The OsNRAMP1 iron transporter is involved in Cd accumulation in rice

Cadmium (Cd) is a heavy metal toxic to humans and the accumulation of Cd in the rice grain is a major agricultural problem, particularly in Asia. The role of the iron transporter OsNRAMP1 in Cd uptake and transport in rice was investigated here. An OsNRAMP1:GFP fusion protein was localized to the plasma membrane in onion epidermal cells. The growth of yeast expressing OsNRAMP1 was impaired in the presence of Cd compared with yeast transformed with an empty vector. Moreover, the Cd content of OsNRAMP1-expressing yeast exceeded that of the vector control. The expression of OsNRAMP1 in the roots was higher in a high Cd-accumulating cultivar (Habataki) than a low Cd-accumulating cultivar (Sasanishiki) regardless of the presence of Cd, and the amino acid sequence of OsNRAMP1 showed 100% identity between Sasanishiki and Habataki. Over-expression of OsNRAMP1 in rice increased Cd accumulation in the leaves. These results suggest that OsNRAMP1 participates in cellular Cd uptake and Cd transport within plants, and the higher expression of OsNRAMP1 in the roots could lead to an increase in Cd accumulation in the shoots. Our results indicated that OsNRAMP1 is an important protein in high-level Cd accumulation in rice.     

Analysis of field-moist Cd contaminated paddy soils during rice grain fill allows reliable prediction of grain Cd levels

Research undertaken over the last 40 years has confirmed that the long-term consumption of cadmium (Cd) contaminated rice contributes to human Cd disease. Rice is the staple of millions throughout South and Southeast Asia. Therefore, the ability to accurately assess the risk of rice grain Cd uptake in areas of elevated soil Cd would be a pre-requisite to protecting public health and regional export security. During 2001–2002, 308 concomitant soil and rice grain samples were collected from a Cd/Zn co-contaminated site in Western Thailand and determined for aqua regia digested soil Cd and rice grain Cd. No significant relationship was observed between total soil Cd and rice grain Cd (r ² = 0.117). This intuitively is to be expected since total soil Cd bears no relationship to phyto-available Cd. Similarly no relationship was observed between 0.005 M DTPA extractable soil (air-dry) Cd and rice grain Cd (r ² = 0.165). Again this result could have been predicted as the phyto-availability of Cd in paddy soils is a function of the complex interaction between soil pH, redox conditions and the presence of competing ions. Consequently, in 2003 a further study was undertaken to assess the effectiveness of commonly utilized soil extractants namely, 0.1, 0.05 and 0.01 M CaCl₂ solutions at a soil extractant ratio of 1:5 and 1 M NH₄NO₃ for 2 h or 4 h extractions times at a soil/extractant ratio of 1:2.5. Soil samples were collected at the critical rice grain fill stage and sub-divided into Portion A which was subjected to conventional air-drying and sample preparation procedures and Portion B which was maintained at Field Condition (FC) and stored at <4°C until extractions were undertaken. Concomitant rice grain samples were collected at maturity. The results indicate that air-dried soil samples subjected to conventional soil preparation procedures were totally ineffective at predicting the uptake of Cd by rice stem, leaf or grain, regardless of extractant. Further, the results indicate that the Stepwise Regression model incorporating 0.1 M CaCl₂ extractable Cd and soil pH_w determined on field moist samples accounts for 63.8% of the variability in rice grain Cd.     

Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth

Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::β-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.     

Application of Aspergillus aculeatus to rice roots reduces Cd concentration in grain

Plant and Soil - ‘Cd toxicity in rice’ events have resulted in vast public concern and uncertainty. Effective bioremediation could be accomplished via applying microbes that are capable...     

Identification and characterization of the major mitochondrial Fe transporter in rice

