Chloroplast Protein Synthesis Elongation Factor Research Articles

Independent and Combined Effects of High Temperature and Drought Stress During Grain Filling on Plant Yield and Chloroplast EF-Tu Expression in Spring Wheat

High temperature and drought stress are among the two most important environmental factors influencing crop growth, development and yield processes. These two stresses commonly occur in combination. Objectives of this research were to investigate the independent and combined effects of high temperature and drought stress during grain filling on physiological, vegetative and yield traits and expression of a chloroplast protein synthesis elongation factor (EF-Tu) of wheat (Triticum aestivum L.). Two spring wheat cultivars (Pavon-76 and Seri-82) were grown at control temperatures (CT; day/night, 24/14 � C; 16/ 8 h photo/dark period) from sowing to heading. Thereafter, one half of the plants were exposed to high temperature stress (HT; 31/18 � C in Exp. 1 and 34/ 22 � C in Exp. 2), drought stress (withholding water), or a combination of both HT and drought stress. There were significant influences of HT and/or drought stress on physiological, growth and yield traits. There was no cultivar or cultivar by temperature or cultivar by drought interaction effects on most traits. The decreases in leaf photosynthesis were greater at HT compared with drought alone throughout the stress period, and the combination of HT and drought had the lowest leaf photosynthetic rates. Overall, HT or drought had similar effects (about 48‐56 % decrease) on spikelet fertility, grain numbers and grain yield. High temperature decreased grain numbers (by 56 % averaged across both experiments) and individual grain weight (by 25 %), while, respective decreases due to drought were 48 % and 35 %. This suggests that the grain numbers were more sensitive to HT and grain weights to drought for the range of temperatures tested in this research. The interaction between HT and drought stress was significant for total dry weights, harvest index and spikelet fertility, particularly when HT stress was severe (34/22 � C). The combined effects of HT and drought were greater than additive effects of HT or drought alone for leaf chlorophyll content, grain numbers and harvest index. High temperature stress and the combination of HT and drought stress but not drought stress alone resulted in the overexpression of EF-Tu in both spring wheat cultivars.

Analysis of differential protein expression during the early stage of in vitro tuberization in taro

Tuberization is a complex and multilevel developmental process. Many important metabolic changes in the early stage of tuberization are crucial to the tuber differentiation and development. In this study, we attempted to identify proteins differentially expressed in the early stage of in vitro tuberization in taro ( Colocasia esculenta var. antiquorum) by two-dimensional polyacrylamide gel electrophoresis (2-DE) and mass spectrometry (MS). Protein samples from shoot tips cultured in 8% sucrose media at 0, 2, 4, 6 and 8 d were separated with 2-DE. A total of 13 differentially expressed proteins were analyzed with MS. Four proteins via, glyceraldehyde-3-phosphate dehydrogenase, alcohol dehydrogenase, chloroplast protein synthesis elongation factor (EF-Tu), and ankyrin repeat protein HBP1 were successfully identified during in vitro tuberization of taro. This implies that important metabolic changes, including sucrose metabolism, signal transduction and cell defense, occurred in the early stage of in vitro tuberization in taro.

Role of Phosphatidic Acid in High Temperature Tolerance in Maize

Maize (Zea mays L.) germplasm exhibits large genetic variations in tolerance to high temperature (HT) stress under field conditions, but the mechanisms underling this variation are largely unknown. Based on many years of field observation, maize inbred line B76 consistently displays better tolerance to HT than B106. Heat waves during growing season cause leaf firing in developing leaves and tassel blasting in B106 but not in B76. The difference in HT tolerance between the two inbred lines was confirmed in growth chambers under controlled conditions. The two inbred lines showed similar level in the induction of heat shock proteins and a maize chloroplast protein synthesis elongation factor (EF‐Tu), two mechanisms known to involve in HT tolerance in maize. A drastic decrease in photosynthetic system II (PSII) quantum efficiency occurred at 34 to 35°C in B106; in B76, it occurred at a much higher temperature (>38°C). Cell membranes of B76 appeared to be more stable under HT than those of B106 based on electrolyte leakage analysis. Lipid profiles of young developing leaves by lipidomics showed that, among all lipids detected, only phosphatidic acid (PA) exhibited significant higher level in B76 than in B106 under both normal and HT stressed conditions (p < 0.02). Moreover, PA was the only lipid that was significantly increased by HT treatment (p < 0.05). Our results suggest that membrane thermostability is essential to HT tolerance and that PA may play an important role in imparting membrane thermostability, and, hence, HT tolerance in maize.

Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: segregation pattern, expression and effects of the transgene

We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation following exposure to heat stress (18 h at 45 degrees C) [Fu et al. in Plant Mol Biol 68:277-288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45 degrees C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.

Fine mapping and candidate gene analysis of a green-revertible albino gene gra(t) in rice

Green-revertible albino is a novel type of chlorophyll deficiency in rice (Oryza sativa L.), which is helpful for further research in chlorophyll synthesis and chloroplast development to illuminate their molecular mechanism. In the previous study, we had reported a single recessive gene, gra(t), controlling this trait on the long arm of chromosome 2. In this paper, we mapped the gra(t) gene using 1,936 recessive individuals with albino phenotype in the F(2) population derived from the cross between themo-photoperiod-sensitive genic male-sterile (T/PGMS) line Pei'ai 64S and the spontaneous mutant Qiufeng M. Eventually, it was located to a confined region of 42.4 kb flanked by two microsatellite markers RM2-97 and RM13553. Based on the annotation results of RiceGAAS system, 11 open reading frames (ORFs) were predicted in this region. Among them, ORF6 was the most possible gene related to chloroplast development, which encoded the chloroplast protein synthesis elongation factor Tu in rice. Therefore, we designated it as the candidate gene of gra(t). Sequence analysis indicated that only one base substitution C to T occurred in the coding region, which caused a missense mutation (Thr to Ile) in gra(t) mutant. These results are very valuable for further study on gra(t) gene.

Impact of Nighttime Temperature on Physiology and Growth of Spring Wheat

Climate models predict greater increases in nighttime temperature in the future. The impacts of high nighttime temperature on wheat (Triticum aestivum L.) are not well understood. Objectives of this research were to quantify the impact of high nighttime temperatures during reproductive development on phenology, physiological, vegetative, and yield traits of wheat. Two spring wheat cultivars (Pavon‐76 and Seri‐82) were grown at optimum temperatures (day/night, 24/14°C; 16/8 h light/dark photoperiod) from sowing to booting. Thereafter, plants were exposed to four different nighttime temperatures (14, 17, 20, 23°C) until maturity. The daytime temperature was 24°C across all treatments. There were significant influences of high nighttime temperatures on physiological, growth, and yield traits, but no cultivar or cultivar by temperature interactions were observed. High nighttime temperatures (>14°C) decreased photosynthesis after 14 d of stress. Grain yields linearly decreased with increasing nighttime temperatures, leading to lower harvest indices at 20 and 23°C. High nighttime temperature (≥20°C) decreased spikelet fertility, grains per spike, and grain size. Compared to the control (14°C), grain filling duration was decreased by 3 and 7 d at night temperatures of 20 and 23°C, respectively. High nighttime temperature increased the expression of chloroplast protein synthesis elongation factor in both cultivars suggesting possible involvement of this protein in plant response to stress.

Chloroplast protein synthesis elongation factor, EF-Tu, reduces thermal aggregation of rubisco activase

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize. The recombinant precursor of this protein, pre-EF-Tu, has been found to exhibit chaperone activity and protect heat-labile proteins, such as citrate synthase and malate dehydrogenase, from thermal aggregation. Chloroplast EF-Tu is highly conserved and it is possible that the chaperone activity of this protein is not species-specific. In this study, we investigated the effect of native wheat pre-EF-Tu on thermal aggregation of rubisco activase. Additionally, we investigated the effect of native and recombinant maize pre-EF-Tu on activase aggregation. Activase was chosen because it displays an exceptional sensitivity to thermal aggregation and constrains photosynthesis at high temperature. The native precursors of both wheat and maize EF-Tu displayed chaperone activity, as shown by the capacity of both proteins to reduce thermal aggregation of rubisco activase in vitro. Similarly, the recombinant maize pre-EF-Tu protected activase from thermal aggregation. This is the first report on chaperone activity of native pre-EF-Tu and the first evidence for thermal protection of a photosynthetic enzyme by this putative chaperone. The results are consistent with the hypothesis that chloroplast EF-Tu plays a functional role in heat tolerance by acting as a molecular chaperone.

