Isolation of New Gravitropic Mutants under Hypergravity Conditions.

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 (eal1) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis.

Mechanism of higher plant gravity sensing.

Higher plants have developed statocytes, specialized tissues or cells for gravity sensing, and subsequent signal formation. Root and shoot statocytes commonly harbor a number of amyloplasts, and amyloplast sedimentation in the direction of gravity is a critical process in gravity sensing. However, the molecular mechanism underlying amyloplast-dependent gravity sensing is largely unknown. In this review, we mainly describe the molecular basis for the gravity sensing mechanism, i.e., the molecules and their functions involved in amyloplast sedimentation. Several analyses of statocyte images in living plant organs have implied differences in the regulation of amyloplast movements between root and shoot statocytes. Amyloplasts in shoot statocytes display not only sedimentable but upward, saltatory movements, but the latter are rarely observed in root statocytes. A series of genetic studies on shoot gravitropism mutants of Arabidopsis thaliana has revealed that two intracellular components, the vacuolar membrane (VM) and actin microfilaments (AFs), within the shoot statocyte play important roles in amyloplast dynamics. Flexible VM structures surrounding the amyloplasts seem to allow them to freely sediment toward the bottom of cells. In contrast, long actin cables mediate the saltatory movements of amyloplasts. Thus, amyloplasts in shoot statocytes undergo a dynamic equilibrium of movement, and a proper intracellular environment for statocytes is essential for normal shoot gravitropism. Further analyses to identify the molecular regulators of amyloplast dynamics, including sedimentation, may contribute to an understanding of the gravity sensing mechanism in higher plants.

Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis.

Phototropism is an adaptation response, through which plants grow towards the light. It involves light perception and asymmetric distribution of the plant hormone auxin. Here we identify a crucial part of the mechanism for phototropism, revealing how light perception initiates auxin redistribution that leads to directional growth. We show that light polarizes the cellular localization of the auxin efflux carrier PIN3 in hypocotyl endodermis cells, resulting in changes in auxin distribution and differential growth. In the dark, high expression and activity of the PINOID (PID) kinase correlates with apolar targeting of PIN3 to all cell sides. Following illumination, light represses PINOID transcription and PIN3 is polarized specifically to the inner cell sides by GNOM ARF GTPase GEF (guanine nucleotide exchange factor)-dependent trafficking. Thus, differential trafficking at the shaded and illuminated hypocotyl side aligns PIN3 polarity with the light direction, and presumably redirects auxin flow towards the shaded side, where auxin promotes growth, causing hypocotyls to bend towards the light. Our results imply that PID phosphorylation-dependent recruitment of PIN proteins into distinct trafficking pathways is a mechanism to polarize auxin fluxes in response to different environmental and endogenous cues.

Gravity-induced PIN transcytosis for polarization of auxin fluxes in gravity-sensing root cells.

Auxin is an essential plant-specific regulator of patterning processes that also controls directional growth of roots and shoots. In response to gravity stimulation, the PIN3 auxin transporter polarizes to the bottom side of gravity-sensing root cells, presumably redirecting the auxin flux toward the lower side of the root and triggering gravitropic bending. By combining live-cell imaging techniques with pharmacological and genetic approaches, we demonstrate that PIN3 polarization does not require secretion of de novo synthesized proteins or protein degradation, but instead involves rapid, transient stimulation of PIN endocytosis, presumably via a clathrin-dependent pathway. Moreover, gravity-induced PIN3 polarization requires the activity of the guanine nucleotide exchange factors for ARF GTPases (ARF-GEF) GNOM-dependent polar-targeting pathways and might involve endosome-based PIN3 translocation from one cell side to another. Our data suggest that gravity perception acts at several instances of PIN3 trafficking, ultimately leading to the polarization of PIN3, which presumably aligns auxin fluxes with gravity vector and mediates downstream root gravitropic response.

Role of PIN-mediated auxin efflux in apical hook development of Arabidopsis thaliana.

