Conservation of structure and cold-regulation of RNA-binding proteins in cyanobacteria: probable convergent evolution with eukaryotic glycine-rich RNA-binding proteins

The rbp gene family of the cyanobacterium Anabaena variabilis strain M3 consists of eight members that encode small RNA-binding proteins containing a single RNA recognition motif (RRM). Similar genes are found in the genomes of Synechocystis sp. PCC6803, Helicobacter pylori and Treponema pallidum, but are absent from the other completely sequenced prokaryotic genomes. The expression of the rbp genes of Anabaena is induced by low temperature, with the exception of the rbpD gene. We found four stretches of conserved sequences in the 5'-untranslated region of the cyanobacterial rbp genes that are known to be induced by low temperature. The cold-regulated Rbp proteins contain a short C-terminal glycine-rich domain. In this respect, these proteins are similar to plant and mammalian glycine-rich RNA-binding proteins (GRPs), which also contain a single RRM domain with a C-terminal glycine-rich domain and are highly expressed at low temperature. Detailed phylogenetic analysis showed, however, that the cyanobacterial Rbp proteins and the eukaryotic GRPs do not belong to a single lineage, but that the glycine-rich domains are likely to have been added independently. The cold-regulation of both types of proteins is also likely to have evolved independently. Furthermore, the chloroplast RNA-binding proteins are not likely to have originated from the Rbp proteins of endosymbiont cyanobacterium, but are supposed to have diverged from the GRPs. These results suggest that the cyanobacterial Rbp proteins and the eukaryotic GRPs are similar in both structure and regulation, but that this apparent similarity has resulted from convergent evolution.     

Zebrafish cerebrospinal fluid mediates cell survival through a retinoid signaling pathway

Cerebrospinal fluid (CSF) includes conserved factors whose function is largely unexplored. To assess the role of CSF during embryonic development, CSF was repeatedly drained from embryonic zebrafish brain ventricles soon after their inflation. Removal of CSF increased cell death in the diencephalon, indicating a survival function. Factors within the CSF are required for neuroepithelial cell survival as injected mouse CSF but not artificial CSF could prevent cell death after CSF depletion. Mass spectrometry analysis of the CSF identified retinol binding protein 4 (Rbp4), which transports retinol, the precursor to retinoic acid (RA). Consistent with a role for Rbp4 in cell survival, inhibition of Rbp4 or RA synthesis increased neuroepithelial cell death. Conversely, ventricle injection of exogenous human RBP4 plus retinol, or RA alone prevented cell death after CSF depletion. Zebrafish rbp4 is highly expressed in the yolk syncytial layer, suggesting Rbp4 protein and retinol/RA precursors can be transported into the CSF from the yolk. In accord with this suggestion, injection of human RBP4 protein into the yolk prevents neuroepithelial cell death in rbp4 loss‐of‐function embryos. Together, these data support the model that Rbp4 and RA precursors are present within the CSF and used for synthesis of RA, which promotes embryonic neuroepithelial survival. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 75–92, 2016     

Down regulation of extramacrochaetae mRNA by a Drosophila neural RNA binding protein Rbp9 which is homologous to human Hu proteins.

Rbp9 is an RNA binding protein expressed mainly in the central nervous system of adult Drosophilamelanogaster. Rbp9 shares a high degree of sequence similarity with human neural proteins referred to as Hu antigens. Hu antigens bind to U-rich mRNA destabilizing elements with a high affinity and, thus, have been implicated as regulators of mRNA stability. Using in vitro RNA binding assays, we found that Rbp9 binds strongly to poly U sequences. We then employed a Selex system to identify a consensus Rbp9 binding site (UUUXUUUU). Information obtained from the Selex results allowed the detection of two repeats of the Rbp9 consensus binding sequence in the 3' untranslated region of extramacrochaetae mRNA. UV crosslinking experiments demonstrated that Rbp9 interactsspecifically with emc mRNA. The requirement of Rbp9 protein in the down regulation of emc mRNA was confirmed by northern (RNA) analysis, which revealed that the level of emc mRNA increased 10-fold in rbp9 mutant flies. Taken together with the in vitro RNA binding results, the genetic evidence obtained strongly supports the hypothesis that Rbp9 functions as a regulator of RNA stability.     

