A dehydrin cognate protein from pea (Pisum sativum L.) with an atypical pattern of expression

Dehydrins are a family of proteins characterised by conserved amino acid motifs, and induced in plants by dehydration or treatment with ABA. An antiserum was raised against a synthetic oligopeptide based on the most highly conserved dehydrin amino acid motif, the lysine-rich block (core sequence KIKEK-LPG). This antiserum detected a novel M _r 40 000 polypeptide and enabled isolation of a corresponding cDNA clone, pPsB61 (B61). The deduced amino acid sequence contained two lysine-rich blocks, however the remainder of the sequence differed markedly from other pea dehydrins. Surprisingly, the sequence contained a stretch of serine residues, a characteristic common to dehydrins from many plant species but which is missing in pea dehydrin.The expression patterns of B61 mRNA and polypeptide were distinctively different from those of the pea dehydrins during seed development, germination and in young seedlings exposed to dehydration stress or treated with ABA. In particular, dehydration stress led to slightly reduced levels of B61 RNA, and ABA application to young seedlings had no marked effect on its abundance.The M _r 40 000 polypeptide is thus related to pea dehydrin by the presence of the most highly conserved amino acid sequence motifs, but lacks the characteristic expression pattern of dehydrin. By analogy with heat shock cognate proteins we refer to this protein as a dehydrin cognate.     

Novel conserved segments are associated with differential expression patterns for Pinaceae dehydrins

Dehydrins are thought to play an essential role in the response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins have been classified into five groups according to the presence of conserved and repeated motifs in their amino acid sequence. Due to their putative functions in the response to stress, dehydrins have been often used as candidate genes in studies on population variability and local adaptation to environmental conditions. However, little is still known regarding the differential role played by such groups or the mechanism underlying their function. Based on the sequences corresponding to dehydrins available in public databases we have isolated eight different dehydrins from cDNA of Pinus pinaster. We have obtained also their genomic sequences and identified their intron/exon structure. Quantitative RT-PCR analysis of their expression pattern in needles, stems and roots during a severe and prolonged drought stress, similar to the ones trees must face in nature, is also reported. Additionally, we have identified two amino acid motifs highly conserved and repeated in Pinaceae dehydrins and absent in angiosperms, presumably related to the divergent expression profiles observed.     

Thylakoid proteome variation of Eutrema salsugineum in response to drought and salinity combined stress

It is well known that plant responses to stress involve different events occurring at different places of the cell/leaf and at different time scales in relation with the plant development. In fact, the organelles proteomes include a wide range of proteins that could include a wide range of proteins showing a considerable change in cellular functions and metabolism process. On this basis, a comparative proteomics analysis and fluorescence induction measurements were performed to investigate the photosynthetic performance and the relative thylakoid proteome variation in Eutrema salsugineum cultivated under salt stress (200 mM NaCl), water deficit stress (PEG) and combined treatment (PEG + NaCl) as a hyperosmotic stress. The obtained results showed a significant decrease of plant growth under drought stress conditions, with the appearance of some toxicity symptoms, especially in plants subjected to combined treatment. Application of salt or water stress alone showed no apparent change in the chlorophyll a fluorescence transients, primary photochemistry (fluorescence kinetics of the O-J phase), the PQ pool state (J-I phase changes), (Fv/Fm) and (Fk/Fj) ratios. However, a considerable decrease of all these parameters was observed under severe osmotic stress (PEG + NaCl). The thylakoid proteome analysis revealed 58 proteins showing a significant variation in their abundance between treatments (up or down regulation). The combined treatment (PEG + NaCl) induced a decrease in the expression of the whole PSII core subunit (D1, D2, CP43, CP47, PsbE and PsbH), whereas the OEC subunits proteins remained constant. An increase in the amount of PsaD, PsaE, PsaF, PsaH, PsaK and PsaN was detected under drought stress (PEG5%). No significant change in the accumulation of Cyt b6 and Cyt f was observed. Some regulated proteins involved in cellular redox homeostasis were detected (glutamine synthetase, phosphoglycerate kinase, transketolase), and showed a significant decrease under the combined treatment. Some oxidative stress related proteins were significantly up-regulated under salt or drought stress and could play a crucial role in the PSI photoprotection and the control of ROS production level.     

