Modifications of a conserved regulatory network involving INDEHISCENT controls multiple aspects of reproductive tissue development in Arabidopsis.

Disrupting pollen tube growth and fertilization in Arabidopsis plants leads to reduced seed set and silique size, providing a powerful genetic system with which to identify genes with important roles in plant fertility. A transgenic Arabidopsis line with reduced pollen tube growth, seed set and silique growth was used as the progenitor in a genetic screen to isolate suppressors with increased seed set and silique size. This screen generated a new allele of INDEHISCENT (IND), a gene originally identified by its role in valve margin development and silique dehiscence (pod shatter). IND forms part of a regulatory network that involves several other transcriptional regulators and involves the plant hormones GA and auxin. Using GA and auxin mutants that alter various aspects of reproductive development, we have identified novel roles for IND, its paralogue HECATE3, and the MADS box proteins SHATTERPROOF1/2 in flower and fruit development. These results suggest that modified forms of the regulatory network originally described for the Arabidopsis valve margin, which include these genes and/or their recently evolved paralogs, function in multiple components of GA/auxin-regulated reproductive development.

GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis.

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.

A novel MYB-related gene from Arabidopsis thaliana expressed in developing anthers.

A novel MYB-like gene (AtMYB103) was isolated from a genomic library of Arabidopsis. Plants transgenic for chimeric AtMYB103 promoter/GUS genes expressed the enzyme in early anthers. In situ hybridization of flower sections showed a high level of AtMYB103 mRNA in the tapetum and middle layer of developing anthers.

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH.

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pHi) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pHi, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152-163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1.

Detection and characterization of an activity which aligns mesodermal cells into parallel arrays.

A cell line of mesodermal origin, FS9, was found to release a Cell Orienting Factor into its culture medium. In contrast with the random migration of controls, the orienting activity causes migrating mesenchymal cells to form an orderly "halo' surrounding tissue explants; individual cells and their cytoskeletons are elongated and parallel to each other but at right angle to the explant. No effect on the rate of cell movement was apparent. The orienting activity could be quantified by counting the number of cells found within strings radiating at right angles to a single tissue explant in the presence of FS9 conditioned medium or by using NIH image analysis. A dose dependent relationship with half maximal activity occurring at a 25% dilution of conditioned medium was observed. Cells that migrated randomly in the absence of conditioned medium became oriented within 4 h of exposure to 50% conditioned medium. Conversely, when the conditioned medium was removed, parallel alignment was rapidly lost. The orienting activity was found in conditioned media from a variety of mesodermal derivatives. Transformation of Balb/c 3T3 cells using EJ-ras oncogene led to augmented production of the activity. Furthermore, insulin was required in serum-free medium to support its production, Laminin, fibronectin and collagen and a range of pure cytokines, neither promoted nor inhibited orientation. Cell alignment was also unaffected by treatments which interfered with cell-substrate interactions and motility including the addition of the RGD peptide or anti-integrin beta 1 and beta 3 antibodies. A protein is likely to be involved since the activity was heat and trypsin sensitive and non-dialysable. The possibility is discussed that the orienting activity is a novel protein(s) which alters intercellular interactions to promote the formation of an aligned pattern by migrating mesenchymal cells.

Promoter and expression studies on an Arabidopsis thaliana dehydrin gene.

A genomic clone of a group 2 lea/rab/dehydrin gene from Arabidopsis thaliana, Xero2/lti30, was cloned and sequenced. Promoter-GUS fusions were introduced into plants to analyse the promoter and determine expression patterns. Using root cultures, GUS expression was found to be moderately stimulated by abscisic acid (ABA), wounding, cold and dehydration. Results with an ABA-deficient mutant suggested endogenous ABA is required for these responses. Promoter deletion studies indicated multiple cis-acting elements are involved in the induction of the gene. GUS expression occurred in desiccated seeds, in all tissues of young seedlings and in roots (with the exception of the root tip), desiccated pollen grains, trichomes and the vascular tissues of leaves and stems in mature plants.

A novel myb-related gene from Arabidopsis thaliana.

