, the biosynthetic rate of ZRMP was determined to be 18 ng h(-1) g(-1) fresh weight, giving a turnover time of 25 h. A method for the mass isotopomer abundance analysis of the cytokinins in the zeatin family, based on selective reaction monitoring, was also developed to gain further sensitivity. Use of this technique showed that there was a higher level of enrichment in zeatin nucleotide than in the corresponding nucleoside, in agreement with the hypothesis that cytokinin nucleotides are primary products in this pathway.

Arabidopsis↗

Multiple mediators of plant programmed cell death: interplay of conserved cell death mechanisms and plant-specific regulators.

Programmed cell death (PCD) is a process aimed at the removal of redundant, misplaced, or damaged cells and it is essential to the development and maintenance of multicellular organisms. In contrast to the relatively well-described cell death pathway in animals, often referred to as apoptosis, mechanisms and regulation of plant PCD are still ill-defined. Several morphological and biochemical similarities between apoptosis and plant PCD have been described, including DNA laddering, caspase-like proteolytic activity, and cytochrome c release from mitochondria. Reactive oxygen species (ROS) have emerged as important signals in the activation of plant PCD. In addition, several plant hormones may exert their respective effects on plant PCD through the regulation of ROS accumulation. The possible plant PCD regulators discussed in this review are integrated in a model that combines plant-specific regulators with mechanisms functionally conserved between animals and plants.

Apoptosis↗

Spatiotemporal asymmetric auxin distribution: a means to coordinate plant development.

The plant hormone auxin plays crucial roles in regulating plant growth development, including embryo and root patterning, organ formation, vascular tissue differentiation and growth responses to environmental stimuli. Asymmetric auxin distribution patterns have been observed within tissues, and these so-called auxin gradients change dynamically during different developmental processes. Most auxin is synthesized in the shoot and distributed directionally throughout the plant. This polar auxin transport is mediated by auxin influx and efflux facilitators, whose subcellular polar localizations guide the direction of auxin flow. The polar localization of PIN auxin efflux carriers changes in response to developmental and external cues in order to channel auxin flow in a regulated manner for organized growth. Auxin itself modulates the expression and subcellular localization of PIN proteins, contributing to a complex pattern of feedback regulation. Here we review the available information mainly from studies of a model plant, Arabidopsis thaliana, on the generation of auxin gradients, the regulation of polar auxin transport and further downstream cellular events.

Arabidopsis↗

MAP kinases in plant signal transduction.

Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. Distinct MAPK pathways are regulated by different extracellular stimuli and are implicated in a wide variety of biological processes. In plants there is evidence for MAPKs playing a role in the signaling of abiotic stresses, pathogens and plant hormones. The large number and divergence of plant MAPKs indicates that this ancient mechanism of bioinformatics is extensively used in plants and may provide a new molecular handle on old questions.

Amino Acid Sequence↗

Ethylene gas: it's not just for ripening any more!

Hormones play a central role in regulating plant growth and development. There are five well-characterized plant hormones, the chemically simplest of which is the gaseous olefin ethylene. Molecular genetic studies of Arabidopsis are beginning to reveal the mechanisms controlling ethylene biosynthesis, perception and signal transduction.

Arabidopsis↗

Auxin distribution and plant pattern formation: how many angels can dance on the point of PIN?

The plant hormone auxin is central in patterning diverse plant tissues. The direction of auxin flow and the distribution of auxin within tissues are regulated by auxin efflux transporters that are polarly localized in cells. Feedback regulation between auxin and its transporters establishes homeostatic patterns of auxin accumulation but allows dynamic repatterning in response to developmental or environmental cues.

Arabidopsis Proteins↗

Structure prediction, evolution and ligand interaction of CHASE domain.

Cytokinins are plant hormones involved in the essential processes of plant growth and development. They bind with receptors known as CRE1/WOL/AHK4, AHK2, and AHK3, which possess histidine kinase activity. Recently, the sensor domain cyclases/histidine kinases associated sensory extracellular (CHASE) was identified in those proteins but little is known about its structure and interaction with ligands. Distant homology detection methods developed in our laboratory and molecular phylogeny enabled the prediction of the structure of the CHASE domain as similar to the photoactive yellow protein-like sensor domain. We have identified the active site pocket and amino acids that are involved in receptor-ligand interactions. We also show that fold evolution of cytokinin receptors is very important for a full understanding of the signal transduction mechanism in plants.

