ABSTRACTA modification of the ‘cold plaque’ screening technique (Hodge et al., Plant Journal1992, 2, 257–260) was used to screen a cDNA library constructed from drought‐stressed leaf tissue of the desiccation tolerant (‘resurrection’) grass Sporobolus stapfianus. This technique allowed a large number of clones representing genes expressed at low abundance to be isolated. An examination of expression profiles revealed that several of these genes are induced in desiccation‐tolerant tissue experiencing severe drought stress. Further characterization indicated that the gene products encoded include an eIF1 protein translation initiation factor and a glycine‐ and proline‐rich protein which have not previously been associated with drought stress. In addition, genes encoding a serine/threonine phosphatase type 2C, a tonoplast‐intrinsic protein (TIP) and an early light‐inducible protein (ELIP) were isolated. A number of these genes are expressed differentially in desiccation‐tolerant and desiccation‐sensitive tissues, suggesting that they may be associated with the desiccation tolerance response of S. stapfianus. The results indicate that there may be unique gene regulation processes occurring during induction of desiccation tolerance in resurrection plants which allow different drought‐responsive genes to be selectively expressed at successive levels of water loss.

In order to identify genes involved in expression of desiccation tolerance in the foliage of the grass Sporobolus stapfianus, a cDNA library was constructed from desiccated leaf tissue of S. stapfianus. Differential screening resulted in the isolation of a number of clones which detect transcripts whose abundance alters during drought stress and the associated induction of desiccation tolerance. The characteristics of 6 of these cDNA clones are presented here. Transcripts represented by three of the cDNA clones accumulate during drying and following treatment of leaves with abscisic acid (ABA). A fourth cDNA clone detects a transcript which also accumulates following application of ABA but the transcript level fluctuates during drying. The remaining two cDNA clones are not responsive to ABA but transcript levels are present throughout the drying process. Characterisation of these cDNAs has led to the identification of the encoded proteins. Some have similarity to proteins which are known to be involved in the protection of cellular organelles and detoxification processes and they include dehydrin, LEA group 3 and thiol proteases which have been identified in other systems and shown to be induced by water stress. In addition one clone showed similarity to glyoxalase I, an enzyme involved in the removal of toxic byproducts produced during glycolysis, which has been shown to be induced by salt stress in tomato.

The rare African grass Sporobolus stapfianus is capable of surviving total air-dryness. Little is known about the genetic factors associated with this remarkable trait. Several genes have been isolated from drought-stressed leaf tissue of S. stapfianus, including genes encoding a glycine-rich protein, another with similarity to a yeast glyoxalase I gene and one cDNA which does not show any similarity with known genes. Some of the genes have not previously been linked to desiccation tolerance while some have previously been reported as being upregulated in response to drought stress or expressed throughout all stages of desiccation [Blomstedt, C., et al., Plant Growth Regulation 24, 219–228 (1998)]. To provide insight into changes in gene expression which are important in drought resistance the transcript levels in both desiccation-tolerant and desiccation-sensitive tissues, in response to varying degrees of drought stress, have been analysed. Genes whose expression decreases in leaf tissue in response to desiccation were also characterised, such as those encoding chlorophyll a/b binding protein and catalase. This study indicates the complexity of the drought stress response in the resurrection grass, S. stapfianus, which involves co-ordinated positive and negative regulation of several genes throughout the dehydration process.

