Inhibition of endosomal trafficking by brefeldin A interferes with long-distance interaction between chloroplasts and plasma membrane transporters

The huge internodal cells of the characean green algae are a convenient model to study long-range interactions between organelles via cytoplasmic stream- ing. It has been shown previously that photometabolites and reactive oxygen species released by illuminated chloroplasts are transmitted to remote shaded regions where they interfere with photosynthetic electron transport and the differential activity of plasma membrane transporters, and recent findings indi- cated the involvement of organelle trafficking pathways. In the present study, we applied pulse amplitude-modulated microscopy and pH-sensitive elec- trodes to study the effect of brefeldin A (BFA), an inhibitor of vesicle trafficking, on long-distance interactions in Chara australis internodal cells. These data were compared with BFA-induced changes in organelle number, size and dis- tribution using fluorescent dyes and confocal laser scanning microscopy. We found that BFA completely and immediately inhibited endocytosis in intern- odal cells and induced the aggregation of organelles into BFA compartments within 30–120 min of treatment. The comparison with the physiological data suggests that the early response, the arrest of endocytosis, is related to the attenuation of differences in surface pH, whereas the longer lasting forma- tion of BFA compartments is probably responsible for the acceleration of the cyclosis-mediated interaction between chloroplasts. These data indicate that intracellular turnover of membrane material might be important for the circulation of electric currents between functionally distinct regions in illumi- nated characean internodes and that translational movement of metabolites is delayed by transient binding of the transported substances to organelles.

Introduction
The characean internodal cells generate alternating patterns of low and high pH along their surface (pHo; Fig. S1A; reviewed by Beilby and Bisson 2012 and Beilby 2016). The pH banding pattern correlates withthe photosynthetic activity of the anchored chloroplasts being high at the acidic regions because of enhanced availability of carbon dioxide (Fig. S1B; Plieth et al. 1994, Bulychev and Vredenberg 2003). The acid- ification of the external medium is because of the activity of plasma membrane located H+ ATPases thatpump cytoplasmic protons outward. To maintain pH homeostasis of the cytoplasm, outward directed pro- ton current is balanced by H+ influx or OH− efflux (high pH channels; Bisson and Walker 1980). Previous studies suggest that the high pH channels are acti- vated by metabolites released from photosynthetically active chloroplasts (Eremin et al. 2007, Bulychev and Komarova 2014).There is evidence that the intracellular turnover of membrane material might be important for the circu- lation of electric currents between functionally distinct regions in illuminated characean internodes and for long-distance signaling within the cell. For example, an inhibitor of cytoplasmic vesicle trafficking, wortmannin suppressed the pH banding in Chara and eliminated a minor component in the cyclosis-mediated changes of chlorophyll (Chl) fluorescence (Bulychev and Foiss- ner 2017). The action of other inhibitors of vesicular transport, e.g. brefeldin A (BFA), on proton flows and Chl fluorescence changes is of interest. The alteration in the abundance or turnover rates of cytoplasmic vesi- cles may restrict the intracellular mobility of laterally transported solutes through binding and the release of these solutes to membrane vesicles. Boot et al. (2012) observed the polar movement of indole-3-acetic acid (IAA) along Chara internodes at velocities compara- ble to or lower than the rate of cytoplasmic stream- ing. However, IAA transport was insensitive to inhibition of cyclosis, unlike metabolite signaling detected with pulse amplitude-modulated microscopy (PAM) fluorom- etry (Bulychev and Foissner 2017).The interactions between the endosomal trafficking, lateral transmission of metabolites and the formation of banding pattern are hardly investigated to date. Recent studies revealed similarities and distinctions in the effects of BFA and wortmannin on structure and func- tion of plant cells (Bulychev et al. 2018).