The apical hook of dark-grown Arabidopsis seedlings is a simple structure that develops soon after germination to protect the meristem tissues during emergence through the soil and that opens upon exposure to light. Differential growth at the apical hook proceeds in three sequential steps that are regulated by multiple hormones, principally auxin and ethylene. We show that the progress of the apical hook through these developmental phases depends on the dynamic, asymmetric distribution of auxin, which is regulated by auxin efflux carriers of the PIN family. Several PIN proteins exhibited specific, partially overlapping spatial and temporal expression patterns, and their subcellular localization suggested auxin fluxes during hook development. Genetic manipulation of individual PIN activities interfered with different stages of hook development, implying that specific combinations of PIN genes are required for progress of the apical hook through the developmental phases. Furthermore, ethylene might modulate apical hook development by prolonging the formation phase and strongly suppressing the maintenance phase. This ethylene effect is in part mediated by regulation of PIN-dependent auxin efflux and auxin signaling.

Dynamic aspects of ion accumulation by vesicle traffic under salt stress in Arabidopsis.

The intracellular membrane dynamics of Arabidopsis cells under high salt treatment were investigated. When Arabidopsis was treated with high levels of NaCl in hydroponic culture, root tip cells showed rapid changes in the vacuolar volume, a decrease in the number of small acid compartments, active movement of vesicles and accumulation of Na(+) both in the central vacuole and in the vesicles around the main vacuole observed with the Na(+)-dependent fluorescence of Sodium Green. Detailed observation of Arabidopsis suspension-cultured cells under high salt treatment showed a similar pattern of response to that observed in root tip cells. Immunostaining of suspension-cultured cells with antibodies against AtNHX1 clearly showed the occurrence of dotted fluorescence in the cytoplasm only under salt treatment. We also confirmed the existence of AtNHX1 in the vacuolar membrane isolated from suspension-cultured cells with immunofluorescence. Knockout of the vacuolar Q(a)-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein VAM3/SYP22 caused an increase in salt tolerance. In mutant plants, the distribution of Na(+) between roots and shoots differed from that of wild-type plants, with Na(+) accumulating more in roots and less in the shoots of the mutant plants. The role of vesicle traffic under salt stress is discussed.

A genetic framework for the control of cell division and differentiation in the root meristem.

Plant growth and development are sustained by meristems. Meristem activity is controlled by auxin and cytokinin, two hormones whose interactions in determining a specific developmental output are still poorly understood. By means of a comprehensive genetic and molecular analysis in Arabidopsis, we show that a primary cytokinin-response transcription factor, ARR1, activates the gene SHY2/IAA3 (SHY2), a repressor of auxin signaling that negatively regulates the PIN auxin transport facilitator genes: thereby, cytokinin causes auxin redistribution, prompting cell differentiation. Conversely, auxin mediates degradation of the SHY2 protein, sustaining PIN activities and cell division. Thus, the cell differentiation and division balance necessary for controlling root meristem size and root growth is the result of the interaction between cytokinin and auxin through a simple regulatory circuit converging on the SHY2 gene.

A C2H2-type zinc finger protein, SGR5, is involved in early events of gravitropism in Arabidopsis inflorescence stems.

Plants can sense the direction of gravity and change the growth orientation of their organs. To elucidate the molecular mechanisms of gravity perception and the signal transduction of gravitropism, we have characterized a number of shoot gravitropism (sgr) mutants of Arabidopsis. The sgr5-1 mutant shows reduced gravitropism in the inflorescence stem but its root and hypocotyl have normal gravitropism. SGR5 encodes a zinc finger protein with a coiled-coil motif. The SGR5-GFP fusion protein is localized in the nucleus of Arabidopsis protoplasts, suggesting that SGR5 may act as a transcription factor. Analysis of GUS expression under the control of the SGR5 promoter revealed that SGR5 is mainly expressed in the endodermis, the gravity-sensing tissue in inflorescence stems. Furthermore, the observation that endodermis-specific expression of SGR5 using the SCR promoter in the sgr5-1 mutant restores shoot gravitropism indicates that it could function in the gravity-sensing endodermal cell layer. In contrast to other sgr mutants reported previously, almost all amyloplasts in the endodermal cells of the sgr5-1 mutant sedimented in the direction of gravity. Taken together, our results suggest that SGR5 may be involved in an early event in shoot gravitropism such as gravity perception and/or a signaling process subsequent to amyloplast sedimentation as a putative transcription factor in gravity-perceptive cells.

