CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

Phototropin phosphorylation of ROOT PHOTOTROPISM 2 and its role in mediating phototropism, leaf positioning, and chloroplast accumulation movement in Arabidopsis.

Directional movements impact the ability of plants to respond and adjust their growth accordingly to the prevailing light environment. The plasma-membrane associated protein, ROOT PHOTOTROPISM 2 (RPT2) is a key signalling component involved in chloroplast accumulation movement, leaf positioning, and phototropism, all of which are regulated redundantly by the ultraviolet/blue light-activated AGC kinases phototropin 1 and 2 (phot1 and phot2). We recently demonstrated that members of the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)/RPT2-like (NRL) family in Arabidopsis thaliana, including RPT2, are directly phosphorylated by phot1. However, whether RPT2 is a substrate for phot2, and the biological significance of phot phosphorylation of RPT2 remains to be determined. Here, we show that RPT2 is phosphorylated by both phot1 and phot2 at a conserved serine residue (S591) within the C-terminal region of the protein. Blue light triggered the association of 14-3-3 proteins with RPT2 consistent with S591 acting as a 14-3-3 binding site. Mutation of S591 had no effect on the plasma membrane localization of RPT2 but reduced its functionality for leaf positioning and phototropism. Moreover, our findings indicate that S591 phosphorylation within the C-terminus of RPT2 is required for chloroplast accumulation movement to low level blue light. Taken together, these findings further highlight the importance of the C-terminal region of NRL proteins and how its phosphorylation contributes to phot receptor signalling in plants.

A kinesin-like protein, KAC, is required for light-induced and actin-based chloroplast movement in Marchantia polymorpha.

Chloroplasts accumulate on the cell surface under weak light conditions to efficiently capture light but avoid strong light to minimize photodamage. The blue light receptor phototropin regulates the chloroplast movement in various plant species. In Arabidopsis thaliana, phototropin mediates the light-induced chloroplast movement and positioning via specialized actin filaments on the chloroplasts, chloroplast-actin filaments. KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC) and CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) are pivotal for chloroplast-actin-based chloroplast movement and positioning in land plants. However, the mechanisms by which KAC and CHUP1 regulate chloroplast movement and positioning remain unclear. In this study, we characterized KAC and CHUP1 orthologs in the liverwort Marchantia polymorpha, MpKAC and MpCHUP1, respectively. Their knockout mutants, Mpkack° and Mpchup1k°, impaired the light-induced chloroplast movement. Although Mpchup1k° showed mild chloroplast aggregation, Mpkack° displayed severe chloroplast aggregation, suggesting the greater contribution of MpKAC to the chloroplast anchorage to the plasma membrane. Analysis of the subcellular localization of the functional MpKAC-Citrine indicated that MpKAC-Citrine formed a punctate structure on the plasma membrane. Structure-function analysis of MpKAC revealed that a deletion of the conserved C-terminal domain abrogates the targeting to the plasma membrane and its function. A deletion of the N-terminal motor domain retained the plasma membrane targeting but abrogates the formation of punctate structure and showed severe defect in the light-induced chloroplast movement. Our findings suggest that the formation of the punctate structure on the plasma membrane of MpKAC is essential for chloroplast movement.

Phototropin2 Contributes to the Chloroplast Avoidance Response at the Chloroplast-Plasma Membrane Interface.

Blue-light-induced chloroplast movements play an important role in maximizing light utilization for photosynthesis in plants. Under a weak light condition, chloroplasts accumulate to the cell surface to capture light efficiently (chloroplast accumulation response). Conversely, chloroplasts escape from strong light and move to the side wall to reduce photodamage (chloroplast avoidance response). The blue light receptor phototropin (phot) regulates these chloroplast movements and optimizes leaf photosynthesis by controlling other responses in addition to chloroplast movements. Seed plants such as Arabidopsis (Arabidopsis thaliana) have phot1 and phot2. They redundantly mediate phototropism, stomatal opening, leaf flattening, and the chloroplast accumulation response. However, the chloroplast avoidance response is induced by strong blue light and regulated primarily by phot2. Phots are localized mainly on the plasma membrane. However, a substantial amount of phot2 resides on the chloroplast outer envelope. Therefore, differentially localized phot2 might have different functions. To determine the functions of plasma membrane- and chloroplast envelope-localized phot2, we tethered it to these structures with their respective targeting signals. Plasma membrane-localized phot2 regulated phototropism, leaf flattening, stomatal opening, and chloroplast movements. Chloroplast envelope-localized phot2 failed to mediate phototropism, leaf flattening, and the chloroplast accumulation response but partially regulated the chloroplast avoidance response and stomatal opening. Based on the present and previous findings, we propose that phot2 localized at the interface between the plasma membrane and the chloroplasts is required for the chloroplast avoidance response and possibly for stomatal opening as well.

Regulation of Photosynthetic Carbohydrate Metabolism by a Raf-Like Kinase in the Liverwort Marchantia polymorpha.

