Chloroplast Avoidance Research Articles

CHUP1 restricts chloroplast movement and effector-triggered immunity in epidermal cells.

Chloroplast Unusual Positioning 1 (CHUP1) plays an important role in the chloroplast avoidance and accumulation responses in mesophyll cells. In epidermal cells, prior research showed silencing CHUP1-induced chloroplast stromules and amplified effector-triggered immunity (ETI); however, the underlying mechanisms remain largely unknown. CHUP1 has a dual function in anchoring chloroplasts and recruiting chloroplast-associated actin (cp-actin) filaments for blue light-induced movement. To determine which function is critical for ETI, we developed an approach to quantify chloroplast anchoring and movement in epidermal cells. Our data show that silencing NbCHUP1 in Nicotiana benthamiana plants increased epidermal chloroplast de-anchoring and basal movement but did not fully disrupt blue light-induced chloroplast movement. Silencing NbCHUP1 auto-activated epidermal chloroplast defense (ECD) responses including stromule formation, perinuclear chloroplast clustering, the epidermal chloroplast response (ECR), and the chloroplast reactive oxygen species (ROS), hydrogen peroxide (H2O2). These findings show chloroplast anchoring restricts a multifaceted ECD response. Our results also show that the accumulated chloroplastic H2O2 in NbCHUP1-silenced plants was not required for the increased basal epidermal chloroplast movement but was essential for increased stromules and enhanced ETI. This finding indicates that chloroplast de-anchoring and H2O2 play separate but essential roles during ETI.

Hyperspectral imaging for chloroplast movement detection.

We employed hyperspectral imaging to detect chloroplast positioning and assess its influence on common vegetation indices. In low blue light, chloroplasts move to cell walls perpendicular to the direction of the incident light. In high blue light, chloroplasts exhibit the avoidance response, moving to cell walls parallel to the light direction. Irradiation with high light results in significant changes in leaf reflectance and the shape of the reflectance spectrum. Using mutants with disrupted chloroplast movements, we found that blue-light-induced changes in the reflectance spectrum are mostly due to chloroplast relocations. We trained machine learning methods in the classification of leaves according to the chloroplast positioning, based on the reflectance spectra. The convolutional network showed low levels of misclassification of leaves irradiated with high light even when different species were used for training and testing, suggesting that reflectance spectra may be used to detect chloroplast avoidance in heterogeneous vegetation. We also examined the correlation between chloroplast positioning and values of indices of normalized-difference type for various combinations of wavelengths and identified an index sensitive to chloroplast positioning. We found that values of some of the vegetation indices, including those sensitive to the carotenoid levels, may be altered due to chloroplast rearrangements.

Enhancement of the CRISPR/Cas9-Based Genome Editing System in Lettuce (Lactuca sativa L.) Using the Endogenous U6 Promoter.

The CRISPR/Cas9 system has been widely applied as a precise gene-editing tool for studying gene functions as well as improving agricultural traits in various crop plants. Here, we optimized a gene-editing system in lettuce (Lactuca sativa L.) using the endogenous U6 promoter and proved that the PHOT2 gene is a versatile target gene. We isolated the LsU6-10 promoter from 10 U6 snRNA genes identified from the lettuce genome database for comparison with the AtU6-26 promoter that has been used to drive sgRNAs in lettuce. Two CRISPR/Cas9 vectors were constructed using the LsU6-10 and AtU6-26 promoters to drive sgRNA361 to target the PHOT2 gene. The chloroplast avoidance response was defective in lettuces with biallelic mutations in the targeted PHOT2 gene, as in the Arabidopsis phot2 mutant. The PHOT2 gene mutations were stably heritable from the R0 to R2 generations, and the high gene-editing efficiency enabled the selection of transgene-free lines in the R1 generation and the establishment of independent phot2 mutants in the R2 generation. Our results suggest that the LsU6-10 promoter is more effective than the AtU6-26 promoter in driving sgRNA for the CRISPR/Cas9 system in lettuce and that PHOT2 is a useful target gene to verify gene editing efficiency without any detrimental effects on plant growth, which is often a consideration in conventional target genes.

