Novel FixL homologues in Chlamydomonas reinhardtii bind heme and O(2).

Genome inspection revealed nine putative heme-binding, FixL-homologous proteins in Chlamydomonas reinhardtii. The heme-binding domains from two of these proteins, FXL1 and FXL5 were cloned, expressed in Escherichia coli, purified and characterized. The recombinant FXL1 and FXL5 domains stained positively for heme, while mutations in the putative ligand-binding histidine FXL1-H200S and FXL5-H200S resulted in loss of heme binding. The FXL1 and FXL5 [Fe(II), bound O(2)] had Soret absorption maxima around 415 nm, and weaker absorptions at longer wavelengths, in concurrence with the literature. Ligand-binding measurements showed that FXL1 and FXL5 bind O(2) with moderate affinity, 135 and 222 µM, respectively. This suggests that Chlamydomonas may use the FXL proteins in O(2)-sensing mechanisms analogous to that reported in nitrogen-fixing bacteria to regulate gene expression.

Evolutionary significance of an algal gene encoding an [FeFe]-hydrogenase with F-domain homology and hydrogenase activity in Chlorella variabilis NC64A.

[FeFe]-hydrogenases (HYDA) link the production of molecular H(2) to anaerobic metabolism in many green algae. Similar to Chlamydomonas reinhardtii, Chlorella variabilis NC64A (Trebouxiophyceae, Chlorophyta) exhibits [FeFe]-hydrogenase (HYDA) activity during anoxia. In contrast to C. reinhardtii and other chlorophycean algae, which contain hydrogenases with only the HYDA active site (H-cluster), C. variabilis NC64A is the only known green alga containing HYDA genes encoding accessory FeS cluster-binding domains (F-cluster). cDNA sequencing confirmed the presence of F-cluster HYDA1 mRNA transcripts, and identified deviations from the in silico splicing models. We show that HYDA activity in C. variabilis NC64A is coupled to anoxic photosynthetic electron transport (PSII linked, as well as PSII-independent) and dark fermentation. We also show that the in vivo H(2)-photoproduction activity observed is as O(2) sensitive as in C. reinhardtii. The two C. variabilis NC64A HYDA sequences are similar to homologs found in more deeply branching bacteria (Thermotogales), diatoms, and heterotrophic flagellates, suggesting that an F-cluster HYDA is the ancestral enzyme in algae. Phylogenetic analysis indicates that the algal HYDA H-cluster domains are monophyletic, suggesting that they share a common origin, and evolved from a single ancestral F-cluster HYDA. Furthermore, phylogenetic reconstruction indicates that the multiple algal HYDA paralogs are the result of gene duplication events that occurred independently within each algal lineage. Collectively, comparative genomic, physiological, and phylogenetic analyses of the C. variabilis NC64A hydrogenase has provided new insights into the molecular evolution and diversity of algal [FeFe]-hydrogenases.

Identification of protein interaction regions of VINC/NEAT1/Men epsilon RNA.

The virus inducible non-coding RNA (VINC) was detected initially in the brain of mice infected with Japanese encephalitis virus (JEV) and rabies virus. VINC is also known as NEAT1 or Men epsilon RNA. It is localized in the nuclear paraspeckles of several murine as well as human cell lines and is essential for paraspeckle formation. We demonstrate that VINC interacts with the paraspeckle protein, P54nrb through three different protein interaction regions (PIRs) one of which (PIR-1) is localized near the 5' end while the other two (PIR-2, PIR-3) are localized near the 3' region of VINC. Our studies suggest that VINC may interact with P54nrb through a novel mechanism which is different from that reported for protein coding RNAs.

Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

Gravity signal transduction in primary roots.

AIMS: The molecular mechanisms that correlate with gravity perception and signal transduction in the tip of angiosperm primary roots are discussed. SCOPE: Gravity provides a cue for downward orientation of plant roots, allowing anchorage of the plant and uptake of the water and nutrients needed for growth and development. Root gravitropism involves a succession of physiological steps: gravity perception and signal transduction (mainly mediated by the columella cells of the root cap); signal transmission to the elongation zone; and curvature response. Interesting new insights into gravity perception and signal transduction within the root tip have accumulated recently by use of a wide range of experimental approaches in physiology, biochemistry, genetics, genomics, proteomics and cell biology. The data suggest a network of signal transduction pathways leading to a lateral redistribution of auxin across the root cap and a possible involvement of cytokinin in initial phases of gravicurvature. CONCLUSION: These new discoveries illustrate the complexity of a highly redundant gravity-signalling process in roots, and help to elucidate the global mechanisms that govern auxin transport and morphogenetic regulation in roots.

Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass state transition.

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.

Non-enzymatic protein modification by the Maillard reaction reduces the activities of scavenging enzymes in Vigna radiata.

The non-enzymatic modification of proteins through the Maillard reaction plays an important role in the loss of seed viability during seed storage. In the present study we examined whether the Maillard reaction reduces the activities of scavenging enzymes in Vigna radiata (mung bean) seeds during storage. Seeds were stored under various conditions for different duration. Maillard products were monitored by measuring protein fluorescence, and the activities of glutathione reductase (GR), superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POX) were determined. The accumulation of Maillard products in seed axes increased during storage with increasing moisture content and temperature, and was correlated with the decline in seed vigour. The activities of GR, CAT and APX decreased in proportion to the increase in Maillard products at all the moisture contents and temperatures tested. These enzymatic changes were also correlated with seed vigour. However, the activities of SOD and POX remained unchanged and appeared to be less sensitive to the Maillard reaction.

Protein modification by Amadori and Maillard reactions during seed storage: roles of sugar hydrolysis and lipid peroxidation.

The non-enzymatic modifications of proteins through Amadori and Maillard reactions play an important role in the loss of seed viability during storage. In the present study, the contribution of sugar hydrolysis and lipid peroxidation to Amadori and Maillard reactions, and to seed deterioration was investigated in mung-bean (Vigna radiata Wilczek). The contents of glucose and lipid peroxidation products in seed axes increased significantly during storage. The accumulation of Amadori products in seed axes was correlated to the lipid peroxidation, whereas the accumulation of Maillard products was closely correlated to sugar hydrolysis. The rate of accumulation of Maillard products was not well correlated to the content of Amadori products in both seed axes and protein/glucose model system, reflecting the complex nature of Amadori and Maillard reactions. The content of Amadori products in seed axes increased during the early stages of seed ageing, whereas the content of Maillard products increased steadily during the entire period of storage. The accumulation of Maillard products in seed axes was associated with the decline of seed vigour. These data suggest that, during seed ageing, sugar hydrolysis and lipid peroxidation are coupled with non-enzymatic protein modification through Amadori and Maillard reactions.