Amino Acid Sequence↗

Cell expansion in roots.

Cell expansion in roots is crucial for the exploration and exploitation of the soil substrate and the plethora of activities that roots engage in. Expansion requires the coordinated activities of many cell processes. Central to this is the control of ion transport during vacuolar growth, which mediates the increase in cell size and the concomitant production of new wall and membrane at the surface of growing cells. The cytoskeleton plays an important role in growth and the control of growth direction. Evidence is accumulating to show that plant hormones also coordinate cell expansion throughout the plant by controlling the activities of growth-regulating DELLA proteins.

Arabidopsis↗

OsBLE3, a brassinolide-enhanced gene, is involved in the growth of rice.

Brassinosteroids (BRs) are a group of plant hormones involved in a wide range of plant growth and developmental processes. To investigate the mechanism of BR action in monocots, a brassinolide (BL) upregulated gene designated OsBLE3 was identified, cloned and characterized in rice. It was mainly expressed in roots and leaf sheaths with levels of expression directly dependent on the dose of BL. In situ hybridization detected OsBLE3 mRNA in the shoot apical meristem, organ primordia and vascular tissue. Furthermore, its expression was enhanced by co-treatment with BL and low concentrations of IAA. These results, and the existence of auxin response elements in the 5'-flanking region of the OsBLE3 gene, indicate that OsBLE3 expression is under control of both BR and auxin. The GUS reporter gene driven by a 2277 bp OsBLE3 putative promoter was mainly expressed in vascular tissues, branch root primordia and was responsive to exogenous BL treatment. OsBLE3 transcript levels were greatly reduced in brd1 plants, a BL deficient mutant, compared to the wild type control. In OsBRI1 antisense transgenic rice and OsBLE3, the BR-insensitive mutant expression of OsBLE3 in response to exogenous BL treatment was significantly lower compared to that in control plants transformed with a vacant vector. Reduced OsBLE3 expression and growth retardation was also observed in OsBLE3 antisense transgenic rice plants. Internode cell length of the OsBLE3 antisense transgenic lines was about 70% of that in the vacant vector transformed control lines. These results suggest that OsBLE3 is involved in cell elongation in rice through dual regulation by BL and IAA.

Amino Acid Sequence↗

Cytokinin receptor: just another histidine kinase.

The cytokinin family of plant hormones is involved in diverse aspects of plant growth and development in vivo and in culture. Two groups have recently shown that a two-component histidine kinase functions as a cytokinin receptor specifically required for vascular development.

Arabidopsis↗

Interactions of PIN and PGP auxin transport mechanisms.

Polarized transport of the plant hormone auxin influences multiple growth processes in plants and is regulated by plasma-membrane-localized efflux and uptake carriers. The PGP (P-glycoprotein) ABC transporters (ATP-binding-cassette transporters), PIN (pin-formed) subfamily of major facilitator proteins and members of AUX/LAX families have been shown to independently transport auxin both in planta and in heterologous systems. However, PIN- and PGP-mediated transport in heterologous systems exhibits decreased substrate specificity and inhibitor-sensitivity compared with what is seen in plants and plant cells. To determine whether PIN-PGP interactions enhance transport specificity, we analysed interactions of the representative auxin-transporting PGPs with PIN1 and AUX1 in planta and in heterologous systems. Here, we provide evidence that PINs and PGPs interact and function both independently and co-ordinately to control polar auxin transport and impart transport specificity and directionality. These interactions take place in protein complexes stabilized by PGPs in detergent-resistant microdomains.

ATP-Binding Cassette Transporters↗

PINOID-mediated signaling involves calcium-binding proteins.

The plant hormone auxin is a central regulator of plant development. In Arabidopsis, the PINOID (PID) protein serine/threonine kinase is a key component in the signaling of this phytohormone. To further investigate the biological function of PID, we performed a screen for PID-interacting proteins using the yeast two-hybrid system. Here, we show that PID interacts with two calcium-binding proteins: TOUCH3 (TCH3), a calmodulin-related protein, and PID-BINDING PROTEIN 1 (PBP1), a previously uncharacterized protein containing putative EF-hand calcium-binding motifs. The interaction between PID and the calcium-binding proteins is significant because it is calcium dependent and requires an intact PID protein. Furthermore, the expression of all three genes (PID, TCH3, and PBP1) is up-regulated by auxin. TCH3 and PBP1 are not targets for phosphorylation by PID, suggesting that these proteins act upstream of PID. PBP1 was found to stimulate the autophosphorylation activity of PID, and calcium influx and calmodulin inhibitors where found to enhance the activity of PID in vivo. Our results indicate that TCH3 and PBP1 interact with the PID protein kinase and regulate the activity of this protein in response to changes in calcium levels. This work provides the first molecular evidence for the involvement of calcium in auxin-regulated plant development.