A novel myb-like gene (Atmyb5) has been isolated from a genomic library of Arabidopsis thaliana. The gene contains a single intron in the region coding for the Myb domains. The Myb domains are highly homologous to other animal and plant Myb proteins. Arabidopsis plants transgenic for a chimeric Atmyb5 promoter/GUS gene expressed the enzyme in a developmentally controlled and tissue specific manner. The GUS activity was detected in developing leaf trichomes, stipules, epidermal cells on the margins of young rosette and cauline leaves, and in immature seeds. Atmyb5 mRNA appears between fertilization and the 16 cell stage of embryo development and persists beyond the heart stage.

Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products.

Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library. The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli. The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies. The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS). The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS. Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS. This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS. The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo. The third alpha-helix and three adjacent amino acids in the third repeat (R3) of c-Myb was replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein. This chimeric protein bound to PBS with a low affinity but failed to bind to MBS. Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6. This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding.

Studies on the promoter of the Arabidopsis thaliana cdc2a gene.

The 5' flanking (promoter) region of the Arabidopsis thaliana cdc2a gene was cloned and sequenced. A number of putative regulatory motifs were identified including one Myc and three Myb protein binding sequences plus one abscisic acid and two auxin responsive elements. One of the three Myb protein binding sequences is positioned within an auxRE. Promoter-GUS fusions were introduced into plants to study the role of two promoter regions in regulating gene expression. Absence of one Myb binding sequence and the auxRE containing a Myb binding sequence resulted in a significant reduction in expression levels as did a deletion involving the Myc and the third Myb binding sequences along with the second auxRE. However, no changes in expression patterns were observed. The results were quantified using transgenic root cultures.

A monoclonal antibody that interferes with the post-aggregation adhesion of Dictyostelium discoideum cells.

A monoclonal antibody that interferes with the EDTA-resistant adhesion of Dictyostelium discoideum slug cells recognised a carbohydrate epitope on four major antigens (95, 90, 35 and 30 kDa) in slug cells. The 35 and 30 kDa antigens were specific for stalks and spores, respectively. The 30 kDa antigen was identified as the cell surface glycoprotein, PsA. Cyclic AMP, acting via cell surface receptors, induced only the 90 kDa slug cell antigen. Slug cell adhesion proteins may be involved in cell-sorting and the glycosylation of the 95 and 90 kDa antigens appeared to be abnormal in a mutant defective in cell-sorting. Previously, a 150 kDa glycoprotein has been strongly implicated in slug cell adhesion and the present work suggests that additional glycoprotein(s) are involved.

Polysaccharides influence the aggregation of Dictyostelium discoideum cells and bind to developmentally regulated cell surface proteins.

Six of ten anionic polysaccharides studied were found to significantly reduce the adhesion of growth-phase Dictyostelium discoideum cells. However, only hyaluronic acid, chondroitin-4-sulfate and chondroitin-6-sulfate interfered with the adhesion of aggregation-competent cells. Neither EDTA-stable nor EDTA-sensitive adhesion of postaggregation cells were affected by the polyanions. The two chondroitin sulfates influenced the aggregation of cells in submerged cultures, long and broad aggregation streams being formed and the broad sheets of cells eventually building multilayered aggregates. Radioiodination of cell surface proteins followed by cellulose fiber affinity chromatography identified the same nine proteins bound by hyaluronic acid and the chondroitin sulfates, six of which were regulated during development. Protease-resistant anionic material isolated from cells bound the same surface proteins as the three glycosaminoglycans. Discoidin I bound to the uncoupled cellulose fibers, suggesting a structural role for the lectin in the extracellular slime sheath. Anionic polysaccharides and cell surface lectins that bind them may be involved in the cell recognition, cell aggregation, and the cell sorting that occurs during pattern formation.

Changes in cytosolic pH and calcium of guard cells precede stomatal movements.