The uptake, translocation and compartmentalization of Fe are essential for plant cell function and life cycle. Despite rapid progress in our understanding of Fe homeostasis in plants, Fe transport from the cytoplasm to mitochondria was, until recently, poorly understood. The screening of 3,993 mutant lines for symptoms of Fe deficiency resulted in the identification and characterization of a major mitochondrial Fe transporter (MIT) in rice. MIT was found to localize to mitochondria and to complement the growth of a yeast strain defective in mitochondrial Fe transport. The knockout of MIT resulted in a lethal phenotype, and in knock-down plants, several agronomic characteristics were compromised, such as plant height, average number of tillers, days to flower, fertility and yield. Changes in the expression of genes involved in Fe transport suggested a disturbance of cellular Fe transport. Furthermore, the mitochondrial Fe concentration and the activity of the mitochondrial Fe-S enzyme aconitase were significantly reduced compared with wild-type plants. The identification of MIT is a significant advance in the field of plant Fe nutrition and should facilitate the cloning of paralogs from other plant species.Key words: aconitase, iron, mitochondria, mitochondrial iron transporter, Oryza sativaIron (Fe) is an integral cofactor for several proteins, as its redox state can be readily changed. In plants, Fe is essential for chlorophyll biosynthesis and the synthesis of heme. In mitochondria, including those of plants, Fe is essential for electron transport as well as the synthesis of heme and cytoplasmic Fe-sulfur (Fe-S) cluster-containing proteins.^1–3 Thus, when the mitochondrial Fe supply is limited, the metabolic and respiratory activities of this organelle are impaired, and the supply of Fe-S proteins to the cytoplasm is disrupted.^4,5 However, excess Fe is toxic due to the production of reactive oxygen species (ROS). In mitochondria, where free ROS are generated as a side reaction of electron transport, excess Fe can be particularly harmful.The synthesis of Fe chelators, the regulation of Fe-associated enzymes, and the properties of transporters involved in the absorption and translocation of Fe are well described in references ^6–11; however, the protein responsible for transporting Fe into the mitochondria of plants was only recently identified. We screened a rice T-DNA library consisting of 3,993 independent lines for typical symptoms of Fe deficiency despite the presence of Fe-sufficient conditions. Screening resulted in the identification of a line harboring a T-DNA insertion in the second exon of the mitochondrial Fe transporter (MIT).¹² MIT belongs to the mitochondrial solute carrier (MSC) family, whose members localize to the mitochondrial inner membrane and transport a wide range of substrates, including Fe.¹³ The yeast (Mrs3-Mrs4 ¹⁴) and zebrafish (mitoferrin¹⁵) mitochondrial Fe transporters also belong to the MSC family. Given that the rice genome contains several members of the MSC family, it would have been difficult to identify the mitochondrial Fe transporter through homology search; thus, a screening approach was used. In addition to MIT in rice, we identified putative mitochondrial Fe transporters in Arabidopsis, maize, barley, oat, grapes and castor bean (Fig. 1). Interestingly, unlike yeast, zebra fish and Arabidopsis, rice has only one MIT gene.Open in a separate windowFigure 1Phylogeny of putative plant mitochondrial Fe transporters. ZmMIT 1 (Zea mays): AC {"type":"entrez-protein","attrs":{"text":"F87311","term_id":"25288411","term_text":"pir||F87311"}}F87311; ZmMIT 2: AC {"type":"entrez-nucleotide","attrs":{"text":"G39656","term_id":"3358865","term_text":"G39656"}}G39656; VvMIT (Vitis vinifera): ACA {"type":"entrez-protein","attrs":{"text":"O21380","term_id":"75277206","term_text":"O21380"}}O21380; AtMIT 1: {"type":"entrez-protein","attrs":{"text":"NP_172184","term_id":"15222270","term_text":"NP_172184"}}NP_172184; AtMIT 2: {"type":"entrez-protein","attrs":{"text":"NP_180577","term_id":"15227718","term_text":"NP_180577"}}NP_180577; HVMIT (Hordeum vulgare): {"type":"entrez-protein","attrs":{"text":"BAJ88514","term_id":"326522937","term_text":"BAJ88514"}}BAJ88514; SbMIT (Sorghum bicolor): {"type":"entrez-protein","attrs":{"text":"XP_002468019","term_id":"1205924179","term_text":"XP_002468019"}}XP_002468019; Rc (Ricinus