A phylogenetically conserved APETALA2/ETHYLENE RESPONSE FACTOR, ERF12, regulates Arabidopsis floral development.

KEY MESSAGE: Arabidopsis ETHYLENE RESPONSE FACTOR12 (ERF12), the rice MULTIFLORET SPIKELET1 orthologue pleiotropically affects meristem identity, floral phyllotaxy and organ initiation and is conserved among angiosperms. Reproductive development necessitates the coordinated regulation of meristem identity and maturation and lateral organ initiation via positive and negative regulators and network integrators. We have identified ETHYLENE RESPONSE FACTOR12 (ERF12) as the Arabidopsis orthologue of MULTIFLORET SPIKELET1 (MFS1) in rice. Loss of ERF12 function pleiotropically affects reproductive development, including defective floral phyllotaxy and increased floral organ merosity, especially supernumerary sepals, at incomplete penetrance in the first-formed flowers. Wildtype floral organ number in early formed flowers is labile, demonstrating that floral meristem maturation involves the stabilisation of positional information for organogenesis, as well as appropriate identity. A subset of erf12 phenotypes partly defines a narrow developmental time window, suggesting that ERF12 functions heterochronically to fine-tune stochastic variation in wild type floral number and similar to MFS1, promotes meristem identity. ERF12 expression encircles incipient floral primordia in the inflorescence meristem periphery and is strong throughout the floral meristem and intersepal regions. ERF12 is a putative transcriptional repressor and genetically opposes the function of its relatives DORNRÖSCHEN, DORNRÖSCHEN-LIKE and PUCHI and converges with the APETALA2 pathway. Phylogenetic analysis suggests that ERF12 is conserved among all eudicots and appeared in angiosperm evolution concomitant with the generation of floral diversity.

DORNRÖSCHEN, DORNRÖSCHEN-LIKE, and PUCHI redundantly control floral meristem identity and organ initiation in Arabidopsis.

The biphasic floral transition in Arabidopsis thaliana involves many redundant intersecting regulatory networks. The related AP2 transcription factors DORNRÖSCHEN (DRN), DORNRÖSCHEN-LIKE (DRNL), and PUCHI individually execute well-characterized functions in diverse developmental contexts, including floral development. Here, we show that their combined loss of function leads to synergistic floral phenotypes, including reduced floral merosity in all whorls, which reflects redundant functions of all three genes in organ initiation rather than outgrowth. Additional loss of BLADE-ON-PETIOLE1 (BOP1) and BOP2 functions results in the complete conversion of floral meristems into secondary inflorescence shoots, demonstrating that all five genes define an essential regulatory network for establishing floral meristem identity, and we show that their functions converge to regulate LEAFY expression. Thus, despite their largely discrete spatiotemporal expression domains in the inflorescence meristem and early floral meristem, PUCHI, DRN, and DRNL interdependently contribute to cellular fate decisions. Auxin might represent one potential non-cell-autonomous mediator of their gene functions, because PUCHI, DRN, and DRNL all interact with auxin transport and biosynthesis pathways.

Arabidopsis floral phytomer development: auxin response relative to biphasic modes of organ initiation.

In the Arabidopsis inflorescence meristem (IM), auxin is considered a prepatterning signal for floral primordia, whereas a centripetal mode of positional information for floral organ identity is inherent to the ABCE model. However, spatio-temporal patterns of organ initiation in each whorl at the earliest initiation stages are largely unknown. Evidence suggests that initial flower development occurs along an abaxial/adaxial axis and conforms to phytomer theory. Use of the founder cell marker DORNRÖSCHEN-LIKE (DRNL) as a tool in leafy, puchi, and apetala 1 cauliflower mutant backgrounds suggests that bract founder cells are marked at the IM periphery. The DRNL transcription domain in the wild-type IM is spatially discrete from DR5 expression, suggesting that bract initiation is independent of canonical auxin response. When bracts develop in lfy and puchi mutant floral primordia the initiation of lateral sepals precedes the specification of medial sepals compared with wild type, showing an interplay between bract and abaxial sepal founder cell recruitment. In the perianthia (pan) mutant background, DRNL expression indicates that a radial outer whorl arrangement derives from splitting of sepal founder cell populations at abaxial and adaxial positions. This splitting of incipient sepal primordia is partially dependent on PRESSED FLOWER (PRS) activity and implies that sepal specification is independent of WUSCHEL and CLAVATA3 expression, as both marker genes only regain activity in stage-2 flowers, when patterning of inner floral organs switches to a centripetal mode. The transition from an initially abaxial/adaxial into a centripetal patterning programme, and its timing represent an adaptive trait that possibly contributes to variation in floral morphology, especially unidirectional organ initiation.

