Alterations in Membrane Protein-Profile during Cold Treatment of Alfalfa

Changes in pattern of membrane proteins during cold acclimation of alfalfa have been examined. Cold acclimation for 2 to 3 days increases membrane protein content. Labeling of membrane proteins in vivo with [³⁵S]methionine indicates increases in the rate of incorporation as acclimation progresses. Cold acclimation induces the synthesis of about 10 new polypeptides as shown by SDS-PAGE and fluorography of membrane proteins labeled in vivo.     

Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings

Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P³DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.     

Extractant Influence on the Relationship between Extractable Proteins and Cold Tolerance of Alfalfa

The influence of ionic composition and pH of extractant on the relationship between the extracted proteins and the cold tolerance of Vernal and Arizona Common alfalfa (Medicago sativa L.) was examined. Five environments were used to induce different tolerance levels. The quantity of protein extracted from plants was influenced by the hardening environment, cultivar, and ionic composition and pH of 29 extractants. Extractants with a pH below 6 generally extracted less protein.     

Oryzalin-Induced Changes in Water Status and Cytoskeleton Proteins of Winter Wheat Seedlings upon Cold Acclimation and ABA Treatment

The effect of oryzalin (a specific inhibitor of tubulin polymerization in plant cells) on water retention by the leaves and roots of winter wheat (Triticum aestivum L.) seedlings was studied. The cultivars differing in their frost resistance were compared after their acclimation to low temperature (3°C for 3 or 7 days) and after treatment with ABA. In control untreated plants, oryzalin reduced the water-retaining capacity (WRC) of leaves and roots. Both hardening and ABA lowered the effect of the inhibitor on WRC in leaves, whereas their effects on water retention by roots were opposite, i.e., hardening weakened and ABA intensified the effect of oryzalin. Oryzalin-induced reduction of WRC decreased in the following sequence of cultivars: weakly frost resistant moderately frost resistant highly frost resistant. It was more pronounced in the leaves than in the roots, the latter being characterized by the lower WRC and lower frost resistance. After three-day-long hardening of plants, an additive effect of hypothermia and ABA on oryzalin-induced decrease in WRC of leaves and the lack of such effect in the roots were observed. The immunochemical analysis of the composition and content of cytoskeletal proteins with Western blotting showed that in the leaves the actin/tubulin ratio was higher than in the roots. The treatment of nonacclimated plants with ABA lowered the content of - and -tubulins and actin in roots but did not affect the level of actin in leaves. Hardening negated the effects of ABA on cytoskeletal proteins. Oryzalin produced the greatest inhibitory effect on WRC and an increase in frost resistance in ABA-treated plants in the experiments with leaves of the weakly frost resistant cultivar before and after hardening. Organ- and cultivar-specific and ABA-mediated dependence of WRC on cytoskeletal proteins and microtubules and microfilaments formed by them is supposed to result from their effect on the state of intracellular water and water permeability of the plasma membrane. In the course of cold acclimation of plants and upon their treatment with ABA, this dependence was more distinctly expressed in leaves than in roots, and especially in the plants of the weakly frost resistant cultivar.     

Basic Proteins of Plant Nuclei during Normal and Pathological Cell Growth

Histone proteins were studied by microphotometry of plant tissue sections stained with fast green at pH 8.1. For comparative purposes the Feulgen reaction was used for deoxyribose nuclei acid (DNA); the Sakaguchi reaction for arginine; and the Millon reaction for estimates of total protein. Analysis of Tradescantia tissues indicated that amounts of nuclear histone fell into approximate multiples of the gametic (egg or sperm) quantity except in dividing tissues, where amounts intermediate between multiples were found. In differentiated tissues of lily, corn, onion, and broad bean, histones occurred in constant amounts per nucleus, characteristic of the species, as was found also for DNA. Unlike the condition in several animal species, the basic proteins of sperm nuclei in these higher plants were of the histone type; no evidence of protamine was found. In a plant neoplasm, crown gall of broad bean, behavior of the basic nuclear proteins closely paralleled that of DNA. Thus, alterations of DNA levels in tumor tissues were accompanied by quantitatively similar changes in histone levels to maintain the same Feulgen/fast green ratios found in homologous normal tissues.     