Amino Acid Sequence↗

The ethylene signal transduction pathway in plants.

Ethylene (C2H4), the chemically simplest plant hormone, is among the best-characterized plant growth regulators. It participates in a variety of stress responses and developmental processes. Genetic studies in Arabidopsis have defined a number of genes in the ethylene signal transduction pathway. Isolation of two of these genes has revealed that plants sense this gas through a combination of proteins that resemble both prokaryotic and eukaryotic signaling proteins. Ethylene signaling components are likely conserved for responses as diverse as cell elongation, cell fate patterning in the root epidermis, and fruit ripening. Genetic manipulation of these genes will provide agriculture with new tools to prevent or modify ethylene responses in a variety of plants.

Amino Acid Sequence↗

On the trail of a new regulatory system in plants. Polyomines as modulators of plant development sponsored by Fundación Juan March, Madrid, Spain, February 4-6, 1991.

Investigations into the physiology and biochemistry of polyamines in plants have virtually exploded in the last decade. While not yet accepted by plant physiologists as plant hormones or even as major regulators of plant growth and development, the polyamines require consideration in such varied fields as cell division, organ differentiation, senescence, and stress. We are sure to see more conferences on this subject in the near future.

Adaptation, Physiological↗

The PS-IAA4/5-like family of early auxin-inducible mRNAs in Arabidopsis thaliana.

The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts in Arabidopsis thaliana. The corresponding genes, IAA1 to IAA14, are homologs of PS-IAA4/5 and PS-IAA6 from pea, Aux22 and Aux28 from soybean, ARG3 and ARG4 from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cycloheximide. Cycloheximide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cycloheximide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 microM IAA. However, individual genes respond differentially between 10 nM and 5 microM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2 and aux1. The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spacers vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative beta alpha alpha-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic beta-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development.

Amino Acid Sequence↗

Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.

Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low-density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM. To quantify the mitogenic activity of CM, we examined conditions for the assay system using mechanically dispersed mesophyll cells of Asparagus officinalis L. and established a highly sensitive bioassay method. By use of this method, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat-stable, susceptible to pronase digestion, and resistant to glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 10(7)-fold by column chromatography, and two factors named phytosulfokine-alpha and -beta (PSK-alpha and PSK-beta) were obtained. By amino acid sequence analysis and mass spectrometry, the structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) and sulfated tetrapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH). PSK-alpha and PSK-beta were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors, confirming the structure for PSK-alpha and PSK-beta mentioned above. This is the first elucidation of the structure of a conditioned medium factor required for the growth of low-density plant cell cultures.

Amino Acid Sequence↗

An early auxin-responsive Aux/IAA gene from wheat (Triticum aestivum) is induced by epibrassinolide and differentially regulated by light and calcium.

The plant hormone auxin plays a central role in regulating many aspects of plant growth and development. This largely occurs as a consequence of changes in gene expression. The Aux/IAA genes are best characterized among the early auxin-responsive genes, which encode short-lived transcriptional repressors. In most plants examined, including Arabidopsis, soybean, and rice, the Aux/IAA genes constitute a large gene family. By screening the available databases, at least 15 expressed sequence tags (ESTs) have been identified from wheat (Triticum aestivum), which exhibit high sequence identity with Aux/IAA homologues in other species. One of these Aux/IAA genes, TaIAA1, harbouring all the four conserved domains characteristic of the Aux/IAA proteins, has been characterized in detail. The expression of TaIAA1 is light-sensitive, tissue-specific, and is induced within 15-30 min of exogenous auxin application. Also, the TaIAA1 transcript levels increase in the presence of a divalent cation, Ca(2+), and this effect is reversed by the calcium-chelating agent, EGTA. The TaIAA1 gene qualifies as the primary response gene because an increase in its transcript levels by auxin is unaffected by cycloheximide. In addition to auxin, the TaIAA1 gene is also induced by brassinosteroid, providing evidence that interplay between hormones is crucial for the regulation of plant growth and development.

Blotting, Southern↗