A nitrite transporter associated with nitrite uptake by higher plant chloroplasts

Chloroplasts take up cytosolic nitrite during nitrate assimilation. In this study we identified a nitrite transporter located in the chloroplasts of higher plants. The transporter, CsNitr1-L, a member of the proton-dependent oligopeptide transporter (POT) family, was detected during light-induced chloroplast development in de-etiolating cucumber seedlings. We detected a CsNitr1-L-green fluorescent protein (GFP) fusion protein in the chloroplasts of leaf cells and found that an immunoreactive 51 kDa protein was present in the isolated inner envelope membrane of chloroplasts. CsNitr1-L has an isoform, CsNitr1-S, with an identical 484 amino acid core sequence; however, in CsNitr1-S the 120 amino acid N-terminal extension is missing. Saccharomyces cerevisiae cells expressing CsNitr1-S absorbed nitrite from an acidic medium at a slower rate than mock-transformed control cells, and accumulated nitrite to only one-sixth the concentration of the control cells, suggesting that CsNitr1-S enhances the efflux of nitrite from the cell. Insertion of T-DNA in a single CsNitr1-L homolog (At1g68570) in Arabidopsis resulted in nitrite accumulation in leaves to more than five times the concentration found in the wild type. These results show that it is possible that both CsNitr1-L and CsNitr1-S encode efflux-type nitrite transporters, but with different subcellular localizations. CsNitr1-L may possibly load cytosolic nitrite into chloroplast stroma in the chloroplast envelope during nitrate assimilation. The presence of genes homologous to CsNitr1-L in the genomes of Arabidopsis and rice indicates that facilitated nitrite transport is of general physiological importance in plant nutrition.     

Contribution of gibberellin deactivation by AtGA2ox2 to the suppression of germination of dark-imbibed Arabidopsis thaliana seeds

Gibberellin levels in imbibed Arabidopsis thaliana seeds are regulated by light via phytochrome, presumably through regulation of gibberellin biosynthesis genes, AtGA3ox1 and AtGA3ox2, and a deactivation gene, AtGA2ox2. Here, we show that a loss-of-function ga2ox2 mutation causes an increase in GA(4) levels and partly suppresses the germination inability during dark imbibition after inactivation of phytochrome. Experiments using 2,2-dimethylGA(4), a GA(4) analog resistant to gibberellin 2-oxidase, in combination with ga2ox2 mutant seeds suggest that the efficiency of deactivation of exogenous GA(4) by AtGA2ox2 is dependent on light conditions, which partly explains phytochrome-mediated changes in gibberellin effectiveness (sensitivity) found in previous studies.     

Wall ingrowths in epidermal transfer cells of Vicia faba cotyledons are modified primary walls marked by localized accumulations of arabinogalactan proteins

Despite the importance of transfer cells in enhancing nutrient transport in plants, little is known about how deposition of the complex morphology of their wall ingrowths is regulated. We probed thin sections of mature cotyledon epidermal transfer cells of Vicia faba with affinity probes and antibodies specific to polysaccharides and glycoproteins, to determine the distribution of these components in their walls. Walls of these transfer cells consist of the pre-existing primary wall, a uniformly deposited wall layer and wall ingrowths which are comprised of two regions; an electron-opaque inner region and an electron-translucent outer region. The primary wall reacted strongly with antibodies against esterified pectin, xyloglucan, the side chains of rhamnogalaturonan-1 and a cellulase-gold affinity probe. The electron-opaque inner region of wall ingrowths displayed a similar labeling pattern to that of the primary wall, showing strong cross-reactivity with all antibodies tested, except those reacting against highly de-esterified pectins. The electron-opaque outer layer of developmentally more mature wall ingrowths reacted strongly with anti-callose monoclonal and polyclonal antibodies, but showed no reaction for pectin or xyloglucan antibodies or the cellulase-gold affinity probe. The plasma membrane-wall interface was labeled strongly with anti-arabinogalactan protein (AGP) antibodies, with some AGP-reactive antibodies also labeling the electron-translucent zone. Nascent wall ingrowths were labeled specifically with AGPs but not anti-callose. A reduction in wall ingrowth density was observed when developing transfer cells were exposed to beta-d-glucosyl Yariv reagent compared with controls. Our results indicate that wall ingrowths of transfer cells are primary wall-like in composition and probably require AGPs for localized deposition.     