Sinorhizobium meliloti Mutants Deficient in Phosphatidylserine Decarboxylase Accumulate Phosphatidylserine and Are Strongly Affected during Symbiosis with Alfalfa

Sinorhizobium meliloti contains phosphatidylglycerol, cardiolipin, phosphatidylcholine, and phosphatidylethanolamine (PE) as major membrane lipids. PE is formed in two steps. In the first step, phosphatidylserine synthase (Pss) condenses serine with CDP-diglyceride to form phosphatidylserine (PS), and in the second step, PS is decarboxylated by phosphatidylserine decarboxylase (Psd) to form PE. In this study we identified the sinorhizobial psd gene coding for Psd. A sinorhizobial mutant deficient in psd is unable to form PE but accumulates the anionic phospholipid PS. Properties of PE-deficient mutants lacking either Pss or Psd were compared with those of the S. meliloti wild type. Whereas both PE-deficient mutants grew in a wild-type-like manner on many complex media, they were unable to grow on minimal medium containing high phosphate concentrations. Surprisingly, the psd-deficient mutant could grow on minimal medium containing low concentrations of inorganic phosphate, while the pss-deficient mutant could not. Addition of choline to the minimal medium rescued growth of the pss-deficient mutant, CS111, to some extent but inhibited growth of the psd-deficient mutant, MAV01. When the two distinct PE-deficient mutants were analyzed for their ability to form a nitrogen-fixing root nodule symbiosis with their alfalfa host plant, they behaved strikingly differently. The Pss-deficient mutant, CS111, initiated nodule formation at about the same time point as the wild type but did form about 30% fewer nodules than the wild type. In contrast, the PS-accumulating mutant, MAV01, initiated nodule formation much later than the wild type and formed 90% fewer nodules than the wild type. The few nodules formed by MAV01 seemed to be almost devoid of bacteria and were unable to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 were yellowish, indicating that the plants were starved for nitrogen. Therefore, changes in lipid composition, including the accumulation of bacterial PS, prevent the establishment of a nitrogen-fixing root nodule symbiosis.Rhizobia are soil bacteria able to form a symbiosis with legume plants, which leads to the formation of nitrogen-fixing root nodules. The establishment and functioning of this symbiosis are based on the recognition of signal molecules, which are produced by both the bacterial and plant partners. Known recognition factors of the bacterial endosymbiont include nodulation (Nod) factors, extracellular polysaccharides, lipopolysaccharides, K antigens, and cyclic glucans (24, 53). These factors are required for nodule formation, the infection process, and the colonization of the root nodule. Recently it was demonstrated that adequate levels of phosphatidylcholine (PC) are also required in order to allow the formation of a fully functional symbiosis between Bradyrhizobium japonicum and its soybean host plant (35). Under conditions of phosphate limitation, Sinorhizobium meliloti replaces the majority of its phospholipids with phosphorus-free membrane lipids, such as sulfolipids, ornithine-containing lipids, and diacylglyceryl-N,N,N-trimethylhomoserine lipids (20). Rhizobial mutants lacking the ability to form any one of these phosphorus-free membrane lipids or all three lipids at the same time form effective nitrogen-fixing root nodules (30, 31), demonstrating that not all major bacterial membrane lipids are required for the onset of a successful symbiosis.Escherichia coli is the prokaryote with the best-studied membrane lipid biosynthesis. In E. coli, three major membrane phospholipids, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL), are present. Certain functions have been defined for specific membrane phospholipids in E. coli. Anionic phospholipids (PG and CL) were shown to be involved in the initiation of DNA replication (60) and in the translocation of outer membrane precursor proteins (27). The zwitterionic PE is essential for a proper functioning of the electron transfer chain (34), for the assembly and functionality of lactose permease (4, 5), and for motility and chemotaxis (47). Certain specific functions have also been shown for other membrane lipids. Recently PC has been shown to be required for pathogenesis of Legionella pneumophila, Brucella abortus, and Agrobacterium tumefaciens on their hosts (7, 8, 9, 59). The cationic membrane lipid lysyl-phosphatidylglycerol is involved in conferring resistance to cationic antimicrobial peptides of the host''s innate immune system to Staphylococcus aureus (40), and the presence of LPG in Rhizobium tropici also increases resistance to the cationic peptide polymyxin B (52).In the initial step of the pathway leading to PE formation, phosphatidylserine (PS) synthase (Pss) is responsible for the formation of PS from CDP-diacylglycerol and serine (EC 2.7.8.8) (Fig. ​(Fig.1).1). In the subsequent step, PS is decarboxylated by PS decarboxylase (Psd) (EC 4.1.1.65) to yield PE (17, 58). In S. meliloti, PE is a substrate for the enzyme phospholipid N-methyltransferase (PmtA) (15), leading to the formation of PC. A gene coding for the Pss enzyme (pssA) has been found and cloned from prokaryotes (11, 19, 38, 51), lower eukaryotes, such as Saccharomyces cerevisiae (28, 37), and plants (12). In a previous work we described the construction and characterization of an S. meliloti mutant deficient in Pss (51).Open in a separate windowFIG. 1.Biosynthesis of phospholipids in Sinorhizobium meliloti. SAM, S-adenosylmethionine; SAHC, S-adenosylhomocysteine; PgsA, phosphatidylglycerolphosphate synthase; Pgp, phosphatidylglycerolphosphate phosphatase; Cls, cardiolipin synthase; Pss, phosphatidylserine synthase; Psd, phosphatidylserine decarboxylase; PmtA, phospholipid N-methyltransferase; Pcs, phosphatidylcholine synthase.Psds have been described and characterized for a wide range of organisms, including bacteria, such as E. coli (22, 23, 29) and Bacillus subtilis (32), lower eukaryotes, such as S. cerevisiae (6, 54-56) and Plasmodium falciparum (1), plants, such as Arabidopsis thaliana (36, 41), and mammals (CHO [Chinese hamster ovary] cells) (26). All Psd sequences identified so far seem to be phylogenically related (see Fig. S1A in the supplemental material). Interestingly, S. meliloti lacks a good homologue to any of the above-mentioned Psds.Here we describe the identification and characterization of the sinorhizobial psd gene coding for Psd. The mutant MAV01, in which the sinorhizobial psd gene is deleted, accumulated PS to about 20% of total lipids when grown in complex growth medium. We compared the mutant MAV01 to a sinorhizobial mutant deficient in Pss (CS111) (51) under free-living conditions and during symbiosis. The Pss-deficient mutant, CS111, forms about 30% fewer nodules than the wild type on its alfalfa host plant, whereas the PS-accumulating mutant, MAV01, forms 90% fewer nodules than the wild type. Nodule formation in the mutant MAV01 sets in about 20 days later than that in the wild type. The few nodules formed by the psd-deficient mutant seem to be almost devoid of bacteria and are not able to fix nitrogen. Leaves of alfalfa plants inoculated with the mutant MAV01 are yellowish, indicating that the plants are starved for nitrogen. The accumulation of PS, therefore, although allowing wild-type-like growth in different growth media, strongly interferes with nodule development.     