communis): {"type":"entrez-protein","attrs":{"text":"XP_002516353","term_id":"255550607","term_text":"XP_002516353"}}XP_002516353.MIT knock-out results in a lethal phenotype, with defects in several growth characteristics also observed in heterozygous plants. Hydroponically grown MIT knock-down (mit-2) plants accumulated 41 and 51% more Fe than wild-type (WT) plants in their roots and shoots, respectively, and their mitochondrial Fe content was lower. Changes in gene expression, determined through microarray analysis, were consistent with a disruption of cellular Fe homeostasis in mit-2 plants. These results suggested that changes in mitochondrial Fe alter cellular Fe homeostasis. The growth of mit-2 plants was significantly weaker than that of WT plants in terms of chlorophyll content as well as the length and dry weight of the roots and shoots. Furthermore, compared with WT plants, there was a decrease in the activity of the Fe-S protein aconitase in the roots (data not shown), shoots and purified mitochondria of mit-2 plants, suggesting significant effects on Fe-S cluster synthesis in the knock-down plants.¹² The growth of mit-2 plants in soil was also significantly compromised in terms of average number of tillers, plant height, delayed flowering and fertility, such that the yield of mit-2 plants was only 34% of that obtained with WT plants.MIT localized to mitochondria and in yeast was able to complement the growth defect of the Δmrs3Δmrs4 mutant, confirming the protein''s function as a mitochondrial Fe transporter. To understand the role of MIT during germination and seed development in rice, the MIT promoter was used to drive the expression of β-glucuronidase (GUS). GUS was expressed during germination and at all stages of seed development. Gene expression was also observed in the callus, around the basal part of the leaf sheath, and in all leaf tissues except vascular tissue (Fig. 2). A search of the microarray database (RiceXPro;¹⁶ ricexpro.dna.affrc.go.jp/) revealed that MIT expression is not diurnally regulated in the leaf blade, roots or stem. Instead, steady-state expression was observed in those tissues as well as in the anther, ovary, embryo, endosperm and pistil. In the leaf blade, expression slightly decreased from 27 to 76 days after transplantation (DAT) and then increased at 125 DAT (Fig. 3A).Open in a separate windowFigure 2MIT promoter-driven GUS expression. (A) Callus; (B) longitudinal section at the basal part of the leaf sheath; (C–E) leaf transverse sections.Open in a separate windowFigure 3Expression of MIT during the different growth stages of rice. (A) Leaf blade: 27, 76 and 125 days after transplantation (DAT); (B) root: 27 and 76 DAT ; (C) stem: 83 and 90 DAT ; Normalized signal intensity for MIT, derived from spatiotemporal profiling of various tissues and organs (RXP_0001) generated through ricexpro.dna.affrc.go.jp/RXP_0003/index.php. Error bars represent the SD. n = 3. Dotted line: samples collected at midnight; continuous line: samples collected at noon.As Fe is involved in vital mitochondrial processes,^1–3 it is not surprising that a mutation in the mitochondrial Fe transporter is lethal. In zebrafish, a mutation in the mitoferrin gene results in severely hypochromic erythrocytes,¹⁵ whereas defective mitochondrial Fe homeostasis in Arabidopsis is lethal.¹⁷ Moreover, knock-out plants for the mitochondrially synthesized Fe-S cluster exporter exhibit chlorosis.¹⁸It is still not clear in which form Fe is transported by MIT. Arabidopsis ferric chelate reductase 7 (FRO7) localizes to chloroplasts, where it is important for Fe homeostasis.¹⁹ The two members of the Arabidopsis FRO family localize to mitochondria,²⁰ raising the possibility that, as in the chloroplast, Fe(III) is reduced to Fe²⁺ before being transported to mitochondria. However, rice has only two FRO family genes, and in silico analysis revealed that neither localizes to mitochondria.²¹ Thus, the redox state of Fe transported to rice mitochondria remains to be determined.The lethal phenotype of MIT knockout plants and the significantly impaired growth of mit-2 plants highlight the importance of mitochondrial Fe transport in plant growth and development. Not only is the identification of MIT a significant advance in understanding cellular Fe homeostasis in rice, but it may also lead to improved Fe content in this essential grain. Moreover, it will facilitate the identification of mitochondrial Fe transporters in other plant species.     