Stomatal opening is induced by indoleacetic acid (IAA), cytokinins, and fusicoccin (FC), whereas stomatal closure is induced by abscisic acid (ABA). To test the effect of these growth regulators on guard cell cytosolic Ca2+ ([Ca2+]cyt) and pH (pHcyt), epidermal strips were taken from the lower side of leaves of the orchid Paphiopedilum tonsum and were loaded with acetomethoxy-esterified forms of the Ca2+ indicator fluo-3 or the pH indicator 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein. Basal [Ca2+]cyt ranged from 0.05 to 0.3 M and was 0.22 +/- 0.015 (n = 21). Increases in both [Ca2+]cyt and pHcyt were observed in guard cells after application of 10-100 M ABA to open stomata, and these preceded stomatal closure. The increase in [Ca2+]cyt ranged from 1.5- to 3-fold and was seen in 7 of 10 experiments. Guard cell alkalinization began within 2 min of ABA treatment and continued for the next 8 min. The increase ranged from 0.04 to 0.3 pH unit and was seen in 13 of 14 experiments. Guard cell [Ca2+]cyt increased, whereas pHcyt decreased after treatment of closed stomata with IAA, kinetin, or FC. In response to 50-100 M IAA, [Ca2+]cyt increased 1.5- to 2-fold in all cases, and pHcyt decreased 0.2-0.4 unit within 5 min in 7 experiments. Within 12 min, 10-100 M kinetin caused [Ca2+]cyt to increase in 28 of 34 experiments (1.3- to 2.5-fold) and pHcyt fell 0.1-0.4 unit in 15 of 17 treatments. The response to 10-50 M FC was similar in both time and magnitude. These results show that stomatal opening is accompanied by an increase in [Ca2+]cyt and cytosolic acidification in the guard cells, whereas stomatal closure is preceded by an increase in [Ca2+]cyt and cytosolic alkalinization in the guard cells. The order of these events is still uncertain, but changes in pHcyt are correlated with stomatal movement, and these changes may be an important factor in the regulation of guard cell movement.

Effects of auxin and abscisic acid on cytosolic calcium and pH in plant cells.

Dark-grown corn coleoptiles and parsley hypocotyls and their roots were loaded with acetoxymethyl esterified forms of the Ca2+ indicator fluo-3, and the pH indicator 2',7'-bis (2-carboxyethyl)-5(and-6)-carboxyfluorescein. These tissues were treated with the plant growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin analogue, or abscisic acid (ABA), and the cytosolic pH (pHcyt) and cytosolic Ca2+ ([Ca2+]cyt) changes were monitored by confocal scanning optical microscopy. Over a period of 4 min pHcyt decreased 0.1-0.2 pH unit and [Ca2+]cyt increased from 280 to 380 nM in response to 2,4-D. ABS, on the other hand, induced cytosolic alkalinization of 0.05-0.1 pH unit with a concomitant increase in [Ca2+]cyt from 240 to 320 nM over a 4-min period. Responses similar to these were observed in all the tissues tested. We suggest that pHcyt profoundly influences signaling by[Ca2+]cyt, possibly by regulating Ca2+-protein binding, and that the divergent effects of auxin and ABA on pHcyt underlie their mutual antagonism.

Phototropism and geotropism in maize coleoptiles are spatially correlated with increases in cytosolic free calcium.

Phototropism and gravitropism in the shoots and roots of higher plants are the result of asymmetric growth. This is explained by the redistribution of growth regulators following exposure to gravity or unilateral light (the Cholodny-Went hypothesis). The positive phototropism and the negative geotropism of grass seedling coleoptiles are believed to result from lateral movement of auxin from the irradiated to the shaded side and from the upper to the lower side, respectively. Many physiological processes in plants, including auxin-induced cell elongation, are reported to be under the control of calcium. Added auxin triggers oscillations in cytosolic free calcium ([Ca2+]cyt) and cytosolic pH (pHcyt) in epidermal cells of maize coleoptiles. Until recently, it has not been possible to visualize these changes spatially with the commonly used fluorescent cation indicators. Using a scanning laser confocal microscope, a new visible wavelength Ca2+ probe fluo-3 and the fluorescent pH indicator BCECF, we have recorded rapid light-induced increases in [Ca2+]cyt and a lowering of pHcyt of cells on the shaded side of maize coleoptiles. In horizontally orientated coleoptiles, [Ca2+]cyt increases and pHcyt decreases in the more rapidly elongating cells on the lower side. For the first time, rapid changes in [Ca2+]cyt and pHcyt are correlated directly with increases in cell elongation stimulated by light and gravity.