Induction of polyunsaturated fatty-acid synthesis enhances tolerance of a cyanobacterium, Cylindrospermopsis raciborskii, to low-temperature photoinhibition

Acyl-lipid desaturation introduces double bonds (unsaturated bonds) at specifically defined positions of fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. Desaturation pattern of the glycerolipids of Cylindrospermopsis raciborskii (C. raciborskii), a filamentous cyanobacterial strain, was determined in cells grown at 35 degrees C and 25 degrees C. The lowering of the growth temperature from 35 degrees C to 25 degrees C resulted in a considerable accumulation of polyunsaturated octadecanoic fatty acids in all lipid classes. Lipid unsaturation of C. raciborskii was also compared to Synechocystis PCC6803. In C. raciborskii cells, a shift in growth temperature induced a much more pronounced alteration in the desaturation pattern of all lipid classes than in Synechocystis PCC6803. The tolerance to low-temperature photoinhibition of the C. raciborskii cells grown at 25 degrees C and 35 degrees C was also compared to the tolerance of Synechocystis cells grown at the same temperatures. Lower growth temperature increased the tolerance of C. raciborskii cells but not that of Synechocystis cells. These results strengthen the importance of polyunsaturated glycerolipids in the tolerance to environmental stresses and may give a physiological explanation for the determinative role of C. raciborskii strain in algal blooming in the Lake Balaton (Hungary).     

Alpha-tocopherol is essential for acquired chill-light tolerance in the cyanobacterium Synechocystis sp. strain PCC 6803

Unlike Escherichia coli, the cyanobacterium Synechocystis sp. strain PCC 6803 is insensitive to chill (5°C) in the dark but rapidly losses viability when exposed to chill in the light (100 μmol photons m⁻² s⁻¹). Preconditioning at a low temperature (15°C) greatly enhances the chill-light tolerance of Synechocystis sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of α-tocopherol after exposure to chill-light stress. Mutants unable to synthesize α-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P_petE controlled the level of α-tocopherol and ACLT. We conclude that α-tocopherol is essential for ACLT of Synechocystis sp. strain PCC 6803. The role of α-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.     

Acquisition of thermotolerance in Saccharomyces cerevisiae without heat shock protein hsp 104 and in the absence of protein synthesis.

Acquisition of thermotolerance in response to a preconditioning heat treatment at 40 degrees C was studied in mutants of the yeast Saccharomyces cerevisiae lacking a specific heat shock protein or the ability to synthesize proteins at 40 degrees C. A mutant carrying a deletion of heat shock protein hsp 104 and the corresponding wildtype strain were both highly sensitive to heat stress at 50.4 degrees C without preconditioning but both acquired almost the same level of thermotolerance after 60 min of preconditioning. Both strains showed equal induction of trehalose-6-phosphate synthase and accumulated equal levels of trehalose during the treatment. The conditional mutant ts--187 synthesized no proteins during the preconditioning heat treatment but nevertheless acquired thermotolerance, albeit to a lesser degree than the corresponding wildtype strain. Induction of trehalose-6-phosphate synthase and accumulation of trehalose were reduced to a similar extent. These results show that acquisition of thermotolerance and accumulation of trehalose are closely correlated during heat preconditioning and are modulated by protein synthesis but do not require it.     