FKF1 Interacts with CHUP1 and Regulates Chloroplast Movement in Arabidopsis.

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, and even cell death (avoidance). Arabidopsis blue light receptors phot1 and phot2 (phototropins) have been reported to regulate chloroplast movement. This study discovered that another blue light receptor, FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), regulates chloroplast photorelocation by physically interacting with chloroplast unusual positioning protein 1 (CHUP1), a critical component of the chloroplast motility system. Leaf cross-sectioning and red-light transmittance results showed that overexpression of FKF1 compromised the avoidance response, while the absence of FKF1 enhanced chloroplast movements under high light. Western blot analysis showed that CHUP1 protein abundance is altered in FKF1 mutants and overexpression lines, indicating a potential regulation of CHUP1 by FKF1. qPCR results showed that two photorelocation pathway genes, JAC1 and THRUMIN1, were upregulated in FKF1-OE lines, and overexpression of FKF1 in the THRUMIN1 mutant weakened its accumulation and avoidance responses, indicating that JAC1 and THRUMIN1 may play a role in the FKF1-mediated chloroplast avoidance response. However, the precise functional roles of JAC1 and THRUMIN1 in this process are not known.

Combined effects of high irradiance and temperature on the photosynthetic and antioxidant responses of Thalassia hemprichii and Halophila ovalis

Abstract During midday low tides, tropical intertidal seagrasses are challenged by high irradiance and high temperature. This study assessed photosynthetic and oxidative stress responses of Thalassia hemprichii and Halophila ovalis exposed to 150 and 1000 μmol photons m−2 s−1 and 30 and 40 °C for 3 h. High temperature (40 °C) significantly decreased the maximum quantum yield of both seagrasses and this heat-induced photoinhibition was exacerbated by high irradiance (1000 μmol photons m−2 s−1). High irradiance also aggravated the effects of high temperature on the effective quantum yield of T. hemprichii. Non-photochemical quenching (NPQ) of T. hemprichii was induced by both stressors with no additive effects. In contrast, NPQ of H. ovalis was induced under high irradiance at 30 °C but was inhibited at 40 °C. Nevertheless, antioxidant enzyme activity and reactive oxygen species content did not differ among treatments in either seagrass. Monitoring chloroplast distribution in H. ovalis revealed a partial inhibitory effect of high temperature on chloroplast avoidance movement under high irradiance. Our results suggest that warming events may cause detrimental impacts on shallow water seagrasses. Halophila ovalis may be more vulnerable than T. hemprichii as its photoprotection, i.e. NPQ and chloroplast avoidance movement, was hindered at high temperature.

The endoplasmic reticulum membrane-bending protein RETICULON facilitates chloroplast relocation movement in Marchantia polymorpha.

Plant cells alter the intracellular positions of chloroplasts to ensure efficient photosynthesis, a process controlled by the blue light receptor phototropin. Chloroplasts migrate toward weak light (accumulation response) and move away from excess light (avoidance response). Chloroplasts are encircled by the endoplasmic reticulum (ER), which forms a complex network throughout the cytoplasm. To ensure rapid chloroplast relocation, the ER must alter its structure in conjunction with chloroplast relocation movement, but little is known about the underlying mechanism. Here, we searched for interactors of phototropin in the liverwort Marchantia polymorpha and identified a RETICULON (RTN) family protein; RTN proteins play central roles in ER tubule formation and ER network maintenance by stabilizing the curvature of ER membranes in eukaryotic cells. Marchantia polymorpha RTN1 (MpRTN1) is localized to ER tubules and the rims of ER sheets, which is consistent with the localization of RTNs in other plants and heterotrophs. The Mprtn1 mutant showed an increased ER tubule diameter, pointing to a role for MpRTN1 in ER membrane constriction. Furthermore, Mprtn1 showed a delayed chloroplast avoidance response but a normal chloroplast accumulation response. The live cell imaging of ER dynamics revealed that ER restructuring was impaired in Mprtn1 during the chloroplast avoidance response. These results suggest that during the chloroplast avoidance response, MpRTN1 restructures the ER network and facilitates chloroplast movement via an interaction with phototropin. Our findings provide evidence that plant cells respond to fluctuating environmental conditions by controlling the movements of multiple organelles in a synchronized manner.