Protein synthesis in vivo was studied with two-dimensional SDS-PAGE of extracts of leaves on intact drying plants of Sporobolus stapfianus (a desiccation-tolerant grass) and S. pyramidalis (a desiccation-sensitive species). Protein complements were also studied in dried, detached leaves of S. stapfianus (detached leaves are also desiccation-sensitive). Extensive changes in in vivo proteins were observed in S. stapfianus plants drying intact: 25 novel proteins, 10 proteins augmented, 13 proteins decreased and 7 proteins disappeared. Two main phases could be distinguished in leaves as plants dry. In the first phase (85-51% relative water content (RWC) range), 10 novel proteins appeared and 2 proteins increased in content. In the second phase (37-3.5% RWC), 15 novel proteins appeared and 2 proteins increased in content. Some changes in protein patterns were also observed in desiccation- sensitive leaves, i.e. in (a) S. stapfianus leaves drying detached and in (b) drying S. pyramidalis leaves attached. These changes were fewer than those in desiccation-tolerant leaves, i.e. in (c) S. stapfianus leaves drying on intact plants; that is, (a) and (b) differed from each other and from (c), which indicates that there is no common injury-related pattern of protein change. These results are consistent with the view that changes in protein complements accompany the induction of desiccation tolerance in drying plants of S. stapfianus.

AnEscherichia coli strain containing a recombinant plasmid encoding the pyruvate decarboxylase and alcohol dehydrogenase genes fromZymomonas mobilis metabolized glucose and xylose to near theoretical yields of ethanol. Enzyme activity measurements indicate high expression levels of both plasmid-encodedZymomonas proteins in the recombinantE. coli. The expression inE. coli is under the control of a promoter in theZymomonas sequence upstream of the pyruvate decarboxylase gene. The maximum ethanol level, using 4% glucose as substrate, was 1.8% (w/v) in anaerobic conditions. In aerobic conditions the natural repression ofE. coli alcohol dehydrogenase results in less ethanol production from clones expressing onlyZymomonas pyruvate decarboxylase.

Pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis purified to homogeneity by using dye-ligand and ion-exchange chromatography. Antibodies produced against the enzyme and the amino-terminal sequence obtained for the pure enzyme were used to select and confirm the identity of a genomic clone encoding the enzyme selected from a genomic library of Z. mobilis DNA cloned into pUC9. The genomic fragment encoding the enzyme expressed high levels of pyruvate decarboxylase in Escherichia coli. Possible RNA polymerase and ribosome-binding sites have been identified in the 5'-untranslated region of the pyruvate decarboxylase gene.

Pyruvate decarboxylase (EC 4.1.1.1), the penultimate enzyme in the alcoholic fermentation pathway of Zymomonas mobilis, converts pyruvate to acetaldehyde and carbon dioxide. The complete nucleotide sequence of the structural gene encoding pyruvate decarboxylase from Zymomonas mobilis has been determined. The coding region is 1704 nucleotides long and encodes a polypeptide of 567 amino acids with a calculated subunit mass of 60,790 daltons. The amino acid sequence was confirmed by comparison with the amino acid sequence of a selection of tryptic fragments of the enzyme. The amino acid composition obtained from the nucleotide sequence is in good agreement with that obtained experimentally.

The two alcohol dehydrogenases found in Zymomonas mobilis have each been purified using dye-ligand chromatography and affinity elution with nucleotides. The isoenzyme with lower electrophoretic mobility (ZADH-1) is a zinc enzyme with properties essentially similar to preparations described elsewhere. The faster isoenzyme (ZADH-2) accounted for some 90% of the ethanol-oxidizing activity in freshly prepared extracts and corresponded to the iron-activated enzyme previously described. This enzyme was inactivated by zinc; activity could only be retained during purification by including either ferrous ions or cobaltous ions in the buffers. ZADH-2 has relatively low acetaldehyde reductase activity; consequently ZADH-1 is responsible for about half of the physiological activity (acetaldehyde reduction) in Zymomonas cells. Kinetic studies showed that ZADH-2 is activated by ethanol in both reaction directions; a hypothesis for the mechanism of activation is presented. Metal ion analyses of ZADH-2 prepared in the presence of iron or cobalt indicated one atom of the relevant metal per subunit, with no significant zinc content. N-terminal sequence analyses showed that the ZADH-1 has some homology with the Bacillus stearothermophilus enzyme, whereas ZADH-2 resembles the yeast enzyme more closely.