ИСТИНА

Pollen grain is a 2- or 3-cellular organism specified for fertilization in plants. Two sperms or their progenitor cell are located within the large vegetative cell. Mature pollen that is released from stamen is dehydrated and dormant. After landing on a pistil of a flower, it hydrates, activates metabolism and vegetative cell forms a pollen tube, which grows with an amazing speed and brings sperms to the ovule. The first microscopic observations of pollen were described in the middle of XVII century. Early studies were focused on pollen morphology and the role of pollen grain in fertilization. In recent works spotlight is shifted towards fundamental mechanisms of cell polarization and polar growth. The least studied is the process of pollen release from the dormant state and its activation, due to the massive pollen wall, which causes significant methodological limitations. Our investigations have been focused on the role of inorganic ions in germination. Earlier it has been demonstrated that calcium and protons form an intracellular gradient with the peak concentration of calcium close to the pollen tube tip [1] and an alkaline band in subapical region [2]. The role of anions remained a mystery. We studied the role of anion channels in pollen activation and the regulation of pollen tube growth with qualitative and quantitative fluorescence microscopy. One of the most important parameters of physiological activity is plasmalemma membrane potential. We used two fluorescent dyes for detection of membrane potential shifts - slow potential-dependent dye DiBAC4(3) and fast voltage-sensitive indicator Di-4-ANEPPS. Both dyes hadn`t been previously used for plant cell studies. Along with that, to assess the distribution and movement of organelles in the cytoplasm, we used widely applied dies for mitochondria (NAO) and membrane traffic (FM 4-64). We found that during pollen activation membrane hyperpolarization of the vegetative cell plasmalemma occurs (Figure 1). Anion channel blocker NPPB caused membrane depolarization in pollen (Figure 1) and totally blocked germination, reflecting the key role of anion channels in the induction of this process [3]. A novel technique of membrane voltage mapping was applied to pollen tubes, revealing a longitudinal gradient along the pollen tube, which is maintained predominantly by NPPB-sensitive anion channels and, to a lesser extent, by H+-ATPase. In normal (growing) tubes subapical region was relatively depolarized and the shank of the tube was hyperpolarized (Figure 2); NPPB completely dissipated the gradient in both regions and arrested the tube growth [4]. An important feature of tip-growing cells is polar organelle distribution. It can be clearly seen in double-stained pollen tubes: the tip is tightly packed with membrane vesicles travelling towards apical membrane and forming a cone (Figure 3a). Subapical region contains mitochondria, moving along a reversed fountain trajectory. We found that NPPB severely disrupted this pattern, with tip-adjacent cone dissipating and mitochondria entering the apical zone (Figure 3b). Obtained results demonstrate the role of anion channels in pollen germination and a tight connection between ionic regulation of polar growth and morphological mechanisms underlying the process.