Guard-Cell-Specific Expression of Phototropin2 C-Terminal Fragment Enhances Leaf Transpiration.

Phototropins (phot1 and phot2) are plant-specific blue light receptors that mediate chloroplast movement, stomatal opening, and phototropism. Phototropin is composed of the N-terminus LOV1 and LOV2 domains and the C-terminus Ser/Thr kinase domain. In previous studies, 35-P2CG transgenic plants expressing the phot2 C-terminal fragment–GFP fusion protein (P2CG) under the control of 35S promoter showed constitutive phot2 responses, including chloroplast avoidance response, stomatal opening, and reduced hypocotyl phototropism regardless of blue light, and some detrimental growth phenotypes. In this study, to exclude the detrimental growth phenotypes caused by the ectopic expression of P2C and to improve leaf transpiration, we used the PHOT2 promoter for the endogenous expression of GFP-fused P2C (GP2C) (P2-GP2C) and the BLUS1 promoter for the guard-cell-specific expression of GP2C (B1-GP2C), respectively. In P2-GP2C plants, GP2C expression induced constitutive phototropin responses and a relatively dwarf phenotype as in 35-P2CG plants. In contrast, B1-GP2C plants showed the guard-cell-specific P2C expression that induced constitutive stomatal opening with normal phototropism, chloroplast movement, and growth phenotype. Interestingly, leaf transpiration was significantly improved in B1-GP2C plants compared to that in P2-GP2C plants and WT. Taken together, this transgenic approach could be applied to improve leaf transpiration in indoor plants.

Phototropin 1 Mediates High-Intensity Blue Light-Induced Chloroplast Accumulation Response in a Root Phototropism 2-Dependent Manner in Arabidopsis phot2 Mutant Plants.

Phototropins, namely, phototropin 1 (phot1) and phototropin 2 (phot2), mediate chloroplast movement to maximize photosynthetic efficiency and prevent photodamage in plants. Phot1 primarily functions in chloroplast accumulation process, whereas phot2 mediates both chloroplast avoidance and accumulation responses. The avoidance response of phot2-mediated chloroplasts under high-intensity blue light (HBL) limited the understanding of the function of phot1 in the chloroplast accumulation process at the HBL condition. In this study, we showed that the phot2 mutant exhibits a chloroplast accumulation response under HBL, which is defective when the root phototropism 2 (RPT2) gene is mutated in the phot2 background, mimicking the phenotype of the phot1 phot2 double mutant. A further analysis revealed that the expression of RPT2 was induced by HBL and the overexpression of RPT2 could partially enhance the chloroplast accumulation response under HBL. These results confirmed that RPT2 also participates in regulating the phot1-mediated chloroplast accumulation response under HBL. In contrast, RPT2 functions redundantly with neural retina leucine zipper (NRL) protein for chloroplast movement 1 (NCH1) under low-light irradiation. In addition, no chloroplast accumulation response was detected in the phot2 jac1 double mutant under HBL, which has been previously observed in phot2 rpt2 and phot1 phot2 double mutants. Taken together, our results indicated that phot1 mediates the HBL-induced chloroplast accumulation response in an RPT2-dependent manner and is also regulated by j-domain protein required for chloroplast accumulation response 1 (JAC1).

Green light attenuates blue-light-induced chloroplast avoidance movement in Arabidopsis and Landoltia punctata.

