Isaac Kurek

A transgenic rice cell lineage expressing the oat arginine decarboxylase (adc) cDNA constitutively accumulates putrescine in callus and seeds but not in vegetative tissues

We introduced the oat adc cDNA into rice under the control of the constitutive maize ubiquitin 1 promoter. We studied molecularly and biochemically sixteen independent transgenic plant lines. Significant increases in mRNA levels, ADC enzyme activity and polyamines were measured in transgenic callus. These increases were not maintained in vegetative tissue or seeds in regenerated plants, with the exception of one lineage. This particular lineage showed very significant increases in putrescine preferentially in seeds (up to 10 times compared to wild type and controls transformed with the hpt selectable marker alone). We have demonstrated that in cereals such as rice, over-expression of the oat adc cDNA results in increased accumulation of polyamines at different stages of development. We have also demonstrated that strong constitutive promoters, such as the maize ubiquitin 1 promoter, are sufficient to facilitate heritable high-level polyamine accumulation in seed. Our results demonstrate that by screening adequate numbers of independently derived transgenic plants, it is possible to identify those individuals which express a desired phenotype or genotype.

Overexpression of the Wheat FK506-Binding Protein 73 (FKBP73) and the Heat-Induced Wheat FKBP77 in Transgenic Wheat Reveals Different Functions of the Two Isoforms

The FK506-binding proteins (FKBPs) belong to the peptidyl prolyl cis-trans isomerase (PPIase) family, and catalyse the rotation of the peptide bond preceding a proline. They are conserved in organisms from bacteria to man. In order to understand the function of plant FKBP isoforms, we have produced transgenic wheat plants overexpressing each of the two wheat FKBPs: wFKBP73 (which is expressed in young vegetative and reproductive tissues under normal growth conditions) and wFKBP77 (which is induced by heat stress). Transgenic lines overexpressing wFKBP77 at 25°C showed major morphological abnormalities, specifically relating to height, leaf shape, spike morphology and sterility. In these plants, the levels of hsp90 mRNA were over two fold higher than in controls, indicating a common regulatory pathway shared between wFKBP77 and Hsp90. Transgenic lines overexpressing wFKBP73 showed normal vegetative morphology, but the grain weight and composition was altered, corresponding to changes in amylase activity during seed development.

Deletion of the C-terminal 138 amino acids of the wheat FKBP73 abrogates calmodulin binding, dimerization and male fertility in transgenic rice.

Wheat FKBP73 (wFKBP73) belongs to the FK506-binding protein (FKBP) family which, in common with the cyclophilin and parvulin families, possesses peptidyl prolylcis-trans isomerase (PPIase) activity. Wheat FKBP73 has been shown to contain three FKBP12-like domains, a tetratricopeptide repeat (TPR) via which it binds heat shock protein 90 and a calmodulin-binding domain (CaMbd). In this study we investigated: (1) the contribution of the N-terminal and C-terminal moieties of wFKBP73 to its biological activity by over-expression of the prolyl isomerase domains in transgenic rice, and (2) the biochemical characteristics of the C-terminal moiety. The recombinant wFKBP73 was found to bind calmodulin via the CaMbd and to be present mainly as a dimer in solution. The dimerization was abrogated when 138 amino acids from the C-terminal half were deleted. Expression of the full-length FKBP73 produced fertile rice plants, whereas the expression of the peptidyl prolyl cis-trans isomerase domains in transgenic rice resulted in male-sterile plants. The male sterility was expressed at various stages of anther development with arrest of normal pollen development occurring after separation of the microspores from the tetrads. Although the direct cause of the dominant male sterility is not yet defined, we suggest that it is associated with a novel interaction of the prolyl isomerase domains with anther specific target proteins.