In this study, we examined the influence of BFA on light-dependent proton flows across the plasma membrane and on the cyclosis-mediated regulation of photosystem (PS) II activity in Chara internodal cells. Long-distance signaling was investigated by the application of local illumination and measuring Chl fluorescence at non-irradiated downstream areas (Fig. S1C). Such experiments have previously revealed that chloroplast electron transport can be modified by reducing equiva- lents released from illuminated regions and transported to non-illuminated chloroplasts via the streaming endo- plasm (Fig. S1C; Bulychev et al. 2013, Bulychev and Komarova 2015, 2017).BFA is a macrocyclic lactone produced by a range of fungi belonging to different genera (Wang et al. 2002) and responsible for inducing leaf spot disease insusceptible plants (Tietjen et al. 1985). The interest in BFA initially focused on its antiviral and antitumor activ- ities (Tamura et al. 1968) which were, however, insuffi- cient for clinical application. Later, it became clear that BFA is a useful tool for investigating vesicle trafficking in animal (Takatsuki and Tamura 1985) and plant cells (Satiat-Jeunemaitre and Hawes 1992a, b). Since then, BFA has become widely used in the study of membrane pathways toward and away from the plasma membrane. The target of BFA are guanine-nucleotide exchange fac- tors which activate ADP-ribosylation factor proteins, small GTPases, involved in the recruitment of membrane coats required for cargo sorting and for the release of vesicles (see Singh and Jürgens 2018 for review). A com- mon feature of BFA treatment is the formation of BFA compartments or BFA bodies (Satiat-Jeunemaitre and Hawes 1992b).

They usually consist of agglomerations of Golgi bodies and/or trans-Golgi network (TGN), or of their remnants, and may include aggregates of endo- plasmic reticulum (ER) cisternae (Staehelin and Driouich 1997, Nebenführ et al. 2002, Ritzenthaler et al. 2002, Robinson et al. 2008). In most plant cells investigated so far, BFA predominantly inhibits the release of Golgi and TGN vesicles and their fusion with the plasma mem- brane (exocytosis; Naramoto et al. 2010). In Nicotiana BY2 cells, BFA has been reported to arrest the release of endosomes from the plasma membrane (endocytosis; Jelinkova et al. 2015).In the present study, we investigated the effect of BFA on the multicellular green alga Chara. We found that BFA completely and immediately inhibited endocytosis in internodal cells and induced the formation of BFA com- partments within 30–120 min of treatment. The compar- ison with the physiological data suggests that the early response, the arrest of endocytosis, is related to changes in pH banding, whereas the longer lasting agglomer- ation and fusion of organelles is probably responsible for the acceleration of the cyclosis-mediated interac- tion between chloroplasts, owing to reduced binding of metabolites to membrane surfaces.Thalli of Chara australis R.Br. were grown as described previously (Foissner et al. 2016). Internodal cells were isolated from the main axis with a small pair of scissors and left in artificial fresh water (AFW; 10−3 M NaCl, 10−4 M KCl and 10−4 M CaCl2) until use.BFA (BFA; Sigma Aldrich) was dissolved in dimethylsulfoxide (DMSO) at a concentration of 70 mM. Thestock solution was diluted with AFW and the control solutions contained the appropriate amount of DMSO.Alkaline and acid bands were identified with tip-sensitive antimony pH microelectrodes as described by Bulychev et al. (2001).Chl fluorescence measurementsChl fluorescence was measured on microscopic cell regions (∼100 μm in diameter) with a Microscopy PAM fluorometer (Walz) combined with an Zeiss Axiovert25 CFL inverted microscope. Weak measuring light from the blue light-emitting diode (LED) of the PAM directed through the microscope optical path excitedminimal fluorescence (Fo) measured in dark-adapted cells and fluorescence (F′, actual Chl fluorescence in acell exposed to actinic light) observed under dim back- ground illumination. Maximal Chl fluorescence yields in dark-adapted cells (Fm) and in cells exposed to actinic light (Fm′) were induced by saturating light pulses. The signal from the photomultiplier was processed with WinControl-3 software (Walz). It was also digitized with a PCI-6024E AD-converter (National Instruments) and displayed on a computer. Data points were sampled at intervals of ∼51 ms.

The whole cell was continuously exposed to dim background light (BGL). This light was directed from the upper illuminator of the Axiovert 25 CFL micro- scope and passed through a blue glass filter (SZS-22,