Live imaging of DORNRÖSCHEN and DORNRÖSCHEN-LIKE promoter activity reveals dynamic changes in cell identity at the microcallus surface of Arabidopsis embryonic suspensions.

KEY MESSAGE : Transgenic DRN::erGFP and DRNL::erGFP reporters access the window from explanting Arabidopsis embryos to callus formation and provide evidence for the acquisition of shoot meristem cell fates at the microcalli surface. The DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) genes encode AP2-type transcription factors, which are activated shortly after fertilisation in the zygotic Arabidopsis embryo. We have monitored established transgenic DRN::erGFP and DRNL::erGFP reporter lines using live imaging, for expression in embryonic suspension cultures and our data show that transgenic fluorophore markers are suitable to resolve dynamic changes of cellular identity at the surface of microcalli and enable fluorescence-activated cell sorting. Although DRN::erGFP and DRNL::erGFP are both activated in surface cells, their promoter activity marks different cell identities based on real-time PCR experiments and whole transcriptome microarray data. These transcriptome analyses provide no evidence for the maintenance of embryogenic identity under callus-inducing high-auxin tissue culture conditions but are compatible with the acquisition of shoot meristem cell fates at the surface of suspension calli.

Floral meristem initiation and emergence in plants.

Plant development and architecture is regulated by meristems that initiate lateral organs on their flanks. The gene regulatory networks that govern the transition of a vegetative shoot apical meristem into an inflorescence meristem (IM), together with those necessary to specify floral meristem (FM) identity have been elucidated in Arabidopsis thaliana and are highly complex and redundant. FMs are initiated in the axils of cryptic bracts and evidence suggests that FMs emerge and differentiate along an abaxial/adaxial axis, in contrast to existing models of centroradial positional information within FMs. Real-time imaging has revealed dynamic cell division and gene expression patterns associated with incipient primordia in the IM. This review, however, outlines how little is known concerning the identity of these primordia, the timing of FM specification and commitment in relation to the establishment of FM identity, and the interplay between bract and FM founder cell recruitment and development.

The effect of minoxidil on keratocyte proliferation in cell culture.

PURPOSE: To determine if minoxidil inhibits keratocyte proliferation in a nontoxic manner. METHODS: Rabbit keratocytes were cultured in Eagle's minimum essential medium supplemented with fetal bovine serum. Minoxidil varying in concentration from 10(0) to 10(3) micrograms/ml was added to the culture medium and incubated for 7 days. The cultures were inspected for morphologic appearance and the cell number was determined at 1, 3 and 7 days after the addition of minoxidil. After 7 days of incubation, minoxidil was withdrawn from the cell culture medium and the cells were examined 3 and 7 days thereafter. In addition, a nonradioactive cytotoxic assay was performed to determine if toxicity is associated with the presence of minoxidil. RESULTS: Minoxidil inhibited keratocyte proliferation in a dose-dependent fashion. 29% of control growth was achieved when keratocytes were cultured for 7 days in 10(3) micrograms/ml, whereas 82% control growth was achieved when keratocytes were cultured in 10(2) micrograms/ml of minoxidil. Intermediate concentrations between 10(2) and 10(3) micrograms/ml produced a linear decline in cell counts in a dose-dependent fashion. The concentration of minoxidil required for 50% control growth at 7 days extrapolated from the dose-response curve was 600 micrograms/ml. Upon withdrawal of minoxidil, cell counts returned to baseline for concentrations of 10(2) micrograms/ml or less. Phase contrast microscopy revealed that the presence of minoxidil was associated with intercellular separation, enlargement of cell bodies and elongated processes. After the withdrawal of minoxidil, the cells in all media reassumed the morphological features of normal keratocytes which included a regular fusiform shape and extensive intercellular contact. The nonradioactive cytotoxic assay revealed the lack of cytotoxicity at all concentrations of minoxidil based on a lack of lactate dehydrogenase release. CONCLUSIONS: Minoxidil inhibits keratocyte proliferation by a nontoxic mechanism. It might be particularly useful for modulating corneal wound healing following excimer laser photorefractive keratectomy.