Floral development of an asexual and female-like mutant carrying two deletions in gynoecium-suppressing and stamen-promoting functional regions on the Y chromosome of the dioecious plant Silene latifolia

Sexual dimorphism is controlled by genes on the Y chromosome in the dioecious plant Silene latifolia. K034 is the first mutant with female flowers and asexual flowers in one individual. Its stamens are suppressed completely, and its gynoecium exhibits two suppression patterns. One gynoecium resembles a thin rod, as in wild-type males (asexual flower); the other is imperfectly suppressed, having 1-3 carpels (female-like flower). The ratio of these patterns was 9 : 1. To exclude the possibility of chimerism in K034, we crossed a female-like flower of K034 with a wild-type male. Progeny obtained from this crossing had asexual and female-like flowers in one individual. This two-flower-type phenotype was inherited without separating. To examine the identity of flower organs in K034, we analyzed the development of asexual and female-like flowers using scanning electron microscopy and in situ hybridization with SLM1 and SLM2 (orthologs of AGAMOUS and PISTILLATA, respectively) as probes. Mitotic spreads of root tip chromosomes from hairy root cultures showed that K034 had 25 chromosomes. Fluorescent in situ hybridization analysis, using a subtelomeric repetitive sequence (KpnI subfamily) as a probe, indicated that K034 possessed two X chromosomes and one Y chromosome (Y(d)), of which Y(d) had been rearranged to lose the pseudoautosomal region (PAR). PCR analysis using Y-specific sequence-tagged site (STS) markers clarified that Y(d) of K034 had two other deletions in gynoecium-suppressing and stamen-promoting regions. It is reasonable to suggest that these sex chromosomal abnormalities resulted in two abnormal sexual phenotypes: the asexual and imperfect female (female-like) flowers in K034.     

Intra-vacuolar reserves of membranes during stomatal closure: the possible role of guard cell vacuoles estimated by 3-D reconstruction

Stomatal apertures are regulated by morphological changes in guard cells which have been associated with guard cell vacuolar structures. To investigate the contribution of guard cell vacuoles to stomatal movement, we examined the dynamics of vacuolar membrane structures in guard cells and evaluated the changes in vacuolar volumes and surface areas during stomatal movement. Using a transgenic Arabidopsis line expressing green fluorescent protein (GFP)-AtVAM3, we have found that the guard cell vacuolar structures became complicated during stomatal closure with the appearance of numerous intra-vacuolar membrane structures. A three-dimensional (3-D) reconstruction using our originally developed software, REANT (reconstructor and analyzer of 3-D structure), and photobleaching analysis revealed the continuity of the vacuolar structures, even when they appeared to be compartmented in confocal images of closed stomata. Furthermore, calculations of the surface area by REANT revealed an increase in vacuolar surface area during stomatal closure but a decrease in the surface area of the guard cells. Movement of a vital staining dye, FM4-64, to the vacuolar membrane was accelerated during ABA-induced stomatal closure in Vicia faba. These results suggest that the guard cell vacuoles store some portion of the excess membrane materials produced during stomatal closure as intra-vacuolar structures.     

Disruption of actin filaments by latrunculin B affects cell wall construction in Picea meyeri pollen tube by disturbing vesicle trafficking

The involvement of actin filaments (AFs) in vesicle trafficking, cell wall construction and tip growth was investigated during pollen tube development of Picea meyeri. Pollen germination and tube elongation were inhibited in a dose-dependent manner by the latrunculin B (LatB) treatment. The fine AFs were broken down into disorganized fragments showing a tendency to aggregate. FM4-64 labeling revealed that the dynamic balance of vesicle trafficking was perturbed due to F-actin disruption and the fountain-like cytoplasmic pattern changed into disorganized Brownian movement. The configuration and/or distribution of cell wall components, such as pectins, callose and cellulose, as well as arabinogalactan proteins changed in obvious ways after the LatB application. Fourier transform infrared (FTIR) analysis further established significant changes in the chemical composition of the wall material. Our results indicate that depolymerization of AFs affects the distribution and configuration of cell wall components in Picea meyeri pollen tube by disturbing vesicle trafficking.     

