Avihai Perl

Enhanced oxidative-stress defense in transgenic potato expressing tomato Cu,Zn superoxide dismutases.

SummaryThe two cDNAs coding for the cytosolic (cyt) and the chloroplast-located (chl) Cu,Zn superoxide dismutases (SODs) of tomato (Perl-Treves et al. 1988) were cloned into respective binary vectors and mobilized into Agrobacterium strains. Potato tuber discs were infected with either of the two agrobacterial strains and cultured on selective medium containing kanaymcin. The integration of either of the cyt or the chl SOD transgenes was verified by Southern-blot hybridization. The enzymatic activity of the additional tomato chl Cu,Zn SOD could be distinguished from endogenous SOD activity since the latter isozyme migrated faster on SOD-activity gels. Several transgenic potato lines harboring either the cyt or the chl SOD genes of tomato showed elevated tolerance to the superoxide-generating herbicide paraquat (methyl viologen). After exposure of shoots to paraquat, tolerance was recorded either by scoring symptoms visually or by measurements of photosynthesis using the photoacoustic method. Root cultures from transgenic lines that harbored the additional cyt Cu,Zn SOD gene of tomato were tolerant to methyl viologen up to 10-5 M; a lower tolerance was recorded in roots of transgenic lines that expressed the additional chl Cu,Zn SOD of tomato.

Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures.

SummaryGenetic transformation of cereals by direct DNA delivery via microprojectile bombardment has become an established procedure in recent years. But the derivation of functional transgenic plants, especially in wheat, is still problematic, mainly due to low efficiency of DNA delivery and the reduced regeneration capability of microprojectile-bombarded tissue. We focussed on these two aspects and found that the regeneration of scutellar calli of wheat can be rendered highly efficient and considerably accelerated by a liquid culture phase in screen rafts. We also found that the expression of a reporter gene following DNA delivery by microprojectile can be improved by maintaining the scutellar calli in 0.25 M mannitol before and after bombardment, by bombardment in the presence of silver thiosulfate and Ca(NO3)2 (rather than CaCl2) and by the elimination of spermidine from the DNA/microprojectile mixture. A protocol that includes all these features leads to several-fold higher transient expression of the reporter gene than have previously published procedures.

Regulation of the leucoanthocyanidin dioxygenase gene expression in Vitis vinifera

Abstract The control of expression of the leucoanthocyanidin dioxygenase (LDOX) gene was studied in grape. LDOX converts leucoanthocyanidins to anthocyanidins in the anthocyanins pathway. In the present study, the promoter of the ldox gene was cloned and fused to the uidA reporter gene, and the expression of this fusion was analyzed in transformed plants and in transformed red fruit cell suspensions. The ldox promoter-uidA gene fusion was expressed in all the plant organs. White light, calcium and sucrose induced the ldox gene expression. The induction by both light and calcium under light suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the ldox gene. The induction of the ldox gene by light and by sucrose under light indicates a close interaction between the sucrose and the light signaling pathways. Analysis of the ldox promoter sequence revealed the existence of several putative DNA binding motifs.

Ethylene and in vitro culture of potato: suppression of ethylene generation vastly improves protoplast yield, plating efficiency and transient expression of an alien gene

Ethylene release by potato shoots cultured in closed boxes was suppressed by the addition of silver thiosulfate to the culture medium. Shoots cultured in the presence of silver thiosulfate produced appreciably more tissue and the yield of protoplasts per unit tissue mass was vastly increased, resulting in an 8 fold increase of protoplast yield per shoot. Exposure of pricked leaves to macerating enzymes facilitated ethylene generation. Leaves of shoots which were previously cultured in silver thiosulfate containing medium generated much less ethylene than leaves from control shoots and this generation could be further reduced by the addition of acetylsalicylic acid during maceration. The capability of polyethylene glycol treated potato protoplasts to produce microcalli was vastly increased by the addition of silver thiosulfate during exposure of protoplasts to Ca(NO3)2 following the polyethylene glycol treatment. Similarly, when a plasmid (pCAP212) containing an expressible gene for chloramphenicol acetyltransferase was introduced into potato protoplasts through a polyethylene glycol treatment, the transient expression of acetyltransferase was very much increased by the addition of a short incubation of the protoplasts with silver thiosulfate.