Structure and function of the vp1 gene homologue from the resurrection plant Craterostigma plantagineum Hochst.

A gene that is homologous to the vp1 genes from maize and rice, the Arabidopsis abi3 gene and PvAlf from Phaseolus vulgaris was isolated from the resurrection plant, Craterostigma plantagineum Hochst, The C. plantagineum gene (epvp1) encodes a protein of 688 amino acids and contains five introns at positions identical to those in the Arabidopsis, maize and rice homologues. The cpvp1 transcript is present in mature seeds and in young seedlings up to 8 days following germination. The ability of the cpvp1 gene product to activate target genes was demonstrated by transient expression experiments in tobacco protoplasts using reporter gene constructs containing the beta-glucuronidase (GUS) gene fused to the promoter of two late embryogenesis abundant (LEA)-like genes, CDeT27-45 and CDeT6-19 from C. plantagineum, or the promoter of a maize gene encoding a 22-kDa zein seed storage protein.

Analysis of cDNA clones encoding sucrose-phosphate synthase in relation to sugar interconversions associated with dehydration in the resurrection plant Craterostigma plantagineum Hochst.

Sucrose-phosphate synthase (SPS) is a key enzyme in the regulation of sucrose metabolism, being responsible for the synthesis of sucrose 6-phosphate from fructose 6-phosphate and uridine 5'-diphosphate-glucose. We report on the isolation and characterization of cDNA clones encoding SPS from Craterostigma plantagineum Hochst., a resurrection plant in which the accumulation of sucrose is considered to play an important role in tolerance to severe protoplastic dehydration. Two distinct classes of cDNAs encoding SPS were isolated from C. plantagineum, and are represented by the clones Cpsps1 and Cpsps2. The transcripts corresponding to both cDNAs decrease to very low levels in dehydrating leaves of C. plantagineum. Only the Cpsps1 transcript occurs in the roots, where it is present at a higher level than in leaves and increases upon dehydration of the plant. Higher enzymatic activities have been determined in protein extracts of dehydrated tissues compared with untreated tissues, which correlates with an increase in protein levels. It is suggested that the overall regulation of SPS is strongly influenced by the changing composition of the cytoplasm in C. plantagineum leaves during the dehydration-rehydration cycle.

Polyhexamethylene biguanide (PHMB) in the treatment of experimental Fusarium keratomycosis.

PURPOSE: We wanted to determine whether topical polyhexamethylene biguanide (PHMB) 0.02% was effective in the treatment of experimental Fusarium keratomycosis in rabbits. METHODS: Fusarium solani keratomycosis was induced in the eyes of 12 New Zealand white rabbits. The rabbits were treated with PHMB 0.02% in one eye and placebo in the other eye for 6 days. The rabbits were evaluated in a masked fashion using a standardized system for clinical progression of the disease. Then the corneas were trephined and growth of F. solani in colony-forming units per milliliter (CFU/ml) determined. RESULTS: Clinical evaluation demonstrated no significant mean difference (p > 0.10) in clinical scores between treated and control eyes on day 6 (0.583 +/- 2.503). There was a significant mean CFU difference (p = 0.06) between treated eyes and control eyes (182.5 +/- 314.44). Seven of 12 eyes (58%) in the PHMB group exhibited no growth, whereas two of 12 (17%) eyes reported no growth in the control group. One of 12 eyes (8%) reported > 100 CFU in the PHMB group, whereas seven of 12 eyes (58%) reported > 100 CFU in the control group. CONCLUSIONS: PHMB 0.02% was effective in significantly reducing the fungal growth in our rabbit model of Fusarium keratomycosis. The future role of PHMB in the treatment of Fusarium keratitis needs to be further evaluated.

Ketorolac, prednisolone, and dexamethasone for postoperative inflammation.

