Collection and Chemical Composition of Pure Phloem Sap from Zea mays L

Many obscure points remain to be resolved with respect to the transport of photosynthates from source to sink through sieve tubes. It is very important to be able to collect and analyze the phloem sap if an attempt is to be made to explain the phloem transport system. The insect laser technique (Kawabe et al. 1980) has been shown to be useful method for the study of this problem. The insect laser technique has been used for detailed determination of the chemical composition of phloem sap from C3 plants, such as wheat and rice (Fukumorita and Chino 1982, Hayashi and Chino 1986). Fisher and Frame (1984) collected phloem sap from Zea mays L. using an aphid technique, but its chemical composition has not been reported. The present report provides the first description of the chemical composition of pure phloem sap from maize. In addition, the concentration of nucleotides in phloem sap has been precisely determined by HPLC, and the adenylate energy charge has been calculated. The maize plants were grown under natural light conditions in a greenhouse at 25°C, with a supply of a complete nutrient solution that contained 0.75 mM NaNO3, 0.38 mu NH4NO3, 0.07 mM KH2PO4, 0.48 mM K2SO4, 0.58 mM CaCl2, 0.04 mM EDTA-NaFe, 0.50 mM H3BO3, 0.004 mM MnSO4, 0.004 mM ZnSO4, 0.001 mM CuSO4, 0.001 mM Na2MoO4, and 0.42 mM MgSO4. The solution was renewed every 3 days, and the pH was adjusted to 5.5 daily. Phloem sap was collected from the 4th or 6th leaf sheath of maize plants at the 6th to 8th leaf stage in a collecting room (25,000 lux at plant height, 25°C) by the insect laser technique (Kawabe et al. 1980). Small brown planthoppers (Loadephax striatellus Fallen) were used and the stylets of insects were cut by a YAG laser beam. The exudate was easily collected by placing microcapillaries (Drummond Scientific Co., Broomall, Penn., U.S.A.) of lfi\ or 0.5//1 capacity over the cut end of stylets with a micromanipulator. Collected phloem sap was diluted with 99/il of distilled water and stored in a freezer at —20°C until analysis. The amount of phloem sap collected from one stylet was between 0.09 and 1.86 (A. The rate of exudation of maize phloem sap was generally lower than that of rice and wheat. Anions and cations were analyzed with an ion-chromatoanalyzer (Model IC-100, Yokogawa Electric Co., Tokyo, Japan). Other compounds, such as amino acids, nucleotides and sugars, were determined by HPLC (Model L6200, Hitachi Co., Tokyo, Japan). The cations, anions and amino acid were analyzed under the same conditions as those described previously (Hayashi and Chino 1986). The conditions for the analysis of sugars by HPLC were as follows; column, Gelpack GL-C610 (Hitachi-kasei Co., Tokyo, Japan); column temperature, 60°C; mobile phase, H2O; flow rate, l.Oml/min; detector, refractive index monitor (Model 655A-30, Hitachi Ltd.). The nucleotides were determined by HPLC as described by Nieman and Clark (1984). The collection of phloem sap from maize was found to be more difficult than that from rice and wheat because the insects scarcely fed on the maize plants. All the data shown in the Tables are from the analysis of representative samples of phloem sap which were exuded from 6th leaf sheath of maize at the stage of the 8.4th leaf during the course of about 4 h under 25,000 lux at 25°C, in the collecting room. The rate of exudation of the sap was 0.07 ftl/h. Table 1 shows the concentrations of each of the solu-