Tissue-Specific Expression of the Tobacco Mosaic Virus Movement Protein in Transgenic Potato Plants Alters Plasmodesmal Function and Carbohydrate Partitioning

Transgenic potato (Solanum tuberosum) plants expressing the movement protein (MP) of tobacco mosaic virus (TMV) under the control of the promoters from the class I patatin gene (B33) or the nuclear photosynthesis gene (ST-LS1) were employed to further explore the mode by which this viral protein interacts with cellular metabolism to change carbohydrate allocation. Dye-coupling experiments established that expression of the TMV-MP alters plasmodesmal function in both potato leaves and tubers when expressed in the respective tissues. However, whereas the size-exclusion limit of mesophyll plasmodesmata was increased to a value greater than 9.4 kD, this size limit was smaller for plasmodesmata interconnecting tuber parenchyma cells. Starch and sugars accumulated in potato leaves to significantly lower levels in plants expressing the TMV-MP under the ST-LS1 promoter, and rate of sucrose efflux from petioles of the latter was higher compared to controls. It is interesting that this effect was expressed only in mature plants after tuber initiation. No effect on carbohydrate levels was found in plants expressing this protein under the B33 promoter. These results are discussed in terms of the mode by which the TMV-MP exerts its influence over carbon metabolism and photoassimilate translocation, and the possible role of plasmodesmal function in controlling these processes.

In vitro tuberization in transgenic potatoes harboring ß-glucoronidase linked to a patatin promoter: effects of sucrose levels and photoperiods

Abstract Sections of axenic potato shoots containing one leaf were found to synchronically produce a microtuber in their single leaf axil 7 days after transfer to inductive medium containing 8% sucrose, kinetin and ancymidol (a gibberellin-biosynthesis inhibitor). Shoots of transgenic Solanum tuberosum harboring a chimeric gene containing a patatin promoter sequence followed by the gene coding for s-glucuronidase as a reporter protein, were utilized. Microtubers were initiated in inductive medium under either long-day (LD) or short-day (SD) photoperiods. The minimal duration of culture in 8% (w/v) sucrose (followed by culture in 2% (w/v) sucrose) is 1 or 3 days, when the shoots were subsequently maintained (for 6 or 4 days) in LD or SD, respectively. Changes in patatin transcription were evaluated by following s-glucuronidase activity. Strong increase in s-glucuronidase activity was observed in tuber initials 5 days after transfer to tuber-induction medium. This increase paralleled the accumulation of radioactivity in the tuber initials of shoots cultured in radioactive sucrose. s-Glucuronidase activity analyses at various periods after induction and in different tissues of the in vitro cultured shoot-sections as well as the elimination of one of the obligatory induction conditions, suggested that the requirement for high sucrose levels does not represent an energy demand but rather an induction signal. Day-length has apparently a decisive role, after exposure to high-levels of sugar, in maintaining the tuberization signal.

Lysine and threonine metabolism are subject to complex patterns of regulation in Arabidopsis.

To study the regulation of lysine and threonine metabolism in plants, we have transformed Arabidopsis thaliana with chimeric genes encoding the two bacterial enzymes dihydrodipicolinate synthase (DHPS) and aspartate kinase (AK). These bacterial enzymes are much less sensitive to feedback inhibition by lysine and threonine than their plant counterparts. Transgenic plants expressing the bacterial DHPS overproduced lysine, but lysine levels were quite variable within and between transgenic genotypes and there was no direct correlation between the levels of free lysine and the activity of DHPS. The most lysine-overproducing plants also exhibited abnormal phenotypes. However, these phenotypes were detected only at early stages of plant growth, while at later stages, new buds emerged that looked completely normal and set seeds. Wild-type plants exhibited relatively high levels of free threonine, suggesting that in Arabidopsis AK regulation may be more relaxed than in other plants. This was also supported by the fact that expression of the bacterial AK did not cause any dramatic elevation in this amino acid. Yet, the relaxed regulation of threonine synthesis in Arabidopsis was not simply due to a reduced sensitivity of the endogenous AK to feedback inhibition by lysine and threonine because growth of wild-type plants, but not of transgenic plants expressing the bacterial AK, was arrested in media containing these two amino acids. The present results, combined with previous studies from our laboratory, suggest that the regulation of lysine and threonine metabolism is highly variable among plant species and is subject to complex biochemical, physiological and environmental controls. The suitability of these transgenic Arabidopsis plants for molecular and genetic dissection of lysine and threonine metabolism is also discussed.