Chloroplast movement to the anticlinal walls in excess light, referred to as chloroplast avoidance movement, is one strategy to prevent high light damage. Chloroplast avoidance movement is mediated by the blue-light photoreceptor phototropin. Since some blue-light effects are reversed by green light, we investigated the effect of green wavelengths on chloroplast avoidance. Chloroplast position was visualized via microscopy and by transmission of red light through the leaves of Arabidopsis thaliana and Landoltia punctata (duckweed). Green light reduced blue-light-induced chloroplast avoidance movement but only when green light was presented simultaneously with blue light. Green light alone had no effect on chloroplast position. An action spectrum for green-light attenuation of chloroplast avoidance in duckweed revealed peaks at 510, 550, and 590 nm. Blue-light-induced chloroplast avoidance movement in three Arabidopsis mutants with reduced nonphotochemical quenching, npq1, npq4, and npq7 was not affected by green light. The action spectrum does not conform to any known photoreceptor. The lack of a green-light response in the npq mutants of Arabidopsis suggests a possible role for the xanthophyll cycle or a signal from the chloroplast in control of chloroplast avoidance movement.

Impaired chloroplast positioning affects photosynthetic capacity and regulation of the central carbohydrate metabolism during cold acclimation

Photosynthesis and carbohydrate metabolism of higher plants need to be tightly regulated to prevent tissue damage during environmental changes. The intracellular position of chloroplasts changes due to a changing light regime. Chloroplast avoidance and accumulation response under high and low light, respectively, are well known phenomena, and deficiency of chloroplast movement has been shown to result in photodamage and reduced biomass accumulation. Yet, effects of chloroplast positioning on underlying metabolic regulation are less well understood. Here, we analysed photosynthesis together with metabolites and enzyme activities of the central carbohydrate metabolism during cold acclimation of the chloroplast unusual positioning 1 (chup1) mutant of Arabidopsis thaliana. We compared cold acclimation under ambient and low light and found that maximum quantum yield of PSII was significantly lower in chup1 than in Col-0 under both conditions. Our findings indicated that net CO2 assimilation in chup1 is rather limited by biochemistry than by photochemistry. Further, cold-induced dynamics of sucrose phosphate synthase differed significantly between both genotypes. Together with a reduced rate of sucrose cycling derived from kinetic model simulations our study provides evidence for a central role of chloroplast positioning for photosynthetic and metabolic acclimation to low temperature.

Blue Light Acclimation Reduces the Photoinhibition of Phalaenopsis aphrodite (Moth Orchid).

The moth orchid is an important ornamental crop. It is very sensitive to high light irradiation due to photoinhibition. In this study, young orchid tissue culture seedlings and 2.5” potted plants pretreated under blue light (BL, λmax = 450 nm) at 100 µmol m−2 s−1 for 12 days (BL acclimation) were found to have an increased tolerance to high light irradiation. After BL acclimation, orchids had an increased anthocyanin accumulation, enhanced chloroplast avoidance, and increased chlorophyll fluorescence capacity whenever they were exposed to high light of 1000 μmol m−2 s−1 for two weeks (HL). They had higher Fv/Fm, electron transport rate (ETR), chlorophyll content, catalase activity and sucrose content when compared to the control without BL acclimation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that transcript levels of phototropins, D1, RbcS, PEPCK, Catalase and SUT2 were upregulated in the BL-acclimated orchids. Consequently, BL acclimation orchids had better growth when compared to the control under long-term high light stress. In summary, this study provides a solution, i.e., BL acclimation, to reduce moth orchid photoinhibition and enhance growth before transplantation of the young tissue culture seedlings and potted plants into greenhouses, where they usually suffer from a high light fluctuation problem.