Heat-induced accumulation of chloroplast protein synthesis elongation factor, EF-Tu, in winter wheat

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum). In this study, we assessed heat tolerance and determined the relative levels of EF-Tu in mature plants (at flowering stage) of 12 cultivars of winter wheat experiencing a 16-d-long heat treatment (36/30 degrees C, day/night temperature). In addition, we also investigated the expression of EF-Tu in young plants experiencing a short-term heat shock (4h at 43 degrees C). Heat tolerance was assessed by examining the stability of thylakoid membranes, measuring chlorophyll content, and assessing plant growth traits (shoot dry mass, plant height, tiller number, and ear number). In mature plants, relative levels of EF-Tu were determined after 7 d of heat stress. High temperature-induced accumulation of EF-Tu in mature plants of all cultivars, and a group of cultivars that showed greater accumulation of EF-Tu displayed better tolerance to heat stress. Young plants of all cultivars but one did not show significant increases in the relative levels of EF-Tu. The results of the study suggest that EF-Tu protein may play a role in heat tolerance in winter wheat.

Expression of chloroplast protein synthesis elongation factor, EF-Tu, in two lines of maize with contrasting tolerance to heat stress during early stages of plant development

Maize chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance, and previous studies have shown that under heat stress this protein accumulates in 14-d-, 17-d-, and 21-d-old plants of maize genotypes with increased tolerance to stress. In the present study, we investigated the expression of EF-Tu genes in heat tolerant, ZPBL 1304, and heat sensitive, ZPL 389, maize lines during early stages of their development (5-21-d-old plants) under both control and heat stress conditions. We also investigated the expression of EF-Tu in mature plants of these lines under field conditions and assessed heat tolerance in young seedlings at different stages of their development. The expression of EF-Tu was studied by determining the relative levels of EF-Tu protein and the steady state levels of EF-Tu mRNA. Chloroplast EF-Tu showed differential expression during early stages of plant development, and the heat tolerant and the heat sensitive line differed in the expression of EF-Tu under heat stress. In ZPBL 1304, plants of all ages (except 5-d-old shoots) showed heat-induced accumulation of both EF-Tu transcript and EF-Tu protein. In contrast, in ZPL 389, only plants up to 14d of age displayed increased accumulation of EF-Tu under heat stress. The increase in the relative level of EF-Tu in ZPL 389 was not preceded by an increase in the steady state level of EF-Tu mRNA. Under heat stress, the relative levels of EF-Tu correlated positively with plant heat tolerance. The results are consistent with the hypothesis that maize EF-Tu plays a role in heat tolerance and suggest that under heat stress conditions, the regulation of expression of EF-Tu may be different in the heat tolerant and heat sensitive maize lines.

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 degrees C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.

A maize mutant with decreased capacity to accumulate chloroplast protein synthesis elongation factor (EF-Tu) displays reduced tolerance to heat stress

A chloroplast protein synthesis elongation factor, EF-Tu, accumulates in some maize ( Zea mays L.) genotypes that show increased tolerance to heat stress [Planta 212 (2001) 359]. Previous studies, have suggested that maize EF-Tu may play a role in the development of heat tolerance [Plant Sci. 163 (2002) 1075]. In the present study, we identified a maize mutant that has decreased capacity to synthesize and accumulate EF-Tu under heat stress and tested the ability of the mutant to tolerate heat stress. Experiments were conducted in the laboratory using 17-day old plants. Stress tolerance was assessed by examining heat stability of photosynthetic membranes (thylakoids), thermal aggregation of chloroplast stromal proteins, and plant growth after exposure to stress. Compared to wild type (non-mutant), the mutant showed reduced tolerance to heat stress. Importantly, chloroplast stromal proteins from the EF-Tu-deficient mutant displayed greater aggregation during exposure to high temperature than chloroplast stromal proteins from the wild type. The results strongly support the hypothesis that maize EF-Tu plays a role in the development of heat tolerance by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal aggregation.