EPR study of thylakoid membrane dynamics in mutants of the carotenoid biosynthesis pathway of Synechocystis sp. PCC6803

EPR spectroscopy using 5-doxylstearic acid (5-SASL) and 16-doxylstearic acid (16-SASL) spin probes was used to study the fluidity of thylakoid membranes. These were isolated from wild type Synechocystis and from several mutants in genes encoding selected enzymes of the carotenoid biosynthesis pathway and/or acyl-lipid desaturases. Cyanobacteria were cultivated at 25°C and 35°C under different light regimes: photoautotrophically (PAG) and/or in light-activated heterotrophic conditions (LAHG). The relative fluidity of membranes was estimated from EPR spectra based on the empirical outermost splitting parameter in a temperature range from 15°C to 40°C. Our findings demonstrate that in native thylakoid membranes the elimination of xanthophylls decreased fluidity in the inner membrane region under optimal growth conditions (25°C) and increased it under sublethal heat stress (35°C). This indicated that the overall fluidity of native photosynthetic membranes in cyanobacteria may be influenced by the ratio of polar to non-polar carotenoid pools under different environmental conditions.     

CrbpI modulates glucose homeostasis and pancreas 9-cis-retinoic acid concentrations

Cellular retinol-binding protein type I (CrbpI), encoded by Rpb1, serves as a chaperone of retinol homeostasis, but its physiological effects remain incompletely understood. We show here that the Rbp1(-/-) mouse has disrupted retinoid homeostasis in multiple tissues, with abnormally high 9-cis-retinoic acid (9cRA), a pancreas autacoid that attenuates glucose-stimulated insulin secretion. The Rbp1(-/-) pancreas has increased retinol and intense ectopic expression of Rpb2 mRNA, which encodes CrbpII: both would contribute to increased β-cell 9cRA biosynthesis. 9cRA in Rbp1(-/-) pancreas resists postprandial and glucose-induced decreases. Rbp1(-/-) mice have defective islet expression of genes involved in glucose sensing and insulin secretion, as well as islet α-cell infiltration, which contribute to reduced glucose-stimulated insulin secretion, high glucagon secretion, an abnormally high rate of gluconeogenesis, and hyperglycemia. A diet rich in vitamin A (as in a standard chow diet) increases pancreas 9cRA and impairs glucose tolerance. Crbp1 attenuates the negative impact of vitamin A (retinol) on glucose tolerance, regardless of the dietary retinol content. Rbp1(-/-) mice have an increased rate of fatty acid oxidation and resist obesity when fed a high-fat diet. Thus, glucose homeostasis and energy metabolism rely on Rbp1 expression and its moderation of pancreas retinol and of the autacoid 9cRA.     

Effect of inoculation with the endophyte Clavibacter sp. strain Enf12 on chilling tolerance in Chorispora bungeana

Endophytic bacteria have been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. The objective of this study was to evaluate the effect of an endophytic bacterium, Clavibacter sp. strain Enf12, in regenerated plantlets of Chorispora bungeana subjected to chilling stress (0°C). Aerial biomass and physiological markers for chilling stress, such as electrolyte leakage, lipid peroxidation, reactive oxygen species (ROS) accumulation, proline content and activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), guaiacol peroxidase (EC 1.11.1.7) and ascorbate peroxidase (EC 1.11.1.11), were assessed. We demonstrated that Clavibacter sp. strain Enf12 was capable of colonizing internal tissues of regenerated plantlets of C. bungeana and maintained stable population densities under both normal (20°C) and chilling (0°C) conditions. Inoculation enhanced plantlet growth under both conditions and significantly attenuated the chilling-induced electrolyte leakage, lipid peroxidation and ROS accumulation. The endophyte significantly increased the activities of antioxidant enzymes and proline content in C. bungeana plantlets under chilling stress. These findings suggest that Clavibacter sp. strain Enf12 inoculation stimulates the growth of C. bungeana plantlets and improves their tolerance to chilling stress through enhancing the antioxidant defense system.     

Detection of sugar accumulation and expression levels of correlative key enzymes in winter wheat (Triticum aestivum) at low temperatures

Carbohydrate accumulation is common in frost-resistant plants, and many enzymes participate in this process. The sugar content and expression levels of metabolic enzymes related to sugar biosynthesis in response to drops in temperature were measured in two cultivars of winter wheat (Triticum aestivum) with different cold tolerances. The results indicate that the two cultivars examined, Dongnongdongmai 1 and Jimai 22, accumulated high levels of carbohydrate before November 4 (above 0°C), and that accumulation decreased as temperatures fell. However, this decrease was more modest in Dongnongdongmai 1, which had a higher sugar content. Sucrose and fructose were the main soluble sugars, indicating an important role in freezing tolerance. Gene expression studies revealed that expression of the genes encoding chloroplastic enzymes was significantly upregulated in the tillering nodes. Expression upregulation of TaSS and TaTPT may be helpful for sugar accumulation before November 4.     