Amyloplasts and vacuolar membrane dynamics in the living graviperceptive cell of the Arabidopsis inflorescence stem.

We developed an adequate method for the in vivo analysis of organelle dynamics in the gravity-perceptive cell (endodermis) of the Arabidopsis thaliana inflorescence stem, revealing behavior of amyloplasts and vacuolar membranes in those cells. Amyloplasts in the endodermis showed saltatory movements even before gravistimulation by reorientation, and these movements were confirmed as microfilament dependent. From our quantitative analysis in the wild type, the gravity-oriented movement of amyloplasts mainly occurred during 0 to 3 min after gravistimulation by reorientation, supporting findings from our previous physiological study. Even after microfilament disruption, the gravity-oriented movement of amyloplasts remained. By contrast, in zig/sgr4 mutants, where a SNARE molecule functioning in vacuole biogenesis has been disrupted, the movement of amyloplasts in the endodermis is severely restricted both before and after gravistimulation by reorientation. Here, we describe vacuolar membrane behavior in these cells in the wild-type, actin filament-disrupted, and zig/sgr4 mutants and discuss its putatively important features for the perception of gravity. We also discuss the data on the two kinds of movements of amyloplasts that may play an important role in gravitropism: (1) the leading edge amyloplasts and (2) the en mass movement of amyloplasts.

The VTI family of SNARE proteins is necessary for plant viability and mediates different protein transport pathways.

The Arabidopsis genome contains a family of v-SNAREs: VTI11, VTI12, and VTI13. Only VTI11 and VTI12 are expressed at appreciable levels. Although these two proteins are 60% identical, they complement different transport pathways when expressed in the yeast vti1 mutant. VTI11 was identified recently as the mutated gene in the shoot gravitropic mutant zig. Here, we show that the vti11 zig mutant has defects in vascular patterning and auxin transport. An Arabidopsis T-DNA insertion mutant, vti12, had a normal phenotype under nutrient-rich growth conditions. However, under nutrient-poor conditions, vti12 showed an accelerated senescence phenotype, suggesting that VTI12 may play a role in the plant autophagy pathway. VTI11 and VTI12 also were able to substitute for each other in their respective SNARE complexes, and a double-mutant cross between zig and vti12 was embryo lethal. These results suggest that some VTI1 protein was necessary for plant viability and that the two proteins were partially functionally redundant.

Arabidopsis thaliana: A Model for the Study of Root and Shoot Gravitropism.

UNLABELLED: For most plants, shoots grow upward and roots grow downward. These growth patterns illustrate the ability for plant organs to guide their growth at a specified angle from the gravity vector (gravitropism). They allow shoots to grow upward toward light, where they can photosynthesize, and roots to grow downward into the soil, where they can anchor the plant as well as take up water and mineral ions.Gravitropism involves several steps organized in a specific response pathway. These include the perception of a gravistimulus (reorientation within the gravity field), the transduction of this mechanical stimulus into a physiological signal, the transmission of this signal from the site of sensing to the site of response, and a curvature-response which allows the organ tip to resume growth at a predefined set angle from the gravity vector.The primary sites for gravity sensing are located in the cap for roots, and in the endodermis for shoots. The curvature response occurs in the elongation zones for each organ. Upon gravistimulation, a gradient of auxin appears to be generated across the stimulated organ, and be transmitted to the site of response where it promotes a differential growth response. Therefore, while the gravity-induced auxin gradient has to be transmitted from the cap to the elongation zones in roots, there is no need for a longitudinal transport in shoots, as sites for gravity sensing and response overlap in this organ.A combination of molecular genetics, physiology, biochemistry and cell biology, coupled with the utilization of Arabidopsis thaliana as a model system, have recently allowed the identification of a number of molecules involved in the regulation of each phase of gravitropism in shoots and roots of higher plants. In this review, we attempt to summarize the results of these experiments, and we conclude by comparing the molecular and physiological mechanisms that underlie gravitropism in these organs. ABBREVIATIONS: GSPA: gravitational set point angle; IAA: indole-3-acetic acid; NAA: 1-naphthalene acetic acid; NPA: 1-N-naphthylphthalamic acid; 2,4-D: 2,4-dichlorphenoxy acetic acid; TIBA: 2,3,5-triiodobenzoic acid.