Two poplar methyl salicylate esterases display comparable biochemical properties but divergent expression patterns

Two genes encoding proteins of 98% sequence identity that are highly homologous to tobacco methyl salicylate (MeSA) esterase (SABP2) were identified and cloned from poplar. Proteins encoded by these two genes displayed specific esterase activities towards MeSA to produce salicylic acid, and are named PtSABP2-1 and PtSABP2-2, respectively. Recombinant PtSABP2-1 and PtSABP2-2 exhibited apparent Km values of 68.2 ± 3.8 μM and 24.6 ± 1 μM with MeSA, respectively. Structural modeling using the three-dimensional structure of tobacco SABP2 as a template indicated that the active sites of PtSABP2-1 and PtSABP2-2 were highly similar to that of tobacco SABP2. Under normal growing conditions, PtSABP2-1 showed the highest level of expression in leaves and PtSABP2-2 was most highly expressed in roots. In leaf tissues of poplar plants under stress conditions, the expression of PtSABP2-1 was significantly down-regulated by two stress factors, whereas the expression of PtSABP2-2 was significantly up-regulated by four stress factors. The plausible mechanisms leading to these two highly homologous MeSA esterase genes involved in divergent biological processes in poplar are discussed.     

Comparison of adenosine metabolism in leaves of several mangrove plants and a poplar species

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8-¹⁴C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia candel and Sonneratia alba, 56–92% of [8-¹⁴C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina, a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8-¹⁴C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa, only limited amounts of [8-¹⁴C]adenosine were metabolised (< ca. 30% taken up by leaf disks), but synthesis of ATP and ADP was stimulated by salt stress. In Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8-¹⁴C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.     

Molecular cloning and expression of a 2-Cys peroxiredoxin gene in the crustacean Eurypanopeus depressus induced by acute hypo-osmotic stress

Peroxiredoxins (Prxs) are a family of ubiquitous proteins that help minimize the harmful effects of oxidative stress by catalyzing the reduction of hydrogen peroxide (H₂O₂) and organic hydroperoxides to less harmful forms. A full-length cDNA corresponding to a 2-Cys Prx gene was isolated from the flatback mud crab Eurypanopeus depressus and designated as EdPrx-1 (GenBank accession no. EU684547). EdPrx-1 has a major open-reading frame of 594 bp and is capable of encoding a polypeptide of 198 amino acid residues. Like other 2-Cys Prxs, EdPrx-1 protein possesses two conserved cysteine residues that play an essential role for the antioxidant activity of the proteins. The EdPrx-1 protein, as deduced from the cDNA sequence, shows a high level (74–93%) of sequence similarity to the 2-Cys Prxs from other crustaceans as well as those from many arthropod species (73–76% similarity). It shares about 70% sequence similarity with homologs from mammalian species. EdPrx-1 gene is expressed at low level in the gill, hypodermis, and hepatopancreas tissues of the crab under non-stressed condition; however, its expression is elevated about three-fold in the gills under hypo-osmotic stress. This suggests a possible role in protecting against oxidative stress caused by the increased metabolic activities associated with hyperosmoregulation.     

Pea dehydrins: identification,characterisation and expression

An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species.The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI=6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum.A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins.B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water.During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.     

Molecular cloning and characterization of a novel gene encoding zinc finger protein from <Emphasis Type="Italic">Medicago sativa</Emphasis> L.

A suppression subtraction hybridization (SSH) cDNA library had been constructed to identify differentially expressed genes. Based on the sequence of an expressed sequence tag (EST) homologous to Pisum sativum zinc finger protein mRNA (Accession number: AF160911), the full-length cDNA of 1,676 nucleotides was cloned from alfalfa by rapid amplification of cDNA ends (RACE). It was designated as MsZFN, encoding a protein of 418 amino acids. The amino acid sequence compared by blast revealed high homology with zinc finger protein of other plants. Sequence comparison showed that there were five conserved typical zinc finger motifs, and one sugar transfer protein signature. The calculated molecular weight of the MsZFN protein was 45.8 k Da, and theoretical isoelectric point was 8.13. The MsZFN localized in nucleus. Under normal growth conditions, differential expression of MsZFN exhibited that the expression was the highest in leaf and the lowest in root. MsZFN was quickly and transiently induced by NaCl treatment and reached its maximum at 30 min.     

