A heterotrofic streptomycin insensitive cell line of bean (Fhaseolus vulgaris) cell suspension cultures was selected. Growth of bean cells in liquid culture occurred m the presence of extracellular sucrose, glucose or fructose. For all sub- sequent metabolic studies, bean cells were cultured on a medium containing sucrose as only carbohydrate source. Exogenous su- crose was rapidly hydrolyzed to stoichiometric amounts of hexose. Glucose was taken up preferentially over fructose, which was not significantly metabolized until all glucose was removed from the medium. Apparantly the stationary phase is reached when the carbohydrate in the culture medium is depleted.
Sucrose, glucose and fructose pools are labelled in vivo from h of incubation in labelled sucrose and glucose, only 3.4% and 10.5% of the total” label ‘taken up, respectively remained in the free sugars sucrose, glucose and fructose. Whereas 60% and 49% of the total radioactivity taken up were released as carbon dioxide from labelled sucrose and glucose, respectively.
Unlabelled glucose inhibited uptake from labelled sucrose and unlabelled sucrose inhibited uptake from labelled glucose.
p-chloromercuribenzenesulfonic acid inhibited sucrose uptake by 80%, whereas hexose accumulation was unaffected.
Activities of key enzymes involved in sucrose metabolism i.e. sucrose synthase, invertase, ATP-dependent phosphofructokinse (PFK), PPi-dependent i->hosphof ructokinase (PFP) and uridine 5′- diphosphate glucose pyrophosphorylase (UDPG PPase), were pre- sent at all physiological growth phases in bean cell suspension cultures. Invertase activity was selectively inhibited, whereas sucrose synthase and PFP were selectively induced, from the dormant/latent period to the active growing period of cell growth in liquid medium. UDPG PPase and PFK remained es- sentially unchanged throughout the growth cycle. High activities of UDPG PPase were detected.
Only sucrose synthase increased when additional sucrose was added extracellularly in the mid-logarithmic phase of growth.
Sucrose, glucose and fructose levels of bean cells in the mid- logarithmic phase of growth decreased by 96%, 77% and 75%, respectively, when starved for 38h. However, activity of PFP, PFK, UDPG PPase, sucrose synthase, acid and alkaline invertase remained essentially unchanged during the first 24h of starvation. When cells were starved for 14h and then transferred to a sucrose containing medium, no intracellular increase was observed for sucrose, glucose or fructose within the subsequent 24h.
Cells in the mid-logarithmic phase of growth before exposure to water stress were less sensitive to polyethelene glycol-induced water stress, than cells in the lag phase of growth.
Activity of sucrose synthase and UDPG PPase, was selectively inhibited by water stress in lag phase cells and mid-logarithmic phase cells exposed to water stress. However, invertase ac- tivity was selectively induced from the latent/dormant period to the active growing phase, and remained essentially unchanged in mid-logarithmic phase cells subjected to water stress.
This is the first report of the levels of some of the enzymes involved in glycolysis and sucrose degradation in bean cells at different phases of growth in liquid medium. Furthermore, it is the first report of the effect of water stress on activity of key enzymes involved in sucrose degradation.
|Degree Type||Masters degree|