FtsZ1/FtsZ2 Turnover in Chloroplasts and the Role of ARC3

Abstract

Chloroplast division requires filamentation temperature-sensitive Z (FtsZ), a tubulin-like GTPase of cyanobacterial endosymbiotic origin. Plants and algae possess two distinct FtsZ protein families, FtsZ1 and FtsZ2 that co-assemble into a ring (Z-ring) at the division site. Z-ring assembly and disassembly and division site positioning is controlled by both positive and negative factors via their specific interactions with FtsZ1 and FtsZ2. Here we present the in planta analysis of Arabidopsis FtsZ1 and FtsZ2 turnover in the context of a native chloroplast division machinery. Fluorescence recovery after photobleaching analysis was conducted using fluorescently tagged FtsZ at wild-type (WT)-like levels. Rapid photobleaching, low signal-to-noise ratio, and phototropic movements of chloroplasts were overcome by (i) using progressive intervals in time-lapse imaging, (ii) analyzing epidermal rather than stromal chloroplasts, and (iii) employing image stack alignment during postprocessing. In plants of WT background, fluorescence recovery half-times averaged 117 and 325 s for FtsZ1 and FtsZ2, respectively. In plants lacking ARC3, the key negative regulator of FtsZ assembly, the turnover was threefold slower. The findings are discussed in the context of previous results conducted in a heterologous system.

In situ structure of FtsZ mini-rings in Arabidopsis chloroplasts

“…Perhaps, the propensity of FtsZ to curve is predicated on the features summarized above. On the other hand, these features could also echo the dynamic remodeling that FtsZ assemblies are constantly undergoing [19,20]. White and green arrowheads are separated by 360° each, i.e., one turn along the central axis.…”

“…Negative control plants were untransformed ftsZ2-1/2-2 double knockout plants [10]. The AtFtsZ2-mYFP construct has been described previously [20]. Plants were grown in Redi-Earth (SunGro Horticulture, Bellevue, WA, USA) substrate in a rooftop greenhouse at a temperature of 72 °C and a relative humidity of 62 %.…”

“…For structured illumination microscopy, leaf tissue was formaldehyde fixed under microwave irradiation, a process that preserves both the structural detail and the fluorescent protein signal [20,34]. The tissue was vacuum infiltrated with 3 % formaldehyde in phosphatebuffered saline (PBS, 0.14 M NaCl, 2.7 mM KCl, 6.5 mM Na 2 HPO 4 , 5 mM KH 2 PO 4 , 3.0 mM NaN 3 ; pH = 7.3) and incubated at room temperature for 30 min.…”

“…Extract from approximately 1-mg fresh tissue was loaded per lane. Immunodetection with affinity-purified goat anti-peptide antibody, recognizing the residues 168 through 184 in AtFtsZ2-1 [35] was performed as described in Johnson et al [20]. Equal loading was confirmed by Ponceau S staining of the RuBisCO band on membranes (0.1 % (w/v) Ponceau S in 5 % (v/v) acetic acid).…”

“…FtsZ1 and FtsZ2 have differential guanosine triphosphatase (GTPase) activity [17,18] and are capable of linear coassembly to form heteropolymers [17]. FtsZ1 and FtsZ2 exhibit dynamic turnover in vivo [19] that is promoted by the ARC3 protein [20]. How the individual FtsZ filaments assemble into higher-order structures to form the chloroplast dividing Z-ring is not well understood.…”