Molekularbiologische und biochemische Charakterisierung pflanzlicher Hexokinasen in Tabak
Final Report Abstract
The allosteric enzyme hexokinase (HXK) is present in virtually all living organisms and catalyzes the key step in carbohydrate metabolism, the ATP-dependant phosphorylation of Glucose (Glc). Its main product, the glucose-6-phosphate (G6P), is a substrate for the oxidative pentose phosphate pathway (OPPP), the NDP-glucose pathway and glycolysis. The popularity of plant HXKs rose in the course of the discovery, that HXKs mediate sugar sensing and signaling in plant cells. HXKs acting as Glc-sensors are involved in growth promotion, plant development and probably senescence. The nuclear localization enables sugar perception and subsequent modulation of relevant gene expressions, in particular of photosynthetic genes, by interaction with other nuclear proteins. HXK genes were isolated by PCR and cloning techniques demonstrating that many plant species comprised up to ten HXK genes. The expression of multiple HXK isoforms with different kinetic attributes and subcellular localizations indicate that each individual isoform has a well-defined physiological role within the plant. The tobacco (Nicotiana tabacum) HXK gene family consists of at least 10 members. I found by quantitative real-time reverse-transcription PCR (qRT-PCR) that one isoform, NtHXK1, is expressed predominantly in all aerial organs. Furthermore, silencing of NtHXK1 produced a distinct phenotype with strong growth defects, leaf chlorosis and stunted leaf development. The determination of soluble sugars and starch in mature leaves revealed that NtHXK1 suppression generated a starch-excess phenotype, which is defined by retaining high levels of starch in photosynthetic cells after the night phase. High accumulations of the starch breakdown products maltose and Glc are responsible for the excess starch accumulation, most likely by a feedback inhibition of starch breakdown. Furtheremore, it could be demonstrated that NtHXK1 is able to replace AtHXK1 and restore wild type features, like glucose sensitivity or growth promotion, by employing complementation experiments in gin2-1(glucose insensitive; AtHXK1 null mutant). Hereby, it was confirmed that NtHXK1 functions as a plant Glc sensor and is involved in sugar sensing and signaling. Based on the observed results, it could be concluded that in leaves NtHXK1 is the major isoform amongst other HXK isoforms that primarily metabolizes Glc deriving from transitory starch breakdown in order to supply continuous sucrose synthesis and leaf respiration, thus performing the pivotal role as the connection link between the starch breakdown pathway and the glycolytic pathway. Furthermore, it can be proposed that the observed phenotypes produced by silencing of NtHXK1 are most likely the result of the losses of both functions, the catalytic and the sensing function.