The CorA Mg2+ transporter does not transport Fe2+

corA encodes the constitutively expressed primary Mg2+ uptake system of most eubacteria and many archaea. Recently, a mutation in corA was reported to make Salmonella enterica serovar Typhimurium markedly resistant to Fe2+-mediated toxicity. Mechanistically, this was hypothesized to be from an ability of CorA to mediate the influx of Fe2+. Consequently, we directly examined Fe2+ transport and toxicity in wild-type versus corA cells. As determined by direct transport assay, CorA cannot transport Fe2+ and Fe2+ does not potently inhibit CorA transport of 63Ni2+. Mg2+ can, relatively weakly, inhibit Fe2+ uptake, but inhibition is not dependent on the presence of a functional corA allele. Although excess Fe2+ was slightly toxic to S. enterica serovar Typhimurium, we were unable to elicit a significant differential sensitivity in a wild-type versus a corA strain. We conclude that CorA does not transport Fe2+ and that the relationship, if any, between iron toxicity and corA is indirect.     

The timing of grain Cd accumulation in rice plants: the relative importance of remobilisation within the plant and root Cd uptake post-flowering

The pathways by which Cd is accumulated in rice grain are not well understood, in particular the components attributable to direct transfer from the root, and to remobilisation of Cd previously accumulated in other plant parts. In order to observe the timing of Cd accumulation in rice plants and determine the major period for accumulation of Cd which can be translocated to the grain, Cd was supplied to the roots of rice plants grown under static hydroponic conditions at a non-toxic, environmentally relevant concentration (50 nM), according to three different timing regimes: (1) Pre-flowering Cd, (2) Post-flowering Cd, or (3) Continuous Cd. The rate of accumulation of Cd in the developing grain was monitored by harvesting immature rice panicles at four time points prior to a final harvest. Nearly all grain Cd was accumulated within 16 days of anthesis and the contribution of post-flowering Cd uptake was evident from 7 days after flowering. It was estimated that 60% of the final grain Cd content was remobilised from that accumulated by the plant prior to flowering and the other 40% came from uptake during grain maturation. This study shows that Cd uptake from the root to the grain in rice is indeed possible post-flowering and it is an important source of grain Cd.     

The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants

Using expression analysis, the role of the sucrose transporter OsSUT1 during germination and early growth of rice seedlings has been examined in detail, over a time-course ranging from 1 d to 7 d post-imbibition. Unlike the wheat orthologue, TaSUT1, which is thought to be directly involved in sugar transfer across the scutellar epithelium, OsSUT1 is not expressed in the scutellar epithelial cell layer of germinating rice and is, therefore, not involved in transport of sugars across the symplastic discontinuity between the endosperm and the embryo. OsSUT1 expression was also absent from the aleurone cells, indicating it is not involved in the transport of sucrose in this cell layer during germination. However, by 3 d post-imbibition, OsSUT1 was present in the companion cells and sieve elements of the scutellar vascular bundle, where it may play a role in phloem loading of sucrose for transport to the developing shoot and roots. This sucrose is most likely sourced from hexoses imported from the endosperm. In addition, sucrose may be remobilized from starch granules which are present at a high density in the scutellar ground tissues surrounding the vasculature and at the base of the shoot. OsSUT1 was also present in the coleoptile and the first and second leaf blades, where it was localized to the phloem along the entire length of these tissues, and was also present within the phloem of the primary roots. OsSUT1 may be involved in retrieval of sugars from the apoplasm in these tissues.     

The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains

Key message

Rice OsYSL9 is a novel transporter for Fe(II)-nicotianamine and Fe(III)-deoxymugineic acid that is responsible for internal iron transport, especially from endosperm to embryo in developing seeds.

Abstract

Metal chelators are essential for safe and efficient metal translocation in plants. Graminaceous plants utilize specific ferric iron chelators, mugineic acid family phytosiderophores, to take up sparingly soluble iron from the soil. Yellow Stripe 1-Like (YSL) family transporters are responsible for transport of metal-phytosiderophores and structurally similar metal-nicotianamine complexes. Among the rice YSL family members (OsYSL) whose functions have not yet been clarified, OsYSL9 belongs to an uncharacterized subgroup containing highly conserved homologs in graminaceous species. In the present report, we showed that OsYSL9 localizes mainly to the plasma membrane and transports both iron(II)-nicotianamine and iron(III)-deoxymugineic acid into the cell. Expression of OsYSL9 was induced in the roots but repressed in the nonjuvenile leaves in response to iron deficiency. In iron-deficient roots, OsYSL9 was induced in the vascular cylinder but not in epidermal cells. Although OsYSL9-knockdown plants did not show a growth defect under iron-sufficient conditions, these plants were more sensitive to iron deficiency in the nonjuvenile stage compared with non-transgenic plants. At the grain-filling stage, OsYSL9 expression was strongly and transiently induced in the scutellum of the embryo and in endosperm cells surrounding the embryo. The iron concentration was decreased in embryos of OsYSL9-knockdown plants but was increased in residual parts of brown seeds. These results suggested that OsYSL9 is involved in iron translocation within plant parts and particularly iron translocation from endosperm to embryo in developing seeds.