Differing photosynthetic responses to excess irradiance in the two coexisting seagrasses, Halophila ovalis and Halophila decipiens: Chloroplast avoidance movement, chlorophyll fluorescence, and leaf optical properties

Seagrasses cope with excess irradiance using a suit of photoprotective mechanisms, however, Halophila species are the only seagrasses known to exhibit chloroplast avoidance movement. Here we examined this phenomenon together with concurrent change in photosynthetic and leaf optical properties in H. ovalis and H. decipiens. Although co-existing, these two seagrasses displayed different light use characteristics derived from rapid light curves; H. ovalis and H. decipiens showed features of sun- and shade-adapted plants, respectively. Experimentally the seagrass leaves were exposed to low, medium, and high irradiance (80, 200 and 800 μmol photons m−2 s−1, respectively) for 60 min, followed by 60 min in darkness. Chloroplast movement occurred in H. ovalis exposed to medium or high irradiance and in H. decipiens exposed to low or medium irradiance. Chloroplast gathering was observed after 20 min of exposure and chloroplast dispersal was observed after 20 min in darkness, dependent on treatment and species. Increasing irradiance lowered the effective quantum yield and increased the non-photochemical quenching (NPQ). Despite stronger up-regulation in NPQ, photoinhibition was detected in H. decipiens. The absorption factor and the Soil-Plant Analysis Development value were modulated by chloroplast movement rather than a change in chlorophyll content, except for H. decipiens exposed to high irradiance where photodamage of chlorophylls may have occurred. Chloroplast movement in both seagrasses was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, indicating regulation by photosynthesis. Our results suggest that H. ovalis has a higher tolerance to excess irradiance than H. decipiens and that this is assisted partly by chloroplast avoidance movement.

Chloroplast avoidance movement: a novel paradigm of ROS signalling.

The damaging effects of supra-optimal irradiance on plants, often turning to be lethal, may be circumvented by chloroplast avoidance movement which realigns chloroplasts to the anticlinal surfaces of cells (parallel to the incident light), essentially minimizing photon absorption. In angiosperms and many other groups of plants, chloroplast avoidance movement has been identified to be a strong blue light (BL)-dependent process being mediated by actin filaments wherein phototropins are identified as the photoreceptor involved. Studies through the last few decades have identified key molecular mechanisms involving Chloroplast Unusual Positioning 1 (CHUP1) protein and specific chloroplast-actin (cp-actin) filaments. However, the signal transduction pathway from strong BL absorption down to directional re-localization of chloroplasts by actin filaments is complex and ambiguous. Being the immediate cellular products of high irradiance absorption and having properties of remodelling actin as well as phototropin, reactive oxygen species (ROS) deemed to be more able and prompt than any other signalling agent in mediating chloroplast avoidance movement. Although ROS are presently being identified as fundamental component for regulating different plant processes ranging from growth, development and immunity, its role in avoidance movement have hardly been explored in depth. However, few recent reports have demonstrated the direct stimulatory involvement of ROS, especially H2O2, in chloroplast avoidance movement with Ca2+ playing a pivotal role. With this perspective, the present review discusses the mechanisms of ROS-mediated chloroplast avoidance movement involving ROS-Ca2+-actin communication system and NADPH oxidase (NOX)-plasma membrane (PM) H+-ATPase positive feed-forward loop. A possible working model is proposed.

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.

The importance of chloroplast movement, nonphotochemical quenching, and electron transport rates in light acclimation and tolerance to high light in Arabidopsis thaliana.

While essential for photosynthesis, excess light can damage plants. We investigated how growth light conditions affect two photoprotective strategies, chloroplast movement and nonphotochemical quenching (NPQ), as well as electron transport rates (ETR), and the relative importance of these processes in the short-term stress tolerance of Arabidopsis thaliana. We grew wild-type (WT) and mutant plants with impaired chloroplast movement (phot1, phot2, phot1 phot2, chup1) or NPQ (npq1) at low (160 μmol photons m-2 s-1 ) or intermediate light (400 μmol photons m-2 s-1 ) before quantifying transmission changes due to chloroplast movement, NPQ, ETR, and the ability to recover from a short-term high-light treatment. Plants with impaired chloroplast avoidance movement (phot2, phot1 phot2, chup1) did not recover as well from a short-term high light treatment as the WT or npq1 and phot1 mutants. Plants grown at intermediate light recovered more completely from the same stress treatment regardless of their genotype and despite reduced degrees of transmission changes due to chloroplast movement. This result was due in part to all genotypes having up to a 2-fold increase in ETRmax and a slight increase in NPQmax . Growth light conditions affect which mechanisms are important in dealing with short-term high-light stress. The chloroplast avoidance response is important for low-light-grown plants, while increases in ETRmax and NPQmax allow plants grown at intermediate light intensities to avoid being damaged.