Wide-ranging effects of eight cytochalasins and latrunculin A and B on intracellular motility and actin filament reorganization in characean internodal cells

Numerous forms of cytochalasins have been identified and, although they share common biological activity, they may differ considerably in potency. We investigated the effects of cytochalasins A, B, C, D, E, H and J and dihydrocytochalasin B in an ideal experimental system for cell motility, the giant internodal cells of the characean alga Nitella pseudoflabellata. Cytochalasins D (60 microM) and H (30 microM) were found to be most suited for fast and reversible inhibition of actin-based motility, while cytochalasins A and E arrested streaming at lower concentrations but irreversibly. We observed no clear correlation between the ability of cytochalasins to inhibit motility and the actual disruption of the subcortical actin bundle tracks on which myosin-dependent motility occurs. Indeed, the actin bundles remained intact at the time of streaming cessation and disassembled only after one to several days' treatment. Even when applied at concentrations lower than that required to inhibit cytoplasmic streaming, all of the cytochalasins induced reorganization of the more labile cortical actin filaments into actin patches, swirling clusters or short rods. Latrunculins A and B arrested streaming only after disrupting the subcortical actin bundles, a process requiring relatively high concentrations (200 microM) and very long treatment periods of >1 d. Latrunculins, however, worked synergistically with cytochalasins. A 1 h treatment with 15 nM latrunculin A and 4 microM cytochalasin D induced reversible fragmentation of subcortical actin bundles and arrested cytoplasmic streaming. Our findings provide insights into the mechanisms by which cytochalasins and latrunculins interfere with characean actin to inhibit motility.     

Roles for the N- and C-terminal domains of phytochrome B in interactions between phytochrome B and cryptochrome signaling cascades

Plants fine-tune light responses through interactions betweenphotoreceptors. We have previously reported that the greeningof Arabidopsis thaliana roots is regulated synergistically byphytochromes and cryptochromes. In the present study, we investigatedthe functions of the N- and C-terminal domains of phytochromeB (phyB) in the interactions between phyB and cryptochrome signalingcascades. Transgenic Arabidopsis expressing the phyB N-terminaldomain fused to green fluorescent protein (GFP), ß-glucuronidase(GUS) and the nuclear localization signal (NLS) showed intenseroot greening under blue light, indicating that the C-terminaldomain was dispensable for the synergistic interaction in theinduction of root greening. However, root greening under redlight was substantially reduced in the absence of the C-terminaldomain. This effect was opposite to the previous observationthat removal of the C-terminal domain enhanced the signalingactivity of phyB in the inhibition of hypocotyl elongation.In addition, we found that overexpression of the isolated C-terminaldomain of phyB enhanced the blue light response not only forroot greening but also for the inhibition of hypocotyl elongation.Analysis of this activity on various photoreceptor mutant backgroundsdemonstrated that the isolated C-terminal domain enhanced cryptochromesignaling. In summary, these results demonstrate that differentdomains of phyB can play various roles which are dependent onlight conditions as well as on the specific physiological response.     

Changes in plant mitochondrial electron transport alter cellular levels of reactive oxygen species and susceptibility to cell death signaling molecules

Transgenic tobacco (Nicotiana tabacum) lacking mitochondrial alternative oxidase (AOX) have been compared with wild-type (Wt) tobacco using two different systems, either suspension cell cultures or leaves. In both systems, a lack of AOX was accompanied by an increase in some anti-oxidant defenses, consistent with the hypothesis that a lack of AOX increases the mitochondrial generation of reactive oxygen species (ROS). In most cases, this increase in anti-oxidant defenses could more than offset the presumed increased rate of ROS generation, resulting paradoxically in a lower steady-state level of ROS than was found in Wt leaves or suspension cells. We also found that the amount of cell death induced by salicylic acid or nitric oxide correlated strongly with the level of ROS (irrespective of the level of AOX), while death induced by azide was dependent upon the presence or absence of AOX. These results suggest that susceptibility to cell death by signaling molecules (salicylic acid and nitric oxide) is dependent upon the steady-state cellular level of ROS and that AOX levels clearly contribute to this steady state, perhaps by influencing the rate of mitochondrial-generated ROS and hence the cellular level of anti-oxidant defenses.     

The plant growth-promoting fungus Penicillium simplicissimum GP17-2 induces resistance in Arabidopsis thaliana by activation of multiple defense signals

