Moving the green

Abstract

Chloroplast movement as a response of plants to light variations is presented as an example in each classical textbook, showing that these organelles accumulate in response to low light and avoid high light irradiation. In sharp contrast to the morphological discovery of the phenomenon, which dates back more than a century, the molecular understanding of this effect is just at its beginning and only recently first components of the signal cascade initiating this process were described. Among these, a protein termed CHUP1 was identified. This protein is present in the outer membrane of chloroplasts and thereby discussed as the first component of a possible ‘moving ensemble’ assembling at the ‘moved cargo’. The protein is able to interact with actin and profilin—and even more, is able to regulate this interaction in vitro. Thereby, today it can be stated that actin filament reformation and chloroplast repositioning are coordinated if not dependent on each other.

Profilin as Key for Chloroplast Movement?

The light dependence of the positioning of chloroplasts is known since the fifties of the 19^th century¹ and in the fifties of the 20^th its relation to the photosynthetic activity was formulated² leading to the today accepted model that chloroplasts change their position in order to optimize their activity. Several different modes were postulated over time to explain the mechanisms of movement including a model of “pulling fibers” proposed in 1914, which in part can be seen as the first explanation of the cytoskeletal function,³ which was subsequently confirmed.⁴ Astonishingly, in contrast to the long history of descriptive investigations, only recently the first chloroplast protein involved in movement was discovered, the 112 kDa protein CHUP1 (Chloroplast unusual positioning 1).^5–7 Deletion of CHUP1 function results in loss of chloroplast accumulation and avoidance response, even though Oikawa and coworkers stated that the actin cytoskeleton remains unaffected.⁶ However, this interpretation has to be taken with care as GFP-mTalin was used, which is recently discussed to not stain all actin arrays and which can cause artificial aggregation of actin.⁸ Strikingly, the chup1 mutation does not reveal a large alteration of the global expression profile under normal growth conditions, and most genes found to be upregulated encode proteins generally found to function in stress response.⁷ The expression of these genes was also found to be upregulated in the wild type after light treatment, but did not significantly change in the mutant plants after light treatment.⁷ Hence, it has to be suggested that for plants carrying a knock out of chup1 light stress is induced at a lower dosage.

The protein CHUP1 itself is localized at the outer envelope of chloroplasts via its hydrophobic N-terminus.^6,7 CHUP1 contains an actin binding domain.⁹ Indeed, an interaction between actin and CHUP1, independent of the polymerization state of actin, was demonstrated in vitro.^6,7 This is in line with an idea that CHUP1 would act as a platform regulating the actin polymerization.^4,6 One known modulator of actin polymerization is profilin.¹⁰ Strikingly, CHUP1 contains a putative PRM1 profilin-binding motif^6,11 besides the actin binding domain. An interaction between both proteins, CHUP1 and profilin, independent of the phosphorylation status of profilin, as well as a direct influence of CHUP1 on the formation of actin-profilin-complexes was observed in vitro.⁷ The interaction of profilin and CHUP1 in a cellular environment can also be visualized when expressing CHUP1 without its N-terminal membrane anchor as GFP fusion⁷ and profilin as RFP fusion. In the absence of ΔN-CHUP1, profilin is evenly distributed within the cytoplasm (and in part in the nucleus), which is in line with its function as an actin filament regulating protein (Fig. 1A, bottom). In the presence of ΔN-CHUP1, profilin-RFP fluorescence co-localizes with the previously observed punctured pattern of ΔN-CHUP1-GFP (Fig. 1A, top, compare RFP and GFP channel).⁷ Hence, it can be concluded that profilin interacts with CHUP1 in a cellular environment.

Figure 1.

Profilin and Chup1 interact in native environment. (A) Protoplasts isolated and transformed with indicated constructs were analysed by fluorescence microscopy as described.⁷ Shown is the chlorophyll autofluorescence (left), the RFP fluorescence (middle) or the GFP fluorescence (right) of a representative cell transformed with profilin-RFP (AT4G29350) (bottom) or with profilin-RFP and ΔN-CHUP1-GFP (top, middle). (B) Model for chloroplast movement, showing the recruitment of profilin and actin to the chloroplast by the CHUP1 protein and initiation of actin polymerisation by the addition of actin monomers to the growing filament, regulated by profilin action.

Arabidopsis encodes five profilin isoforms: three constitutively expressed genes (PRF1-3, expressed in leaf tissues¹²) and two genes with pollen specific expression (PRF4, 5).¹³ Profilin makes up to 0.3% of total proteins in rosette leafs¹⁴ and thereby has to be considered as a global player for many actin-filament dependent cellular functions including the positioning of chloroplasts. The observed interaction between CHUP1 and both, actin and profilin, confirms the initially proposed model,¹⁵ which can now be refined (Fig. 1B) as CHUP1 not only binds the two proteins, but also stimulates the formation of profilactin. However, the finding also raises one question:

The Good Old Days of Chloroplast Movement Aren't Over Yet?

The model proposed here (Fig. 1B) and elsewhere¹⁵ pictures CHUP1 as a ‘work bench’ and may be considered as a docking site for actin filaments. Is that already the key for the movement of the organelle? Indeed, such polymerization dependent movement was recently identified for vesicles.^16–18 Another example is the actinbased- profilin-dependent movement of the intracellular pathogen Listeria monocytogenes.¹⁹ However, the pushing force, resulting from the comet tails formed by polymerizing actin, is not observed in the vicinity of chloroplasts, but the principle could still be similar. Furthermore, profilin positively modulates the polymerization at barbed ends rather than on pointed ends, at least in yeast,²⁰ which is in line with the proposed model (Fig. 1B). Hence, if chloroplasts indeed move by actin polymerization one has to suggest that movement is driven by the speed of polymerization of actin filaments. Chloroplasts move with a speed of 1–1.5 µm/min.²¹ The rate of actin filament growth determined e.g., by the velocity of actin-patches can, however, be 10 times higher.²² Therefore, it is tempting to speculate that CHUP1 is directly involved in movement of the organelle, but at this stage we can only manifest a function in actin polymerization, while the final link to movement awaits its investigation.

Addendum to: Schmidt von Braun S, Schleiff E. The chloroplast outer membrane protein CHUP1 interacts with actin and profilin. Planta. 2008;227:1151–1159. doi: 10.1007/s00425-007-0688-7.