Inactivation of a low temperature-induced RNA helicase in Synechocystis sp. PCC 6803: physiological and morphological consequences

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.     

Low night temperatures have a differential effect on the diurnal cycling of gene expression in cold–sensitive and tolerant tomatoes*

Abstract. Comparisons were made between the changes in mRNA levels induced by low night temperatures in the cold–sensitive tomato and two altitudinal ecotypes of the wild species L. hirsutum. Changes in mRNA levels were detected by resolving in vitro translation products of poly(A)⁺ RNA by 2-D PAGE. The treatment was applied by first growing plants in a thermoperiod of 25/18°C and then switching to 25/6°C. All tomatoes displayed a diurnal cycling in which a set of mRNAs accumulated at the end of the 18°C nights, whereas another accumulated at the end of the 25°C days. The accumulation of night specific mRNAs was inhibited by 6°C nights in the cold sensitive tomatoes while that of the tolerant one was only marginally affected. All tomatoes showed a similar reduction in the apparent turnover rate of the day specific mRNAs during the 6°C nights. Finally, low night temperatures induced the accumulation of six to eight mRNAs in all genotypes. This number increased by 15 in L. esculentum after the seventh night and are likely involved in stress response rather than acclimation/tolerance. The tomato is proposed as a genetic model to discriminate genes involved in acclimation/tolerance from those involved in stress response.     

Acclimation of entomopathogenic nematodes to novel temperatures: trehalose accumulation and the acquisition of thermotolerance

The effect of thermal acclimation on trehalose accumulation and the acquisition of thermotolerance was studied in three species of entomopathogenic nematodes adapted to either cold or warm temperatures. All three Steinernema species accumulated trehalose when acclimated at either 5 or 35 degrees C, but the amount of trehalose accumulation differed by species and temperature. The trehalose content of the cold adapted Steinernema feltiae increased by 350 and 182%, of intermediate Steinernema carpocapsae by 146 and 122% and of warm adapted Steinernema riobrave by 30 and 87% over the initial level (18.25, 27.24 and 23.97 microg trehalose/mg dry weight, respectively) during acclimation at 5 and 35 degrees C, respectively. Warm and cold acclimation enhanced heat (40 degrees C for 8h) and freezing (-20 degrees C for 4h) tolerance of S. carpocapsae and the enhanced tolerance was positively correlated with the increased trehalose levels. Warm and cold acclimation also enhanced heat but not freezing tolerance of S. feltiae and the enhanced heat tolerance was positively correlated with the increased trehalose levels. In contrast, warm and cold acclimation enhanced the freezing but not heat tolerance of S. riobrave, and increased freezing tolerance of only warm acclimated S. riobrave was positively correlated with the increased trehalose levels. The effect of acclimation on maintenance of original virulence by either heat or freeze stressed nematodes against the wax moth Galleria mellonella larvae was temperature dependent and differed among species. During freezing stress, both cold and warm acclimated S. carpocapsae (84%) and during heat stress, only warm acclimated S. carpocapsae (95%) maintained significantly higher original virulence than the non-acclimated (36 and 47%, respectively) nematodes. Both cold and warm acclimated S. feltiae maintained significantly higher original virulence (69%) than the non-acclimated S. feltiae (0%) during heat but not freezing stress. In contrast, both warm and cold acclimated S. riobrave maintained significantly higher virulence (41%) than the non-acclimated (14%) nematodes during freezing, but not during heat stress. Our data indicate that trehalose accumulation is not only a cold associated phenomenon but is a general response of nematodes to thermal stress. However, the extent of enhanced thermal stress tolerance conferred by the accumulated trehalose differs with nematode species.     