Arabidopsis thaliana grown in soil amended with barley grain inocula of Penicillium simplicissimum GP17-2 or receiving root treatment with its culture filtrate (CF) exhibited clear resistance to Pseudomonas syringae pv. tomato DC3000 (Pst). To assess the contribution of different defense pathways, Arabidopsis genotypes implicated in salicylic acid (SA) signaling expressing the NahG transgene or carrying disruption in NPR1 (npr1), jasmonic acid (JA) signaling (jar1) and ethylene (ET) signaling (ein2) were tested. All genotypes screened were protected by GP17-2 or its CF. However, the level of protection was significantly lower in NahG and npr1 plants than it was in similarly treated wild-type plants, indicating that the SA signaling pathway makes a minor contribution to the GP17-2-mediated resistance and is insufficient for a full response. Examination of local and systemic gene expression revealed that GP17-2 and its CF modulate the expression of genes involved in both the SA and JA/ET signaling pathways. Subsequent challenge of GP17-2-colonized plants with Pst was accompanied by direct activation of SA-inducible PR-2 and PR-5 genes as well as potentiated expression of the JA-inducible Vsp gene. In contrast, CF-treated plants infected with Pst exhibited elevated expression of most defense-related genes (PR-1, PR-2, PR-5, PDF1.2 and Hel) studied. Moreover, an initial elevation of SA responses was followed by late induction of JA responses during Pst infection of induced systemic resistance (ISR)-expressing plants. In conclusion, we hypothesize the involvement of multiple defense mechanisms leading to an ISR of Arabidopsis by GP17-2.     

Reliability of an electrophoretic and image processing analysis of human skeletal muscle taken from m. vastus lateralis

The relative content of myosin heavy chain (MHC) isoforms IIb, IIa and I in human skeletal muscle taken from the m. vastus lateralis of 30 healthy male subjects was analysed using mini-gel electrophoresis. Repeated electrophoretic gels utilizing the same methods were produced for all subjects and the determination of MHC protein bands was performed using a digital scanner and National Institutes of Health (NIH) Image software and laser densitometry. A comparison between the NIH Image processing technique and laser densitometry revealed differences of 6.47%, 6.35% and 6.84% between these measurement techniques for MHC-IIb, -IIa and -I isoforms, respectively. The percentage technical error of measurement (TEM%) between electrophoretic gels was shown to be 19.1%, 17.8% and 14.2%, with regard to percentage of occurrence of MHC-IIb, -IIa and -I isoforms respectively. The variation in electrophoretic gel analyses was shown to be 5.7%, 7.3% and 5.5%, with regard to the percentage of MHC-IIb, -IIa and -I isoforms respectively. Intra-class correlations comparing NIH Image and laser densitometry produced r values in the range 0.38–0.63. Comparisons between and within gel analyses produced r values in the range 0.59–0.94 and 0.93–0.98, respectively. Analyses of variance revealed no significant differences (P < 0.05) between analysis techniques, between␣gels or within gels for the measurement of MHC-IIb, -IIa and -I isoforms. The inter-gel error between fibre subgroups was moderate for the two type-II MHC populations and less for type-I MHC; the intra-individual error in the measuring technique used for classifying the MHC-IIb, -IIa and -I protein bands was small. The results obtained in this investigation showed consistent trends which may reflect a false classification of the type-II MHC populations for the inter-gel and intra-individual analyses. The NIH Image software and digitizing process was shown to be a valid and reliable method for distinguishing between MHC protein bands of human skeletal tissue as separated by mini-gel electrophoretic techniques. Accepted: 6 January 1997     

Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.     

Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.     

Elicitor-induced cytoskeletal rearrangement relates to vacuolar dynamics and execution of cell death: in vivo imaging of hypersensitive cell death in tobacco BY-2 cells

Disintegration of the vacuolar membrane (VM) has been proposed to be a crucial event in various types of programmed cell death (PCD) in plants. However, its regulatory mechanisms are mostly unknown. To obtain new insights on the regulation of VM disintegration during hypersensitive cell death, we investigated the structural dynamics and permeability of the VM, as well as cytoskeletal reorganization during PCD in tobacco BY-2 cells induced by a proteinaceous elicitor, cryptogein. From sequential observations, we have identified the following remarkable events during PCD. Stage 1: bulb-like VM structures appear within the vacuolar lumen and the cortical microtubules are disrupted, while the cortical actin microfilaments are bundled. Simultaneously, transvacuolar strands including endoplasmic microtubules and actin microfilaments are gradually disrupted and the nucleus moves from the center to the periphery of the cell. Stage 2: cortical actin microfilament bundles and complex bulb-like VM structures disappear. The structure of the large central vacuole becomes simpler, and small spherical vacuoles appear. Stage 3: the VM is disintegrated and a fluorescent dye, BCECF, leaks out of the vacuoles just prior to PCD. Application of an actin polymerization inhibitor facilitates both the disappearance of bulb-like vacuolar membrane structures and induction of cell death. These results suggest that the elicitor-induced reorganization of actin microfilaments is involved in the regulation of hypersensitive cell death via modification of the vacuolar structure to induce VM disintegration.