This 6-week, partially masked, three-arm, multicenter study was conducted to evaluate the postoperative anti-inflammatory efficacy of ketorolac, a cyclooxygenase inhibitor. The study setting was the clinical practice of six ophthalmic surgeons. The study enrolled 157 candidates for routine extracapsular cataract extraction or phaco-emulsification and posterior-chamber intraocular lens implantation. Patients who received any glucocorticoid or cyclooxygenase inhibitor within 1 week of surgery were excluded. All patients were treated with solutions of 0.5% ketorolac, 1% prednisolone acetate, or 0.1% dexamethasone instilled into the operative eye three times daily from 1 day before surgery to 4 weeks after surgery. Efficacy variables included the signs of anterior-segment inflammation, primarily cells and flare in the anterior chamber, as observed by slit-lamp biomicroscopy; fluorescein leakage across the blood-aqueous barrier as measured by fluorophotometry; and the rating of efficacy by the investigator. No significant differences were seen between ketorolac and either glucocorticoid in cells and flare. No significant differences were found in other signs of inflammation, except conjunctival hyperemia and Descemet's folds at week 2. Ketorolac showed significantly greater efficacy than the glucocorticoids against blood-aqueous barrier breakdown at day 5 and week 2, as demonstrated by the difference in fluorescein concentration between the operated and nonoperated eyes. Investigators did not detect any significant difference in rating for overall effectiveness and acceptability. These findings support the use of ketorolac as an alternative to glucocorticoids for the treatment of postoperative inflammation.

Localization of endothelin-like immunoreactivity and endothelin binding sites in rabbit eyes.

Immunohistochemistry and in vitro autoradiographic techniques were used to investigate the localization of endothelin (ET)-like immunoreactivity and [125I]ET-1 binding sites in sections of ocular tissues of normal rabbits. ET-like immunoreactivity was localized to the corneal endothelium and the pre-epithelial layer of the cornea, which was specifically inhibited by addition of exogenous ET-1. Immunoreactivity for ET was also observed in the retinal ganglion layer and periocular fat tissues. These, however, were not inhibited in the presence of ET-1. Autoradiography showed binding sites for ET-1 in the iris and ciliary body. Emulsion-dipped slides of sections of rabbit eyes incubated with [125I]ET-1 showed specific labeling for binding sites over structures that were identified from serial sections stained with hematoxylineosin. These were the anterior stroma of the iris root, the walls of capillaries, the circular arteries, and the veins of the ciliary body and extraocular muscles. No binding sites were found in the cornea or conjunctiva. The difference in regional distributions of ET storage and binding sites in rabbit ocular tissue suggests that the peptide may have specific physiologic functions.

Dynamic shifts in corneal topography after radial and transverse keratotomy.

PURPOSE: The authors aimed to quantitate the dynamic patterns of change in corneal topography after multistaged radial and transverse keratotomy using digitized video-keratography. METHODS: Single and paired radial and transverse keratotomies, with videokeratoscopy between each stage and at the end of the procedure, were performed on fresh animal cadaver eyes using an artificial orbit system. RESULTS: All incisions led to central flattening. A single radial keratotomy caused flattening adjacent to the incision, and steepening 180 degrees away. A paired radial keratotomy caused increased flattening in the meridian of the incisions, and less flattening 90 degrees away. A single transverse incision caused steepening adjacent to the incision and diffuse flattening elsewhere. A paired transverse incision caused flattening near the optical center along the meridian bisecting the incisions and steepening 90 degrees away. CONCLUSION: The authors have demonstrated that computerized videokeratography can be used successfully to systematically quantitate dioptric shifts in multiple hemimeridians and measurement zone diameters after refractive surgery.

Localization and characterization of binding sites for endothelin in Harder's gland in rabbits.

