A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions.

Low phosphorous availability, a common condition of many soils, is known to stimulate phosphatase activity in plants; however, the molecular details of this response remain mostly unknown. We purified and sequenced the N-terminal region of a phosphate starvation induced acid phosphatase (AtACP5) from Arabidopsis thaliana, and cloned its cDNA and the corresponding genomic DNA. The nucleotide sequence of the cDNA predicted that AtACP5 is synthesised as a 338 amino acid-long precursor with a signal peptide. AtACP5 was found to be related to known purple acid phosphatases, especially to mammal type 5 acid phosphatases. Other similarities with purple acid phosphatases, which contain a dinuclear metal centre, include the conservation of all residues involved in metal ligand binding and resistance to tartrate inhibition. In addition, AtACP5, like other type 5 acid phosphatases, displayed peroxidation activity. Northern hybridisation experiments, as well as in situ glucuronidase (GUS) activity assays on transgenic plants harbouring AtACP5:GUS translational fusions, showed that AtACP5 is not only responsive to phosphate starvation but also to ABA and salt stress. It is also expressed in senescent leaves and during oxidative stress induced by H2O2, but not by paraquat or salicylic acid. Given its bifunctionality, as it displays both phosphatase and peroxidation activity, we propose that AtACP5 could be involved in phosphate mobilisation and in the metabolism of reactive oxygen species in stressed or senescent parts of the plant.

Heterologous expression of a plant small heat-shock protein enhances Escherichia coli viability under heat and cold stress.

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17. 5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37 degrees C to 50 degrees C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4 degrees C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stress.

Analysis of xylem formation in pine by cDNA sequencing.

Secondary xylem (wood) formation is likely to involve some genes expressed rarely or not at all in herbaceous plants. Moreover, environmental and developmental stimuli influence secondary xylem differentiation, producing morphological and chemical changes in wood. To increase our understanding of xylem formation, and to provide material for comparative analysis of gymnosperm and angiosperm sequences, ESTs were obtained from immature xylem of loblolly pine (Pinus taeda L.). A total of 1,097 single-pass sequences were obtained from 5' ends of cDNAs made from gravistimulated tissue from bent trees. Cluster analysis detected 107 groups of similar sequences, ranging in size from 2 to 20 sequences. A total of 361 sequences fell into these groups, whereas 736 sequences were unique. About 55% of the pine EST sequences show similarity to previously described sequences in public databases. About 10% of the recognized genes encode factors involved in cell wall formation. Sequences similar to cell wall proteins, most known lignin biosynthetic enzymes, and several enzymes of carbohydrate metabolism were found. A number of putative regulatory proteins also are represented. Expression patterns of several of these genes were studied in various tissues and organs of pine. Sequencing novel genes expressed during xylem formation will provide a powerful means of identifying mechanisms controlling this important differentiation pathway.

Site-directed mutagenesis of active site residues in a class I endochitinase from chestnut seeds.

Despite the intensive research on plant chitinases, largely bolstered by their antifungal properties, little is known at present about the structure-activity relationships of these enzymes. Here we report the identification of essential active site residues in endochitinase Ch3, a class I enzyme abundant in chestnut seeds. Knowledge-based protein modeling as well as structural and sequence comparisons were performed to identify potential catalytic residues. Different mutated proteins were then generated by site-directed mutagenesis, expressed in Escherichia coli , and characterized for their chitinolytic activity. Glu124 and Glu146, the only carboxylic residues properly located into the active site cleft to participate in catalysis, were both mutated to Gln and Asp. Our results suggest that Glu124 functions as the general acid catalyst whereas Glu146 is likely to act as a general base. Other mutations involving three highly conserved active site residues, Gln173, Thr175, and Asn254, also impaired the chitinolytic activity of Ch3. The effects of these variants on the fungus Trichoderma viride revealed that catalysis is not necessary for antifungal activity. Similarly to its homologous nonenzymatic polypeptides hevein and stinging nettle lectin, the N-terminal chitin-binding domain of Ch3 appears to interfere itself with hyphal growth.

Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons.

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.

Electrophoretic analysis of seed storage proteins from gymnosperms.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pore gradient gel electrophoresis (PGGE) followed by SDS-PAGE and Western blot analysis were used to characterize the seed storage proteins from seven gymnosperm species from the families Pinaceae (Abies alba, Cedrus atlantica and Picea abies), Cupressaceae (Biota orientalis, Chamaecyparis lawsoniana and Cupressus arizonica), and Taxaceae (Taxus baccata). SDS-PAGE and PGGE X SDS-PAGE indicate the presence of proteins with characteristics similar to the 7S globulins in all the species studied. Antibodies to a 7S globulin subunit from Pinus pinaster cross-reacted with homologous polypeptides from Pinaceae species, but not with corresponding subunits from species belonging to other families. Also detected in each of the studied species, with the exception of A. alba and T. baccata, were those of C. atlantica may be extracted by saline buffer, while the remainder require a dissociating agent. Antibodies raised against the small subunit from P. pinaster 11S protein recognized only the corresponding polypeptides from Pinaceae species. Overall, these results help clarify our knowledge of gymnosperm seed storage proteins.

2S Arginine-rich proteins from Pinus pinaster seeds.

Low molecular weight globulins, which are abundant proteins in the Pinus pinaster Ait. megagametophyte, were purified and characterized. They showed a dimeric structure formed of one large and one small subunit linked by disulfide bridges. They were characterized by a high Arg and Glx content and by a relatively high Cys content. A comparison of their characteristics with those of angiosperm 2S proteins suggests that there is homology between them.

Biochemical genetics of a 7S globulin-like protein from Pinus pinaster seed.

The megagametophytes of seeds of Pinus pinaster Ait. contain two types of oligomeric globulins of approximately 175 and 190 kDa that are comprised of 47-kDa and 27- and 22-kDa, monomers, respectively, joined by weak interactions. The 27- and 22-kDa components were purified and their N-terminal sequences determined. Both polypeptides were inherited as if they were coded by a single unit of recombination. The results obtained suggest that these two polypeptides originate from a single protein that undergoes proteolytic processing. The characteristics of this P. pinaster globulin indicate that it is a member of the 7S globulin family of seed storage proteins.