Genetic variation and correlations among responses to five insecticides within natural populations of Drosophila melanogaster (Diptera: Drosophilidae).

To investigate the genetic basis of cross-resistance to insecticides, natural populations of Drosophila melanogaster (Meigen) were first collected from four different locations in Japan. After 10-80 isofemale lines of each population had been established in a laboratory, the susceptibility of each line to each of the insecticides permethrin, malathion, fenitrothion, prothiophos, and DDT was examined. Broad ranges of continuous variation in susceptibility to all the chemicals were observed within each natural population as a whole. In addition, highly significant correlations among responses to organophosphates were observed. However, based on the coefficients of determination, about less than half of variation in responses to one insecticide could be explained by variation in responses to another insecticide, suggesting that not only a common resistance factor but also other factors could be involved in a natural population. Genetic analyses by using resistant and susceptible inbred lines from the same natural population demonstrated that resistance to organophosphates in some resistant lines could be due to a single or tightly linked factors, and that resistance in the other line may be due to more than one major factor. These observations could suggest that several resistance factors may be involved within each natural population, and that some of major factors could contribute to correlations among responses to organophosphates. These major factors could then contribute to the broad ranges of continuous variation observed at the level of the populations.

Correlation of magnetic resonance imaging and histological findings in a large basilar tip aneurysm after coil embolization--case report.

A 73-year-old female with a large basilar tip aneurysm underwent endovascular coil embolization with interlocking detachable coils (IDCs). The patient subsequently died of pneumonia 25 days after the embolization, and the aneurysm specimen was obtained at autopsy. Histological examination showed that the intraaneurysmal structure consisted of three layers. The outer layer was mildly organized thrombus surrounding the coils, the middle layer was disorganized clot, and the inner layer consisted of fresh blood clot. Gradient-echo magnetic resonance (MR) imaging taken before death had demonstrated a central region of high intensity and a peripheral low intensity region corresponding to the inner and middle-outer layers of the aneurysm, respectively. Intraaneurysmal placement of IDCs leads to the formation of a clot surrounding the coils. However, clot formation may be inadequate where the packing of the IDCs is incomplete. Comparison of the MR imaging and histological findings in this case show that gradient-echo MR imaging can assess thrombus and residual blood flow within the aneurysm.

Seasonal fluctuation in susceptibility to insecticides within natural populations of Drosophila melanogaster: empirical observations of fitness costs of insecticide resistance.

To investigate genetic variation and seasonal fluctuation in susceptibility to insecticides, natural populations of Drosophila melanogaster were collected from Katsunuma in mid summer and late fall for two consecutive years. After isofemale lines of each population collected in each season had been established in a laboratory, the susceptibility of each line to five insecticides, including permethrin, malathion, prothiophos, fenitrothion, and DDT, was examined. Lines of each population exhibited the broad ranges of variation in susceptibility to all chemicals. Comparison between populations in different seasons indicated that genetic variation in susceptibility to organophosphates fluctuated in consistency with the population size, in which the susceptibility increased in fall. In addition, highly significant correlations were observed among responses to organophosphates, and the correlations also fluctuated with seasons. On the other hand, genetic variation in susceptibility to permethrin and DDT was less fluctuated. These results suggest that not only a common resistance factor for organophosphate resistance but also different resistance factor(s) for each insecticide could be involved within a natural population, and that the fluctuation observed in the susceptibility to organophosphates could be associated with fitness costs of organophosphate resistance factor(s).

