Extracellular ATP Signaling is Linked to Endocytic Vesicle Recycling in Root Apex

Abstract

Plants are sessile organisms, the roots of which exudate a large number of chemical compounds into the rhizosphere that contain several chemicals and microbes. It is known that at the root apex, the transition zone is located between the apical meristem and basal elongation zone. The transition zone plays a role as an important environmental sensor and controller of the motoric outputs. The high endocytic vesicle recycling found in this root apex region is essential to translocate PIN proteins (PINs) that, presumed auxin efflux carriers. PINs are reported to be major players for many root tropic growth responses, including root gravitropism and phototropism. Therefore, investigations of the transition zone may provide a comprehensive understanding of plants, and especially their adaptation to the sessile life. The aim of this thesis is to investigate the dynamics and activities of endocytic vesicle recycling in the root apex transition zone in response to such environmental stimuli as light, root cultivation medium components (MES), and solvents (DMSO and ethanol) <i>via</i> extracellular (eATP).<br /><b>Chapter 1</b> describes the activity of endocytic vesicle recycling and PIN2 localization in root cells grown at different durations of light exposure. In this study, dark-grown seedlings showed lower rates of endocytic recycling activities in cells of root apex transition zones, compared to the light-grown roots. Interestingly, light-promoted endocytic recycling activity was attenuated to a level equivalent to dark-grown roots by an additional 24 hours of dark treatment. PIN2-GFP was shown to accumulate in vacuoles both in dark-grown and 24-hour dark treatment seedlings. Moreover, the PIN2-GFP signal found in 24-hour dark-treated roots was stronger than in the dark-grown sample. Here, I am proposing a model for dynamic regulation of PIN2 localization regulated by endocytic vesicle recycling in the transition zone according to light circumstances, which might be important for roots to prepare for upcoming unfavorable light.<br /><b>Chapter 2</b> describes the DMSO and EtOH impacts in <i>Arabidopsis</i> root on endocytic vesicle recycling and cellular F-actin polarities. These are closely related to membrane conditions. In this study, DMSO and EtOH showed growth inhibition, interrupted endocytic vesicle recycling and disturbance of F-actin polarization in<i>Arabidopsis</i> root. Distortion of the plasma membrane shape was shown in plasmolyzed root epidermal cells in the presence of these chemicals. These results suggest that both DMSO and EtOH, in the range known as experimentally effective concentrations, may modify plasma membrane properties, thereby affecting endocytic vesicle recycling and cellular polarity in living cells.<br /><b>Chapter 3</b> describes the effects of different concentrations of the MES buffer using growing root apices of <i>Arabidopsis</i>. The results show that 1% of MES significantly inhibits root growth, the number of root hairs and the length of the meristem, whereas 0.1% promotes root growth in the root apex area (region spanning from the root tip up to the transition zone). Furthermore, superoxide generation in the root apex disappeared at 1% of MES. These results suggest that MES disturbs normal root morphogenesis by changing the reactive oxygen species’ (ROS) homeostasis in the root apex.<br /><b>Chapter 4</b> describes the impact of eATP as a signaling molecule on root growth. In this study, the light-grown seedlings showed inhibited root growth with 1 mM eATP, whereas the dark-grown seedlings showed no inhibition. Moreover, BFA treatments indicate that eATP modify activity of endocytic vesicle recycling in root cells. eATP-induced inhibition of root growth and endocytic vesicle recycling requires ROS generation/signaling by NADPH oxidase (AtRBOHC), which was confirmed using the loss-of-function mutant line <i>rhd2-4</i>.<br /><b>Chapter 5</b> describes the mechanism of the inhibition of root gravitropic response and growth by eATP using confocal microscopy. Five minutes of ATP treatment enhanced the endocytic vesicle recycling, whereas a treatment longer than five min inhibited it. Moreover, eATP-induced inhibition of root elongation and endocytic vesicle recycling require eATP receptor, DORN1, as shown using the point-mutated line <i>dorn1-1</i>. DORN1 is relevant for the plasma membrane (PM) rigidity as documented with plasmolysis using mannitol. The PM rigidity is known to be involved in control of the endocytic recycling activity. Next, pH changes were monitored after eATP application to roots of the pHusion (apoplastic pH indicator) transgenic <i>Arabidopsis</i> line. As a result, eATP lowered the pH value in the root tip. Moreover, the highest expression level of <i>DORN1</i> (At5G60300) was shown at the root apex transition zone. These findings suggest that eATP disturbs the pH value and endocytic recycling activity in the root apex, resulting in inhibition of root growth and gravitropic response. <br /> In conclusion, obtained results indicate that the root apex transition zone responds to environmental stimuli by alteration of the activities of endocytic recycling, ROS generation, membrane rigidity, and root apex zonation. These studies provide the first insights for an understanding of eATP signaling in plant cell physiology, and also have relevance for other research fields, such as agriculture and potentially also pharmaceutical or medical studies.