UV-B Induces Chloroplast Movements in a Phototropin-Dependent Manner.

We examined the impact of UV-B irradiation on chloroplast movements in Arabidopsis leaves. Directional chloroplast movements induced by blue light have been described in multiple plant species. In weak light, chloroplasts accumulate at periclinal cell walls to increase light capture. In strong light, chloroplasts exhibit the avoidance response, as they move towards anticlinal walls to protect the photosynthetic apparatus from light-induced damage. In Arabidopsis, chloroplast movements are triggered by phototropins, phot1 and phot2, which are known as blue/UV-A photoreceptors. We found that irradiation with UV-B of 3.3 µmol·m−2·s−1 induced chloroplast accumulation in wild-type plants. UV-B-triggered accumulation was dependent on the presence of phototropins, especially phot1, but not on UVR8 (the canonical UV-B photoreceptor). Irradiation with strong UV-B of 20 µmol·m−2·s−1 did not induce substantial chloroplast relocations in wild-type leaves. However, in the jac1 mutant, which is defective in chloroplast accumulation, strong UV-B elicited chloroplast avoidance. This indicated that UV-B can also activate signaling to the avoidance response. To assess the possibility of indirect effects of UV-B on chloroplast movements, we examined the impact of UV-B on the actin cytoskeleton, which serves as the motile system for chloroplast movements. While irradiation with UV-B of 3.3 µmol·m−2·s−1 did not affect the actin cytoskeleton, strong UV-B disrupted its structure as shown using an Arabidopsis line expressing Lifeact-green fluorescent protein (GFP). In wild-type plants, pretreatment with strong UV-B attenuated chloroplast responses triggered by subsequent blue light irradiation, further indicating that this UV-B intensity also indirectly affects chloroplast movements. Taken together, our results suggest that the effect of UV-B on chloroplast movement is twofold: it directly induces phototropin-mediated movements; however, at higher intensities, it attenuates the movements in a nonspecific manner.

Light stress-induced chloroplast movement and midday depression of photosynthesis in sorghum leaves

ABSTRACTPlants are exposed to high light intensity, high leaf temperatures and high air-to-leaf water vapor pressure deficit (ALVPD) during the day. These environmental stresses cause stomatal closure and photoinhibitory damage, leading to midday depression of photosynthesis. Chloroplast positioning is essential for the efficient operation of photosynthesis. However, chloroplast behavior before, during, and even after the midday depression of photosynthesis remains unknown. We investigated changes in the intracellular positioning of chloroplasts and photosynthetic traits under a diurnal pattern of light. Sorghum leaves were exposed to a 12-h regime of light mimicking the natural light environment, with constant leaf temperature and ALVPD. Net photosynthetic rate (Pn) showed a diurnal pattern, and midday depression in Pn was observed at 3.8 h of irradiation. Depression in Pn was attributed to stomatal limitation because the decrease in Pn was in accordance with the decrease in stomatal conductance. The maximum efficiency of photosystem II decreased with the increase in light intensity and remained low after 12 h of irradiation. Bundle sheath chloroplasts swelled after 8 h of irradiation, representing the accumulation of starch. Conversely, mesophyll chloroplasts exhibited avoidance response after 4 h of irradiation, and the avoidance position was maintained during the remainder of the daytime. These data suggest that chloroplasts are subject to light stress during and after the midday depression of photosynthesis. The intensity of natural light is excessive for most of the day and this light stress induces chloroplast avoidance response and depression of photosynthesis.