Identification of Differentially Expressed Proteins and Phosphorylated Proteins in Rice Seedlings in Response to Strigolactone Treatment

Strigolactones (SLs) are recently identified plant hormones that inhibit shoot branching and control various aspects of plant growth, development and interaction with parasites. Previous studies have shown that plant D10 protein is a carotenoid cleavage dioxygenase that functions in SL biosynthesis. In this work, we used an allelic SL-deficient d10 mutant XJC of rice (Oryza sativa L. spp. indica) to investigate proteins that were responsive to SL treatment. When grown in darkness, d10 mutant seedlings exhibited elongated mesocotyl that could be rescued by exogenous application of SLs. Soluble protein extracts were prepared from d10 mutant seedlings grown in darkness in the presence of GR24, a synthetic SL analog. Soluble proteins were separated on two-dimensional gels and subjected to proteomic analysis. Proteins that were expressed differentially and phosphoproteins whose phosphorylation status changed in response to GR24 treatment were identified. Eight proteins were found to be induced or down-regulated by GR24, and a different set of 8 phosphoproteins were shown to change their phosphorylation intensities in the dark-grown d10 seedlings in response to GR24 treatment. Analysis of these proteins revealed that they are important enzymes of the carbohydrate and amino acid metabolic pathways and key components of the cellular energy generation machinery. These proteins may represent potential targets of the SL signaling pathway. This study provides new insight into the complex and negative regulatory mechanism by which SLs control shoot branching and plant development.     

Accumulation of Dehydrins and ABA-Inducible Proteins in Wheat Seedlings during Low-Temperature Acclimation

Using one-dimensional SDS-PAGE and immunochemical methods, we detected the presence and estimated the content of dehydrins and ABA-inducible (RAB) polypeptides in etiolated seedlings of four spring and three winter wheat (Triticum aestivum L.) cultivars differing in frost hardiness. We hardened three-day-old seedlings at 4°C for nine days or grew them at 22°C for a day (control seedlings). We established that heat-stable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD, which are characteristic of hardened wheat seedlings, were homologous to polypeptides from a dehydrin family and polypeptides with mol wts of 209, 196, 66, 50, and 41 kD were immunologically related to RAB-proteins. We supposed that these COR polypeptides were involved in the prevention of local protein dehydration and denaturation during hypothermia. Analysis of the relative content of COR proteins revealed a close correlation between the cultivar frost hardiness and the concentration of these proteins. It seems evident that different accumulation of dehydrins and RAB polypeptides in different cultivars of a single species is one of the causes for different plant frost hardiness.     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

Polymorphism of Thermostable Proteins in Soft Wheat Seedlings during Low-Temperature Acclimation

