The plasma membrane and tonoplast PIP and TIP aquaporins abundance during the elongation growth of coleoptile cells of rice seedlings with different growth rates under normoxia and submergence

Coleoptiles of cereals are characterized by a rather limited time interval of development and perform a protective function. In the hydrophytic cereal rice (Oryza sativa L.), the ability of coleoptile cells to grow by elongation protects the seedling from oxygen deficiency during flooding. Sharp Cell elongation is accompanied by intense vacuolization. This process is due to increased water transport through the plasma membrane and tonoplast, which is carried out by special transporters of the aquaporin group. In the presented work, for the first time, data were obtained that made it possible to reveal a crosslink between changes in the amount of plasma membrane aquaporins of the PIP1 and TIP1 subfamilies and the intensity of cell elongation in coleoptiles of rice seedlings both under normoxia and hypoxia and to reveal the unequal functional significance of plasma membrane and tonoplast aquaporins during cell elongation. The aim of the work was to evaluate the dynamics of the content of aquaporins with predominant localization in the plasma membrane and tonoplast of coleoptile cells during elongation growth in rice under aeration and oxygen deficiency. Seeds of two rice varieties of domestic selection were used: Amethyst (slow-growing) and Kuban 3 (fast-growing). The seeds were surface sterilized and germinated (50 pcs.) under hydroponic conditions in the dark at 29 °С on glass walkways (control plants) or flooded in 750 ml jars using 4 % Knop nutrient solution. Micropreparations were made to determine the length of the cells in the central part of the coleoptiles. They were photographed using a Leica DM 2500 light microscope and a Leica DFC450 C camera (Leica Microsystems GmbH, Germany) at a magnification of 20×. The total microsomal fraction was obtained from coleoptile cells of seedlings at a temperature of 4 °С. The protein content in the samples was determined by the Bradford micromethod using the Coumassie Brilliant Blue G250 dye. The absorption of the dye adsorbed on the proteins was measured on a SPECTROstar Nano plate spectrophotometer (BMG Labtech, Germany) at λ = 595 nm. The protein concentration in the samples was determined based on a calibration graph constructed using a solution of egg albumin in 150 mM sucrose and 10 mM Tris-MES. The proteins were separated by denaturing electrophoresis in 10% polyacrylamide gel. The content of aquaporins PIP1 with predominant localization on in the plasma membrane and TIP1 with predominant localization in the tonoplast in the total microsomal fraction was estimated by immunoblot analysis using specific polyclonal antibodies. Microscopic analysis of the length of cells in the central part of coleoptiles revealed the completion of growth in the seedlings of the Kuban 3 variety on the 5th day of development under aerobic conditions. Elongation of coleoptile cells in the Ametist variety was low intense throughout the tested period. When flooded, coleoptiles of the Kuban 3 variety showed almost 2-fold elongation of cells already on the 3rd day of germination compared to the control. More intensive growth under flooding persisted until the 7th day of germination. In the Ametist variety, the differences revealed on the 3rd day were not as great as in the Kuban 3 variety, but by the 5th day the cell length increased almost 3 times. During cell elongation, the protein content in the fraction of cellular endomembranes changed. Due to a significant increase in cell size on the 5th day after germination, there was an almost 3-fold decrease in the concentration of protein in the microsomal fraction. This process continued, although not so intensively, further, by the 7th day of seedling development. A decrease in protein content was noted for both the Kuban 3 and Ametist varieties. Under oxygen deficiency (flooding), it was less pronounced. Using immunoblotting, unequal dynamics of accumulation of proteins of the PIP1 and TIP1 subfamilies aquaporins was shown both during aerobic development of rice seedlings and under oxygen deficiency. The accumulation of PIP1 aquaporins corresponded to a change in the intensity of coleoptile cell elongation. The effect was more visible in the Kuban 3 variety seedlings. However, regardless of the variety and amount of oxygen during germination, the content of vacuolar aquaporins TIP1 gradually increased. The obtained data indicate differences in the functional significance of aquaporins of the plasmalema membrane and tonoplast in the process of elongation growth of coleptile cells under normoxia and oxygen deficiency conditions.