Growth irradiance affects ureide accumulation and tolerance to photoinhibition in Eutrema salsugineum (Thellungiella salsuginea)

Plants are able to acclimate to their growth light environments by utilizing a number of short- and long-term mechanisms. One strategy is to prevent accumulation of excess reactive oxygen species that can lead to photoinhibition of photosynthesis. Ureides, generated from purine degradation, have been proposed as antioxidants and involved in certain abiotic stress responses. Eutrema salsugineum (Thellungiella salsuginea) is an extremophilic plant known to exhibit a high degree of tolerance to a variety of abiotic stresses that invariably generate reactive oxygen species. In the present study we have investigated the possible role of the ureide metabolic pathway during acclimation to growth irradiance and its conference of tolerance to photoinhibition in Eutrema. Ureide accumulation was greater under high light growth which also conferred tolerance to photoinhibition at low temperature as measured by the maximal quantum yield of PSII photochemistry. This may represent an adaptive plastic response contributing to the extreme tolerance exhibited by this plant. Our results would provide evidence that ureide accumulation may be involved in abiotic stress as another defence mechanism in response to oxidative stress.     

Mitochondrial ROS production and subsequent ERK phosphorylation are necessary for temperature preconditioning of isolated ventricular myocytes

Hypothermia and hypothermic preconditioning are known to be profoundly cardioprotective, but the molecular mechanisms of this protection have not been fully explained. In this study, temperature preconditioning (16 °C) was found to be cardioprotective in isolated adult rat ventricular myocytes, enhancing contractile recovery and preventing calcium dysregulation after oxidative stress. Hypothermic preconditioning preserved mitochondrial function by delaying the pathological opening of the mitochondrial permeability transition pore (mPTP), whereas transient mPTP flickering remained unaltered. For the first time, reactive oxygen species (ROS) from the mitochondria are shown to be released exclusively during the hypothermic episodes of the temperature-preconditioning protocol. Using a mitochondrially targeted ROS biosensor, ROS release was shown during the brief bursts to 16 °C of temperature preconditioning. The ROS scavenger N-(2-mercaptopropionyl) glycine attenuated ROS accumulation during temperature preconditioning, abolishing the protective delay in mPTP opening. Temperature preconditioning induces ROS-dependant phosphorylation of the prosurvival kinase extracellular signal-regulated kinase (ERK)1/2. ERK1/2 activation was shown to be downstream of ROS release, as the presence of a ROS scavenger during temperature preconditioning completely blocked ERK1/2 activation. The cardioprotective effects of temperature preconditioning on mPTP opening were completely lost by inhibiting ERK1/2 activation. Thus, mitochondrial ROS release and ERK1/2 activation are both necessary to signal the cardioprotective effects of temperature preconditioning in cardiac myocytes.     

Accumulation of some nitrogen compounds in response to salt stress and their relationships with salt tolerance in rice (Oryza sativa L.) seedlings

The salt-induced accumulation of some nitrogen compounds (free amino acids, ammonium and urea) in shoots of eight rice cultivars differing in salt tolerance was investigated. Salt treatment (100 mM, 6 days) significantly increased the proline content of shoots but this appeared to be a reaction to stress damage and not associated with salt tolerance, because proline contents were higher in the more sensitive cultivars. Besides proline, some other free amino acids also accumulated leading to a significant increase in the total amino acid content of the stressed seedlings. High levels of free ammonium also accumulated under conditions of stress; this was highly correlated with the accumulation of Na⁺ in the shoots and negatively correlated with salt tolerance. The accumulation of ammonium was positively correlated with the accumulation of many free amino acids, and also associated with the production of urea in the stressed seedlings. Results from the present investigations suggest that an increase in the concentration of some free amino acids including proline, may be a result of the reassimilation of the stress-induced ammonium. A high capacity to assimilate ammonium may be an important factor in alleviating the consequence of stress because ammonium can be toxic at high concentrations.