Expression analysis of a stress-modulated transcript in drought tolerant and susceptible cultivars of sorghum (Sorghum bicolor)

The present study reports the cloning of a 581 bp sequence, designated as SbEST8, from the osmotically stressed germinated seeds of a drought tolerant cultivar of sorghum (Sorghum bicolor). The SbEST8, which shows no homology with the reported gene sequences, is present in multiple copies and lacks restriction fragment length polymorphism among different sorghum cultivars. The expression of SbEST8 in the germinating seeds of sorghum was modulated by different abiotic stresses. Kinetic studies revealed that imposition of osmotic stress after 8h resulted in maximum levels of SbEST8 mRNA in the germinating seeds of cv. ICSV-272, with further stress causing a decline to undetectable levels by 16 h. However, relieving the stress after 12h resulted in an enhancement of SbEST8 mRNA levels for at least another 4h following which it declined. The decrease in SbEST8 mRNA levels in the leaves at 30 DAS in response to drought stress was observed only in the drought susceptible cultivar (CSV-216), whereas its expression was either increased substantially or remained unaffected in the tolerant cultivars, thus suggesting its role in water stress tolerance.     

Evidence for the role of wheat eukaryotic translation initiation factor 3 subunit g (TaeIF3g) in abiotic stress tolerance

The gene encoding eIF3g (TaeIF3g), one of the 11 subunits of eukaryotic translation initiation factor 3 (eIF3), was cloned from wheat for carrying out its functional analysis. Transgenic expression of TaeIF3g enhanced the tolerance of TaeIF3g-overexpressing parental yeast cells and Arabidopsis plants under different abiotic stress conditions. Compared to untransformed plants, TaeIF3g-overexpressing Arabidopsis thaliana plants exhibited significantly higher survival rate, soluble proteins and photosynthetic efficiency, and enhanced protection against photooxidative stress under drought conditions. This study provides first evidence that TaeIF3g imparts stress tolerance and could be a potential candidate gene for developing crop plants tolerant to abiotic stress.     

Caspase-mediated cleavage and DNase activity of the translation initiation factor 3, subunit G (eIF3g)

Eukaryotic translation initiation factor 3 is composed of 13 subunits (eIF3a through eIF3m) and plays an essential role in translation. During apoptosis, several caspases rapidly down-regulate protein synthesis by cleaving eIF4G, -4B, -3j, and -2α. In this study, we found that the activation of caspases by cisplatin in T24 cells induces the cleavage of subunit G of the eIF3 complex (eIF3g). The cleavage site (SLRD²²⁰G) was identified, and we found that the cleaved N-terminus was translocated to the nucleus, activating caspase-3, and that it also showed a strong DNase activity. These data demonstrate the important roles of eIF3g in the translation initiation machinery and in DNA degradation during apoptosis.     

Malignant transformation by the eukaryotic translation initiation factor 3 subunit p48 (eIF3e)

Several components of translation, e.g. eIF4E and PKR, are implicated in cancer. The e-subunit (p48) of mammalian initiation factor 3 is encoded by the Int6 gene, a common site for integration of the mouse mammary tumor virus genome, leading to the production of a truncated eukaryotic initiation factor-3e (eIF3e). Stable expression of a truncated eIF3e in NIH 3T3 cells causes malignant transformation by four criteria: foci formation; anchorage independent growth; accelerated growth; and lack of contact inhibition. Stable expression of full-length eIF3e does not cause transformation. The truncated eIF3e also inhibits the onset of apoptosis caused by serum starvation.     

Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8)

The P56S mutation in VAPB (vesicle-associated membrane protein-associated protein B) causes autosomal dominant motoneuronal diseases. Although it was reported that the P56S mutation induces localization shift of VAPB from endoplasmic reticulum (ER) to non-ER compartments, it remains unclear what the physiological function of VAPB is and how the P56S mutation in VAPB causes motoneuronal diseases. Here we demonstrate that overexpression of wild type VAPB (wt-VAPB) promotes unfolded protein response (UPR), which is an ER reaction to suppress accumulation of misfolded proteins, and that small interfering RNA for VAPB attenuates UPR to chemically induced ER stresses, indicating that VAPB is physiologically involved in UPR. The P56S mutation nullifies the function of VAPB to mediate UPR by inhibiting folding of VAPB that results in insolubility and aggregate formation of VAPB in non-ER fractions. Furthermore, we have found that expression of P56S-VAPB inhibits UPR, mediated by endogenous wt-VAPB, by inducing aggregate formation and mislocalization into non-ER fractions of wt-VAPB. Consequently, the P56S mutation in a single allele of the VAPB gene may diminish the activity of VAPB to mediate UPR to less than half the normal level. We thus speculate that the malfunction of VAPB to mediate UPR, caused by the P56S mutation, may contribute to the development of motoneuronal degeneration linked to VAPB/ALS8.     