Wheat FKBP73 functions in vitro as a molecular chaperone independently of its peptidyl prolyl cis-trans isomerase activity

Abstract. Peptidyl-prolyl cis-trans isomerases (PPIases) catalyse protein folding by accelerating the slow step of cis-trans isomerisation of peptidyl-prolyl bonds. Wheat (Triticum aestivum L.) FKBP73 (wFKBP73) is a peptidyl-prolyl cis-trans isomerase belonging to the FK506-binding protein (FKBP) family. It comprises three FKBP12-like domains, tetratricopeptide repeats and a calmodulin-binding domain (CaMbd). In vitro studies indicated that wFKBP73 possesses PPIase activity, binds calmodulin and forms a heterocomplex with mammalian p23 and wheat Hsp90 in wheat-germ lysate. To further study the role of wFKBP73 we have analysed its chaperone properties. Using the thermal unfolding and aggregation of citrate synthase (CS) as a model system, we have shown that the plant wFKBP73 exhibits chaperone activity, being able to suppress CS aggregation independently of its PPIase activity. The wFKBP73 interacts transiently with non-native CS and slows down its inactivation kinetics, whereas the mammalian homologue, hFKBP52 binds tightly to CS and does not affect its rate of inactivation. Hence, the first plant FKBP shown to function as a molecular chaperone has a mode of action different from that of the mammalian FKBP52.

Studies on the expression of the wheat prolyl isomerase FKBP73 during plant development

Abstract Prolyl isomerases are ubiquitous proteins which accelerate the slow steps of protein folding by isomerisation of peptide bonds in which proline participates. We have isolated and characterised a wheat prolyl isomerase belonging to the FK506 binding protein family (FKBP) named wFKBP73. By using polyclonal antibodies raised against the recombinant wheat FKBP, its expression in wheat organs, during seed imbibition and caryopsis maturation was studied. The highest level of the wFKBP73 expression was found in imbibed embryos and 2 day old seedlings, whereas the protein could not be detected in mature green leaves. When young leaves and roots were dissected into segments, the highest expression was associated with the meristematic region. The wFKBP73 accumulated during seed maturation and was present in dry seeds. Its presence in dry seeds may suggest its importance in assisting correct folding of newly synthesized proteins and maintaining the native structure of certain macromolecules. The wFKBP73 was determined to be a stable protein with a half life of 41 h. The polyclonal antibodies raised against the recombinant protein detected cross reacting proteins in barley, corn, rice, and pea suggesting that the FKBP73 is a conserved protein in plants. The wFKBP locus Xtav1926(Fkbp) was mapped on the short arm of the wheat group seven chromosome.

Novel Wheat Molecular Chaperones Belonging to the PPIase-FKBP Family: Studies on Their Regulation and Tissue Specificity

Protein folding in vivo is mediated by an array of proteins that act either as molecular chaperones or foldases or can have both functions. The molecular chaperones were originally defined as a group of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structures (Ruddon, Bedows, 1997). They occur ubiquitously and many of them are classified as stress proteins, although they have essential functions under normal growth (Hartl, 1996; Buchner, 1996). When cells are exposed to elevated temperature or other environmental stresses, heat shock proteins belonging to several gene families are induced. Many heat shock proteins function as chaperones and play important roles in normal growth as well as in stress tolerance.

Studies on Wheat Prolyl Isomerase in Transgenic Plants

Peptidyl prolyl isomerases catalyse the interconversion between cis and trans forms of the peptide bond preceding proline residues in proteins. Three distinct families of prolyl isomerases have been identified: the cyclophilins, the FKBPs and the parvulins (Schmid 1997; Dolinski, Heitman 1997). Cyclophilins and FKBPs have attracted attention as the cellular receptors for the immunosuppresants cyclosporin A, FK506 and rapamycin, but this role in drug action is distinct from the enzymatic activity. Members of the three prolyl isomerase families are highly conserved, abundant and expressed in multiple cellular compartments, suggesting that these enzymes play a critical role in cell physiology. Because prolyl isomerases can accelerate slow protein refolding steps in vitro, it has been speculated that this is what they do in vivo. A number of proteins physically associated with different prolyl isomerases such as kinases and transcription factors have been identified but the physiological significance of these associations remains to be established. The prolyl isomerases have been implicated in various cellular functions (independent of their prolyl isomerase activity) such as DNA degradation activity and RNA maturation.