Chlorophyll fluorescence parameters to assess utilization of excitation energy in photosystem II independently of changes in leaf absorption

Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 μmol m−2 s−1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73–81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.

Phototropins Mediate Chloroplast Movement in Phalaenopsis aphrodite (Moth Orchid).

Chloroplast movement is important for plants to avoid photodamage and to perform efficient photosynthesis. Phototropins are blue light receptors in plants that function in chloroplast movement, phototropism, stomatal opening, and they also affect plant growth and development. In this study, full-length cDNAs of two PHOTOTROPIN genes, PaPHOT1 and PaPHOT2, were cloned from a moth orchid Phalaenopsis aphrodite, and their functions in chloroplast movement were investigated. Phylogenetic analysis showed that PaPHOT1 and PaPHOT2 orthologs were highly similar to PHOT1 and PHOT2 of the close relative Phalaenopsis equestris, respectively, and clustered with monocots PHOT1 and PHOT2 orthologs, respectively. Phalaenopsis aphrodite expressed a moderate level of PaPHOT1 under low blue light of 5 μmol�m-2�s-1 (BL5) and a high levels of PaPHOT1 at >BL100. However, PaPHOT2 was expressed at low levels at <BL50 but expressed at high levels at > BL100. Analysis of light-induced chloroplast movements using the SPAD method indicated that orchid accumulated chloroplasts at <BL10. The chloroplast avoidance response was detectable at >BL25 and significant chloroplast avoidance movement was observed at >BL100. Virus-induced gene silencing of PaPHOTs in orchids showed decreased gene expression of PaPHOTs and reduced both chloroplast accumulation and avoidance responses. Heterologous expression of PaPHOT1 in Arabidopsis phot1phot2 double mutant recovered chloroplast accumulation response at BL5, but neither PaPHOT1 nor PaPHOT2 was able to restore mutant chloroplast avoidance at BL100. Overall, this study showed that phototropins mediate chloroplast movement in Phalaenopsis orchid is blue light-dependent but their function is slightly different from Arabidopsis which might be due to gene evolution.

Functional characterization of GhPHOT2 in chloroplast avoidance of Gossypium hirsutum

Chloroplast movement mediated by the plant-specific phototropin blue light photoreceptors is crucial for plants to cope with fluctuating light conditions. While chloroplasts accumulate at weak light-illuminated areas, chloroplast avoidance response mediated primarily by the phototropin2 (phot2) receptor is induced by strong light illumination. Although extensive studies have been performed on phot2-mediated chloroplast avoidance in the model plant Arabidopsis, little is known on the role of the corresponding PHOT2 orthologs in chloroplast movement in cotton. In this study, we found that chloroplast avoidance movement also occurs in the tetraploid G. hirsutum and two diploid species, G. arboreum and G. raimondii, albeit with distinct features. Further bioinformatics and genetic analysis identified the cotton PHOT2 ortholog, GhPHOT2-1, which retained a conserved role in plant chloroplast avoidance movement under strong blue light. Ghphot2-1was localized in the plasma membrane and formed aggregates after high blue light irradiation. Constitutive expression of GhPHOT2-1 restored chloroplast avoidance and accumulation response, as well as phototropism, and leaf flattening characteristics of the Arabidopsis phot2 or phot1 phot2 mutants. On the contrary, silencing of GhPHOT2-1 by virus-induced gene silencing (VIGS) disrupted high blue light-induced chloroplast avoidance movement and caused photo damage in cotton leaves. Taken together, these findings demonstrated that GhPHOT2-1 is a conserved PHOT2 ortholog in regulating chloroplast avoidance and the other aforementioned phot2-mediated responses, implicating its potential role for improving high light tolerance in cotton cultivars.