High purity preparations of higher plant vacuolar H+-ATPase reveal additional subunits. Revised subunit composition

A fast protein liquid chromatography procedure for purification of the V-type H+-ATPase from higher plant vacuolar membrane to yield near-homogeneous enzyme with a specific activity of 20-25 mumol/mg.min is described. When precautions are taken to ensure the quantitative recovery of protein before sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the preparation is found to be constituted of seven major polypeptides of 100, 67, 55, 52, 44, 32, and 16 kDa, respectively, and two minor components of 42 and 29 kDa. The 52-, 44-, and 32-kDa polypeptides do not cross-react with antisera raised to the 67- and 55-kDa subunits of the enzyme, and two independent sample preparation procedures yield the same apparent subunit composition. The additional polypeptides are not breakdown products or aggregates of the previously identified subunits of the ATPase. The ATPase of tonoplast vesicles is subject to MgATP-dependent cold inactivation, and the conditions for inactivation are identical to those for the bovine chromaffin granule H+-ATPase (Moriyama, Y., and Nelson, N. (1989) J. Biol. Chem. 264, 3577-3582). Cold inactivation is accompanied by the detachment of five major polypeptides of 67, 55, 52, 44, and 32 kDa from the membrane, and all five components co-migrate with the corresponding polypeptides of the purified ATPase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 100- and 16-kDa polypeptides of the ATPase are not removed from the membrane during cold inactivation, but the latter can be purified to homogeneity by chloroform:methanol extraction of the fast protein liquid chromatography-purified enzyme. It is concluded that the tonoplast H+-ATPase is constituted of 6-7 major polypeptides organized into a peripheral sector comprising the 67-, 55-, 52-, 44-, and 32-kDa components and an integral sector consisting of the 100- and 16-kDa polypeptides. The V-type H+-ATPase from animal endomembranes and higher plant vacuolar membranes therefore have remarkably similar subunit compositions and gross topographies.     

TaER Expression Is Associated with Transpiration Efficiency Traits and Yield in Bread Wheat

ERECTA encodes a receptor-like kinase and is proposed as a candidate for determining transpiration efficiency of plants. Two genes homologous to ERECTA in Arabidopsis were identified on chromosomes 6 (TaER2) and 7 (TaER1) of bread wheat (Triticum aestivum L.), with copies of each gene on the A, B and D genomes of wheat. Similar expression patterns were observed for TaER1 and TaER2 with relatively higher expression of TaER1 in flag leaves of wheat at heading (Z55) and grain-filling (Z73) stages. Significant variations were found in the expression levels of both TaER1 and TaER2 in the flag leaves at both growth stages among 48 diverse bread wheat varieties. Based on the expression of TaER1 and TaER2, the 48 wheat varieties could be classified into three groups having high (5 varieties), medium (27 varieties) and low (16 varieties) levels of TaER expression. Significant differences were also observed between the three groups varying for TaER expression for several transpiration efficiency (TE)- related traits, including stomatal density (SD), transpiration rate, photosynthetic rate (A), instant water use efficiency (WUEi) and carbon isotope discrimination (CID), and yield traits of biomass production plant^-1 (BYPP) and grain yield plant^-1 (GYPP). Correlation analysis revealed that the expression of TaER1 and TaER2 at the two growth stages was significantly and negatively associated with SD (P<0.01), transpiration rate (P<0.05) and CID (P<0.01), while significantly and positively correlated with flag leaf area (FLA, P<0.01), A (P<0.05), WUEi (P<0.05), BYPP (P<0.01) and GYPP (P<0.01), with stronger correlations for TaER1 than TaER2 and at grain-filling stage than at heading stage. These combined results suggested that TaER involved in development of transpiration efficiency -related traits and yield in bread wheat, implying a function for TaER in regulating leaf development of bread wheat and contributing to expression of these traits. Moreover, the results indicate that TaER could be exploitable for manipulating important agronomical traits in wheat improvement.     

THE DISTRIBUTION AND DIFFERENTIATION OF LYMPH-BORNE IMMUNOBLASTS AFTER INTRAVENOUS INJECTION INTO SYNGENEIC RECIPIENTS

Thoracic duct lymph from inbred, hooded rats was collected 3–5 days after antigenic stimulation of the caudal lymph nodes. During this period the lymph contained 10–15% of large, basophilic lymphoid blast cells (immunoblasts). By incubating the lymph cells at 38.C with radioactive DNA precursors, either ³H-thymidine or ¹²⁵I-deoxyuridine, the immunoblasts became labelled but the small lymphocytes did not. The lymph cells were then washed and injected intravenously into syngeneic recipients which were killed after various intervals up to 24 hr so that the radioactivity of their organs could be assayed by scintillation counting and autoradiography.
The main finding was that in animals killed after 4 or more hours the small gut always contained most of the recoverable activity and autoradiographs showed that this was because the injected cells had infiltrated the lamina propria in large numbers. Earlier, many of the injected cells were retained temporarily in the lungs, liver and spleen but many of them soon left those organs and entered the lamina propria of the small gut.
An electron microscope study of autoradiographs showed that 24 hr after injection the cells which entered the lamina propria of the gut had differentiated into plasma cells so that they displayed abundant, lamellar endoplasmic reticulum.