     

The relative exclusion of zinc and iron from rice grain in relation to rice grain cadmium as compared to soybean: Implications for human health

During 2000–2002, diagnostic rice and soybean plant samples and concurrent soil samples were collected from cultivated fields within a geo-physically unique Zn/Cd co-contaminated location in Thailand. For the fields sampled, aqua regia-digested Zn and Cd concentrations ranged from 2.91–284 and 254–8036 mg kg^–1, respectively. In comparison, rice and soybean Cd concentrations ranged from 0.02–5.00 and 1.08–1.71 mg kg^–1, respectively. Further, the results indicate that grain Cd, Zn and Fe concentrations are in the order rice^Gr=soybean^Gr, soybean^Gr>rice^Gr, soybean^Gr>rice^Gr, respectively. However, and critically from a human health perspective, Cd:Zn and Cd:Fe ratios are in the order rice^Gr>soybean^Gr. In addition, the rice^Gr Cd:Fe ratio is an order of magnitude higher than that determined for soybean^Gr. The results of this study, clearly demonstrate that compared to rice stalk (rice^St) and rice leaf (rice^L), rice^Gr accumulates comparatively higher Cd than Zn and Fe thus resulting in the high rice^Gr Cd:Zn and Cd:Fe ratios. This is in direct contrast to the results observed for soybean.     

RNAi mediated silencing of lipoxygenase gene to maintain rice grain quality and viability during storage

Lipoxygenase (LOX) is a common enzyme which catalyzes lipid peroxidation of seeds and consequently enhances seed quality deterioration and decreases seed viability. During seed storage, peroxidation of unsaturated fatty acids occur due to enhancement of LOX activity which directly leads to reduction in seed vigour and deterioration of grain nutritional quality. This study was undertaken to overcome these problem during rice seed storage by attenuating LOX activity using RNAi technology. To improve seed storage stability, we down regulated LOX gene activity by using a functional fragment of the LOX gene under the control of both constitutive (CaMV35S) and aleurone-specific (Oleosin-18) promoter separately. To understand the storage stability, RNAi–LOX seeds and non-transgenic control seeds were subjected to accelerated aging at 45 °C and 85 % relative humidity for 14 days. Our studies demonstrate that down regulation of LOX activity reduces the seed quality deterioration under storage condition. In addition GC–MS analysis revealed that reduction of fatty acid level in non-transgenic seeds during storage was higher when compared with that of transgenic rice seeds. Furthermore, the transgenic rice seeds with reduced LOX activity exhibited enhanced seed germination efficiency after storage than that of non-transgenic rice seeds. This study will have direct impact on nutritional stability of quality rice grains.     

In vivo analysis of metal distribution and expression of metal transporters in rice seed during germination process by microarray and X-ray Fluorescence Imaging of Fe, Zn, Mn, and Cu

To investigate the flow of the metal nutrients iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) during rice seed germination, we performed microarray analysis to examine the expression of genes involved in metal transport. Many kinds of metal transporter genes were strongly expressed and their expression levels changed during rice seed germination. We found that metal transporter genes such as ZIP family has tendency to decrease in their expressions during seed germination. Furthermore, imaging of the distribution of elements (Fe, Mn, Zn, and Cu) was carried out using Synchrotron-based X-ray microfluorescence at the Super Photon ring-8 GeV (SPring-8) facility. The change in the distribution of each element in the seeds following germination was observed by in vivo monitoring. Iron, Mn, Zn, and Cu accumulated in the endosperm and embryos of rice seeds, and their distribution changed during rice seed germination. The change in the patterns of mineral localization during germination was different among the elements observed.     