Localization and abundance of chloroplast protein synthesis elongation factor (EF-Tu) and heat stability of chloroplast stromal proteins in maize

Chloroplasts from a line of maize with high levels of chloroplast protein synthesis elongation factor (EF-Tu), ZPBL 1304, display greater heat stability than chloroplasts from a line with lower levels of EF-Tu, ZPL 389. We hypothesize that the greater heat stability of chloroplasts from ZPBL 1304 line may partly be attributed to EF-Tu, which may be protecting chloroplasts from heat injury. In this study, we investigated the subcellular distribution of EF-Tu and the heat stability of chloroplast stromal proteins in ZPBL 1304 and ZPL 389. Immunogold labeling and transmission electron microscopy (TEM) revealed that the chloroplast EF-Tu is localized mostly in the stroma and that under normal conditions agranal chloroplasts have a higher relative level of this protein than granal chloroplasts. Light scattering experiments with chloroplast stromal extracts showed that stromal proteins from the maize line with higher levels of EF-Tu, ZPBL 1304, display greater heat stability than stromal proteins from the line with lower levels of EF-Tu, ZPL 389. The results support the hypothesis that maize EF-Tu plays a role in the development of heat tolerance, possibly by acting as a molecular chaperone and protecting chloroplast stromal proteins from thermal aggregation.

Heterologous expression of maize chloroplast protein synthesis elongation factor (EF-Tu) enhances Escherichia coli viability under heat stress

A heat-tolerant maize line, ZPBL 1304, synthesizes increased amounts of chloroplast protein synthesis elongation factor (EF-Tu) under heat stress conditions. Previous studies have suggested that maize EF-Tu may be involved in the development of heat tolerance [Planta 212 (2001) 359; J. Plant Physiol. 153 (1998) 497]. In this study, we tested the hypothesis that overexpression of maize EF-Tu enhances the viability of Escherichia coli under heat stress. The approach was to expose E. coli transformed with a maize EF-Tu expression vector (pTrcHis2A-Zmeftu1) to 55 °C and assess viability at 37 °C. Western blots showed E. coli overproduced recombinant EF-Tu protein, and the protein seemed to be in a highly soluble form. E. coli overexpressing maize EF-Tu showed increased viability after exposure to heat stress, demonstrating that the maize EF-Tu is involved in the development of heat tolerance.

Heat-stress induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize line

A heat-tolerant maize (Zea mays L.) line, ZPBL 1304, synthesizes a unique set of five heat-shock polypeptides of 45 kDa. Previous studies suggested that these polypeptides might play a role in the development of thermotolerance in maize (Ristic et al., 1996, J. Plant Physiol. 149:424-432; Ristic et al., 1998, J. Plant Physiol. 153:497-505). In the present study, we isolated these polypeptides, sequenced them, and investigated their subcellular distribution and origin. Of the five polypeptides of 45 kDa, three polypeptides, including the two most abundant ones, yielded amino acid sequences similar to the chloroplast and bacterial protein synthesis elongation factor (EF-Tu). This was further confirmed using an antibody raised against maize EF-Tu, which showed a very strong reaction with the 45-kDa heatshock protein(s). Studies on subcellular distribution and origin revealed that the 45-kDa polypeptides were localized to the chloroplasts, and were likely of nuclear origin. A full-length maize EF-Tu cDNA (Zmeftu1), previously isolated from the B73 line of maize, was used as a probe for northern blot analysis of RNA extracted from the ZPBL 1304 maize line (the nucleotide and deduced amino acid sequences of Zmeftu1 are 88% identical to the rice EF-Tu sequence). Northern blots showed a 1.85-fold increase in steady-state levels of EF-Tu mRNA during heat stress. An increase in EF-Tu transcript levels during heat stress was accompanied by increased levels of the EF-Tu protein. Isolated chloroplasts from heat-stressed plants also had higher levels of EF-Tu as compared to control chloroplasts. The maize EF-Tu polypeptides showed > 80% sequence similarity with the bacterial EF-Tu, which has recently been shown to function as a molecular chaperone and to play a role in the protection of other proteins from thermal denaturation (Caldas et al., 1998, J. Biol. Chem. 273:11478-11482). It is hypothesized that chloroplast EF-Tu of the ZPBL 1304 maize line plays an important role in the development of thermotolerance.