A highly conserved binding site in vesicle-associated membrane protein-associated protein (VAP) for the FFAT motif of lipid-binding proteins

A variety of lipid-binding proteins contain a recently described motif, designated FFAT (two phenylalanines in an acidic tract), which binds to vesicle-associated-membrane protein-associated protein (VAP). VAP is a conserved integral membrane protein of the endoplasmic reticulum that contains at its amino terminus a domain related to the major sperm protein of nematode worms. Here we have studied the FFAT-VAP interaction in Saccharomyces cerevisiae, where the VAP homologue Scs2 regulates phospholipid metabolism via an interaction with the FFAT motif of Opi1. By introducing mutations at random into Scs2, we found that mutations that abrogated binding to FFAT were clustered in the most highly conserved region. Using site-directed mutagenesis, we identified several critical residues, including two lysines widely separated in the primary sequence. By examining all other conserved basic residues, we identified a third residue that was moderately important for binding FFAT. Modeling VAP on the known structure of major sperm protein showed that the critical residues form a patch on a positively charged face of the protein. In vivo functional studies of SCS22, a second SCS2-like gene in S. cerevisiae, showed that SCS2 was the dominant gene in the regulation of Opi1, with a minor contribution from SCS22. We then established that reduction in the affinity of Scs2 mutants for FFAT correlated well with loss of function, indicating the importance of these residues for binding FFAT motifs. Finally, we found that human VAP-A could substitute for Scs2 but that it functioned poorly, suggesting that other factors modulate the binding of Scs2 to proteins with FFAT motifs.     

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.     

Kinetic and thermodynamic properties of wall bound invertase in heat tolerant and susceptible cultivars of wheat

We have identified two types of invertases, one bound ionically and the other covalently to the particulate fraction in grains of heat tolerant C 306 and heat susceptible WH 542 cultivars of wheat (Triticum aestivum L.). The cell walls contained a high level of invertase activity, of which 79.2–72.8% was extractable by 2 M NaCl and 14.9–21.1% by 0.5% EDTA in C 306 and WH 542, respectively. The NaCl-released invertase constituted the predominant fraction. Using 5–100 mM sucrose and pH range of 4.0–7.0, the apparent Michaelis constant (K _m, enzyme substrate affinity measure) of enzyme ranged from 5.73 to 16.06 mM for C 306 and from 6.08 to 19.86 mM for WH 542. The V _max (maximum catalytic rate) values at these pH were higher in C 306 (0.63–11.04 μg sucrose hydrolysed min⁻¹) than WH 542 (0.51–8.73 μg sucrose hydrolysed min⁻¹). By employing photo-oxidation and by studying the effect of pH on K _m and V _max, the involvement of histidine and α-carboxyl groups at the active site of the enzyme was indicated. The two cultivars also showed differential response in terms of thermodynamic properties of the enzyme i.e. energy of activation (E _a), enthalpy change (ΔH) and entropy change (ΔS). NaCl-released invertase showed differential response to metal ions in two cultivars suggesting their distinctive nature. Mn²⁺, Cu²⁺, Hg²⁺, Mg²⁺, Zn²⁺ and Cd²⁺ were strong inhibitors in WH 542 as compared to C 306 while K⁺, Ca²⁺ were stimulators in both the cultivars. Overall the results suggest that genetic differences exist in wall bound invertase properties of wheat grains as evident in its altered kinetic behaviour.     

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.     

On the functions of the h subunit of eukaryotic initiation factor 3 in late stages of translation initiation

Background  

The eukaryotic translation initiation factor 3 (eIF3) has multiple roles during the initiation of translation of cytoplasmic mRNAs. How individual subunits of eIF3 contribute to the translation of specific mRNAs remains poorly understood, however. This is true in particular for those subunits that are not conserved in budding yeast, such as eIF3h.     

An integrative proteome analysis of different seedling organs in tolerant and sensitive wheat cultivars under drought stress and recovery

Roots, leaves, and intermediate sections between roots and leaves (ISRL) of wheat seedlings show different physiological functions at the protein level. We performed the first integrative proteomic analysis of different tissues of the drought‐tolerant wheat cultivar Hanxuan 10 (HX‐10) and drought‐sensitive cultivar Chinese Spring (CS) during a simulated drought and recovery. Differentially expressed proteins (DEPs) in the roots (122), ISRLs (146), and leaves (163) showed significant changes in expression in response to drought stress and recovery. Numerous DEPs associated with cell defense and detoxifications were significantly regulated in roots and ISRLs, while in leaves, DEPs related to photosynthesis showed significant changes in expression. A significantly larger number of DEPs related to stress defense were upregulated in HX‐10 than in CS. Expression of six HSPs potentially related to drought tolerance was significantly upregulated under drought conditions, and these proteins were involved in a complex protein–protein interaction network. Further phosphorylation analysis showed that the phosphorylation levels of HSP60, HSP90, and HOP were upregulated in HX‐10 under drought stress. We present an overview of metabolic pathways in wheat seedlings based on abscisic acid signaling and important protein expression patterns.     

Expression analysis of dehydrin multigene family across tolerant and susceptible barley (Hordeum vulgare L.) genotypes in response to terminal drought stress

Dehydrins are one of the characteristic families of plant proteins that usually accumulate in response to drought. In the present study, gene expressions of dehydrin multigene family (13 genes) were examined in flag leaves of tolerant (Yousef) and susceptible (Moroco) barley varieties under terminal drought to characterize the involvement of dehydrins in the adaptive processes. The stomatal conductance, RWC, and Chl a, b contents had more reduction in Moroco than the Yousef which has more elevated osmotic adjustment. Drought stress increased significantly MDA and electrolyte leakage levels, but greater in Moroco, indicating a poor protection of cell and cytoplasmic membrane in this variety. Yousef variety had no reduction in grain yield under drought condition. Five genes (Dhn1, Dhn3, Dhn5, Dhn7 and Dhn9) were exclusively induced in Yousef under drought stress. In the stress condition, relative gene expression of Dhn3, Dhn9 had the direct correlations (P < 0.05) with Chl a, b contents, osmotic adjustment, stomatal conductance, plant biomass and grain yield, and the negative correlations (P < 0.05) with MDA and electrolyte leakage levels. The results supported the impending functional roles of dehydrin K_n and particularly Y_nSK_n types in dehydration tolerance of barley during the reproductive stage.