ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.     

Ethylene biosynthesis in a chilling-sensitive<Emphasis Type="Italic">Arabidopsis</Emphasis> mutant,<Emphasis Type="Italic">chs4-2</Emphasis>

We investigated chilling-induced changes in ethylene levels in Arabidopsis to find plants with distinct patterns of ethylene production in the cold-related biosynthetic pathway. The sensitive mutants identified here includedchs1-2,chs4-2, andchs6-2. Among these, plants of thechs4-2 mutant produced more ethylene than did the wild type after both were transferred from 4°C or 10°C to 22°C. This mutant also showed less freezing tolerance and more electrolyte leakage than the wild-type plants. Our results suggest a relationship between ethylene biosynthesis and chilling sensitivity in the mutant To determine which of the enzymes involved in ethylene biosynthesis were induced by chilling, we tested the activities of ACC synthase and ACC oxidase in both mutant and wild-type plants, and found greater activity by ACC synthase as well as a higher ACC content in the mutants after all the plants were transferred from 10°C to 22°C. However, ACC oxidase activity did not differ between mutant and wild-type plants in response to chilling treatment Therefore, we conclude thatchs4-2 mutants produce more ethylene than do other mutants or the wild type during their recovery from chilling conditions. Furthermore, we believe that ACC synthase is the key enzyme involved in this response.     

Does Ethylene Mediate Cluster Root Formation under Iron Deficiency?

Casuarina glauca develops proteoid (cluster) roots in response to Fe deficiency. This study set out to investigate the possible involvement of ethylene in the initiation and/or the morphogenesis of cluster roots (CR). For this purpose, the effect of Ag+ added as silver thiosulfate, an inhibitor of ethylene action has been studied in plants growing hydroponically. No CR formation was observed in these growth conditions. Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine, 1- aminoisobutyric acid, aminoxyacetic acid or cobalt chloride also eliminated the positive effect of Fe deficiency on CR formation in C. glauca. CR were not formed in Fe- deficient roots in the presence of ethylene inhibitors, suggesting a role for ethylene in the morphological responses to Fe deficiency. Interestingly, treatment of Casuarina plants with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid stimulated significantly the formation of CR, even if plants are supplied with Fe. However, this stimulation did not reach the level of CR obtained in Fe-deficient plants. These results suggest that an ethylene-mediated signalling pathway is involved in CR formation process in C. glauca.     

NMDA受体参与小鼠的前额扣带回的神经突触传递

谷氨酸性突触是哺乳动物神经系统的主要兴奋性突触。在正常条件下,大多数的突触反应是由谷氨酸的AMPA受体传递的。NMDA受体在静息电位下为镁离子抑制。在被激活时,NMDA受体主要参与突触的可塑性变化。但是,许多NMDA受体拮抗剂在全身或局部注射时能产生行为效应,提示NMDA受体可能参与静息状态的生理功能。此文中,我们在离体的前额扣带回脑片上进行电生理记录,发现NMDA受体参与前额扣带回的突触传递。在重复刺激或近于生理性温度时,NMDA受体传递的反应更为明显。本文直接显示了NMDA受体参与前额扣带回的突触传递,并提示NMDA受体在前额扣带回中起着调节神经元兴奋的重要作用。     

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.     

A naturally enhanced green fluorescent protein from magnificent sea anemone (Heteractis magnifica) and its functional analysis

A novel fluorescent protein termed hmGFP homologous to the green fluorescent protein (GFP) from Aequorea victoria was cloned from the tentacles of sea anemone Heteractis magnifica by EST sequencing and analysis of cDNA library and followed by using RT-PCR. The sequence analysis suggested that the chromophore, consensus amino acids, and secondary structure of 11 beta-strands of hmGFP were similar to those of GFP from other species. The recombinant hmGFP protein with high purity was obtained by the fusion expression of pETTRX-hmGFP in Escherichia coli and subsequent purification. The pH sensitivity and fluorescence spectroscopy of recombinant hmGFP were characterized. The excitation spectrum of recombinant hmGFP has a rather wide major peak with a maximum at 490 nm and a shoulder at 420 nm, and its emission spectrum at 510 nm. The expression of hmGFP and the chimera IPL through hmGFP in CHO cells has shown that the fusion protein IPL through hmGFP has retained the normal membrane targeting of the IPL from Dasyatis akajei, as well as maintaining fluorescent properties similar to those of native hmGFP, suggesting a promising prospect of the application in biotechnology research for the new protein.     

A cofactor-dependent phosphoglycerate mutase homolog from Bacillus stearothermophilus is actually a broad specificity phosphatase

The distribution of phosphoglycerate mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram-positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.     

The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth

Sibling neurons in the embryonic central nervous system (CNS) of Drosophila can adopt distinct states as judged by gene expression and axon projection. In the NB4-2 lineage, two even-skipped (eve)-expressing sibling neuronal cells, RP2 and RP2sib, are formed in each hemineuromere. Throughout embryogenesis, only RP2, but not RP2sib, maintains eve expression. In this report, we describe a P-element induced mutation that alters the expression pattern of EVE in RP2 motoneurons in the Drosophila embryonic CNS. The mutation was mapped to a Drosophila homolog of human AF10/AF17 leukemia fusion genes (alf), and therefore named Dalf. Like its human counterparts, Dalf encodes a zinc finger/leucine zipper nuclear protein that is widely expressed in embryonic and larval tissues including neurons and glia. In Dalf mutant embryos, the RP2 motoneuron no longer maintains EVE expression. The effect of the Dalf mutation on EVE expression is RP2-specific and does not affect other characteristics of the RP2 motoneuron. In addition to the embryonic phenotype, Dalf mutant larvae are retarded in their growth and this defect can be rescued by the ectopic expression of a Dalf transgene under the control of a neuronal GAL4 driver. This indicates a requirement for Dalf function in the nervous system for maintaining gene expression and the facilitation of normal growth.     

Oxygen control of ethylene biosynthesis during seed development in Arabidopsis thaliana (L.) Heynh

An unforeseen side-effect on plant growth in reduced oxygen is the loss of seed production at concentrations around 25% atmospheric (50 mmol mol-1 O2). In this study, the model plant Arabidopsis thaliana (L.) Heynh. cv. 'Columbia' was used to investigate the effect of low oxygen on ethylene biosynthesis during seed development. Plants were grown in a range of oxygen concentrations (210 [equal to ambient], 160, 100, 50 and 25 mmol mol-1) with 0.35 mmol mol-1 CO2 in N2. Ethylene in full-sized siliques was sampled using gas chromatography, and viable seed production was determined at maturity. Molecular analysis of ethylene biosynthesis was accomplished using cDNAs encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase in ribonuclease protection assays and in situ hybridizations. No ethylene was detected in siliques from plants grown at 50 and 25 mmol mol-1 O2. At the same time, silique ACC oxidase mRNA increased three-fold comparing plants grown under the lowest oxygen with ambient controls, whereas ACC synthase mRNA was unaffected. As O2 decreased, tissue-specific patterning of ACC oxidase and ACC synthase gene expression shifted from the embryo to the silique wall. These data demonstrate how low O2 modulates the activity and expression of the ethylene biosynthetic pathway during seed development in Arabidopsis.