[Irinotecan (topoisomerase-I inhibitor) for the treatment of recurrent primary intracranial malignant lymphoma].

Primary intracranial malignant lymphoma is a fetal disease with poor prognosis, and there is no effective treatment against recurrent primary intracranial malignant lymphomas. We report 3 cases of malignant lymphoma treated with irinotecan (topoisomerase-I inhibitor, camptothecin derivatives), an aromatic drug extracted from camptotheca acuminata. After the initial diagnosis, surgical resection followed by radiation therapy was performed for one cerebral, and two cerebellar malignant lymphomas. The tumors recurred 1 month, 18 months, and 18 months after the initial treatment, respectively. The former two cases were treated with additional radiation therapy and/or radiosurgery for the recurrent tumors; however, the tumors recurred again. All cases were treated finally with a combination therapy of irinotecan and cis-platinum followed by a maintenance therapy with irinotecan only. All cases showed a sharp roentgenographical response to the chemotherapy even after cumulative recurrences. One patient died of systemic infection, and another died of intracranial tumor recurrence 11 and 29 months after the initial diagnosis, respectively. Autopsies revealed multiple tumor recurrences in both these cases. The other patient died 31 months after the initial diagnosis, also due to intracranial tumor recurrences. These results indicate the usefulness of irinotecan for the treatment of recurrent primary intracranial malignant lymphoma; however, further investigation is necessary to establish a better protocol for irinotecan treatment against primary intracranial malignant lymphoma.

Graphic analysis of microscopic tumor cell infiltration, proliferative potential, and vascular endothelial growth factor expression in an autopsy brain with glioblastoma.

BACKGROUND: Growth of brain tumors requires tumor-cell attachment to adjacent structures, degradation of surrounding matrixes, migration of tumor cells, proliferation of vasculature, and tumor cell proliferation. Comparison of the findings on neuroimaging, degrees and patterns of tumor invasion, regional tumor cell viability detected by Ki-67 immunohistochemistry, and regional vascular endothelial growth factor (VEGF) expression in whole-brain specimen of glioblastoma therefore is of great interest, and will facilitate study of the host reaction against the glioblastoma. METHODS: We graphically analyzed microscopic tumor-cell infiltration, regional differences in Ki-67 labeling indices (LI), and immunohistochemical expression of VEGF in an autopsy brain with glioblastoma. RESULTS: Glioblastoma cells infiltrated the brain far beyond the gross limits of the tumor and the areas with high signal intensity on T2-weighted magnetic resonance images. A wide range of histologic malignancy was apparent from hematoxylin-eosin staining and the Ki-67 labeling indices. VEGF was highly expressed in normal astrocytes located outside the tumor. CONCLUSION: Graphic analysis of histologic and immunohistochemical patterns is a useful method of investigating the mechanisms of glioma growth, tumor cell infiltration in the brain, and the host reaction of the brain against neoplasms.

Magnetic resonance imaging findings in cerebral fat embolism: correlation with clinical manifestations.