Arabidopsis thaliana MTP1 is a Zn transporter in the vacuolar membrane which mediates Zn detoxification and drives leaf Zn accumulation

The Arabidopsis thaliana metal tolerance protein 1 (MTP1) of the cation diffusion facilitator family of membrane transport proteins can mediate the detoxification of Zn in Arabidopsis and yeast. Xenopus laevis oocytes expressing AtMTP1 accumulate more Zn than oocytes expressing the AtMTP1(D94A) mutant or water-injected oocytes. An AtMTP1-GFP fusion protein localizes to the vacuolar membrane in root and leaf cells. The analysis of Arabidopsis transformed with a promoter-GUS construct suggests that AtMTP1 is not produced throughout the plant, but primarily in the subpopulation of dividing, differentiating and expanding cells. RNA interference-mediated silencing of AtMTP1 causes Zn hypersensitivity and a reduction in Zn concentrations in vegetative plant tissues.     

Hormonal changes in the grains of rice subjected to water stress during grain filling

Lodging-resistant rice (Oryza sativa) cultivars usually show slow grain filling when nitrogen is applied in large amounts. This study investigated the possibility that a hormonal change may mediate the effect of water deficit that enhances whole plant senescence and speeds up grain filling. Two rice cultivars showing high lodging resistance and slow grain filling were field grown and applied with either normal or high amount nitrogen (HN) at heading. Well-watered and water-stressed (WS) treatments were imposed 9 days post anthesis to maturity. Results showed that WS increased partitioning of fixed (14)CO(2) into grains, accelerated the grain filling rate but shortened the grain filling period, whereas the HN did the opposite way. Cytokinin (zeatin + zeatin riboside) and indole-3-acetic acid contents in the grains transiently increased at early filling stage and WS treatments hastened their declines at the late grain filling stage. Gibberellins (GAs; GA(1) + GA(4)) in the grains were also high at early grain filling but HN enhanced, whereas WS substantially reduced, its accumulation. Opposite to GAs, abscisic acid (ABA) in the grains was low at early grain filling but WS remarkably enhanced its accumulation. The peak values of ABA were significantly correlated with the maximum grain filling rates (r = 0.92**, P < 0.01) and the partitioning of fixed (14)C into grains (r = 0.95**, P < 0.01). Exogenously applied ABA on pot-grown HN rice showed similar results as those by WS. Results suggest that an altered hormonal balance in rice grains by water stress during grain filling, especially a decrease in GAs and an increase in ABA, enhances the remobilization of prestored carbon to the grains and accelerates the grain filling rate.     

The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth

Summary The involvement of exogenous calcium ions in the regulation of pollen tube formation has been investigated in Haemanthus albiflos L. and Oenothera biennis L. by following the changes that occur in pollen germination, tube growth, and ⁴⁵⁺Ca²⁺ uptake and distribution upon application of Verapamil (an inhibitor of calcium channels), lanthanum (a Ca²⁺ substitute), and ruthenium red (believed to raise the intracellular calcium level). It was found that exogenous Ca²⁺ takes part in the formation of the calcium gradient present in germinating pollen grains and growing pollen tubes. Ca²⁺ ions enter the cells through calcium channels. Raising or reducing ⁴⁵Ca²⁺ uptake causes disturbances in the germination of the pollen grains and in the growth of the pollen tubes.     

The pollen receptor kinase LePRK2 mediates growth-promoting signals and positively regulates pollen germination and tube growth

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.     

System xc- and glutamate transporter inhibition mediates microglial toxicity to oligodendrocytes

Elevated levels of extracellular glutamate cause excitotoxic oligodendrocyte cell death and contribute to progressive oligodendrocyte loss and demyelination in white matter disorders such as multiple sclerosis and periventricular leukomalacia. However, the mechanism by which glutamate homeostasis is altered in such conditions remains elusive. We show here that microglial cells, in their activated state, compromise glutamate homeostasis in cultured oligodendrocytes. Both activated and resting microglial cells release glutamate by the cystine-glutamate antiporter system xc-. In addition, activated microglial cells act to block glutamate transporters in oligodendrocytes, leading to a net increase in extracellular glutamate and subsequent oligodendrocyte death. The blocking of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors or the system xc- antiporter prevented the oligodendrocyte injury produced by exposure to LPS-activated microglial cells in mixed glial cultures. In a whole-mount rat optic nerve, LPS exposure produced wide-spread oligodendrocyte injury that was prevented by AMPA/kainate receptor block and greatly reduced by a system xc- antiporter block. The cell death was typified by swelling and disruption of mitochondria, a feature that was not found in closely associated axonal mitochondria. Our results reveal a novel mechanism by which reactive microglia can contribute to altering glutamate homeostasis and to the pathogenesis of white matter disorders.