Regulation of lysine synthesis in transgenic potato plants expressing a bacterial dihydrodipicolinate synthase in their chloroplasts

The essential amino acid lysine is synthesized in higher plants by a complex pathway that is predominantly regulated by feedback inhibition of two enzymes, namely aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS). Although DHPS is thought to play a major role in this regulation, the relative importance of AK is not known. In order to study this regulation, we have expressed in the chloroplasts of transgenic potato plants a DHPS derived from Escherichia coli at a level 50-fold above the endogenous DHPS. The bacterial enzyme is much less sensitive to lysine inhibition than its potato counterpart. DHPS activity in leaves, roots and tubers of the transgenic plants was considerably higher and more resistant to lysine inhibition than in control untransformed plants. Furthermore, this activity was accompanied by a significant increase in level of free lysine in all three tissues. Yet, the extent of lysine overproduction in potato leaves was significantly lower than that previously reported in leaves of transgenic plants expressing the same bacterial enzyme, suggesting that in potato, AK may also play a major regulatory role in lysine biosynthesis. Indeed, the elevated level of free lysine in the transgenic potato plants was shown to inhibit the lysine-sensitive AK activity in vivo. Our results support previous reports showing that DHPS is the major rate-limiting enzyme for lysine synthesis in higher plants, but they suggest that additional plant-specific regulatory factors are also involved.

Establishment of long-term embryogenic cultures of seedless Vitis vinifera cultivars — a synergistic effect of auxins and the role of abscisic acid

Abstract A regeneration protocol, that will enable a transfer from a differentiated stage to a dedifferentiated stage and vice versa, is highly important for the development of genetic transformation systems for grape. Somatic embryogenesis and subsequent diploid plants have been obtained from anthers of Vitis vinifera commercial seedless cultivars. So far these cultivars were considered as recalcitrant to embryogenesis and long-term maintenance of their embryogenic cell lines was never reported. Anthers produce embryogenic calli when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Embryos were formed and maintained upon transfer to a MS medium supplemented with indole-3-aspartic acid and 2-naphthoxyacetic acid. A synergistic effect was observed while combining these two auxins. Recallusing was achieved when a single embryo was transferred to MS medium supplemented with indole-3-aspartic acid and 2,4-dichlorophenoxyacetic acid. Abscisic acid was found to play a major role in the long-term maintenance of this callus. Germination and plantlet formation was characterized by a high frequency of abnormal vitrified plants. Conversion into morphological normal plants was achieved by rooting the abnormal plantlets on MS medium supplemented with α-naphthalenacetic acid.

Nuclear-organelle interaction in Solanum : interspecific cybridizations and their correlation with a plastome dendrogram

SummaryAlloplasmic compatibility, namely the functional interaction between the nuclear genome of a given species with plastomes and chondriomes of alien species, is of considerable relevance in plant biology. The genus Solanum encompasses a wide spectrum of species and is therefore suitable for a study of this compatibility. We thus chose the nuclear genome of Solanum tuberosum (potato) and organelles (chloroplast and mitochondria) from 14 other Solanum species to initiate an investigation of intrageneric nucleus/organelle interactions. An assessment of the diversity of the chloroplast DNAs from these 15 species resulted in the construction of a plastome dendrogram (phylogenetic tree). In parallel we extended a previous study and performed ten additional fusion combinations by the “donor-recipient protoplast fusion” procedure, using potato protoplasts as recipients and protoplasts from any of ten other Solanum species as donors. We found that two fusion combinations did not yield cybrids and that the chloroplasts of S. polyadenium and the mitochondria (or mitochondrial components) from S. tarijense could not be transferred to cybrids bearing potato nuclei. In general, there is a correlation, albeit not perfect, between the cybridization data and the plastome dendrogram. These results furnish valuable information toward future transfer of plasmoneencoded breeding traits from wild Solanum species into potato. This information should also be useful for the planning of asymmetric protoplast fusion between potato and wild accessions for the improvement of pathogen and stress resistance of potato cultivars.