Comparative mapping of QTLs for Al tolerance in rice and identification of positional Al-induced genes

Aluminum (Al) toxicity is the major factor limiting crop productivity in acid soils. In this study, a recombinant inbreed line (RIL) population derived from a cross between an Al sensitive lowland indica rice variety IR1552 and an Al tolerant upland japonica rice variety Azucena, was used for mapping quantitative trait loci (QTLs) for Al tolerance. Three QTLs for relative root length (RRL) were detected on chromosome 1, 9, 12, respectively, and 1 QTL for root length under Al stress is identical on chromosome 1 after one week and two weeks stress. Comparison of QTLs on chromosome 1 from different studies indicated an identical interval between C86 and RZ801 with gene(s) for Al tolerance. This interval provides an important start point for isolating genes responsible for Al tolerance and understanding the genetic nature of Al tolerance in rice. Four Al induced ESTs located in this interval were screened by reverse Northern analysis and confirmed by Northern analysis. They would be candidate genes for the QTL.

A CHASE domain containing protein kinase OsCRL4, represents a new AtCRE1-like gene family in rice

AtCRE1 is known to be a cytokinin receptor in Arabidopsis. The AtCRE1 protein contains CHASE domain at the N-terminal part, followed by a transmitter (histidine kinase) domain and two receiver domains. The N-terminal CHASE domain of AtCRE1 contains putative recognition sites for cytokinin. Five CHASE domains containing proteins were found in rice, OsCRL1a, OsCRL1b, OsCRL2, OsCRL3, and OsCRL4. OsCRL1a, OsCRL1b, OsCRL2 and OsCRL3 contain the four domains existing in CRE1, whereas OsCRL4 only contains the CHASE domain and a putative Ser/Thr protein kinase domain. The authors cloned the encoding gene OsCRL4 and found that it represents a new member of the cytokinin receptor protein in rice.

Purification, Biochemical and Immunological Characterization of Acid Invertases from Apple Fruit

: The soluble acid invertase (SAI) and cell wall-bound invertase (CWI) were purified from apple fruit to apparent electrophoretic homogeneity. Based on sequencing, substrate specificity, and immunoblotting assay, the purified enzymes were identified to be two isoforms of acid invertase (β-fructosidase; EC 3.2.1. 26). The SAI and CWI have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kDa composed of 50 kDa subunits. The SAI has a lower Km value for sucrose and higher Km for raffinose compared with CWI. These acid invertases differ from those in other plants in some of their biochemical properties, such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose, and a mixed form of inhibition by fructose to their activity. The antibodies directed against the SAI and CWI recognized, from the crude extract, three polypeptides with a molecular mass of 50, 68, and 30 kDa, respectively. These results provide a substantial basis for the further studies of the acid invertases in apple fruit.

Salt tolerance conferred by over-expression of OsNHX1 gene in Poplar 84K

OsNHX1 gene (Na+/H+ antiporter gene of Oryza sativa L.) was introduced into Poplar 84K with Agrobacte- rium tumefaciens-mediated transformation. PCR, Southern and Northern blot analysis showed that OsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 200 mmol/L NaCl, while the Na+ content in the leaves of the transgenic plants grown at 200 mmol/L NaCl was significantly higher than that in plants grown at 0 mmol/L NaCl. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.

Prokaryotic expression and characterization of a pea actin isoform (PEAc1) fused to GFP

Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells,it is difficult to purifyeach actin isoform in sufficient quantities for analysing itsphysicochemical properties. In the present study, apea(pisum Sativum L.)actin isoform (PEAc1)fused to His-tag at its amino terminus and GFP(green fluorescent protein)atits Carboxyl terminus were expressed in E. coli in inclusionbodies. The fusion protein (PEAc1-GFP)was highly purifiedwith the yield of above 2 mg/L culture by dissolving inclu-sions in 8 mol/L urea,renaturing by dialysis in a gradient of urea,and affinity binding to Ni-resin. The purified mono-meric PEAc1-GFP could efficiently bind on DNase I andinhibit the latter抯 enzyme activity. PEAc1-GFP could po-lymerise into green fluorescent filamentous structures(F-PEAc1-GFP),which could be labelled byTRITC-phalloidin,a specific agent for observing microfila-ments. The PEAc1-GFP polymerlzation curve was identicalwith that of chicken skeletal muscle actin. The critical con-centration for PEAc1-Gfp to polymerise into filaments is 0.24 μmol/L.The F-PEAc1-GFP could stimulate myosinMg-ATPase activity in a protein concentration dependantmanner (about 4 folds at 1 mg/mL F-PEAc1-GFP). The re-sults above show that the PEAc1 fused to GFP retained theassembly characteristic of actin, indicating that gene fusion,prokaryotic expression, denaturation and renaturation,andaffinity chromatography is a useful strategy for obtainingplant actin isoform proteins in a large amount.

Dimethyl Sulfoxide Is Feasible for Plant Tubulin Assembly In vitro： A Comprehensive Analysis

Abstract: It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro. However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro. In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2 mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 °C. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 °C, or following the addition to or the removal of Ca2+ from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-) carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in vitro.

Protein tyrosine phosphatasesinvolved in signaling of the ABA-induced H_2O_2 generationin guard cells of Vicia faba L.

