Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5-flanking sequence contains heat shock elements and other potential regulatory sequences.     

Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine

A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85% homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93% homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.     

Molecular characterization and physiological role of ascorbate peroxidase from halotolerant Chlamydomonas sp. W80 strain

A cDNA clone encoding an ascorbate peroxidase was isolated from the cDNA library from halotolerant Chlamydomonas W80 by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 282 amino acids with a calculated molecular mass of 30,031 Da, preceded by the chloroplast transit peptide consisting of 37 amino acids. In fact, ascorbate peroxidase was localized in the chloroplasts of Chlamydomonas W80 cells; the activity was detected in the stromal fraction but not in the thylakoid membrane. The deduced amino acid sequence of the cDNA showed 54 and 49% homology to chloroplastic and cytosolic ascorbate peroxidase isoenzymes of spinach leaves, respectively. The enzyme from Chlamydomonas W80 cells was purified to electrophoretic homogeneity. The molecular properties of the purified enzyme were similar to those of the other algal ascorbate peroxidases rather than those of ascorbate peroxidases from higher plants. The enzyme was relatively stable in ascorbate-depleted medium compared with the chloroplastic ascorbate peroxidase isoenzymes of higher plants. The presence of NaCl (3%) as well as of beta-d-thiogalactopyranoside was needed for the expression of Chlamydomonas W80 ascorbate peroxidase in Escherichia coli.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein.

A nearly full-length cDNA clone isolated from the rat pheochromocytoma cell line, PC12, revealed extensive nucleotide sequence similarity between the rat cDNA and the Drosophila melanogaster hsp70 gene. The rat recombinant clone encodes a 71,000-dalton protein that is 70% identical with the dipteran hsp70 protein. Remarkably, a truncated segment of this cDNA clone was originally isolated by immunoreactivity with antisera raised to catecholamine-synthesizing enzymes, suggesting that this heat shock protein and these catecholamine enzymes shared antigenic determinants. The rat hsp70-related mRNA is responsible for the production of a constitutive hsp70 protein, because it is present in abundant amounts in various tissues at normal growth temperatures and is only minimally induced by hyperthermia. The rat hsp70-related sequence is part of a multigene family that extends across species to mice and humans.     

Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts.

The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.     

Nucleoside diphosphate kinase from pea chloroplasts: purification,cDNA cloning and import into chloroplasts

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.Abbreviations NDPK nucleoside diphosphate kinase - preNDPK precursor NDPK - ps-NDPK cDNA coding for Pisum sativum NDPK II We thank Dr. Schmidt, University Göttingen, Germany, for doing the protein sequencing. This work was supported in part by grants from the Deutsche Forschungsgemeinschaft.     

Purification and characterization of pea cytosolic ascorbate peroxidase

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.     

Structure and Light-Induced Expression of a Small Heat-Shock Protein Gene of Pharbitis nil

To isolate genes that are regulated by a photoperiod that promotes flowering in Pharbitis nil, a cDNA library representing mRNA of induced cotyledons was screened by differential hybridization. The DNA sequence of one cDNA clone isolated by this approach, clone 12L, showed homology to plant small heat-shock protein (hsp) genes. P. nil genomic clones hybridizing to clone 12L were isolated, and the DNA sequences of two P. nil small hsp (shsp) genes, shsp-1 and shsp-2, were determined. The derived amino acid sequences of shsp-1 and shsp-2 showed maximum homology to the 17.9-kD soybean hsp, a member of the class II cytoplasmic hsps found in plants. A study of the expression of shsp-1 and shsp-2 genes by RNase protection assay indicated that shsp-1 is induced by photoperiod, by light treatment of dark-grown P. nil seedlings, and by heat shock, and that shsp-2 is induced only by heat shock. Analysis of the sequences of the nontranscribed region indicates that both genes contain multiple heat-shock elements. The shsp-1 gene, in addition, contains sequences homologous to the GT-1-binding site, which may play a role in its light-regulated expression.     

Cloning and nucleotide sequencing of three heat shock protein genes (hsp90, hsc70, and hsp19.5) from the diamondback moth, Plutella xylostella (L.) and their expression in relation to developmental stage and temperature

Heat shock protein genes, hsp90, hsc70, and hsp19.5, were cloned and sequenced from the diamondback moth, Plutella xylostella (L.) by RT-PCR and RACE method. The cDNA sequence analysis of hsp90 and hsp19.5 revealed open reading frames (ORFs) of 2,151 and 522 bp in length, which encode proteins with calculated molecular weights of 82.4 and 19.5 kDa, respectively. Analysis of cDNA from hsc70 revealed an ORF of 1,878 bp coding a protein with a calculated molecular weight of 69.3 kDa. Furthermore, the analysis of genomic DNA from hsc70 confirmed the presence of introns while no introns were apparent in hsp90 and hsp19.5. Southern blot analysis suggested the presence of multiple copies of each gene family in the DBM genome. Detectable expression of hsp19.5 was observed at the pupal stage while expression of hsp90 and hsc70 was detected at both pupal and adult stages. At adult stage, females showed a higher expression of hsp90 and hsc70 than males. An increased expression was observed in all three genes after exposure to a high temperature in both sexes. These results suggest that in addition to a heat shock response, these HSP genes might be involved in other functions during the course of development in DBM.     

Oxaloacetate translocator in plant mitochondria

(1) Mitochondria were prepared from leaves of spinach, green and etiolated seedlings and roots of pea, potato tuber and rat liver and heart. In the case of leaf mitochondria, an improved isolation procedure resulted in high respiratory rates (460–510 nmol/mg protein per min) and good respiratory control ratio (6.8–9.8) with glycine as substrate. (2) In these mitochondria oxaloacetate transport was studied either by following the inhibitory effect of oxaloacetate on the respiration of NADH-linked substrates or by determining the consumption of [4-¹⁴C]oxaloacetate. (3) Studies of the competition by other carboxylates and effect of inhibitors on the oxaloacetate transport demonstrate that mitochondria from spinach leaves, green pea seedlings, etiolated pea seedlings and pea roots contain a specific translocator for oxaloacetate with a very high affinity to its substrate (K_m = 3–7 μM) and an even higher sensitivity to its competitive inhibitor phthalonate (K_i = 3–5 μM). The V_max values ranged from 150 to 180 nmol/mg protein per min for mitochondria from etiolated pea seedlings and pea roots and from 550 to 570 nmol/mg protein per min for mitochondria from spinach leaves and green pea seedlings. In mitochondria from potato tuber, the K_m was about one order of magnitude higher (V_max = 450 nmol/mg protein per min). In mitochondria from rat liver and rat heart, a specific translocator for oxaloacetate was not found. (4) The oxaloacetate translocator enables the functioning of a malate-oxaloacetate shuttle for the transfer of reducing equivalents across the inner mitochondrial membrane. (5) This malate-oxaloacetate shuttle appears to play a role in the photorespiratory cycle in catalyzing the transfer of reducing equivalents generated in the mitochondria during glycine oxydation to the peroxysomal compartment for the reduction of β-hydroxypyruvate. (6) Interaction between the mitochondrial and the chloroplastic malate oxaloacetate shuttles would make it possible for surplus-reducing equivalents, generated by photosynthetic electron transport, to be oxidized by mitochondrial electron transport.