The characterization and localization of binding sites for endothelin-1 (ET-1) labeled with iodine 125I were investigated in homogenized tissues and sections of Harder's glands of normal rabbits. The membrane of Harder's glands was harvested and incubated with 125I-ET-1 (0.25-1 nmol/L) in 20 +/- 4 mg of protein per 0.25 mL at 37 degrees C for 90 min in the presence of protease inhibitors. Specific labeling was assessed by coincubating unlabeled ET-1, ET-2, ET-3 and other unrelated cytokines. The tissue labeled with 125I-ET-1 was collected by filtration and counted in a gamma counter. For an in vitro autoradiography study, 15 microns cryostat sections were incubated with 125I-ET-1 (0.1 nmol/L). They were fixed, dipped in liquid emulsion and kept for 6 days before development. Membrane counting showed that the binding of 125I-ET-1 to Harder's gland was saturable. Scatchard data analysis revealed one class of binding with a dissociation constant (Kd) of 0.33 nmol/L and a maximal density of binding (Bmax) of 794 attomole/mg of protein. The binding was inhibited most by ET-1, followed by ET-2 and then ET-3 but not by unrelated peptides. Emulsion-dipped slides with sections showed specific high-density labeling mainly over structures identified from serial sections stained by hematoxylin-eosin as the walls of capillaries, arterioles, arteries, and veins of the glands. Less dense binding was found in both white and pink lobes of the gland. No binding was found in fat and connective tissues. The distribution of endothelin action sites in the glandular blood vessels and Harder's gland suggests that the peptide may have a role in the regulation of blood circulation and glandular secretion in the normal rabbit.

Interferon-alpha and interferon-gamma induced modulation of proteins in human corneal fibroblasts.

Little is known about the effects of interferon (IFN) on cell function in the eye. We have analyzed the effect of INF-alpha and IFN-gamma on the expression of proteins in cultured human corneal fibroblasts. Treatment with IFN-alpha increased the synthesis of proteins of 84, 76, 52, and 28 kD and decreased the synthesis of a 72-kD protein. Treatment with IFN-gamma increased the synthesis of proteins of 83, 66, 64, 54, and 47 kD. The effect of IFN-alpha and IFN-gamma were first detected at 5-9 h and 9 h, respectively, after the addition of the IFNs and were maximal at 17 and 24 h, respectively. Most of the changes were seen at doses of 1 x 10(1) to 1 x 10(2) U/ml of IFN-alpha or IFN-gamma and were maximal at 1 x 10(2) to 1 x 10(3) U/ml. Thus, each IFN induced distinct proteins based on apparent molecular weight and isoelectric point. These results show that IFN-alpha and IFN-gamma affect the synthesis of small groups of distinct proteins in human corneal fibroblasts.

Photosynthesis and nutrient supply in needles of Sitka spruce [Picea sitchensis (Bong.) Carr.].

Growth and photosynthetic development were measured for currently developing needles of young trees of Sitka spruce [Picea sitchensis (Bong.) Carr.] throughout their fourth growth season. Treatments included trees that were fully fertilized (control), trees deficient in phosphorus (-P), or nitrogen (-N), and trees initially deficient but then supplied with phosphorus (-PR) or nitrogen (-NR). Growth was measured in terms of needle projected area, and the photosynthetic components measured were pigment concentration, net photosynthetic rate, (PN ), activity of ribulose-1,5-bisphosphate carboxylase, (RuBPC), stomatal conductance to CO2 , (Gs ), and the intercellular partial pressure of CO2 , (Ci ). Needle growth was rapid, beginning in early May and being complete by the end of the month or early in June. Free growth occurred in the -NR treatment. Photosynthesis increased throughout the season, reaching a peak in August, with some variables subsequently showing a decrease in value. PN increased more rapidly during needle expansion than either chlorophyll concentration or RuBPC activity. Phosphorus deficiency led to a reduction in RuBPC activity, which was restored to the control value following Refertilization with P. Nitrogen deficiency severely reduced values of all variables studied, except Ci , which was higher than for the controls. Refertilization of -N trees caused a very rapid increase in values of all variables, with an increase in Ci , representing a larger increase in mesophyll conductance to CO2 , (GM ), than GS , PN and RuBPC activity were significantly correlated with total chlorophyll concentration for all treatments, but PN was not correlated with GS or RuBPC activity.

Conjunctivitis of the newborn.

Infectious conjunctivitis of the newborn is caused by a wide variety of microorganisms. The ocular findings may be part of a widespread systemic infection. Clinical presentations are not diagnostic of the cause, and a microbiologic work-up with cytology, cultures, and microbial sensitivities is mandatory. The selection of specific antimicrobial therapy is based on the findings of laboratory studies. Prophylaxis with silver nitrate solution, 1.0% tetracycline, or 0.05% erythromycin ointment is effective for the prevention of gonococcal and chlamydial conjunctivitis in the newborn.