To optimize growth and development, plants monitor photosynthetic activities and appropriately regulate various cellular processes. However, signaling mechanisms that coordinate plant growth with photosynthesis remain poorly understood. To identify factors that are involved in signaling related to photosynthetic stimuli, we performed a phosphoproteomic analysis with Marchantia polymorpha, an extant bryophyte species in the basal lineage of land plants. Among proteins whose phosphorylation status changed differentially between dark-treated plants and those after light irradiation but failed to do so in the presence of a photosynthesis inhibitor, we identified a B4-group Raf-like kinase, named PHOTOSYNTHESIS-RELATED RAF (MpPRAF). Biochemical analyses confirmed photosynthesis-activity-dependent changes in the phosphorylation status of MpPRAF. Mutations in the MpPRAF gene resulted in growth retardation. Measurement of carbohydrates demonstrated both hyper-accumulation of starch and reduction of sucrose in Mppraf mutants. Neither inhibition of starch synthesis nor exogenous supply of sucrose alleviated the growth defect, suggesting serious impairment of Mppraf mutants in both the synthesis of sucrose and the repression of its catabolism. As a result of the compromised photosynthate metabolism, photosynthetic electron transport was downregulated in Mppraf mutants. A mutated MpPRAF with a common amino acid substitution for inactivating kinase activity was unable to rescue the Mppraf mutant defects. Our results provide evidence that MpPRAF is a photosynthesis signaling kinase that regulates sucrose metabolism.

Light-induced chloroplast movements in Oryza species.

Light-induced chloroplast movements control efficient light utilization in leaves, and thus, are essential for leaf photosynthesis and biomass production under fluctuating light conditions. Chloroplast movements have been intensively analyzed using wild-type and mutant plants of Arabidopsis thaliana. The molecular mechanism and the contribution to biomass production were elucidated. However, the knowledge of chloroplast movements is very scarce in other plant species, especially grass species including crop plants. Because chloroplast movements are efficient strategy to optimize light capture in leaves and thus promote leaf photosynthesis and biomass, analysis of chloroplast movements in crops is required for biomass production. Here, we analyzed chloroplast movements in a wide range of cultivated and wild species of genus Oryza. All examined Oryza species showed the blue-light-induced chloroplast movements. However, O. sativa and its ancestral species O. rufipogon, both of which are AA-genome species and usually grown in open condition where plants are exposed to full sunlight, showed the much weaker chloroplast movements than Oryza species that are usually grown under shade or semi-shade conditions, including O. officinalis, O. eichingeri, and O. granulata. Further detailed analyses of different O. officinalis accessions, including sun, semi-shade, and shade accessions, indicated that the difference in chloroplast movement strength between domesticated rice plants and wild species might result from the difference in habitat, and the shape of mesophyll chlorenchyma cells. The findings of this study provide useful information for optimizing Oryza growth conditions, and lay the groundwork for improving growth and yield in staple food crop Oryza sativa.

Chloroplast Accumulation Response Enhances Leaf Photosynthesis and Plant Biomass Production.

Under high light intensity, chloroplasts avoid absorbing excess light by moving to anticlinal cell walls (avoidance response), but under low light intensity, chloroplasts accumulate along periclinal cell walls (accumulation response). In most plant species, these responses are induced by blue light and are mediated by the blue light photoreceptor, phototropin, which also regulates phototropism, leaf flattening, and stomatal opening. These phototropin-mediated responses could enhance photosynthesis and biomass production. Here, using various Arabidopsis (Arabidopsis thaliana) mutants deficient in chloroplast movement, we demonstrated that the accumulation response enhances leaf photosynthesis and plant biomass production. Conspicuously, phototropin2 mutant plants specifically defective in the avoidance response but not in other phototropin-mediated responses displayed a constitutive accumulation response irrespective of light intensities, enhanced leaf photosynthesis, and increased plant biomass production. Therefore, our findings provide clear experimental evidence of the importance of the chloroplast accumulation response in leaf photosynthesis and biomass production.

RPT2/NCH1 subfamily of NPH3-like proteins is essential for the chloroplast accumulation response in land plants.

In green plants, the blue light receptor kinase phototropin mediates various photomovements and developmental responses, such as phototropism, chloroplast photorelocation movements (accumulation and avoidance), stomatal opening, and leaf flattening, which facilitate photosynthesis. In Arabidopsis, two phototropins (phot1 and phot2) redundantly mediate these responses. Two phototropin-interacting proteins, NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT PHOTOTROPISM 2 (RPT2), which belong to the NPH3/RPT2-like (NRL) family of BTB (broad complex, tramtrack, and bric à brac) domain proteins, mediate phototropism and leaf flattening. However, the roles of NRL proteins in chloroplast photorelocation movement remain to be determined. Here, we show that another phototropin-interacting NRL protein, NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), and RPT2 redundantly mediate the chloroplast accumulation response but not the avoidance response. NPH3, RPT2, and NCH1 are not involved in the chloroplast avoidance response or stomatal opening. In the liverwort Marchantia polymorpha, the NCH1 ortholog, MpNCH1, is essential for the chloroplast accumulation response but not the avoidance response, indicating that the regulation of the phototropin-mediated chloroplast accumulation response by RPT2/NCH1 is conserved in land plants. Thus, the NRL protein combination could determine the specificity of diverse phototropin-mediated responses.

Ferns, mosses and liverworts as model systems for light-mediated chloroplast movements.

Light-induced chloroplast movement is found in most plant species, including algae and land plants. In land plants with multiple small chloroplasts, under weak light conditions, the chloroplasts move towards the light and accumulate on the periclinal cell walls to efficiently perceive light for photosynthesis (the accumulation response). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response). In most plant species, blue light induces chloroplast movement, and phototropin receptor kinases are the blue light receptors. Molecular mechanisms for photoreceptors, signal transduction and chloroplast motility systems are being studied using the model plant Arabidopsis thaliana. However, to further understand the molecular mechanisms and evolutionary history of chloroplast movement in green plants, analyses using other plant systems are required. Here, we review recent works on chloroplast movement in green algae, liverwort, mosses and ferns that provide new insights on chloroplast movement.