Although protein tyrosine phosphatases (PTPases) play an important role in signal transduction in animal cells, little is known about the function of PTPases in higher plants. Hydrogen peroxide (H2O2) and mito- gen-activated protein kinases (MAPKs) are the critical com- ponents of ABA signaling pathway in guard cells. PTPase is an important regulator of MAPK, which is believed to medi- ate ABA-induced H2O2 generation in guard cells of Vicia faba L. Here, we investigate the possible role of PTPases in stomatal movement process. Phenylarsine oxide (PAO), a specific inhibitor of PTPases, could prevent ABA or H2O2-induced stomatal closure of Vicia faba L; furthermore, it could promote opening of the stomata closed by ABA or H2O2. The activity of PTPases can be effectively inhibited by PAO and H2O2. DTT had no effect on the PAO-induced inhi- bition of PTPases activity, but it could relieve the inhibition of H2O2 on PTPases activity. PAO could also inhibit the ABA-induced H2O2 generation in guard cells of Vicia faba L. These results suggested that PTPases is a critical signaling component in ABA-induced stomatal closure, and serve as targets for H2O2 lying on the signaling pathways downstream of ABA induced H2O2 generation.

Protein Tyrosine Phosphatases Mediate the Signaling Pathway of Stomatal Closure of Vicia faba L.

: The regulation of stomatal movement is one of the most important signaling networks in plants. The H+-ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, while there are some conflicting results regarding the effectiveness of the plasma membrane H+-ATPase inhibitor, vanadate, in inhibiting stomata opening. We observed that 2 mmol/L vanadate hardly inhibited light-stimulated stomata opening in epidermal peels of Vicia faba L., but significantly inhibited dark- and ABA-induced stomatal closure. These results cannot be explained with the previous findings that H+-ATPase was inhibited by vanadate. In view of the fact that vanadate is an inhibitor of protein tyrosine phosphatases (PTPases), we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase. As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Typical PTPase activity was found in guard cells of V. faba; moreover, the phosphatase activity could be inhibited by both vanadate and PAO. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.

A monosaccharide transporter is localized to sieve plate and plasmodesmal channel in developing apple fruit

Two major classes of plant sugar transporters, sucrose and monosaccharide transporters, may be localized to tonoplast or plasma membrane. The monosaccharide transporters may also be localized in plastid. However, whether these transporters reside in other subcellular compartments remains unclear. We recently detected in apple fruit a 52 kD plasma membrane-localized monosaccharide transporter, and showed that this transporter may be functional in phloem unloading in the fruit. In this paper, we report that this monosaccharide transporter is also localized to sieve plate and plasmodesmal channel in apple fruit. The amount of this sieve plate- and plasmodesma-associated transporter changes during fruit development. This amount of the transporter expression may be altered in the phloem sieve elements but not in the parenchyma cells by a photoassimilate deficiency applied by the shoot girdling treatment, suggesting that the monosaccharide transporter of the special sub-cellular localization may be of biological significance.

Constitutive activation of AtMEK5,a MAPK kinase,induces salicylic acid- independent cell death in Arabidopsis thaliana

AtMEK5 DD is an active mutant of AtMEK5, a MAP kinase kinase in Arabidopsis. Induction of AtMEK5 DD expression in transgenic plants leads to activation of 44 and 48 kD MAPKs and causes a rapid cell death. To compare the cell death induced by the expression of AtMEK5 DD with the HR-cell death induced by avirulence pathogen infection, we analyzed the activation of downstream MAP Kinase and induction of PR genes expression in permanent transgenic Arabidopsis plants. In-gel kinase activity assay revealed that the infection of Pseudomonas syringae DC3000 harboring Avr Rpt2 gene also lead to activation of 44 and 48 kD MAPKs. PAL, PR1 and PR5 were strongly induced in plants undergo- ing HR-cell death caused by the infection of P. syringae DC3000, while only the expression of PR5 was strongly in- duced in transgenic plants expressing AtMEK5 DD protein. NahG protein in AtMEK5 ×NahG plants cannot suppress DD the cell death induced by AtMEK5 . And AtMEK5 DD DD pro- tein expressed AtMEK5 ×NahG plants showed no signifi- DD cant change in salicylic acid (SA) level. All these suggest that the cell death induced by the activation of AtMEK5 is sali- cylic acid-independent.

Identification and function analysis of spectrin-like protein in pollen tubes of lily (Lilium davidii Duch)

The elongation of pollen tube is an important process of sex- ual reproduction in higher plant. Cytoskeleton plays a major regulatory role in the elongation of pollen tubes. But whether membrane skeleton is involved in the pollen tube elongation is not clear. In this study, imuno- chemical detection of spectrin-like protein has been carried out in pollen tubes. By use of 2-dimensional electrophoresis(2DE) and western blotting, two spectrin-like proteins are found, one is 150 kD, and the other is 105 kD, with pI being 4.54 and 4.39, respectively. 150 kD spec- trin-like protein is located in plasma membrane of pollen tube and 105 kD spectrin-like protein is located in cytoplasm, probably functioning as a subunit to form a dimmer (210 kD) in vivo. The elongation of pollen tubes is inhibited after spectrin antibody was injected into a growing pollen tube. These results suggest that spectrin-like proteins exist in pollen tube and play an important regulating role in the elongation proc- ess of pollen tubes from lily.

Ca^(2+) is involved in muscarineacetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

Acetyicholine (ACh) is an important neuro-chemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytusolic Ca^2+ with fluorescent probe Fiuo-3 AM under the confocal microscopy, we found that exogenous ACh increased cytosolic Ca^2+ concentration of guard cells of Vicia faba L. Muscarlne, an agonist of muscarine acetyicholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca^2+ in guard cells. This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca^2+ or ruthenium red to block Ca^2+ released from vacuole respectively, the results showed that the increased cytosolic Ca^2+ mainly come from intracellular Ca^2+ store. The evidence supports that Ca^2+ is involved in guard-cell response to ACh and that Ca^2+ sigual is coupled to mAChRs in ACh signal transduction in guard cells.