Purification and N-Terminal Sequence-Analysis of Pea Chloroplast Protein Synthesis Factor EF-G

Chloroplast protein synthesis elongation factor G (chlEF-G) has been purified from whole-cell extracts of light-induced pea ( Pisum sativum) seedlings. The first step in the purification scheme relies on the affinity of organellar EF-G for Escherichia coli ribosomes in the presence of the antibiotic, fusidic acid. A complex between organellar EF-G, E. coli ribosomes, GDP, and fusidic acid was isolated by high-speed centrifugation. The largest major protein eluted from this complex by high salt has an apparent molecular weight of 86,000 and is only a minor component of similar preparations from dark-grown seedlings. The same polypeptide copurifies with EF-G activity upon size exclusion HPLC on a Waters Protein-Pak 200SW column. The N-terminal amino acid sequence of chlEF-G has been determined by direct sequencing of gel-purified protein. Like many proteins that are processed upon import into chloroplasts, it has an N-terminal alanine residue. Part of the putative chlEF-G gene has been amplified using oligonucleotides corresponding to the N-terminal amino acid sequence of the purified protein and to highly conserved sequences within the GTP-binding domains of other elongation factors. The deduced amino acid sequence displays high sequence identity to the corresponding region of the chloroplast EF-G gene product from soybean, somewhat less similarity to bacterial EF-Gs, and only low homology to mitochondrial EF-G and to eukaryotic cytoplasmic EF-2 genes. The chlEF-G gene appears to be encoded by a two-copy gene family in pea and a single-copy gene in Arabidopsis thaliana.

Euglena gracilis chloroplast elongation factor Tu. Purification and initial characterization.

The chloroplast protein synthesis elongation factor Tu (EF-Tuchl) has been purified to near homogeneity from Euglena gracilis. Chromatography of the postribosomal supernatant of light-induced Euglena on DEAE-Sephadex reveals two forms of EF-Tuchl. Further purification has shown that one species consists of a complex between EF-Tuchl and a factor that stimulates its activity. The other species consists of free EF-TUchl. The factor has been purified from both chromatographic forms by taking advantage of the molecular weight shift that occurs upon disruption of the complex between EF-Tuchl and the stimulatory factor. EF-Tuchl consists of a single polypeptide chain with a molecular weight of about 50,000. EF-Tuchl is as active on Escherichia coli ribosomes as it is on its homologous ribosomes but displays no detectable activity on eukaryotic cytoplasmic ribosomes. It is stimulated in polymerization by E. coli EF-Ts and will form a complex with the prokaryotic factor that can be isolated by gel filtration chromatography. Like E. coli EF-Tu, it is sensitive to modification by N-ethylmaleimide and is inhibited by the antibiotic kirromycin. Thus, the chloroplast factor has many features that reflect the close relationship between prokaryotic and chloroplast translational systems.

Purification of Euglena gracilis chloroplast elongation factor G and comparison with other prokaryotic and eukaryotic translocases.

Euglena gracilis chloroplast protein synthesis elongation factor G (EF-Gchl) has been purified to about 80% homogeneity by a two-step procedure which removes all traces of the cytoplasmic and mitochondrial translocases (EF-2 and EF-Gmt). The purification scheme generally results in approximately 130-fold purification with 20% recovery of the total EF-G activity present in whole cell extracts. The Euglena EF-Gchl is a monomeric protein with a molecular weight of approximately 85,000. As observed for all translocases to date, the activity of Euglena EF-Gchl is inhibited by treatment with low concentrations of N-ethylmaleimide, suggesting that a free sulfhydryl group is required for catalytic activity. Treatment with 3 microM fusidic acid results in a 50% inhibition of Euglena EF-Gchl activity and of the EF-G activity present in Chlamydomonas reinhardtii. About 10-fold higher concentrations of this antibiotic are required to inhibit the mitochondrial EF-G of Euglena and Escherichia coli EF-G to the same extent. Yeast mitochondrial EF-G is clearly distinguishable from the other organellar translocases tested, requiring 1 mM fusidic acid for 50% inhibition. Fusidic acid also inhibits the cytoplasmic translocases from yeast, wheat germ, and Euglena, although a wide range of sensitivities is observed. When antiserum raised against highly purified Euglena EF-Gchl is used to inhibit enzymatic translocation, a low degree of cross-reaction of the antiserum with Chlamydomonas EF-G and with E. coli EF-G is observed. The EF-G activity present in spinach is very slightly inhibited by the antiserum, whereas that of yeast is not affected. The mitochondrial and cytoplasmic translocases of Euglena are also unaffected by the antiserum against EF-Gchl. The evolutionary implications of these observations are discussed.