Intracellular reorganization and ionic signaling of the Phycomyces stage I sporangiophore in response to gravity and touch.

Unicellular zygomycete fungus Phycomyces blakesleeanus exhibits a typical apical growth that depends on a complex interaction of different physiological processes. Sensitivity to the light, wind, barriers, touch and gravity of Phycomyces sporangiophore implicate the existence of the cross-talk between different signaling pathways in the same cell. Touch and gravity in Phycomyces sporangiophore seem to share some common elements of Ca(2+) and H(+) ion-based signal transduction systems. Apoplastic Ca(2+) and H(+) ionic fluxes are important for establishing cell polarity in tip growing sporangiophore both in vertical and tilted position. Upon gravistimulation sporangiophores display asymmetric ionic distribution and intracellular reorganization leading to change in the growth pattern.

Intracellular Reorganization upon Gravistimulation

Although all living organisms on the Earth are continuously exposed to physical signal of the gravity, cellular mechanism of the gravitropism is far from being clear. Unicellular tip-growing cells are excellent experimental systems for dissecting the mechanisms of gravitropic signaling because cell extension, gravity sensing and gravity response are all placed in the apical zone of the single cell. Phycomyces sporangiophore is a suitable fungal model cell for investigation of the gravitropic signaling. As this vertically growing unbranched hypha is able to respond to light, wind, barriers, touch and gravity, it is reasonable to assume that all these responses must be highly integrated to provide an appropriate growth response. Phycomyces sporangiophores grow in the air and display negative gravitropic bending upon displacement from vertical position., Buoyant lipid globules in subapical region and sedimenting octahedral crystals as statoliths in central vacuole play an essential role in gravitropism in immature sporangiophore cell (stage I).- It has been earlier reported that density differences of the vacuoles and the cytoplasm may play a role for gravisensing of sporangiophores. In this work we have observed the spatial relationship between the vacuole and the protoplasm of a living sporangiophore and revealed intracellular reorganization of sporangiophore upon vertical displacement. Sporangiophores responded to gravistimulation by bending growth, gradual dissolving lipid globules and sedimentation of octahedral crystals from central vacuole (Figs. 1 and 2). Bending growth is a very slow process which is accompanied by appearance of intensive vacuolization in the apical and subapical region of sporangiophores that could be associated with gravitropic response and/or with regular developmental processes (Fig. 1). Sporangiophores kept in vertical position showed gradual moving central vacuole to apical part of sporangiophore, but without significant vacuolization. These small vacuoles might be involved in preparation of some material for the next developmental stage and/or the site where the octahedral crystals are formed. Moreover, we have observed “Spitzenkörper like body” in the apical zone of Phycomyces sporangiophore, as previously also detected in apical region of Phycomyces sporangiophores (Fig. 2). The appearance of accumulation of wall vesicles in the form of an organized apical body or “Spitzenkörper” just behind the apex is characteristic for almost all tip growing cells like hyphae of septate fungi,, pollen tubes,, root hairs and algal rhizoids. Although the exact role of these high-organized apical vesicles is still unclear, it might be speculated that they play an important role in directing growth and maintaining cell polarity. Developmental processes of sporangiophore could interfere with gravity-triggered processes, so it is still unclear whether these processes are related or taking place in parallel. Thus, developmental changes would incidentally coincide with gravitropic response. Further ultrastructural investigation would be required in order to clarify whether the appearance of intensive vacuolization would be associated with gravisensing mechanisms.

Figure 1. Original images of an intact wild-type strain Phycomyces sporangiophore (stage I) taken during measurements of net ion fluxes at different positions of Ca2+ and H+ ion-selective microelectrode near the surface of the cell kept in vertical orientation for 24 min and subsequently tilted by 45° for 2 h 25 min. The numbers in left corner of images denote the real time when the images were taken. Apical part of sporangiophore displayed bending growth in tilted position. The appearance of intensive vacuolization in apical and subapical regions of sporangiophore was observed after 1h of gravistimulation. CV, central vacuole; LGs, -lipid globules; Vs, vacuoles. Bar marker = 50 μm.

Figure 2. Original images of an intact wild-type strain Phycomyces sporangiophore (stage I) kept in horizontal position (3.44 p.m.−10.28 p.m.). The gradual intracellular reorganization was observed upon displacement from vertical position: the position of lipid globules was changed, intensive vacuolization in apical and subapical regions, and “Spitzenkörper like body” (vesicle clusters) in the apical zone of the sporangiophore (arrow) appeared. The numbers in right corner of images denote the real time when the images were taken. CV, central vacuole; LGs, lipid globules; Vs, vacuoles. Bar marker = 100 μm.

Ionic Signaling in Response to Touch and Gravity

The thigmo responses are found over the entire range of the plant kingdom, but there is still unclear how the thigmo signal is transmitted to the interior of the cell and what the components are included in this signal transduction chain. The sensitivity of Phycomyces sporangiophores to touch as mechanical stimulus is additional adaptive mechanism for redirecting growth due to obstacle. Phycomyces sporangiophores responded to touch stimulus as other filamentous fungi where hyphal growing points usually serve as sensors. The application of touch stimulus on Phycomyces sporangiophore caused transient influxes of Ca2+ and H+ ions into the cell which appear likely to be important for avoiding obstacle by transiently increasing growth rate by inward-directed ion fluxes. (Fig. 3). These transient apoplastic ionic changes along sporangiophores implicate that thigmo signals are probably received at the cell wall and transmitted through the plasma membrane via opening stretch-activated Ca2+ channels. Ionic signaling has been proposed to transduce both touch and gravity perception in plants., The presence of stretch-activated channels as well as calcium-induced calcium release channels in plants suggests the possibility that mechano-perturbations cause increases in intracellular Ca2+ due to gating of these channels.- For highly polarized cells, such as root hairs and pollen tubes, control of expansion-related mechanical stresses seems to be a fundamental aspect of growth regulation.- The touch may result in perturbation of connections among the cell wall, plasma membrane and the cytoskeleton, thus conducting the extracellular stimulus into an intracellular signal. Touch and gravity in Phycomyces sporangiophore seem to share some common elements of Ca2+ and H+ ion-based signal transduction systems, as previously also reported for the plants., It appears likely that in Phycomyces sporangiophores Ca2+ channels detected in plasma membrane of growing zone are also involved in mechanosensing mechanisms. Although increase of cytosolic Ca2+ via Ca2+ channels in the plasma membrane in response to touch is well demonstrated,, the molecular basis for perception of a mechanical stimulus remains to be identified.

Figure 3. Representative records of touch-induced transient changes of net Ca2 and +H+ ion fluxes (outward-efflux as positive, and inward-influx as negative values) recorded from intact Phycomyces sporangiophores. Thigmo stimulus caused transient alkalization at the surface of sporangiophore and influxes of both ions measured.