Hagai Karchi

THE LYSINE-DEPENDENT STIMULATION OF LYSINE CATABOLISM IN TOBACCO SEED REQUIRES CALCIUM AND PROTEIN PHOSPHORYLATION

The accumulation of free lysine in tobacco seed triggers the stimulation of lysine-ketoglutarate reductase, an enzyme that acts in lysine catabolism. The mechanism of lysine-ketoglutarate reductase stimulation was studied in two different systems: (1) developing seeds of wild-type plants in which the low basal lysine-ketoglutarate reductase activity can be stimulated by the exogenous addition of lysine; and (2) developing seeds of transgenic tobacco plants expressing a bacterial dihydrodipicolinate synthase in which lysine-ketoglutarate reductase activity is stimulated by endogenous lysine overproduction. In both systems, the stimulation of lysine-ketoglutarate reductase activity was significantly reduced when treated with the Ca2+ chelator EGTA. Moreover, the inhibitory effect of EGTA was overcome by the addition of Ca2+ but not Mg2+, suggesting that the lysine-dependent activation of lysine-ketoglutarate reductase requires Ca2+. This was further confirmed by a significant stimulation of lysine-ketoglutarate reductase activity following the treatment of wild-type seeds with ionomycin (an ionophore that increases Ca2+ flow into the cytoplasm). In addition, treatment of wild-type seeds with the protein phosphatase inhibitor okadaic acid triggered a significant induction in lysine-ketoglutarate reductase activity, whereas treatment of the transgenic seeds with the protein kinase inhibitor K-252a caused a significant reduction in its activity. Thus, we conclude that the stimulation of lysine-ketoglutarate reductase activity by lysine in tobacco seed operates through an intracellular signaling cascade mediated by Ca2+ and protein phosphorylation.

Assembly and Transport of Wheat Storage Proteins

Summary Following sequestration into the endoplasmic reticulum (ER), the storage proteins of wheat ( Triticum aestivum L.) may either be retained and packaged into protein bodies (PB) inside the organelle or be exported to the Golgi complex. To unravel the signals and mechanisms regulating the assembly and sorting of these proteins within the ER, we expressed wild type and mutant forms of a γ type gliadin in Xenopus oocytes. A considerable amount of the wild type γ-gliadin was secreted via the Golgi into the medium, while still a significant proportion was retained within the ER of the oocytes where it assembled into dense PB. A deletion mutant of the γ-gliadin, encoding only the N-terminal region, which is composed of tandem repeats of a consensus PQQPFPQ sequence, was entirely retained within the oocytes, while another deletion mutant encoding only the C-terminal unique-sequence region of this protein was entirely secreted. Retention of the γ-gliadin within the ER could not be explained by rapid precipitation or assembly into insoluble deposits inasmuch as protein could diffuse rather efficiently within the organelle for several hours. In contrast, mutants of the γ-gliadin, lacking specific conserved cysteines in the C-terminal region, were entirely retained within the oocytes and were unable to diffuse within the ER. We thus hypothesize that the assembly and sorting of wheat gliadins within the ER are determined by concerted interactions between the Nand C-terminal regions of these proteins with ER resident proteins.

Molecular genetic dissection and potential manipulation of lysine metabolism in seeds

Summary Lysine is among the most important essential amino acids in the diet of human and livestock because it is presented in severely limiting amounts in cereals and other important crops. Attempts to increase lysine levels in plants were made by reducing the sensitivity of the key enzyme in lysine biosynthesis, namely dihydrodipicolinate synthase, to feedback inhibition by lysine. However, these studies showed that in plant seeds, lysine accumulation is determined not only by the rate of its synthesis, but also by the rate of its catabolism via the α-amino adipic acid pathway. Our laboratory is currently studying the regulation of lysine catabolism in plants in order to explore potentials of reducing lysine catabolic fluxes in transgenic plants. In plants, like animals, lysine is catabolized via saccharopine by two consecutive enzymes, lysine-ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH), which are linked on a single bifunctional polypeptide. Yet SDH activity of the LKR/SDH polypeptide may be limiting in vivo because of its non-physiological pH optimum of activity. In some plants, like Arabidopsis and canola, this is overcome by the presence of an additional monofunctional SDH enzyme, which is encoded by the same locus that encodes the bifunctional LKR/SDH enzyme. Results from our, and other laboratories imply that attempts to generate high-lysine crop plants should take into account a seed-specific reduction of lysine catabolism.

Regulation of Lysine Catabolism in Plants

Seeds of many crop plants are considered as a poor nutritional value food source, due to very low levels of the essential amino acid lysine. Attempts to increase lysine content in seeds are hampered by the presence of an active catabolic process that degrades lysine via saccharopine into other metabolites. The first enzyme in this catabolic process is lysine ketoglutarate reductase (LKR). We have recently obtained evidence that strongly suggest that lysine finely controls its own level in seeds by maintaining LKR activity balanced using two separate complementary mechanisms. I) LKR activity is post-translationally stimulated by lysine via a Ca+2 dependent intracellular signaling cascade that ends in the phosphorylation of the enzyme, thus, preventing lysine to accumulate to high levels. II) subsequent binding of lysine to the active site of the enzyme may expose phosphate residues on the surface of LKR rendering them more accessible to dephosphorylation, thus allowing lysine to accumulate to a sufficient level needed for protein synthesis.

T-DNA and “gain of function” tobacco mutants with altered threonine metabolism

A transferred DNA (T-DNA) tagging vector, containing four enhancers of the 35S gene promoter (Waiden, 94), was used to obtain “gain of function” tobacco mutants with altered threonine metabolism. From 150 million transferred protoplasts, 17 plants were regenerated whose growth was resistant to a high level of threonine and to its toxic analog hydroxynorvaline. The majority of these plants contained a single T-DNA insert, genetically co-segregating with the threonine resistance. The mutants consisted of two categories: threonine overproducers and threonine non-overproducers. The overproducer mutants were probably connected with regulation of threonine biosynthesis while the non-overproducers mutants may be results of altered threonine sequestration.