The accumulation of thermostable stress proteins during hardening was studied in etiolated seedlings of spring (cvs. Rollo, Drott, Angara-86, and Tyumenskaya-80) and winter (moderately frost-resistant cv. Bezostaya-1 and highly frost-resistant cvs. Irkutskaya ozimaya and Zalarinka) wheat using one-dimensional SDS-PAGE. Hardening was performed at 4°C for nine days. Seedling tolerance to low subzero temperatures was estimated from electrolyte leakage and seedling survival after freezing. Hardening of all wheat genotypes tested resulted in the accumulation of thermostable cold-regulated (COR) polypeptides with mol wts of 209, 196, 169, 66, 50, and 41 kD. A densitometric analysis demonstrated a close correlation between the cultivar frost tolerance and the relative content of COR proteins, which evidently indicated the protective functions of the latter. These results led us to suggest that the level of specific protective agents, thermostable high-molecular-weight COR polypeptides in particular, determines the degree of plant frost resistance within a particular plant species.     

Ribosomal Changes during Induction of Cold Hardiness in Black Locust Seedlings

Protein synthesis has been implicated in the cold-hardening process. Ribosomes from cold hardy and nonhardy black locust (Robinia pseudoacacia L.) seedlings were compared to determine if cold acclimation is related to alteration of ribosomal structure. Ribosomal structure, as indicated by thermal melting profiles, appears to be altered during induction of hardiness. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicates at least 17 proteins from hardy seedlings that are different from those of nonhardy seedlings. These different proteins may be partially responsible for the different thermal melting profiles observed.     

Cold and Warm Denaturation of Proteins

We introduce a simplified protein model where the water degrees of freedom appear explicitly (although in an extremely simplified fashion). Using thismodel we are able to recover both the warm and the cold protein denaturation within a single framework, while addressing important issues about the structure of model proteins.     

Loop Folds in Proteins and Evolutionary Conservation of Folding Nuclei

Abstract

We show that loops of close contacts involving hydrophobic residues are important in protein folding. Contrary to Berezovsky and Trifonov (J. Biomol. Struct. Dyn. 20, 5–6, 2002) the loops important in protein folding usually are much larger in size than 23–31 residues, being instead comparable to the size of the protein for single domain proteins. Additionally what is important are not single loop contacts, but a highly interconnected network of such loop contacts, which provides extra stability to a protein fold and which leads to their conservation in evolution.     

Evolutionarily Conserved Interaction between the Phosphoproteins and X Proteins of Bornaviruses from Different Vertebrate Species

Bornavirus, a non-segmented, negative-strand RNA viruses, is currently classified into several genetically distinct genotypes, such as Borna disease virus (BDV) and avian bornaviruses (ABVs). Recent studies revealed that bornavirus genotypes show unique sequence variability in the putative 5′ untranslated region (5′ UTR) of X/P mRNA, a bicistronic mRNA for the X protein and phosphoprotein (P). In this study, to understand the evolutionary relationship among the bornavirus genotypes, we investigated the functional interaction between the X and P proteins of four bornavirus genotypes, BDV, ABV genotype 4 and 5 and reptile bornavirus (RBV), the putative 5′ UTRs of which exhibit variation in the length. Immunofluorescence and immunoprecipitation analyses using mammalian and avian cell lines revealed that the X proteins of bornaviruses conserve the ability to facilitate the export of P from the nucleus to the cytoplasm via interaction with P. Furthermore, we showed that inter-genotypic interactions may occur between X and P among the genotypes, except for X of RBV. In addition, a BDV minireplicon assay demonstrated that the X and P proteins of ABVs, but not RBV, can affect the polymerase activity of BDV. This study demonstrates that bornaviruses may have conserved the fundamental function of a regulatory protein during their evolution, whereas RBV has evolved distinctly from the other bornavirus genotypes.     

苜蓿幼苗对铝胁迫的生理响应及综合评价

为探讨铝胁迫对苜蓿(Medicago sativa)幼苗生长和生理特性的影响,对铝胁迫下苜蓿地上和地下生物量、光合色素及根尖胼胝质含量进行测定,并对根尖结构进行观察,最后采用隶属函数分析法对苜蓿耐铝性进行评价。结果表明,随着铝胁迫的增加,苜蓿地上、地下部分生物量呈降低趋势,低浓度和高浓度铝胁迫使苜蓿生物量显著下降(P0.05);苜蓿的叶绿素含量呈下降趋势,胼胝质积累量增多;中、高浓度铝胁迫使根尖胼胝质含量显著上升。随铝胁迫浓度升高,根尖横切面细胞发生较大变化,尤其在高浓度时,细胞干瘪且排列紊乱。隶属函数分析结果表明,No. 12和No. 18苜蓿材料的耐铝性较好,可在南方酸性富铝化土壤中推广应用。