36003:

Maize (Zea mays L.) endosperm slices may be used for in vivo studies of starch biosynthesis.

Authors:	Huang, Cindy Xin(A)Liu, Kang-Chien(A)Shannon, Jack, C.(A)
Affiliations:	(A): The Pennsylvania State Univ., Horticulture Dept.
Presenter:	Shannon, Jack C., jshannon@psu.edu

We recently reported that ADP-Glc synthesized in the cytosol of maize endosperm cells is transported into amyloplasts by a membrane specific translocator, BT1, where it is metabolized to starch (Pl Phy 117: 1235). We also reported that maize endosperm slices are permeable to sugars (Pl Phy 94: 996). We hypothesized that if cells of maize endosperm slices are permeable to ADP-Glc then they may be used for semi-in vivo starch biosynthesis studies. We now report that maize endosperm slices take up [14C]ADP-Glc and rapidly convert it into starch. [14C]ADP-Glc conversion to starch was linear for 120 min when the endosperm slices were incubated in a medium containing 3 mM ADP-Glc, but ADP-Glc was limiting at lower concentrations. An incubation solution containing 20 mM Bicine, pH 8.5, 2 mM EDTA, 2 mM MgCl2, 2 mM GSH, 10 mM KC2H3O2 and 3 mM [14C]ADP-Glc (specific activity of 0.333 mCi/mmol) was selected for subsequent studies. Amyloplast membranes of the brittle-1 (bt1) maize mutant are deficient in the adenylate transporter. To access amyloplast membrane intactness we compared [14C]starch synthesis in bt1 slices with those prepared from wx. Endosperm slices from bt1 were only 39% as active in starch synthesis as slices from wx. FSBA, an adenosine analog, reduced uptake and conversion of ADP-Glc to starch in wx slices. These studies provide evidence that the plasma membrane is permeable to the applied ADP-Glc but that most of the amyloplast membranes are intact, and a functional adenylate translocator (BT1) is required for the transport of ADP-Glc into the amyloplasts. After incubation of wx endosperm slices in medium containing 3 mM [14C]ADP-Glc for 5, 15 and 30 min, 15%, 33% and 49% of the extractable radioactivity was recovered in starch with 39%, 31% and 20% still in ADP-Glc, respectively. There was little metabolism of ADP-Glc to Glc, Fru and Suc, but in slices incubated for 5 min, 20% of the radioactivity was recovered in a component of the alcohol soluble sugar extract which moved to a position between the origin and sucrose during paper chromatography and 18% was in the 10% ethanol soluble (WSP) fraction. Radioactivity in these fractions declined with incubation as that in starch increased. The identity of these rapidly labeled components will be determined and their relationship to the starch biosynthetic pathway discussed. We suggest that maize endosperm slices are a useful semi-in vivo system for studying the early events of starch biosynthesis.