OBJECTIVES: Cerebral fat embolism (CFE) is a serious complication after fracture of long bones. The mortality rate of CFE may be high. However, recent progress in treatment may decrease the mortality. We studied the validity of magnetic resonance imaging (MRI) to detect and grade severity of CFE in 11 patients with CFE. METHODS: Glasgow Coma Scale score, PaO2, PaCO2 at the onset, and minimal hemoglobin and platelet levels were monitored, and phagocytes in bronchoalveolar lavage fluid were counted. Brain computed tomographic and MRI scans were performed serially. MRI findings were graded into four categories according to the severity of T2-weighted images. RESULTS: High-intensity T2 signals were identified in the various brain regions as early as 4 hours after onset of CFE. The maximum MRI grade significantly correlated with Glasgow Coma Scale score at the onset of CFE (p < 0.01). High-intensity T2 signal lesions fused and enlarged with time. In most cases, they diminished within 2 weeks. Three patients had persistent morbidity. CONCLUSION: MRI-T2-weighted imaging seems to be the most sensitive imaging technique for diagnosing CFE, and correlates well with the clinical severity of brain Injury. With the aid of proper treatment for pulmonary fat embolism, CFE is a potentially reversible disease that can have a good outcome.

Fluorescence automatic cell sorter and immunohistochemical investigation of CD68-positive cells in meningioma.

Infiltration of brain neoplasms by mononuclear cells including monocytes/macrophages has attracted little attention since they have marked morphological heterogeneity. Twenty-seven meningiomas were studied by anti-CD68 antibody-gated flow cytometry and by immunohistochemical analysis using the anti-CD68 antibodies. Flow cytometric analysis divided cells contained within tumor tissues into CD68-positive and -negative cells. In addition, eight gliomas, eight metastatic brain tumor, and 12 pituitary adenomas were investigated in the same way to compare meningiomas. The mean contents of CD68-positive cells were 24.0 +/- 3.7% in meningiomas, 4.4 +/- 1.4% in gliomas, 9.5 +/- 3.9% in metastatic brain tumors, and 4.5 +/- 1.8% in pituitary adenomas. Immunohistochemically, CD68-positive cells showed significant heterogeneity and were detected as round, rod-shaped, ameboid and ramified cells in meningiomas. Although the infiltrated mononuclear cells in gliomas have been investigated to some degree and showed that they express cytokines and/or growth factors, these infiltrated cells in meningioma have barely been studied. The CD68-positive cells detected in this study are likely to be monocytes, macrophages and microglias, and are presumed to be in various functional stages and to play important roles in growth regulation in meningioma.

[Changes in QOL in patients with brain tumors measured by mood changes during and after treatment].

UNLABELLED: Patients with brain tumors are exposed to severe stress which may have an influence on their quality of life (QOL). To measure QOL in those patients, we measure their mood state during and after the treatments. MATERIALS: 16 patients who were admitted to our department for treatment of brain tumors, were included in the study. The tumors included 5 gliomas, 6 meningiomas and others. They were 7 males and 9 females, and age distributed from 19 to 74 years old. All patients presented more than 90% of the Karnofsky performance score (KPS) on discharge, so they were expected to return to their previous social life. METHOD: The self-answering tests were performed on admission (Pre), on discharge (Post 1), and at more than 5 months after the discharge (Post 2). The tests included an original questionnaire asking consent to admission and treatment, and concerning the feeling of disability to cope with conditions of living, Cornell Medical Index (CMI) which measured the neurosis, and Profile of mood state (POMS) which measured the 6 subscales of patients mood states including tension-anxiety (TA), depression (D), anger-hostility (AH), vigor (V), fatigue (F), and confusion (C). RESULTS: The questionnaire showed that the patients feel satisfied with having consented to the treatment. The feeling of disability to cope with living became stronger in Post 2 than in Pre. CMI showed a borderline of neurosis in three patients on admission, and in four patients in Post 2. Only the TA of POMS subscale improved significantly in Post 2. However, other subscales were unchanged. It is characteristic that all of the subscales of POMS showed less disturbance on discharge compared with that on admission, but, they returned to the Prelevel after they returned to social life. CONCLUSIONS: Patients with brain tumors have satisfactory consent to the treatment, however, they feel disability to cope with social life. On discharge, they showed a better mood state compared to that on admission, but the mood state turned for the worse again during the follow-up period. It is evident that patients with brain tumors are exposed to severe stress even after the completion of the treatment. The results necessitate our taking patients' mental health into consideration for our treatment protocol.