Confocal imaging of ionised calcium in living plant cells.

Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively.

A mycoplasmal protein influences tumour cell invasiveness and contact inhibition in vitro.

Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 mouse sarcoma cells, were previously found to inhibit the highly invasive behaviour of FS9 cells in vitro. We show that p37 originates from Mycoplasma hyorhinis. Infecting various cell lines with the mycoplasma consistently increased their invasiveness when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. Conversely, removing the mycoplasmas or blocking p37 with specific Fab fragments reduced invasiveness. Analysis of individual cell collisions using time-lapse filming showed that the addition of Fab fragments to cells infected with M. hyorhinis greatly increased the level of contact inhibition. The antibody also reduced the invasiveness of transformed cells that did not express the p37 antigen. Hence, a cellular protein or proteins that are structurally related to p37 apparently influence invasive behavior.

The effects of transcription on the nucleosome structure of four Dictyostelium genes.

Micrococcal nuclease digestion of Dictyostelium discoideum nuclei from various developmental stages was used to investigate transcription-related changes in the chromatin structure of the coding region of four genes. Gene activity was determined by Northern blotting and nuclear run on experiments. During strong transcription of the developmentally regulated cysteine proteinase I gene, a smear superimposed on a nucleosomal ladder was observed, indicating perturbation of nucleosomal structure was occurring. However, two other developmentally regulated genes, discoidin I and pSC253, showed only slight nucleosome disruption during high levels of transcription. The chromatin structure of a fourth gene (pCZ22) was disrupted throughout development, even at those stages where transcription was greatly reduced. We suggest that although nucleosome structure can be transiently perturbed by the passage of the transcription complex in vivo, the degree of perturbation and the speed with which nucleosomes reassemble is also influenced by the DNA sequence.

The upstream limit of nuclease-sensitive chromatin in Dictyostelium rRNA genes neighbors a topoisomerase I-like cluster.

In the chromatin of Dictyostelium ribosomal RNA (rRNA) genes, the coding and upstream flanking regions are sensitive to endonucleases. This sensitivity stops about 2.3 x 10(3) bases upstream from the transcription start, at a point we call the structural boundary. Upstream from the boundary an 850 base-pair region is strongly protected against micrococcal nuclease cleavage, particularly in rapidly transcribing vegetative cells, and upstream from this the pattern of nuclease protection suggests that positioned nucleosomes are present. On the gene side of the structural boundary nucleosomes are known to be absent in vegetative cells but present in differentiating slug cells where the rRNA synthesis rate is lower. We show that in slugs these nucleosomes are randomly distributed, in contrast to those upstream from the boundary. Close to the gene side of the boundary is a duplication of the putative promoter located 29 base-pairs distant from four clustered topoisomerase I recognition sequences, which are cleaved by endogenous topoisomerase I-like activity. An additional topoisomerase I recognition sequence found upstream from the structural boundary is not cleaved in chromatin. The possible significance of these sequences and structures in transcription is discussed.

A mycoplasma high-affinity transport system and the in vitro invasiveness of mouse sarcoma cells.

FS9 mouse sarcoma cells were previously shown to be highly invasive when confronted with chicken heart fibroblasts using Abercrombie's confronted explant technique. This invasion could be inhibited by addition to the assay of Fab fragments of a monoclonal antibody directed against p37, a protein associated with the surface of FS9 cells. We have cloned and sequenced the gene for p37. We show that it originates from Mycoplasma hyorhinis and that UGA is a tryptophan codon in this organism. We present evidence that the p37 gene is part of an operon encoding two additional proteins which are highly similar to components of the periplasmic binding-protein-dependent transport systems of Gram-negative bacteria, and we suggest that p37 is part of a homologous, high-affinity transport system in M. hyorhinis, a Gram-positive bacterium. We discuss the influence of p37 and M. hyorhinis on contact inhibition of locomotion of mammalian cells.