BackgroundPlant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.ResultsHere, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.ConclusionsWe show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.

Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII-light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.

Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.

The LIL3 protein of Arabidopsis (Arabidopsis thaliana) belongs to the light-harvesting complex (LHC) protein family, which also includes the light-harvesting chlorophyll-binding proteins of photosystems I and II, the early-light-inducible proteins, PsbS involved in nonphotochemical quenching, and the one-helix proteins and their cyanobacterial homologs designated high-light-inducible proteins. Each member of this family is characterized by one or two LHC transmembrane domains (referred to as the LHC motif) to which potential functions such as chlorophyll binding, protein interaction, and integration of interacting partners into the plastid membranes have been attributed. Initially, LIL3 was shown to interact with geranylgeranyl reductase (CHLP), an enzyme of terpene biosynthesis that supplies the hydrocarbon chain for chlorophyll and tocopherol. Here, we show another function of LIL3 for the stability of protochlorophyllide oxidoreductase (POR). Multiple protein-protein interaction analyses suggest the direct physical interaction of LIL3 with POR but not with chlorophyll synthase. Consistently, LIL3-deficient plants exhibit substantial loss of POR as well as CHLP, which is not due to defective transcription of the POR and CHLP genes but to the posttranslational modification of their protein products. Interestingly, in vitro biochemical analyses provide novel evidence that LIL3 shows high binding affinity to protochlorophyllide, the substrate of POR. Taken together, this study suggests a critical role for LIL3 in the organization of later steps in chlorophyll biosynthesis. We suggest that LIL3 associates with POR and CHLP and thus contributes to the supply of the two metabolites, chlorophyllide and phytyl pyrophosphate, required for the final step in chlorophyll a synthesis.

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Chlorophyll is indispensable for life on Earth. Dynamic control of chlorophyll level, determined by the relative rates of chlorophyll anabolism and catabolism, ensures optimal photosynthesis and plant fitness. How plants post-translationally coordinate these two antagonistic pathways during their lifespan remains enigmatic. Here, we show that two Arabidopsis paralogs of BALANCE of CHLOROPHYLL METABOLISM (BCM) act as functionally conserved scaffold proteins to regulate the trade-off between chlorophyll synthesis and breakdown. During early leaf development, BCM1 interacts with GENOMES UNCOUPLED 4 to stimulate Mg-chelatase activity, thus optimizing chlorophyll synthesis. Meanwhile, BCM1's interaction with Mgdechelatase promotes degradation of the latter, thereby preventing chlorophyll degradation. At the onset of leaf senescence, BCM2 is up-regulated relative to BCM1, and plays a conserved role in attenuating chlorophyll degradation. These results support a model in which post-translational regulators promote chlorophyll homeostasis by adjusting the balance between chlorophyll biosynthesis and breakdown during leaf development.

Oxygenic photosynthesis requires chlorophyll (Chl) for the absorption of light energy, and charge separation in the reaction center of photosystem I and II, to feed electrons into the photosynthetic electron transfer chain. Chl is bound to different Chl-binding proteins assembled in the core complexes of the two photosystems and their peripheral light-harvesting antenna complexes. The structure of the photosynthetic protein complexes has been elucidated, but mechanisms of their biogenesis are in most instances unknown. These processes involve not only the assembly of interacting proteins, but also the functional integration of pigments and other cofactors. As a precondition for the association of Chl with the Chl-binding proteins in both photosystems, the synthesis of the apoproteins is synchronized with Chl biosynthesis. This review aims to summarize the present knowledge on the posttranslational organization of Chl biosynthesis and current attempts to envision the proceedings of the successive synthesis and integration of Chl into Chl-binding proteins in the thylakoid membrane. Potential auxiliary factors, contributing to the control and organization of Chl biosynthesis and the association of Chl with the Chl-binding proteins during their integration into photosynthetic complexes, are discussed in this review.

The assembly of light-harvesting chlorophyll-binding proteins (LHCPs) is coordinated with chlorophyll biosynthesis during chloroplast development. The ATP-independent chaperone known as chloroplast signal recognition particle 43 (cpSRP43) mediates post-translational LHCP targeting to the thylakoid membrane, and also participates in tetrapyrrole biosynthesis (TBS). How these distinct actions of cpSRP43 are controlled has remained unclear. Here, we demonstrate that cpSRP43 effectively protects several TBS proteins from heat-induced aggregation and enhances their stability during leaf greening and heat shock. While the substrate-binding domain (SBD) of cpSRP43 is sufficient for chaperoning LHCPs, stabilization of TBS clients requires the chromodomain 2 of the protein. Strikingly, cpSRP54 -which activates cpSRP43's LHCP-targeted function -inhibits the chaperone activity of cpSRP43 towards TBS proteins. High temperature weakens the interaction of cpSRP54 with cpSRP43, thus freeing cpSRP43 to interact with and protect the integrity of TBS proteins. Our data indicate that the temperature-sensitivity of the cpSRP43-cpSRP54 complex enables cpSRP43 to serve as an autonomous chaperone for thermoprotection of TBS proteins.