Sequence composition versus sequence order in the cryoprotective function of an intrinsically disordered stress‐response protein

Intrinsically disordered stress proteins have been shown to act as chaperones, protecting proteins from damage caused by stresses such as freezing and thawing. Dehydration proteins (dehydrins) are intrinsically disordered stress proteins that are found in almost all land plants. They consist of a variable number of the short, semi‐conserved, Y‐, S‐, and K‐segments, with longer stretches of poorly conserved sequences in between. Previous studies have provided conflicting views on the details of the dehydrin cryoprotective mechanism of enzymes. Experiments with polyethylene glycol (PEG) have shown that PEG cryoprotective efficiency is the same as dehydrins of the same hydrodynamic radius, suggesting that the protein's disordered and polar nature is important, rather than the specific order of the residues. To further elucidate the mechanism, we created scrambled variants of the wild grape dehydrins K₂ and YSK₂ and tested their ability to protect lactate dehydrogenase and yeast frataxin homolog‐1 from freeze/thaw damage. The results show that for preventing aggregation, it is the sequence composition and the size of the dehydrin that is the most important factor in protection, while for freeze/thaw damage causing loss of secondary structure, it is the sequence composition that is most significant.     

Cloning and characterization of calreticulin and its association with salinity stress in P. trituberculatus

Calreticulin (CRT) is a highly conserved and multifunctional endoplasmic reticulum (ER) chaperone protein and plays important roles in salinity stress response. Portunus trituberculatus is a commercially important fishery species, and water salinity conditions influence its commercial farming significantly. In order to research the function of calreticulin under salinity stress, the full-length cDNA sequence of calreticulin from P. trituberculatus (PtCRT) was firstly cloned and characterized. The complete cDNA sequence of PtCRT is 1676 bp with 1218 bp open reading frame (ORF), encoding a polypeptide of 405 amino acids. Multiple sequence alignments showed that the deduced acid amino sequences of PtCRT shared the highest homology to CRT of Fenneropenaeus chinensis (89 %). Fluorescent quantitative real-time PCR analysis indicated that PtCRT was expressed in all detected tissues and showed the highest expression level in hepatopancreas. In addition, salinity challenge significantly influenced the expression level of PtCRT in gill. Six single nucleotide polymorphisms (SNPs) were detected in cDNA sequence of PtCRT, and one SNP was associated with the salt tolerant trait. All results indicated that PtCRT plays an important role in mediating the salinity adaption of P. trituberculatus.     

Genetic manipulation of polyamine metabolism in poplar II: effects on ethylene biosynthesis

Possible competition between polyamine and ethylene metabolisms was studied in two types of transgenic poplar (Populus nigra × maximowiczii) cells: (a) constitutively expressing a mouse ornithine decarboxylase (ODC, EC 4.1.1.17) cDNA under the control of double 35S cauliflower mosaic virus (CaMV) promoter (cell line 2E), and (b) constitutively expressing a Datura S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA under the control of a single 35S CaMV promoter (line PS-18). The 2E cells contained significantly higher putrescine (Put) as well as spermidine (Spd) contents than the non-transgenic (NT) cells. The PS-18 cells contained three- to five-fold lower amounts of Put than the NT cells; their Spd content was either comparable to NT cells (at 3 d of culture) or it was higher than the NT cells (at 6 d of culture). The production of ethylene in the 2E cells was generally higher than in the NT cells throughout the 7-d culture period. Ethylene production in the PS-18 cells was comparable to NT cells. The cellular content of 1-aminocyclo-propane-1-carboxylic acid in the NT and 2E cells was quite similar, while it was slightly lower in the PS-18 cells. It is concluded that in poplar cells the cellular pool of S-adenosylmethionine is probably large enough to satisfy the demand for both polyamine and ethylene production and no competition between the two pathways is apparent.