A chloroplast protein atlas reveals punctate structures and spatial organization of biosynthetic pathways.

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,034 candidate chloroplast proteins using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of poorly characterized proteins; identify novel components of nucleoids, plastoglobules, and the pyrenoid; and reveal widespread protein targeting to multiple compartments. We discovered and further characterized cellular organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and unexpected spatial distributions of enzymes within the chloroplast. We also used machine learning to predict the localizations of other nuclear-encoded Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to accelerate studies of chloroplast architecture and functions.

WUSCHEL-responsive At5g65480 interacts with CLAVATA components in vitro and in transient expression.

The CLAVATA (CLV) signaling pathway is essential for shoot meristem homeostasis in Arabidopsis. CLV acts to limit the expression domain of the stem cell-promoting gene WUSCHEL (WUS). The closely related receptor-kinases CLV1 and BAM1 are key components in this pathway; however, the downstream factors that link the receptors to WUS regulation are poorly understood. The Arabidopsis gene At5g65480 was recently identified as a direct transcriptional target up-regulated by WUS. We have independently identified this gene which we term CCI1 as a CLV1 and BAM1 interacting protein in vitro and in transient expression. CCI1 has phosphatidylinositide-binding activity in vitro and localizes to the plasma membrane in transient expression. Furthermore, CLV signaling components and CCI1 both partition to detergent-resistant membrane microdomains characterized as lipid rafts.

CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification.

CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor kinase CLV1 binds to the CLV3-derived CLE ligand. The biochemical role of the receptor-like protein CLV2 has remained largely unknown. Although genetic analysis suggested that CLV2, together with the membrane kinase CRN, acts in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here, we report detection of distinct CLV2-CRN heteromultimeric and CLV1-BAM multimeric complexes in transient expression in tobacco and in Arabidopsis meristems. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane-localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3-derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 overexpression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand-binding receptor complexes controlling stem cell specification in Arabidopsis.

Post-translational regulation of the Arabidopsis circadian clock through selective proteolysis and phosphorylation of pseudo-response regulator proteins.

The circadian clock controls the period, phasing, and amplitude of processes that oscillate with a near 24-h rhythm. One core group of clock components in Arabidopsis that controls the pace of the central oscillator is comprised of five PRR (pseudo-response regulator) proteins whose biochemical function in the clock remains unclear. Peak expression of TOC1 (timing of cab expression 1)/PRR1, PRR3, PRR5, PRR7, and PRR9 are each phased differently over the course of the day and loss of any PRR protein alters period. Here we show that, together with TOC1, PRR5 is the only other likely proteolytic substrate of the E3 ubiquitin ligase SCF(ZTL) within this PRR family. We further demonstrate a functional significance for the phosphorylated forms of PRR5, TOC1, and PRR3. Each PRR protein examined is nuclear-localized and is differentially phosphorylated over the circadian cycle. The more highly phosphorylated forms of PRR5 and TOC1 interact best with the F-box protein ZTL (ZEITLUPE), suggesting a mechanism to modulate their proteolysis. In vivo degradation of both PRR5 and ZTL is inhibited by blue light, likely the result of blue light photoperception by ZTL. TOC1 and PRR3 interact in vivo and phosphorylation of both is necessary for their optimal binding in vitro. Additionally, because PRR3 and ZTL both interact with TOC1 in vivo via the TOC1 N terminus, taken together these data suggest that the TOC1/PRR3 phosphorylation-dependent interaction may protect TOC1 from ZTL-mediated degradation, resulting in an enhanced amplitude of TOC1 cycling.

ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light.

The circadian clock is essential for coordinating the proper phasing of many important cellular processes. Robust cycling of key clock elements is required to maintain strong circadian oscillations of these clock-controlled outputs. Rhythmic expression of the Arabidopsis thaliana F-box protein ZEITLUPE (ZTL) is necessary to sustain a normal circadian period by controlling the proteasome-dependent degradation of a central clock protein, TIMING OF CAB EXPRESSION 1 (TOC1). ZTL messenger RNA is constitutively expressed, but ZTL protein levels oscillate with a threefold change in amplitude through an unknown mechanism. Here we show that GIGANTEA (GI) is essential to establish and sustain oscillations of ZTL by a direct protein-protein interaction. GI, a large plant-specific protein with a previously undefined molecular role, stabilizes ZTL in vivo. Furthermore, the ZTL-GI interaction is strongly and specifically enhanced by blue light, through the amino-terminal flavin-binding LIGHT, OXYGEN OR VOLTAGE (LOV) domain of ZTL. Mutations within this domain greatly diminish ZTL-GI interactions, leading to strongly reduced ZTL levels. Notably, a C82A mutation in the LOV domain, implicated in the flavin-dependent photochemistry, eliminates blue-light-enhanced binding of GI to ZTL. These data establish ZTL as a blue-light photoreceptor, which facilitates its own stability through a blue-light-enhanced GI interaction. Because the regulation of GI transcription is clock-controlled, consequent GI protein cycling confers a post-translational rhythm on ZTL protein. This mechanism of establishing and sustaining robust oscillations of ZTL results in the high-amplitude TOC1 rhythms necessary for proper clock function.

Formation of an SCF(ZTL) complex is required for proper regulation of circadian timing.

The circadian timing system involves an autoregulatory transcription/translation feedback loop that incorporates a diverse array of factors to maintain a 24-h periodicity. In Arabidopsis a novel F-box protein, ZEITLUPE (ZTL), plays an important role in the control of the free-running period of the circadian clock. As a class, F-box proteins are well-established components of the Skp/Cullin/F-box (SCF) class of E3 ubiquitin ligases that link the target substrates to the core ubiquitinating activity of the ligase complex via direct association with the Skp protein. Here we identify and characterize the SCFZTL complex in detail. Yeast two-hybrid tests demonstrate the sufficiency and necessity of the F-box domain for Arabidopsis Skp-like protein (ASK) interactions and the dispensability of the unique N-terminal LOV domain in this association. Co-immunoprecipitation of full-length (FL) ZTL with the three known core components of SCF complexes (ASK1, AtCUL1 and AtRBX1) demonstrates that ZTL can assemble into an SCF complex in vivo. F-box-containing truncated versions of ZTL (LOV-F and F-kelch) can complex with SCF components in vivo, whereas stably expressed LOV or kelch domains alone cannot. Stable expression of F-box-mutated FL ZTL eliminates the shortened period caused by mild ZTL overexpression and also abolishes ASK1 interaction in vivo. Reduced levels of the core SCF component AtRBX1 phenocopy the long period phenotype of ztl loss-of-function mutations, demonstrating the functional significance of the SCFZTL complex. Taken together, our data establish SCFZTL as an essential SCF class E3 ligase controlling circadian period in plants.